Quantification of Phytochemicals from Commercial Spirulina ...
14
Research Article Quantification of Phytochemicals from Commercial Spirulina Products and Their Antioxidant Activities Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia Correspondence should be addressed to Naif Abdullah Al-Dhabi; [email protected] and Mariadhas Valan Arasu; [email protected] Received 21 July 2015; Revised 20 November 2015; Accepted 25 November 2015 Academic Editor: G. K. Jayaprakasha Copyright © 2016 N. A. Al-Dhabi and M. Valan Arasu. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e present study aimed to profile the polyunsaturated fatty acids, sugars, free amino acids, and polyphenols in 37 varieties of Spirulina commonly available in the market using gas chromatography and high performance liquid chromatography. In addition, the biological potentials of the Spirulina samples were evaluated by analysing the in vitro antioxidant activities using various analytical techniques. e analyses revealed the presence of 13 polyunsaturated fatty acids, 18 amino acids, 7 sugars, and polyphenols. e polyunsaturated fatty acids contents were varied between Spirulina samples. e total polyunsaturated fatty acids amount was 4.25 mg/100 g, and the average among of sapienic acid detected was 2.25 mg/100 g, which was followed by linoleic acid (16.7%) and -linolenic acid (14%). Among the 7 sugars, the hexose levels were the highest (73.85%). e total amino acids contents ranged from 11.49 to 56.14 mg/100 g, and the individual essential amino acids accounted for 17% to 39.18%. e “natural” tablets exhibited the highest polyphenols levels (24 mg/g). All of the Spirulina samples expressed dose-dependent antioxidant activities. e polyunsaturated fatty acids, sugars, free amino acids, and polyphenols contents varied widely, and the variations in these compounds between the Spirulina samples were significant. 1. Introduction Cyanobacteria also include unicellular organisms and all of them are not spiral shaped (Spirulina is spiral shaped). ey grow naturally in the water of warm climates and are primarily cultivated in ponds and small lakes. In contrast to other living organisms, microalgae or Spirulina is one of the microalgae and is not the only microalga which do not require organic, inorganic, nutrient, and other carbon sources for growth and can survive in higher alkaline pHs and in greater bicarbonate and carbonate concentrations. Beginning in the sixteenth century, cyanobacteria have been used as a traditional food source for African and Mexican people. Among the microalgae, Spirulina maxima (Arthrospira max- ima), Spirulina platensis (Arthrospira platensis), and Spirulina fusiformis (Arthrospira fusiformis) are the most widely culti- vated species around the world and are widely used as health foods, food additives, and potential sources of high value chemicals and pharmaceutical metabolites [1]. Each year, more than three thousand tons of Spirulina are cultivated around the world for human nutrition and the production of other fine commodity chemicals [2]. Several similar studies with market samples of Spirulina have been carried out earlier and data are available [3]. In recent years, people have been interested in consuming Spirulina in tablet and powder forms due to its relatively high contents of protein (58%), carbohydrates (30%), fat (8%), dietary fibres (3%), sugars (3%), vitamins (<1%), and phytochemicals [4, 5]. Spirulina contains fatty acids such as linoleic acid, docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid, and stearidonic acid, respectively. Spirulina also contains moderate amounts of vitamins such as vitamin A, vitamin C, vitamin E, vitamin B12, thiamine, nicotinamide, pyridoxine, riboflavin, and folic acid and beneficial pigments, such as chlorophyll-a, zeax- anthin, diatoxanthin, 3 -hydroxyechinenone, echinenone, beta-carotene, xanthophyll, canthaxanthin, c-phycocyanin, Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2016, Article ID 7631864, 13 pages http://dx.doi.org/10.1155/2016/7631864
Transcript of Quantification of Phytochemicals from Commercial Spirulina ...
Research ArticleQuantification of Phytochemicals from Commercial SpirulinaProducts and Their Antioxidant Activities
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu
Department of Botany and Microbiology Addiriyah Chair for Environmental Studies College of ScienceKing Saud University PO Box 2455 Riyadh 11451 Saudi Arabia
Correspondence should be addressed to Naif Abdullah Al-Dhabi naldhabiksuedusaand Mariadhas Valan Arasu mvalanarasugmailcom
Received 21 July 2015 Revised 20 November 2015 Accepted 25 November 2015
Academic Editor G K Jayaprakasha
Copyright copy 2016 N A Al-Dhabi and M Valan Arasu This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
The present study aimed to profile the polyunsaturated fatty acids sugars free amino acids and polyphenols in 37 varieties ofSpirulina commonly available in the market using gas chromatography and high performance liquid chromatography In additionthe biological potentials of the Spirulina samples were evaluated by analysing the in vitro antioxidant activities using variousanalytical techniquesThe analyses revealed the presence of 13 polyunsaturated fatty acids 18 amino acids 7 sugars and polyphenolsThe polyunsaturated fatty acids contents were varied between Spirulina samples The total polyunsaturated fatty acids amount was425mg100 g and the average among of sapienic acid detected was 225mg100 g which was followed by linoleic acid (167)and 120574-linolenic acid (14) Among the 7 sugars the hexose levels were the highest (7385) The total amino acids contentsranged from 1149 to 5614mg100 g and the individual essential amino acids accounted for 17 to 3918 The ldquonaturalrdquo tabletsexhibited the highest polyphenols levels (24mgg) All of the Spirulina samples expressed dose-dependent antioxidant activitiesThepolyunsaturated fatty acids sugars free amino acids and polyphenols contents variedwidely and the variations in these compoundsbetween the Spirulina samples were significant
1 Introduction
Cyanobacteria also include unicellular organisms and allof them are not spiral shaped (Spirulina is spiral shaped)They grow naturally in the water of warm climates and areprimarily cultivated in ponds and small lakes In contrastto other living organisms microalgae or Spirulina is one ofthe microalgae and is not the only microalga which do notrequire organic inorganic nutrient and other carbon sourcesfor growth and can survive in higher alkaline pHs and ingreater bicarbonate and carbonate concentrations Beginningin the sixteenth century cyanobacteria have been used asa traditional food source for African and Mexican peopleAmong the microalgae Spirulina maxima (Arthrospira max-ima) Spirulina platensis (Arthrospira platensis) and Spirulinafusiformis (Arthrospira fusiformis) are the most widely culti-vated species around the world and are widely used as healthfoods food additives and potential sources of high value
chemicals and pharmaceutical metabolites [1] Each yearmore than three thousand tons of Spirulina are cultivatedaround the world for human nutrition and the production ofother fine commodity chemicals [2] Several similar studieswith market samples of Spirulina have been carried outearlier and data are available [3] In recent years people havebeen interested in consuming Spirulina in tablet and powderforms due to its relatively high contents of protein (58)carbohydrates (30) fat (8) dietary fibres (3) sugars(3) vitamins (lt1) and phytochemicals [4 5] Spirulinacontains fatty acids such as linoleic acid docosahexaenoicacid eicosapentaenoic acid arachidonic acid and stearidonicacid respectively Spirulina also contains moderate amountsof vitamins such as vitamin A vitamin C vitamin E vitaminB12 thiamine nicotinamide pyridoxine riboflavin and folicacid and beneficial pigments such as chlorophyll-a zeax-anthin diatoxanthin 31015840-hydroxyechinenone echinenonebeta-carotene xanthophyll canthaxanthin c-phycocyanin
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 7631864 13 pageshttpdxdoiorg10115520167631864
2 Evidence-Based Complementary and Alternative Medicine
beta-cryptoxanthin myxoxanthophyll oscillaxanthin phy-cobiliproteins and allophycocyanin [5 6] However thenutritional contents of Spirulina depend on the cultivationconditions and the processing methods The nutritionalcomponents and other phytochemicals in Spirulina primarilyexhibit anti-inflammatory antioxidant antidiabetic neuro-protective hepatoprotective and anticancer activities [7]The regular consumption of Spirulina ameliorates the symp-toms of premenstrual cycles in women and the symptomsof amyotrophic lateral sclerosis Spirulina prevents allergicreactions and aids in the removal of metals from the body Arecent study suggested that Spirulinahelps to bind radioactiveelements and is useful for protecting the human body fromexposure to radiation therapy The phenolic compoundspresent in the Spirulina are primarily involved in the redoxmechanism and act as hydrogen donors reducing agentsmetal chelator singlets and oxygen quenchers [8] Thereforephenolic compounds can prevent the formation of ROSand reactive nitrogen species which include free radicalssuch as hydroxyl and superoxide anions and nitric oxideand nonfree radical species such as hydrogen peroxide andnitrous acid The development of phenolic compounds asantioxidants for the treatment of various human diseaseshas increased Therefore there is an urgent need to identifynovel antioxidant molecules with fewer side effects and sig-nificant hepatoprotective effects [9] To overcome disordersthe regular consumption of natural health-promoting foodssuch as Spirulina tablets or powders is advised In vitrostudies demonstrated that the Spirulina and Nestoc specieshave several therapeutic properties due to their ability toscavenge superoxide and hydroxyl radicals and inhibit lipidperoxidation [10 11] Therefore the present study aimed toinvestigate the metabolite profiles and antioxidant propertiesof 37 commercially available Spirulina samples
2 Materials and Methods
21 Chemicals and Solvents Standard methyl esters offatty acids were obtained from Supelco (37 ComponentFAME Mix) Triglyceride (IS C110 triundecanoin) waspurchased from Nu-Chek Prep (Elysian MN USA) BF3-methanol (10 ww) was procured from Supelco (BellefontePA USA) Analytical grade diethyl ether (DE) pyrogal-lol petroleum ether (PE) chloroform and ethanol werepurchased from Sigma-Aldrich Chemical Co (St LouisMo USA) Thirty-seven Spirulina samples with differentcountries of origin in the forms of tablets and capsules wereprocured from specialist shops (Table 1) All the studiedSpirulina samples were procured from different markets inthe world The details of the samples were mentioned inTable 1
22 Extraction of Lipids from the Spirulina Samples The totallipids in the Spirulina samples were extracted according tothe following modified method of Mossoba et al (2003)[12] Briefly one gram of finely powdered sample with 2mLpyrogallol solution (in ethanol 95 50mgmL) and 1mLtriglyceride internal standard solution (IS C110 triunde-canoin 5mgmL in iso-octane) was transferred into a 50mL
tube After proper mixing 100mL 83MHCl was added intothe tube which was then incubated in a shaking water bathat 70ndash80∘C for 2 h During the incubation the contents of thetubes were intermittently mixed to release the fat from thewalls of the tubes After incubation the samples were allowedto cool at room temperature and mixed with 15mL diethylether (DE) The DE layer was then separated and filteredin the column using Na
2SO4and petroleum ether (PE)
Subsequently the collected PE layers were slowly evaporatedusing a nitrogen streamandused for the extraction of the fattyacids
23 Extraction and Quantification of the Fatty Acids Thetotal lipids in the Spirulina were extracted according themethod of Mossoba et al (2003) with modifications [12]Briefly the extracted lipids were saponified with 05N NaOHin methanol (15mL) for 5min at 100∘C and cooled at roomtemperature After cooling the samples were treated with2mL of BF3-methanol and incubated at 100∘C for 10minand allowed to cool at room temperature The samples werethen thoroughly vortexed with 2mL of isooctane and 1mL ofsaturated NaCl solution for 10min Next the upper isooctanelayer was carefully transferred into tubes and injected intoa Hewlett-Packard 6890 series gas chromatograph (GC)equipped with an autoinjector and a flame-ionization detec-tor (Agilent Technologies Little Falls Del USA) The fattyacids were separated in a fused-silica capillary column (SP-2560 100m times 025 nm times 02 120583m film thickness SupelcoUSA) The GC oven was heated to 100∘C and held for 4minand then further increased to 240∘C at a rate of 3∘Cminand held at 240∘C for 15min The injector and detectortemperatures were set at 225∘C and 285∘C respectively Themobile gas (helium) applied at a flow rate of 075mLminTheconcentrations of the individual fatty acids were calculatedbased on the relative retention times of the standardmixturesThe conversion of FAMEs to corresponding fatty acids areshown in Table 2
The response factor (119877119894) of each fatty acid was calculated
as follows
119877119894=119875119904119894
119875119904C110times119882C110119882119894
(1)
where 119875119904119894is peak area of individual fatty acid in mixed
FAMEs standard solution 119875119904C110 is peak area of C110 fattyacid in mixed FAMEs standard solution119882C110 is weight ofinternal standard in mixed FAMEs standard solution and119882119894is weight of individual FAME in mixed FAMEs standard
solutionThe amounts of the individual compounds in the test
samples were calculated as follows
119882FAME119894 =119875119905119894times119882119905C110 times 10067
119875119905C110 times 119877119894 (2)
where 119875119905119894is peak area of the fatty acid 119894 in the test portion
119882119905C110 is weight of C110 in the internal standard added totest portion g 10067 is conversion of the internal standardfrom triglyceride to FAME and 119875119905C110 is peak area of C110in the internal standards in the test portion
Evidence-Based Complementary and Alternative Medicine 3
Table1Spiru
linaprod
uctslistsandtheirc
ountry
oforigin
Snu
mber
Prod
ucttype
Manufacturin
gcompany
Cou
ntry
oforigin
Web
address
1Tablets
TAAU
Australia
PvtL
tdNT
Australia
httpwwwaustralianspirulin
acom
au
2Ca
psules
GeneralNutritionCorpP
ittsburgh
USA
httpwwwgn
ccom
3
Capsules
Naturersquos
Way
Prod
ucts
Inc
Sprin
gvilleUtah
USA
httpwwwnaturesw
ayco
m4
Tablets
Goo
dlsquoN
NaturalN
ewYo
rkUSA
httpwwwgood
andn
aturalsto
reco
m5
Tablets
Now
Food
sBloo
mingdale
USA
httpwwwno
wfood
scom
6Tablets
NatureP
ureInc
LarkspurC
alifo
rnia
USA
7Tablets
Source
NaturalsInc
SantaC
ruzCa
lifornia
USA
httpwwwsourcenaturalscom
8Tablets
Jarrow
Form
ulasL
osAngele
sCA
USA
httpwwwjarrow
com
9Tablets
Earthrise
NutritionalsLL
CIrvineC
AUSA
httpearthrise
com
10Tablets
NutrexHaw
aiiIncKa
ilua-Ko
naH
awaii
USA
httpwwwnu
trex-hawaiicom
11Ca
psules
Pure
PlanetProd
ucts
Inc
Long
BeachCA
USA
httpsw
wwpu
replanetco
m12
Tablets
PuritanrsquosPrideInc
Oakdale
New
York
USA
httpwwwpu
ritanco
m13
Capsules
21stCentury
Health
CareIncAriz
ona
USA
httpwww21stc
enturyvitaminsc
om14
Tablets
JapanAlgae
CoLtdTo
kyo
Japan
httpwwwsp100com
15Tablets
AllSeason
sHealth
Ham
pshire
UnitedKingdo
mhttpwwwcareho
mec
ouk
16
Capsules
FushiW
ellbeing
LtdL
ondo
nUnitedKingdo
mhttpwwwfushicouk
17Tablets
BioveaL
ondo
nUnitedKingdo
mhttpwwwbioveaco
m18
Capsules
ParryNutraceuticalsCh
ennai
India
httpwwwparrynutraceuticalsc
om19
Tablets
Lifestream
InternationalLtdNorthcoteA
uckland
New
Zealand
httpwwwlifestre
amco
nz
20Tablets
Green
Health
Auckland
New
Zealand
httpwwwgreenh
ealth
conz
21Tablets
RBCLifeSciencesIncBu
rnabyBritish
Colum
bia(
BC)
Canada
httpwwwrbclifesciencesco
m22
Tablets
Swiss
HerbalR
emediesL
tdRichmon
dHillO
ntario
Canada
httpwwwsw
issnaturalcom
23Ca
psules
HerbalSele
ctG
uelphOntario
Canada
httpwwwherbalselectca
24Ca
psules
Gou
rmetNutritionFB
IncST
E-Julie
(Quebec)
Canada
httpwwwgourmetfbco
m25
Capsules
TerraV
itaFine
Who
leHerbsB
rampton
Ontario
Canada
httpdsldnlm
nihgov
26Ca
psules
DXN
marketin
gMalaysia
httpwwwdx
nmalaysia
com
27Ca
psules
Hydrolin
aBiotech
PvtIndia
India
httpwwwhydrolinabiotechco
m28
Capsules
Prim
eHealth
Labo
ratorie
sLtd
Austr
alia
Not
available
29Tablets
LaurelHerbalprodu
cts
India
Not
available
30Tablets
IMOCon
trolP
vtLtd
India
httpwwwim
ocon
trolin
31Ca
psules
Acum
enPh
armaceutic
alsP
vtIndia
httpwwwacum
enph
armco
m32
Capsules
Bio-LifeOrganicSpiru
lina
Malaysia
httpshealth
review
2uwordp
ressco
m20100915
biolifespirulin
a33
Tablets
Dharain
Pharmaceutic
als
India
httpwwwmihnaticom
34Tablets
21stCentury
Health
CareIncAriz
ona
USA
httpw
ww21stc
enturyvitaminsc
om
35Tablets
Zuellig
BharmaS
DWB
HD
Malaysia
httpwwwzuelligph
armac
om36
Tablets
Kordels
Spiru
lina
India
httpwwwraku
tencom
37Tablets
ElkenCh
ewable
Malaysia
httpselken4m
rtwordp
ressco
m
4 Evidence-Based Complementary and Alternative Medicine
Table 2 Fatty acids contents (mg100 g in different Spirulina)
Sample
FA
Total1400 1600 161 180 181t 181(n-9)c 181(n-7)c 182t 182(n-6)c 2000 183(n-6) 2002 203n6119891FA119894
09421 09481 09477 09530 09527 09524 095201 09517 09524 09570 0952 09565 095571 001 188 022 029 000 009 002 001 065 001 067 001 001 252 000 207 010 009 000 009 002 001 032 001 016 001 001 2363 001 197 011 034 001 011 005 004 075 001 067 000 000 2554 001 255 027 006 000 005 001 001 085 000 073 001 001 2945 001 189 017 030 001 007 002 001 062 000 053 000 001 2446 001 190 031 013 001 009 004 001 073 001 070 001 002 2457 001 238 017 079 000 006 002 001 055 001 046 001 001 3428 002 281 014 085 000 015 002 001 080 001 050 001 001 3989 001 200 018 011 000 014 007 001 065 001 040 001 001 24510 001 280 014 045 000 009 001 001 087 001 073 001 001 3511 001 210 023 014 000 016 006 001 060 001 037 001 001 26512 001 212 025 006 000 014 007 001 078 001 052 001 001 25913 001 236 027 040 000 011 002 001 080 001 082 001 001 31614 001 248 024 005 000 013 002 002 099 001 082 001 001 29215 001 178 028 004 000 014 001 001 062 001 058 000 000 22616 001 295 015 213 001 011 003 002 067 002 055 000 000 53717 002 191 010 006 001 015 003 003 070 001 057 000 001 22518 002 195 015 017 000 008 003 003 064 001 060 000 000 23819 002 204 012 016 001 014 003 003 068 001 051 000 001 24920 002 212 012 025 001 012 004 004 071 001 066 000 001 26421 001 181 026 021 001 005 002 001 065 001 062 001 001 23522 000 143 022 004 000 012 002 001 054 000 053 001 001 18123 001 327 014 111 000 018 007 001 076 002 044 001 001 47324 001 258 028 006 000 007 002 002 074 001 059 001 001 30125 001 249 026 006 000 010 003 001 072 001 058 001 001 29326 001 173 023 005 000 010 003 001 044 000 039 000 001 21227 002 217 025 006 001 012 006 001 075 001 060 001 001 26328 001 298 023 135 000 013 003 002 090 002 052 001 001 47229 001 158 011 007 000 017 004 001 053 001 049 001 001 19530 002 236 014 005 001 017 003 003 083 001 065 000 000 27531 001 188 015 005 000 019 003 001 054 000 040 001 001 22832 001 175 019 007 000 008 001 001 059 000 049 001 001 2133 001 229 033 006 000 010 003 001 078 001 070 001 001 2834 004 395 021 015 002 023 008 007 137 001 124 001 001 45935 002 210 031 008 000 016 012 001 055 001 060 002 001 26836 001 262 015 008 000 018 004 003 104 001 058 001 001 30537 002 214 014 005 001 011 004 004 077 000 077 000 001 246Numbers 1 to 37 were the sample names (Table 1) Total fatty acids were considered only for PUFA 1400 myristic acid 1600 sapienic acid 161 palmitoleicacid 180 stearic acid 181t elaidic acid 181(n-9)c oleic acid 181(n-7)c vaccenic acid 182t linolelaidic acid 182(n-6)c linoleic acid 2000 eicosenoic acid183(n-6) 120574-linolenic acid 2002 eicosadienoic acid and 203n6 dihomo-gamma-linolenic acid
The weight of the fatty acid (119882119894) was determined as
follows
119882119894= 119882FAME119894 times 119891FA119894 (3)
where 119891FA119894 is conversion factors for the conversion of theFAMEs to their corresponding fatty acids
24 Extraction and Quantification of Sugars Using HPLCThe carbohydrates present in the Spirulina samples werequantified according to the following the standard method[14] Briefly a 100mg portion of the powdered sample wasmixed with 10mL sterile distilled water and boiled at 100∘Cfor one hour After heating the debris was separated bycentrifugation at 10000 rpm for 10min Next the debris-free
Evidence-Based Complementary and Alternative Medicine 5
solution was mixed with 10 5 trichloroacetic acid (TCA)and incubated at room temperature for 10min Subsequentlythe samples were centrifuged at 10000 rpm for 10min andthen filtered through a 045120583mPTFE syringe filter for HPLCanalysis (HP1100 Agilent Co USA) The filtered sampleswere separated through a 300 times 78mm Aminex HPX-87H (Bio-Rad Hercules CA USA) column at 60∘C usinga 50mM H
2SO4monophasic solvent system with a flow
rate of 05mLmin and a column wavelength of 220 nmThe injection sling was 10 120583L The quantifications of theindividual sugars were based on the peak areas and calculatedas equivalents of standard compounds
25 Extraction and Quantification of the Free Amino AcidsUsing HPLC Individual free amino acids were extractedand quantified according to the method of Park et al(2014) with modifications [15] Briefly a 100mg portionof a fine powdered sample was mixed with 12mL of 5trichloroacetic acid (TCA) in a 2mL Eppendorf tube andvigorously shaken for 5minThe slurry sample was incubatedat room temperature for 60min and the upper layer wasthen separated by centrifugation The collected sampleswere diluted with 01M HCl and then filtered through a045 120583m PTFE syringe filter The filtrate was then analysedby HPLC (Agilent Technologies Palo Alto CA) The HPLCanalyses of free amino acids were conducted according to theldquorapid accurate sensitive and reproducible HPLC analysisof amino acids analysisrdquo method with Zorbax Eclipse-AAAcolumns using an Agilent 1100 HPLC system Briefly theseparation of the free amino acidswas performed on aZorbaxEclipse AAA analytical columnThe oven temperature of thecolumn was set at 40∘C and the detection wavelength wasset a 338 nm The injection volume was 10 120583L The mobilephase consisted of a mixture of 40mM NaH
2PO4(pH 78
solvent A) and solvent B (ACN MeOH and water at a45 45 10 vvv ratio) was passed at a rate of 20mLminThe HPLC separation parameters were as follows 0min0 B 0ndash19min 0 B 19ndash211min 57 B 211ndash216min100 B 216ndash25min 100 B 25ndash251min 0 B and 251ndash30min 0 B A sample with an amino acid content of50 pmoL120583L was used as the standard The quantifications ofthe different amino acids were based on the peak areas andwere calculated as equivalents of the standard compoundsAll contents are expressed as milligrams lowast gramfresh weight(FW)
26 Total Polyphenol Analysis The total polyphenols wereestimated according to the method of Folin-Ciocalteu (mod-ified from Lin and Tang 2006) [16] Briefly 10mg of thesample was dissolved in 1mL ofmethanol with 2mL of Folin-Denis reagent and 35 sodium carbonate (Na
2CO3) The
mixture was stored at room temperature for 30min Theabsorbancewasmeasuredwith anUV-Vis spectrophotometerat 750 nm The total polyphenols were calculated as gallicacid equivalents based on a calibration curve for gallic acid(0 25 50 and 100 120583gmL) using the following equation thatwas based on the calibration curve 119910 = minus09706119909 + 38935(1198772 = 09992)
27 In Vitro Antioxidant Assays271 Preparation of the Spirulina One gram of the finepowder sample was mixed with 5mL of ethanol in a screw-cap tube by vortexing for 5min and then kept in an orbitalshaker at 150 rpm for 24 h at room temperature for thoroughextraction After incubation the samples were centrifuged at13000 rpm for 15min at 4∘C The resulting supernatant wasvacuum evaporated at 30∘C and the resulting extract wasused for the antioxidant assays
272 Reducing Power Activity Assay The reducing powerassay was performed according to the method of Oyaizu(1986) [17] Volume of 100 120583L of various concentrations(20ndash100120583gmL) of the samples was mixed with phosphatebuffer (25mL) and 1 potassium ferricyanide (25mL) andincubated at 50∘C for 20 minutes After incubation 25mLof 10 trichloroacetic acid was added and the samples werecentrifuged at 3000 rpm for 10min The upper layer of thesolution (25mL) was mixed with distilled water (25mL)and a freshly prepared 01 ferric chloride solution (05mL)and measured at an absorbance at 700 nm The control wasprepared in a similar manner but the sample was excludedVitamin C at various concentrations was used as a standardIncreases in the absorbance of the reaction mixture indicatedincreases in reducing power
273 DPPH Radical Scavenging Activity Assay The DPPHradical scavenging assay performed according to the methodof Hatano et al (1988) [18] Briefly 100 120583L of the sampleand vitamin C (concentration 100ndash500120583gmL) was mixedwith 200120583L of freshly prepared DPPH solution (1mgmL inmethanol) and incubated at room temperature in the darkfor 30 minutes The controls included only deionized waterand the DPPH solution The absorbances of the resultingsolutions were measured in triplicate at 517 nm followingcentrifugation at 12000 rpm for 10min
The scavenging activity was calculated as follows
scavenging activity () = [1 minus(1198600minus 1198601)
1198602
] times 100 (4)
where1198600is absorbance of sample119860
1is absorbance of blank
and 1198602is absorbance of control
274 Hydroxyl Radical-Scavenging Activity Assay The hy-droxyl radical-scavenging assay was performed accordingto the method of Elizabeth and Rao (1990) with slightmodification [19]The reagents for the assay were freshly pre-pared Briefly one millilitre samples of the working solutionsthat consisted of different ratios of the extract were mixedwith 100mL of 28mM 2-deoxy-2-ribose in phosphate buffer(pH 74) EDTA (104mM 1 1 vv) 100mL H
2O2(1mM)
200mL of FeCl3(200mM) and 100mL ascorbic acid (1mM)
The resulting solutions were mixed evenly and the reactionmixtures were incubated at 37∘C for 1 h The degradation ofdeoxyribose was determined by reading the absorbance at532 nm against the blank solution using a microplate reader(BioRad) Vitamin C was used as a positive control The
6 Evidence-Based Complementary and Alternative Medicine
experiments were conducted in triplicate The scavengingactivities were calculated according to (4)
3 Results and Discussion
31 Variations in the Fatty Acid Contents The total lipidcontents were extracted from the Spirulina samples and theindividual fatty acid compositions of the samples were anal-ysedwith gas chromatography A gas chromatograph coupledwith a flame ionization detector guided the identificationof the following 10 unsaturated and fatty acids (sapienicacid palmitoleic acid elaidic acid oleic acid vaccenic acidlinolelaidic acid linoleic acid eicosenoic acid 120574-linolenicacid and dihomo-gamma-linolenic acid) and three saturatedand fatty acids (myristic acid stearic acid and eicosadienoicacid) (Figure 1 and Table 2) The individual PUFA contentswere quantified by comparing the standard fatty acids withtheir indices The PUFA contents in the Spirulina samplesranged from 301 (DXN Marketing capsules) to 741 g100 g(21st Century HealthCare Inc Arizona tablets Table 4)Interestingly there were comparatively lower amounts of 120574-linolenic acid in the studied Spirulina samples this acidaccounted for an average of 14 of the total PUFAs Theamounts of 120574-linolenic acid ranged from 016 g100 g (Gen-eral Nutrition Corp Pittsburgh capsules) to 124 g100 g(21st Century HealthCare Inc Arizona tablets) HoweverMuhling et al (2005) reported palmitic acid (C160) noted asthe dominant fatty acid in wild Spirulina samples [20] In ourstudy sapienic acid been noted as the major fatty acids Manyin vitro studies have confirmed that 120574-linolenic acid can beused to effectively lower cholesterol and treat atopic eczemabreast cancer and premenstrual disorder [21ndash25] RecentlySajilata et al (2008) extracted and purified S platensisactive components via lipid fractionation silica gel columnpurification and thin-layer chromatographic methods [26]It has been reported that 120572-linolenic acid and 120574-linolenic acidare required for the survival of animals and humans Patilet al (2007) [27] Patil et al (2007) profiled the individualPUFAs fromBacillariophyceae Cyanophyceae RhodophyceaeXanthophyceae Cryptophyceae Prymnesiophyceae Eustig-matophyceae and Chlorophyceae microalgae and suggestedthat the cultivation conditions particularly light intensityand other nutritional components exert important effectson the PUFA compositions [27] Many companies processmicroalgae and supply the results in the forms of capsulesand tables to the market Recently the interest in the use ofSpirulina tablets as energy foods has been renewed due to therelatively high contents of protein phytochemicals and othernutrients in such tablets Efforts should be made to analysethemetabolite profiles of the commercially available Spirulinaproducts because in a previous study we confirmed thattrace amounts of heavymetals that could cause serious healthproblems for consumers are present in some commerciallyavailable Spirulina samples (Table 3) [13]
32 Quantification of Individual Sugars by HPLC Hexose(ie glucose fructose galactose and rhamnose) pentose(ie xylose and ribose) and disaccharide sugars were
35
30
25
(pA
)
20
15
10
FID1 A (FC140730SIG10006D)
20 30 40 50 60 70 80
40
110
120
140
160
161
180
181
t18
1(n
-9)c
181
(n-7
)c18
2t
182
(n-6
)c18
3t
183
(n-6
)c18
3(n
-3)
202
203
n620
4(n
-6)
200
40
1100
11000
1111
120
140
00016
000016
0016
06
006
0006
016
06
016
0061666666666611111111
161
180
181
t18
1(n
-9)c
181
(n1(18
1(n(
181
(nn1(nn
181
(n1(n
181
(n1(n
1(n
181
(n18
1(n
181
(n((1(((((1(18
1(18
1118
111818111111111111
-7)c18
18
182
t111111111
182
(nnn(nn18
2(nnnnnn((((
182(
-6)c)6)6)6)))6))666666666666666666666666
183
t1111111111
183
(nnn18
3(n((((
183((
183(
-6)c6))6)6)))6)6)6))6)6)666666666666666
(18
3(n
88888818818888888111)
-3)
202
20202000020020202002222222222222222222222222 203
n620
4(n
-6)
200
200
200
200
222222222222
Figure 1 Gas chromatograms of the fatty acids identified in theSpirulina samples The peaks numbers refer to the individual fattyacids listed in Table 2
extracted from the 37 Spirulina samples and clearly base-line eluted by HPLC The quantitative results revealed thatglucose fructose and sucrose were present in the greatestamounts followed by xylose ribose galactose and rhamnoseThe total sugar contents of the Spirulina samples ranged from309 to 122167mg100 g (Table 4) Together glucose fructosegalactose and rhamnose accounted for an average of 7385of the total sugar contents Among the major individualsugars glucose accounted for an average of 351mg100 g and52 of the total sugar contents Similarly Chaiklahan et al(2013) reported that rhamnose and glucose account for 53and 13 of the total sugars respectively [28] The rhamnosecontents varied from 8 to 58mg100 g of the total sugarsaccounting for an average of 65 [29] The results indicatedthat among the pentose sugars xylose (average 908) andribose (average 475) were the major components in theSpirulina samples The final outcome of this study is thatthe variations in the individual sugar contents between theSpirulina samples were acceptable due to the processing con-ditions of the each commercial Spirulina product Moreovera literature stated that the extraction of total polysaccharidesand other monosaccharides from Spirulina followed by thequantification of the individual sugar molecules identifiedrhamnose as the predominant sugar followed by glucose andfructose [29]
33 Quantifications of the Individual Amino Acids by HPLCHPLC analyses were used to quantify 22 free amino acidsincluding aspartate asparagine serine glutamine histidineglycine threonine arginine alanine 120574-aminobutyric acid(GABA) tyrosine valine cystine methionine tryptophanphenylalanine isoleucine leucine and lysine in the Spirulinasamples but the separation profiles revealed that only 18 freeamino acids were detectable in the samples but the otherfree amino acids did not detected which may be not presentin the samples (Table 5 and Figure 2) Since the sampleswere marketed by the trademark of different companies andthe nutrient profile of each sample would be varied theseresults revealed that the amounts of total free amino acidsin the 37 Spirulina samples ranged from 1149mg100 g to5614mg100 g The essential amino acid content averagesranged from 206 to 3172mg100 g and contributed averagesthat ranged from 170 to 3918 of the total amino acidsAmong the essential amino acids leucine was identified
Evidence-Based Complementary and Alternative Medicine 7
Table 3 Content of heavy metals in the Spirulina samples available in the market
Sample name Amount mgKg dry weightNickel Zinc Mercury Platinum Magnesium Manganese Total
1 0211 0533 0002 0001 0002 0076 08252 4672 5627 0028 001 003 0587 109543 2016 2397 0017 0008 0018 0603 50594 2147 1628 002 0011 0026 0436 42685 2199 1235 0017 0008 0014 117 46436 3726 6225 0022 0009 0028 0007 100177 2601 2817 0018 0011 0028 0007 54828 3577 1871 0023 0012 0031 0309 58239 3519 3267 0021 0011 003 0008 685610 2442 2041 0017 0008 0021 0137 466611 3785 3007 0019 0008 0028 0008 685512 3597 2859 0019 0008 0024 0006 651313 2857 2901 0019 0008 0026 1643 745414 2852 2114 0026 0008 0042 0011 505315 2437 1568 002 0008 0028 0008 406916 2712 2434 0019 0009 0033 1777 698417 2948 2513 0021 0009 0028 18 731918 2633 1876 0016 0009 0023 1328 588519 3731 3184 0017 0009 0036 2248 922520 2225 1548 0008 0009 0019 1132 494121 1618 1478 0014 0006 002 0005 314122 1589 4626 0016 0007 0023 0009 62723 3272 4428 002 0008 0018 0008 775424 3558 3733 0017 0009 0034 1433 878425 2319 2586 0017 0008 0024 0006 496Numbers 1 to 37 were the sample names (Table 1) Samples 26ndash37 were not analyzed [13]
1
10
2
34 56
7
89 11
1213
14
1516
17 181920 21
22
Inte
nsity
(mAU
)
0
05
1
15
2
0 2 4 6 8Retention time (min)
10 12 14 16 18
Figure 2 HPLC chromatogram of the standard free amino acidsThe peaks numbers refer to the free amino acids listed in Table 5Peaks numbers 1 aspartate 2 glutamate 3 asparagine 4 serine 5 S-methylmethionine (vitamin U) 6 glutamine 7 histidine 8 glycine9 threonine 10 arginine 11 alanine 12 gamma-aminobutyricacid (GABA) 13 tyrosine 14 cystine 15 valine 16 methionine17 norvaline 18 tryptophan 19 phenylalanine 20 isoleucine 21leucine 22 lysine
as predominant (053 to 759mg100 g) and accounted formore than 30 of the essential amino acids The Spirulinatablets marketed in India under the brand name ldquoDharainPharmaceuticalsrdquo exhibited the greatest essential amino acid
contents However the brand name products exhibited valuesthat were comparatively lower than the maximum observedvalues Vitamin U methionine norvaline and tryptophanwere not observed in the samples The essential aminoacid compositions of the microalgae were very similar tothe reported protein contents [30] Clement et al (1967)determined the individual and total amino acid contentsin S maxima [31] This study reported that aspartate wasdominant in the Spirulina samples and that histidine cystinetryptophan and methionine were observed at the lowestlevels This report found a level of aspartate that was similarto that of another report In general many companies marketSpirulina samples as nutraceutical food however there is anurgent need to know the nutritional compositions of each ofthese Spirulina products [32] This study confirmed that theamino acid compositions of Spirulina samples varied with thecompanies that produced them Therefore amino acid-richsamples should be consumed by humans to maintain theirhealth
34 Determination of the Total Phenolic Compounds Figure 3shows the total phenolic compounds calculated as equivalentsto gallic acidThe results revealed that the distributions of thetotal phenolic compounds varied between the commercialproducts The products ranged from 24mgg (21st Century
8 Evidence-Based Complementary and Alternative Medicine
Table 4 Individual sugar contents (mg100 g) in different Spirulina
Sample Sugar contents (mg100 g)Glucose Fructose Xylose Galactose Ribose Sucrose Rhamnose Total
1 172 plusmn 14 38 plusmn 345 25 plusmn 150 155 plusmn 056 18 plusmn 125 54 plusmn 56 25 plusmn 00 3425 plusmn 4332 71 plusmn 1389 44 plusmn 15 88 plusmn 422 78 plusmn 312 5 plusmn 000 65 plusmn 024 18 plusmn 023 363 plusmn 5193 22933 plusmn 2759 59 plusmn 505 55 plusmn 160 7 plusmn 071 45 plusmn 300 54 plusmn 345 35 plusmn 089 46611 plusmn 15774 5133 plusmn 416 68 plusmn 501 21 plusmn 111 54 plusmn 201 7 plusmn 003 29 plusmn 203 19 plusmn 11 24977 plusmn 0385 34733 plusmn 643 15 plusmn 124 46 plusmn 34 8 plusmn 023 25 plusmn 025 84 plusmn 00 58 plusmn 32 58111 plusmn 1926 26 plusmn 346 26 plusmn 28 32 plusmn 1 9 plusmn 017 16 plusmn 000 72 plusmn 130 45 plusmn 1 222 plusmn 3467 14833 plusmn 1041 34 plusmn 178 37 plusmn 403 45 plusmn 312 18 plusmn 187 80 plusmn 915 25 plusmn 028 36644 plusmn 18088 15633 plusmn 1629 15 plusmn 121 39 plusmn 545 11 plusmn 102 31 plusmn 164 45 plusmn 745 14 plusmn 002 33311 plusmn 18869 253 plusmn 520 29 plusmn 598 65 plusmn 84 24 plusmn 20 22 plusmn 003 62 plusmn 605 35 plusmn 096 46333 plusmn 230910 25533 plusmn 1002 78 plusmn 74 98 plusmn 12 NA NA 34 plusmn 510 NA 52844 plusmn 146211 12833 plusmn 289 51 plusmn 089 45 plusmn 71 23 plusmn 185 24 plusmn 007 86 plusmn 688 18 plusmn 112 37711 plusmn 15412 36166 plusmn 66 49 plusmn 104 25 plusmn 090 14 plusmn 22 16 plusmn 008 95 plusmn 00 8 plusmn 032 58955 plusmn 18013 48233 plusmn 58 35 plusmn 208 45 plusmn 503 18 plusmn 103 54 plusmn 109 NA 11 plusmn 078 73077 plusmn 13414 59233 plusmn 615 26 plusmn 10 85 plusmn 523 44 plusmn 29 22 plusmn 107 29 plusmn 215 16 plusmn 047 81144 plusmn 24915 24866 plusmn 8 59 plusmn 68 48 plusmn 49 11 plusmn 22 15 plusmn 03 67 plusmn 168 24 plusmn 49 47089 plusmn 15416 21033 plusmn 95 72 plusmn 56 95 plusmn 012 10 plusmn 062 24 plusmn 13 49 plusmn 90 17 plusmn 063 47244 plusmn 42317 517 plusmn 24 93 plusmn 405 25 plusmn 52 12 plusmn 062 26 plusmn 42 52 plusmn 051 28 plusmn 103 75433 plusmn 11518 88466 plusmn 265 15 plusmn 025 22 plusmn 07 18 plusmn 215 28 plusmn 412 58 plusmn 152 38 plusmn 108 105989 plusmn 32719 33833 plusmn 125 44 plusmn 114 14 plusmn 09 27 plusmn 312 11 plusmn 000 49 plusmn 302 26 plusmn 00 50977 plusmn 03820 752 plusmn 526 51 plusmn 46 16 plusmn 012 9 plusmn 025 22 plusmn 08 67 plusmn 307 45 plusmn 28 98733 plusmn 219321 96866 plusmn 273 50 plusmn 254 18 plusmn 059 58 plusmn 812 24 plusmn 014 84 plusmn 101 19 plusmn 51 120722 plusmn 125222 260 plusmn 229 43 plusmn 63 94 plusmn 126 65 plusmn 474 26 plusmn 295 55 plusmn 078 54 plusmn 290 599 plusmn 17323 10733 plusmn 709 24 plusmn 40 25 plusmn 045 22 plusmn 012 25 plusmn 005 78 plusmn 184 28 plusmn 105 30311 plusmn 53824 15166 plusmn 76 52 plusmn 265 63 plusmn 040 45 plusmn 294 11 plusmn 032 37 plusmn 000 47 plusmn 001 40222 plusmn 38525 10266 plusmn 16 68 plusmn 43 24 plusmn 145 48 plusmn 2 7 plusmn 005 81 plusmn 028 35 plusmn 025 36189 plusmn 32726 7933 plusmn 1115 94 plusmn 50 25 plusmn 209 56 plusmn 623 45 plusmn 110 55 plusmn 612 45 plusmn 17 39977 plusmn 03827 60466 plusmn 53 12 plusmn 137 26 plusmn 423 44 plusmn 256 24 plusmn 000 84 plusmn 071 37 plusmn 032 83055 plusmn 09628 90166 plusmn 76 15 plusmn 07 45 plusmn 23 25 plusmn 045 10 plusmn 007 56 plusmn 183 34 plusmn 000 109555 plusmn 7629 798 plusmn 1311 45 plusmn 524 58 plusmn 112 14 plusmn 157 32 plusmn 118 101 plusmn 1102 36 plusmn 188 110133 plusmn 15030 14416 plusmn 623 48 plusmn 568 48 plusmn 525 60 plusmn 212 22 plusmn 005 28 plusmn 520 28 plusmn 025 39272 plusmn 126231 155 plusmn 83 25 plusmn 262 25 plusmn 236 18 plusmn 45 21 plusmn 007 79 plusmn 623 46 plusmn 069 38966 plusmn 178932 47233 plusmn 135 18 plusmn 20 65 plusmn 425 9 plusmn 012 45 plusmn 108 56 plusmn 102 55 plusmn 568 73477 plusmn 125133 534 plusmn 31 44 plusmn 154 77 plusmn 323 28 plusmn 204 24 plusmn 102 48 plusmn 013 28 plusmn 047 79433 plusmn 98134 158 plusmn 346 58 plusmn 403 49 plusmn 140 34 plusmn 350 58 plusmn 004 21 plusmn 232 47 plusmn 512 44566 plusmn 178935 353 plusmn 256 27 plusmn 245 58 plusmn 10 25 plusmn 326 21 plusmn 179 55 plusmn 00 55 plusmn 011 598 plusmn 34636 798 plusmn 1311 49 plusmn 307 55 plusmn 09 45 plusmn 018 40 plusmn 64 48 plusmn 036 41 plusmn 002 1092 plusmn 138537 17133 plusmn 15 78 plusmn 50 24 plusmn 045 11 plusmn 001 14 plusmn 034 75 plusmn 045 25 plusmn 205 39944 plusmn 10Numbers 1 to 37 were the sample names (Table 1)
HealthCare Inc Arizona) to 244mgg (source NaturalsInc Santa Cruz California) The highest level of totalpolyphenol contents (24mgg) was observed in the tabletsprocured from source Naturals Inc Santa Cruz Califor-nia Miranda et al (1998) claimed that the main phenoliccompounds namely chlorogenic acid synaptic acid salicylicacid trans-cinnamic acid and caffeic acid were commonlypresent in Spirulina [33] The present study also coincideswith the report of Miranda et al (1998) [33] However themetabolic pathways for the formation of phenolics com-pounds in Spirulina and their importance are still unknown
[34]Thepolyphenols contained the ideal chemical structuresand different bioactivities that included anti-inflammatoryantiviral antioxidant antithrombotic vasodilatory and anti-carcinogenic properties [35] Wu et al (2005) demonstratedthat the presence of total phenolic components and othermetabolites are related to antioxidant properties [36]
35 Antioxidant Properties The results revealed that theantioxidant properties of the Spirulina samples were dose-dependent (Figures 4ndash6) The DPPH assay and hydroxylscavenging assay results revealed that all the Spirulina extracts
Evidence-Based Complementary and Alternative Medicine 9
Table5Free
aminoacid
contents(m
g100g
fresh
wt)
indifferent
Spiru
lina
(a)
Num
ber
Aminoacids
RT(m
in)
Molecular
weight
12
34
56
78
910
1112
1314
1516
171
Aspartate
148
13310
137
137
203
266
524
134
366
124
247
097
355
405
333
089
106
169
170
2Glutamate
277
14713
ND(a)
ND(a)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
025
025
ND
092
039
ND
051
407
032
042
027
017
030
023
028
043
046
4Serin
e603
10509
169
169
049
117
114
129
159
202
158
137
092
140
057
161
186
212
118
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
064
064
ND
189
325
055
146
066
ND
053
212
ND
109
ND
065
ND
098
7Histidine
715
15515
ND
ND
ND
072
ND
043
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8Glycine
736
7507
253
253
051
351
128
145
177
230
198
305
ND
159
102
204
236
271
228
9Th
reon
ine
762
11912
154
154
045
212
098
078
171
128
124
145
192
148
084
107
108
163
208
10Arginine
847
17420
281
281
187
187
282
400
304
220
512
470
155
281
159
231
248
512
302
11Alanine
897
8909
ND
ND
ND
1168
ND
ND
ND
ND
ND
ND
ND
812
ND
ND
901
1612
1294
12GABA
926
10312
038
038
021
154
ND
032
035
126
100
084
ND
072
046
032
034
105
132
13Ty
rosin
e1043
18119
208
208
046
304
069
170
181
143
ND
254
ND
166
ND
124
150
219
128
14Cy
stine
1226
24030
ND
ND
ND
145
ND
ND
ND
ND
130
ND
045
ND
086
ND
ND
ND
052
15Va
line
1256
11715
213
213
072
534
076
192
251
ND
ND
107
ND
268
ND
142
120
402
319
16Methion
ine
1282
1492
1ND
ND
ND
080
ND
ND
ND
ND
ND
ND
093
ND
ND
ND
043
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19Ph
enylalanine
1437
16519
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
20Isoleucine
1460
13117
198
198
044
431
069
169
223
113
201
062
039
207
061
089
077
279
200
21Leucine
1543
13117
277
277
054
596
114
268
333
121
263
088
053
287
074
115
140
383
329
22Lysin
e1605
14619
168
168
038
404
079
000
244
163
190
221
071
170
072
138
145
295
163
Total
2186
2186
809
5304
1918
1816
2641
2044
2156
2065
1334
3132
1213
1455
2587
4666
3788
(b)
Num
berAminoacids
RT(m
in)
Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
1As
partate
148
13310
276
321
144
288
145
071
275
046
110
187
198
156
056
121
116
162
128
049
194
102
2Glutamate
277
14713
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
089
096
042
097
ND
ND
022
039
021
ND
ND
032
ND
035
ND
024
126
ND
052
037
4Serin
e603
10509
112
191
092
104
217
111
056
069
114
112
196
101
062
102
101
111
317
079
174
115
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
131
152
098
187
ND
ND
107
ND
ND
ND
ND
053
ND
ND
057
ND
ND
ND
ND
ND
7Histidine
715
15515
048
056
ND
091
ND
ND
ND
ND
ND
ND
ND
ND
ND
030
ND
ND
053
ND
039
031
8Glycine
736
7507
265
313
196
395
175
169
108
204
227
219
337
141
146
224
231
151
633
147
537
203
9Th
reon
ine
762
11912
283
363
184
234
130
072
073
058
094
157
161
109
069
210
171
104
174
070
134
119
10Arginine
847
17420
252
312
198
235
537
247
174
164
219
266
358
266
134
280
301
241
846
188
263
291
11Alanine
897
8909
927
1357
860
1280
1168
904
ND
803
926
984
1289
436
760
1530
1157
651
2385
603
1634
624
12GABA
926
10312
126
183
099
135
048
179
020
174
113
163
436
044
672
116
057
044
077
023
235
097
13Ty
rosin
e1043
18119
233
136
121
330
222
144
064
151
184
193
326
136
109
117
171
163
445
113
220
182
10 Evidence-Based Complementary and Alternative Medicine
(b)Con
tinued
Num
berAminoacids
RT(m
in)Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
14Cy
stine
1226
24030
130
080
065
164
038
ND
ND
ND
ND
ND
ND
ND
ND
076
ND
ND
057
ND
078
ND
15Va
line
1256
11715
434
538
243
605
297
149
070
129
169
265
305
119
162
360
342
167
1612
ND
441
144
16Methion
ine
1282
1492
1069
035
ND
098
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
043
ND
ND
064
ND
069
ND
19Ph
enylalanine
1437
16519
276
253
113
336
142
083
ND
042
046
038
098
048
062
165
087
059
293
062
173
074
20Isoleucine
1460
13117
295
343
129
398
207
095
044
092
105
150
170
085
106
192
267
111
ND
068
ND
ND
21Leucine
1543
13117
474
582
241
659
399
181
071
103
121
147
195
124
161
332
335
162
759
096
314
123
22Lysin
e1605
14619
288
301
108
447
248
163
065
162
161
121
270
116
078
168
140
136
627
108
274
159
Total
470556
1429
3560
8339
7425681149223426
1030
0143
38196525774100353322858597160848
312301
Num
bers1to37
werethe
samplen
ames
(Table1)(a)NDnot
detected
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Evidence-Based Complementary and Alternative Medicine
beta-cryptoxanthin myxoxanthophyll oscillaxanthin phy-cobiliproteins and allophycocyanin [5 6] However thenutritional contents of Spirulina depend on the cultivationconditions and the processing methods The nutritionalcomponents and other phytochemicals in Spirulina primarilyexhibit anti-inflammatory antioxidant antidiabetic neuro-protective hepatoprotective and anticancer activities [7]The regular consumption of Spirulina ameliorates the symp-toms of premenstrual cycles in women and the symptomsof amyotrophic lateral sclerosis Spirulina prevents allergicreactions and aids in the removal of metals from the body Arecent study suggested that Spirulinahelps to bind radioactiveelements and is useful for protecting the human body fromexposure to radiation therapy The phenolic compoundspresent in the Spirulina are primarily involved in the redoxmechanism and act as hydrogen donors reducing agentsmetal chelator singlets and oxygen quenchers [8] Thereforephenolic compounds can prevent the formation of ROSand reactive nitrogen species which include free radicalssuch as hydroxyl and superoxide anions and nitric oxideand nonfree radical species such as hydrogen peroxide andnitrous acid The development of phenolic compounds asantioxidants for the treatment of various human diseaseshas increased Therefore there is an urgent need to identifynovel antioxidant molecules with fewer side effects and sig-nificant hepatoprotective effects [9] To overcome disordersthe regular consumption of natural health-promoting foodssuch as Spirulina tablets or powders is advised In vitrostudies demonstrated that the Spirulina and Nestoc specieshave several therapeutic properties due to their ability toscavenge superoxide and hydroxyl radicals and inhibit lipidperoxidation [10 11] Therefore the present study aimed toinvestigate the metabolite profiles and antioxidant propertiesof 37 commercially available Spirulina samples
2 Materials and Methods
21 Chemicals and Solvents Standard methyl esters offatty acids were obtained from Supelco (37 ComponentFAME Mix) Triglyceride (IS C110 triundecanoin) waspurchased from Nu-Chek Prep (Elysian MN USA) BF3-methanol (10 ww) was procured from Supelco (BellefontePA USA) Analytical grade diethyl ether (DE) pyrogal-lol petroleum ether (PE) chloroform and ethanol werepurchased from Sigma-Aldrich Chemical Co (St LouisMo USA) Thirty-seven Spirulina samples with differentcountries of origin in the forms of tablets and capsules wereprocured from specialist shops (Table 1) All the studiedSpirulina samples were procured from different markets inthe world The details of the samples were mentioned inTable 1
22 Extraction of Lipids from the Spirulina Samples The totallipids in the Spirulina samples were extracted according tothe following modified method of Mossoba et al (2003)[12] Briefly one gram of finely powdered sample with 2mLpyrogallol solution (in ethanol 95 50mgmL) and 1mLtriglyceride internal standard solution (IS C110 triunde-canoin 5mgmL in iso-octane) was transferred into a 50mL
tube After proper mixing 100mL 83MHCl was added intothe tube which was then incubated in a shaking water bathat 70ndash80∘C for 2 h During the incubation the contents of thetubes were intermittently mixed to release the fat from thewalls of the tubes After incubation the samples were allowedto cool at room temperature and mixed with 15mL diethylether (DE) The DE layer was then separated and filteredin the column using Na
2SO4and petroleum ether (PE)
Subsequently the collected PE layers were slowly evaporatedusing a nitrogen streamandused for the extraction of the fattyacids
23 Extraction and Quantification of the Fatty Acids Thetotal lipids in the Spirulina were extracted according themethod of Mossoba et al (2003) with modifications [12]Briefly the extracted lipids were saponified with 05N NaOHin methanol (15mL) for 5min at 100∘C and cooled at roomtemperature After cooling the samples were treated with2mL of BF3-methanol and incubated at 100∘C for 10minand allowed to cool at room temperature The samples werethen thoroughly vortexed with 2mL of isooctane and 1mL ofsaturated NaCl solution for 10min Next the upper isooctanelayer was carefully transferred into tubes and injected intoa Hewlett-Packard 6890 series gas chromatograph (GC)equipped with an autoinjector and a flame-ionization detec-tor (Agilent Technologies Little Falls Del USA) The fattyacids were separated in a fused-silica capillary column (SP-2560 100m times 025 nm times 02 120583m film thickness SupelcoUSA) The GC oven was heated to 100∘C and held for 4minand then further increased to 240∘C at a rate of 3∘Cminand held at 240∘C for 15min The injector and detectortemperatures were set at 225∘C and 285∘C respectively Themobile gas (helium) applied at a flow rate of 075mLminTheconcentrations of the individual fatty acids were calculatedbased on the relative retention times of the standardmixturesThe conversion of FAMEs to corresponding fatty acids areshown in Table 2
The response factor (119877119894) of each fatty acid was calculated
as follows
119877119894=119875119904119894
119875119904C110times119882C110119882119894
(1)
where 119875119904119894is peak area of individual fatty acid in mixed
FAMEs standard solution 119875119904C110 is peak area of C110 fattyacid in mixed FAMEs standard solution119882C110 is weight ofinternal standard in mixed FAMEs standard solution and119882119894is weight of individual FAME in mixed FAMEs standard
solutionThe amounts of the individual compounds in the test
samples were calculated as follows
119882FAME119894 =119875119905119894times119882119905C110 times 10067
119875119905C110 times 119877119894 (2)
where 119875119905119894is peak area of the fatty acid 119894 in the test portion
119882119905C110 is weight of C110 in the internal standard added totest portion g 10067 is conversion of the internal standardfrom triglyceride to FAME and 119875119905C110 is peak area of C110in the internal standards in the test portion
Evidence-Based Complementary and Alternative Medicine 3
Table1Spiru
linaprod
uctslistsandtheirc
ountry
oforigin
Snu
mber
Prod
ucttype
Manufacturin
gcompany
Cou
ntry
oforigin
Web
address
1Tablets
TAAU
Australia
PvtL
tdNT
Australia
httpwwwaustralianspirulin
acom
au
2Ca
psules
GeneralNutritionCorpP
ittsburgh
USA
httpwwwgn
ccom
3
Capsules
Naturersquos
Way
Prod
ucts
Inc
Sprin
gvilleUtah
USA
httpwwwnaturesw
ayco
m4
Tablets
Goo
dlsquoN
NaturalN
ewYo
rkUSA
httpwwwgood
andn
aturalsto
reco
m5
Tablets
Now
Food
sBloo
mingdale
USA
httpwwwno
wfood
scom
6Tablets
NatureP
ureInc
LarkspurC
alifo
rnia
USA
7Tablets
Source
NaturalsInc
SantaC
ruzCa
lifornia
USA
httpwwwsourcenaturalscom
8Tablets
Jarrow
Form
ulasL
osAngele
sCA
USA
httpwwwjarrow
com
9Tablets
Earthrise
NutritionalsLL
CIrvineC
AUSA
httpearthrise
com
10Tablets
NutrexHaw
aiiIncKa
ilua-Ko
naH
awaii
USA
httpwwwnu
trex-hawaiicom
11Ca
psules
Pure
PlanetProd
ucts
Inc
Long
BeachCA
USA
httpsw
wwpu
replanetco
m12
Tablets
PuritanrsquosPrideInc
Oakdale
New
York
USA
httpwwwpu
ritanco
m13
Capsules
21stCentury
Health
CareIncAriz
ona
USA
httpwww21stc
enturyvitaminsc
om14
Tablets
JapanAlgae
CoLtdTo
kyo
Japan
httpwwwsp100com
15Tablets
AllSeason
sHealth
Ham
pshire
UnitedKingdo
mhttpwwwcareho
mec
ouk
16
Capsules
FushiW
ellbeing
LtdL
ondo
nUnitedKingdo
mhttpwwwfushicouk
17Tablets
BioveaL
ondo
nUnitedKingdo
mhttpwwwbioveaco
m18
Capsules
ParryNutraceuticalsCh
ennai
India
httpwwwparrynutraceuticalsc
om19
Tablets
Lifestream
InternationalLtdNorthcoteA
uckland
New
Zealand
httpwwwlifestre
amco
nz
20Tablets
Green
Health
Auckland
New
Zealand
httpwwwgreenh
ealth
conz
21Tablets
RBCLifeSciencesIncBu
rnabyBritish
Colum
bia(
BC)
Canada
httpwwwrbclifesciencesco
m22
Tablets
Swiss
HerbalR
emediesL
tdRichmon
dHillO
ntario
Canada
httpwwwsw
issnaturalcom
23Ca
psules
HerbalSele
ctG
uelphOntario
Canada
httpwwwherbalselectca
24Ca
psules
Gou
rmetNutritionFB
IncST
E-Julie
(Quebec)
Canada
httpwwwgourmetfbco
m25
Capsules
TerraV
itaFine
Who
leHerbsB
rampton
Ontario
Canada
httpdsldnlm
nihgov
26Ca
psules
DXN
marketin
gMalaysia
httpwwwdx
nmalaysia
com
27Ca
psules
Hydrolin
aBiotech
PvtIndia
India
httpwwwhydrolinabiotechco
m28
Capsules
Prim
eHealth
Labo
ratorie
sLtd
Austr
alia
Not
available
29Tablets
LaurelHerbalprodu
cts
India
Not
available
30Tablets
IMOCon
trolP
vtLtd
India
httpwwwim
ocon
trolin
31Ca
psules
Acum
enPh
armaceutic
alsP
vtIndia
httpwwwacum
enph
armco
m32
Capsules
Bio-LifeOrganicSpiru
lina
Malaysia
httpshealth
review
2uwordp
ressco
m20100915
biolifespirulin
a33
Tablets
Dharain
Pharmaceutic
als
India
httpwwwmihnaticom
34Tablets
21stCentury
Health
CareIncAriz
ona
USA
httpw
ww21stc
enturyvitaminsc
om
35Tablets
Zuellig
BharmaS
DWB
HD
Malaysia
httpwwwzuelligph
armac
om36
Tablets
Kordels
Spiru
lina
India
httpwwwraku
tencom
37Tablets
ElkenCh
ewable
Malaysia
httpselken4m
rtwordp
ressco
m
4 Evidence-Based Complementary and Alternative Medicine
Table 2 Fatty acids contents (mg100 g in different Spirulina)
Sample
FA
Total1400 1600 161 180 181t 181(n-9)c 181(n-7)c 182t 182(n-6)c 2000 183(n-6) 2002 203n6119891FA119894
09421 09481 09477 09530 09527 09524 095201 09517 09524 09570 0952 09565 095571 001 188 022 029 000 009 002 001 065 001 067 001 001 252 000 207 010 009 000 009 002 001 032 001 016 001 001 2363 001 197 011 034 001 011 005 004 075 001 067 000 000 2554 001 255 027 006 000 005 001 001 085 000 073 001 001 2945 001 189 017 030 001 007 002 001 062 000 053 000 001 2446 001 190 031 013 001 009 004 001 073 001 070 001 002 2457 001 238 017 079 000 006 002 001 055 001 046 001 001 3428 002 281 014 085 000 015 002 001 080 001 050 001 001 3989 001 200 018 011 000 014 007 001 065 001 040 001 001 24510 001 280 014 045 000 009 001 001 087 001 073 001 001 3511 001 210 023 014 000 016 006 001 060 001 037 001 001 26512 001 212 025 006 000 014 007 001 078 001 052 001 001 25913 001 236 027 040 000 011 002 001 080 001 082 001 001 31614 001 248 024 005 000 013 002 002 099 001 082 001 001 29215 001 178 028 004 000 014 001 001 062 001 058 000 000 22616 001 295 015 213 001 011 003 002 067 002 055 000 000 53717 002 191 010 006 001 015 003 003 070 001 057 000 001 22518 002 195 015 017 000 008 003 003 064 001 060 000 000 23819 002 204 012 016 001 014 003 003 068 001 051 000 001 24920 002 212 012 025 001 012 004 004 071 001 066 000 001 26421 001 181 026 021 001 005 002 001 065 001 062 001 001 23522 000 143 022 004 000 012 002 001 054 000 053 001 001 18123 001 327 014 111 000 018 007 001 076 002 044 001 001 47324 001 258 028 006 000 007 002 002 074 001 059 001 001 30125 001 249 026 006 000 010 003 001 072 001 058 001 001 29326 001 173 023 005 000 010 003 001 044 000 039 000 001 21227 002 217 025 006 001 012 006 001 075 001 060 001 001 26328 001 298 023 135 000 013 003 002 090 002 052 001 001 47229 001 158 011 007 000 017 004 001 053 001 049 001 001 19530 002 236 014 005 001 017 003 003 083 001 065 000 000 27531 001 188 015 005 000 019 003 001 054 000 040 001 001 22832 001 175 019 007 000 008 001 001 059 000 049 001 001 2133 001 229 033 006 000 010 003 001 078 001 070 001 001 2834 004 395 021 015 002 023 008 007 137 001 124 001 001 45935 002 210 031 008 000 016 012 001 055 001 060 002 001 26836 001 262 015 008 000 018 004 003 104 001 058 001 001 30537 002 214 014 005 001 011 004 004 077 000 077 000 001 246Numbers 1 to 37 were the sample names (Table 1) Total fatty acids were considered only for PUFA 1400 myristic acid 1600 sapienic acid 161 palmitoleicacid 180 stearic acid 181t elaidic acid 181(n-9)c oleic acid 181(n-7)c vaccenic acid 182t linolelaidic acid 182(n-6)c linoleic acid 2000 eicosenoic acid183(n-6) 120574-linolenic acid 2002 eicosadienoic acid and 203n6 dihomo-gamma-linolenic acid
The weight of the fatty acid (119882119894) was determined as
follows
119882119894= 119882FAME119894 times 119891FA119894 (3)
where 119891FA119894 is conversion factors for the conversion of theFAMEs to their corresponding fatty acids
24 Extraction and Quantification of Sugars Using HPLCThe carbohydrates present in the Spirulina samples werequantified according to the following the standard method[14] Briefly a 100mg portion of the powdered sample wasmixed with 10mL sterile distilled water and boiled at 100∘Cfor one hour After heating the debris was separated bycentrifugation at 10000 rpm for 10min Next the debris-free
Evidence-Based Complementary and Alternative Medicine 5
solution was mixed with 10 5 trichloroacetic acid (TCA)and incubated at room temperature for 10min Subsequentlythe samples were centrifuged at 10000 rpm for 10min andthen filtered through a 045120583mPTFE syringe filter for HPLCanalysis (HP1100 Agilent Co USA) The filtered sampleswere separated through a 300 times 78mm Aminex HPX-87H (Bio-Rad Hercules CA USA) column at 60∘C usinga 50mM H
2SO4monophasic solvent system with a flow
rate of 05mLmin and a column wavelength of 220 nmThe injection sling was 10 120583L The quantifications of theindividual sugars were based on the peak areas and calculatedas equivalents of standard compounds
25 Extraction and Quantification of the Free Amino AcidsUsing HPLC Individual free amino acids were extractedand quantified according to the method of Park et al(2014) with modifications [15] Briefly a 100mg portionof a fine powdered sample was mixed with 12mL of 5trichloroacetic acid (TCA) in a 2mL Eppendorf tube andvigorously shaken for 5minThe slurry sample was incubatedat room temperature for 60min and the upper layer wasthen separated by centrifugation The collected sampleswere diluted with 01M HCl and then filtered through a045 120583m PTFE syringe filter The filtrate was then analysedby HPLC (Agilent Technologies Palo Alto CA) The HPLCanalyses of free amino acids were conducted according to theldquorapid accurate sensitive and reproducible HPLC analysisof amino acids analysisrdquo method with Zorbax Eclipse-AAAcolumns using an Agilent 1100 HPLC system Briefly theseparation of the free amino acidswas performed on aZorbaxEclipse AAA analytical columnThe oven temperature of thecolumn was set at 40∘C and the detection wavelength wasset a 338 nm The injection volume was 10 120583L The mobilephase consisted of a mixture of 40mM NaH
2PO4(pH 78
solvent A) and solvent B (ACN MeOH and water at a45 45 10 vvv ratio) was passed at a rate of 20mLminThe HPLC separation parameters were as follows 0min0 B 0ndash19min 0 B 19ndash211min 57 B 211ndash216min100 B 216ndash25min 100 B 25ndash251min 0 B and 251ndash30min 0 B A sample with an amino acid content of50 pmoL120583L was used as the standard The quantifications ofthe different amino acids were based on the peak areas andwere calculated as equivalents of the standard compoundsAll contents are expressed as milligrams lowast gramfresh weight(FW)
26 Total Polyphenol Analysis The total polyphenols wereestimated according to the method of Folin-Ciocalteu (mod-ified from Lin and Tang 2006) [16] Briefly 10mg of thesample was dissolved in 1mL ofmethanol with 2mL of Folin-Denis reagent and 35 sodium carbonate (Na
2CO3) The
mixture was stored at room temperature for 30min Theabsorbancewasmeasuredwith anUV-Vis spectrophotometerat 750 nm The total polyphenols were calculated as gallicacid equivalents based on a calibration curve for gallic acid(0 25 50 and 100 120583gmL) using the following equation thatwas based on the calibration curve 119910 = minus09706119909 + 38935(1198772 = 09992)
27 In Vitro Antioxidant Assays271 Preparation of the Spirulina One gram of the finepowder sample was mixed with 5mL of ethanol in a screw-cap tube by vortexing for 5min and then kept in an orbitalshaker at 150 rpm for 24 h at room temperature for thoroughextraction After incubation the samples were centrifuged at13000 rpm for 15min at 4∘C The resulting supernatant wasvacuum evaporated at 30∘C and the resulting extract wasused for the antioxidant assays
272 Reducing Power Activity Assay The reducing powerassay was performed according to the method of Oyaizu(1986) [17] Volume of 100 120583L of various concentrations(20ndash100120583gmL) of the samples was mixed with phosphatebuffer (25mL) and 1 potassium ferricyanide (25mL) andincubated at 50∘C for 20 minutes After incubation 25mLof 10 trichloroacetic acid was added and the samples werecentrifuged at 3000 rpm for 10min The upper layer of thesolution (25mL) was mixed with distilled water (25mL)and a freshly prepared 01 ferric chloride solution (05mL)and measured at an absorbance at 700 nm The control wasprepared in a similar manner but the sample was excludedVitamin C at various concentrations was used as a standardIncreases in the absorbance of the reaction mixture indicatedincreases in reducing power
273 DPPH Radical Scavenging Activity Assay The DPPHradical scavenging assay performed according to the methodof Hatano et al (1988) [18] Briefly 100 120583L of the sampleand vitamin C (concentration 100ndash500120583gmL) was mixedwith 200120583L of freshly prepared DPPH solution (1mgmL inmethanol) and incubated at room temperature in the darkfor 30 minutes The controls included only deionized waterand the DPPH solution The absorbances of the resultingsolutions were measured in triplicate at 517 nm followingcentrifugation at 12000 rpm for 10min
The scavenging activity was calculated as follows
scavenging activity () = [1 minus(1198600minus 1198601)
1198602
] times 100 (4)
where1198600is absorbance of sample119860
1is absorbance of blank
and 1198602is absorbance of control
274 Hydroxyl Radical-Scavenging Activity Assay The hy-droxyl radical-scavenging assay was performed accordingto the method of Elizabeth and Rao (1990) with slightmodification [19]The reagents for the assay were freshly pre-pared Briefly one millilitre samples of the working solutionsthat consisted of different ratios of the extract were mixedwith 100mL of 28mM 2-deoxy-2-ribose in phosphate buffer(pH 74) EDTA (104mM 1 1 vv) 100mL H
2O2(1mM)
200mL of FeCl3(200mM) and 100mL ascorbic acid (1mM)
The resulting solutions were mixed evenly and the reactionmixtures were incubated at 37∘C for 1 h The degradation ofdeoxyribose was determined by reading the absorbance at532 nm against the blank solution using a microplate reader(BioRad) Vitamin C was used as a positive control The
6 Evidence-Based Complementary and Alternative Medicine
experiments were conducted in triplicate The scavengingactivities were calculated according to (4)
3 Results and Discussion
31 Variations in the Fatty Acid Contents The total lipidcontents were extracted from the Spirulina samples and theindividual fatty acid compositions of the samples were anal-ysedwith gas chromatography A gas chromatograph coupledwith a flame ionization detector guided the identificationof the following 10 unsaturated and fatty acids (sapienicacid palmitoleic acid elaidic acid oleic acid vaccenic acidlinolelaidic acid linoleic acid eicosenoic acid 120574-linolenicacid and dihomo-gamma-linolenic acid) and three saturatedand fatty acids (myristic acid stearic acid and eicosadienoicacid) (Figure 1 and Table 2) The individual PUFA contentswere quantified by comparing the standard fatty acids withtheir indices The PUFA contents in the Spirulina samplesranged from 301 (DXN Marketing capsules) to 741 g100 g(21st Century HealthCare Inc Arizona tablets Table 4)Interestingly there were comparatively lower amounts of 120574-linolenic acid in the studied Spirulina samples this acidaccounted for an average of 14 of the total PUFAs Theamounts of 120574-linolenic acid ranged from 016 g100 g (Gen-eral Nutrition Corp Pittsburgh capsules) to 124 g100 g(21st Century HealthCare Inc Arizona tablets) HoweverMuhling et al (2005) reported palmitic acid (C160) noted asthe dominant fatty acid in wild Spirulina samples [20] In ourstudy sapienic acid been noted as the major fatty acids Manyin vitro studies have confirmed that 120574-linolenic acid can beused to effectively lower cholesterol and treat atopic eczemabreast cancer and premenstrual disorder [21ndash25] RecentlySajilata et al (2008) extracted and purified S platensisactive components via lipid fractionation silica gel columnpurification and thin-layer chromatographic methods [26]It has been reported that 120572-linolenic acid and 120574-linolenic acidare required for the survival of animals and humans Patilet al (2007) [27] Patil et al (2007) profiled the individualPUFAs fromBacillariophyceae Cyanophyceae RhodophyceaeXanthophyceae Cryptophyceae Prymnesiophyceae Eustig-matophyceae and Chlorophyceae microalgae and suggestedthat the cultivation conditions particularly light intensityand other nutritional components exert important effectson the PUFA compositions [27] Many companies processmicroalgae and supply the results in the forms of capsulesand tables to the market Recently the interest in the use ofSpirulina tablets as energy foods has been renewed due to therelatively high contents of protein phytochemicals and othernutrients in such tablets Efforts should be made to analysethemetabolite profiles of the commercially available Spirulinaproducts because in a previous study we confirmed thattrace amounts of heavymetals that could cause serious healthproblems for consumers are present in some commerciallyavailable Spirulina samples (Table 3) [13]
32 Quantification of Individual Sugars by HPLC Hexose(ie glucose fructose galactose and rhamnose) pentose(ie xylose and ribose) and disaccharide sugars were
35
30
25
(pA
)
20
15
10
FID1 A (FC140730SIG10006D)
20 30 40 50 60 70 80
40
110
120
140
160
161
180
181
t18
1(n
-9)c
181
(n-7
)c18
2t
182
(n-6
)c18
3t
183
(n-6
)c18
3(n
-3)
202
203
n620
4(n
-6)
200
40
1100
11000
1111
120
140
00016
000016
0016
06
006
0006
016
06
016
0061666666666611111111
161
180
181
t18
1(n
-9)c
181
(n1(18
1(n(
181
(nn1(nn
181
(n1(n
181
(n1(n
1(n
181
(n18
1(n
181
(n((1(((((1(18
1(18
1118
111818111111111111
-7)c18
18
182
t111111111
182
(nnn(nn18
2(nnnnnn((((
182(
-6)c)6)6)6)))6))666666666666666666666666
183
t1111111111
183
(nnn18
3(n((((
183((
183(
-6)c6))6)6)))6)6)6))6)6)666666666666666
(18
3(n
88888818818888888111)
-3)
202
20202000020020202002222222222222222222222222 203
n620
4(n
-6)
200
200
200
200
222222222222
Figure 1 Gas chromatograms of the fatty acids identified in theSpirulina samples The peaks numbers refer to the individual fattyacids listed in Table 2
extracted from the 37 Spirulina samples and clearly base-line eluted by HPLC The quantitative results revealed thatglucose fructose and sucrose were present in the greatestamounts followed by xylose ribose galactose and rhamnoseThe total sugar contents of the Spirulina samples ranged from309 to 122167mg100 g (Table 4) Together glucose fructosegalactose and rhamnose accounted for an average of 7385of the total sugar contents Among the major individualsugars glucose accounted for an average of 351mg100 g and52 of the total sugar contents Similarly Chaiklahan et al(2013) reported that rhamnose and glucose account for 53and 13 of the total sugars respectively [28] The rhamnosecontents varied from 8 to 58mg100 g of the total sugarsaccounting for an average of 65 [29] The results indicatedthat among the pentose sugars xylose (average 908) andribose (average 475) were the major components in theSpirulina samples The final outcome of this study is thatthe variations in the individual sugar contents between theSpirulina samples were acceptable due to the processing con-ditions of the each commercial Spirulina product Moreovera literature stated that the extraction of total polysaccharidesand other monosaccharides from Spirulina followed by thequantification of the individual sugar molecules identifiedrhamnose as the predominant sugar followed by glucose andfructose [29]
33 Quantifications of the Individual Amino Acids by HPLCHPLC analyses were used to quantify 22 free amino acidsincluding aspartate asparagine serine glutamine histidineglycine threonine arginine alanine 120574-aminobutyric acid(GABA) tyrosine valine cystine methionine tryptophanphenylalanine isoleucine leucine and lysine in the Spirulinasamples but the separation profiles revealed that only 18 freeamino acids were detectable in the samples but the otherfree amino acids did not detected which may be not presentin the samples (Table 5 and Figure 2) Since the sampleswere marketed by the trademark of different companies andthe nutrient profile of each sample would be varied theseresults revealed that the amounts of total free amino acidsin the 37 Spirulina samples ranged from 1149mg100 g to5614mg100 g The essential amino acid content averagesranged from 206 to 3172mg100 g and contributed averagesthat ranged from 170 to 3918 of the total amino acidsAmong the essential amino acids leucine was identified
Evidence-Based Complementary and Alternative Medicine 7
Table 3 Content of heavy metals in the Spirulina samples available in the market
Sample name Amount mgKg dry weightNickel Zinc Mercury Platinum Magnesium Manganese Total
1 0211 0533 0002 0001 0002 0076 08252 4672 5627 0028 001 003 0587 109543 2016 2397 0017 0008 0018 0603 50594 2147 1628 002 0011 0026 0436 42685 2199 1235 0017 0008 0014 117 46436 3726 6225 0022 0009 0028 0007 100177 2601 2817 0018 0011 0028 0007 54828 3577 1871 0023 0012 0031 0309 58239 3519 3267 0021 0011 003 0008 685610 2442 2041 0017 0008 0021 0137 466611 3785 3007 0019 0008 0028 0008 685512 3597 2859 0019 0008 0024 0006 651313 2857 2901 0019 0008 0026 1643 745414 2852 2114 0026 0008 0042 0011 505315 2437 1568 002 0008 0028 0008 406916 2712 2434 0019 0009 0033 1777 698417 2948 2513 0021 0009 0028 18 731918 2633 1876 0016 0009 0023 1328 588519 3731 3184 0017 0009 0036 2248 922520 2225 1548 0008 0009 0019 1132 494121 1618 1478 0014 0006 002 0005 314122 1589 4626 0016 0007 0023 0009 62723 3272 4428 002 0008 0018 0008 775424 3558 3733 0017 0009 0034 1433 878425 2319 2586 0017 0008 0024 0006 496Numbers 1 to 37 were the sample names (Table 1) Samples 26ndash37 were not analyzed [13]
1
10
2
34 56
7
89 11
1213
14
1516
17 181920 21
22
Inte
nsity
(mAU
)
0
05
1
15
2
0 2 4 6 8Retention time (min)
10 12 14 16 18
Figure 2 HPLC chromatogram of the standard free amino acidsThe peaks numbers refer to the free amino acids listed in Table 5Peaks numbers 1 aspartate 2 glutamate 3 asparagine 4 serine 5 S-methylmethionine (vitamin U) 6 glutamine 7 histidine 8 glycine9 threonine 10 arginine 11 alanine 12 gamma-aminobutyricacid (GABA) 13 tyrosine 14 cystine 15 valine 16 methionine17 norvaline 18 tryptophan 19 phenylalanine 20 isoleucine 21leucine 22 lysine
as predominant (053 to 759mg100 g) and accounted formore than 30 of the essential amino acids The Spirulinatablets marketed in India under the brand name ldquoDharainPharmaceuticalsrdquo exhibited the greatest essential amino acid
contents However the brand name products exhibited valuesthat were comparatively lower than the maximum observedvalues Vitamin U methionine norvaline and tryptophanwere not observed in the samples The essential aminoacid compositions of the microalgae were very similar tothe reported protein contents [30] Clement et al (1967)determined the individual and total amino acid contentsin S maxima [31] This study reported that aspartate wasdominant in the Spirulina samples and that histidine cystinetryptophan and methionine were observed at the lowestlevels This report found a level of aspartate that was similarto that of another report In general many companies marketSpirulina samples as nutraceutical food however there is anurgent need to know the nutritional compositions of each ofthese Spirulina products [32] This study confirmed that theamino acid compositions of Spirulina samples varied with thecompanies that produced them Therefore amino acid-richsamples should be consumed by humans to maintain theirhealth
34 Determination of the Total Phenolic Compounds Figure 3shows the total phenolic compounds calculated as equivalentsto gallic acidThe results revealed that the distributions of thetotal phenolic compounds varied between the commercialproducts The products ranged from 24mgg (21st Century
8 Evidence-Based Complementary and Alternative Medicine
Table 4 Individual sugar contents (mg100 g) in different Spirulina
Sample Sugar contents (mg100 g)Glucose Fructose Xylose Galactose Ribose Sucrose Rhamnose Total
1 172 plusmn 14 38 plusmn 345 25 plusmn 150 155 plusmn 056 18 plusmn 125 54 plusmn 56 25 plusmn 00 3425 plusmn 4332 71 plusmn 1389 44 plusmn 15 88 plusmn 422 78 plusmn 312 5 plusmn 000 65 plusmn 024 18 plusmn 023 363 plusmn 5193 22933 plusmn 2759 59 plusmn 505 55 plusmn 160 7 plusmn 071 45 plusmn 300 54 plusmn 345 35 plusmn 089 46611 plusmn 15774 5133 plusmn 416 68 plusmn 501 21 plusmn 111 54 plusmn 201 7 plusmn 003 29 plusmn 203 19 plusmn 11 24977 plusmn 0385 34733 plusmn 643 15 plusmn 124 46 plusmn 34 8 plusmn 023 25 plusmn 025 84 plusmn 00 58 plusmn 32 58111 plusmn 1926 26 plusmn 346 26 plusmn 28 32 plusmn 1 9 plusmn 017 16 plusmn 000 72 plusmn 130 45 plusmn 1 222 plusmn 3467 14833 plusmn 1041 34 plusmn 178 37 plusmn 403 45 plusmn 312 18 plusmn 187 80 plusmn 915 25 plusmn 028 36644 plusmn 18088 15633 plusmn 1629 15 plusmn 121 39 plusmn 545 11 plusmn 102 31 plusmn 164 45 plusmn 745 14 plusmn 002 33311 plusmn 18869 253 plusmn 520 29 plusmn 598 65 plusmn 84 24 plusmn 20 22 plusmn 003 62 plusmn 605 35 plusmn 096 46333 plusmn 230910 25533 plusmn 1002 78 plusmn 74 98 plusmn 12 NA NA 34 plusmn 510 NA 52844 plusmn 146211 12833 plusmn 289 51 plusmn 089 45 plusmn 71 23 plusmn 185 24 plusmn 007 86 plusmn 688 18 plusmn 112 37711 plusmn 15412 36166 plusmn 66 49 plusmn 104 25 plusmn 090 14 plusmn 22 16 plusmn 008 95 plusmn 00 8 plusmn 032 58955 plusmn 18013 48233 plusmn 58 35 plusmn 208 45 plusmn 503 18 plusmn 103 54 plusmn 109 NA 11 plusmn 078 73077 plusmn 13414 59233 plusmn 615 26 plusmn 10 85 plusmn 523 44 plusmn 29 22 plusmn 107 29 plusmn 215 16 plusmn 047 81144 plusmn 24915 24866 plusmn 8 59 plusmn 68 48 plusmn 49 11 plusmn 22 15 plusmn 03 67 plusmn 168 24 plusmn 49 47089 plusmn 15416 21033 plusmn 95 72 plusmn 56 95 plusmn 012 10 plusmn 062 24 plusmn 13 49 plusmn 90 17 plusmn 063 47244 plusmn 42317 517 plusmn 24 93 plusmn 405 25 plusmn 52 12 plusmn 062 26 plusmn 42 52 plusmn 051 28 plusmn 103 75433 plusmn 11518 88466 plusmn 265 15 plusmn 025 22 plusmn 07 18 plusmn 215 28 plusmn 412 58 plusmn 152 38 plusmn 108 105989 plusmn 32719 33833 plusmn 125 44 plusmn 114 14 plusmn 09 27 plusmn 312 11 plusmn 000 49 plusmn 302 26 plusmn 00 50977 plusmn 03820 752 plusmn 526 51 plusmn 46 16 plusmn 012 9 plusmn 025 22 plusmn 08 67 plusmn 307 45 plusmn 28 98733 plusmn 219321 96866 plusmn 273 50 plusmn 254 18 plusmn 059 58 plusmn 812 24 plusmn 014 84 plusmn 101 19 plusmn 51 120722 plusmn 125222 260 plusmn 229 43 plusmn 63 94 plusmn 126 65 plusmn 474 26 plusmn 295 55 plusmn 078 54 plusmn 290 599 plusmn 17323 10733 plusmn 709 24 plusmn 40 25 plusmn 045 22 plusmn 012 25 plusmn 005 78 plusmn 184 28 plusmn 105 30311 plusmn 53824 15166 plusmn 76 52 plusmn 265 63 plusmn 040 45 plusmn 294 11 plusmn 032 37 plusmn 000 47 plusmn 001 40222 plusmn 38525 10266 plusmn 16 68 plusmn 43 24 plusmn 145 48 plusmn 2 7 plusmn 005 81 plusmn 028 35 plusmn 025 36189 plusmn 32726 7933 plusmn 1115 94 plusmn 50 25 plusmn 209 56 plusmn 623 45 plusmn 110 55 plusmn 612 45 plusmn 17 39977 plusmn 03827 60466 plusmn 53 12 plusmn 137 26 plusmn 423 44 plusmn 256 24 plusmn 000 84 plusmn 071 37 plusmn 032 83055 plusmn 09628 90166 plusmn 76 15 plusmn 07 45 plusmn 23 25 plusmn 045 10 plusmn 007 56 plusmn 183 34 plusmn 000 109555 plusmn 7629 798 plusmn 1311 45 plusmn 524 58 plusmn 112 14 plusmn 157 32 plusmn 118 101 plusmn 1102 36 plusmn 188 110133 plusmn 15030 14416 plusmn 623 48 plusmn 568 48 plusmn 525 60 plusmn 212 22 plusmn 005 28 plusmn 520 28 plusmn 025 39272 plusmn 126231 155 plusmn 83 25 plusmn 262 25 plusmn 236 18 plusmn 45 21 plusmn 007 79 plusmn 623 46 plusmn 069 38966 plusmn 178932 47233 plusmn 135 18 plusmn 20 65 plusmn 425 9 plusmn 012 45 plusmn 108 56 plusmn 102 55 plusmn 568 73477 plusmn 125133 534 plusmn 31 44 plusmn 154 77 plusmn 323 28 plusmn 204 24 plusmn 102 48 plusmn 013 28 plusmn 047 79433 plusmn 98134 158 plusmn 346 58 plusmn 403 49 plusmn 140 34 plusmn 350 58 plusmn 004 21 plusmn 232 47 plusmn 512 44566 plusmn 178935 353 plusmn 256 27 plusmn 245 58 plusmn 10 25 plusmn 326 21 plusmn 179 55 plusmn 00 55 plusmn 011 598 plusmn 34636 798 plusmn 1311 49 plusmn 307 55 plusmn 09 45 plusmn 018 40 plusmn 64 48 plusmn 036 41 plusmn 002 1092 plusmn 138537 17133 plusmn 15 78 plusmn 50 24 plusmn 045 11 plusmn 001 14 plusmn 034 75 plusmn 045 25 plusmn 205 39944 plusmn 10Numbers 1 to 37 were the sample names (Table 1)
HealthCare Inc Arizona) to 244mgg (source NaturalsInc Santa Cruz California) The highest level of totalpolyphenol contents (24mgg) was observed in the tabletsprocured from source Naturals Inc Santa Cruz Califor-nia Miranda et al (1998) claimed that the main phenoliccompounds namely chlorogenic acid synaptic acid salicylicacid trans-cinnamic acid and caffeic acid were commonlypresent in Spirulina [33] The present study also coincideswith the report of Miranda et al (1998) [33] However themetabolic pathways for the formation of phenolics com-pounds in Spirulina and their importance are still unknown
[34]Thepolyphenols contained the ideal chemical structuresand different bioactivities that included anti-inflammatoryantiviral antioxidant antithrombotic vasodilatory and anti-carcinogenic properties [35] Wu et al (2005) demonstratedthat the presence of total phenolic components and othermetabolites are related to antioxidant properties [36]
35 Antioxidant Properties The results revealed that theantioxidant properties of the Spirulina samples were dose-dependent (Figures 4ndash6) The DPPH assay and hydroxylscavenging assay results revealed that all the Spirulina extracts
Evidence-Based Complementary and Alternative Medicine 9
Table5Free
aminoacid
contents(m
g100g
fresh
wt)
indifferent
Spiru
lina
(a)
Num
ber
Aminoacids
RT(m
in)
Molecular
weight
12
34
56
78
910
1112
1314
1516
171
Aspartate
148
13310
137
137
203
266
524
134
366
124
247
097
355
405
333
089
106
169
170
2Glutamate
277
14713
ND(a)
ND(a)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
025
025
ND
092
039
ND
051
407
032
042
027
017
030
023
028
043
046
4Serin
e603
10509
169
169
049
117
114
129
159
202
158
137
092
140
057
161
186
212
118
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
064
064
ND
189
325
055
146
066
ND
053
212
ND
109
ND
065
ND
098
7Histidine
715
15515
ND
ND
ND
072
ND
043
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8Glycine
736
7507
253
253
051
351
128
145
177
230
198
305
ND
159
102
204
236
271
228
9Th
reon
ine
762
11912
154
154
045
212
098
078
171
128
124
145
192
148
084
107
108
163
208
10Arginine
847
17420
281
281
187
187
282
400
304
220
512
470
155
281
159
231
248
512
302
11Alanine
897
8909
ND
ND
ND
1168
ND
ND
ND
ND
ND
ND
ND
812
ND
ND
901
1612
1294
12GABA
926
10312
038
038
021
154
ND
032
035
126
100
084
ND
072
046
032
034
105
132
13Ty
rosin
e1043
18119
208
208
046
304
069
170
181
143
ND
254
ND
166
ND
124
150
219
128
14Cy
stine
1226
24030
ND
ND
ND
145
ND
ND
ND
ND
130
ND
045
ND
086
ND
ND
ND
052
15Va
line
1256
11715
213
213
072
534
076
192
251
ND
ND
107
ND
268
ND
142
120
402
319
16Methion
ine
1282
1492
1ND
ND
ND
080
ND
ND
ND
ND
ND
ND
093
ND
ND
ND
043
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19Ph
enylalanine
1437
16519
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
20Isoleucine
1460
13117
198
198
044
431
069
169
223
113
201
062
039
207
061
089
077
279
200
21Leucine
1543
13117
277
277
054
596
114
268
333
121
263
088
053
287
074
115
140
383
329
22Lysin
e1605
14619
168
168
038
404
079
000
244
163
190
221
071
170
072
138
145
295
163
Total
2186
2186
809
5304
1918
1816
2641
2044
2156
2065
1334
3132
1213
1455
2587
4666
3788
(b)
Num
berAminoacids
RT(m
in)
Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
1As
partate
148
13310
276
321
144
288
145
071
275
046
110
187
198
156
056
121
116
162
128
049
194
102
2Glutamate
277
14713
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
089
096
042
097
ND
ND
022
039
021
ND
ND
032
ND
035
ND
024
126
ND
052
037
4Serin
e603
10509
112
191
092
104
217
111
056
069
114
112
196
101
062
102
101
111
317
079
174
115
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
131
152
098
187
ND
ND
107
ND
ND
ND
ND
053
ND
ND
057
ND
ND
ND
ND
ND
7Histidine
715
15515
048
056
ND
091
ND
ND
ND
ND
ND
ND
ND
ND
ND
030
ND
ND
053
ND
039
031
8Glycine
736
7507
265
313
196
395
175
169
108
204
227
219
337
141
146
224
231
151
633
147
537
203
9Th
reon
ine
762
11912
283
363
184
234
130
072
073
058
094
157
161
109
069
210
171
104
174
070
134
119
10Arginine
847
17420
252
312
198
235
537
247
174
164
219
266
358
266
134
280
301
241
846
188
263
291
11Alanine
897
8909
927
1357
860
1280
1168
904
ND
803
926
984
1289
436
760
1530
1157
651
2385
603
1634
624
12GABA
926
10312
126
183
099
135
048
179
020
174
113
163
436
044
672
116
057
044
077
023
235
097
13Ty
rosin
e1043
18119
233
136
121
330
222
144
064
151
184
193
326
136
109
117
171
163
445
113
220
182
10 Evidence-Based Complementary and Alternative Medicine
(b)Con
tinued
Num
berAminoacids
RT(m
in)Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
14Cy
stine
1226
24030
130
080
065
164
038
ND
ND
ND
ND
ND
ND
ND
ND
076
ND
ND
057
ND
078
ND
15Va
line
1256
11715
434
538
243
605
297
149
070
129
169
265
305
119
162
360
342
167
1612
ND
441
144
16Methion
ine
1282
1492
1069
035
ND
098
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
043
ND
ND
064
ND
069
ND
19Ph
enylalanine
1437
16519
276
253
113
336
142
083
ND
042
046
038
098
048
062
165
087
059
293
062
173
074
20Isoleucine
1460
13117
295
343
129
398
207
095
044
092
105
150
170
085
106
192
267
111
ND
068
ND
ND
21Leucine
1543
13117
474
582
241
659
399
181
071
103
121
147
195
124
161
332
335
162
759
096
314
123
22Lysin
e1605
14619
288
301
108
447
248
163
065
162
161
121
270
116
078
168
140
136
627
108
274
159
Total
470556
1429
3560
8339
7425681149223426
1030
0143
38196525774100353322858597160848
312301
Num
bers1to37
werethe
samplen
ames
(Table1)(a)NDnot
detected
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 3
Table1Spiru
linaprod
uctslistsandtheirc
ountry
oforigin
Snu
mber
Prod
ucttype
Manufacturin
gcompany
Cou
ntry
oforigin
Web
address
1Tablets
TAAU
Australia
PvtL
tdNT
Australia
httpwwwaustralianspirulin
acom
au
2Ca
psules
GeneralNutritionCorpP
ittsburgh
USA
httpwwwgn
ccom
3
Capsules
Naturersquos
Way
Prod
ucts
Inc
Sprin
gvilleUtah
USA
httpwwwnaturesw
ayco
m4
Tablets
Goo
dlsquoN
NaturalN
ewYo
rkUSA
httpwwwgood
andn
aturalsto
reco
m5
Tablets
Now
Food
sBloo
mingdale
USA
httpwwwno
wfood
scom
6Tablets
NatureP
ureInc
LarkspurC
alifo
rnia
USA
7Tablets
Source
NaturalsInc
SantaC
ruzCa
lifornia
USA
httpwwwsourcenaturalscom
8Tablets
Jarrow
Form
ulasL
osAngele
sCA
USA
httpwwwjarrow
com
9Tablets
Earthrise
NutritionalsLL
CIrvineC
AUSA
httpearthrise
com
10Tablets
NutrexHaw
aiiIncKa
ilua-Ko
naH
awaii
USA
httpwwwnu
trex-hawaiicom
11Ca
psules
Pure
PlanetProd
ucts
Inc
Long
BeachCA
USA
httpsw
wwpu
replanetco
m12
Tablets
PuritanrsquosPrideInc
Oakdale
New
York
USA
httpwwwpu
ritanco
m13
Capsules
21stCentury
Health
CareIncAriz
ona
USA
httpwww21stc
enturyvitaminsc
om14
Tablets
JapanAlgae
CoLtdTo
kyo
Japan
httpwwwsp100com
15Tablets
AllSeason
sHealth
Ham
pshire
UnitedKingdo
mhttpwwwcareho
mec
ouk
16
Capsules
FushiW
ellbeing
LtdL
ondo
nUnitedKingdo
mhttpwwwfushicouk
17Tablets
BioveaL
ondo
nUnitedKingdo
mhttpwwwbioveaco
m18
Capsules
ParryNutraceuticalsCh
ennai
India
httpwwwparrynutraceuticalsc
om19
Tablets
Lifestream
InternationalLtdNorthcoteA
uckland
New
Zealand
httpwwwlifestre
amco
nz
20Tablets
Green
Health
Auckland
New
Zealand
httpwwwgreenh
ealth
conz
21Tablets
RBCLifeSciencesIncBu
rnabyBritish
Colum
bia(
BC)
Canada
httpwwwrbclifesciencesco
m22
Tablets
Swiss
HerbalR
emediesL
tdRichmon
dHillO
ntario
Canada
httpwwwsw
issnaturalcom
23Ca
psules
HerbalSele
ctG
uelphOntario
Canada
httpwwwherbalselectca
24Ca
psules
Gou
rmetNutritionFB
IncST
E-Julie
(Quebec)
Canada
httpwwwgourmetfbco
m25
Capsules
TerraV
itaFine
Who
leHerbsB
rampton
Ontario
Canada
httpdsldnlm
nihgov
26Ca
psules
DXN
marketin
gMalaysia
httpwwwdx
nmalaysia
com
27Ca
psules
Hydrolin
aBiotech
PvtIndia
India
httpwwwhydrolinabiotechco
m28
Capsules
Prim
eHealth
Labo
ratorie
sLtd
Austr
alia
Not
available
29Tablets
LaurelHerbalprodu
cts
India
Not
available
30Tablets
IMOCon
trolP
vtLtd
India
httpwwwim
ocon
trolin
31Ca
psules
Acum
enPh
armaceutic
alsP
vtIndia
httpwwwacum
enph
armco
m32
Capsules
Bio-LifeOrganicSpiru
lina
Malaysia
httpshealth
review
2uwordp
ressco
m20100915
biolifespirulin
a33
Tablets
Dharain
Pharmaceutic
als
India
httpwwwmihnaticom
34Tablets
21stCentury
Health
CareIncAriz
ona
USA
httpw
ww21stc
enturyvitaminsc
om
35Tablets
Zuellig
BharmaS
DWB
HD
Malaysia
httpwwwzuelligph
armac
om36
Tablets
Kordels
Spiru
lina
India
httpwwwraku
tencom
37Tablets
ElkenCh
ewable
Malaysia
httpselken4m
rtwordp
ressco
m
4 Evidence-Based Complementary and Alternative Medicine
Table 2 Fatty acids contents (mg100 g in different Spirulina)
Sample
FA
Total1400 1600 161 180 181t 181(n-9)c 181(n-7)c 182t 182(n-6)c 2000 183(n-6) 2002 203n6119891FA119894
09421 09481 09477 09530 09527 09524 095201 09517 09524 09570 0952 09565 095571 001 188 022 029 000 009 002 001 065 001 067 001 001 252 000 207 010 009 000 009 002 001 032 001 016 001 001 2363 001 197 011 034 001 011 005 004 075 001 067 000 000 2554 001 255 027 006 000 005 001 001 085 000 073 001 001 2945 001 189 017 030 001 007 002 001 062 000 053 000 001 2446 001 190 031 013 001 009 004 001 073 001 070 001 002 2457 001 238 017 079 000 006 002 001 055 001 046 001 001 3428 002 281 014 085 000 015 002 001 080 001 050 001 001 3989 001 200 018 011 000 014 007 001 065 001 040 001 001 24510 001 280 014 045 000 009 001 001 087 001 073 001 001 3511 001 210 023 014 000 016 006 001 060 001 037 001 001 26512 001 212 025 006 000 014 007 001 078 001 052 001 001 25913 001 236 027 040 000 011 002 001 080 001 082 001 001 31614 001 248 024 005 000 013 002 002 099 001 082 001 001 29215 001 178 028 004 000 014 001 001 062 001 058 000 000 22616 001 295 015 213 001 011 003 002 067 002 055 000 000 53717 002 191 010 006 001 015 003 003 070 001 057 000 001 22518 002 195 015 017 000 008 003 003 064 001 060 000 000 23819 002 204 012 016 001 014 003 003 068 001 051 000 001 24920 002 212 012 025 001 012 004 004 071 001 066 000 001 26421 001 181 026 021 001 005 002 001 065 001 062 001 001 23522 000 143 022 004 000 012 002 001 054 000 053 001 001 18123 001 327 014 111 000 018 007 001 076 002 044 001 001 47324 001 258 028 006 000 007 002 002 074 001 059 001 001 30125 001 249 026 006 000 010 003 001 072 001 058 001 001 29326 001 173 023 005 000 010 003 001 044 000 039 000 001 21227 002 217 025 006 001 012 006 001 075 001 060 001 001 26328 001 298 023 135 000 013 003 002 090 002 052 001 001 47229 001 158 011 007 000 017 004 001 053 001 049 001 001 19530 002 236 014 005 001 017 003 003 083 001 065 000 000 27531 001 188 015 005 000 019 003 001 054 000 040 001 001 22832 001 175 019 007 000 008 001 001 059 000 049 001 001 2133 001 229 033 006 000 010 003 001 078 001 070 001 001 2834 004 395 021 015 002 023 008 007 137 001 124 001 001 45935 002 210 031 008 000 016 012 001 055 001 060 002 001 26836 001 262 015 008 000 018 004 003 104 001 058 001 001 30537 002 214 014 005 001 011 004 004 077 000 077 000 001 246Numbers 1 to 37 were the sample names (Table 1) Total fatty acids were considered only for PUFA 1400 myristic acid 1600 sapienic acid 161 palmitoleicacid 180 stearic acid 181t elaidic acid 181(n-9)c oleic acid 181(n-7)c vaccenic acid 182t linolelaidic acid 182(n-6)c linoleic acid 2000 eicosenoic acid183(n-6) 120574-linolenic acid 2002 eicosadienoic acid and 203n6 dihomo-gamma-linolenic acid
The weight of the fatty acid (119882119894) was determined as
follows
119882119894= 119882FAME119894 times 119891FA119894 (3)
where 119891FA119894 is conversion factors for the conversion of theFAMEs to their corresponding fatty acids
24 Extraction and Quantification of Sugars Using HPLCThe carbohydrates present in the Spirulina samples werequantified according to the following the standard method[14] Briefly a 100mg portion of the powdered sample wasmixed with 10mL sterile distilled water and boiled at 100∘Cfor one hour After heating the debris was separated bycentrifugation at 10000 rpm for 10min Next the debris-free
Evidence-Based Complementary and Alternative Medicine 5
solution was mixed with 10 5 trichloroacetic acid (TCA)and incubated at room temperature for 10min Subsequentlythe samples were centrifuged at 10000 rpm for 10min andthen filtered through a 045120583mPTFE syringe filter for HPLCanalysis (HP1100 Agilent Co USA) The filtered sampleswere separated through a 300 times 78mm Aminex HPX-87H (Bio-Rad Hercules CA USA) column at 60∘C usinga 50mM H
2SO4monophasic solvent system with a flow
rate of 05mLmin and a column wavelength of 220 nmThe injection sling was 10 120583L The quantifications of theindividual sugars were based on the peak areas and calculatedas equivalents of standard compounds
25 Extraction and Quantification of the Free Amino AcidsUsing HPLC Individual free amino acids were extractedand quantified according to the method of Park et al(2014) with modifications [15] Briefly a 100mg portionof a fine powdered sample was mixed with 12mL of 5trichloroacetic acid (TCA) in a 2mL Eppendorf tube andvigorously shaken for 5minThe slurry sample was incubatedat room temperature for 60min and the upper layer wasthen separated by centrifugation The collected sampleswere diluted with 01M HCl and then filtered through a045 120583m PTFE syringe filter The filtrate was then analysedby HPLC (Agilent Technologies Palo Alto CA) The HPLCanalyses of free amino acids were conducted according to theldquorapid accurate sensitive and reproducible HPLC analysisof amino acids analysisrdquo method with Zorbax Eclipse-AAAcolumns using an Agilent 1100 HPLC system Briefly theseparation of the free amino acidswas performed on aZorbaxEclipse AAA analytical columnThe oven temperature of thecolumn was set at 40∘C and the detection wavelength wasset a 338 nm The injection volume was 10 120583L The mobilephase consisted of a mixture of 40mM NaH
2PO4(pH 78
solvent A) and solvent B (ACN MeOH and water at a45 45 10 vvv ratio) was passed at a rate of 20mLminThe HPLC separation parameters were as follows 0min0 B 0ndash19min 0 B 19ndash211min 57 B 211ndash216min100 B 216ndash25min 100 B 25ndash251min 0 B and 251ndash30min 0 B A sample with an amino acid content of50 pmoL120583L was used as the standard The quantifications ofthe different amino acids were based on the peak areas andwere calculated as equivalents of the standard compoundsAll contents are expressed as milligrams lowast gramfresh weight(FW)
26 Total Polyphenol Analysis The total polyphenols wereestimated according to the method of Folin-Ciocalteu (mod-ified from Lin and Tang 2006) [16] Briefly 10mg of thesample was dissolved in 1mL ofmethanol with 2mL of Folin-Denis reagent and 35 sodium carbonate (Na
2CO3) The
mixture was stored at room temperature for 30min Theabsorbancewasmeasuredwith anUV-Vis spectrophotometerat 750 nm The total polyphenols were calculated as gallicacid equivalents based on a calibration curve for gallic acid(0 25 50 and 100 120583gmL) using the following equation thatwas based on the calibration curve 119910 = minus09706119909 + 38935(1198772 = 09992)
27 In Vitro Antioxidant Assays271 Preparation of the Spirulina One gram of the finepowder sample was mixed with 5mL of ethanol in a screw-cap tube by vortexing for 5min and then kept in an orbitalshaker at 150 rpm for 24 h at room temperature for thoroughextraction After incubation the samples were centrifuged at13000 rpm for 15min at 4∘C The resulting supernatant wasvacuum evaporated at 30∘C and the resulting extract wasused for the antioxidant assays
272 Reducing Power Activity Assay The reducing powerassay was performed according to the method of Oyaizu(1986) [17] Volume of 100 120583L of various concentrations(20ndash100120583gmL) of the samples was mixed with phosphatebuffer (25mL) and 1 potassium ferricyanide (25mL) andincubated at 50∘C for 20 minutes After incubation 25mLof 10 trichloroacetic acid was added and the samples werecentrifuged at 3000 rpm for 10min The upper layer of thesolution (25mL) was mixed with distilled water (25mL)and a freshly prepared 01 ferric chloride solution (05mL)and measured at an absorbance at 700 nm The control wasprepared in a similar manner but the sample was excludedVitamin C at various concentrations was used as a standardIncreases in the absorbance of the reaction mixture indicatedincreases in reducing power
273 DPPH Radical Scavenging Activity Assay The DPPHradical scavenging assay performed according to the methodof Hatano et al (1988) [18] Briefly 100 120583L of the sampleand vitamin C (concentration 100ndash500120583gmL) was mixedwith 200120583L of freshly prepared DPPH solution (1mgmL inmethanol) and incubated at room temperature in the darkfor 30 minutes The controls included only deionized waterand the DPPH solution The absorbances of the resultingsolutions were measured in triplicate at 517 nm followingcentrifugation at 12000 rpm for 10min
The scavenging activity was calculated as follows
scavenging activity () = [1 minus(1198600minus 1198601)
1198602
] times 100 (4)
where1198600is absorbance of sample119860
1is absorbance of blank
and 1198602is absorbance of control
274 Hydroxyl Radical-Scavenging Activity Assay The hy-droxyl radical-scavenging assay was performed accordingto the method of Elizabeth and Rao (1990) with slightmodification [19]The reagents for the assay were freshly pre-pared Briefly one millilitre samples of the working solutionsthat consisted of different ratios of the extract were mixedwith 100mL of 28mM 2-deoxy-2-ribose in phosphate buffer(pH 74) EDTA (104mM 1 1 vv) 100mL H
2O2(1mM)
200mL of FeCl3(200mM) and 100mL ascorbic acid (1mM)
The resulting solutions were mixed evenly and the reactionmixtures were incubated at 37∘C for 1 h The degradation ofdeoxyribose was determined by reading the absorbance at532 nm against the blank solution using a microplate reader(BioRad) Vitamin C was used as a positive control The
6 Evidence-Based Complementary and Alternative Medicine
experiments were conducted in triplicate The scavengingactivities were calculated according to (4)
3 Results and Discussion
31 Variations in the Fatty Acid Contents The total lipidcontents were extracted from the Spirulina samples and theindividual fatty acid compositions of the samples were anal-ysedwith gas chromatography A gas chromatograph coupledwith a flame ionization detector guided the identificationof the following 10 unsaturated and fatty acids (sapienicacid palmitoleic acid elaidic acid oleic acid vaccenic acidlinolelaidic acid linoleic acid eicosenoic acid 120574-linolenicacid and dihomo-gamma-linolenic acid) and three saturatedand fatty acids (myristic acid stearic acid and eicosadienoicacid) (Figure 1 and Table 2) The individual PUFA contentswere quantified by comparing the standard fatty acids withtheir indices The PUFA contents in the Spirulina samplesranged from 301 (DXN Marketing capsules) to 741 g100 g(21st Century HealthCare Inc Arizona tablets Table 4)Interestingly there were comparatively lower amounts of 120574-linolenic acid in the studied Spirulina samples this acidaccounted for an average of 14 of the total PUFAs Theamounts of 120574-linolenic acid ranged from 016 g100 g (Gen-eral Nutrition Corp Pittsburgh capsules) to 124 g100 g(21st Century HealthCare Inc Arizona tablets) HoweverMuhling et al (2005) reported palmitic acid (C160) noted asthe dominant fatty acid in wild Spirulina samples [20] In ourstudy sapienic acid been noted as the major fatty acids Manyin vitro studies have confirmed that 120574-linolenic acid can beused to effectively lower cholesterol and treat atopic eczemabreast cancer and premenstrual disorder [21ndash25] RecentlySajilata et al (2008) extracted and purified S platensisactive components via lipid fractionation silica gel columnpurification and thin-layer chromatographic methods [26]It has been reported that 120572-linolenic acid and 120574-linolenic acidare required for the survival of animals and humans Patilet al (2007) [27] Patil et al (2007) profiled the individualPUFAs fromBacillariophyceae Cyanophyceae RhodophyceaeXanthophyceae Cryptophyceae Prymnesiophyceae Eustig-matophyceae and Chlorophyceae microalgae and suggestedthat the cultivation conditions particularly light intensityand other nutritional components exert important effectson the PUFA compositions [27] Many companies processmicroalgae and supply the results in the forms of capsulesand tables to the market Recently the interest in the use ofSpirulina tablets as energy foods has been renewed due to therelatively high contents of protein phytochemicals and othernutrients in such tablets Efforts should be made to analysethemetabolite profiles of the commercially available Spirulinaproducts because in a previous study we confirmed thattrace amounts of heavymetals that could cause serious healthproblems for consumers are present in some commerciallyavailable Spirulina samples (Table 3) [13]
32 Quantification of Individual Sugars by HPLC Hexose(ie glucose fructose galactose and rhamnose) pentose(ie xylose and ribose) and disaccharide sugars were
35
30
25
(pA
)
20
15
10
FID1 A (FC140730SIG10006D)
20 30 40 50 60 70 80
40
110
120
140
160
161
180
181
t18
1(n
-9)c
181
(n-7
)c18
2t
182
(n-6
)c18
3t
183
(n-6
)c18
3(n
-3)
202
203
n620
4(n
-6)
200
40
1100
11000
1111
120
140
00016
000016
0016
06
006
0006
016
06
016
0061666666666611111111
161
180
181
t18
1(n
-9)c
181
(n1(18
1(n(
181
(nn1(nn
181
(n1(n
181
(n1(n
1(n
181
(n18
1(n
181
(n((1(((((1(18
1(18
1118
111818111111111111
-7)c18
18
182
t111111111
182
(nnn(nn18
2(nnnnnn((((
182(
-6)c)6)6)6)))6))666666666666666666666666
183
t1111111111
183
(nnn18
3(n((((
183((
183(
-6)c6))6)6)))6)6)6))6)6)666666666666666
(18
3(n
88888818818888888111)
-3)
202
20202000020020202002222222222222222222222222 203
n620
4(n
-6)
200
200
200
200
222222222222
Figure 1 Gas chromatograms of the fatty acids identified in theSpirulina samples The peaks numbers refer to the individual fattyacids listed in Table 2
extracted from the 37 Spirulina samples and clearly base-line eluted by HPLC The quantitative results revealed thatglucose fructose and sucrose were present in the greatestamounts followed by xylose ribose galactose and rhamnoseThe total sugar contents of the Spirulina samples ranged from309 to 122167mg100 g (Table 4) Together glucose fructosegalactose and rhamnose accounted for an average of 7385of the total sugar contents Among the major individualsugars glucose accounted for an average of 351mg100 g and52 of the total sugar contents Similarly Chaiklahan et al(2013) reported that rhamnose and glucose account for 53and 13 of the total sugars respectively [28] The rhamnosecontents varied from 8 to 58mg100 g of the total sugarsaccounting for an average of 65 [29] The results indicatedthat among the pentose sugars xylose (average 908) andribose (average 475) were the major components in theSpirulina samples The final outcome of this study is thatthe variations in the individual sugar contents between theSpirulina samples were acceptable due to the processing con-ditions of the each commercial Spirulina product Moreovera literature stated that the extraction of total polysaccharidesand other monosaccharides from Spirulina followed by thequantification of the individual sugar molecules identifiedrhamnose as the predominant sugar followed by glucose andfructose [29]
33 Quantifications of the Individual Amino Acids by HPLCHPLC analyses were used to quantify 22 free amino acidsincluding aspartate asparagine serine glutamine histidineglycine threonine arginine alanine 120574-aminobutyric acid(GABA) tyrosine valine cystine methionine tryptophanphenylalanine isoleucine leucine and lysine in the Spirulinasamples but the separation profiles revealed that only 18 freeamino acids were detectable in the samples but the otherfree amino acids did not detected which may be not presentin the samples (Table 5 and Figure 2) Since the sampleswere marketed by the trademark of different companies andthe nutrient profile of each sample would be varied theseresults revealed that the amounts of total free amino acidsin the 37 Spirulina samples ranged from 1149mg100 g to5614mg100 g The essential amino acid content averagesranged from 206 to 3172mg100 g and contributed averagesthat ranged from 170 to 3918 of the total amino acidsAmong the essential amino acids leucine was identified
Evidence-Based Complementary and Alternative Medicine 7
Table 3 Content of heavy metals in the Spirulina samples available in the market
Sample name Amount mgKg dry weightNickel Zinc Mercury Platinum Magnesium Manganese Total
1 0211 0533 0002 0001 0002 0076 08252 4672 5627 0028 001 003 0587 109543 2016 2397 0017 0008 0018 0603 50594 2147 1628 002 0011 0026 0436 42685 2199 1235 0017 0008 0014 117 46436 3726 6225 0022 0009 0028 0007 100177 2601 2817 0018 0011 0028 0007 54828 3577 1871 0023 0012 0031 0309 58239 3519 3267 0021 0011 003 0008 685610 2442 2041 0017 0008 0021 0137 466611 3785 3007 0019 0008 0028 0008 685512 3597 2859 0019 0008 0024 0006 651313 2857 2901 0019 0008 0026 1643 745414 2852 2114 0026 0008 0042 0011 505315 2437 1568 002 0008 0028 0008 406916 2712 2434 0019 0009 0033 1777 698417 2948 2513 0021 0009 0028 18 731918 2633 1876 0016 0009 0023 1328 588519 3731 3184 0017 0009 0036 2248 922520 2225 1548 0008 0009 0019 1132 494121 1618 1478 0014 0006 002 0005 314122 1589 4626 0016 0007 0023 0009 62723 3272 4428 002 0008 0018 0008 775424 3558 3733 0017 0009 0034 1433 878425 2319 2586 0017 0008 0024 0006 496Numbers 1 to 37 were the sample names (Table 1) Samples 26ndash37 were not analyzed [13]
1
10
2
34 56
7
89 11
1213
14
1516
17 181920 21
22
Inte
nsity
(mAU
)
0
05
1
15
2
0 2 4 6 8Retention time (min)
10 12 14 16 18
Figure 2 HPLC chromatogram of the standard free amino acidsThe peaks numbers refer to the free amino acids listed in Table 5Peaks numbers 1 aspartate 2 glutamate 3 asparagine 4 serine 5 S-methylmethionine (vitamin U) 6 glutamine 7 histidine 8 glycine9 threonine 10 arginine 11 alanine 12 gamma-aminobutyricacid (GABA) 13 tyrosine 14 cystine 15 valine 16 methionine17 norvaline 18 tryptophan 19 phenylalanine 20 isoleucine 21leucine 22 lysine
as predominant (053 to 759mg100 g) and accounted formore than 30 of the essential amino acids The Spirulinatablets marketed in India under the brand name ldquoDharainPharmaceuticalsrdquo exhibited the greatest essential amino acid
contents However the brand name products exhibited valuesthat were comparatively lower than the maximum observedvalues Vitamin U methionine norvaline and tryptophanwere not observed in the samples The essential aminoacid compositions of the microalgae were very similar tothe reported protein contents [30] Clement et al (1967)determined the individual and total amino acid contentsin S maxima [31] This study reported that aspartate wasdominant in the Spirulina samples and that histidine cystinetryptophan and methionine were observed at the lowestlevels This report found a level of aspartate that was similarto that of another report In general many companies marketSpirulina samples as nutraceutical food however there is anurgent need to know the nutritional compositions of each ofthese Spirulina products [32] This study confirmed that theamino acid compositions of Spirulina samples varied with thecompanies that produced them Therefore amino acid-richsamples should be consumed by humans to maintain theirhealth
34 Determination of the Total Phenolic Compounds Figure 3shows the total phenolic compounds calculated as equivalentsto gallic acidThe results revealed that the distributions of thetotal phenolic compounds varied between the commercialproducts The products ranged from 24mgg (21st Century
8 Evidence-Based Complementary and Alternative Medicine
Table 4 Individual sugar contents (mg100 g) in different Spirulina
Sample Sugar contents (mg100 g)Glucose Fructose Xylose Galactose Ribose Sucrose Rhamnose Total
1 172 plusmn 14 38 plusmn 345 25 plusmn 150 155 plusmn 056 18 plusmn 125 54 plusmn 56 25 plusmn 00 3425 plusmn 4332 71 plusmn 1389 44 plusmn 15 88 plusmn 422 78 plusmn 312 5 plusmn 000 65 plusmn 024 18 plusmn 023 363 plusmn 5193 22933 plusmn 2759 59 plusmn 505 55 plusmn 160 7 plusmn 071 45 plusmn 300 54 plusmn 345 35 plusmn 089 46611 plusmn 15774 5133 plusmn 416 68 plusmn 501 21 plusmn 111 54 plusmn 201 7 plusmn 003 29 plusmn 203 19 plusmn 11 24977 plusmn 0385 34733 plusmn 643 15 plusmn 124 46 plusmn 34 8 plusmn 023 25 plusmn 025 84 plusmn 00 58 plusmn 32 58111 plusmn 1926 26 plusmn 346 26 plusmn 28 32 plusmn 1 9 plusmn 017 16 plusmn 000 72 plusmn 130 45 plusmn 1 222 plusmn 3467 14833 plusmn 1041 34 plusmn 178 37 plusmn 403 45 plusmn 312 18 plusmn 187 80 plusmn 915 25 plusmn 028 36644 plusmn 18088 15633 plusmn 1629 15 plusmn 121 39 plusmn 545 11 plusmn 102 31 plusmn 164 45 plusmn 745 14 plusmn 002 33311 plusmn 18869 253 plusmn 520 29 plusmn 598 65 plusmn 84 24 plusmn 20 22 plusmn 003 62 plusmn 605 35 plusmn 096 46333 plusmn 230910 25533 plusmn 1002 78 plusmn 74 98 plusmn 12 NA NA 34 plusmn 510 NA 52844 plusmn 146211 12833 plusmn 289 51 plusmn 089 45 plusmn 71 23 plusmn 185 24 plusmn 007 86 plusmn 688 18 plusmn 112 37711 plusmn 15412 36166 plusmn 66 49 plusmn 104 25 plusmn 090 14 plusmn 22 16 plusmn 008 95 plusmn 00 8 plusmn 032 58955 plusmn 18013 48233 plusmn 58 35 plusmn 208 45 plusmn 503 18 plusmn 103 54 plusmn 109 NA 11 plusmn 078 73077 plusmn 13414 59233 plusmn 615 26 plusmn 10 85 plusmn 523 44 plusmn 29 22 plusmn 107 29 plusmn 215 16 plusmn 047 81144 plusmn 24915 24866 plusmn 8 59 plusmn 68 48 plusmn 49 11 plusmn 22 15 plusmn 03 67 plusmn 168 24 plusmn 49 47089 plusmn 15416 21033 plusmn 95 72 plusmn 56 95 plusmn 012 10 plusmn 062 24 plusmn 13 49 plusmn 90 17 plusmn 063 47244 plusmn 42317 517 plusmn 24 93 plusmn 405 25 plusmn 52 12 plusmn 062 26 plusmn 42 52 plusmn 051 28 plusmn 103 75433 plusmn 11518 88466 plusmn 265 15 plusmn 025 22 plusmn 07 18 plusmn 215 28 plusmn 412 58 plusmn 152 38 plusmn 108 105989 plusmn 32719 33833 plusmn 125 44 plusmn 114 14 plusmn 09 27 plusmn 312 11 plusmn 000 49 plusmn 302 26 plusmn 00 50977 plusmn 03820 752 plusmn 526 51 plusmn 46 16 plusmn 012 9 plusmn 025 22 plusmn 08 67 plusmn 307 45 plusmn 28 98733 plusmn 219321 96866 plusmn 273 50 plusmn 254 18 plusmn 059 58 plusmn 812 24 plusmn 014 84 plusmn 101 19 plusmn 51 120722 plusmn 125222 260 plusmn 229 43 plusmn 63 94 plusmn 126 65 plusmn 474 26 plusmn 295 55 plusmn 078 54 plusmn 290 599 plusmn 17323 10733 plusmn 709 24 plusmn 40 25 plusmn 045 22 plusmn 012 25 plusmn 005 78 plusmn 184 28 plusmn 105 30311 plusmn 53824 15166 plusmn 76 52 plusmn 265 63 plusmn 040 45 plusmn 294 11 plusmn 032 37 plusmn 000 47 plusmn 001 40222 plusmn 38525 10266 plusmn 16 68 plusmn 43 24 plusmn 145 48 plusmn 2 7 plusmn 005 81 plusmn 028 35 plusmn 025 36189 plusmn 32726 7933 plusmn 1115 94 plusmn 50 25 plusmn 209 56 plusmn 623 45 plusmn 110 55 plusmn 612 45 plusmn 17 39977 plusmn 03827 60466 plusmn 53 12 plusmn 137 26 plusmn 423 44 plusmn 256 24 plusmn 000 84 plusmn 071 37 plusmn 032 83055 plusmn 09628 90166 plusmn 76 15 plusmn 07 45 plusmn 23 25 plusmn 045 10 plusmn 007 56 plusmn 183 34 plusmn 000 109555 plusmn 7629 798 plusmn 1311 45 plusmn 524 58 plusmn 112 14 plusmn 157 32 plusmn 118 101 plusmn 1102 36 plusmn 188 110133 plusmn 15030 14416 plusmn 623 48 plusmn 568 48 plusmn 525 60 plusmn 212 22 plusmn 005 28 plusmn 520 28 plusmn 025 39272 plusmn 126231 155 plusmn 83 25 plusmn 262 25 plusmn 236 18 plusmn 45 21 plusmn 007 79 plusmn 623 46 plusmn 069 38966 plusmn 178932 47233 plusmn 135 18 plusmn 20 65 plusmn 425 9 plusmn 012 45 plusmn 108 56 plusmn 102 55 plusmn 568 73477 plusmn 125133 534 plusmn 31 44 plusmn 154 77 plusmn 323 28 plusmn 204 24 plusmn 102 48 plusmn 013 28 plusmn 047 79433 plusmn 98134 158 plusmn 346 58 plusmn 403 49 plusmn 140 34 plusmn 350 58 plusmn 004 21 plusmn 232 47 plusmn 512 44566 plusmn 178935 353 plusmn 256 27 plusmn 245 58 plusmn 10 25 plusmn 326 21 plusmn 179 55 plusmn 00 55 plusmn 011 598 plusmn 34636 798 plusmn 1311 49 plusmn 307 55 plusmn 09 45 plusmn 018 40 plusmn 64 48 plusmn 036 41 plusmn 002 1092 plusmn 138537 17133 plusmn 15 78 plusmn 50 24 plusmn 045 11 plusmn 001 14 plusmn 034 75 plusmn 045 25 plusmn 205 39944 plusmn 10Numbers 1 to 37 were the sample names (Table 1)
HealthCare Inc Arizona) to 244mgg (source NaturalsInc Santa Cruz California) The highest level of totalpolyphenol contents (24mgg) was observed in the tabletsprocured from source Naturals Inc Santa Cruz Califor-nia Miranda et al (1998) claimed that the main phenoliccompounds namely chlorogenic acid synaptic acid salicylicacid trans-cinnamic acid and caffeic acid were commonlypresent in Spirulina [33] The present study also coincideswith the report of Miranda et al (1998) [33] However themetabolic pathways for the formation of phenolics com-pounds in Spirulina and their importance are still unknown
[34]Thepolyphenols contained the ideal chemical structuresand different bioactivities that included anti-inflammatoryantiviral antioxidant antithrombotic vasodilatory and anti-carcinogenic properties [35] Wu et al (2005) demonstratedthat the presence of total phenolic components and othermetabolites are related to antioxidant properties [36]
35 Antioxidant Properties The results revealed that theantioxidant properties of the Spirulina samples were dose-dependent (Figures 4ndash6) The DPPH assay and hydroxylscavenging assay results revealed that all the Spirulina extracts
Evidence-Based Complementary and Alternative Medicine 9
Table5Free
aminoacid
contents(m
g100g
fresh
wt)
indifferent
Spiru
lina
(a)
Num
ber
Aminoacids
RT(m
in)
Molecular
weight
12
34
56
78
910
1112
1314
1516
171
Aspartate
148
13310
137
137
203
266
524
134
366
124
247
097
355
405
333
089
106
169
170
2Glutamate
277
14713
ND(a)
ND(a)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
025
025
ND
092
039
ND
051
407
032
042
027
017
030
023
028
043
046
4Serin
e603
10509
169
169
049
117
114
129
159
202
158
137
092
140
057
161
186
212
118
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
064
064
ND
189
325
055
146
066
ND
053
212
ND
109
ND
065
ND
098
7Histidine
715
15515
ND
ND
ND
072
ND
043
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8Glycine
736
7507
253
253
051
351
128
145
177
230
198
305
ND
159
102
204
236
271
228
9Th
reon
ine
762
11912
154
154
045
212
098
078
171
128
124
145
192
148
084
107
108
163
208
10Arginine
847
17420
281
281
187
187
282
400
304
220
512
470
155
281
159
231
248
512
302
11Alanine
897
8909
ND
ND
ND
1168
ND
ND
ND
ND
ND
ND
ND
812
ND
ND
901
1612
1294
12GABA
926
10312
038
038
021
154
ND
032
035
126
100
084
ND
072
046
032
034
105
132
13Ty
rosin
e1043
18119
208
208
046
304
069
170
181
143
ND
254
ND
166
ND
124
150
219
128
14Cy
stine
1226
24030
ND
ND
ND
145
ND
ND
ND
ND
130
ND
045
ND
086
ND
ND
ND
052
15Va
line
1256
11715
213
213
072
534
076
192
251
ND
ND
107
ND
268
ND
142
120
402
319
16Methion
ine
1282
1492
1ND
ND
ND
080
ND
ND
ND
ND
ND
ND
093
ND
ND
ND
043
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19Ph
enylalanine
1437
16519
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
20Isoleucine
1460
13117
198
198
044
431
069
169
223
113
201
062
039
207
061
089
077
279
200
21Leucine
1543
13117
277
277
054
596
114
268
333
121
263
088
053
287
074
115
140
383
329
22Lysin
e1605
14619
168
168
038
404
079
000
244
163
190
221
071
170
072
138
145
295
163
Total
2186
2186
809
5304
1918
1816
2641
2044
2156
2065
1334
3132
1213
1455
2587
4666
3788
(b)
Num
berAminoacids
RT(m
in)
Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
1As
partate
148
13310
276
321
144
288
145
071
275
046
110
187
198
156
056
121
116
162
128
049
194
102
2Glutamate
277
14713
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
089
096
042
097
ND
ND
022
039
021
ND
ND
032
ND
035
ND
024
126
ND
052
037
4Serin
e603
10509
112
191
092
104
217
111
056
069
114
112
196
101
062
102
101
111
317
079
174
115
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
131
152
098
187
ND
ND
107
ND
ND
ND
ND
053
ND
ND
057
ND
ND
ND
ND
ND
7Histidine
715
15515
048
056
ND
091
ND
ND
ND
ND
ND
ND
ND
ND
ND
030
ND
ND
053
ND
039
031
8Glycine
736
7507
265
313
196
395
175
169
108
204
227
219
337
141
146
224
231
151
633
147
537
203
9Th
reon
ine
762
11912
283
363
184
234
130
072
073
058
094
157
161
109
069
210
171
104
174
070
134
119
10Arginine
847
17420
252
312
198
235
537
247
174
164
219
266
358
266
134
280
301
241
846
188
263
291
11Alanine
897
8909
927
1357
860
1280
1168
904
ND
803
926
984
1289
436
760
1530
1157
651
2385
603
1634
624
12GABA
926
10312
126
183
099
135
048
179
020
174
113
163
436
044
672
116
057
044
077
023
235
097
13Ty
rosin
e1043
18119
233
136
121
330
222
144
064
151
184
193
326
136
109
117
171
163
445
113
220
182
10 Evidence-Based Complementary and Alternative Medicine
(b)Con
tinued
Num
berAminoacids
RT(m
in)Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
14Cy
stine
1226
24030
130
080
065
164
038
ND
ND
ND
ND
ND
ND
ND
ND
076
ND
ND
057
ND
078
ND
15Va
line
1256
11715
434
538
243
605
297
149
070
129
169
265
305
119
162
360
342
167
1612
ND
441
144
16Methion
ine
1282
1492
1069
035
ND
098
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
043
ND
ND
064
ND
069
ND
19Ph
enylalanine
1437
16519
276
253
113
336
142
083
ND
042
046
038
098
048
062
165
087
059
293
062
173
074
20Isoleucine
1460
13117
295
343
129
398
207
095
044
092
105
150
170
085
106
192
267
111
ND
068
ND
ND
21Leucine
1543
13117
474
582
241
659
399
181
071
103
121
147
195
124
161
332
335
162
759
096
314
123
22Lysin
e1605
14619
288
301
108
447
248
163
065
162
161
121
270
116
078
168
140
136
627
108
274
159
Total
470556
1429
3560
8339
7425681149223426
1030
0143
38196525774100353322858597160848
312301
Num
bers1to37
werethe
samplen
ames
(Table1)(a)NDnot
detected
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Evidence-Based Complementary and Alternative Medicine
Table 2 Fatty acids contents (mg100 g in different Spirulina)
Sample
FA
Total1400 1600 161 180 181t 181(n-9)c 181(n-7)c 182t 182(n-6)c 2000 183(n-6) 2002 203n6119891FA119894
09421 09481 09477 09530 09527 09524 095201 09517 09524 09570 0952 09565 095571 001 188 022 029 000 009 002 001 065 001 067 001 001 252 000 207 010 009 000 009 002 001 032 001 016 001 001 2363 001 197 011 034 001 011 005 004 075 001 067 000 000 2554 001 255 027 006 000 005 001 001 085 000 073 001 001 2945 001 189 017 030 001 007 002 001 062 000 053 000 001 2446 001 190 031 013 001 009 004 001 073 001 070 001 002 2457 001 238 017 079 000 006 002 001 055 001 046 001 001 3428 002 281 014 085 000 015 002 001 080 001 050 001 001 3989 001 200 018 011 000 014 007 001 065 001 040 001 001 24510 001 280 014 045 000 009 001 001 087 001 073 001 001 3511 001 210 023 014 000 016 006 001 060 001 037 001 001 26512 001 212 025 006 000 014 007 001 078 001 052 001 001 25913 001 236 027 040 000 011 002 001 080 001 082 001 001 31614 001 248 024 005 000 013 002 002 099 001 082 001 001 29215 001 178 028 004 000 014 001 001 062 001 058 000 000 22616 001 295 015 213 001 011 003 002 067 002 055 000 000 53717 002 191 010 006 001 015 003 003 070 001 057 000 001 22518 002 195 015 017 000 008 003 003 064 001 060 000 000 23819 002 204 012 016 001 014 003 003 068 001 051 000 001 24920 002 212 012 025 001 012 004 004 071 001 066 000 001 26421 001 181 026 021 001 005 002 001 065 001 062 001 001 23522 000 143 022 004 000 012 002 001 054 000 053 001 001 18123 001 327 014 111 000 018 007 001 076 002 044 001 001 47324 001 258 028 006 000 007 002 002 074 001 059 001 001 30125 001 249 026 006 000 010 003 001 072 001 058 001 001 29326 001 173 023 005 000 010 003 001 044 000 039 000 001 21227 002 217 025 006 001 012 006 001 075 001 060 001 001 26328 001 298 023 135 000 013 003 002 090 002 052 001 001 47229 001 158 011 007 000 017 004 001 053 001 049 001 001 19530 002 236 014 005 001 017 003 003 083 001 065 000 000 27531 001 188 015 005 000 019 003 001 054 000 040 001 001 22832 001 175 019 007 000 008 001 001 059 000 049 001 001 2133 001 229 033 006 000 010 003 001 078 001 070 001 001 2834 004 395 021 015 002 023 008 007 137 001 124 001 001 45935 002 210 031 008 000 016 012 001 055 001 060 002 001 26836 001 262 015 008 000 018 004 003 104 001 058 001 001 30537 002 214 014 005 001 011 004 004 077 000 077 000 001 246Numbers 1 to 37 were the sample names (Table 1) Total fatty acids were considered only for PUFA 1400 myristic acid 1600 sapienic acid 161 palmitoleicacid 180 stearic acid 181t elaidic acid 181(n-9)c oleic acid 181(n-7)c vaccenic acid 182t linolelaidic acid 182(n-6)c linoleic acid 2000 eicosenoic acid183(n-6) 120574-linolenic acid 2002 eicosadienoic acid and 203n6 dihomo-gamma-linolenic acid
The weight of the fatty acid (119882119894) was determined as
follows
119882119894= 119882FAME119894 times 119891FA119894 (3)
where 119891FA119894 is conversion factors for the conversion of theFAMEs to their corresponding fatty acids
24 Extraction and Quantification of Sugars Using HPLCThe carbohydrates present in the Spirulina samples werequantified according to the following the standard method[14] Briefly a 100mg portion of the powdered sample wasmixed with 10mL sterile distilled water and boiled at 100∘Cfor one hour After heating the debris was separated bycentrifugation at 10000 rpm for 10min Next the debris-free
Evidence-Based Complementary and Alternative Medicine 5
solution was mixed with 10 5 trichloroacetic acid (TCA)and incubated at room temperature for 10min Subsequentlythe samples were centrifuged at 10000 rpm for 10min andthen filtered through a 045120583mPTFE syringe filter for HPLCanalysis (HP1100 Agilent Co USA) The filtered sampleswere separated through a 300 times 78mm Aminex HPX-87H (Bio-Rad Hercules CA USA) column at 60∘C usinga 50mM H
2SO4monophasic solvent system with a flow
rate of 05mLmin and a column wavelength of 220 nmThe injection sling was 10 120583L The quantifications of theindividual sugars were based on the peak areas and calculatedas equivalents of standard compounds
25 Extraction and Quantification of the Free Amino AcidsUsing HPLC Individual free amino acids were extractedand quantified according to the method of Park et al(2014) with modifications [15] Briefly a 100mg portionof a fine powdered sample was mixed with 12mL of 5trichloroacetic acid (TCA) in a 2mL Eppendorf tube andvigorously shaken for 5minThe slurry sample was incubatedat room temperature for 60min and the upper layer wasthen separated by centrifugation The collected sampleswere diluted with 01M HCl and then filtered through a045 120583m PTFE syringe filter The filtrate was then analysedby HPLC (Agilent Technologies Palo Alto CA) The HPLCanalyses of free amino acids were conducted according to theldquorapid accurate sensitive and reproducible HPLC analysisof amino acids analysisrdquo method with Zorbax Eclipse-AAAcolumns using an Agilent 1100 HPLC system Briefly theseparation of the free amino acidswas performed on aZorbaxEclipse AAA analytical columnThe oven temperature of thecolumn was set at 40∘C and the detection wavelength wasset a 338 nm The injection volume was 10 120583L The mobilephase consisted of a mixture of 40mM NaH
2PO4(pH 78
solvent A) and solvent B (ACN MeOH and water at a45 45 10 vvv ratio) was passed at a rate of 20mLminThe HPLC separation parameters were as follows 0min0 B 0ndash19min 0 B 19ndash211min 57 B 211ndash216min100 B 216ndash25min 100 B 25ndash251min 0 B and 251ndash30min 0 B A sample with an amino acid content of50 pmoL120583L was used as the standard The quantifications ofthe different amino acids were based on the peak areas andwere calculated as equivalents of the standard compoundsAll contents are expressed as milligrams lowast gramfresh weight(FW)
26 Total Polyphenol Analysis The total polyphenols wereestimated according to the method of Folin-Ciocalteu (mod-ified from Lin and Tang 2006) [16] Briefly 10mg of thesample was dissolved in 1mL ofmethanol with 2mL of Folin-Denis reagent and 35 sodium carbonate (Na
2CO3) The
mixture was stored at room temperature for 30min Theabsorbancewasmeasuredwith anUV-Vis spectrophotometerat 750 nm The total polyphenols were calculated as gallicacid equivalents based on a calibration curve for gallic acid(0 25 50 and 100 120583gmL) using the following equation thatwas based on the calibration curve 119910 = minus09706119909 + 38935(1198772 = 09992)
27 In Vitro Antioxidant Assays271 Preparation of the Spirulina One gram of the finepowder sample was mixed with 5mL of ethanol in a screw-cap tube by vortexing for 5min and then kept in an orbitalshaker at 150 rpm for 24 h at room temperature for thoroughextraction After incubation the samples were centrifuged at13000 rpm for 15min at 4∘C The resulting supernatant wasvacuum evaporated at 30∘C and the resulting extract wasused for the antioxidant assays
272 Reducing Power Activity Assay The reducing powerassay was performed according to the method of Oyaizu(1986) [17] Volume of 100 120583L of various concentrations(20ndash100120583gmL) of the samples was mixed with phosphatebuffer (25mL) and 1 potassium ferricyanide (25mL) andincubated at 50∘C for 20 minutes After incubation 25mLof 10 trichloroacetic acid was added and the samples werecentrifuged at 3000 rpm for 10min The upper layer of thesolution (25mL) was mixed with distilled water (25mL)and a freshly prepared 01 ferric chloride solution (05mL)and measured at an absorbance at 700 nm The control wasprepared in a similar manner but the sample was excludedVitamin C at various concentrations was used as a standardIncreases in the absorbance of the reaction mixture indicatedincreases in reducing power
273 DPPH Radical Scavenging Activity Assay The DPPHradical scavenging assay performed according to the methodof Hatano et al (1988) [18] Briefly 100 120583L of the sampleand vitamin C (concentration 100ndash500120583gmL) was mixedwith 200120583L of freshly prepared DPPH solution (1mgmL inmethanol) and incubated at room temperature in the darkfor 30 minutes The controls included only deionized waterand the DPPH solution The absorbances of the resultingsolutions were measured in triplicate at 517 nm followingcentrifugation at 12000 rpm for 10min
The scavenging activity was calculated as follows
scavenging activity () = [1 minus(1198600minus 1198601)
1198602
] times 100 (4)
where1198600is absorbance of sample119860
1is absorbance of blank
and 1198602is absorbance of control
274 Hydroxyl Radical-Scavenging Activity Assay The hy-droxyl radical-scavenging assay was performed accordingto the method of Elizabeth and Rao (1990) with slightmodification [19]The reagents for the assay were freshly pre-pared Briefly one millilitre samples of the working solutionsthat consisted of different ratios of the extract were mixedwith 100mL of 28mM 2-deoxy-2-ribose in phosphate buffer(pH 74) EDTA (104mM 1 1 vv) 100mL H
2O2(1mM)
200mL of FeCl3(200mM) and 100mL ascorbic acid (1mM)
The resulting solutions were mixed evenly and the reactionmixtures were incubated at 37∘C for 1 h The degradation ofdeoxyribose was determined by reading the absorbance at532 nm against the blank solution using a microplate reader(BioRad) Vitamin C was used as a positive control The
6 Evidence-Based Complementary and Alternative Medicine
experiments were conducted in triplicate The scavengingactivities were calculated according to (4)
3 Results and Discussion
31 Variations in the Fatty Acid Contents The total lipidcontents were extracted from the Spirulina samples and theindividual fatty acid compositions of the samples were anal-ysedwith gas chromatography A gas chromatograph coupledwith a flame ionization detector guided the identificationof the following 10 unsaturated and fatty acids (sapienicacid palmitoleic acid elaidic acid oleic acid vaccenic acidlinolelaidic acid linoleic acid eicosenoic acid 120574-linolenicacid and dihomo-gamma-linolenic acid) and three saturatedand fatty acids (myristic acid stearic acid and eicosadienoicacid) (Figure 1 and Table 2) The individual PUFA contentswere quantified by comparing the standard fatty acids withtheir indices The PUFA contents in the Spirulina samplesranged from 301 (DXN Marketing capsules) to 741 g100 g(21st Century HealthCare Inc Arizona tablets Table 4)Interestingly there were comparatively lower amounts of 120574-linolenic acid in the studied Spirulina samples this acidaccounted for an average of 14 of the total PUFAs Theamounts of 120574-linolenic acid ranged from 016 g100 g (Gen-eral Nutrition Corp Pittsburgh capsules) to 124 g100 g(21st Century HealthCare Inc Arizona tablets) HoweverMuhling et al (2005) reported palmitic acid (C160) noted asthe dominant fatty acid in wild Spirulina samples [20] In ourstudy sapienic acid been noted as the major fatty acids Manyin vitro studies have confirmed that 120574-linolenic acid can beused to effectively lower cholesterol and treat atopic eczemabreast cancer and premenstrual disorder [21ndash25] RecentlySajilata et al (2008) extracted and purified S platensisactive components via lipid fractionation silica gel columnpurification and thin-layer chromatographic methods [26]It has been reported that 120572-linolenic acid and 120574-linolenic acidare required for the survival of animals and humans Patilet al (2007) [27] Patil et al (2007) profiled the individualPUFAs fromBacillariophyceae Cyanophyceae RhodophyceaeXanthophyceae Cryptophyceae Prymnesiophyceae Eustig-matophyceae and Chlorophyceae microalgae and suggestedthat the cultivation conditions particularly light intensityand other nutritional components exert important effectson the PUFA compositions [27] Many companies processmicroalgae and supply the results in the forms of capsulesand tables to the market Recently the interest in the use ofSpirulina tablets as energy foods has been renewed due to therelatively high contents of protein phytochemicals and othernutrients in such tablets Efforts should be made to analysethemetabolite profiles of the commercially available Spirulinaproducts because in a previous study we confirmed thattrace amounts of heavymetals that could cause serious healthproblems for consumers are present in some commerciallyavailable Spirulina samples (Table 3) [13]
32 Quantification of Individual Sugars by HPLC Hexose(ie glucose fructose galactose and rhamnose) pentose(ie xylose and ribose) and disaccharide sugars were
35
30
25
(pA
)
20
15
10
FID1 A (FC140730SIG10006D)
20 30 40 50 60 70 80
40
110
120
140
160
161
180
181
t18
1(n
-9)c
181
(n-7
)c18
2t
182
(n-6
)c18
3t
183
(n-6
)c18
3(n
-3)
202
203
n620
4(n
-6)
200
40
1100
11000
1111
120
140
00016
000016
0016
06
006
0006
016
06
016
0061666666666611111111
161
180
181
t18
1(n
-9)c
181
(n1(18
1(n(
181
(nn1(nn
181
(n1(n
181
(n1(n
1(n
181
(n18
1(n
181
(n((1(((((1(18
1(18
1118
111818111111111111
-7)c18
18
182
t111111111
182
(nnn(nn18
2(nnnnnn((((
182(
-6)c)6)6)6)))6))666666666666666666666666
183
t1111111111
183
(nnn18
3(n((((
183((
183(
-6)c6))6)6)))6)6)6))6)6)666666666666666
(18
3(n
88888818818888888111)
-3)
202
20202000020020202002222222222222222222222222 203
n620
4(n
-6)
200
200
200
200
222222222222
Figure 1 Gas chromatograms of the fatty acids identified in theSpirulina samples The peaks numbers refer to the individual fattyacids listed in Table 2
extracted from the 37 Spirulina samples and clearly base-line eluted by HPLC The quantitative results revealed thatglucose fructose and sucrose were present in the greatestamounts followed by xylose ribose galactose and rhamnoseThe total sugar contents of the Spirulina samples ranged from309 to 122167mg100 g (Table 4) Together glucose fructosegalactose and rhamnose accounted for an average of 7385of the total sugar contents Among the major individualsugars glucose accounted for an average of 351mg100 g and52 of the total sugar contents Similarly Chaiklahan et al(2013) reported that rhamnose and glucose account for 53and 13 of the total sugars respectively [28] The rhamnosecontents varied from 8 to 58mg100 g of the total sugarsaccounting for an average of 65 [29] The results indicatedthat among the pentose sugars xylose (average 908) andribose (average 475) were the major components in theSpirulina samples The final outcome of this study is thatthe variations in the individual sugar contents between theSpirulina samples were acceptable due to the processing con-ditions of the each commercial Spirulina product Moreovera literature stated that the extraction of total polysaccharidesand other monosaccharides from Spirulina followed by thequantification of the individual sugar molecules identifiedrhamnose as the predominant sugar followed by glucose andfructose [29]
33 Quantifications of the Individual Amino Acids by HPLCHPLC analyses were used to quantify 22 free amino acidsincluding aspartate asparagine serine glutamine histidineglycine threonine arginine alanine 120574-aminobutyric acid(GABA) tyrosine valine cystine methionine tryptophanphenylalanine isoleucine leucine and lysine in the Spirulinasamples but the separation profiles revealed that only 18 freeamino acids were detectable in the samples but the otherfree amino acids did not detected which may be not presentin the samples (Table 5 and Figure 2) Since the sampleswere marketed by the trademark of different companies andthe nutrient profile of each sample would be varied theseresults revealed that the amounts of total free amino acidsin the 37 Spirulina samples ranged from 1149mg100 g to5614mg100 g The essential amino acid content averagesranged from 206 to 3172mg100 g and contributed averagesthat ranged from 170 to 3918 of the total amino acidsAmong the essential amino acids leucine was identified
Evidence-Based Complementary and Alternative Medicine 7
Table 3 Content of heavy metals in the Spirulina samples available in the market
Sample name Amount mgKg dry weightNickel Zinc Mercury Platinum Magnesium Manganese Total
1 0211 0533 0002 0001 0002 0076 08252 4672 5627 0028 001 003 0587 109543 2016 2397 0017 0008 0018 0603 50594 2147 1628 002 0011 0026 0436 42685 2199 1235 0017 0008 0014 117 46436 3726 6225 0022 0009 0028 0007 100177 2601 2817 0018 0011 0028 0007 54828 3577 1871 0023 0012 0031 0309 58239 3519 3267 0021 0011 003 0008 685610 2442 2041 0017 0008 0021 0137 466611 3785 3007 0019 0008 0028 0008 685512 3597 2859 0019 0008 0024 0006 651313 2857 2901 0019 0008 0026 1643 745414 2852 2114 0026 0008 0042 0011 505315 2437 1568 002 0008 0028 0008 406916 2712 2434 0019 0009 0033 1777 698417 2948 2513 0021 0009 0028 18 731918 2633 1876 0016 0009 0023 1328 588519 3731 3184 0017 0009 0036 2248 922520 2225 1548 0008 0009 0019 1132 494121 1618 1478 0014 0006 002 0005 314122 1589 4626 0016 0007 0023 0009 62723 3272 4428 002 0008 0018 0008 775424 3558 3733 0017 0009 0034 1433 878425 2319 2586 0017 0008 0024 0006 496Numbers 1 to 37 were the sample names (Table 1) Samples 26ndash37 were not analyzed [13]
1
10
2
34 56
7
89 11
1213
14
1516
17 181920 21
22
Inte
nsity
(mAU
)
0
05
1
15
2
0 2 4 6 8Retention time (min)
10 12 14 16 18
Figure 2 HPLC chromatogram of the standard free amino acidsThe peaks numbers refer to the free amino acids listed in Table 5Peaks numbers 1 aspartate 2 glutamate 3 asparagine 4 serine 5 S-methylmethionine (vitamin U) 6 glutamine 7 histidine 8 glycine9 threonine 10 arginine 11 alanine 12 gamma-aminobutyricacid (GABA) 13 tyrosine 14 cystine 15 valine 16 methionine17 norvaline 18 tryptophan 19 phenylalanine 20 isoleucine 21leucine 22 lysine
as predominant (053 to 759mg100 g) and accounted formore than 30 of the essential amino acids The Spirulinatablets marketed in India under the brand name ldquoDharainPharmaceuticalsrdquo exhibited the greatest essential amino acid
contents However the brand name products exhibited valuesthat were comparatively lower than the maximum observedvalues Vitamin U methionine norvaline and tryptophanwere not observed in the samples The essential aminoacid compositions of the microalgae were very similar tothe reported protein contents [30] Clement et al (1967)determined the individual and total amino acid contentsin S maxima [31] This study reported that aspartate wasdominant in the Spirulina samples and that histidine cystinetryptophan and methionine were observed at the lowestlevels This report found a level of aspartate that was similarto that of another report In general many companies marketSpirulina samples as nutraceutical food however there is anurgent need to know the nutritional compositions of each ofthese Spirulina products [32] This study confirmed that theamino acid compositions of Spirulina samples varied with thecompanies that produced them Therefore amino acid-richsamples should be consumed by humans to maintain theirhealth
34 Determination of the Total Phenolic Compounds Figure 3shows the total phenolic compounds calculated as equivalentsto gallic acidThe results revealed that the distributions of thetotal phenolic compounds varied between the commercialproducts The products ranged from 24mgg (21st Century
8 Evidence-Based Complementary and Alternative Medicine
Table 4 Individual sugar contents (mg100 g) in different Spirulina
Sample Sugar contents (mg100 g)Glucose Fructose Xylose Galactose Ribose Sucrose Rhamnose Total
1 172 plusmn 14 38 plusmn 345 25 plusmn 150 155 plusmn 056 18 plusmn 125 54 plusmn 56 25 plusmn 00 3425 plusmn 4332 71 plusmn 1389 44 plusmn 15 88 plusmn 422 78 plusmn 312 5 plusmn 000 65 plusmn 024 18 plusmn 023 363 plusmn 5193 22933 plusmn 2759 59 plusmn 505 55 plusmn 160 7 plusmn 071 45 plusmn 300 54 plusmn 345 35 plusmn 089 46611 plusmn 15774 5133 plusmn 416 68 plusmn 501 21 plusmn 111 54 plusmn 201 7 plusmn 003 29 plusmn 203 19 plusmn 11 24977 plusmn 0385 34733 plusmn 643 15 plusmn 124 46 plusmn 34 8 plusmn 023 25 plusmn 025 84 plusmn 00 58 plusmn 32 58111 plusmn 1926 26 plusmn 346 26 plusmn 28 32 plusmn 1 9 plusmn 017 16 plusmn 000 72 plusmn 130 45 plusmn 1 222 plusmn 3467 14833 plusmn 1041 34 plusmn 178 37 plusmn 403 45 plusmn 312 18 plusmn 187 80 plusmn 915 25 plusmn 028 36644 plusmn 18088 15633 plusmn 1629 15 plusmn 121 39 plusmn 545 11 plusmn 102 31 plusmn 164 45 plusmn 745 14 plusmn 002 33311 plusmn 18869 253 plusmn 520 29 plusmn 598 65 plusmn 84 24 plusmn 20 22 plusmn 003 62 plusmn 605 35 plusmn 096 46333 plusmn 230910 25533 plusmn 1002 78 plusmn 74 98 plusmn 12 NA NA 34 plusmn 510 NA 52844 plusmn 146211 12833 plusmn 289 51 plusmn 089 45 plusmn 71 23 plusmn 185 24 plusmn 007 86 plusmn 688 18 plusmn 112 37711 plusmn 15412 36166 plusmn 66 49 plusmn 104 25 plusmn 090 14 plusmn 22 16 plusmn 008 95 plusmn 00 8 plusmn 032 58955 plusmn 18013 48233 plusmn 58 35 plusmn 208 45 plusmn 503 18 plusmn 103 54 plusmn 109 NA 11 plusmn 078 73077 plusmn 13414 59233 plusmn 615 26 plusmn 10 85 plusmn 523 44 plusmn 29 22 plusmn 107 29 plusmn 215 16 plusmn 047 81144 plusmn 24915 24866 plusmn 8 59 plusmn 68 48 plusmn 49 11 plusmn 22 15 plusmn 03 67 plusmn 168 24 plusmn 49 47089 plusmn 15416 21033 plusmn 95 72 plusmn 56 95 plusmn 012 10 plusmn 062 24 plusmn 13 49 plusmn 90 17 plusmn 063 47244 plusmn 42317 517 plusmn 24 93 plusmn 405 25 plusmn 52 12 plusmn 062 26 plusmn 42 52 plusmn 051 28 plusmn 103 75433 plusmn 11518 88466 plusmn 265 15 plusmn 025 22 plusmn 07 18 plusmn 215 28 plusmn 412 58 plusmn 152 38 plusmn 108 105989 plusmn 32719 33833 plusmn 125 44 plusmn 114 14 plusmn 09 27 plusmn 312 11 plusmn 000 49 plusmn 302 26 plusmn 00 50977 plusmn 03820 752 plusmn 526 51 plusmn 46 16 plusmn 012 9 plusmn 025 22 plusmn 08 67 plusmn 307 45 plusmn 28 98733 plusmn 219321 96866 plusmn 273 50 plusmn 254 18 plusmn 059 58 plusmn 812 24 plusmn 014 84 plusmn 101 19 plusmn 51 120722 plusmn 125222 260 plusmn 229 43 plusmn 63 94 plusmn 126 65 plusmn 474 26 plusmn 295 55 plusmn 078 54 plusmn 290 599 plusmn 17323 10733 plusmn 709 24 plusmn 40 25 plusmn 045 22 plusmn 012 25 plusmn 005 78 plusmn 184 28 plusmn 105 30311 plusmn 53824 15166 plusmn 76 52 plusmn 265 63 plusmn 040 45 plusmn 294 11 plusmn 032 37 plusmn 000 47 plusmn 001 40222 plusmn 38525 10266 plusmn 16 68 plusmn 43 24 plusmn 145 48 plusmn 2 7 plusmn 005 81 plusmn 028 35 plusmn 025 36189 plusmn 32726 7933 plusmn 1115 94 plusmn 50 25 plusmn 209 56 plusmn 623 45 plusmn 110 55 plusmn 612 45 plusmn 17 39977 plusmn 03827 60466 plusmn 53 12 plusmn 137 26 plusmn 423 44 plusmn 256 24 plusmn 000 84 plusmn 071 37 plusmn 032 83055 plusmn 09628 90166 plusmn 76 15 plusmn 07 45 plusmn 23 25 plusmn 045 10 plusmn 007 56 plusmn 183 34 plusmn 000 109555 plusmn 7629 798 plusmn 1311 45 plusmn 524 58 plusmn 112 14 plusmn 157 32 plusmn 118 101 plusmn 1102 36 plusmn 188 110133 plusmn 15030 14416 plusmn 623 48 plusmn 568 48 plusmn 525 60 plusmn 212 22 plusmn 005 28 plusmn 520 28 plusmn 025 39272 plusmn 126231 155 plusmn 83 25 plusmn 262 25 plusmn 236 18 plusmn 45 21 plusmn 007 79 plusmn 623 46 plusmn 069 38966 plusmn 178932 47233 plusmn 135 18 plusmn 20 65 plusmn 425 9 plusmn 012 45 plusmn 108 56 plusmn 102 55 plusmn 568 73477 plusmn 125133 534 plusmn 31 44 plusmn 154 77 plusmn 323 28 plusmn 204 24 plusmn 102 48 plusmn 013 28 plusmn 047 79433 plusmn 98134 158 plusmn 346 58 plusmn 403 49 plusmn 140 34 plusmn 350 58 plusmn 004 21 plusmn 232 47 plusmn 512 44566 plusmn 178935 353 plusmn 256 27 plusmn 245 58 plusmn 10 25 plusmn 326 21 plusmn 179 55 plusmn 00 55 plusmn 011 598 plusmn 34636 798 plusmn 1311 49 plusmn 307 55 plusmn 09 45 plusmn 018 40 plusmn 64 48 plusmn 036 41 plusmn 002 1092 plusmn 138537 17133 plusmn 15 78 plusmn 50 24 plusmn 045 11 plusmn 001 14 plusmn 034 75 plusmn 045 25 plusmn 205 39944 plusmn 10Numbers 1 to 37 were the sample names (Table 1)
HealthCare Inc Arizona) to 244mgg (source NaturalsInc Santa Cruz California) The highest level of totalpolyphenol contents (24mgg) was observed in the tabletsprocured from source Naturals Inc Santa Cruz Califor-nia Miranda et al (1998) claimed that the main phenoliccompounds namely chlorogenic acid synaptic acid salicylicacid trans-cinnamic acid and caffeic acid were commonlypresent in Spirulina [33] The present study also coincideswith the report of Miranda et al (1998) [33] However themetabolic pathways for the formation of phenolics com-pounds in Spirulina and their importance are still unknown
[34]Thepolyphenols contained the ideal chemical structuresand different bioactivities that included anti-inflammatoryantiviral antioxidant antithrombotic vasodilatory and anti-carcinogenic properties [35] Wu et al (2005) demonstratedthat the presence of total phenolic components and othermetabolites are related to antioxidant properties [36]
35 Antioxidant Properties The results revealed that theantioxidant properties of the Spirulina samples were dose-dependent (Figures 4ndash6) The DPPH assay and hydroxylscavenging assay results revealed that all the Spirulina extracts
Evidence-Based Complementary and Alternative Medicine 9
Table5Free
aminoacid
contents(m
g100g
fresh
wt)
indifferent
Spiru
lina
(a)
Num
ber
Aminoacids
RT(m
in)
Molecular
weight
12
34
56
78
910
1112
1314
1516
171
Aspartate
148
13310
137
137
203
266
524
134
366
124
247
097
355
405
333
089
106
169
170
2Glutamate
277
14713
ND(a)
ND(a)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
025
025
ND
092
039
ND
051
407
032
042
027
017
030
023
028
043
046
4Serin
e603
10509
169
169
049
117
114
129
159
202
158
137
092
140
057
161
186
212
118
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
064
064
ND
189
325
055
146
066
ND
053
212
ND
109
ND
065
ND
098
7Histidine
715
15515
ND
ND
ND
072
ND
043
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8Glycine
736
7507
253
253
051
351
128
145
177
230
198
305
ND
159
102
204
236
271
228
9Th
reon
ine
762
11912
154
154
045
212
098
078
171
128
124
145
192
148
084
107
108
163
208
10Arginine
847
17420
281
281
187
187
282
400
304
220
512
470
155
281
159
231
248
512
302
11Alanine
897
8909
ND
ND
ND
1168
ND
ND
ND
ND
ND
ND
ND
812
ND
ND
901
1612
1294
12GABA
926
10312
038
038
021
154
ND
032
035
126
100
084
ND
072
046
032
034
105
132
13Ty
rosin
e1043
18119
208
208
046
304
069
170
181
143
ND
254
ND
166
ND
124
150
219
128
14Cy
stine
1226
24030
ND
ND
ND
145
ND
ND
ND
ND
130
ND
045
ND
086
ND
ND
ND
052
15Va
line
1256
11715
213
213
072
534
076
192
251
ND
ND
107
ND
268
ND
142
120
402
319
16Methion
ine
1282
1492
1ND
ND
ND
080
ND
ND
ND
ND
ND
ND
093
ND
ND
ND
043
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19Ph
enylalanine
1437
16519
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
20Isoleucine
1460
13117
198
198
044
431
069
169
223
113
201
062
039
207
061
089
077
279
200
21Leucine
1543
13117
277
277
054
596
114
268
333
121
263
088
053
287
074
115
140
383
329
22Lysin
e1605
14619
168
168
038
404
079
000
244
163
190
221
071
170
072
138
145
295
163
Total
2186
2186
809
5304
1918
1816
2641
2044
2156
2065
1334
3132
1213
1455
2587
4666
3788
(b)
Num
berAminoacids
RT(m
in)
Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
1As
partate
148
13310
276
321
144
288
145
071
275
046
110
187
198
156
056
121
116
162
128
049
194
102
2Glutamate
277
14713
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
089
096
042
097
ND
ND
022
039
021
ND
ND
032
ND
035
ND
024
126
ND
052
037
4Serin
e603
10509
112
191
092
104
217
111
056
069
114
112
196
101
062
102
101
111
317
079
174
115
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
131
152
098
187
ND
ND
107
ND
ND
ND
ND
053
ND
ND
057
ND
ND
ND
ND
ND
7Histidine
715
15515
048
056
ND
091
ND
ND
ND
ND
ND
ND
ND
ND
ND
030
ND
ND
053
ND
039
031
8Glycine
736
7507
265
313
196
395
175
169
108
204
227
219
337
141
146
224
231
151
633
147
537
203
9Th
reon
ine
762
11912
283
363
184
234
130
072
073
058
094
157
161
109
069
210
171
104
174
070
134
119
10Arginine
847
17420
252
312
198
235
537
247
174
164
219
266
358
266
134
280
301
241
846
188
263
291
11Alanine
897
8909
927
1357
860
1280
1168
904
ND
803
926
984
1289
436
760
1530
1157
651
2385
603
1634
624
12GABA
926
10312
126
183
099
135
048
179
020
174
113
163
436
044
672
116
057
044
077
023
235
097
13Ty
rosin
e1043
18119
233
136
121
330
222
144
064
151
184
193
326
136
109
117
171
163
445
113
220
182
10 Evidence-Based Complementary and Alternative Medicine
(b)Con
tinued
Num
berAminoacids
RT(m
in)Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
14Cy
stine
1226
24030
130
080
065
164
038
ND
ND
ND
ND
ND
ND
ND
ND
076
ND
ND
057
ND
078
ND
15Va
line
1256
11715
434
538
243
605
297
149
070
129
169
265
305
119
162
360
342
167
1612
ND
441
144
16Methion
ine
1282
1492
1069
035
ND
098
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
043
ND
ND
064
ND
069
ND
19Ph
enylalanine
1437
16519
276
253
113
336
142
083
ND
042
046
038
098
048
062
165
087
059
293
062
173
074
20Isoleucine
1460
13117
295
343
129
398
207
095
044
092
105
150
170
085
106
192
267
111
ND
068
ND
ND
21Leucine
1543
13117
474
582
241
659
399
181
071
103
121
147
195
124
161
332
335
162
759
096
314
123
22Lysin
e1605
14619
288
301
108
447
248
163
065
162
161
121
270
116
078
168
140
136
627
108
274
159
Total
470556
1429
3560
8339
7425681149223426
1030
0143
38196525774100353322858597160848
312301
Num
bers1to37
werethe
samplen
ames
(Table1)(a)NDnot
detected
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 5
solution was mixed with 10 5 trichloroacetic acid (TCA)and incubated at room temperature for 10min Subsequentlythe samples were centrifuged at 10000 rpm for 10min andthen filtered through a 045120583mPTFE syringe filter for HPLCanalysis (HP1100 Agilent Co USA) The filtered sampleswere separated through a 300 times 78mm Aminex HPX-87H (Bio-Rad Hercules CA USA) column at 60∘C usinga 50mM H
2SO4monophasic solvent system with a flow
rate of 05mLmin and a column wavelength of 220 nmThe injection sling was 10 120583L The quantifications of theindividual sugars were based on the peak areas and calculatedas equivalents of standard compounds
25 Extraction and Quantification of the Free Amino AcidsUsing HPLC Individual free amino acids were extractedand quantified according to the method of Park et al(2014) with modifications [15] Briefly a 100mg portionof a fine powdered sample was mixed with 12mL of 5trichloroacetic acid (TCA) in a 2mL Eppendorf tube andvigorously shaken for 5minThe slurry sample was incubatedat room temperature for 60min and the upper layer wasthen separated by centrifugation The collected sampleswere diluted with 01M HCl and then filtered through a045 120583m PTFE syringe filter The filtrate was then analysedby HPLC (Agilent Technologies Palo Alto CA) The HPLCanalyses of free amino acids were conducted according to theldquorapid accurate sensitive and reproducible HPLC analysisof amino acids analysisrdquo method with Zorbax Eclipse-AAAcolumns using an Agilent 1100 HPLC system Briefly theseparation of the free amino acidswas performed on aZorbaxEclipse AAA analytical columnThe oven temperature of thecolumn was set at 40∘C and the detection wavelength wasset a 338 nm The injection volume was 10 120583L The mobilephase consisted of a mixture of 40mM NaH
2PO4(pH 78
solvent A) and solvent B (ACN MeOH and water at a45 45 10 vvv ratio) was passed at a rate of 20mLminThe HPLC separation parameters were as follows 0min0 B 0ndash19min 0 B 19ndash211min 57 B 211ndash216min100 B 216ndash25min 100 B 25ndash251min 0 B and 251ndash30min 0 B A sample with an amino acid content of50 pmoL120583L was used as the standard The quantifications ofthe different amino acids were based on the peak areas andwere calculated as equivalents of the standard compoundsAll contents are expressed as milligrams lowast gramfresh weight(FW)
26 Total Polyphenol Analysis The total polyphenols wereestimated according to the method of Folin-Ciocalteu (mod-ified from Lin and Tang 2006) [16] Briefly 10mg of thesample was dissolved in 1mL ofmethanol with 2mL of Folin-Denis reagent and 35 sodium carbonate (Na
2CO3) The
mixture was stored at room temperature for 30min Theabsorbancewasmeasuredwith anUV-Vis spectrophotometerat 750 nm The total polyphenols were calculated as gallicacid equivalents based on a calibration curve for gallic acid(0 25 50 and 100 120583gmL) using the following equation thatwas based on the calibration curve 119910 = minus09706119909 + 38935(1198772 = 09992)
27 In Vitro Antioxidant Assays271 Preparation of the Spirulina One gram of the finepowder sample was mixed with 5mL of ethanol in a screw-cap tube by vortexing for 5min and then kept in an orbitalshaker at 150 rpm for 24 h at room temperature for thoroughextraction After incubation the samples were centrifuged at13000 rpm for 15min at 4∘C The resulting supernatant wasvacuum evaporated at 30∘C and the resulting extract wasused for the antioxidant assays
272 Reducing Power Activity Assay The reducing powerassay was performed according to the method of Oyaizu(1986) [17] Volume of 100 120583L of various concentrations(20ndash100120583gmL) of the samples was mixed with phosphatebuffer (25mL) and 1 potassium ferricyanide (25mL) andincubated at 50∘C for 20 minutes After incubation 25mLof 10 trichloroacetic acid was added and the samples werecentrifuged at 3000 rpm for 10min The upper layer of thesolution (25mL) was mixed with distilled water (25mL)and a freshly prepared 01 ferric chloride solution (05mL)and measured at an absorbance at 700 nm The control wasprepared in a similar manner but the sample was excludedVitamin C at various concentrations was used as a standardIncreases in the absorbance of the reaction mixture indicatedincreases in reducing power
273 DPPH Radical Scavenging Activity Assay The DPPHradical scavenging assay performed according to the methodof Hatano et al (1988) [18] Briefly 100 120583L of the sampleand vitamin C (concentration 100ndash500120583gmL) was mixedwith 200120583L of freshly prepared DPPH solution (1mgmL inmethanol) and incubated at room temperature in the darkfor 30 minutes The controls included only deionized waterand the DPPH solution The absorbances of the resultingsolutions were measured in triplicate at 517 nm followingcentrifugation at 12000 rpm for 10min
The scavenging activity was calculated as follows
scavenging activity () = [1 minus(1198600minus 1198601)
1198602
] times 100 (4)
where1198600is absorbance of sample119860
1is absorbance of blank
and 1198602is absorbance of control
274 Hydroxyl Radical-Scavenging Activity Assay The hy-droxyl radical-scavenging assay was performed accordingto the method of Elizabeth and Rao (1990) with slightmodification [19]The reagents for the assay were freshly pre-pared Briefly one millilitre samples of the working solutionsthat consisted of different ratios of the extract were mixedwith 100mL of 28mM 2-deoxy-2-ribose in phosphate buffer(pH 74) EDTA (104mM 1 1 vv) 100mL H
2O2(1mM)
200mL of FeCl3(200mM) and 100mL ascorbic acid (1mM)
The resulting solutions were mixed evenly and the reactionmixtures were incubated at 37∘C for 1 h The degradation ofdeoxyribose was determined by reading the absorbance at532 nm against the blank solution using a microplate reader(BioRad) Vitamin C was used as a positive control The
6 Evidence-Based Complementary and Alternative Medicine
experiments were conducted in triplicate The scavengingactivities were calculated according to (4)
3 Results and Discussion
31 Variations in the Fatty Acid Contents The total lipidcontents were extracted from the Spirulina samples and theindividual fatty acid compositions of the samples were anal-ysedwith gas chromatography A gas chromatograph coupledwith a flame ionization detector guided the identificationof the following 10 unsaturated and fatty acids (sapienicacid palmitoleic acid elaidic acid oleic acid vaccenic acidlinolelaidic acid linoleic acid eicosenoic acid 120574-linolenicacid and dihomo-gamma-linolenic acid) and three saturatedand fatty acids (myristic acid stearic acid and eicosadienoicacid) (Figure 1 and Table 2) The individual PUFA contentswere quantified by comparing the standard fatty acids withtheir indices The PUFA contents in the Spirulina samplesranged from 301 (DXN Marketing capsules) to 741 g100 g(21st Century HealthCare Inc Arizona tablets Table 4)Interestingly there were comparatively lower amounts of 120574-linolenic acid in the studied Spirulina samples this acidaccounted for an average of 14 of the total PUFAs Theamounts of 120574-linolenic acid ranged from 016 g100 g (Gen-eral Nutrition Corp Pittsburgh capsules) to 124 g100 g(21st Century HealthCare Inc Arizona tablets) HoweverMuhling et al (2005) reported palmitic acid (C160) noted asthe dominant fatty acid in wild Spirulina samples [20] In ourstudy sapienic acid been noted as the major fatty acids Manyin vitro studies have confirmed that 120574-linolenic acid can beused to effectively lower cholesterol and treat atopic eczemabreast cancer and premenstrual disorder [21ndash25] RecentlySajilata et al (2008) extracted and purified S platensisactive components via lipid fractionation silica gel columnpurification and thin-layer chromatographic methods [26]It has been reported that 120572-linolenic acid and 120574-linolenic acidare required for the survival of animals and humans Patilet al (2007) [27] Patil et al (2007) profiled the individualPUFAs fromBacillariophyceae Cyanophyceae RhodophyceaeXanthophyceae Cryptophyceae Prymnesiophyceae Eustig-matophyceae and Chlorophyceae microalgae and suggestedthat the cultivation conditions particularly light intensityand other nutritional components exert important effectson the PUFA compositions [27] Many companies processmicroalgae and supply the results in the forms of capsulesand tables to the market Recently the interest in the use ofSpirulina tablets as energy foods has been renewed due to therelatively high contents of protein phytochemicals and othernutrients in such tablets Efforts should be made to analysethemetabolite profiles of the commercially available Spirulinaproducts because in a previous study we confirmed thattrace amounts of heavymetals that could cause serious healthproblems for consumers are present in some commerciallyavailable Spirulina samples (Table 3) [13]
32 Quantification of Individual Sugars by HPLC Hexose(ie glucose fructose galactose and rhamnose) pentose(ie xylose and ribose) and disaccharide sugars were
35
30
25
(pA
)
20
15
10
FID1 A (FC140730SIG10006D)
20 30 40 50 60 70 80
40
110
120
140
160
161
180
181
t18
1(n
-9)c
181
(n-7
)c18
2t
182
(n-6
)c18
3t
183
(n-6
)c18
3(n
-3)
202
203
n620
4(n
-6)
200
40
1100
11000
1111
120
140
00016
000016
0016
06
006
0006
016
06
016
0061666666666611111111
161
180
181
t18
1(n
-9)c
181
(n1(18
1(n(
181
(nn1(nn
181
(n1(n
181
(n1(n
1(n
181
(n18
1(n
181
(n((1(((((1(18
1(18
1118
111818111111111111
-7)c18
18
182
t111111111
182
(nnn(nn18
2(nnnnnn((((
182(
-6)c)6)6)6)))6))666666666666666666666666
183
t1111111111
183
(nnn18
3(n((((
183((
183(
-6)c6))6)6)))6)6)6))6)6)666666666666666
(18
3(n
88888818818888888111)
-3)
202
20202000020020202002222222222222222222222222 203
n620
4(n
-6)
200
200
200
200
222222222222
Figure 1 Gas chromatograms of the fatty acids identified in theSpirulina samples The peaks numbers refer to the individual fattyacids listed in Table 2
extracted from the 37 Spirulina samples and clearly base-line eluted by HPLC The quantitative results revealed thatglucose fructose and sucrose were present in the greatestamounts followed by xylose ribose galactose and rhamnoseThe total sugar contents of the Spirulina samples ranged from309 to 122167mg100 g (Table 4) Together glucose fructosegalactose and rhamnose accounted for an average of 7385of the total sugar contents Among the major individualsugars glucose accounted for an average of 351mg100 g and52 of the total sugar contents Similarly Chaiklahan et al(2013) reported that rhamnose and glucose account for 53and 13 of the total sugars respectively [28] The rhamnosecontents varied from 8 to 58mg100 g of the total sugarsaccounting for an average of 65 [29] The results indicatedthat among the pentose sugars xylose (average 908) andribose (average 475) were the major components in theSpirulina samples The final outcome of this study is thatthe variations in the individual sugar contents between theSpirulina samples were acceptable due to the processing con-ditions of the each commercial Spirulina product Moreovera literature stated that the extraction of total polysaccharidesand other monosaccharides from Spirulina followed by thequantification of the individual sugar molecules identifiedrhamnose as the predominant sugar followed by glucose andfructose [29]
33 Quantifications of the Individual Amino Acids by HPLCHPLC analyses were used to quantify 22 free amino acidsincluding aspartate asparagine serine glutamine histidineglycine threonine arginine alanine 120574-aminobutyric acid(GABA) tyrosine valine cystine methionine tryptophanphenylalanine isoleucine leucine and lysine in the Spirulinasamples but the separation profiles revealed that only 18 freeamino acids were detectable in the samples but the otherfree amino acids did not detected which may be not presentin the samples (Table 5 and Figure 2) Since the sampleswere marketed by the trademark of different companies andthe nutrient profile of each sample would be varied theseresults revealed that the amounts of total free amino acidsin the 37 Spirulina samples ranged from 1149mg100 g to5614mg100 g The essential amino acid content averagesranged from 206 to 3172mg100 g and contributed averagesthat ranged from 170 to 3918 of the total amino acidsAmong the essential amino acids leucine was identified
Evidence-Based Complementary and Alternative Medicine 7
Table 3 Content of heavy metals in the Spirulina samples available in the market
Sample name Amount mgKg dry weightNickel Zinc Mercury Platinum Magnesium Manganese Total
1 0211 0533 0002 0001 0002 0076 08252 4672 5627 0028 001 003 0587 109543 2016 2397 0017 0008 0018 0603 50594 2147 1628 002 0011 0026 0436 42685 2199 1235 0017 0008 0014 117 46436 3726 6225 0022 0009 0028 0007 100177 2601 2817 0018 0011 0028 0007 54828 3577 1871 0023 0012 0031 0309 58239 3519 3267 0021 0011 003 0008 685610 2442 2041 0017 0008 0021 0137 466611 3785 3007 0019 0008 0028 0008 685512 3597 2859 0019 0008 0024 0006 651313 2857 2901 0019 0008 0026 1643 745414 2852 2114 0026 0008 0042 0011 505315 2437 1568 002 0008 0028 0008 406916 2712 2434 0019 0009 0033 1777 698417 2948 2513 0021 0009 0028 18 731918 2633 1876 0016 0009 0023 1328 588519 3731 3184 0017 0009 0036 2248 922520 2225 1548 0008 0009 0019 1132 494121 1618 1478 0014 0006 002 0005 314122 1589 4626 0016 0007 0023 0009 62723 3272 4428 002 0008 0018 0008 775424 3558 3733 0017 0009 0034 1433 878425 2319 2586 0017 0008 0024 0006 496Numbers 1 to 37 were the sample names (Table 1) Samples 26ndash37 were not analyzed [13]
1
10
2
34 56
7
89 11
1213
14
1516
17 181920 21
22
Inte
nsity
(mAU
)
0
05
1
15
2
0 2 4 6 8Retention time (min)
10 12 14 16 18
Figure 2 HPLC chromatogram of the standard free amino acidsThe peaks numbers refer to the free amino acids listed in Table 5Peaks numbers 1 aspartate 2 glutamate 3 asparagine 4 serine 5 S-methylmethionine (vitamin U) 6 glutamine 7 histidine 8 glycine9 threonine 10 arginine 11 alanine 12 gamma-aminobutyricacid (GABA) 13 tyrosine 14 cystine 15 valine 16 methionine17 norvaline 18 tryptophan 19 phenylalanine 20 isoleucine 21leucine 22 lysine
as predominant (053 to 759mg100 g) and accounted formore than 30 of the essential amino acids The Spirulinatablets marketed in India under the brand name ldquoDharainPharmaceuticalsrdquo exhibited the greatest essential amino acid
contents However the brand name products exhibited valuesthat were comparatively lower than the maximum observedvalues Vitamin U methionine norvaline and tryptophanwere not observed in the samples The essential aminoacid compositions of the microalgae were very similar tothe reported protein contents [30] Clement et al (1967)determined the individual and total amino acid contentsin S maxima [31] This study reported that aspartate wasdominant in the Spirulina samples and that histidine cystinetryptophan and methionine were observed at the lowestlevels This report found a level of aspartate that was similarto that of another report In general many companies marketSpirulina samples as nutraceutical food however there is anurgent need to know the nutritional compositions of each ofthese Spirulina products [32] This study confirmed that theamino acid compositions of Spirulina samples varied with thecompanies that produced them Therefore amino acid-richsamples should be consumed by humans to maintain theirhealth
34 Determination of the Total Phenolic Compounds Figure 3shows the total phenolic compounds calculated as equivalentsto gallic acidThe results revealed that the distributions of thetotal phenolic compounds varied between the commercialproducts The products ranged from 24mgg (21st Century
8 Evidence-Based Complementary and Alternative Medicine
Table 4 Individual sugar contents (mg100 g) in different Spirulina
Sample Sugar contents (mg100 g)Glucose Fructose Xylose Galactose Ribose Sucrose Rhamnose Total
1 172 plusmn 14 38 plusmn 345 25 plusmn 150 155 plusmn 056 18 plusmn 125 54 plusmn 56 25 plusmn 00 3425 plusmn 4332 71 plusmn 1389 44 plusmn 15 88 plusmn 422 78 plusmn 312 5 plusmn 000 65 plusmn 024 18 plusmn 023 363 plusmn 5193 22933 plusmn 2759 59 plusmn 505 55 plusmn 160 7 plusmn 071 45 plusmn 300 54 plusmn 345 35 plusmn 089 46611 plusmn 15774 5133 plusmn 416 68 plusmn 501 21 plusmn 111 54 plusmn 201 7 plusmn 003 29 plusmn 203 19 plusmn 11 24977 plusmn 0385 34733 plusmn 643 15 plusmn 124 46 plusmn 34 8 plusmn 023 25 plusmn 025 84 plusmn 00 58 plusmn 32 58111 plusmn 1926 26 plusmn 346 26 plusmn 28 32 plusmn 1 9 plusmn 017 16 plusmn 000 72 plusmn 130 45 plusmn 1 222 plusmn 3467 14833 plusmn 1041 34 plusmn 178 37 plusmn 403 45 plusmn 312 18 plusmn 187 80 plusmn 915 25 plusmn 028 36644 plusmn 18088 15633 plusmn 1629 15 plusmn 121 39 plusmn 545 11 plusmn 102 31 plusmn 164 45 plusmn 745 14 plusmn 002 33311 plusmn 18869 253 plusmn 520 29 plusmn 598 65 plusmn 84 24 plusmn 20 22 plusmn 003 62 plusmn 605 35 plusmn 096 46333 plusmn 230910 25533 plusmn 1002 78 plusmn 74 98 plusmn 12 NA NA 34 plusmn 510 NA 52844 plusmn 146211 12833 plusmn 289 51 plusmn 089 45 plusmn 71 23 plusmn 185 24 plusmn 007 86 plusmn 688 18 plusmn 112 37711 plusmn 15412 36166 plusmn 66 49 plusmn 104 25 plusmn 090 14 plusmn 22 16 plusmn 008 95 plusmn 00 8 plusmn 032 58955 plusmn 18013 48233 plusmn 58 35 plusmn 208 45 plusmn 503 18 plusmn 103 54 plusmn 109 NA 11 plusmn 078 73077 plusmn 13414 59233 plusmn 615 26 plusmn 10 85 plusmn 523 44 plusmn 29 22 plusmn 107 29 plusmn 215 16 plusmn 047 81144 plusmn 24915 24866 plusmn 8 59 plusmn 68 48 plusmn 49 11 plusmn 22 15 plusmn 03 67 plusmn 168 24 plusmn 49 47089 plusmn 15416 21033 plusmn 95 72 plusmn 56 95 plusmn 012 10 plusmn 062 24 plusmn 13 49 plusmn 90 17 plusmn 063 47244 plusmn 42317 517 plusmn 24 93 plusmn 405 25 plusmn 52 12 plusmn 062 26 plusmn 42 52 plusmn 051 28 plusmn 103 75433 plusmn 11518 88466 plusmn 265 15 plusmn 025 22 plusmn 07 18 plusmn 215 28 plusmn 412 58 plusmn 152 38 plusmn 108 105989 plusmn 32719 33833 plusmn 125 44 plusmn 114 14 plusmn 09 27 plusmn 312 11 plusmn 000 49 plusmn 302 26 plusmn 00 50977 plusmn 03820 752 plusmn 526 51 plusmn 46 16 plusmn 012 9 plusmn 025 22 plusmn 08 67 plusmn 307 45 plusmn 28 98733 plusmn 219321 96866 plusmn 273 50 plusmn 254 18 plusmn 059 58 plusmn 812 24 plusmn 014 84 plusmn 101 19 plusmn 51 120722 plusmn 125222 260 plusmn 229 43 plusmn 63 94 plusmn 126 65 plusmn 474 26 plusmn 295 55 plusmn 078 54 plusmn 290 599 plusmn 17323 10733 plusmn 709 24 plusmn 40 25 plusmn 045 22 plusmn 012 25 plusmn 005 78 plusmn 184 28 plusmn 105 30311 plusmn 53824 15166 plusmn 76 52 plusmn 265 63 plusmn 040 45 plusmn 294 11 plusmn 032 37 plusmn 000 47 plusmn 001 40222 plusmn 38525 10266 plusmn 16 68 plusmn 43 24 plusmn 145 48 plusmn 2 7 plusmn 005 81 plusmn 028 35 plusmn 025 36189 plusmn 32726 7933 plusmn 1115 94 plusmn 50 25 plusmn 209 56 plusmn 623 45 plusmn 110 55 plusmn 612 45 plusmn 17 39977 plusmn 03827 60466 plusmn 53 12 plusmn 137 26 plusmn 423 44 plusmn 256 24 plusmn 000 84 plusmn 071 37 plusmn 032 83055 plusmn 09628 90166 plusmn 76 15 plusmn 07 45 plusmn 23 25 plusmn 045 10 plusmn 007 56 plusmn 183 34 plusmn 000 109555 plusmn 7629 798 plusmn 1311 45 plusmn 524 58 plusmn 112 14 plusmn 157 32 plusmn 118 101 plusmn 1102 36 plusmn 188 110133 plusmn 15030 14416 plusmn 623 48 plusmn 568 48 plusmn 525 60 plusmn 212 22 plusmn 005 28 plusmn 520 28 plusmn 025 39272 plusmn 126231 155 plusmn 83 25 plusmn 262 25 plusmn 236 18 plusmn 45 21 plusmn 007 79 plusmn 623 46 plusmn 069 38966 plusmn 178932 47233 plusmn 135 18 plusmn 20 65 plusmn 425 9 plusmn 012 45 plusmn 108 56 plusmn 102 55 plusmn 568 73477 plusmn 125133 534 plusmn 31 44 plusmn 154 77 plusmn 323 28 plusmn 204 24 plusmn 102 48 plusmn 013 28 plusmn 047 79433 plusmn 98134 158 plusmn 346 58 plusmn 403 49 plusmn 140 34 plusmn 350 58 plusmn 004 21 plusmn 232 47 plusmn 512 44566 plusmn 178935 353 plusmn 256 27 plusmn 245 58 plusmn 10 25 plusmn 326 21 plusmn 179 55 plusmn 00 55 plusmn 011 598 plusmn 34636 798 plusmn 1311 49 plusmn 307 55 plusmn 09 45 plusmn 018 40 plusmn 64 48 plusmn 036 41 plusmn 002 1092 plusmn 138537 17133 plusmn 15 78 plusmn 50 24 plusmn 045 11 plusmn 001 14 plusmn 034 75 plusmn 045 25 plusmn 205 39944 plusmn 10Numbers 1 to 37 were the sample names (Table 1)
HealthCare Inc Arizona) to 244mgg (source NaturalsInc Santa Cruz California) The highest level of totalpolyphenol contents (24mgg) was observed in the tabletsprocured from source Naturals Inc Santa Cruz Califor-nia Miranda et al (1998) claimed that the main phenoliccompounds namely chlorogenic acid synaptic acid salicylicacid trans-cinnamic acid and caffeic acid were commonlypresent in Spirulina [33] The present study also coincideswith the report of Miranda et al (1998) [33] However themetabolic pathways for the formation of phenolics com-pounds in Spirulina and their importance are still unknown
[34]Thepolyphenols contained the ideal chemical structuresand different bioactivities that included anti-inflammatoryantiviral antioxidant antithrombotic vasodilatory and anti-carcinogenic properties [35] Wu et al (2005) demonstratedthat the presence of total phenolic components and othermetabolites are related to antioxidant properties [36]
35 Antioxidant Properties The results revealed that theantioxidant properties of the Spirulina samples were dose-dependent (Figures 4ndash6) The DPPH assay and hydroxylscavenging assay results revealed that all the Spirulina extracts
Evidence-Based Complementary and Alternative Medicine 9
Table5Free
aminoacid
contents(m
g100g
fresh
wt)
indifferent
Spiru
lina
(a)
Num
ber
Aminoacids
RT(m
in)
Molecular
weight
12
34
56
78
910
1112
1314
1516
171
Aspartate
148
13310
137
137
203
266
524
134
366
124
247
097
355
405
333
089
106
169
170
2Glutamate
277
14713
ND(a)
ND(a)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
025
025
ND
092
039
ND
051
407
032
042
027
017
030
023
028
043
046
4Serin
e603
10509
169
169
049
117
114
129
159
202
158
137
092
140
057
161
186
212
118
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
064
064
ND
189
325
055
146
066
ND
053
212
ND
109
ND
065
ND
098
7Histidine
715
15515
ND
ND
ND
072
ND
043
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8Glycine
736
7507
253
253
051
351
128
145
177
230
198
305
ND
159
102
204
236
271
228
9Th
reon
ine
762
11912
154
154
045
212
098
078
171
128
124
145
192
148
084
107
108
163
208
10Arginine
847
17420
281
281
187
187
282
400
304
220
512
470
155
281
159
231
248
512
302
11Alanine
897
8909
ND
ND
ND
1168
ND
ND
ND
ND
ND
ND
ND
812
ND
ND
901
1612
1294
12GABA
926
10312
038
038
021
154
ND
032
035
126
100
084
ND
072
046
032
034
105
132
13Ty
rosin
e1043
18119
208
208
046
304
069
170
181
143
ND
254
ND
166
ND
124
150
219
128
14Cy
stine
1226
24030
ND
ND
ND
145
ND
ND
ND
ND
130
ND
045
ND
086
ND
ND
ND
052
15Va
line
1256
11715
213
213
072
534
076
192
251
ND
ND
107
ND
268
ND
142
120
402
319
16Methion
ine
1282
1492
1ND
ND
ND
080
ND
ND
ND
ND
ND
ND
093
ND
ND
ND
043
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19Ph
enylalanine
1437
16519
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
20Isoleucine
1460
13117
198
198
044
431
069
169
223
113
201
062
039
207
061
089
077
279
200
21Leucine
1543
13117
277
277
054
596
114
268
333
121
263
088
053
287
074
115
140
383
329
22Lysin
e1605
14619
168
168
038
404
079
000
244
163
190
221
071
170
072
138
145
295
163
Total
2186
2186
809
5304
1918
1816
2641
2044
2156
2065
1334
3132
1213
1455
2587
4666
3788
(b)
Num
berAminoacids
RT(m
in)
Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
1As
partate
148
13310
276
321
144
288
145
071
275
046
110
187
198
156
056
121
116
162
128
049
194
102
2Glutamate
277
14713
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
089
096
042
097
ND
ND
022
039
021
ND
ND
032
ND
035
ND
024
126
ND
052
037
4Serin
e603
10509
112
191
092
104
217
111
056
069
114
112
196
101
062
102
101
111
317
079
174
115
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
131
152
098
187
ND
ND
107
ND
ND
ND
ND
053
ND
ND
057
ND
ND
ND
ND
ND
7Histidine
715
15515
048
056
ND
091
ND
ND
ND
ND
ND
ND
ND
ND
ND
030
ND
ND
053
ND
039
031
8Glycine
736
7507
265
313
196
395
175
169
108
204
227
219
337
141
146
224
231
151
633
147
537
203
9Th
reon
ine
762
11912
283
363
184
234
130
072
073
058
094
157
161
109
069
210
171
104
174
070
134
119
10Arginine
847
17420
252
312
198
235
537
247
174
164
219
266
358
266
134
280
301
241
846
188
263
291
11Alanine
897
8909
927
1357
860
1280
1168
904
ND
803
926
984
1289
436
760
1530
1157
651
2385
603
1634
624
12GABA
926
10312
126
183
099
135
048
179
020
174
113
163
436
044
672
116
057
044
077
023
235
097
13Ty
rosin
e1043
18119
233
136
121
330
222
144
064
151
184
193
326
136
109
117
171
163
445
113
220
182
10 Evidence-Based Complementary and Alternative Medicine
(b)Con
tinued
Num
berAminoacids
RT(m
in)Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
14Cy
stine
1226
24030
130
080
065
164
038
ND
ND
ND
ND
ND
ND
ND
ND
076
ND
ND
057
ND
078
ND
15Va
line
1256
11715
434
538
243
605
297
149
070
129
169
265
305
119
162
360
342
167
1612
ND
441
144
16Methion
ine
1282
1492
1069
035
ND
098
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
043
ND
ND
064
ND
069
ND
19Ph
enylalanine
1437
16519
276
253
113
336
142
083
ND
042
046
038
098
048
062
165
087
059
293
062
173
074
20Isoleucine
1460
13117
295
343
129
398
207
095
044
092
105
150
170
085
106
192
267
111
ND
068
ND
ND
21Leucine
1543
13117
474
582
241
659
399
181
071
103
121
147
195
124
161
332
335
162
759
096
314
123
22Lysin
e1605
14619
288
301
108
447
248
163
065
162
161
121
270
116
078
168
140
136
627
108
274
159
Total
470556
1429
3560
8339
7425681149223426
1030
0143
38196525774100353322858597160848
312301
Num
bers1to37
werethe
samplen
ames
(Table1)(a)NDnot
detected
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Evidence-Based Complementary and Alternative Medicine
experiments were conducted in triplicate The scavengingactivities were calculated according to (4)
3 Results and Discussion
31 Variations in the Fatty Acid Contents The total lipidcontents were extracted from the Spirulina samples and theindividual fatty acid compositions of the samples were anal-ysedwith gas chromatography A gas chromatograph coupledwith a flame ionization detector guided the identificationof the following 10 unsaturated and fatty acids (sapienicacid palmitoleic acid elaidic acid oleic acid vaccenic acidlinolelaidic acid linoleic acid eicosenoic acid 120574-linolenicacid and dihomo-gamma-linolenic acid) and three saturatedand fatty acids (myristic acid stearic acid and eicosadienoicacid) (Figure 1 and Table 2) The individual PUFA contentswere quantified by comparing the standard fatty acids withtheir indices The PUFA contents in the Spirulina samplesranged from 301 (DXN Marketing capsules) to 741 g100 g(21st Century HealthCare Inc Arizona tablets Table 4)Interestingly there were comparatively lower amounts of 120574-linolenic acid in the studied Spirulina samples this acidaccounted for an average of 14 of the total PUFAs Theamounts of 120574-linolenic acid ranged from 016 g100 g (Gen-eral Nutrition Corp Pittsburgh capsules) to 124 g100 g(21st Century HealthCare Inc Arizona tablets) HoweverMuhling et al (2005) reported palmitic acid (C160) noted asthe dominant fatty acid in wild Spirulina samples [20] In ourstudy sapienic acid been noted as the major fatty acids Manyin vitro studies have confirmed that 120574-linolenic acid can beused to effectively lower cholesterol and treat atopic eczemabreast cancer and premenstrual disorder [21ndash25] RecentlySajilata et al (2008) extracted and purified S platensisactive components via lipid fractionation silica gel columnpurification and thin-layer chromatographic methods [26]It has been reported that 120572-linolenic acid and 120574-linolenic acidare required for the survival of animals and humans Patilet al (2007) [27] Patil et al (2007) profiled the individualPUFAs fromBacillariophyceae Cyanophyceae RhodophyceaeXanthophyceae Cryptophyceae Prymnesiophyceae Eustig-matophyceae and Chlorophyceae microalgae and suggestedthat the cultivation conditions particularly light intensityand other nutritional components exert important effectson the PUFA compositions [27] Many companies processmicroalgae and supply the results in the forms of capsulesand tables to the market Recently the interest in the use ofSpirulina tablets as energy foods has been renewed due to therelatively high contents of protein phytochemicals and othernutrients in such tablets Efforts should be made to analysethemetabolite profiles of the commercially available Spirulinaproducts because in a previous study we confirmed thattrace amounts of heavymetals that could cause serious healthproblems for consumers are present in some commerciallyavailable Spirulina samples (Table 3) [13]
32 Quantification of Individual Sugars by HPLC Hexose(ie glucose fructose galactose and rhamnose) pentose(ie xylose and ribose) and disaccharide sugars were
35
30
25
(pA
)
20
15
10
FID1 A (FC140730SIG10006D)
20 30 40 50 60 70 80
40
110
120
140
160
161
180
181
t18
1(n
-9)c
181
(n-7
)c18
2t
182
(n-6
)c18
3t
183
(n-6
)c18
3(n
-3)
202
203
n620
4(n
-6)
200
40
1100
11000
1111
120
140
00016
000016
0016
06
006
0006
016
06
016
0061666666666611111111
161
180
181
t18
1(n
-9)c
181
(n1(18
1(n(
181
(nn1(nn
181
(n1(n
181
(n1(n
1(n
181
(n18
1(n
181
(n((1(((((1(18
1(18
1118
111818111111111111
-7)c18
18
182
t111111111
182
(nnn(nn18
2(nnnnnn((((
182(
-6)c)6)6)6)))6))666666666666666666666666
183
t1111111111
183
(nnn18
3(n((((
183((
183(
-6)c6))6)6)))6)6)6))6)6)666666666666666
(18
3(n
88888818818888888111)
-3)
202
20202000020020202002222222222222222222222222 203
n620
4(n
-6)
200
200
200
200
222222222222
Figure 1 Gas chromatograms of the fatty acids identified in theSpirulina samples The peaks numbers refer to the individual fattyacids listed in Table 2
extracted from the 37 Spirulina samples and clearly base-line eluted by HPLC The quantitative results revealed thatglucose fructose and sucrose were present in the greatestamounts followed by xylose ribose galactose and rhamnoseThe total sugar contents of the Spirulina samples ranged from309 to 122167mg100 g (Table 4) Together glucose fructosegalactose and rhamnose accounted for an average of 7385of the total sugar contents Among the major individualsugars glucose accounted for an average of 351mg100 g and52 of the total sugar contents Similarly Chaiklahan et al(2013) reported that rhamnose and glucose account for 53and 13 of the total sugars respectively [28] The rhamnosecontents varied from 8 to 58mg100 g of the total sugarsaccounting for an average of 65 [29] The results indicatedthat among the pentose sugars xylose (average 908) andribose (average 475) were the major components in theSpirulina samples The final outcome of this study is thatthe variations in the individual sugar contents between theSpirulina samples were acceptable due to the processing con-ditions of the each commercial Spirulina product Moreovera literature stated that the extraction of total polysaccharidesand other monosaccharides from Spirulina followed by thequantification of the individual sugar molecules identifiedrhamnose as the predominant sugar followed by glucose andfructose [29]
33 Quantifications of the Individual Amino Acids by HPLCHPLC analyses were used to quantify 22 free amino acidsincluding aspartate asparagine serine glutamine histidineglycine threonine arginine alanine 120574-aminobutyric acid(GABA) tyrosine valine cystine methionine tryptophanphenylalanine isoleucine leucine and lysine in the Spirulinasamples but the separation profiles revealed that only 18 freeamino acids were detectable in the samples but the otherfree amino acids did not detected which may be not presentin the samples (Table 5 and Figure 2) Since the sampleswere marketed by the trademark of different companies andthe nutrient profile of each sample would be varied theseresults revealed that the amounts of total free amino acidsin the 37 Spirulina samples ranged from 1149mg100 g to5614mg100 g The essential amino acid content averagesranged from 206 to 3172mg100 g and contributed averagesthat ranged from 170 to 3918 of the total amino acidsAmong the essential amino acids leucine was identified
Evidence-Based Complementary and Alternative Medicine 7
Table 3 Content of heavy metals in the Spirulina samples available in the market
Sample name Amount mgKg dry weightNickel Zinc Mercury Platinum Magnesium Manganese Total
1 0211 0533 0002 0001 0002 0076 08252 4672 5627 0028 001 003 0587 109543 2016 2397 0017 0008 0018 0603 50594 2147 1628 002 0011 0026 0436 42685 2199 1235 0017 0008 0014 117 46436 3726 6225 0022 0009 0028 0007 100177 2601 2817 0018 0011 0028 0007 54828 3577 1871 0023 0012 0031 0309 58239 3519 3267 0021 0011 003 0008 685610 2442 2041 0017 0008 0021 0137 466611 3785 3007 0019 0008 0028 0008 685512 3597 2859 0019 0008 0024 0006 651313 2857 2901 0019 0008 0026 1643 745414 2852 2114 0026 0008 0042 0011 505315 2437 1568 002 0008 0028 0008 406916 2712 2434 0019 0009 0033 1777 698417 2948 2513 0021 0009 0028 18 731918 2633 1876 0016 0009 0023 1328 588519 3731 3184 0017 0009 0036 2248 922520 2225 1548 0008 0009 0019 1132 494121 1618 1478 0014 0006 002 0005 314122 1589 4626 0016 0007 0023 0009 62723 3272 4428 002 0008 0018 0008 775424 3558 3733 0017 0009 0034 1433 878425 2319 2586 0017 0008 0024 0006 496Numbers 1 to 37 were the sample names (Table 1) Samples 26ndash37 were not analyzed [13]
1
10
2
34 56
7
89 11
1213
14
1516
17 181920 21
22
Inte
nsity
(mAU
)
0
05
1
15
2
0 2 4 6 8Retention time (min)
10 12 14 16 18
Figure 2 HPLC chromatogram of the standard free amino acidsThe peaks numbers refer to the free amino acids listed in Table 5Peaks numbers 1 aspartate 2 glutamate 3 asparagine 4 serine 5 S-methylmethionine (vitamin U) 6 glutamine 7 histidine 8 glycine9 threonine 10 arginine 11 alanine 12 gamma-aminobutyricacid (GABA) 13 tyrosine 14 cystine 15 valine 16 methionine17 norvaline 18 tryptophan 19 phenylalanine 20 isoleucine 21leucine 22 lysine
as predominant (053 to 759mg100 g) and accounted formore than 30 of the essential amino acids The Spirulinatablets marketed in India under the brand name ldquoDharainPharmaceuticalsrdquo exhibited the greatest essential amino acid
contents However the brand name products exhibited valuesthat were comparatively lower than the maximum observedvalues Vitamin U methionine norvaline and tryptophanwere not observed in the samples The essential aminoacid compositions of the microalgae were very similar tothe reported protein contents [30] Clement et al (1967)determined the individual and total amino acid contentsin S maxima [31] This study reported that aspartate wasdominant in the Spirulina samples and that histidine cystinetryptophan and methionine were observed at the lowestlevels This report found a level of aspartate that was similarto that of another report In general many companies marketSpirulina samples as nutraceutical food however there is anurgent need to know the nutritional compositions of each ofthese Spirulina products [32] This study confirmed that theamino acid compositions of Spirulina samples varied with thecompanies that produced them Therefore amino acid-richsamples should be consumed by humans to maintain theirhealth
34 Determination of the Total Phenolic Compounds Figure 3shows the total phenolic compounds calculated as equivalentsto gallic acidThe results revealed that the distributions of thetotal phenolic compounds varied between the commercialproducts The products ranged from 24mgg (21st Century
8 Evidence-Based Complementary and Alternative Medicine
Table 4 Individual sugar contents (mg100 g) in different Spirulina
Sample Sugar contents (mg100 g)Glucose Fructose Xylose Galactose Ribose Sucrose Rhamnose Total
1 172 plusmn 14 38 plusmn 345 25 plusmn 150 155 plusmn 056 18 plusmn 125 54 plusmn 56 25 plusmn 00 3425 plusmn 4332 71 plusmn 1389 44 plusmn 15 88 plusmn 422 78 plusmn 312 5 plusmn 000 65 plusmn 024 18 plusmn 023 363 plusmn 5193 22933 plusmn 2759 59 plusmn 505 55 plusmn 160 7 plusmn 071 45 plusmn 300 54 plusmn 345 35 plusmn 089 46611 plusmn 15774 5133 plusmn 416 68 plusmn 501 21 plusmn 111 54 plusmn 201 7 plusmn 003 29 plusmn 203 19 plusmn 11 24977 plusmn 0385 34733 plusmn 643 15 plusmn 124 46 plusmn 34 8 plusmn 023 25 plusmn 025 84 plusmn 00 58 plusmn 32 58111 plusmn 1926 26 plusmn 346 26 plusmn 28 32 plusmn 1 9 plusmn 017 16 plusmn 000 72 plusmn 130 45 plusmn 1 222 plusmn 3467 14833 plusmn 1041 34 plusmn 178 37 plusmn 403 45 plusmn 312 18 plusmn 187 80 plusmn 915 25 plusmn 028 36644 plusmn 18088 15633 plusmn 1629 15 plusmn 121 39 plusmn 545 11 plusmn 102 31 plusmn 164 45 plusmn 745 14 plusmn 002 33311 plusmn 18869 253 plusmn 520 29 plusmn 598 65 plusmn 84 24 plusmn 20 22 plusmn 003 62 plusmn 605 35 plusmn 096 46333 plusmn 230910 25533 plusmn 1002 78 plusmn 74 98 plusmn 12 NA NA 34 plusmn 510 NA 52844 plusmn 146211 12833 plusmn 289 51 plusmn 089 45 plusmn 71 23 plusmn 185 24 plusmn 007 86 plusmn 688 18 plusmn 112 37711 plusmn 15412 36166 plusmn 66 49 plusmn 104 25 plusmn 090 14 plusmn 22 16 plusmn 008 95 plusmn 00 8 plusmn 032 58955 plusmn 18013 48233 plusmn 58 35 plusmn 208 45 plusmn 503 18 plusmn 103 54 plusmn 109 NA 11 plusmn 078 73077 plusmn 13414 59233 plusmn 615 26 plusmn 10 85 plusmn 523 44 plusmn 29 22 plusmn 107 29 plusmn 215 16 plusmn 047 81144 plusmn 24915 24866 plusmn 8 59 plusmn 68 48 plusmn 49 11 plusmn 22 15 plusmn 03 67 plusmn 168 24 plusmn 49 47089 plusmn 15416 21033 plusmn 95 72 plusmn 56 95 plusmn 012 10 plusmn 062 24 plusmn 13 49 plusmn 90 17 plusmn 063 47244 plusmn 42317 517 plusmn 24 93 plusmn 405 25 plusmn 52 12 plusmn 062 26 plusmn 42 52 plusmn 051 28 plusmn 103 75433 plusmn 11518 88466 plusmn 265 15 plusmn 025 22 plusmn 07 18 plusmn 215 28 plusmn 412 58 plusmn 152 38 plusmn 108 105989 plusmn 32719 33833 plusmn 125 44 plusmn 114 14 plusmn 09 27 plusmn 312 11 plusmn 000 49 plusmn 302 26 plusmn 00 50977 plusmn 03820 752 plusmn 526 51 plusmn 46 16 plusmn 012 9 plusmn 025 22 plusmn 08 67 plusmn 307 45 plusmn 28 98733 plusmn 219321 96866 plusmn 273 50 plusmn 254 18 plusmn 059 58 plusmn 812 24 plusmn 014 84 plusmn 101 19 plusmn 51 120722 plusmn 125222 260 plusmn 229 43 plusmn 63 94 plusmn 126 65 plusmn 474 26 plusmn 295 55 plusmn 078 54 plusmn 290 599 plusmn 17323 10733 plusmn 709 24 plusmn 40 25 plusmn 045 22 plusmn 012 25 plusmn 005 78 plusmn 184 28 plusmn 105 30311 plusmn 53824 15166 plusmn 76 52 plusmn 265 63 plusmn 040 45 plusmn 294 11 plusmn 032 37 plusmn 000 47 plusmn 001 40222 plusmn 38525 10266 plusmn 16 68 plusmn 43 24 plusmn 145 48 plusmn 2 7 plusmn 005 81 plusmn 028 35 plusmn 025 36189 plusmn 32726 7933 plusmn 1115 94 plusmn 50 25 plusmn 209 56 plusmn 623 45 plusmn 110 55 plusmn 612 45 plusmn 17 39977 plusmn 03827 60466 plusmn 53 12 plusmn 137 26 plusmn 423 44 plusmn 256 24 plusmn 000 84 plusmn 071 37 plusmn 032 83055 plusmn 09628 90166 plusmn 76 15 plusmn 07 45 plusmn 23 25 plusmn 045 10 plusmn 007 56 plusmn 183 34 plusmn 000 109555 plusmn 7629 798 plusmn 1311 45 plusmn 524 58 plusmn 112 14 plusmn 157 32 plusmn 118 101 plusmn 1102 36 plusmn 188 110133 plusmn 15030 14416 plusmn 623 48 plusmn 568 48 plusmn 525 60 plusmn 212 22 plusmn 005 28 plusmn 520 28 plusmn 025 39272 plusmn 126231 155 plusmn 83 25 plusmn 262 25 plusmn 236 18 plusmn 45 21 plusmn 007 79 plusmn 623 46 plusmn 069 38966 plusmn 178932 47233 plusmn 135 18 plusmn 20 65 plusmn 425 9 plusmn 012 45 plusmn 108 56 plusmn 102 55 plusmn 568 73477 plusmn 125133 534 plusmn 31 44 plusmn 154 77 plusmn 323 28 plusmn 204 24 plusmn 102 48 plusmn 013 28 plusmn 047 79433 plusmn 98134 158 plusmn 346 58 plusmn 403 49 plusmn 140 34 plusmn 350 58 plusmn 004 21 plusmn 232 47 plusmn 512 44566 plusmn 178935 353 plusmn 256 27 plusmn 245 58 plusmn 10 25 plusmn 326 21 plusmn 179 55 plusmn 00 55 plusmn 011 598 plusmn 34636 798 plusmn 1311 49 plusmn 307 55 plusmn 09 45 plusmn 018 40 plusmn 64 48 plusmn 036 41 plusmn 002 1092 plusmn 138537 17133 plusmn 15 78 plusmn 50 24 plusmn 045 11 plusmn 001 14 plusmn 034 75 plusmn 045 25 plusmn 205 39944 plusmn 10Numbers 1 to 37 were the sample names (Table 1)
HealthCare Inc Arizona) to 244mgg (source NaturalsInc Santa Cruz California) The highest level of totalpolyphenol contents (24mgg) was observed in the tabletsprocured from source Naturals Inc Santa Cruz Califor-nia Miranda et al (1998) claimed that the main phenoliccompounds namely chlorogenic acid synaptic acid salicylicacid trans-cinnamic acid and caffeic acid were commonlypresent in Spirulina [33] The present study also coincideswith the report of Miranda et al (1998) [33] However themetabolic pathways for the formation of phenolics com-pounds in Spirulina and their importance are still unknown
[34]Thepolyphenols contained the ideal chemical structuresand different bioactivities that included anti-inflammatoryantiviral antioxidant antithrombotic vasodilatory and anti-carcinogenic properties [35] Wu et al (2005) demonstratedthat the presence of total phenolic components and othermetabolites are related to antioxidant properties [36]
35 Antioxidant Properties The results revealed that theantioxidant properties of the Spirulina samples were dose-dependent (Figures 4ndash6) The DPPH assay and hydroxylscavenging assay results revealed that all the Spirulina extracts
Evidence-Based Complementary and Alternative Medicine 9
Table5Free
aminoacid
contents(m
g100g
fresh
wt)
indifferent
Spiru
lina
(a)
Num
ber
Aminoacids
RT(m
in)
Molecular
weight
12
34
56
78
910
1112
1314
1516
171
Aspartate
148
13310
137
137
203
266
524
134
366
124
247
097
355
405
333
089
106
169
170
2Glutamate
277
14713
ND(a)
ND(a)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
025
025
ND
092
039
ND
051
407
032
042
027
017
030
023
028
043
046
4Serin
e603
10509
169
169
049
117
114
129
159
202
158
137
092
140
057
161
186
212
118
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
064
064
ND
189
325
055
146
066
ND
053
212
ND
109
ND
065
ND
098
7Histidine
715
15515
ND
ND
ND
072
ND
043
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8Glycine
736
7507
253
253
051
351
128
145
177
230
198
305
ND
159
102
204
236
271
228
9Th
reon
ine
762
11912
154
154
045
212
098
078
171
128
124
145
192
148
084
107
108
163
208
10Arginine
847
17420
281
281
187
187
282
400
304
220
512
470
155
281
159
231
248
512
302
11Alanine
897
8909
ND
ND
ND
1168
ND
ND
ND
ND
ND
ND
ND
812
ND
ND
901
1612
1294
12GABA
926
10312
038
038
021
154
ND
032
035
126
100
084
ND
072
046
032
034
105
132
13Ty
rosin
e1043
18119
208
208
046
304
069
170
181
143
ND
254
ND
166
ND
124
150
219
128
14Cy
stine
1226
24030
ND
ND
ND
145
ND
ND
ND
ND
130
ND
045
ND
086
ND
ND
ND
052
15Va
line
1256
11715
213
213
072
534
076
192
251
ND
ND
107
ND
268
ND
142
120
402
319
16Methion
ine
1282
1492
1ND
ND
ND
080
ND
ND
ND
ND
ND
ND
093
ND
ND
ND
043
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19Ph
enylalanine
1437
16519
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
20Isoleucine
1460
13117
198
198
044
431
069
169
223
113
201
062
039
207
061
089
077
279
200
21Leucine
1543
13117
277
277
054
596
114
268
333
121
263
088
053
287
074
115
140
383
329
22Lysin
e1605
14619
168
168
038
404
079
000
244
163
190
221
071
170
072
138
145
295
163
Total
2186
2186
809
5304
1918
1816
2641
2044
2156
2065
1334
3132
1213
1455
2587
4666
3788
(b)
Num
berAminoacids
RT(m
in)
Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
1As
partate
148
13310
276
321
144
288
145
071
275
046
110
187
198
156
056
121
116
162
128
049
194
102
2Glutamate
277
14713
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
089
096
042
097
ND
ND
022
039
021
ND
ND
032
ND
035
ND
024
126
ND
052
037
4Serin
e603
10509
112
191
092
104
217
111
056
069
114
112
196
101
062
102
101
111
317
079
174
115
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
131
152
098
187
ND
ND
107
ND
ND
ND
ND
053
ND
ND
057
ND
ND
ND
ND
ND
7Histidine
715
15515
048
056
ND
091
ND
ND
ND
ND
ND
ND
ND
ND
ND
030
ND
ND
053
ND
039
031
8Glycine
736
7507
265
313
196
395
175
169
108
204
227
219
337
141
146
224
231
151
633
147
537
203
9Th
reon
ine
762
11912
283
363
184
234
130
072
073
058
094
157
161
109
069
210
171
104
174
070
134
119
10Arginine
847
17420
252
312
198
235
537
247
174
164
219
266
358
266
134
280
301
241
846
188
263
291
11Alanine
897
8909
927
1357
860
1280
1168
904
ND
803
926
984
1289
436
760
1530
1157
651
2385
603
1634
624
12GABA
926
10312
126
183
099
135
048
179
020
174
113
163
436
044
672
116
057
044
077
023
235
097
13Ty
rosin
e1043
18119
233
136
121
330
222
144
064
151
184
193
326
136
109
117
171
163
445
113
220
182
10 Evidence-Based Complementary and Alternative Medicine
(b)Con
tinued
Num
berAminoacids
RT(m
in)Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
14Cy
stine
1226
24030
130
080
065
164
038
ND
ND
ND
ND
ND
ND
ND
ND
076
ND
ND
057
ND
078
ND
15Va
line
1256
11715
434
538
243
605
297
149
070
129
169
265
305
119
162
360
342
167
1612
ND
441
144
16Methion
ine
1282
1492
1069
035
ND
098
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
043
ND
ND
064
ND
069
ND
19Ph
enylalanine
1437
16519
276
253
113
336
142
083
ND
042
046
038
098
048
062
165
087
059
293
062
173
074
20Isoleucine
1460
13117
295
343
129
398
207
095
044
092
105
150
170
085
106
192
267
111
ND
068
ND
ND
21Leucine
1543
13117
474
582
241
659
399
181
071
103
121
147
195
124
161
332
335
162
759
096
314
123
22Lysin
e1605
14619
288
301
108
447
248
163
065
162
161
121
270
116
078
168
140
136
627
108
274
159
Total
470556
1429
3560
8339
7425681149223426
1030
0143
38196525774100353322858597160848
312301
Num
bers1to37
werethe
samplen
ames
(Table1)(a)NDnot
detected
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 7
Table 3 Content of heavy metals in the Spirulina samples available in the market
Sample name Amount mgKg dry weightNickel Zinc Mercury Platinum Magnesium Manganese Total
1 0211 0533 0002 0001 0002 0076 08252 4672 5627 0028 001 003 0587 109543 2016 2397 0017 0008 0018 0603 50594 2147 1628 002 0011 0026 0436 42685 2199 1235 0017 0008 0014 117 46436 3726 6225 0022 0009 0028 0007 100177 2601 2817 0018 0011 0028 0007 54828 3577 1871 0023 0012 0031 0309 58239 3519 3267 0021 0011 003 0008 685610 2442 2041 0017 0008 0021 0137 466611 3785 3007 0019 0008 0028 0008 685512 3597 2859 0019 0008 0024 0006 651313 2857 2901 0019 0008 0026 1643 745414 2852 2114 0026 0008 0042 0011 505315 2437 1568 002 0008 0028 0008 406916 2712 2434 0019 0009 0033 1777 698417 2948 2513 0021 0009 0028 18 731918 2633 1876 0016 0009 0023 1328 588519 3731 3184 0017 0009 0036 2248 922520 2225 1548 0008 0009 0019 1132 494121 1618 1478 0014 0006 002 0005 314122 1589 4626 0016 0007 0023 0009 62723 3272 4428 002 0008 0018 0008 775424 3558 3733 0017 0009 0034 1433 878425 2319 2586 0017 0008 0024 0006 496Numbers 1 to 37 were the sample names (Table 1) Samples 26ndash37 were not analyzed [13]
1
10
2
34 56
7
89 11
1213
14
1516
17 181920 21
22
Inte
nsity
(mAU
)
0
05
1
15
2
0 2 4 6 8Retention time (min)
10 12 14 16 18
Figure 2 HPLC chromatogram of the standard free amino acidsThe peaks numbers refer to the free amino acids listed in Table 5Peaks numbers 1 aspartate 2 glutamate 3 asparagine 4 serine 5 S-methylmethionine (vitamin U) 6 glutamine 7 histidine 8 glycine9 threonine 10 arginine 11 alanine 12 gamma-aminobutyricacid (GABA) 13 tyrosine 14 cystine 15 valine 16 methionine17 norvaline 18 tryptophan 19 phenylalanine 20 isoleucine 21leucine 22 lysine
as predominant (053 to 759mg100 g) and accounted formore than 30 of the essential amino acids The Spirulinatablets marketed in India under the brand name ldquoDharainPharmaceuticalsrdquo exhibited the greatest essential amino acid
contents However the brand name products exhibited valuesthat were comparatively lower than the maximum observedvalues Vitamin U methionine norvaline and tryptophanwere not observed in the samples The essential aminoacid compositions of the microalgae were very similar tothe reported protein contents [30] Clement et al (1967)determined the individual and total amino acid contentsin S maxima [31] This study reported that aspartate wasdominant in the Spirulina samples and that histidine cystinetryptophan and methionine were observed at the lowestlevels This report found a level of aspartate that was similarto that of another report In general many companies marketSpirulina samples as nutraceutical food however there is anurgent need to know the nutritional compositions of each ofthese Spirulina products [32] This study confirmed that theamino acid compositions of Spirulina samples varied with thecompanies that produced them Therefore amino acid-richsamples should be consumed by humans to maintain theirhealth
34 Determination of the Total Phenolic Compounds Figure 3shows the total phenolic compounds calculated as equivalentsto gallic acidThe results revealed that the distributions of thetotal phenolic compounds varied between the commercialproducts The products ranged from 24mgg (21st Century
8 Evidence-Based Complementary and Alternative Medicine
Table 4 Individual sugar contents (mg100 g) in different Spirulina
Sample Sugar contents (mg100 g)Glucose Fructose Xylose Galactose Ribose Sucrose Rhamnose Total
1 172 plusmn 14 38 plusmn 345 25 plusmn 150 155 plusmn 056 18 plusmn 125 54 plusmn 56 25 plusmn 00 3425 plusmn 4332 71 plusmn 1389 44 plusmn 15 88 plusmn 422 78 plusmn 312 5 plusmn 000 65 plusmn 024 18 plusmn 023 363 plusmn 5193 22933 plusmn 2759 59 plusmn 505 55 plusmn 160 7 plusmn 071 45 plusmn 300 54 plusmn 345 35 plusmn 089 46611 plusmn 15774 5133 plusmn 416 68 plusmn 501 21 plusmn 111 54 plusmn 201 7 plusmn 003 29 plusmn 203 19 plusmn 11 24977 plusmn 0385 34733 plusmn 643 15 plusmn 124 46 plusmn 34 8 plusmn 023 25 plusmn 025 84 plusmn 00 58 plusmn 32 58111 plusmn 1926 26 plusmn 346 26 plusmn 28 32 plusmn 1 9 plusmn 017 16 plusmn 000 72 plusmn 130 45 plusmn 1 222 plusmn 3467 14833 plusmn 1041 34 plusmn 178 37 plusmn 403 45 plusmn 312 18 plusmn 187 80 plusmn 915 25 plusmn 028 36644 plusmn 18088 15633 plusmn 1629 15 plusmn 121 39 plusmn 545 11 plusmn 102 31 plusmn 164 45 plusmn 745 14 plusmn 002 33311 plusmn 18869 253 plusmn 520 29 plusmn 598 65 plusmn 84 24 plusmn 20 22 plusmn 003 62 plusmn 605 35 plusmn 096 46333 plusmn 230910 25533 plusmn 1002 78 plusmn 74 98 plusmn 12 NA NA 34 plusmn 510 NA 52844 plusmn 146211 12833 plusmn 289 51 plusmn 089 45 plusmn 71 23 plusmn 185 24 plusmn 007 86 plusmn 688 18 plusmn 112 37711 plusmn 15412 36166 plusmn 66 49 plusmn 104 25 plusmn 090 14 plusmn 22 16 plusmn 008 95 plusmn 00 8 plusmn 032 58955 plusmn 18013 48233 plusmn 58 35 plusmn 208 45 plusmn 503 18 plusmn 103 54 plusmn 109 NA 11 plusmn 078 73077 plusmn 13414 59233 plusmn 615 26 plusmn 10 85 plusmn 523 44 plusmn 29 22 plusmn 107 29 plusmn 215 16 plusmn 047 81144 plusmn 24915 24866 plusmn 8 59 plusmn 68 48 plusmn 49 11 plusmn 22 15 plusmn 03 67 plusmn 168 24 plusmn 49 47089 plusmn 15416 21033 plusmn 95 72 plusmn 56 95 plusmn 012 10 plusmn 062 24 plusmn 13 49 plusmn 90 17 plusmn 063 47244 plusmn 42317 517 plusmn 24 93 plusmn 405 25 plusmn 52 12 plusmn 062 26 plusmn 42 52 plusmn 051 28 plusmn 103 75433 plusmn 11518 88466 plusmn 265 15 plusmn 025 22 plusmn 07 18 plusmn 215 28 plusmn 412 58 plusmn 152 38 plusmn 108 105989 plusmn 32719 33833 plusmn 125 44 plusmn 114 14 plusmn 09 27 plusmn 312 11 plusmn 000 49 plusmn 302 26 plusmn 00 50977 plusmn 03820 752 plusmn 526 51 plusmn 46 16 plusmn 012 9 plusmn 025 22 plusmn 08 67 plusmn 307 45 plusmn 28 98733 plusmn 219321 96866 plusmn 273 50 plusmn 254 18 plusmn 059 58 plusmn 812 24 plusmn 014 84 plusmn 101 19 plusmn 51 120722 plusmn 125222 260 plusmn 229 43 plusmn 63 94 plusmn 126 65 plusmn 474 26 plusmn 295 55 plusmn 078 54 plusmn 290 599 plusmn 17323 10733 plusmn 709 24 plusmn 40 25 plusmn 045 22 plusmn 012 25 plusmn 005 78 plusmn 184 28 plusmn 105 30311 plusmn 53824 15166 plusmn 76 52 plusmn 265 63 plusmn 040 45 plusmn 294 11 plusmn 032 37 plusmn 000 47 plusmn 001 40222 plusmn 38525 10266 plusmn 16 68 plusmn 43 24 plusmn 145 48 plusmn 2 7 plusmn 005 81 plusmn 028 35 plusmn 025 36189 plusmn 32726 7933 plusmn 1115 94 plusmn 50 25 plusmn 209 56 plusmn 623 45 plusmn 110 55 plusmn 612 45 plusmn 17 39977 plusmn 03827 60466 plusmn 53 12 plusmn 137 26 plusmn 423 44 plusmn 256 24 plusmn 000 84 plusmn 071 37 plusmn 032 83055 plusmn 09628 90166 plusmn 76 15 plusmn 07 45 plusmn 23 25 plusmn 045 10 plusmn 007 56 plusmn 183 34 plusmn 000 109555 plusmn 7629 798 plusmn 1311 45 plusmn 524 58 plusmn 112 14 plusmn 157 32 plusmn 118 101 plusmn 1102 36 plusmn 188 110133 plusmn 15030 14416 plusmn 623 48 plusmn 568 48 plusmn 525 60 plusmn 212 22 plusmn 005 28 plusmn 520 28 plusmn 025 39272 plusmn 126231 155 plusmn 83 25 plusmn 262 25 plusmn 236 18 plusmn 45 21 plusmn 007 79 plusmn 623 46 plusmn 069 38966 plusmn 178932 47233 plusmn 135 18 plusmn 20 65 plusmn 425 9 plusmn 012 45 plusmn 108 56 plusmn 102 55 plusmn 568 73477 plusmn 125133 534 plusmn 31 44 plusmn 154 77 plusmn 323 28 plusmn 204 24 plusmn 102 48 plusmn 013 28 plusmn 047 79433 plusmn 98134 158 plusmn 346 58 plusmn 403 49 plusmn 140 34 plusmn 350 58 plusmn 004 21 plusmn 232 47 plusmn 512 44566 plusmn 178935 353 plusmn 256 27 plusmn 245 58 plusmn 10 25 plusmn 326 21 plusmn 179 55 plusmn 00 55 plusmn 011 598 plusmn 34636 798 plusmn 1311 49 plusmn 307 55 plusmn 09 45 plusmn 018 40 plusmn 64 48 plusmn 036 41 plusmn 002 1092 plusmn 138537 17133 plusmn 15 78 plusmn 50 24 plusmn 045 11 plusmn 001 14 plusmn 034 75 plusmn 045 25 plusmn 205 39944 plusmn 10Numbers 1 to 37 were the sample names (Table 1)
HealthCare Inc Arizona) to 244mgg (source NaturalsInc Santa Cruz California) The highest level of totalpolyphenol contents (24mgg) was observed in the tabletsprocured from source Naturals Inc Santa Cruz Califor-nia Miranda et al (1998) claimed that the main phenoliccompounds namely chlorogenic acid synaptic acid salicylicacid trans-cinnamic acid and caffeic acid were commonlypresent in Spirulina [33] The present study also coincideswith the report of Miranda et al (1998) [33] However themetabolic pathways for the formation of phenolics com-pounds in Spirulina and their importance are still unknown
[34]Thepolyphenols contained the ideal chemical structuresand different bioactivities that included anti-inflammatoryantiviral antioxidant antithrombotic vasodilatory and anti-carcinogenic properties [35] Wu et al (2005) demonstratedthat the presence of total phenolic components and othermetabolites are related to antioxidant properties [36]
35 Antioxidant Properties The results revealed that theantioxidant properties of the Spirulina samples were dose-dependent (Figures 4ndash6) The DPPH assay and hydroxylscavenging assay results revealed that all the Spirulina extracts
Evidence-Based Complementary and Alternative Medicine 9
Table5Free
aminoacid
contents(m
g100g
fresh
wt)
indifferent
Spiru
lina
(a)
Num
ber
Aminoacids
RT(m
in)
Molecular
weight
12
34
56
78
910
1112
1314
1516
171
Aspartate
148
13310
137
137
203
266
524
134
366
124
247
097
355
405
333
089
106
169
170
2Glutamate
277
14713
ND(a)
ND(a)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
025
025
ND
092
039
ND
051
407
032
042
027
017
030
023
028
043
046
4Serin
e603
10509
169
169
049
117
114
129
159
202
158
137
092
140
057
161
186
212
118
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
064
064
ND
189
325
055
146
066
ND
053
212
ND
109
ND
065
ND
098
7Histidine
715
15515
ND
ND
ND
072
ND
043
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8Glycine
736
7507
253
253
051
351
128
145
177
230
198
305
ND
159
102
204
236
271
228
9Th
reon
ine
762
11912
154
154
045
212
098
078
171
128
124
145
192
148
084
107
108
163
208
10Arginine
847
17420
281
281
187
187
282
400
304
220
512
470
155
281
159
231
248
512
302
11Alanine
897
8909
ND
ND
ND
1168
ND
ND
ND
ND
ND
ND
ND
812
ND
ND
901
1612
1294
12GABA
926
10312
038
038
021
154
ND
032
035
126
100
084
ND
072
046
032
034
105
132
13Ty
rosin
e1043
18119
208
208
046
304
069
170
181
143
ND
254
ND
166
ND
124
150
219
128
14Cy
stine
1226
24030
ND
ND
ND
145
ND
ND
ND
ND
130
ND
045
ND
086
ND
ND
ND
052
15Va
line
1256
11715
213
213
072
534
076
192
251
ND
ND
107
ND
268
ND
142
120
402
319
16Methion
ine
1282
1492
1ND
ND
ND
080
ND
ND
ND
ND
ND
ND
093
ND
ND
ND
043
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19Ph
enylalanine
1437
16519
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
20Isoleucine
1460
13117
198
198
044
431
069
169
223
113
201
062
039
207
061
089
077
279
200
21Leucine
1543
13117
277
277
054
596
114
268
333
121
263
088
053
287
074
115
140
383
329
22Lysin
e1605
14619
168
168
038
404
079
000
244
163
190
221
071
170
072
138
145
295
163
Total
2186
2186
809
5304
1918
1816
2641
2044
2156
2065
1334
3132
1213
1455
2587
4666
3788
(b)
Num
berAminoacids
RT(m
in)
Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
1As
partate
148
13310
276
321
144
288
145
071
275
046
110
187
198
156
056
121
116
162
128
049
194
102
2Glutamate
277
14713
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
089
096
042
097
ND
ND
022
039
021
ND
ND
032
ND
035
ND
024
126
ND
052
037
4Serin
e603
10509
112
191
092
104
217
111
056
069
114
112
196
101
062
102
101
111
317
079
174
115
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
131
152
098
187
ND
ND
107
ND
ND
ND
ND
053
ND
ND
057
ND
ND
ND
ND
ND
7Histidine
715
15515
048
056
ND
091
ND
ND
ND
ND
ND
ND
ND
ND
ND
030
ND
ND
053
ND
039
031
8Glycine
736
7507
265
313
196
395
175
169
108
204
227
219
337
141
146
224
231
151
633
147
537
203
9Th
reon
ine
762
11912
283
363
184
234
130
072
073
058
094
157
161
109
069
210
171
104
174
070
134
119
10Arginine
847
17420
252
312
198
235
537
247
174
164
219
266
358
266
134
280
301
241
846
188
263
291
11Alanine
897
8909
927
1357
860
1280
1168
904
ND
803
926
984
1289
436
760
1530
1157
651
2385
603
1634
624
12GABA
926
10312
126
183
099
135
048
179
020
174
113
163
436
044
672
116
057
044
077
023
235
097
13Ty
rosin
e1043
18119
233
136
121
330
222
144
064
151
184
193
326
136
109
117
171
163
445
113
220
182
10 Evidence-Based Complementary and Alternative Medicine
(b)Con
tinued
Num
berAminoacids
RT(m
in)Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
14Cy
stine
1226
24030
130
080
065
164
038
ND
ND
ND
ND
ND
ND
ND
ND
076
ND
ND
057
ND
078
ND
15Va
line
1256
11715
434
538
243
605
297
149
070
129
169
265
305
119
162
360
342
167
1612
ND
441
144
16Methion
ine
1282
1492
1069
035
ND
098
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
043
ND
ND
064
ND
069
ND
19Ph
enylalanine
1437
16519
276
253
113
336
142
083
ND
042
046
038
098
048
062
165
087
059
293
062
173
074
20Isoleucine
1460
13117
295
343
129
398
207
095
044
092
105
150
170
085
106
192
267
111
ND
068
ND
ND
21Leucine
1543
13117
474
582
241
659
399
181
071
103
121
147
195
124
161
332
335
162
759
096
314
123
22Lysin
e1605
14619
288
301
108
447
248
163
065
162
161
121
270
116
078
168
140
136
627
108
274
159
Total
470556
1429
3560
8339
7425681149223426
1030
0143
38196525774100353322858597160848
312301
Num
bers1to37
werethe
samplen
ames
(Table1)(a)NDnot
detected
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Evidence-Based Complementary and Alternative Medicine
Table 4 Individual sugar contents (mg100 g) in different Spirulina
Sample Sugar contents (mg100 g)Glucose Fructose Xylose Galactose Ribose Sucrose Rhamnose Total
1 172 plusmn 14 38 plusmn 345 25 plusmn 150 155 plusmn 056 18 plusmn 125 54 plusmn 56 25 plusmn 00 3425 plusmn 4332 71 plusmn 1389 44 plusmn 15 88 plusmn 422 78 plusmn 312 5 plusmn 000 65 plusmn 024 18 plusmn 023 363 plusmn 5193 22933 plusmn 2759 59 plusmn 505 55 plusmn 160 7 plusmn 071 45 plusmn 300 54 plusmn 345 35 plusmn 089 46611 plusmn 15774 5133 plusmn 416 68 plusmn 501 21 plusmn 111 54 plusmn 201 7 plusmn 003 29 plusmn 203 19 plusmn 11 24977 plusmn 0385 34733 plusmn 643 15 plusmn 124 46 plusmn 34 8 plusmn 023 25 plusmn 025 84 plusmn 00 58 plusmn 32 58111 plusmn 1926 26 plusmn 346 26 plusmn 28 32 plusmn 1 9 plusmn 017 16 plusmn 000 72 plusmn 130 45 plusmn 1 222 plusmn 3467 14833 plusmn 1041 34 plusmn 178 37 plusmn 403 45 plusmn 312 18 plusmn 187 80 plusmn 915 25 plusmn 028 36644 plusmn 18088 15633 plusmn 1629 15 plusmn 121 39 plusmn 545 11 plusmn 102 31 plusmn 164 45 plusmn 745 14 plusmn 002 33311 plusmn 18869 253 plusmn 520 29 plusmn 598 65 plusmn 84 24 plusmn 20 22 plusmn 003 62 plusmn 605 35 plusmn 096 46333 plusmn 230910 25533 plusmn 1002 78 plusmn 74 98 plusmn 12 NA NA 34 plusmn 510 NA 52844 plusmn 146211 12833 plusmn 289 51 plusmn 089 45 plusmn 71 23 plusmn 185 24 plusmn 007 86 plusmn 688 18 plusmn 112 37711 plusmn 15412 36166 plusmn 66 49 plusmn 104 25 plusmn 090 14 plusmn 22 16 plusmn 008 95 plusmn 00 8 plusmn 032 58955 plusmn 18013 48233 plusmn 58 35 plusmn 208 45 plusmn 503 18 plusmn 103 54 plusmn 109 NA 11 plusmn 078 73077 plusmn 13414 59233 plusmn 615 26 plusmn 10 85 plusmn 523 44 plusmn 29 22 plusmn 107 29 plusmn 215 16 plusmn 047 81144 plusmn 24915 24866 plusmn 8 59 plusmn 68 48 plusmn 49 11 plusmn 22 15 plusmn 03 67 plusmn 168 24 plusmn 49 47089 plusmn 15416 21033 plusmn 95 72 plusmn 56 95 plusmn 012 10 plusmn 062 24 plusmn 13 49 plusmn 90 17 plusmn 063 47244 plusmn 42317 517 plusmn 24 93 plusmn 405 25 plusmn 52 12 plusmn 062 26 plusmn 42 52 plusmn 051 28 plusmn 103 75433 plusmn 11518 88466 plusmn 265 15 plusmn 025 22 plusmn 07 18 plusmn 215 28 plusmn 412 58 plusmn 152 38 plusmn 108 105989 plusmn 32719 33833 plusmn 125 44 plusmn 114 14 plusmn 09 27 plusmn 312 11 plusmn 000 49 plusmn 302 26 plusmn 00 50977 plusmn 03820 752 plusmn 526 51 plusmn 46 16 plusmn 012 9 plusmn 025 22 plusmn 08 67 plusmn 307 45 plusmn 28 98733 plusmn 219321 96866 plusmn 273 50 plusmn 254 18 plusmn 059 58 plusmn 812 24 plusmn 014 84 plusmn 101 19 plusmn 51 120722 plusmn 125222 260 plusmn 229 43 plusmn 63 94 plusmn 126 65 plusmn 474 26 plusmn 295 55 plusmn 078 54 plusmn 290 599 plusmn 17323 10733 plusmn 709 24 plusmn 40 25 plusmn 045 22 plusmn 012 25 plusmn 005 78 plusmn 184 28 plusmn 105 30311 plusmn 53824 15166 plusmn 76 52 plusmn 265 63 plusmn 040 45 plusmn 294 11 plusmn 032 37 plusmn 000 47 plusmn 001 40222 plusmn 38525 10266 plusmn 16 68 plusmn 43 24 plusmn 145 48 plusmn 2 7 plusmn 005 81 plusmn 028 35 plusmn 025 36189 plusmn 32726 7933 plusmn 1115 94 plusmn 50 25 plusmn 209 56 plusmn 623 45 plusmn 110 55 plusmn 612 45 plusmn 17 39977 plusmn 03827 60466 plusmn 53 12 plusmn 137 26 plusmn 423 44 plusmn 256 24 plusmn 000 84 plusmn 071 37 plusmn 032 83055 plusmn 09628 90166 plusmn 76 15 plusmn 07 45 plusmn 23 25 plusmn 045 10 plusmn 007 56 plusmn 183 34 plusmn 000 109555 plusmn 7629 798 plusmn 1311 45 plusmn 524 58 plusmn 112 14 plusmn 157 32 plusmn 118 101 plusmn 1102 36 plusmn 188 110133 plusmn 15030 14416 plusmn 623 48 plusmn 568 48 plusmn 525 60 plusmn 212 22 plusmn 005 28 plusmn 520 28 plusmn 025 39272 plusmn 126231 155 plusmn 83 25 plusmn 262 25 plusmn 236 18 plusmn 45 21 plusmn 007 79 plusmn 623 46 plusmn 069 38966 plusmn 178932 47233 plusmn 135 18 plusmn 20 65 plusmn 425 9 plusmn 012 45 plusmn 108 56 plusmn 102 55 plusmn 568 73477 plusmn 125133 534 plusmn 31 44 plusmn 154 77 plusmn 323 28 plusmn 204 24 plusmn 102 48 plusmn 013 28 plusmn 047 79433 plusmn 98134 158 plusmn 346 58 plusmn 403 49 plusmn 140 34 plusmn 350 58 plusmn 004 21 plusmn 232 47 plusmn 512 44566 plusmn 178935 353 plusmn 256 27 plusmn 245 58 plusmn 10 25 plusmn 326 21 plusmn 179 55 plusmn 00 55 plusmn 011 598 plusmn 34636 798 plusmn 1311 49 plusmn 307 55 plusmn 09 45 plusmn 018 40 plusmn 64 48 plusmn 036 41 plusmn 002 1092 plusmn 138537 17133 plusmn 15 78 plusmn 50 24 plusmn 045 11 plusmn 001 14 plusmn 034 75 plusmn 045 25 plusmn 205 39944 plusmn 10Numbers 1 to 37 were the sample names (Table 1)
HealthCare Inc Arizona) to 244mgg (source NaturalsInc Santa Cruz California) The highest level of totalpolyphenol contents (24mgg) was observed in the tabletsprocured from source Naturals Inc Santa Cruz Califor-nia Miranda et al (1998) claimed that the main phenoliccompounds namely chlorogenic acid synaptic acid salicylicacid trans-cinnamic acid and caffeic acid were commonlypresent in Spirulina [33] The present study also coincideswith the report of Miranda et al (1998) [33] However themetabolic pathways for the formation of phenolics com-pounds in Spirulina and their importance are still unknown
[34]Thepolyphenols contained the ideal chemical structuresand different bioactivities that included anti-inflammatoryantiviral antioxidant antithrombotic vasodilatory and anti-carcinogenic properties [35] Wu et al (2005) demonstratedthat the presence of total phenolic components and othermetabolites are related to antioxidant properties [36]
35 Antioxidant Properties The results revealed that theantioxidant properties of the Spirulina samples were dose-dependent (Figures 4ndash6) The DPPH assay and hydroxylscavenging assay results revealed that all the Spirulina extracts
Evidence-Based Complementary and Alternative Medicine 9
Table5Free
aminoacid
contents(m
g100g
fresh
wt)
indifferent
Spiru
lina
(a)
Num
ber
Aminoacids
RT(m
in)
Molecular
weight
12
34
56
78
910
1112
1314
1516
171
Aspartate
148
13310
137
137
203
266
524
134
366
124
247
097
355
405
333
089
106
169
170
2Glutamate
277
14713
ND(a)
ND(a)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
025
025
ND
092
039
ND
051
407
032
042
027
017
030
023
028
043
046
4Serin
e603
10509
169
169
049
117
114
129
159
202
158
137
092
140
057
161
186
212
118
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
064
064
ND
189
325
055
146
066
ND
053
212
ND
109
ND
065
ND
098
7Histidine
715
15515
ND
ND
ND
072
ND
043
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8Glycine
736
7507
253
253
051
351
128
145
177
230
198
305
ND
159
102
204
236
271
228
9Th
reon
ine
762
11912
154
154
045
212
098
078
171
128
124
145
192
148
084
107
108
163
208
10Arginine
847
17420
281
281
187
187
282
400
304
220
512
470
155
281
159
231
248
512
302
11Alanine
897
8909
ND
ND
ND
1168
ND
ND
ND
ND
ND
ND
ND
812
ND
ND
901
1612
1294
12GABA
926
10312
038
038
021
154
ND
032
035
126
100
084
ND
072
046
032
034
105
132
13Ty
rosin
e1043
18119
208
208
046
304
069
170
181
143
ND
254
ND
166
ND
124
150
219
128
14Cy
stine
1226
24030
ND
ND
ND
145
ND
ND
ND
ND
130
ND
045
ND
086
ND
ND
ND
052
15Va
line
1256
11715
213
213
072
534
076
192
251
ND
ND
107
ND
268
ND
142
120
402
319
16Methion
ine
1282
1492
1ND
ND
ND
080
ND
ND
ND
ND
ND
ND
093
ND
ND
ND
043
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19Ph
enylalanine
1437
16519
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
20Isoleucine
1460
13117
198
198
044
431
069
169
223
113
201
062
039
207
061
089
077
279
200
21Leucine
1543
13117
277
277
054
596
114
268
333
121
263
088
053
287
074
115
140
383
329
22Lysin
e1605
14619
168
168
038
404
079
000
244
163
190
221
071
170
072
138
145
295
163
Total
2186
2186
809
5304
1918
1816
2641
2044
2156
2065
1334
3132
1213
1455
2587
4666
3788
(b)
Num
berAminoacids
RT(m
in)
Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
1As
partate
148
13310
276
321
144
288
145
071
275
046
110
187
198
156
056
121
116
162
128
049
194
102
2Glutamate
277
14713
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
089
096
042
097
ND
ND
022
039
021
ND
ND
032
ND
035
ND
024
126
ND
052
037
4Serin
e603
10509
112
191
092
104
217
111
056
069
114
112
196
101
062
102
101
111
317
079
174
115
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
131
152
098
187
ND
ND
107
ND
ND
ND
ND
053
ND
ND
057
ND
ND
ND
ND
ND
7Histidine
715
15515
048
056
ND
091
ND
ND
ND
ND
ND
ND
ND
ND
ND
030
ND
ND
053
ND
039
031
8Glycine
736
7507
265
313
196
395
175
169
108
204
227
219
337
141
146
224
231
151
633
147
537
203
9Th
reon
ine
762
11912
283
363
184
234
130
072
073
058
094
157
161
109
069
210
171
104
174
070
134
119
10Arginine
847
17420
252
312
198
235
537
247
174
164
219
266
358
266
134
280
301
241
846
188
263
291
11Alanine
897
8909
927
1357
860
1280
1168
904
ND
803
926
984
1289
436
760
1530
1157
651
2385
603
1634
624
12GABA
926
10312
126
183
099
135
048
179
020
174
113
163
436
044
672
116
057
044
077
023
235
097
13Ty
rosin
e1043
18119
233
136
121
330
222
144
064
151
184
193
326
136
109
117
171
163
445
113
220
182
10 Evidence-Based Complementary and Alternative Medicine
(b)Con
tinued
Num
berAminoacids
RT(m
in)Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
14Cy
stine
1226
24030
130
080
065
164
038
ND
ND
ND
ND
ND
ND
ND
ND
076
ND
ND
057
ND
078
ND
15Va
line
1256
11715
434
538
243
605
297
149
070
129
169
265
305
119
162
360
342
167
1612
ND
441
144
16Methion
ine
1282
1492
1069
035
ND
098
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
043
ND
ND
064
ND
069
ND
19Ph
enylalanine
1437
16519
276
253
113
336
142
083
ND
042
046
038
098
048
062
165
087
059
293
062
173
074
20Isoleucine
1460
13117
295
343
129
398
207
095
044
092
105
150
170
085
106
192
267
111
ND
068
ND
ND
21Leucine
1543
13117
474
582
241
659
399
181
071
103
121
147
195
124
161
332
335
162
759
096
314
123
22Lysin
e1605
14619
288
301
108
447
248
163
065
162
161
121
270
116
078
168
140
136
627
108
274
159
Total
470556
1429
3560
8339
7425681149223426
1030
0143
38196525774100353322858597160848
312301
Num
bers1to37
werethe
samplen
ames
(Table1)(a)NDnot
detected
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 9
Table5Free
aminoacid
contents(m
g100g
fresh
wt)
indifferent
Spiru
lina
(a)
Num
ber
Aminoacids
RT(m
in)
Molecular
weight
12
34
56
78
910
1112
1314
1516
171
Aspartate
148
13310
137
137
203
266
524
134
366
124
247
097
355
405
333
089
106
169
170
2Glutamate
277
14713
ND(a)
ND(a)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
025
025
ND
092
039
ND
051
407
032
042
027
017
030
023
028
043
046
4Serin
e603
10509
169
169
049
117
114
129
159
202
158
137
092
140
057
161
186
212
118
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
064
064
ND
189
325
055
146
066
ND
053
212
ND
109
ND
065
ND
098
7Histidine
715
15515
ND
ND
ND
072
ND
043
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8Glycine
736
7507
253
253
051
351
128
145
177
230
198
305
ND
159
102
204
236
271
228
9Th
reon
ine
762
11912
154
154
045
212
098
078
171
128
124
145
192
148
084
107
108
163
208
10Arginine
847
17420
281
281
187
187
282
400
304
220
512
470
155
281
159
231
248
512
302
11Alanine
897
8909
ND
ND
ND
1168
ND
ND
ND
ND
ND
ND
ND
812
ND
ND
901
1612
1294
12GABA
926
10312
038
038
021
154
ND
032
035
126
100
084
ND
072
046
032
034
105
132
13Ty
rosin
e1043
18119
208
208
046
304
069
170
181
143
ND
254
ND
166
ND
124
150
219
128
14Cy
stine
1226
24030
ND
ND
ND
145
ND
ND
ND
ND
130
ND
045
ND
086
ND
ND
ND
052
15Va
line
1256
11715
213
213
072
534
076
192
251
ND
ND
107
ND
268
ND
142
120
402
319
16Methion
ine
1282
1492
1ND
ND
ND
080
ND
ND
ND
ND
ND
ND
093
ND
ND
ND
043
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19Ph
enylalanine
1437
16519
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
20Isoleucine
1460
13117
198
198
044
431
069
169
223
113
201
062
039
207
061
089
077
279
200
21Leucine
1543
13117
277
277
054
596
114
268
333
121
263
088
053
287
074
115
140
383
329
22Lysin
e1605
14619
168
168
038
404
079
000
244
163
190
221
071
170
072
138
145
295
163
Total
2186
2186
809
5304
1918
1816
2641
2044
2156
2065
1334
3132
1213
1455
2587
4666
3788
(b)
Num
berAminoacids
RT(m
in)
Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
1As
partate
148
13310
276
321
144
288
145
071
275
046
110
187
198
156
056
121
116
162
128
049
194
102
2Glutamate
277
14713
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3As
paragine
583
13212
089
096
042
097
ND
ND
022
039
021
ND
ND
032
ND
035
ND
024
126
ND
052
037
4Serin
e603
10509
112
191
092
104
217
111
056
069
114
112
196
101
062
102
101
111
317
079
174
115
5Vitamin
U659
1997
0ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6Glutamine
684
14615
131
152
098
187
ND
ND
107
ND
ND
ND
ND
053
ND
ND
057
ND
ND
ND
ND
ND
7Histidine
715
15515
048
056
ND
091
ND
ND
ND
ND
ND
ND
ND
ND
ND
030
ND
ND
053
ND
039
031
8Glycine
736
7507
265
313
196
395
175
169
108
204
227
219
337
141
146
224
231
151
633
147
537
203
9Th
reon
ine
762
11912
283
363
184
234
130
072
073
058
094
157
161
109
069
210
171
104
174
070
134
119
10Arginine
847
17420
252
312
198
235
537
247
174
164
219
266
358
266
134
280
301
241
846
188
263
291
11Alanine
897
8909
927
1357
860
1280
1168
904
ND
803
926
984
1289
436
760
1530
1157
651
2385
603
1634
624
12GABA
926
10312
126
183
099
135
048
179
020
174
113
163
436
044
672
116
057
044
077
023
235
097
13Ty
rosin
e1043
18119
233
136
121
330
222
144
064
151
184
193
326
136
109
117
171
163
445
113
220
182
10 Evidence-Based Complementary and Alternative Medicine
(b)Con
tinued
Num
berAminoacids
RT(m
in)Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
14Cy
stine
1226
24030
130
080
065
164
038
ND
ND
ND
ND
ND
ND
ND
ND
076
ND
ND
057
ND
078
ND
15Va
line
1256
11715
434
538
243
605
297
149
070
129
169
265
305
119
162
360
342
167
1612
ND
441
144
16Methion
ine
1282
1492
1069
035
ND
098
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
043
ND
ND
064
ND
069
ND
19Ph
enylalanine
1437
16519
276
253
113
336
142
083
ND
042
046
038
098
048
062
165
087
059
293
062
173
074
20Isoleucine
1460
13117
295
343
129
398
207
095
044
092
105
150
170
085
106
192
267
111
ND
068
ND
ND
21Leucine
1543
13117
474
582
241
659
399
181
071
103
121
147
195
124
161
332
335
162
759
096
314
123
22Lysin
e1605
14619
288
301
108
447
248
163
065
162
161
121
270
116
078
168
140
136
627
108
274
159
Total
470556
1429
3560
8339
7425681149223426
1030
0143
38196525774100353322858597160848
312301
Num
bers1to37
werethe
samplen
ames
(Table1)(a)NDnot
detected
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 Evidence-Based Complementary and Alternative Medicine
(b)Con
tinued
Num
berAminoacids
RT(m
in)Molecular
weight
1819
2021
2223
2425
2627
2829
3031
3233
3435
3637
14Cy
stine
1226
24030
130
080
065
164
038
ND
ND
ND
ND
ND
ND
ND
ND
076
ND
ND
057
ND
078
ND
15Va
line
1256
11715
434
538
243
605
297
149
070
129
169
265
305
119
162
360
342
167
1612
ND
441
144
16Methion
ine
1282
1492
1069
035
ND
098
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17Norvalin
e1323
11715
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
18Tryptoph
an1388
20433
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
043
ND
ND
064
ND
069
ND
19Ph
enylalanine
1437
16519
276
253
113
336
142
083
ND
042
046
038
098
048
062
165
087
059
293
062
173
074
20Isoleucine
1460
13117
295
343
129
398
207
095
044
092
105
150
170
085
106
192
267
111
ND
068
ND
ND
21Leucine
1543
13117
474
582
241
659
399
181
071
103
121
147
195
124
161
332
335
162
759
096
314
123
22Lysin
e1605
14619
288
301
108
447
248
163
065
162
161
121
270
116
078
168
140
136
627
108
274
159
Total
470556
1429
3560
8339
7425681149223426
1030
0143
38196525774100353322858597160848
312301
Num
bers1to37
werethe
samplen
ames
(Table1)(a)NDnot
detected
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 11
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0102030
Gal
lic ac
id
equi
vale
nt (m
gg)
Figure 3 Determination of the total phenolic compounds of the 37varieties (Table 1) of Spirulina (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
activ
ity (
)
Figure 4 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by DPPH radical scavenging activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
020406080
100
Ant
ioxi
dant
ac
tivity
()
Figure 5 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by reducing power activity assay (119899 = 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Spirulina samples
0255075
100125
Ant
ioxi
dant
ac
tivity
()
Figure 6 Antioxidant activities of the 37 varieties (Table 1) ofSpirulina by hydroxyl radical-scavenging activity assay (119899 = 3)
showed the activity in a concentration-dependent mannerNumbers of antioxidant metabolites are present in plants andSpirulina The scavenging abilities and reductive propertiesof the Spirulina samples exhibited dose-dependent activitiesMetabolites especially those having the phenolic functionalgroup in their chemical structure have been reported toshow many useful properties including anti-inflammatoryactivity oestrogenic activity enzyme inhibition antiallergicactivity antioxidant activity vascular activity and cytotoxicantitumour activityThe results from three antioxidant assaysof the 37 samples were not correlated with the total phe-nolic compounds or the other determined compounds Theresults indicated that the antioxidant potentials were notsignificantly correlated with their total phenolic compounds(data were not shown) because the antioxidant activity of the
sampleswas not directly proportional with respect to the totalphenolic compounds results The regular consumption ofantioxidant-containing food additives helps to slow oxidativestress andminimize the spread of oxidative stress-related dis-eases [37] The antioxidant compounds such as phycobilinsand phycocyanins that are present in Spirulina exert theiractions by scavenging free radicals by acting as hydrogen per-oxyl radical and peroxynitrite acceptors These antioxidantcompounds also inhibit the activities of catalytic enzymessuch as lipoxygenase and cyclooxygenase or enhance theactivity of enzymes such as glutathione peroxidase catalaseand superoxide dismutase [38]Wu et al (2005) reported thatSpirulina extracts exhibited greater antioxidant propertiesdue to the presence of various phenolic compounds [36] Anumber of cyanobacteria especially the species of Chlorellaare believed to be useful as excellent food sources withantioxidant activities by modern researchers [39] Due to itsrich vitamin protein phenolic compound polyunsaturatedfatty acid and other microelement contents Spirulina couldbe used as a better nutrient food by consumers
4 Conclusions
Thirteen unsaturated fatty acids 19 free amino acids 7 sugarsand the total polyphenolic components were separated andidentified from 37 Spirulina samples using GC and HPLCmethods The contents of each metabolite were quantifiedand remarkable variations in the individual metabolites wereobserved between the different varieties Specifically theSpirulina tablets distributed by 21st CenturyHealthCare Incwere relatively suitable due to their abundance of fatty acidssugars amino acids and polyphenolsThe in vitro antioxidantactivity results confirmed that the activities were dose-dependent The Spirulina products that are available on themarket are rich in antioxidant polyphenolic components andare suitable choices for regular consumption The presenceof individual phenolic compounds in the different productsshould be studied because these metabolites are used forthe treatment of stress-related diseases and cardiovasculardisorders
Conflict of Interests
The authors declare no conflict of interests
Authorsrsquo Contribution
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-ceived the study and designed the experiments and also areinvolved in writing the paper All authors read and approvedthe final version of the paper Naif Abdullah Al-Dhabi andMariadhas Valan Arasu contributed equally to this work
Acknowledgment
The Project was full financially supported by King SaudUniversity through Vice Deanship of Research Chairs
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
12 Evidence-Based Complementary and Alternative Medicine
References
[1] C Jimenez B R Cossıo and F X Niell ldquoRelationship betweenphysicochemical variables and productivity in open ponds forthe production of Spirulina a predictive model of algal yieldrdquoAquaculture vol 221 no 1ndash4 pp 331ndash345 2003
[2] L Brennan and P Owende ldquoBiofuels from microalgaemdashareview of technologies for production processing and extrac-tions of biofuels and co-productsrdquo Renewable and SustainableEnergy Reviews vol 14 no 2 pp 557ndash577 2010
[3] J J Ortega-Calvo C Mazuelos B Hermosin and C Saiz-Jimenez ldquoChemical composition of Spirulina and eukaryoticalgae food products marketed in Spainrdquo Journal of AppliedPhycology vol 5 no 4 pp 425ndash435 1993
[4] K Chopra and B Mahendra ldquoAntioxidant profile of Spirulinaa blue-green microalgardquo in Spirulina in Human Nutrition andHealth M E Gershwin and A Belay Eds pp 101ndash119 CRCPress London UK 2008
[5] O Tokusoglu and M K Unal ldquoBiomass nutrient profiles ofthree microalgae Spirulina platensis Chlorella vulgaris andIsochrisis galbanardquo Journal of Food Science vol 68 no 4 pp1144ndash1148 2003
[6] A S Babadzhanov N Abdusamatova F M Yusupova NFaizullaeva L GMezhlumyan andM KMalikova ldquoChemicalcomposition of Spirulina platensis cultivated in UzbekistanrdquoChemistry of Natural Compounds vol 40 no 3 pp 276ndash2792004
[7] A Ramamoorthy and S Premakumari ldquoEffect of supplementa-tion of Spirulina on hypercholesterolemic patientsrdquo Journal ofFood Science and Technology vol 33 no 2 pp 124ndash128 1996
[8] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997
[9] J Ferreira ldquoEffect of butylated hydroxyanisole on electrontransport in rat livermitochondriardquo Biochemical Pharmacologyvol 40 no 4 pp 677ndash684 1990
[10] M Khan J C Shobha I K Mohan et al ldquoProtective effectof Spirulina against doxorubicin-induced cardiotoxicityrdquo Phy-totherapy Research vol 19 no 12 pp 1030ndash1037 2005
[11] D S Lee D S Jeon S G Park et al ldquoEffect of cold storage onthe contents of glucosinolates in Chinese cabbage (Brassica rapaL ssp pekinensis)rdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 38ndash42 2015
[12] MMossoba J Kramer P DelmonteM Yurawecz and J RaderAOACOfficial Method 99606 Fat (Total Saturated and Unsat-urated in Foods) Hydrolytic Extraction Gas ChromatographicMethod First Action 1996 Revised 2001 AOCS Press UrbanaIll USA 2003
[13] N A Al-Dhabi ldquoHeavy metal analysis in commercial Spirulinaproducts for human consumptionrdquo Saudi Journal of BiologicalSciences vol 20 no 4 pp 383ndash388 2013
[14] MValanArasuMW JungDHKim et al ldquoIdentification andphylogenetic characterization of novel Lactobacillus plantarumspecies and their metabolite profiles in grass silagerdquo Annals ofMicrobiology vol 65 no 1 pp 15ndash25 2015
[15] S Park M V Arasu M-K Lee et al ldquoAnalysis and metaboliteprofiling of glucosinolates anthocyanins and free amino acidsin inbred lines of green and red cabbage (Brassica oleracea L)rdquoLWTmdashFood Science and Technology vol 58 no 1 pp 203ndash2132014
[16] J-Y Lin and C-Y Tang ldquoDetermination of total phenolic andflavonoid contents in selected fruits and vegetables as well as
their stimulatory effects on mouse splenocyte proliferationrdquoFood Chemistry vol 101 no 1 pp 140ndash147 2006
[17] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986
[18] T Hatano H Kagawa T Yasuhara and T Okuda ldquoTwonew flavonoids and other constituents in licorice root theirrelative astringency and radical scavenging effectsrdquo Chemicaland Pharmaceutical Bulletin vol 36 no 6 pp 2090ndash2097 1988
[19] K Elizabeth and M W A Rao ldquoOxygen radical scavengingactivity of curcuminrdquo International Journal of Pharmaceuticsvol 58 pp 237ndash240 1990
[20] M Muhling A Belay and B A Whitton ldquoScreeningArthrospira (Spirulina) strains for heterotrophyrdquo Journal ofApplied Phycology vol 17 no 2 pp 129ndash135 2005
[21] D R Reddy V S S V Prassas and U N Das ldquoIntratumouralinjection of gamma leinolenic acid in malignant gliomasrdquoJournal of Clinical Neuroscience vol 5 no 1 pp 36ndash39 1998
[22] V A Ziboh ldquoBiochemical basis for the anti-inflammatoryaction of gamma-linolenic acidrdquo in Omega-6 Essential FattyAcids Pathophysiology and Roles in Clinical Medicine D FHorrobin Ed pp 187ndash201 Alan Liss NewYork NY USA 1989
[23] A Bordoni P L Biagi M Masi et al ldquoEvening primrose oil(Efamol) in the treatment of children with atopic eczemardquoDrugs under Experimental and Clinical Research vol 14 no 4pp 291ndash297 1988
[24] D F Horrobin ldquoThe role of essential fatty acids andprostaglandins in the premenstrual syndromerdquo The Journal ofReproductive Medicine vol 28 pp 465ndash468 1983
[25] T Ishikawa Y Fujiyama O Igarashi et al ldquoEffects of gam-malinolenic acid on plasma lipoproteins and apolipoproteinsrdquoAtherosclerosis vol 75 no 2-3 pp 95ndash104 1989
[26] M G Sajilata R S Singhal andM Y Kamat ldquoFractionation oflipids and purification of 120574-linolenic acid (GLA) from Spirulinaplatensisrdquo Food Chemistry vol 109 no 3 pp 580ndash586 2008
[27] V Patil T Kallqvist E Olsen G Vogt and H R GisleroslashdldquoFatty acid composition of 12 microalgae for possible use inaquaculture feedrdquo Aquaculture International vol 15 no 1 pp1ndash9 2007
[28] R Chaiklahan N Chirasuwan P Triratana V Loha S Tiaand B Bunnag ldquoPolysaccharide extraction from Spirulina spand its antioxidant capacityrdquo International Journal of BiologicalMacromolecules vol 58 pp 73ndash78 2013
[29] J-B Lee T Hayashi K Hayashi et al ldquoFurther purification andstructural analysis of calcium spirulan from Spirulina platensisrdquoJournal of Natural Products vol 61 no 9 pp 1101ndash1104 1998
[30] M R Brown C D Garland S W Jeffrey I D Jameson andJ M Leroi ldquoThe gross and amino acid compositions of batchand semi-continuous cultures of Isochrysis sp (clone TISO)Pavlova lutheri andNannochloropsis oculatardquo Journal of AppliedPhycology vol 5 pp 285ndash296 1993
[31] G Clement C Giddey and RMenzi ldquoAmino acid compositionand nutritive value of the alga Spirulina maximardquo Journal of theScience of Food and Agriculture vol 18 no 11 pp 497ndash501 1967
[32] A Richmond and E W Becker ldquoTechnological aspects of masscultivationmdasha general outlinerdquo inHandbook of Microalgal MassCulture A Richmond Ed pp 245ndash263 CRC Press BocaRaton Fla USA 1984
[33] M S Miranda R G Cintra S B M Barros and J Mancini-Filho ldquoAntioxidant activity of themicroalga SpirulinamaximardquoBrazilian Journal of Medical and Biological Research vol 31 no8 pp 1075ndash1079 1998
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 13
[34] L M Colla C Oliveira Reinehr C Reichert and J A VCosta ldquoProduction of biomass and nutraceutical compoundsby Spirulina platensis under different temperature and nitrogenregimesrdquo Bioresource Technology vol 98 no 7 pp 1489ndash14932007
[35] C Manach A Mazur and A Scalbert ldquoPolyphenols andprevention of cardiovascular diseasesrdquo Current Opinion inLipidology vol 16 no 1 pp 77ndash84 2005
[36] L-C Wu J-A A Ho M-C Shieh and I-W Lu ldquoAntioxidantand antiproliferative activities of Spirulina and chlorella waterextractsrdquo Journal of Agricultural and Food Chemistry vol 53 no10 pp 4207ndash4212 2005
[37] S Shukla A Mehta V K Bajpai and S Shukla ldquoIn vitroantioxidant activity and total phenolic content of ethanolicleaf extract of Stevia rebaudiana Bertrdquo Food and ChemicalToxicology vol 47 no 9 pp 2338ndash2343 2009
[38] V B Bhat and K M Madyastha ldquoScavenging of peroxynitriteby phycocyanin and phycocyanobilin from Spirulina platensisprotection against oxidative damage to DNArdquo Biochemical andBiophysical Research Communications vol 285 no 2 pp 262ndash266 2001
[39] E Christaki P Florou-Paneri and E Bonos ldquoMicroalgae anovel ingredient in nutritionrdquo International Journal of FoodSciences and Nutrition vol 62 no 8 pp 794ndash799 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
