Research ArticleKainari a Unique Greek Traditional Herbal Tea fromthe Island of Lesvos Chemical Analysis and Antioxidant andAntimicrobial Properties

Evangelia Bampali Konstantia Graikou Nektarios Aligiannis and Ioanna Chinou

Department of Pharmacognosy and Chemistry of Natural Products Faculty of Pharmacy School of Health SciencesNational and Kapodistrian University of Athens Panepistimiopolis-Zografou 15771 Athens Greece

Correspondence should be addressed to Ioanna Chinou ichinoupharmuoagr

Received 1 September 2017 Accepted 16 November 2017 Published 26 February 2018

Academic Editor Yoshiji Ohta

Copyright copy 2018 Evangelia Bampali et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The chemical composition as well as the total phenolic content (TPC) and the potential antioxidant and antimicrobial activityof three Kainari-herbal tea samples from different areas of Lesvos Island (Greece) was evaluated The rich aroma of the mixtureswas studied throughGC-MS as well as throughHeadspace Solid-PhaseMicroextraction (HS-SPME)GC-MS analyses Cinnamonclove nutmeg pepper and ginger were identified asmain ingredients while throughout the chemical analysis of the volatiles of oneselected sample several secondary metabolites have been isolated and identified on the basis of GC-MS as well as spectral evidenceas eugenol cinnamic aldehyde and myristicin cinnamyl alcohol alpha-terpinyl acetate and 120573-caryophyllene Furthermore twofood dyes azorubine and amaranth were also isolated and identified from the infusions The total phenolic content was estimatedand the free radical scavenging activity was determined byDPPH andABTS assays and the antimicrobial activity of the extracts wastested showing a very interesting profile against all the assayed microorganisms Due to its very pleasant aroma and taste propertiesas well as to its bioactivities Kainari-herbal tea could be further proposed as functional beverage

1 Introduction

For over thousand years herbs and plants have played amajorrole in traditional and herbal based medicines Herbal infu-sions and plants are the major source of phenolic compoundsin our diet It is known that phenolic compounds have manybiological activities such as antioxidant and antimicrobial [1]Due to the increasing popularity of a whole market for herbaldietary supplements and traditional medicines we present inthis study the chemical analysis and potential bioactivities ofa unique herbal tea combination named ldquoKainarirdquo

Kainari is a combination (mixture) of several spices inpowder form which is used as herbal tea usually with anintense red color traditionally prepared in the Greek islandof Lesvos and used in folkmedicine as a warming beverage inwinter Historically it was brought to the island from Greekemigrants fromAsiaMinor in early 19th century In Turkey asimilar herbal tea called ldquokaynarrdquo (means in Turkish boiled)or Lohusa Serbeti (convalescence serbet) is used traditionally

in childbirth to give energy to the new mothers and to boosttheir lactation [2]

The tradition of making ldquoLohusa Serbetirdquo after a birthprobably is derived from Byzantine birth tradition (custom)where what was called ldquolochozemardquo a nourishing broth wasgiven to new mothers [2 3] It is mentioned in ceremonialbooks that after the birth of an imperial child ldquoPorphyro-genitusrdquo and in one-week-long period of celebration thepreparation of ldquolochozemardquo a nutritious ldquochildbed soup likerdquobroth was offered in Constantinoplersquos main streets Then asbirth custom it was given to all new mothers to aid milkproduction and recovery [4 5]

The exact history of ldquoLohusa Serbetirdquo is not known butthe drink was certainly popular during the latter part of theOttoman Empire (1299ndash1922) when the drink was given tonew mothers people that visited the new mother and withjugs of the drink also sent to the motherrsquos friends family andcleric as a way of announcing the birth of her child Afterdrinking ldquoLohusa Serbetirdquo everybody present had to say a

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 6802753 7 pageshttpsdoiorg10115520186802753

2 Evidence-Based Complementary and Alternative Medicine

prayer asking God to bless the new mother with a plentifulsupply of breast milk [2] A fifteenth-century Turkish poemby Suleyman Celebi describes how Amine mother of theprophet Muhammed is given serbet to quench her thirstduring labour [6]

It is also important the red color of the drink as it is asymbol in Turkey for good luck and joy [2] but also hasa symbolic imperial importance [7] Moreover red color isconsidered apotropaic by which is meant that red color isthought to ward off evil so by giving a red drink to a newlactating mother protect both the mother and her baby [2]

To prepare ldquoLohusa Serbetirdquo water cinnamon sticks andcloves and ldquolohusardquo sugar (pink-red in color natural redcolor or red food dye) are boiled together to be served hotin winter or cold during summer [2 8 9]

ldquoKainarirdquo as already referred to arrived to Lesvos Islandafter 1922 and it is used as a pleasant aromatic tonic warm-ing beverage The composition of the mixture differentiatesfrom ldquoLohusa Serbetirdquo and in our days Kainari infusion isprepared by adding hot water to half spoon of Kainari mix-ture which is produced by mixing powders of several spiceswhile the exact composition ofmixture is kept secret Some ofthe ingredients that are usually part of the herbal tea combi-nation are mainly cinnamon and clove and in smaller quanti-ties other spices such as ginger nutmeg galangal and pepper

The objective of this research was to study for the firsttime to the best of our knowledge the composition of threedifferent Kainari samples from three villages of Lesvos Island(Greece) as well as to evaluate the total phenolic content(TPC) and the potential antioxidant and antimicrobial activ-ity Six volatile compounds and two azo food dyes havebeen isolated and identified The total phenolic content wasestimated by Folin-Ciocalteu method and the free radicalscavenging activity was determined by DPPH and ABTSassays Moreover the antimicrobial activity of the sampleswas tested against six Gram-positive and Gram-negativebacteria two oral pathogens and three pathogenic fungi

2 Materials and Methods

21 Materials Three samples of Kainari were obtained fromdifferent areas of Lesvos Island Mytilene (KN1) Paleochori(KN2) andAgiassos (KN3) Analytical grade solvents (pentanemethanol and water) were used for the extractions and thechromatographic techniquesThe chemicals used throughoutthis study such as 11-diphenyl-2-picrylhydrazyl (DPPH) 22-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)Folin-Ciocalteu reagent gallic acid caffeic acid and Troloxpowder silica RP-18 and silica gel plates (Kieselgel 60 F25420 times 20 cm) were purchased from Sigma-Aldrich and Merck

22 Methods

221 Preparation of Extracts

(i) Preparation of Herbal Teas (KN1 Aq KN2 Aq and KN3Aq) 04500 g of each of the Kainari samples was extractedusing 200mL of boiled distilled water left for 45min at 25∘CThe filtrates were freeze-dried (Alpha I5 Christ Osterode amHarz Germany) and stored in desiccator

(ii) Preparation of Methanol Extracts (KN1Meth KN2Meth andKN3Meth) 09750g of each of theKainari sampleswas extract-ed using 400mL of methanol for 2 hours at 25∘C and after fil-tration was evaporated in a rotary evaporator (R-200 BuchiFlawil Switzerland) and stored at minus20∘C until further analysis

(iii) Preparation of Pentane Extracts 04500 g of each of theKainari samples was extracted using 200mL of pentane for2 hours at 25∘C and after filtration was evaporated (R-200Buchi Flawil Switzerland) and stored as previously

222 Isolation of Compounds(i) Isolation of Volatile CompoundsThe essential oil of sampleKN2 was received by Clevenger distillation using 1500 g ofsample KN2 The distillate A (35mL) consisting of essentialoil and aromatic water was collected in a flask while 2mlof pure essential oil B was collected separately and analyzedthrough GC-MS

In the distillate A was added 2 g of NaCl and the solutionwas cooled in ice water and transferred to a separatoryfunnel with 50mL of hexane The aqueous layer A1 wasdiscarded and the organic layer A2 was evaporated via rotaryevaporation (R-200 Buchi Flawil Switzerland) to yield anoily extract C In the extract C 15mL hexane and 25mL of5 NaOH were added for further liquid-liquid separationThe aqueous layer C1 contained the sodium salt of eugenolwhile the eugenol acetate 120573-caryophyllene myristicin andcinnamic aldehyde remained in the organic layer C2

Layer C1 was then evaporated and cooled on ice while50mLof 6MHClwas added to the solution ensuring that thesolution was acidic and it was extracted with 15mL hexaneyielding eugenol which was identified through NMR GC-MS and comparison with literature data [10]

The organic layer C2 was chromatographed throughpreparative thin layer chromatography on silica gel platesusing cyclohexanedichloromethane 3070 vv as mobilephase yielding cinnamic aldehyde myristicin cinnamylalcohol alpha-terpinyl acetate and 120573-caryophyllene Allcompounds were identified through NMR andor GC-MSand literature data [11 12]

(ii) Isolation of Azo Dyes The freeze-dried herbal teas ofKN1 and KN2 (454mg) which presented the same chro-matographic profile and the freeze-dried herbal tea of KN3(450mg) were separately further purified using a mediumpressure liquid chromatographyMPLC (Borosilikat 33 CodeNumber 28147 Buchi Flawil Switzerland) packed withreversed phase silica gel (RP18) and two Series 1 pumpswith flow speed 8mLmin eluted with decreasing polaritiesH2OMeOH (1000 to 0100) Azorubine was isolated and

identified from the water fraction of KN1-KN2 and amaranthfrom KN3 The isolated compounds were identified by NMRand literature data [13]

223 Identification of Compounds(i) Headspace Solid-Phase Microextraction (HS-SPME) Therich aroma of the Kainari samples was studied through HS-SPMEGC-MS analyses [14] The HS-SPME was performedwith carboxenpolydimethylsiloxane coated fiber (75120583mcoating 57330-U Supelco Bellefonte PA USA) attached in

Evidence-Based Complementary and Alternative Medicine 3

a manual SPME fiber holder (Supelco) For SPME extraction10mg of each sample in a glass vial (15mL) closed with PTFEcoated silicone rubber septum was used The temperature inour experiment was set at 70ndash75∘C and the vial with samplewas placed on the hot-plate for 15minutes After that time thefiber was exposed for 5minutes and then it was transferred toperform GC-MS analysis

(ii) GC-MS Analysis The analyses were performed on aHP 6890 GC with 5973 MSD (Hewlett-Packard Germany)with ionization energy 70 eV The GC is equipped with a110 splitsplitless injector and a 30m long DP5 capillarycolumn 025mm internal diameter and 025mm thicknessThe temperature in the injection sample was 200∘C andgas was He and flow rate is 07mLmin Identification wasmade using the Wiley275 library and bibliographic data Thetemperature programs that have been used are the following

Temperature Program I The initial temperature is 100∘C andthen rises at a rate of 4∘Cmin up to amaximum temperatureof 300∘C Total analysis time was 52min This program wasused to analyze methanol and pentane extracts

Temperature Program II The initial column temperature is50∘C which is maintained for 3 minutes and then increasedat a rate of 30∘Cmin to 150∘C From 150∘C it rises at a rateof 3∘Cmin up to 250∘C where it remains for 10min It wasused for the HS-SPME analyses [10]

Temperature Program III The initial column temperature is60∘C and then increases at a rate of 3∘Cmin to a maximumtemperature of 280∘C Total analysis time was 93min It wasused for the analysis of the essential oil B

(iii) Nuclear Magnetic Resonance (NMR) 1H-NMR spectrawere obtained on a Bruker DRX 400 instrument (400MHz)using CD

3OD CDCl

3 and DMSO-d6 as solvents and TMS

as an internal standard

224 Antioxidant Properties

(i) Total Phenolic Content (TPC) It was determined by usingFolin-Ciocalteu method with slight modifications [15] Asreferred to literature 10mg of each crudemethanol extract orlyophilized herbal tea was dissolved in 1mL of correspondingextracting solvent to produce the stock of sample solutionThe lower concentrations of samplewere prepared by diluting100 120583L of the stock sample solution with 900120583L methanolor distilled water Then 50 120583L of sample was put in atest tube and mixed with 60120583L of Folin-Ciocalteu reagentfollowed by an addition of 120 120583L of 35 sodium carbonateafter 3 minutes Then the resulting mixture was incubatedin dark at room temperature for an hour and absorbancewas measured at 725 nm (UV-1700 Shimadzu CorporationKyoto Japan) after incubation Caffeic acid was used ascalibrated standard and results were expressed as milligramCaffeic Acid Equivalent per gram of dry extract (mg CAEgdry extract) The content of phenolics for each extract wasdetermined in triplicate

Alongside 25120583L of extracts or standard solution of gallicacid (25 to 100 120583gmL) in DMSO was added to 125 120583Lof a Folin-Ciocalteu solution (10 vv) followed by 100120583L

of sodium carbonate (75wv) in a 96-well plate Thereagents were mixed and incubated for 30 minutes at roomtemperature protected from light and the absorbance wasmeasured at 765 nm Total phenolic content was expressedas mg Gallic Acid Equivalents per gram of dry extract (mgGAEg dry extract)The content of phenolics for each extractwas determined in triplicate

(ii) 11-Diphenyl-2-picrylhydrazyl (DPPH) Assay The DPPHradical scavenging assay was performed according to apreviously described method [15] The stock DPPH solution(314 120583M) was prepared in absolute ethanol and kept in darkat room temperature until its use Gallic acid was used asa positive control (IC50 45 120583gmL) and the total extractswere diluted in DMSO at appropriate concentrations Brieflyin a 96-well plate 190 120583L of the DPPH solution and 10120583Lof gallic acid or samples were incubated for 30min at roomtemperature protected from light and the absorbance wasmeasured at 517 nm A negative control containing 10 120583LDMSO and 190 120583L DPPH was performed each time as wellas blanks containing 10 120583L sample and 190120583L EtOH

(iii) 22-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic Acid)(ABTS) Assay TheABTS radical cation scavenging assay wasperformed using themethod reported by Re et al (1999) withslight modifications [16] In brief a stock ABTS aqua solution(7mM) was reacted with potassium persulfate aqua solution(245mM) and kept overnight in dark to yield a dark coloredsolution containing ABTS∙+ radical cation Trolox was usedas a positive control (IC50 83 120583gmL) and the total extractswere diluted in DMSO at appropriate concentrations Priorto use in the assay the ABTS∙+ radical cation was dilutedwith distilled water for an initial absorbance of about 0700(plusmn002) at 734 nm Afterwards in a 96-well plate 100 120583l ofABTS∙+ radical cation solution and 50 120583l of Trolox or sampleswere added incubated for 10min at room temperature andprotected from light and the absorbance was measured at734 nm A negative control containing 50 120583L DMSO and100 120583L ABTS was performed each time as well as blankscontaining 50 120583L sample and 100 120583L dist H

2O

The percentage of DPPH and ABTS scavenging wasestimated by the following equation

119860119860 = [(119860 minus 119861) minus (119862 minus 119863)](119860 minus 119861) times 100 (1)

119860 is the control (without sample) 119861 is the blank (withoutsample without DPPHABTS) 119862is the sample and 119863 isthe blank sample (without DPPHABTS) IC50 values wereestimated for the most active extracts

For both experiments which referred to the free radicalscavenging all samples were analyzed in triplicate Measure-ments were performed using a TECAN Infinite M200 PROmultimode reader (Tecan Group Mannedorf Switzerland)

225 Antimicrobial Bioassay Themethanol extracts and theherbal teas of Kainari were evaluated for their in vitro antimi-crobial activities using the standard antibiotics netilmicinamoxicillin and clavulanic acid in order to evaluate thesensitivity of the tested bacteria while 5-fluorocytosineand amphotericin were used as sensitivity controls of the

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Comparative table for the powders of KN1 KN2 and KN3 with HS-SPME

Compound area KN1 area KN2 area KN3 Possible contained plantCinnamic aldehyde 988 1000 1519 Cinnamomum cassiaSafrole lt1 lt1 - Myristica fragrans Piper nigrumCamphene lt1 278 - Myristica fragrans Piper nigrumEugenol 3450 3018 2271 Syzygium aromaticum Cinnamomum cassia Myristica fragrans Piper nigrum120572-Copaene 188 416 1081 Syzygium aromaticum Piper nigrum

120573-Caryophyllene 1561 3018 3316 Cinnamomum cassia Syzygium aromaticum Piper nigrum Myristica fragransElettaria cardamomum

120572-Humulene 250 377 359 Syzygium aromaticum Piper nigrum Elettaria cardamomumAr-Curcumene 194 166 - Zingiber officinaleZingiberene 113 126 - Zingiber officinaleMyristicin 334 - - Myristica fragrans

tested fungi [17 18] A total of eleven microorganisms wereassayed amongwhich therewere fourGram-positive bacteriaStaphylococcus aureus (ATCC 25923) Staphylococcus epider-midis (ATCC 12228) Streptococcus mutans and Streptococcusviridans and four Gram-negative bacteria Escherichia coli(ATCC 25922) Enterobacter cloacae (ATCC 13047)Klebsiellapneumoniae (ATCC 13883) and Pseudomonas aeruginosa(ATCC 227853) as well as three pathogen fungi Candidaalbicans (ATCC 10231) C tropicalis (ATCC 13801) and Cglabrata (ATCC 28838)

3 Results and Discussion

31 Results from HS-SPMEGC-MS Analyses All three sam-ples of ldquoKainarirdquo from Mytilini Paleochori and Agiassos inLesvos were studied for the first time to our knowledge Therich aroma of the mixture was studied through HeadspaceSolid-Phase Microextraction (HS-SPME)GC-MS analysesFurthermore the pentane and methanol extracts were alsostudied through GC-MS with respective results The majorconstituents that have been determined through GC-MSanalyses were cinnamic aldehyde eugenol myristicin 120573-caryophyllene curcumene and zingiberene which can berelated to the possible contained spices as shown in Table 1

Through analyses of all extracts of the three samples(herbal teas methanol and pentane extracts) and the simul-taneous use of bibliography we conclude that the maincomponents in all three samples are cinnamon (sim610) andclove (sim310) and the remaining part (110) corresponds toboth nutmeg and ginger

It is remarkable that according to the analyses of the volat-iles in KN3 they were not identified as the main componentsof ginger (Zingiber officinale) and consequently we assumethat ginger is not included in the mixture or it is included insmall quantities As for the presence of cardamomandpepperdue to the lack of someof theirmainmetabolites (someof themexist in other contained plants at the same time) we concludethat if they are present they will be in small quantities

32 Isolation and Determination of Compounds Six com-pounds were isolated from the essential oil of sample ΚN2 byapplying a series of liquid-liquid extractions based on acid-base chemical reactions as well as with preparative TLCeugenol cinnamic aldehyde andmyristicin were determinedby NMR spectroscopy and GC-MS analysis while cinnamyl

Table 2 Total phenolic content of the herbal teas of KN1 KN2 andKN3

mg CAEgr dry extract mg GAEgr dry extractSample KN1 aq 30829 1152Sample KN2 aq 32031 1492Sample KN3 aq 38341 1845

Table 3 inhibition at DPPH and ABTS assay of herbal teas andmethanol extracts

DPPH ABTS inhibition50 120583gmL

inhibition10 120583gmL

KN1 aq 374 plusmn 01 284 plusmn 01KN2 aq 502 plusmn 14 388 plusmn 13KN3 aq 647 plusmn 03 469 plusmn 09KN1 meth 646 plusmn 03 482 plusmn 07KN2 meth 409 plusmn 07 269 plusmn 07KN3 meth 614 plusmn 01 403 plusmn 14

alcohol alpha-terpinyl acetate and 120573-caryophyllene weredetermined only by GC-MS analysis

Also two food dyeswere isolated and identified as parts ofthe family of azo colors Samples KN1 and KN2 contained thesame food color azorubine [19] while sample KN3 containedamaranth [11]

The azo colors are compounds bearing the functionalgroup R-N = N-R1015840 where R and R1015840 may be either arylor alkyl Azo dyes are one of the most important chemicalcategories of dyes which are used for the coloring of naturaland synthetic fibers candies and cosmetics but also foodand beverages [20 21] Many azo dyes are nontoxic althoughsome of them have been accused for allergic reactions andincreasing hyperactivity in children [22]

The two isolated compounds are being referred to EFSAas amaranth (E123) and azorubine or carmoisine (Ε122) andare well-known food dyes [19 23]

33 Antioxidant Properties The total phenolic content (TPC)of the three samples was notably high probably due tothe known rich phenolic concentration of all spices thatthey contain [24 25] The sample KN3 revealed the higherconcentration of phenolic content (Table 2)

The free radical scavenging activities of herbal teasand methanol extracts were determined by DPPH andABTS assays (Table 3) All extracts showed significant

Evidence-Based Complementary and Alternative Medicine 5

Table4Antim

icrobialactiv

ityof

herbalteas

andmethano

lextracts

Samples

Saureus

Sepidermidis

Paeruginosa

Eclo

acae

Kpn

eumoniae

Ecoli

Smutan

sSvirid

ans

Calbicans

Ctro

picalis

Cgla

brata

Kainari

KN1a

q16009

17004

14013

14015

13010

15017

13030

14039

12040

13039

13040

KN1m

eth

11070

11073

10090

10093

10089

10095

ntnt

11042

13033

12031

KN2aq

17004

17005

15010

15013

15009

15012

15015

16016

12043

13040

13042

KN2meth

10072

11068

10086

10087

10092

09098

ntnt

10053

11044

12042

KN3aq

18002

19001

16006

16005

16005

15008

17007

18003

13039

13031

13029

KN3meth

10072

11068

10086

10087

10092

09098

ntnt

12038

13033

13034

5-Fluo

rocytosin

ent

ntnt

ntnt

ntnt

nt01sdot10minus31sdot10minus310sdot10minus3

Amph

otericin

ntnt

ntnt

ntnt

ntnt

1sdot10minus305sdot10minus304sdot10minus3

Amoxicillin

2sdot10minus3

2sdot10minus3

24sdot10minus322sdot10minus328sdot10minus32sdot10minus3

ntnt

ntnt

ntNetilm

icin

4sdot10minus3

4sdot10minus3

88sdot10minus38sdot10minus3

8sdot10minus3

10sdot10minus3

ntnt

ntnt

ntClavulanicacid2sdot10minus3

2sdot10minus3

24sdot10minus322sdot10minus328sdot10minus32sdot10minus3

ntnt

ntnt

nt

6 Evidence-Based Complementary and Alternative Medicine

antioxidant activity performing 30ndash64 inhibition at DPPHassay (50 120583gmL) as well as 26ndash48 inhibition at ABTSassay (10 120583gmL) The herbal tea KN3 performed the higherinhibition at DPPH (647) and at ABTS (469) which is inaccordance with the high TPC Also themethanol extracts ofKN1 and KN3 showed high inhibition

34 Antimicrobial Bioassay According to in vitro antimicro-bial tests (Table 4) against two Gram-positive bacteria (Saureus and S epidermidis) and four Gram-negative ones (Ecoli E cloacae K pneumoniae and P aeruginosa) as well asagainst oral pathogens (S mutans and S viridans) and threehuman pathogenic fungi (Candida albicans C tropicalis andC glabrata) the herbal teas exhibited a broad spectrum ofstrong antimicrobial activity while KN3 herbal tea appearedto be themost active against all testedmicroorganisms whichcan be explained by the high percentage of cinnamic aldehydeand 120573-caryophyllene that appears in herbal tea of KN3 asshown in Table 1

4 Conclusion

In conclusion the chemical composition as well as thetotal phenolic content (TPC) and the potential antioxidantand antimicrobial activity of three Kainari-herbal teas fromdifferent areas of Lesvos Island (Greece) was evaluated Allsamples exerted very broad and strong antimicrobial activ-ities against almost all Gram-positive and Gram-negativehuman pathogenic bacteria as well as strong antioxidantactivities

According to the determined chemical analysis and totalphenolic content it could be concluded that ldquoKainarirdquo herbalinfusion except its very pleasant taste could be also furtherused as a functional herbal beverage due to its beneficialproperties strong antioxidant and antimicrobial activitiesfor consumers Certainly more studies along the potentialfurther bioactivities as well as the entire process of theproduction of stable batches of ldquoKainarirdquo combination atindustrial level have to be designed in future

Disclosure

A preliminary version of this study entitled ldquoKainari ndash AGreek Traditional Herbal Tea from Lesvos Island ChemicalAnalysis Antioxidant and Antimicrobial Propertiesrdquo by thesame authors has been subjected and accepted as posterpresentation in ICNPU-2017 3rd International Conferenceon Natural Products Utilization which was held in BanskoBulgaria 18ndash21 October 2017 (PP24)

Conflicts of Interest

The authors declare they have no conflicts of interest for thiswork

References

[1] I C Arts and P C Hollman ldquoPolyphenols and disease risk inepidemiologic studiesrdquo American Journal of Clinical Nutritionvol 81 no 1 pp 317Sndash325S 2005

[2] V Williams Celebrating Life Customs around the World FromBaby Showers to Funerals [3 volumes] ABC-CLIO 2016

[3] A Papaconstantinou andA-MMaffry Talbot Becoming Byzan-tine children and childhood in Byzantium vol 1 HarvardUniversity Press 1 2009

[4] P Koukoules Speculum vol IV Π120572120587120572120577119906120590120578120589 1951[5] GDagronEmperor and priestThe imperial office in Byzantium

Cambridge University Press 2003[6] O A Talat Eski Turk Edebiyatında Mazmunlar ve Izahı Akcag

Yayınları Ankara 2000[7] K Albala Food Cultures of theWorld Encyclopedia Bloomsbury

Publishing 2011[8] Binnurrsquos Turkish Cookbook ldquoLohusa Serbetrdquo httpwwwturk-

ishcookbookcom201003lohusa-sherbetphp[9] ldquoLohusa Serbeti A Special Baby Sherbetrdquo A Seasonal Cook in

Turkey June 15 2011rdquo httpseasonalcookinturkeycomlohusa-serbeti-special-baby-sherbet

[10] ldquoIsolation of Eugenol Organic Chemistry 241 Lab ColbyCollegerdquo

[11] C X You H Y Jiang W J Zhang et al ldquoContact toxicityand repellency of the main components from the essential oilof Clausena anisum-olens against two stored product insectsrdquoJournal of Insect Science vol 15 no 1 2015

[12] Trans-Cinnamaldehyde ldquoHanHong Webrdquo httpwwwhan-honggroupcomnmrnmr enB34317html

[13] E M Elgendy and N A Al-Zahrani ldquoComparative Study ofNatural and Synthetic Food Additive Dye Amaranth throughPhotochemical Reactionsrdquo Indian Journal of Science Researchvol 4 pp 827ndash832 2015

[14] K G Miller C F Poole and T M P Pawlowskı ldquoClassificationof the botanical origin of cinnamonby solid-phasemicroextrac-tion and gas chromatographyrdquo Chromatographia vol 42 no 11-12 pp 639ndash646 1996

[15] A Bampouli K Kyriakopoulou G Papaefstathiou et al ldquoEval-uation of total antioxidant potential of Pistacia lentiscus varchia leaves extracts using UHPLC-HRMSrdquo Journal of FoodEngineering vol 167 article no 7970 pp 25ndash31 2015

[16] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[17] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 pp 493ndash496 1966

[18] I Wiegand K Hilpert and R E W Hancock ldquoAgar and brothdilution methods to determine the minimal inhibitory concen-tration (MIC) of antimicrobial substancesrdquo Nature Protocolsvol 3 no 2 pp 163ndash175 2008

[19] ldquoScientific Opinion on the re-evaluation of AzorubineCarmoi-sine (E 122) as a food additiverdquo EFSA Journal vol 7 no 11 p1332 2009

[20] R L M Allen Colour Chemistry Thomas Nelson amp SonsLondon UK 1979

[21] K Venkataraman The Chemistry of Synthetic Dyes AcademicPress London UK 1978

[22] FDA Background Document for the Food Advisory CommitteeCertified Color Additives in Food and Possible Association withAttention Deficit Hyperactivity Disorder in Children 2011

[23] ldquoScientific Opinion on the re-evaluation of Amaranth (E 123) asa food additiverdquo EFSA Journal vol 8 no 7 p 1649 2010

Evidence-Based Complementary and Alternative Medicine 3

a manual SPME fiber holder (Supelco) For SPME extraction10mg of each sample in a glass vial (15mL) closed with PTFEcoated silicone rubber septum was used The temperature inour experiment was set at 70ndash75∘C and the vial with samplewas placed on the hot-plate for 15minutes After that time thefiber was exposed for 5minutes and then it was transferred toperform GC-MS analysis

(ii) GC-MS Analysis The analyses were performed on aHP 6890 GC with 5973 MSD (Hewlett-Packard Germany)with ionization energy 70 eV The GC is equipped with a110 splitsplitless injector and a 30m long DP5 capillarycolumn 025mm internal diameter and 025mm thicknessThe temperature in the injection sample was 200∘C andgas was He and flow rate is 07mLmin Identification wasmade using the Wiley275 library and bibliographic data Thetemperature programs that have been used are the following

Temperature Program I The initial temperature is 100∘C andthen rises at a rate of 4∘Cmin up to amaximum temperatureof 300∘C Total analysis time was 52min This program wasused to analyze methanol and pentane extracts

Temperature Program II The initial column temperature is50∘C which is maintained for 3 minutes and then increasedat a rate of 30∘Cmin to 150∘C From 150∘C it rises at a rateof 3∘Cmin up to 250∘C where it remains for 10min It wasused for the HS-SPME analyses [10]

Temperature Program III The initial column temperature is60∘C and then increases at a rate of 3∘Cmin to a maximumtemperature of 280∘C Total analysis time was 93min It wasused for the analysis of the essential oil B

(iii) Nuclear Magnetic Resonance (NMR) 1H-NMR spectrawere obtained on a Bruker DRX 400 instrument (400MHz)using CD

3OD CDCl

3 and DMSO-d6 as solvents and TMS

as an internal standard

224 Antioxidant Properties

(i) Total Phenolic Content (TPC) It was determined by usingFolin-Ciocalteu method with slight modifications [15] Asreferred to literature 10mg of each crudemethanol extract orlyophilized herbal tea was dissolved in 1mL of correspondingextracting solvent to produce the stock of sample solutionThe lower concentrations of samplewere prepared by diluting100 120583L of the stock sample solution with 900120583L methanolor distilled water Then 50 120583L of sample was put in atest tube and mixed with 60120583L of Folin-Ciocalteu reagentfollowed by an addition of 120 120583L of 35 sodium carbonateafter 3 minutes Then the resulting mixture was incubatedin dark at room temperature for an hour and absorbancewas measured at 725 nm (UV-1700 Shimadzu CorporationKyoto Japan) after incubation Caffeic acid was used ascalibrated standard and results were expressed as milligramCaffeic Acid Equivalent per gram of dry extract (mg CAEgdry extract) The content of phenolics for each extract wasdetermined in triplicate

Alongside 25120583L of extracts or standard solution of gallicacid (25 to 100 120583gmL) in DMSO was added to 125 120583Lof a Folin-Ciocalteu solution (10 vv) followed by 100120583L

of sodium carbonate (75wv) in a 96-well plate Thereagents were mixed and incubated for 30 minutes at roomtemperature protected from light and the absorbance wasmeasured at 765 nm Total phenolic content was expressedas mg Gallic Acid Equivalents per gram of dry extract (mgGAEg dry extract)The content of phenolics for each extractwas determined in triplicate

(ii) 11-Diphenyl-2-picrylhydrazyl (DPPH) Assay The DPPHradical scavenging assay was performed according to apreviously described method [15] The stock DPPH solution(314 120583M) was prepared in absolute ethanol and kept in darkat room temperature until its use Gallic acid was used asa positive control (IC50 45 120583gmL) and the total extractswere diluted in DMSO at appropriate concentrations Brieflyin a 96-well plate 190 120583L of the DPPH solution and 10120583Lof gallic acid or samples were incubated for 30min at roomtemperature protected from light and the absorbance wasmeasured at 517 nm A negative control containing 10 120583LDMSO and 190 120583L DPPH was performed each time as wellas blanks containing 10 120583L sample and 190120583L EtOH

(iii) 22-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic Acid)(ABTS) Assay TheABTS radical cation scavenging assay wasperformed using themethod reported by Re et al (1999) withslight modifications [16] In brief a stock ABTS aqua solution(7mM) was reacted with potassium persulfate aqua solution(245mM) and kept overnight in dark to yield a dark coloredsolution containing ABTS∙+ radical cation Trolox was usedas a positive control (IC50 83 120583gmL) and the total extractswere diluted in DMSO at appropriate concentrations Priorto use in the assay the ABTS∙+ radical cation was dilutedwith distilled water for an initial absorbance of about 0700(plusmn002) at 734 nm Afterwards in a 96-well plate 100 120583l ofABTS∙+ radical cation solution and 50 120583l of Trolox or sampleswere added incubated for 10min at room temperature andprotected from light and the absorbance was measured at734 nm A negative control containing 50 120583L DMSO and100 120583L ABTS was performed each time as well as blankscontaining 50 120583L sample and 100 120583L dist H

2O

The percentage of DPPH and ABTS scavenging wasestimated by the following equation

119860119860 = [(119860 minus 119861) minus (119862 minus 119863)](119860 minus 119861) times 100 (1)

119860 is the control (without sample) 119861 is the blank (withoutsample without DPPHABTS) 119862is the sample and 119863 isthe blank sample (without DPPHABTS) IC50 values wereestimated for the most active extracts

For both experiments which referred to the free radicalscavenging all samples were analyzed in triplicate Measure-ments were performed using a TECAN Infinite M200 PROmultimode reader (Tecan Group Mannedorf Switzerland)

225 Antimicrobial Bioassay Themethanol extracts and theherbal teas of Kainari were evaluated for their in vitro antimi-crobial activities using the standard antibiotics netilmicinamoxicillin and clavulanic acid in order to evaluate thesensitivity of the tested bacteria while 5-fluorocytosineand amphotericin were used as sensitivity controls of the

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Comparative table for the powders of KN1 KN2 and KN3 with HS-SPME

Compound area KN1 area KN2 area KN3 Possible contained plantCinnamic aldehyde 988 1000 1519 Cinnamomum cassiaSafrole lt1 lt1 - Myristica fragrans Piper nigrumCamphene lt1 278 - Myristica fragrans Piper nigrumEugenol 3450 3018 2271 Syzygium aromaticum Cinnamomum cassia Myristica fragrans Piper nigrum120572-Copaene 188 416 1081 Syzygium aromaticum Piper nigrum

120573-Caryophyllene 1561 3018 3316 Cinnamomum cassia Syzygium aromaticum Piper nigrum Myristica fragransElettaria cardamomum

120572-Humulene 250 377 359 Syzygium aromaticum Piper nigrum Elettaria cardamomumAr-Curcumene 194 166 - Zingiber officinaleZingiberene 113 126 - Zingiber officinaleMyristicin 334 - - Myristica fragrans

tested fungi [17 18] A total of eleven microorganisms wereassayed amongwhich therewere fourGram-positive bacteriaStaphylococcus aureus (ATCC 25923) Staphylococcus epider-midis (ATCC 12228) Streptococcus mutans and Streptococcusviridans and four Gram-negative bacteria Escherichia coli(ATCC 25922) Enterobacter cloacae (ATCC 13047)Klebsiellapneumoniae (ATCC 13883) and Pseudomonas aeruginosa(ATCC 227853) as well as three pathogen fungi Candidaalbicans (ATCC 10231) C tropicalis (ATCC 13801) and Cglabrata (ATCC 28838)

3 Results and Discussion

31 Results from HS-SPMEGC-MS Analyses All three sam-ples of ldquoKainarirdquo from Mytilini Paleochori and Agiassos inLesvos were studied for the first time to our knowledge Therich aroma of the mixture was studied through HeadspaceSolid-Phase Microextraction (HS-SPME)GC-MS analysesFurthermore the pentane and methanol extracts were alsostudied through GC-MS with respective results The majorconstituents that have been determined through GC-MSanalyses were cinnamic aldehyde eugenol myristicin 120573-caryophyllene curcumene and zingiberene which can berelated to the possible contained spices as shown in Table 1

Through analyses of all extracts of the three samples(herbal teas methanol and pentane extracts) and the simul-taneous use of bibliography we conclude that the maincomponents in all three samples are cinnamon (sim610) andclove (sim310) and the remaining part (110) corresponds toboth nutmeg and ginger

It is remarkable that according to the analyses of the volat-iles in KN3 they were not identified as the main componentsof ginger (Zingiber officinale) and consequently we assumethat ginger is not included in the mixture or it is included insmall quantities As for the presence of cardamomandpepperdue to the lack of someof theirmainmetabolites (someof themexist in other contained plants at the same time) we concludethat if they are present they will be in small quantities

32 Isolation and Determination of Compounds Six com-pounds were isolated from the essential oil of sample ΚN2 byapplying a series of liquid-liquid extractions based on acid-base chemical reactions as well as with preparative TLCeugenol cinnamic aldehyde andmyristicin were determinedby NMR spectroscopy and GC-MS analysis while cinnamyl

Table 2 Total phenolic content of the herbal teas of KN1 KN2 andKN3

mg CAEgr dry extract mg GAEgr dry extractSample KN1 aq 30829 1152Sample KN2 aq 32031 1492Sample KN3 aq 38341 1845

Table 3 inhibition at DPPH and ABTS assay of herbal teas andmethanol extracts

DPPH ABTS inhibition50 120583gmL

inhibition10 120583gmL

KN1 aq 374 plusmn 01 284 plusmn 01KN2 aq 502 plusmn 14 388 plusmn 13KN3 aq 647 plusmn 03 469 plusmn 09KN1 meth 646 plusmn 03 482 plusmn 07KN2 meth 409 plusmn 07 269 plusmn 07KN3 meth 614 plusmn 01 403 plusmn 14

alcohol alpha-terpinyl acetate and 120573-caryophyllene weredetermined only by GC-MS analysis

Also two food dyeswere isolated and identified as parts ofthe family of azo colors Samples KN1 and KN2 contained thesame food color azorubine [19] while sample KN3 containedamaranth [11]

The azo colors are compounds bearing the functionalgroup R-N = N-R1015840 where R and R1015840 may be either arylor alkyl Azo dyes are one of the most important chemicalcategories of dyes which are used for the coloring of naturaland synthetic fibers candies and cosmetics but also foodand beverages [20 21] Many azo dyes are nontoxic althoughsome of them have been accused for allergic reactions andincreasing hyperactivity in children [22]

The two isolated compounds are being referred to EFSAas amaranth (E123) and azorubine or carmoisine (Ε122) andare well-known food dyes [19 23]

33 Antioxidant Properties The total phenolic content (TPC)of the three samples was notably high probably due tothe known rich phenolic concentration of all spices thatthey contain [24 25] The sample KN3 revealed the higherconcentration of phenolic content (Table 2)

The free radical scavenging activities of herbal teasand methanol extracts were determined by DPPH andABTS assays (Table 3) All extracts showed significant

Evidence-Based Complementary and Alternative Medicine 5

Table4Antim

icrobialactiv

ityof

herbalteas

andmethano

lextracts

Samples

Saureus

Sepidermidis

Paeruginosa

Eclo

acae

Kpn

eumoniae

Ecoli

Smutan

sSvirid

ans

Calbicans

Ctro

picalis

Cgla

brata

Kainari

KN1a

q16009

17004

14013

14015

13010

15017

13030

14039

12040

13039

13040

KN1m

eth

11070

11073

10090

10093

10089

10095

ntnt

11042

13033

12031

KN2aq

17004

17005

15010

15013

15009

15012

15015

16016

12043

13040

13042

KN2meth

10072

11068

10086

10087

10092

09098

ntnt

10053

11044

12042

KN3aq

18002

19001

16006

16005

16005

15008

17007

18003

13039

13031

13029

KN3meth

10072

11068

10086

10087

10092

09098

ntnt

12038

13033

13034

5-Fluo

rocytosin

ent

ntnt

ntnt

ntnt

nt01sdot10minus31sdot10minus310sdot10minus3

Amph

otericin

ntnt

ntnt

ntnt

ntnt

1sdot10minus305sdot10minus304sdot10minus3

Amoxicillin

2sdot10minus3

2sdot10minus3

24sdot10minus322sdot10minus328sdot10minus32sdot10minus3

ntnt

ntnt

ntNetilm

icin

4sdot10minus3

4sdot10minus3

88sdot10minus38sdot10minus3

8sdot10minus3

10sdot10minus3

ntnt

ntnt

ntClavulanicacid2sdot10minus3

2sdot10minus3

24sdot10minus322sdot10minus328sdot10minus32sdot10minus3

ntnt

ntnt

nt

6 Evidence-Based Complementary and Alternative Medicine

antioxidant activity performing 30ndash64 inhibition at DPPHassay (50 120583gmL) as well as 26ndash48 inhibition at ABTSassay (10 120583gmL) The herbal tea KN3 performed the higherinhibition at DPPH (647) and at ABTS (469) which is inaccordance with the high TPC Also themethanol extracts ofKN1 and KN3 showed high inhibition

34 Antimicrobial Bioassay According to in vitro antimicro-bial tests (Table 4) against two Gram-positive bacteria (Saureus and S epidermidis) and four Gram-negative ones (Ecoli E cloacae K pneumoniae and P aeruginosa) as well asagainst oral pathogens (S mutans and S viridans) and threehuman pathogenic fungi (Candida albicans C tropicalis andC glabrata) the herbal teas exhibited a broad spectrum ofstrong antimicrobial activity while KN3 herbal tea appearedto be themost active against all testedmicroorganisms whichcan be explained by the high percentage of cinnamic aldehydeand 120573-caryophyllene that appears in herbal tea of KN3 asshown in Table 1

4 Conclusion

In conclusion the chemical composition as well as thetotal phenolic content (TPC) and the potential antioxidantand antimicrobial activity of three Kainari-herbal teas fromdifferent areas of Lesvos Island (Greece) was evaluated Allsamples exerted very broad and strong antimicrobial activ-ities against almost all Gram-positive and Gram-negativehuman pathogenic bacteria as well as strong antioxidantactivities

According to the determined chemical analysis and totalphenolic content it could be concluded that ldquoKainarirdquo herbalinfusion except its very pleasant taste could be also furtherused as a functional herbal beverage due to its beneficialproperties strong antioxidant and antimicrobial activitiesfor consumers Certainly more studies along the potentialfurther bioactivities as well as the entire process of theproduction of stable batches of ldquoKainarirdquo combination atindustrial level have to be designed in future

Disclosure

A preliminary version of this study entitled ldquoKainari ndash AGreek Traditional Herbal Tea from Lesvos Island ChemicalAnalysis Antioxidant and Antimicrobial Propertiesrdquo by thesame authors has been subjected and accepted as posterpresentation in ICNPU-2017 3rd International Conferenceon Natural Products Utilization which was held in BanskoBulgaria 18ndash21 October 2017 (PP24)

Conflicts of Interest

The authors declare they have no conflicts of interest for thiswork

References

[1] I C Arts and P C Hollman ldquoPolyphenols and disease risk inepidemiologic studiesrdquo American Journal of Clinical Nutritionvol 81 no 1 pp 317Sndash325S 2005

[2] V Williams Celebrating Life Customs around the World FromBaby Showers to Funerals [3 volumes] ABC-CLIO 2016

[3] A Papaconstantinou andA-MMaffry Talbot Becoming Byzan-tine children and childhood in Byzantium vol 1 HarvardUniversity Press 1 2009

[4] P Koukoules Speculum vol IV Π120572120587120572120577119906120590120578120589 1951[5] GDagronEmperor and priestThe imperial office in Byzantium

Cambridge University Press 2003[6] O A Talat Eski Turk Edebiyatında Mazmunlar ve Izahı Akcag

Yayınları Ankara 2000[7] K Albala Food Cultures of theWorld Encyclopedia Bloomsbury

Publishing 2011[8] Binnurrsquos Turkish Cookbook ldquoLohusa Serbetrdquo httpwwwturk-

ishcookbookcom201003lohusa-sherbetphp[9] ldquoLohusa Serbeti A Special Baby Sherbetrdquo A Seasonal Cook in

Turkey June 15 2011rdquo httpseasonalcookinturkeycomlohusa-serbeti-special-baby-sherbet

[10] ldquoIsolation of Eugenol Organic Chemistry 241 Lab ColbyCollegerdquo

[11] C X You H Y Jiang W J Zhang et al ldquoContact toxicityand repellency of the main components from the essential oilof Clausena anisum-olens against two stored product insectsrdquoJournal of Insect Science vol 15 no 1 2015

[12] Trans-Cinnamaldehyde ldquoHanHong Webrdquo httpwwwhan-honggroupcomnmrnmr enB34317html

[13] E M Elgendy and N A Al-Zahrani ldquoComparative Study ofNatural and Synthetic Food Additive Dye Amaranth throughPhotochemical Reactionsrdquo Indian Journal of Science Researchvol 4 pp 827ndash832 2015

[14] K G Miller C F Poole and T M P Pawlowskı ldquoClassificationof the botanical origin of cinnamonby solid-phasemicroextrac-tion and gas chromatographyrdquo Chromatographia vol 42 no 11-12 pp 639ndash646 1996

[15] A Bampouli K Kyriakopoulou G Papaefstathiou et al ldquoEval-uation of total antioxidant potential of Pistacia lentiscus varchia leaves extracts using UHPLC-HRMSrdquo Journal of FoodEngineering vol 167 article no 7970 pp 25ndash31 2015

[16] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[17] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 pp 493ndash496 1966

[18] I Wiegand K Hilpert and R E W Hancock ldquoAgar and brothdilution methods to determine the minimal inhibitory concen-tration (MIC) of antimicrobial substancesrdquo Nature Protocolsvol 3 no 2 pp 163ndash175 2008

[19] ldquoScientific Opinion on the re-evaluation of AzorubineCarmoi-sine (E 122) as a food additiverdquo EFSA Journal vol 7 no 11 p1332 2009

[20] R L M Allen Colour Chemistry Thomas Nelson amp SonsLondon UK 1979

[21] K Venkataraman The Chemistry of Synthetic Dyes AcademicPress London UK 1978

[22] FDA Background Document for the Food Advisory CommitteeCertified Color Additives in Food and Possible Association withAttention Deficit Hyperactivity Disorder in Children 2011

[23] ldquoScientific Opinion on the re-evaluation of Amaranth (E 123) asa food additiverdquo EFSA Journal vol 8 no 7 p 1649 2010

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Comparative table for the powders of KN1 KN2 and KN3 with HS-SPME

Compound area KN1 area KN2 area KN3 Possible contained plantCinnamic aldehyde 988 1000 1519 Cinnamomum cassiaSafrole lt1 lt1 - Myristica fragrans Piper nigrumCamphene lt1 278 - Myristica fragrans Piper nigrumEugenol 3450 3018 2271 Syzygium aromaticum Cinnamomum cassia Myristica fragrans Piper nigrum120572-Copaene 188 416 1081 Syzygium aromaticum Piper nigrum

120573-Caryophyllene 1561 3018 3316 Cinnamomum cassia Syzygium aromaticum Piper nigrum Myristica fragransElettaria cardamomum

120572-Humulene 250 377 359 Syzygium aromaticum Piper nigrum Elettaria cardamomumAr-Curcumene 194 166 - Zingiber officinaleZingiberene 113 126 - Zingiber officinaleMyristicin 334 - - Myristica fragrans

tested fungi [17 18] A total of eleven microorganisms wereassayed amongwhich therewere fourGram-positive bacteriaStaphylococcus aureus (ATCC 25923) Staphylococcus epider-midis (ATCC 12228) Streptococcus mutans and Streptococcusviridans and four Gram-negative bacteria Escherichia coli(ATCC 25922) Enterobacter cloacae (ATCC 13047)Klebsiellapneumoniae (ATCC 13883) and Pseudomonas aeruginosa(ATCC 227853) as well as three pathogen fungi Candidaalbicans (ATCC 10231) C tropicalis (ATCC 13801) and Cglabrata (ATCC 28838)

3 Results and Discussion

31 Results from HS-SPMEGC-MS Analyses All three sam-ples of ldquoKainarirdquo from Mytilini Paleochori and Agiassos inLesvos were studied for the first time to our knowledge Therich aroma of the mixture was studied through HeadspaceSolid-Phase Microextraction (HS-SPME)GC-MS analysesFurthermore the pentane and methanol extracts were alsostudied through GC-MS with respective results The majorconstituents that have been determined through GC-MSanalyses were cinnamic aldehyde eugenol myristicin 120573-caryophyllene curcumene and zingiberene which can berelated to the possible contained spices as shown in Table 1

Through analyses of all extracts of the three samples(herbal teas methanol and pentane extracts) and the simul-taneous use of bibliography we conclude that the maincomponents in all three samples are cinnamon (sim610) andclove (sim310) and the remaining part (110) corresponds toboth nutmeg and ginger

It is remarkable that according to the analyses of the volat-iles in KN3 they were not identified as the main componentsof ginger (Zingiber officinale) and consequently we assumethat ginger is not included in the mixture or it is included insmall quantities As for the presence of cardamomandpepperdue to the lack of someof theirmainmetabolites (someof themexist in other contained plants at the same time) we concludethat if they are present they will be in small quantities

32 Isolation and Determination of Compounds Six com-pounds were isolated from the essential oil of sample ΚN2 byapplying a series of liquid-liquid extractions based on acid-base chemical reactions as well as with preparative TLCeugenol cinnamic aldehyde andmyristicin were determinedby NMR spectroscopy and GC-MS analysis while cinnamyl

Table 2 Total phenolic content of the herbal teas of KN1 KN2 andKN3

mg CAEgr dry extract mg GAEgr dry extractSample KN1 aq 30829 1152Sample KN2 aq 32031 1492Sample KN3 aq 38341 1845

Table 3 inhibition at DPPH and ABTS assay of herbal teas andmethanol extracts

DPPH ABTS inhibition50 120583gmL

inhibition10 120583gmL

KN1 aq 374 plusmn 01 284 plusmn 01KN2 aq 502 plusmn 14 388 plusmn 13KN3 aq 647 plusmn 03 469 plusmn 09KN1 meth 646 plusmn 03 482 plusmn 07KN2 meth 409 plusmn 07 269 plusmn 07KN3 meth 614 plusmn 01 403 plusmn 14

alcohol alpha-terpinyl acetate and 120573-caryophyllene weredetermined only by GC-MS analysis

Also two food dyeswere isolated and identified as parts ofthe family of azo colors Samples KN1 and KN2 contained thesame food color azorubine [19] while sample KN3 containedamaranth [11]

The azo colors are compounds bearing the functionalgroup R-N = N-R1015840 where R and R1015840 may be either arylor alkyl Azo dyes are one of the most important chemicalcategories of dyes which are used for the coloring of naturaland synthetic fibers candies and cosmetics but also foodand beverages [20 21] Many azo dyes are nontoxic althoughsome of them have been accused for allergic reactions andincreasing hyperactivity in children [22]

The two isolated compounds are being referred to EFSAas amaranth (E123) and azorubine or carmoisine (Ε122) andare well-known food dyes [19 23]

33 Antioxidant Properties The total phenolic content (TPC)of the three samples was notably high probably due tothe known rich phenolic concentration of all spices thatthey contain [24 25] The sample KN3 revealed the higherconcentration of phenolic content (Table 2)

The free radical scavenging activities of herbal teasand methanol extracts were determined by DPPH andABTS assays (Table 3) All extracts showed significant

Evidence-Based Complementary and Alternative Medicine 5

Table4Antim

icrobialactiv

ityof

herbalteas

andmethano

lextracts

Samples

Saureus

Sepidermidis

Paeruginosa

Eclo

acae

Kpn

eumoniae

Ecoli

Smutan

sSvirid

ans

Calbicans

Ctro

picalis

Cgla

brata

Kainari

KN1a

q16009

17004

14013

14015

13010

15017

13030

14039

12040

13039

13040

KN1m

eth

11070

11073

10090

10093

10089

10095

ntnt

11042

13033

12031

KN2aq

17004

17005

15010

15013

15009

15012

15015

16016

12043

13040

13042

KN2meth

10072

11068

10086

10087

10092

09098

ntnt

10053

11044

12042

KN3aq

18002

19001

16006

16005

16005

15008

17007

18003

13039

13031

13029

KN3meth

10072

11068

10086

10087

10092

09098

ntnt

12038

13033

13034

5-Fluo

rocytosin

ent

ntnt

ntnt

ntnt

nt01sdot10minus31sdot10minus310sdot10minus3

Amph

otericin

ntnt

ntnt

ntnt

ntnt

1sdot10minus305sdot10minus304sdot10minus3

Amoxicillin

2sdot10minus3

2sdot10minus3

24sdot10minus322sdot10minus328sdot10minus32sdot10minus3

ntnt

ntnt

ntNetilm

icin

4sdot10minus3

4sdot10minus3

88sdot10minus38sdot10minus3

8sdot10minus3

10sdot10minus3

ntnt

ntnt

ntClavulanicacid2sdot10minus3

2sdot10minus3

24sdot10minus322sdot10minus328sdot10minus32sdot10minus3

ntnt

ntnt

nt

6 Evidence-Based Complementary and Alternative Medicine

antioxidant activity performing 30ndash64 inhibition at DPPHassay (50 120583gmL) as well as 26ndash48 inhibition at ABTSassay (10 120583gmL) The herbal tea KN3 performed the higherinhibition at DPPH (647) and at ABTS (469) which is inaccordance with the high TPC Also themethanol extracts ofKN1 and KN3 showed high inhibition

34 Antimicrobial Bioassay According to in vitro antimicro-bial tests (Table 4) against two Gram-positive bacteria (Saureus and S epidermidis) and four Gram-negative ones (Ecoli E cloacae K pneumoniae and P aeruginosa) as well asagainst oral pathogens (S mutans and S viridans) and threehuman pathogenic fungi (Candida albicans C tropicalis andC glabrata) the herbal teas exhibited a broad spectrum ofstrong antimicrobial activity while KN3 herbal tea appearedto be themost active against all testedmicroorganisms whichcan be explained by the high percentage of cinnamic aldehydeand 120573-caryophyllene that appears in herbal tea of KN3 asshown in Table 1

4 Conclusion

In conclusion the chemical composition as well as thetotal phenolic content (TPC) and the potential antioxidantand antimicrobial activity of three Kainari-herbal teas fromdifferent areas of Lesvos Island (Greece) was evaluated Allsamples exerted very broad and strong antimicrobial activ-ities against almost all Gram-positive and Gram-negativehuman pathogenic bacteria as well as strong antioxidantactivities

According to the determined chemical analysis and totalphenolic content it could be concluded that ldquoKainarirdquo herbalinfusion except its very pleasant taste could be also furtherused as a functional herbal beverage due to its beneficialproperties strong antioxidant and antimicrobial activitiesfor consumers Certainly more studies along the potentialfurther bioactivities as well as the entire process of theproduction of stable batches of ldquoKainarirdquo combination atindustrial level have to be designed in future

Disclosure

A preliminary version of this study entitled ldquoKainari ndash AGreek Traditional Herbal Tea from Lesvos Island ChemicalAnalysis Antioxidant and Antimicrobial Propertiesrdquo by thesame authors has been subjected and accepted as posterpresentation in ICNPU-2017 3rd International Conferenceon Natural Products Utilization which was held in BanskoBulgaria 18ndash21 October 2017 (PP24)

Conflicts of Interest

The authors declare they have no conflicts of interest for thiswork

References

[1] I C Arts and P C Hollman ldquoPolyphenols and disease risk inepidemiologic studiesrdquo American Journal of Clinical Nutritionvol 81 no 1 pp 317Sndash325S 2005

[2] V Williams Celebrating Life Customs around the World FromBaby Showers to Funerals [3 volumes] ABC-CLIO 2016

[3] A Papaconstantinou andA-MMaffry Talbot Becoming Byzan-tine children and childhood in Byzantium vol 1 HarvardUniversity Press 1 2009

[4] P Koukoules Speculum vol IV Π120572120587120572120577119906120590120578120589 1951[5] GDagronEmperor and priestThe imperial office in Byzantium

Cambridge University Press 2003[6] O A Talat Eski Turk Edebiyatında Mazmunlar ve Izahı Akcag

Yayınları Ankara 2000[7] K Albala Food Cultures of theWorld Encyclopedia Bloomsbury

Publishing 2011[8] Binnurrsquos Turkish Cookbook ldquoLohusa Serbetrdquo httpwwwturk-

ishcookbookcom201003lohusa-sherbetphp[9] ldquoLohusa Serbeti A Special Baby Sherbetrdquo A Seasonal Cook in

Turkey June 15 2011rdquo httpseasonalcookinturkeycomlohusa-serbeti-special-baby-sherbet

[10] ldquoIsolation of Eugenol Organic Chemistry 241 Lab ColbyCollegerdquo

[11] C X You H Y Jiang W J Zhang et al ldquoContact toxicityand repellency of the main components from the essential oilof Clausena anisum-olens against two stored product insectsrdquoJournal of Insect Science vol 15 no 1 2015

[12] Trans-Cinnamaldehyde ldquoHanHong Webrdquo httpwwwhan-honggroupcomnmrnmr enB34317html

[13] E M Elgendy and N A Al-Zahrani ldquoComparative Study ofNatural and Synthetic Food Additive Dye Amaranth throughPhotochemical Reactionsrdquo Indian Journal of Science Researchvol 4 pp 827ndash832 2015

[14] K G Miller C F Poole and T M P Pawlowskı ldquoClassificationof the botanical origin of cinnamonby solid-phasemicroextrac-tion and gas chromatographyrdquo Chromatographia vol 42 no 11-12 pp 639ndash646 1996

[15] A Bampouli K Kyriakopoulou G Papaefstathiou et al ldquoEval-uation of total antioxidant potential of Pistacia lentiscus varchia leaves extracts using UHPLC-HRMSrdquo Journal of FoodEngineering vol 167 article no 7970 pp 25ndash31 2015

[16] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[17] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 pp 493ndash496 1966

[18] I Wiegand K Hilpert and R E W Hancock ldquoAgar and brothdilution methods to determine the minimal inhibitory concen-tration (MIC) of antimicrobial substancesrdquo Nature Protocolsvol 3 no 2 pp 163ndash175 2008

[19] ldquoScientific Opinion on the re-evaluation of AzorubineCarmoi-sine (E 122) as a food additiverdquo EFSA Journal vol 7 no 11 p1332 2009

[20] R L M Allen Colour Chemistry Thomas Nelson amp SonsLondon UK 1979

[21] K Venkataraman The Chemistry of Synthetic Dyes AcademicPress London UK 1978

[22] FDA Background Document for the Food Advisory CommitteeCertified Color Additives in Food and Possible Association withAttention Deficit Hyperactivity Disorder in Children 2011

[23] ldquoScientific Opinion on the re-evaluation of Amaranth (E 123) asa food additiverdquo EFSA Journal vol 8 no 7 p 1649 2010

Evidence-Based Complementary and Alternative Medicine 5

Table4Antim

icrobialactiv

ityof

herbalteas

andmethano

lextracts

Samples

Saureus

Sepidermidis

Paeruginosa

Eclo

acae

Kpn

eumoniae

Ecoli

Smutan

sSvirid

ans

Calbicans

Ctro

picalis

Cgla

brata

Kainari

KN1a

q16009

17004

14013

14015

13010

15017

13030

14039

12040

13039

13040

KN1m

eth

11070

11073

10090

10093

10089

10095

ntnt

11042

13033

12031

KN2aq

17004

17005

15010

15013

15009

15012

15015

16016

12043

13040

13042

KN2meth

10072

11068

10086

10087

10092

09098

ntnt

10053

11044

12042

KN3aq

18002

19001

16006

16005

16005

15008

17007

18003

13039

13031

13029

KN3meth

10072

11068

10086

10087

10092

09098

ntnt

12038

13033

13034

5-Fluo

rocytosin

ent

ntnt

ntnt

ntnt

nt01sdot10minus31sdot10minus310sdot10minus3

Amph

otericin

ntnt

ntnt

ntnt

ntnt

1sdot10minus305sdot10minus304sdot10minus3

Amoxicillin

2sdot10minus3

2sdot10minus3

24sdot10minus322sdot10minus328sdot10minus32sdot10minus3

ntnt

ntnt

ntNetilm

icin

4sdot10minus3

4sdot10minus3

88sdot10minus38sdot10minus3

8sdot10minus3

10sdot10minus3

ntnt

ntnt

ntClavulanicacid2sdot10minus3

2sdot10minus3

24sdot10minus322sdot10minus328sdot10minus32sdot10minus3

ntnt

ntnt

nt

6 Evidence-Based Complementary and Alternative Medicine

antioxidant activity performing 30ndash64 inhibition at DPPHassay (50 120583gmL) as well as 26ndash48 inhibition at ABTSassay (10 120583gmL) The herbal tea KN3 performed the higherinhibition at DPPH (647) and at ABTS (469) which is inaccordance with the high TPC Also themethanol extracts ofKN1 and KN3 showed high inhibition

34 Antimicrobial Bioassay According to in vitro antimicro-bial tests (Table 4) against two Gram-positive bacteria (Saureus and S epidermidis) and four Gram-negative ones (Ecoli E cloacae K pneumoniae and P aeruginosa) as well asagainst oral pathogens (S mutans and S viridans) and threehuman pathogenic fungi (Candida albicans C tropicalis andC glabrata) the herbal teas exhibited a broad spectrum ofstrong antimicrobial activity while KN3 herbal tea appearedto be themost active against all testedmicroorganisms whichcan be explained by the high percentage of cinnamic aldehydeand 120573-caryophyllene that appears in herbal tea of KN3 asshown in Table 1

4 Conclusion

In conclusion the chemical composition as well as thetotal phenolic content (TPC) and the potential antioxidantand antimicrobial activity of three Kainari-herbal teas fromdifferent areas of Lesvos Island (Greece) was evaluated Allsamples exerted very broad and strong antimicrobial activ-ities against almost all Gram-positive and Gram-negativehuman pathogenic bacteria as well as strong antioxidantactivities

According to the determined chemical analysis and totalphenolic content it could be concluded that ldquoKainarirdquo herbalinfusion except its very pleasant taste could be also furtherused as a functional herbal beverage due to its beneficialproperties strong antioxidant and antimicrobial activitiesfor consumers Certainly more studies along the potentialfurther bioactivities as well as the entire process of theproduction of stable batches of ldquoKainarirdquo combination atindustrial level have to be designed in future

Disclosure

A preliminary version of this study entitled ldquoKainari ndash AGreek Traditional Herbal Tea from Lesvos Island ChemicalAnalysis Antioxidant and Antimicrobial Propertiesrdquo by thesame authors has been subjected and accepted as posterpresentation in ICNPU-2017 3rd International Conferenceon Natural Products Utilization which was held in BanskoBulgaria 18ndash21 October 2017 (PP24)

Conflicts of Interest

The authors declare they have no conflicts of interest for thiswork

References

[1] I C Arts and P C Hollman ldquoPolyphenols and disease risk inepidemiologic studiesrdquo American Journal of Clinical Nutritionvol 81 no 1 pp 317Sndash325S 2005

[2] V Williams Celebrating Life Customs around the World FromBaby Showers to Funerals [3 volumes] ABC-CLIO 2016

[3] A Papaconstantinou andA-MMaffry Talbot Becoming Byzan-tine children and childhood in Byzantium vol 1 HarvardUniversity Press 1 2009

[4] P Koukoules Speculum vol IV Π120572120587120572120577119906120590120578120589 1951[5] GDagronEmperor and priestThe imperial office in Byzantium

Cambridge University Press 2003[6] O A Talat Eski Turk Edebiyatında Mazmunlar ve Izahı Akcag

Yayınları Ankara 2000[7] K Albala Food Cultures of theWorld Encyclopedia Bloomsbury

Publishing 2011[8] Binnurrsquos Turkish Cookbook ldquoLohusa Serbetrdquo httpwwwturk-

ishcookbookcom201003lohusa-sherbetphp[9] ldquoLohusa Serbeti A Special Baby Sherbetrdquo A Seasonal Cook in

Turkey June 15 2011rdquo httpseasonalcookinturkeycomlohusa-serbeti-special-baby-sherbet

[10] ldquoIsolation of Eugenol Organic Chemistry 241 Lab ColbyCollegerdquo

[11] C X You H Y Jiang W J Zhang et al ldquoContact toxicityand repellency of the main components from the essential oilof Clausena anisum-olens against two stored product insectsrdquoJournal of Insect Science vol 15 no 1 2015

[12] Trans-Cinnamaldehyde ldquoHanHong Webrdquo httpwwwhan-honggroupcomnmrnmr enB34317html

[13] E M Elgendy and N A Al-Zahrani ldquoComparative Study ofNatural and Synthetic Food Additive Dye Amaranth throughPhotochemical Reactionsrdquo Indian Journal of Science Researchvol 4 pp 827ndash832 2015

[14] K G Miller C F Poole and T M P Pawlowskı ldquoClassificationof the botanical origin of cinnamonby solid-phasemicroextrac-tion and gas chromatographyrdquo Chromatographia vol 42 no 11-12 pp 639ndash646 1996

[15] A Bampouli K Kyriakopoulou G Papaefstathiou et al ldquoEval-uation of total antioxidant potential of Pistacia lentiscus varchia leaves extracts using UHPLC-HRMSrdquo Journal of FoodEngineering vol 167 article no 7970 pp 25ndash31 2015

[16] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[17] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 pp 493ndash496 1966

[18] I Wiegand K Hilpert and R E W Hancock ldquoAgar and brothdilution methods to determine the minimal inhibitory concen-tration (MIC) of antimicrobial substancesrdquo Nature Protocolsvol 3 no 2 pp 163ndash175 2008

[19] ldquoScientific Opinion on the re-evaluation of AzorubineCarmoi-sine (E 122) as a food additiverdquo EFSA Journal vol 7 no 11 p1332 2009

[20] R L M Allen Colour Chemistry Thomas Nelson amp SonsLondon UK 1979

[21] K Venkataraman The Chemistry of Synthetic Dyes AcademicPress London UK 1978

[22] FDA Background Document for the Food Advisory CommitteeCertified Color Additives in Food and Possible Association withAttention Deficit Hyperactivity Disorder in Children 2011

[23] ldquoScientific Opinion on the re-evaluation of Amaranth (E 123) asa food additiverdquo EFSA Journal vol 8 no 7 p 1649 2010

6 Evidence-Based Complementary and Alternative Medicine

antioxidant activity performing 30ndash64 inhibition at DPPHassay (50 120583gmL) as well as 26ndash48 inhibition at ABTSassay (10 120583gmL) The herbal tea KN3 performed the higherinhibition at DPPH (647) and at ABTS (469) which is inaccordance with the high TPC Also themethanol extracts ofKN1 and KN3 showed high inhibition

34 Antimicrobial Bioassay According to in vitro antimicro-bial tests (Table 4) against two Gram-positive bacteria (Saureus and S epidermidis) and four Gram-negative ones (Ecoli E cloacae K pneumoniae and P aeruginosa) as well asagainst oral pathogens (S mutans and S viridans) and threehuman pathogenic fungi (Candida albicans C tropicalis andC glabrata) the herbal teas exhibited a broad spectrum ofstrong antimicrobial activity while KN3 herbal tea appearedto be themost active against all testedmicroorganisms whichcan be explained by the high percentage of cinnamic aldehydeand 120573-caryophyllene that appears in herbal tea of KN3 asshown in Table 1

4 Conclusion

In conclusion the chemical composition as well as thetotal phenolic content (TPC) and the potential antioxidantand antimicrobial activity of three Kainari-herbal teas fromdifferent areas of Lesvos Island (Greece) was evaluated Allsamples exerted very broad and strong antimicrobial activ-ities against almost all Gram-positive and Gram-negativehuman pathogenic bacteria as well as strong antioxidantactivities

According to the determined chemical analysis and totalphenolic content it could be concluded that ldquoKainarirdquo herbalinfusion except its very pleasant taste could be also furtherused as a functional herbal beverage due to its beneficialproperties strong antioxidant and antimicrobial activitiesfor consumers Certainly more studies along the potentialfurther bioactivities as well as the entire process of theproduction of stable batches of ldquoKainarirdquo combination atindustrial level have to be designed in future

Disclosure

A preliminary version of this study entitled ldquoKainari ndash AGreek Traditional Herbal Tea from Lesvos Island ChemicalAnalysis Antioxidant and Antimicrobial Propertiesrdquo by thesame authors has been subjected and accepted as posterpresentation in ICNPU-2017 3rd International Conferenceon Natural Products Utilization which was held in BanskoBulgaria 18ndash21 October 2017 (PP24)

Conflicts of Interest

The authors declare they have no conflicts of interest for thiswork

References

[1] I C Arts and P C Hollman ldquoPolyphenols and disease risk inepidemiologic studiesrdquo American Journal of Clinical Nutritionvol 81 no 1 pp 317Sndash325S 2005

[2] V Williams Celebrating Life Customs around the World FromBaby Showers to Funerals [3 volumes] ABC-CLIO 2016

[3] A Papaconstantinou andA-MMaffry Talbot Becoming Byzan-tine children and childhood in Byzantium vol 1 HarvardUniversity Press 1 2009

[4] P Koukoules Speculum vol IV Π120572120587120572120577119906120590120578120589 1951[5] GDagronEmperor and priestThe imperial office in Byzantium

Cambridge University Press 2003[6] O A Talat Eski Turk Edebiyatında Mazmunlar ve Izahı Akcag

Yayınları Ankara 2000[7] K Albala Food Cultures of theWorld Encyclopedia Bloomsbury

Publishing 2011[8] Binnurrsquos Turkish Cookbook ldquoLohusa Serbetrdquo httpwwwturk-

ishcookbookcom201003lohusa-sherbetphp[9] ldquoLohusa Serbeti A Special Baby Sherbetrdquo A Seasonal Cook in

Turkey June 15 2011rdquo httpseasonalcookinturkeycomlohusa-serbeti-special-baby-sherbet

[10] ldquoIsolation of Eugenol Organic Chemistry 241 Lab ColbyCollegerdquo

[11] C X You H Y Jiang W J Zhang et al ldquoContact toxicityand repellency of the main components from the essential oilof Clausena anisum-olens against two stored product insectsrdquoJournal of Insect Science vol 15 no 1 2015

[12] Trans-Cinnamaldehyde ldquoHanHong Webrdquo httpwwwhan-honggroupcomnmrnmr enB34317html

[13] E M Elgendy and N A Al-Zahrani ldquoComparative Study ofNatural and Synthetic Food Additive Dye Amaranth throughPhotochemical Reactionsrdquo Indian Journal of Science Researchvol 4 pp 827ndash832 2015

[14] K G Miller C F Poole and T M P Pawlowskı ldquoClassificationof the botanical origin of cinnamonby solid-phasemicroextrac-tion and gas chromatographyrdquo Chromatographia vol 42 no 11-12 pp 639ndash646 1996

[15] A Bampouli K Kyriakopoulou G Papaefstathiou et al ldquoEval-uation of total antioxidant potential of Pistacia lentiscus varchia leaves extracts using UHPLC-HRMSrdquo Journal of FoodEngineering vol 167 article no 7970 pp 25ndash31 2015

[16] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[17] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 45 no 4 pp 493ndash496 1966

[18] I Wiegand K Hilpert and R E W Hancock ldquoAgar and brothdilution methods to determine the minimal inhibitory concen-tration (MIC) of antimicrobial substancesrdquo Nature Protocolsvol 3 no 2 pp 163ndash175 2008

[19] ldquoScientific Opinion on the re-evaluation of AzorubineCarmoi-sine (E 122) as a food additiverdquo EFSA Journal vol 7 no 11 p1332 2009

[20] R L M Allen Colour Chemistry Thomas Nelson amp SonsLondon UK 1979

[21] K Venkataraman The Chemistry of Synthetic Dyes AcademicPress London UK 1978

[22] FDA Background Document for the Food Advisory CommitteeCertified Color Additives in Food and Possible Association withAttention Deficit Hyperactivity Disorder in Children 2011

[23] ldquoScientific Opinion on the re-evaluation of Amaranth (E 123) asa food additiverdquo EFSA Journal vol 8 no 7 p 1649 2010

Evidence-Based Complementary and Alternative Medicine 7

[24] L Su J-J Yin D Charles K Zhou J Moore and L Yu ldquoTotalphenolic contents chelating capacities and radical-scavengingproperties of black peppercorn nutmeg rosehip cinnamon andoregano leafrdquo Food Chemistry vol 100 no 3 pp 990ndash997 2007

[25] D F Cortes-Rojas C R F de Souza and W P OliveiraldquoClove (Syzygium aromaticum) A precious spicerdquoAsian PacificJournal of Tropical Biomedicine vol 4 no 2 pp 90ndash96 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom