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CHAPTER 2

LITERATURE REVIEW

2.1 SPIRULINA AS A FOOD AND MEDICINE

Microalgae are capable of producing valuable metabolites, such as

pigments, proteins and vitamins for feed addition, pharmaceutical and

nutraceutical purposes [42-44, 40]. It is the cheap source of protein and other

essential nutritional requirement for human when compared to the fruits and

vegetables available in the market. Many experimental studies have been

conducted for protein production, by microorganisms for food and feed

purposes. Among these, the micro alga, Spirulina platensis presents advantages

due to high protein content (60 to 70%) with low nucleic acids concentration

and the amino acids content similar to the FAO’s recommendation [45,12].

In addition, there is a possibility of obtaining other products like

pigments such as carotenoids, phycocyanin and chlorophyll, vitamins and

polyunsaturated fatty acids including omega-3 fatty acid [46, 47]. Another

advantage over Chlorella and Scenedesmus (used as food supplements for

human being) is its easy digestibility due to lack of cellulosic membrane [48].

The S.platensis could be an alternative source of protein for human food and

feed purposes [49]. The biomass can be introduced directly in the diet and it

can also be used in case of malnutrition.

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The occurrence, isolation, morphology and most importantly the

uniqueness and prospects of Spirulina as a food source was cited in the review

of Ciferri [16].

2.2 CULTIVATION

Spirulina can be easily cultivated by providing basic salts in the

medium under laboratory conditions. Spirulina has been cultivated generally in

a synthetic medium developed by Zarrouk. Zarrouk’s medium, rich in

bicarbonate, has been successfully served as a common culture medium for

Spirulina culture over years [39]. Zarrouk’s medium is a comprehensive yet

elaborate and expensive for large scale algal production. Several formulae for

algal media are available in literature and supports growth rate compared with

standard synthetic media.

2.2.1 Supplements.

Seshadri and Thomas [41] simplified Spirulina cultivation by utilizing

certain rural wastes such as bone meal and effluents from biogas digesters.

They used biogas effluent (cow dung as substrate) to replace most of the

chemicals in the original medium developed for the cultivation of Spirulina.

Researchers already tried with waste water, sewage mixture, cow manure,

fertilizer etc., for mass cultivation of Spirulina. An attempt was made to

cultivate Spirulina platensis using human urine directly. Their objective was to

achieve biomass production and oxygen evolution, for potential application to

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nutrient regeneration and air revitalization in life-support system. The growth

of Spirulina platensis was lower in diluted human urine, when compared to

Zarrouk’s medium [50]. Growth and pigment production of Spirulina platensis

were stimulated at low concentration of Pb2+(1 mg/l). The biosynthesis of

pigment seems to be decreasing with increasing concentration of Pb2+ [51].

The kitchen waste water (KW) and oil-extracted fermented

soybean water (SW) for cultivation of S.platensis was evaluated. The water

quality, biomass production and pigment content of S.platensis were

determined every 5 days for a period of 15 days. The maximum biomass

production was achieved in 5% SW was 0.90 g/l and high phycocaynin

(21.27mg/g) in 10% SW was obtained [52]. Spirulina cultivation was achieved

in swine waste water and swine dung extract medium [53,54]. The biomass

thus obtained can be added to fish, cattle and poultry feed for meat production.

Spirulina platensis was cultured in CFTRI medium with Petha waste

(Benincasa hispida) supplement at a concentration ranging from 10% - 60%

with 12/12 light and dark cycle for a period of 27 days. The growth was

measured in the form of dry mass. High biomass production was found in

lower concentrations whereas at higher concentrations there was decline in

biomass yield [55].

The Spirulina platensis was cultivated in laboratory, under

controlled conditions (30ºC, photoperiod of 12 hours light/dark provided by

fluorescent lamps at a light intensity of 140 �mol photons.m-2.s-1 and constant

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bubbling air) in three different culture media: (1) Paoletti medium (control), (2)

Paoletti supplemented with 1 g/1 NaCl (salinated water) and (3) Paoletti

medium prepared with desalinator’s wastewater. The effects of these

treatments on growth, protein content and amino acid profile were measured.

The cell concentrations observed in Paoletti medium was 2.587g/l, Paoletti

supplemented with salinated water was3.545 g/l and with desalinator’s

wastewater was 4.954 g.L-1 [56].

Water from Mangueira Lagoon located at Rio Grande do Sul State,

Brazil was used as a culture medium to cultivate Spirulina [57], as the water

contains high levels of carbonates and has high pH. The effect of

un-supplemented Mangueira Lagoon Water (MLW) or MLW supplemented

with 1.125 or 2.250 mg/L of urea or 42 mg/L of sodium bicarbonate, on the

growth of S.platensis in fed-batch culture system was tested. It was found that,

the addition of 1.125 mg/L of urea resulted in 2.67 fold increase in the final

biomass concentration of S. plantensis.

Pelizer [58] determined the inoculum’s age and size of the

S. platensis. Cultivation was carried out in Erlenmeyer’s flasks and in mini

tanks with four different periods of incubation as 3, 6, 10 and 14 days with four

different concentrations of 50,100,150 and 200 mg/L were experimented. They

concluded that the best inoculum’s age was six days with 50 mg/L of

concentration.

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The influence of temperature and nitrogen concentration in the

medium on the production of biomass and composition of protein, lipids and

phenolic compounds were analyzed [59]. They found that growth at 35 °C had

shown negative effect on biomass production and positive effect on the

production of protein, lipids and phenolics. The highest level of these

compounds was obtained in Zarrouk’s medium containing 1.875 or 2.5 (g / l-1)

sodium nitrate. Higher biomass densities and productivity were obtained at

30 °C than at 35 °C, but nitrogen concentration appeared to have no effect on

the amount of protein, lipids or phenolics, indicating that at 30 °C the

concentration of sodium nitrate in Zarrouk’s medium (2.50 g/l-1) can be

reduced without loss of productivity.

The factors influencing growth and C-phycocyanin (CPC) yield of

Spirulina platensis were evaluated. The cell mass grown with MgCl2 showed

maximum CPC concentration upto 0.413 mg/ml and minimum of 0.356 mg/ml

with MgCO3 and with MgNO3 0.404 mg/ml respectively [60].

The study conducted to ascertain the suitability of spent wash (distillery

effluent) as a medium to grow the Spirulina platensis [61] proved its utility.

The spent wash alone at the concentration of 7.5% was found to be optimum

and the pH of 9.2 favored the growth of the organism. It was concluded that

the spent wash could be effectively utilized to grow Spirulina, and thereby this

attempt could be sure in effect with the disposal of the spent wash without

creating any environmental hazard. The study conducted using swine dung

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extract in cultivating Spirulina platensis proved that the swine dung medium

could be an alternative and cheap medium for Spirulina platensis [62].

To obtain high yield of Spirulina platensis biomass, it is necessary to

define an optimum medium composition consisting of mineral salts and

organic complex derived from low cost resources. This study is in search of

cheap resources for the enhanced yield of Spirulina platensis biomass and thus

to define media composition for a good growth. Four different supplements

were incorporated at four different concentrations to the basal medium [39].

They are:

1. Multivitamins

2. Tender coconut water

3. Cow urine and

4. Cow dung extract

Multivitamin is a preparation intended to supplement human diet with

vitamins, dietary minerals and other nutritional elements. Such preparations are

available in the form of tablets, capsules, pastilles, powders, liquids and

injectable formulations. Multivitamins are recognized by the Codex

Ailmentarius Commission (the United Nations authority on food standards) as

a category of food. Multivitamins are commonly provided in combination with

minerals. Basic commercial multivitamin supplement products often contain

the following ingredients: vitamin C,B1, B2, B3, B6, B9(folic acid), B12,

B5(pantothenate), H(biotin), A, E, D3,K1, potassium iodide, cupric sulfate

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anhydrous, picolinate, cupric sulfate monohydrate, cupric trioxide,

selenomethionine, borates, zinc, calcium, magnesium, chromium, manganese,

molybdenum, beta carotenes and iron(Wikipedia).

The Tender coconut water, liquid endosperm, is the most nutritious

wholesome beverage that the nature has provided for the people of the tropics

to overcome the sultry heat. It has calorific value of 17.4 calories/100gm. “It is

unctuous, sweet, increases semen, promotes digestion and clears the urinary

path”, says Ayurveda on tender coconut water. The major chemical

constituents of coconut water are sugar and minerals and minor constituents

are fat and nitrogenous substances. Tender coconut water contains both

ascorbic acid and vitamins of B group. The concentration of ascorbic acid

ranges from 2.2 to 3.7/ml, which gradually diminishes as the kernel

surrounding the water begins to harden (Wikipedia).

The cow dung contains the following chemical components; Nitrogen,

Phosphorous, Potassium, Calcium, Sulphur, Zinc, Copper, Manganese and

Iron. The fresh cow dung contains 18.8% dry matter and 1.83% of total

Nitrogen on dry matter basis.

Cow urine, as the Ayurvedic texts denotes, is a great elixir, proper diet,

pleasing to heart, provides mental and physical strength and enhances

longevity. It balances bile, mucous and airs, cures heart diseases and has effect

on poison. In Ayurveda where ever urine is mentioned, it is to be understood as

cow urine. The cow urine has the following chemical composition:

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Nitrogen(N2,NH2), Sulphur, Ammonia(NH3), Copper(Cu), Iron(Fe),

Urea(CO(NH2)2), Uric acid(C5H4N4O3), Phosphate(P), Sodium(Na),

Potassium(K), Manganese(Mn), Carbolic acid(HCOOH), Calcium(Ca),

Salt(NaCl), Vitamins (A,B,C,D,E), Lactose, Hipuric acid (CgNgNox),

Creatinin (C4HgN2O2), Aurum hydroxide (AuOH), Enzymes and other

Minerals(Wikipedia).

2.3 EXTRACTION OF C-PHYCOCYANIN

Spirulina, photosynthetic cyanobacteria possesses pigments for the

absorption of light energy. The photosynthetic pigments and electron transport

chain are located in thylakoid membrane. The photosynthetic pigments are

chlorophyll-a, chlorophyll-b, carotenoids and phycobilisomes

(Allophycocyanin, C-phycocyanin and Phycoerythrin) [33, 34]. The

C-phycocyanin of spirulina constitutes major portion and the other two –

allophycocyanin and phycoerythrin holds the minor. C-Phycocyanin is

composed of two dissimilar � and � protein subunits of 17000 and 19500 KDa

respectively, having one bilin chromophore attached to the �-subunit (�-84)

and other to the �-subunit (�-84, �-155). C-Phycocyanin is found as trimers (�,

�) 3, hexamers (�,�)6 and stacks of hexamers. The absorbance maxima are

615-620nm and at A620/A280 is indication of purity [36].

The phycocyanin of spirulina has been used mainly as food pigment,

however small quantities are included as biochemical tracers in Immunoassays

due to their fluorescent properties [37]. Recently, it has been observed that

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phycocyanin has also shown anti-inflammatory and anti-cancer properties [38].

The cyanobacteria Spirulina platensis is an excellent source of phycocyanin.

The protein fraction of Spirulina may contain upto 20% phycocyanin [37].

The drying methods adopted for the processing of Spirulina leads to

approximately 50% loss of phycocyanin. Sarada [63] found that fresh biomass

was suitable for phycocyanin extraction. Phycocyanin was stable over a pH

range of 5-7.5 at 9 ± 1°C, whereas temperature beyond 40°C caused total

instability. A simple and efficient method for the separation and purification

of C-phycocyanin and allophycocyanin from Spirulina platensis is by

ammonium sulphate precipitation and purification by ion exchange

chromatography and gel filtration chromatography was elucidated. The purity

of C-phycocyanin was 5.06 and allophycocyanin was 5.34. [64]. Phycocyanin

from the micro alga Spirulina maxima was extracted by supercritical extraction

using CO2. The biomass was crushed by cutting mills and then manually

ground with dry ice. Phycocyanin, being insoluble in CO2, thus indirectly

separated. The total extraction yield of about 3 % / mass co-solvent

corresponds to the average lipid content of 3.27% in alga Spirulina maxima

[65].

A study conducted [66] on the efficiency of phycocyanin

extraction from Spirulina platensis using ultra sonic irradiation revealed the

fact that phycocyanin could be extracted with higher purity at fu =28 kHz than

at fu =20 kHz. This study endorsed that rapid and selective extraction of

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phycocyanin from S.platensis is possible if an optimized ultrasonic application

is developed for a given suspension.

An improved drying method and efficient procedure for the optimum

extraction of phycocyanin from Spirulina was developed [67]. When Spirulina

biomass was dried at 25ºC under shadow by air circulation and extraction of

phycocyanin at 40ºC for 24hrs in phosphate buffer at pH7.0 (0.1M), yielded

maximum phycocyanin (80mg/g). This also showed relatively highest purity

ratio of 1.8, in contrast to purity ratio of 0.45 to 1.34 obtained with other

methods. Extraction of phycocyanin in hydrochloric acid (2.0N to 10.0N)

showed the contamination of chlorophyll in the phycocyanin extract. The

proposed method of air-drying and extraction suggested that this method

showed only 5 to 7% loss of phycocyanin during drying process. The effects of

the temperature and the biomass-solvent ratio on phycocyanin concentration

and the purity of phycocyanin were evaluated [68]. Accordingly, optimum

conditions for the extraction of phycocyanin from S.platensis are the highest

biomass solvent ratio, 0.08g/L, and a temperature of 25oC. Under these

conditions, it was possible to obtain an extract of phycocyanin with a

concentration of 3.68 mg mL-1 and purity ratio (A615/A280) of 0.46.

An optimized method was developed to obtain maximum extraction of

C-phycocyanin. The extraction was performed with distilled water and 1%

CaCl2 solution and the cell was disrupted by ultra sound using a sonicator.

A concentration of 0.3116mg/ml of phycocyanin was obtained in CaCl2

extraction and 0.299mg/ml in distilled water [69].

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2.4 PURIFICATION OF C-PHYCOCYANIN

Phycobiliproteins are the major photosynthetic pigments in

cyanobacteria. They are water-soluble proteins having covalently attached

tetrapyrroles. C-phycocyanin (CPC) is the major component of

Phycobiliproteins family. C-phycocyanin exhibits a strong red fluorescence

when it is present in native and concentrated form. It is used as nutrient,

ingredients and natural color for food and cosmetics [70], potential therapeutic

agent in oxidative stress – induced diseases [71] and as fluorescent markers in

biomedical researches [72]. The Phycobiliproteins consists of two dissimilar

polypeptide chain, � and � of approximately 17 and 18Kda respectively. Each

polypeptide chain carries one or more covalently attached bilins [73,74]. The

purity of C-phycocyanin is generally evaluated based on the absorbance ratio

of A 620 /A 280. The C-phycocyanin purity of 0.7 is considered as food grade,

3.9 as reactive grade and greater than 4.0 as analytical grade [75]. Various

researchers have developed several methods for the purification of

C-phycocyanin [76-80]. Almost all the methods of purification of

C-phycocyanin involve number of steps, where precipitation, purification,

dialysis are used in the initial purification, while ion-exchange chromatography

and gel filtration chromatography in the final purification.

A method was developed for the purification of C- phycocycanin [81].

The cell-free extracts have been precipitated with 50%-saturated ammonium

sulphate and centrifuged to remove cell debris. The pellet was subjected to

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further fractionation with ammonium sulphate at 35% and 50% saturation. The

resulting pellet was enriched with respect to C-Phycocyanin, and by treating

the supernatant to produce 50% saturation, a fraction containing

allophycocyanin was isolated. The partially purified extracts were dialyzed

against 25mM-sodium phosphate buffer, at pH6-8, and subjected to

fractionation at 40%, 45% and 50% saturation with ammonium sulphate. The

precipitated fraction was resuspended in 1-5ml of 25mM-sodium phosphate

buffer, at pH6-8, and further purified by gel filtration with Sephadex G-200

column (bed volume 50ml). The eluted fractions, that had E654/E620 ratios

greater than 1-57 (allophycocyanin) and less than 0-25 (C-Phycocyanin), were

retained, and after treatment with ammonium sulphate at 50% saturation, the

biliprotein was subjected to G200 sephadox column chromatography and

protein was identified by SDS-PAGE and HPLC method.

C–phycocyanin was also purified by a multi - step treatment of the

crude extract with rivanol in the ratio 10:1 (v/v), followed by 40% saturation

with ammonium sulfate [36]. After removal of rivanol by gel filtration on

Sephadex G-25, the pigment solution was saturated to 70% with ammonium

sulphate. The purified C- phycocyanin had an emission and absorption maxima

at 620 and 650 nm respectively, and absorbance ratio A 620 / A 280 of 4.3

which are specific for pure biliprotien. Homogeneity was demonstrated by

Sodium Deodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE).

Two bands of molecular weight 17,000 and 19,500 Kda, corresponding to

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� and � subunits of the pigment respectively were obtained. The yield of

C- phycocyanin was 46% from its content in the crude extract.

Purification of phycobiliprotiens from phycoerythrin (PE) rich strains

of Nostoc muscorum was carried out. Salting out with 55 % (NH4)2SO4

precipitates >80% of each class of phycobiliprotein. Chromatography on

DEAE cellulose-52 column gives pure phycoerythrin (A562/A280 = 8.12) while

rechromatography of the blue coloured fraction from this column using

hydroxyl apatite column yields pure phycocyanin (PC)(A615/A280 = 3.89).

SDS - PAGE analysis of purified PE and PC yields two polypeptides (~19.4

and 16.9 kDa). PE is found to be stable at the pH range of 5-9. This protocol

helps to get highly pure PE (72%) and PC (39%) [82].

An experiment was carried out to recover high C-phycocyanin with

purity from crude phycocyanin of S.platensis. The effect of ammonium

sulphate concentration, volume and pH of resuspension were evaluated. The

best purification conditions with ammonium sulphate fractionation at

0-20% / 20-50%, in relation to a resuspension volume/initial volume of 0.52 in

a 7.0 pH buffer. Under this condition the purity increased to 70% with an

83.8% recovery [83]. Aqueous two phase extraction was employed for the

purification of C-phycocyanin from S.platensis. The influence of various

parameters such as aqueous two phase systems type, phase forming salt,

molecular weight of the phase forming polymer, system pH, phase

composition, phase volume ratio, its type and concentration of neutral salts on

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differential partitioning of C-phycocyanin was evaluated. Desirable conditions

for the purification of C-phycocyanin to 3.52 from initial purity of 1.18 was

achieved at pH 6, tie line length of 35.53% with a phase volume ratio of 0.8 in

a single step of aqueous two phase extraction. Multiple extractions resulted in

further increase in the purity of C-phycocyanin without losing the yield and a

maximum purity of 4.05 is achieved in third aqueous two phase extraction

[84].

Some important parameters were evaluated for the purification of

phycocyanin, using Ion Exchange Chromatography [68]. The influence of pH

and temperature on the equilibrium partition coefficient was investigated. The

equilibrium isotherm for the phycocyanin-resin system was also determined.

The separation of phycocyanin using Q-Sepharose ion exchange resin was

evaluated in terms of pH and elution volume that improved the purity recovery.

The highest partition coefficients were obtained in the pH range from 7.5 to 8.0

at 250C. Under these conditions, the equilibrium isotherm for phycocyanin

adsorption was Qm of 22.7 mg/ ml and Kd of 3.1 x 10-2 mg/ml. The best

conditions for phycocyanin purification, using Ion Exchange Column was at

pH 7.5 with an elution volume of 36ml, obtaining 77.3% recovery and a 3.4

fold increase in purity.

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2.5 ANTIMICROBIAL ACTIVITY

The microbial infection causes high rate of mortality in human

population and aquaculture organisms. Nowadays, the use of antibiotics has

increased significantly due to heavy infections and the pathogenic bacteria

becoming resistant to drugs are common due to indiscriminate use of

antibiotics. It has become a great problem of giving treatment against resistant

pathogenic bacteria [85]. Hence, there is dire need for alternatives. Many

workers revealed that the crude extracts of Indian seaweeds are active against

Gram-positive bacteria [86]. The Spirulina, as a whole, has been known only

for its nutritional value, but the antimicrobial property of the C-phycocyanin

has not been studied in detail in Indian context.

The functional bioactive compounds and biological activities of lipids in

S.platensis were studied. The polar fractions were generally characterized by

high percentage of tocopherols where �-tocopherol constitutes about 73% of

total tocopherols present, the rest being �-tocopherol. Total lipids and lipid

classes inhibited the growth of different microorganisms except Gram negative

bacteria. At high concentrations the tested lipids appeared more effective

against A.niger [87].

The studies so far conducted on antimicrobial or antiviral were based

on lipids associated with various algal strains. This may be because of ease in

conventionally designed organic solvent extraction (components from algae)

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which inevitably resulted in lipid components or of carbohydrates primarily

polysaccharides.

2.5.1 Antibacterial activity

Antibacterial activity of 6 cyanobacterial strains (Anabaena variabilis,

Oscillatoria sp, Chroococcus sp, Nostoc sp, Plectonema boryanum and

cytonema sp) were evaluated against Staphylococcus epidermidis. Out of 2

extracts (Hexane and methanol) at the concentration of 25µl/disc, the hexane

extract showed maximum activity [88]. The hexane, chloroform and ethanol

extracts of six marine macro algae from the North Eastern Brazilian coast were

tested for antimicrobial activity by the single disc method [89]. Best results

were shown by the hexane extracts of Amansia multifida against enteric Gram

negative strain such as Enterobacter aerogenes, Klebsiella pneumoniae,

Pseudomonas aeruginosa, Salmonella typhi, Salmonella cholerasuis, Serratia

marcescens, Vibrio cholerae and the Gram positive bacteria Bacillus subtilis

and Staphylococcus aureus (14.5 to 18.5mm). in-vitro screening of organic

solvent extracts of three marine algae viz., Gracilaria corticata, Ulva fasciata

and Enteromorpha compressa and five mangroves viz., Aegiceras

corniculatum, Aegialitis rotundifolia, Aglaia cucullata, Cynometra iripa and

Xylocarpus granatum to the growth of six virulent strains of fish bacterial

pathogens were viz., Edwardsiella tarda, Vibrio alginolyticus, Psuedomonas

fluorescens, Pseudomonas aeruginosa and Aeromonas hydrophila (2 strains)

were evaluated [90]. The extract showed species specific activity in inhibiting

the growth of six virulent strains of bacteria pathogenic to fish

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viz., Edwardsiella tarda, Vibrio alginolyticus, Pseudomonas fluorescens,

Pseudomonas aeruginosa and Aeromonas hydrophila (2 strains). Three

methanol extracts of C.iripa were active against all the six pathogens, where as

A.corniculatum and A.cucullata was active against four of the pathogens. The

chromatographic fraction of active extracts of A. corniculatum, C.iripa and

G.corticata resulted in enriched fractions with wide spectrum activity and

lower values of minimum inhibitory concentration.

The methanol, acetone, diethyl ether, and ethanol extracts were

obtained in 11 seaweed species from the coast of Urla-Izmir, Turkey [91]. The

extracts were tested for their antibacterial activities, against Enterococcus

faecalis, Staphylococcus aureas, Streptococcus epidermidis, Pseudomonas

aeruginosa and Eschericia coli by the disc diffusion method. Diethyl ether was

found to be the best solvent, for extracting the effective antibacterial materials,

from the algal species, with the exception of D.linearis. Ethanol was found to

be the most effective solvent to extract the antibacterial principle from

D.linearis. Diethyl ether extracts of fresh Cystoseira mediterranea,

Enteromorpha linza, Ulva rigida, Gracilaria gracilis and Ectocarpus

siliculosus showed effective results (7-15mm) against all test organisms.

However, diethyl ether extracts of some species, such as Padina pavonica,

Colpomenia sniosa, Dictyopteris membranacea, Ceramium rubrum and

Acathophora nojadiformis gave different results. There was no significant

difference in antibacterial activity with acetone and methanol extracts of each

alga. A comparative study of dried and fresh extract for antimicrobial activity

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revealed that, all the test organisms were sensitive to fresh extracts of algae.

Although fresh extracts of G.gracilis, D.linearis, and E.siliculosus inhibited the

test bacteria and, their dried extracts had no inhibition activity on either Gram-

negative or Gram-positive bacteria.

Various fractions of ethanol extracts from Bacopa monnieri aerial

parts were obtained. The ethyl acetate and the N-butanol fractions were

screened for antibacterial and anti fungal activities by zone inhibition study

and determination of MIC. The ethyl acetate fraction was found to be more

potent than the N-butanol fraction, though both of them were endowed with

antimicrobial activity [92]. The influence of culture conditions, nitrogen

concentrations in growth medium on characterization of some nutraceutical

compounds from the alga Spirulina maxima were studied [93]. Antioxidant

and antibacterial activities, as well as chemical composition of organic

extracts, obtained from different cultures, were examined. The amount of total

carotenoids, chlorophyll derivatives and phenolic compounds were associated

inversely with concentration of nitrogen in the growth media. The antibacterial

activity results showed that all S.maxima extracts exhibited effective

antibacterial activity against six bacterial strains (Bacillus subtilis, Bacillus

cereus, Staphylococcus aureus, Micrococcus luteus, Klebsiella pneumoniae,

Serratia marcescence) and the zone ranges from 7-18mm and MIC values

ranges from 30 - 40µg/ml. In addition, all S.maxima extracts possessed potent

antioxidant properties, compared to commercial antioxidants. The

chromatographic analysis of Spirulina organic extracts with TLC and HPLC

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showed that carotenoids, chlorophyll derivatives and phenolic compounds

were present as main constituents and their quantity were significantly changed

depending on cultural conditions. Thus, Spirulina is useful for the production

of bioactive compounds with antioxidant and antimicrobial activity apart from

being a natural pigment.

2.5.2 Antifungal activity

The antifungal property of omega-3 fatty acid obtained from three

microalgae-Chlorella minutissima, Haematococcus pluvialis and Tetraselmis

suecica were tested against Candida albicans [94]. The extraction of fatty acid

was performed by solvents like chloroform, methanol and hexane and the

activity was determined by the agar dilution method. The growth of the fungus

was not inhibited up to the concentration of 2mg/ml. The methanol, acetone,

diethyl ether and ethanol extracts of 11 species of seaweed had been tested for

their antifungal activity [91] by disc diffusion method (25µl/disc). Diethyl

ether was found to be the best solvent, for extracting the effective antifungal

principle from the algae species, with the exception of D.linearis. Ethanol was

the most effective solvent to extract the antifungal material from D.linearis.

Diethyl ether extracts of fresh Cystoseira mediterranea, Enteromorpha linza,

Ulva rigida, Gracilaria gracilis and Ectocarpus siliculosus showed effective

results (10-15mm) against the test organisms. However, diethyl ether extracts

of some species, such as Padina pavonica, Colpomenia sniosa, Dictyopteris

membranacea, Ceramium rubrum and Acathophora nojadiformis gave

different results (7-15mm). A comparative study of dried and fresh extract for

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antifungal activity revealed that, the test organism was sensitive to fresh

extracts of algae.

The Antifungal property of Chroococcus disperses isolated from paddy

field in Iran was characterized [95]. Of the supernatant, methanolic and hexane

extracts tested on four strains of fungi – Candida kefyr, Candida albicans,

Aspergillus niger and Aspergillus fumigatus, the hexane extract did not inhibit

the growth of the fungi, whereas the supernatants and methanolic extracts of

Chroococcus sps showed a good activity against C.kefyr and minimum activity

against C.albicans (10-20µg/ml). The antifungal activity of the three

Cyanobacteria-Anabaena oryzae, Tolyphothrix ceytonica and Spirulina

platensis and two green microalgae- Chlorella pyrenoidosa and Scenedesmus

quadricauda were evaluated [96]. They used ethanol, acetone, diethyl ether,

and methanol as solvent to extract the antimicrobial principle and tested on

Aspergillus niger, Aspergillus flavus, penicillium herquei, Fusarium

moniliforme, Helminthosporium Sp, Alternaria brassica, Saccharomyces

cerevisiae and Candida albicans by agar well diffusion method (100µl/well).

They found that Spirulina platensis and Anabaena oryzae had the highest

antifungal activity.

The functional bioactive lipid compounds extracted from Spirulina

platensis were tested against four fungal species [87]. The recovered lipids

were characterized with high percentage of tocopherols, where �-tocopherol

constitutes about 73% of total tocopherols present, and the rest being

�-tocopherol. The antifungal activity was determined by agar well diffusion

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method for Aspergillus niger, Aspergillus flavus, Saccharomyces cerevisiae

and Candida albicans. Wells were loaded with 20,40,60,80 and 100µl of the

extract and incubated for 48-72hrs. The total lipids were more active against

fungi and yeasts than the lipid classes. The maximum inhibition zone obtained

with 100µl/well was by Aspergillus niger (28.6±1.53mm).

2.5.3 Antiviral activity:

Novel bioactive compounds isolated from blue-green alga were

found to have antitumor, antiviral, and immunomodulatory properties. Thus,

these compounds and their derivatives can be used to treat human and animal

tumors, inhibit viral growth, and provide immunomodulatory activity [97]. The

Chlorella ellipsoids was found to have antiviral substances, which inactivated

Arboviruses as well as Vesicular Stomatitis virus [98]. Partial characterization

of the active component reveals that it was a substance soluble in a number of

organic solvents and appeared to act photo-dynamically on the viruses.

Extracts from two species of marine red algae, Cryptosyphonia

woodii and Farlowia mollis, specifically inhibited the replication of Herpes

simplex virus in-vitro [99]. The preliminary reports described the inhibition of

type-1and 2 Herpes simplex virus (HSV-1 and HSV-2), replication in cell

monolayer, pretreated with extracts, derived from two related species of

Rhodophyta – Cryptosyphonia woodii and Farlowia mollis. The criteria for

antiviral activity were a reduction in virus infectivity between treated and

control monolayers of at least 2 log10. This degree of inhibition was observed

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only with HSV-1 and HSV-2. Nevertheless, eleven other viruses tested were

not inhibited, viz, coxsackie-B5, echo-6, polio-2, rhino-2, respiratory syncytial,

adeno-7, vaccinia, vesicular stomatitis, Japanese encephalitis, and Western

Equine Encephalitis and sindbis viruses.

Extracts of two species of marine algae, Constantinea simplex and

Farlowia mollis, were assayed for its antiviral activity in tissue culture and in

experimental infections [100]. The treatment of confluent mouse embryo

fibroblast cell monolayer with either compound before viral inoculation was

effective in inhibiting the replication of Herpes simplex virus type-1 and 2,

Vaccinia virus, and Vesicular Stomatitis virus, but not on

Encephalomyocarditis virus, Semliki Forest virus, or Murine Cytomegalovirus.

Prophylactic administration of these extracts were effective in reducing final

mortality or prolonging the mean death day of animals inoculated by the

intraperitoneal, intracerebral, or intranasal routes with Herpes simplex virus

type-2. When therapy was initiated after viral inoculation or at a site other than

that of viral inoculation, no significant effect on mortality or on mean death

day was observed. Neither preparation was effective neither in mice inoculated

intraperitoneally with Encephalomyocarditis virus, Semliki Forest virus, or

Murine Cytomegalovirus nor in animals infected intravaginally with Herpes

simplex virus type-2. The prophylactic but not therapeutic antiviral activity of

these preparations, seriously limits their potential use in human Herpes simplex

virus infections.

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A new reverse transcriptase (RT) inhibitor was extracted and

purified from the red alga Schizymenia pacifica and studied on HIV [101]. The

algal extract obtained has selectively inhibited Human Immunodeficiency

Virus (HIV) RT and replication in-vitro. When MT-4 cells were treated with

more than 10(4) inhibitory units (IU) of algal extract per ml after HIV

infection, significant inhibition of viral antigen synthesis were observed.

Further, more than 90% of cells were viable in the cultures exposed to 4 X

10(4) to 8 X 10(4) IU of algal extract per ml, while almost all the MT-4 cells in

the control culture had died by 10 days after HIV infection. The inhibitory

effect of algal extract on HIV replication was confirmed by plaque reduction

assays.

A novel virucidal protein, named cyanovirin-N (CV-N) isolated from

blue-green alga Nostoc ellipsosporum has the molecular weight of 11kDa

[102]. This compound irreversibly inactivated diverse T-lymphocyte-tropic

(T-tropic), laboratory adapted strains of HIV type-1, HIV type-2 and Simian

Immunodeficiency Virus (SIV). A water based extract Aphanizomenon

flosaquae containing high concentration of phycocyanin inhibited the invitro

growth of one out of four tumour cell lines. Phormidium tenue containing

several diacyl-glycerol compounds effectively inhibited chemically induced

skin tumours in mice. Studies on whole blue algae in humans, mice, rats, cats,

and chickens have demonstrated their effect on phagocytosis NK cell function

and inflammation. Blue-green algae, in general, contain a significant amount

of carotenoids, namely beta carotene, lycopene and lutein, providing it good

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antioxidant properties. By their quenching action on reactive oxygen species,

antioxidants carry intrinsic anti-inflammatory properties. Macrolides are one of

the structural types often seen in Streptomyces-metabolites, and several major

antibiotics belong to this class. Macrolides were also produced by blue-green

algae that include Scytophycins from Scytonema sp. and Tplytoxin from

Tolypothrix conglutinate var. colorata.

The in-vivo antiviral activity of �-interferon production promoting

fraction from the marine brown alga, Sargassum hemiphyllum (Turner)

C.agardh was studied [103]. They examined the antiviral efficacy of the

extract that markedly promoted the production of �-interferon in MG-63 cells

in culture. The partially purified fraction from the methanol extract of

S.hemiphyllum (Turner) C.agardh MC-9 was most effective at a concentration

of 1µg/ml enhancing the productivity approximately 14 fold and two fold in

the presence of polyinosinic-polycytidylic acid and the Sendai virus,

respectively. Antiviral tests of MC-9 revealed the therapeutic efficacy in mice

infected with Aujeszky’s disease virus. This result suggests that MC-9

manifests its antiviral activity by modulating host-immunodefence systems.

Carrageenan, a class of sulphated polysaccharides extracted from

marine red algae was shown to inhibit the infectivity of genital Human

Papiloma Virus in-vitro, with nearly a thousand fold greater potency than

Heparin. Carrageenan is widely used commercially as a thickening agent for a

variety of foods and cosmetics products. Carrageenan acts primarily by

preventing the binding of HPV virions to the cells. The finding was consistent

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with the fact that Carrageenan resembles Heparin sulfate, which is regarded as

a highly effective model HPV inhibitor. In a study Carrageenan was also

shown to block HPV infection through a second, post attachment Heparan

sulphate-independent effect [104].

The hot water extract of a brown marine alga Cystoseira myrica, from

the Persian Gulf was an effective antiviral compound against KOS strain of

HSV-1 in cell culture [105]. The extract exhibited antiviral activity against

Herpes simplex virus type 1(HSV-1) not only during adsorption of virus to the

cells but also on post attachment stages of viral replication. The water extract

of C.myrica was sterilized by filtration and autoclaving, respectively. The IC50

of filtered extract was 99µg/ml and the IC50 of autoclaved extract was

125µg/ml. based on results of selectivity index (SI) values of the extracts,

which were 33.4 and 28.2 for filtered and autoclaved extracts, respectively.

The antiviral compound in the water extract of C. myrica was found to be heat

stable. Also the SI values for inhibition of the post attachment stages of HSV-1

replication were 23.1 and 21.7 for filtered and autoclaved extracts,

respectively. The IC50 in this phase of study were 143 and 162µg/ml for

filtered and autoclaved extracts, respectively. Therefore, C. myrica could be a

good candidate as a natural source for isolation of anti-HSV-1 compounds.

The effects of phycocyanin on HIV virus, Cancer and the immune

system was reviewed [106]. Water extract of Spirulina inhibits HIV-1

replication in human derived T-cell lines and in human peripheral blood

mononuclear cells. Calcium spirulin is a polymerized sugar molecule, obtained

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from Spirulina. Hamsters treated with this water soluble extracts had better

recovery rates when infected with lethal Herpes Virus. Red micro algal

polysaccharides significantly inhibited the production of retroviruses (Murine

Leukemia Virus- MuLV) and cell transformation by Murine Sarcoma Virus

(MuSV-124) in cell culture.

The molecular mechanism of the human immune potentiating

capacity of Spirulina was identified by analyzing blood cells of volunteers with

pre and post oral administration of hot water extract of Spirulina [107]. Natural

Killer (NK) cell functions represented by �-Interferon (IFN) production and

cytolysis were enhanced after administration of Spirulina in more than 50%

subjects. From their observations, it was concluded that in human beings

Spirulina acts directly on myeloid lineages either directly or indirectly on NK

cells. The presence of co-operative Interleukin (IL)-12 and 18 is critically

important for NK-mediated �-IFN production.

In a study [108] a protein-bound pigment, allophycocyanin purified

from blue- green algae was reported for the first time to exhibit anti-

enterovirus-71 activities. Allophycocyanin neutralized the Enterovirus-71-

induced cytopathic effect in both Human Rhabdomyosarcoma cells and

African green monkey kidney cells. The 50% inhibitory concentration was

approximately 0.045±0.012 µM in Green monkey kidney cells. The cytotoxic

concentration of allophycocyanin for Rhabdomyosarcoma cells and African

Green monkey kidney cells were 1.653±0.003µM and 1.521±0.012µM,

respectively. A plaque reduction assay showed that the concentrations of

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allophycocyanin for reducing plaque formation by 50% were approximately

0.056±0.007µM and 0.101±0.032µM, when allophycocyanin were added at the

stage of viral adsorption and post adsorption respectively. Antiviral activity

was more efficient in cultures treated with allophycocyanin before viral

infection compared with that in the cultures treated after infection.