3.0. chapter 1 extraction and characterisation...

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3.0. CHAPTER 1

EXTRACTION AND CHARACTERISATION OF POLYSACCHARIDES FROM

BROWN SEAWEED S. WIGHTII

3.1. INTRODUCTION

Marine environment is a major potential source of functional materials,

including Omega-3 oils, essential minerals, vitamins, antioxidants, peptides, enzymes

and polysaccharides (Anon, 2008a). All these materials are extracted from different

marine living organisms including microbes, plants and animals. Among these

seaweeds or marine macroalgae are one of the important sources and they are a part of

staple diet from time immemorial in the orient as they are nutritionally rich materials

(Dawczynski et al., 2007). They are excellent source of vitamins, dietary fibres,

minerals and proteins (Lee et al., 2008). In addition, some hydrocolloid products

derived from seaweeds are used in several industries like cosmetics, pharmaceuticals,

food industries, etc. (Chandini et al., 2008a,b). The total global seaweed production in

the year 2005 was around 1.3 million tones by natural harvest and 14.8 million tones by

culture production (FAO, 2007). In 2009, more than 15 million tones of seaweeds were

produced from global capture and aquaculture (FAO, 2012).

Increased research attention is being paid to seaweeds as sources of major

bioactive compounds including carotenoids, fatty acids, polysaccharides and

phytosterols, as they have been reported to possess several beneficial effects including

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antioxidant, anticoagulant, antitumor and anti-cancerous properties (Nagai and

Yukimoto, 2003; Chandini et al., 2008a; Lee et al., 2008). They have also been paid

much attention in a bid to develop new drugs and health foods. Brown seaweeds are

known to contain more bioactive components than either green or red seaweeds (Anon,

2008b). Some of the bioactive compounds identified from brown seaweeds include

phylopheophylin, phlorotannins, fucoxanthin, polysaccharides and various other

metabolites (Hosakawa et al., 2006). Sargassum wightii is one of the brown algae with

wide pharmacological actions (Anthony et al., 2007).

Fucoidans, one of the sulfated polysaccharides extracted from brown seaweeds,

first isolated by Kylin and it contains substantial percentages of L-fucose and sulfate

ester groups (Berteau and Mulloy, 2003). The fucoidan has been extensively studied

due to its numerous biological activities including anticoagulant and antithrombotic,

antitumor, antiviral, anti-complement and anti-inflammatory activities (Beress et al.,

1993; Patankar et al., 1993; Blondin et al., 1996; Haroun-Bouhedja et al., 2000;

Bojakowski et al., 2001; Marais and Joseleau, 2001). Fucoidans are a-L-fucose

polysaccharides containing sulphate groups and minor monosaccharides such as D-

galactose, D-xylose, D-glucose, D-mannose and D-uronic acid. Their structures are

complex, the polymers are heterogeneous and no defined regularity has been observed

(Kusaykin et al., 2008). Variations in the fucoidan structure are observed between

species which have an impact on the determination of the polysaccharide structure.

Fucoidan extracted from Ascophyllum nodosum is mainly composed of fucose linked in

(1,3) and (1,4) glycoacidic linkage (Chevolot et al., 1999; Daniel et al., 1999; Chevolot

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et al., 2001; Daniel et al., 2001; Marais and Joseleau, 2001). Lateral chains are

composed of single or several fucosyl units with branching in position 4. For Fucus

vesiculosus, Percival and McDowell (1967) have proposed two possible structures. The

first one consists of fucose linked in (1,2) with sulphate in position 4. The second one is

similar to the first structure proposed, but in this case, the fucose units are linked in

(1,3). Patankar et al. (1993) have studied the fucoidan extracted from F. vesiculosus and

found 1, 3 - linkages between fucose. The ending fucose units were also found to hold

branching with (1,2) or (1,4)-linkages.

Alginates are other polysaccharides naturally present in the cell wall of brown

seaweeds (Kloareg and Quatrano, 1988). These polysaccharides show interesting

rheological properties: they enable to enhance aqueous solutions viscosity at low

concentration, and to form gels or thin films. They are widely used in various fields of

industries such as textile, food, paper, cosmetics, pharmaceuticals, etc. (Pérez et al.,

1992). Alginate is the major structural polysaccharide of marine brown algae; it has

combined feature of abundant resources with a linear copolymers of L-guluronic acid

and D-mannurunic acid units (Xu et al., 2006). The major structural polysaccharide of

brown seaweeds in alginic acid, a linear copolymer of (1→4) linked β-D-

mannopyranuronic acid (m) and (1→4) linked α-L-gulopyramuronic acid (G) residues,

arranged in heteropolymeric and homo polymeric blocks (Painter, 1983; Larsen et al.,

2003). The content of uronic acids with species and tissue types, and partial hydrolysis

of alginic acid allows the preparation of fractions enriched in water and homopolymeric

blocks (Haug et al., 1974; Craigie et al., 1984). Considering the importance of the

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above, the present study was undertaken to extract and characterize the polysaccharides

(fucoidan, sodium alginate and alginic acid) from brown seaweed S. wightii with the

following key objectives

Objectives

1. To collect the brown seaweed S. wighti from the rockey shore regions of

Kanyakumari coast.

2. To extract the polysaccharides such as fucoidan, sodium alginate and alginic

acid from the collected brown seaweed and to calculate their yield.

3. To determine the purity of individual polysaccharide by phytochemical analysis.

4. To analyze the physical and chemical characteristics of fucoidan, sodium

alginate and alginic acid, individually.

5. To characterize the fucoidan, sodium alginate and alginic acid through UV,

FT-IR and NMR analysis.

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3.2. MATERIALS AND METHODS

3.2.1. Collection of seaweed

The brown seaweed S. wightii was collected from the rockey shore regions of

Kanyakumari coast, Tamilnadu, India (Plate 3.1). The taxonomical position of S. wighti

is given below

Division : Phaeophyta

Class : Phaeophyceae

Order : Fucales

Family : Sargassaceae

Genus : Sargassum

Species : wightii

3.2.2. Extraction of fucoidan

The collected seaweed was washed thoroughly and dried under shade at room

temperature. The dried seaweed was ground well by using mixer grinder and sieved

using a nylon sieve in order to remove seaweed fibre. The fucoidan was extracted by

the method proposed by Yang et al. (2008). 20g of milled seaweed was treated with 1

litre of ethanol with constant mechanical stirring for 12h at room temperature to remove

pigments and proteins. Then washed with acetone and centrifuged at 1800g for 10min.

Then the residue was dried at room temperature. From this dried biomass, 5g was taken

and extracted with 100ml of distilled water at 65°C with stirring for 1h. The extraction

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was conducted twice and the extracts were combined. The extract was centrifuged at

18500xg for 10min and the supernatant was collected. The supernatant was mixed with

1% CaCl2 and the solution was kept at 4°C for overnight to precipitate alginic acid. The

solution was centrifuged at 18500xg for 10min and the supernatant was collected.

Ethanol (99%) was added in to the supernatant to obtain the final ethanol concentration

of 30% and the solution was placed at 4°C for 4h. Then the solution was centrifuged at

18500xg for 10min and the supernatant was collected. More ethanol (99%) was added

into the supernatant to obtain the final concentration of 70% and the solution was placed

at 4°C overnight. The fucoidan was obtained by the filtration of the solution with a

nylon membrane (0.45µm pore size) and the product was washed with ethanol (99%)

and acetone. Then the fucoidan was dried at room temperature overnight. The dried

fucoidan was packed in an airtight container until further use (Plate 3.2). The yield of

fucoidan was calculated by the following formula.

Weight of fucoidan

Yield of fucoidan (%) = x 100

Weight of milled seaweed

3.2.3. Extraction of sodium alginate

The sodium alginate was extracted from the brown seaweed S. wightii by the

modified method of Torres et al. (2007). 100g of milled seaweed sample was weighed

and soaked in 2% formaldehyde taken in air tight conical flask for 24h. After 24h, the

formaldehyde solution was filtered out and the residue was washed with distilled water

for 2 to 3 times. Then 0.2 M HCl solution was added to the residue and kept at room

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temperature for 24h. After 24h, the solution was removed and the residue was washed

with distilled water for 2 to 3 times. The residue was extracted with 2% sodium

carbonate for overnight. The extract was filtered through muslin cloth bag and the

filtrate was bleached with 2.5% sodium hypo chloride. Then the solution was

evaporated and dried at 60ºC in a hot air oven. The final product was scraped from the

beaker and made into powder (Plate 3.3). The powdered product was weighed to

calculate sodium alginate yield.

Weight of sodium alginate

Yield of sodium alginate (%) = x 100


3.2.4. Extraction of alginic acid

The alginic acid was extracted from the brown seaweed S. wightii by the

modified method of Torres et al. (2007). 100g of milled seaweed sample was weighed

and soaked in 2% formaldehyde taken in air tight conical flask for 24h. After 24h, the

formaldehyde solution was filtered out and the residue was washed with distilled water

for 2 to 3 times. Then 0.2 M HCl solution was added to the residue and kept at room

temperature for 24h. After 24 h, the solution was removed and the residue was washed

with distilled water for 2 to 3 times. The residue was extracted with 2% sodium

carbonate for overnight. The extract was filtered through muslin cloth bag. Then 5%

HCl was added to the filtrate for precipitation of alginic acid. Then the precipitate was

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separated by centrifugation method. Then the product was dried and made in to powder

(Plate 3.4). The powdered product was weighed to calculate alginic acid yield.

Weight of alginic acid

Yield of alginic acid (%) = x 100


3.2.5. Determination of purity of seaweed polysaccharides (phytochemical analysis)

To determine the purity of seaweed polysaccharides, tests for alkaloids,

carbohydrates, flavonoids, steroids, terpins, saponins, tannins and phenols were carried

out (Harborne, 1973; Trease and Evans, 1989; Sofowora, 1993).

3.2.5.1. Alkaloids

0.5g each of fucoidan, sodium alginate and alginic acid were individually

dissolved in 10ml of diluted HCl (0.1N) and filtered. The filtrate was used to test the

presence of alkaloids. Dragendroff’s reagent was added to the filtrate, formation of red

colored precipitate indicated the presence of alkaloids.

3.2.5.2. Saponins

0.5g each of fucoidan, sodium alginate and alginic acid were dissolved

individually in 5 ml of distilled water in a test tube. The solution was shaken vigorously

and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive

oil and shaken vigorously. An appearance of creamy mass of small bubbles indicated

the presence of saponins.

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3.2.5.3. Tannins

0.5 g each of fucoidan, sodium alginate and alginic acid were individually boiled

with 10 ml of distilled water in a test tube and then filtered. A few drops of 0.1% ferric

chloride was added and observed for brownish green or a blue-black coloration

indicating the presence of tannins.

3.2.5.4. Phlobatannins

0.5 g each of fucoidan, sodium alginate and alginic acid were individually

dissolved in 5ml of distilled water and filtered. The filtrate was boiled with 2% HCl

solution. Red precipitate indicated the presence of phlobatannins.

3.2.5.5. Flavonoids


dissolved in diluted NaOH and then HCl was added. A yellow solution that turns

colorless indicated the presence of flavonoids.

3.2.5.6. Steroids

2ml of acetic anhydride was added to 0.5 g each of individual polysaccharides

such as fucoidan, sodium alginate and alginic acid with 2 ml of H2SO4. The colour

changed from violet to blue or green in samples indicating the presence of steroids.

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3.2.5.7. Terpenoids (Salkowski method)

0.5 g each of fucoidan, sodium alginate and alginic acid were individually added

to 2 ml of chloroform. Then 3ml of concentrated H2SO4 was carefully added to form a

layer. A reddish brown coloration of the interface indicated the presence of terpenoids.

3.2.5.8. Cardiac glycosides (Keller-Killiani test)


dissolved in 5 ml of distilled water. Then 2 ml of glacial acetic acid containing one drop

of ferric chloride solution was added. This was underplayed with 1 ml of concentrated

sulphuric acid. A brown ring at the interface indicated the presence of deoxysugar

characteristic of cardenolides. A violet ring may appear below the brown ring, while in

the acetic acid layer a greenish ring may form just above the brown ring and gradually

spread throughout this layer.

3.2.5.9. Phenolics

100mg each of fucoidan, sodium alginate and alginic acid were individually

boiled with 1ml of distilled water and filtered. Then 2ml of filtrate was taken and 2ml

of 1% ferric chloride solution was added in a test tube. Formation of bluish black color

indicated the presence of Phenolic nucleus.

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3.2.5.10. Carbohydrates (Molisch’s test)


dissolved in 5ml of distilled water and filtered. The filtrate was used to test the presence

of carbohydrates. To 1ml of filtrate, 2 drops of Molisch’s reagent was added in a test

tube and 2ml of Conc H2So4 was added carefully along the side of the test tube.

Formation of violet ring at the junction indicated the presence of carbohydrates.

3.2.6. Physical characteristics of polysaccharides

The physical properties such as organoleptic characters, pH, particle size,

moisture content, boiling point and solubility of polysaccharides such as fucoidan,

sodiuim alginate and alginic acid were analyzed individually by the following

methodologies.

3.2.6.1. Organoleptic evaluation

The organoleptic characters such as color, odour, taste and texture of fucoidan,

sodium alginate and alginic acid were evaluated individually based on the

methodologies described by Kumar et al. (2011).

3.2.6.2. Estimation of pH (Schofield and Taylor, 1955)

1g each of fucoidan, sodium alginate and alginic acid were individually

dissolved in 100ml of distilled water. The pH of individual polysaccharide was then

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determined with the help of a digital pH meter (Digital pH meter, model 2001, Digisum

electronics system).

3.2.6.3. Particle size measurement

The ocular micrometer was used to measure the size of individual particle of

polysaccharides by using a microscope. The powdered sample of fucoidan, sodium

alginate and alginic acid were individually dispersed in glycerin and a smear of the

dispersion was made on a glass slide and examined under microscope. The size of 500

particles was measured using a calibrated ocular micrometer (Kumar et al., 2011).

3.2.6.4. Moisture content

Moisture content is the individual loss in mass of fucoidan, sodium alginate and

alginic acid on heating at 105 ± 1o C under operating conditions specified.

Apparatus

Metal dishes 7-8cm diameter and 2.3cm width provided with tight slip on covers.

Procedure

0.1g each of fucoidan, sodium alginate and alginic acid were weighed separately

and kept in previously dried and tarred dishes. Then the individual polysaccharide was

heated in an oven at 105 ± 1o C for 1h. Then the dish was taken out from the oven and

immediately closed the lid and cooled in a desicator containing phosphorus pentoxide.

Then the weight was taken and again heated the dish with sample in oven for a further

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period of 1h, cooled and reweighed. Repeated this process until change in weight

between two successive observations did not exceed 1mg. The experiment was carried

out in triplicate to determine the mean value.

Moisture and volatile matter present by weight = W1 x 100

W

Where, W1 = Loss in g of material on drying

W = Weight in g of the material taken for test

3.2.6.5. Solubility of polysaccharides

Solubility of fucoidan, sodium alginate and alginic acid was checked

individually with different organic solvents and acids. For this, 0.1g each of the

individual polysaccharide was dissolved in 1ml each of individual solvents such as

water, ethanol, methanol, Dimethylsulfoxide (DMSO), acetone, ether, chloroform,

Dichloromethane (DCM) and acids such as hydrochloric acid (HCl) and sulphuric acid

(H2SO4). Then the solubility was observed.

3.2.7. Biochemical constituents of polysaccharides

The biochemical components such as protein, carbohydrate, lipid, fucose,

sulfate, total ash, acid insoluble ash and water soluble ash of fucoidan, sodium alginate

and alginic acid were estimated individually by the following procedures.

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3.2.7.1. Protein estimation (Lowry et al., 1951)

Principle

The carbonyl group of protein reacts with the copper ion present in the alkali

solution and then this complex reacts with phosphomolybdic acid present in folin

phenol reagent and gets reduced with tyrosine and tryptophan.

Reagents

80% Ethanol

80ml of ethanol was dissolved in 20ml of distilled water

NaOH (0.1 N)

For 0.1 N NaOH solution, 400mg of NaOH was dissolved in 100ml of distilled

water.

NaOH (1N)

For the preparation of 1N NaOH solution, 4g of NaOH was dissolved in 100ml

of distilled water

Solution A

Solution A was prepared by dissolving 2g of sodium carbonate in 100ml of

0.1 N NaOH.

Solution B

Solution B was prepared by dissolving 500mg of copper sulphate in 1% sodium

potassium tartarate (1g of sodium potassium tartarate in 100ml distilled water).

Solution C

Solution C was prepared by mixing 50ml of solution A with 1ml solution B

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Folin phenol reagent

Folin phenol reagent was prepared by mixing 1ml of folin phenol with 1ml

distilled water.

Blank

For blank, 0.5ml of 1N NaOH, 5ml of solution C and 0.5ml of folin phenol were

taken.

Procedure

A known amount of individual polysaccharide was taken and ground it well with

80% ethanol. Then it was centrifuged at 5000 rpm for 15 min. Then the precipitate was

taken and dissolved in 1N NaOH and made up to 5ml. From this, 0.5ml was taken,

followed by 5ml of solution C was added and kept it for 10min. Finally, 0.5 ml of folin

phenol reagent was added and the intensity of colour developed was read at 640 nm in a

spectrophotometer (Techcomp 8500).

Calculation

Protein present in OD of the sample

the sample (%) = x Concentration of the std x 100

OD of the standard

Weight of the sample

3.2.7.2. Carbohydrate estimation (Seifter et al., 1950)

Principle

Carbohydrates are first hydrolyzed into simple sugars using dilute hydrochloric

acid. In hot acidic medium, glucose is dehydrated to hydroxyl methyl furfurol. This

compound forms green coloured products with Anthrone reagent.

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Reagents

Anthrone reagent

200mg of Anthrone was dissolved in 100ml of ice cold 95% sulphuric acid.

Stock standard glucose

100 mg of glucose was dissolved in 100ml of distilled water.

Working standard

10 ml of stock standard glucose was diluted in 100 ml of distilled water.

Procedure

The carbohydrate content of individual polysaccharide was estimated by the

anthrone method. To an aliquot of polysaccharide sample, 4ml of anthrone reagent was

added and incubated in a boiling water bath for 15min. Tubes were then cooled to room

temperature at dark condition. Then the optical density was measured at 750 nm by

using spectrophotometer. Here glucose (100 mg / 100 ml distilled water) was used as

the standard.

Calculation

Carbohydrate present in OD of the sample


OD of the standard


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3.2.7.3. Lipid estimation (Folch et al., 1957)

Principle

Quantitative determination of sulpho-phosphovanillin method depends on the

reaction of lipid extracted from the polysaccharides using chloroform methanol with

sulphuric acid, phosphoric acid and vanillin to give a red color complex.

Reagents

Chloroform methanol (2:1)

This reagent was prepared by mixing 200 ml of chloroform with 100 ml

methanol

Sodium chloride (0.9%)

900 mg of NaCl was dissolved in 100 ml distilled water.

Sulpho-phosphovannilin reagent

200 mg of vannilin powder was dissolved in 100ml of 80% orthophosphoric

acid.

Standard

8 mg of cholesterol was dissolved in 4 ml of chloroform methanol mixture (2:1).

Blank

5ml of chloroform methanol (2:1) was taken as a blank.

Procedure

Known weight of individual polysaccharide sample was taken and homogenized

well with 4ml of chloroform methanol (2:1) mixture. After mixing well, 0.2 ml of 0.9%

sodium chloride was added and kept the mixture overnight at room temperature. The

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lower layer of lipid was collected carefully and dried in vacuum desiccator. The dried

total lipid was dissolved by using concentrated sulphuric acid by keeping in boiling

water bath for 10min. From the above prepared total lipid sample, 0.2 ml was taken in

test tube and 5ml of sulpho-phosphovannilin reagent was added, shaken well and kept

for 30 min. The intensity of red color was measured at 520nm by using

spectrophotometer.

Calculation

Lipid present in OD of the sample


OD of the standard


3.2.7.4. Estimation of Fucose content

The fucose content of fucoidan, sodium alginate and alginic acid was

determined by phenol sulphuric acid method proposed by Dubois et al. (1956).

Reagent preparation

1. 89% phenol: 80g of phenol was dissolved in 20ml distilled water and dissolved

at 50o C.

2. Concentrated sulphuric acid.

Procedure

20 mg of individual polysaccharide sample was dissolved in 2 ml of distilled

water and taken in a test tube. Then 0.10 ml of 89% phenol reagent was added to the

test-tube. Then 6.0ml of concentrated sulfuric acid was added to the test tube and

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mixed well. Then the solution was kept at room temperature for 10min and the optical

density was measured at 490nm by using a spectrophotometer. Simultaneously a blank

was also set by using distilled water instead of sample. For fucose analysis, the

commercial L-fucose was used as the standard.

Fucose content of OD of the sample


OD of the standard


3.2.7.5. Ash content

The ash content of individual polysaccharide was determined by incinerating 1g

of the product taken in a silica crucible and kept in a muffle furnace at 6000C. After

incineration, the net content was cooled and weighed and expressed in terms of

percentage.

Ash content (%) = (W1 – W2)/W x 100

W1 = Fresh sample and weight of silica crucible

W2 = Incinerated sample and silica crucible

W = Weight of the sample

a. Acid insoluble ash

The ash obtained as described above was boiled with 25 ml of 2N HCl for five

minutes. The insoluble ash was collected on an ash less filter paper and washed with hot

water. The insoluble ash was transferred into a silica crucible, ignited and weighed. The

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procedure was repeated to get a constant weight. The percentage of acid insoluble ash

was calculated with reference to the air-dried sample.

b. Water-soluble ash

The ash obtained as described for the determination of total ash was boiled for 5

min with 25 ml of water. The insoluble matter was collected on ash less filter paper and

washed with hot water. The insoluble ash was then transferred into silica crucible,

ignited for 15 min, and weighed. The procedure was repeated to get a constant weight.

The weight of insoluble matter was subtracted from the weight of the total ash. The

difference of weight was considered as water-soluble ash. The percentage of water-

soluble ash was calculated with reference to the air dried sample.

3.2.7.6. Estimation of sulfate content (Dodgson and Price, 1962)

Estimation of the sulphate content of polysaccharide involves acid hydrolysis,

followed by determination of liberated inorganic sulphate by turbidometric method. In

this method sulphate is estimated turbidometrically as barium sulphate, light absorption

at 360nm being measured and gelatin being used as a cloud stabilizer.

Reagents preparation

1. Barium chloride reagent: 2g of gelatin was dissolved in 400ml of hot water

(60-70o C) and allowed to stand at 4

o for 6h. Then 2g of barium chloride was

dissolved in the semi gelatinous fluid and the reagent was stored at 4o C for

overnight before use.

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2. 3% trichloroacetic acid: 3g of trichloroacetic acid was dissolved in 100ml

distilled water.

Procedure

A known amount of individual polysaccharide was dissolved in sufficient

amount of N- hydrochloric acid in glass test tube. Then it was sealed in an oxygas flame

and kept in an oven at a temperature of 105 - 110o C for 5h. After being cooled, the

content of the tube was mixed before opening. From this, 0.2ml was taken in a test tube

containing 3.8ml of 3% trichloroacetic acid. Then 1 ml of barium chloride- gelatin

reagent was added and mixed well. Then the tube was kept at room temperature for 15-

20min and the optical density was measured at 360nm by using spectrophotometer.

Simultaneously a blank was also set by using 0.2ml of hydrochloric acid instead of

sample. For sulphate analysis, the commercial K2SO4 was used as a standard.

Sulfate content of OD of the sample


OD of the standard


3.2.8. Purification of polysaccharides


dissolved in 25ml of distilled water and heated at reflux with 0.75 ml of 3.0M HCl for

3h. After cooling, the mixture was centrifuged at 3000xg and the supernatant solution

was neutralized with 1.0M NaOH and poured over 100ml of ethanol. Then the

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precipitate was dissolved in distilled water and freeze dried (Fraction). Then the freeze

dried polysaccharides was hydrolyzed by the method proposed by Rioux et al. (2007).

3.2.9. Hydrolysis of polysaccharides

3.2.9.1. Hydrolysis of fucoidan

In order to convert the polysaccharide into monosaccharide, the purified

fucoidan was subjected for hydrolysis. For this, 20 mg of fucoidan was hydrolyzed with

2 M H2SO4 at 100o C for 30 min. Then the material was neutralized with 6 M NaOH.

Then the sample was freeze dried and kept for further analysis (Nagaoka et al., 1999).

3.2.9.2. Hydrolysis of sodium alginate and alginic acid

In order to reduce the viscosity of the sample as well as to convert the

polysaccharide into monosaccharide, the purified sodium alginate as well as alginic acid

were individually subjected for hydrolysis. For this, 20mg each of individual product

was dissolved in 5ml of distilled water and heated at 90o C for 1h. Then 1ml of 0.1 N

HCl was added to the samples and heated at 90o C for 2h. Then the samples were freeze

dried individually and kept for further analysis (Marais and Joseleau, 2001).

3.2.10. UV-vis spectral analysis

The individual hydrolyzed polysaccharide was scanned in the range of 190 –

1100nm in UV spectrophotometer (Techcomp, UV VIS 8500) for determining

maximum absorbance (λ max).

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3.2.11. FT-IR analysis

The qualitative analysis of the active principles of individual polysaccharide was

done by Fourier Transmission Infra Red (FTIR) method, described by Kemp (1991).

Procedure

Preparation of KBr discs

KBr discs were prepared by grinding the individual polysaccharide (fucoidan,

sodium alginate and alginic acid) (0.1 – 2.0 by weight) with KBr and compressing the

whole into a transparent wafer or disc. The KBr was dried, and it is an advantage to

carry out grinding under an infrared lamp to avoid condensation of atmospheric

moisture, which gives to broad absorption at 3500 cm -1

.

o The particle size of grinding was achieved by grinding KBr sample complex to

< 2 µm to avoid wavelength scaling.

o Given high pressure to KBr disc to condense fairly to 13 mm in diameter and 0.3

mm in thickness.

Infra Red analysis

The frequency of the spectra set to analysis was between 4000 - 400 cm-1

wave

number and the vibration spectrum was recorded as graphical chart. The instrument

used to FTIR analysis was Shimadzu, Japan.

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Applications of IR Spectroscopy to organic molecules

Organic functional groups differ from one another both in the strength of the

bond(s) involved, and in the masses of the atoms involved. For instance, the O – H and

C = O functional groups each contain atoms of different masses connected by bonds of

different strengths. According to the above, expect the O – H and C = O groups to

absorb IR radiation at different positions in the spectrum. The presence of a strong,

broad band between 3200 and 3400 cm-1

indicates the presence of an O – H group in the

molecule, while the presence of a strong band around 1700 cm-1

confirms the presence

of a C = O group.

For organic molecules, the infrared spectrum can be divided into three regions.

Absorptions between 4000 and 1300 cm-1

are primarily due to specific functional

groups and bond types. Those between 1300 and 909 cm-1

, the fingerprint region, are

primarily due to more complex interactions in the molecules, and those between 909

and 650 cm-1

are usually associated with the presence of benzene rings in the molecules.

Some particularly important regions are indicated below in the table as vibrational

frequencies for organic molecules.

Vibrational frequencies for organic molecules

Bond type Specific context V1 cm-1

C – H Csp3-H 2800 – 3000

Csp2-H 3000 – 3100

Cont…….

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Csp-H 3300

C-C C-C 1250 – 1450

C=C 1600 – 1670

CºC 2100 – 2260

C-N C-N 1030 – 1230

C=N 1640 – 1690

CºN 2210 – 2260

C-O C-O 1020 – 1275

C=O 1650 – 1800

C-X C-F 1000 – 1350

C-Cl 800 – 850

C-Br 500 – 680

C-I 200 – 500

N-H RNH2, R2NH 3400 – 3500 (Two)

RNH3+, R2NH2

+,

R3NH+

2250 – 3000

RCONH2, RCONHR’ 3400 – 3500

O-H ROH 3610 – 3640 (free)

3200 – 3400 (H-bonded)

RCO2H 2500 – 3000

N-O RNO2 1350 – 1560

Cont……

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RONO2

1620 – 1640

1270 – 1285

RN=O 1500 – 1600

RO-N=O

1610 – 1680 (Two)

750 – 815

C=N-OH 930 – 960

R3N-O+ 950 – 970

R2SO 1040 – 1060

R2S(=O)O

1310 – 1350

1120 – 1160

R-S(=O)2-OR’

1330 – 1420

1145 – 1200

Cumulated systems C=C=C 1950

C=C=O 2150

R2C=N=N 2090 – 3100

RN=C=CO 2250 – 2275

RN=N=N 2120 – 2160

Out of plane bending

vibrations

Alkynes CºC-H 600 – 700

Alkenes RCH=CH2 910, 990

R2C=CH2 890

Cont……

43

Trans-RCH=CHR 970

Cis-RCH=CHR 725, 675

R2C=CHR 790 – 840

mono- 730 – 770, 690 – 710 (two)

o- 735 – 770

m- 750 – 810, 690 – 710 (two)

p- 810 – 840

1,2,3- 760 – 780, 705 – 745 (two)

1,3,5- 810 – 865, 675 – 730 (two)

1,2,4- 805 – 825, 870, 885 (two)

1,2,3,4- 800 – 810

1,2,4,5- 855 – 870

1,2,3,4 840 – 850

Penta- 870

Carbonyl stretching

frequencies

Aldehydes RCHO 1725

C=CCHO 1685

ArCHO 1700

Ketones R2C=O 1715

C=C-C=O 1675

Ar-C=O 1690

Cont……

44

Four-membered cyclic 1780

Five- membered cyclic 1745

Six- membered cyclic 1715

RCOOH 1760 (Monomer), 1710 (Dimer)

1720 (Monomer), 1690 (Dimer)

RCO2 1550-1610, 1400 (two)

Esters RCOOR 1735

C=C-COOR 1720

ArCOOR 720

g-lactone 1770

d-lactone 1735

Amides RCONH2

1690 (free)

1650 (associated)

RCONHR’

1680 (free)

1655 (associated

RCONR2 1650

b-lactam 1745

g- lactam 1700

d- lactam 1640

Acid anhydrides 1820, 1760 (two)

Acyl halides 1800

45

3.2.12. 13

C and 1H NMR analysis

After hydrolysis, the individual polysaccharide sample was dissolved in 0.5ml

D2O (Deutrium dioxide), and the proton number and carbon number of individual

polysaccharide were identified and confirmed by 1H and

13C NMR experiments using a

Bruker Biospin Avance 400 NMR spectrometer (1H frequency = 400.13 MHz,

13C

frequency = 100.62 MHz) at 298 K using 5-mm broad band inverse probe head

equipped with shielded z-gradient and XWIN-NMR software version 3.5 using TMS as

an internal reference. One-dimensional 1H and

13C spectra were obtained using one

pulse sequence. One-dimensional 13

C spectra using Spin Echo Fourier Transform

(SEFT) and Quaternary Carbon Detection (QCD) 42 sequences were also performed to

aid the structure identification (Jayaprakash and Kalaiselvi, 2007).

3.2.13. Statistical analysis

The data obtained in the present study were expressed as Mean ± SD and were

analyzed using one way ANOVA test and students’t’ test with a post hoc multiple

comparison of SNK test as a significant level of 5% using a computer software

STATISTICA 06 (Statosoft, Bedford, UK).

46

3.3. RESULTS

The present study was carried out to extract and characterize the polysaccharides

such as fucoidan, sodium alginate and alginic acid of brown seaweed S. wightii. The

detailed results are given below.

3.3.1. Percentage yield of polysaccharides

The yield of individual polysaccharide extracted from the brown seaweed

S. wightii is given in Table 3.1. Among the three extracted polysaccharides, sodium

alginate represented the maximum yield of 16.352%, whereas the yield of fucoidan and

alginic acid was 2.832 and 3.932%, respectively. The one way ANOVA revealed that

the variation between the yield of different polysaccharides was statistically more

significant (F= 198.9252; P< 0.0001) (Table 3.1a).

3.3.2. Determination of purity of polysaccharides by phytochemical analysis

The purity of the three polysaccharides such as fucoidan, sodium alginate and

alginic acid was determined through phytochemical tests. The result indicated that in all

the three polysaccharides, the phytochemical constituents such as alkaloids, saponins,

tannins, phlobatannins, flavonoids, steroids, terpenoids, cardiac glycosides and phenols

were absent. Only carbohydrate as well as sugar derivative of saponins were found to be

present in fucoidan, sodium alginate and alginic acid, which confirmed the better purity

of the same (Table 3.2).

47

3.3.6. Physical properties of polysaccharides

The results on physical properties such as organoleptic characters, pH, particle

size, moisture content, boiling point and solubility of fucoidan, sodium alginate and

alginic acid are given in the Table 3.3.

3.3.6.1. Organoleptic evaluation

One of the organoleptic characters, colour was varied in all the three products.

For instance, the colour of fucoidan was dark brown, whereas it was whitish yellow and

white to yellowish brown colour in sodium alginate and alginic acid, respectively. All

the three products were odour less, however fucoidan represented tasteless and the other

two products were salty taste. The texture of all the three products indicated powder

form (Table 3.3).

3.3.6.2. pH of polysaccharides

The pH of the polysaccharides was detected after preparation of 1% solution.

The pH of 1% fucoidan, sodium alginate and alginic acid solutions was 6.2, 9.21 and

2.35, respectively.

3.3.6.3. Particle size of polysaccharides

From each polysaccharide products, totally 500 particles were individually

measured by using ocular micrometer. The particles were grouped into different size

ranges (0-10µm to >70µm). Among these, a maximum of 25 and 23% of particles of

48

fucoidan and sodium alginate, respectively were within the size range between 11 and

20µm. 28% of particles of alginic acid were within the size range between 21 and

30µm, whereas minimum of 3% particles of fucoidan and alginic acid were within the

size range between 61 and 70µm. Similarly, only 3% of particles of alginic acid alone

were within the size range of >70µm (Table 3.3).

3.3.6.4. Moisture content of polysaccharides

The level of moisture content observed in fucoidan, sodium alginate and alginic

acid was 8.0 ± 0.961, 16.0 ± 1.12 and 18.0 ± 1.07 %, respectively (Table 3.3).

3.3.6.5. Solubility of polysaccharides

The solubility behavior of fucoidan, sodium alginate and alginic acid indicated

that, they were readily soluble in distilled water and sulphuric acid, but they were

insoluble in all the tested organic solvents such as ethanol, methanol, DMSO, acetone,

ether, chloroform, dichloromethane and n- butanol. However, fucoidan and sodium

alginate were partially soluble in hydrochloric acid, except alginic acid (Table 3.3).

3.3.7. Biochemical composition of polysaccharides

The result on biochemical composition of polysaccharides is given in Table 3.4.

The major component of fucoidan, sodium alginate and alginic acid was carbohydrate

(57.25 ± 1.815, 46.13 ± 2.1 and 44.36 ± 1.42 %), with little amount of protein (4.51 ±

0.79, 5.21 ± 0.927 and 5.82 ± 0.72 %) and lipid (3.77 ± 0.421, 4.13 ± 0.364 and 4.06 ±

49

0.341 %) contents, respectively. The fucose content of fucoidan, sodium alginate and

alginic acid were 70.61 ± 2.18, 29.19 ± 1.36 and 28.99 ± 1.09 %, respectively. The ash

values such as total ash, acid insoluble ash and water soluble ash were 0.7 ± 0.012,

0.042 ± 0.0036 and 0.392 ± 0.028 in fucoidan, 1.92 ± 0.052, 0.134 ± 0.0082 and 1.036

± 0.042% in sodium alginate and 1.41 ± 0.032, 0.098 ± 0.0064 and 0.676 ± 0.052 in

alginic acid, respectively. The sulphate content of sodium alginate was

14.57 ± 0.560 %.

The statistical student‘t’ test revealed that the variation between fucoidan and

sodium alginate containing biochemical constituents such as carbohydrate, fucose, total

ash, acid insoluble ash, water soluble ash and sulphate were statistically significant

(Carbohydreate: t = 6.801085; P< 0.01; fucose: t= 28.62799; P< 0.0001; total ash: t=

39.9054; P< 0.0001; acid insoluble ash: t= 20.9235; P< 0.0001; water soluble ash: t=

23.526; P< 0.0001 and sulphate: t= 14.89913; P< 0.0001). Invariably, the variation of

biochemical constituents such as protein and lipid between fucoidan and sodium

alginate were statistically non significant (Protein: t= 0.96612; P> 0.05 and lipid t =

1.72938; P> 0.05). Similarly, the variation of biochemical constituents such as

carbohydrate, fucose, total ash, acid insoluble ash, water soluble ash and sulphate

between fucoidan and alginic acid were statistically significant (Carbohydreate: t =

9.222841; P< 0.001; fucose: t= 29.83021; P< 0.0001; total ash: t= 36.6805; P< 0.0001;

acid insoluble ash: t= 14.4591; P< 0.0001; water soluble ash: t= 9.82039; P< 0.001 and

sulphate: t= 9.418501; P< 0.0001). But the variation of biochemical constituents such as

protein and lipid between fucoidan and alginic acid were statistically non significant

50

(protein: t= 1.98061; P> 0.05; lipid: t= 1.56891; P> 0.05). Likewise, the variation of

biochemical constituents such as total ash, acid insoluble ash, water soluble ash and

sulphate between sodium alginate and alginic acid were statistically significant (total

ash: t= 14.46748; P< 0.0001; acid insoluble ash: t= 6.763222; P< 0.01; water soluble

ash: t= 10.1298; P< 0.001 and sulphate: t= 28.782; P< 0.0001). But the variation of

biochemical constituents such as protein, carbohydrate, lipid and fucose content

between sodium alginate and alginic acid were statistically nonsignificant (protein: t=

0.85072; P>0.05; carbohydrate: t= 1.187946; P> 0.05; lipid: t= 0.310473; P> 0.05;

fucose: t= 0.221766; P> 0.05) (Table 3.4a).

3.3.8. UV spectral analysis

3.3.8.1. Fucoidan

The result on UV spectral analysis of purified fucoidan is given in Fig. 3.1. The

spectral analysis showed the presence of fucoidan at the absorption maxima of 371,

393, 405, 408, 411, 415, 417, 419, 421 and 425nm, respectively.

3.3.8.2. Sodium alginate

The UV spectral analysis of purified sodium alginate indicated the presence of

uronic acid and manuronic acid at the absorption maxima of 204, 206, 299 and 699nm,

respectively (Fig. 3.2).

51

3.3.8.3. Alginic acid

The UV spectral analysis of purified alginic acid showed the presence of uronic

acid and manuronic acid at the absorption maxima of 299, 318 and 392nm, respectively

(Fig. 3.3).

3.3.9. FT-IR analysis

3.3.9.1. Fucoidan

The result on the FT-IR analysis of fucoidan of S. wightii is given in the Table

3.5 and Fig. 3.4. In the region of 3600–1600 cm-1

, three bands appeared with a broad

band centered at 3450.48cm-1

assigned to hydrogen bonded O–H stretching vibrations,

the weak signal at wavelength 2146.37 cm-1

indicated the presence of C=C=O and the

asymmetric stretching of carboxylate O–C–O vibration at 1604.82 cm-1

. The band at

1422.70 cm-1

may be due to C–OH deformation vibration with contribution of O–C–O

symmetric stretching vibration of carboxylate group. The weak band at 1253.00cm-1

indicated the presence of S=O stretching vibration of sulphate group. The band at

1037.21 cm-1

may also be due to C–O stretching vibrations. The spectrum showed a

band at 891.62 cm-1

assigned to the C1–H deformation vibration of b-mannuronic acid

residues. The band at 818.88 cm-1

seems to be characteristic of C-O-S stretching of

sulphate group. The band at 618.68cm-1

may be due to C≡C-H stretching vibration.

52


The FT-IR analysis of sodium alginate of S. wightii represented that in 3600–

1600 cm-1

region, four bands appeared with a broad band centered at 3416.38cm-1

. It

was assigned to hydrogen bond (O–H) stretching vibrations, the weak signal at 2934.04

cm-1

due to C–H stretching vibrations, the wavelength at 2143.14 cm-1

indicated the

presence of C=C=O and the asymmetric stretching of carboxylate O–C–O vibration at

1609.70 cm-1

. The band at 1415.03 cm-1

may be due to C–OH deformation vibration

with contribution of O–C–O symmetric stretching vibration of carboxylate group. The

weak bands at 1088.66 cm-1

may be assigned to C–O stretching, and C–O and C–C

stretching vibrations of pyranose rings; the band at 1034.89 cm-1

may also be due to

C–O stretching vibrations. The spectrum showed a band at 949.42 cm-1

, which was

assigned to the C–O stretching vibration of uronic acid residues, and one at 891.62 cm-1

assigned to the C1–H deformation vibration of b-mannuronic acid residues. The band at

816.33 cm-1

seems to be characteristics of mannuronic acid residues. The band at

622.49cm-1

may be due to C≡C-H stretching vibration (Table 3.6 and Fig. 3.5).


The FT-IR result indicated that in the region of 3600–1600 cm-1

, five bands

appeared with a broad band centered at 3414.74 cm-1

. It was assigned to hydrogen bond

(O–H) stretching vibrations, the weak signal at 2929.30 cm-1

due to C–H stretching

vibrations, the wavelength at 2360.18 cm-1

indicated the presence of R2C=N=N and the

asymmetric stretching of carboxylate O–C–O vibration at 1611.26 cm-1

. The band at

53

1417.64 cm-1

may be due to C–OH deformation vibration with contribution of O–C–O

symmetric stretching vibration of carboxylate group. The weak bands at 1038.05 cm-1

may be assigned to C–O stretching, and C–O and C–C stretching vibrations of pyranose

rings. The spectrum showed a band at 887.25 cm-1

assigned to the C1–H deformation

vibration of b-mannuronic acid residues. The band at 812.91cm-1

seems to be

characteristic of mannuronic acid residues (R=C=CHR). The band at 618.68cm-1

may

be due to C≡C-H stretching vibration (Table 3.7 and Fig. 3.6).

3.3.10. 13

C and 1H NMR analysis

3.3.10.1. Fucoidan

The results on 13

C and 1H NMR spectral analysis of purified fucoidan are given

in the Fig. 3.7 and 3.8. The 13

C NMR spectrum showed sharp absorptions

corresponding to a (1-6)-β-D-linked galacton at ppm 101.6 (C-1), 75.62 (C-5), 72.8 (C-

3), 70.77 (C-2), 69.82 (C-6) and 69.17 (C-4). Similarly, the 1H NMR spectrum showed

the correlation of these signals with the ppm of 4.464 (H-1), 3.901 (H-5), 3.682 (H-3),

3.556 (H-2), 4.075/ 3.959 (H-6/H-60) and 4.075 (H-4), respectively. The 13

C absorption

at ppm 101.6, 99.94, 96.12 correlated with 1H absorption at ppm 5.684, 5.107 and 5.097

are corresponding to a terminal α-L fucose, 3- linked α-L fucose and 3,4 distribution of

α-L-fucose, which suggested the presence of 3 sulfated 4 linked and 4 sulfated 3-linked

α- L-fucose. The 13

C NMR spectrum showed absorptions corresponding to a β-D

mannuronic acid at ppm 101.6 (C-1), 71.78 (C-2), 72.83 (C-3), 77.69 (C-4), 75.62 (C-5)

and 175.26 (C-6). Similarly, the 1H NMR spectrum showed the correlation of these

54

signals with the ppm of 5.09 (H-1), 3.901 (H-2), 3.74 (H-3), 3.95 (H-4), 3.79 (H-5) and

1.11 (H-6), respectively.


The 13

C NMR spectrum showed absorptions corresponding to a β-D mannuronic

acid at ppm 99.92 (C-1), 66.88 (C-2), 69.80 (C-3), 77.72 (C-4), 75.53 (C-5) and 175.25

(C-6). Similarly, the 1H NMR spectrum showed the correlation of these signals with the

ppm of 4.248 (H-1), 3.899 (H-2), 3.693 (H-3), 3.991 (H-4), 3.765 (H-5) and 0.967 (H-

6), respectively. Similarly the 13

C NMR spectrum showed sharp absorptions

corresponding to a guluronic acid at ppm 16.62, 57.27, 64.77, 87.73 and 166.19

respectively for C1, C2, C3, C4, C5 and C6. The 1H NMR spectrum showed the

correlation of these signals with the ppm of 0.932, 3.394, 3.413, 3.430 and 3.528

respectively for H1, H2, H3, H4 and H5, respectively (Fig. 3.9 and 3.10).


The result on 13

C and 1H NMR spectral analysis of purified alginic acid are

given in the Fig. 3.11 and 3.12. The 13

C NMR spectrum showed absorptions

corresponding to a β-D mannuronic acid at ppm 99.89 (C-1), 66.88 (C-2), 69.79 (C-3),

79.97 (C-4), 75.53 (C-5) and 175.34 (C-6). Similarly, the 1H NMR spectrum showed

the correlation of these signals with the ppm of 4.248 (H-1), 3.830 (H-2), 3.693 (H-3),

3.987 (H-4), 3.721 (H-5) and 1.005 (H-6), respectively. Similarly the 13

C NMR

spectrum showed sharp absorptions corresponding to a guluronic acid at ppm 16.58,

55

57.11, 64.62, 131.17 and 165.53 respectively for C1, C2, C3, C4, C5 and C6. The 1H

NMR spectrum showed the correlation of these signals with the ppm of 0.970, 3.432,

3.450, 3.467 and 3.485 respectively for H1, H2, H3, H4 and H5, respectively.

56

3.4. DISCUSSION

Seaweeds have always been of greater interest in Asian culture as marine food

sources (Fleurence, 1999). Seaweeds contain few lipids and are a good source of

proteins, vitamins and minerals (Ito and Hori, 1989). Also, seaweeds contain a large

array of nutraceutical components, including antioxidant and bioactive polysaccharides

such as fucoidan, laminaran and alginates (Plaza et al., 2008). The polysaccharide

composition of macro algae differs significantly to terrestrial biomass. Brown algae

contain a high carbohydrate fraction dominated by alginates, laminarin, manntitol and

fucoidan depending on the species. In the present study the polysaccharides such as

fucoidan, sodium alginate and alginic acid were extracted from brown seaweed

S. wightii and characterized through FT-IR and NMR analysis. The fucoidan was

extracted and the yield observed was 2.832 ± 0.204 %. Usually the fucoidan content of

various seaweeds are varied much. Chotigeat et al. (2004) have reported the yield of

fucoidan extracted from S. polycystum was 2.74 ± 1.18g per 100g dry weight, which is

almost same as that extracted from Pelvetica canaliculata by HCl method by Colliec

et al. (1994). Yang et al. (2008) have extracted fucoidan polymer from the brown

seaweed Undaria pinnatifida and the yield observed was 8.8%. Several methods were

adopted to extract fucoidan from different species. Dietrich et al. (1995) have used the

enzyme maxatase for fucoidan extraction from certain seaweed species such as Dictyota

mertensis, Radina gymnospora and S. vulgare. Similarly, Pereira et al. (1999) have

tried to extract fucoidan from the seaweed Fucus vesiculosus, Laminaria brasiliensis

and Ascophyllum nodosum by using papain. Likewise, Hoshino et al. (1998) used 10%

57

TCA to extract fucoidan from S. horneri and used HCl to get fucoidan from

P. canaliculata and S. polycystum. In all the above methods, the yield of fucoidan was

varied widely, which could be concluded the method of extraction as well as seaweed

species.

The yield of sodium alginate and alginic acid observed in the present study was

16.352 ± 1.42% and 3.932 ± 0.423%, respectively. Usually the alginate content of

various seaweeds is varied much. Torres et al. (2007) reported the yield of alginate

extracted from S. vulgare was 16.9%. Davis et al. (2004) found the yield within the

range of 21.1 – 24.5% in S. fluitans and 16.3 – 20.5% in S. oligocystum with variations

being depended on the alginate extraction methods used. Davis et al. (2003) have

studied the yield of alginate extracted from S. dentifolium, S. asperifolium and

S. latifolium as 3.25, 12.4 and 17.7%, respectively.

In the present study, the phytochemical characteristics of fucoidan, sodium

alginate and alginic acid were individually assessed to determine their purity. The

results indicated that the polysaccharides are pure form and they have no alkaloids,

tannins, phlobatannins, flavonoids, steroids, terpenoids, cardiac glycosides, phenols, but

the only element carbohydrate and its derivative saponins were present in all the three

products, which confirm the purity. Similarly, Kumar et al. (2011) have reported the

purity of tamarind seed polysaccharide by phytochemical analysis, which indicated the

absence of alkaloids, steroids, flavonoids, saponins, tannins and phenols, however only

58

carbohydrate was found to be present, which confirms the purity of tamarind seed

polysaccharide.

The organoleptic and other physical characters such as color, odour, taste,

texture, pH, moisture content and solubility of fucoidan were in the order of dark

brown, odourless, tasteless, powder or slippery form, 6.2 and 8%, respectively.

Similarly, Takara Bio Inc., Japan, has produced Takara Kombu fucoidan from seaweeds

and they studied the colour, texture, pH and moisture content and they were in the order

of light greenish brown colour, powder form, 6.7 and 6.0%, respectively. Umi No

Shizuku, Kamerycah (Hong Kong) Limited, Kowloon, Hong Kong, had reported that

the colour and texture of fucoidan of Cladosiphon okamuranus was dark brown colour

and powder form. Santa Cruz Biotechnology Inc., Germani, has extracted the fucoidan

from Fucus vesiculosis and the colour and texture of this fucoidan were yellow to

brown and powder form.

In the present study, the organoleptic and physical characteristic features of

sodium alginate were analyzed. The color, odor, taste, texture, pH and moisture content

of sodium alginate were in the order of whitish yellow color, odourless, salty taste,

powder form, 9.21 and 16%, respectively. Xiamen JieJing Biology Technology Co.,

Ltd., Xiamen, China, has extracted the sodium alginate from marine alga and observed

the organoleptic characters. It stated that the colour, odour, texture and taste of sodium

alginate were white or light yellow, odorless, vagiform powder and tasteless,

respectively. Likewise, Suqian Broad Seaweed Industry Co., Ltd, China, has extracted

59

and characterized the sodium alginate from seaweeds. This industry reported that

colour, taste, texture, pH and moisture content of sodium alginate were white or light

yellow colour, tasteless, granule or powder, 6.0 – 8.0 and ≤ 15%, respectively.

According to FAO (1995) the colour, texture and moisture content of sodium alginate of

seaweeds were white to yellowish brown, filamentous, grainy, granular or powder

forms and 15%, respectively. Science Lab.com, Inc., Humble, United States (Chemical

and Laboratory equipment), has published the physical properties of sodium alginate

and quoted that the color, odor and taste of sodium alginate were white to off white

color, odourless and tasteless, respectively.

The organoleptic and other physical properties of alginic acid were in the order

of white to yellowish brown color, odourless, salty taste, powder form, pH 2.35 and

moisture content of 18%, respectively. Similarly, Cyber colloids Ltd., Ireland (E400

Alginic acid) has reported the color, odour, texture, pH and moisture content of alginic

acid were white to yellowish brown color, nearly odorless, granular or powder, 2 to 3

and 15%, respectively. The Good Scent Company, East Montana Avenue, Oak Creek,

USA, has studied the physical parameters of alginic acid of seaweed and observed the

color, odor, taste and texture of alginic acid were white to pale yellow color, odorless

and tasteless, respectively. FMC BioPolymer, Philadelphia, USA, has reported that the

color, odor, texture, taste and pH of alginic acid was white to yellowish color, odorless,

tasteless, free flowing powder, 1.5 - 3.5 (in 3% aqueous dispersion), respectively.

60

The particle size of individual polysaccharide was determined by using ocular

micrometer. Totally 500 particles of individual polysaccharide with different sizes were

measured. Among these 3 to 25%, 7 to 23% and 3 to 28% particles were in the size

range of 0 – 10 to >70µm, respectively in fucoidan, sodium alginate and alginic acid.

Similarly, Kumar et al. (2011) measured 500 particles of tamarind seed polysaccharide

by ocular micrometer method. They reported that 2% of particles were with in the size

range of 0 - 30µm, 10.4% of particles were within the size range of 30 - 60µm, 51.6%

of paticles were within the size range of 60-90µm, 34% of particles were within the size

range of 90-120µm and only 3.6% of particles were in the size group of >120µm. FMC

Biopolymer, Philadelphia, USA, has reported the size of sodium alginate after extracted

from brown seaweed and observed the size range between 75 and 250µm.

In the present study, the solubility behavior of fucoidan indicated that it is

readily soluble in distilled water and sulphuric acid, but insoluble in certain organic

solvents like ethanol, methanol, DMSO, acetone, ether, chloroform, dichloromethane

and n- butanol. Similarly, Santa Cruz Biotechnology Inc., Germani, has extracted the

fucoidan from F. vesiculosis and it was readily soluble in water. Buenavisata

(Biopolymer and seperation technologies), South Buena Vista Street, Burbank, United

States, has reported that the fucoidan of brown seaweed Undaria pinnatifida was

readily soluble in water. In the present study, it was also assessed that the solubility

behavior of sodium alginate indicated that it is readily soluble in distilled water and

sulphuric acid, but insoluble in the above organic solvents. According to FAO (1995)

report, the solubility behavior of sodium alginate is that it is slowly soluble in water,

61

forming a viscous solution and insoluble in ethanol and ether. Science Lab.com, Inc.,

Humble, United States (Chemical and Laboratory equipment), has published the

solubility behavior of sodium alginate, as it is readily soluble in cold and hot water and

insoluble in diethyl ether.

The solubility behavior of alginic acid indicated that it was readily soluble in

distilled water and sulphuric acid, but insoluble in certain organic solvents such as

ethanol, methanol, DMSO, acetone, ether, chloroform, Dichloromethane, n- butanol and

hydrochloric acid. Similarly, Cyber colloids Ltd., Ireland (E400 Alginic acid) has

reported that the alginic acid was insoluble in water and organic solvents and slowly

soluble in solutions of sodium carbonate, sodium hydroxide and trisodium phosphate.

The Good Scent Company, East Montana Avenue, Oak Creek, USA, has studied that

the alginic acid was soluble in alkaline water solution. FMC BioPolymer , Philadelphia,

USA, has pointed out that alginic acid was insoluble in water.

In the present study, the biochemical components such as protein, carbohydrate,

lipid, fucose, total ash, acid insoluble ash, water soluble ash and sulphate contents of

fucoidan were in the order of 4.51, 57.25, 3.77, 70.61, 0.7, 0.042, 0.392 and 45.06 %,

respectively. Similarly, Yang et al. (2008) have reported that the fucoidan of U.

pinnatifida consisted of mostly carbohydrates (54.9%), fucose (78.8%), galactose

(21.2%) and sulphates (41.5%), with a small amount of proteins (2.8%). The

biochemical component of fucoidan of brown seaweeds such as Ecklonia stolonifera,

Ascophyllum nodosum, Fucus vesiculosus, Adenocystis utricularisk and F. serratus has

62

been reported that the comparable amount (42 – 66%) of carbohydrates with smaller

amount (11.5 – 34.2%) of sulphates and protein contents varied from 0 to 12.4% (Lee et

al., 1995; Marais and Joseleau, 2001; Rupérez et al., 2002; Ponce et al., 2003; Bilan et

al., 2006; Rioux et al., 2007). In contrast, some of the researchers stated that the

biochemical composition of fucoidan is significantly different depending on species,

anatomical regions, growing conditions, extraction procedures and analytical methods

(Lee et al., 1995; Chizhov et al., 1999; Duarte et al., 2001; Marais and Joseleau, 2001;

Bilan et al., 2002, 2004, 2006; Ponce et al., 2003).

The biochemical composition of alginates was estimated. The biochemical

components such as protein, carbohydrate, lipid, fucose, total ash, acid insoluble ash,

water soluble ash and sulphate contents recorded were 5.21, 46.13, 4.13, 29.19, 1.92,

0.134, 1.036 and 14.57 %, respectively in sodium alginate and 5.82, 44.36, 4.06, 28.99,

1.41, 0.098, 0.676 and 25.91 %, respectively in alginic acid. Similarly, Torres et al.

(2007) explained the biochemical constituents of alginate extracted from S. vulgare.

They reported that the protein values determined were 1.1% for S. vulgare low-viscosity

alginate (SVLV) and 1.0% for S. vulgare high-viscosity alginate (SVHV). The Moisture

and ash contents of SVLV and SVHV were 14 & 16% and 2 & 1%, respectively. Larsen

et al. (2003) have reported the biochemical composition of alginates of algae harvested

from the Egyptian Red Sea coast. They observed that total carbohydrate and fucose

contents were 74.93 and 11.63%, respectively in C. trinode, 57.87 and 5.62%,

respectively in S. dentifolium, 32.16 and 4.15%, respectively in S. asperifolium and

42.26 and 8.24%, respectively in S. latifolium.

63

In the present study, the UV vis spectral analysis showed the presence of

fucoidan at the absorption maxima of 371, 393, 405, 408, 411, 415, 417, 419, 421 and

425nm, respectively and FT- IR analysis revealed the presence of sulphate group (S=O

stretching) at 1253.00cm-1

. The spectrum showed a band at 891.62 cm-1

assigned to the

C1–H deformation vibration of mannuronic acid residues. The band at 818.88 cm-1

seems to be characteristic of C-O-S stretching of sulphate group. Similarly, Duarte et al.

(2001) have reported the FT-IR analysis of fucoidan from the brown seaweed

S. stenophyllum. They pointedout that the fucoidan fraction showed the band at 817.8–

821.6 cm-1

, suggesting the presence of equatorial sulfate groups on the C-2 and C-3

positions or sulfate groups linked to the primary C-6 position. Chandıa and Matsuhiro

(2008) studied the FT-IR analysis of fucoidan fraction from Lessonia vadosa. They

suggested that the IR spectrum of the polysaccharide showed the characteristic band of

S-O stretching vibration at 1259 cm-1

and a band at 849.5 cm-1

due to C–O–S vibration

which was assigned to sulfate group linked to axial secondary alcoholic group. No band

was found around 1700 cm-1

due to carbonyl group of uronic acids. The asymmetric

deformation of O–S–O group absorption at 582.1 cm-1

confirmed the presence of

significant amount of sulfate groups. Bilan et al. (2006) studied that the FT- IR

analsysis of fucoidan extracted from the brown seaweed Fucus serratus. They pointed

out that the IR-spectrum of fucoidan contained an intense absorption band at 1240 cm-1

(S=O) common to all the sulfate esters. An additional sulfate absorption band at 824

cm-1

(C–O–S, secondary equatorial sulfate) and a band at 844 cm-1

(C–O–S, secondary

64

axial sulfate) indicated the majority of sulfate groups occupy positions 2 and/or 3, and

only a minor part of sulfate is located at position 4 of fucopyranose residues.

The UV spectral analysis of purified sodium alginate showed the presence of

uronic acid and manuronic acid at the absorption maxima of 204, 206, 299 and 699nm

and the FT-IR analysis revealed the presence of uronic acid (C-O stretching) at the

wave length of 949.42 cm-1

and the vibration at 891.62 and 816.33 cm-1

indicated the

presence of b-mannuronic acid and mannuronic acid residues, respectively. The UV

spectral analysis of purified alginic acid showed the presence of uronic acid and

manuronic acid at the absorption maxima of 299, 318 and 392nm and the FT-IR

analysis revealed the vibration at 887.25 and 812.91 cm-1

indicated the presence of b-

mannuronic acid and mannuronic acid residues. In accordance with these, Leal et al.

(2008) have reported the FT-IR analysis of alginate in three species of brown seaweeds.

They observed a spectral band at 948.5 cm-1

, which was assigned to be the C–O

stretching vibration of uronic acid residues, and one at 888.3 cm-1

assigned to the C1–H

deformation vibration of b-mannuronic acid residues and the band at 820.0 cm-1

seems

to be characteristic of mannuronic acid residues. Zhang et al. (2008) have reported the -

OH groups present in alginate are clearly seen at 3400 cm-1

. They also suggested that

the peaks attributed to the -CH2 groups present at 2931 cm-1

and 2926 cm-1

in alginate

and some distinct peaks such as carboxyl group showed strong absorption bands at 1614

cm-1

, 1416 cm-1

and 1306 cm-1

, respectively due to carboxyl anions. The band at 1648

cm-1

is attributed to the absorption band of the carbonyl (-HC=O) stretching. The other

65

band at 1041 cm-1

that was assigned to the stretching vibration of (CH-OH) appeared at

1643cm-1

and 1045 cm-1

for the composite gel beads.

1H NMR and

13C spectroscopy are the reliable methods for the determination of

the composition and also the block structures of fucoidan molecules (Larsen et al.,

2003). In the present study, the 13

C and 1H NMR analysis were performed after

hydrolysis of fucoidan fraction. The result of 13

C and 1H NMR indicated the presence of

carbons and anomeric protons of (1,6)-β-D-linked galacton, α-L- fucose and

β-D-manuronic acid in purified fucoidan. Similarly, Bilan et al. (2006) have studied the

NMR structure of fucoidan of brown seaweed Fucus serratus, they observed the NMR

spectra contained correlation peaks at 5.08/3.94 and 5.08/4.00 ppm corresponding to H-

10/H-3 and H-10 /H-4 interactions usual for (1-3)- linked fucobioside fragments as well

as peak at 4.98 / 3.87 and 4.98 / 1.30 ppm corresponding to H-10/H-4 and H-10/H-6

interactions for (1-4)-linked fucobioside fragments. Bilan et al. (2008) have reported

that the sulfated glucuronofucan containing both fucofuranose and fucopyranose

residues from the brown alga Chordaria flagelliformis. They suggested that the main

fucoidan chain was built up of (1, 3)-linked a-L-fucopyranose residues. The mode of

substitution in this chain was proved by the downfield location of C-3B at 76.0 ppm and

was confirmed by the presence of correlation peak 5.10 / 4.05ppm in 1H NMR spectrum

corresponding to inter-residue H-1B/H-3B and H-1B/H-4B interactions typical of (1,

3)-linked a-L-fucopyranoside chains. Similarly, Chizhov et al. (1999) studied the NMR

structure of fucoidan from the brown seaweed Chorda filum. They reported that the

acetyl groups are attached at O2 in two different a-(1, 3)-linked fucose residues (C-1:

66

94.7 and 93.4 ppm, H-1: 5.25 and 5.32 ppm, respectively). a - (1, 3 and 1, 2)-fucose

residues give signals of anomeric carbons at 93.4 and 92.9 ppm; this splitting possibly

arises due to the effect of the acetate attached to a neighboring fucose residue.

In the present study, the 13

C and 1H NMR analysis were also performed in

sodium alginate and alginic acid fractions after hydrolysis of the same. The result of the

13C and

1H NMR structure indicated the presence of carbons and anomeric protons of

gluronic acid and manuronic acid. In accordance with these, Torres et al. (2007) pointed

out that the NMR structure of S. vulgare alginate composed of guluronic acid anomeric

proton (G-1) at 5.06 ppm; guluronic acid H-5 (G-5) at 4.4 ppm; mannuronic acid

anomeric proton (M-1) and the C-5 of alternating blocks (GM-5) overlapped at 4.7

ppm. Similarly, Larsen et al. (2003) have studied the NMR spectrum of alginates from

algae harvested at the Egyptian Red Sea coast. They reported that the presence of

gluronic acid and manuronic acid protons at 3.50 – 3.56 and 3.80 – 4.80ppm,

respectively and the carbon at 71.2 – 72.4 and 93.4 – 94.8ppm in S. asperifolium

alginate The results presented here indicated that the brown seaweed S. wightii has a

main source of fucose, gluronic acid and manuronic acid containing polysaccharides

such as fucoidan, sodium alginate and alginic acid.

67

3.5. SUMMARY

The present study was undertaken to extract and characterize the

polysaccharides from brown seaweed S. wightii. The highlights of the study are

summarized below.

· An initial investigation was carried out to extract the polysaccharides such as

fucoidan, sodium alginate and alginic acid from brown seaweed S. wightii and

the yield observed was 2.832, 16.352 and 3.932%, respectively.

· The purity of polysaccharides was determined by preliminary phytochemical

analysis. The phytochemical result indicated that all the three polysaccharides

have only the presence of carbohydrates and its derivative saponins.

· The organoleptic characters of fucoidan indicated: dark brown colour, odourless,

tasteless, powder or slippery form. Like wise, the physical properties indicated

that it has 6.2 pH and 8 % moisture content. The particle size of the grains of

fucoidan was ranged from 1 to 70µm. It was readily soluble in water and

sulphuric acid.

· The organoleptic characters of sodium alginate indicated: whitish yellow color,

odourless, salty taste and the texture was in powder form. The physical

properties indicated that it has 9.21 pH and 16% moisture content. The particle

size of the grains of sodium alginate was inbetween 1 and 70µm. It was readily

soluble in water and sulphuric acid.

68

· The organoleptic characters of alginic acid indicated: white to yellowish brown

colour, odourless, salty taste, powder form. The physical properties indicated

that it has 2.35 pH and 18% moisture content. The particle size of the alginic

acid was ranged from 1 to >70µm. It was readily soluble in water and sulphuric

acid.

· The biochemical components such as protein, carbohydrate, lipid, fucose, total

ash, acid insoluble ash, water soluble ash and sulphate contents of fucoidan were

4.51, 57.25, 3.77, 70.61, 0.7, 0.042, 0.392 and 45.06 %, respectively. Similarly,

all the above biochemical components were in the order of 5.21, 46.13, 4.13,

29.19, 1.92, 0.134, 1.036 and 14.57 % in sodium alginate and 5.82, 44.36, 4.06,

28.99, 1.41, 0.098, 0.676 and 25.91 % in alginic acid, respectively.

· UV vis spectral analysis and FT-IR analysis were carried out to assigned the

functional groups of the purified polysaccharides after hydrolysis. These

observations indicated the presence of uronic acid and sulphate residues in

fucoidan sample. Whereas these results indicated the presence of uronic acid and

manuronic acid residues in sodium alginate sample, but the presence of gluronic

acid and manuronic acid in alginic acid.

· The result of 13

C and 1H NMR analysis indicated the presence of carbon and

anomeric proton of (1,6)-β-D linked galacton, α-L-fucose and β-D-manuronic

acid in purified fucoidan. The result of 13

C and 1H NMR analysis indicated the

69

presence of carbons and anomeric protons of guluronic acid and manuronic acid

in purified sodium alginate and alginic acid, respectively.

3.0. chapter 1 extraction and characterisation...

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