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PART-I
CHEMICAL INVESTIGATION OF THE POLYSACCHARIDES OF SOME BROWN SEAWEED SPECIES

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Chapter-I.1
Chemical studies on the alginate of brown seaweed species: Sargassum tenerrimum, Sargassum wightii, Cystoseira indica and Padina tetrastromatica

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Chemical studies on the alginate of brown seaweed species: Sargassum tenerrimum, Sargassum wightii, Cystoseira indica and Padina tetrastromatica

I.1.1 INTRODUCTION I.1.2 MATERIALS AND METHODS I.1.2.1 I.1.2.2 I.1.2.3 I.1.2.3.1 I.1.2.3.2 I.1.2.3.3 I.1.2.3.4 I.1.2.3.5 I.1.2.3.6 I.1.2.3.7 I.1.2.3.8 I.1.2.3.9 Collection of seaweeds Extraction of alginate from seaweeds Characterization of alginate Determination M/G ratio Determination of M and G blocks Viscosity measurement Molecular weight determination (GPC) FT-IR spectroscopy
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C NMR spectroscopy

Optical rotation and Circular dichroism (CD) spectroscopy Scanning electron microscope (SEM) X-ray diffraction analysis

I.1.2.3.10 Rheological determinations I.1.3 RESULTS AND DISCUSSION I.1.3.1 I.1.3.2 I.1.3.3 I.1.3.4 I.1.3.5 I.1.3.6 I.1.3.7 I.1.3.8 Physicochemical results Molecular weight determination (GPC) FT-IR spectroscopy
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C NMR spectroscopy

Optical rotation and Circular dichroism (CD) spectroscopy Scanning electron microscope (SEM) X-ray diffraction analysis Rheological measurements

I.1.4 CONCLUSION I.1.5 REFERENCES

32 I.1.1 INTRODUCTION Seaweeds are the only natural source of agar, carrageenan and alginates, the industrially important polysaccharides. The former two phycocolloids are synthesized by red seaweeds, and the latter being produced exclusively by their brown counterparts except one recent report of alginate from a red calcareous alga Corallina pilulifera Usov et al. (1995). It is noteworthy that alginates are also produced by bacteria viz. Azotobacter vinelandii and several Pseudomonas species Skjak-Braek et al. (1986). Alginates are hydrocolloids, water soluble biopolymers extracted from brown seaweed. They were first investigated in the late 19th century by a British chemist E.C. Stanford. (www.fao.org). Alginate is a collective term for a family of polysaccharides produced by brown algae (Painter 1983). It is a polysaccharide consisting of linear co-polymer of -1,4-D-mannuronic acid and -1,4-L-guluronic acid Haug et al. (1966) (Figure I.1.1). The average lengths of the blocks being about 20 units and the proportions of the two acids have been found to vary in the extracts of different species and even in different parts of the same plant. Alginate is present as salts of different metals, primarily Na+ and Ca+2. It occurs in both the intercellular regions and the cell walls and it is considered to be the principal skeletal material of all brown seaweeds (Haug et al.1967; Ji et al. 1981; Cora et al. 1992; Nishide et al. 1996). The main brown seaweeds that are processed commercially worldwide for the alginate are Macrocystis pyrifera, Laminaria spp., Ascophyllum spp., Ecklonia spp., Lessonia spp. and Padina gymnospora. None of them occur in the Indian waters except for some other Padina species. Extraction of alginic acid/alginate from brown algae and characterization has been reported by many authors (Haug & Larsen 1962; Haugh, Larsen & Smidsrod, 1974; Nishide et al. 1987; Andrade et al. 2004). The worldwide annual industrial production of alginate is estimated to be 30,000 metric tons, which is probably less than 10% of the biosynthesized material in crops of macroalgae. These figures allow us to consider such a polysaccharide as an unlimited and renewable resource even for a steadily growing industry (Draget et al. 2005). In addition, alginate production by fermentation is also technically possible, although it dose not meet the requirement of economic feasibility. The Indian production of alginate is mainly from Sargassum spp. There exist a few reports on alginate from Indian waters (Alankararao, 1988; Mody et al. 1992; Redekar and Raje 2000 and Ganesan et al. 2001). There are, however, no reports on the detailed systematic chemical investigation of alginates of Indian brown seaweeds.

33 Therefore, Sargassum tenerrimum, Sargassum wightii (Family: Sargassaceae), Cystoseira indica (Family: Cystoseiraceae) and Padina tetrastromatica (Family: Dictyotaceae) were selected for detailed chemical studies for their alginates. The main industrial applications of alginate as a natural polymeric material are linked to its stabilizing, viscosifying, gelling properties and its ability to retain water. Alginate is largely used as a viscosifier in textile printing because of its shear-thinning characteristics. Its use has also been reported to increase color yield and brightness. In addition, various applications of alginate in the food industry are currently being exploited. The ability of this polysaccharide to stabilize aqueous mixtures, dispersions and emulsions together with its gel-forming and viscosifying properties represent key features for the use of alginate in food applications. Several alginate-based restructured products (pet food, reformed meet, onion rings, crab sticks, to name few) are available on the market for large-scale distribution (Cotterll and Kovacs 1980; Littlecott 1982; Sime 1990). In addition, alginate is widely used as an additive for the production of low-sugar jam, jellies and fruit fillings (Toft et al. 1986). In these applications, synergistic interactions with proteins and other polysaccharide are exploited. Alginates well meet all the requirements for their use in pharmaceutical and medical applications. They have been largely used in wound dressings, dental impression and formulations for preventing gastric reflux. However, the most advanced biotechnological and biomedical applications of alginate resides in its use as a hydrogel for cell immobilization for applications ranging from production of ethanol from yeast cells and of antibiotics or steroids (Smidsrod and Skjak-Breaek 1990) to transplantation and cell therapy (Lim and Sun 1980). In the latter case, alginate gel is used as a selective immune barrier to protect the transplanted cells from the host immune system. There has been a notable increase in the number of alginate applications in recent years and the possibility of using such a polysaccharide for advanced biomedical therapies requires very detailed knowledge of its molecular characteristics. In fact, a clear understanding of the structure-function relationships is crucial for successful preparation of refined alginates for those applications where specific biochemical and physicochemical features have to be met. In addition, engineering of alginate molecules, by tailor-making their composition and properties or by introducing cellspecific signals, represents an important step forward for future novel applications in biotechnology field.

34 I.1.2 MATERIALS AND METHODS I.1.2.1 Collection of seaweeds

The seaweeds of this investigation were collected from the Indian coasts and the details are: Sargassum tenerrimum was collected during March 2008 to April 2008 from Veraval (20o 54.875 N, 70o 20.832 E), Diu (20o 42.727 N, 70o 55.487 E), from the inter-tidal zone on the west coast of India. Sargassum weightii used in this study was collected during December 2007 to February 2008 from Hare island (09o 11.749 N, 79o 03.31 E) from the inter-tidal zone on the southeast coast. Cystoseira indica used in this study was collected in January, 2008 from Diu (20o 42.727 N, 70o 55.487 E), from the inter-tidal zone of west Coast of India. Padina tetrastromatica used in this study was collected during January-February 2008 from Okha (22o 28.580 N, 69o 04.254 E) from the inter-tidal zone on the west coast and Valai island (09o 10.445 N, 78o 55.55 E) from the inter-tidal zone on the southeast coast of India (Oza and Zaidi, 2001; www.algaebase.org). Herbaria specimens of all the seaweed species were submitted with the CSMCRI Herbarium. All the seaweeds were washed with tap water to remove the solid impurities from the plants and were dried in the shade and powdered in a rotating ball mill and stored in separate plastic containers. Methanol (MeOH), formalin, sodium carbonate (Na2CO3), sodium hydroxide (NaOH), hydrochloric acid (HCl), sulphuric acid (H2SO4) of LR grade were used and were purchased from Ranbaxy Fine Chemicals Ltd., Mohali, Punjab (India). Sigma (A2158-250G) alginate was used as a reference sample for benchmarking. I.1.2.2 Extraction of alginate from seaweeds

Pretreatment: Dried algal powder was defatted with repeated extraction with MeOH (100ml X 4) in a percolator for four days at room temperature. Then 10g of each algal material were treated with 90ml of 0.1% formalin overnight (Dea, 1986). The residual formalin solution was drained off. Then the algal material was washed with water thrice and dried at room temperature and then at 40oC. Alginate was extracted by the method described by Nishide et al. (1996). A 10g sample of pretreated dry algal fronds were first soaked in warm distilled water (60oC, 300ml) for 1h and then filtered through nylon cloth. After homogenization of the algal material, it was extracted with 200ml of Na2CO3 solution (0.17N) under stirring condition at 75oC for 4h. Distilled water (800ml) was then added and the solution was

35 separated from the residual algal powder by filtration through a bed of Celite-545 and was acidified with 10% HCl to pH 1.00 to give a gelatinous precipitate. The gel was allowed to stand at room temperature for 3h and was collected by centrifugation at 5000rpm for 20min. Then 200ml of 50% MeOH were added to the gel and the mixture was neutralized with 10% NaOH with constant stirring. After standing overnight, the mixture was filtered through a layer of cotton cloth to separate the neutralized gel. The gel was washed successively with 60% MeOH, 95% MeOH and finally with acetone and was dried at 30o for12h. The alginate obtained from the treated algal material of S. tenerrimum, S. wightii, C. indica and P. tetrastromatica were designated as STAL (yield 18%), SWAL (20%), CIAL (10%) and PTAL (14%), respectively. I.1.2.3 I.1.2.3.1 Characterization of alginate Determination of M/G ratio

Mannuronic acid to guluronic acid ratios (M/G) of these alginates were determined by a rapid one-pot method of hydrolysis of sodium alginate using microwave irradiation (Chhatbar et al. 2009). Alginate (5g) was dissolved in 125ml of 0.15M oxalic acid or 0.25M H2SO4 and 25ml portions of the alginate solution were exposed to 100% microwave power for five different exposure durations (15min). The experiment was repeated with 0.05, 0.15, 0.25 and 0.5M oxalic acid/H2SO4. After microwave irradiation Polymannuronic acid (PMA) and polyguluronic acid (PGA) were isolated from the hydrolyzed samples using pH dependent method (Chanda et al. 2001; Sakugawa et al. 2004) (Figure I.1.2). The mixture was neutralized with CaCO3 and filtered and the solution was passed through Dowex 1-X8 anion exchange resin column (100-200 mesh; 20 x 2cm) in acetate form and fractionated by gradient elution with 0.5-2.0N acetic acid. Carbohydrate of each fraction was monitored by the Phenol sulfuric acid method (Dubois et al. 1956). Two fractions were obtained, former being guluronic acid (G) and the latter mannuronic acid (M). Both the pooled M and G fractions were evaporated under vacuum to a small volume and passed through a cation exchange resin column (IR-120) to eliminate the cations. The effluent was again evaporated to a syrup and this treatment was repeated 2-3 times in order to expel the acetic acid. Upon adding 30ml of water, a small amount of anion exchange resin (OH- form) was added to remove the color and residual anions. After filtration, the filtrate was treated with NaOH (pH 8) to sufficiently transform the lactones to sodium uronate. The solution was allowed to pass through a cation exchange resin column and the uronic acid liberated was titrated (after making up the effluent to 50ml or 100ml) against 0.1N NaOH to confirm the exact concentration of

36 each uronic acid. The M/G ratios measured by this improved method (with 0.15M oxalic acid/ 0.25M H2SO4 under 100% MW power for 4min) were comparable with that obtained from conventionally hydrolyzed sodium alginate (with 80% H2SO4 at 20oC for 18h and then 2N H2SO4 at 100oC for 6h), which was reported by Haug and Larsen (1962) and Ji et al. (1981). I.1.2.3.2 Determination of M and G blocks

The different uronic acid blocks of alginate were determined according to the method of Haug et al. (1974). Partial hydrolysis was carried out by suspending 0.5g of alginate in 100 parts of 0.3M HCl for 2h at 100oC. After 2h the suspension was cooled and centrifuged to give the MG-block fraction in the supernatant. The amount of material in the supernatant was determined by the method reported Knutson and Jeans (1968) (vide section I.2.1.2.4). The residue was suspended in water and dissolved by careful neutralization. The volume was adjusted to give an alginate concentration of 1%, NaCl was added to 0.1M concentration, and the solution was mixed with 25mM HCl to pH 2.8-3.0. The precipitate (GG-block fraction) was collected by centrifugation, suspended in water and solubilised by neutralization. The amounts of uronic acids in the precipitate and in the supernatant (MM-block) were determined (vide section I.2.1.2.4). I.1.2.3.3 Viscosity measurement

Apparent viscosity of alginate samples e.g. STAL, SWAL, CIAL and PTAL (1.5% in distilled water) were measured using a Brookfield Viscometer (DV-II +Pro) at 27oC. Spindle SC4-18 was used for apparent viscosity measurement at 60 rpm. I.1.2.3.4 Molecular weight determination (GPC)

For the determination of molecular weights (Mn, Mw and Mz) of alginate samples STAL, SWAL, CIAL, PTAL and Sigma (A2158-250G) gel permeable chromatography (GPC) was carried out on Waters alliance HPLC, with Waters 2695 separation modules and 2414 refractive index detector. Two columns Ultra hydrogel 120 and Ultra hydrogel 500 were used, column length and diameter was 300mm and 7.8mm, respectively. The columns were eluted with 0.1M NaNO3 solution at a flow rate of 0.5ml/min. The oven temperature was maintained at 45oC. Dextran standards with Mp value ranges 4.01 x 105; 1.96 x 105; 4.35 x 104; 4.4 x 103Da. (MW: 6.68 x 105; 2.73 x 105; 4.86 x 104; 5.2 x 103 Da, respectively) were used for preparation of calibration curve (Figure I.1.3). The molecular weight of alginate samples STAL, SWAL, CIAL

37 and PTAL, poly dispersity and area measurements were calculated using standard calibration curve by Empower2 software, USA provided with the instrument. The polysaccharide sample (50mg) was dissolved in 100ml of HPLC grade water using Tomy ES-315 autoclave (110oC, 30min.), filtered through nylon membrane filter (Whatman 0.45m) and 0.5ml of sample was injected in column. The molecular weights of standards and samples were determined by GPC according to the method described by Li et al. (2008). I.1.2.3.5 FT-IR spectroscopy

Infrared spectra of the alginate samples STAL, SWAL, CIAL and PTAL were recorded on a Perkin-Elmer Spectrum GX FT-IR system (USA), by taking 10.0mg of sample in 600mg KBr. All spectra were average of two counts with 10 scans each and a resolution of 5 cm-1. I.1.2.3.6
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C NMR spectroscopy

Noise-decoupled 13C NMR spectra were recorded on a Bruker Avance-II 500 (Ultrashield) Spectrometer, Switzerland, at 125 MHz. Alginate of SWAL and their respective oligomers PMA and PGA were dissolved in NaOH+D2O mixture (50mg/ml) and the spectra were recorded at 35oC with 50005200 accumulations, pulse duration 5.9 s, acquisition time 1.2059s and relaxation delay 6s using DMSO as internal standard (ca. 39.5). I.1.2.3.7 Optical rotation and Circular dichroism (CD) spectroscopy

Optical rotations were measured for all alginate samples of SWAL, STAL, CIAL and PTAL (0.250g/100ml at 27oC at 589nm) on a Rudolph Digi pol-781 Polarimeter (Rudolph Instruments Inc, NJ, USA). Circular dichroism (CD) spectra of alginates (Sigma and S. wightii), PMAs (Sigma and S. wightii alginates) and PGAs (Sigma and S. wightii alginates) were recorded on a Jasco model J-815 CD Spectrometer, using measurement range of 190250nm and at a concentration 0.8 mg/ml (800 ppm). Molar ellipticity values [] are reported in mdeg. The ratio of peak height to trough depth was calculated using Eq. (1), described by Morris et al. (1980). Peak/trough ratio = (trough peak/ trough) ---- (1)

38 I.1.2.3.8 Scanning electron microscopy (SEM)

The surface morphology of the alginate samples was analysed on a Carl-Zeiss Leo VP 1430 scanning electron microscope (SEM) applying an accelerating voltage of 10 or 20 kV and magnification 1 to 38 K respectively. Vacuum oven dried samples of SWAL and their respective oligomeric samples were mounted on a sample holder and coated with gold under vacuum prior to the studies. I.1.2.3.9 X-ray diffraction analysis

Powder X-ray diffraction studies of SWAL and their respective oligomers (i.e. PGA and PMA) were done on a Philips Xpert MPD X-ray powder diffractometer using 2 = 10o to 60o. I.1.2.3.10 Rheological determinations

The flow curves for STAL, SWAL, CIAL, PTAL and Sigma alginate in 0.1 M NaCl aqueous solutions (1.0%) were obtained on a Anton Paar, Physica MCR 301 rheometer, cone-plate model C-CC27/T200/SS, at 25oC. I.1.3 RESULTS AND DISCUSSION I.1.3.1 Physicochemical results

Sulit & Juan, (1955) first studied the alginate contents of several Sargassum species. There are many literature reports on the extraction of alginate, wherein different extraction conditions and scales have been employed e.g. on laboratory and pilot plant scales using various brown seaweed species of Indian waters e.g. Turbinaria ornata, Hormophysa triquetra, Cystophyllum muricatum and Sargassum swartzii (Doshi et al. 1984; Sai Krishnamurthy 2000), Sargassum tenerrimum and Sargassum wightii were reported by (Redekar and Raje et al. 2000) and (Ganeshan et al. 2001), respectively. There exists, however, no report on the detailed systematic chemical investigation of alginates of Indian brown seaweeds. The members belonging to order Fucales (Cystoseira indica) and Dictyotales (Padina tetrastromatica) are little studied for their polysaccharide contents. There exists no report of studies on alginate from Padina tetrastromatica and Cystoseira indica of Indian waters. Therefore, Sargassum tenerrimum, Sargassum wightii, Cystoseira indica and Padina tetrastromatica were selected for detailed chemical studies for their alginates.

39 The yields alginates of S. tenerrimum, S. wightii, C. indica and P. tetrastromatica were given as STAL (yield 18%), SWAL (20%), CIAL (10%) and PTAL (14%) in the Table I.1.1. Yields were calculated on the basis of as received seaweeds. In the present investigation the viscosities of the alginates obtained from S. tenerrimum (STAL, 120 cps) was found to be higher than those of S. wightii, C. indica and P. tetrastromatica (SWAL, 110 cps; CIAL, 80 cps and PTAl, 95 cps respectively). Uronic acid sequence e.g M and G block was determined of the samples of alginate and results were depicted in Table I.1.1 Table I.1.1 Analytical data of the alginates Sargassum tenerrimum (STAL) 18 120 Alginates of Sargassum Cystoseira wightii indica (SWAL) (CIAL) 20 10 110 80 Sigma alginate Padina tetrastromatica (A2158250G) (PTAL) 14 95 200

Yielda (%) Viscosity (cps) (c 1.5 wt%) at 27oC

MG (%) 30 25 22 MM (%) 28 20 20 GG (%) 42 55 58 a Yields were calculated on the basis of as received seaweeds

24 36 40

28 32 40

The M/G ratios and % weights of PGA and PMA obtained under optimized microwave (0.15M Oxalic acid or 0.25M H2SO4, 4min) conditions in the present method for the alginate of STAL, SWAL, CIAL, PTAL and Sigma were comparable to the ones obtained by conventional method reported by Haug and Larsen (1962) and Ji et al. (1981) (Table I.1.2). M/G ratios obtained with 0.15M Oxalic acid or 0.25M H2SO4 and 0.5M Oxalic or 0.5M H2SO4 acids were also comparable (Table I.1.2). This investigation demonstrated that sodium alginate can be hydrolyzed under microwave irradiation, using mild hydrolytic conditions, as opposed the reported methods using hydrolytic reaction with high acid concentration requiring longer reaction time. The M/G ratio of alginate obtained from C. indica (CIAL) were found to be lower (0.32) then those of S. tenerrimum, S. wightii, P. tetrastromatica and Sigma alginate (STAL, 0.61; SWAL, 0.39; PTAl, 0.53 and Sigma 0.78 respectively). I.1.3.2 Molecular weight determination (GPC)

40 The standard dextran calibration curve and chromatograms of alginate samples of Sigma, STAL, SWAL, CIAL and PTAL were shown in Figure I.1.4. The molecular weights (Mn, Mw, Mp and MZ) and poly dispersity were depicted in Table I.1.3. I.1.3.3 FT-IR Spectroscopy

The FT-IR spectrum of the alginates (STAL, SWAL, CIAL and PTAL) are depicted in (Figure I.1.5). All the spectra appeared to be identical and the prominent bends were in the range: max (KBr) (cm-1) 3430-3440 (O-H str, br, s), 2928-2930 (C-H str, w), 1604-1625 (asymmetric -C=O str, s), 1420-1462 (C-H bending, w), 1027-1031 (C-O-C str, s), the fingerprint, or anomeric, region (950750 cm-1) is the most discussed in carbohydrates (cf. Tulchinsky et al. 1976; Mathlouthi & Koenig 1986; Silverstein et al. 1991; Leal et al. 2008) [br = broad, s = strong, m = medium, w = weak, str = stretching]. The band at 948.5 cm-1 was assigned to the CO stretching vibration of uronic acid residues, and the one at 888.3 cm-1 to the C1H deformation vibration of -mannuronic acid residues. The band at 820.0 cm-1 was presumably due to mannuronic acid residues (Chanda et al. 2001 & 2004). I.1.3.4 The
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C NMR spectroscopy

C NMR spectra of the alginate of SWAL and their respective constituting

oligomers (e.g. PGA and PMA) have been presented in Figure I.1.6. The chemical shifts are in good agreement with those reported in the literature (Tako et al. 2000; Zhang et al. 2004; Sakugawa et al. 2004; Leroux et al. 2004) and are furnished in Table I.1.4. The carbonyl carbons (C-6 position) of mannuronic (M) and guluronic (G) acids appeared at 176.75 (G6) and 177.25 (M6) ppm (cf. Grasdalen et al. 1981), and the corresponding anomeric carbons appeared at 101.45 (G1) and 100.97 (M1) ppm. The remaining carbon resonances were attributed to as follows: 66.48 (G2), 71.50 (M2), 70.53 (G3), 72.90 (M3), 81.55 (G4), 79.35 (M4), 68.52 (G5), 77.30 (M5) ppm. Very similar carbon resonances were obtained in the spectra of sodium alginate derived from S. tenerrimum (STAL), C. indica (CIAL) and P. tetrastromatica (PTAL) and their respective oligomers (e.g. PGA & PMA) (Figures and data for these are not shown). I.1.3.5 Optical rotation and circular dichroism (CD) spectroscopy

The specific rotation of the aqueous solution (0.25%) of the alginate samples SWAL, STAL, CIAL and PTAL were measured and results were presented in the Table I.1.5. All the alginate samples showed negative optical rotation values.

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Table I.1.2 Comparison of M/G ratios of sodium alginate determined by conventional methoda with those of the present study using microwave heating. Reaction conditions STAL PGAb PMAb M/G (%) (%) ratio 38.5 23.1 0.60 SWAL CIAL PTAL PGAb PMAb M/G (%) (%) ratio 45.6 24.2 0.53 Alginate of Sigma (A2158-250G) PGAb PMAb M/G (%) (%) ratio 46.7 35.5 0.76

Conventional method (80% H2SO4 (20oC, 18 h) and then 2N H2SO4 (100oC, 6h) MW (0.15 M Oxalic 44.3 27.0 0.61 61.8 24.1 0.39 61.2 19.6 0.32 48.2 25.5 0.53 52.3 40.8 acid, 4 min) c MW (0.25 M 42.5 26.0 0.61 63.5 24.8 0.39 60.4 19.3 0.32 50.6 26.8 0.53 51.5 40.2 H2SO4, for 4 min) c MW (0.5 M Oxalic 43.6 25.3 0.58 60.4 22.2 0.36 62.8 22.6 0.36 43.8 23.6 0.54 47.3 34.5 acid, 4 min) c MW (0.5 M H2SO4, 40.0 22.8 0.57 57.9 21.5 0.37 59.4 20.8 0.35 44.0 22.0 0.50 45.8 34.3 for 4 min) c a Haug and Larsen (1962) and Ji et al. (1981); b % of PGA and PMA were calculated by UV-VIS spectroscopy as described by Haug and Larsen (1962) and Ji et al. (1981); cMW=Microwave heating in the present study

PGAb PMAb M/G PGAb PMAb M/G (%) (%) ratio (%) (%) ratio 55.20 21.40 0.387 58.4 19.3 0.33

0.78 0.78 0.73 0.75

42 Table I.1.3 Gel permeation chromatographic data of alginates Samples Retention Time (min) 13.8 13.0 13.5 13.8 13.9 Molecular weights (Da) Mw Mp Mz 315668 225066 119687 57007 57514 72895 158637 72212 38909 32435 694348 624652 297615 129614 123132 Polydispersity 1.10 1.23 1.56 1.34 2.49

Mn 285785 182394 76722 42542 23053

Sigma (A2158-250G) STAL SWAL CIAL PTAL

Table I.1.4 13C NMR shifts, observed for sodium alginate, poly-guluronic acid (PGA) and poly-mannuronic acid (PMA) recorded in D2O with DMSO as internal standard Components Sodium alginate a ppm 100.97 (100.97) 71.50 (70.83) 72.90 (72.29) 79.35 (78.94) 77.30 (77.01) 176.25 (175.58) 101.45 (101.44) 66.48 (66.04) 70.53 (70.01) 81.55 (80.71) 68.52 (68.18) 176.68 (175.75) 100.11 (100.37) 68.11 (68.78) 71.43 (71.20) 79.09 (78.16) 76.83 (76.21) 175.24 (174.3) Poly-guluronic acid b 101.03 (101.54) C-1 of GG 66.61 (66.04) C-2 of GG 70.27 (69.2) C-3 of GG 81.09 (80.40) C-4 of GG 67.00 (67.16) C-5 of GG 175.16 (173.85) C-6 of GG a b Values in the brackets (Tako, et al. 2000); Values in the brackets (Zhang, et al. 2004; Sakugawa, et al. 2004; Leroux, et al. 2004) Assignment of carbon C- 1 of MM C-2 of MM C-3 of MM C- 4 of MM C- 5 of MM C-6 of MM C- 1 of GG C- 2 of GG C- 3 of GG C-4 of GG C- 5 of GG C- 6 of GG C-1 C-2 C-3 C-4 C-5 C-6 of MM of MM of MM of MM of MM of MM

Poly-mannuronic acid b

43 Circular dichroism (CD) spectra of alginate (Sigma and S. wightii), PMA (Sigma and S. wightii alginates) and PGA (Sigma and S. wightii alginates) are shown in Figures I.1.7a-c. The CD spectrum of poly-L-guluronate (PGA) showed entirely negative peak and polyD-mannuronate (PMA) had a strong positive peak indicating clear separation of PGA and PMA after acid hydrolysis of the sodium alginate, while CD spectrum of alginate showed intermediate behavior, with a peak at ca. 200 nm, and a trough at ca. 215 nm (Figure I.1.7a). The relative amounts of D-mannuronate and L-guluronate residues were also observed from the ratio of peak height to trough depth. The peak/trough ratios were less than 1 for all the alginate samples, indicating high content of poly-guluronate residue in it (Morris, et al. 1980). If the peak/trough were < 1, the overall composition of any compound will show negative CD spectrum. When the spectrum crosses the baseline, then the overall composition shows entirely positive spectrum (i.e., peak/trough >1) (Morris et al. 1975; Morris et al. 1980; Dentini et al. 2006). The peak/trough ratios of alginate samples were calculated and results were presented in the Table I.1.5. Very similar results were found with the CD spectra of sodium alginate from S. tenerrimum (STAL), C. indica (CIAL) and P. tetrastromatica (PTAL) (Figures are not shown). Table I.1.5 Specific rotations and peak/trough ratios of alginate samples Properties Optical rotation []D (c 0.25 in H2O, 27oC)a Peak/trough ratio
a

SWAL -25.67o 0.59

STAL -68.12o 0.71

CIAL -45.25o 0.55

PTAL -53.17o 0.48

Alginate of Sigma (A2158-250G) -26.98 o 0.63

[]D (0.3% w/v) -0.098o (10oC); -0.110o (at 60oC) (Tako, et al. 2000); []D (1.0% w/v) 113o (Bi, et al. 2007) , used wavelength 589nm. I.1.3.6 Scanning electron microscopy (SEM)

The SEM images of sodium alginate (SWAL), PGA and PMA have been given in Figure I.1.8. The SEM image of the alginate showed mixture of PGA and PMA, while PGA and PMA SEM images showed different morphologies indicating separation of PGA and PMA (Figure I.1.8).

44 I.1.3.7 X-ray diffraction

X-ray diffraction patterns exhibited that the sodium alginate (SWAL) was crystalline in nature, while PGA and PMA appeared in amorphous and crystalline forms, respectively (Figure I.1.9). X-ray patterns of PGA and PMA confirmed that PMA in the alginate was crystalline in nature, may be due to the ordered structure and regular geometric arrangement of the molecules in the PMA of alginate (Figure I.1.9). Very similar X-ray diffraction patterns were found with sodium alginates from S. tenerrimum (STAL), C. indica (CIAL) and P. tetrastromatica (PTAL) and their representative oligomers (e.g. PGA & PMA) (Figures are not shown). I.1.3.8 Rheological measurements

Rheological behavior is an important parameter for the application of polysaccharides in the food industry. Flow curves for sodium alginate samples STAL, SWAL, CIAL, PTAL and sigma in aqueous solution were performed in the concentration 1.0% in 0.1M NaCl (Figures I.1.10). In the Power-Law model, the solution behavior can be calculated using Eq. (2) described by Torres et al. (2007). mn ----- (2)

Where m is the consistency index (Pa s), n the flow behavior index, the shear stress and is the shear rate. The parameters m and n can be obtained by linear regression analysis (Marcotte et al. 2001; Torres et al. 2007). The n values for all these samples were in the range 0.8-0.95. For Newtonian fluids the flow behavior indices are closer to 1. No variations in the viscosity of all alginate samples at different shear rate were obtained, demonstrating that all these alginates behaved as Newtonian fluid in solution. I.1.4 CONCLUSION Alginates were extracted from the brown seaweeds Sargassum tenerrimum, Sargassum wightii, Cystoseira indica and Padina tetrastromatica of Indian waters. The yields and viscosity of the alginates of Sargassum tenerrimum and Cystoseira indica were the highest and the lowest respectively in this series. A rapid new microwave assisted method was developed wherein alginates were hydrolyzed to yield M- and G-acids. M/G ratios determined by this method were in good agreement with those obtained by the

45 conventional heating method (Chhatbar et al. 2009). The M/G ratio of the alginate of Sargassum tenerrimum was higher than that of Cystoseira indica alginate this was in line with the viscosity values obtained. This constitutes the first systematic chemical studies on alginate of Indian brown seaweeds, which will be useful in the works on the biodiversity of alginophytes.

I.1.5 REFERENCES Alankararao, G.S.J.G., Prasad, R.Y. and Rao, R.K., Phykos, 1988, 27, 174-176. Andrade, L.R., Salgado, L.T., Farina, M, Pereira, M.S. and Paulo, A.S., Journal of Structural Biology, 2004, 145,216225. Bi, F., Mahmood, S.J., Arman, M., Taj, N. and Iqbal, S., Physics and Chemistry of Liquids, 2007, 45, 453-461. Chanda, N.P., Matsuhiro, B., Mejas, E. and Moenne, A., Journal of Applied Phycology, 2004, 6, 127-133. Chanda, N.P., Matsuhiro, B. and Vasquez, A.E., Carbohydrate Polymers, 2001, 46, 81-87. Chhatbar, M., Meena, R., Prasad, K. and Siddhanta A.K., Carbohydrate Polymers, 2009, 76, 650-656. Cora, A.S., John, M.B., Heinz, G.F. and Robert, M.M., Carbohydrate Research, 1992, 225, 11-26. Cottrell, I.W. and Kovacs, P., Alginates. In: Crawford, H.B., Williams, J. (eds) Handbook of water soluble gums and resins, McGraw-Hill, Auckland, 1980, pp 21-43. Dea, I.C.M., The impact of biotechnology and advanced methodologies. In Industrial polysaccharides, Stivala, S.S., Crescenzi, V. and Dea, I.C.M. (eds), Gordon and Breach, New York, 1986, p 367.

46 Dentini, M., Rinaldi, G., Barbetta, A., Risica, D. and Skjak-Brk, G., Carbohydrate Polymers, 2006, 63, 519. Doshi, Y.A., Parekh, R.G., Chauhan, V.D. and Taqui Khan, M.M., Industrial carbohydrate conference, Ahmedabad, January 28-29: 286-302, 1984. Draget, K.I., Smidsrod, O. and Skjak-Braek, G., Alginates from algae. In: Steinbuchel, A., Rhee, S.K. (eds) Polysaccharides and polyamides in the food industry: properties, production and patents. Wiley, Weinheim, 2005, pp 1-30. Dubois, M., Gilles, k.A., Hamilton, J.K., Rebers, P.A. and Smith, F. Analytical Chemistry, 1956, 28,350. Ganesan, M., Mairh, O.P. and Subba Rao, P.V., Indian Journal of Marine Science, 2001, 30, 108-110. Grasdalen, H., Larsen, B. and Smidsrod, O., Carbohydrate Research, 1981, 89, 179-191. Haug A. and Larsen B., Acta Chemica Scandinavica, 1962, 16, 1908-1918. Haug, A., Larsen, B. and Smidsord, O., Acta Chemica Scandinavica, 1966, 20, 183-190. Haug, A., Larsen, B. and Smidsrod, O., Carbohydrate Research, 1974, 32, 217-225. Haug, A. and Smidsrod, O., Nature, 1967, 215, 757. Ji, M.C.W. and Lijun, H., Oceanologia et Limnologia Sinica, 1981, 12, 240-248. Knutson, C. A. and Jeanes, A. Analytical Biochemistry, 1968, 24, 470. Leal, D., Matsuhiro, B., Rossib, M. and Caruso, F., Carbohydrate Research, 2008, 343, 308316. Leroux, F., Gachon, J. and Besse, J-P., Journal of Solid State Chemistry, 2004, 1771, 245-250.

47 Li Bi, Ye., Jingsong, Z., Xi Jun, Ye., Qing Jeu, T.,Yan Fang, Lin., Chun Yu, G., Xin Jui, D.,Ying Jie, P., Carbohydrate Research, 2008, 343, 746. Lim, F. and Sun, A.M., Science, 1980, 210, 908-910. Littlecott, G.W., Food Technology of Australian, 1982, 34, 412-418. Marcotte, M., Hoshahili, R.A.T. and Ramaswamy, S.H., Food Research International, 2001, 34, 695703. Mathlouthi, M. and Koenig, J.L., Advances in Carbohydrate Chemistry & Biochemistry, 1986, 44, 766. Mody, I.C. and Chauhan, V.D., Research Industry, 1992, 37, 118-120. Morris, E.R., Rees, D.A. and Thom, D., Carbohydrate Research, 1980, 81, 305-314. Morris, E.R., Rees, D.A., Sanderson, G.R. and Thom, D., Journal of the Chemical Society Perkin Transactions, 1975, 2, 1418. Nishide E. H., Anzai N. U. and Kazutosi N., Hydrobiologia, 1996, 326/327, 515-518. Nishide E., Anzai H., Uchida N., Hydrobiologia, 1987, 151/152, 551555. Oza, R.M., Zaidi, S.H., A revised checklist of Indian marine algae, CSMCRI, Bhavnagar, Gujarat, India, 2001. Painter, T.J., Algal polysaccharies. In: Aspinall, G.O., (ed) The polysaccharides. Academic, New York, 1983, pp 196-286. Redekar, P.D. and Raje, P.C., Seaweed Research Utilization, 2000, 22, 4143. Sai Krishnamurthy, A., Chemical investigation of Indian marine algae with special reference to water soluble metabolites. Ph D Thesis,Bhavnagar University, 2000.

48 Sakugawa, K., Ikeda, A., Takemura, A., and Ono, H., Journal of Applied Polymer Science, 2004, 93, 1372-1377. Schimdt, E. and Vocke, F., Chemische Berichte, 1926, 59, 1585-1588. Silverstein, R.M., Clayton Bassier, G. and Morrill, T.C., Spectrometric Identification of Organic Compounds; Wiley: New York, 1991. Sime, W., Alginates in: harris, P., (ed) Food gels. Elsevier, London, 1990, pp 53-78. Smidsrod, O. and Skjak-Breaek, G., Trends in Biotechnology, 1990, 8, 71-78. Skjak-Braek, G., Grasdalen, H. and Larsen, B., Carbohydrate Research, 1986,154 239250. Sulit, J.I. and San Juan, R.G., Philipp. J. Fish, 1995, 3, 47. Tako, M., Yoza, E. and Tohma, S., Botanica Marina, 2000, 43, 393-398. Toft, K., Grasdalen, H. and Smidsrod, O., ACS Symposium Series, 1986, 310, 117-132. Torres, M.R., Sousa, A.P.A., Silva Filho, E.A.T., Melo, D.F., Feitosa, J.P.A., de Paula, R. C.M., and Lima, M.G.S., Carbohydrate Research, 2007, 342, 20672074. Tulchinsky, V.M., Zurabyan, Z.E., Asankoshoev, K.A., Kogan, G.A. and Khorlin, A.Y., Carbohydrate Research, 1976, 51, 18. Usov, A.I., Bilan, M.I. and Klochkova, N.G., Botanica Marina., 1995, 38, 43-51. www.algaebase.org Zhang, Z., Yu, G., Guan, H., Zhao, X., Du, Y. and Jiang, X., Carbohydrate Research, 2004, 339, 1475-1481.

49

L-Guluronic acid

D-Mannuronic acid

Figure I.1.1 Repeating units of MG block of alginate

Sodium alginate (Sargassum sp or Sigma)

Partial acid hydrolysis (0.3 N Oxalic/ 0.5 N H2SO4 for 4 min, MW)

Precipitate (alginic acid)

Filtrate

Precipitate dissolved in 0.1 M Na2CO3 pH 2.85, by 0.1 N HCL

Precipitate (Poly-guluronic acid, PGA) Precipitate (Poly-mannuronic acid, PMA)

Filtrate pH 1.0, by 0.1 N HCL

Filtrate

Figure I.1.2 Procedure of polymanuuronic acid (PMA) and polyguluronic acid (PGA) isolation from sodium alginate.

50

Figure I.1.3 GPC calibration curve of Dextran standards with different molecular weight

51

Figure I.1.4 Gel permeable chromatogram of Sigma alginate (a), STAL (b), SWAL (c), CIAL (d) and PTAL (e).

52

2163 2340 947

a
2932 1416 3436 2144 1610

1299 1092 1034

888 818 779 717

621

492 419

890 948 817 672 620 420

b
2928

2342 1304 1421

%T

3434

1622

1099 1031 891 949 817

c
2854 2923 3439

2358 1093 1032 1414 1626 2146 2365 1607 950 816 1413 2934 1093 1034 893

623

620 419

d
4000.0 3600 3200

2800

2400

2000

1800 1600 cm-1

1400

1200

1000

800

600

400.0

Figure I.1.5 FT-IR spectra of sodium alginate (a) STAL (b) SWAL (c) CIAL and (d) PTAL

53

13

C NMR in D2O; DMSO as internal standard

13

C NMR in D2O; DMSO as internal standard

13

C NMR in D2O; DMSO as internal standard

Figure I.1.6 13C NMR of sodium alginate, PMA and PGA of S. wightii

54

40 30 20 10

CD[mdeg]

0 -10 -20 -30 -40 -50 190

(SWAL) alginate Sigma alginate (Reference)

200

210

220

230

240

Wavelength(nm)
50 40 30 20

b
20

c
poly-mannuronic acid (PMA) SWAL
CD[mdeg]
PMA (Sigma alginate)

CD[medg]

10 0 -10 -20
-20

poly-guluronic acid (PGA) SWAL

PGA (Sigma alginate)

-30 -40 190 200 210 220 230 240


190 250 200 210 220 230 240 250

Wavelength(nm)

wavelngth(nm)

Figure I.1.7 Circular Dichroism (CD) of (a) Alginate (Sigma, using as reference and SWAL, prepared in this study), (b & c) PGA and PMA of Sigma and Sargassum (SWAL) alginates.

55

Sodium alginate

PGA

PMA

Figure I.1.8 Scanning electron microscopy (SEM) images of sodium alginate as well as PGA and PMA

56

(a)

(b)

(c)

Figure I.1.9 X-ray diffraction patterns of (a) sodium alginate (b) PMA and (c) PGA.

57

80

40000 35000

80

40000 35000

70

STAL
30000

SWAL
60 30000

Viscosity [mPa.s]

Viscosity [mPa.s]

60

Shear stress [mPa]

25000 20000 15000

25000 20000 15000 10000 20 5000 0 0 0 200 400 600 800 1000

Shear stress [mPa]

50

40

40 10000 30 5000 0 20 0 200 400 600 800 1000

Shear rate [1/s]

Shear rate [1/s]

40 35

10000

50 45

30000

CIAL
30

8000
40

PTAL

25000

20000

Shear stress [mPa]

Shear stress [mPa]

Viscosity [mPa.s]

25 20

6000

Viscosity [mPa.s]

35 15000 30 25 20 15 10000

4000 15 10 5 0 0 200 400 600 800 1000 2000

5000

0
10 100 200 300 400 500 600 700 800 900

0 1000 1100

Shear rate [1/s]

Shear rate [1/s]

Figure I.1.10 Effect of shear rate on the viscosity of STAL, SWAL, CIAL and PTAL solutions in 0.1M NaCl at 25oC.