Certificate of Guide and Candidateshodhganga.inflibnet.ac.in/bitstream/10603/9159/8/08_chapter...

58

Chapter I.2

Chemical studies on the polysaccharides of the

brown seaweed species Cystoseira indica,

Padina tetrastromatica and Sargassum tenerrimum

Cystoseira indica

Sargassum tenerrimum

Padina tetrastromatica

59

I.2.1 INTRODUCTION

I.2.2 MATERIALS AND METHODS

I.2.2.1 Collection of seaweed

I.2.2.2 Extraction

I.2.2.3 Fractionation and purification

I.2.2.4 General Methods and spectral analyses

I.2.2.5 Spectroscopic studies

I.2.3 RESULTS AND DISCUSSION

I.2.3.1 Physicochemical results

I.2.3.2 Spectral data

I.2.3.3 Molecular weight determination (GPC)

I.2.3.4 Gas chromatography-mass spectrometry

I.2.3.5 Linkage analysis of CFCIsps

I.2.3.6 Linkage analysis of CFPTsps

I.2.3.7 Linkage analysis of CFSTsps

I.2.4 SUMMARY

I.2.5 REFERENCES

2 Chemical studies on the polysaccharides of the brown

seaweed species Cystoseira indica, Padina tetrastromatica and


60

I.2.1 INTRODUCTION

Sulphated fucans, polysaccharides containing substantial percentages of L-fucose and

sulphated ester groups, are constituents of brown algae and some marine invertebrates

(Painter, 1983; Vilela-Silva et al. 2002; Berteau & Mulloy, 2003). They consist of

(1→3)- and/or (1→4)-linked fucosyl backbones that are partially substituted at C-2

and/or C-4 with sulphate groups and fucosyl residues (Patankar et al. 1993; Chevelot

et al. 1999; Kariya et al. 2004). Fucoidans have been extensively studied due to their

diverse biological activities, since they are potent anticoagulant (Nagumo and

Nishino, 1996; Chaubet et al. 2000), antitumor (Itoh et al. 1993) and antiviral agents

(Baba et al. 1988; McClure et al. 1992). Fairly wide varieties of bioactivities have

been reviewed from the brown algal polysaccharides (Siddhanta & Sai

Krishnamurthy, 2001). Commercially important gelling seaweed polysaccharides e.g.

agar, carrageenan and alginates, as well as various bioactive ones such as blood

anticoagulant, anti-viral, anti-cancer etc. have been reviewed

(Siddhanta, 1999;

Siddhanta et al. 2005; Meena and Siddhanta, 2006; Siddhanta et al. 2006). Structural

studies on fucoidans from the brown seaweed Sargassum stenophyllum, is reported by

Duarte et al. (2001). Fucoidan having backbone built up of alternating (1→3) and

(1→4) linked α-L-fucopyranose residues occurred commonly in both the brown

seaweed species Ascophyllum nodosum and Fucus evanescens reported by Chevolot

et al. (2001) and Bilan et al. (2002) respectively. Chemical modifications of these

fucoidans have not been reported so far, which are central theme of this work.

All seaweed species are abundantly available in Indian waters, Cystoseira indica

belonging to the Division-Phaeophyta (Class-Phaeophyceae, Order-Fucales, Family-

Cystoseiraceae, Genus-Cystoseira, and Species-indica), Padina tetrastromatica

belonging to the phylum-Phaeophyta (Class-Phaeophyceae, Order-Dictyotales,

Family- Dictyotaceae, Genus-Padina, Species-tetrastromatica) and Sargassum

tenerrimum belonging to the Division-Phaeophyta (Class-Phaeophyceae, Order-

Fucales, Family- Sargassaceae, Genus-Sargassum and Species-tenerrimum)

(www.algaebase.org). Structural features and antiviral activity of sulphated fucans

from the brown seaweeds Cystoseira indica and Padina tetrastromatica have been

reported by (Mandal et al. 2007 and Karmakar et al. 2009). The latter reports came

out very recently from the same research group, when the work described in this

dissertation on this polysaccharide was completed. Chemical studies on the sulphated

polysaccharide of Sargassum tenerrimum has not been reported in the literature.

Sulphated polysaccharide of Sargassum tenerrimum consist of 4-linked, 3,4-linked,

2,3-linked and 2,3,4-linked fucose; 3,4-inked and 2,3,4,6-linked galactose residues

61

(GC-MS,13

C NMR). Structure of the basic units of this polysaccharide (CFSTsps) is

depicted in Figure I.2.1.

Therefore, it was decided to carry out detailed chemical studies on polysaccharides of

brown seaweed Cystoseira indica, Padina tetrastromatica and Sargassum tenerrimum

of Indian waters. In this chapter I.2 structural features and physico-chemical

properties of the sulphated polysaccharides of these brown seaweed species have been

described.

I.2.2 MATERIALS AND METHODS

I.2.2.1 Collection of seaweed

Cystoseira indica (AL-II-114-03) used in this study was collected in January, 2008

from Diu (20o 42.727’ N, 70

o 55.487

’ E), the inter-tidal zone of West Coast of India,

Padina tetrastromatica (AL-II-120-05 and AL-II-129-06) was collected during

January-February 2008 from Okha (22o 28.580’ N, 69

o 04.254

’ E) from the inter-tidal

zone in the west coast and Valai island (09o 10.445’ N, 78

o 55.55

’ E) from the inter-

tidal zone in the south east coast of India and Sargassum tenerrimum (AL-II-114-03)

was collected during March 2008 to April 2008 from Veraval (20o 54.875’ N, 70

o

20.832’ E), Diu (20

o 42.727’ N, 70

o 55.487

’ E), from the inter-tidal zone in the west

coast of India (Oza and Zaidi, 2001, www.algaebase.org).

Collection of seaweed sample was made by hand picking during low tide. The plants

were pulled out from their attachment. After the collection, the seaweed was washed

with clear sea water to get rid of mud, dirt and sand from the samples. Freshly

collected sample was dried in the shade and preserved at ambient temperature. The

algal sample was powdered in the rotating boll mill prior to the extraction. Safety

precautions have been taken during the collection of seaweeds. Specimen samples

have been deposited with the CSMCRI Herbarium, Bhavnagar, India for referencing.

Cystoseira indica



62

I.2.2.2 Extraction

Cold and hot water soluble sulphated polysaccharides were extracted from each

seaweed species following the standard method reported by Siddhanta et al. (2001).

Cold water extraction

Dried algal powder was first depigmented by repeated extractions with methanol in a

percolator. For the Cold water extract (CWE), depigmented dried algal powder (50g)

was soaked overnight in 20 volumes (w/v) of DM water at 10oC. CWE was filtered

through muslin cloth followed by vacuum filtration over Celite bed on a Buchner

funnel. CWE obtained after filtration was again clarified by centrifugation at 8000

rpm for 15 minute to obtain clear brown colored supernatant which was concentrated

up to 1/3 of its volume on a rotavapor under reduced pressure. Concentrated CWE

was precipitated with isopropyl alcohol (CWE: IPA - 1:2 v/v) and precipitate was

recovered by centrifugation. Precipitated CWE was dissolved in minimum volume of

distilled water and dialyzed (Sigma dialysis tubing, molecular weight cut off (MWCO

12000 Dalton) against tap water for 24h and then against distilled water till it became

chloride free (AgNO3 test). Salt-free CWE was lyophilized (VirTis Freeze Dryer,

USA) to yield dried crude CWE containing sulphated polysaccharide (cf. Siddhanta et

al. 2001). The yield of crude CWE was ca. 4.0% with respect to as received dried

seaweed.

Hot water extraction

An algal residue obtained after the cold water extraction was further extracted with

1000 ml of DM water at 80oC for 3 h. (x2) on a water bath. Hot water extract (HWE)

was filtered through muslin cloth followed by vacuum filtration over Celite bed on a

Buchner funnel. HWE was obtained following the method described above (for

CWE). The yield of crude HWE was ca. 6.0% with respect to as received dried

seaweed.

I.2.2.3 Fractionation and purification

Charged and neutral polysaccharides were separated from hot water extract (HWE) of

brown algae Cystoseira indica (CIsps) and Padina tetrastromatica (PTsps) following

the method described by Sen et al. (2002).

Dried crude polysaccharides 1.0g was dissolved in 100ml of water and treated with

4% cetyltriammonium bromide (CTAB) containing 0.01% sodium sulphate, light

brown colored precipitates of the cetyltriammonium (CTA) salt of the sulphated

63

polysaccharide was obtained. The treated polysaccharide solution was kept overnight

to achieve complete precipitation of charged polysaccharides. However, while a major

part of the sulphated polysaccharide precipitated and filtered, possibly neutral fraction

would in the filtrate along with excess CTAB. Neutral fraction was recovered by

precipitation of excess CTAB with NaI followed by filtration. Neutral fraction was

precipitated with IPA (Filtrate: IPA-1:2 v/v). Precipitated neutral fraction was

dissolved in minimum amount of distilled water and lyophilized. CTA salt of

sulphated polysaccharides was dissolved in 400ml. of 4M NaCl by stirring at ambient

temperature for 36h. After the dissolution of the CTA salt, the CTAB was removed by

repeated extraction with n-butyl alcohol in a separating funnel. Aqueous layer was

concentrated up to ¼ of its volume on a rotavapor and dialyzed against tap water, and

finally against distilled water for the complete removal of salts. After dialysis,

aqueous layer was concentrated up to ½ of its volume and the charged fraction was

precipitated with IPA (1:2 v/v), which was dissolved in minimum amount of distilled

water and lyophilized.

Preparation of DEAE cellulose ion exchange column

10g of diethyl amino ethyl (DEAE) cellulose (Cl- form) (DE 11, Sisco Research

Laboratory Pvt. Ltd. Mumbai, India) was soaked in 100 ml of distilled water and left

overnight and filtered through nylon cloth. Then it was suspended in 0.5M HCl

(100ml) and deaerated with stirring under vacuum for about 20 minute and then

without vacuum for another 20 minute. The DEAE cellulose was filtered off and

washed with distilled water to make it acid free and thereafter suspended in 0.5M

NaOH (100ml). The alkaline suspension was treated in the same way as it was done

with the acid suspension. These operations were repeated twice. The treated DEAE

cellulose was loaded in the column (15 x 2 cm), equilibrated with 0.5M NaCl

solution, washed with distilled water till chloride free and then it was used after

equilibration with the appropriate eluent.

Anion exchange chromatography

Anion exchange chromatography of crude STsps sample obtained from HWE of

Sargassum tenerriumum was carried out on a DEAE cellulose (chloride form) column

(15 x 2 cm) prepared as above. Elution of the column was done with a stepwise

gradient of aqueous NaCl solution ranging from 0.0 to 2M at 25oC at a flow rate of 25

ml/h. Elution of the sulphated polysaccharide (SPS) from column was monitored by

measuring the sugar concentration by phenol-sulphuric acid method (Dubois et al.,

1956). Fractions were collected and concentrated on a Buchi rotavapor and the

64

concentrate was dialyzed against tap water (12h) using Sigma dialysis tubing (D-

0655; MWCO 12,000 Dalton) and then against distilled water (24h) to remove SPS

having molecular weight lesser than 12,000 Dalton. Dialyzed product was freeze-

dried. The major product was obtained from 0.5M and 1M NaCl elutes. The 1.5 and

2.0M elutes did not contain any dissolved polysaccharide residue.

I.2.2.4 General Methods and spectral analysis

Estimation of total sugar

Total sugar was estimated by Dubois method (Dubois et al. 1956) using glucose as

standard (Range 10- 100µg). 8-10mg of sample was dissolved in 10ml of distilled

water and used.

Reagents: 1. Glucose solution (AR), 50µg/ml; 2. Concentrated H2SO4 (AR); 3. 5%

phenol (AR) solution (in distilled water).

Method: Sample aliquots were taken up to 2ml (100µg) and volume of each aliquot

was adjusted to 2ml with distilled water. 1ml of 5% phenol solution was added to

each aliquot followed by rapid addition of 5ml H2SO4, in ice- bath. In blank, the

sample aliquot was replaced by 2ml of water. All the tubes containing sample and

blank solutions were incubated at room temperature for 30 minute. UV absorption

was measured at 485 nm. A standard curve was used for a given set of reagents.

Estimation of uronic acid

Uronic acid content was estimated by Knutson and Jeanes method (1968).

Galacturonic acid (sigma) was used as a standard. Rang of the method is 10-50µg.

SPS sample 10mg was hydrolyzed with 5ml of 1N H2SO4 at 100oC for 12h,

hydrolyzate was centrifuged at 5000 rpm for 20 minute and supernatant was used.

Reagents: 1. Concentrated H2SO4 (AR); 2. Borate stock solution ( 24.74g of boric

acid was dissolved in 45ml of 4.0M KOH and the volume was made to 100ml with

distilled water, therefore, the final concentration of borate was 4.0M); 3. Sulphuric

acid:borate reagent (1.0ml of borate solution was mixed with 40ml of concentrated

H2SO4); 4. Carbazole solution: 0.1% carbazole in ethanol was prepared and 5.

Galacturonic acid (AR) solution: 100µg/ml was prepared.

65

Method: Sample aliquots were added up to 0.7ml in 6ml of sulphuric acid:borate

reagent in ice bath and volume was adjusted with distilled water to 6.7ml, shaken well

and incubated in ice bath for 10 minute. Carbazole solution 0.2ml was added (green

color developed) and warmed up at 55oC in water bath for 30 minute (color changed

green to pink). Each aliquot had its own blank; both experiments and blanks had same

volume of sample aliquots, but in blank sample, distilled water was added instead of

hydrolysate sample. Standard curve was obtained by using the same set of aliquots for

which blank was prepared with distilled water mixing with 0.2ml carbazole. The

reading was taken at 530 nm. Varian CARY 500 Scan, UV-visible spectrophotometer

instrument was used for the above mentioned estimations.

Estimation of sulphate

Sulphate content of the SPS sample was estimated by turbidimetric method (Dodgson

et al. 1962) using K2SO4 as a standard. Range of the method is 20-200µg. SPS sample

was hydrolyzed in 1.0N HCl at 110oC for 17h in a sealed tube, hydrolysate was

centrifuges at 5000 rpm for 20 minute and supernatant was used for analysis.

Reagents: 1. Stock solution: 360mg K2SO4 in 100ml distilled water (working

solution: 1:2 dilution of stock solution gives approximately 99µg of sulphate per

0.1ml); 2. Barium chloride-Gelatin solution: 100ml of 0.5% gelatin solution was

prepared and kept at 4oC overnight and 0.5g of BaCl2 was added to that and allowed

to stand for 3-4h before use; 3. 4% trichloroacetic acid (TCA) in distilled water.

Method

Aliquots of K2SO4 working solution were taken up-to 0.2ml and volume of each

aliquot was adjusted with distilled water to 0.2ml, 3.8ml of 4% TCA was added to

each aliquot followed by addition of 1ml BaCl2-gelatin solution. Blank was prepared

in the same way replacing K2SO4 solution with 0.2ml distilled water. After incubation

for 15-20 minute at room temperature, UV absorbency was measured at 360 nm. In

the first set of experiment (A), each experimental solution was prepared with

particular aliquots of SPS samples and volume was made up to 0.2ml with 1N HCl

and TCA (3.8ml) was added followed by gelatin-BaCl2 solution (1ml). The UV

absorbency was measured (A) at 360 nm against a blank solution containing 1N HCl

(0.2ml), TCA (3.8ml) and gelatin- BaCl2 solution (1ml). In the second set of

experiment (B), each experiment solution was prepared with same sample aliquots

volume which was used for set ‘A’ experiment and volume was made up to 0.2ml

with 1N HCl and addition of 3.8ml TCA followed by 1ml of gelatin solution. The UV

66

absorbencies were measured (B) against a blank solution containing 1N HCl (0.2ml),

TCA (3.8ml) and gelatin solution (1ml) absorption due to sulphate is equal to (A-B),

B denotes the absorption quantum arising out of the UV- active components in the

hydrolyzed SPS samples other than sulphate.

Moisture

Moisture contents of SPS samples were considered as the losses in mass from a

sample (1g) after drying at 100oC 2

oC (Moisture analyzer SARTORIUS A G

GOTTINGEN MA30-000V3, 12201154, GERMANY)

Determination of ash content

Sulphated polysaccharide (SPS) samples were ignited at (800o 10

oC for 6h) and

percentage of ash contents was calculated based on the weights of the oven dried SPS

samples.

Metal analysis

Metal ion analyses were carried out after ignition of known weight of SPS and

resultant ash was digested using acid solution. Volume of digested samples adjusted

up to 100ml with distilled water. Metal ions (Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Na, Ni,

Pb, As, B, Zn) were measured by inductively coupled plasma (ICP)

spectrophotometry on a Perkin-Elmer ICP-OES Optima 2000DV machine, following

the method described by Wolnik (1988).

Estimation of Nitrogen

Total Nitrogen (N) was estimated by Kjeldahl method on a KEL PLUS-KES 20l

Digestion unit attached to KEL PLUS- CLASSIC DX Distillation unit (M/s

PELICAN Equipments, Chennai, India).

Specific rotation

Optical rotations were measured for SPS (0.25% (0.250g/100ml at 30oC used

wavelength 589 nm) on a Rudolph Digi pol-781 Polarimeter (Rudolph Instruments

Inc, NJ, USA).

Viscosity

67

Apparent viscosity of SPS (1% in DM water) was measured using a Brookfield

Viscometer (DV-II +Pro) at 27oC. Spindle SC4-18 was used for apparent viscosity

measurement at speed of 60 rpm.

Molecular weight determination (GPC)

Same method has been followed as described in section (I.1.2.3.4) for the

determination of molecular weights (Mn, Mw and Mz) of the charge fraction of CIsps,

PTsps and STsps. The molecular weights of standards and samples were determined by

GPC according to the method described by Li et al. (2008).

Preparation of IR-120 ion (cation) exchange column

A 20ml volume of IR-120 resin was taken in a glass column (length of resin column

20cm, ID 1.3cm) and soaked in distilled water overnight. For charging of the resin

material, 100 ml 0.1N NaOH was passed through the column (elution rate 5ml/min.).

Column was washed with distilled water till it become alkali free and then 100ml of

0.1N HCl was passed through the column at the same rate stated above. The column

was washed with distilled water till it become acid free. The whole process was

repeated thrice to ensure the complete conversion of resin in to H+ form. After use,

the column was regenerated by washing with 80ml 0.1N HCl and rate was adjusted so

that all volume was eluted in 20 minute and again column was washed with distilled

water till acid free and used for next sample.

Preparation of alditol acetate of polysaccharide

Alditol acetates of SPS were prepared by following the method of Siddhanta et al.

(2001). Polysaccharide sample (50mg) was hydrolyzed with 5ml of 2M H2SO4 at

100oC for 6h in a sealed tube (Usov et al. 1983). The hydrolysate was centrifuged and

neutralized with BaCO3 after adding the 25ml DM water in to the supernatant. The

neutralized solution was filtered and evaporated up to 5-10ml under reduced pressure,

a pinch of NaBH4 was added and kept at room temperature for 6h for the reduction.

The reduced solution was passed through the IR-120 (H+) column to remove sodium

ion of NaBH4. The column was eluted with twice the bed volume of the column with

distilled water to ensure complete elution and elute was co-distilled with methanol to

remove the borate present in the solution and finally evaporated to dryness to yield

alditol. Alditol was dried over blue silica in a vacuum desiccator and acetylated with

pyridine/acetic anhydride (1:1) at 100oC for 20 minute. The reaction mixture was

poured in to the ice-water and extracted with ethyl acetate. The extract was washed

68

with distilled water, saturated solution of Na2CO3 and CuSO4 to remove excess acetic

acid and pyridine respectively, dried over anhydrous Na2SO4 and then evaporated to

dryness. The residue was dissolved in dichloromethane and subjected to GC-MS

analysis.

Gas chromatography-Mass spectroscopy (GC-MS) method for carbohydrate profiling

GC-MS analysis of the alditol acetates of sulphated polysaccharide samples (crude,

charged and neutral fractions) as well as standard sugar samples were carried out on a

Shimadzu GC-MS-QP2010 machine, using a SGE BP-225 capillary column (25 m,

0.25µm, 0.22mm), employing temperature programming (160oC to 230

oC @10

oC per

min) using helium as a carrier gas at a constant flow rate of 1ml/min. The electron

impact (EI) mass-spectra were recorded at 70 eV. The retention time and mass spectra

of sulphated polysaccharide samples (crude, charged and neutral fractions) were

compared with those of alditol acetate of individual standard sugars (Figure I.2.9 to

Figure I.2.19).

Methylation of polysaccharide

The SPS (100mg) was suspended in round bottom flask (RBF, tightly fitted with

rubber septum) containing 15ml of dried dimethyl sulphoxide (DMSO) and 1.0g

powdered NaOH at 5 to 10oC under stirring. Methyl iodide (CH3I) was added in three

parts (1ml x 3) each interval of 30 min. and reaction mixture was stirred for over night

to achieve greatest methylation of polysaccharide sample. After that, the nitrogen gas

stream was purged into the reaction mixture to remove unreacted methyl iodide. The

reaction mixture was then dialyzed against tap water and followed by DM water to

remove solvent and unreacted reagents. After dialysis, the product was freeze dried

and used for the subsequent preparation of partially methylated alditol acetates

(PMAA) as described in above method. PMAA was dissolved in dichloromethane

(CH2Cl2) and subjected to GC-MS analysis for glycosidic linkage study.

GC-MS method for partially methylated alditol acetate (PMAA)

GC-MS analysis of the partially methylated alditol acetate of SPS sample was carried

out on a Shimadzu GC-MS-QP2010 machine, using a SGE BP-225 capillary column

(25m, 0.25µm, 0.22mm), employing temperature programming [50oC (hold for 2min.)

to 215oC @40

oC per min.] using helium as a carrier gas at a constant flow rate of 1

ml/min. The electron impact (EI) mass-spectra were recorded at 70 eV. The PMAA

69

products of SPS were characterized by GC-MS on the basis of their mass

fragmentation patterns and retention times (Figure I.2.20 to Figure I.2.22).

Linkage analysis of SPS

Partially methylated alditol acetate (PMAA) sample of SPS was prepared according to

the method reported by Ciucanu et al. (1984) and the product was characterized by

GC-MS. The mass fragmentation patterns of the PMAA of SPS sample were

compared and validated with that of PMAA of individual standard sugars provided by

CCRC (Complex Carbohydrate Research Centre) data bank (www.ccrc.uga.edu) as

well as the ones reported by Sassaki et al. (2005), Mandal et al. (2007). The sugar

residues in the sample were identified by comparing the relative retention time with

that of the respective standard sugar alditol acetate as well as by comparison of their

mass fragmentation pattern (www.ccrc.uga.edu). This led to the conclusion that the

sugars present are pyranose, as furanose structure could not explain the mass

fragmentation patterns.

I.2.2.5 Spectroscopic studies

Infrared spectroscopy

Infrared spectra of the fractions of SPS were recorded on FT-IR using a Perkin-Elmer

Spectrum GX FT-IR system, by taking 10.0mg of sample in 600mg KBr. All spectra

were average of two counts with 10 scans each and a resolution of 5cm-1

.

13

C NMR spectroscopy

Noise-decoupled 13

C NMR spectra were recorded on a Bruker Avance-II 500 (Ultra

shield) Spectrometer, Switzerland, at 125 MHz. SPS sample was dissolved in D2O

(60mg/ml) and the spectra was recorded at 70oC, using DMSO as internal standard

(ca. δ 39.5ppm).

I.2.3 RESULTS AND DISCUSSION

I.2.3.1 Physicochemical results

The polysaccharides (crude HWEs) of Cystoseira indica (CIsps), Padina

tetrastromatica (PTsps) and Sargassum tenerrimum (STsps) were fractionated as

described above in Section I.2.2.3. The charged (CF) and neutral (NF) fractions

obtained from the crude HWEs were freeze dried and the products were isolated and

characterized. The respective fractions CFCIsps, CFPTsps and CFSTsps were major in

70

quantity (5.4%, 5.8% and 3.8% respectively), and therefore, these were selected for

physicochemical analyses e.g. total sugar, sulphate, uronic acid contents, protein,

viscosity, ash contents, moisture were determined. The results along with those of the

CWEs are given in Table I.2.1. The sulphate content viscosity and total sugar of the

respective charged fractions were greater than those of CWEs (Table I.2.1). The

uronic acid was found higher in CWEs of CIsps (4.2%) and STsps (4.5%) than those of

the charged fractions (CFCIsps 1.4% and CFSTsps 0.5%). The reverse was the case

with PTsps, i.e., 2.8% (CWE) vs 4.5% (CFPTsps) (Table I.2.1) [vide 13

C NMR data

below exhibiting carbonyl group at 176.0 ppm in CFPTsps]. Although, this sort of

anomaly cannot be explained at present, this observation would be of great

biosynthetic significance in the polysaccharide biochemistry in plants. The CFCIsps,

CFPTsps and CFSTsps had negative specific rotation of varied quanta [α]D27

-13.102o, -

59.24o and -48.24

o respectively (c 0.25%, H2O, 27

oC).

Table I.2.1 Yield and physicochemical data of sulphated polysaccharides (SPS)

Extracts Yield*

(%)

Moisture

(%)

Total

sugar

(%)

Uronic

acid

(%)

N

(%)

Proteina

(%)

Ash

(%)

Sulphate

(%)

Viscosityb

(cP)

Cystoseria indica

CWE CIsps 4.0 9.3 38.6 4.2 0.49 3.06 5.2 4.2 1.8

HWE CIsps 6.2 11.4 ND ND ND ND ND ND ND

CFCIsps 5.4 10.6 42.8 1.4 0.3 1.87 11.3 11.5 2.3


CWE PTsps 5.0 9.5 35.8 2.8 0.23 1.43 6.3 5.2 1.7

HWE PTsps 6.5 10.4 ND ND ND ND ND ND ND

CFPTsps 5.8 11.6 38.6 4.5 0.4 2.5 8.7 11.3 2.0


CWE STsps 5.0 8.5 36.7 4.5 0.35 2.18 6.3 4.6 1.7

HWE STsps 7.5 12.2 ND ND ND ND ND ND ND

CFSTsps

(1M NaCl

fraction)

3.8 7.6 40.5 0.5 0.24 1.5 7.5 14.3 2.4

*Yield was calculated on the basis of as received dry weight of seaweed; other constituents in % weight of

respective sulphated polysaccharides; a

values of protein contents were calculated multiplying the estimated N2

content values with the factor 6.25 (cf; Marks et al. 1985); b

Viscosity was measured in 1% conc. at 27oC ; ND=

Not determined.

Metal analysis

Metal ion contents of SPS were measured by inductively coupled plasma (ICP)

spectrophotometry and the results are presented in Table I.2.2. The absence/negligible

content of some prominent toxic metal ions e.g. Cd, Pb, Cr and As in these

polysaccharides suggested that the polysaccharide would be suitable for ingestible

applications.

71

Table I.2.2 Metal ion contents in the of SPS

Elements CFCIsps (ppm) CFPTsps (ppm) CFSTsps (ppm)

B 0.352 0.335 0.371

Na 6.242 4.242 3.542

Mg 5.024 9.024 6.024

K 30.94 28.94 30.55

Ca 4.471 9.471 7.481

Cr Nil 0.004 Nil

Mn 0.038 0.038 0.042

Fe 0.735 0.735 0.835

Co Nil Nil Nil

Ni 0.008 Nil Nil

Cu 0.016 0.016 0.013

Zn 0.056 0.210 0.240

As Nil Nil Nil

Pb Nil Nil Nil

Cd Nil Nil Nil

I.2.3.2 Spectral data

FT-IR spectroscopy

FT-IR spectra of HWE CIsps, CFCIsps, and the neutral fraction NFCIsps, HWE PTsps,

CFPTsps, and the neutral fraction NFPTsps, HWE STsps, CFSTsps and the neutral

fraction NFSTsps are depicted in the Figure Nos. I.2.2a-c, I.2.3a-c and I.2.4a-c,

respectively. The prominent bands appeared in the range, υmax (KBr) (cm-1

): 3411-3494

(O-H str, br, s), 2923-2935 (C-H str, w), 1615-1641 (bound H2O, s), 1417-1462 (C-H

bending, w), 1250-1263 (>S=O str, s), 1000-1100 (C-O-C str, s) (cf. Lloyd et al.

1961; Turvey & Williams 1962; Hirst et al. 1965; Mollet et al. 1998). The additional

sulphate absorption bands at 845-850 cm−1

(C-O-S, secondary axial sulfate position at

C-4 of the fucopyranose residue. (cf. Patankar et al. 1993; Chizhov et al. 1999; Duarte

et al. 2001; Bilan et al. 2002) [br = broad, s = strong, m = medium, w = weak, str =

stretching]. FT-IR spectra of the charged fractions showed the absorption band of

sulphate esters in the range 1250-1263 cm-1

while the neutral fraction did not have

sulphate ester band. The presence of sulphate esters band in charged fraction (Figures

72

I.2.2b, I.2.3b and I.2.4b) and absence in neutral fraction (Figures I.2.2c, I.2.3c and

I.2.4c) indicated that charged and neutral fraction was successfully separated.

13

C-NMR spectroscopy

The assignments of carbon-13 chemical shifts (Figures I.2.5 to I.2.7) observed were

done on the basis of comparison with those reported in the literature. The δ values

that were reported for sugar residues having different linkage patterns in the sulphated

polysaccharides of brown seaweeds. The anomeric carbons of the major sugar

residues in CFCIsps viz. fucopyranose, xylose and ribose moieties, appeared at 102.49,

104.68 and 106.11 ppm, respectively (Table I.2.3 and Figure I.2.5). In CFPTsps the

anomeric carbons of α-L-fucopyranose, galactose, and xylose moieties appeared at

101.54, 103.85 and 102.73 ppm respectively (Table I.2.4 and Figure I.2.6), while in

CFSTsps those of fucopyranose and galactopyranose moieties appeared at 100.41 and

103.91 ppm, respectively (Table I.2.5 and Figure I.2.7). Assignments of chemical

shifts were found to be in good agreement with linkage patterns that were deduced by

GC-MS. These conclusions fitted well on the pyranose structures of sugar residues in

the biosynthesized copolymer by this brown seaweed species (cf. Chizhov et al. 1999;

Chevolot et al. 2001; Duarte et al. 2001; Bilan et al. 2002; Li et al. 2006).

Table I.2.3 13

C NMR shift, observed for CFCIsps recorded in D2O with DMSO as

internal standard

No. Sugar residues 13

C NMR chemical shifts

Assigned carbon

(δ values in ppm)

1 →4)-Fucp (1→, →3)- Fucp (1→, →2)- Fucp (1→,

→3,4)-Fucp (1→, →2,3)- Fucp (1→,

→2,3,4)-Fucp (1→

C-1(102.06), C-5(67.92),

C-6(16.5)

2 →2)-Fucp (1→, →2,3)-Fucp (1→ C-2(75.56)

3 →3)- Fucp (1→, →3,4)-Fucp (1→,

→2,3)-Fucp (1→

C-3(77.17)

4 →4)-Fucp (1→, →3,4)-Fucp (1→ C-4(81.98)

4 →3)-Xylp (1→, →2,3,4)-Xylp (1→ C-1(104.02), C-5(69.95),

C-6(66.34)

6 →2,3,4)-Xylp (1→ C-2(74.19), C-4(83.64)

7 →2,3,4)-Xylp (1→, →3)-Xylp (1→ C-3 (77.78),

9 D-Ribp (1→, →2,4)-Ribp (1→

→2,3,4)-Galp (1→

C-1(105.68), C-5(70.67),

C-6(66.34), C-2(73.15),

C-4(79.45),

10 →6)-Galp (1→ C-6 (62.02)

73

Table I.2.4 13

C NMR shift, observed for CFPTsps recorded in D2O with DMSO as

internal standard



Assigned carbon ( δ values

CFPTsps, in ppm)

1 →4)- L-Fucp (1→, →2)- L-Fucp (1→,

→3,4)- L-Fucp (1→, →2,3)- L-Fucp (1→,

→2,3,4)- L-Fucp (1→

C-1(101.54), C-5 (66.49),

C-6 (16.69)

2 →2)- L-Fucp (1→

→2,3)- L-Fucp (1→, →2,3,4)- L-Fucp (1→

C-2 (75.24)

3 →3,4)- L-Fucp (1→

→2,3)- L-Fucp (1→

C-3 (77.54)

4 →4)- L-Fucp (1→

→3,4)- L-Fucp (1→

C-4 (82.48)

5 →4)-D-Galp (1→, →3,4)- D-Galp (1→,

→2,3,4)- D-Galp (1→, →3,4)- D-Manp (1→,

→2,3,4,6)-D-Manp (1→

C-4 (79.48)

6 →3,4)- D-Galp (1→,→3,4)- D-Manp (1→,

→3,6)- D-Manp (1→,→2,3,4)- D-Galp (1→,

→3,4,6)- D-Galp (1→,→2,3,6)- D-Galp (1→

C-3 (82.48)

7 →4)- D-Galp (1→,→3,4)- D-Galp (1→,

→3,4)- D-Manp (1→,→3,6)- D-Manp (1→,

→2,3,4)- D-Galp (1→,→3,4,6)- D-Galp (1→,

→2,3,6)- D-Galp (1→, →2,3,4,6)-D-Manp (1→

C-1 (103.85), C-5 (74.31)

8 D-Xylp (1→, →3)-D-Xylp (1→

→3,4)- D-Manp (1→, →2,3,4,6)-D-Manp (1→

C-1(102.73), C-5 (72.92)

9 →4)- D-Galp (1→

C-2 (71.47)

10 → 3,6)- D-Manp (1→, →4,6)-D-Manp (1→

C-6 (70.45)

11 →4)- D-Galp (1→, →6)- D-Galp (1→

→4)- D-GlcpA (1→, →4)- D-Manp (1→

C-3 (72.53)

12 →6)- D-Galp (1→

→ 3,6)-D-Manp (1→

C-4 (68.25)

13 →6)- D-Galp (1→, →4)-D-Galp (1→,

→2,3,4)- D-Galp (1→, →3,4,6)- D-Galp (1→,

→2,3,6)- D-Galp (1→

C-6 (62.28)

14 Carboxyl carbon of uronic acid

176.0

74

Table I.2.5 13

C NMR shift, observed for CFSTsps recorded in D2O with DMSO as

internal standard



Assigned carbon

( δ values CFSTsps, in ppm)

1 →4)- L-Fucp (1→, →3,4)- L-Fucp (1→,

→2,3)- L-Fucp (1→, →2,3,4)- L-Fucp(1→

C-1(100.42), C-5(69.53),

C-6(17.37)

2 →2,3)- L-Fucp (1→

→2,3,4)- L-Fucp(1→

C-2(74.67)

3 →3,4)- L-Fucp (1→

→2,3)- L-Fucp (1→

C-3(77.37)

5 →4)- L-Fucp (1→

→3,4)- L-Fucp (1→

C-4(79.25)

7 →3,4)- D-Galp (1→,

→2,3,4,6)- D-Galp (1→

C-1 (103.91), C-5(74.67)

8 →2,3,4,6)- D-Galp (1→ C-2(71.72), C-6(62.64)

9 →3,4)- D-Galp (1→ C-3(86.36), C-4(79.25)

I.2.3.3 Molecular weight determination (GPC)

The gel permeable chromatograms of CFCIsps, CFPTsps and CFSTsps are depicted in

Figure I.2.8a-c. The molecular weights (Mn, Mw, Mp and MZ) and polydispersity are

given in Table I.2.6. The high polydispersity indices indicated the highly branched,

non-homogeneous structure for the polysaccharides unlike synthetic polymers.

Table I.2.6 Gel permeation chromatographic data of SPS

I.2.3.4 Gas chromatography-Mass spectrometry

The retention times (in minutes) of alditol acetates of standard sugars were: rhamnitol

acetate 10.40; fucitol acetate 10.54; ribitol acetate 11.03; arabinitol acetate 11.26;

xylitol acetate 12.09; mannitol acetate 15.71; galactitol acetate 16.38; glucitol acetate

16.98 (Figure I.2.9). The sugar residues in the SPS samples (crude, charged and

neutral fractions) were identified by comparing the relative retention time with that of

Samples R. Time

(Min.)

Molecular weights (Da) Poly

dispersity Mn Mw Mp Mz

CFCIsps 24.97 88160 320167 225522 759154 3.63

CFPTsps 24.27 160399 710214 353234 1839807 4.428

CFSTsps 24.47 107538 355526 305551 769948 3.30

75

the respective standard sugar alditol acetates as well as by comparison of their mass

fragmentation patterns (Figure I.2.10 to Figure I.2.19).

Natural sugar composition of the HWE CIsps, and its charged (≥95%) and neutral

(≤5%) fractions were determined by comparing the GC-MS profile of the alditol

acetates (Figure I.2.10, Figure I.2.11 and Figure I.2.12) with those of the standard

sugars (Figure I.2.9). The HWE CIsps contained six monosaccharide units and charged

fraction (CFCIsps) contained the same set of five monosaccharide units except

arabinose, while the neutral fraction (NFCIsps) contained six units (Table I.2.7) (cf.

Siddhanta et al., 2001). Xylose was not found in neutral fraction, on the other hand

the charged fraction did not contain arabinose and mannose (Table I.2.7). This finding

indicated that fucose, xylose and galactose sugar residues would be sulphated in CIsps

charged fraction structure. Mandal et al, (2007) described the crude hot water extract

of Cystoseira indica containing fucose in major amount with minor presence of

galactose, xylose, mannose and glucose. However, in the present investigation

fucose, ribose, arabinose, xylose, mannose and galactose units were detected in the

crude polysaccharide sample.

Natural sugar composition of the HWE PTsps, its charged (≥95%) and neutral (≤5%)

fractions were determined by comparing the GC-MS profile of the alditol acetates of

the PTsps samples (Figure I.2.13; Figure I.2.14; Figure I.2.15) with those of the

standard sugars (Figure I.2.9). The HWE PTsps contained rhamnose, fucose, xylose,

mannose, galactose and glucose monosaccharide units. Their charged fractions

(CFPTsps) contained fucose, xylose, mannose and galactose, and the neutral fractions

(NFPTsps) contained rhamnose, xylose, mannose, galactose and glucose units (Table

I.2.7). Karmakar et al. (2009) reported that polysaccharide of Padina tetrastromatica

contained fucose in major amount and in this purified fucan contained significant

amounts of xylose and galactose residues as branch points. In the present

investigation, mannose was detected along with fucose, xylose and galactose units.

The sugar composition of the HWE STsps, and its charged (≥95%) and neutral (≤5%)

fractions were determined by comparing the GC-MS profile of the alditol acetates of

the STsps samples (Figure I.2.16; Figure I.2.17; Figure I.2.18 and Figure I.2.19) with

those of the standard sugars (Figure I.2.9). The GC-MS analysis of the alditol acetates

of the HWE STsps, and its charged (CFSTsps) and neutral fractions (NFSTsps) revealed

the presence of different carbohydrate moieties in varied proportions, which is

presented in Table I.2.7. Examination of Table I.2.7 showed that the CFSTsps (1M

NaCl eluate) contained only fucose and galactose units. The HWE STsps contained

rhamnose, fucose, ribose, arabinose, xylose, mannose, galactose and glucose

76

monosaccharide units. This indicated that rhamnose, ribose, arabinose, xylose,

mannose and glucose residues were not sulphated.

Table I.2.7 Carbohydrate profile (GC-MS) of crude sulphated polysaccharides

(HWEs) and fractions

Carbohydrate

moieties

Rha Fuc Rib Ara Xyl Mann Gal Glu

HWE CIsps (%

area)

ND 29.63 19.08 7.29 6.55 4.43 33.01 ND

CFCIsps ND 80.25 7.08 ND 6.24 ND 4.16 2.28

NFCIsps ND 28.23 21.95 13.86 ND 7.79 2.84 6.27

HWE PTsps (%

area)

1.19 13.81 ND ND 6.56 27.21 17.17 34.07

CFPTsps ND 32.65 ND ND 9.16 14.53 43.66 ND

NFPTsps 48.24 ND ND ND 7.59 7.30 15.48 9.89

HWE STsps (%

area)

2.19 28.56 3.46 3.94 21.38 12.46 21.12 6.89

CFSTsps (0.5M

NaCl)

6.47 41.34 ND 1.89 7.82 11.63 23.35 7.49

CFSTsps

(1M NaCl)

ND 70.26 ND ND ND ND 29.74 ND

NFSTsps ND ND ND ND ND 9.24 30.52 60.23

ND=Not detected; HWE=Hot water extract; CIsps=Sulphated polysaccharide of Cystoseira indica;

CF=Charged fraction; NF=Neutral fraction; PTsps=Sulphated polysaccharide of Padina tetrastromatica;

STsps= Sulphated polysacharide of Sargassum tenerrimum

I.2.3.5 Linkage analysis of CFCIsps

The partially methylated alditol acetates (PMAA) of CFCIsps were characterized by

GC-MS on the basis of their retention times and fragmentation patterns (Figure

I.2.20). Linkage analysis of the CIsps charged fraction was carried out by GC-MS and

the results are shown in Table I.2.8. Results of linkage analysis revealed that

backbone structure of the polysaccharide were probably made of two major sugar

residues e.g. fucose and xylose. Five different types of linkage patterns could be

identified in two major sugar residues fucose and xylose, while galactose and glucose

showed possible single linkage patterns e.g. →6)- D-Galp (1→ and →2,3,4)- D-Galp

(1→ , respectively. The greatest amount (30.54%) of 1,3,5-tri-O-acetyl-6-deoxy-2,4-

di-O-methyl galactitol indicated the presence of mainly 3-linked fucopyranose as the

backbone of the polysaccharide structure. This sulphated hetero-polysaccharide was

constituted of a highly branched structure due to the presence of →2,3,4)-Fucp(1→,

→2,3,4)-Xylp(1→, →2,3,4)-Galp(1→, →3,4)-Fucp(1→,→2,3)-Fucp(1→,→2,4)-

Ribp(1→ linkage patterns identified in the sugar residues. The sulphation pattern

77

could not be deduced on the basis of these data leaving one to conclude that it

definitely existed on any position of the xylofucan backbone, with a possible

participation of the other sulphated sugar residues.

Table I.2.8 Linkage analysis of CFCIsps by GC-MS

Sugar PMAA of sugar Deduced linkage Mol (%) Molar

ratio

Base

peak

(m/z)

L-Fucose

(Total Mol %

= 77.11)

1,5-Di-O-acetyl--6-deoxy-

2,3,4-tri-O-methyl galactitol

L-Fucp-(1→

(Terminal)

6.84 4.15 101

1,3,5-Tri-O-acetyl-6-deoxy-

2,4-di-O-methyl galactitol

→3)- L-Fucp (1→ 30.54 18.54 117



→4)- L-Fucp (1→ 4.51 2.74 117



→2)- L-Fucp (1→ 6.16 3.74 131

1,3,4,5-Tetra-O-acetyl-6-

deoxy-2-O-methyl galactitol

→3,4)- L-Fucp (1→ 8.18 4.97 117



→2,3)- L-Fucp (1→ 9.43 5.72 131

1,2,3,4,5-Penta-O-acetyl

galactitol

→2,3,4)- L-Fucp (1→ 11.45 6.95 128

D-

Xylose(Total

Mol % =

9.19)


2,3,4-tri-O-methyl xylitol

D-Xylp (1→

(Terminal)

1.65 1 117

1,3,5-Tri-O-acetyl-2,4-di-O-

methyl xylitol

→3)- D-Xylp (1→ 3.74 2.27 117


xylitol

→2,3,4)- D-Xylp (1→ 3.80 2.31 115

D-Ribose

(Total Mol %

=6.64)


2,3,4-tri-O-methyl ribitol

D-Ribp (1→

(Terminal)

4.88 2.96 117

1,2,4,5-Tetra-O-acetyl--6-

deoxy-3-O-methyl ribitol

→2,4)- D-Ribp (1→ 1.76 1.1 115

D-Galactose

(Total Mol %

= 5.08)

1,5,6-Tri-O-acetyl-2,3,4-tri-

O-methyl galactitol

→6)- D-Galp (1→ 5.08 3.1 117

D-Glucose

(Total Mol %

= 1.98


galactitol

→2,3,4)- D-Galp (1→ 1.98 1.2 129

Note: PMAA= Partially methylated alditol acetate; m/z = mass values of ion fragments

I.2.3.6 Linkage analysis of CFPTsps

The PMAA of CFPTsps was characterized by GC-MS on the basis of the retention

times and fragmentation patterns (Figure I.2.21). Linkage analysis of the PTsps

charged fraction was carried out by GC-MS and the results are shown in Table I.2.9.

Results of linkage analysis revealed that back bone structure of the polysaccharide

was probably made of two major sugar residues e.g. fucose and galactose. The

greatest amount (13.6%) of 1,2,3,5-Tetra-O-acetyl-6-deoxy-4-O-methyl galactitol

indicated the presence of mainly 2,3-linked fucopyranose residues as the backbone of

the polysaccharide structure. This sulphated hetero-polysaccharide existed as a highly

branched structure due to the presence →2,3,4,6)-Manp(1→, →2,3,4)-Fucp(1→,

78

→2,3,4)-Galp(1→, →3,4,6)-Galp(1→, →2,3,6)-Galp(1→, →3,4)-Fucp(1→,→2,3)-

Fucp(1→, →3,6)-Manp(1→, →3,4)-Manp (1→, →4,6)-Manp(1→, →3,4)-Galp(1→

sugar residues as obtained from the linkage pattern analysis. The sulphation pattern

could not be deduced on the basis of these data leaving one to infer that it definitely

existed on any position of the galactofucan backbone, with a possible participation of

the other sulphated sugar residues.

Table I.2.9 Linkage analysis of CFPTsps by GC-MS

Sugar PMAA of sugar Deduced linkage Mol (%) Molar

ratio

Base

peak

(m/z)

L-Fucose

(Total Mol %

= 48.60)


2,3-di-O-methyl-L-galactitol

→4)- L-Fucp (1→ 9.4 10.4 117



→2)- L-Fucp (1→ 4.6 5.1 131


deoxy-2-O-methyl-L-

galactitol

→3,4)- L-Fucp (1→ 7.6 8.4 117


deoxy-4-O-methyl-L-

galactitol

→2,3)- L-Fucp (1→ 13.6 15.1 131

1,2,3,4,5-Penta-O-acetyl-D-

galactitol

→2,3,4)- L-Fucp (1→ 13.4 14.9 115

D-

Xylose(Total

Mol % =

9.19)



D-Xylp (1→

(Terminal)

12.4 13.8 101


methyl-D-xylitol

→3)-D-Xylp (1→ 5.2 5.8 117

D-Mannose

(Total Mol %

= 15.7)

1,3,5,6-Tetra-O-acetyl-2,4-

di-O-methyl-D-mannitol

→3,6)- D-Manp (1→ 7.7 8.5 117



→3,4)- D-Manp (1→ 1.3 1.4 117

1,4,5,6- Tetra -O-acetyl-2,3-


→4,6)-D-Manp (1→ 1.7 1.9 117

1,2,3,4,5,6-Hexa-O-acetyl-D-

mannitol

→2,3,4,6)-D-Manp (1→ 5.0 5.5 115

D-Galactose

(Total Mol %

= 18.10)


O-methyl-D-galactitol

→4)-D-Galp (1→ 4.5 5.0 117



→6)-D-Galp (1→ 4.3 4.8 117


di-O-methyl-D-galactitol

→3,4)- D-Galp (1→ 3.9 4.3 117

1,2,3,4,5-Penta-O-acetyl-6-


→2,3,4)- D-Galp (1→ 0.9 1 129



→3,4,6)- D-Galp (1→ 1.9 2.1 117



→2,3,6)- D-Galp (1→ 2.6 2.9 129


I.2.3.7 Linkage analysis of CFSTsps

The PMAA of CFSTsps was characterized by GC-MS on the basis of the retention

times and fragmentation patterns (Figure I.2.22). Linkage analysis of the STsps

79

charged 1M NaCl fraction was carried out by GC-MS and the results are shown in

Table I.2.10. Results of linkage analysis revealed that backbone structure of the

polysaccharide was probably consisted of two major sugar residues e.g. fucose and

galactose. The largest amount (55.7%) of 1,2,3,5-Tetra-O-acetyl-6-deoxy-4-O-methyl

galactitol indicated the presence of mainly 2,3-linked fucopyranose residues,

constituting the backbone of the polysaccharide. The formation of 1,4,5-tri-O-acetyl-

6-deoxy-2,3-di-O-methyl galactitol, 1,3,4,5-tetra-O-acetyl-6-deoxy-2-O-methyl

galactitol, and 1,2,3,4,5-penta-O-acetyl galactitol indicated that the fucose would be

4-, 3,4- and 2,3,4-linked in the respective residues. The presence of 1,3,4,5-tri-O-

acetyl-2,6-di-O-methyl-D-galactitol and 1,2,3,4,5,6-hexa-O-acetyl D-galactitol

indicated that galactose was 3,4- and 2,3,4,6- linked, respectively.

Table I.2.10 Linkage analysis of CFSTsps by GC-MS

Sugar PMAA of sugar Deduced linkage Mol

(%)

Molar

ratio

Base

peak

(m/z)

L-Fucose

(Total Mol

% = 94.0)

1,4,5-Tri-O-acetyl-6-

deoxy-2,3-di-O-methyl

galactitol

→4)- L-Fucp (1→ 6.5 3.82 117


deoxy-2-O-methyl

galactitol

→3,4)- L-Fucp (1→ 10.3 6.05 117


deoxy-4-O-methyl

galactitol

→2,3)- L-Fucp (1→ 55.7 32.76 131


galactitol

→2,3,4)- L-Fucp (1→ 21.5 12.65 115

D-Galactose

(Total Mol

% = 6.0)

1,3,4,5-Tri-O-acetyl-2,6-

di-O-methyl-D-galactitol

→3,4)- D-Galp (1→ 4.3 2.53 117

1,2,3,4,5,6-Hexa-O-acetyl

D-galactitol

→2,3,4,6)- D-Galp (1→ 1.7 1 115


I.2.4 SUMMARY

Structural features and physico-chemical properties of the sulphated polysaccharides

of brown seaweed species Cystoseira indica, Padina tetrastromatica and Sargassum

tenerrimum have been studied. The hot water soluble sulphated polysaccharides were

extracted, fractionated and physico-chemical properties were studied. Metal analysis

showed the negligible values of some prominent toxic metal ions e.g. Cr, Ni, Cd, Pb

and As in CFCIsps, CFPTsps and CFSTsps suggested that the polysaccharide may be

suitable for ingestible applications. The charged fraction CFCIsps, CFPTsps and

CFSTsps contained ca. 11.5%, 11.3% and 14.3% sulphate, respectively (cf. Table

I.2.1). Major sulphation occurred on fucose and xylose sugar residues in CFCIsps,

80

fucose and galactose sugar residues in CFPTsps and CFSTsps (GC-MS of alditol

acetate), however, the position of sulphate group could not be determined. The

glycosidic linkage analysis indicated that the charged fractions were highly branched

which led to the formation of respective non-homogeneous entities. Thus these

polysaccharides present a classical example of sulphated heteropolysaccharides

encompassing all the naturally occurring sugar residues in varied proportions.

I.2.5 REFERENCES

Baba, M., Snoeck, R., Pauwels, R., DeClercq, E., Antimicrobial Agents and

Chemotherapy, 1988, 32, 1742-1745.

Berteau, O & Mulloy, B., Glycobiology, 2003, 13, 29–40.

Bilan, M.I., Grachev, A.A., Ustuzhanina, N.E., Shashkov, A.S., Nifantiev, N.E.,

Usov, A.I., Carbohydrate Research, 2002, 337, 719-730.

Chaubet F., Chevolot L., Jozefonvicz J., Durand P., Boisson-Vidal C., In: Bioactive

Carbohydrate Polymers. Paulsen B.S., (Ed.), Relationships Between Chemical

Characteristics and Anticoagulant Activity of Low Molecular Weight Fucans from

Marine Algae, Kluwer Academic: Netherlands, 2000, pp. 59-84.

Chevolot, L., Foucault, A., Chaubet, F., Kervarec, N., Sinquin, C., Fishe, A., Boisson-

Vidal, C., Carbohydrate Research, 1999, 319, 154-165.

Chevolot, L., Mulloy, B., Ratiskol, J., Foucault, A., Colliec-Jouault, S., Carbohydrate

Research, 2001, 330, 529-535.

Chizhov, A.O., Dell, A., Morris, H.R., Haslam, S.M., McDowell, R.A., Shashkov,

A.S., Nifant’ev, N.E., Khatuntseva, E.A., Usov, A.I., Carbohydrate. Research, 1999,

320, 108-119.

Ciucanu, I., Kerek, F., Journal of Chromatography, 1984, 286, 179.

Dodgson, K. S., Price, R.G., Biochemical Journal, 1962. 84, 106-110.

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. Smith, F. Analytical

Chemistry, 1956, 28, 350-356.

81

Duarte, M.E.R., Cardoso, M.A., Noseda, M.D., Cerezo, A.S. Carbohydrate Research,

2001, 333, 281-293.

Hirst, E., Mackie, W., Percival, E. Journal of the Chemical Society, 1965, 2958-2967.

Itoh, H., Noda, H., Amano, H., Zhuang, C., Mizuno, T., Ito, H., Anticancer Research,

1993, 13, 2045-2052.

Kariya, Y, Mulloy, B, Imai, K, Tominaga, A, Kaneko, T, Asari, A, Suzuki, K,

Masuda, H, Kyogashima, M & Ishii, T., Carbohydrate Research 2004, 339, 1339-1346.

Karmakar, P., Ghosh, T., Sinha, S., Saha, S., Mandal, P., Ghosal, P.K. and Ray, B.,

Carbohydrate Polymers, 2009, 78 (3), 416-421.

Knutson, C.A. and Jeanes, A. Analytical Biochemistry, 1968, 24, 470-481.

Li, B., Wei, X., Sun, J., Xu, S. Carbohydrate Research, 2006, 341, 1135-1146.

Li Bi, Ye., Jingsong, Z., Xi Jun, Ye., Qing Jeu, T.,Yan Fang, Lin., Chun Yu, G., Xin

Jui, D.,Ying Jie, P. Carbohyderate Research, 2008, 343, 746-752.

Lloyd, A.G., Dodgson, K.S., Price, R.B., & Rose, F.A. Biochimica et Biophysica

Acta, 1961, 46, 108-116.

Mandal, P., Mateu, C.G., Chattopadhyay, K., Pujol, C.A., Damonte, E.B., and Ray,

B., Antiviral chemistry & chemotherapy, 2007, 18(3), 153-162.

Marks, D.L., Buchsbaum, R., Swain, T., Analytical Biochemistry, 1985, 147, 136–143.

McClure, M.O., Moore, J.P., Blanc, D.F., Scotting, P., Cook, G.M.W., Keynes, R.J.,

Weber, J.N., Davies, D., Weiss, R.A., AIDS Res. Human Retrovir. 1992, 8, 19-26.

Meena, R. and Siddhanta, A.K., Agar and Value Addition of Indian Agarophytes. In:

A. Tewari (Ed.), Recent Advances on Applied Aspects of Indian Marine Algae with

Reference to Global Scenario, Vol. 2; CSMCRI Bhavnagar, India, 2006, pp. 172-184.

Mollet, J.C., Rahaoui, A., Lamoine, Y. Journal of Applied Phycology, 1998, 10, 59-66.

82

Nagumo, T. and Nishino, T. In: Polysaccharides in Medicinal Applications. Dumitriu

S., (Ed.), Fucan Sulfates and Their Anticoagulant Activities, Marcel Dekker, New

York, 1996, pp. 545-574.

Oza, R.M., Zaidi, S.H. A revised checklist of Indian marine algae, CSMCRI,

Bhavnagar, Gujarat, India, 2001.

Painter, T.J., Algal polysaccharides. In Edited by GO Aspinall. London: Academic

Press The Polysaccharides, 1983, vol. 2, pp. 195–285.

Patankar, M.S, Oehninger S, Barnett T,Williams R.L & Clark G.F, Journal of

Biological Chemistry 1993, 268, 21770–21776.

Sassaki, G.L., Gorin, P.A.J., Souza, L.M., Czelusniak, P.A., Iacomini, M.,

Carbohydrate Research, 2005, 340, 731-739.

Sen Sr, A.K., Das, A.K., Sarkar, K.K., Siddhanta, A.K., Takano, R., Kamei, K., Hara,

S., Botanica. Marina, 2002, 45, 331-338.

Siddhanta, A.K., Indian Hydrobiology, 7 Supplements, 2005, 29.

Siddhanta, A.K., Goswami, A.M., Ramavat, B.K., Mody, K.H., Maihr, O.P., Indian

Journal of Marine Science, 2001, 30, 166-172.

Siddhanta, A.K., Meena, R., Prasad, K., Sai Krishna Murthy, A., Seaweed

polysaccharides, their bioactivity and value addition-The Indian perspective. In: A.

Tewari (Ed.), Recent Advances on Applied Aspects of Indian Marine Algae with

Reference to Global Scenario, Vol. 2; CSMCRI Bhavnagar, India, 2006, pp. 229.

Siddhanta, A.K. and Sai Krishna Murthy, A., Journal of Indian Chemical Society,

2001, 78, 431-437.

Siddhanta, A.K., Shanmugam, M., Mody, K.H., Goswami, A. M., Ramavat, B. K.

International Journal of Biological Macromolecules, 1999, 26, 151-154.

Turvey, J.R., Williams, T.P., Journal of the Chemical Society, 1962, 2119-2122.

Usov, A.I., Ivanova, E.G., Shashkov, A.S., Botanica Marina, 1983, 16, 285-294.

83

Usov, A.I., Mirodhnikova, L.I., kochetkor, N.K., Zhurnal Obshcheii Khimii, 1972, 42,

945.

Vilela-Silva A.C.E.S, Castro M.O, Valente A.P, Biermann C.H & Mourao P.A.S,

Journal of Biological Chemistry, 2002, 277, 379–387.

Wolnik, K.A., Enzymology, 1988, 158, 190-205.

www.algaebase.org.

www.ccrc.uga.edu

84

Figures I.2.1 Structures of (A) fucopyranose (B) galactopyranose

A

B

Figure I.2.2 FT-IR spectra of (a) HWE CIsps (b) CFCIsps and (c) NFCIsps of Cystoseira

indica (CIsps)

a

b

c

cm-1

% T

85

Figure I.2.3 FT-IR spectra of (a) HWE PTsps (b) CFPTsps and (c) NFPTsps of Padina

tetrastromatica (PTsps)

4000 3500 3000 2500 2000 1500 1000 500

1417

16232930

3411

cm-1

1100

845

1054

1252

14181635

3438

% T

850

10911251

1424

1627

2935

2923

3394

a

b

c

86

Figure I.2.4 FT-IR spectra of (a) HWE STsps (b) CFSTsps and (c) NFSTsps of Sargassum

tenerrimum (STsps)

4000 3500 3000 2500 2000 1500 1000 500

10781421

163629293431

cm-1

850

10541263

16402930

3440% T

1044

1250

14221615

29323417

a

b

c

87

ppm

Figure I.2.5 13

C-NMR spectrum of CFCIsps

CFCIsps 13

C NMR in D2O; DMSO as

internal standard

88

ppm

Figure I.2.6 13

C-NMR spectrum of CFPTsps

CFPTsps 13


internal standard

89

ppm

Figure I.2.7 13

C-NMR spectrum of CFSTsps

CFSTsps 13


internal standard

90

Figure I.2.8 Gel permeable chromatograms of polysaccharides of (a) CFCIsps, (b) CFPTsps

and (c) CFSTsps

a

b

c

91

STD_MIX_Sugars

Rha Fuc Rib Ara Xyl Man Gal Glu

R.Time (min.) 10.400 10.542 11.033 11.267 12.092 15.717 16.383 16.983

% of sugar 33.95 5.64 12.19 4.84 10.83 18.87 6.32 7.36

Ratio 1.00 0.16 0.35 0.14 0.31 0.55 0.18 0.21

R.Time:10.542(Scan#:786)

R.Time:11.033(Scan#:845)

R.Time:10.400(Scan#:769)

Contd.

92

R.Time:12.092(Scan#:972)

R.Time:15.717(Scan#:1407)

R.Time:11.267(Scan#:873)

STD_MIX_Sugars


R.Time (min.) 10.400 10.542 11.033 11.267 12.092 15.717 16.383 16.983

% of sugar 33.95 5.64 12.19 4.84 10.83 18.87 6.32 7.36

Ratio 1.00 0.16 0.35 0.14 0.31 0.55 0.18 0.21

93

R.Time:16.983(Scan#:1559)

Figure I.2.9 GC-MS spectra for standard sugars

R.Time:16.383(Scan#:1487)

STD_MIX_Sugars


R.Time (min.) 10.400 10.542 11.033 11.267 12.092 15.717 16.383 16.983

% of sugar 33.95 5.64 12.19 4.84 10.83 18.87 6.32 7.36

Ratio 1.00 0.16 0.35 0.14 0.31 0.55 0.18 0.21

94

Cystoseira indica_HWE_crude PS


R.Time (min.) ------ 10.54 11.03 11.26 12.10 15.73 16.40 -------

% of sugar ------ 29.63 19.08 7.29 6.55 4.43 33.01 -------

Ratio ------ 0.90 0.59 0.22 0.20 0.13 1.00

-------

R.Time:11.03 (Scan#:845)

R.Time:10.54 (Scan#:786)

R.Time:11.26 (Scan#:873)

Contd

95

R.Time:15.73 (Scan#:1409)

R.Time:16.40 (Scan#:1489)

R.Time:12.10 (Scan#:973)

Figure I.2.10 GC-MS spectra for the sugar unit of HWE CIsps

96

Cystoseira indica_HWE_charged PS


R.Time (min.) ------ 10.53 10.99 ------ 12.07 ------- 16.33 16.93

% of sugar ------ 80.25 7.08 ------ 6.24 ------- 4.16 2.28

Ratio ------ 1.00 0.088 ------ 0.077 ------- 0.052 0.028

R.Time:10.99 (Scan#:840)

R.Time:10.53 (Scan#:784)

R.Time:12.07 (Scan#:969)

Contd

97

R.Time:16. 93 (Scan#:1533)

R.Time:16.34 (Scan#:1481)

Figure I.2.11 GC-MS spectra for the sugar unit of CFCIsps

98

Cystoseira indica_HWE_nutral PS


R.Time (min.) ------ 10.49 10.99 11.34 ------- 15.66 16.33 16.96

% of sugar ------ 28.23 21.95 13.86 ------- 7.79 2.84 6.27

Ratio ------ 1.00 0.77 0.49 ------- 0.27 0.10

-------

R.Time:10.99 (Scan#:840)

Ribose

R.Time:10.49 (Scan#:780)

R.Time:11.34 (Scan#:882)

Contd

99

R.Time:16.33 (Scan#:1481)

R.Time:16.95 (Scan#:1556)

R.Time:15.66 (Scan#:1400)

Figure I.2.12 GC-MS spectra for the sugar unit of NFCIsps

100

Contd

Padina tetrastromatica_crude PS


R.Time (min.) 10.25 10.41 ----- ----- 11.92 15.44 16.08 16.68

% of sugar 1.19 13.81 ----- ----- 6.56 27.21 17.17 34.07

Ratio 0.035 0.40 ----- ----- 0.19 0.80 0.50

1.00

R.Time:10.41 (Scan#:829)

R.Time:10.25 (Scan#:810)

R.Time:11.92 (Scan#:1011)

101

R.Time:16.08 (Scan#:1511)

R.Time:16.68 (Scan#:1582)

R.Time:15.44 (Scan#:1434)

Figure I.2.13 GC-MS spectra for the sugar unit of HWE PTsps

102

Padina tetrastromatica_HWE_charged PS


R.Time (min.) ------ 10.42 ----- ------ 11.96 15.49 16.15 -------

% of sugar ------ 32.65 ----- ------ 9.16 14.53 43.66 -------

Ratio ------ 0.75 ----- ------ 0.21 0.33 1.00

-------

R.Time:11.96 (Scan#:1016)

R.Time:10.42 (Scan#:832)

R.Time:15.49 (Scan#:1441)

Contd

103

R.Time:16.15 (Scan#:1519)

Figure I.2.14 GC-MS spectra for the sugar unit of CFPTsps

104

Contd

Padina tetrastromatica_HWE_nutral PS


R.Time (min.) 10.43 ----- ----- ------ 11.96 15.50 16.16 16.74

% of sugar 49.76 ----- ----- ------ 7.82 7.74 15.97 10.20

Ratio 1.00 ----- ----- ------ 0.16 0.15 0.32 0.20

R.Time:11.96 (Scan#:1016)

R.Time:10.43 (Scan#:833)

R.Time:15.50 (Scan#:1441)

105

R.Time:16.74 (Scan#:1590)

R.Time:16.16 (Scan#:1520)

Figure I.2.15 GC-MS spectra for the sugar unit of NFPTsps

106

Sargasum tenerrimum_HWE_Crude PS


R.Time (min.) 10.38 10.53 11.01 11.24 12.07 15.67 16.34 16.94

% of Area 2.19 28.56 3.46 3.94 21.38 12.46 21.12 6.89

Ratio 0.076 1 0.12 0.14 0.74 0.43 0.74 0.24

R.Time:10.53 (Scan#:785)

R.Time:11.01(Scan#:843)

Contd.

R.Time:10.38(Scan#:768)

107

R.Time:16.94 (Scan#:1554)

R.Time:16.34 (Scan#:1482)

R.Time:11.24 (Scan#:871)

R.Time:12.07(Scan#:970)

R.Time:15.67(Scan#:1402)

Figure I.2.16 GC-MS spectra for the sugar unit of HWE STsps

108

Sargassum tenerrimum_HWE_0.5M NaCl fraction PS


R.Time (min.) 10.28 10.43 ------ 11.14 11.96 15.51 16.16 16.75

% of Area 6.47 41.34 ------ 1.89 7.82 11.63 23.35 7.49

Ratio 0.15 1 ------ 0.045 0.18 0.28 0.56 0.18

R.Time:10.43 (Scan#:833)

R.Time:10.28 (Scan#:815)

R.Time:11.14 (Scan#:918)

Contd.

109

R.Time:11.96 (Scan#:1016)

R.Time:15.51 (Scan#:1442)

R.Time:16.16 (Scan#:1521)

R.Time:16.75 (Scan#:1591)

Figure I.2.17 GC-MS spectra for the sugar unit of 0.5M NaCl fraction of

CFSTsps

110

Sargassum tenerrimum_HWE_1.0M NaCl fraction PS


R.Time (min.) ----- 10.44 ------ ----- ----- ----- 16.17 -----

% of Area ----- 70.26 ------ ----- ----- ----- 29.74 -----

Ratio ----- 1 ------ ----- ----- ----- 0.42 -----

R.Time:16.18 (Scan#:1522)

R.Time:10.44 (Scan#:834)

Figure I.2.18 GC-MS spectra for the sugar unit of 1.0M NaCl fraction of CFSTsps

111

Sargassum tenerrimum_HWE _neutral PS


R.Time (min.) -------- -------- ----- ----- ------ 15.43 16.1 16.67

% of sugar -------- -------- ------ ------ ------ 9.24 30.52 60.23

Ratio -------- -------- ------ ------ ------- 0.15 0.50 1

R.Time:15.43 (Scan#:1433)

R.Time:16.67 (Scan#:1582)

R.Time:16.10 (Scan#:1509)

Figure I.2.19 GC-MS spectra for the sugar unit of NFSTsps

112

Cystoseira indica CIsps-PMAA

PMAA sugar residue Rt Area

%

Molar

ratio

m/z Mode of linkage


2,3,4-tri-O-methyl galactitol

6.303 6.02 4.15 203,161,143,131,117,

101,89

L-Fucp-(1→

(Terminal)



7.026 29.46 18.54 161,131,117,101,87 →3)- L-Fucp (1→


xylitol

7.304 4.15 2.74 157,144,115,100,87 →2,3,4)-D-Xylp

(1→


galactitol

7.377 2.42 3.74 129,115,102,99,87 →2,3,4)-D-Galp

(1→



7.846 1.38

4.97 204,143,117,101,86 D-Xylp (1→

(Terminal)



8.147 4.35 5.72 203,143,131,117,101,89 →4)- L-Fucp (1→


methyl xylitol

8.204 3.45 6.95 117,99,85 →3)-D-Xylp (1→



8.313 5.94 1 189,131,115,99,89 →2)- L-Fucp (1→



9.058 8.58 2.27 173,129,117,99,87 →3,4)- L-Fucp

(1→



9.688 9.89 2.31 261,201,159,143,131,11

3,99,89

→2,3)- L-Fucp

(1→


galactitol

10.184 12.97 2.96 289,231,170,145,128,11

5,99,85

→2,3,4)- L-Fucp

(1→


2,3,4-tri-O-methyl ribitol

10.568 4.09 1.1 233,161,131,117,116,

101,99,87

D-Ribp (1→

(Terminal)


O-methyl galactitol

11.608 5.36 3.1 189,161,129,117,101,99

,87

→6)- D-Galp (1→

1,2,4,5-Tetra-O-acetyl--6-

deoxy-3-O-methyl ribitol

18.562 1.92 1.2 201,129,112,99,87 →2,4)-D-Ribp

(1→

R.Time:6.303 (Scan#:491)

Contd.

113

R.Time: 7.026 (Scan#:578)

R.Time: 7.304 (Scan#:611)

R.Time: 7.377 (Scan#:620)

Contd.

R.Time:7.846 (Scan#:676)

R.Time: 8.145 (Scan#:712)

114

R.Time:8.204 (Scan#:719)

R.Time: 8.313 (Scan#:732)

Contd.

R.Time:9.058 (Scan#:822)

R.Time: 9.688 (Scan#:897)

R.Time:10.184 (Scan#:957)

115

R.Time: 10.568 (Scan#:1003)

R.Time:11.608 (Scan#:1128)

R.Time: 18.562 (Scan#:1962)

Figure I.2.20 GC-MS spectra for the methylated sugar unit of CFCIsps

116

Padina tetrastromatica PTsps-PMAA


%

Molar

ratio

m/z Mode of linkage



7.54 9.91 10.4 161,129,117,101,87 D-Xylp-(1→

(Terminal)



8.96 8.59 5.1 203,143,129,117,101,87 →4)- L-Fucp (1→



9.12 4.21 8.4 189,131,115,99,89 →2)- L-Fucp (1→

1,3,5,6-Tetra-O-acetyl-2,4-di-

O-methyl-D-mannitol

9.25 8.35 15.1 189,161,129,117,101,87 →3,6)-D-Manp(1→


deoxy-2-O-methyl-L-

galactitol

9.71 7.56

14.9 173,129,117,99,87 →3,4)-L-Fucp (1→


deoxy-4-O-methyl-L-

galactitol

10.14 13.47 13.8 261,201,159,143,131,113

,99,85

→2,3)-L-Fucp (1→

1,2,3,4,5-Penta-O-acetyl-D-

galactitol

10.44 14.45 5.8 289,231,170,145,129,115

,99,85

→2,3,4)-L-Fucp

(1→



10.57 4.50 8.5 233,173,127,117,99,87 →4)-D-Galp (1→


methyl-D-xylitol

10.71 4.44 1.4 233,129,117,99,87 →3)-D-Xylp (1→



11.32 4.35 1.9 189,161,129,117,101,99,

87

→6)-D-Galp (1→



11.61 4.18 5.5 185,129,117,87 →3,4)- D-Galp (1→


O-methyl-D-mannitol

11.78 1.47 5.0 129,117,87 →3,4)-D-Manp

(1→

1,2,3,4,5-Penta-O-acetyl-6-O-

methyl-D-galactitol

12.72 1.08 4.8 286,189,129,117,99,87 →2,3,4)- D-Galp

(1→

1,4,5,6- Tetra -O-acetyl-2,3-


12.94 1.88 4.3 261,127,117,111,101,85 →4,6)-D-Manp

(1→


methyl-D-galactitol

13.87 2.24 1 139,117,97 →3,4,6)- D-Galp

(1→


methyl-D-galactitol

14.95 3.05 2.1 261,189,129,115,99,87 →2,3,6)- D-Galp

(1→

1,2,3,4,5,6-Hexa-O-acetyl-D-

mannitol

15.51 6.27 2.9 289,217,187,170,145,139

,115,103,97

→2,3,4,6)-D-Manp

(1→

Contd.

R.Time:7.54 (Scan#:642)

117

R.Time: 8.96 (Scan#:812)

R.Time: 9.12 (Scan#:832)

R.Time: 9.25 (Scan#:847)

Contd.

R.Time:7.54 (Scan#:642)

R.Time: 8.96 (Scan#:812)

118

R.Time: 9.12 (Scan#:832)

R.Time: 9.25 (Scan#:847)

Contd.

R.Time:9.71 (Scan#:902)

R.Time: 10.14 (Scan#:954)

R.Time:10.44 (Scan#:990)

119

R.Time:10.71 (Scan#:1022)

R.Time: 11.32 (Scan#:1095)

R.Time:11.61 (Scan#:1130)

R.Time: 11.77 (Scan#:1150)

Contd.

R.Time: 10.57 (Scan#:1005)

120

Figure I.2.21 GC-MS spectra for the methylated sugar unit of CFPTsps

R.Time:12.72 (Scan#:1263)

R.Time: 12.94 (Scan#:1290)

R.Time:13.87 (Scan#:1401)

R.Time: 14.95 (Scan#:1531)

R.Time: 15.51 (Scan#:1598)

121

Contd.

Sargassum tenerrimum_1.0M NaCl fraction_PMAA.

STsps


%

Molar

ratio

m/z Mode of linkage



9.16 5.8 3.82 203,143,117,101,87 →4)- L-Fucp (1→



9.71 10.1 6.05 173,129,117,99,87 →3,4)- L-Fucp (1→



10.15 54.6 32.76 261,201,189,143,131,

113,99,89

→2,3)- L-Fucp (1→


galactitol

10.45 22.8 12.65 289,231,187,170,145,

128,115,99,85

→2,3,4)- L-Fucp (1→

1,3,4,5-Tri-O-acetyl-2,6-di-


11.61 4.6 2.53 305,143,129,117,87 →3,4)- D-Galp (1→

1,2,3,4,5,6-Hexa-O-acetyl

galactitol

16.16 2.1 1 289,259,217,187,170,1

57,145,139,115,103,85

→2,3,4,6)-D-Galp (1→

R.Time: 9.16 (Scan#:836)

R.Time: 9.71 (Scan#:902)

122

R.Time: 10.15 (Scan#:956)

R.Time: 10.45 (Scan#:991)

R.Time: 11.61 (Scan#:1130)

R.Time: 16.16 (Scan#:1677)

Figure I.2.22 GC-MS Profile of PMAA of CFSTsps

Date post:	09-Jun-2018
Category:	Documents
Upload:	duongbao
View:	215 times
Download:	0 times

Certificate of Guide and Candidateshodhganga.inflibnet.ac.in/bitstream/10603/9159/8/08_chapter...

Documents