Indian Journal o f Experimen tal Biology Vo l. 39, April 2001. pp. 365-370

Blood anticoagulant sulphated polysaccharides of the marine green algae Codium dwarkense (Boergs.) and C. tomentosum (Huds.) Stackh.

M. Shanmugam, K. H. Mody & A. K. Siddhanta* Marine Algae & Marine Environment Di scipl ine Central Salt and Marine Chemical s Research Institute

Bhavnagar 364 002, India

Phone o . 0278-567760; Fax No. 0278-566970/567562 E- mail : salt @csir.res. in

Received II May 2000; revised 13 D ecember 2000

Cold water extracts of marine green algae Codillm dwarkellse and C. IOmelllOS11II1 were precipitated with different mo­lar concentrations of KCI and were subjected to anion exchange and gel filtration chromatography. Both the speci es yielded sulphated arabi nan th rough bioassay-guided purification and both were chemically identified as a po lymer of a-L­arabinofuran ose. Products were assayed fo r their blood anticoagulant activity using PT, APTr and IT tests and found that they differed in the potency of acti vity though they are chemicall y identical. Bioassay-guided purification of cold water ex­tract of C. lomelllOSllII1 yi elded sulphated arabi nan and sulphated arabinogalactan .

Anticoagulant ac tivity of marine algae was first reported in 19361

• There exist many reports of an ticoagulant activity associated with su lphated polysaccharides (SPS) of red and brown algae, but very few does concerning those of green algae. Anticoagulant acti vity associated with green algae was first reported in 19852

. High molecular weight proteoglycans and low molecular weight sulphated polysaccharides from C. fragile ssp. a(!alllicuI1/3.4, su lphated rhamnan of MOllOslroll/a lIilidu/115 and su lphated arabinan of C. laluII/6 and C. dwarkellse7

were reported to possess anticoagulant activity. It has been observed that KCI precipitated crude polysaccharides afforded acti ve components with fewer steps of purification and in higher yields, with respect to those involved in the purification without KCI precipitation. In this paper we report the results of the investigation on KCI precipitated polysaccharide products of C. dwarkense and C. IOll/en(osulII. The products were purified and the molecular enti ty responsible for activity was identified. Alongside, the cold water extract of C. IOlllell{OSUII/ was also studied for its blood anticoagulant ac tivity.

Materials and Methods CodiulI/ dwarkellse Boergs. was collected in March

*Correspondent au th o r

1996 from Okha (Lat. 22° 28' N, Long. 69° OS' E) in the west coast of India, from lower inter-tidal zone. C. tomentoswn (Huds.) Stackh. was collected from Krusadai Island (Lat. 09° 16' N, Long. 79° 12' E) in March 1996 from inter-tidal rock-pools. Algal materials were thoroughly cleaned, quick-washed with tap water, dried in shade, powdered and depigmented by methanol in a Soxhlet apparatus. 100g of depigmented powder was soaked in 20 times more volume (w/v) of distilled water and kept overnight at 4 to 5°C and cold water extract (CWE) was prepared as reported by Siddhanta et af.

Fractionation alld purification Known concentration of KCI solution was added to

1% SPS solution (CWE) to have the final molar concentration in the range of 0.1 M to 3.0 M KCI. Precipitates were dialysed and freeze-dried. Anion exchange chromatography was carried out on a DEAE cellulose (CI- form) column (45 x 3cm) eluted with a stepwise gradient of distilled water to 3.0 M NaCI solution at 25°C at a flow rate of 25mllhr. Elution was monitored by measuring the sugar concentration using Phenol-sulphuric acid method8

. Products were obtained by dialysis and Iyophi lisation. Molecular weight based fractionation of SPS was performed using gel filtration chromatography on a Seralose CL - 4B gel column (Sisco Research Laboratory (S RL), India; 45 x 2.5 cm). The sample was loaded and eluted with 0.2 M NaCI containing 0.02% of sodium

366 INDIAN J EX? SIOL. APRIL 2001

azide at 25°C at a flow rate of 5.8mllh. Fractions (2.5 to 4.0ml) were collected monitoring their carbohydrate contents. The fractions were pooled and the products obtained after dialysis and Iyophi lisation.

Evaluation of blood allticoagulallt activity-­Blood was collected from healthy volunteers and normal human plasma was prepared in the following manner: Blood was collected using a disposable polypropylene syringe and anticoagulated using 3.8% tri-sodium citrate in a polypropylene container (9 parts of blood to I part of tri-sodium citrate solution) and it was centrifuged immediately at 4000rpm for 10-15min. Algal SPS sample was prepared in normal saline (0.85% NaCl) solution. Concentrations of samples were taken from l).lg to I mg/ml based on their purity , potency and the method used . Heparin (sodium salt, SRL, India; 140.3 units/mg) was used as standard for comparative study. Three techniques, viz. Activated Partial Thromboplast in Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT)9 tests were used to evaluate the activity. Results of the clotting tests were expressed as a Clotting Time Ratio (CT ratio) which was calculated by dividing the clotting time achieved with test sample, by the time achieved under simi lar conditions with normal saline solution .

Physicohell1ical analysis -Total sugarS, sulphate lO,

. lid' 'd l ' f d protem an urOIllC act - contents 0 extracte polysaccharide samples were determined by spectrophometric methods using Shimadzu UV -160A UV -Visible Spectrophotometer. eutral sugar composition of the SPS were determined after hydrolysis with 2ml of 2 M H2S04 at 100°C for 6hr13

,

reduction 14 and acetylation 15. Alditol acetates were chromatographed on a BP-225 capillary column, (50% cyanopropyl coated, 25M, 00 0.33mm, ID 0.22mm, SGE, Australia) running isothermall y at

2l0°C using hel ium as carrier gas at the flow rate of 30mllsec (Inj. temp. 250°C, Oet. temp. 280°C) on a Hewlett-Packard 5890 Series II GC con nected to 5971 Series MSO machine. The results were compared with the GC profile of alditol acetates of the standard sugars. Infrared spectra were recorded on KBr pellets on a Bio-Rad IR machine, FTS-40, USA. I Hand 13C NMR spectra of KCI precipitated products were recorded at 30°C in 0 20 and that of the desulphated product (DPS) was recorded in OMSO-d6 at 70° at 200 MHz on a Bruker Digital NMR, Avance OPX 200 machine. Oesulphation of sulphated polysaccharides was done using melhanolic hydrogen chloride method 16. Molecular weight of the sample was determined from the standard curve drawn u. inQ. different molecu lar weight markers I( 1.95 x 10'1 to 2 ~ 106 daltons) and their elution volumes . Molecular homogeneity of the purified products was assessed by electrophoresis. Cellulose acetate electrophoresis was conducted at a constant current of ImA/cm with O.IM HCI solution for 2hr. The strip (16x2cm) was developed by dipping into 0.5% toluidine blue reagent in 3% acetic acid.

Resul ts

Chemical composition of sulphated polysaccharides of both cold water extracts (CWE) and hot water extracts (HWE) or C. tOlllelltoSl/1II were studied. CWE contained higher sugar and sulphate and lower protein and uronic acid contents than the hot water extracts, i.e. 33.92%, 31.49%, 8.43%, 0.73% in CWE and 22.06%, 17.14%, 16.10% and 2.03% in HWE respectively . Blood anticoagulant activity of both the extracts was estimated by PT test using 2mg/ml concentration, eWE showing higher blood anticoagulant activity than HWE. Therefore, CWE

Table I--Chemical composition and anticoagulant activity (PT test) of chromatographic fractions of colc.l wa ter extract of C. 10 111 (, IIiOS 1/111

Fractions Yield Protein Uronic acid Sulphate Sugar com~osition (%) CT ratio (%) (%) (%) (%) Arabinose Galactose Soollg/ml 1000llg/mi

0 SO.30 4.22 tr 39.02 7!l.69 21.31 n S.25

E 8.:n tr tr 32.53 n n n 3.76

01 11.40 tr a 28.5S n n 1.00 2.52

02 29.00 a a 36.33 86.37 13.63 2.29 > 10

03 11.10 a a 30.28 n n 2.00 7.64

04 IUD a a 3 1.88 n n 1.00 2.58

1)2a 28.00 a a 40.82 100 0.0 >10 > 10

02b 39.00 a a jO.61 60.4~ j9.St'i 3.46 6.60

a. absen t; n. not done; tr . trace.

SHANMUGAM et al. : BLOOD ANTICOAGULANT FROM MARINE GREE ALGAE 367

was purified to identify precisely the molecular species responsib le for eliciting potent blood anticoagulant activi ty. It was achieved by anion exchange chromatography and gel filt ration techniques. It yie lded two components, su lph ated arabi nan (02a), a high molecular weight component (MW 1.9 x 106

), exhibiting the highest activity and sulphated arabinogalactan (02b), a lower MW component (2.7 to 3.0 x 105

) having lower activity. Chem ical analysis, neutral sligar composition and BAC activity of all the fractions of CWE of C. tOlll entosLllI1 are presented in Table I. It was observed that the molecular species responsible for activi ty is same a that repo rted for C. dwarkellse7

, i.e . a polymer of sulphated a-L­arabinofuranose.

Purification of KCI-precipitated eWEs of C. dll'arkellse and C. tOlllelltosulII--Cold water ex tracts were precipitated with 0.06 to 3.0 M KCI. Fractions were analysed and found that trace amounts of uronic

acid and protein were present in all the fract ions. Both precipitates and supernatant were tested for their activity and supernatants were found to be inactive. All the precipitates, particularly 0.12 M KCI­precipitate (fraction J) showed the highest acti vity in C. dwarkense and 0.5 M KCI precipitate (fraction K) was the most active in the ca e of that of C. tOlllentoslllll . Sulphate contents of all the precipitates ranged from 28.9 to 39.9% and those of supernatants were lesser than 20%. Analyses of alditol acetate of fraction J and K by GC-MS revealed that the fractions contained predominantly arabinose (J: 95.47~

arabinose and 4.53% galactose; K: 87.55% arabinose and 12.45% galactose). Sulphate contents were ca. 37.0% in both the fractions (Table 2).

Fractions J and K were chromatographed on OEAE cellulose column using 0.1 to 1.0 M NaCI solutions. In both the cases 0.2 M fraction (named fraction J I & K I respectively) showed high anticoagul ant activity and it contained 100% arabinose (Table 3). Traces of

Table 2---Chemical composition and blood anti coagulant activity of KCI -precipitated fractions of CWE of C. dwarkel/se and C. tOlllel/lOSlII1l

C. dll'arkel/ se

(M) KCI Sulphate Sugar composition (0/0 )

Fracti on (0/0) Arabin ose Galactose

0.06 37.4

0. 12(1) 37.0 95.47 4.53

0 . 18 39.9

0.24 39.3

0.30 34.9

0.40 32.1

0.50 30.4

1.0 3 1.0

2.0 28.9

3.0 29 .2

*CT ratio obtain ed in PT te s t.

CT rati o* (M)KC I

500flg/ml Fraction

1. 84 0.1

2.69 0.2

2.38 0.3

2.07 0.4

2.07 0.5 ( K)

2. 10 1.0

2. 15 2.0

1.90 3.0

1.92

1.67

Sulphate

(0/0)

38 .7

31.5

33.3

35.1

37. 1

33.1

35.2

30.2

C. tOlll ellt O.VlIIIl

Sugar composi ti o n ('Yo) CT rati o*

Arabin ose Galacto se 500flg/ml

2.44

87.55 12.45

3.05

3.02

2.38

3. 17

2.70

2 .64

1. 88

Table 3---Chemical composition and anticoagulant ac ti vi ty of fract ions of J and K

C. d\V(/rkel/se C. IOlIlelllO.1"II1Il

(M) aCI Yield Sulphate Arabinose CT rati o* (M) aCI Yield Sulphate Arabinose CT ratio* Fractions (%) ('Yo) (%) (500~tg/ml ) Frac ti ons ('Yo ) ('Yo) ('Yo) (500flg/ml)

0.10 0.61 6.7 1.1 2 0. 10 0.8 1 5.65 1.2 1

0.15 6.30 32.0 1 6.25 0.20 (K I: 33.91 41.18 100 6.25

0.20 (J I) 18.46 32.09 100 6.86 0.30 8.78 40.75 2.68

0.25 10.76 35.05 5.93 0.40 3.51 38.63 2. 12

0.30 10.0 33.80 5. 12 0.45 2.36 38.19 2.06

0.40 10.0 32.71 4.68 0.50 1.41 28.28 1.93

0.50 4.0 38.fi9 4.37 0.60 0.33 28.0 1.20

1.0 4.61 27.33 1.7 1 1.0 0.30 27.40 1.00

*CT ratio obtai ned in PT test.

368 INDI AN J EXP BIOL. APRIL 200 1

Table 4--(:o lllparison of anticoagulant activities of fractions J I a and K I a with heparin*

Conc. PT AP'IT TT

(~lg/llll ) Jla Ki a Heparin Jl a Kia Heparin Jl a Kia Heparin

I 1.0 1.0 1.0 1.0 1.0 1.0 1.35 1.0 1.23

2 1.0 1.0 1.0 1.0 1.0 1.0 1.82 2.0 1.7

3 1.0 1.0 1.0 1.0 1.0 1.0 2.82 2.5 3.76

4 1.0 1.0 1.0 1.0 1.0 2.05 4.05 3.43 >10

5 1.0 1.0 1.0 1.34 1.2 2.54 6. 11 4.37 >10

10 1.0 1.0 1.0 2.06 1.68 >10 >10 >10 >10

20 1.0 1.0 1.0 2.87 4.35 >10 >10 >10 >10

30 1.0 1.0 1.0 3.0 >10 >10 >10 >10 >10

40 1.0 1.0 1.0 6.6 >10 > 10 >10 >10 >10

50 1.32 1.57 1.0 >10 >10 >10 >10 >10 >10

100 1.60 1.73 1.6 >10 >10 >10 >10 >10 >10

250 2.73 3.06 2. 1 >10 >10 >10 >10 >10 >10

500 > 10 >10 6.6 >10 > 10 >10 >10 >10 >10

1000 >10 >10 >10 >10 >10 >10 >10 >10 >10

*Anticoagulant ac ti vities are expressed as CT ratio in PT, APTT and TT tests.

pro tein and uronic acid were detected in J 1 and K I. 100mg of each of J I and K I were refractionated on gel column and fraction s were collected monitoring their sugar contents. A single peak was e luted in both the ca es and they were di stributed over the e lution volume range 28 to 96ml. The products obtained from C. dll'arkense and C. tOlllenlOslI1II named J I a and K I a re pecti ve ly. Both the fracti ons J I a and K I a contained solely arabinose and sulphate content were 37.5% and 38.85 respecti vely. The estimated MW of the both the frac tions are 1.9 x 106

. It is reported that KCI precipitated sulphated polysaccharides yie ld active fraction in a fewer steps of purification with higher yield . Both the frac tions Jla and K I a migrated toward the cathode in cellulose acetate strip electrophore is as a s ingle band indi cating the ir homogeneity in term s of molecular size.

Bioassay--Blood anticoagulant activity of fracti ons J I a and K I a were evaluated using PT, APT[ and IT tests. The e ffect of increasi ng concentrati on of algal an ticoagulant incorporated into clotting assays are described here. From the Table 4, it is evident that the sensitivities of the three techniques are di fferent. The sensitivity o f the method decreases in the order of IT, APIT and PT and this observation is in consonance wi th that reported for proteoglycans and sulphated polysaccharides5

. Anticoagulant activity was found to be associated with higher arabinose content of SPS component incorporated into the assays. Higher anticoagulant activity was associated with higher arabinose and sulphate content. CT ratio

> I 0 was observed at 4)lg/ml and 10)lg/ml in J I a and K I a respectively in T[ test. The increase in anticoagulant activity with the increase in arabinose and sulphate contents was also demonstrated in the APTI and PT tests . In APT[ tests, CT rati o > 10 was observed at 50 and 30)..lg/ml in J la and Ki a re pecti vely and in PT test both afforded CT ratio> I 0 at 500)lg/ml concentration (Table 4).

Characterisation of fractions J J a and K J a-J I a: IR spectrum frequencies were 3950(w), 3848(w), 3478(br s), 2978(sh), 2602(w), 2354(s), 23 18(s), 2138(w) , 1833(w), I 776(m), 1641 (br s), 1563(m), 1532(w), 1503(w), 1443(m), 1399(m), 1229(s) , 1141 (s), 1058(s), 102 1(s), 956(m), 921(sh), 885(s), 854(sh) and 8 18(m) em-I. [s = strong, w = weak, sh = shou lder, br = broad, m = medium]. Typical absorbance type A (sYIII/IIetrical ring breathing ji-eqllency) , types B and C (stretching I/ /Odes for slIbstitll ents) and type D (C- H deJor/llation Jor all H­atolll attached to a C-ato/ll that is directly attached to a rillg oxygen atolll) for a furanose ring system6

.17

were observed at 921, 885, 854 and 8 18 cm-I, respectively. The presence of sulphate was indicated by a strong band at 1229 cm-I (S=O stretching). The anomeric proton appeared at 85.40, which is in agreement with the reported o nes for a-L­configuration of arabinofuranosel 8

. The proton resonances of desulphated form o f J 1 a appeared at 04.93, s (anomeri c proton); 04.55 br s; 04.06, d , ]= 11.2 Hz; 83.95, s; 83.81, s; 83.53, d , J= 11.2 Hz.

SHANMUGAM el al. : BLOOD ANTICOAGULA T FROM MARINE GREE ALGAE 369

The anomeric carbon resonance appeared at 895.91(C-I) and other chemical shifts arc 74.69 (C-4),66.46 (C-2), 64.79 (C-3) and 62.12 (C-5).

KJa-IR spectrum frequencies were: 3870(w), 3576(w), 3462(br s), 2676(sh), 2526(w) , 2326(w), 2274(w), 1813(w), 1787(w), 1654(br s), 1540(m), 1238(s), 1141(s), 1060(s), 1005(s), 921(w), 885(m), 852(m) and 8 15(m) cm-I. Typical absorbance type A, types Band C and type 0 for a furanose ring system were observed at 921, 885, 852, and 8 15 cm-I, respectively . The presence of sulphate was indicated by a strong band at 1238 cm·I(S=O stretchi ng). The anomeric proton appeared at 85.40. Proton resonances of desulphated product: 84.92, s (anomeric proton); 84.58 br s; 84.19, s; 84.06, d, J= I 0.8 Hz; 83.95, s; 83.81 , s; 83.52, d, J= 10.8 Hz. Chemical shi fts in the 13C NMR spectrum were assigned as follows: 895.94 (C-I), 74.69(C-4) , 66.49(C-2) , 64.8 1 (C-3) and 62.20(C-5). Therefore, anticoagulant product (K I a) isolated from C. /OlllellloSlIIlI too is constituted of sulphated a-L-arabinofuranose units. These data are in agreement with the reported values for a-L­arabi nan isolated from root of PillllS dellsiflora llJ

,

seeds of Samalia lIIalabaria20 and Cadilllll lallllll of Japane. e waters6

.

Discussion

A polymer of sulphated a-L-arabinofuranose was found to be responsible for the anticoagulan t activity of C. dllarkellse as well as C. l alll ellloSlllll . The CT ratios of 100% arabi nan-containing fractions of both the CWEs, in terms of PT, are in excess of lOin the case of C. IOlll elllaSlIlII (Table I) a well as C. dwarkellse

7. It is well known that sulphate moiety has

a very important role to play in exhibi ting blood . I . . 2 I-? 3 TI I antlcoagu ant actiVi ty -' . le su phate content

ranged from 5 to 38.85% in all the crude, semipurified and purified SPS fractions of both the plant species. Minimum anticoagulant activity was observed in the fractions with sulphate content in the range of 15 to 20%. Fractions with more than 30% sulphate showed high activity. Cold water extracts of C. dwarkellse and C. IOlllelllosulII have been shown to contain on ly one type of molecular spec ies (GC-MS) which exh ibited the highe. t anticoagulant activity. The cst imated MWs of J I a and K I a were in the order of 1.9 x 106

.

Although compounds having blood anticoagulant activity with such high MW is not common, high MW proteoglycans of some Codilllll species4

.5 as well as

SPS of red algae have, however, been reported to

exhibit anticoagulant activitl4-26

. The desulphated products of Jla and Kia showed no activity . Complete desulphation was done with methanolic hydrogen chloride, and it was ensured that the desulphated polysaccharide gave negative BaCi2-

gelatin test. Desu lphated arabi nan was less soluble in water than the su lphated one, presumably due to an increase in intermolecular hydrogen bonding in the desulphated species, whereas in the case of a marine green alga Bryopsis lIIaxillla was assumed to be due to the interaction of the (1---7 3)-/3-D-xylan and cellulose in the cell-wall polysaccharide27

.

Both products (J I a and K I a) showed higher activity than heparin in 50llgiml concentrati on and above in PT test, and lower activity in APTT and TT tests (Table 4). At 250llg/ml K I a is more active than J I a. In APTT test, at 20llg/ml and above K I a was more active than J 1 a. In TT tests, however, at and above 4Ilg/ml, Jla is more active than Kia. Although both the products (J I a and K I a) contain the same chemical entity (sulphated a-L-arabinofuranose), they showed blood anticoagulant activit ies in various degrees . The reason for thi s may be attributed to the variations in the finer structures of the polymer vi::. . vanatlOns in C-sulphation, linkages and conformational geometry which were demonstrated by the fact that the most active KCI-precipitated product of C. dwarkellse (J I a) was obtained with O.12M KCl whereas in the case of C. IOlllen/OSIIIII the product (K I a) with highest activity was obtained by 0.5M KCI, the quanta of activities being different (Table 2).

The su lphated polysaccharides extrac ted from C. /OlllellloSIlIll and C. d IVa rkellse exh i bi t poten t anticoagulant activity. Bioassay-guided purification of CWE of C. IOlll elllOSlI1II yie lded two components, su lphated arabi nan , a high molecul ar weight component exhibiting the highest activity and a su lphated arabinogalactan, a relatively low molecular weight component having lower activity. KCI -precipitated products of both the species yielded only su lphated arabinan, th e most active component in a fewer steps of purification ; the biological activities of sulphated polysaccharides being dependent on their molecular weight, sulphate content and sugar compositions . The SPS with a mixture of arabinose and galactose and relatively low sulphate content showed lower activity . The hi gh molecular we igh t fraction of SPS with hi gh

370 INDIAN J EXP BIOL, APRIL 2001

sulphate and exclusive ly arabinose contents showed s trong anticoagul ant activity and might be a po tent anticoagulant agent.

Acknowledgement The authors are g rateful to Dr P. K. Ghosh,

Director, CSMCRI for hi s valuable suggestions. They are thankful to Mr A. Tewari and Dr. O . P. Mairh for their kind help and encouragement. One of the authors (MS) is grateful to the Department of Ocean Development, New Delhi for an award of Senior Research Fellowship under a ational Programme (DOD/9-DS/I/96-2110; dated 16/1211996).

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