Indian Journal of Experimental Biology Vol. 42, May 2004, pp 529-532

Extraction of heparin and heparin-like substance from marine mesogastropod mollusc Turritella attenuata (Lamarck, 1779)

M Arumugam & A Shanmugam*

Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502, India

Received 26 Novell/ber 2002; revised 5 February 2004

Heparin was extracted from marine gastropod T. allelllla/a through the sequential precipitation with methanol and ethanol. The metachromatic dye method using toluid ine blue was used to estimate colorimetrically the amoun t of heparin present in the sample. The anticoag ulant activity of the sample was ca lculatcd as per United States of Pharmacopoeia stan­dard procedure using sheep blood . After the purification. sa mpl es were analyzed, for the presence of heparin , with agarose­gel electrophoresis and HPLC and the mobility o f the sample and the peak respective ly were co mpared with standard hepa­rin. The results of the present study shall help in finding out alternate source.

Key words: Heparin , Mesogastropod moll usc, Turri/ella allelllla/a

IPC Code: Int. CI 7 A61 K

The sulfated glycosaminoglycan, heparin is a well­known anticoagulant l used to prevent or limit clotting and thrombus formation in cardiovascular diagnostic and surgical procedures, as with in-dwelling venous catheters, cardiopulmonary catheters, surgery of the heart and vessels, metal and plastic prostheses and extracorporeal circulation artificial organs and trans­plants2

. Heparin binds with consensus peptide in Hu­man Immunodeficiency Yirus (HIY), blocking its replication ill vitro3

. Lot of heparin-like materials with high blood anticoagulant activity have been extracted from marine flora and fauna4

.5

. During the present investigation a substance, which may be chemically similar to heparin, was obtained from the common marine meso gastropod Turritella atten.uata.

Heparin or heparin-like polysaccharides occur not only in mammals but also in lower vertebrate species, as well as in certain other invertebrates6. Unlike heparan sulfate, heparin was only found in some spe­cies of invertebrates, e.g., molluscs and crustaceans7

.

Molluscs show extensive species diversity and their by-products have received much attention from the beginning of 20th century. Among the molluscs, some have pronounced pharmacological activities or other properties useful in the biomedical arena. It is sur-

*Corresponden t author Phone: 04144-243070 Ext. 212 Fax : 041 44-243555 E-mail: shanpappu @yahoo.com

prisi ng that some of these pharmacological activities are attributed to the presence of sulfated polysaccha­rides8,9 .

Though the presence of such anticoagulants in various molluscs has been reported, the exact function in mollusc is still not known. It is not possible for this compound to act as anticoagul ant, si nce unlike mam­mal s molluscs are devoid of coagulation systems. From the data available on glycosaminoglycans, par­ticularly heparin and heparan sulfate, it is understood that they mostly associated with organisms of great complexity, as represented by the presence of skele­ton and complex circulatory and digestive systems 10.

Therefore, in the present study, isolation of heparin from marine mesogastropod Turritella attenuata has been attempted. The isolated compound was puri­fied II and was further subjected to HPLC separation for the purity. The isolated product was subjected to agarose gel electrophoresis using standard heparin as control, quantified using metachromatic method 12 and studied for its anticoagulant activit/ 3

.

Materials and Methods Isolation.-The animals (Turritella attenuata) were

collected from the Parangipettai coast (Lat. 11°29'N; Long,79°46'E), east coast of India. The whole animal viscera was blended and defatted with acetone, fil­tered and further defatted with petroleum ether fol­lowing the standard method 14. The defatted tissues

S30 INDIAN J EXP BIOL, MAY 2004

were ai r dried at room temperature. Dried defatted tissue (SO g) was ground and mixed with SOO ml of 0.4 M sodium sulphate (Na2S04)' The mixture was incubated at Ss oC for 90 min. The pH was maintained at 11.5. After incubation aluminium-di-sulphate {Alz (504)3} crystals were added to bring down the pH to 7.7 and heated to 9SoC for 1 hI'. The sample was cooled and centrifuged. After centrifugation the su­pernatant was collected and treated with cetyl pyri­dinium chloride (CPC) for further extraction. To the collected supernatant 70 ml of 3% CPC in 0.8 M NaC I was added. The mixture was stirred until the precipi­tate formation was completed. This suspension was incubated at 37°C for 24 hr and centrifuged at 4°C for 90 min. in a refrigerated centrifuge to collect the crude heparin complex. The precipitate was redis­solved in 2M NaCI to remove pyridinium salts from heparin and centrifuged for 30 minutes at 2S00 rpm in refrigerated centrifuge. The supernatant was collected and 3 volumes of 99.9% methanol was added to the precipitated crude heparin. The heparin was collected by centrifugation at 3000 rpm at 4°C for 30 min in refrigerated centrifuge. The precipitate was also washed with 99.9% ethanol and then air-dried.

Purification-The purification was also done for the crude sample by using standard procedure I I. Briefly, S g crude sample, obtained after the extrac­tion using CPC, was dissolved in 200 ml of distilled water. To the solution, 10 g barium acetate was added slowly with stirring and the pH was adjusted to 6.S. The mixture was heated to 60°C and then left at SoC for 24 hI' till the precipitate was formed. The precipi­tate was collected by centrifugation. The supernatant was again kept at SoC for 24 hr. To the supernatant, 2 volume of methanol was added, and then the mixture was incubated at SoC for 12 hr. Again the precipitate was collected by centrifugation. This precipitate was dissolved in 12S ml of O.lM di-sodium hydrogen phosphate at pH 8.8. The mixture was heated and fil­tered at 60°C. To the filtrate, 2 volume of methanol was added and the mixture was maintained at the SoC for 2 hr. The precipitate was washed twice with methanol and dried under vacuum (Fraction I). Re­maining supernatant was collected and then 1 volume of methanol was added, developed precipitate was collected and washed with methanol and vacuum dried (Fraction II) .

Estimation of heparin in the sample-The yield and activity of heparin were estimated following the metachromatic dye '2 method and again the yield and

anticoagulant activity of the samples were studied using US pharmacopeia method ' 3. The presence of heparin in sample was detected using the method of agarose gel electrophoresis reported by Volpi et al. ls

.

The presence of heparin signals in the sample was recorded using HPLC analysis (Shimadzu, model LC9A) on CI8 column, distilled water as eluent, at a flow rate of lml/min. 200 fll each of standard heparin (Sigma) solution (1 mg/ml) and the crude extract (2S mg/ml) were injected.

Results and Discussion The total yield of heparin and heparin-like com­

pounds and its activity calculated by metachromatic method were found to be as 73,600 units/kg of wet tissue and 16 IU/mg of the sample respectively in T. attenuata. The two fractions (F I and F II) obtained after the purification were tested for the yield and ac­tivity of the heparin and heparin-like compounds, which were calculated as 19.104X 106 and 7.08 X 105 units/kg and 96 IU and 12 IU/mg of each sample re­spectively. In the crude sample of scallop, cod and flounder viscera the heparin and heparin-like com­pounds were reported lesser (8000-9000 units/kg wet weight) than found in marine gastropod in the present study 16.

The yield and anticoagulant activity of the crude sample was calculated as 43.66x106 units/kg of wet tissue and 37 USP units/mg respectively as per the method recommended by US Pharmacopeia, whereas the yield and anticoagulant activity of those two frac­tions (F I and F II) were estimated as IS.S2 X 106 and 4.72 x 105 units/kg of wet tissue and 78 and 8 IU/mg of sample respectively.

The anticoagulant activity of heparin differs from species to species due to their interaction with the en­zymes and inhibitors of the coagulation system9

. The anticoagulant activity of T. attenuata (l5.S2X 106

units/kg of wet tissue) is 10 to 15 times more than that in virginea ocean clam (1,72,400 IU/kg); but at the same time the anticoagulant activity of virginea ocean clam was found to be 6 to 40 more than that of the scallop viscera (5836 IU/kg of wet tissue)'4.

In the present study, the anticoagulant activity (37, 78 and 8 USP units/mg of crude sample, Fraction I alld II respectively) was higher than that of Spisula solidisima and Cypraea islandica in which the antico­agulant activity in the crude sample ranged from 70 to 12 USP units/mg6

. At the same time, the anticoagulant activity of T. attenuata was lesser when compared to

ARUMUGAM & SHANMUGAM: EXRACTION OF HEPARIN ETC. FROM MARINE GASTROPOD, TURRITELLA 531

that of the same animals (130 to 150 USP units/mg) when studied after purification l7

• The probable reason for such difference in the activity of heparin is the function of the amount of non-anticoagulant impurity carried over to the isolated product. Nevertheless, it is possible to increase their potency by purifying it through repeated fractionation as evidenced from the study in sheep and pork in which the heparin antico­agulant activity was reported as 59 and 81 USP/mg respectively. But heparin with high anticoagulant po­tency of 170 units/mg has been isolated from the same species after purification. Thus it is reasonable to as­sume that heparin with high biological potency could be obtai ned by repeated and exhaustive fractionation, resulting in less impurities, than material of consider­able less potencl. The difference in heparin activity may be a function on non-anticoagulant impurity, which carried over into the isolated product. Thus, it could be expected that the repeated fractionation of T. attenuata heparin would also result in high antico­agulant activity.

Dietrich et al. 18 reported a high anticoagulant ac­tivity (320 IU/mg) in the mollusc Anomalocardia bra­silialla, the other molluscs like Tivela l1lactroides and Donax strialus exhibited the anticoagulant potency of 220 and 180 IU/mg respectively. The heparin isolated from those molluscs is similar to mammalian heparin and the difference was fo und in the molecular weight of the compound. The sample with high molecular weight showed more anticoagulant activity than low molecular weight heparin ls

. Somasundaram isolated heparin from the bivalve mollusc K.opima which showed the anticoagulant activity of about 92 units/mg l9

.

The agarose gel electrophoresis carried out for the commercial heparin (sodium salt), i.e. standard, and sample prepared from T. attenuata showed the pre­sence of a single major component in the purified fractions I and II (Fig. 1). The overall chemical analy­sis showed less impurity of the sample as visualized from the F I strong band whose migration was similar to that of heparin standard (S); whereas the fraction II did not form a clear band but the other related poly­saccharides residues moved in the same direction in a diffused manner. The electrophoretic migration of the standard heparin and the purified Fraction I extracted from T. attenuata showed the same electrophoretic mobility like that of the other studies in A. brasiliana, D. striatus and T. mactroides l 8

, Pomacea sp., T. gibbus and A. brasiliana7

.

In the chromatogram obtained for the standard heparin and the sample prepared from T. attenuata, three peaks were observed at 3.044, 5.779 and 8.001 rtm . Out of these three peaks, the third peak was seen at the same retention time of the standard hepari n (Figs ~ : lllll ~)

....

J

Fig. I-Agarosc ~LI electrophoresis of , landaI'd hepar in ~llld sam-ples [S - Standard heparin, FI - Fraction I, FH - Fracti on III

0

'" p 0

<1- ' 0 : u; :

/'- ' .... ' '"':

Ir3 .-'.0

If> /'- '

~ ~: .,; ~:

,. .

~. :

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'I)

;!

~

_ . __ 1.- __ __ I.. ....... __ __ 1.. ____ \. .•• _.1.... ___ 1.. ___ _ 1.. __ _

, I' "

~ ~ ~ 1<'> ~ !Xl :0- :-~ ..;, ~ N '" OJ ~ '" ~ ;- ~ ~

Fig, 2-HPLC chromatograph of standard heparin

532 INDIAN J EXP BIOL, MA Y 2004

I'

' I{) 'r--. ",

. ",

JO iLl)

0 <0

' OJ ' 0

:'" :'"

, r-- . (,-- , Il), -.

I • • , • •

[""~ .";"~ .;.~ .;.;."~ .. "~"" , I

_ _ ___ J .. _________ \.. __ __ I . ____ , _ ____ _ __ _ I ____ J.. ____ 1. _ ........

Fig. 3-HPLC chromatograph of sa mpl e

The su lphated g lycosam inoglycans- in Fl obtai ned from T. attenuato described in the present study meet all of the criteria for defi ning it as they are heparin­like compounds or even 'heparin' which needs further investigation including the determination of molecul ar weight and NMR studies.

The studies also revealed the performance of the heparin ex tracted from T. attenuata to be good and resembles with that of the commercial heparin . There­fore this marine mesogastropod mollusc could be con­sidered as a good alternative source fo r extraction of this sulfated mucopolysaccharide.

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and physical and physical compari son of heparin fro m d if­ferent mammalian speci es, IJiochelll Biophys Acta, 83 ( 1964) 272

2 Bradshaw R A & Wessler S, Heparin struc ture, function s and clin ical implicat ions in Advallces ill experilllelllal lIledicine alld clinical ill/plicatiolls ed ited by R A Bradshaw and S Wess ler (Plenu m, New York), 1975,52

3 Baba M, Pauwels R, Balazarini J, AI nour J , Desmyter J & De C lercq E, Mechani sm of inhibitory effect of dextran sul­fate and heparin on replication of human immuno deficiency virus ill vitro , Proc Natl Acad Sci USA, 85 (1988) 6132

4 Hovingh P, Piepkorn M & Linker, A, Isolated heparin from so mc vertebra tes , Biochelll Journal, 237 (1986) 573

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lioll alld clinical applicatiolls, ed ited by N M Mc Duffi (Academ ic Press, New York) 1979,3

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7 Nader H B, C hava nte S F, Santos E A, O li veira F W, de­Paiva J F, Jeronimo S M B, Medeiros G F, de-Abreu L R D, Leite E L, de-Sousa-Filho J F, Castro R A B, Toma L. Ter­sariol 1 L S, Porcionatto M A & Dietrich C P, Heparan sul-

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II Dietrich C P, Paiva J F, Moraes C T, Takahashi H K, Por­c ionatlo M A & Nader H 8, Isola tion and characterizatio n o f a heparin witil high ant icoagulant activ ity from Alloll/a/ocar­dia brasiliana, Biochilllica et Biophysica Acta, 843 ( 1985) I

12 Smith P K, Mallia A K & Hermanson G T, Colorimetric method for the assay of heparin content in immobili zed hepa rin preparatio!ls Allalytical Bioahelll, 109 (1980) 466

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17 Burson S L, Fahrenbach M J, Frommhagen L H, Riccardi B A, Brown R A, Brockman J A, Lewry H V & Stockstad E L R, Isolation and purification of mactins, heparin like anti­coagulants from mollusca, JAm Chelll Soc, 78 (1956) 5874

18 Dietrich C P, Nader H B, Paiva J F, Santos E A, Holme K R & Perlin A S, Heparin in mollusk: Chemical enzy matic deg­radation and super (l3)c and super (I) H NMR spectroscopial evidence for maintenance of the structure th rough evolution , II/t J Bioi MacrOlllol, II (1989) 36 1

19 SOlllasundararn M S T, Hepari n from Katelysia opillla, Ph D thesis, CAS in marine Biology, Annamalai U niversity, Portonovo, India, 1990, 74