Anticoagulant activity of marine green and brown algae collected from Jeju Island in Korea

Bioresource Technology 98 (2007) 1711–1716

Anticoagulant activity of marine green and brown algae collected from Jeju Island in Korea Yasantha Athukorala a, Ki-Wan Lee a, Se-Kwon Kim b, You-Jin Jeon

a,¤

a

b

Faculty of Applied Marine Science, Cheju National University, Jeju 690-756, South Korea Department of Chemistry, Pukyong National University, Daeyon-3-Dong, Nam-Gu, Busan 608-737, Republic of Korea Received 14 February 2006; received in revised form 6 July 2006; accepted 7 July 2006 Available online 14 September 2006

Abstract Twenty-two algal species were evaluated for their potential anticoagulant activities. Hot water extracts from selected species, Codium fragile and Sargassum horneri showed high activated partial thromboplastin time (APTT). UltraXo extract of C. fragile and S. horneri exhibited the most potent anticoagulant activity. Furthermore, in both algal species, active compounds were mainly concentrated in >30 kDa faction. The crude polysaccharide fraction (>30 kDa; CpoF) of C. fragile composed of »80% carbohydrate and »19% of protein; the crude polysaccharide fraction (>30 kDa; CpoF) of S. horneri was composed of 97% of carbohydrate and »2% of protein. Therefore, most probably the active compound, or compounds of the algal species were related to high molecular weight polysaccharide, or a complex form with carbohydrate and protein (proteoglycan). © 2006 Elsevier Ltd. All rights reserved. Keywords: Algae; Enzymatic hydrolysis; Anticoagulant activity; Codium fragile; Sargassum horneri

1. Introduction Jeju Island is located in the southwest sea of the Korean peninsula and is highlighted for its uniqueness. Especially, in the coastal area of this Island the seawater level Xuctuates rapidly. Therefore, the algal species present along the shoes of Jeju Island may require high endogenous biological protection as an adaptative response to this especial environment. Recently several biologically important seaweed species from Jeju Island have been reported (Athukorala et al., 2003, 2005; Siriwardhana et al., 2003; Heo et al., 2005; Karawita et al., 2005). However, yet there are few or less systematically studied reports regarding the potential anticoagulant activity of Jeju Island seaweeds. In 1913, scientists investigated blood anticoagulant properties of marine brown algae (Killing, 1913). Even if it is diYcult to elucidate the exact structure of the anticoagu*

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lant polysaccharides isolated from algae, the research interest to isolate anticoagulant compounds from marine seaweeds is continuously increased in the Weld of pharmaceutical industry. Heparin is the drug of the choice in prevention of thromboembolic disorders. But recently alternative drugs for heparin are in high demand due to its bad and long-term side eVects. Therefore, as an alternative source, seaweed polysaccharides gain much attention in the pharmaceutical industry to develop better and safe drugs with low or less side eVects. Recently, there was a case study on the changes of the haemorrhage, plasma cholesterol and albumin and clinical eVects in 36 children with refractory nephrosis after treatment with fucans. The results of that study suggest that fucan might be used in the anticoagulant treatment of refractory nephrosis (Shanmugam and Mody, 2000). Therefore, algal anticoagulants in future may add a new dimension in vascular disorders. Hence, the aim of this study was to screen Jeju Island seaweeds for their potential anticoagulant activities, which might expand the possibility to Wnd better anticoagulant drug.

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2. Methods

2.4. Blood coagulation assay

2.1. Materials

Normal pooled plasma was made from ten individual healthy donors, without history of bleeding or thrombosis. Nine parts of blood collected by venipuncture ware drawn into one part of 3.8% sodium citrate. Blood was centrifuged for 20 min at 2400 £ g, and the plasma was stored at ¡60 °C until use. All coagulation assays were performed with four individual replicates using dual-channel clot-2, (SEAC, Italy) and mean values were taken. For activated partial thromboplastin time (APTT) assay, citrated normal human plasma (90 l) was mixed with a solution of algal extract (10 l) and incubated for 1 min at 37 °C, then APTT reagent (100 l) was added to the mixture and incubated for 5 min at 37 °C. Thereafter clotting was induced by adding 0.025 M CaCl2 (100 l) and clotting time was recorded. In prothrombin time (PT) assay, citrated normal human plasma (90 l) was mixed with a solution of algal extract (10 l) and incubated for 10 min. Then, PT (200 l) pre-incubated for 10 min at 37 °C was added and clotting time was recorded. For thrombin time (TT) measurement, citrated normal human plasma (190 l) was mixed with a solution of algal extract (10 l) and incubated for 2 min. Then pre-incubated TT reagent (10 min, at 37 °C) was added (100 l) into the mixture and clotting time was recorded. All algal extracts including heparin were dissolved in water and in the control group, only saline was used.

Marine green and brown algae used in this study were collected along the shores of Jeju Island in Korea. Salt, sand and epiphytes were removed using tap water. Finally, seaweed samples were rinsed with fresh water and freezedried at ¡20 °C for further experiments. APTT (ellagic + bovine phospholipid) and CaCl2 solution were obtained from International Reagents Corporation (Japan), PT (rabbit thromboplastin) and TT reagents were purchased from Fisher ScientiWc Company (USA). Carbohydrases such as Viscozyme L (a multi-enzyme complex containing a wide range of carbohydrases, including arabanase, cellulase, beta-glucanase, hemicellulase and xylanase), Celluclast 1.5L FG (catalyzing the breakdown of cellulose into glucose, cellobiose and higher glucose polymers), AMG 300L (an exo 1,4-alpha-D-glucosidase), Termamyl 120L (a heat stable alpha-amylases), UltraXo L (a heat stable multi-active betaglucanase) and Wve proteases such as Protamax (hydrolysis of food proteins), Kojizyme 500MG (boosting of the Soya sauce fermentation), Neutrase 0.8L (an endoprotease), Flavourzyme 500MG (containing both endopeptidase and exopeptidase activities), Alcalase 2.4 L FG (a endoprotease) were obtained from Novo Co. (Novozyme Nordisk, Bagsvaerd, Denmark). Heparin was purchased from Sigma and all the other chemicals used in this study had 90%, or grater purity. 2.2. Water extracts from algae One gram of the ground algal powder was mixed with 50 ml of water and placed in shaking incubator for 12 h at 70 °C. The mixtures were centrifuged at 3500 rpm for 20 min at 4 °C and Wltered with Whatman Wlter paper. Finally, each supernatant was subjected for anticoagulant assay. 2.3. Enzymatic extracts from algae The preparation of enzymatic extracts was followed as previously reported (Heo et al., 2003). Dried alga sample was ground (MFC SI mill, Janke and Kunkel Ika–Wreck, Staufen, Germany) and sieved through a 50 standard testing sieve. A hundred gram of algae sample was homogenized with water (2L), and then 1 g or, 1 ml enzyme was mixed. The enzymatic hydrolytic reactions were performed for 12 h to achieve optimum degree of the hydrolysis. Before the digestion pH of the homogenate was adjusted to its optimal pH value. As soon as the enzymatic reactions complete, the digests were boiled for 10 min at 100 °C to inactivate the enzyme. Each sample was clariWed by centrifugation (3000 rpm, for 20 min at 4 °C) to remove the residue. All samples were kept in ¡20 °C for further experiments.

2.5. Crude polysaccharide separation The enzymatic extract (240 ml) was mixed well with 480 ml of 99.5% ethanol. Then, the mixture was allowed to stand for 30 min at a room temperature and then crude polysaccharides were collected by centrifugation at 10,000 £ g for 20 min at 4 °C (Matsubara et al., 2000; Kuda et al., 2002). Hereafter, the collected precipitate was referred to as crude polysaccharide fraction (CpoF) and the resultant supernatant was referred to as crude phenolic fraction (CphF). CpoF and CphF were concentrated separately under vacuum at 40 °C and removed all ethanol, and then samples were dissolved in water for further experiments. 2.6. Molecular weight fractionation of algal extract Molecular weight fraction of the enzymatic extracts from algae was conducted by our previous method (Athukorala et al., 2006a,b). Algal extract solution was passed through micro-Wltration membranes (5, 10 and 30 kDa) using Millipore’s Lab scale TFF system (Millipore Corporation, Bedford, Massachusetts, USA) to obtain diVerent molecular weight fractions. Finally, all the fractions (>30, 30 » 10, 10 » 5 and <5 kDa) were separately processed to evaluate anticoagulant activity.

Y. Athukorala et al. / Bioresource Technology 98 (2007) 1711–1716

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2.7. Determination of carbohydrate, protein and total phenolic content

Table 2 Anticoagulant activity (APTT) of selected algal species after EtOH precipitation

The carbohydrate content of the alga sample was determined by the phenol-H2SO4 method in association of oYcial analytical chemists (AOAC, 1990). The amount of crude protein was determined by the Lowry method (1951), using bovine serum albumin as the calibration standard. Total phenolic content of the crude polysaccharide fraction was determined according to a protocol similar to that of Chandler and Dodds (1993).

Sample

Monostroma nitidum Codium fragile Laminaria ochotensis Hizikia fusiformis Sargassum horneri Sargassum siliquastrum Myagropsis myagroides

APTT (s) Supernatant (CphF)

Precipitate (CpoF)

37.1 40.6 37.2 47.2 38.1 37.1 37.1

220 >300 225 250 >300 200 180

Results are expressed as means of three determinations. Sample concentration is 100 g/ml.

3. Results and discussion Various anticoagulant polysaccharide compounds have been isolated and characterized from marine algae (Matsubara, 2004). As reported previously, hot water was used for the extraction of anticoagulant compounds from marine brown and green algae (Shanmugam and Mody, 2000). The extracts were subjected to APTT assay to estimate their anticoagulant eYcacy. As shown in Table 1, M. nitidum, C. fragile, L. ochotensis, H. fusiformis, S. horneri, S. siliquastrum and M. myagroides showed high anticoagulant potential among the tested algal species. The green alga C. fragile showed the highest activity. Many algal species exert their anticoagulant action through the sulphated polysaccharides (Church et al., Table 1 Anticoagulant activities of hot water extracts from some green and brown algal species collected from Jeju Island in Korea ScientiWc name

Collected area

APTT (s)

Green algae Monostroma nitidum Enteromorpha compress Enteromorpha intestinalis Enteromorpha linza Ulva pertusa Codium fragile

Jocheon Jocheon Dodu Jocheon Jocheon Kimnyung

200 66 62 75 55 250

Brown algae Ishige okamurai Ishige sinicola Colpomenia sinuosa Undaria pinnatiWda Laminaria ochotensis Padina arborescens Myelophycus simplex Hizikia fusiformis Sargassum coreanum Sargassum fulvellum Sargassum horneri Sargassum siliquastrum Sargassum thunbergii Scytosiphon lomentaria Peltaronia bighamiae Myagropsis myagroides

Jocheon Seongsan Seongsan Seongsan Seongsan Seongsan Dodu Jocheon Jocheon Seongsan Seongsan Seongsan Jocheon Jocheon Seongsan Seongsan

63 76 57 48 150 37 63 160 70 75 170 170 85 89 53 165

Results are expressed as means of three determinations. Sample concentration is 100 g/ml, concentration of the samples were adjusted according to the dry weight.

1989). But, some algal species trigger anticoagulant activity through protein, or glycoprotein-like compounds (Yasuda et al., 2004). Therefore, in this study, as a preliminary trial, water extracts of the selected algal samples were subjected to ethanol precipitation and the resultant crude polysaccharide fraction (CpoF) and crude polyphenol fraction (CphF) were evaluated for their potential anticoagulant activities; the results are shown in Table 2. According to the results, in all tested samples the activity was observed in crude polysaccharide fraction. Among the tested algal species, the CpoFs of C. fragile and S. horneri showed the highest APTT activity (>300 s). Crude polysaccharide fractions of L. ochotensis, M. nitidum, H. fusiformis and S. siliquastrum showed over 200 s APTT activity while CpoF of M. myagroides showed about 180 s APTT activity. Therefore, C. fragile and S. horneri were selected for the next anticoagulant experiments. Bio-active compounds from plant material can be extracted by various techniques. Among these, enzymatic hydrolysis of plant material oVered advantages over other techniques (Bejerano et al., 1991; Gaur et al., 2006). Enzymes can convert water-insoluble materials into water soluble materials, also this method do not adapt any toxic chemicals. Interestingly, this technique leads relatively higher yield in bioactive compound extraction and shows enhanced biological activity in comparison with water and organic solvent extraction (Athukorala et al., 2006a,b). But, only few studies have been conducted to utilize enzymes to extract bioactive compounds from algal biomass. The degree of enzymatic hydrolysis was diVerent with the treated enzymes (Heo et al., 2003; Vlasenko et al., 1997). Enzymes are able to digest linkages of the plant cell wall materials. The rate of hydrolysis depends on the type of linkage and on chain length of the polysaccharide sample. Therefore, the digestion of alga by enzymes increases the releasing of extractable polysaccharide. In this study, C. fragile and S. horneri were enzymatically digested with cabohydrases and proteases to prepare water-soluble extracts and their potential anticoagulant activities were evaluated. The basic characteristics of those enzymes are summarized in Table 3. All the tested enzymatic extracts of C. fragile showed good

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Table 3 Characterization and optimum hydrolysis conditions of particular enzymes Enzyme

Enzyme characterization

Optimum conditions pH

Temperature

Carbohydrases Viscozyme Celluclast AMG Termamyl UltraXo

Arabanase, cellulase, -glucanase, hemi-cellulase and xylanase Catalyzing the breakdown of cellulose into glucose cellobiose and higher glucose polymer An exo-1, 4--D-glucosidase A heat stable -amylase A heat stable multi-active -glucanase

4.5 4.5 4.5 6.0 7.0

50 50 60 60 60

Proteases Protamex Kojizyme Neutrase Flavourzyme Alcalase

Hydrolysis of food protein Boosting of the soya sauce fermentation An endoprotease Containing both endoprotease and exopeptodase activities A endo protease

6.0 6.0 6.0 7.0 8.0

40 40 50 50 50

anticoagulant activities as evident from APTT and TT assays (Table 4). However, all the extracts were devoid of PT activity. Comparatively, carbohydrase extracts of alga showed better activities than those of protease extracts. Of the tested enzymatic extracts, UltraXo extract showed the highest APTT (>300 s) and TT (80 s) activities, respectively. Except Termamyl extract, the extracts by other carbohydrases showed > 200 (s) of APTT activities. The results of the enzymatic extracts of S. horneri are shown in Table 4. All the tested extracts showed considerable anticoagulant activity on APTT and TT assays. Taken together, the enzymatic extracts prepared by carbohydrases showed relatively higher anticoagulant potential than those by proteases. Interestingly, the digestion of the alga sample by carbohydrases resulted almost two times higher activity than those of protease extracts. Among carbohydrate digestive enzymes, the extract obtained after hydrolysis of S. horneri with UltraXo gave the highest anticoagulant activity. Especially, the above fraction prolonged both APTT and TT eVectively but it was devoid of PT activity. Table 4 Anticoagulant activities of C. fragile and S. horneri hydrolyzed with carbohydrases and proteases Sample

Carbohydrases Viscozyme extract Celluclast extract AMG extract Termamyl extract UltraXo extract Proteases Protamex extract Kojizyme extract Neutrase extract Flavourzyme extract Alcalase extract Control

APTT (s)

TT (s)

PT (s)

A

B

A

B

A

B

278 252 257 181 >300

226 252 187 181 >300

50 63 60 58 80

70 71 70 53 77

13 13 13 13 13

13 13 13 13 13

204 184 216 191 101

104 84 116 91 90

50 55 48 30 60

58 60 58 73 60

13 13 13 13 13

13 13 13 13 13

37

26

13

A – C. fragile; B – S. horneri; Results are expressed as means of two determinations. Sample concentration is 100 (g/ml).

According to previous reports, the anticoagulant activity of the sulphated polysaccharide depended on its molecular weight (Pereira et al., 1999). Normally, sulphated polysaccharides (sulphated fucoidan and galactofucans) with 50– 100,000 Da are considered as potential anticoagulants, whereas fractions with >850,000 Da usually demonstrate low anticoagulant activities (Shanmugam and Mody, 2000). However, sulphated polysaccharide isolated from Fucus vesiculosus, with molecular weight of 7.4 £ 104 Da showed 60–80% of the activity of heparin in the recalciWcation tests and 15–18% heparin activity in the whole human blood. Therefore, in order to get an idea of the molecular weight of the active fraction, the UltraXo extracts of C. fragile and S. horneri were passed through ultra-Wltration membranes (5, 10 and 30 kDa) and relevant molecular weight cut-oV fractions (>30, 30 » 10, 10 » 5 and <5 kDa) were separated and evaluated for their anticoagulant activity. Molecular weight fractionation results of C. fragile and S. horneri are shown in Table 5. From UltraXo extracts of both species, the most potent activity was recorded from >30 kDa fraction. The highest molecular weight fraction strongly prolonged the clotting times in APTT and TT assay, but had very slight activity on PT assay. However, no potential activity was reported from the other fractions of UltraXo extract. According to molecular weight fractionation results in both algal species the active compound/ compounds were concentrated in > 30 kDa fraction. ThereTable 5 Anticoagulant activities of molecular weight fractions from UltraXo extract of C. fragile and S. horneri Sample (100 g/ml)

APTT (s) A

B

A

B

A

B

Above 30 kDa fraction 30 » 10 kDa fraction 10 » 5 kDa fraction Below 5 kDa fraction Water

>300 35 40 32

>300 37 37 35

126 26 26 26

78 26 26 26

20 13 15 13

13 13 13 13

32

TT (s)

26

PT (s)

13

A – C. fragile; B – S. horneri; Results are expressed as means of two determinations.

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fore, >30 kDa fraction of each algal species was subjected to ethanol precipitation to obtain the crude polysaccharide fraction (CpoF). A dose-dependent anticoagulant activity of the crude polysaccharide fraction of the C. fragile is shown in Fig. 1. As expected, APTT activity of crude polysaccharide fraction increased with the sample concentration. The crude polysaccharide fraction of UltraXo extract of S. horneri also showed a similar dose-dependent anticoagulant potential to that of C. fragile (Fig. 1). Under the same conditions, the CpoF of C. fragile showed higher anticoagulant activity than that of S. horneri. C. fragile crude polysaccharide fraction at the concentration of 60 g/ml showed » 350 (s) in APTT assay, whereas that of S. horneri at the same concentration showed » 250 (s) APTT value. Therefore, UltraXo hydrolysate of C. fragile had higher anticoagulant potential than that of S. horneri. However, both samples showed less activity than that of heparin. In this study, crude polysaccharide fractions of the both algal species eVectively prolonged APTT and TT. Normally high clotting time of anticoagulants in APTT is due to the inhibition of the intrinsic and/or common pathway, whereas prolongation of TT indicates inhibition of thrombin activity or Wbrin polymerization. To get an idea about the composition of the active compounds, approximate chemical composition of the crude polysaccharide samples was studied. CpoF of C. fragile composed of » 80% carbohydrate, » 19% of protein and 55 mg of phenolic compounds/100 ml of extract, therefore, the active compound of the C. fragile appeared to be high molecular weight polysaccharide, or a complex form with

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carbohydrate and protein (proteoglycans). The CpoF of S. horneri was composed of 97% of carbohydrate, » 2% of protein and 102 mg of phenolic compounds/100 ml of extract. According to previous reports, most probably the active compound of these brown algae might be due to a sulphated polysaccharide with high molecular weight. The anticoagulant principle of Sargassum cinctum was a sulphated polysaccharide; it mainly contained galactose and a trace amount of fucose with sulphate (Mody et al., 1997). Therefore, a similar kind of compound could be associated with the activity of S. horneri. Most of green algae exert their anticoagulant activity through polysaccharide, or proteoglycan. In many cases, arabinose and its sulphate derivatives elicit anticoagulant activity of green algae. Sulphated arabinan isolated from Codium latum showed 12.6 times more antithrombin activity when standard heparin was taken as one (Uehara et al., 1992). The anticoagulant activity of most green seaweeds are due to sulphated arabinan, however, sulphated arabinogalactan also have been reported as possible anticoagulant compound in green algae (Siddhanta et al., 1999). Therefore, arabinan and sulphate groups of the isolated compounds play a crucial role in prolonging anticoagulant activity of these algal species. The carbohydrate hydrolysing enzyme, UltraXo L, produced by Humicola insolens, was the best enzyme preparation in releasing arabinose, from both soluble and insoluble arabinoxylan and ferulic acid from insoluble arabinoxylan (Sorensen et al., 2003). In this study, both UltraXo extracts of C. fragile and S. horneri showed much better activities than

Fig. 1. Comparison of APTT activity of the crude polysaccharide fractions (>30 kDa; CpoF) of C. fragile ( ) and S. horneri ( ) with that of heparin. All coagulation assays were performed with two replicates.

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other respective enzymatic hydrolysates. UltraXo digestion release mainly arabinose and its derivatives to the extraction media. Sometimes, this might be the reason for the high anticoagulant activity of the UltraXo extract of C. fragile. Acknowledgements This research was supported by a grant (p-2004-02) from the Marine Bioprocess Research Center of the Marine Bio 21 Center, funded by the Ministry of Maritime AVairs and Fisheries, Republic of Korea. References AOAC., 1990. OYcial methods of analysis. 16 Ed., Assoc. OYce. AgrChemists, Washington, D.C., pp. 69–74, 487–491. Athukorala, Y., Lee, K.W., Shahidi, F., Heu, M.S., Kim, H.T., Lee, J.S., Jeon, Y.J., 2003. Antioxidant eYcacy of extracts of an edible red alga (Grateloupia Wlicina) in linoleic acid and Wsh oil. J. Food Lipids 10, 313– 327. Athukorala, Y., Lee, K.W., Park, E.J., Heo, M.S., Yeo, I.K., Lee, Y.D., Jeon, Y.J., 2005. Reduction of lipid peroxidation and H2O2 mediated DNA damage by a red alga (Grateloupia Wlicina) methanolic extract. J. Sci. Food Agric. 85, 2341–2348. Athukorala, Y., Jung, W.K., Jeon, Y.J., 2006a. An anticoagulative polysaccharide from an enzymatic extract of Ecklonia cava. Carbohydr. Polym., doi:10.1016/j.carbpol.2006.03.002. Athukorala, Y., Kim, K.N., Jeon, Y.J., 2006b. Antiproliferative and antioxidant properties of an enzymatic hydrolysate from brown alga, Ecklonia cava. Food Chem. Toxicol. 44, 1065–1074. Bejerano, R.N., Moppes, D.V., Sivan, A., Arad, S., 1991. Potential production of protoplasts from Porphyridium sp. using an enzymatic extract of its predator Gymnodinium sp. Bioresour. Technol. 38, 127–131. Chandler, S.F., Dodds, J.H., 1993. The eVect of phosphate, nitrogen and sucrose on the production of phenolics and solasidine in callus cultures of Solanum laciniatum. Plant Cell Rep. 34, 105–110. Church, F.C., Meade, J.B., Treanor, E.R., Whinna, H.C., 1989. Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III and thrombin. J. Biol. Chem. 264, 3618– 3623. Gaur, R., Sharm, A., Khare, S.K., Gupta, M.N., 2006. A novel process for extraction of edible oils enzyme assisted three phase partitioning (EATPP). Bioresour. Technol., doi:10.1016/j.biortech.2006.01.023. Heo, S.J., Lee, K.W., Song, C.B., Jeon, Y.J., 2003. Antioxidant activity of enzymatic extracts from brown seaweeds. Algae 18, 71–81.

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