Structural diversity of fucoidans and their radioprotective effect.

Fucoidans are biologically active sulfated polysaccharides of brown algae. They have a great structural diversity and a wide spectrum of biological activity. This review is intended to outline what is currently known about the structures of fucoidans and their radioprotective effect. We classified fucoidans according to their composition and structure, examined the structure of fucoidans of individual representatives of algae, summarized the available data on changes in the yields and compositions of fucoidans during algae development, and focused on information about underexplored radioprotective effect of these polysaccharides. Based on the presented in the review data, it is possible to select algae, which are the sources of fucoidans of desired structures and to determine the best time to harvest them. The use of high purified polysaccharides with established structures increase the value of studies of their biological effects and the determination of the dependence "structure - biological effect".

The structure of fucoidan from Sargassum oligocystum and radiosensitizing activity of galactofucans from some algae of genus Sargassum.

The aim of this study was to establish the fine structure of fucoidan from Sargassum oligocystum and to study the radiosensitizing effect of fucoidans from three algae of genus Sargassum (S. oligocystum, S. duplicatum, and S. feldmannii) with different structures. The fucoidan SoF2 from S. oligocystum was sulfated (32%) galactofucan (Fuc:Gal = 2:1), with a Mw of 183 kDa (Mw/Mn = 2.0). Its supposed structure was found to be predominantly 1,3-linked fucose as the main chain, with branching points at C2 and C4. The branches could be single galactose and/or fucose short chains with terminal galactose residues. Sulfate groups were found at positions C3, C2, and/or C4 of fucose residues and at C2 and/or C4 of galactose residues. The radiosensitizing effect of galactofucans from S. oligocystum, S. duplicatum, and S. feldmannii against human melanoma SK-MEL-28, colon HT-29, and breast MDA-MB-231 cancer cells was investigated. The influence of all investigated polysaccharides treatments with/without X-ray radiation on colony formation of human melanoma cells SK-MEL-28 was weak. Fucoidan from S. feldmannii has been shown to be the most promising radiosensitizing compound against human colon HT-29 and breast MDA-MB-231 cancer cells.

Composition of polysaccharides and radiosensitizing activity of native and sulfated laminarans from the Tаuуа basicrassa Kloczc. et Krupn.

Polysaccharide fractions of alginate, laminarans and fucoidans were obtained from the brown alga Tauya basicrassa. Yields of alginate and laminarans were large (19.7 % and 5.62 %, respectively), whereas the content of fucoidans (0.52 %) was not significant. Alginate and laminarans had typical structures for those substances. Fucoidans were low- and medium-sulfated heterogeneous polysaccharides. The fucoidan fraction 1TbF1 was sulfated fucogalactan containing a backbone from 1,6-linked residues of ß-d-galactopyranose with branches at C3 and C4, terminal fucose and galactose residues and fragments from 1,3-; 1,4-; and 1,2-fucose residues. Sulfate groups were found at positions 2 and 4 of fucose, and positions 2, 3 and 4 of galactose residues. Laminaran 2TbL was subjected to a sulfation to obtain the derivative 2TbLS with partial sulfation (46 %) at C2, C4 and C6. It was shown that 2TbL and 2TbLS inhibited colony formation of sensitize-tested colon cancer cells HT-29 and HCT-116 to X-ray radiation.

Sulfated steroids of Halichondriidae family sponges - Natural inhibitors of polysaccharide-degrading enzymes of bacterium Formosa algae, inhabiting brown alga Fucus evanescens.

Inhibiting effects of sulfated steroids from marine sponges of Halichondriidae family: halistanol sulfate, topsentiasterol sulfate D and chlorotopsentiasterol sulfate D were investigated on three different types of enzymes degrading polysaccharides of brown algae: endo-1,3-ß-d-glucanase GFA, fucoidan hydrolase FFA2 and bifunctional alginate lyase ALFA3 from marine bacterium Formosa algae KMM 3553T, inhabiting thalli of brown alga Fucus evanescens. This is the first research, devoted to influence of a marine natural compound on three functionally related enzymes that make up the complex of enzymes, necessary to degrade unique carbohydrate components of brown algae. Alginic acid, 1,3-ß-D-glucan (laminaran) and fucoidan jointly constitute practically all carbohydrate biomass of brown algae, so enzymes, able to degrade such polysaccharides, are crucial for digesting brown algae biomass as well as for organisms surviving and proliferating on brown algae thalli. Halistanol sulfate irreversibly inhibited native endo-1,3-ß-D-glucanases of marine mollusks, but reversibly competitively inhibited recombinant endo-1,3-ß-d-glucanase GFA. This fact indicates that there are significant structural differences between the enzymes of practically the same specificity. For alginate lyase and fucoidan hydrolase halistanol sulfate was irreversible inhibitor. Topsentiasterol sulfate D was less active inhibitor whereas chlorotopsentiasterol sulfate D was the strongest inhibitor of enzymes under the study. Chlorotopsentiasterol sulfate D caused 98% irreversible inhibition of GFA. Chlorotopsentiasterol sulfate D also caused reversible and 100% inhibition of ALFA3, which is unusual for reversible inhibitors. Inhibition of FFA2 was complete and irreversible in all cases.

Fucoidan Sulfatases from Marine Bacterium Wenyingzhuangia fucanilytica CZ1127T.

Fucoidans belong to a structurally heterogeneous class of sulfated polysaccharides isolated from brown algae. They have a wide spectrum of biological activities. The complex structures of these polysaccharides hinder structure-activity relationships determination. Fucoidan sulfatases can make useful tools for the determination of the fine chemical structure of fucoidans. In this study, identification and preparation of two recombinant sulfatases able to catalyze the cleavage of sulfate groups from fragments of fucoidan molecules is described for the first time. Two genes of sulfatases swf1 and swf4 of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T were cloned and the proteins were produced in Escherichia coli cells. Sulfatases SWF1 and SWF4 are assigned to S1_17 and S1_25 subfamilies of formylglycine-dependent enzymes of S1 family (SulfAtlas). Some molecular and biochemical characteristics of recombinant fucoidan sulfatases have been studied. Detailed specificity and catalytic features of sulfatases were determined using various sulfated fucooligosaccharides. Structures of products produced by SWF1 and SWF4 were established by nuclear magnetic resonance (NMR) spectroscopy. Based on the obtained data, the enzymes are classified as fucoidan exo-2O-sulfatase (SWF1) and fucoidan exo-3O-sulfatase (SWF4). In addition, we demonstrated the sequential action of sulfatases on 2,3-di-O-sulfated fucooligosacchrides, which indicates an exolitic degradation pathway of fucoidan by a marine bacterium W. fucanilytica CZ1127T.

A novel sulfated fucan from Vietnamese sea cucumber Stichopus variegatus: Isolation, structure and anticancer activity in vitro.

In the present study, three sulfated polysaccharides, two fractions of fucosylated chondroitin sulfates, and one sulfated fucan were isolated from the body wall of the Vietnamese sea cucumber Stichopus variegatus. The structure of the sulfated fucan fraction SvF3 from S. variegatus was investigated for the first time. According to NMR spectroscopy data, the sulfated fucan SvF3 contained 1,2- and 1,3-linked α-l-fucopyranose residues. Sulfate groups were found at the 2 and/or 4 positions. The structural analysis of fucoidan was assisted by tandem mass spectrometry; the recently-developed technique of autohydrolysis in heavy­oxygen water for the obtaining of selectively labeled fucoidan fragments was applied. The labeling (+2 Da mass shift at the reducing end) allowed us to assign MS/MS data unambiguously, and thus to confirm the NMR data and revealed minor sulfation at position 3. It was shown that the sulfated fucan SvF3 was not cytotoxic to human breast cancer T-47D and MDA-MB-231 cell lines, and it inhibited colony formation of those cells in vitro. SvF3 also possessed slight activity against migration of MDA-MB-231 cells in vitro.

Structural characteristics and anticancer activity in vitro of fucoidan from brown alga Padina boryana.

The sulfated and acetylated fucoidan fraction, containing fucose, galactose, mannose, glucose and uronic acid residues, was isolated from the brown alga Padina boryana. The structure of galactofucan part was studied after different modifications by NMR spectroscopy and mass spectrometry. It was shown that galactofucan contained the main chain of alternating 1,4-linked α-l-fucopyranose and 1,3-linked ß-d-Galactopyranose. Single fucose residues were found as branches at C4 of galactose residues. Also, fucoidan contained 1,3- or 1,4-linked Fuc-Fuc and Gal-Gal fragments. The sulfate groups occupied positions C2, C3 and C4 of both fucose and galactose residues, which was shown by tandem mass spectrometry of fragments, labeled with heavy-oxygen. The anticancer effect of native and modified fucoidan fractions was studied in vitro on the colorectal carcinoma cells DLD-1 and HCT-116. All fucoidans had no cytotoxicity under 400 µg/mL and inhibited colony formation of cancer cells at concentration of 200 µg/mL.

Polysaccharides from brown algae Sargassum duplicatum: the structure and anticancer activity in vitro.

The laminaran SdL and fucoidan SdF were isolated from brown algae Sargassum duplicatum. SdL was 1,3;1,6-ß-d-glucan (1,3:1,6=6:1) with a main chain, represented by 1,3-linked glucose residues, due to NMR spectroscopy data. Single glucose residues could form branches at C6. Unusual structure of fucoidan SdF was studied by chemical and enzymatic methods, NMR spectroscopy of desulfated and deacetylated polysaccharide and mass spectrometry of fucoidan fragments labeled with 18O. Fucoidan was sulfated (31.7%) and acetylated galactofucan (Fuc:Gal∼1:1) with a main chain of 1,4-linked alternating α-l-fucose and ß-d-galactose residues. Side chains were represented by extensive (DP≥5) 1,3-linked 2,4-disulfated α-l-fucose residues with branching points at C2. Fucose residues in the main chain were sulfated at C2 and less at C3, while galactose residues were sulfated at C2, C3, and less at C4, C6. The fucoidan SdF was effective against colony formation of colon cancer cells in vitro.

The comparison of structure and anticancer activity in vitro of polysaccharides from brown algae Alaria marginata and A. angusta.

Laminaran and three fucoidan fractions were obtained from the brown alga Alaria marginata. Alaria angusta, studied earlier by us, has the same polysaccharide composition. Galactofucan AmF3 from A. marginata has a main chain of →3)-α-l-Fucp-(2,4-SO3(-))-(1→residues, similar to galactofucan from A. angusta. However, the structure of the branches in fucoidan AmF3 can differ from those in the fucoidan from A. angusta. The following fragments were identified in AmF3: HexA-(1→2)-Fuc, HexA-(1→2)-Gal, Gal-(1→4)-HexA, Fuc-(1→2)-Gal-6-SO3(-), Fuc-4-SO3(-)-(1→6)-Gal, Gal-(1→2)-Gal-2-SO3(-), Gal-4-SO3(-)-(1 →6)-Gal, Gal-4-SO3(-)-(1→3)-Fuc-(1→3)-Fuc, Fuc-4-SO3(-)-(1→6)-Gal-(1→4)-Gal, Gal-(1→4)-Gal-(1→3)-Fuc, Gal-2-SO3(-)-(1→4)-Gal-(1→4)-Gal, Gal-(1→4)-Gal-6-SO3(-)-(1→2)-Gal. Chains of galactose residues (DP up to 9) were found in AmF3 fucoidan. The laminarans, galactofucans and their derivatives from both algae exhibited no cytotoxicity in vitro. Polysaccharides from A. angusta were more effective against colony formation of HT-29 cells, while those from A. marginata had a greater effect on T-47D cells. Sulfated and desulfated fucoidans possessed weak antitumor activity using SK-MEL-28 cells.

Structural elucidation of polysaccharide fractions from the brown alga Coccophora langsdorfii and in vitro investigation of their anticancer activity.

Laminaran, fucoidan, and alginate were isolated from the brown alga Coccophora langsdorfii collected in the Japan Sea. The structural characteristics of polysaccharides were investigated by NMR spectroscopy. The laminaran was determined as ß-d-glucan, which consisted of 80% of 1,3- and 20% of 1,6-linked residues and was terminated with mannitol. The alginate was a guluronic acid-rich polysaccharide (M/G=0.85). Fucoidan, sulfated α-l-fucan, contained a linear backbone of alternating (1→3)- and (1→4)- linked α-l-fucopyranose residues with sulfate at C2 and C4 of (1→3)-α-l-fucopyranose residues. Anticancer activity of this fucoidan was investigated in comparison with activity of fucoidan having similar linear backbone from the brown alga Fucus evanescens. The fucoidan from C. langsdorfii significantly inhibited colony formation of SK-MEL-5 and SK-MEL-28 melanoma cells (the percentage of inhibition was 28 and 76, respectively) and weakly inhibited colony formation of breast adenocarcinoma cells MDA-MB-231 (the percentage of inhibition was about 5). Similar results were obtained for fucoidan from F. evanescens; the percentage of inhibition of colony formation of SK-MEL-5 and SK-MEL-28 melanoma cells was 54 and 56, respectively. The inhibition of colony formation of breast adenocarcinoma cells MDA-MB-231 was weak. We suppose that other sulfated and partially acetylated fucoidans consisting of (1→3)- and (1→4)-linked α-l-fucopyranose residues may suppress progression of melanoma cell colony formation similar to fucoidans of C. langsdorfii and F. evanescens.

Structure and anticancer activity in vitro of sulfated galactofucan from brown alga Alaria angusta.

Laminaran and three fractions of fucoidan were isolated from brown alga Alaria angusta. The laminaran AaL was characterized as a typical 1,3;1,6-ß-D-glucan (ratio of bonds 1,3:1,6 = 10:1). Fucoidans AaF1 and AaF2 are sulfated heteropolysaccharides, containing fucose, galactose, mannose and xylose. The fraction AaF3 is sulfated and acetylated galactofucan with the main chain represented by a repeating unit → 3)-α-L-Fucp-(2,4-SO3(-))-(1 →. According the data of methylation analysis, AaF3 contains mainly 1,3-linked fucose, less 1,4-linked and 1,4,6-linked galactose residues. The autohydrolysis (37 °C) of fucoidan AaF3 allowed to obtain selectively 2-desulfaled polysaccharide fraction, built up of fucose only, and low molecular weight (LMW) fraction. The negative-ion tandem mass spectrometry of LMW fraction, further hydrolyzed by acid hydrolysis identified the following fragments: Gal-2-SO3(-)-(1 → 4)-Gal, Gal-4-SO3(-)-(1 → 4)-Gal, Gal-(1 → 2)-Gal-4-SO3(-), Fuc-2-SO3(-)-(1 → 4)-Gal, Gal-2-SO3(-)-(1 → 3)-Fuc-(1 → 3)-Fuc, Fuc-2-SO3(-)-(1 → 3)-Fuc-(1 → 4)-Gal. The laminaran AaL and the fucoidan AaF3 exhibited no cytotoxicity in vitro for HT 29, T-47D, and SK-MEL-28 cell lines. The AaF3 fraction suppressed colony formation of HT 29 and T-47D cells, AaL-only HT 29 cells.

Further studies on structure of fucoidan from brown alga Saccharina gurjanovae.

A sulfated galactofucan SgF (MW 123kDa) was purified from the brown alga Saccharina gurjanovae. Polysaccharide was depolymerized by autohydrolysis at 25 and 60°C, and products were studied by mass spectrometry and (13)C NMR spectroscopy. According to results of investigation, the main chain of this polysaccharide is built of a repeating units →3)-α-L-Fucp-(2,4-OSO3(-))-(1→. Fucose chains could be sometimes terminated by (1→3)-linked galactose residues. Shorter (1→4)- and/or (1→6)-linked sulfated galactose chains are attached at positions C-2, C-3 of fucose residues. Sulfate groups can occupy positions C-2 and/or sometimes C-3 of Gal residues, but a sulfation at C-4 of the galactofucan could not be excluded. The SgF-AH25-H preparation (71kDa) was obtained by autohydrolysis of SgF at 25°C, which leaded to a selective desulfation at C-2 and, probably, to a cleavage of galactose chains, since structure of SgF-AH25-H represented a repeating unit →3)-α-l-Fucp-(4-OSO3(-))-(1→, which was definitely established by (13)C NMR spectroscopy. Galactofucan SgF and its derivative SgF-AH25-H exhibited no cytotoxic activity and leaded to about the same colony formation inhibition in colon cancer DLD-1 cells. Hence, structural simplification of SgF by lowering its molecular weight, desulfation at C-2 and removing of galactose residues by autohydrolysis at 25°C did not decrease its anticancer activity. This procedure allows obtaining standardized products which can be used as medical.

Structure, enzymatic transformation and anticancer activity of branched high molecular weight laminaran from brown alga Eisenia bicyclis.

The structure of high molecular weight laminaran from brown alga Eisenia bicyclis was investigated by chemical and enzymatic methods, NMR spectroscopy and mass spectrometry. The laminaran from E. bicyclis was characterized as 1,3;1,6-ß-D-glucan with the high content of 1,6-linked glucose residues (ratio of bonds 1,3:1,6=1.5:1), which are both in the branches and in the main chain of the laminaran. The degree of polymerization of fragments, building from 1,3-linked glucose residues with single glucose branches at C-6 or without it, was no more than four glucose residues. The main part of 1,3-linked glucose blocks was builded from disaccharide fragments. 1,6-Linked glucose residues were localized basically on non-reduced ends of molecules. The degree of polymerization of 1,6-linked blocks was not greater than three glucose residues. Laminaran contained laminarioligosaccharides, gentiobiose, gentiotriose and single glucose residues in the branches at the C-6. Laminaran and its products of enzymatic hydrolysis inhibited a colony formation of human melanoma SK-MEL-28 and colon cancer DLD-1 cells. It was shown that decreasing the molecular weight of native laminaran to a determined limit (degree of polymerization 9-23) and increasing the content of 1,6-linked glucose residues increased the anticancer effect. Therefore, they may be perspective antitumor agents.

Isolation of fucoxanthin and highly unsaturated monogalactosyldiacylglycerol from brown alga Fucus evanescens C Agardh and in vitro investigation of their antitumor activity.

Fucoxanthin (FX) and highly unsaturated monogalactosyldiacylglycerol (MGDG) were isolated from the ethanol extract of brown alga Fucus evanescens. Their structures were identified by nuclear magnetic resonance, complemented by electrospray ionization mass spectrometry (ESIMS). MGDG was identified as 1-O-(5Z,8Z,11Z,14Z,17Z-eicosapentanoyl)-2-O-(6Z,9Z,12Z,15Z-octadecatetraenoyl)-3-O-ß-D-galactopiranosyl-sn-glycerol. Antitumor activity of these compounds was tested on human melanoma (SK-MEL-28) cells. MGDG and FX inhibited the growth of human melanoma cells in a dose-dependent manner. IC50 values for growth inhibition were 104 and 114 µM, correspondently.

Structural characteristics and anticancer activity of fucoidan from the brown alga Sargassum mcclurei.

Three different fucoidan fractions were isolated and purified from the brown alga, Sargassum mcclurei. The SmF1 and SmF2 fucoidans are sulfated heteropolysaccharides that contain fucose, galactose, mannose, xylose and glucose. The SmF3 fucoidan is highly sulfated (35%) galactofucan, and the main chain of the polysaccharide contains a →3)-α-L-Fucp(2,4SO3⁻)-(1→3)-α-L-Fucp(2,4SO3⁻)-(1→ motif with 1,4-linked 3-sulfated α-L-Fucp inserts and 6-linked galactose on reducing end. Possible branching points include the 1,2,6- or 1,3,6-linked galactose and/or 1,3,4-linked fucose residues that could be glycosylated with terminal ß-D-Galp residues or chains of alternating sulfated 1,3-linked α-L-Fucp and 1,4-linked ß-D-Galp residues, which have been identified in galactofucans for the first time. Both α-L-Fucp and ß-D-Galp residues are sulfated at C-2 and/or C-4 (and some C-6 of ß-D-Galp) and potentially the C-3 of terminal ß-D-Galp, 1,4-linked ß-D-Galp and 1,4-linked α-L-Fucp residues. All fucoidans fractions were less cytotoxic and displayed colony formation inhibition in colon cancer DLD-1 cells. Therefore, these fucoidan fractions are potential antitumor agents.

Comparative study of polysaccharides from reproductive and sterile tissues of five brown seaweeds.

Sterile and reproductive tissues of five brown algae (Sargassum pallidum, Silvetia babingtonii, Fucus evanescens, Saccharina japonica, and Alaria ochotensis) from Russian Far East seas were compared for the content and monosaccharide composition of fucoidans as well as the content of laminarans and alginic acids. It was proved that reproduction has an apparent effect on fucoidan content and its monosaccharide composition. Fucoidan content in fertile tissues was shown to be 1.3-1.5 times as high as in sterile ones. Based on the present and previously reported data, we believe that fucoidan accumulation during the development of reproductive structure is a general trend for brown seaweeds. Reproduction also caused changes in the monosaccharide composition. As a result, fertile plants synthesized fucoidan with less heterogeneous monosaccharide composition in comparison with sterile ones. Structural changes of this polysaccharide are species-specific and perhaps depend on the type of the synthesized polysaccharide. The fertile plants of S. babingtonii yielded the highest fucoidan content [25% dry weight (dw)] among the tested species, whereas the maximum alginic acid content was found for the sterile tissues of S. japonica-up to 43% dw. There was no general trend in alginic acid content variation during sporulation. The alginic acid content was much higher in the sterile tissues of S. japonica in comparison with the fertile ones. On the contrary, the fertile tissues of A. ochotensis contained more alginic acid than the sterile ones.

Molecular characterization and therapeutic potential of a marine bacterium Pseudoalteromonas sp. KMM 701 alpha-galactosidase.

An alpha-galactosidase capable of converting B red blood cells into the universal blood type cells at the neutral pH was produced by a novel obligate marine bacterium strain KMM 701 (VKM B-2135 D). The organism is heterotrophic, aerobic, and halophilic and requires Na+ ions and temperature up to 34 degrees C for its growth. The strain has a unique combination of polysaccharide-degrading enzymes. Its single intracellular alpha-galactosidase exceeded other glycoside hydrolases in the level of expression up to 20-fold. The alpha-galactosidase was purified to determine the N-terminal amino acid sequences and new activities. It was found to inhibit Corynebacterium diphtheria adhesion to host buccal epithelium cell surfaces with high effectiveness. The nucleotide sequence of the homodimeric alpha-galactosidase indicates that its subunit is composed of 710 amino acid residues with a calculated Mr of 80,055. This alpha-galactosidase shares structural property with 36 family glycoside hydrolases. The properties of the enzyme are likely to be highly beneficial for medicinal purposes.

Effect of 1,3;1,6-beta-D-glucans on developing sea urchin embryos.

The effect of 1,3;1,6-beta-D-glucooligo- and polysaccharides with different structures (from 1 to 10 kDa of molecular mass; from 10-25% of beta-1,6-linked glucose residues content) on the developing embryos of sea urchin, Strongylocentrotus intermedius, was evaluated for the screening of potential positive stimulants. 1,3;1,6-beta-D-glucans with a molecular mass of between 6-10 kDa and at concentrations of 0.05-0.25 mg/ml shown the best modulator effect on the sea urchin embryos. 1,3;1,6-beta-D-glucans increased the survival of the sea urchin embryos up to 2.5-fold compared with the control animals.

The rolC gene induces expression of a pathogenesis-related beta-1,3-glucanase in transformed ginseng cells.

The Agrobacterium rhizogenes rolC oncogene is capable of stimulating production of secondary metabolites in transformed plant cells that suggest its possible involvement in plant defense reactions. We tested whether the gene could also affect production of pathogenesis-related proteins. Using a well-known group of PR-proteins, such as beta-1,3-glucanases, we observed a 10-fold increase in total beta-1,3-glucanase activity in rolC-transformed Panax ginseng cells compared with normal cells. The increase was due to the production of a salicylic acid-activated beta-1,3-glucanase isoform. We isolated cDNA of the corresponding beta-1,3-glucanase gene (Pg-glu1), which shared 38-60% sequence identity with previously reported sequences of plant beta-1,3-glucanases at the protein level. Levels of Pg-glu1 mRNA transcripts were tightly correlated with expression of the rolC gene. Our data, together with previously reported information, indicate that A. rhizogenes can activate plant defense reactions via expression of T-DNA oncogenes.

Brown seaweed protein as an inhibitor of marine mollusk endo-(1-->3)-beta-D-glucanases.

Aqueous ethanol extracts from brown seaweed were found to contain substances inhibiting endo-(1-->3)-beta-D-glucanases, the digestive enzymes of marine mollusks. The inhibitors were detected in 70% of the brown seaweeds investigated. An irreversible protein inhibitor with high specificity for endo-(1-->3)-beta-D-glucanases of marine mollusks was isolated from the brown seaweed, Laminaria cichorioides. As determined by gel filtration, the molecular weight of the inhibitor was 46 kDa. The value of [I]50 (10(-8) M) for the inhibitor was comparable with the corresponding value for natural alpha-amylase inhibitors from terrestrial plants. Chemical modification results indicated that tryptophan, dicarboxylic acid, histidine and probably tyrosine residues of inhibitor molecule are important for interaction of the inhibitor with the enzyme.