Comp. Biochem. Physiol. Vol. 73C, No. 1, pp. 41 to 43, 1982 0306-4492/82/050041-03503.00/0 Printed in Great Britain © 1982 Pergamon Press Ltd

A COMPARATIVE STUDY IN ACTIVITY OF TRITERPENE

INVERTEBRATES OF

VITRO OF PHYSIOLOGICAL GLYCOSIDES OF MARINE

ECHINODERM TYPE

T. A. KUZNETSOVA, M. M. ANISIMOV, A. M. POPOV, S. I. BARANOVA, SH. SH. AFIYATULLOV, I. I. KAPUSTINA, A. S. ANTONOV and G. B. ELYAKOV

Pacific Institute of Bio-organic Chemistry, Far East Science Centre, Academy of Sciences of the U.S.S.R., Vladivostok, 690022, U.S.S.R.

(Received 16 October 1981)

Abstract 1. The cytostatic and antimicrobial activity of triterpene glycosides of 19 holothurian species of the Pacific tropical zone has been studied.

2. It has been demonstrated that yeast and tumor cells display a comparable sensibility to the action of triterpene oligosides of sea cucumbers.

3. Gram-positive and Gram-negative bacteria are not sensible to the action of glycosides in doses to 500 mkg/ml.

4. Triterpene glycoside~stichoposides, thelothurins and oligosides of Holothuria of genus Bohadschia are the most active in relation to fungal, yeast microflora and tumor cells.

INTRODUCTION

It has been ascertained that triterpene glycosides of marine echinoderms possess a diverse physiological activity (Nigrelli, 1952; Nigrelli & Zahl, 1952; Shi- mada, 1969; Pettit et al., 1967; Anisimov et al., 1972). The data obtained in this field show that a search for new sources of glycosides in marine invertebrates and further investigation of biomedicinal properties of these compounds is promising.

The results are given in this paper on the studies of antimicrobial and cytostatic activity of triterpene oli- gosides of sea cucumbers inhabiting the sublittoral zone of islands of the Great Barrier Reef and Western Samoa. The work was conducted during the expedi- tion aboard the research vessel Professor Bogorov in the tropical zone from December 1979 till March 1980.

MATERIALS AND METHODS

The determination of holothurian species was conducted by Dr Jane Marshall (Australia) and Dr V. Kiselyov (U.S.S.R.).

Isolation qJ'91ycoside ji'actions ~]' Holothuria

Glycoside fractions were obtained from aqueous alcoho- lic extracts of animals. After evaporation of ethanol, the aqueous residue was left for 15 hr at +4°C, the precipitate being separated and the supernatant was subjected to extraction times with benzoic and then with butanol. The glycosides Stichopus chloronotus. St. variegatus, Stichopus sp., Holothuria leucospilota, H. discrepans, H. atra pass into the butanol. In those cases when at the cooling of the aqueous residue, sedimentation was not observed (H. nobilis, H. impotiens, H. hilla, H. edulis, Holothuria sp., Actinopyya ohesa, Bohad.~chia .qraefjbi, Act. marmorata, St. horrens), the solution was successively extracted with ben- zole followed by butanol.

Determination o[ physioloqical activity of glycoside fractions The cytostatic effect of glycoside fractions was studied in

accord with a modified Shrek method permitting a differ-

entiation of dead and live cells by coloration of tumorous cells with eosine (Bogoslavaskaya et al., 1976). The cells of sarcoma-37 taken from mice on the seventh day of the tumor development were used in the experiment. To 0.9 ml of the suspension of tumorous cells (1.5-2.0 x l 0 6 cells/ml of medium), for the preparation of which Henksa medium was used (Research Institute of Vaccines and Sera, Tomsk), 0.1 ml of aqueous or aqueous-alcoholic solution of the studied glycoside fractions was added in different concen- trations. The ethanol concentration in the incubation medium did not exceed 0.5~o. The cells were incubated for 1 hr at 37'C. After incubation in 1:2 ratio to the suspen- sion of cells, a 0.1% eosine solution was added and the quantity of coloured and uncoloured cells was determined under the microscope.

The anti-yeast activity of glycoside fractions was studied in vitro on yeast cells Saccharomyces carlsbergensis. The incubation medium was composed of 0.9 ml cell suspension (about 2 x 106 cells in 1 ml of nutrient medium) and 0.1 ml of aqueous or aqueous-alcoholic solution of glycoside to be tested. The cells were incubated for 18 hr at 30C. As a nutrient, medium of the following composition was used in the experiments (g/l): glucose -50; KH2PO4 1.0; KEHPO4_0.1 ; MgSO4"7HzO 0.7; KCI---3; dry hydroly- sate of whale meat 15; 100#/ml of benzyl penicillin; 100 mkg/ml of streptomycine sulphate. Activity of glyco- side fractions was determined by the turbidimetric method. The cells grown on solid nutrient medium containing 1% of agar in the medium of the above-mentioned compo- sition served as an oculum.

A study of the antifungal and antibacterial action was carried out on the cells of Candida albicans, Staphylococcus aureus and Escherichia coil after the previously described method (Shcheglov et al., 1979).

RESULTS AND DISCUSSION

The data on the physiological activity of glycosides are given in Tables 1 and 2. As seen from the results given in Table 1, the glycosides isolated from the sea cucumbers of genus Bohadschia, family Holothurii- dae, have the greatest activity. Some glycosides of this type have already been described (Kitagawa et al.,

41

42 T. A. KUZNETSOVA et al.

Table 1. Data on physiological activity of glycoside fractions of holothurians of family Holothuriidae and Stichopodidae

Effective doses (mkg/ml) inhibiting by 100'~,,

Type of Source of Yeast Dead tumorous glycoside extraction proliferation cells (':{;)

Holothurin A

Holothurin B

Mixture of holothurins A and B

Glycosides of bivittosides type

Mixture of stichoposides C and D

Stichoposides of unknown structure

Mixture of stichoposide D and less polar glycoside

Mixture of thelothurins A and B

Other thelothurins

Holothuria atra 12.5 25.0 H. impatiens 100.0 100.0 Actinopy,qa ohesa 6.20 12.5 H. coluber 25.0 12.5 A. mauritiana 25.0 25.0 Bohadschia graeffei 1.5 6.2 H. atra 25.0 50.0

H. atra 100.0 100.0 H. hilla 50.0 50.0 H. edulis 3.1 6.2 H. leucospilota 50.0 25.0 H. di,screpans 12.5 6.2 Bohadschia marmorata 3. I 12.5 B. argus 3.1 12.5 B. vitiensis 3.1 25.0

Stichopus chloronotus 6.2 12.5 St. varie,qatus 1.5 6.2 Stichopus sp. 1.5 6.2

St. horrens 25.0 50.0

Thelenota ananas 3.1 6.2

Thelenota anax 6.2 25.0

1981). These compounds display an inhibit ing effect in doses of 1.5-3.1 mkg/ml. Their lytic concentra t ions when acting on the suspension of tumorous cells are somewhat higher from 6.2 to 12.5mkg/ml. F rom compar ison with physiological activity of other glyco- sides given in Table 1 we may infer that Holo thur ins A obtained from sea cucumbers Act. obesa, Act . maur- itiana, H. coluber, H. atra, B. graeffei and the mixture of Holo thur ins A and B of sea cucumbers H. discre- pans and H. edulis in sufficiently low concentra t ions inhibit the growth of yeast and tumorous cells. Holo thur in A obtained from different animals differs rather greatly in degree of inhibit ing effect on yeast and tumorous cells. Their effective inhibi t ing doses

vary from 1.5 to 100mkg/ml. The structures of Holothur ins A and B of some species of sea cucumbers are described (Kitagawa et al., 1974, 1979, 1980; Stonik et al., 1979; Kuznetsova et al., 1981).

When studying the lityc action of Holothur ins A and B of H. atra differing only in the quant i ty of monosaccharides, a decrease in activity of glycosides was observed in t ransi t ion from Holo thur in A to Holo thur in B. This confirms a supposit ion of the fact that the carbohydra te chain length influences the manifestat ion of physiological activity of oligosides.

Glycoside fractions of St. variegatus and Thelenota ananas have the most inhibit ing effect among glyco- sides of sea cucumbers of family Stichopodidae. Their

Table 2. Antimicrobial spectrum of action of glycoside fractions of holothurians of family Holothuriidae and Stichopodidae

Effective doses inhibiting growth of micro-organisms by 100,°~i; (mkg/ml)

Candida Staphylococcus Escherichia Type of glycoside Source of extraction albicans aureus coli

Holothurin A Actinopyga mauritiana 6.25 > 500 > 500 Glycosides of bivittosides type Bohadschia vitiensi,s 12.5 > 500 > 500 Mixture of holothurins H. discrepans 50.0 > 500 > 500

A and B H. hilla t00.0 > 500 > 500 Mixture of stichoposides St. r'ariegatus 12.5 > 50t) > 500

C and D Mixture of stichoposides St. horrens 50.0 > 500 > 500

D and less polar glycoside Stichoposides of Stichopus sp. 12.5 > 500 > 500

unknown structure

Triterpene glycosides of marine echinoderms 43

effective doses inhibiting the yeast proliferation by 100% are 1.5 and 3.2 mkg/ml. In relation to tumorous cells the doses are somewhat h igher--6 .2mkg/ml . Chemical structures of stichoposides and thelothurins typical of this family of animals are described (Kale- corn et al., 1976; Stonik et al., 1981).

The antimicrobial spectrum of some obtained gly- coside fractions has been studied by us (Table 2). Gly- cosides of Holothurin A type (Act. maurit iana) and glycosides of B. vitiensis displayed an inhibiting effect when acting on pathogenic fungi Candida albicans in concentrations of 6.25 and 12.5 mkg/ml, respectively. Among glycosides of sea cucumbers of the family Sti- chopodidae the most active are stichoposides of St. variegatus and St ichopus sp., the effective doses of which are equal to 12.5 mkg/ml. The other glycoside fractions of sea cucumbers of both families showed a slight activity in relation to pathogenic fungi.

Gram-posit ive and Gram-negative bacteria were found to be insensitive to the action of glycosides even in doses of 500 mkg/ml.

Acknowledgement--The authors express their gratitude to the Governments of Australia and Western Samoa, also to the local authorities of these states for affording a possi- bility to work in their territorial waters.

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