Triterpene glycosides from sea cucumber Stichopus japonicus Selenka

Comp. Biochem. Physiol. Vol. 78B, No. 2, pp. 421-426, 1984

0305-0491/84 $3.00 + 0.00 '~) 1984 Pergamon Press Ltd

Printed in Great Britain

TRITERPENE GLYCOSIDES FROM SEA CUCUMBER S T I C H O P U S JAPONICUS SELENKA I. I. MALTSEV, V. A. STONIK,A. I. KALINOVSKY a n d G. B. ELYAKOV Pacific Institute of Bioorganic Chemistry, Far East Science Centre, USSR Academy of Sciences, Vladivostok--22, USSR

(Received 18 July 1983)

Abstract--1. Triterpenoidal oligoglycosides from the far eastern sea cucumber Stitchopus japonicus Selenka collected in the Posiet Bay, Japan Sea were investigated. The glycoside fraction was shown to consist of two main components, holotoxins A~ and B~ which have been isolated by chromatography on silica gel columns. 2. The chemical structures of the holotoxins were elucidated on the basis of chemical and physicochemical evidence as 3-O-{[2'-(3-O-methyl-D-glucopyranosyl-(l~ 3)-fl-o-xylopyranosyl-(1-+4)-fl-D-quinovopyranosyl)]-[4'-(3-O-methyl-D-glucopyranosyl-(l-+ 3)-fl-D-glucopyranosyl)]-fl-D-xylopyrano-syl}-holosta-9(l 1), 25(26)-dien-3fl-ol-16-one and 3-O-{[2'-(3-O-methyl-D-glucopyranosyl-(1--+3)-/~-D-xylopyranosyl-(l--+4)-

~-D-quin~v~pyran~sy~)]-[ 4~-(D-g~uc~pyran~syl-( ~-* 3)-[3-D-g~uc~pyran~sy~)]-~-D- xyl~pyran~syl }-h~sta9(1 l),25(26)-dien-3fl-ol-16-one respectively. 3. The proposed structures were compared with close related but not identical structures of holotoxin A and B from other collection of the same holothurian. The probable reasons of the distinction were discussed. 4. The previously-established difference in glycoside structures between Stichopus japonicus and other representatives of the family Stichopodidae was corroborated.

Chromatographic analyses

INTRODUCTION The various species o f sea c u c u m b e r s (Echinod e r m a t a , H o l o t h u r o i d e a ) have been k n o w n to contain holostane-type triterpene glycosides (Elyakov et al., 1973). These substances have interesting physiological activity including cytotoxic (Anisimov et al., 1980) a n d antifungal properties (Shimada, 1969). Some sea c u c u m b e r glycosides were suggested to use as antimycosic drugs (Shimada, 1969). The great interest has developed in studying triterpenoidal holostane-type oligoglycosides of the far eastern sea c u c u m b e r Stichopusjaponicus Selenka (far eastern trepang) since Elyakov et al. (1968) reported the isolation of three glycosides from this species. Later K i t a g a w a et al. (1978) clarified the structures of two m a i n glycoside c o m p o n e n t s n a m e d holotoxins A (1) and B (2) from S. japonicus collected near the J a p a n shore. In a recent short c o m m u n i c a t i o n (Elyakov et al., 1983) we reported different structure for the m a j o r glycoside, holotoxin A 1 (3), from S. japonicus collected in the Posiet Bay, J a p a n Sea. In this work we wish to report the structure elucidations of holotoxin A~ and second m a j o r glycoside holotoxin B~ (4) from the same collection of S. japonicus. The c o m p a r i s o n of these structures with previously described related c o m p o u n d s from sea c u c u m b e r s is also the aim o f the paper. MATERIALS AND METHODS

Animals Sea cucumbers were caught in shallow waters of the Posiet Bay near Vladivostok in June 1982. 421

Thin-layer chromatography (TLC) of glycosides was carried out on plates of silica gel KSK using the solvent system: CHC13-MeOH-H20, 65:25:4 (system A). Holotoxins A l and B 1 were purified by repeated chromatography on silica gel columns (silica gel L, 40/100, Czechoslovakia) using solvent system: CHC13-MeOH-H20, 75:25:1 (system B). The aldononitrile peracetates and acetates of partially methylated methyl glycosides were analyzed by gas-liquid chromatography (GLC). The conditions used for the analyses were: glass columns 5 ft x 81in. (o.d.) with 3~o OF-1 on Chromaton N-AW-HMDS, Ar as the carrier gas, a flow rate of 60ml/min, temperature 110-210°C, 4°C/min (Zvet 110 Chromatograph). GLG-mass-spectrometry study was carried out with LKB 9000S gas chromatograph-mass spectrometer, He was used as the carrier gas with a rate 50 ml/min. The column used was 10ft × ~in. (o.d.) packed with 1.5~o QF-I on Chromaton N-AW-HMDS. The conditions selected for analyses were: injection port 275°C, molecular separator 265°C, ion source 255°C, column 110-210°C, 4c'C/min, ionizing voltage 70 eV.

Preparation of glycosides The ground-up animals (10 kg) were covered with 70~o EtOH and kept 24 hr. The extract was filtered and evaporated to dryness in vacuo. The dry residue was dissolved in n-BuOH (200 ml). Complex of glycoside fraction with cholesterol was obtained by addition of cholesterol (100 mg of cholesterol in n-BuOH per 1 g of dry extract residue) to the solution. The resultant precipitate was separated by centrifugation and dissolved in pyridine to dissociate (5 ml of pyridine per 0.1 g of the complex). Glycoside fraction was isolated after dilution of the solution with ether (5 v/v) by filtration. The glycoside precipitate was washed with ether and subjected to repeated column chromatography on silica gel L in the system B.

422

I. 1. MALTSEV el a].

Glycosides prepared were recrystallized from EtOH. Holotoxin A~ (3), m.p. 258-260'C, [:~]~J= - 6 9 . 2 (c ~ 1.2, pyridine). Holotoxin B 1 (41, m.p. 223 224 C, [~l~~- - 74 ( c - 1.0, pyridine).

tfydrolysis o! g@cosides and progenhTs A glycoside or progenin (2 rag) was dissolved in 12~'0 HC1 (I ml) and heated in a boiling water bath for 2 hr. The acid hydrolysate was neutralized with ion-exchange resin {Dowex - HCO~). Monosaccharides were eluted from the resin with water. The eluate was concentrated in vacuo to give a residue, which was treated with N H 2 O H HC1 (2 mg) and pyridine (1 ml). The mixture was heated at 100 C for I hr. A solution obtained was treated with Ac,O (1 ml) at 100C for I hr and evaporated to dryness under reduced pressure. The aldononitrile peracetates were analysed by means of G[,C and GLC-MS. Methylation of holotoxins and progenins .lbllowed by methalIol)'si~' To a solution of a glycoside or progenin (10rag) in DMSO (1 ml) the dimsyl carbanion solution (2 ml) (prepared from 250mg Nail and 8ml of DMSO, Ar atmosphere, 4(~6OC, l hr) was added the solution was kept stirring under Ar atmosphere for 1 hr. The reaction mixture was treated with CH)J (2 ml), kept stirring for further I hr at 25 C, poured into cold water (5 ml) and extracted with CH3J (3 x 2 ml). The combined extract was washed with saturated Na~SeO3 solution (2 ml) and water (3 x 2 ml). dried and concentrated to give a per-O-methyl derivative. A solution of the derivative in anhydrous 10"i; ttC1/MeOH (2ml) was heated at 7 0 C for 2hr, diluted with MeOH (5 ml) and evaporated in cacuo. The residue was treated with pyridine AceO (l:l) mixture (2 ml) at 100 C for 1 hr. concentrated and analyzed by GLC and GLC-MS methods. Catalytic hydrogenation ol holotoxm A A solution of the glycoside (200 rag) in water (50 nal) was hydrogenated over PtO, (20 mg) at room temperature for 24hr, filtered and concentrated under reduced pressure to give dihydroderivative (5) (195rag). Recrystallization of the product from EtOH furnished 5, m.p. 254-256 C, [:l]~c~= - 61.7 ( c - 1.0, pyridine). Periodic oxidation qf glycosides Glycoside was dissolved in water (2 ml) and NaJO4 (5 rag) was added to the solution. The mixture was kept at 5 C for 12 hr. The reaction products were extracted with n-BuOH (2 x 1 hal) and after evaporation of the solvent hydrolysed as above. Smilh degradalion 01 dehydroderivative (5) The derivative 5 (200 mg) was dissolved in water (50 ml) and N a J Q (100 mg) was added to the solution at 5 C. After 12 hr the solution was filtered with the aid of Polychrome-1 (10 g) (USSR). Polychrome was washed with water (50 ml). The product of oxidation was eluted from the sorbent with 50"i; EtOH (50ml). The extract was concentrated under reduced pressure to dryness and the residue was dissolved in anhydrous EtOH (50ml) and treated with NaBH 4 ( 100 mg) for 10 rain. The reaction was terminated by adding a few drops of acetic acid (pH = 5). The solution was evaporated, residue was dissolved in water (50m|) and chromatographed over Polychrome-l, solvents: water and alter it, 50~{; EtOH. The ethanolic eluale was concentrated to dryness and treated by 0.5'~, HCI (50 ml) for 30 rain. The solution was separated by reversed phase chromatography (Polychromc-1, eluents: water and after it, 5if?{; EtOH). The aqueous eluate was neutralized with Dowex I × 10 resin (HCO~), evaporated in racuo and chromatographed over silica gel L column in the system B to give 10rag of 6, amorphous, [ ~ ] ~ = - 115 (c ,-1.0, water). The alcoholic

eluate was concentrated under reduced pressure, and the residue was recrystallized from ethanol to give progenin 7, m.p. 247 249 C. [~]~'~57 tc-'- /.0. pyridine). In analogous manner. Smith degradation of 7 gave dihydroholotoxinogenin (9), m.p. 270 C, [:~]~ = 88 (c-= 1.0. chloroform). ~C NMR spectrum ,aas identical to one published previousiy (Kalinovsk} c; at_ 19801. ~tt N M R spectrum (CDCI~I: 1.40s (21-CH0. 1.20s (19-CH 0, 1.00s (31-CH0, 0.89d, J2t~/¢ 1.01tz 132-CH0, 0.87d (26,27-('t1~), 0.85 s (30-C[10- 5.30 m I 11-1H), 3.23 dd t3-H:~ 1. 3.11m (8-H), 2.52m (12-2}t, 17-H). 227 Add ~1~,~5~;= 15.3Hz. i t s e l f - 1 . 2 H z (15x-H). 2.08 B br.d. 115fl-H); J,<12x 2..1 He. J,,12/; ~ 5.4 Uz. Jl, s :: 2.0 n z . J[Cxl2/i = 17.7 [tz, J/.'~ s - 3.1 Hz. J:t., 2.0 Hz (measured in C~,D~,). It was consistent with the spectrum published by ('. Djerassi fi)r holost-9(lI)-en-25.3fl-diol-16-one (Tan c* al., 19751.

Enqt'matic h l:th'o@s'i.s O/ holotoxin A I and B E A glycoside (200mg) was dissolved in water (50ml1 and cellulase (50mg, Boehringer Mannheim GmbH) was added. The total mixture was kept stirring at 37 C [\~r 100 hr, concentrated under reduced pressure to dryness, and the residue was extracted with 20 ml CHCIv-MeOH (1:1). The extract was evaporated and chromatographed over silica gel k column in the system B to giw," corresponding progenin. Progenin 8, m.p. 220 C, [~]~¢~'= --50.4 (c 0.5, pyridine). Progenin 10, m.p. 281 282 : [x]~~= 66.7 (c 1.0, pyridine). 5;pectro~raphic equipment The NMR spectra were recorded on Bruker HX-90E and WM-250 spectrometers in pyridine or DMSO with tetramethyls)lane 18 = 0) as an internal standard, IR spectra were taken on Specord IR-75 instrument (CHCI0. Optical rotations were measured on Perk)n- Elmer Model 141 using a 10cm microccll. Melting points were determined on a Boetius apparatus and are reported uncorrected.

RESUI.TS T w o new oligoglycosides, h o l o t o x i n A 1 (3) a n d h o l o t o x i n B~ (4), were isolated f r o m the sea c u c u m b e r Sfich@us japonicus collected near V l a d i v o s t o k . T h e third m i n o r glycoside c o m p o n e n t we c o u l d n o t isolate in sufficient q u a n t i t y for the s t r u c t u r e identification. S o m e structural peculiarities tk~r o b t a i n e d c o m p o u n d s were e s t a b l i s h e d by '~C N M R s p e c t r o s c o p y (Table 1 ). T a k i n g into a c c o u n t these d a t a a n d the p r e p a r a t i o n o f d i h y d r o h o l o ~ o x i n o g e n i n (9) alter two step S m i t h d e g r a d a t i o n o f d i h y d r o h o l o t o x i n A, (5), the h o l o s t a 9(11),25(261-dien-3fi-ol-16-one s t r u c t u r e (111 can be p r o p o s e d for the g e n u i n e a g l y c o n e o f h o l o t o x i n s At a n d B~ (3 a n d 4 c o r r e s p o n d i n g l y ) . T h e s a m e s t r u c t u r e (11) was p o s t u l a t e d for the geniuine a g l y c o n e o f fiolotoxins A a n d B (1 a n d 2) f r o m J a p a n e s e collection o f Stichopus japonicus by K i t a g a w a et al. (1978). Recently, this c o m p o u n d has been o b t a i n e d after a mild acid h y d r o l y s i s o f the glycoside f r a c t i o n f r o m Peter G r e a t Bay c o l l e c t i o n o f Stichopus japonicus ( S h a r i p o v et al., 19801. T h e r e f o r e h o l o t o x i n s A~ a n d B, have identical a g l y c o n e moieties with these r e p o r t e d by K i t a g a w a et al. (1978) ['or h o l o t o x i n s A a n d B. O n the o t h e r h a n d f r o m i n s p e c t i o n o f the s p e c t r a for h o l o t o x i n s A, a n d B~ no simple c o n c l u s i o n can be d r a w n a b o u t their c o m p l e t e identity with the h o l o t o x i n s previously d e s c r i b e d by K i t a g a w a et aL (1976b, 1978). In fact signals o f the c a r b o h y d r a t e p a r t

Triterpene glycosides

423

Table 1. 13C NMR spectra of aglycone parts of holotoxins A t (3) (CsDsN, 333°K) and BI (4) [(CD3)2SO, 290°K] 3 C~ C2 C3 C4 C5 C6 C7

C8 C9 Ci0 *These

4

3

36.5 35.9 27.0 26.6 88.6 88.3 39.9 * 53.2 52.5 21.2 20.5 28.6 28.0 39.0 38.2 151.5 150.6 39.9 * signals were covered by

4

C. CI2 Ci3 C14 Cls Ci6 CL7 CI8

111.3 32.4 55.9 42.2 52.1 212.6 61.7 175.8 Ci9 22.1 C2o 83.0 that of solvent.

110.9 31.2 55.1 41.6 51.7 213.7 60.2 176.0 21.7 83.0

__j/

:

,, , I

(~H2OHI i ,

OHI J

I ~..

',

CH2OH

o

OH a

R'

CH3

O

.o-- I OH

I ~t.

', .O" ~ f . ..¢-

5) I'-"xhOH

.....

1~/

--

I

-

"o

~

v

/

,._

,

:

/

-I1R=

(~

2R:-

(

// ,'

~ R'=CH:OH,R"=H

O ] --"~7

R=

3 R = - - - - ~ , R' = H, R"= CH3 "-'-~,

]

I('CH3

4 R=

o

--I OH

/R

R'=H A T . - t . A . A . '

IoX~____( :Holx~.__~ , ~ ~ / .......

4 26.6 37.8 21.7 37.6 145.3 110.6 22.3 16.3 27.7 20.5

O~'-.,./O . ~ - - - ~

CH20HI t

[ O R " 6"]1 ~

3 27.0 38.5 22.2 38.1 145.5 110.5 22.5 16.9 28.4 20.7

2,4,6- tri- O- methyl- 3-O- acetylglucopyranoside, flmethyl 3- O- methyl- 2,4- di- O- acetylxylopyranoside and ~t-methyl 2,4,6-tri-O-methyl-3-O-acetylglucopyranoside (in order of the increase of retention time at GLC) identified by GLC and GLC-MS. The values of chemical shifts of anomeric carbons [105.3(4C), 104.9, 102.9 ppm] in 13C NMR spectrum of 3 indicated the fl-configuration for all glycoside bonds (Shashkov et al., 1976). The sole progenin (8) was obtained by enzymatic hydrolysis of holotoxin AL with cellulase. When hydrolyzed, it yielded xylose, quinovose and 3-0methylglucose (2:l:l). After methylation of 8 followed by methanolysis and acetylation, the following compounds were identified: fl- and s-methyl 2,3,4,6tetra-O-methylglucopyranosides, s-methyl 3,4-di-Omethyl-2-O-acetylxylopyranoside, fl- and or-methyl

carbons of holotoxin A~ (Table 2) were practically identical with corresponding signals of the stichoposide C and astichoposide C spectra (Stonik et al., 1982a), i.e. the glycosides with other monosaccharide composition, than it was established for holotoxin A (Kitagawa et al., 1978). Holotoxin A1 gave in agreement with the spectral data the following monosaccharides (ratio): D-glucose, o-quinovose, o-xylose and 3-O-methyl-Dglucose (1 : 1: 2: 2). Methylation of 3 at Hakomori conditions followed by methanolysis and acetylation led to the formation of fl- and ~t-methyl 2,3,4,6-tetra-O-methylglucopyranosides, fl- and ~t-methyl 2,3-di-O-methyl-4-Oacetylquinovopyranosides, fl- and a-methyl 2,4-diO-methyl-3-O-acetylxylopyranosides, s-methyl 3-0methyl- 2,4- di - O- acetylxylopyranoside, fl- methyl

' ,

C2] C22 C23 C, 4 C~5 C26 C27 C30 C31 C32

OH

CH3 I CH2OH

~HOH

,~O r,,_ r----O, V - - - - O - - C H /OCH3~4"L~-3~ [

OH

OH

6 Scheme 1.

R ' = H , R"=H

424

I . I . MALTSEV et al. Table 2. ~3C N M R spectra of carbohydrate parts of glycosides 3, 7, 8 (CsDsN, 333 K), compound 6 (D20, 290 K), glycosides 4 and 10 [(CD3)2SO, 290 K] (,E C~ C~ C~ C j, C~t C~ C~ C] C{ C~ C~ C! C] C4 C~ C~ Ca C4 C] C 4, C46 OMe C~ C~ C~ C] C~ C~ C} C2 C~ C] C~ C~ OMe

3

4

105.3 83.0 75.4* 78. I 64.0 105.3 76.3 75.7* 85.7 71.8 18,1 104.9 73.4* 87.7 69.0 66.4 105.3 74.9 87.7 70.8 78.1 62.4 60.4 102.9 73.2* 88. I 70.0 78. I 62.4 105.3 74.9 87.7 70.9 78. ] 62.5 60.4

104.2 82.0 74.3 76.5 63.4 104.2 75.5 74.8 84.8 70.8 17.6 103.4 72.3 87.5 68.5 65,5 104.2 73,8 86. I 69.7 76.9* 61.2 60.4 101.1 72.3 87.5 68.9 76.6* 61.2 104.2 74.1 77.2* 70.5 77.2* 61.2

6

7

8

10

106.8 75.(I 76. I 78.1 64.3

ii)5.5 83.9 77.9 71.0 66.5 105.5 76.5 75.7 86.0 71.9 18.1 104.9 73.5 87.8 69.2 66.5 105.5 75.0 87.8 71.0 78.2 62.6 60.4

104.2 82.0 74.1 76.2 63.3 104.2 75.2 74.8 84.8 70.6 17.5 103,4 72.1 87.4 68.2 65.3 1(14.2 73.8 86.1 69.6 77.0" 61.2 60.2 101.7 73,0 77.2* 7(I.3 76.6* 61.2

63.5 85.2 67.9 18.4 103.9" 73.8 85.2 68.9 65.6 103.7" 73,8 86.1 70.0 78.1 61.6 60.8 103.0 73.2 88.2 70.0 78. I 62.5 105.5 75,0 87.8 70.9 78. [ 62.5 60.4

*These assignments may be interchangeable.

2,3-di-O-methyl-4-O-acetylquinovopyranosides, /.Cand :t- methyl 2,4- di - O- methyl - 3 - O- acetylxylo pyranosides. The '3C N M R spectrum of its carbohydrate part correlated with our supposition for the structure of 8: signals of anomeric carbons were observed at 105.5 (3C), 104.9 ppm C-4 signal in the first xylose residue was shifted in comparison with 3 from 78.1 to 71.0ppm as a result of the elimination of the glucose-3-O-methylglucose fragment from this position. In order to finally solve the structure elucidation, Smith degradation was carried out with the dihydroderivative of holotoxin A~ (5) (which has more stable aglycone to an acid treatment). The only quinovose was broken by treatment 5 with periodate. The progenin 7 and bioside of deoxyerythritol (6) were obtained by Smith degradation of 5. Monosaccharide analysis of 6 showed that it contains equimolecular mixture of xylose and 3-0methylglucose. After methylation followed by methanolysis and acetylation, it gave 2,4-di-O-methyl3-O-acetyl-l-deoxyerythritol, fl- and :t-methyl 2,3,4,6-tetra-O-methylglucopyranosides, fl- and ~methyl 2,4-di-O-methyl-3-O-acetylxylopyranosides. The t3C N M R spectrum of 6 is presented in Table 2. Therefore, the structure of 6 has become unequivocal. Progenin 7 contained one mole each of xylose, glucose and 3-O-methylglucose. After methylation of 7 followed by methanolysis and acetylation were identified: [~- and :t-methyl 2,3,4,6-tetra-O-methyl-

glucopyranosides, /3- and 7-methyl 2,3-di-Omethyl-4-O-acetylxylopyranosides, /3- and :t-methyl 2,4,6-tri-O-methyl-3-O-acetylglucopyranosides. Smith degradation of 7 (the xylose residue was decomposed) gave earlier known genin 9 (Kitagawa et al., 1976a). Based on the accumulated evidence, the structure of holotoxin A~ has been established as 3-0{[T-O-O-methyl - D - glucopyranosyl-( 1~ 3)-/3- D- xylopyranosyl-(1 ~ 4)-/3-o-quinovopyranosyl)]-[4'-(3-Omethyl-D-glucopyranosyl-(l ~ 3)-/3-D-glucopyranosyl)]-fl - D - xylopyranosyl} -holosta-9(11 ), 25(26)-dien3/?-ol- 16-one. Methylation of holotoxin B~ (4) foUowed by methanolysis and acetylation led to the formation of the same compounds as the holotoxin A~ (3) gave at similar treatment. The ~3C N M R spectrum of holotoxin B~ was closely related to one of 3 (Table 2). On the other hand acid hydrolysis of 4 gave the other ratio of monosaccharides in mixture as compared with 3, namely: D-glucose, D-quinovose, D-xylose and 3-O-methyl-D-glucose (2:1:2:1). In our further investigation of the glycoside structure we studied progenins (8, 10) isolated after enzymatic hydrolysis of 4 with cellulase. Progenin 10 lost one glucose residue as compared with holotoxin B~. Hence this glucose residue occupied the terminal position in 4. Methylation of 10 followed by methanolysis and acetylation afforded fland :t-methyl 2,3,4.6-tetra-O-methylglucopyranosides, /3- and ~-methyl 2,3-di-O-methyl-4-O-

425

Triterpene glycosides acetylquinovopyranosides, fl- and a-methyl 2,4-diO-methyl-3-O-acetylxylopyranosides, fl- and ormethyl 3-O-methyl-2,4-di-O-acetylxylopyranosides. The other progenin obtained after enzymatic hydrolysis of holotoxin B1 was identified as 8 isolated earlier from holotoxin At after the same procedure. (Comparison of ~3C N M R spectra, constants, acid hydrolysis products, and compounds which were obtained after methylation followed by methanolysis and acetylation.) Consequently, the structure of holotoxin B~ has been established as 3-O-{[2'-(3-O-methyl-D-glucopyranosyl-(1 ~ 3)-fl-D-xylopyranosyl-(1 --* 4)-fl-Dquinovopyranosyl)]-[4'-(D-glucopyranosyl-(1 ~ 3)-flD - glucopyranosyl)] - fl - D - xylopyranosyl } - holosta 9(11),25(26)-dien-3fl-ol- 16-one. DISCUSSION The holotoxins isolated from our collection had the distinguished carbohydrate parts compared with holotoxins A and B of Kitagawa et al. (1978) while the sapogenol moieties were unchanged. It was supposed (Kitagawa et al., 1978) that the carbohydrate ingredients in the Stichopus japonicus oligoglycosides are able to vary if the habitat (the locality, etc) of the animal differs. This is one of the possible reasons for observed distinction of two S. japonicus collections. On the other hand as has been noticed in our studies on the glycosides of the Pacific, Indo-Pacific and Atlantic sea cucumbers and in analyses of literature data, the glycoside fractions from the animals collected in the different areas (although the species is identical) consist of the same components. However relative quantity of these components within a fraction would possibly vary. Moreover, there are a definite regularity in the carbohydrate part structures of sea cucumbers and a resemblance or identity of such structures for related species. Actually, the comparison of the carbohydrate chain structure for holotoxin At (3) with those of the major glycosides from related species showed their identity. For example, the major glycosides from the Indo-Pacific S. chloronotus (Kitagawa et al., 1981, Stonik et al., 1982a), S. variegatus (Stonik et al., 1982a) and Caribbean Astichopus multifidus (Stonik et al., 1982a) possess the same structures of the carbohydrate moiety as 3. Thelenotoside A from the glycoside fraction of Thelenota ananas (Stonik et al., 1982b) (other genus of Stichopodidae family) includes the same oligoside fragment (12) which is present in holotoxins At and BL. Similar fragment (13) which was found in holotoxins A and B seems to be more characteristic for Holothuriidae glycosides than for Stichopodidae. OH H 0 CHaOH

o

.o

I OH

CH3 I o

R

o

.o

I OH

/ OH

Scheme 2.

12 R=H

13 R = CH~I-I

This fragment (13) was found in the glycosides of "holothurin A" type. Chanley and his group (1959, 1969a,b) proposed the name "holothurin A" for the glycoside fraction from Actinopyga agassizzi which includes the components possessing 12ct-hydroxyholost-9(11)-ene system in aglycone moiety and four monosaccharides (glc, xyl, qui, 3-O-Me-glc). Later similar glycosides were identified in a number of sea cucumbers belonging to Holothuria and Actinopyga genera (family Holothuriidae). The acid hydrolyses of these fractions gave 22,25-oxidoholothurinogenin, 17-dehydroxyholothurinogenin, holothurinogenin and other related artifact sapogenols. It was clear that the sapogenols were derived from corresponding glycoside components of "holothurin A" (Elyakov et al., 1973, 1975). Some of these glycoside components of holothurin A" have been isolated. Holothurin A from Pacific Holothuria leucospilota (Kitagawa et al., 1979), holothurin At from Caribbean H. fioridana (Oleynikova et al., 1982), echinoside A from Pacific Actinopyga echinites (Kitagawa et al., 1980), holothurin A: (=echinoside A) from the Indo-Pacific Bohadschia graeffei (Kalinin and Stonik, 1982) possess the identical structure of carbohydrate chain (13, sulfated at C-4 of xylose). So far the inconstancy of carbohydrate chain structures in different collection was described only for holotoxins from all known oligoglycosides of sea cucumbers (Kitagawa et al., 1976b; Kitagawa et al., 1978; present paper). Therefore, the presence or absence of variation in a carbohydrate chain of holotoxins depending on habitat of the animal seems to require detailed reinvestigation. This may be marked that glycosides from the majority of Stichopodidae species differ in aglycone part from those of S. japonicus and possibly S. californicus. Among a series of questions which can be asked about the biogenesis of holotoxins such as following: are there the principal distinction between carbohydrate moieties of holotoxins on the other hand and glycosides of the rest Stichopodidae species on the other hand? May oligoside parts of holotoxins be originated from the diet of the animal? Further investigations must reply to these questions.

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