Quantification of a saponin steroid from the starfish Asterias Vulgaris

Comp. Biochem. Physiol., 1976, Vol. 54a, pp. 561 to 563. Pergamon Press. Printed in Great Britain

QUANTIFICATION OF A SAPONIN STEROID FROM THE STARFISH ASTERIAS V ULGARIS MICHAEL W. GILGAN 1, RICHARD K. PIKE2 AND JOHN W. APSIMON3 1Fisheries and Marine Service, Halifax Laboratory, P.O. Box 429, Halifax, Nova Scotia, Canada B3J 2R3, 2Department of Pharmaceutical Chemistry, Health Protection Branch, National Health and Welfare, Tunney's Pasture, Ottawa, Ontario, Canada K1A 0L2 and 3Department of Chemistry, Carleton University, Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6 (Received 18 June 1975)

Abstract--1. Solvolysis of crude saponin was found to be preferable to mineral acid hydrolysis. 2. XAD-2 Resin was found to be superior to n-butanol in extracting saponin from starfish. 3. Whole body extraction leads to greater yields of saponin than did "milking". 4. The seasonal and limited geographical variation of the asterone content in starfish was studied. et al. (1973), or by chopping and soaking them in distilled water (2-3 hr). These aqueous extracts were filtered, with Studies on the toxic principles in starfish began late the aid of Celite 545, under reduced pressure through filter in the fifties at the University of Tokyo, Hashimoto paper (Whatman No. 42) and passed through a column of washed (see manufacturers' recommendations) Amber& Yasumoto (1960). These workers explored the distribution of saponin in echinoderms and also the sea- IRe XAD-2 resin (B.D.H.) (70 em 3. bed vol/200 ml aqueous solution) at a rate of 4 bed vol/hr. The column was washed sonal variation of saponin concentration in starfish, with water (dist. and degassed, 1 bed vol) and methanol Yasumoto et al. (1966). The quantification of the (degassed, 1 bed vol). The combined eluates containing the saponin present in the starfish studied (Asterias amurcrude saponins were dried on a rotary evaporator at 40°C. ensis) was made by assaying the haemolytic activity The dried residue of crude saponin was used for solvolysis of the saponin. Reports of investigations into the and gas chromatography. nature of the saponin aglycones from starfish began For mineral acid hydrolysis crude saponin solution to appear in the early 1970's, Ikegami et al. (1973); (4 ml) in water (10mg/ml) were made up to a vol of 8 ml to given concentrations of hydrochloric acid. Samples were Kamiya et al. (1974); Shimizu (1972); Smith et al. then heated at 100°C for the specified lengths of time. After (1973), and up to the present the aglycones isolated cooling the acidic solution was extracted with chloroform have been steroids of relatively similar structure. The (4 ml x 2). This organic layer was washed with sodium hyone most significant to this reportl 5~,pregn-9(ll)drogen carbonate solution (saturated) and then sodium ene-3~,6~diol-20-one, referred to by the trivial name chloride solution (saturated)~ The chloroform solution was asterone in this paper, has been shown to be present dried over calcium sulphate, filtered and evaporated to give in the common East Coast starfish, ApSimon et al. a syrup containing the aglycones. The residue was dis(1973). solved in aqueous methanol (70%) which was washed with Since asterone is closely related in structure to hexane (1/10 vol x 2). The aglycone was extracted from the aqueous methanol with dichloromethane (equal vol x 2), many of the vertebrate steroid hormones and might evaporation of this organic layer gave a residue containing provide a commercial source for potential conversion to the corticosteroids, Gurst et al. (1973), it seemed the steroidal aglycone. Dried aliquots of crude saponin were also solvolyzed desirable to establish the abundance of this compound in starfish on a seasonal and limited geo- by dissolution in tetrahydrofuran (T.H.F., refluxed and distilled from potassium hydroxide pellets) containing 70~o graphical basis. The previous studies of this nature perchloric acid (1 ml/70ml T.H.F.). The saponin suspenhave relied upon the haemolytic activity of the sion in T.H.F. (1 mg/ml) was heated at the desired temperasaponin to establish its concentration, a procedure ture for varying lengths of time (routinely 50°C for 4 hr) which is sensitive to all the saponins present. Since in screw-capped culture tubes. The solvolysis was stopped by adding saturated aqueous sodium bicarbonate (equal we were primarily interested in the presence of a parvol). Ethyl acetate was then added (equal vol) and the comticular saponin a quantification method was required bined organic phase was washed with water (0.5 vol x 2) which was specific to this saponin. and evaporated. The dried residue was dissolved in a measured volume of ethyl acetate (usually 1 ml) for GLC analysis. MATERIALS AND METHODS Aliquot samples of extracted asterone in ethyl acetate Duplicate samples of Asterias vulgaris (600-1400 g) were were dried in vacuo over Drierite. The residue in 0.3 mi collected from three areas on the Atlantic Coast of Nova reacti-vials (Pierce Chemical Co.) was dissolved in anhyScotia, Sober Island, Sambro Bay (Nr. Halifax) and Sandy drous pyridine (10 #1) treated with N,O-bis(trimethylsilyl)Cove. The outlying collection points were about 100 miles trifluoroacetamide (10/A BSTFA, Pierce Chemical Co.) in opposite directions of Halifax. The samples were stored and allowed to react (0.5 hr room temp) before GLC analyat -30°F and subsequently analyzed by the scheme de- sis. Routine analyses were performed with a Hewlett-Packard model 402 gas chromatograph equipped with a scribed. Preparation of aqueous extracts of starfish was made 4ft x 4mm (i.d.) glass column packed with 1 or 3% either by "milking" them as previously described, ApSimon OV-101 on Chromosorb WHP (Pierce Chemical Co.). The 561

INTRODUCTION

562

MICHAEL W. GILGAN, RICHARD K. PIKE AND JOHN W. APSIMON 0.05

Table 1. The efficiency of saponin removal from aqueous solution by XAD-2

0'04

Fraction

m Ist cycle

0'03 o

/

2nd cycle

0"02

-/

o n-

• 0.SN HCt • O.I N HCt

/

•

0"01

Methanol

48.2

68.9

--

trace

50% Methanol

10.5

2.3

Methanol

29.1

1.5

116

2.6

* Average of duplicate estimations.

0.30

0"20

Table 2. Comparison of saponin extraction with XAD-2 and with the "classical" n-butanol method

I

4

8

I

12

I

I

16

[

20

I

24

Time,

28

I

i

32

I

36

40

hr

Fig. I. Hydrolysis of saponin in hydrochloric acid. oven temperature was 240°C with the helium flow of ca. 60 ml/min. Detections were always by hydrogen flame ionization. Peak areas were estimated by weighing peak cutouts of either the original chart paper or of Xerox copies. An internal standard (n-hexacosane) was routinely used. RESULTS AND DISCUSSION Since there were no published results which indicated optimal conditions for the commonly used hydrochloric acid hydrolysis of saponin, aliquots of a crude saponin preparation were subjected to hydrolysis at 100°C by aqueous acid from concentrations of 0.05-5 N. Concentrations of 1-5 normal were found to largely or wholly destroy asterone while progressively lower concentrations (0.5-0.05 N) yielded progressively higher amounts of asterone (Fig. 1). As a result an attempt was made to achieve hydrolysis by the milder conditions developed for urinary steroid conjugate hydrolysis, Burnstein & Lieberman (1958); Burnstein et al. (1960). A comparison of the asterone yield with time and temperature is shown in Fig. 2. The release of asterone was essentially complete after 4 hr at 50°C and was significantly greater than that by hydrochloric acid hydrolysis. The stable plateau indicated that no significant asterone destruction occurred. This solvolytic procedure (see Materials and Methods) was the one routinely used. 0"50

-

-

0,0

./.j'/'""

°/

.~

(%) 24.8

The frequently used extraction procedure for saponins using n-butanol, ApSimon et al. (1973), was found to be tedious and lead us to develop a scheme using reversed phase adsorption on a resin, AmberliteXAD-2. Table 1 shows that an XAD-2 column efficiently (ca. 90%) removed saponin (measured as asterone) from an aqueous solution. Recycling the aqueous solution a second time through the resin removed only another 4% of the total saponin content. A direct comparison on equal weights (1.1 kg) of starfish was made, one extracted using the n-butanol technique the other extracted using the XAD-2 resin technique. The results of this comparison, shown in Table 2 indicate that the XAD-2 reversed phase adsorption technique achieved slightly more than twice the yield of saponin as did the n-butanol method. Finally a comparison of the "milking" technique and whole body extraction was made. From Table 3 it can be seen that the yield of asterone via the "milking" technique (35%) was not sufficient to warrant use of this method for asterone estimation. However the product saponin must have been purer than that obtained from the whole body extracts (131 #g asterone/mg crude saponin for the former compared to 84 #g for the latter). As a result of these foregoing studies the following sequence of procedure was adopted as a standard for subsequent studies: 1. Preparation of whole body extracts. 2. Removal of saponin by XAD-2 resin. 3. Solvolysis of crude saponin by perchloric acid in tetrahydrofuran. 4. GLC separation and quantification of the TMS ether of asterone. This scheme was used to estimate the asterone content of starfish from three areas over the span of a year. These results shown graphically in Fig. 3 indicate the variation in asterone abundance. It is not clear as to why there is variation but it may either

I

0

-~,

(mg) 13.4

Aqueous Solution after two cycles

O.05N HCI

Percent of Total RecoveredAsterone in Fractions*

50% Methanol Water Wash

o

Weight of Dried Fractions

50"C

OqO

Method

Total Sapontn Dry Weight (rag)

Asterone Content of Sapontn (pg/~)

Total Asterone Content (mg)

1660 1670

3g 42

64 70

787

41

32

20"C l

•

I

J

i

l

2

3

4

5

Time,

,,j

q 6

hr

Fig. 2. Solvolysis of saponin in HC10~I'HF.

-

XAD-2 n-Butanol

Saponin steroid from A. vulgaris Table 3. Comparison of "milking" and whole-body extraction for saponin recovery by XAD-2 Total Weight Dry Saponln (~)

Hethod

Total Asterone Percentof Content (mg) Total Asterone (%)

96.1

Milking Carcasses after milking

273

12.6

35

22.9

65

reflect water temperature which is locally very variable but highest is September or it may reflect diet. SUMMARY The quantitative usefulness of saponin isolation schemes, saponin hydrolysis and subsequently the quantification of free asterone were assessed. A study of the seasonal and limited geographical variation of saponin (measured as asterone) in starfish was made; this assay procedure would appear to be pertinent

IO E

/

• Sober island

]

• Sandy Cove

~ 6

o

P 2 o

0

I

t

I

t

I

I

I

I

I •1

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I

Jan. Feb. Mar. Apr. May JuneJuly AurJ.Sep.Oct. Nov. Dec.

Collection date

Fig. 3. Seasonal and geographical variation of asterone.

563

to investigations of all starfish saponins, saponins in general.

if not

Acknowledgements--This work was made possible through the generous financial assistance of the National Research Council (PRAI grant). We are also indebted to the staff of the Fisheries and Marine Services, Halifax, without whose willing help this study could not have been made. REFERENCES APSIMON J. W., BUCCINI J. A. & BADRlPERSAUDS. (1973) Marine organic chemistry--I. Isolation of 3fl,6ct-dihydroxy-5~t-pregn-9(11)-one-20-one from the saponins of the starfish Asteriasforbesi. A rapid method for extracting starfish saponins. Can. J. Chem. 51, 850-855. • BURNSTEIN S. & LIEBERMAN S. (1958) Kinetics and mechanism of solvolysis of steroid hydrogen sulphates. J. Ant chem. Soc. 80, 5235-5239. BURNSTEIN S., JACOBSOHNG.. M. & LIEBERMANS. (1960) The cleavage of androsterone fl-D-glucopyranosiduronic acid in organic media. J. Am. chem. Soc. 82, 1226-1230. GURST J. E., SHEIKH Y. M. & DJERASSI C. (1973) Synthesis of corticosteroids from marine sources. J. Am. chem. Soc. 95, 628-629. HASHIMOTO Y. & YASUMOTOT. (1960) Confirmation of saponin as a toxic principle of starfish. Bull. Jap. Soc. scient. Fish. 26, 1132-1138. I ~ G A ~ S., KAMIVA Y. & TAMURA S. (1973) Structures of two C-27 steroids constituting asterosaponins A and B. Agri. biol. Chem. 37, 367-370. KAMIYAY., IKeGAM1S. & TAMURAS. (1974) A novel steroid 3fl,6~-15,24-tetrahydroxy-5-cholestane from asterosaponins. Tetrahedron Lett. g. 655-658. Smmzu Y. (1972) Characterization of an acid hydrolysis product of starfish toxins as a 5~ pregnane derivative. J. Am. chem. Soc. 94, 4051-4052. S~n3"H D. S. H., TURNER A. B. & MACmE A. M. (1973) Marine steroids. Part 1. Structures of the principal aglycones from the saponins of the starfish, Marthasterias glaciales. J. chem. Soc. 1745-1754. YASUMOTOT., TANAKAM. & HASHIMOTOY. (1966) Distribution of saponin in echinoderms. Bull. Jap. Soc. scient. Fish. 32. 673-676.