ANALYTICAL

BIOCHEMISTRY

81,

196-208 ( 1977)

Estimation of a Steroid Released from the Crude Saponins of the Starfish, Asterias vulgaris, by Solvolysis: Seasonal and Geographic Abundance
B. G. BURNS,* M. W. GILGAN,* V. H. LOGAN,* J. BunNELL,? AND J. W. APSIMONJT
*Department of the En~Yronment. Fisheries and Marine Service. P.O. Box 429, Halifax, Nova Scotia Canada B3J 2R3; and tDepartment of Chemistry. Carleton University, Ottawa. Ontario KIS 5B6, Canada Received December 20, 1976; accepted April 12. 1977 Conditions were optimized for the quantitative extraction and isolation, by reversed-phase adsorption, of a saponin, from the common or purple starfish, Asterias vulgaris. and for the subsequent solvolytic release and quantitation of a steroid aglycone, Sa-pregn-9tl l)-ene-3P,6a-diol-20-one. With the method developed, the abundance of the aglycone was found to vary seasonally and was greatest from April to June at two locations on the Atlantic coast of Nova Scotia. When several locations were examined during October, the abundance of the aglycone was also found to vary geographically.

The toxic component of many starfish was found to be saponin (1). From the saponins of starfish, several workers have isolated a number of steroid aglycones [cf. Ref. (2)]. Of these, 5a-pregn-9(1 l)-ene-3P,6adiol-20-one (here referred to as asterone) was of particular interest, as it could be the starting material for the synthesis of steroid pharmaceuticals (4). This compound has now been isolated from acid hydrolysates of the saponins of several starfish (5-9). Shimizu (13) suggested that asterone was generated from a hypothetical parent steroid by acid hydrolysis. The recent work of Kitagawa et nl. (14) with saponins of Acanthasterplanci supported Shimizus hypothesis, since they were able to isolate a sapogenol which would yield asterone in the presence of acid. The validity of the hypothesis for Asterias saponins remains to be determined. A knowledge of the nature and abundance of the starfish saponins would be of potential value in two ways. First, the aglycones mentioned above might make useful chemicals. Second, if the residual body mass of the starfish could be sufficiently detoxified by removal of the saponin, it might be valuable as a protein supplement for animal feed. To obtain information on both of these factors, we wished to determine
196
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ESTIMATION

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the seasonal and geographic distribution of the saponin which yields asterone in the locally abundant common or purple starfish, Asteriru r~@a~is, and, if possible, to develop saponin extraction procedures which might allow usable protein recovery. Previous studies showed that the saponin could be usefully recovered from aqueous extracts by adsorption on a resin, and that the desired aglycone was released most efficiently by solvolysis (3,3). We now report further development of the procedure, permitting analysis of more samples and, hence. more reliable results. The methods developed should be applicable to related saponins as well. MATERIALS AND METHODS

To facilitate rapid analysis, the solvolysis procedure was optimized under various conditions of temperature, time. and acid concentration. Dried samples of a crude saponin preparation, obtained by Amberlite XAD-2 resin adsorption as reported previously (3), were dispersed in purified tetrahydrofuran (refluxed over KOH pellets and distilled under anhydrous conditions)-perchloric acid solutions (THF-PCA) at 1.0 mg of crude saponin/ml. The THF-PCA solutions contained 10. 20, or 40 ~1 of 60% perchloric acid/ml. Samples of each preparation were heated at 50, 60, or 70C. At 1, 2. 3, or 4 h under each condition, a portion (200 ~1) of each was removed and analyzed for asterone content as described in the general procedure. The results are presented graphically in Fig. 1.

To determine the adsorptive ability of resins from different sources. replicate samples of a standard crude saponin preparation (as above) were processed with the resins. and the recoveries of saponin-containing asterone (asterone-saponin) were determined. Recoveries were compared with the asterone content of the standard saponin. The resin samples, 1.5 g dry weight of either the Drug-Skreen resin (Brinkmann Instruments, Ltd.. Canada) or Chromosorb 102, 100/120 mesh (Applied Science Laboratories, Inc.). were poured into empty Drug-Skreen columns ( I .O cm i.d. x 6 cm) equipped with glass-wool plugs and were washed as in the general procedure for small columns. Replicate l.O-mg crude saponin samples were dissolved in 10.0 ml of distilled water and were extracted with the resins as in the general procedure (Table 1). To determine losses due to handling alone, crude saponin samples were dissolved in methanol water and were then treated as though they had been eluted from columns (Handling-loss controls. Table 1). To determine

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ET AL.

PCA/ml

THF 0 50" A 60" 0 70"

2O.Opl

PCA/ml

THF

40.0

pl PCA/ml

THF

SOL~OdlS

DURATION
FIG. 1. The asterone yield from crude temperature. time, and acid concentration. saponin by solvolysis

(HR.!?)
under varying conditions of

the effect of solvent washes, fresh Chromosorb 102-resin columns were washed by two procedures prior to use with standard saponin samples: (i) acetone (2 x 10 ml), methanol (2 x 10 ml), water (2 x 10 ml); (ii) acetone (2 x 10 ml), 0.1 N NaOH in isopropanol (10 ml), isopropanol (10 ml), methanol (10 ml). and water (2 x 10 ml). Similarly, the adsorptive ability of fresh Chromosorb 102 was compared with that of Chromosorb 102 which had been used for an extraction and then washed by procedure (ii) prior to use (Table 1). Crude Extract Preparutim Aqueous extrcrcts. The starfish were coarsely ground with a meat grinder and then were blended to a uniform paste with a high-speed blender (Lourdes, Multi-Mixer Model MM-IA) without additional water. The homogenates were suspended in water (1.0 g/1.0 ml), and samples (10.0 ml) were removed and stored at -30C. For extraction with the adsorptive columns, thawed samples were first centrifuged at low speed to remove coarse solids and then were centrifuged through a Celite 545 (Johns Manville)-Cellulosepulver MN 300 HR (Macherey, Nagel and Co.) column (1 cm i.d. x 1 cm Celite/l cm Cellulosepulver/l cm Celite) to remove fine solids. Columns and vessels were rinsed with small volumes (2 ml) of distilled water. The filtrates were

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THE Ael~rru OF AMBERLITE XAD-2 FROM Two ASTERONE-SAPONIN AND THE EFFECTIVENESS

SOURCES TO RECOVER CRUDE OF WASHING THE RESIN Average recovery tri) 24.9 3.3 78.7 72. I

Resin Brinkman Drug-Skreen

Chromosorb

101

Chromosorb

103. washed

as in procedure

x0.2 87.6 86.7 97.0 X2.6 8l.J 76.9 70.7 6X.6 122 I08 99

Chromosorb Chromosorb

102. washed IO?, fresh

as in procedure

ii

Chromosorb

102, used and washed

Handling-loss

control

processed with Chromosorb 102 columns as described in the general procedure to determine the effectiveness of recovery of the asteronein a similar manner (blanks, saponin. Water samples were processed Table 2). Samples (1.0 ml) of crude filtrates, produced as above. were diluted to 10.0 ml with malic acid buffer (0.01 M, pH 3.5) and were centrifuged to remove the protein precipitate. Similarly, crude filtrate samples (1.0 ml) were diluted to 10.0 ml with distilled water. Again. the recoverable saponin was collected by resin adsorption, and the asterone content was determined as in the general procedure. Methrzolic estr-acts. The samples (2.00 g) of the starfish homogenate prepared for the aqueous extracts were suspended in methanol (2.3 ml/l g of homogenate) in centrifuge tubes and were mixed thoroughly. The precipitate was removed by centrifugation, the supernatant fluid was collected, and the residue was washed with 70% methanoliwater (4.0 ml). The methanolic extracts were combined and extracted with

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benzene (2.0 ml, discarded), and the methanol was evaporated with a rotary vacuum evaporator (maximum bath temperature: 40C). The aqueous residue was diluted to 10.0 ml with distilled water. A sample of this (2.5 ml) was further diluted to 10.0 ml with water and was processed with a resin column to determine the recoverable saponin. Since low concentrations of methanol (less than 10%) should not have interfered with saponin recovery, samples were prepared for dilution trials in the same manner as above. However, instead of the methanolic extract being evaporated after benzene extraction, it was diluted to 100.0 ml with distilled water. Samples (10.0 ml) were then analyzed for asterone as usual. The results of trials with extraction procedures are summarized in Table 2.
Generul Procedure

The following procedure was adapted from that reported in previous studies (2,3) in ways indicated by the aforementioned experiments. Starjisll collection and storuge. Starfish were collected by either dipnet, entanglement in weighted string mops, or SCUBA diving. In the first two cases, the animals were always obtained at low tide in 2 m or less of water. Samples obtained at Fergusons Cove were always obtained by diving in water up to 20 m deep. Generally, only a single species was present. However, due to their very similar habits and appearance, some A. forbesi may have been collected as A. vulgaris. Samples of the collected animals (four to seven per sample) were placed in plastic bags and stored on ice until they reached the laboratory. They were then weighed, tagged, and stored at -30C until analysis.
TABLE
COMPARISON OF METHODS

2
ASTERONE-SAPONIN

FOR EXTRACTING

Starfish homogenate extraction procedure Blank Aqueous extract Acidified aqueous extract Diluted aqueous extract 70% Methanol extracts Methanol evaporated Methanol diluted Triplicate determinations. Mean 2 standard deviation.

Yield of asterone from isolated saponin (fig/S g of live starfish) 0 142 t 15.0 189 2 7.5 295 + 34.3 372 + 49.3 247 5 50.9

ESTIMATION

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To determine the seasonal variation in asterone content of the starfish saponins, samples were collected at two locations, when possible, throughout the year: Fergusons Cove (near Halifax, Nova Scotia) and Sober Island (ca 100 miles northeast of Halifax). To obtain an estimate of the potential place-to-place variation in asterone content, samples were obtained from fishermen from scattered locations throughout Nova Scotia and from one location in New Brunswick during October (see Table 3). Homogcnute prrptrrution. The frozen starfish samples were blended as described for aqueous extracts. The homogenates were then placed in plastic weighing boats, covered tightly with aluminuim foil. and stored at
-30C.
TABLE
VARI,XTIO~* IN ESTIMATED ASI ERONE Sample Sample Pugwash. Halifax area N.S. Harbor. N.S. date (1975) October October IO 2I YIELD

3
or STARFISH of dry (pg./g FROM SEVERAL SII t-s Asterone content dry weight) 291 I40 275 I72 I I4 1% IX8 239 246 415 378 300 I92 240 X0 154 IY3 14-1 I0 IiX I53

Percentage

solids in fresh sample IO.7 IX.1 17.4 3.7 72.6 23.1

Deer

Island.

N.B.

October

29

Sober

Island.

N.S.

October

I7

21.7 I.1 17.4 9.5 17. I 17.X

Fergusons

Cove,

N.S.

October

I8

Annapolis

Basin.

N.S.

October

71)

2O.Y 3.4 20.5

Bay

of Fundy Briar

transect Island.

October

I3

2x.2 72.7 31.3

neajN.S. Bay

of Fundy Meteghan,

transect

October

I3

35.6 34. I 74.7

near N.S. I N.S..

Nova

Scotia:

N.B.,

New

Brunswick

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Methnnolic estraction und retversed-phase adsorption. The portions (2.00 g) of starfish homogenate were prepared for adsorptive extraction by the procedure described for methanolic extracts. Empty Brinkman Drug-Skreen columns were repacked with 1.5 g of washed (procedure ii) Chromosorb 102, and the resin was equilibrated with water. The sample prepared above was passed quickly through the column three times. The column was washed with water (10 ml) and eluted with 50% methanol-water (10 ml) and methanol (10 ml). The combined methanolic eluates were evaporated to dryness with a rotary vacuum evaporator, and the residue was redissolved in water (1.0 ml). An appropriate-sized sample (usually 200 yl) was removed and evaporated to dryness with a stream of nitrogen. Crude suponin solvolysis and mterone estimation. Crude saponin samples were dried in IYKIIO over Drierite and were solvolysed in 0.50 ml of THF, containing 10 ~1 of 60% perchloric acid/ml, at 70C for lh. The reaction mixture was cooled, and 0.10 ml of saturated sodium bicarbonate solution and 2.5 ml of chloroform were added. The aqueous phase was removed, and the chloroform solution containing the liberated aglycones was washed with water (3 x 0.5 ml). Alycone samples were dried for at least 0.5 hr over Drierite in IYZCUO, with or without cholesterol internal standard. Dried samples were dissolved in anhydrous pyridine (10.0 ~1) and BSTFA (10 ~1, N,O-bis(trimethylsilyl)-trifluoroacetamide, Pierce Chemical Co.) and were heated to 50C for 10 min. Immediately after reaction, samples were analyzed as before (3) with a gas chromatograph (Hewlett-Packard, Model 5730A) utilizing flame ionization detection. Samples were injected into a glasslined injection port and were passed into a column (265C, N, at 60 ml/min) of 3% OV-101 on Chromosorb W-HP, 80- 100 mesh (Pierce Chemical Co.), packed in a silanized glass column (1.83 m x 4 mm i.d.). The eluted peak areas were integrated with a digital integrator (Hewlett-Packard, Model 3380A). The column and detectors were standardized with authentic asterone and cholesterol treated as above. Standardization plots for both compounds gave straight lines with slopes (-m) of 0.868 for cholesterol and 1.02 for asterone using the equation v = --171x+ c (v = relative peak height, s = micrograms of standard injected). The intercept ( was zero in both cases. RESULTS

The previously reported solvolytic procedure (3) on which the present work was based was adapted from the mild-hydrolysis procedures for

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the conjugates of hydrolysis-sensitive vertebrate steroids. While the procedure proved to be satisfactory, the relatively stable asterone should have permitted more harsh conditions and, hence, more rapid analysis. The effect of temperature and acid concentration on asterone recovery were, therefore, examined. The relatively constant maximum yield of asterone obtained under different PCA concentration and solvolysis conditions (Fig. 1) indicates that this was the maximum obtainable yield. This yield could be obtained under several combinations of conditions. The combination which gave the maximum yield most quickly with the lowest acid concentration was 10 ~1 of PCAiml of THF at 70C and, hence, was used in subsequent studies. Under the conditions selected, neither the tempeature nor the acid concentration was critical.
Asterone -Saponin Isolation by Resin Adsorption

Previous studies had shown that saponin adsorption on Amberlite XAD-2 might be a desirable method of isolating starfish saponin from aqueous extracts (3). However, these trials were conducted on a scale impractical for use with a large number of samples, and, hence, it was necessary to reduce the sizes of the samples and columns. To ensure that the column, resins, and sample form were adequate, a series of experiments were conducted utilizing previously isolated crude saponin as the source material so that recoveries could be evaluated. It was felt that the Amberlite XAD-2 previously used was too coarse (20-50 mesh) to provide efficient adsorption with small columns. Therefore, the effectiveness of the finer resin preparation in Brinkman DrugSkreen columns and that of the same columns repacked with Chromosorb 102 resin (1001120 mesh) were compared. Since it was apparent that the efficiency of the XAD-2 varied depending on its source (Table l), the effect of washing on fresh resin was also compared (Table I). First, fresh resin was washed by two procedures to determine if there would be any pronounced residual solvent effect. Then, resin which had been used for saponin recovery previously was rewashed, and its adsorptive ability was compared to that of fresh material (Table 1). It was apparent that XAD-2 resin varied in its ability to adsorb the saponin. While it was assumed that the probable cause for the failure of the Drug-Skreen resin was that it was not wetted thoroughly, the preliminary rinses with water and acetone or methanol were intended to avoid this problem and were the same as those used for Chromosorb 102. Other unreported trials with Drug-Skreen gave similar low recoveries (average: 25.1%). It is also apparent from Table 1 that XAD-2 must be reused with caution, since it probably is not completely regenerated by

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ETAL.

washing (procedure ii). While XAD-2 did give reasonably effective recovery, it was decided that all subsequent isolation be accompanied by standard crude saponin samples as controls to allow correction for variation in the adsorptive efficiency and warning should it fail. Differences between tests and recoveries of less than 100% due to handling losses (on glassware or from foaming) were not likely since the handling-loss control (Table 1) did not show any loss of the desired saponin compared to that of the untreated standard.
Crude Extract Preparation

Formerly, water extracts of starfish were found to give good recoveries of asterone-saponin (3) from large samples when used in conjunction with Amberlite XAD-2. However, such preparations presented handling difficulties, particularly with small samples, since they tended to clog the fine adsorbant columns. Presumably, the columns were clogged due to both the presence of particulates (clogged during sample passage) and the adsorption of undesirable materials such as proteins (clogged during attempts to elute them with methanol-water). Attempts to avoid this problem by centrifuging the samples through Celite 545 or microcrystalline cellulose were of limited success. Coagulation of the protein by heating (6oC), with or without acidfication, was only partially successful, since the precipitates formed were not easily removable by the lowspeed centrifugation available. Saponin recoveries after these procedures were only fair. Subsequently, several more successful procedures were compared. Table 2 summarizes the yield of asterone from a single starfish homogenate by: (a) the original water extraction procedure; (b) acidification of the water extract; (c) extensive dilution of the homogenate: (d) extraction with 70% methanol, followed by evaporation of the alcohol prior to adsorptive extraction: and (e) extraction with 70% methanol, followed by extensive dilution to less than 10% methanol (which does not elute saponin from XAD-2). When one compares the results listed in Table 2, it is apparent that 70% methanol extraction allows the best yield of asterone-saponin. It is not apparent why it was necessary to evaporate; rather than dilute, the methanol. Perhaps, the presence of even dilute methanol reduced the Chromosorb 102 adsorptive efficiency. It is not clear why the concentrated aqueous extract permitted only poor recoveries. Perhaps, the saponin associated with the protein strongly enough so that it was not completely adsorbed by the resin. Dilution of the extract may have shifted the equilibrium so that more saponin was in true solution and thereby, available for adsorption by diluting the salts present as well as the saponin. The methanolic extracts did not significantly clog resin columns. While the diluted and acidified aqueous extracts showed considerable improve-

ESTIMATION

OF

A SAPONIN

FROM

STARFISH

0 FURGUSONs

COVE

0 J&N FES M&R APR MAY

L JN JL AUG SEPT OCT NO DE0

SAMPLE

TIME

FIG. 1. Seasonal abundance of the asterone-saponin in starfish (A. ~.r,/~tr~Y.sl from Fergusons Cove and Sober Island. Nova Scotia (1974- 1975). Values -t one standard deviation were averages of three or more hamples. Points without indicated variation were single determinations. Sea temperatures were determined at Fergusons Cove.

ment over the original aqueous extracts, they still caused some clogging. For this reason and because many trials using procedures not reported here gave rather erratic results, these similar procedures were not considered further. When standard saponin was added to starfish homogenate samples and was re-extracted by the 70% methanol procedure as above, 105% (average of three estimates) was recovered. It was concluded that the aqueous methanol extraction gave a sufficiently quantitative saponin extraction for use in analysis.

The results summarized in Fig . 2 show that there is seasonal variation in the asterone-saponin content of the starfish, A. \s/r/~rlri.s. Both Sober Island and Fergusons Cove samples showed higher asterone levels in the late spring. The very high asterone yield for two of the three June samples from Sober Island (918. 761, and 466 pglg dry weight) indicates that there may be a brief spring content maximum. Unfortunately, only a single sample was obtainable in mid-June (Fig. 2). but it would ap-

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AL.

pear that the high levels had already dissipated by then. The June Fergusons Cove value was an average of seven samples and the July 5 value was an average of five, but none showed the very high values of the two Sober Island samples. Since each estimate was of a pool of several starfish, it would seem likely that the results are real, even though their significance to the starfish is not clear. The maximum may be so brief that it was missed by the Fergusons Cove sampling. The Sober Island samples were always obtained in very shallow water and at low tide. This fact may be reflected in the behavior of the animal and, hence, in the saponin level, should the level be related to special activities (such as spawning or gonadal maturation). The data suggest that the asterone-saponin levels were lowest when the water temperatures were lowest. The Fergusons Cove samples showed the least seasonal variation and reflect a more constant temperature, salinity, and, perhaps, diet. Sober Island water temperatures were not recorded, as they would have been very variable and relatively meaningless due to the extreme tidal flushing at the site. The average values would be expected to be similar to those recorded at Fergusons Cove.
Geographic Vuriation

garis during October

The results of the geographic distribution of asterone-saponin in A. ~lul1975 are listed in Table 3. The values have not been averaged in order to illustrate typical variation. The Pugwash sample was so small that it did not permit more than one pooled sample, nevertheless, the asterone estimate was very similar to those of the others. A comparatively low value was obtained from the Deer Island transect and the two Bay of Fundy transects which suggests that A. \julgaris from the Bay of Fundy may generally have lower asterone-saponin contents. Alternatively, frothing can be observed on the water when the starfish have been disturbed, presumably due to saponin release; therefore, the frequent disturbance which occurred during shipment and capture by dragging (transects) may have resulted in a reduced saponin yield. The apparent seasonal variation just considered limits the meaningfulness ofthe geographic distribution values, since it is not known what determines the observed variations. The consistency of the asteronesaponin content of September and October samples from Sober Island in 1974 and 1975 (Fig. 2) indicates that the seasonal results should be consistent from one year to the next. However, the cyclic variation may be different at different locations (Fig. 2). Therefore, geographic distribution data will have limited meaning until the cause of the variation has been determined.
DISCUSSION

The modifications of the procedure formerly used (2,3) to that of the general procedure adopted here do not represent any unexpected changes,

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but are optimizations of conditions for use with small samples suitable for analytical work. None of the procedures required critical conditions. It was necessary to use standard saponin references to check the functional efficiency of the XAD-2 adsorbant and to allow correction for losses. While XAD-2 did vary greatly in performance between sources, little variation was found when a specific preparation was obtained and the general procedure was followed closely. The procedure was not as simple and efficient as desired. but the alternate classical procedure of n-butanol extraction. followed by dialysis (8), would have presented even greater difficulty and lower precision. None of the modifications attempted (not all reported here, since they were not eventually used) achieved the yields obtained after methanol/water extraction (Table 2). Methanol/water (ca. 70%) was selected as the extracting solvent instead of methanol, because of crude saponins apparently low solubility in methanol, but good solubility in water, and also to minimize the extraction of undesirable nonpolar lipids which might affect the XAD-2 adsorption. While we have routinely used the nonpolar OV-101 stationary phase for glc analysis, we have also employed the more polar OV-17. On this polar stationary phase, the order of one of the constituents (half-asterone, 2) reverses. eluting before the equivalent asterone-TMS rather than afterward. An artifact formed by perchloric acid with THF which we have not been able to eliminate has a retention time close to that of asterone-TMS. With both OV- 17 and OV- 101. the column temperature must be selected to avoid artifact interference. Formation of acetates could be usefully substituted for TMS derivatization and would permit the use of ethyl acetate as a solvolytic solvent (which produces partial acetate exchange). However. the acetate peaks tend to tail during glc separation and, therefore. were not used for routine analysis. The material which gas-chromatographed like authentic asterone. its acetate, and asteroneTMS has been isolated by liquid chromatography (Chloroform: ethyl acetate) on Porasil A (60) and behaves like authentic asterone [synthesized by ApSimon and co-workers, Ref. (IO)] and its derivatives in gc and tic. The seasonal variation in the abundance of asterone in A. ~~ulgrrris is quite different from that oftotal saponin in A. rrmurensis ( 11) which showed a marked summer maximum. The saponins of the seacucumber, Stichoprs japonictrs. also show a summer maximum (12). This may indicate that the saponin parent of asterone does not serve the same function as the general saponins. The very great difference between the climatic conditions for the animals in this study and those for the animals in the other two studies (Japanese waters) limits a reasonable comparison. Previous studies by some of the authors yielded similar but more variable results (3.3). The present results indicate that the asterone yield would vary substantially between starfish samples.

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REFERENCES
1. Hashimoto. Y., and Yasumoto, T. (1960) &t/l. Jupun. Sock. Sci. Fish. 26, 1132- 1138. 2. Pike, R.. Gilgan. M. W., and ApSimon. J. W. (1974) in Proceedings, Food-Drugs from the Sea, pp. 375-387. 3. Gilgan. M. W.. Pike, R. K., and ApSimon. J. W. (1976) Camp. Bioclrrm. Pilysiol. 54B, 561-563. 4. Gurst. J. E., Sheikh, Y. M., and Djerassi. C. (1973) J. Amer. Chrm. Sot. 95. 628-629. 5. Sheikh. Y. M.. Tursch. B. M., and Djerassi. C. (1972) J. Amer. Chrm. SM. 94, 3278. 6. Shimizu, Y. (1972) J. Amer. Chem. SW. 94, 4051. 7. Sheikh, Y. M.. Kaisin, M.. and Djerassi, C. (1973) Steroids 22, X35-850. 8. ApSimon. J. W., Buccini. J. A.. and Badripersaud. S. (1973) Canud. J. C/rem. 51, 850-855. 9. Fleming. W. J.. Salathe. R.. Wyllie, S. G., and Howden. M. E. H. (1976) Camp. Biochrm. Physiol. 53B. 267-272. 10. ApSimon. J. W., and Eenkhoom. J. A. (1974) Cunnd. J. C/rem. 52, 4113-4116. II. Yasumoto. T., Tanaka, M.,and Hasimoto. Y. (1966) BuI/. Japan. SM. Sci. Fish. 32, 673-676. 12. Matsuno, T.. Sakushima, A.. and Ishida. T. (1973)B/1/I. Jrrpun. SW. Sci. Fish. 39, 307310. 13. Shimizu. Y. (1972) in Proceedings, Food-Drugs from the Sea, pp. 291-297. 14. Kitagawa. I.. Kobayashi, M., Sugawara. T.. and Yosioka. I. (1975) Terruhedron Left. 1975, 967-970.