Studies of Luffa echinata III. The Oil and the Saponin

Studies of Lzcffu echinutu 111 The Oil and the Saponin By M. L. KHORANA and K. H. RAISINGHANI The oil of the fruits of Luffa echinata Roxb. is found to deposit the hydrocarbon, hentriacontane, Cz1H? Saponin has been isolated in partially purified form, and the sapogenin is identified as gypsogenin. On reduction with sodium borohydride it gave hederagenin. Reduction of its methyl ester by a modified Wolff-Kishner method gave oleanolic acid. Glucose, rhamnose, and xylose have been identified among the sugars present. They seem to occur in the molecular ratio of glucose-xylose-rhamnose 3: 2: 1.

Roxb., family Cucurbitaceae, is a plant commonly used in India as a medicine (1). Various therapeutic properties have been attributed to this drug (1, 2 ) . ,%me studies on the oil (3, 4), an ether extract containing cucurhitacins ( 5 ) , and a saponin with diuretic activity (6, 7) have been reported earlier The present work reports further studies on the oil and the saponin. Hentriacontane has been isolated from the oil obtained by the extraction of the fruits with petroleum ether and identified by mixed melting point. The saponin could not be obtained in pure or crystalline form. However, partially purified saponin, when hydrolyzed with 5 per cent hydrochloric acid, gave gypsogenin and a mixture of sugars. The aglucone (gypsogenin) melted at 268269" after sintering a t 240'. It gave a positive test with Liebermann-Burchard reagent and with commercial thionyl chloride. Elemental analysis and molecular weight determination showed it to be CBoH4,,04.Infrared spectra showed it to contain hydroxyl, carboxyl, and carbonyl groups (absorption at 2.9,5.8,and 5 9 p ) . Reduction of the methyl ester of the aglucone by a modified Wolff-Kishner method (8) gave oleanolic acid: confirmed by mixed melting point and infrared spectra as well as mixed melting points of the esters. Reduction of the aglucone with sodium borohydride gave hedeiagenin, which was confitmed by mixed melting points. To identify the sugais present in the glycoside, the aglycone was removed from the hydrolysate, and the filtrate, after neutralization with barium carbonate, was chromatographed using n-butanol, UFFA ECHINATA

Recaved October 21. 1960, from The Department of Chemical Technology. University of Bombay, Uombay 19. India Accepted for publication November 4 , 1960 Part I1 in press The auihors are grateful t o May and Baker Ltd for the financial support given for this work Our thanks are ako due t o Dr T. S Gore for the microanalysis

acetic acid, water mixture (4: 1:5) for development. Staining with aniline hydrogen phthalate showed the presence of glucose, xylose, and rhaninose. Quantitative estimation by the modified Somogyi procedure (9, 10) showed the respective sugars to be present in the ratio of :3 :2 : 1. EXPERIMENTAL

echinata fruits obtained from Extractives.-Luffa the local market and pharmacognostically identified were powdered to 16 mesh. The powdered drug (9 Kg.) was exhausted by percolation, successively, with petroleum ether ( S a - S O O ) , ether, and alcohol (70%). Removal of the solvent from the extracts, using vacuum when necessary, gave the extractives: petroleum ether 4.0y0, ether 0.3y0, alcohol 3.0%. The Oil.-The oil, obtained by evaporation of petroleum ether, on keeping for three months at room temperature deposited a greenish oily solid, which after separation by decantation, washing with petroleum ether, and two crystallizations from petroleum ether, gave a compound, m. p. 66-68", yield 50 mg. Further yields of the compound were obtained by pouring the mother liquor from the crystallization over an alumina column (60 Gm.) and eluting with three 100-ml. portions of petroleum ether. Fractions two and three gave a compound melting at 68-69"; total yield 1.2 Gm. This as well as product obtained by crystallization were identified as henby mixed melting point, 68". triacontane, C~IHM, The authentic sample of hentriacontane was obtained from Citrullus colocynthis (11). Isolation of Saponin.-Alcohol (70%) was used to exhaust the marc left after the ether extraction. The solvent was removed, and the green color was extracted from 200 Gm. of the residual (270 Grn.) by refluxing it four times, each time with 1 L. of absolute alcohol for two hours. The residue was dissolved by refluxing in 70% alcohol and precipitated with ether. On repeating this treatment seven times, a yellow amorphous precipitate which turned yellowish brown to brown on standing was obtained; yield 165 Gm. The product which darkened at 160° and decomposed at 225-230" is referred t o as "partially purified saponin." The Sapogenin.-When the partially purified saponin (80 Gm.) was refluxed with 5% alcoholic hydrochloric acid for four hours on a water bath,

687

Journal of Pharmaceutical Sciences

688 a dark brown precipitate of sapogenin separated. After removing the alcohol under partial vacuum, the residual liquor was diluted with water and the crude sapogenin that precipitated after being filtered and washed with water was dark brown in color; yield 0.19%. When the crude sapogenin was repeatedly extracted with ether (5 X 75 ml.) and the combined extracts shaken with 5% aqueous potassium hydroxide, a yellow, semisolid precipitate of potassium salt separated in the aqueous layer. The precipitate, collected on a sintered-glass funnel, was transferred to a flask, covered with ether, and decomposed with dilute hydrochloric acid. The separated ethereal layer was washed with water till free from acid, dried over exsiccated sodium sulfate, and treated with Norite. The sapogenin obtained on evaporation of the ether, was a white amorphous powder; yield, 0.037y0; it sintered at 240°, melted a t 264-268'; [a]Y+91.08" (in ethanol). The white amorphous powder (1.1 Gm.), dissolved in 25 ml. of chloroform, was adsorbed on an alumina column and eluted f i s t with 100 ml. of chloroform (fraction I), then with two 100-ml. portions of chloroform-ethanol mixture (95: 5) (fractions I1 and 111), and finally with 100 ml. of absolute alcohol (fraction IV). Fraction I gave a product melting below 200'. The residues from fractions 11, 111, and IV, m . p. 268-271", total yield 750 mg., were mixed and recrystallized from aqueous methanol, t o obtain a compound melting a t 268-269", [a]: +90.YS0 (in ethanol), yield, 200 mg. Anal.-Calcd. for C30H4604: C, 76.55; H, 9.85. Found: C, 76.4; H, 9.95. Reaction of the compound with LiebermannBurchard reagent as well as with commercial thionyl chloride gave a red-violet color changing t o brown. Infrared spectra of the compound in Nujol, sodium chloride prism, showed absorption in the region of 2.9, 5.8, and 5.9p, indicating the presence of hydroxy, carbonyl, and carboxyl groups. Methyl Ester of the Supogenin.-Other methylating agents having failed, the carboxyl group was methylated with ethereal diazomethane. Diazomethane in ether (400 ml.) was added to 650 mg. of sapogenin dissolved in 350 ml. of sodium-dried ether. After allowing the solution t o stand for five hours, the ether and excess diazomethane were distilled off. Recrystallization from aqueous methanol gave white microscopic needles (620 mg.), m. p. 184185O. Reduction of the Ester.-Reduction of the methyl ester (500 mg.) by the Wolff-Kishner method modified by Huang-Minlon for steroids gave a compound, which on recrystallization from methanol, melted a t 302-303", [a]? $78.42" (in chloroform), yield, 180 mg. I t was identified as oleanolic acid by mixed melting point with an authentic sample and by its infrared spectra. Reduction of the Supogenin.-To 50 mg. of the sapogenin in 20 ml. of methanol was added 1 ml. of 2 N sodium hydroxide solution, the solution was cooled and 10 mg. of sodium borohydride in 10 ml. of methanol was added. The mixture was kept in an ice bath for fifty hours and then allowed t o come to room temperature. Dilute sulfuric acid was added t o decompose the excess of sodium borohydride. The filtered and washed precipitate crystallized from aqueous methanol as colorless prisms, yield 22 mg., m. p. 328-330" (decompn.);

mixed m. p. with an authentic sample of hederagenin, 330-331". The methyl ester prepared with ethereal diazomethane gave m. p. 239-241" and mixed m. p. with an authentic sample, 238-240O.l Sugars of the Saponin.-To avoid sugar loss during hydrolysis, the saponin (10 Gm.) was only partially hydrolyzed by refluxing for three and onehalf hours with 5% alcoholic sulfuric acid. Alcohol was removed under vacuum and the residue diluted with water t o precipitate the sapogenin. The filtrate was neutralized with barium carbonate, centrifuged in tubes for thirty minutes, and the decanted solution, being still acidic, was again neutralized with barium carbonate (Congo red) and centrifuged. Qualitative Paper Chromatography.-Spots of the hydrolysate on Whatman No. 1filter paper (10 X 19 inches) were developed by the descending method, with n-butanol-acetic acid-water (4: 1:5 upper phase) for fourteen hours. The chromatogram was dried a t room temperature for six hours and sprayed with aniline hydrogen phthalate in n-butanol saturated with water. On drying the chromatogram a t 110' for two minutes, three brown spots and one pink spot were revealed. Cochromatographing with authentic samples showed that the sugars in the hydrolysate were glucose, xylose, and rhamnose; their Rf values, average of three observations, are reported in Table I along with those of authentic samples of the sugars. TABLE I.--KELATIVERf VALUESOF SUGARS

Sugar

Hydrolysate 30 =t2'

Glucose Xylose Rhamnose Fourth spota

0.19 0.29 0.37 0.51

Authentic Sample 30 f Z 0

Reported at 20°

0.19 0.29 0.37

0.18 0.28 0.37

..

..

a The fourth spot did not reduce Fehling's solution

Relative Proportion of Sugars. The technique of Somogyi (9), extended by Flood, Hirst, and Jones (10) for synthetics and hydrolyzed polysaccharides of plant extracts was followed. The same solution of the hydrolysate as used in qualitative work was used for quantitative estimation. Two Whatman filter papers (No. 1, 10 X 19 inches) were spotted a t intervals of 2 inches with a fine capillary, delivering solutions of the order of 0.1 ml.; the spots were 4 inches from the end of the paper. The chromatogram was developed as previously described. With the help of one strip cut from the main sheet and sprayed with aniline hydrogen phthalate, positions of the sugars on the unsprayed chromatograms were marked and cut out. Similar pieces cut from fresh paper served as blanks. Each cut piece was extracted three times with small quantities of water a t 80" and the extract made up t o 5 ml. Solutions of known sugars (0.1 mg. sugar in 5 ml. of distilled water) taken in similar bottles were used as standards. Addition of the reagents and other treatment of bottles was done as per standard procedure (Somogyi, loc. cit.). The iodine liberated was titrated against 0.01 N sodium thiosulfate, 1 Thanks are due to Prof, D. H. R. Barton for authentic samples of hederagenin and its methyl ester.

Vol. 50, No. 8, August 1961 using starch indicator. Average results of two readings showed that the molecular proportion of glucose-xylose-rhamnose was 2.92: 1.95: 1 which may be taken as 3:2:1. REFERENCES (1) Kirtikar, K. R . . and Basu, B. D., "Indian Medicinal Plants," Vol. 2, 2nd ed.. Lalit Mohan Basu, Allahabad, India, 1933, p. 1125. (2) Nadkarni, A. K . , "Indian Materia Medica," Vol. 1,3rd ed., G. R. Bhatkal; Bombay, India, 1954, p. 753. (3) Pandya, ,K.C., Nigam, R. G. S., and Tayal. J. N., Current Sci. Indta, 18. 451 (1949).

689 (4) Bhatt, R. H., and Khorana, M. L., Indian J . Pharm., 19,208(1957). (5) Lavie, D., Shfrvo, Y.,Gottlieb, 0. R . , Desai, R. B., and Khorana, M. L., in press. ( 6 ) Bhatt R. H. Khorana M. L. Gaitonde B. B Raikar K. P. P k e l J.'R. and Kebre 'M. L. J . ' J - G&p of Ho&itals And G r h t Medical College,' 3 , llO('1958). ( 7 ) Bhatt, R. H., Khorana,. M. L., Patel, J. R., Gaitonde, B. B. and Kebre. M. S., fndaan J . Phystol. Pharmacol., 2 , 309(lb58). (8) Huang-Minlon, J . A m . Chem. Soc., 7 1 , 3301(1949). (9) Somogyi M. J . Biol. Chcm. 160 61(1945). (10)Flood E: Hirst, E. L.: and Jones, J. K. N., J . Chcm. Soc.,'1948,1679. (11) Power, F. B., and Moore, C. W., ibid., 97,1099(19??). (12) Block R. J. Darrum E. L. and Zeweig G. Manual of P&r Chiomalogradhy and i'apcr Elrctro&or&?' Academic Press Inc.. New York, N. Y., 1958, pp. 185-189.

A.

Investigation of the Free Amino Acids and Amanita Toxins in Amanita Species By P. CATALFOMO and V. E. TYLER, Jr. Chromatographicdetermination of the free amino acid content of numerous collections of several species of the genus Amanita and the closely related genus Vaginata revealed the presence of several amino acids that were common to all species. Differences in amino acid patterns were due to the presence or absence of proline, phenylalanine, and 7-aminobutyric acid or the occurrence of several unidentified ninhydrin-positive spots. The applicability of these data as an aid to the systematic classification of mushrooms is considered. The same species were also examined for the presence of amanita toxins. No evidence for the presence of these toxins was found, but bufotenine was detected chromatographically in A. Porphyria. HE GENUS Amanita has been the subject of Tconsiderable physiological and chemical investigation. Interest in this genus may be attributed to the severe toxicity demonstrated by several of its species and the difficulties encountered in efforts to elucidate the chemical nature of the active constituents. The bulk of the chemical investigations conducted up to the present time have been concerned with the nature of the toxic agents in those members of the genus exhibiting the most pronounced physiological actions. Especially significant were the findings of Wieland and Wieland (1) which estahlished the presence of five toxic principles, designated amanita toxins, in A . phalloides. Block, zt al. ( 2 ) , surveyed several genera of mushrooms, including thirteen species of -4 manitiz, for the presence of amanita toxins. However, relatively little is known about the general chemical composition of the members of the genus. The occurrence in western Washington of a considerable number of species of Amanita,

Received November 2, 1980, from the College of Pharmacy, University of Washington, Seattle 5. Accepted for publication December 8, 19tiO. Abstracted from a dissertation submitted to the Graduste School of the University of Washington by P. Catalfomo in partial fullillment of the requirements for the degree of Master of Science. This investigation was supported o the State of Washington Initiative 171 Funds for Researcx in Biology -. and Medicine.

which for the most part had not been previously investigated, prompted the present study. The primary object of this investigation was to identify the free amino acids contained in the various species of Amanita and the closely related genus Vagiitata and to determine the occurrence or absence of amanita toxins in them. A secondary objectivc was to determine the applicability of these data in the systematic classification of these mushrooms. Considering that the characteristics employed to distinguish between species of dmaiiita and Vaginatu, as described in the kevs of Smith (3) and Singer (4),include spore size, the amyloid reaction, pileus color, the form of the volva, and the presence or absence of warts or annuli, all of which are variable and difficult to interpret on specimens of different ages, i t is evident that species delineation is fraught with difficulty, and the establishment of more absolute characters is highly desirable. Data on chemical composition should be of considerable value as an object.ive criterion for the identification of Amanitu species. EXPERIMENTAL Amanita Species Examined.-All specimens of the naturally occurring carpophores used in this investigation were collected in western Washington