1979 Rev. latinoamer. Quim. 10, pp.171-173

by A. Ulubelen, U. Sankawa
Momordica charantia are well known for their pharmacological activities. The seeds of M. charantia have been used against diabetes mellitus…

Steroids an Hydrocarbons of the Leaves of Momordica charantia

Author: A. Ulubelen, U. Sankawa

Type of Publication: Pre-Clinical

Date of Publication: 1979

Publication: Rev. latinoamer. Quim. 10, pp.171-173, 1979

Organization: Faculty of Pharmacy, University of Istanbul, Faculty of Pharmaceutical Sciences, University of Tokyo

Abstract: The alcohol extract of the dried leaves of Momordica charantia yielded 7-stigmasten-Bol and 7,25-stigmastadien-3-ol as a mixture and 5,25-stigmastadien-3-ol and glucoside as well as n-octacosan (C₂₈ H₅₄), triacontanol (C₁₀ H₅₂ O) and a new phytosphingosin (C₃₃ H₅ O₆ n). The compounds were identified by spectral methods.

INTRODUCTION

Momordica charantia are well known for their pharmacological activities. The seeds of M. charantia have been used against diabetes mellitus in Puerto Rico (1,2), and an insulin-like hypoglycaemic action was shown to be prtesent in the seeds of the plant (3). This activity was attributed to a steroidal compound, cahrantin 94,5), which was later found to be a mixture of sitosteryl-3-D- glucoside and a new compound 5,25-stigmastadien-3-ol D-glucoside (6).

Tissue cultures of the unripe fruits of M. charantia yielded diosgenin and 7-stigmasten-ol (7) and 5-stigmasten-3, 25-diol (8), 5,25-stigmastadien-3-ol and 7,25-stigmastadien-3-ol were detected in the fruits themselves (9).

From the aqueous extract of the seeds of M. charantia a protein with oncostatic activity was obtained (10). Antihelmintic activity was also reported for the same plant (11,12). In 1936 Diaz mentioned alkaloid-like substances in the fruits of M. charantia (13). From the petroleum ether fractions of M. charantia and M. dioica a substance with alkaloid properties was later isolated (14). A non-quaternary alkaloid with antinicotinic and antimuscarinic activity was found in M. foetida (15). An aqueous extract of the plant showed abortive effects on pregnant rabbits (16). 5,25-Stimastadien-3-ol D-glucoside was also found in the same plant (17).

In the present study the alcoholic extract of the leaves of Momordica charantia was examined. Preliminary tests indicated the presence of a group of tertiary alkaloids. After separation of these alkaloids by 10% acetic acid extraction, the remaining extract was evaporated to dryness and subjected to column chromatography. The hydrocarbons n-octacosan, tricontanol, and the steroids, 7-stigmasten-3ol, 7,25-stigmastadien-3-ol and 5,24-stigmastadien-3-ol glucoside were isolated along with a new phytosphingosin (C₄₈ H₈₃ O₆ N). The latter compounds is the first of this molecular-weight phytosphingo lipids are known from yeast molds and mushrooms (18) and a mixture of phthiocerols (C₃₆ H₇₆ O₃) and (C₃₂ H₇₀ O₃) was obtained from tubercle bacilli (19-21). The structures of the phthiocerols from tubercle bacilli were determined by mass spectral interpretation. In the determination of the partial structure of the new phytospingosin, the MS of the known phthiocerols and phytosphingosin were useful (21, 22).

Experimental

The spectra were recorded with the following instruments: IR, Jasco, DS 701: NMR, Joel 100 MHZ, Joel-Ol SG-2; GC, Hewlett-Parkard 402 A (3% OV-25, 270⁰, He 100 ml/min). Melting points were taken in a micro melting point apparatus and were not corrected.

The leaves of M. charantia (Cucurbitaceae) were collected from the Botanical Garden of the University of Istanbul. About 2 kg of the powdered leaves were macerated, then percolated with 95% ethanol. Upon evaporation under vacuum 160 g of a dark green residue was obtained. Preliminary tests (Dragendorff and Mayer’s) showed the presence of alkaloids. In order to separate the alkaloids the green residue was dissolved in chloroform, and the insoluble part was separated by centrifugation (50 g) (mainly inorganic compounds). The chloroform solution was extracted exhaustively with 10% aqueous acetic acid to remove the alkaloids (Mayer’s reagent). The acetic extract was made alkaline with ammonia and extracted with chloroform; about 1.2 g of a crude alkaloid mixture was obtained. The crude mixture showed two main and a few smaller alkaloid spots on TLC plates. (The determination of these alkaloids will be the subject of another study).

After removal of the alkaloids, the remaining solution was evaporated to dryness, yielding 20 g of residue. The material fractioned elutions yielded 147.8 mg of a hydrocarbon, crystallized from chloroform-methanol mixture (1:1) and determined to be n-octacosan by spectral methods, later fractions with ethyl acetate concentrations increasing to 70% yielded a hydrocarbon alcohol which was found to be triacontanol; 10% ethyl acetate yielded a steroidal mixture (A₃) which was later found to contain 7-stigmasten-3-ol and 7,25-stigmastadien-3-ol by GC-MS separation. 5% methanol in chloroform eluted the new phytosphingosin, and 10% methanol yielded stigmast-5,25-dien-3-ol D-glucoside.

n-OCTACOSAN (C₂₆ H₅₁): m.p. 59-60^o, Lit. 61-62⁰ (23, p.17). IR (in KBr) showed a long-chain hydrocarbon, NMR (in CDCI₃, TMS) corroborated this findings.

Analytical calculations: Found C, 84.97; H, 16.67

Calculated for C₂₆ H₅₈ C, 85.27; H, 16.72

TRIACONTANOL (C₁₀ H₆₂ O): m.p. 84-86⁰, lit. 86-86.5⁰ (23, p. 49); IR (in KBr) and NMR (in CDCI₃ TMS) showed a long chain hydrocarbon alcohol.

Analytical calculations: Found C, 78.19; H, 13.67

Calculated for C₃₀ H₆₂ O. H₂O C, 78.60; H, 13.97

7-STIGMASTEN-3-OL and 7,25-STIGMASTADIEN-3-Ol (A₃). A₃ gave a single spot on TLC and argentized plates, m.p. 168-169⁰; IR (in KBr) showed an exocyclic methylene group (1635, 835 cm ^–1), NMR (in CDCI₃, TMS) 0.58 (CH₃ – 18,s) (indicated ∆-double bond), 0.80 (CH₃ – 19,s), 0.86 (CH₃ – 29, d,J= 7Hz), 1.00 (CH₃-21,d,J=6Hz), 3.5 (CH-O, m), 4.58 and 4.66 (1 H each, br. D, = CH₂); 5.08 (br.d, ∆ ⁷H) (integration of this signal indicated almost two protons). The mass fragments was similar to that given by Sucrow (9), except for a peak at 414 in addition to a molecular ion at 412. These data together with the d 5.08 signal in the NMR spectrum, suggested the presence of a mixture. Gas chromatographic separation showed the presence of two compounds (3:2 ratio ^a with retention times of 4.2 and 5.0 minutes respectively. MS of 7-stigmasten-3-ol, M 414, at m/z 399 (M-CH₃), 299, 271 (M-side chain-2H), 255, 246, 212 (base peak): MS of 7,25-stigmastadien-3-ol, M 412 at m/z 397 (M-CH₃), 328, 314, 299, 271, 254, 245, 211.

Direct comparison with standard samples confirmed the above given findings, namely that these compounds are 7-stigamasten-3-ol and 7,25-stigmastadien-3-ol. 5,25-STIGMASTADIEN-3-OL D-GLUCOSIDE: m.p. 275-280⁰ (degradation); IR (in KBr) showed exocyclic methylene (1635, 885 cm^-1) and the presence of sugar (broad band at 3600 and 1075, 1050, 1025 cm^-1); MS, M 576, at m/z 412 (aglycone), 396 (agly- H₂O) (base peak), 328, 314, 302, 254, 231.

Acetylation in pyridine anhydride at room temperature gave an acetate, m.p. 160-164⁰, IR (in KBr) showed that no free hydroxyl group was left. NMR (in CDCI₃, TMS) 0.72 (CH₃-C = ,s), 1.98, 2.00, 2.04, 2.08 (4 acetyl singlet), other peaks are at 3.6 (m), 4.2 (m), 4.6 (t), 4.9 (br. d), 5.3 (br. d) ppm.

MS, M^- 742, at m/z 421 (aglycone), 394 (agly-H₂O) other peaks are essentially the same as that of the glycoside.

Hydrolysis of the compound utilized 6N HCl under a reflux for 6 hr. The sugar was found to be glucose by comparison with a standard sample (TLC and PC). The aglycone was found to be 5,25-stigmastadien-3-ol on the basis of spectral findings and comparison to the literature values (6).

NEW PHYTOSPHINGOSIN (C₄₃ H₈₃ O₆N): m.p. 143-145⁰; IR (in KBr) 3350 ()H), 3200 (NH₂), 2910, 2845, 1615 (unsaturation), 1545, 1465, 1065, 1015 (C-O), 962 (N-H), 720 [(CH₃)n] cm^-1; NMR is taken at 50⁰ C (in CDCI₃, TMS) 0.92 (3H, br.s), 1.3 [(CH₂0 n,s], 2.55 (OH₁ D₂O exchange), 3.62 (OCH₃,s), 4.0 (br.t), 5.4 (vinylic protons, br.s. Acetylation of the compounds was performed in the usual manner, m.p. 58⁰ , IR (in KBr) 2920, 2850, 1745, 1735 (acetyl) 1660 (unsaturation), 1530, 1465, 1375, 1265, 1235, 1220 (acety C-O), 1140, 965, 720 [(CH₃)n] cm^-1 NMR (in CDCI₃, TMS) 0.9 (3H,s), 1.25 [(CH₂)n,s], 2.04, 2.06, 2.10, 2.18 (acetyl), 3.62 (OCH₃), 4.3 (m), 5.00 (m), 5.3 (m), UV (in MeOH)  max 209 nm (no conjugation).

Analytical calculations: Found C, 73.18; H, 12.31; N, 2.05

Calculated for C₄₃ H₆₃ O₆N C, 72.77; H, 11.70; N, 1.97

MS, at m/z 691 (M-H₂ O) this peak is characterized for hydrocarbon alcohols. The predeuteriomethyl derivatives of the compound gave a small molecular ion peak at m/z 792 ().3%) which indicated the perdeuteriomethylation of four hydroxyls and one amino group (5 X CD₃). These data together with NMR results, suggested the presence of a methoxyl group in the molecule.

When the mass spectral data were compared to those given in the literature for other phtiocerols (21), many similarities were observed; for example the strong peaks at m/z 337; 339 (for derivatized compound at m/z 354; 356) indicated that a part of the molecule should be: CH₃- (CH₂)₂₁-CH- Furthermore, the following peaks up to m/z

║

OH

422 show that the following units at m/z 351, 365, 379, 393, 407, and 421 differ successively by 14 mass units, in accord with the cleavage of a (CH₂)n chain. Strong ions for C₁₀ H₃₈ and C₁₀ H₄₀, typical for phtiocerols 921), were also present at m/z 278; 280. The base peak at m/z 43 represents a-CH-CH-group, and a strong peak

║

OH

at m/z 57 (80) indicates a – CH-Ch-CH₂ group. The ion at m/z 60

║

OH

(30%) corresponds to the –CH-CH₂NH₂ group. In the derivatized

║

OH

compounds corresponding peaks were found at m/z 61 and m/z 76. The base peak in ther derivatives is derived from the end group of the molecule, namely –CH-CH₃-N-CD₃ (a), m/z 61 ion being (a-NHCD₃)  

OCD₃ H

And the latter corresponding (a-CD₃). The small peak at m/z 663 (M-H₂O-CHNH₂) in the underivatized compound is also indicative for the proposed end group. The double bonds are not conjugated as seen in the Uv spectrum of the molecule. The partial stucture of the molecule is given in Figure1.

CH₃ –(CH₂)₂₁-CH-(CH₂-[C₁₂H₁₈O₄] –CH-CH₂NH₂

 

OH OH

Figure 1

[ 3 double bonds 3 hydroxyl groups 1 methoxyl]

The complete of the new phytosphingosin will further investigation.

ACKNOWLEDGEMENTS

One of the authors (AU) would like to express her gratitude to JSPS for a three months grant to be able to work at the University of Tokyo.

We would like to thank Prof. W. Sucrow for some of the standard samples.

REFERENCES

G. Rivera, Am. J. Pharm., 13, 281 (1941).

J. A. Pons and D. S. Stevenson, Puerto Rico J. Pub. Health and Trop. Med., 19, 196 (1943); C. A. 38, 2117.

G. Rivera, Am. J. Pharm., 114, 72 (1942).

M. M. Lolitkar and M. R. Rajarama Rao, J. Univ. Bombay, 29, 223 (1962); C. A. 58, 9537d.

M. M. Lolitkar and M. R. Rajarama Rao, Indian J. Pharm., 28, 129 (1966).

W. Sucrow, Tetrahedron Lett. 26, 2217 (1965).

P. Khonna and S. Mohan, Indian J. Experimental. Biol., 11, 58 (1973): C. A. 79, 7588h.

W. Sucrow, Chem. Ber. 99, 2765 (1966).

W. Sucrow, ibid, 99, 3359 (1966).

T. Tun and Y. Rin, Japan Kokai 7.667.714 (C.I.A 61 K 35/78); C. A. 85P, 198150e.

F. M. Friese, Apoth, Ztg. 44, (1929); C.A. 24, 684.

J. Lal, S. Chandra, V. Raviprakash, M. Sabir, Ind. J. Phsiol., Pharmacol., 20, 64 (1976); C. A. 85, 104621.

L. R. Diaz, Puerto Rico J. Pub. Health and Trop. Med., 11, 812 (1936); C. A. 30, 6133.

J. W. Airan and N. D. Ghatge, Current Sci. (India)19, 19 (1950); C. A. 44, 5139.

A. A. Olaniyi and V. O. Marquis, J. Pharm (Yaba, Niger.) 6, 117 (1975); C. A. 84, 84360 n.

D .G. Steym, Onderstepoort, J. Vet. Sci. Animal Ind., 9, 107 (1937); C. A. 32, 4279.

A. A. Olaniyi, Lloydia, 38, 361 (1975).

H. E. Carter, W. D. Celmer, W. E. M. Lands, K. L. Mueller and H. H. Tomizawa, J. Biol Chem, 206, 613 (1954).

L. Ahquist, R. Ryhage, E. Stenhagen and E. Von Sydow, Arkiv for Kemi, 14, 211 (1959).

R. Ryhage, S. Stallberg-Stenhagen and E. Stenhagen, ibid, 14, 247 (1959).

R. Ryhage, S. Stallberg-Stenhagen and E. Stenhagen, Ibid, 14, 259 (1959).

J. H. Cardellina and R. E. Moore, Phytochem. 17, 554 (1978).

W. Karrer, Konstitution und Vorkmmen der Organischen Planzenstoffe, Brik Hauser Verlag Basel (1958).