New Cucurbitane Triterpenoids and Steroidal Glycoside from Momordica charantia

Three new cucurbitane triterpenoids 1–3 and one new steroidal glycoside 4, were isolated together with ten known compounds from Momordica charantia. The structures of new compounds were determined to be 19(R)-n-butanoxy-5β,19-epoxycucurbita-6,23-diene-3β,25-diol 3-O-β-glucopyranoside (1), 23-O-β-allopyranosyle-cucurbita-5,24-dien-7α,3β,22(R),23(S)-tetraol 3-O-β-allopyranoside. (2), 23(R),24(S),25-trihydroxycucurbit-5-ene 3-O-{[β-glucopyranosyl(1→6)]-O-β-glucopyranosyl}-25-O-β-glucopyranoside (3), and 24(R)-stigmastan-3β,5α,6β-triol-25-ene 3-O-β-glucopyranoside (4), respectively. Their structures were elucidated by the combination of mass spectrometry (MS), one and two-dimensional NMR experiments and chemical reactions.


Introduction
The plant Momordica charantia L (cucurbitaceae) is cultivated in Asian countries.Its fruit, called kugua in Chinese and bitter melon in English, is a popular vegetable in Asian countries and is becoming a popular food supplement to lower blood glucose worldwide [1].So far, more than 100 compounds, mainly cucurbitane-and oleanene-type triterpenes, have been isolated from the fruits, OPEN ACCESS seeds, leaves, canes and roots of this genus.Recently, studies have discovered that the triterpenes from this genus showed biological activities such as antidiabetic [2], anti-HIV [3], anticancer [4] properties.Based on our continued interest in the discovery of sugar-reducing and anticancer compounds from M. charantia, we have examined the methanolic extracts of M. charantia from Chengjiang County, Yunnan Province, China.Herein, we report the isolation and structural elucidation of four new compounds from this plant (Figure 1).Notes: glc = glucopyranosyl; all = allopyranosyl.
The 13 C-NMR of 2 (Tables 1 and 2) showed 30 aglycone carbon signals and 12 signals from two sugars, which indicated that 2 was a triterpene saponin.Acid hydrolysis of 2 liberated allose, which was identified by TLC comparison with an authentic sample.The 13 C-NMR data of 2 bore a resemblance to those of karaviloside V [5], with the exception of the signal of the hydroxy group at C-7 in 2 instead of an OMe group in karaviloside V.This deduction was in accordance with the evidence that C-7 in 2 was upshifted by 10.2 ppm when compared with C-7 in karaviloside V. Further analysis of the HMBC spectra of 2 proved the above deduction (Figure 3).The stereostructure of the aglycone moiety was characterized by ROESY experiment (Figure 3), which were observed between H-7 and 3H-19β, H-8β; H-8 and 3H-18β; 3H-30α and H-16α.Thus, the structure of 2 was confirmed as 23-O-β-allopyranosyle-cucurbita-5,24-dien-7α,3β,22(R),23(S)-tetraol 3-O-β-allopyranoside.  1 and 2) showed the presence of seven tertiary methyls, one secondary methyl, a trisubstituted double bond, six quaternary carbons, seven aglycone methylenes, and two sugar methylenes, as well as three anomeric carbons, which indicated that a triterpenoid glycoside with two sugar moieties.Acid hydrolysis of 3 released glucose, which was identified by TLC comparison with an authentic sample.Carbon signals from 13      3) showed signals for two tertiary methyl groups at δ 0.72 and 1.52 (each 3H, s), a secondary methyl at δ 0.96 (3H, d, J = 6.4 Hz), one allylic methyl group at 1.59 (3H, s), one third methyl at δ 0.83 (3H, t, J = 7.4 Hz) and two anomeric olefinic protons at δ 4.85 (1H, s), 4.78 (1H, d, J = 2.1 Hz) as well as one anomeric proton at δ 4.95 (1H, d, J = 7.7 Hz).The 13 C-NMR data indicated that the presence of a steroidal glycoside bearing five methyls and a sugar unit.Acid hydrolysis of 4 afforded a glucose which was confirmed by TLC comparison with an authentic sample.All above data suggested that it was almost same as 24(R)-stigmastan-3β,5α,6β-triol 3-O-β-glucopuranoside [13], exception of a double bond between C-25 and C-26 in 4 instead of a methine group at C-25 and a methyl group at C-26.In the corresponding HMBC spectrum (Figure 5), the correlations between H-26 and C-24, 27 as well as between H-29 to C-24, 28 were observed.Thus, compound 4 was identified as 24(R)stigmastan-3β,5α,6β-triol-25-ene 3-O-β-glucopyranoside.

Plant Material
The fresh fruits of M. charantia were purchased from in Chengjiang County, Yuxi City, Yunnan Province, P.R. China, in August 2008, and identified by Prof. Shukun Chen.

Conclusion
We have isolated three new triterpenes and one new steroidal glycoside from the fruits of M. charantia, together with ten known compounds karaviloside II, karaviloside III, momordicoside K, kuguaglycoside B, momordicoside L, momordicoside M, momordicoside N, momordicoside B, momordicoside S, and momordicoside A. Although we researched on the same specie from different localities [3,7], the chemical constitutions of them were different.So, for better using the fruits of M. charantia as a food supplement or a plant extract to lower blood glucose, we had better pay attention to their chemical constitutions in different localities.

Figure 1 .
Figure 1.Structures of the new compounds from M. charantia.
C-NMR data were superimposable on those of momordicoside S [2], except for those around the C-22 position.The signal of C-22 was upshifted by 29 ppm, the C-20, C-23 were upshifted by 10.5 ppm, 4.8 ppm, and the C-21, C-24 signals were downshifted by 3.6 ppm, 4.4 ppm, respectively, which suggested that a hydroxyl group attached to C-22 of 3 was disappeared compared with momordicoside S. The 1 H-NMR and 13 C-NMR spectra of C-20, 21, 22, 23, 24, and 25 of 3 were in good agreement with those of momordicoside R [2].