Benzyl Glycosides from the Aerial Parts of Gynostemma laxum and Their NF-κB Inhibitory Activity in HepG2 Cells

Abstract

Nuclear transcription factor-κB (NF-κB) represents a family of Rel domain-containing proteins including RelA, RelB, c-Rel, NF-κB1, and NF-κB2. The activation of NF-κB has been linked to multiple pathophysiological conditions such as cancer, arthritis, asthma, inflammatory bowel disease, and other inflammatory conditions. NF-κB can be activated by various stimuli, such as microbial and viral products, cytokines, DNA damage and noxious chemicals. The induction of a number of pro-inflammatory mediators occurs as the results of increased inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activities. Therefore, suppression of iNOS and COX-2 activities can be an important approach to preventing inflammation in organs. NF-κB and the signaling pathways that regulate many physiological processes, including the innateand adaptiveimmune responses, cell death, and inflammation have become a focal point for intense drug discovery and development efforts. Indeed, growing evidences has validated NF-κB as a target for anti-inflammatory and anticancer agents. Gynostemma genus (Cucurbitaceae) is a climbing vine, attaching itself to supports using tendrils. Over 30 species of Gynostemma are known to grow throughout China, mostly in the South-west. In Vietnam, there are only two species of the Gynostemma genus recorded to date, Gynostemma pentaphyllum (Thunb.) Makino and Gynostemma laxum (Wall.) Cogn. Of these, G. laxum is distributed throughout Vietnam from the plains to mountainous areas. The aerial parts of G. laxum have been used as a bitter stomachic, a laxative, anti-inflammatory as well as anti-obesity. Up to date, no report has been reported about chemical components from this plant. In our screening project for antiinflammatory agents from natural sources, we found G. laxum to possess anti-inflammatory effects. As a part of our continuing research to elucidate the anti-inflammatory compounds, we report herein the isolation, structural elucidation, and evaluation of the inhibitory effects on NFκB activation and iNOS and COX-2 expression of a new benzyl glycoside, 2,4-dihydroxybenzyl-O-α-L-rhamnopyranoside (1), along with nine known compounds from G. laxum. The dried aerial parts of G. laxum were extracted with MeOH and partitioned with n-hexane and ethyl acetate, and water. From these fractions and subsequently separation, one new and nine known compounds were isolated (see Figure 1). Compound 1 was obtained as a white amorphous powder and its molecular formula, C13H18O7, determined on the basis of ESI MS at m/z 287 [M+H] (positive) and HR ESI MS at m/z 287.1147 [M+H] (calcd C13H19O7 for 287.1131). The H-NMR spectrum of 1 (DMSO-d6) (Table 1) showed the following signals: one secondary methyl group at δH 1.03 (d, J = 6.0 Hz), assigned to H-6 of the rhamnose; one anomeric proton at δH 4.72 (br s); one oxygenated methylene (δH 4.42 and 4.50); and one 1,2,4-trisubstituted aromatic ring with ABX coupling patterns [δH 7.20 (d, J = 8.0 Hz), 7.25 (d, J = 8.0 Hz), and 7.96 (s)]. The C-NMR and DEPT spectra (Table 1) revealed 13 carbon signals, of which, 7 signals were assigned to a benzyl moiety and 6 signals belonged to rhamnose moiety. The Hand C-NMR data of 1 (Table 1) were similar to those of benzyl-O-α-L-rhamnopyranoside except for an appearance of two hydroxyl groups at aromatic ring. In the HMBC spectrum (see Figure 2), two proton signals at δH 4.45 and 4.50 of oxygenated methylene group correlated with carbons C-1 (δC 126.3), C-2 (δC 148.1), and C-6 (δC 126.4); one proton signal at δH 7.25 (H-6) correlated with carbons C-2 (δC 148.1), C-4 (δC 154.0), and C-7 (δC