A New Megastigmane Glucoside and Other Constituents from Desmodium gangeticum

A new megastigmane glycoside, gangeticoside ( 1 ), and three known compounds leonuriside A ( 2 ), methyl benzoate 2-O- β -D-glucopyranoside ( 3 ), and tortoside A ( 4 ) were isolated from the aerial part of Desmodium gangeticum. Their structures were determined by 1D and 2D NMR spectra. The isolated compounds were evaluated for their inhibitory eﬀect on NO production in LPS-stimulated RAW264.7 cells. Among them, compounds 1 , 2, and 3 exhibited strong eﬀect with the IC 50 values of 22.3, 15.6, 7.3 μ M, respectively.


Introduction
Desmodium gangeticum (L.) DC. is a woody perennial herb plant, which distributed wildly in the mountainous area in Vietnam, or several regions in Asia and Africa [1,2]. D. gangeticum has been used for the treatment of asthma, stomatitis, arthritis, eczema, or some other diseases [1]. Previous studies proved the contents of flavonoids, alkaloids, and sterols in D. gangeticum with hepatoprotective, antiinflammatory, antileishmanial, immunomodulatory, cardioprotective, antinociceptive wound healing, antidiabetic, and antiulcer activities [3][4][5][6]. Nitric oxide (NO) has been known to involve in the regulation of various physiological processes in mammals and the overproduction of NO is responsible for the pathological development of inflammation, cancer, and diabetes [7,8]. erefore, inhibitors of NO production have potential therapeutic value as anti-inflammatory agents [9]. In this study, we reported the isolation of a new megastigmane glycoside 1 and three known compounds from D. gangeticum (Figure 1). e isolated compounds were evaluated for their inhibition of nitric oxide (NO) production in LPS-stimulated RAW264.7 cells.

Extractions and Isolation.
e air-dried powdered aerial parts of Desmodium gangeticum were extracted with methanol (6 L × 3 times) at room temperature in 24 hours. e combined extracts were concentrated to obtain a methanol (MeOH) crude residue (180 g), which was suspended in water and then successively partitioned with ethyl acetate. Evaporation of solvents under reduced pressure gave ethyl acetate residues (87.5 g). e water layer was adsorbed on a Diaion HP-20 column and then eluted with water, methanol 50%, and 100% to collect W, M50, and M100 fractions, respectively. Fraction M100 was subjected to a silica gel chromatography column with gradient mixtures of CH 2 Cl 2 -MeOH (1/0-0/1, v/v) to yield seven fractions, B1-B7. Fraction B2 was separated on a Sephadex LH-20 column eluted with methanol 50% to obtain two subfraction B2.1 and B2.2. e fraction B2.1 was purified on a YMC-C18 chromatography column eluted with methanol 67% to yield compound 1 (5.2 mg). e fraction B2.2 was isolated on a silica gel chromatography column with CH 2 Cl 2 -MeOH (6/ 1, v/v) elution to obtain compound 2 (10.3 mg). Compound 3 (6.1 mg) was separated from the fraction B4 using a YMC-C18 chromatography column eluted with methanol 50%. Fraction B6 was isolated on a Sephadex LH-20 column eluted with methanol 60%, followed by a YMC RP-C18 column eluted with methanol 50% to collect compound 4 (6.6 mg).

Acid Hydrolysis and Sugar
Identification. Compound 1 (1 mg) was heated in 1N HCl (500 μL) at 80°C for 2 h, then the solution was extracted with ethyl acetate (1 ml × 3). e aqueous layer was neutralized with NH 4 OH and then dried under reduced pressure. e obtained residue was redissolved in 150 μL pyridine containing 10 μmol of L-cysteine methyl ester and heated at 80°C for 1 h. 6 µl o-tolyl isothiocyantate was added, and the solution was heated for another hour. e reaction solution was then analyzed by HPLC using Cosmosil 5C18-MS-II column (4.6 × 150 mm), mobile phase of 20% acetonitrile in 0.2% TFA water, UV detection at 254 nm. e sugars were identified as D-glucose (t R 9.05 min).  Journal of Chemistry

Assay for Inhibition of NO Production.
e anti-inflammatory effects of the isolated compounds were evaluated via the inhibition of NO production in LPS-stimulated RAW264.7 cells [10]. Briefly, RAW264.7 cells were seeded in 96-well plates at 2 × 105 cells/well and incubated for 24 h. e plate were pretreated with various concentrations of test samples for 30 min and then incubated for another 24 h with or without 1 μg/mL LPS. As a parameter of NO synthesis, nitrite concentration in the culture supernatant was measured by the Griess method. 100 μL of the culture supernatant was transferred to other 96-well plate and 100 μL of Griess reagent were added. e absorbance of the reaction solution was read at 570 nm with a microplate reader. e remaining cell solutions in cultured 96-well plates were used to evaluate cell viability by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cardamonin was used as a positive control. e 13 C-NMR and HSQC spectra ( Figures S3 and S4) of 1 indicated the presence of 21 signals including three methyl, four methylene, nine methine, and five quaternary carbon groups. A glucopyranose was recognized by six characteristic signals at δ C 103.1 (C-1′′), 75.1 (C-2′′), 78.0 (C-3′′), 71.6 (C-4′′), 77.9 (C-5′′), and 62.7 (C-6′′). Acid hydrolysis followed by HPLC analysis allowed confirming the D-glucose. e remaining 15 carbon signals suggested the presence of a megastigmane structure. e positions of the two double bonds at C-7 and C-9 were assigned by HMBC ( Figure S5), correlations of H-10 to C-8, C-9, and the carboxyl group, H-7 to C-6, C-8, and C-9, and H-8 to C-6, C-7, C-9, and C-10. e position of glucose was elucidated by the correlation between H-1′ and C-3. e positions of three methyls were assigned by HMBC correlations between H-11 to C-1, H-13 to C-5, and H-14 to C-9 ( Figure 2). e compound 1 showed similar structural properties to dihydrophaseic acid, the other megastigmane in the previous study [11]. However, the presence of the quaternary carbon C-1 and the HMBC correlation between H-12 to the carboxyl group revealed the structure of a lactone, rather than a carboxylic acid. As can be seen from Figure 2, the NOE correlation between H-7/H-12 and no correlation between H-7/H-8 indicated E geometry of the double bond at C-7, whereas the correlation between H-10/ H-14 suggested Z-geometry of C-9 double bond ( Figure S6). Based on the above analysis, the structure of compound 1 was elucidated as a new megastigmane glycoside, which was named gangeticoside. ree known compounds were elucidated by comparing their NMR data to the previous reports, including leonuriside A (2) [12], methyl benzoate 2-O-β-D-glucopyranoside (3) [13], and tortoside A (4) [14]. Compounds 1, 2, and 3 exhibited significant anti-inflammatory activity with NO inhibition IC 50 at 22.3, 15.6, 7.3 µM, respectively, while compound 4 was inactive (Table 1). e MTT assay showed that those compounds had no significant toxicity to RAW264.7 cells up to 50 μM (data not shown), indicating that the inhibitory effect on NO production was not due to cytotoxicity.

Results and Discussion
It is reported that the extracts of Desmodium gangeticum exhibited significant anti-inflammatory effect in vitro as well as in animal models [15,16]    Journal of Chemistry (17Z,20Z)-hexacosa-17, 20-dien-9-one and gangenoid dose-dependently inhibited proinflammatory cytokines TNF-α and IL-6 in LPS-stimulated RAW264.7 cells [17]. e pterocarpenoid gangetin exhibited potent anti-inflammatory properties against the carrageenininduced paw edema in rats [18]. Consistently, the present study identified other components in D. gangeticum responsible for the antiinflammation effect of this plant.

Conclusions
In conclusion, a new megastigmane glycoside, gangeticoside (1), and three known compounds leonuriside A (2), methyl benzoate 2-O-β-D-glucopyranoside (3), and tortoside A (4) were isolated from the aerial part of Desmodium gangeticum. Data Availability e data used in the study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.