One new triterpenoids from Momordica charantia L

Abstract Momordica charantia L. (M. charantia) is an annual climbing herb in Cucurbitaceae. It is not only a food, but also a drug with a long history of application. This study aims to isolate and identify the chemical constituents form M. charantia and evaluate their inhibiting effect on Hcy-induced proliferation of VSMCs. Silica column chromatography, ODS silica column chromatography, Sephadex LH-20 column chromatography and semi-preparative HPLC were used to obtain one new compound (1). The inhibition on Hcy-induced proliferation of VSMCs 1 was tested through MTT method. As a result, 1 could partially rescue Hcy-induced proliferation of VSMCs at both 5 and 25 μM. GRAPHICAL ABSTRACT


Introduction
Momordica charantia L. (M.charantia), whose fruit is also called bitter melon, is an annual climbing herb in Cucurbitaceae (Jia et al. 2017) which native to tropical East India.M. charantia has a long history of cultivation in China with a Chinese name "kugua"."Kugua" means bitter melon, which comes from its special taste.At first, M. charantia existed as an ornamental plant for vertical greening in the courtyard (Chen 2010) and was not eaten.At present, M. charantia has appeared on the table all over the world as a common ingredient.M. charantia is also an important medicinal material in many ethnopharmacy.It has the effects of clearing heat, nourishing blood, tonifying kidney, invigorating spleen, nourishing liver and brightening eyes in the theory of traditional Chinese medicine.
M. charantia has a long history of being used as medicine.It has been recorded as medicine in many Asian countries and regions.Modern research shows that M. charantia contains a variety of effective chemical components, and has a variety of pharmacological activities, such as hypoglycemic effect (Cai et al. 2008;Cortez-Navarrete et al. 2018;Peter et al. 2019), antioxidation (Chen et al. 2021), anti-inflammation (Chao et al. 2014), improving immunity (Chen et al. 2019), anti-HIV (Fang and Ng 2011) and anti-tumor (Sun et al. 2016).
In this research, one new triterpenoid (1) was isolated from M. charantia and its structure was identified.Subsequently, the inhibitory effect of 1 on the Hcy-induced proliferation of VSMCs was detected.The results of this study will provide a preliminary theoretical basis for the application of M. charantia, and further improve the M. charantia research system..85, 0.86, 0.88, 1.26 (each 3H, s), and 1.53 (6H, s)], implying the potential for 1 to be a triterpenoid.In addition to a group of crotonyl signals [d C 166.1, 123.9, 144.7, 17.7; d H 7.00 (1H, dq, J ¼ 15.5, 7.0), 5.77 (1H, 1dd, J ¼ 15.5, 1.5), 1.58 (dd, J ¼ 7.0, 1. HMBC spectra, correlations from H-19 to C-5, C-8, C-9, C-10, C-11 and from the methoxyl proton (d H 3.47) to C-19 were detected, suggesting the existence of C-9-O-C-5 fragment and thereby establishing of the extra ring moiety (Figure 1A).Furthermore, HMBC correlation from H-3 to C-1 0 of the crotonyl determined that the crotonyl was linked to C-3 of the triterpenoid skeleton.Thus, the planner structure of 1 was established as shown (Figure 1A).The stereo-configuration of 1 was determined via investigation on NOE correlations.In the NOESY spectrum, correlations of H-19/H-8/CH 3 -18 and H-3/H-28/H-10/CH 3 -30/H-17 were given, suggesting the stereo-configuration of 1 was as shown.Finally, the structure of 1 was elucidated as shown (Figure 1A).

Inhibition on the Hcy-induced proliferation of VSMCs
Recently, the fruits of M. charantia has been reported to attenuate atherosclerosis in the mouse model (Zeng et al. 2018).Therefore, in this study, the effect of isolated compounds on vascular smooth muscle cells (VSMCs) was investigated.
The proliferation of VSMCs plays an important role in the pathogenesis of atherosclerosis.Homocysteine (Hcy) is a sulfur-containing amino acid and an intermediate product of methionine metabolism.It is shown that Hcy is no less harmful than hyperlipidemia, and capable of promoting proliferation of VSMCs, inducing them to secrete proteases and promoting the phenotypic transformation of VSMCs (Zhang et al. 2012).
Therefore, the inhibitory effect of 1 on the Hcy-induced proliferation of VSMCs was tested.As a result, 1 could partially rescue Hcy-induced proliferation of VSMCs at both 5 and 25 lM, suggesting the potential usage of 1 in the treatment of atherosclerosis.As far as we know, it is also the first report of a natural product rescuing Hcy-induced proliferation of VSMCs.

Apparatus and reagents
NMR spectrum was performed on an Bruker AVANCE 600 FT-NMR spectrometer with trimethylsilyl used as a reference.HR-ESI-MS spectrum was performed on an Agilent 6550-Q-TOF LC-MS spectrometer.Semi-preparative HPLC was performed on an Agilent 1200 HPLC spectrometer with ultraviolet detector.The ODS column (Ultimate XB-C18, 10 Â 250, 5 lm) for semi-preparative HPLC was purchased from Shanghai Welch Materials Co., Ltd.Silica gel for chemical separation was purchased from Qingdao Marine Chemical Inc., ODS silica gel and Sephadex LH-20 was purchased from Thermo Fisher Scientific Inc., the other chemical reagents of HPLC and analytical grade were from Tianjin Kermel Chemical Co., Ltd.

Plant material
Mature fruits of M. charantia were collected in Weifang city, Shandong Province, P.R. China.The material was authenticated by Dr. Jian Wu from Harbin Uniersity of Commerce.A voucher specimen (NO.20180710) has been deposited at the College of Pharmacy, Harbin University of Commerce.

Extraction and isolation
Crushed M. charantia (10 kg) was reflux extracted three times with 95% ethanol (8 times volume), and the extract solution was dried and concentrated under vacuum to yield the extract (2.3 kg).The extract was suspended in water and partitioned successively with petroleum ether, dichloromethane and n-butanol.The dichloromethane protion (266.3 g) was then performed on a silica gel column chromatography and eluted with a gradient of dichloromethane-methanol and obtaining 10 fractions (Fr.1-10).Fraction 4 was further subjected on an ODS silica gel column chromatography and eluted with a gradient of MeOH-H 2 O to give 6 fractions (Fr.4.1-4.6).Fraction 4.3 was further subjected on a Sephadex LH-20 column chromatography eluted with MeOH-H 2 O, and further purified by HPLC using 80% as eluent to obtain compound 1 (19.5 mg, RT ¼ 28.9 min).

Inhibition on the Hcy-induced proliferation of VSMCs
Cell viability was determined by the method of MTT accordting to the method with minor modification (Zhang et al. 2020).Briefly, Human VSMCs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (1% penicillin/ streptomycin) in an incubator with 5% CO 2 at 37 C.The cells were subcultured when 70-80% synchronized.Cells between passages 3 and 6 were used in the test.VSMCs were subjected to stimulation with Hcy for 48 h before test.Cells stimulated with Hcy were seeded in 96-well plates at a concentration of 6 Â 10 3 cells/well for cell viability test.After the cells adhered to the wall, the cells were cultured in serum-free medium for 24 h to synchronize the cells.The cells were cultured at 37 C in 5% CO 2 incubator for 48 h with test compound (1).After the culture, 20 lL MTT solution was added to each well.Continue incubation at 37 C for 4 $ 6 h, terminate the culture, carefully discard the culture supernatant, add 150 lL DMSO to each well, shake for 10 min, and measure the absorbance (OD value) of each hole at the wavelength of 490 nm by the microplate reader.
Compound 1 was isolated as a white amorphous powder.The HR-ESIMS of 1 afforded a pseudo-molecular ion peak [M þ H] þ at m/z 555.4041 (calcd for [C 35 H 55 O 5 ] þ m/z 555.4044), suggesting the molecular formula of 1 was C 35 H 54 O 5 with nine degrees of unsaturation.The 1 H NMR spectrum of 1 revealed six angular methyl signals [d H 0 Figure 1.The structure of compounds 1.