Two new triterpenoids preventing the hydrocortisone-induced injury in HMEC-1 cells from Momordica charantia L.

Abstract Momordica charantia L. (M. charantia) is an annual climbing herb in Cucurbitaceae. As a medicinal and edible homologous plant, it has a long history of application. This study aims to isolate and identify the chemical constituents from M. charantia and evaluate their prevention effect on hydrocortisone-induced injury in HMEC-1 cells. 10 kg of M. charantia was extracted with 95% ethanol for three times and partitioned with petroleum ether, dichloromethane and n-butanol. The dichloromethane part was performed by silica, ODS silica, Sephadex LH-20 column chromatography and semi-preparative HPLC to obtain two new compounds. The prevention effect on hydrocortisone-induced injury in HMEC-1 cells of these two compounds was determined by the method of CCK-8. The cell viability of HMEC-1 cells treated with 2 (25 μM) was 85.85% ± 4.39%. The results indicated that 2 exhibited significantly prevention effect on hydrocortisone-induced injury in HMEC-1 cells but 1 exhibited no this activity in vitro. Graphical Abstract


Introduction
Momordica charantia L. (M. charantia), an annual climbing herb in Cucurbitaceae, is widely cultivated in temperate and tropical regions all over the world Jia et al. 2017). M. charantia has a long cultivation history in China as a food material because of its strong growth potential, easy cultivation, few diseases and pests (Han et al. 2008). M. charantia contains glycosides with special taste which shows a unique bitter taste. In particular, when cooking with other food materials, M. charantia will not spread its bitterness to the others. So, M. charantia become one of the most favorite foods in China.
As a medicinal and edible homologous plant, M. charantia is also widely used as herb medicine (Mishra et al. 2022). Its pharmacological effects, including hypoglycemic effect (Kulkarni et al. 2021;Cortez-Navarrete et al. 2018;Peter et al. 2019), anti-tumor (Sun et al. 2016), anti-virus (Fang and Ng 2011), anti-parasite, improving immunity, improving immune function (Chen et al. 2019) and others (Chen et al. 2019;Chen et al. 2021;Chao et al. 2014;Yaldız et al. 2015), have been confirmed by relevant studies. Chemical investigation indicates that polysaccharides, lipids, terpenoids, saponins, phenolics and sterols are the main bioactive ingredients in M. charantia. (Jia et al. 2017). In this research, two new triterpenoids were isolated from M. charantia and their structures were identified. Subsequently, the prevention effect of the components on hydrocortisone-induced injury in HMEC-1 cells was preliminarily detected.

Structural identification
Compound 1 was obtained as a white amorphous powder whose HR-ESI-MS spectrum gave a pseudo-molecular ion peak at m/z 525.3181 [M þ Na] -19a)] was also indicated. In the 13 C-NMR spectrum, 30 carbon signals, including those for two carbonyls (d C 165.7 and 178.5) and one double bond (d C 128.5 and 139.1) were revealed. All the NMR data of 1 were similar to those of schisanlactone H (Zhou et al. 2009), except for disappearance of one methoxyl and one pair of double carbon signals and appearance of the aforementioned three-membered ring signals. Subsequently, HMBC correlations from H-19 to C-1 and C-11 led to the determination of the location of the three-membered ring and correlations from H-27 to C-24 and C-26 confirmed the presence of D 24 . Taking other HMBC correlations into account, the planar structure of 1 was assembled as an undescribed 3,4-secocycloartane triterpenoid as shown (Fig. 1). The relative stereochemistry of 1 was established based on the NOESY correlations and the biogenetic consideration with respect to lanostane triterpenes. Moreover, compound 1 showed a positive cotton effect at 250 nm in the experimental ECD spectrum, suggesting the R-configuration of C-22 (Zhou et al. 2009;Liu et al. 2017;Yu et al. 2016). Accordingly, the structure of 1 was established as shown.
Compound 2 was also obtained as a white amorphous powder. The HR-ESI-MS spectrum of 2 indicated a protonated ion peak at m/z 501. Additionally, evident discrepancy of carbon signals of C-20 to C-23 (d C 20.8, 25.7, 75.5, 83.3 for 1 and d C 19.8, 33.6, 44.1, 66.7 for 2) and C-26 (d C 165.7 for 1 and d C 171.5 for 2) were also observed in the 13 C-NMR spectrum of 2, suggesting a different substitution pattern for C-20 to C-27. HMBC spectrum of 2 gave the long-rang interactions from H-27 to C-26 and C-24 and from 21-CH 3 to H-17 and H-22 determined the substitution pattern for C-20 to C-27 and correlations from 30-CH 3 to C-5 and C-29 and from the -OCH 3 to C-3 led to the elucidation of the location of the methoxyl and terminal double bound. In conjunction with other HMBC correlations, the planar structure of 2 was assembled as shown in Figure 1. The relative stereochemistry of 1 was established based on the NOESY correlations and the biogenetic consideration regarding lanostane triterpenes. The coupling constant (J ¼ 8.3 Hz) between H-24 and H-23, together with the negative cotton effect at 305 nm in the ECD spectrum of 2 suggested the R-configuration of C-23 (Yang et al. 2010;Hasegawa et al. 1985). Therefore, the structure of 2 was established as shown.

Prevention of hydrocortisone-induced injury in HMEC-1 cells
HMEC-1 cell line is a kind of human microvascular endothelial cells. Microvascular endothelial cells (MEC) play a very important role in inflammatory response, tumor growth and wound healing. In the field of wound healing, due to the maturity of in vitro culture technology of epidermal cells and dermal fibroblasts, a lot of in-depth studies have been carried out on these two kinds of cells. In contrast, dermal microvascular endothelial cells, another major cell involved in wound healing, are limited by the difficulty of isolation and culture technology in vitro. Therefore, the prevention of 1 and 2 on the hydrocortisoneinduced injury in HMEC-1 cells was tested. The results indicated that 2 could prevent the hydrocortisone-induced injury in HMEC-1 cells in a dose-dependent manner. The cell viability of HMEC-1 cells treated with 2 at a concentration of 25 lM was 85.85% ± 4.39%, but 1 exhibited no prevention on the hydrocortisone-induced injury in HMEC-1 cells.

Apparatus and reagents
NMR spectrum was performed on a Bruker AVANCE 600 FT-NMR spectrometer with trimethylsilyl as a reference. HR-ESI-MS spectrum was performed on a Waters Xevo G2-XS QTof LC-MS spectrometer. Silica gel for column chromatography was purchased from Qingdao Marine Chemical Inc., ODS silica gel for column chromatography was purchased from Shanghai Welch Materials Co., Ltd. and Sephadex LH-20 was purchased from Thermo Fisher Scientific Inc. Semi-preparative HPLC was performed on an Agilent 1100 HPLC with ultraviolet detector. The ODS column for semi-preparative HPLC was purchased from Agilent Technologies, Inc. The other chemical reagents of HPLC and analytical grade were from Tianjin Damao Reagent Factory.

Plant material
Momordica charantia was obtained from Shenyang, China, and was identified by Dr. Bing Liu from Harbin University of Commerce, preserving the plant specimen (PVJ20130821) in the herbarium.

Extraction and isolation
Crushed M. charantia (10 kg) was reflux extracted three times with 95% ethanol (10 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 part (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 5 was further subjected on an ODS silica gel column chromatography and eluted with a gradient of MeOH-H 2 O to give 5 fractions (Fr. 5.1-5.5). Fraction 5.2 was further subjected on a Sephadex LH-20 column chromatography eluted with MeOH-H 2 O, and purified by semi-preparative HPLC to obtain compound 1 (23.5 mg) and 2 (20.8 mg).

Prevention of hydrocortisone-induced injury in HMEC-1 cells
Cell viability was determined by the CCK-8 method. Briefly, inoculate 100 lL cell suspension per well in 96 well plates. The plates were pre cultured in an incubator for 24 h (37 C, 5% CO 2 ). Add a certain amount of compounds 1 and 2 to the culture plate. Incubate the plates in the incubator for 12 h. Add 10 lL CCK-8 solution to each hole and avoid bubbles in the hole. Incubate the plates in the incubator for 1 h. The absorbance at 450 nm was determined by microplate reader.

Statistical analysis
SPSS 20.0 was used for statistical and values were expressed as means ± SD. Origin 2021 was used for creating the histogram.

Conclusions
The chemical constituents of M. charantia were studied, and two new triterpenoid compounds were isolated. 2 could partially prevent the hydrocortisone-induced injury in HMEC-1 cells in a dose-dependent manner, but 1 exhibited no prevention on the hydrocortisone-induced injury in HMEC-1 cells. M. charantia has become a mature cash crop, and its medicinal effect has been concerned by the field of pharmacy. This research results further promoted the research progress of pharmacological effects and composition of M. charantia and provided a driving force for the development of M. charantia industry.

Disclosure statement
The authors report no declarations of interest.

Funding
The author(s) reported there is no funding associated with the work featured in this article.