Novel Cucurbitane Triterpenes from the Tubers of Hemsleya amabilis with Their Cytotoxic Acitivity

Chemical research of the medicinal plant Hemsleya amabilis (Cucurbitaceae) yielded five new cucurbitane-type triterpenes hemslelis A–E (1–5) by silica gel column, ODS column, and semi-HPLC techniques. Their structures were determined by spectroscopic analysis and examined alongside existing data from prior studies. Compounds 1–5 were evaluated for their cytotoxic activities against three human tumor cell lines, Hela, HCT-8, and HepG-2, with the IC50 ranging from 5.9 to 33.9 μM compared to Cisplatin.


Introduction
Hemsleya, a genus of Cucurbitaceae family, is comprised of more than thirty species in tropical and subtropical regions of China [1]. Most tubers of the plants in this genus have been used as traditional medicine in ethnic minority areas of China. Previous phytochemical evaluations on this genus have disclosed the presence of abundant compounds, such as diterpenes, oleanane, and cucurbitane-type triterpenes [2][3][4]. Among these components, cucurbitane triterpenes have shown potent cytotoxic activity. Hemslecin A (also called cucurbitacin IIa) was reported to suppress cancer cell growth in vitro and reduce tumor size on mouse H22 liver cancer [5]. Some evaluations have indicated that the mechanisms of cucurbitacins' activities includes the disruption of the Jaks-Stat (Janus kinase-signal transducer and activator of transcription) signaling pathway, and especially the STAT3 signaling pathway [6][7][8][9][10].
Hemsleya amabilis, a species of the genus Hemsleya, commonly known as "xue dan" in Yunnan province of China, has been long used as a part of "Dai" medicine and dispensed for bacillary dysentery, tuberculosis, stomachache, whooping cough, and bile duct infection (The Pharmacopoeia Commission of PRC, 2005). For the purpose of finding new bioactive cucurbitane triterpenes from this medicinal plant, we examined the ethanol extract of H. amabilis and isolated five new cucurbitane triterpenes, hemslelis A-E (1-5) (Figure 1). In this paper, we reported the isolation and structure elucidation of the new compounds as well as their cytotoxic activity.
Compound 3 was obtained as an amorphous white powder, and its molecular formula was established as C30H48O5 on the basis of the positive molecular ion peak at m/z 511.3328 [M + Na] + in the HRESMS. Its 1 H and 13 C-NMR data (Table 1) were close to those of 2, with the exception of the lack of one carbonyl ketone bond and the emergence of one oxygenated methine signal. In comparison with 2, the signal for C-7 revealed a powerful upfield shift to δC 66.5 (−133.1 ppm), which indicated that the ketone group at C-7 in 2 was reduced to hydroxyl group in 3. Taken together with 1 H-1 H COSY, HSQC, HMBC, and NOE spectra, the structure of compound 3 was determined to be 3β, 7β, 26, 27-tetrahydroxycucurbita-5, 24-dien-11-one, and named hemslelis C.
Compound 5 was isolated as a white amorphous powder. Its molecular formula was established as C35H52O9 by HRESIMS (observed m/z 639.3522 [M + Na]+, Calcd for 639.3509), requiring ten degrees of unsaturation. The NMR data of 5 were similar to those of compound 4, except for the missing carbon of C-26 and hydroxyl group at C-20 in 5 which were confirmed by the 13 C-NMR data (δC 36.4, C-20) and HMBC correlations from δH 2.26 (H-27) to δC 198.2 (C-25). The sugar moiety was located at C-3 on the basis of the correlation between the proton signal at δH 3.71 (H-3) and anomeric carbon at δC 107.6. The type and absolute configuration of the sugar was Taken together with the NOESY spectrum, the structure of 2 was established as 3β, 26, 27-trihydroxycucurbita-5, 24-dien-7,11-dione, and given the trivial name hemslelis B.
Compound 3 was obtained as an amorphous white powder, and its molecular formula was established as C 30 H 48 O 5 on the basis of the positive molecular ion peak at m/z 511.3328 [M + Na] + in the HRESMS. Its 1 H-and 13 C-NMR data (Table 1) were close to those of 2, with the exception of the lack of one carbonyl ketone bond and the emergence of one oxygenated methine signal. In comparison with 2, the signal for C-7 revealed a powerful upfield shift to δ C 66.5 (−133.1 ppm), which indicated that the ketone group at C-7 in 2 was reduced to hydroxyl group in 3. Taken together with 1 H-1 H COSY, HSQC, HMBC, and NOE spectra, the structure of compound 3 was determined to be 3β, 7β, 26, 27-tetrahydroxycucurbita-5, 24-dien-11-one, and named hemslelis C.
Compound 5 was isolated as a white amorphous powder. Its molecular formula was established as C 35 H 52 O 9 by HRESIMS (observed m/z 639.3522 [M + Na] + , calcd. for 639.3509), requiring ten degrees of unsaturation. The NMR data of 5 were similar to those of compound 4, except for the missing carbon of C-26 and hydroxyl group at C-20 in 5 which were confirmed by the 13 C-NMR data (δ C 36.4, C-20) and HMBC correlations from δ H 2.26 (H-27) to δ C 198.2 (C-25). The sugar moiety was located at C-3 on the basis of the correlation between the proton signal at δ H 3.71 (H-3) and anomeric carbon at δ C 107.6.

Cytotoxic Activity
Compounds 1-5 were tested for their cytotoxic activities against three human tumor cell lines, Hela, HCT-8, and HepG-2, with the IC 50 ranging from 5.9 to 33.9 µM compared to Cisplatin, the positive control (Table 2). It should be noted that none of the isolated compounds showed any selectivity in their cytotoxic activities. Compounds 2 and 3 displayed moderate activities towards HCT-8 human tumor cell lines, with the IC 50 values of 5.9 and 6.1 µM, respectively. While, compound 4 was inactive against HCT-8, and compound 5 were inactive against both Hela and HepG-2. From the biological results, it can be noted that the oxidation of the fatty chain at C-17 in 5 may have decreased the activity. It was previously reported that some cucurbitane triterpenes that had similar structures to the compounds showed potent cytotoxic activities against several cancer cell lines

Discussion
Cucurbitane triterpenes, with the characteristics of a tetracyclic system and a fatty chain, were isolated from the tubers of H. amabilis, which are widely distributed in genus of Hemsleya. Ethnobotanically, the tubers of plant known as "xue dan" in the Yunnan province of China, and have been long used as a part of "Dai" medicine. Cucurbitane triterpenes have shown potent cytotoxic activity. As a result, we investigated all the isolated compounds for their cytotoxic activity. Compared with the cisplatin positive control group, all compounds displayed a measure of cytotoxic activities against human tumor cell lines, with the IC 50 ranging from 5.9 to 33.9 µM. Further analysis of the data showed that compounds 2 and 3 displayed moderate activities towards HCT-8 human tumor cell lines over other compounds, while compound 5 was inactive against both Hela and HepG-2, which indicated that the oxidation of the fatty chain at C-17 in 5 may decreased the activity.

General Experimental Procedures
Optical rotation data were obtained using a Perkin-Elmer 341 digital polarimeter (Perkin-Elmer, Waltham, MA, USA). IR data were recorded using a Shimadzu FTIR-8400S spectrophotometer (Shimadzu, Kyoto, Japan). NMR data were obtained with a Bruker AV III 600 NMR spectrometer (Bruker, Billerica, MA, USA) (chemical shift values are presented as δ values with TMS as the internal standard) using the solvent Pyrridine-d 5 as references. HRESIMS data were acquired using a LTQ-Obitrap XL spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). HPLC was performed using a Lumiere K-1001 pump, a Lumiere K-2501 single λ absorbance detector, and an YMC-Pack ODS-A column (5 µm, 10 × 250 mm, YMC, Kyoto, Japan). Silica gel (100-200 mesh) and silica gel GF254 sheets (0.20-0.25 mm) (Qingdao Marine Chemical Plant, Qingdao, China) were used for column chromatography and TLC, respectively. TLC spots were visualized under UV light and by dipping into 5% H 2 SO 4 in EtOH followed by heating. All solvents used were of analytical grade (Beijing Chemical Works). The cell lines were obtained from ATCC.

Plant Material
The tubers of H. amabilis were collected in Chongqing, Sichuan Province, People's Republic of China, in August 2017, and were authenticated by Prof. Si-Rong Yi. A voucher specimen (CS170802) has been deposited at the Hebei University of Chinese Medicine.

Extraction and Isolation
The tubers of H. amabilis (1.2 kg) were powdered and extracted three times with 95% EtOH. The combined extract was concentrated under reduced pressure to furnish a dark brown residue (75.4 g), which was suspended in H 2 O and partitioned with petroleum ether, CH 2 Cl 2 , and EtOAc, respectively. The EtOAc fraction (13.3 g) was subjected to silica gel (100-200 mesh, 10 × 80 cm) column chromatography using a CH 2 Cl 2 -MeOH gradient

Acid Hydrolysis of 5
Compound 5 (2.0 mg) was heated in 3 mol/L CF 3 COOH (4 mL) for 3 h in a water bath. Each mixture was then extracted with EtOAc. The aqueous layer was evaporated to dryness with ethanol in vacuo at 50 • C until neutral. The residues were determined in comparison with D-Glucose using TLC (CHCl 3 :MeOH:H 2 O = 3:2:0.2, visualization with ethanol-5% H 2 SO 4 spraying). Furthermore, the absolute configurations of the sugars were determined by gas chromatography according to a method previously described [17,18]. By this method, L-cysteine methyl ester hydrochloride (0.06 mol/L) and hexamethyldisilazane-trimethylchlorosilane (HMDS-TMCS, 3:1) were added to the aqueous residue for derivatization. The solution was then centrifuged and the precipitate removed. After these processes, n-hexane was used to extract derivate, which was then analyzed by GC. D-Glucose (t R = 24.3 min) was detected by comparing with authentic monosaccharide.

Cytotoxic Bioassays
The cytotoxic activities of compounds 1-5 were evaluated using the MTT procedure with human cancer cell lines Hela (ATCC CCL-2), HCT-8 (ATCC CCL-244), and HepG-2 (ATCC HB-8065). The cells were incubated in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum and cultured at a density of 1.2 × 10 4 cells/mL in a 96-well microtiter plate. Five different concentrations of each agent dissolved in dimethyl sulfoxide (DMSO) were then put in the wells. Each concentration was evaluated three times. After incubation under 5% CO 2 at 37 • C for 48 h, 10 µL of MTT (4 mg/mL) was placed into each well, and the cells were incubated for an additional 4 h. Then, the liquid was taken out, and DMSO (200 µL) was put into the wells. The absorbance was documented with a microplate reader at a wavelength of 570 nm.