Identification of 16,25-O-diacetyl-cucurbitane F and 25-O-acetyl-23,24-dihydrocucurbitacin F as novel anti-cancer chemicals

Seven new cucurbitane glucosides, hemslepensides J-P (1–7), and two known compounds, 16,25-O-diacetyl-cucurbitane F (8) and 25-O-acetyl-23,24-dihydrocucurbitacin F (9), were isolated from the tubers of Hemsleya pengxianensis var. jinfushanensis. The structures of 1–7 were elucidated using infrared absorption spectroscopy, nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The treatment of HT29 cells, human colon cancer cells, with compounds 8 and 9 inhibited cell proliferation. Further study demonstrated that compounds 8 and 9 induced F-actin aggregation, G2/M phase cell cycle arrest and cell apoptosis in HT29 cells. In summary, the present study enriched the chemical composition research of H. pengxianensis, and suggested that the compounds 8/9 treatment may be a potentially useful therapeutic option for colon cancer.


Introduction
Tetracyclic triterpenes are a diverse group of natural products consisting of four rings and 30 carbon atoms. grosvenorii (Cucurbitaceae) or Momordica Linn. (Cucurbitaceae), tetracyclic triterpenes have been reported with multiple biological activities including anti-inflammatory function, cytotoxicity and anti-cancer, preventive and curative effects against CCl 4 -induced hepatotoxicity and anti-fertility activities [1][2][3]. The genus Hemsleya (Cucurbitaceae) includes approximately 30 species, distributed in the subtropical to temperate regions in Asia, eastern India and northern Vietnam. In China, it is mainly distributed in the southwest to south central regions. Tetracyclic triterpenes were identified as the main chemical constituents in the tubers of Hemsleya pengxianensis var. jinfushanensis [4 -8].
As previously reported, our studies had identified 16 new compounds with cytotoxic activity against the human cancer cell lines [6][7][8]. Recently, further investigation of H. pengxianensis var. jinfushanensis resulted in the discovery of another seven new cucurbitane-type triterpenoids, named hemslepensides J-P (1-7), together with two known aglycones (8-9) (figure 1). In this paper, we elucidated the isolation and structure of seven new saponins. Moreover, based on our research results and previous published data [4][5][6][7][8], the relationships between the nuclear magnetic resonance (NMR) characteristics and the common substituents of cucurbitane tetracyclic triterpenes were discussed.
Compound 8 was first isolated in our laboratory and its cytotoxic activity has been screened in H460, SW620 and DU145 cell lines [6]. As the main component of H. pengxianensis var. jinfushanensis, compound 9 has been shown to inhibit human lung adenocarcinoma cell line A549 growth both in vitro and in vivo [9,10]. Although compounds 8 and 9 exhibit potent cytotoxicity, the underlying mechanism in colon cancer cells remains to be elucidated. In previous experiments, we screened the cytotoxicity of compounds 8 and 9. The results revealed that these two compounds were more effective than cisplatin against colon cancer HT29 cells. So, the anti-colon cancer mechanism of compounds 8 and 9 was studied targeting the HT29 cell line.

Extraction and isolation
The air-dried rhizomes (     no.

Enzymatic hydrolysis of 1 -7 and determination of the absolute configuration of the monosaccharides
To each solution of compounds 1 -7 (4 mg) in H 2 O (4 ml) was added cellase (each 20 mg) and stirred at 408C for 120 h. The reaction mixture was partitioned between EtOAc and H 2 O and the EtOAc layer was separated. The aqueous layer was evaporated under reduced press to afford solid saccharide mixture. The authentic samples of D-(þ)-glucose and L-(2)-glucose and the saccharide mixture were dissolved in 1 ml of H 2 O and mixed with 1 ml of EtOH to which (S)-(2)-a-methylbenzylamine (7 ml) and NaBH 3 CN (6.75 mg) were added, respectively. The mixture was stirred at 408C for 4 h followed by the addition of glacial acetic acid (0.2 ml) and evaporated under reduced pressure to afford solid mixture. Then, the cells per well were treated with 10 ml of MTT solution (5 mg ml 21 in PBS) and reincubated for 4 h at 378C. The medium was removed and 150 ml well 21 DMSO was replaced. The plates were oscillated gently for 5-10 min to dissolve fully in a 96-well plate oscillator. The OD value of each well was measured at 570 nm using the Victor 1420 instrument, and the half-maximal inhibitory concentration (IC 50 ) values were calculated using the SPSS statistical software. Cisplatin served as a positive control. At least, three independent experiments were performed.

Flow cytometric analysis for cell cycle distribution
The 3 Â 10 5 well 21 HT29 cells were seeded into six-well plates in 2 ml of medium and incubated at 378C overnight. Then, the HT29 cells were treated with DMSO (2‰), compounds 8 and 9 (0, 0.1 or 1 mM, respectively) for 24 h. Afterwards, the cells were collected by trypsinization and fixed in 70% ice-cold ethanol at 48C for another 24 h. The cells were then washed twice with PBS and stained with fluorochrome solution including PI and RNAse in the dark for 30 min at 378C. The DNA content was analysed by flow cytometer. At least three independent experiments were performed.

Cell apoptosis detection
The HT29 cells were plated at a concentration of 3 Â 10 5 cells per well in 2 ml of medium in six-well plates overnight. Then, the cells were treated with either 0.1 or 1 mM compounds 8 and 9, respectively, for 24 h. Following harvesting by trypsinization, the cells were washed once with PBS and then were resuspended in 195 ml of Annexin V-FITC binding buffer. Afterwards, 5 ml of Annexin V-FITC and 10 ml of PI were added in turn followed by incubating in the dark for 15 min at room temperature (20 -258C). The cells were analysed using flow cytometer. At least three independent experiments were performed.

Statistical analysis
All statistical comparisons were made by Statistical Product and Service Solutions (SPSS, v. 20.0). The differences were described as statistically significant if p , 0.05.  1 and 2) with those of delavanoside A indicated that these two compounds have the same aglycone and they just differ in the attachment point of the sugar residue [11]. The sugar residue was attached to C-26 (d C 73.5) in 1, which was confirmed by the HMBC correlations from d    Hemslepenside L (3) was isolated as a white solid. The molecular formula C 42 H 70 O 13 was determined via its HRESIMS data (m/z 805.4714 [MþNa] þ , calcd. for 805.4714), which suggested that 3 and hemslepenside H were a pair of isomers [8]. The 13 C NMR data of 3 (table 2) and hemslepenside H were almost identical with the exception of the b-D-glucose link location. Two b-D-glucoses were linked to C-3 and C-26, respectively, in hemslepenside H, whereas two b-D-glucoses composed a disaccharide unit, then linked to C-27 in 3, which could be confirmed by HMBC correlations from d The molecular weight of 6 was 162 mass units more than that of jinfushanoside B [4], which suggested an additional hexose unit existed in 6. The additional b-D-glucose was connected to C-6 0 , which could be deduced from the HMBC correlations from d H 4.41 (1H, d, J ¼ 7.8 Hz, H-1 00 ) to d C 70.0 (C-6 0 ). The same NOESY and HMBC correlations for the 26,27-dihydroxycucurbita-11-one skeleton as those in jinfushanoside B were found. Thus, the structure of 6 was assigned as 3b,26,27-trihydroxycucurbita-5,

Results and discussion
Hemslepenside P (7) was isolated as a white amorphous powder. The elemental formula for 7 was assigned as C 42 H 68 O 14 from the ion peak [MþNH 4 ] þ at m/z 814.4964 (calcd. for 814.4953) in the positivemode HRESIMS. In the 13 C NMR and DEPT spectra, the significant difference between 7 and carnosifloside I came from the great downfield of C-20 (from d C 35.9 in carnosifloside I to 75.8 in 7), which indicated the attachment of a hydroxyl to C-20 in 7 [12]. The above deduction was confirmed by its 16 mass units more than that of carnosifloside I and supported by HMBC correlations from d  table 3. Compounds 8 and 9 displayed significant cytotoxicity; however, except compound 2, the other six new compounds had no cytotoxicity. The results suggest that the cytotoxicity of cucurbitane tetracyclic triterpenes sapogenins is generally more potent than that of saponins based on previous reports of our group [6][7][8].

The effects of compounds 8 and 9 on the G 2 /M phase of the cell cycle in HT29 cells
To investigate the underlying mechanism of the proliferation inhibitory effects of compounds 8 and 9 in HT29 cells, the cell cycle check points were examined by flow cytometry. HT29 cells were treated with 0.1 and 1 mM of compounds 8 and 9 for 24 h in the experimental group, while the control group was treated with the DMSO (2‰). As shown in figure 4, treatment with DMSO (2‰) had no effect on the distribution of cell cycle. Compared with the control group, the percentage of cells in the G 2 /M phase increased significantly with the increasing dose of 8 and 9. After treatment with 1 mM compound 8 for 24 h, the percentage of cells in the G 2 /M phase increased from 4.1% to 13.1%. Compound 9 showed similar effects to compound 8. These results suggested that induction of G 2 /M cell cycle arrest may contribute to the inhibitory effects of compounds 8 and 9 on cell proliferation.

The effects of compounds 8 and 9 on apoptotic induction in HT29 cells
To determine if apoptosis is involved in the two compounds' induced inhibition of cell proliferation, we employed Annexin V/PI double staining to examine the effect of compounds 8 and 9 on apoptosis. The results indicated that treating with 0.1 and 1 mM compounds 8 or 9 for 24 h induced a significant increase in the percentage of apoptotic and necrotic cells when compared with the control group. As shown in figure 5, DMSO (2‰) did not induce cell apoptosis, while compounds 8 and 9 significantly increased the percentage of apoptotic and necrotic cells in a dose-dependent manner. These results suggested that induction of cell apoptosis may be involved in the inhibitory effects of compounds 8 and 9 on cell proliferation in HT29 cells.

Induction of cell morphological changes by compounds 8 and 9 in HT29 cells
As the cytoskeleton is very important for maintaining the native morphology and function of cells, we investigated that the microfilaments change by staining F-actin after the treatment with compounds 8 and 9. Negative control HT29 cells exhibited F-actin that mainly surrounded the edge of cells, especially in the contact area of cells ( figure 6). Meanwhile, the formation of lamellipodia was observed at the leading edge (indicated by yellow arrows in figure 6). The microfilaments in solvent control cells (2‰ DMSO) were as intact as that of negative control, whereas destruction of cytoskeleton was observed in cells treated with compounds 8 and 9. The distribution of F-actin was diminished significantly in area near the surface of the cells ( figure 6). Overall, the F-actin aggregation (indicated by white arrows in figure 6) was increased with increasing concentration of