Cytotoxic Polyhydroxylated Oleanane Triterpenoids from Cissampelos pareira var. hirsuta

Three new polyhydroxylated oleanane triterpenoids, cissatriterpenoid A−C (1−3), along with one known analogue (4), were isolated from the whole plant of Cissampelos pareira var. hirsuta. Their chemical structures were elucidated by extensive spectroscopic data (IR, HR-ESI-MS, 1H-NMR, 13C-NMR, DEPT, 1H-1H COSY, HSQC, HMBC, NOESY) and the microhydrolysis method. The isolation of compounds 1–4 represents the first report of polyhydroxylated oleanane triterpenoids from the family Menispermaceae. All isolated compounds were evaluated for their cytotoxicity against five human cancer cell lines, and the inhibitory activity against NO release in LPS-induced RAW 264.7 cells. Compound 3 showed the most potent cytotoxic activities against the A549, SMMC-7721, MCF-7, and SW480 cell lines, with IC50 values of 17.55, 34.74, 19.77, and 30.39 μM, respectively, whereas three remaining ones were found to be inactive. The preliminary structure–activity relationship analysis indicated that the γ-lactone ring at C-22 and C-29, and the olefinic bond at C-12 and C-13 were structurally required for the cytotoxicity of polyhydroxylated oleanane triterpenoids against these four cell lines. Based on lipid-water partition coefficients, compound 3 is less lipophilic than 1 and 4, which agrees with their cytotoxic activities. This confirms the potential of C. pareira var. hirsuta in the tumor treatment.


Introduction
So far, cancer has become the most common disease threatening human health and life worldwide. Natural products, characteristic of low toxicity and high efficiency have played an irreplaceable role in cancer chemotherapy and chemoprevention in the last half-century. Over 60% of clinical anticancer drugs come from natural products, such as paclitaxel, etoposide, camptothecin, and vincristine, etc [1]. Oleanane triterpenoids are phyrophosphate oligomers of six isopentenyl structure units. They possess extensive and important biological activities, such as cardio-, hepato-, and gastro-protective, anti-inflammatory, antiviral, antidiabetic, antimicrobial, antiparasitic, analgesic and wound-healing effects, as well as inducing apoptosis in cancer cells [2]. Among these biological properties, their anticancer effects are most attractive as they are confirmed in various pharmacological models in vitro and in vivo [3]. Oleanane triterpenoids exhibit cytotoxic effects in many cancer cell lines, such as oral, esophageal, liver, brain, colorectal, ovary, breast, lung, skin cancers, and leukemia [4]. They regulate multiple cellular signaling pathways, including nuclear factor-KB, AKT, signal transducer and activator of transcription 3, mammalian target of rapamycin, caspases, intercellular adhesion molecule 1, vascular endothelial growth factor, and poly (ADP-ribose) polymerase in cancer cells [5]. As promising anti-cancer agents, they inhibit the viability and proliferation of human cancer cells, prevent their migration and metastasis, and induce their apoptosis [6]. So, oleanane triterpenoids will be a significant source of new anticancer-drug development.
Cissampelos pareira L. var. hirsuta (Buch. ex DC.) Forman is a perennial climbing shrub of Menispermaceae family, which is widely distributed in the Southwest of China, India. Its whole plant has been commonly used for the treatment of stomachache, cardiodynia, fever, skin disease, and swelling pain, etc [7]. However, very few researches on its chemical compositions have been reported. Previous phytochemical investigation of the genus Cissampelos showed the presence of alkaloids [8], flavonoids [9], terpenes [10], and pregnane glycosides [11]. Our previous chemical and pharmacological investigations on C. pareira var. hirsuta revealed the presence of polyhydroxylated pregnane glycosides, and their cytotoxic properties [11]. As our ongoing research for potential cytotoxic natural products from traditional Chinese medicines, three new polyhydroxylated oleanane triterpenoids, cissatriterpenoid A−C (1−3), and one known analogue (4), were obtained from the dried whole plant of C. pareira var. hirsuta ( Figure 1). Detailed isolation, structure elucidation, and cytotoxicity, and NO inhibitory activity of those isolates are reported herein. pathways, including nuclear factor-КB, AKT, signal transducer and activator of transcription 3, mammalian target of rapamycin, caspases, intercellular adhesion molecule 1, vascular endothelial growth factor, and poly (ADP-ribose) polymerase in cancer cells [5]. As promising anti-cancer agents, they inhibit the viability and proliferation of human cancer cells, prevent their migration and metastasis, and induce their apoptosis [6]. So, oleanane triterpenoids will be a significant source of new anticancer-drug development. Cissampelos pareira L. var. hirsuta (Buch. ex DC.) Forman is a perennial climbing shrub of Menispermaceae family, which is widely distributed in the Southwest of China, India. Its whole plant has been commonly used for the treatment of stomachache, cardiodynia, fever, skin disease, and swelling pain, etc [7]. However, very few researches on its chemical compositions have been reported. Previous phytochemical investigation of the genus Cissampelos showed the presence of alkaloids [8], flavonoids [9], terpenes [10], and pregnane glycosides [11]. Our previous chemical and pharmacological investigations on C. pareira var. hirsuta revealed the presence of polyhydroxylated pregnane glycosides, and their cytotoxic properties [11]. As our ongoing research for potential cytotoxic natural products from traditional Chinese medicines, three new polyhydroxylated oleanane triterpenoids, cissatriterpenoid A−C (1−3), and one known analogue (4), were obtained from the dried whole plant of C. pareira var. hirsuta ( Figure 1). Detailed isolation, structure elucidation, and cytotoxicity, and NO inhibitory activity of those isolates are reported herein.

Results and Discussion
The 95% EtOH extract of the whole plant of C. pareira var. hirsuta was partitioned between petroleum ether (PE), CH2Cl2, n-BuOH and water, respectively. The CH2Cl2 layer was fractionated and purified by repeated column chromatography, allowing for the isolation of three new polyhydroxylated oleanane triterpenoids, cissatriterpenoid A−C (1−3), along with one known analogue (4). By comparing their physical and spectroscopic data with the literature values, the known metabolite was identified as chichipegenin (4) [12].

Results and Discussion
The 95% EtOH extract of the whole plant of C. pareira var. hirsuta was partitioned between petroleum ether (PE), CH 2 Cl 2 , n-BuOH and water, respectively. The CH 2 Cl 2 layer was fractionated and purified by repeated column chromatography, allowing for the isolation of three new polyhydroxylated oleanane triterpenoids, cissatriterpenoid A−C (1−3), along with one known analogue (4). By comparing their physical and spectroscopic data with the literature values, the known metabolite was identified as chichipegenin (4) [12].
By the MTS method, the cytotoxic activities of all isolated compounds were evaluated against HL-60, A549, SMMC-7721, MCF-7, and SW480 cell lines. Compound 3 showed the most potent cytotoxic activities against A549, SMMC-7721, MCF-7, and SW480 cell lines, with IC 50 values of 17.55, 34.74, 19.77, and 30.39 µM, respectively, whereas the three remaining isolates were found to be inactive. By ChemBio 3D Ultra, the lipid-water partition coefficients of compounds 1−4 were determined as 7.57, 4.23, 4.65, and 6.45, respectively. Compounds 1−4 were tested against NO release effect in LPS-induced RAW 264.7 cells. Unfortunately, they showed no obvious inhibitory activity against NO production.

Plant Material
The whole plants of C. pareira var. hirsuta were collected in Yunnan province, China, in July 2018, and identified by Prof. Cheng-Ming Dong at School of Pharmacy, Henan University of Chinese Medicine, where a voucher specimen (SE 20180705) was deposited. Fresh whole plants were dried in the sun.

Extraction and Isolation
The dried whole plants of C. pareira var. hirsuta (50.7 kg) were powdered and refluxed with 95% EtOH (3 × 300 L) at 90 • C, and the combined solution was evaporated to dryness under reduced pressure to produce a crude extract (2.7 kg). The extract was dispersed in water (9 L) and successively partitioned with petroleum ether (PE, 9 L × 3), CH 2 Cl 2 (9 L × 3), and n-BuOH

Microhydrolysis of Compound 1
Compound 1 (10 mg) was dissolved in 1.0 mL of MeOH, then 0.3 mL of 28% sodium methylate solution was added dropwise under an N 2 atmosphere. After 4 h stirring at room temperature, the reaction mixture was neutralized by dilute HCl, and extracted with CH 2 Cl 2 . The extract was concentrated under reduced pressure, and isolated by preparative HPLC eluted with CH 3 CN-H 2 O (60:40) at a flow rate of 6 mL min -1 to obtain chichipegenin (tR 42.2 min) [12].

Cytotoxicity Asssay
Using the MTS method previously described [15], the cytotoxic activities of compounds 1-4 were evaluated against human myeloid leukemia HL-60, lung cancer A-549, hepatocellular carcinoma SMMC-7721, breast cancer MCF-7, and colon cancer SW-480 cell lines. All cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) at 37 • C under 5% CO 2 in a humidified atmosphere. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). In brief, 100 µL of adherent cells was seeded into each well in a 96-well cell culture plate and kept for 24 h for adherence. Each tumor cell line was exposed to various concentrations of the test compound in triplicate for 48 h. After incubation, MTS (20 µL) was added to each well, and the incubation continued for 4 h at 37 • C. The absorbance was measured at 492 nm in a 96-well microtiter plate reader. The IC 50 value of each compound was calculated by the Reed-Muench's method. Cisplatin was used as positive control.

NO Inhibitory Activity
NO production in the murine monocytic RAW 264.7 macrophage was evaluated by the previously reported protocol [16]. L-NG-Monomethyl arginine (LNMMA) was used as a positive control. The RAW 264.7 macrophages were cultured in DMEM medium containing 10% FBS and penicillin-streptomycin (100 U/mL) at 37 • C under 5% CO 2 . The test compounds were dissolved in DMSO and then diluted to different concentrations by medium. Cells were precultured in 96-well plates (2 × 10 5 cells/well) for 24 h, and then incubated with serial dilutions of the test compounds and LPS (1.0 µg/mL) for 18 h. Then, 100 µL of Griess reagent and 100 µL of culture supernatant were mixed, and incubated for 5 min. The optical density of the mixture was read at 570 nm by an automated microplate reader. The NO inhibitory rate was determined via a comparison with the control group.