Absolute Configuration and Biological Evaluation of Novel Triterpenes as Possible Anti-Inflammatory or Anti-Tumor Agents

Two new compounds, ardisiapunine B (1) and ardisiapunine C (2), were isolated from Ardisia lindleyana D. Dietr. Their structures were examined using HR–ESI–MS, IR, (1D, 2D) NMR spectroscopic analyses, single–crystal X–ray diffraction, and ECD calculation. It was found that the two new compounds belong to unusual oleanane-type triterpenes, with compound 1 bearing an acetal unit and a C–13–C–18 double bond, and compound 2 bearing a C–28 aldehyde group and a C–18–C–19 double bond. The anti-inflammatory properties of compounds 1 and 2 were tested on NO production and cellular morphology using RAW264.7 cells, and their anti-tumor properties were tested on cytotoxic activities, cellular morphology, cell apoptosis, and cell cycle. The results showed that compound 1 exhibited a potent cytotoxicity against HepG2 cell lines with an IC50 of 12.40 μM. Furthermore, it is possible that compound 1 inhibits cell proliferation by blocking the cell G2/M phase and promoting cell apoptosis. Compound 2 exhibited a potential anti-inflammatory activity by decreasing the production of NO in LPS–stimulated RAW264.7 cells. Comparative analysis of the structures of compounds 1 and 2 revealed that the acetal structure and double bond positions were the main differences between them, and these are presumed to be the main reasons for the extreme differences in their cytotoxicity and anti-inflammatory activities. From these new findings, two promising lead compounds were identified for the future development of potential anti–inflammatory or anti–tumor agents.


Introduction
Ardisia lindleyana D. Dietr, a kind of evergreen dwarf shrub, belongs to the family of Myrsinaceae. It is mainly distributed across tropical America, the Pacific Islands, the eastern Indian Peninsula, Eastern Asia, and Southern Asia [1], and is grown for use in traditional Miao Minority medicine and as an ornamental shrub [2]. Previous research on the chemical constituents of this plant has led to the isolation of isocoumarins [3], quinones [4,5], and triterpenoids [3], some of which were found to have various bioactivities, including antiinflammatory and anti-tumor activities.
In preliminary studies, we reported that the ethanol extract of the plant had a significant inhibitory effect on the proliferation of multiple tumor cells in vitro, disclosing that this plant may contain natural products with anti-tumor properties [6]. In this paper, we report the results of our further chemical investigation of Ardisia lindleyana D. Dietr, which led to the isolation of two new oleanane-type triterpenes, one with a diacetal moiety (1) and another with an aldehyde group (2). Their structures, including absolute configurations, were characterized by one-and two-dimensional NMR spectroscopic analyses, including DEPT, HSQC, HMBC, 1 H-1 H COSY, and NOESY experiments, single-crystal X-ray diffraction, and ECD calculation. Details of the structural elucidations for compounds 1 and 2 are shown in Figure 1A. The structures of the new compounds 1 and 2, both oleanane-type triterpenes, are novel, with compound 1 bearing an acetal unit and a C-13-C-18 double bond, and compound 2 bearing a C-28 aldehyde group and a C-18-C-19 double bond. The characterization of compounds 1 and 2 add not only to the diversity of the chemical constituents of Ardisia lindleyana D. Dietr, but also to the diversity of oleanane-type triterpenes. Triterpenes are common in nature and engage in various biological activities, such as anti-tumor activity, anti-inflammatory activity, antibacterial activity, and antiviral activity. Among these, the anti-tumor activity of triterpenes is measured by their ability to induce apoptosis, block nuclear factor-kappaB activation, inhibit signal transducer, inhibit topoisomerases, and activate transcription and angiogenesis [7]. Hence, compounds 1 and 2 were evaluated for their cytotoxicity against B16F10, A549, H460, MCF-7, HepG2, Hela, and U87 cell lines in vitro, and evaluated for their anti-inflammatory properties using the Griess reaction and the results of screening the production of NO by LPS-stimulated murine macrophage cell lines RAW264.7.
The process of derivatization of compound 2 to compound 2-DNP was as follows: A stirred solution of compound 2 (28 mg, 59 µmoL) was dissolved in a stirred solution of dichloromethane: methanol (1:1, v/v, 25 mL). Acetic acid (0.2 mL) and DNPH (18 mg, 91 µmoL) were added to the mixture at a temperature of 60 • C, then the mixture was stirred at the same temperature for 30 min.  (Table 1) revealed the disappearance of carbon signals at 205.9 ppm (C=O), as well as the appearance of six peaks at δ C 116.7 ppm, 122.7 ppm (overlapped), 129.0 ppm, 129.6 ppm, 137.2 ppm, and 145.6 ppm, and three signals at δ H 8.12 ppm, 8.19 ppm, and 9.04 ppm in the 1 H NMR spectra (Table 1), which were attributed to a benzene group. Moreover, the presence of hydrazone moiety (C=N-NH) was confirmed by the singlet peaks at 11.8 ppm and 157.4 ppm in the 1 H NMR and 13 C NMR spectra, respectively.
In this work, two new oleanane-type triterpenes were obtained from Ardisia lindleyana D. Dietr. This is the first report about oleanane-type triterpenes with double bonds at C-13-C-18 or C-18-C-19 obtained from Ardisia lindleyana D. Dietr, which has broadened our understanding of the structural diversity of this genus of metabolites. To the best of our knowledge, although an oleanane-type triterpene containing an acetal moiety had been reported [11], there are no reports of one with the properties of compound 1, bearing an acetal and a C-13-C-18 double bond, being obtained from natural sources. It is worth mentioning that such compounds, with a double bond at this position, are very special and rare. The structure of compound 2, bearing a C-28 aldehyde group and a C-18-C-19 double bond, also appears to be unique.     Table 3). The IC 50 values for the different tumor cells were as follows: HepG2 < B16F10 < MCF-7 < Hela < U87 < A549 < H460. However, compound 2 did not exhibit any cytotoxic activities on any of the seven cell lines. Comparative analysis of the structures of compounds 1 and 2 revealed that the acetal structure and double bond positions were the main differences between them, and these are presumed to account for the differences in cytotoxic activities. In other words, we believe that a C-30 acetal unit and a C-13-C-18 double bond are essential features for producing cytotoxic activities.

Cell Morphology
To assess whether compound 1 could induce any morphological changes, HepG2 cells were incubated with different concentrations of compound 1. The cells were then observed and photographed using a fluorescence microscope after being stained with 3,3 -dioctadecyloxacarbocyanine perchlorate (Dio) and Hoechst 33342 [12,13]. As shown in Figure 4A, compound 1 altered the cellular morphology considerably regardless of the dose. The untreated HepG2 cells (control group) exhibited a regular appearance, intensive growth, integral cell membrane, and clear nucleolus. After treatment with compound 1, cell membrane and nuclear condensation can be observed, and the cells were eventually destroyed, showing elongated or irregular shapes, condensation of chromatin, and cell shrinkage, which revealed that compound 1 could induce apoptosis in vitro.

Cell Apoptosis and Cell Cycle
In order to further explore the mechanism of compound 1 against tumors, HepG2 cells were incubated with different concentrations of compound 1 to observe its effect on cell apoptosis and cell cycle. The results showed that compound 1 could increase the cells in the G2/M phase from 17.53% to 22.05% (3.13 µM), 26.03% (6.25 µM), and 36.48% (12.5 µM) ( Figure 4B,C), and the apoptosis rate from 14.31% to 24.00% (3.13 µM), 30.06% (6.25 µM), and 54.27% (12.5 µM) ( Figure 4D,E) in a dose-dependent manner. Compared with the control group, compound 1 at concentrations of 6.25 µM and 12.5 µM significantly increased both the cells in the G2/M phase and the apoptosis rate (p < 0.01 or p < 0.001). Therefore, we speculated that compound 1 might inhibit cell proliferation by blocking the cell G2/M phase and promoting cell apoptosis.

Anti-Inflammatory Activity against NO Production
The anti-inflammatory assays of compounds 1 and 2 were evaluated in LPS-stimulated RAW264.7 macrophages with dexamethasone as the positive control. The results showed that compound 1 produced a cytotoxic effect on RAW264.7 cells. Therefore, the antiinflammatory activity of compound 1 has not been tested. The results also showed that compound 2 did not affect cell viability on RAW264.7 cells at a concentration of 50 µM to 6.25 µM ( Figure 5B). As shown in Figure 5C, the results showed that compound 2 exhibited good inhibitory activity against NO production in a dose-dependent manner. In addition, we used a microscope to observe the effect of compound 2 on the cell morphology of LPS-stimulated RAW264.7 cells in order to more accurately evaluate its anti-inflammatory properties. As can be seen in Figure 5A, the cells in the control group were plump and round, but the cells undergoing LPS stimulation became flat and irregular, with slender tentacles. However, the LPS-stimulated cell morphology returned to normal when compound 2 intervened. The above results demonstrate that compound 2 exhibits a potential anti-inflammatory activity by decreasing the production of NO in LPS-stimulated RAW264.7 cells. In the cell apoptosis assay, all the cells were collected, and 100,000 cells were taken and incubated with Annexin V-FITC and PI for 20 min at room temperature without light. In the cell cycle assay, all the cells were collected and fixed with precooled 70% ethanol for 12 h at 4 • C. The cells were then washed with PBS and incubated with PI for 30 min at 37 • C without light. Finally, all the cells were detected using a flow cytometer. Data are presented as mean ± SD, n = 3. One way ANOVA, ** p < 0.001, *** p < 0.001, compared with control group. Dexamethasone was used as a positive control. In the cell morphology assay, the cells were observed and photographed using a microscope. Cell viability was determined using the CCK-8 method by measuring the absorbance with a microplate photometer at 450 nm. In (B,C), the abscissa groups from left to right are the control group (without LPS), model group, compound 1 at a concentration of 6.25 µM group, compound 1 at a concentration of 25 µM group, compound 1 at a concentration of 50 µM group and positive control group, respectively. In the NO production assay, the supernatants were collected for the measurement of NO production using the Griess reagent. Data are presented as mean ± SD, n = 3. One way ANOVA, *** p < 0.001, compared with LPS group.

Plant Materials
The roots of Ardisia lindleyana D. Dietr were collected from Guangxi (104 • 26 -112 • 04 E, 20 • 54 -26 • 24 N at an altitude of about 300 m) in China, and identified by Prof. Bin Li, Beijing Institute of Radiation Medicine. A voucher specimen (Voucher # 2018-0808) was deposited in the specimen cabinet of the Beijing Institute of Radiation Medicine.

Isolations of Compounds
The root bark (60 kg) was extracted with 80% EtOH. Upon concentration with a rotary evaporator, the root bark yielded a dark, brownish black residue (11.8 kg). This residue was then dispersed in deionized water and partitioned successively with petroleum ether, EtOAc, and n-BuOH. The EtOAc extract (290 g) was subjected to a silica gel column using Dichloromethane-MeOH

Effect of Isolated Compounds on Cytotoxicity of Seven Cancer Cell Lines
Seven cancer cell lines, the B16F10, A549, H460, MCF-7, HepG2, Hela, and U87 cancer cell lines, were utilized in the cytotoxic activities assay. The MCF-7, HepG2, Hela, and U87 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA), and The B16F10, A549, and H460 cell lines were cultured in RPMI 1640 medium (RPMI 1640, SIGMA, U.S.), in each case supplemented with 10% fetal calf serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin in an atmosphere containing 5% CO 2 at 37 • C. Cells were seeded in 96-well plates with a density of 5 × 10 4 cells/mL and treated with different concentrations of the test compounds for 48 h. The cytotoxicity assay was performed using the Cell Counting Kit-8 (CCK-8, Applygen technologies Co., Ltd., Beijing, China) assay.

Effect of Compound 1 on Cell Morphology, Cell Cycle, and Apoptosis of HepG2 Cells
HepG2 cells were dispensed in a 12-well plate at a density of 5 × 10 4 cells/mL, incubated at 37 • C for 24 h, and treated with different concentrations of compound 1 for 48 h.

Cell Morphology Assay
After removing the supernatant, the cells were washed twice with 1.0 mL PBS and fixed with 4% Paraformaldehyde fix solution (Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China) for 2 h. Fixed cells were then washed twice with 1.0 mL PBS and incubated with Dio (Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China) at 37 • C in darkness. After 20 min of incubation, the cells were washed twice with 1.0 mL PBS. Then, the cells were stained with Hoechst 33342 staining solution (Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China) for 10 min and washed twice with 1.0 mL PBS. The stained cells in PBS were then observed and photographed using a fluorescence microscope.

Cell Apoptosis and Cell Cycle Assay
In the cell apoptosis assay, all the cells were collected, and 100,000 cells were taken and incubated with Annexin V-FITC and PI for 20 min at room temperature without light. In the cell cycle assay, all the cells were collected and fixed with precooled 70% ethanol for 12 h at 4 • C. The cells were then washed with PBS and incubated with PI for 30 min at 37 • C without light. All the cells were detected using a flow cytometer.

Effect of Isolated Compounds on NO Production
A RAW264.7 murine macrophage cell line (American Type Culture Collection, ATCC No. TIB-71) was purchased from the Cancer Institute and Hospital of the Chinese Academy of Medical Sciences. Cells were cultured in DMEM medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin in an atmosphere containing 5% CO 2 at 37 • C. The cells were cultured for 3-5 days to reach the logarithmic phase and then used for experiments. The cells were treated with tested compounds and a positive control (dexamethasone) at different concentrations and then stimulated with LPS (1 µg/mL) for 24 h.
3.6.1. Cell viability Assay on RAW264.7 Cells The cytotoxic effects of compounds 1 and 2 on RAW264.7 cells were evaluated using a CCK-8 assay. RAW264.7 cells were dispensed in a 96-well plate at a density of 1 × 10 5 cell per well, incubated at 37 • C for 24 h, and treated with the tested agents for the indicated periods of time. Then, 10 µL of cell counting kit-8 solution was added to each well, followed by 1 h incubation. The absorbance at 450 nm of each well was measured with a Multiskan MK-3 microplate reader (Thermo Fisher Co., Waltham, MA, USA). Compound 2 showed no cytotoxic effect on RAW264.7 cells at different concentrations (6.25, 25, 50 µM). In addition, cell morphology was observed under an OLYMPUS CKX53 microscope (Olympus Co., Tokyo, Japan).

Determination of the NO Content
NO production was determined using a nitric oxide assay Kit (Applygen technologies Co., Ltd., Beijing, China) based on the Griess reaction principle [14,15]. A quantity of 50 microliters of culture supernatant was transferred to 96-well plates and mixed with 50 microliters of Griess R1 reagent after incubation at room temperature for 5 min. The samples were then mixed with an equal volume Griess R2 reagent for another 5 min. The absorbance of each mixture at 540 nm was measured using a microplate reader [16].

Cell Morphology Assay
Without any post-processing, the cells were observed and photographed using a microscope.

Conclusions
In this study, we identified two new compounds, including ardisiapunine B (1) and ardisiapunine C (2), which we isolated from Ardisia lindleyana D. Dietr. The structures of the two new compounds were elucidated using 1D and 2D NMR spectroscopic analyses, HR-ESI-MS, single-crystal X-ray diffraction, and ECD calculation. It was found that the two new compounds are unusual oleanane-type triterpenes, with compound 1 bearing an acetal unit and a C-13-C-18 double bond, and compound 2 bearing a C-28 aldehyde group and a C-18-C-19 double bond. Activity assay results showed that compound 1 exhibited a potent cytotoxicity against HepG2 and B16F10 cell lines, and that, furthermore, it might inhibit cell proliferation by blocking the cell G2/M phase and promoting cell apoptosis. These new findings provide a framework for the further exploration of compound 1, which exhibits potential anti-tumor activity by inducing cell cycle arrest and apoptosis. Compound 2 had moderate inhibitory effects on the production of NO in LPS-stimulated RAW264.7 cells. Comparative analysis of the structures of compounds 1 and 2 revealed that the acetal structure and different double bond positions were the main differences between them, and these are presumed to be the main reasons for the extreme differences in their cytotoxicity and anti-inflammatory activities. From these new findings, two promising lead compounds were identified for the future development of potential anti-inflammatory or anti-tumor agents.

Conflicts of Interest:
The authors declare no conflict of interest.