A new oleanane-type triterpene from Ardisia lindleyana D.Dietr and its cytotoxic activity

Abstract A new oleanane-type triterpene, ardisiapunine A (1), together with eight known compounds were isolated from the roots of Ardisia lindleyana D.Dietr. Their chemical structures were determined by means of spectroscopic methods including HR-ESI-MS and (1 D, 2 D) NMR data. The absolute configuration of compound 1 was established by a single-crystal X-ray diffraction experiment. The new compound is an unusual oleanane-type triterpene bearing an acetal and a C-13—C-18 double bond. The cytotoxicity of all isolated compounds were evaluated using four human cancer cell lines, including A549, HepG2, HeLa and U87. The new compound 1 and compound 2 were weakly active but the known compound 6 exhibited a high cytotoxicity compared to cisplatin. Graphical Abstract


Introduction
The genus Ardisia Sw. is the largest member in the family Primulaceae (Zhao and Liu 1999;Wang and Xia 2013). More than 700 species of Ardisia are found throughout tropical and subtropical regions of the world (Xie et al. 2021). In China, 65 species and 12 varieties of Ardisia distribute in the south of the Yangtze River Basin (Wang and Xia 2013). Ardisia lindleyana D.Dietr, a kind of evergreen dwarf shrub, is a member of genus Ardisia and mainly distributes in areas of the south China including Guangdong, Guangxi, Zhejiang, Jiangxi, Fujian and Hunan (Li et al. 2006a). It is an important traditional Miao Minority medicine for promoting blood circulation to restore menstrual flow, expelling wind and removing dampness (Li et al. 2006a). Besides, it has been widely planted as an ornamental shrub. In the past few decades, Ardisia had been reported to contain various chemical constituents such as quinones (Tian et al. 1987;Podolak et al. 2021), coumarins (Jia et al. 1995;Liu et al. 2010Liu et al. , 2020, flavonoids (Hu et al. 2015;Zeng et al. 2019), triterpenoid saponins (Chang et al. 2007;Zheng et al. 2008;Qiang et al. 2010;Liu et al. 2011Liu et al. , 2016 with different biological activity. However, there were few reports (Li et al. 2006a(Li et al. , 2006bMa et al. 2012) about the chemical constituents of Ardisia lindleyana. Through the preliminary screening of anti-tumor activity in vitro, we found that the ethanol extract of Ardisia lindleyana had a significant inhibitory effect on the proliferation of tumor cells. Consequently, it is necessary to dig out the anti-tumor active ingredients in Ardisia lindleyana D.Dietr.
All obtained compounds (1-9) were evaluated for their cytotoxicity against A549, HepG2, HeLa and U87 cell lines using the cck8 method (Table 1). Compounds 1 and 2 were weakly active but 6 showed a high cytotoxicity compared to the control drug cisplatin. Compounds 1 and 2 are oleanane-type triterpene, the vast majority of this type compounds can inhibit the proliferation of tumor cells, which are consistent with the results of this paper. Compound 6 is a highly potent non-competitive hypoxanthine nucleotide dehydrogenase inhibitor, which is the active component of mycophenolate ester after metabolism in vivo. It has good antiviral, anti-fungal, anti-bacterial, anti-tumor and immunosuppressive activities, and has been clinically used in the treatment of organ transplantation rejection and immune diseases. While other compounds were not found to inhibit the proliferation of tumor cells.

Plant material
The collection of Ardisia lindleyana D.Dietr (Root parts) was done in March 2019 from Guangxi (the collection location is 104 26 0 -112 04 0 E, 20 54 0 -26 24 0 N with an altitude of about 300 meters) in China, and identified by Prof. Bin Li, Beijing Institute of Radiation Medicine. The plant was identified through three steps: (1) observing its morphology; (2) checking the literature; (3) checking specimens by comparison with authentic samples and microscopic anatomy. Voucher Specimen (Voucher # 2018-0808) was deposited in specimen cabinet of Beijing Institute of Radiation Medicine.

Cytotoxic assays
Four human cancer cell lines, A549 cell (human lung cancer cell) line, HepG2 cell (human liver cancer cell) line, HeLa cell (Human cervical cancer cell) line and U87 cell (human glioma cell) line were utilized in the cytotoxic activity assay. HepG2 cell, HeLa cell and U87 cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA), and A549 cell line were maintained in RPMI 1640 medium (RPMI 1640, SIGMA, USA), all that supplemented with 10% fetal calf serum (Gibco, USA), 100 U/mL penicillin, and 100ug/mL streptomycin in an atmosphere containing 5% CO 2 at 37 C. The cell counting kit-8 (cck8) cell cytotoxicity test was used in the cytotoxicity test. Cells were seeded in 96-well plates with a density of 5 Â 10 4 cells/mL and treated with different concentrations of test compounds for 48 h, then 10 lL cck8 solution were added to all the wells. After 1 h incubation, the absorbance at 450 nm of each well was measured in a multiscan photometer. The IC 50 values were calculated using GraphPad Prism 8 software and listed in Table 1. Data were represented as mean ± SD of three independent experiments.

Conclusion
In summary, the chemical analysis of Ardisia lindleyana D.Dietr led to the identification of one new oleanane-type triterpene and eight known compounds. The new triterpenoid bearing a double bond at C13-C18 and an acetal moiety was different with other triterpenoid reported in this genus before, which has been rarely reported in the plant kingdom. The observed inhibition of cancer cell growth by compounds 1, 2 and 6 suggests that they are anticancer active ingredients.

Disclosure statement
No potential conflict of interest was reported by the authors.

Supplementary material
1 H and 13 C NMR, 1 H-1 H COSY, HSQC, HMBC, NOESY, HR-ESI-MS and single crystal diffraction spectra of 1 associated with this article are available.

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