Cytotoxic Xanthones from Hypericum stellatum, an Ethnomedicine in Southwest China

Hypericum stellatum, a species endemic to China, is used to treat hepatitis by several ethnic groups in Guizhou Province. This research was inspired by the traditional medicinal usage of H. stellatum, and aims to explore the phytochemistry and bioactivity of H. stellatum to explain why local people in Guizhou widely apply H. stellatum for liver protection. In this study, two new prenylated xanthones, hypxanthones A (8) and B (9), together with seven known compounds, were isolated from the aerial parts of the plant. Spectroscopic data as well as experimental and calculated ECD spectra were used to establish the structures of these compounds. Six xanthones isolated in this study, together with four xanthones previously isolated from H. stellatum, were evaluated for their growth-inhibitory activities against five human liver carcinoma cell lines to analyze the bioactivity and structure-activity relationship of xanthones from H. stellatum. Isojacareubin (6) showed significant cytotoxicity against five human liver carcinoma cell lines, with an IC50 value ranging from 1.41 to 11.83 μM, which was stronger than the positive control cisplatin (IC50 = 4.47–20.62 μM). Hypxanthone B (9) showed moderate cytotoxicity to three of the five cell lines. Finally, structure-activity analysis revealed that the prenyl and pyrano substituent groups of these xanthones contributed to their cytotoxicity.


Introduction
The plants in the genus Hypericum have a long history of use as herbal medicines in China. There are 64 Hypericum species (33 endemic) distributed throughout China [1], and 19 of these species have been used traditionally as medicinal plants with a range of purported benefits, including antidepressant, detoxification, hemostasis, antibacterial, and hepatoprotection activities. Based on ethnobotanical investigations in southeast Guizhou province, eight Hypericum species were found to be used as ethnomedicines by several ethnic groups, including the Shui, Miao, Dong, and She peoples. Previous phytochemical investigations on Hypericum have reported various types of compounds, including naphthodianthrones, flavonoids, prenylated phloroglucinols, and phenols [2][3][4][5]. Furthermore, Hypericum extracts have been found to exhibit various pharmacological activities, including antidepressant, antiviral, antibacterial, and anti-HIV activities [6][7][8][9][10].
Hypericum stellatum is an endemic species to China, primarily distributed in Chongqing and surrounding regions. Our ethnobotanical investigations in Majiang, a county of the Qiandongnan Miao Hypericum stellatum is an endemic species to China, primarily distributed in Chongqing and surrounding regions. Our ethnobotanical investigations in Majiang, a county of the Qiandongnan Miao and Dong Autonomous Prefectures in Guizhou Province, have shown that H. stellatum is used by the local Miao and Buyi people to treat liver diseases such as icterohepatitis. However, no chemical studies had been conducted on this species. In our recent research on H. stellatum, UPLC-Q-TOF-MS was applied to analyze the chemical constituents of the plant. The results showed that H. stellatum was rich in flavonoids, phenolic acids, and polycyclic polyprenylated acylphloroglucinols. H. stellatum possesses strong antioxidant activity and a high content of total phenols. Ten compounds, including flavonoids, xanthones, and phenolic acids, were isolated [11]. To further illuminate the plant′s pharmacological constituents, we used column chromatography (CC), including silica gel, Sephadex LH-20, and recycling preparative HPLC, which yielded two new xanthones and seven known compounds. Six xanthones (1, 2, and 6-9) obtained in this research, together with four previously isolated xanthones (3-5 and 10), were tested for cytotoxicity against five human liver carcinoma cell lines. Herein, we report the isolation, structure elucidation, and bioactivities of these compounds isolated from H. stellatum.

Results and Discussion
A 95% aq. EtOH extract of the aerial part of H. stellatum was suspended in water and extracted successively with petroleum ether, EtOAc, and n-BuOH. The EtOAc extract showed strong antioxidant activity and a high total phenol content. The fraction of EtOAc was repeatedly subjected to silica gel, Sephadex LH-20, and a liquid chromatography loop preparation, yielding two new prenylated xanthones (8 and 9) and seven known compounds. Structures of the new compounds were elucidated using spectroscopic data and calculated via ECD spectra, the spectroscopic data can be seen in Supplementary Materials Figures S1-S16.
Cytotoxic activity tests (Table 2) showed that simply oxygenated xanthone compounds 3 and 5 showed weak cytotoxicity against all five cell lines with IC 50 values > 40 µM. This is probably due to the absence of the groups which are crucial to locking the compound into the domain in the target binding site. Compounds 6, 9 (hypxanthone B), and 10 showed strong cytotoxic activity towards SMMC-7721 cells, with IC 50 values ranging from 1.41 to 8.26 µM, while 2, 7, and 8 (hypxanthone A) showed moderate cytotoxic activity against the same cell line. Compound 6 (IC 50 = 9.09 µM) showed strong cytotoxic activity towards Huh-7 cells, and compounds 9 and 10 showed moderate cytotoxic activity towards Huh-7 cells. Compounds 1, 4, 9, and 10 showed moderate cytotoxic activity towards HepG2 cells, and 6 showed strong cytotoxic activity with an IC 50 value of 2.40 µM. Tests on SK-HEP-1 cells showed that compounds 7 and 10 showed moderate cytotoxic activity, and 6 showed strong cytotoxic activity (IC 50 = 9.20 µM). Compounds 6, 9, and 10 showed moderate cytotoxic activity towards PLC/PRF/5 cells. Cytotoxic activity tests (Table 2) showed that simply oxygenated xanthone compounds 3 and 5 showed weak cytotoxicity against all five cell lines with IC50 values > 40 μM. This is probably due to the absence of the groups which are crucial to locking the compound into the domain in the target binding site. Compounds 6, 9 (hypxanthone B), and 10 showed strong cytotoxic activity towards SMMC-7721 cells, with IC50 values ranging from 1.41 to 8.26 μM, while 2, 7, and 8 (hypxanthone A) showed moderate cytotoxic activity against the same cell line. Compound 6 (IC50 = 9.09 μM) showed strong cytotoxic activity towards Huh-7 cells, and compounds 9 and 10 showed moderate cytotoxic activity towards Huh-7 cells. Compounds 1, 4, 9, and 10 showed moderate cytotoxic activity towards HepG2 cells, and 6 showed strong cytotoxic activity with an IC50 value of 2.40 μM. Tests on SK-HEP-1 cells showed that compounds 7 and 10 showed moderate cytotoxic activity, and 6 showed strong cytotoxic activity (IC50 = 9.20 μM). Compounds 6, 9, and 10 showed moderate cytotoxic activity towards PLC/PRF/5 cells.
As active secondary metabolites, xanthones commonly occur in various herbal medicines; a total of 168 species of herbal plants belonging to 58 genera and 24 families have been found to contain xanthones. Calophyllaceae, Gentianaceae and Clusiaceae (Guttiferae) are the most widely distributed families containing xanthones [23]. In Clusiaceae sensu lato, two genera (Hypericum and Garcinia) are rich in variously oxidized and prenylated xanthones, such as Hypericum uralum [24], Hypericum monogynum [25], Hypericum riparium [26], Garcinia nujiangensis [22], Garcinia cowa [21], and Garcinia mangostana [27]. Many of the compounds isolated from these plants could have remarkable medicinal potential, such as the prenylated xanthone gambogic acid isolated from Garcinia hanburyi. The resin of G. hanburyi, called gamboge in traditional Chinese medicine and ethnomedicine in Asian countries, possesses broad-spectrum anticancer activity and showed safety in Chinese phase II clinical trials carried out in 2009 [28]. Furthermore, our own research, as well as previous studies, has revealed that isojacareubin possesses potential medicinal value. Isojacareubin has been synthesized, and was found to be a potent inhibitor of PKC; these findings identify isojacareubin as a promising lead compound for the development of new antihepatoma agents [29].
Our previous study revealed that the antioxidant ability and total phenol content of EtOAc and n-BuOH extract showed little differences. However, the metabolite profiles of two extractions metabolic profile showed obvious differences. As a result, the n-BuOH extract should likely contain different biomarkers [11]. Further phytochemical research on the n-BuOH extraction will be carried out to search for candidates lead compounds.

Plant Material
The aerial parts of Hypericum stellatum N. Robson were collected from Majiang County, Qiandongnan Miao and Dong Autonomous Prefectures, Guizhou Province, China, in July 2016. The plant was identified by Jun Yang, a taxonomist at the Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. LongCL-060) has been deposited at the Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany.

ECD Calculations
The method used for ECD calculation has been previously reported [30]. The ROESY spectra were used to initially determine the relative configuration of hypxanthone B, followed by the MMFF94s force field (random conformational analysis). The B3LYP/6-31G(d) level of time-dependent density functional theory (TDDFT) was used to optimize the obtained conformers and was followed by ECD calculations via the TDDFT method (B3LYP/6-31+G(d), CPCM model = MeOH), with SpecDis v1.51 (with a half-bandwidth of 0.3 eV) to simulate the Boltzmann-weighted ECD spectra. The Gaussian 09 electronic structure package (version 7.0, Gaussian, Inc., Wallingford, CT, USA) was used to perform all of the calculations.

MTS Assay
Considering its purported medicinal value for the treatment of liver disease, we tested the cytotoxic activities of ten xanthones isolated from H. stellatum against five human liver carcinoma cell lines ( The isolated compounds were tested in vitro for their cytotoxicity to five human liver carcinoma cell lines (SMMC-7721, Huh-7, HepG2, SK-HEP-1, PLC/PRF/5) and the immortalized noncancerous human liver cell (LO2), in a 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)2-(4-sulfopheny)-2H-tetrazolium (MTS; Promega, Beijing, China) assay [31]. In general, cells in the log phase of their cycle were seeded in 96-well plates (4000-5000 cells/well, NEST Biotechnology, Wuxi, China) in a 100-µL volume. After 12 h of incubation at 37 • C, each test compound was added. The cancer cell lines were exposed to the test compounds at five concentrations 1-4 h. The absorbance was measured at the detection wavelength of 490 nm (L1) and the reference wavelength of 680 nm (L2), and cytotoxicity for each compound was expressed as IC 50 values by Reed and Muench s method [32].