Chemical Constituents of Thesium chinense Turcz and Their In Vitro Antioxidant, Anti-Inflammatory and Cytotoxic Activities

Three novel compounds (1–3) along with twenty-six known compounds, two known steroids (4–5) and twenty-four known phenylpropanoids (6–29) were isolated from the whole plant of Thesium chinense Turcz. The structures of the three new compounds were elucidated on the basis of ESI-MS, HR-ESIMS, 1D and 2D NMR, IR, UV spectroscopic data. The absolute stereochemistry of compound 1 was determined by the Gauge-Including Atomic Orbitals (GIAO) method. The in vitro antioxidant, anti-inflammatory and cytotoxic activities of the isolated compounds were evaluated by DPPH radical-scavenging assay, LPS-activated RAW 264.7 cells model and CCK-8 kit, respectively. Compound 11 showed high antioxidant activity with an SC50 value of 16.2 ± 1.6 μM. Compound 21 showed considerable anti-inflammatory activity with an IC50 value of 28.6 ± 3.0 μM. Compounds 4 and 5 displayed potent cytotoxic activity against human NCI-H292, SiHa, A549, and MKN45 cell lines, with the compound 4 having IC50 values of 17.4 ± 2.4, 22.2 ± 1.1, 9.7 ± 0.9, 9.5 ±0.7 μM, and the compound 5 having all IC50 values less than 0.1 μM in vitro.


Introduction
There are approximately 350 species of the genus Thesium (Santalaceae) occurring worldwide in Africa, Europe, Asia, South America and North America [1]. Thesium chinense Turcz, a small perennial and hemi-parasitic plant belonging to the family Santalaceae, distributes in East Asia (China, Japan, Korea, and Mongolia) [2]. The whole plant of T. chinense, commonly called "Bai-Rui-Cao" in China, was first recorded in the ancient medicinal monograph "Tu Jing Ben Cao" in the North Song Dynasty 1000 years ago. In traditional Chinese medicine (TCM), Bai-Rui-Cao is entitled "Botanical Antibiotics" and is mainly used to treat different diseases including mastitis, pulmonitis, tonsillitis, laryngopharyngitis and upper respiratory tract infections. [3]. Previous studies have reported the presence of polysaccharides, flavonoids, alkaloids, terpenoids, D-mannitol, aromatic compounds and aliphatic acids in the plant of T. chinense [4,5]. Modern pharmacological studies have found that T. chinense has diverse activities including anti-inflammation [6], antimicrobial effect [7], analgesic activity [8], antioxidant activity [9] and anti-nephropathy [10].
The chemical and pharmacological investigations into T. chinense are carried out as part of our ongoing work on the discovery of the bioactive compounds from Chinese medicinal herbs. From EtOAc and n-BuOH extracts of T. chinense, three novel compounds (1)(2)(3), along with twenty-six known compounds, two known steroids (4)(5) and twenty-four known phenylpropanoids   (Figure 1), are isolated. The structures of the new compounds (1)(2)(3) are identified on the basis of ESI-MS, HR-ESI-MS, 1D and 2D NMR, IR, UV spectroscopic evidence. To determine the absolute stereochemistry, compound 1 was subjected to the Gauge-Including Atomic Orbitals (GIAO) method. The isolated compounds are evaluated for their antioxidant, anti-inflammatory activities and in vitro cytotoxic activities against four human NCI-H292, SiHa, A549, and MKN45 cancer cell lines by using DPPH radical-scavenging assay, LPS-activated RAW 264.7 cells model and CCK-8 kit, respectively. Herein, we report the isolation, structure determination and activity evaluation, and we preliminarily discuss the structure-activity relationship of the isolates.

Anti-Inflammatory Activity
The in vitro anti-inflammatory activities of compounds 2-29 were evaluated by their inhibitory effects on NO production in LPS-activated RAW 264.7 cell models, and the results showed that most compounds had no anti-inflammatory activities except for ethyl ferulate (21) ( Table 4). Compared with the positive control quercetin (IC 50 = 11.1 ± 1.4 µM), compound 21 displayed considerable anti-inflammatory activity with an IC 50 value of 28.6 ± 3.0 µM. Recent in vivo studies reported that ethyl ferulate (21) also displayed obvious anti-inflammatory effects against LPS-induced acute lung injury in mice [38,39].

Cytotoxic Activity
In the CCK-8 assay, many of the isolated compounds from T. chinense showed moderate or considerable cytotoxic activities against four human cancer cell lines of A549, NCI-H292, SiHa and MKN45 (Table 5). Among them, Scopoletin (28) showed moderate and selective cytotoxic activity against MKN45 with the IC 50 value of 52.8 ± 5.3 µM. Perlplogemn (4) displayed potent cytotoxic activity against NCI-H292, SiHa, A549, and MKN45 with IC 50 values of 17.4 ± 2.4, 22.2 ± 1.1, 9.7 ± 0.9, and 9.5 ±0.7 µM, respectively. It is worth noting that periplocin (5) demonstrated promising cytotoxic activity against the four human cancer cell lines with all the IC 50 values less than 0.1 µM compared to that of the positive control cisplatin (IC 50 = 7.0 ± 0.3 µM). Both perlplogemn (4) and periplocin (5) are known for their cytotoxicity towards different cancer cells, and are now considered as potent leading compounds for several tumors with high drug resistance [40][41][42].

Plant Material
The whole dry herb of T. chinense was collected from Xiangyang city, Hubei Province, China in May 2019. The species was identified by Prof. Kai-Jin Wang at the School of Life Sciences, Anhui University, and a voucher specimen (No. 20190927) was deposited in the School of Pharmacy, Anhui Medical University.

Extraction and Isolation
The air-dried whole plant of T. chinense (10 kg) was extracted with 85% EtOH (3 × 100 L, each 4 h) at 60 • C. The combined EtOH extracts were evaporated at 60 • C using a rotatory evaporator with a vacuum pump to obtain suspended water (9 L), the suspension was successively extracted with petroleum ether, EtOAc, n-BuOH (1:2, v/v, three times each).

DPPH Radical Scavenging Assay
The 2, 2-diphenyl-1-picrylhydrazyl (Aldrich Chem. Co. Ltd, Shanghai, China) radical scavenging activity assay was performed according to the previously published method [43] with some modification. Test samples were dissolved in MeOH to six different concentrations ranging from 3.125 to 100 µM. Then, 100 µL of 100 µM DPPH in MeOH were mixed with 100 µL of samples at different concentrations in the wells of 96-well plates. The reaction mixtures were incubated in the dark at 37 • C for 30 min and then measured at 517 nm. Ascorbic acid was used as the positive control. The scavenging activity (SC) was estimated as follows: % SC = [1 − (A sample − A blank )/A control ] ×100. A sample was the equivalent mixture of test sample and DPPH solution. A blank was the equivalent mixture of test sample and MeOH solution. A control was the equivalent mixture of DPPH and MeOH solution. The SC 50 values were used to evaluate the antioxidant activities of isolated compounds. The experiments were performed in triplicate, and the data were expressed as the means ± SD of three independent experiments.

Anti-Inflammatory Activity
The macrophage RAW 264.7 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). A total of 180 µL of RAW 264.7 cells (1 × 10 4 ) were plated respectively in each well of 96-well plate and cultured for 24 h, then the supernatant of the culture was discarded and 180 µL complete medium with 1 µg/mL LPS and different concentrations of test samples were added into triplicate wells for 24 h. The inhibition effects on LPS-stimulated NO production was evaluated by the Griess reaction assay. The experiments were performed in triplicate, and the data were expressed as the means ± SD of three independent experiments.
The cell viability was determined by the CCK-8 assay method. The cytotoxicity was calculated from the plotted results using untreated cells at 100%.

Cytotoxic Assay (CCK-8 Assay)
Four human cancer cell lines, A549, NCI-H292, SiHa and MKN45, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cancer cells were cultured in 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Sijiqing, Huzhou, China) at 37 • C in a humidified atmosphere with 5% CO 2 . Cisplatin (Abmole Bioscience, Shanghai, China) was used as positive control.
A total of 180 µL of cancer cells (5 × 10 3 ) were plated in each well of 96-well plates and cultured for 24 h, then the supernatant of the culture was discarded and 180 µL complete medium with different concentrations of test samples were added into triplicate wells of each cell line, respectively. Thereafter, A549, NCI-H292, SiHa and MKN45 were incubated for 48 h, respectively. The cell viability was evaluated by Cell Counting Kit-8 (CCK-8, Best Bio., Beijing, China), respectively. The experiments were performed in triplicate, and the data were expressed as the means ± SD of three independent experiments.

Conclusions
In summary, three new compounds (1-3) along with two known steroids (4-5) and twenty-four known phenylpropanoids  were isolated from the EtOAc and n-BuOH extracts from the whole plant of T. chinense. All the compounds were isolated from this species for the first time. The isolates except for compound 1 are evaluated for their antioxidant and anti-inflammatory activities as well as their in vitro cytotoxic activities against four human cancer cell lines. The two new biglycosides (2 and 3) have no activity. Only compound 21 displayed considerable anti-inflammatory activity with an IC 50 value of 28.6 ± 3.0 µM by inhibited LPS-induced NO production in RAW 264.7 cells. Most phenylpropanoids with a phenolic hydroxyl group displayed significant or moderate scavenging activity on DPPH radicals (Table 3). Compared to the positive control ascorbic acid, the three furofuran lignan glycosides (9-11) and tetrahydrofuran lignan glycoside (19) displayed significant scavenging activity on DPPH radicals. The two oxyneoligan 17 and 18 showed moderate activity, and compound 17 displayed much stronger activity than that of 18, which indicated that the methoxy substitution on the benzene ring could enhance the activity. Two phenylethyl derivatives 6 and 7 exhibited weak activity. The phenylpropanoids 8, 12-16 and 29 without phenolic hydroxyl group did not show activity, which suggests that the phenolic hydroxyl group may be one of the key factors in the enhancement of the antioxidant activity.
Two steroid compounds 4 and 5 with α,β-unsaturated lactone ring showed extremely strong cytotoxicity against all four tested cancer cell lines, with IC 50 values ranging from < 0.1 to 22.2 ± 1.1 µM, relative to the positive control, cisplatin (IC 50 values from 7.0 ± 0.3 to 52.8 ± 5.3 µM). In particular, compound 5 showed the strongest cytotoxicity, and its four IC 50 values were less than 0.1 µM. Compound 5 was formed by O-glycosylation at C-3 of 4, which allowed us to reach a preliminary deduction that O-glycosylation at C-3 caused a more positive effect on cytotoxic activity. Compound 28 showed highly selective cytotoxic activity against MKN45 with the IC 50 value of 52.8 ± 5.3 µM among the four human cancer cell lines. Of all the compounds tested, only compounds 4, 5 and 28, with α, β-unsaturated lactone ring, showed certain cytotoxicity, which suggested that α, β-unsaturated lactone ring was an active functional group of anti-tumor, and this research result is consistent with the literature report [44].
This study not only revealed the structural diversity of the chemical components of T. chinense, but also provided lead compounds for the development of antioxidants, anti-inflammatory and antitumor agents, and promoted reasonable use of this herb.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.

Conflicts of Interest:
All authors declare that they have no conflict of interest.