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Chemical constituents from the leaves of Tripterygium wilfordii and their cytotoxic activity

A new flavonol glycoside derivate named kaempferol 3-O-trans-p-coumaroyl-7-O-α-ʟ-rhamnoside (1), together with five known compounds (2–6), was isolated from the EtOAc-soluble extract of the leaves of Tripterygium wilfordii. The structure of new compound was elucidated with spectroscopic and physico-chemical analyses. All the isolates were evaluated for in vitro cytotoxic activity against four human cancer cells including HepG2, Hep3B, A549 and MCF-7. Among them, compound 1 exhibited the significant cytotoxic activities against HepG2 and Hep3B cells with IC50 values of 6.8 ± 1.6 and 4.2 ± 1.3 μM, respectively. The above data demonstrated the potential of 1 for the treatment of liver cancer. In addition, 4 also showed moderate inhibitory activity on HepG2 and Hep3B cells with IC50 values of 16.1 ± 2.3 and 10.7 ± 1.8 μM, respectively.


Introduction
Tripterygium wilfordii Hook. f. (Celastraceae) is a vine shrub, which is widely distributed in China, Korea and Japan. 1 The roots of T. wilfordii, also known as 'Lei-Gong-Teng' in China, were used to treat inflammatory and autoimmune diseases as a traditional Chinese medicine. 2 Pharmacological studies confirmed that T. wilfordii possessed anti-inflammatory, 3 immunosuppressive, antitumour 4 and neuroprotective activities. 5 And a large number of phytochemical investigations on the roots of T. wilfordii revealed the presence of dihydroagarofuran sesquiterpenoids, 6 sesquiterpene alkaloids, 7 abietane diterpenoids, 8 triterpenoids, 9 megastigmane glycosides 10 and a small amount of flavonoids and lignans. 11,12 However, there are less chemical studies on the leaves of T. wilfordii compared with its roots. Thus, a series of studies focused on the leaves of T. wilfordii were carried out. As a result, a new flavonol glycoside derivate named kaempferol 3-O-trans-pcoumaroyl-7-O-α-ʟ-rhamnoside (1), together with five known compounds (2)(3)(4)(5)(6), was isolated. Herein, we report the isolation and structural elucidation of these compounds.
In addition, their cytotoxic activities on HepG2, Hep3B, A549 and MCF-7 cells were also evaluated.

Structural elucidation
Compound 1 was obtained as a yellow amorphous powder, and the molecular formula was established as C 30 H 26 O 12 based on the HRESIMS at m/z 577.1207 for the [M − H] − (Supplemental Figure S5). The IR spectrum showed typical absorption bands of hydroxy and carbonyl groups at 3425 and 1690 cm −1 , respectively. Its 1 H NMR spectrum (Table 1, Supplemental Figure S1 Figure S3 and S4). Although the position of trans-p-coumaroyl moiety could not be determined by the HMBC correlations, two hydroxy signals with larger chemical shifts at δ H 11.1 and 10.9 could indirectly reveal that the transp-coumaroyl moiety was linked with C-3. In addition, the aglycone isolated from the acid hydrolysis assay of 1 was identified as p-coumaroylkaempferol (2) by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods, and it further demonstrated the structure of 1. Thus, the structure of compound 1 was assigned as Moreover, five known compounds (2-6) were obtained from the leaves of T. wilfordii and identified as p-coumaroylkaempferol (2), 14 tripteryol B (3), 15 3β-O-transcoumaroylbetulinic acid (4), 16 camaldulenic acid (5) 17 and crategolic acid (6) 18 based on the NMR data and comparison with literature data (Figure 2).

Analysis of biological activity results
The in vitro cytotoxic activity of compounds 1-6 against HepG2, Hep3B, A549 and MCF-7 cells was evaluated by  the MTT method, and doxorubicin was used as the positive control. As shown in Table 2, compounds 1, 3-6 showed different levels of cytotoxic activity with IC 50 values ranging from 4.2 ± 1.3 to 47.8 ± 2.0 μM. Among these compounds, 1 showed the significant cytotoxic activity on HepG2 and Hep3B cells with IC 50 values of 6.8 ± 1.6 and 4.2 ± 1.3 μM, respectively. In addition, 4 also exhibited moderate cytotoxic activity towards Hep3B cells with IC 50 value of 10.7 ± 1.8 μM. In general, the potential structureactivity relationships could be deduced. The comparison of cytotoxic activity on four cancer cells between 1 and 2 indicated that the increasing cytotoxic activity might be associated with the α-ʟ-rhamnoside moiety which linked with C-7 of 1. Based on the significant cytotoxic activity of 1 on Hep3B cells, the deeply pharmacological investigations on 1 should be further carried out.
wilfordii. All compounds were tested for their in vitro cytotoxic activity against HepG2, Hep3B, A549 and MCF-7 cells. It was noted that compound 1 showed the significant cytotoxic activity on HepG2 and Hep3B cells with IC 50 values of 6.8 ± 1.6 and 4.2 ± 1.3 μM, respectively. This investigation confirmed that p-coumaroyl and caffeoyl moieties could highly increase the cytotoxic effect of alternariol on cancer cells. The previous studies mainly reported the presence of various dihydroagarofuran sesquiterpenoids with potent cytotoxic activity from the roots of T. wilfordii. This study provided a prediction that this kind of phenylpropanoid-conjugated flavonol glycoside might be considered as lead compounds to treat liver cancer. Thus, the in-depth pharmacological investigations on 1 should be carried out.

Plant material
The leaves of T. wilfordii were collected in Maanshan, Anhui province, China, and authenticated by Professor Hongxiang Lou (College of Pharmacy, Shandong University). A voucher specimen of the plant (No. 20200910) was deposited at the Cheeloo College of Medicine, Shandong University, Shandong, China.

Acid hydrolysis of compound 1
The sample of compound 1 (2.0 mg) was added to MeOH solution (3.0 mL) and refluxed with 2 M HCl (3.0 mL) at 100 °C for 4 h. After removal of MeOH under reduced pressure, the reaction mixture was diluted with H 2 O (5.0 mL) and neutralized with NaHCO 3 solution, followed by extraction with EtOAc (3 × 10 mL). Next, the collected organic phase was evaporated and subjected to PTLC using CHCl2-MeOH (5:1) as eluent to yield the aglycone. The water layer was concentrated and submitted to PTLC (CHCl 2 / MeOH/H 2 O = 7:3:0.5) to yield the sugar. The identification of ʟ-rhamnose was carried out by the comparison of Rf values in TLC (CHCl 2 /MeOH/H 2 O = 7:3:0.5) between the obtained sugar and authentic sample. Spot was detected by spraying with vanillin-sulfuric acid followed by heating. In addition, their similar optical rotation could also confirm the type of sugar (

Cytotoxicity assay
The MTT assay was used to determine the cytotoxicity of the compounds against human cancer cell lines according to the method previously described. 20 Briefly, the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% foetal bovine serum, 100 U mL −1 penicillin and 100 µg mL −1 streptomycin in a humidified 37 °C incubator supplied with 5% CO 2 . Then, cells were seeded at 5 × 10 3 cells mL −1 per well onto 96-well flat bottom plate and incubated for 12 h at 37 °C with 5% CO 2 . Compounds 1-6 were dissolved in DMSO to obtain a initial concentration (10 mM) and diluted to obtain a series of concentrations. Next, cells were treated and continuously exposed to different concentrations of compounds for 48 h. MTT (5.0 mg mL −1 ) was added to each well. After 4 h, the crystalline formazan formed was dissolved in DMSO (150 μL), and the absorbance at 570 nm of each well was measured with liquid scintillation counting. All experiments were performed at least three times and the values of IC 50 were measured by GraphPad Prism 8.0 software. DMSO was used as the negative control and doxorubicin was used as a positive control.