Cytotoxic activities of flavonoids from a traditional Mongolian medicinal herb Clematis aethusifolia Turcz.

Abstract In the course of our search for antitumour constituents from the traditional Mongolian medicinal herb Clematis aethusifolia Turcz., 11 flavonoids were isolated for the first time from the dried aerial parts of the plant by flash C18 column chromatography, Sephadex LH-20 and reversed phase preparative HPLC. The planar structures of these flavonoids were established based on 1D and 2D NMR and high-resolution mass spectrometry. Compounds 1, 2, 4 and 5 showed moderate cytotoxicity against a panel of five human solid tumour cell lines, including A-375, a human melanoma cell line; SK-OV-3, a human ovarian cancer cell line; A549, a human lung cancer cell line; HCT-15, a human colorectal adenocarcinoma cell line; and SH-SY5Y, a human neuroblastoma cell line (with IC50 values of 20–70 μM). The obtained cytotoxic apigenin and its derivatives may be useful as standard compounds for the quality control of the crude drug and its preparations.


Introduction
Plants of the Clematis genus have increasingly gained attention because they are widely distributed worldwide and are used as fruits and herb medicines (Rana et al. 2015). Different types of metabolites including saponins (Yan et al. 2009), flavonoids (Xu et al. 2014), alkaloids (Mimaki et al. 2004), coumarins (Hao et al. 2013) and anthocyanins (Hashimoto et al. 2011) have been reported to have various bioactivities. Furthermore, some of these metabolites exhibit antitumour activity, as they were reported to display cytotoxicity or cause apoptosis of cancer cell lines such as the human colon cancer cell line HCT-8 (Yan et al. 2009), liver cancer cell line BeL-7402 (Tian et al. 2013), gastric cancer cell line BGC-823 (Yan et al. 2009), ovarian cancer cell line A-2780 (Kubo et al. 2012), squamous cell carcinoma cell line HSC-2 (Kubo et al. 2012) and human breast cancer cell line MCF-7 (Ding et al. 2009).
The dried aerial part of Clematis aethusifolia Turcz. is used as a traditional Mongolian crude drug and categorised into 'remove lump and stagnation medicine' in Mongolian herbal medicine with functions against tumour, lump, dyspepsia, oedema, wounds and haemorrhoids, among other conditions. The crude drug and one of its preparations 'Zhilouliuwei powder' are widely used clinically and listed in the Ministerial Drug Standards for Mongolian Medicine. Although numerous chemical and bioactive studies of various species of Clematis have been conducted, only few studies have examined C. aethusifolia.
In our search for bioactive metabolites, 11 flavonoids were isolated for the first time from the aerial part of C. aethusifolia, with some showing moderate cytotoxicity against a panel of five human solid tumour cell lines, including A375, SK-OV-3, A549, HCT-15 and SH-SY5Y, using the Cell Counting Kit-8 (CCK-8) assay. Additionally, flavonoids are characteristic components in Clematis species and play an important role in chemotaxonomy. To our knowledge, this is the first study of the chemical constituents and bioactivity of C. aethusifolia Turcz, and will provide scientific basis for quality control and further studies of the drug.

Evaluation of cytotoxicity
Compounds 1-11 were evaluated for cytotoxicity against a panel of five human solid tumour cell lines, including A375, SK-OV-3, A549, HCT-15 and SH-SY5Y, using the CCK-8 assay, in the concentration range 1-200 μM (Table S1) and incubation for 72 h. Compound 1 exhibited moderate cytotoxic activities against A375 and SH-SY5Y with IC 50 values of 57.2 and 20.0 μM, respectively. Compound 2 exhibited moderate cytotoxic activity against SH-SY5Y with an IC 50 value of 46.9 μM. Compound 4 exhibited moderate cytotoxic activities against SK-OV-3 and A549 with IC 50 values of 28.5 and 40.7 μM, respectively. Compound 5 also exhibited moderate cytotoxic activity against SK-OV-3 with an IC 50 value of 70.2 μM. However, compounds 3 and 6-11 showed weak cytotoxic activities against all five human solid tumour cell lines (IC 50 ≥ 100 μM). The results showed that all moderately cytotoxic flavonoid compounds, 1, 2, 4 and 5, involve apigenin and its derivatives, although not all apigenin derivatives show such activities.
The CCK-8 assay is a convenient, sensitive and reliable method with good reproducibility for determining the number of viable cells in a cytotoxicity assay (Kinnunen et al. 2014). In repeated tests, stable cytotoxicity IC 50 values were obtained, supporting the structureactivity relationship. Apigenin and its derivatives showed significantly lower IC 50 values than quercetin derivatives and kaempferol derivatives, suggesting that substitution at position 3 in flavonoids weakens the cytotoxic activity. Apigenin exhibited much higher IC 50 values than its derivatives, including that an O-glycosidic substitution at position 7 would also weaken the cytotoxic activity of flavonoids.

Conclusion
In our study, apigenin and its derivatives from the aerial part of C. aethusifolia showed moderate cytotoxicity against five human solid tumour cell lines. While there have been some reports regarding the cytotoxicity of the 11 isolated flavonoids (Choi et al. 2004;Yu et al. 2007;Minh et al. 2015), there have been no cytotoxic assays of specific human tumour cell lines. However, additional studies are required to elucidate the possible anti-tumour mechanism.
Notably, the numbers of cytotoxic saponins were reported previously from the roots of different species of Clematis, but no saponin has been isolated from C. aethusifolia Turcz. The main constituents from the drug were flavonoids, which play an important role in the chemotaxonomy of Clematis species, and the obtained cytotoxic apigenin and its derivatives may be useful as standard compounds for the quality control of the crude drug and its preparations.

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