One new flavanocoumarin from the thorns of Gleditsia sinensis

Abstract One new flavanocoumarin (1), as well as six known flavonoids (2–7), was isolated from the ethyl acetate extract of the thorns of Gleditsia sinensis. The structures of these compounds were elucidated by extensive spectroscopic measurements and comparison with data reported in literatures. Cytotoxic activities of compounds 1–6 were evaluated against human liver cancer SK-hep-1 cells in vitro by the MTT method, with compound 1 displaying moderate activity (IC50 of 62.53 μM). Furthermore, compound 1 could increase the number of apoptosis cells in a concentration-dependent manner.


Introduction
Gleditsia sinensis Lam. is an arbour of approximately 15 m in height distributed widely in China, such as the Shandong, Hebei and Jiangsu provinces. Its thorns, documented in the Chinese Pharmacopoeia as 'Zao Jiao Ci' , have been used in traditional Chinese medicine for the treatment of various cancers, carbuncle and skin diseases (Zhou, Li, Wang, et al. 2007). Previous studies on the chemical constituents of G. sinensis have led to isolation of limited flavonoids (Zhou, Li, Wang, et al. 2007), triterpenoid saponins (Liu et al. 2015), ellagic acid glycosides (Zhou, Li, Jiang, et al. 2007), and so on. What's more, the earlier pharmacological investigations on the title plant have revealed that the crude flavonoids extracted from the thorns showed significant anticancer activity (Shoemaker et al. 2005). The diversity of flavonoid structures and intriguing bioactivities from this species attracted our attention and prompted us to perform a further chemical investigation. In this paper, we describe the investigation of flavonoids from G. sinensis, activity evaluation against human liver cancer SK-hep-1 tumour cell, as well as apoptosis for cell growth inhibition using a flow cytometry.
Flavonoids 1-6 were evaluated for their cytotoxic activities against human liver cancer SK-hep-1 cell line with cisplatin as positive control in vitro by the MTT method. Compound 1 exhibited moderate cytotoxicity with IC 50 value of 62.53 μM, while the other tested compounds showed none inhibitory activities. and compound 1 could induce apoptotic cell death in SK-hep-1 cells in vitro. after 24 h treatment, compound 1 at 10 μM caused 13.8% early apoptosis and 8.4% late apoptosis, while 14.0% early apoptosis and 15.8% late apoptosis at 30 μM, 10.4% early apoptosis and 26.6% late apoptosis at 50 μM, 12.8% early apoptosis and 38.3% late apoptosis at 70 μM ( Figure 2).

General experimental procedures
CC was undertaken over silica gel (200-300 mesh) and Sephadex LH-20 (Pharmacia Biotech aB, uppsala, Sweden). Preparative HPLC was carried out on Shimadzu LC-6aD, equipped with a SPD-10a detector, and a reversed-phase C 18 column (YMC-Pack ODS-au 20 × 250 mm, 10 μm) was employed. Optical rotations were measured on a Perkin-elmer 241 digital polarimeter at 20 °C. IR spectrum was recorded on a Nicolet 5700 spectrometer. 1D and 2D NMR spectra were taken on a Varian NMR System-600 NMR spectrometer. eSI-MS and HReSI-MS were obtained using an agilent 1100/MSG1946 mass spectrometer. Cell apoptosis was analysed by a BD FaCSaria III flow cytometry. TLC was carried out with glass plate pre-coated silica gel G. Spots were visualised under uV light and by spraying with 10% H 2 SO 4 in 95% etOH, followed by heating at 100 °C. Methanol used in preparative HPLC procedure was in HPLC grade, and other solvents were of analytical grade.  Figure 1. structures of compounds 1-7.

Plant material
The thorns of G. sinensis were collected from Linyi city in Shandong province and authenticated by Prof. Jia Li in Shandong university of Traditional Chinese Medicine. a voucher specimen (No. 20130820) has been deposited in Shandong analysis and Test Center, Shandong academy of Sciences, Jinan, P.R. China.

MTT analysis
Compounds 1-6 were tested for cytotoxic activity against human liver cancer SK-hep-1 cell line, with cisplatin as positive control, by means of the MTT method as described in the literature (Liang et al. 2011).

Cell apoptosis analysis
The Pe annexin V apoptosis detection kit was used according to the manufacturer instructions (BD Biosciences Pharmingen, u.S.a). SK-HeP-1 cells (1 × 10 6 ) were seeded into six-well culture plates and the cells were treated with medium overnight, followed by a treatment of compound 1 (0, 10, 30, 50, 70 μM) and cisplatin (100 μM). after 24 h of treatment, all cells were analysed by flow cytometry within 1 h (BD FaCSaria III, Becton Dickinson, u.S.a).

Conclusion
The present phytochemical investigation of the active extract led to the isolation of seven flavonoids (1-7) including one new flavanocoumarin (1) where the α-pyrone moiety was attached to C-5 and C-6 positions in the flavonoid a-ring. It is noteworthy that compound 1 showed moderate cytotoxicity against human liver cancer SK-hep-1 cell, and induced apoptosis in this cell line, while the other flavonoids showed none inhibitory activities. The pharmacological results suggest that the α-pyrone moiety in 1 might play a key role for cytotoxicity against SK-hep-1 cell. Further research is recommended to establish whether this suggestion is right.

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