Cytotoxic flavonoids from the leaves of Callicarpa nudiflora hook

Abstract A new flavonoid glycoside, luteolin-3′-O-β-D-6″-acetyl glucopyranoside (1), along with six known flavonoids, were isolated from the leaves of Callicarpa nudiflora Hook. The structures of the isolated compounds were established on the basis of extensive spectroscopic analyses. Compound 6 exhibited potent cytotoxicity and compounds 1 and 7 exhibited moderate cytotoxicity against human hepatocellular carcinoma SMMC-7721 cells. Graphical Abstract


Introduction
Callicarpa nudiflora Hook, belonging to Verbenaceae, which was mainly distributed in Guangdong, Guangxi, Hainan of China.C. nudiflora is an authentic medicinal material in Hainan province which is one of the commonly used medicines by Li nationality doctors.In the Li nationality, it is called 'Bu fa' (Flora of China 1982) and mainly distributed in Wuzhishan, Baisha, Lingshui, Chengmai of Hainan province.And is also distributed in India, Vietnam, Malaysia, Singapore and Somewhere else.The leaves of C. nudiflora were used medicinally to stop bleeding and relieve pain, dispel blood stasis and reduce swelling (Zhou et al. 2013;Fu et al. 2017aFu et al. , 2017b)).On the clinical uses, it is used to treat traumatic bleeding, bruises, swelling and pain, rheumatism, tuberculosis, hemoptysis, gastrointestinal bleeding, hepatitis in clinical (Dong et al. 2014;Huang et al. 2014;Luo et al. 2016).Previous phytochemical studies discovered flavonoids, iridoids, lignans and terpenoids as the main chemical constituents of C. nudiflora (Wang et al. 2007;Gao et al. 2010;Luo et al. 2014;Feng et al. 2017).In the present study, we reported the isolation and structural elucidation of a new flavonoid glycoside, luteolin-3 0 -O-b-D-6 00 -acetyl glucopyranoside (1) along with six known flavonoids (Figure 1).In addition, all compounds were evaluated for their cytotoxicity against human hepatocellular carcinoma (HCC) SMMC-7721 cells.connected with C-3 0 .The HMBC correlation from d H 1.89 (H-8 00 ) to d C 170.8 (C-7 00 ) suggested the presence of an acetyl group.Based on the coupling constant (J ¼ 6.8 Hz) of anomeric proton, the configuration of glycosyl can be determined to be b configuration.By comparison the NMR data with the known compound luteolin 3 0 -O-b-D-glucuronide (Okamura et al. 1994), the 1 H and 13 C NMR signals of 1 are basically same with the corresponding signals of luteolin 3 0 -O-b-D-glucuronide except for the acetyl group in compound 1.The HMBC correlations from d H 7.61 (H-6 0 ), 1.89 (H-8 00 ) to d C 170.8 (C-7 00 ) indicated the location of the acetyl group at C-6 00 of the glucosyl group.Therefore, the structure of compound 1 can be deduced as luteolin-3 0 -O-b-D-6 00 -acetyl glucopyranoside.The 1 H and 13 C NMR signals were assigned by COSY, DEPT, HSQC and HMBC spectra experiment.
In addition, all flavonoids were evaluated for their cytotoxicity against human hepatocellular carcinoma SMMC-7721 cells using Cell Counting Kit-8 (CCK8) assay method.The results showed that compound 6 exhibited potent cytotoxicity against SMMC-7721 cells with IC 50 of 0.837 lM, and compound 1 and 7 exhibited moderate cytotoxicity with IC 50 of 42.507 and 67.259 lM, respectively.

General
NMR spectra were recorded on a Bruker AV-500 MHz spectrometer, with tetramethylsilane (TMS) as an internal standard.HR-ESI-MS were recorded on an Agilent Technologies liquid chromatograph connected to a Q-TOF mass spectrometer (Agilent, US).Macroporous resin HPD-100 and ODS-A (YMC-GEL) were used for open column chromatography (CC).Silica gel (Qingdao Haiyang Chemical, Qingdao, China) was used for open column chromatography (CC).TLC were conducted on silica gel GF254 plates (Qingdao Haiyang Chemical, Qingdao, China).Semi-preparative HPLC experiments were carried out using an Agilent 1260 HPLC system with a YMC ODS column (250 Â 4.6 mm, 10 lm).

Plant material
The air-dried leaves of Callicarpa nudiflora were collected from Wuzhishan of Hainan Province, China in October 2015 and identified by Prof. Xi-Feng Sheng (Hunan Normal University, China).A voucher specimen (No.LHZZ-2015) has been deposited in Department of Pharmacy, School of Medicine, Hunan Normal University.

Bioassay
The cytotoxicity against SMMC-7721 cell line was assayed using Cell Counting Kit-8 (CCK8) method.The cells were cultured in DMEM medium with 10% fetal bovine serum (HyClone) at 37 C in a humidifed atmosphere supplied with 5% CO 2 .100 lL of adherent cells was seeded into each well of a 96-well plate and allowed to adhere for 12 h.Then each tumor cell line was treated with the test compounds at various concentrations (0, 5, 10, 20, 40, 80 lM) in triplicate for 48 h.Absorbance was determined at 450 nm in a microplate spectrophotometer.

Conclusions
In conclusion, we reported the isolation, elucidation and cytotoxicity evaluation of flavonoids from the leaves of C. nudiflora.One new flavonoid glycoside along with six known flavonoids were isolated from the leaves of C. nudiflora.In addition, compound 6 (pachypodol) exhibited potent cytotoxicity, and compounds 1 and 7 exhibited moderate cytotoxicity against SMCC-7721 cells.This is the first report of the anti-HCC effects of pachypodol.