A new flavonol glycoside from the florets of Carthamus tinctorius L.

One new flavonol glycoside, 6-hydroxykaempferol-3-O-β-D-glucoside-7-O-β-D-glucuronide (1), together with eight known flavonoids and three known quinochalcones, was isolated from the florets of Carthamus tinctorius L. Their structures were determined by extensive spectroscopic analyses. Their cardioprotective effects against H2O2-induced apoptosis in H9c2 cells were also evaluated; compounds 1, 2, 4–5, 7–10 and 12 provided significant protective effects on H2O2-induced H9c2 cells at the concentration of 25 μg/mL.


Introduction
Carthamus tinctorius L. has been used as a Chinese folk medicine for over 1000 years (Ge et al. 1996). The florets have the function of promoting blood circulation by removing blood stasis . It showed therapeutic potential for coronary heart disease, stroke, gynaecological disease, angina and hypertension (Jiang et al. 2008;Fan et al. 2009). The extract of C. tinctorius L. has been developed as an intravenous injection, and has been extensively applied in hospitals to treat cardiovascular diseases (Fan et al. 2009).
Chemical components include flavonoids, alkaloids, lignans and fatty acids, which were isolated and identified from safflower . Then flavonoids with potent antioxidative effects are the major effective components in traditional herbal medicine used in treating cardiovascular diseases (Sun et al. 2011). Meanwhile, the H9c2 cell line has been widely used in studies investigating cardiomyocyte cellular mechanisms (Hescheler et al. 1991). H 2 O 2 has been extensively used as an inducer of oxidative stress in vitro (Ryter et al. 2007). Thus, this study aimed to research the isolated flavonoids against H 2 O 2 -induced cytotoxicity in cultured H9c2 cells.
All the nine flavonoids and three quinochalcones were evaluated for their anti-oxidative effects against H 2 O 2 -induced apoptosis in cultured H9c2 cells. Among them, compounds 1, 2, 4 -5, 7 -10 and 12 provided significant protective effects on H 2 O 2 -induced H9c2 cells at the concentration of 25 mg/mL (Figure 2). The cardioprotective effects of compounds 7 and 8 were more effective than those of compounds 1-6 which indicated that glycosylation of flavonoids diminished their activity when compared to the corresponding aglycones (Cai et al. 2006). Compounds 1 and 2 exhibited significant cardioprotective effects due to diOH-7, 8 or diOH-3 0 , 4 0 groups (Seyoum et al. 2006). The structure of compound 9 was similar to that of compound 7 (it possesses an additional OH-3 group), which had stronger activity (Amaral et al. 2009). Compound 10 was more active than the other two quinochalcones (compounds 11 and 12) due to the more phenolic hydroxyl groups (Modak et al. 2005).
The results suggested that flavonol aglycones with diOH-7, 8 or diOH-3 0 , 4 0 groups displayed important roles for their protective effects against H 2 O 2 -induced cardiotoxicity. Moreover, the number of the phenolic hydroxyl groups contributed to the activity of quinochalcones. However, further investigations were needed to explain the detailed structure -activity relationships.

Plant material
The florets of C. tinctorius L. were collected from Changji Hui tribe autonomous prefecture of the Xinjiang Uygur Autonomous Region of China in July 2013. The specimen was identified by Zhaoqing Meng, Jiangsu Kanion Pharmaceutical Co. Ltd., Lianyungang, People's Republic of China, where a voucher specimen (No. CKG 20140503G) has been deposited.

Biological assay
The anti-oxidative effects of the isolated flavonoids were evaluated against H 2 O 2 -induced cytotoxicity in cultured H9c2 cells.
3.5.1 Cell culture H9c2 cardiomyocytes were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum at 378C in a water-saturated 5% CO 2 incubator. Reagents for cell cultures were purchased from Sigma (St. Louis, MO, USA).

Cell viability test
H9c2 cells (3000 cells/well) were seeded into 96-well plates after incubation with 50 mM H 2 O 2 for 3 h followed by treatment with compounds 1 -12 (25 mg/mL) for 3 h. At the end of the incubation, CCK-8 reagent was added into each well followed by further incubation for 4 h. The optical density was measured at 450 nm using a multiscan microplate reader (Teken, Beijing, China). Each determination represented the average mean of six replicates.

Statistical analysis
Data were expressed as mean^standard deviation (SD). A post hoc Dunnett's test was used to obtain corrected p-values in group comparisons. Statistical analyses were performed with oneway ANOVA (SPSS version 17.0: Chicago, IL, USA). A p-value of 0.05 or less was considered significant.

Supplementary material
Supplementary figures relating to this article are available online at http://dx.doi.org/10.1080/ 14786419.2015.1045905.

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

Funding
Financial support from the National Science and Technology Major Project 'Key New Drug Creation and Manufacturing Program' [grant number 2013ZX09402203] of the People's Republic of China is gratefully acknowledged.