Two New Quinochalcone C-Glycosides from the Florets of Carthamus tinctorius

Two new quinochalcone C-glycosides, named hydroxysafflor yellow B (1) and hydroxysafflor yellow C (2), along with two known quinochalcone C-glycosides, safflomin C (3) and saffloquinoside C (4), and one known flavanone, (2R)-4',5-dihydroxyl-6,7-di-O-β-d-glucopyranosyl flavanone (5), were isolated from the florets of Carthamus tinctorius. Their structures were determined by extensive spectroscopic (UV, IR, HR-ESI-MS, 1D and 2D NMR) analyses. In addition, these quinochalcone C-glycosides together with hydroxysafflor yellow A and anhydrosafflor yellow B were evaluated for their anti-oxidative effects against H2O2-induced cytotoxicity in cultured H9c2 cells. Among them, compound 2 exhibited significant anti-oxidative effects.

High energy collision induced dissociation MS/MS has been shown to be a practical method for structural verification of C-glycosyl flavonoids [8]. Studies have shown that the stable carbon-carbon bond of C-glycosyl flavonoids is resistant to rupture, so the main cleavages are at the bonds in the sugar moiety, instead of losing a sugar moiety as in O-glycosyl flavonoids [9,10]. In the MS/MS spectra of compounds 1 and 2, both are shown to have lost a glucosyl radical and produce stable radical anions [M − H − 163] −· at m/z 448. In addition, two fragment ions at m/z 119 and 145 could be detected in compounds 1 and 2 ( Figure 3A), which also proved that they have a trans-p-hydroxycinnamoyl group. Meanwhile, fragment ions at m/z 207 and 261 were detected clearly in MS 2 spectra of compounds 1 and 2 ( Figure 3A), which also indicates that they may have a free hydroxyl group at the 4-position [11]. The fragmentation pathway of compound 2 producing fragment ions at m/z 261 and 207 in negative ion mode is proposed in Figure 3B.  The known compounds were readily identified by comparing their spectroscopic data (UV, IR, 1 H-NMR, 13 C-NMR, and HR-ESI-MS) with that reported in the literature as safflomin C (3) [12], saffloquinoside C (4) [13], and (2R)-4',5-dihydroxyl-6,7-di-O-β-D-glucopyranosyl flavanone (5) [14].
The obtained quinochalcone C-glycosides together with HSYA and anhydrosafflor yellow B were evaluated for their anti-oxidative effects against H2O2-induced cytotoxicity in cultured H9c2 cells (Figure 4). Among them, the cardioprotective effects of compound 2 were more robust than those of compounds 1, 3 and 4. On the other hand, HSYA and anhydrosafflor yellow B did not show any significant response. The isolated quinochalcone C-glycosides (60 μg/mL) were pretreated with H9c2 cells for 24 h before the addition of H2O2 (10 mmol/L) for cytotoxicity test as in (A). Vitamin C (1.1 mg/mL) served as a positive control (statistical significance is indicated as * p = 0.031 for hydroxysafflor yellow C vs. safflomin C; ** p = 0.005 for hydroxysafflor yellow C vs. saffloquinoside C; *** p = 0.001 for hydroxysafflor yellow C vs. control).

Discussion
Quinochalcone C-glycosides are the typical constituents of Carthamus tinctorius. Two new quinochalcone C-glycosides (1-2), which add the diversity of these ingredients, were successfully isolated from safflower. The NMR and MS/MS analyses led to elucidation of the structure of compounds 1 and 2 with their partial configurations. Moreover, for D-fructose, the equilibrium concentrations in water are around 57% of β-D-pyranose, 31% of β-D-furanose, 9% of α-D-furanose, and 3% of α-D-pyranose [15]. Coincidentally, we isolated much less of compound 1 than compound 2 from safflower. In the total synthesis of quinochalcone C-glycosides, asymmetric synthesis of carthamin (as the acetate) has been achieved [7]. However, the total syntheses of the other yellow pigments of quinochalcone C-glycosides have not been carried out. The synthesis of analogs in which the glucosyl group or the glucosyl and hydroxyl groups on the chiral carbon were replaced by one or two methyl groups has been achieved for safflomin A, safflomin C, precarthamin, and carthamin [2]. So the study may provide some inspiration for scientists to explore the biogenesis and the total synthesis of quinochalcone C-glycosides.
H2O2 has been extensively used as an inducer of oxidative stress in vitro [16]. Meanwhile, the H9c2 cell line has been widely used in studies investigating cardiomyocyte cellular mechanisms [17,18]. It is worth mentioning that the water extract of Carthamus tinctorius has been developed as an intravenous injection in China and has been extensively applied in hospitals to treat cardiovascular diseases [19]. Thus, it is of interest to determine whether the isolated quinochalcone C-glycosides against H2O2-induced cytotoxicity in cultured H9c2 cells. Of the compounds isolated, the cardioprotective effects of compound 2 were more robust than those of compounds 1, 3 and 4, thereby providing a basis for further studies. On the other hand, HSYA and anhydrosafflor yellow B did not show any significant response.

Plant Material
The florets of Carthamus tinctorius were purchased from Tongling TCM Co., Ltd., Fengyuan, Anhui Province in March 2013 and identified by Professor Chun-Gen Wang. A voucher specimen (No. NJUTCM-20130308) was deposited in the Herbarium of Nanjing University of Chinese Medicine.

Biological Assay
The anti-oxidative effects of the isolated quinochalcone C-glycosides were evaluated against H2O2-induced cytotoxicity in cultured H9c2 cells. The purity of the tested compounds was >98% as identified by NMR and MS.

Cell Culture
Rat pheochromatocytoma H9c2 cell line was obtained from ATCC (Rockefeller, MD, USA). The cells were maintained in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a water-saturated 5.0% CO2 incubator. Reagents for cell cultures were purchased from Sigma (St. Louis, MO, USA).

Cell Viability Test
Cultured H9c2 cells in 96-well-plate (6000 cells/well) were pre-treated with the quinochalcone C-glycosides (60 μg/mL) for 24 h. After being replaced by fresh culture medium, the cultures were treated with 10 mmol/L hydrogen peroxide (H2O2) for 1 h. Cell viability tests were performed with the addition of thiazolyl blue tetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA) in fresh water at a final concentration of 5 mg/mL for three hours. After the solution was removed, the purple preciptate inside the cells was re-suspended in DMSO and then measured at 570 nm absorbance. H2O2 at various concentrations (0, 0.37, 1.1, 3.3 and 10 mmol/L) served as a control for cytotoxicity.

Statistical Analysis
Individual 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 one-way ANOVA (SPSS version 17.0: Chicago, IL, USA). A p value of 0.05 or less was considered significant.

Sample Availability
Samples of the compounds 1 and 2 are available from the authors.

Conclusions
In recent years, C-glycoside chemistry has been one of the main topics in carbohydrate chemistry, not only because of the synthetic challenges posed, but also because C-glycosides have the potential to serve as carbohydrate analogues resistant to metabolic processes. Consequently, C-glycosides are currently receiving much interest as a potential source of therapeutic agents for clinical use [20]. Quinochalcone C-glycosides are regarded as the characteristic compounds that only have been isolated from the florets of Carthamus tinctorius. Recently, this class of compounds was found to have multiple pharmacological activities. Except for the water extract of Carthamus tinctorius, it is worth mentioning that HSYA also has been developed as an intravenous injection in China to treat cardiovascular diseases. In the study, two new quinochalcone C-glycosides, hydroxysafflor yellow B (1) and hydroxysafflor yellow C (2) were isolated and identified from the florets of Carthamus tinctorius. To the best of our knowledge, the C-α-D-fructopyranose is rare in natural quinochalcone C-glycosides. Previous studies have found that quinochalcone C-glycosides are soluble in water, diluted alcohol and practically insoluble in anhydrous ethanol, acetone, diethyl ether, petroleum, and ethyl acetate [21]. However, from our experience, there are large differences in the polarity for this class of compounds, which results in difficulty in NMR experiments. Once the molecular weight of quinochalcone C-glycosides exceeds 1000, simple signal accumulation does not work for 13 C-NMR measurement, which prompted us to develop a better approach and strategy. Anti-oxidative activity evaluation showed that compound 2 was significantly active against H2O2-induced cytotoxicity in cultured H9c2 cells in vitro, which suggests its potential for treatment of cardiovascular diseases.