2.1. Effect of Temperature on the HPLC Resolution of RCAs
An optimum HPLC condition is essential for the qualitative and quantitative analysis of the RCAs extracts. The column temperature is one factor that influences chromatographic retention and selectivity [
20], but it is often overlooked in the analytical method of anthocyanins. In this study, an acidic mobile phase with pH 2.0 was optimized to separate RCAs on a reversed phase HPLC-column at 25 °C at first, as shown in
Figure 1a. Although the separation in the chromatograph seemed acceptable, there were still some shoulder peaks, as shown in the figure. It is interesting to find that the temperature affects the resolution of RCAs in HPLC remarkably. As shown in
Figure 1, the peaks of 9 and 10 and the peaks of 11, 12, and 13 could not be separated at 25 °C. However, as the column temperature increased from 25–45 °C, these peaks became better separated. Generally, as the column temperature increased from 20–45 °C, the retention times of all the RCAs decreased. Consequently, taller and narrower chromatographic peaks could be found in the chromatographic profiles at a higher temperature, as shown in
Figure 1e. With mass spectral characterization, for example, the last peak in
Figure 1a contained three cyanidin-derivatives with different glycosylation patterns (Peaks 11, 12, and 13 were characterized as cyanidin 3-(feruloyl)(feruloyl)-diglucoside-5-glucoside, cyanidin 3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside, and cyanidin 3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside, respectively). These cyanidin-derivatives all had an acetyl diglucoside and a glucoside group, but the only differences at their acetyl groups were feruloyl and sinapoyl, which made them difficult to separate at normal conditions. However, these compounds could be separated linearly with the temperature increased from 25–45 °C. These results show that the column temperature is a critical factor for HPLC separation of RCAs.
It is proposed that the separation of the RCAs by increasing temperatures on the HPLC column be attributed to their different reductions of the elution time with temperature [
20,
21,
22]. The results showed that the retention times of some RCAs were more sensitive to the column temperature. This phenomenon could be explained from a thermodynamic point [
21].
According to Snyder’s theory [
21], the relationship between the retention factor and column temperature for a given solute can be described as:
where kR and TR are the retention factor and (absolute) temperature for a reference condition (e.g., 298 K) and
kT and
T are the retention factor and temperature for the solute at a new temperature,
a being the energy constant. For a given mobile phase and column condition as in this study, the
a value of each RCA is fixed. Hence, by plotting log
kT versus (1/
TR − 1/
T) over the experimental temperature range, the
a value can be obtained from the slope of the plot [
20]. Here, the temperature 25 °C is selected as the reference temperature
TR, and the time at the baseline disturbance (3.0 min) was set as the hold-up time. The retention factor
kT and the
a values for each RCAs are shown in
Table 1.
The value of
a is negatively proportional to the enthalpy for the transfer of the solute from the mobile phase to the stationary phase [
20]. The values of
a were positive for all RCAs, which means that the adsorption process of RCAs was exothermic. The exothermic retention behavior resulted in a decrease in retention with increasing temperature as observed. However, the retention of some anthocyanins may decrease at a different rate, since the slopes of the van’t Hoff curves (Δ
H) for these anthocyanins were not equal [
22]. Therefore, as the temperature increases, the separation between peaks that co-eluted at a low temperature will be much improved. For example, the
a was 1451 and 1235 for Peaks 9 and 10, respectively. The larger value of Peak 9 resulted in more reduction degrees of the retention time and consequently separated Peaks 9 and 10. It is obvious that the larger the difference of the
a values for co-eluted peaks, the more feasible it is to apply the temperature effects to get a good resolution. The difference of the
a values between Peaks 11 and 13 was larger than that between Peaks 12 and 13, which made Peak 11 more easy to separate from Peak 13, as shown in
Figure 1.
2.2. HPLC-ESI-MS Analysis and Quantification of RCAs
Based on the optimum HPLC conditions, the components of the RCAs extracts were determined by ESI-MS, as listed in
Table 1. The anthocyanins profile of red cabbage had been successfully analyzed by means of HPLC-DAD-MS/MS [
3]. According to the results, the derivatives of cyanidin were only found in RCAs [
3]. Therefore, all 13 peaks can be identified based on the comparison of their retention time and the mass spectrum with the published data [
3]. The tentative identification of chemical structures for each peak is listed in
Table 2. The results obtained supported the previous observation that the main structure of anthocyanins in red cabbage was cyanidin-3-diglucoside-5-glucosides, the glycoside chains of which can be nonacylated, monoacylated, and diacylated [
3,
23]. It can be seen that the monoacylated derivatives of cyanidin were predominate in the RCAs. Anthocyanins with acylation have shown good stabilities to light and heating compared to nonacylated anthocyanins [
6]. The purified RCAs contained mainly anthocyanins with acyl chain structures, which are beneficial for their application as a natural colorant in the food industry.
2.3. HPLC Method Validation and Quantification
The HPLC method established above was valid using C3G as a standard anthocyanin reference. The linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and recovery were assessed. For linearity tests, the equation of linear regression was y = 32.51x − 28.36 over the concentration range from 5–60 μg/mL (y is the peak area in mAU*s; x is the concentration in μg/mL; R2 = 0.9962). The LOD and LOQ were 0.080 μg/mL and 0.267 μg/mL determined at S/N of three and 10, respectively. The relative standard deviation (RSD) and recovery were tested and calculated to assess the precision of the method. The RSD values of intra-day variations were ± 0.58% for peak areas and ± 0.43% for retention times. The recovery was 98.71 ± 2.53% based on the C3G addition.
The total anthocyanins in the RCAs extracts were determined with 268 ± 2 μg/mg based on the above method. The concentrations of each RCA in the purified powder are listed in
Figure 2 along with their relative contents (R%). The composition profiles of nonacylated, monoacylated, and diacylated anthocyanins are also shown in
Figure 2. The glycosyl groups of the RCAs were acylated by ferulic acid, sinapic acid,
p-coumaric acid, and caffeic acid. It can be seen that the derivatives of cyanidin in the extracts were distributed mainly into about 19% nonacylated anthocyanins, 51% monoacylated anthocyanins, and 31% diacylated anthocyanins. Wiczkowski et al. [
3] found that nonacylated anthocyanins comprised 27.6% of total RCAs, while monoacylated and diacylated anthocyanins covered 38.4% and 34.1%, respectively. The proportions of each anthocyanin were, however, changed with varietal diversity, maturation time, and cultivation conditions [
24,
25].
2.4. Effect of RCAs against H2O2-Induced Oxidative Stress in HepG2 Cells
Because oxidative stress resulted in inevitable damage during metabolism, we investigated whether treatment with the RCAs represses cell death induced by H
2O
2. As shown in
Figure 3, the cytotoxicity induced by H
2O
2 was significantly increased compared to the control group. On the other hand, treatment of the RCAs at all concentrations suppressed the damage triggered by H
2O
2. Among three concentrations, the RCAs with 1.0 mg/mL showed more inhibitory effects on oxidative stress-induced cytotoxicity. The result indicated that the RCA extracts could provide the first line of defense to HepG2 cells against oxidative stress. It was supposed that the cells reduced oxidative stress-induced apoptosis by directly scavenging reactive oxygen species (ROS) in the cell.
To clarify the effect of the RCAs on HepG2 cells, the activity levels of ROS in cells were measured. As shown in
Figure 3A,C, compared with the control group, the treatment of H
2O
2 significantly increase the ROS levels (
p < 0.05). It is known that H
2O
2 can be readily transported through the lipid bilayer of a cell. This initiates the Fenton reaction with metal ions in the cell to form extremely toxic hydroxyl radicals and cause oxidative stress. Results showed that the addition of the RCAs decreased the fluorescence intensity levels of ROS when compared to the positive control sample. When compared to the negative control sample, the values for ROS production increased by about 34%, 19%, 10%, and 20% at the RCA concentrations of 0, 0.5, 1.0, and 1.5 mg/mL, respectively. The addition of all concentrations of the RCAs decreased the ROS values in HepG2 cells. RCAs treated at 0.5, 1.0, and 1.5 mg/mL inhibited about 11%, 18%, and 10% ROS compared to the positive control sample, respectively. RCAs powder produced the largest inhibition rate at the concentration of 1.0 mg/mL powder, which corresponds to 0.21 mg/mL of anthocyanins. The results clearly demonstrated the potential chemoprotective effect of RCAs against oxidative stress induced by H
2O
2 on human HepG2 cells.
These results were in line with previous reports that polyphenols could give protection against the increase of intracellular ROS and oxidative damage using different cellular models exposed to different oxidative agents [
26,
27,
28]. The intracellular ROS production was closely related to the state of the antioxidant defense systems of the cell. It has been demonstrated that the protective effects of anthocyanins go beyond their simple antioxidant effect, by activating certain molecular pathways related to the antioxidant response, e.g., increase in antioxidant enzyme activity and the protection of mitochondrial functionality [
29,
30]. Shih et al. [
29] found that anthocyanins could induce the activation of phase II enzymes through the antioxidant response element pathway against H
2O
2-induced oxidative stress and apoptosis. Lee et al. [
30] recently reported that berry anthocyanins were able to protect the lipopolysaccharide-stimulated RAW 264.7 macrophages against oxidative damage through the activation of the nuclear factor-erythroid 2-related factor 2 (Nrf2). Therefore, any model or the combined actions of an antioxidant mechanism for the RCAs are possible, e.g., by directly scavenging ROS, or by chelating metal ions, thus inhibiting Fenton reactions, or by enhancing the activity of genes involved in the expression of antioxidant enzymes. However, more detailed determinations including enzyme activities are needed. Overall, the study demonstrated that the RCA extracts obtained markedly reduced intracellular ROS production by H
2O
2 on HepG2 cells and consequently ameliorated cell apoptosis and improved viability.