3.1. Phenolic Composition
Figure 1 shows the chromatography of phenolic compounds of standards and representative “Hangbaiju” samples of ‘DJ2′ and ‘TJ4′. Phenolic acids and flavonoids are two groups of active substances in ‘Hangbaiju’. The contents of individual phenolic compounds and the total mono-caffeoylquinic acid contents (TMAC, the sum of 3-
O-caffeoylquinic acid, 5-
O-caffeoylquinic acid, and 4-
O-caffeoylquinic acid), along with the total di-caffeoylquinic acid contents (TDAC, the sum of 3,5-di-
O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and 3,4-di-
O-caffeoylquinic acid), the total phenolic acid contents (TPAC, the sum of 3-
O-caffeoylquinic acid, 5-
O-caffeoylquinic acid, 4-
O-caffeoylquinic acid, caffeic acid, 3,4-di-
O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, 3,5-di-
O-caffeoylquinic acid) as well as the total flavonoids contents (TFC, the sum of hyperoside, luteoloside, apigenin-7-
O-glucoside, linarin, luteolin, apigenin and acacetin) and three key phenolic components (TKPC, the sum of 5-
O-caffeoylquinic acid, 3,5-di-
O-caffeoylquinic acid and luteoloside), in these samples, are listed in
Table 1 and
Table 2.
The TMAC was found to be in the range of 2552.04 to 7402.83 µg/g DW in 15 ‘Hangbaiju’ samples, in which the ‘Duoju’ samples had the content between 2552.04 and 4352.29 µg/g DW and the ‘Taiju’ samples between 2673.11 and 7402.83 µg/g DW. In comparison of the TMAC among ‘Duoju’, ‘DJ6′ was higher than others (
P < 0.05). The contents of 3-
O-caffeoylquinic acid, 5-
O-caffeoylquinic acid and 4-
O-caffeoylquinic acid in the ‘DJ6′ were found to be 1167.59 µg/g DW, 2392.17 µg/g DW and 792.53 µg/g DW, respectively, which were also significantly higher than the other ‘Duoju’ (
P < 0.05). It is worth noting that 5-
O-caffeoylquinic acid was the dominant mono-caffeoylquinic acid in the ‘Duoju’ samples. These results were consistent with a previous report [
13]. Among the ‘Taiju’ samples, ‘TJ3′ had the highest TMAC (
P < 0.05), with 2844.64 µg/g DW of 3-
O-caffeoylquinic acid, 3139.04 µg/g DW of 5-
O-caffeoylquinic acid and 1365.16 µg/g DW of 4-
O-caffeoylquinic acid, respectively. 5-
O-Caffeoylquinic acid was also the predominant individual mono-caffeoylquinic acid in the ‘Taiju’ samples. Different to ‘Duoju’, the 3-
O-caffeoylquinic acid content in ‘Taiju’ was much higher than 4-
O-caffeoylquinic acid (
Table 1 and
Table 2).
The TDAC in the 6 ‘Duoju’ samples were between 8153.62 µg/g DW and 10,974.94 µg/g DW, and in the 9 ‘Taiju’ samples between 7718.79 to 13,960.39 µg/g DW (
Table 1 and
Table 2). It was observed that 7 out of 9 ‘Taiju’ samples had higher TDAC than the highest content ‘Duoju’ (‘DJ6′, 10,974.94 µg/g DW) sample. Among the 6 ‘Duoju’ samples, the content of 3,4-di-
O-caffeoylquinic acid in the ‘DJ6′ were significantly higher than the rest, whereas the content of 3,5-di-
O-caffeoylquinic acid in the ‘DJ2′ and ‘DJ6′, and content of 4,5-di-
O-caffeoylquinic acid in the ‘DJ5′and ‘DJ6′ were significantly higher than the others (
Table 1). In both ‘Duoju’ and ‘Taiju’ samples, 3,5-di-
O-caffeoylquinic acid was the dominant individual di-caffeoylquinic acid, followed by 4,5-di-
O-caffeoylquinic acid and then 3,4-di-
O-caffeoylquinic acid. ‘TJ5′ contained the highest 3,5-di-
O-caffeoylquinic acid and 4,5-di-
O-caffeoylquinic acid among the tested 15 ‘Hangbaiju’ samples. Regarding caffeic acid content in the ‘Taiju’ samples, ‘TJ3′ possessed the highest content (23.03 µg/g DW), but it was not detected in ‘TJ1′ (
Table 2). Among the ‘Duoju’ samples, the highest caffeic acid content was found in the ‘DJ1′ (10.85 µg/g DW), but it was not detected in ‘DJ4′ and ‘DJ6′ (
Table 1).
The TPAC in ‘Taiju’ was ranged between 10,405.31 µg/g DW and 20,847.76 µg/g DW, and in ‘Duoju’ between 10,709.58 µg/g DW and 15,327.22 µg/g DW, respectively (
Table 1 and
Table 2). It should be noted that the highest and lowest TPAC was all found in ‘Taiju’ samples, and 7 out of 9 ‘Taiju’ samples had higher TPAC than the highest TPAC ‘Duoju’ (DJ6, 15,327.22 µg/g DW) sample. Among the 6 ‘Duoju’ samples, ‘DJ2′ showed significantly higher TPAC than ‘DJ3′ and ‘DJ4′ (
P < 0.05), but no significant differences were observed between ‘DJ2′ with the rest of the ‘Duoju’ samples. Among the ‘Taiju’ samples, ‘TJ3′ had significantly higher TPAC than all of the others (
P < 0.05).
In terms of flavonoids, these ‘Hangbaiju’ samples contained hyperoside, luteoloside, apigenin-7-
O-glucoside, linarin, luteolin, apigenin and acacetin (
Table 1 and
Table 2). The TFC was ranged from 5183.35 to 9792.01 µg/g DW, and ‘Taiju’ had higher TFC than that of ‘Duoju’. Regarding the individual flavonoids, all samples contained hyperoside, luteoloside, apigenin-7-O-glucoside, linarin and luteolin. Specifically, ‘DJ5′ had the highest content of hyperoside (813.68 µg/g DW) and linarin (2605.87 µg/g DW). Both ‘DJ5′ and ‘DJ2′ showed similar content on luteolin (247.09 µg/g DW and 238.37 µg/g DW, respectively), and ‘DJ3′ and ‘DJ4′ contained similar amount of luteoloside (1524.16 µg/g DW and 1476.67 µg/g DW, respectively). ‘DJ4′ showed the highest content of apigenin-7-
O-glucoside among the ‘Duoju’ (
P < 0.05). The contents of luteoloside and linarin in all most ‘Taiju’ were higher than most ‘Duoju’ samples, whereas the contents of luteolin in all ‘Taiju’ were less than all ‘Duoju’ samples (
Table 1 and
Table 2). ‘TJ3′ showed the highest hyperoside content (994.81 µg/g DW), and also showed significant higher luteolin content than other ‘Taiju’ (
P < 0.05). ‘TJ9′ contained the highest content of luteoloside and apigenin-7-
O-glucoside (2416.07 µg/g DW and 3539.55 µg/g DW), whereas the highest content of linarin was found in ‘TJ6′ (3573.51 µg/g DW) and ‘TJ3′ (3470.66 µg/g DW). It should be noted that apigenin was only present in 3 out of 6 ‘Duoju’ samples and 7 out of 9 ‘Taiju’ samples. All ‘Taiju’ did not contain acacetin, which was only found in ‘Duoju’, and ‘DJ6′ possessed the highest content (29.48 µg/g DW).
It has been reported that 5-
O-caffeoylquinic acid, 3,5-di-
O-caffeoylquinic acid and luteoloside were the most crucial phenolic components in the chrysanthemum that influence the antioxidant and medicinal properties [
7,
13]. Therefore, it is important to compare the total content of these three key phenolic components. Among these 15 ‘Hangbaiju’ samples, the TKPC was ranged from 7591.50 µg/g DW to 13,144.12 µg/g DW. In the ‘Duoju’ samples, ‘DJ6′ and ‘DJ2′ contained the highest TKPC. It was observed that 7 ‘Taiju’ samples (TJ1, TJ3, TJ4, TJ5, TJ6, TJ8 and TJ9) had higher TKPC than any of the ‘Duoju’ samples. It should be noticed that ‘TJ3′ and ‘TJ5′ had similar TKPC values and were significantly higher than the other ‘Taiju’ samples (
P < 0.05).
3.2. Antioxidant Activity
DPPH, ABTS and FRAP assays are often used simultaneously to evaluate the in vitro antioxidant activities of extracts or active substances [
20,
21,
22].
Table 3 and
Table 4 show the antioxidant activity of these ‘Hangbaiju’ samples. In the DPPH assay, antioxidants work as hydrogen donors to react with stable DPPH free radical causing its discoloration [
23,
24]. The EC
50 of DPPH of these samples ranged from 1.69 mg/L to 3.04 mg/L. ‘DJ2′ exhibited the highest DPPH scavenging activity among the ‘Duoju’ (
P < 0.05) samples, whereas ‘TJ6′, ‘TJ4′, ‘TJ1′ and ‘TJ8′ had the strongest DPPH scavenging capacity among ‘Taiju’ samples. In the ABTS assay, the stable-colored ABTS radicals are interact with antioxidants and this results in color loss [
14,
24]. In the present study, the ‘Duoju’ samples had the ABTS quenching EC
50 value of 2.13 mg/mL to 2.83 mg/mL, and between 1.82 mg/mL to 2.42 mg/mL for ‘Taiju samples’. The highest ABTS quenching activity of ‘Taiju’ samples was found in ‘TJ4′, whereas ‘TJ1′, ‘TJ5′, ‘TJ6′ and ‘TJ8′ possessed similar ABTS radicals scavenging capacity. FRAP assay is used to estimate the antioxidant activity of a compound through its capacity of reducing ferric into ferrous ions [
14]. In this study, the ‘Hangbaiju’ samples had the FRAP value of 222.28 to 436.51 mg TEAC/g DW, and generally the ‘Taiju’ samples exhibited a higher reduction capacity than did the ‘Duoju’ samples. Among these ‘Duoju’ samples, ‘DJ1′ and ‘DJ6′ had the lowest and highest FRAP reduction capacity, respectively. The highest FRAP reduction ability among ‘Taiju’ was ‘TJ8′ (436.51 mg TEAC/g DW), and the ‘TJ9′ showed the lowest FRAP value.
It has been reported that more hydroxyl groups in the flavonoid molecular structure could enhance the antioxidant capacity [
25]. Additionally, the acylation of caffeoyl group could improve the complexity of the caffeoylquinic acid, resulting in a stronger capacity of scavenging free radicals [
26]. The antioxidant activities of individual phenolic components from these ‘Hangbaiju’ samples were also evaluated (
Table 5). Hyperoside, luteolin, 4,5-di-
O-caffeoylquinic acid, luteoloside, 3,4-di-
O-caffeoylquinic acid, 3,5-di-
O-caffeoylquinic acid, caffeic acid and 5-
O-caffeoylquinic acid show similar DPPH radical scavenge capacity, but they are higher than that of 4-
O-caffeoylquinic acid, 3-
O-caffeoylquinic acid, apigenin, apigenin-7-
O-glucoside and acacetin. However, the DPPH radical scavenging capacity of linarin was not detected.
In terms of ABTS radical scavenging capacity, 4,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, luteolin, 5-O-caffeoylquinic acid, caffeic acid, 4-O-caffeoylquinic acid, hyperoside and luteoloside show similar values, but their ABTS radical scavenging activity was greater than that of 3-O-caffeoylquinic acid, apigenin, apigenin-7-O-glucoside, acacetin and linarin. The strongest FRAP reduction activity was found to be 5-O-caffeoylqunic acid, which was much higher than other phenolic compounds. Additionally, 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid also show great FRAP reduction ability, followed by 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid and caffeic acid. Flavonoids used in the present study show relatively weak FRAP reduction activity, which is in the order of hyperoside > luteolin > luteoloside > apigenin > apigenin-7-O-glucoside > acacetin > linarin.
It has been reported that phenolic compounds play a vital role to the antioxidant activity in
C. morifolium [
3]. A correlation study was conducted between the phenolic compounds and the antioxidant capacities of these ‘Hangbaiju’ samples (
Table 6). It was observed that phenolic compounds in both ‘Duoju’ and ‘Taiju’ exhibit a good correlation with their antioxidant activities. For example, a positive correlation was established between 5-
O-caffeoylquinic acid, TPAC and TKPC with the DPPH and ABTS quenching activity in the ‘Duoju’. However, such a correlation was weak for ‘Taiju’. In the FRAP assay system, TMAC and 5-
O-caffeoylquinic acid showed a weak correlation with the FRAP reducing capacity. This indicates that phenolic acids including 5-
O-caffeoylquinic acid, 3,5-di-
O-caffeoylquinic acid and luteoloside might be the key compounds that contribute to the antioxidant activity of these ‘Hangbaiju’ samples.