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Yang, B. Flavonoids Composition in Astragalus membranaceus. Encyclopedia. Available online: (accessed on 06 December 2023).
Yang B. Flavonoids Composition in Astragalus membranaceus. Encyclopedia. Available at: Accessed December 06, 2023.
Yang, Bao. "Flavonoids Composition in Astragalus membranaceus" Encyclopedia, (accessed December 06, 2023).
Yang, B.(2021, December 09). Flavonoids Composition in Astragalus membranaceus. In Encyclopedia.
Yang, Bao. "Flavonoids Composition in Astragalus membranaceus." Encyclopedia. Web. 09 December, 2021.
Flavonoids Composition in Astragalus membranaceus

Astragalus membranaceus is a valuable medicinal plant species widely distributed in Asia. Its root is the main medicinal tissue rich in methoxylated flavonoids. Origin can highly influence the chemical composition and bioactivity.

antioxidant activity ORAC origin phenolics principal component analysis

1. Introduction

Astragalus membranaceus (Fabaceae) is a well-known traditional Chinese herbal medicine, which is mainly distributed in Asian regions [1]. The root of A. membranaceus is the medicinal tissue widely used due to their beneficial effects to lung health, which has been further proved to exhibit immunomodulatory, antioxidant, antiperspirant, antidiarrheal, and antidiabetic activities [2]. The bioactive compounds in the root of A. membranaceus are complicated. Previous research has revealed that the main bioactivities depend on the presence of non-volatile components, such as polysaccharides, saponins, and phenolics [3][4].
Flavonoids are a natural bioactive compound with C6-C3-C6 skeleton. They are commonly found in plant species, and possess good bioactivities which have been applied in nutraceutical, medicinal, and cosmetic products [5]. Many different subclasses of flavonoids have been described from A. membranaceus including flavone, flavonol, flavanone, flavanonol, chalcone, aurone, isoflavone, and pterocarpan [6]. Phenolics, including flavonoids, have been judged as the marker for quality evaluation and standardization of A. membranaceus and its processed products [7]. Methoxylated flavonoids and their glycosides, such as calycosin and calycosin-7-O-β-D-glucoside, were the major bioactive constituents of A. membranaceus due to their superior bioactivities [8][9]. After glycosylation and methylation, the reactivity and solubility of the flavonoids usually improved and thereby so did their absorption and bioactivity [10]. Many methoxyl flavones and flavonoid glycosides have been proved the superior antioxidant activities [11][12]. Due to the plant origin and extremely low toxicity, they have become the hotspots of natural antioxidants drug discovery and development [13][14].
The accumulation of bioactive compounds depends on geographical location and growing environment, including climate, soil, and fertilizer [15]. It is highly correlated to the health benefits and pharmaceutical activities. Different origins usually lead to different phytochemical profiles, which further relate to the quality of A. membranaceus. However, relevant information regarding this topic is limited. Therefore, in the present study, A. membranaceus from four production origins (Gansu, Shanxi, Inner Mongolia, and Heilongjiang provinces of China) were collected and the flavonoids were identified by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Afterward, the antioxidant activities were investigated by ORAC assay, DPPH radical scavenging activity assay and CAA assay. These results help to understand the qualities of A. membranaceus from different origins.

2. Total Phenolics and Flavonoids

The yield of A. membranaceus extract were 17.11% (Gansu), 16.84% (Shanxi), 14.84% (Heilongjiang), and 10.13% (Inner Mongolia), respectively. Total phenolics and flavonoids contents of A. membranaceus root from four origins are presented in Figure 1. The content of phenolics varied between 135.23–197.40 mg GAE/g extract and in a decreasing order of ‘Inner Mongolia’ (197.40 ± 1.95 mg GAE/g extract), ‘Heilongjiang’ (164.93 ± 2.14 mg GAE/g extract), ‘Shanxi’ (155.62 ± 0.95 mg GAE/g extract), and ‘Gansu’ (135.24 ± 1.66 mg GAE/g extract), respectively. Among them, the ‘Inner Mongolia’ and ‘Heilongjiang’ samples presented significantly (p < 0.05) higher levels of phenolics than the other two origins. Meanwhile, the total flavonoids contents of A. membranaceus were significantly different among the four origins. It ranged from 52.27 to 112.75 mg CE/g extract. Among them, the ‘Inner Mongolia’ samples (112.75 ± 0.77 mg CE/g extract) presented significantly higher (p < 0.05) flavonoids contents than the other three origins, followed by ‘Gansu’ (67.22 ± 3.67 mg CE/g extract) and ‘Shanxi’ (69.12 ± 6.59 mg CE/g extract) samples. Unlike the trend of phenolic compounds, a relatively low flavonoids content was observed in ‘Heilongjiang’ sample (52.27 ± 4.63 mg CE/g extract), which indicated the presence of non-flavonoids phenolics.
Figure 1. Total phenolics and total flavonoids contents of A. membranaceus roots from different origins (Gansu, Inner Mongolia, Shanxi, and Heilongjiang). (A), total phenolics contents; (B), total flavonoids contents. Values in one column with different letters are significantly different (p < 0.05).
It is widely known that a diet rich in fruit and vegetables has a protective effect against cancer insurgence and development. In the presence of an intense stressing event, cells activate specific responses to counteract cell death or senescence, which is known to act as a key task in the onset of age-related pathologies and the loss of tissue homeostasis [16]. Phenolics are generally recognized to be responsible for antioxidant and anti-aging effects, as well other beneficial actions [17]. Previous research showed that the total phenolic content of A. membranaceus from Western Siberia ranged from 100–190 mg GAE/g extract [18], which is consistent with this study. The total phenolics and flavonoids contents of A. membranaceus from Shijiazhuang origin were 27.646 ± 0.11 mg GAE/g extract and 7.048 ± 0.87 mg RE/g extract, respectively [19]. The difference of total phenolics and flavonoids contents distinguished the A. membranaceus materials from different origins clearly.

3. Identification of Bioactive Compounds

The UHPLC–MS/MS analysis of the methanolic extract was performed to characterize the bioactive compounds of A. membranaceus. The total ion chromatogram was presented in positive ion mode. After preliminary comparative analysis by retention time, MS/MS fragments, and the reported data in references or Compound Discoverer, 34 flavonoids were identified, including 13 methoxylated flavonoids, 15 flavonoid glycosides, and 6 flavonols. The compounds identified by UHPLC-MS/MS spectra are summarized in Table 1, and their putative fragmentation pathways are shown in Figure 2. The identification of methoxylated flavonoids and flavonoids glycosides were explained in detail as follows.
Figure 2. The putative fragmentation pathways of isoliquiritigenin (A), vesticarpan (B), and isomucronulatol 7-O-glucoside (C).
Table 1. The identified flavonoids compounds of Astragalus based on the UHPLC-ESI-MS/MS method.
NO. RT (Min) Chemicals [M+H]+ Formula MS2 Fragments (m/z)
1 8.64 Narcissin 625.172 C28H32O16 463.121
2 9.41 Nicotiflorin 595.161 C27H30O15 433.111, 271.059
3 9.58 Flagaloside D 581.196 C26H28O15 563.183, 419.143
4 10.58 Liquiritin 419.129 C21H22O9 257.071
5 10.77 Licoagroside D 449.140 C22H24O10 287.081
6 10.88 Calycosin 7-O-glucoside 447.124 C22H22O10 285.074, 270.051
7 10.98 Odoratin 7-O-glucoside 477.135 C23H24O11 315.085, 300.062
8 11.21 Apigenin 7-O-glucoside 433.108 C21H20O10 401.119, 271.059
9 11.67 Biochanin A 7-O-(6-O-malonyl-glucoside) 533.124 C25H24O13 489.137, 447.126, 285.075, 270.052
10 11.78 Pratensein 7-O-glucoside 463.119 C22H22O11 301.069, 286.050
11 12.54 Ononin 431.130 C22H22O9 269.079, 254.057
12 12.88 Methylinissolin 3-O-glucoside 463.155 C23H26O10 445.149,301.105, 165.054
13 13.06 Isomucronulatol 7-O-glucoside 465.171 C23H28O10 447.164, 303.122, 275.090
14 13.10 7-Hydroxy-2′-methoxy-4′,5′-methylenedioxyisoflavane 301.103 C17H16O5 286.083, 270.087, 123.044
15 13.10 Calycosin 7-O–{6″-[-but-2-enoyl]}-glucoside 515.150 C26H26O11 429.152, 285.075, 270.050
16 13.14 2′, 8-Dihydroxy-4′, 7-dimethoxyisoflavane 303.118 C17H18O5 275.091, 153.054, 123.044
17 13.47 Chrysin 331.078 C17H14O7 316.056, 137.029
18 13.60 Kaempferol 287.090 C15H10O6 271.054, 153.054, 137.023,
19 13.66 Calycosin 285.071 C16H12O5 270.051, 137.023
20 13.70 Methylinissolin 315.201 C18H18O5 300.062, 165.054
21 13.78 Odoratin 315.084 C17H14O6 287.089, 137.059
22 14.37 Vesticarpan 287.089 C16H14O5 272.068, 165.054, 123.044
23 14.52 Apigenin 271.059 C15H10O5 153.017, 137.019
24 14.81 Astragaluquinone 317.099 C17H16O6 302.073, 123.044
25 15.44 Isoliquiritigenin 257.079 C15H12O4 149.061, 137.023, 121.065
26 15.65 Pratensein 301.069 C16H12O6 286.046, 153.018
27 15.66 Pinostrobin 271.086 C16H14O4 256.062, 137.059
28 15.84 Daidzein 255.099 C15H10O4 137.059, 119.044
29 15.87 Formonentin 269.077 C16H12O4 254.056, 137.022
30 16.06 Garbanzol 273.183 C15H12O5 255.173, 137.059
31 16.09 7-Hydroxy-3′,5′-dimethoxyisoflavone 299.088 C17H14O5 284.067, 271.058, 137.058
32 16.65 Butein 273.183 C15H12O5 165.091, 137.059
33 17.81 3′,6-Dihydroxy-4′-methoxy-aurone 285.073 C16 H12 O5 285.075, 270.051, 121.027,
34 18.49 Astragaisoflavane D 603.218 C34H34O10 287.091, 272.068

3.1. Polymethoxylated Flavonoids

Polymethoxylated flavonoids are characteristic compounds distributed in Astragalus species. In positive ion mode, the main feature of the fragmentation of [M+H]+ ions of methoxylated flavonoids is the loss of methyl radical (form the fragment ions of [M+H-nCH3]+). Besides, neutral loss could be observed, such as 28 (CO) and 44 (CO2). The C ring of flavonoids was less stable and prone to be cleaved resulting in various retro-Diels Alder fragments [20]. The even-electron rule could be applied for the identification of methoxylated flavonoids [21]. For example, compound 29 with the retention time of 15.87 min was identified as formononetin due to the quasi-molecular ion peak at m/z 269.077 ([M+H]+). The characteristic MS/MS ion peak at m/z 254.056 ([M+H-CH3]+) was detected. A dominant fragment ion 1,3A+ at m/z 137.022 was presented due to the breakage of C ring. Similarly, vesticarpan (22) at the retention time of 14.37 min had a quasi-molecular ion at m/z 287.089 ([M+H]+). The fragment at 273.068 matched the loss of a methyl residue ([M+H-CH3]+) and yielded secondary fragment ions at m/z 165.054 (6,7A+) and 139.035 (1,3A+). Furthermore, the fragment ion at m/z 123.044 was observed due to the loss of 18 (H2O). The putative fragmentation pathway is shown in Figure 2. Methylinissolin (20) at the retention time of 13.70 min had a base peak at m/z 315.201 ([M+H]+). The typical fragment at m/z 300.062 was detected due to the loss of methyl radical and yielded secondary fragment ions 1,3A+ at m/z 123.044. The A-ring and/or B-ring were easy to produce fragment ions due to the neutral loss of CH4. The peaks with the retention times of 13.10, 13.14, 13.47, 13.66, 13.78, 14.81, 15.65, 15.66, and 16.09 min were assigned to be methoxylated flavonoids with [M+H]+ at m/z 301.103, 303.118, 331.078, 285.071, 315.084, 317.099, 301.069, 271.086, and 299.088, respectively. These fragments were characterized by the losses of methyl radicals and fragment 1,3A+ was the most abundant ion in all the spectra. These compounds were identified as 7-hydroxy-2′-methoxy-4′,5′-methylenedioxyisoflavane (14), 2′,8-dihydroxy-4′,7′-dimethoxyisoflavane (16), chrysin (17), calycosin (19), odoratin (21), astragaluquinone (24), pratensein (26), pinostrobin (27), and 7-hydroxy-3′,5′-dimethoxyisoflavone (31). Additionally, compounds 25 and 32 had the quasi-molecular ion peaks at m/z 257.079 and 273.183 were identified as isoliquiritigenin (25) and butein (32), two principle chalcones, which further formed secondary fragments at m/z 149.061, 137.023, 121.065, and 165.091, 137.059, consistent with literature [22]. These were the characteristic fragment ions produced by chalcone fragmentation pathway (Figure 2A).

3.2. Flavonoid Glycosides

Flavonoid glycosides, including flavonoid C-glycosides and O-glycosides, were two common patterns distributed in plants with multiple bioactivities [23]. In this study, 15 flavonoid O-glycosides were found in A. membranaceus, while 12 of them were identified to bound one or two methyls. After comparing the quasi-molecular ions and fragment ions in MS/MS spectra with those of previously reported literature [24][25], they were identified as narcissin (1), flagaloside D (3), licoagroside D (5), calycosin 7-O-glucopyranoside (6), odoratin 7-O-glucopyranoside (7), biochanin A 7-O-(6-O-malonyl-glucoside) (9), pratensein 7-O-glucopyranoside (10), ononin (11), methylinissolin 3-O-glucoside (12), isomucronulatol 7-O-glucoside (13), respectively. As reported previously, the application of low or medium fragmentation energy results in heterolytic cleavage of their hemiacetal C-O bonds [26]. Besides, the losses of 18 (H2O), 28 (CO), 42 (C2H2O), 44 (CO2) are also characteristic for flavonoid glycoside [27]. In the present work, flavonoid O-glycosides showed similar fragmentation pattern. For instance, compound 13 with the retention time of 13.06 min was identified as isomucronulatol 7-O-glucoside due to the quasi-molecular ion peak at m/z 465.17 ([M+H]+) and the characteristic MS/MS ion at m/z 447.16 ([M+H-H2O]+), and ion at m/z 303.12 ([M-C6H10O5]+) corresponding to the loss of a glucose moiety. The possible fragmentation pattern is proposed in Figure 2C. Furthermore, Compound 11, with parent ion at m/z 412.54, was identified as ononin (C22H22O9), which fragmented into daughter ion at m/z 269.08 due to the loss of a glucose moiety and further yielded the ion at m/z 254.06 ([M-C6H10O5-CH3]+), consistent with the reported data [28]. Compound 6, with the base peak at m/z 447.124 ([M+H]+), matched the ion [Y0]+ at m/z 285.074 with the loss of a glucose residue. The presence of fragment at m/z 270.051 was due to the loss of a methyl radical [Y0-CH3]+. Besides, methylinissolin 3-O-glucoside (12) at the retention time of 12.88 min had a quasi-molecular ion at m/z 463.155 ([M+H]+). The fragments at m/z 445.149 and 301.105 were attributed to [M+H-H2O]+ and [Y0+H]+, respectively. The ion 6,7A+at m/z 165.054 was the dominant fragment ion due to the breakage of C ring. Moreover, the compounds 24, and 8 with the retention times at 9.41, 10.58, and 11.21 min were identified as nicotiflorin, liquiritin, and apigenin 7-O-glucopyranoside with the same fragmentation pattern.

3.3. Multivariate Statistical Analysis

PCA analysis is an unsupervised method usually employed to determine patterns between multivariate samples. The PCA analysis showed a clear tendency of separation among A. membranaceus samples from four origins. Specifically, the first two principal components explained 77.9% of the total variance, in which the first principal component explained 51.2% and the second principal component explained 26.7%. The first principal component was represented by the compounds such as nicotiflorin, liquiritin, ononin, 7-hydroxy-2′-methoxy-4′,5′-methylenedioxyisoflavane, calycosin 7-O-{6″-[-but-2-enoyl]}-glucoside, methylinissolin, isoliquiritigenin, daidzein, and astragaisoflavane D. Most of them presented higher content in the ‘Inner Mongolia’ extract. The supervised discriminant OPLS-DA was performed to classify the samples from four regions and find out the differential compounds. A model with R2X of 0.997, R2Y of 0.998, and Q2 of 0.99 was constructed. R2Y > 0.9 indicated an excellent fitted model, and Q2 > 0.9 suggested a good repeatability and predictability of the model. As shown in Figure 3A, no serious outlier was observed. The outlier is the plot out of the ellipse, which is defined as the Hotelling’s T2 range 95% confidence [29]. The samples from different origins exhibited good separation. The ‘Heilongjiang’ sample and ‘Inner Mongolia’ sample were separated significantly in t [1] direction. ‘Gansu’ sample and ‘Shanxi’ sample located near the centre of the model plane and stayed close to each other, which indicated that their chemical compositions were similar. According to the searching rule of VIP value > 1 and p-value < 0.05, a total of 18 phenolics were regarded as principal metabolites marked in red in Figure 3B. Six methoxylated flavonoids (compounds 7-hydroxy-2′-methoxy-4′,5′-methylenedioxyisoflavane, 2′, 8-Dihydroxy-4′, 7-dimethoxyisoflavane, calycosin, vesticarpan, pratensein, 3′, 6-dihydroxy-4′-methoxy-aurone), eight flavonoid glycosides (compounds flagaloside D, licoagroside D, calycosin 7-O-glucoside, biochanin A 7-O-(6-O-malonyl-glucoside), methylinissolin 3-O-glucoside, calycosin 7-O-{6″-[-but-2-enoyl]}-glucoside, nicotiflorin, apigenin 7-O-glucoside, while the first six of them having one or two methyl groups), and four flavonols (apigenin, isoliquiritigenin, daidzein, butein) were included. Different geographical locations led to the variation of metabolites accumulation.
Figure 3. Multivariate statistical analysis of flavonoids in A. membranaceus from Gansu (GS), Heilongjiang (HLJ), Shanxi (SX), and Inner Mongolia (IMG): (A) OPLS-DA score plot; (B) OPLS-DA loading plot. The important compounds are in red colour. (C) Heatmap of phenolic components in astragalus from different origins. The levels of identified compounds were compared by their peak integration.
The heat map was applied to demonstrate the variation of the identified compounds. As shown in Figure 3C, ‘Inner Mongolia’ extract was rich in flavonoid glycosides and methoxylated flavonoids. Most principal flavonoids including flagaloside D, licoagroside D, calycosin 7-O-glucoside, calycosin 7-O-{6″-[-but-2-enoyl]}-glucoside, apigenin 7-O-glucoside, 7-hydroxy-2′-methoxy-4′,5′-methylenedioxyisoflavane, 2′,8-dihydroxy-4′,7-dimethoxyisoflavane, calycosin, and pratensein all presented high levels in ‘Inner Mongolia’ samples than others. Besides, the principal methoxylated flavonoids such as vesticarpan and 6,3′-dihydroxy-4′-methoxy-auron were higher in the extract of ‘Gansu’ and ‘Heilongjiang’ samples than ‘Shanxi’ samples. Conversely, two principal flavonols (isoliquiritigenin and daidzein), only existed in ‘Shanxi’ sample. It could be used to distinguish ‘Gansu’ and ‘Shanxi’ samples. To compare the chemicals of ‘Heilongjiang’ with the other origins, the flavonoid glycosides and methoxylated flavonoids, such as flagaloside D, calycosin 7-O-glucopyranoside, apigenin 7-O-glucopyranoside, 7-hydroxy-2′-methoxy-4′,5′-methylenedioxyisoflavane, and 8,2′-dihydroxy-7,4′-dimethoxyisoflavane, were presented at relatively lower contents, which were defined as the characteristic compounds of the ‘Heilongjiang’ sample.

4. Antioxidant Activity

4.1. ORAC Value and DPPH Radical Scavenging Activity

ORAC assay is a valid method to evaluate the antioxidant ability through monitoring the inhibition capacity against peroxyl radical [30]. The breakdown of ABAP can provide peroxyl radical and leads to subsequent oxidation. It can be monitored through fluorescent intensity change. As shown in Figure 4A, ‘Inner Mongolia’ (628.94 ± 3.30 μmol TE/g extract) showed significant (p < 0.05) higher ORAC values. The ORAC values of other three samples were in a decreasing order of ‘Gansu’ (553.18 ± 15.28 μmol TE/g extract), ‘Shanxi’ (522.48 ± 21.91 μmol TE/g extract), and ‘Heilongjiang’ (471.29 ± 8.61 μmol TE/g extract). It indicated that ‘Inner Mongolia’ and ‘Gansu’ extracts processed good peroxyl radical inhibition activities.
Figure 4. Antioxidant activities of Astragalus extracts from different origins. (A), ORAC value; (B), DPPH radical scavenging activity; (C), Cytotoxicity; (D), Cellular antioxidant activity. Different letters indicate values have significant differences (p < 0.05).
DPPH test is widely used to evaluate the antioxidant capacity of phenolics [31]. It is based on a stable nitrogen-centred free radical that is characterized by absorbance at 517 nm with a deep violet colour. In the presence of free radical scavenger, the absorbance of DPPH will decrease due to hydrogen donation from antioxidant with a dose-dependent behaviour [32]. As shown in Figure 4B, ‘Heilongjiang’ extract presented a higher DPPH radical scavenging activity with an IC50 value of 8.10 ± 0.54 μmol AA/g extract, followed by ‘Shanxi’ extract (IC50 of 6.63 ± 0.30 μmol AA/g extract) and ‘Inner Mongolia’ extract (IC50 of 6.57 ± 0.40 μmol AA/g extract). The ‘Gansu’ extract possessed the lowest IC50 value of 5.89 ± 0.36 μmol AA/g extract. This order was inconsistent with that of ORAC assay.
The occurrence of oxidation process is correlated with the existence of a surplus of free radicals, which are responsible for multiple diseases [33]. Peroxyl radicals are characterized as free radicals that predominate in the lipid oxidation of biological system [34]. Its inhibition plays an important role in disease prevention. Generally, the radical-trapping antioxidant activity of flavonoids is related to the hydrogen atom transfer to a peroxyl radical [35]. It can be used to explain the different radical-inhibitory activities of four origins samples. A. membranaceus from Lebanon possesses the lowest IC50 value of 102 ± 4.4 μg/mL [36]. Several bioactive compounds have been confirmed. For example, formononetin, calycosin, and calycosin-7-O-glucoside showed superior antioxidant activity and inhibited free radicals generated by DPPH in a dose-dependent manner [37]. Moreover, calycosin can enhance antioxidant enzymatic activities such as glutathione peroxidase, catalase, superoxide dismutase and attenuate H2O2-induced H9C2 cell apoptosis rate in a dose-dependent manner as well [38]. Besides, the chemicals of formononetin from A. membranaceus have evidenced the capacity of inhibiting xanthine oxidase-induced cell injury significantly [39]. They were the major flavonoids in A. membranaceus.

4.2. Cellular Antioxidant Activity (CAA)

HepG2 cells line is a sensitive cell model in the determination of antioxidant biomarkers [40]. In the present work, the cytotoxic effects of A. membranaceus extract at different levels (25, 50, 100, and 200 μg/mL) against HepG2 cells were determined by MTS assay. From the results summarized in Figure 4C, no significant cytotoxicities were observed between extract-treated cells and untreated cells within concentration of 0–50 μg/mL. It indicated that this range could be used for cellular antioxidant activity assay.
The cellular antioxidant activities of A. membranaceus extracts were evaluated by CAA assay, and the results are shown in Figure 4D. It was obvious that the A. membranaceus extracts from four origins could protect HepG2 cells against peroxyl radicals with a dose-dependent effect. Among them, ‘Inner Mongolia’ sample showed a higher CAA value than the others at 3.125 μg/mL, while ‘Gansu’ and ‘Shanxi’ extracts exhibited higher CAA values at 25 μg/mL. Trolox was used as positive control.
CAA assay is performed based on polarity, solubility, and molecular weight of the antioxidant and provides an important tool for the biological assessment of antioxidant activity [41]. In the CAA assay, the DCFH-DA is preloaded into the cell, treated with the intracellular peroxyl radicals generated from ABAP and therefore the fluorescence level is recorded [42]. Nuclear transcription factor, erythroid 2-like 2 (Nrf2) is a central conditioner of antioxidant response elements [43]. Phenolic compounds can protect HepG2 cell against oxidative injury by promoting the Nrf2 translocation, which subsequently attenuates oxidative DNA damage, induce the expression of antioxidant enzymes, and reduce cellular ROS formulation [44][45][46]. The antioxidant activities of phenolics usually depend on the chemical construction of attached functional groups and their permutation. Previous research has mentioned that methoxyl and hydroxyl groups are directly related to the radical-inhibited ability [47]. When the same skeleton was presented, the presence of methoxyl usually brings an enhanced antioxidant activity for phenolics. However, sometimes this substitution by methoxyl can diminish the antioxidant activity [48]. The substitution pattern on the B-ring is important to the antioxidant activity of flavonoids.


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