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 [26]. The even-electron rule could be applied for the identification of methoxylated flavonoids [27]. 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 [28]. These were the characteristic fragment ions produced by chalcone fragmentation pathway (Figure 2A).

Flavonoid glycosides, including flavonoid C-glycosides and O-glycosides, were two common patterns distributed in plants with multiple bioactivities [29]. 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 [30,31], 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 [32]. Besides, the losses of 18 (H₂O), 28 (CO), 42 (C₂H₂O), 44 (CO₂) are also characteristic for flavonoid glycoside [33]. 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 [34]. 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 2, 4, 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.

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 [35]. 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.

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.

ORAC assay is a valid method to evaluate the antioxidant ability through monitoring the inhibition capacity against peroxyl radical [36]. 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.

DPPH test is widely used to evaluate the antioxidant capacity of phenolics [37]. 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 [38]. 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 [39]. Peroxyl radicals are characterized as free radicals that predominate in the lipid oxidation of biological system [40]. 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 [41]. 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 [42]. 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 [43]. 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 [44]. Besides, the chemicals of formononetin from A. membranaceus have evidenced the capacity of inhibiting xanthine oxidase-induced cell injury significantly [45]. They were the major flavonoids in A. membranaceus.

HepG2 cells line is a sensitive cell model in the determination of antioxidant biomarkers [46]. 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 [47]. 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 [48]. Nuclear transcription factor, erythroid 2-like 2 (Nrf2) is a central conditioner of antioxidant response elements [49]. 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 [50,51,52]. 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 [53]. 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 [54]. The substitution pattern on the B-ring is important to the antioxidant activity of flavonoids.