Structural Characterization and Evaluation of the Antioxidant Activity of Phenolic Compounds from Astragalus taipaishanensis and Their Structure-Activity Relationship

Eight phenolic compounds were isolated using bio-guided isolation and purified from the roots of Astragalus taipaishanensis Y. C. Ho et S. B. Ho (A. taipaishanensis) for the first time. Their structures were elucidated by ESI-MS, HR-ESI-MS, 1D-NMR and 2D-NMR as 7,2′-dihydroxy-3′,4′-dimethoxy isoflavan (1), formononetin (2), isoliquiritigenin (3), quercetin (4), kaempferol (5), ononin (6), p-hydroxybenzoic acid (7) and vanillic acid (8). Six flavonoids (compounds 1-6) exhibited stronger antioxidant activities (determined by DPPH, ABTS, FRAP and lipid peroxidation inhibition assays) than those of BHA and TBHQ and also demonstrated noticeable protective effects (particularly quercetin and kaempferol) on Escherichia coli under oxidative stress. Additionally, the chemical constituents compared with those of Astragalus membranaceus and the structure-activity relationship of the isolated compounds were both analyzed. The results clearly demonstrated that A. taipaishanensis has the potential to be selected as an alternative medicinal and food plant that can be utilized in health food products, functional tea and pharmaceutical products.


TPC, TFC and antioxidant activities of extracts and isolated fractions. The TPC and TFC
of the ethanolic extracts (EE) and 4 isolated fractions (PEF, EAF, BF, and WF) of A. taipaishanensis were analyzed ( Table 1). The results demonstrated that the EAF had the highest content of TPC (46.41 ± 0.34 mmol equiv. GAE/100 g) and TFC (185.45 ± 1.04 mmol equiv. QUE/100 g). To identify the fraction with the greatest bioactivity, four in vitro antioxidant activities were observed in each fraction and the most bioactive fraction was then selected for further isolation and purification. The results are displayed in Table 1. For the ABTS •+ and FRAP assay, the EAF had the highest ABTS scavenging activity and reducing ability with values of 994.50 ± 4.21 μ mol trolox/g and 685.67 ± 3.21 μ mol trolox/g, respectively. For the DPPH and lipid peroxidation assays, the IC 50 values of DPPH and lipid peroxidation ranged from 0.059 ± 0.002 mg/mL (EAF) to 0.99 ± 0.004 mg/mL (WF) and 1.95 ± 0.009 mg/mL (EAF) to 6.48 ± 0.039 mg/mL (WF), respectively. In conclusion, the EAF was determined to be the fraction with the greatest antioxidant activity; therefore, later isolation and purification efforts were focused on the EAF. Structure elucidation of isolated compounds. Eight compounds were isolated and identified from the roots of A. taipaishanensis. The NMR data of the compounds are shown in Tables 2 and 3. The structures of compounds 1-8 and the key HMBC correlation of compounds 1 and 2 are illustrated in Figs 1 and 2.
Compound 2 was obtained as white needle crystal. Its molecular formula was established as C 16   based on the ESI-MS and NMR. Compared with compound 2, it had the same isoflavone skeleton. Additionally, the mass spectra and NMR spectra showed signals corresponding to a single sugar unit. The 1 H-NMR spectrum of compound 6 displayed strong signals indicating anomeric protons at δ 5.16 (1H, d, J = 4.8 Hz). The sugar unit was examined by TLC analysis. Acid hydrolysis of compound 6 resulted in a product of D-glucose, which was identified by a comparison with an authentic sample. Based on the coupling constant J = 4.8 Hz greater than 4.0 Hz, the sugar configurations were identified as β -D-glucose. In addition, there was a remarkable upfield shift (Δ δ 1.14) for the C-7 of compound 6 (161.91) compared to compound 2 (163.05), indicating that the sugar unit was attached to C-7. After comparison with published literature data, compound 6 was identified as ononin 24 .
Compound 3 was isolated as a yellow amorphous powder. ESI-MS: m/z 255.49 [M-H] − . The 13 C-NMR and DEPT spectra exhibited signals for nine CH (seven aromatic, two alkene) and six quaternary carbons (one carbonyl, three O-bearing and two aliphatic), respectively. Its molecular formula was determined to    . The 13 C-NMR and DEPT spectra displayed signals for six CH (all aromatic) and nine quaternary carbons (one carbonyl, four O-bearing, four aliphatic). Its molecular formula was determined to be C 15 H 10 O 5 based on the ESI-MS and NMR spectra. Compound 5 was found to have the same skeleton as compound 4. In the 1 H-NMR spectrum, the AA′ BB′ spin-coupling system with signals at δ 6.92 (2H, d, J = 8.5 Hz) and δ 8.04 (1H, d, J = 8.5 Hz) was ascribed to the four protons of the B-ring. The signals at δ 6.19 (1H, d, J = 2.0 Hz) and δ 6.45 (1H, d, J = 2.0 Hz) were meta-substituted protons of the A-ring. When compared with published literature data, compound 5 was identified as kaempferol 26 .
Compound 7 and compound 8 were identified by TLC analysis and a comparison with an authentic sample. Compound 7 was identified as p-hydroxy benzoic acid, and compound 8 was identified as vanillic acid 27 .
To the best of our knowledge, this is the first report of the characterization of phenolic compounds 1-8 isolated from A. taipaishanensis; furthermore, the 2 D-NMR data of compounds 1 and 2 were presented for the first time in this study.
In vitro antioxidant assays of isolated compounds. Phenolic compounds possess biologically beneficial activities and are frequently utilized as antioxidant, antiallergic and antidiabetic reagents 28 . In our study, the compounds 1-6 were tested for antioxidant by using four in vitro antioxidant assays and protective effect on E. coli under peroxide stress. The results showed in the Table 4. For the ABTS assay, all the tested compounds showed strong antioxidant capacity compared to BHA and TBHQ. In particular, compounds 4 and 5 had comparative higher antioxidant capacity with ABTS values over 30 mmol trolox/g. FRAP assay, compounds 4, 5 and 1 had higher antioxidant activity compared to BHA (5.83 ± 0.08 mmol trolox/g) and TBHQ (6.57 ± 0.13 mmol trolox/g), and the values were 14.18 ± 0.13, 14.13 ± 0.16 and 10.77 ± 0.11 mmol trolox/g, respectively. The results of DPPH assay demonstrated that all the tested compounds had stronger DPPH radical-scavenging ability than the standards (BHA and TBHQ). Compounds 4, 5, 1 and 3 in particular had relatively higher antioxidant activity, in which the IC 50 values were lower than 1 μ M. The lipid peroxidation assay was carried out using egg yolk as a lipid-rich medium. For this assay, all the tested compounds exhibited a particular capacity for lipid peroxidation inhibition. In particular, compounds 4, 5 and 1 had comparatively higher lipid peroxidation potential with IC 50 values 1.65 ± 0.08, 1.30 ± 0.09 and 1.87 ± 0.07 μ M, respectively, all of which were lower than 2 μ M.
Protective effect on E. coli under peroxide stress. Six of the isolated compounds were tested on E. coli growth under peroxide stress. We evaluated and compared the specific growth rate (μ ) of E. coli cells by pretreating them with the 6 compounds before subjecting them to oxidative stress damage caused by H 2 O 2 for 30 min. The absorbance (OD 600 ) and μ values are displayed in Figs 3 and 4, respectively. The higher values of the μ and the OD 600 were indicative of a higher antioxidant potential for the compounds. The results demonstrated that all the compounds had the ability to increase the cell growth rate under oxidative stress by 3.86-to 5.56-fold compared to untreated cells subjected to oxidative stress for 30 min. The compound with the highest activity was compound 4, which resulted in a growth rate of 5.56, followed by compound 3 and compound 1, which resulted in growth rates of 4.14 and 4.09, respectively.

Discussion
A. membranaceus and A. membranaceus var. mongholicus are mainly grown in North, Northeast and Northwest China and have been widely used for 2000 years 29 . Flavonoids, triterpene saponins and polysaccharides are the main constituents of this plant. Currently, almost 30 flavonoids have been isolated from A. membranaceus 30,31 , most of them being isoflavones, isoflavans and flavonols. Isoflavones in particular are major active ingredients in A. membranaceus 32 and were selected as marker compounds to evaluate the quality of A. membranaceus. Due to its known multiple health benefits, the natural sources of A. membranaceus are diminishing yearly 33 . In an attempt to address this shortage by exploring alternative plant options, we carried out a study using bio-guided isolation to isolate 8 phenolic compounds from the roots of A. taipaishanensis for the first time, examining and evaluating their bioactivities by in vitro radical scavenging, ferric reducing power and lipid peroxidation in addition to assessing their protective ability on E. coli cells subjected to oxidative stress damage caused by H 2 O 2 .
By comparing the results of four in vitro antioxidant assays, it was evident that quercetin and kaempferol demonstrated comparatively higher levels of antioxidant activity in the assays, whereas the formononetin and ononin had the lowest antioxidant potential, and the 7, 2′ -dihydroxy-3′ ,4′ -dimethoxy isoflavan and isoliquiritigenin had moderate antioxidant capacity. Compared with BHA and TBHQ, all the tested compounds demonstrated higher radical scavenging abilities against the ABTS and DPPH assays, whereas for the lipid peroxidation assay, all the compounds had lower activity than BHA and TBHQ. This result demonstrated that all the compounds isolated from the A. taipaishanensis had good radical scavenging activity. In particular, the results of the antioxidant assays indicated that all the tested compounds had certain antioxidant properties although their activity levels were significantly different. Among the 6 compounds that were tested, we found that the compounds with a flavonol skeleton, such as quercetin (4) and kaempferol (5), always had the highest antioxidant activities. In contrast, 7, 2′ -dihydroxy-3′ ,4′ -dimethoxy isoflavan (1) had a slightly lower antioxidant activity than flavonol followed by chalcone (isoliquiritigenin, 3) and the isoflavone (formononetin, 2), and isoflavone glucoside (ononin, 6) had the lowest antioxidant capacities. These results were consistent with previous reports stating that the number and position of hydroxyl groups and the different flavonoid skeletons would largely influence the radical scavenging ability. Previous reports stated that the 3-OH group would significantly increase the radical scavenging power 34,35 , and among all the tested compounds, only quercetin and kaempferol had the 3-OH group, both of which were found to have the highest anti-radical activities. In contrast, the formononetin and ononin, which had no 3-OH group, had the lowest radical scavenging power 36 ; moreover, the presence of ortho-dihydroxy groups on the B-ring or A-ring could have also enhanced the radical scavenging potential, which is a possible explanation as to why quercetin had slightly higher antioxidant activity in comparison to kaempferol. In addition, the presence of an α, β-double bond in isoliquiritigenin made its anti-radical potential evident 36,37 .
In conclusion, the present study, to the best of our knowledge, was the first time a comprehensive study was carried out on the phytochemicals and antioxidant capacity of the A. taipaishanensis. We isolated 6 flavonoid compounds and 2 phenolic compounds from A. taipaishanensis, all of which were isolated for the first time from this species. Additionally, we found that the main chemical components and the bioactivity of A. taipaishanensis were similar to those found in A. membranaceus, which is recorded in the pharmacopoeia as a medicinal and food plant. The antioxidant activities and structure-activity relationships of these isolated compounds were also evaluated, and the results revealed that all the tested compounds exhibited noticeable antioxidant activity compared to BHA and TBHQ. These findings demonstrate the potential of A. taipaishanensis to serve as a valuable natural resource in the medicinal and food industry and its ability to be used as a possible alternative in response to the diminishing supply of A. membranaceus.

Methods
General. ESI  HPLC with a Zobax Eclipse Plus C 18 column (4.6 × 250 mm, 5 μ m, Agilent). 1 H-and 13 C-spectra were measured with a Bruker AVANCE III spectrometer (500 MHz). Thin layer silica gel and column gel were both purchased from Qingdao Ocean Company (China). Sephadex LH-20 was purchased from GE Healthcare Bioscience AB (Sweden).

Plant material.
Five-year-old roots of A. taipaishanensis were collected on July 2013 at the Taibai Mountains of Shaanxi province, China. The specimen was kept in the herbarium of Northwest A&F University, Yangling, China.
Extraction and isolation. The dried roots of A. taipaishanensis (3.0 kg) were extracted five times each with 95% ethanol (10 L) for 24 h at room temperature. The ethanol extracts (EE, 488 g) were evaporated in vacuum at 45 °C and dried to solid at 35 °C. The EE (420 g) was further partitioned with petroleum ether-water, ethyl acetate-water and n-BuOH-water to obtain the 4 fractions: petroleum ether fraction (PEF, 24 g), ethyl acetate fraction (EAF, 18 g), n-butanol fraction (BF, 40 g) and water fraction (WF, 374 g), respectively. The yield of EE and the four fractions was 16.27%, 5.71%, 4.29%, 9.52% and 80.48%, respectively.
The EAF (18 g) was purified using a silica gel column (200-300 mesh, 1000 × 60 mm) and successively eluted with a petroleum ether/ethyl acetate gradient elution (90:10 to 0:100) to yield 5 fractions   Acid hydrolysis of compound 6. Compound 6 (5.0 mg) was dissolved in 5.0 mL of 2 N HCl, heated at 90 °C for 2 h and then partitioned between ethyl acetate and water. A glycon formononetin was recovered from the ethyl acetate layer and identified by direct comparison with an authentic sample. The water layers were identified by comparison with an authentic sample using TLC analysis 38 . Sugars liberated from compound 6 were identified as glucose.
Total phenolic content. The total phenolic content (TPC) was determined using a modified version of the Folin-Ciocalteau colorimetric method 39 . The phenolic content was calculated as a gallic acid equivalent from the calibration curve of gallic acid standard solutions (20-300 μ g/mL) and was expressed as millimoles of gallic acid equivalent per 100 g of dry weight (mmol equiv. GAE/100 g). The data are reported as the mean ± SD for three replicates.
Total flavonoid content. The total flavonoid content (TFC) was determined using a sodium borohydride/chloranil-based (SBC) assay 40 . A calibration curve was constructed to create a standard using different concentrations of quercetin (0.1-10.0 mM). The total flavonoid content was expressed as millimoles of quercetin equivalents per 100 g of dry weight (mmol equiv. QUE/100 g). The data are reported as the mean ± SD for three replicates.
DPPH radical-scavenging assay. The radical scavenging activity of extracts and compounds were measured by a slightly modified version of the method described by Brand-Williams 41 . All the extracts and compound samples were tested in triplicate. The inhibition rate (I%) of the radical-scavenging capacity was calculated using the following equation: where A DPPH is the absorbance of the DPPH solution, A blank is the absorbance of methanol instead of DPPH, A s-DPPH is the absorbance of the DPPH solution in the presence of the sample, and A s-blank is the absorbance of methanol in the presence of the sample. ABTS •+ radical cation scavenging assay. The ABTS assay was performed as previously described in published literature with some modifications 42 . The results are expressed in terms of micromoles of trolox equivalents per g of dry weight of extracts (μ mol eq. trolox/g) or millimoles of trolox equivalent per gram of dry weight of compounds (mmol eq. trolox/g). All determinations were carried out in triplicate.
Ferric reducing power (FRAP) assay. The FRAP assay used was a modified version of that reported by Benzie and Strain 43 . The FRAP results are expressed in terms of micromoles of trolox equivalents per g of dry weight of extracts (μ mol eq. trolox/g) or millimoles of trolox equivalent per gram of dry weight of compounds (mmol eq. trolox/g). All experiments were carried out in triplicate.
Inhibition of lipid peroxidation. In this assay, a modified thiobarbituric acid reactive species (TBARS) assay was used to measure the amount of lipid peroxide formed. The egg yolk homogenates were used as a lipid-rich medium 44,45 . Percentage inhibition was calculated with the following formula: % = ( − / ) × Inhibition of lipid peroxidation 1 T C 100 where C is the absorbance of fully peroxidized control and T is the absorbance of test sample. BHA and TBHQ were used as reference standards. The IC 50 value was calculated from the regression equation obtained from plotting the sample concentration and rate of inhibition.
Protective effect on H 2 O 2 -induced E. coli. The microbial test system used in the manuscript was an effective method to evaluate the antioxidant properties of medicinal plant extracts and polyphenols. This method is easier in terms of experimental operation, lower in cost compared to cellular antioxidant activity assays and more biological relevant than the popular chemistry antioxidant activity measures. In this study, E. coli (ATCC No. 25922) was used to determine the antioxidant activity of compounds in vivo based on a modified version of the method described by Smirnova 46,47 . Bacteria were incubated in Luria-Bertani medium at 37 °C in a 150-mL flask with shaking at 50 r/min overnight until they reached the mid-log phase. The optical density at 600 nm was used to monitor the cell growth. A 1.5-mL cell suspension was added to 8.5 mL of LB medium and resulted in a final OD 600 of 0.25 ± 0.004. Then, 100 μ L of the compounds (0.1 mM), a 1-mL cell suspension and 8.9 mL of LB medium were mixed and incubated at 37 °C with shaking at 180 r/min for 1.5 h. After the 1.5 h incubation, 6.5 mM of hydrogen peroxide was added and the absorbance at 600 nm was then measured immediately. The sample was then incubated at 37 °C with shaking at 180 r/min for an additional 30 min before the absorbance was measured again. The OD 600 is used to indicate the growth of E. coli, with a higher value of the OD 600 indicating a higher growth rate of E. coli. The isolated compounds have the ability to reduce and eliminate the oxidation of H 2 O 2 and protect the growth of E. coli; therefore, the higher antioxidant ability of the compounds indicates that the specific growth rate will be higher. Specific growth rate was calculated according to the following equation: where μ is the specific growth rate and N 0 and N are the optical density at time zero and t, respectively.

Statistical analysis.
All results are expressed as the mean ± standard deviation (SD). SPSS (Statistical Package for the Social Sciences) software was used to determine significant differences via one-way ANOVA followed by Duncan's test; values < 0.05 were considered to be significant.