Chemical Identification and Antioxidant Screening of Bufei Yishen Formula using an Offline DPPH Ultrahigh-Performance Liquid Chromatography Q-Extractive Orbitrap MS/MS

Wu, Jinyan; Cai, Bangrong; Zhang, Ang; Zhao, Peng; Du, Yan; Liu, Xuefang; Zhao, Di; Yang, Liu; Liu, Xinguang; Li, Jiansheng

doi:https://doi.org/10.1155/2022/1423801

International Journal of Analytical Chemistry

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Abstract Introduction Results and Discussion Conclusion Data Availability Disclosure Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2022 | Article ID 1423801 | https://doi.org/10.1155/2022/1423801

Chemical Identification and Antioxidant Screening of Bufei Yishen Formula using an Offline DPPH Ultrahigh-Performance Liquid Chromatography Q-Extractive Orbitrap MS/MS

Jinyan Wu,^1,2Bangrong Cai,^1,2Ang Zhang,^1,2Peng Zhao,^1,2Yan Du,^1,2Xuefang Liu,^1,2Di Zhao,^1,2Liu Yang,^1,2Xinguang Liu ,^1,2and Jiansheng Li^1,2,3

Academic Editor: Suresh Ponnayyan Sulochana

Received04 Aug 2022

Revised15 Sept 2022

Accepted28 Sept 2022

Published15 Oct 2022

Abstract

Chronic obstructive pulmonary disease (COPD) has high morbidity and mortality and presents a threat to human health worldwide. Numerous clinical trials have confirmed that Bufei Yishen formula (BYF), an herbal medicine, can alleviate the symptoms of COPD by reducing oxidative stress-mediated inflammation. However, the active components of BYF remain unclear. We developed an efficient ultrahigh-performance liquid chromatography Q-Extractive Orbitrap mass spectrometry method to identify the composition of BYF and determine its antioxidant profile through an offline screening strategy based on 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH)-liquid chromatography-mass spectrometry. In total, 189 compounds were identified in BYF extract, including 83 flavonoids, 24 lignans, 20 alkaloids, 15 saponins, 11 terpenoid, 10 saccharides, eight lipids, seven organic acids, two coumarins, two amino acids, and seven other compounds. Among them, 79 compounds were found to have a potential antioxidant activity. In vitro validation indicated that the free radical scavenging activities of rosmarinic acid and calycosin were similar to that of the positive control (DPPH IC₅₀ = 25.72 ± 1.02 and 147.23 ± 25.12 μg/mL, respectively). Furthermore, calycosin had a high content in serum after the oral administration of BYF, indicating that calycosin might be the major antioxidant compound in BYF.

1. Introduction

Chronic obstructive pulmonary disease (COPD) is a major chronic disease with high morbidity and mortality, especially among the elderly and smokers [1]. According to the World Health Authority and Global Initiative for Chronic Obstructive Lung Disease, bronchodilators and corticosteroids along with nonpharmacologic therapies such as pulmonary rehabilitation are frequently used to treat COPD. Although these therapies can reduce exacerbations and alleviate symptoms, there is little evidence to suggest that they can suppress the progression of COPD. Various recent clinical trials have suggested that herbal medicines have the potential to improve symptoms, reduce the frequency of acute exacerbation, and improve the quality of life for COPD patients [1]. Bufei Yishen formula (BYF) is an oral prescription for COPD that has proven clinically effective for COPD control [2]. The use of BYF and BYF combined with other therapies (e.g., acupoint sticking, electroacupuncture, and Tongsai granules) have shown beneficial effects in terms of lung function, clinical symptoms, quality of life, and acute exacerbation frequency in patients with stable COPD [3–5]. In a COPD rat model exposed to cigarette smoke and bacteria, BYF also ameliorated airway inflammation and remodeling [6–11]. We previously demonstrated that the mechanism of BYF against COPD might involve reducing inflammatory cytokines and oxidative stress, regulating immune response and lipid metabolism [12–15], restoring the Th17/Treg balance by activating adenosine 2a receptor [16], modulating the activities of STAT3 and STAT5 in COPD rats [17], and suppressing interleukin expression and/or secretion [18]. However, the effective substances of BYF are not clear at present, which forms a bottleneck problem in the further development of the preparation. It is well known that the ingredients of traditional Chinese medicine are complex, the unclear of effective substances make it difficult to select the biomarkers for quality control.

Oxidative stress triggering sustained inflammatory response is a major contributing factor in COPD [19, 20]. A system analysis integrating transcriptomics, proteomics, and metabolomics showed that the target proteins of BYF against COPD are glutamate-cysteine ligase, glutathione reductase, G6PD, glutathione S-transferase P, glutathione S-transferase A1/2, GSTM1/2, and SOD1, which are predominantly enriched in oxidative stress-related pathways [15]. We speculated that the antioxidant profiling of BYF might help uncover the effective substances of BYF.

At present, “separation-activity verification” is the main strategy for screening antioxidant substances in herbal medicines. In this approach, as many natural products as possible are isolated from the herbal medicine, and their activities are evaluated through antioxidant assays. However, the separation step in this method is time-consuming. Ultrahigh-performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS) has shown great potential for the rapid identification of antioxidants in natural products [21–23]. It is based on the hypothesis that the reaction of antioxidants with 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) will significantly reduce the concentrations of compounds with potential antioxidant activity. Due to the accurate mass measurement provided by UHPLC-HRMS, the antioxidants in herbal medicines can be easily screened and identified. In this work, an efficient UHPLC Q-Extractive Orbitrap MS/MS method was developed to elucidate the chemical composition of BYF. The antioxidants were identified by offline DPPH-UHPLC Q-Extractive Orbitrap MS/MS and their concentrations were detected in rat serum after the oral administration of BYF. Figure 1 shows a schematic diagram of the experimental design. This study combines rapid antioxidant screening based on the chromatography-activity relationship with an evaluation of drug absorption to indicate the antioxidant substances contained in BYF. This allows to quickly identify the antioxidants that really effective in vivo, and the proposed strategy also provides reference for the screening of antioxidants of other traditional Chinese medicines.

2. Experimental Methods

2.1. Reagents and Materials

Methanol (HPLC-grade), acetonitrile (LC-MS grade), and formic acid were purchased from Thermo Fisher Technology Co., Ltd. (Shanghai, China). Ethanol was purchased from Mreda Technology Inc. (Beijing, China). Ultrapure water was produced by a laboratory Milli-Q system (Merck Millipore, Shanghai, China). DPPH and potassium persulfate were purchased from Shanghai Macklin Biochemical Chemical Co. Ltd. (Shanghai, China). The fresh DPPH radical solution was kept away from light. 2,2-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and L-ascorbic acid were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All standard references included in Table 1 were purchased from Shanghai Yuanye Technology Co., Ltd. (Shanghai, China).

2.2. Animals

Adult Sprague-Dawley (SD) male SPF rats weighing 170–200 g were provided by Beijing Weitong Lihua Experimental Animal Company (animal license number SCXK (Yu) 2020-0004). All animals were fed standard feed and were allowed to drink freely for one week at 20°C–25°C. On the day before the experiment, the animals were fasted for 12 h (except for drinking water). All protocols were approved by the Ethics Committee of the Henan University of Chinese Medicine (approval number DWLLGZR202202029).

2.3. Sample Preparation

2.3.1. Preparation of BYF

All medicinal herbs in BYF were obtained from Zhengzhou Ruilong Pharmaceutical Co. (Zhengzhou, China) BYF consists of 12 medicinal materials: Astragali Radix (AR), Fritillariae thunbergii Bulbus (FTB), Pheretima (P), Citri reticulatae Pericarpium (CRP), Ardisia japonicae Herba (AJH), Epimedii folium (EF), Ginseng radix et rhizoma (GRR), Schisandrae chinensis Fructus (SCF), Lycii fructus (LF), Perillae fructus (PF), Corni fructus (CF), and Paeoniae radix Rubra (PRR). Extraction of AR, LF, EF, PRR, and P were conducted by 12 L of water per gram of crude materials by two times. After filtering, the filtrate was concentrated. Reflux extraction of GRR, FTB, CRP, AJH, SCF, PF, and CF were conducted by 10 L of 70% ethanol per gram of crude materials by 2 times. After filtering, the ethanol was recovered from the filtrate and combined with the abovementioned concentrate. The combined concentrate was further concentrated into a thick paste with a relative density of 1.18–1.22 (60°C). Finally, the per-gram dry extract obtained was equivalent to 3.81 g of raw medicinal herb.

BYF extract (0.2 mg) was extracted by ultrasonication with 20 mL of 70% methanol for 30 min. The extract was filtered, and the filtrate was centrifuged at 13000 rpm for 10 min at 4°C. The supernatant was stored at −20°C before analysis. All reference standards and internal standards (IS) were dissolved in methanol at concentrations of 10 μg/mL.

For antioxidant capacity assay, BYF extract, different concentrations of L-ascorbic acid as the positive control (0.5–100 μg/mL) and candidate antioxidants (0.5–1000 μg/mL) were prepared in methanol.

For antioxidant quantification, stock solutions of the standard references and IS were prepared in methanol at concentrations ranging from 0.515 to 1.23 mg/mL. A series of working solutions of mixed reference standards were obtained by further dilution with methanol. Calibration standards were prepared by spiking 10 μL of the standard solutions into 90 μL of blank biological samples to obtain final concentrations of 0.0124–1080 ng/mL. All working solutions were stored at −80°C. Calibration curves were acquired by plotting the peak area ratio (y) of each compound to the IS against the corresponding concentration of each compound (x). The acceptance criterion of a calibration curve was a correlation coefficient (r) of 0.99 or better along with relative errors for each point within ±15%.

2.3.2. Preparation of Serum Samples

The rats were randomly divided into the normal group and BYF group, with six rats in each group. The rats in the BYF group were gavaged twice per day with 18.28 g/kg/d BYF for 7 d. The normal group was given the same amount of normal saline by gavage for 7 d. The rats were fasted for 1 d before blood collection. Blood was collected from the orbital vein at 10 min, 30 min, 1 h, 2 h, and 4 h after the last gavage, and centrifuged at 3000 rpm for 10 min at 4°C. Finally, the serum was separated and stored at −80°C for later use.

To qualitative analyze the BYF components in rat serum after oral administration, we randomly mixed five serum samples from each group collected at different times after administration (20 μL for each sample, 100 μL in total). For the quantitative analysis of BYF components in rat serum, 100 μL of the serum samples collected at different times after BYF administration was taken and analyzed.

A protein precipitation procedure was used to extract BYF components. An 100-μL aliquot of serum was spiked with 300 μL of acetonitrile (containing 200 ng/mL IS and 6 ng/mL ascorbic acid). The mixture was vortex-mixed for 3 min, allowed to stand at 4°C for 5 min, and centrifuged at 4°C and 18534 g for 10 min. Next, 300 μL of the supernatant was transferred into a new tube and evaporated to dryness using an Integrate SpeedVac System (ThermoFisher Scientific Corporation, USA). The residue was redissolved in 50 μL of 50% acetonitrile, and a 35-μL aliquot of the supernatant was collected for analysis.

2.4. Chromatography and MS Conditions

The samples were analyzed by LC-MS/MS using a Dionex Ultimate 3000 UPLC system (ThermoFisher Scientific, Germering, Germany) coupled to a Thermo Scientific Q-Exactive Orbitrap mass spectrometer (ThermoFisher Scientific, Bremen, Germany).

To identify the BYF components in the extract and serum, the samples were loaded onto a Phenomenex Synergi Polar-RP column (2 × 150 mm, 4 μm) at 40°C. Mobile phase A was composed of water and 0.1% formic acid. Mobile phase B was composed of ACN and 0.1% formic acid. The flow rate was 0.3 mL/min. The gradient elution conditions were as follows: 0% B (0–5 min); linear gradient from 0% B to 5% B (5–7 min); 5% B to 20% B (7–10 min); 20% B to 25% B (10–20 min); 25% B to 50% B (20–23 min); 50% B to 100% B (23–40 min); 100% B for 3 min (40–43 min); back to 0% B over 2 minutes; 0% B for 5 min (45–50 min). The injection volume was 5.00 μL.

For the quantitative analysis of antioxidants in rat serum, the samples were loaded onto an Agilent ZORBAX-Extend-C18 LC column (4.6 × 50 mm, 1.8 μm) at 30°C. Mobile phase A was composed of water and 0.1% formic acid. Mobile phase B was composed of acetonitrile and 0.1% formic acid. The flow rate was 0.5 mL/min. The gradient elution condition were as follows: 5% B (0–2 min); linear gradient from 5% B to 23% B (2-3 min); 23% B for 6 min (3–9 min); 23% B to 50% B (9-10 min); 50% B to 68% B (10–15 min); 68% B for 3 min (15–18 min); 68% B to 100% B (18–22 min); 100% B for 2 min (22–24 min); back to 5% B (24-25 min); and 5% B for 2 min (25–27 min). The injection volume was 5 μL.

The mass spectrometer was equipped with a heated electrospray ionization probe. The spray voltage was set at 3500 V for positive ion mode and 2800 V for negative ion mode. The flow rates of the sheath gas and aux gas were 40 and 10 Arb, respectively. The capillary temperature was 325°C, and the aux gas heater temperature was 300°C. Full scans from m/z 100 to 1500 were performed in the Orbitrap at a resolution of 70 K for quantification. The AGC target value was 3 × 10⁶, and the maximum injection time was 200 ms. Parallel reaction monitoring (PRM) mode was used for fragmentation identification and quantification of BYF metabolites. The target MS2 scan in PRM mode was conducted at a resolution of 17.5 K with an isolation width of 4.0 Da, an AGC target value of 2 × 10⁵, and a maximum injection time of 100 ms. The precursor ion/product ions and normalized collision energy for each compound are listed in Table S1 [29].

2.5. Antioxidant Profiling

2.5.1. Offline DPPH-UHPLC Q-Extractive Orbitrap MS/MS

BYF extract (100 µL) was mixed with DPPH solutions of different concentrations (100 µL and 0.5, 1, 2, 5, and 10 mM), and the mixtures were incubated in the dark at room temperature for 30 min. The mixtures were further monitored by UHPLC-Q-Extractive Orbitrap MS/MS. Control experiments in which DPPH solution was replaced by a blank solution were carried out for comparison. The reduction in the peak area compared with the control group indicated the DPPH radical scavenging activity of the compounds in BYF.

2.5.2. Determination of Antioxidant Activities

In order to determine the antioxidant activity of potential antioxidants, DPPH radical scavenging assay, ABTS radical scavenging activity, and ferric-reducing antioxidant power (FRAP) assay were conducted.

(1) DPPH radical scavenging assay. The DPPH radical scavenging assay was performed on a spectrophotometer microplate reader from ThermoFisher Scientific (Vantaa Finland) using multiwell plates as a previously published method described [30]. The DPPH solution was diluted by methanol to 0.1 mM as a working solution. The reaction was initiated by mixing 50 μL of test solution with 150 μL of DPPH working solution and incubated in dark at room temperature for 30 min. Monitoring of the absorbance at 517 nm was carried out after the reaction was completed. The scavenging capacity of samples were calculated by experimental scavenging capacity (ESC) using equation (1) as follows:where Abs_sample is the absorbance value of the sample (DPPH solution plus antioxidant) at each time interval and Abs_blank is the absorbance value of the blank (methanol plus antioxidant(s)). Abs_control is the absorbance value of control (methanol plus DPPH solution).

The value of 50% inhibition (IC₅₀) was calculated by the graph plotting sample concentration and inhibition percentage.

(2) ABTS radical scavenging activity. The ABTS radical scavenging activity of the crude extracts was determined using the method described by Zhou et al. [30] with minor modifications. Aqueous ABTS (7 mM) was mixed with 2.45 mM aqueous potassium persulfate (1 : 1, ), and the solution was left to react for 16 h at room temperature in the dark. The ABTS•+ solution was diluted with absolute ethanol to an absorbance at 734 nm of 0.70 ± 0.02 to obtain an ABTS•+ radical working solution. Then, 160 μL of the ABTS•+ radical working solution was mixed with 40 μL of test solutions, and the mixture was incubated for 6 min. The absorbance of the mixture was measured at 734 nm. The ABTS radical scavenging assay was performed on a spectrophotometer microplate reader. The ABTS radical scavenging activity was calculated according to the following equation:where A sample = the absorbance at 734 nm with sample and A blank = the absorbance at 734 nm without sample. The IC₅₀ value was calculated and represents the concentration necessary to reduce the maximum response of the ABTS by half.

(3) FRAP assay. The FRAP assays were performed by a total antioxidant capacity assay kit with the PRAP method according to manufacturer’s instruction (Beyotime Biotech Inc, Shanghai, China). Briefly, 180 μL FRAP working solution was mixed with 5 μL extract of BYF, or 5 μL distilled water as blank control, or 5 μL 0.15–1.5 mM FeSO₄ standard solution (dissolved in distilled water) as standard curve. The absorbance of the mixture was measured at 593 nm after incubation at 37°C for 3–5 minutes. The total antioxidant capacity of the sample was calculated according to the standard curve. For FRAP method, the total antioxidant capacity of the extract is expressed by the concentration of FeSO₄ standard solution with equivalent antioxidant capacity.

3. Results and Discussion

3.1. Chemical Identification of BYF Components

We developed an UHPLC-Q-Extractive Orbitrap-MS/MS method for the comprehensive characterization of the chemical constituents of BYF extract. The total ion chromatography obtained in positive ion mode is shown in Figure 2(a). First, by consulting literature and the Encyclopedia of Traditional Chinese Medicine, we constructed a MS information database of the components of the materials in BYF. In this library, CRP are the dried pericarps of the ripe fruits of Citrus reticulate Blanco or its cultivars. CRP mainly contain flavonoids, by UHPLC-QTOF MS, Duan et al. identified 75 flavonoids from CRP [24]. PRR are the roots of Paeonia lactiflora and Paeonia anomala subsp. Veitchii, which mainly contain monoterpene glycosides, flavonoids, tannins, phenols and paeonols [25]. GRR are the dry roots and rhizomes of Panax ginseng C. A. Mey. GRR mainly contain triterpene saponins, which are also widely recognized as active components. Qi et al. identified 70 saponins from GRR [27]. PF are the dry ripe fruits of Perilla frutescens (L.) Britt., which mainly contain phenolic acids, triterpenoids, flavonoids and fatty acids [28]. AR are the dry root of Astragalus membranaceus (Fisch.) Bge.var.mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge., which mainly contain triterpene saponins and flavonoids, Chu et al. identified 22 astragalosides from AR [26], and Mei et al. totally identified 47 saponins and 55 flavonoids [31]. FTB are the dry bulb of Fritillaria cirrhosa D. Don, Fritilaria unibracteata Hsiao et K. C. Hsia, Fritillaria przewalskii Maxim., Fritillaria delavayi Franch., Fritillaria taipaiensis P. Y. Li, or Fritillaria unibracteata Hsiao et K. C. Hsiavar. wabuensis (S. Y. Tanget S. C. Yue) Z. D. Liu, S. Wang et S. C. Chen, alkaloids are the main components in FTB, terpenoids and steroids can also be found in FTB [32]. P are the dry body of Pheretima aspergillum, Pheretima vulgaris Chen, Pheretima guillelmi or Pheretima pectinifera Michaelsen, its main components are amino acids and organic acids. Zhang et al. identified 11 free amino acid, 26 organic acids, 11 nucleosides, 5 dipeptides and cyclic dipeptides, and 21 nitrogenous substances from P [33]. AJH are the dry whole herb of Ardisia japonica (Thunb.) Blume. The main components in AJH including benzoquinones, phenols, flavonoids, chromones, triterpenes, and triterpene saponins [34]. EF are the dry leave of Epimedium brevicornu Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim. or Epimedium koreanum Nakai. EF mainly contain flavonoids, in addition, lignans, polysaccharides and alkaloids can also be detected [35, 36]. SCF are the dry ripe fruit of Schisandra chinensis (Turcz.) Baill., SCF mainly contain lignans, and also polysaccharide volatile oil [37]. LF are the fruit of Lycium barbarum L., mainly contain polysaccharides, peptide, alkaloids, flavonoids, terpenes, organic acids, lignans, phenolic amides and carotenoids [38]. CF are the dry ripe sarcocarp of Cornus officinalis Sieb. et Zucc (Cornaceae), include mainly irridoids, organic acids, triterpenes, cornustannins, and carbohydrates [39].

(a)

(b)

(c)

As shown in Table 1, 189 chemical constituents were identified in BYF based on the library; their MS/MS spectra were matched with online databases and/or published references. Structurally, the main components of BYF were flavonoids (83 compounds), lignans (24 compounds), and alkaloids (20 compounds). Other identified components included 15 saponins, 11 terpenoids, 10 saccharides, eight lipids, seven organic acids, two coumarins, two amino acids, and seven other compounds. Among the identified compounds, 37 were identified by comparison with the retention times and MS spectra of standards; the MS/MS spectra of these compounds are shown in Supplementary Materials Figures S1–S37.

3.1.1. Flavonoids

A total of 83 flavonoids were identified in BYF. Among them, 33 flavonoids were from only EF. These flavonoids are mainly flavonoids with isobutenyl at the C-8 position and their glycosides including icariin, epimedin A, and compounds 17, 54, 55, 60, 61, 63, 68, 70, 76, 77, 81, 83, 87, 93, 98, 99, 106, 110, 111, 113, 114, 120, 122, 127, 132, 133, and 166. These flavonoids have the characteristic isobutenyl neutral loss of 56 Da as a diagnostic ion (Table 1). For flavonoid glycosides, the common neutral losses of 162 and 146 Da were due to the presence of glucosyl and rhamnosyl groups. For example, in Figure 3(a), Epimedin B at the retention time of 22.98 min had a positively charged molecular ion ([M + H]⁺) at m/z 809.2841, which yielded secondary fragments at m/z 677.2428 ([M + H-xyl]⁺), 531.1852 ([M + H-xyl-rha]⁺), 369.1324 ([M + H-xyl-rha-glu]⁺), and 313.0698 ([M + H-xyl-rha-glu-isobutenyl]⁺). In addition, 31 flavonoids (mainly flavonoid aglycones) were derived from CRP. The summary of the MS/MS fragments of CRR flavonoids reported by Duan et al. [24] was used for the structural identification of flavonoid aglycones in this work, especially those whose structures were not completely determined. The other flavonoids were mainly from FTB, GRR, LF, PF, and AR.

(a)

(b)

3.1.2. Lignans

A total of 24 lignans were identified in BYF, all of which were from SCF. More than 150 lignans were isolated from SCF, mainly biphenyl cyclooctadienes, spirobenzofuran biphenyl cyclooctadienes, 4-aryltetrahydronaphthalene, 2,3-dimethyl-1,4-diarylbutane, and 2,5-diaryltetrahydrofurans. Among them, biphenyl cyclooctadienes have the most species and the strongest biological activity [40]. Biphenyl cyclooctadienes include schisantherin A, B, and C, gomisin L1, and schisandrin A, B, and C. The characteristic neutral losses of C₄H₆COOH, CH₃OH, CO₂, CO, CH₃, and H₂O were attributed to the presence of 2-methylbutyryl, hydroxymethyl, carboxyl, carbonyl, methyl, and hydroxyl groups in their structures (Table 1). For example, in Figure 3(b), Schisandrin A at the retention time of 30.27 min has a positively charged molecular ion ([M + H]⁺) at m/z 809.2841, which yielded secondary fragments at m/z 402.2029 ([M + H − CH₃]⁺), 386.2079 ([M + H − CH₃ − O]⁺), and 371.1832 ([M + H − 2CH₃ − O]⁺).

3.1.3. Alkaloids

A total of 20 alkaloids were identified in BYF. Among them, 18 alkaloids were from only FTB. FTB mainly contains steroidal alkaloids such as peimisine and peimine [41]. There are few characteristic fragments of steroidal alkaloids, in which only neutral loss of H₂O can be found. Therefore, these structures are confirmed by comparing the retention time with the standard. FTB also contains some alkaloid glycosides such as sibelicin glycoside and yibeinoside A. The common neutral loss of 162 Da was attributed to the presence of a glucosyl group (Table 1).

3.2. Screening of Antioxidant Components Using the Offline DPPH-UHPLC Q-Extractive Orbitrap MS/MS

As mentioned above, BYF can significantly alleviate the symptoms of COPD in clinical practice. We have also done some research on the mechanism of BYF in treating COPD, the most important is BYF treatment could effectively inhibit the inflammatory response of the lungs [12, 13]. In COPD rats, BYF significantly inhibited the expression of IL-1β, IL-6, TNF-α, and sTNFR2 induced by cigarette smoke and bacterial infection exposures. The inhibition of BYF on inflammatory response in rat COPD model may be through restoring the Th17/Treg balance by activating adenosine 2a receptor [16] and modulating the activities of STAT3 and STAT5 [17]. Th17/Treg imbalance is considered to be important of COPD development. In COPD patients, the Th17/Treg cell balance shifts toward Th17 cells, which triggers inflammatory responses in the airways and lungs and exacerbates alveolar destruction by producing interleukin-17 [17]. On the one hand, regulating oxidative stress is also an important mechanism of BYF regulating inflammation. We used transcriptomics and proteomics finding that the target proteins of BYF against COPD are enriched in oxidative stress-related pathways [15], These will further inhibit the inflammatory response related to oxidative stress. Therefore, we studied the antioxidant activity of BYF and its antioxidants in order to reveal its effective substances.

3.2.1. Total Antioxidant Capacity of BYF

We evaluated the total antioxidant capacity of BYF by DPPH, ABTS, and FRAP assays. Table 2 shows that the IC₅₀ values of BYF in the DPPH and ABTS assays were 1136.36 ± 148.03 and 602.35 ± 81.26 μg/mL, respectively. In addition, BYF showed high total antioxidant capacity of FRAP (0.51 ± 0.04 mM). Thus, it is necessary to further screen the active components of BYF.

3.2.2. Antioxidant Screening of BYF

DPPH is a stable free radical with an odd electron. DPPH is commonly used to assess the radical scavenging activity of antioxidants; it is capable of accepting one or more hydrogen atoms from an antioxidant, resulting in an unconjugated structure with reduced MS response, which can be detected by HRMS [42, 43]. Moreover, the use of DPPH saves time and labor compared to other free radicals such as ABTS [44]. This antioxidant screening strategy based on the change in MS signal can be divided into online and offline modes. Online screening requires two HPLC pumps, one for chromatographic separation and the other for delivering DPPH solution. The chromatographic fraction and DPPH react online in the pipeline. This method is rapid but has relatively poor stability [44]. Therefore, the more stable and sensitive offline mode was used in this work. In offline mode, the herbal medicine extract was fully reacted with DPPH, and the reaction solution was injected into the mass spectrometer for antioxidant detection.

In an offline experiment, the concentration ratio of DPPH in the extract will significantly affect the efficiency of antioxidant screening [45]. A relative excess of DPPH will not affect the free radical scavenging ability of the active components. However, when the DPPH concentration is insufficient, the free radical scavenging ability cannot be detected [46]. We optimized the DPPH concentration (Figure 4) and found that 10 mM DPPH was most suitable to screen the free antioxidant components. The components with peak intensity decreased more than 20% were considered as potential antioxidants, which are summarized in Table 3.

3.2.3. Antioxidant Activities of the Potential Antioxidants

To verify the antioxidant activities of the potential antioxidants determined above, we measured the free radical scavenging ability of 13 potential antioxidants with available reference standards by DPPH and ABTS assay. As shown in Table 2, 4 compounds showed high free radical scavenging ability for DPPH and or ABTS. Among them, rosmarinic acid had a strongest scavenging activity in DPPH assay (IC₅₀ = 25.72 ± 1.02 μg/mL), and rosmarinic acid and calycosin both showed strong scavenging activity in ABTS assay (IC₅₀ = 19.00 ± 0.75 and 19.34 ± 5.05 μg/mL, respectively) which superior to ascorbic acid. The results show that phenolic acids and flavonoids in BYF play a major role in the antioxidative activity. Rosmarinic acid has been reported to alleviate oxidative lung damage and airway inflammation based on its strong antioxidant activity [47–49]. Rosmarinic acid can also decrease the population of inflammatory cells; reduce the levels of proinflammatory cytokines such as IL-4, IL-5, and IL-13; upregulate IFN-γ secretion; upregulate the activities of SOD, GPx, and CAT; increase Cu/Zn SOD; and significantly downregulate ROS production and the expressions of NOX-2 and NOX-4 in lung tissues [48]. Calycosin has also shown good antioxidant activity [50] and can ameliorate various lung injuries including sepsis, cecal ligation, and puncture by regulating oxidative stress-mediated inflammation in vivo and augmenting superoxide dismutase and glutathione [51]. Since oxidative stress and inflammation are the main pathogeneses of COPD, we suspect that these components are important antioxidants in BYF for the treatment of COPD.

3.3. Analysis of Antioxidants in Rat Serum

The antioxidants in BYF may not show the expected antioxidant activity in vivo because of their poor absorption after oral administration. To investigate whether the potential antioxidants might be present in vivo, rats were orally administered with a high dosage of BYF extract. For consistency with the efficacy experiment, the serum was collected at 10 min, 30 min, 1 h, 2 h, and 4 h after the last BYF administration (after 7 d of continuous oral administration of BYF extract). Based on the retention time and HRMS spectra, we identified 79 compounds in rat serum after oral BYF administration: 34 flavonoids, 14 lignans, 7 alkaloids, one saponin, three organic acids, four saccharides, three lipids, four terpenoids, two amino acids, one coumarin, and six other compounds. Among them, 26 were identified by comparison with the standard materials (Table S2). The total ion chromatograms of rat serum at 1 h after the administration of BYF extract are shown in Supplementary Materials Figure S38. The 13 main components of BYF in rat serum were quantified; endogenous compounds and compounds with insufficient contents were not measured. The quantitative results are shown in Figure 2(b) and Figure S39, and the standard curves and linear ranges are shown in Table S3. Among the BYF components detected in serum, schisandrin B, schisantherin A, and schisantherin B had the highest contents. Among the validated potential antioxidants (Table 2), hesperidin, and naringenin were detected in rat serum (Table 3 and Table S2); however, they are in trace amounts and the concentrations were not obtained. Rosmarinic acid was not found in serum after oral administrated of BYF, Thus, although rosmarinic acid showed the best antioxidant activity, it may not be the major active component in BYF due to its poor absorption or low content. Calycosin most likely to be responsible for the antioxidant effect of BYF in vivo, because it showed a high content in serum. The serum concentration–time curve of calycosin is shown in Figure 2(c). The serum concentration of calycosin reached its highest level at 10 min after the oral administration of BYF, and calycosin was almost cleared in vivo after 1 h. Based on the above results, the efficacy and mechanism of calycosin in the treatment of COPD in vivo are worthy of further study.

4. Conclusion

In this work, we first identified 189 compounds from the BYF extract. An offline DPPH-UHPLC Q-Extractive Orbitrap MS/MS strategy was developed to rapidly screen the antioxidants in BYF. Rosmarinic acid and calycosin showed high radical scavenging activities in both DPPH and ABTS assays. We detected a high content of calycosin in rat serum after the oral administration of BYF, suggesting that calycosin might be the key antioxidant compound in BYF for the treatment of COPD in vivo.

Data Availability

The other data used in the manuscript are listed in supplementary materials.

Disclosure

Jinyan Wu and Bangrong Cai are co-first author.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Authors’ Contributions

Jinyan Wu conceptualized the study, wrote the original draft, and did the data curation. Bangrong Cai reviewed the article and did the visualization. Ang Zhang did the data curation, investigation and validation of the study. Peng Zhao validated the study. Yan Du did the software and conceptualization. Xuefang Liu investigated the study. Di Zhao gathered resources and conducted the formal analysis. Liu Yang validated the study. Xinguang Liu reviewed the article. Jiansheng Li reviewed the paper. Jinyan Wu and Bangrong Cai contributed equally to this work.

Acknowledgments

This work was supported by National Natural Science Foundation of China (82274274, 81973822); China Postdoctoral Science Foundation (No. 2019M662501, No. 2020M682311); Key R&D and Promotion Projects in Henan Province (No. 202102310170); the Qihuang Scholars Award of The State TCM Academic Leader Program; Qihuang Chief Scientist of The Preservation and Innovative Development of Tcm Talent Project.

Supplementary Materials

The supporting data offered in supplementary materials are as follows: Table S1. Parallel reaction monitoring transitions for metabolites of Bufei Yishen formula included in the assay. Table S2. Chemical composition information of Bufei Yishen Formula Based on UHPLC-Q-Extractive Orbitrap MS. Table S3. Calibration curves, Linear ranges and LLOQs of the Bufei Yishen Formula compounds in serum. Figure S1. The MS/MS spectra of the reference standard of quinic acid. Figure S2. The MS/MS spectra of the reference standard of Pyroglutamic acid. Figure S3. The MS/MS spectra of the reference standard of oxypaeoniflorin. Figure S4. The MS/MS spectra of the reference standard of loganin. Figure S5. The MS/MS spectra of the reference standard of rhoifolin. Figure S6. The MS/MS spectra of the reference standard of Hesperidin. Figure S7. The MS/MS spectra of the reference standard of rosmarinic acid. Figure S8. The MS/MS spectra of the reference standard of diosmin. Figure S9. The MS/MS spectra of the reference standard of Peimisine. Figure S10. The MS/MS spectra of the reference standard of peimine A. Figure S11. The MS/MS spectra of the reference standard of Peiminine B. Figure S12. The MS/MS spectra of the reference standard of Epimedin A (Hexandraside F). Figure S13. The MS/MS spectra of the reference standard of Calycosin. Figure S14. The MS/MS spectra of the reference standard of Epimedin B. Figure S15. The MS/MS spectra of the reference standard of Epimedin C (Baohuside VI). Figure S16. The MS/MS spectra of the reference standard of Icariin. Figure S17. The MS/MS spectra of the reference standard of Ginsenoside Re. Figure S18. The MS/MS spectra of the reference standard of Ginsenoside Rb1. Figure S19. The MS/MS spectra of the reference standard of Perillaldehyde. Figure S20. The MS/MS spectra of the reference standard of astragaloside Iv. Figure S21. The MS/MS spectra of the reference standard of Naringenin. Figure S22. The MS/MS spectra of the reference standard of Apigenin. Figure S23. The MS/MS spectra of the reference standard of Formononetin. Figure S24. The MS/MS spectra of the reference standard of Ginsenoside Rg2. Figure S25. The MS/MS spectra of the reference standard of isosinensetin. Figure S26. The MS/MS spectra of the reference standard of Methyl eugenol. Figure S27. The MS/MS spectra of the reference standard of D-ribo-phytosphingosine. Figure S28. The MS/MS spectra of the reference standard of schisandrol A. Figure S29. The MS/MS spectra of the reference standard of Nobiletin. Figure S30. The MS/MS spectra of the reference standard of tangeretin. Figure S31. The MS/MS spectra of the reference standard of corosolic acid. Figure S32. The MS/MS spectra of the reference standard of Schisantherin B. Figure S33. The MS/MS spectra of the reference standard of Schisantherin A. Figure S34. The MS/MS spectra of the reference standard of Schisandrin A. Figure S35. The MS/MS spectra of the reference standard of Schisandrin B. Figure S36. The MS/MS spectra of the reference standard of Arbutin. Figure S37. The MS/MS spectra of the reference standard of Schisandrin C. Figure S38. A total ion chromatogram of the mix rat serum after administrated of BYF extract at 1 h. Figure S39. The content of Bufei Yishen Formula components in the rat serum. (Supplementary Materials)

References

Z. Justinova, P. Mascia, H. Q. Wu et al., “Reducing cannabinoid abuse and preventing relapse by enhancing endogenous brain levels of kynurenic acid,” Nature Neuroscience, vol. 16, pp. 1652–1661, 2013.
View at: Publisher Site | Google Scholar
J. S. Li and S. Y. Li, ZL.201110117578.1, China Patent, 2011.
Q. Wang, M. Zhang, Y. Ding et al., “Activation of NAD(P)H oxidase by tryptophan-derived 3-hydroxykynurenine accelerates endothelial apoptosis and dysfunction in vivo,” Circulation Research, vol. 114, pp. 480–492, 2014.
View at: Publisher Site | Google Scholar
J. S. Li, S. Y. Li, X. Q. Yu et al., “Bu-Fei Yi-Shen granule combined with acupoint sticking therapy in patients with stable chronic obstructive pulmonary disease: a randomized, double-blind, double-dummy, active-controlled, 4-center study,” Journal of Ethnopharmacology, vol. 141, pp. 584–591, 2012.
View at: Google Scholar
Y. Xie, J. S. Li, X. Q. Yu et al., “Effectiveness of Bufei Yishen granule combined with acupoint sticking therapy on quality of life in patients with stable chronic obstructive pulmonary disease,” Chinese Journal of Integrative Medicine, vol. 19, pp. 260–268, 2013.
View at: Google Scholar
J. S. Li, Y. Li, S. Y. Li et al., “Long-term effects of Tiaobu Feishen therapies on systemic and local inflammation responses in rats with stable chronic obstructive pulmonary disease,” Journal of Chinese Integrative Medicine, vol. 10, pp. 1039–1048, 2012.
View at: Google Scholar
Y. Tian, Y. Li, J. Li et al., “Bufei Yishen granule combined with acupoint sticking improves pulmonary function and morphormetry in chronic obstructive pulmonary disease rats,” BMC Complementary and Alternative Medicine, vol. 15, p. 266, 2015.
View at: Google Scholar
Y. Tian, J. Li, Y. Li et al., “Effects of Bufei Yishen granules combined with acupoint sticking therapy on pulmonary surfactant proteins in chronic obstructive pulmonary disease rats,” BioMed Research International, vol. 2016, Article ID 8786235, 8 pages, 2016.
View at: Publisher Site | Google Scholar
X. Lu, Y. Li, J. Li et al., “Sequential treatments with Tongsai and Bufei Yishen granules reduce inflammation and improve pulmonary function in acute exacerbation-risk window of chronic obstructive pulmonary disease in rats,” Evidence-Based Complementary and Alternative Medicine, vol. 2016, Article ID 1359105, 17 pages, 2016.
View at: Publisher Site | Google Scholar
J. Ma, Y. Tian, J. Li et al., “Effect of bufei yishen granules combined with electroacupuncture in rats with chronic obstructive pulmonary disease via the regulation of TLR-4/NF-kappaB signaling,” Evidence-Based Complementary and Alternative Medicine, vol. 2019, Article ID 6708645, 14 pages, 2019.
View at: Publisher Site | Google Scholar
Y. Tian, Y. Li, J. Li et al., “Bufei Yishen granules combined with acupoint sticking therapy suppress inflammation in chronic obstructive pulmonary disease rats: via JNK/p38 signaling pathway,” Evidence-Based Complementary and Alternative Medicine, vol. 2017, Article ID 1768243, 10 pages, 2017.
View at: Publisher Site | Google Scholar
J. Li, P. Zhao, Y. Li, Y. Tian, and Y. Wang, “Systems pharmacology-based dissection of mechanisms of Chinese medicinal formula Bufei Yishen as an effective treatment for chronic obstructive pulmonary disease,” Scientific Reports, vol. 5, Article ID 15290, 2015.
View at: Google Scholar
J. Li, P. Zhao, L. Yang, Y. Li, Y. Tian, and S. Li, “System biology analysis of long-term effect and mechanism of Bufei Yishen on COPD revealed by system pharmacology and 3-omics profiling,” Scientific Reports, vol. 6, Article ID 25492, 2016.
View at: Google Scholar
L. Yang, J. Li, Y. Li et al., “Identification of metabolites and metabolic pathways related to treatment with bufei yishen formula in a rat COPD model using HPLC Q-TOF/MS,” Evidence-Based Complementary and Alternative Medicine, vol. 2015, Article ID 956750, 9 pages, 2015.
View at: Publisher Site | Google Scholar
P. Zhao, J. Li, L. Yang, Y. Li, Y. Tian, and S. Li, “Integration of transcriptomics, proteomics, metabolomics and systems pharmacology data to reveal the therapeutic mechanism underlying Chinese herbal Bufei Yishen formula for the treatment of chronic obstructive pulmonary disease,” Molecular Medicine Reports, vol. 17, pp. 5247–5257, 2018.
View at: Google Scholar
P. Zhao, X. Liu, H. Dong et al., “Bufei yishen formula restores Th17/Treg balance and attenuates chronic obstructive pulmonary disease via activation of the adenosine 2a receptor,” Frontiers in Pharmacology, vol. 11, p. 1212, 2020.
View at: Google Scholar
P. Zhao, J. Li, Y. Tian et al., “Restoring Th17/Treg balance via modulation of STAT3 and STAT5 activation contributes to the amelioration of chronic obstructive pulmonary disease by Bufei Yishen formula,” Journal of Ethnopharmacology, vol. 217, pp. 152–162, 2018.
View at: Google Scholar
J. Li, L. Yang, Q. Yao et al., “Effects and mechanism of Bufei Yishen formula in a rat chronic obstructive pulmonary disease model,” Evidence-Based Complementary and Alternative Medicinet, vol. 2014, Article ID 381976, 10 pages, 2014.
View at: Publisher Site | Google Scholar
P. J. Barnes, “Cellular and molecular mechanisms of chronic obstructive pulmonary disease,” Clinics in Chest Medicine, vol. 35, pp. 71–86, 2014.
View at: Google Scholar
A. Sunnetcioglu, H. H. Alp, B. Sertogullarindan, R. Balaharoglu, and H. Gunbatar, “Evaluation of oxidative damage and antioxidant mechanisms in COPD, lung cancer, and obstructive sleep apnea syndrome,” Respiratory Care, vol. 61, pp. 205–211, 2016.
View at: Google Scholar
J. Dang, C. Chen, J. Ma et al., “Preparative isolation of highly polar free radical inhibitor from Floccularia luteovirens using hydrophilic interaction chromatography directed by on-line HPLC-DPPH assay,” Journal of chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, vol. 1142, Article ID 122043, 2020.
View at: Google Scholar
B. Han, Z. Xin, S. Ma et al., “Comprehensive characterization and identification of antioxidants in Folium Artemisiae Argyi using high-resolution tandem mass spectrometry,” Journal of chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, vol. 1063, pp. 84–92, 2017.
View at: Google Scholar
X. Wang, G. L. Zuo, C. Y. Wang, H. Y. Kim, S. S. Lim, and S. Q. Tong, “An off-line DPPH-GC-MS coupling countercurrent chromatography method for screening, identification, and separation of antioxidant compounds in essential oil,” Antioxidants, vol. 9, Article ID 32756519, 2020.
View at: Google Scholar
L. Duan, L. Guo, K. Liu, E. H. Liu, and P. Li, “Characterization and classification of seven citrus herbs by liquid chromatography-quadrupole time-of-flight mass spectrometry and genetic algorithm optimized support vector machines,” Journal of Chromatography A, vol. 1339, pp. 118–127, 2014.
View at: Google Scholar
Y. Yang, S. S. Li, J. A. Teixeira da Silva, X. N. Yu, and L. S. Wang, “Characterization of phytochemicals in the roots of wild herbaceous peonies from China and screening for medicinal resources,” Phytochemistry, vol. 174, Article ID 112331, 2020.
View at: Google Scholar
Y. H. Lee, B. Kim, S. Kim et al., “Characterization of metabolite profiles from the leaves of green perilla (Perilla frutescens) by ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry and screening for their antioxidant properties,” Journal of Food and Drug Analysis, vol. 25, pp. 776–788, 2017.
View at: Google Scholar
C. Chu, H. X. Cai, M. T. Ren et al., “Characterization of novel astragaloside malonates from Radix Astragali by HPLC with ESI quadrupole TOF MS,” Journal of Separation Science, vol. 33, pp. 570–581, 2010.
View at: Google Scholar
L. W. Qi, H. Y. Wang, H. Zhang, C. Z. Wang, P. Li, and C. S. Yuan, “Diagnostic ion filtering to characterize ginseng saponins by rapid liquid chromatography with time-of-flight mass spectrometry,” Journal of Chromatography A, vol. 1230, pp. 93–99, 2012.
View at: Google Scholar
D. Shao, X. Liu, J. Wu et al., “Identification of the active compounds and functional mechanisms of Jinshui Huanxian formula in pulmonary fibrosis by integrating serum pharmacochemistry with network pharmacology,” Phytomedicine, vol. 102, Article ID 154177, 2022.
View at: Google Scholar
S. D. Zhou, X. Xu, Y. F. Lin, H. Y. Xia, L. Huang, and M. S. Dong, “On-line screening and identification of free radical scavenging compounds in Angelica dahurica fermented with Eurotium cristatum using an HPLC-PDA-Triple-TOF-MS/MS-ABTS system,” Food Chemistry, vol. 272, pp. 670–678, 2019.
View at: Google Scholar
X. Mei, Y. Wang, Z. Liu et al., “The chemical transformations for Radix Astragali via different alkaline wash conditions by quantitative and qualitative analyses,” Journal of Pharmacy Biomedicine Analytical, vol. 185, Article ID 113164, 2020.
View at: Google Scholar
Q. Q. Wang, Y. Tan, W. W. Liu, and X. Ding, “Advances in studies on chemical constituents pharmacological effects of Fritillaria,” in Proceedings of the 9th National Symposium on Natural Medicinal Material Resources Proceeding and Abstracts, Guangzhou, China, 2011.
View at: Google Scholar
Y. Zhang, W. T. Dong, J. H. Huo, and W. M. Wang, “Analysis on chemical constituents of Pheretima aspergillum by UPLC-Q-TOF-MS,” Chinese Traditional and Herbal Drugs, vol. 48, pp. 252–262, 2017.
View at: Google Scholar
X. L. Chang, W. Li, Z. H. Jia, T. Satou, S. Fushiya, and K. Koike, “Biologically active triterpenoid saponins from Ardisia japonica,” Journal of Natural Products, vol. 70, pp. 179–187, 2007.
View at: Google Scholar
J. Y. Bae, B. Avula, J. Zhao et al., “Analysis of prenylflavonoids from aerial parts of Epimedium grandiflorum and dietary supplements using HPTLC, UHPLC-PDA and UHPLC-QToF along with chemometric tools to differentiate Epimedium species,” Journal of Pharmacy Biomedicine Analytical, vol. 177, Article ID 112843, 2020.
View at: Google Scholar
X. P. Ding, X. T. Wang, L. L. Chen et al., “On-line high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry-chemiluminescence assay of radical scavengers in Epimedium,” Journal of Chromatography A, vol. 1218, pp. 9 1227–1235, 2011.
View at: Google Scholar
Y. L. Liu, S. Fu, L. J. Fan, H. J. Hu, L. F. Lin, and H. Li, “Review on differences in pharmacological, constituents and other aspects between Schisandrae chinensis fructus and Schisandrae sphenantherae fructus,” Chinese Journal of Experimental Traditional Medical Formulae, vol. 23, pp. 228–234, 2017.
View at: Google Scholar
X. Xiao, W. Ren, N. Zhang et al., “Comparative study of the chemical constituents and bioactivities of the extracts from fruits, leaves and root barks of Lycium barbarum,” Molecules, vol. 24, p. 1585, 2019.
View at: Google Scholar
H. Cai, G. Cao, and B. C. Cai, “Rapid simultaneous identification and determination of the multiple compounds in crude Fructus Corni and its processed products by HPLC–MS/MS with multiple reaction monitoring mode,” Pharmaceutical Biology, vol. 51, pp. 273–278, 2013.
View at: Google Scholar
T. Pires, M. I. Dias, L. Barros et al., “Antioxidant and antimicrobial properties of dried Portuguese apple variety (Malus domestica Borkh. cv Bravo de Esmolfe),” Food Chemistry, vol. 240, pp. 701–706, 2018.
View at: Google Scholar
K. R. Nalamolu and S. Nammi, “Antidiabetic and renoprotective effects of the chloroform extract of Terminalia chebula Retz. seeds in streptozotocin-induced diabetic rats,” BMC Complementary and Alternative Medicine, vol. 6, p. 17, 2006.
View at: Google Scholar
X. Song, Y. Wang, and L. Gao, “Mechanism of antioxidant properties of quercetin and quercetin-DNA complex,” Journal of Molecular Modeling, vol. 26, p. 133, 2020.
View at: Google Scholar
J. Zhu, X. Yi, J. Zhang, S. Chen, and Y. Wu, “Chemical profiling and antioxidant evaluation of Yangxinshi tablet by HPLC-ESI-Q-TOF-MS/MS combined with DPPH assay,” Journal of Chromatography B, vol. 1060, pp. 262–271, 2017.
View at: Google Scholar
R. Z. Guo, X. G. Liu, W. Gao et al., “A strategy for screening antioxidants in Ginkgo biloba extract by comprehensive two-dimensional ultra high performance liquid chromatography,” Journal of Chromatography A, vol. 1422, pp. 147–154, 2015.
View at: Google Scholar
Z. Yang, Z. Li, and Z. Li, “DPPH-HPLC-MS assisted rapid identification of endothelial protective substances from Xiao-Ke-An,” Journal of Ethnopharmacology, vol. 211, pp. 188–196, 2018.
View at: Google Scholar
E. Sommella, G. M. Conte, E. Salviati et al., “Fast profiling of natural pigments in different spirulina (Arthrospira platensis) dietary supplements by DI-FT-ICR and evaluation of their antioxidant potential by pre-column DPPH-UHPLC assay,” Molecules, vol. 23, pp. 1–15, 2018.
View at: Google Scholar
M. Alagawany, M. E. Abd El Hack, M. R. Farag et al., “Rosmarinic acid: modes of action, medicinal values and health benefits,” Animal Health Research Reviews, vol. 18, pp. 167–176, 2017.
View at: Google Scholar
Z. Liang, L. Wu, X. Deng et al., “The antioxidant rosmarinic acid ameliorates oxidative lung damage in experimental allergic asthma via modulation of NADPH oxidases and antioxidant enzymes,” Inflammation, vol. 43, pp. 1902–1912, 2020.
View at: Google Scholar
A. Oğuz, A. Böyük, A. Ekinci et al., “Investigation of antioxidant effects of rosmarinic acid on liver, lung and kidney in rats: a biochemical and histopathological study,” Folia Morphologica, vol. 79, pp. 288–295, 2020.
View at: Google Scholar
C. Y. Lu, C. H. Day, C. H. Kuo et al., “Calycosin alleviates H₂O₂-induced astrocyte injury by restricting oxidative stress through the Akt/Nrf2/HO-1 signaling pathway,” Environmental Toxicology, vol. 37, pp. 858–867, 2022.
View at: Google Scholar
Y. Xia, Y. Cao, Y. Sun et al., “Calycosin alleviates sepsis-induced acute lung injury via the inhibition of mitochondrial ROS-mediated inflammasome activation,” Frontiers in Pharmacology, vol. 12, Article ID 690549, 2021.
View at: Google Scholar

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