Profiling, screening, and determination of the antioxidant and anti-inflammatory constituents in Xuanfei Baidu granules by UHPLC-QTOF-MS and UHPLC-DAD

2.1. Sample, chemicals and reagents

XBG was provided by Buchang Pharmaceuticals Co., Ltd. Shandong, China. The 96-well microplates were purchased from Corning Life Sciences (Wujiang) Co., Ltd., Wujiang, China. All reagents of HPLC or analytical grade were purchased from Concord Technology (Tianjin) Co., Ltd., Tianjin, China. 1,1-diphenyl-2-picryhydrazyl (DPPH·), H₂O₂, salicylic acid, L-ascorbic acid, FeSO₄, monosaccharide standards (D-Mannose (Man), L-Rhamnose (Rha), D-Ribose (Rib), D-Glucuronic acid (GlcA), D-Galacturonic acid (GalA), D-Glucose (Glc), D-Galactose (Gal), D-Xylose (Xyl), L-Arabinose (Ara), D-Fucose (Fuc)), Tris-HCl buffer, luteolin-7-O-β-D-glycuronide (≥ 98%, Lot No. 5373-11-5) and acteoside (≥ 98%, Lot No. 61276-17-3) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd, Shanghai, China. COX-2 (human recombinant) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Verbenalin (≥ 98%, Lot No. 548-37-8), liquiritin apioside (≥ 98%, Lot No. 74639-14-8), isoacteoside (≥ 98%, Lot No. 61303-13-7), amygdalin (≥ 98%, Lot No. 29883-15-6), schaftoside (≥ 98%, Lot No. 51938-32-0), rhoifolin (≥ 98%, Lot No. 17306-46-6), isoliquiritin (≥ 98%, Lot No. 5041-81-6), glycyrrhizic acid (≥ 98%, Lot No. 1405-86-3), hastatoside (≥ 98%, Lot No. 50816-24-5), naringin (≥ 98%, Lot No. 10236-47-2), atractylenolide Ⅲ (≥ 98%, Lot No. 73030-71-4), isoschaftoside (≥ 98%, Lot No. 52012-29-0), and polydatin (≥ 98%, Lot No. 65914-17-2) were obtained from Sichuan Weikeqi Biological Technology Co., Ltd, Sichuan, China.

2.2. UHPLC-QTOF-MS analysis

The UHPLC-QTOF-MS analysis of XBG and mixed standard solutions was performed on an Agilent 1260 Infinity II UPLC system coupled with an Agilent 6550 QTOF™ high-resolution mass spectrometer (Agilent Technologies, California, USA). All experimental samples were analyzed employing both positive and negative ionization modes. Detailed sample preparation procedures and QTOF-MS related parameters have been provided in Section S1.

2.3. GNPS-based MS/MS classic molecular networking

Prior to GNPS-based MS/MS MN analysis, raw MS data files (the .d format, Agilent MassHunter) were converted to the .mzXML format using MSConvert 3.0.22085, following the established protocols. After conversion, the files were sent to the GNPS website (https://gnps.ucsd.edu/) using WinSCP 5.17.9 for subsequent processing [8]. In this study, XBG mass spectrometry data were analyzed, with spectrum files designated as follows: G1 (XBG sample) and G6 (blank control). The blank spectrum (G6) was excluded from network construction through pre-filtering. Data were processed in both ionization modes (Positive ion mode: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=c0a3d49bc353482d8d90dc508a035a27, negative ion mode: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=c89461d9afe347c9a88ca09ee9839ca7). Mass tolerance thresholds were set to 0.02 Da for both precursor and fragment ions, the smallest matching cosine value was 0.7, the maximum number of adjacent nodes of a node in the network was 10, and the minimum number of matched fragment ions between two nodes was six. Molecular networks were generated and exported by Cytoscape 3.8.2 for comprehensive network reanalysis in subsequent experiments. Molecular networks were generated and exported by Cytoscape 3.8.2 for comprehensive network analysis in subsequent stages.

2.4. In-house compound library

An in-house compound library was constructed by systematically collating the known chemical constituents from XBG and the 13 TCM components through comprehensive data mining of the online scientific databases (such as SciFinder, ScienceDirect, PubMed, CNKI, and HMDB). The 2D/3D structures, molecular formulas, molecular weights, exact masses, and reported MS fragmentation patterns of over 250 compounds mainly belonging to flavonoids, alkaloids, polysaccharides, saponins, and phenolic acids were included in this library. For preliminary compound identification, the experimental LC-MS data were analyzed using Agilent MassHunter Workstation 10.0 by matching against the in-house compound library through molecular formula comparison and MS/MS spectral searching.

2.5. Chemical composition analysis of XBG polysaccharides

Mix 15 batches of XBG evenly in equal proportions and conduct qualitative analysis on polysaccharides. The amounts of total sugars, protein, and uronic acid were determined through the application of the phenol-sulfuric acid method, the Bradford method, and the m-hydroxy-biphenyl method [9], respectively.

2.6. Antioxidant and anti-inflammatory evaluation of XBG fractions

Fractions of XBG were prepared on a preparative HPLC GX-281 system (GILSON, USA). The HPLCONE^® MCOLUMN-10C18 (50 mm × 250 mm) chromatographic column was utilized. The mobile phase of water (A) and methanol (B) ran at 59 mL/min. The gradient elution program was: 0-60 min, 5%–100% B; 60-70 min, 100% B. Eluents were collected according to the process chart, as shown in Figure S1, and evaporated to remove the solvent to obtain fractions XBG01-XBG27, with proportions of the refined fractions detailed in Table S1.

DPPH· and OH radical scavenging activities and reducing power of the above 27 fractions from XBG were evaluated using the reported method with slight adjustments [11], as detailed in the Section S3.

Further, the anti-inflammatory potentials of those fractions were determined using the Griess method [12] for their impact on the production of NO in LPS-injured RAW 264.7 macrophages, as detailed in Section S4.

2.7. Online DPPH·-UHPLC-DAD analysis

The online DPPH·-UHPLC-DAD analysis was developed based on a previously reported method [8]. The XBG sample was dissolved in 50% methanol-water and prepared as a 5 mg mL^-1 stock solution, while DPPH· was dissolved in methanol and prepared as a 4 mg mL^-1 DPPH· work solution and stored away from light. XBG stock solution (200 μL) and DPPH· work solution (400 μL) were mixed as the DPPH·-pretreated sample, while XBG stock solution (200 μL) mixed with methanol (400 μL) was prepared as the control sample. Both the DPPH·-pretreated sample and the control sample were then incubated in darkness for 30 min and centrifuged at 14,000 rpm for 10 min before injection into the UHPLC-DAD system (Agilent, CA, USA) equipped with Waters ACQUITY UPLC^® BEH C18 (100 mm×2.1 mm, 1.7 μm) column, with chromatographic and detection conditions optimized as follows: chromatographic separation was performed on at 30°C. The mobile phase was 0.1% formic acid in water (A) and methanol (B) at a flow rate of 0.3 mL/min, and the gradient elution program was set as: 0-2 min, 5-10% B; 2-10 min, 10-15% B; 10-17 min, 15-17% B; 17-20 min, 17% B; 20-23 min, 17-33% B; 23-27 min, 33-35% B; 27-31 min, 35-45% B; 31-35 min, 45-95% B; 35-40 min, 95%B; 40-41min, 95-5% B; 41-45min, 5% B. The injection volume was 3 μL. The detection wavelength was set at 254 nm.

2.8. Online COX2-UHPLC-DAD analysis

An online COX2-UHPLC-DAD analysis for discovering the COX-2 inhibitors in XBG was established based on a previously reported method [2]. The XBG sample was dissolved in 50% methanol-water and prepared as a 10 mg/mL stock solution, while COX-2 was dissolved in water and prepared as a 20 U/mL COX-2 work solution. XBG stock solution (40 μL), COX-2 work solution (1000 μL), and methanol (10 μL) were mixed as the COX-2 pretreated sample, while XBG stock solution (40 μL) mixed with water (1000 μL) and methanol (10 μL) was prepared as the control sample. Both the COX-2 pretreated sample and the control sample were then incubated at 37°C for 45 min and centrifuged at 14,000 rpm for 10 min before injection into the UHPLC-DAD system (Agilent, CA, USA). The chromatographic conditions employed in this procedure were consistent with those utilized in the online DPPH·-UHPLC-DAD analysis mentioned above.

2.9. Content determination of the Q-markers of XBG

Both XBG and mixed standard solutions were analyzed on a Waters ACQUITY UPLC^® BEH C18 (100 mm×2.1 mm, 1.7 μm) column, with the injection volume of 1 μL, column temperature at 25°C, and a flow rate of 0.3 mL/min. The elution program was performed simultaneously with 0.1% formic acid-water (A) and acetonitrile (B) as the mobile phases: 0-14 min, 6-8% B; 14-15 min, 8-13% B; 15-17 min, 13-15% B; 17-20 min, 15-17% B; 20-23 min, 17-33% B; 23-27 min, 33% B; 27-31 min, 33-98% B; 31-35 min, 98% B.

Q-markers for XBG were selected, adhering to the acknowledged principles of Q-markers for TCM. And therein, the content determination method of Q-markers in XBG was established on an Agilent UHPLC 1290 liquid system equipped with a DAD detector, after method validation of repeatability, stability, precision, sample recovery, linear range, and limit of detection (LOD) & limit of quantification (LOQ), as detailed in Tables S2-S7.

3.1. Identification of the small molecular component in XBG

Herein, we used a comprehensive analysis method to identify small molecule components in XBG, including UHPLC-QTOF-MS analysis, MS/MS classical MN analysis based on the GNPS, in-house compound library retrieval, and validation of mass spectrometric fragmentation patterns of reference standard compounds.

Firstly, after analyzing XBG samples using the UHPLC-QTOF-MS method, a classical molecular network was created, in which network nodes were automatically annotated with corresponding MS/MS spectra through spectral library search [8]. Information on precursor mass, retention time, and MS/MS fragmentation is concentrated as a node in the molecular network. The clustered network components were nodes with highly similar MS/MS patterns. There were 685 nodes in the molecular network of XBG, of which 200 nodes were successfully clustered (Figures S2-S3), and 71 compounds (Table 1) were identified.

Secondly, we combined an in-house compound library with the HMDB database to identify compounds by matching MS/MS spectral data. Due to incomplete inclusion of compounds in online databases, this step can make the identification results more comprehensive. In addition, the fragmentation pathways of the specific compounds were elucidated by comparing them with standard compounds, thereby identifying other components with similar fragmentation patterns. Combining the determination of fragmentation patterns with the in-house compound library search may be a key method for identifying far more unknown compounds. Herein, we identified another 72 compounds (Table 1) from XBG, including phenylpropanoids, iridoids, flavonoids, bibenzyls, anthraquinones, and triterpenoids. The TIC chromatograms of XBG afforded by UHPLC-QTOF-MS analysis have been supplied in Figures S4-S6.

In one word, we adopted an integrative strategy of UHPLC-QTOF-MS, MN, reference standard comparison, and in-house library retrieval to comprehensively identify the chemical constituents in XBG (Table 1), with the expectation of laying the foundation for further chemical space exploitation of XBG. As a result, 143 constituents were identified by this comprehensive analysis method, far more than those ever reported from XBG or XBF [2], including flavonoids, alkaloids, triterpenoids, anthraquinones, iridoid glycosides, and organic acids.

3.2. Profiling the polysaccharide component of XBG

As shown in Table S8, the contents of total sugar and uronic acid were relatively higher than other components in XBG. Thus, it is of great significance to perform an in-depth profiling of the polysaccharides in XBG to comprehensively elaborate on its chemical composition.

HPLC-ELSD analysis revealed that there were mainly two or more homogeneous polysaccharides in XBG, as shown in Figure 2(a). By comparing the retention times with the dextran standards, it can be concluded that the molecular weight of peak 1 is greater than 670 kDa, and the molecular weight of peak 2 is approximately 5 kDa.

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Figure 2.

Profiles of the XBG polysaccharides. (a) HPLC-ELSD Chromatograms of XBG; (b) HPLC-DAD (254 nm) Chromatograms of the ten mixed monosaccharide standards [mannose (Man, 18.5 min); ribose (Rib, 24.3); rhamnose (Rha, 25.2 min); glucuronic acid (GlcA, 30.8 min); galacturonic acid (GalA, 37.3 min); glucose (Glc, 41.2 min); galactose (Gal, 48.1 min); xylose (Xyl, 50.3 min); arabinose (Ara, 52.2 min); fucose (Fuc, 59.7 min)] (A) and XBG polysaccharides (B) after PMP derivation; (c) ¹H NMR (600 MHz, D₂O) Spectrum of XBG polysaccharides; (d) ¹³C NMR (500 MHz, D₂O) Spectrum of XBG polysaccharides; (e) FT-IR absorbance spectrum of XBG polysaccharides.

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As shown in Figure 2(b), PMP-HPLC analysis of XBG polysaccharides and the ten standard monosaccharides revealed that XBG polysaccharides constituted mainly six common monosaccharides, namely Man, Rha, GalA, Glc, Gal, and Ara, with a content ratio of 1.2: 2.3: 8.5: 36.8: 5.5: 3.8. Proton resonances at δ_H 4.3-4.8 ppm and δ_H 4.8-5.4 ppm in the ¹H NMR spectrum (Figure 2c) showed the presence of β and α-glycosidic bonds in XBG polysaccharides. The ¹³C NMR spectrum (Figure 2d) displayed three main resonance signals at δ_C 90-110 ppm, indicating the presence of at least three glycosidic linkage configurations or types of sugar residues in the XBG polysaccharide component. Specifically, the dominant carbon signal at δ_C 103.25 was typically attributed to the β-configured glycosidic linkages, while the signals at δ_C 101.86 and 99.68 originated from the α-configured glycosidic linkages. Multiple carbon signals at δ_C 52.91–81.10 were assigned to those sugar methines (C2, C3, C4, C5, C6), and the signals at δ_C 18.43 and 13.12 were tentatively assigned to the characteristic methyls of 6-deoxyhexoses such as rhamnose. Further, as shown in Figure 2(e), FT-IR spectrum of XBG polysaccharides showed an intense absorption band at 3365 cm⁻¹ corresponding to the stretching of hydroxyl groups, an absorption at 2934.1 cm⁻¹ attributable to the C–H asymmetric stretching vibration of –CH₂–, a prominent absorption at 1614.4 cm⁻₁, corresponding to C=O asymmetric stretching of carboxylate (COO⁻) moieties in uronic acid residues, and two absorption peaks at 1416.49 cm⁻¹ and 1195.37 cm⁻¹ assignable to the C–O stretching vibrations of carboxyl groups and the O–H deformation vibrations in carboxylic acids (–COOH), respectively. Notably, the absorption bands observed at 1200–1000 cm⁻¹ belonged to the C–O–C bending vibrations (glycosidic linkages) and the C–O–H deformations, confirming the presence of characteristic polysaccharide structural features of XBG, including glycosidic bonds and free hydroxyl groups.

XBG polysaccharides may be derived from GRR, PR, Artemisiae Annuae Herba (AAH), Coicis Semen (CS), Citri Grandis Exocarpium (CGE), EH, and AR, however, it was quite hard to specify which TCM component of XBG the polysaccharides derived from. As is known, EH polysaccharide exerts immune regulatory and anti-hyperlipidemia activities in treatment of autoimmune diseases [13]. AR polysaccharide possesses the effects such as influencing the body’s immune regulatory mechanisms, aiding in the correction of abnormalities in glucose metabolism, and protecting liver [14]. GRR polysaccharide was reported to show biological activities such as antioxidant, anti-inflammatory, and anti-tumor [15]. AAH polysaccharide has immunomodulatory, anti-inflammatory, and antioxidant effects [16]. CGE polysaccharide could reduce oxidative damage, inhibit apoptosis, and reduce the level of NO in brain tissues [17]. PCRR polysaccharide has hypoglycemic and anti-inflammatory potential [18]. CS polysaccharide exerts many pharmacological activities. These include anti-oxidation, enhancing immunity, and lowering blood sugar [19]. PR polysaccharide exhibits immune regulatory effect [20]). Therefore, we can infer that XBG polysaccharides may alleviate the symptoms for COVID-19 patients benefiting from their pharmacological activities such as antioxidation and anti-inflammation.

3.3. Antioxidant and anti-inflammatory activities of XBG fractions and components

Fractions and components of XBG were prepared, and their antioxidant and anti-inflammatory activities were evaluated, as shown in Section S5. The antioxidant and anti-inflammatory potential results of XBG fractions have been presented in Table S9.

As depicted in Figure 3, the DPPH· radical scavenging assay revealed that XBG22 and XBG27 demonstrated the lowest scavenging efficacy, with respective activities of 10.033% and 8.876%. In contrast, XBG03 and XBG07 exhibited the most potent activity, with scavenging rates of 20.463% and 23.124%, respectively. Scavenging rates of the remaining fractions fell within a moderate range of 11.3% to 18.6%. As a result of the OH radical scavenging assay, XBG23 and XBG26 were identified as the least effective fractions, with activities of 9.047% and 4.331%, respectively. Notably, XBG02 stood out with the highest activity at 48.361%, while other fractions displayed a variable range of scavenging rates from 10.411% to 29.186%. In the reducing power assay, XBG01 and XBG16 were found to have the lowest activity, with 9.712% and 9.273%, respectively. XBG02 once again demonstrated the most significant activity at 27.145%, with other fractions showing a range of 10.444% to 15.998% in their reducing capabilities. Collectively, these results underscore the exceptional antioxidant potential of XBG02, which consistently outperformed all assays, highlighting its promising role as a potent antioxidant agent.

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Figure 3.

UHPLC chromatograms and activity bars of XBG fractions. (1) Hastatoside, (2) verbenalin, (3)3,4-dihydroverbenalin, (4) polydatin, (5) acteoside, (6) isoacteoside, and (7) naringin.

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Against the background of in vitro anti-inflammatory activity of macrophages, the NO production rates for fractions XBG11, XBG14, XBG16, XBG18-XBG19, XBG21, and XBG24 were found to be lower than 50%. This observation suggested that these fractions exhibited a relatively diminished capacity to modulate inflammation. Conversely, the NO production rates for the fractions XBG01-XBG05, XBG07-XBG08, XBG10, XBG13, and XBG26 exceeded 70%, indicating a robust anti-inflammatory propensity. These results revealed the potential of the latter fractions as potent modulators of inflammatory responses for XBG.

In a word, fractions XBG01-XBG05, XBG07-XBG08, XBG13, XBG15, XBG17, and XBG20 were found to demonstrate excellent antioxidant and anti-inflammatory activities. They showed superior performances in both scavenging free radicals and NO production, with potential as the key indicators of antioxidant and anti-inflammatory efficacy for XBG. Their activity profiles suggested that these fractions hold significant potential for further exploration in the context of inflammation and oxidative stress-related therapeutic applications. Notably, we found fractions XBG01-XBG05 exhibited obvious antioxidant and anti-inflammatory activities and were composed of polysaccharides. Therefore, we speculated that polysaccharides in XBG possessed valuable antioxidant and anti-inflammatory potentials, which requires further convincing evidence in our future study.

3.4. Online screening of XBG constituents with antioxidant and anti-inflammatory potentials

Then, online DPPH·-UHPLC-DAD and COX2-UHPLC-DAD analyses were applied for screening the antioxidant and anti-inflammatory contributor constituents from XBG. As shown in Figure 4(a), the chromatographic peak areas of 3,4-dihydroverbenalin, polydatin, acteoside, isoacteoside, and naringin decreased significantly after pretreatment with DPPH· radicals, indicating that these five compounds are the main contributors to the DPPH· radicals scavenging activity of XBG. Similarly, as shown in Figure 4(b), acteoside and naringin potently inhibited COX-2 enzyme activity, suggesting that they are two constituents contributing positively to the overall anti-inflammatory activity of XBG.

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Figure 4.

Chromatograms of (a) DPPH·-UHPLC-DAD and (b) COX2-UHPLC-DAD for XBG. (1) Hastatoside, (2) verbenalin, (3) 3,4-dihydroverbenalin, (4) unknown, (5) polydatin, (6) unknown, (7) acteoside, (8) isoacteoside, and (9) naringin.

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As far as we know, among those key constituents from XBG, polydatin alleviated inflammation by reducing NF-κB and mitigated oxidative stress by upregulating glioma-associated oncogene homolog1 (Gli1), Patched-1 (Ptch1) and Superoxide dismutase 1 (SOD1) secretion [21], and acteoside and isoacteoside exhibited their antioxidative and anti-inflammatory activities in vitro (DPPH· assay, TBARS assay on Cu ²⁺-induced oxidized LDL, PGE2 assay) and in vivo (acetic acid injured vascular permeability and carrageenan-injured hind paw edema in rats) [12], and acteoside protected pulmonary endothelial cells against membrane lipid oxidation and free radical-mediated impairment [22]. Further, naringin reduced the expression of inflammatory response-associated signaling factors, e.g., IL-6, IL-8, iNOS, Nrf2, and TNF-α [23], and naringin showed a dose-dependent radical scavenging activity against 1,1-diphenyl-2-picryl-hydrazyl and tetraethylammonium chloride radicals [24]. Herein, our study firstly uncovered that polydatin, acteoside, isoacteoside, and naringin played key roles in the antioxidative activity of XBG, while acteoside and naringin were crucial for XBG to exert its anti-inflammatory effect.

Notably, 3,4-dihydroverbenalin, polydatin, acteoside, isoacteoside, and naringin were found to be present in fractions XBG16, XBG18, XBG19, XBG22, and XBG23, respectively. These fractions exhibited varying degrees of antioxidant and anti-inflammatory activities, which were generally consistent with the abovementioned activity screening results from those fractions and components of XBG.

3.5. Selection of Q-markers for XBG and their content determinations

Although the chemical composition of XBG is relatively clarified [3], Q-marker prediction and evaluation are still needed for its global quality control, allowing further exploitation of its clinical indications and applications. Generally, Q-markers for TCM prescriptions are required to comply with the “five principles” simultaneously, including prescription compatibility, transfer and traceability, specificity, pharmacological effectiveness, and measurability [25].

3.5.5. Measurability

The measurability of chemical components is crucial for global quality control and in-depth efficacy evaluation of a TCM prescription, and a Q-marker needs to be detectable. As is known, the qualitative and quantitative analyses methods and techniques for the polysaccharides in natural medicines are still relatively lacking. However, flavonoids, phenylpropanoids, terpenoids, alkaloids, bibenzyls, and other small molecular constituents in XBG, as exemplified by the main effective constituents such as 3,4-dihydroverbenalin, polydatin, acteoside, isoacteoside, and naringin, could be determined with LC, LC-MS, and other techniques. Therefore, instead of the macromolecular polysaccharides, representative constituents in the small molecular component of XBG were accordingly selected as the Q-markers based on their stability, convenience, and maturity of detection.

Finally, based on the above results, hastatoside, naringin, verbenalin, polydatin, acteoside, glycyrrhizic acid, and isoacteoside could be used as the small molecular Q-markers as the key indicators for quality control of XBG. The content determination of these constituents in 15 batches of XBG was established, as detailed in Table 2, with UHPLC chromatograms of mixed standard substances and XBG, as shown in Figure 5. The contents (%) of hastatoside, verbenalin, polydatin, acteoside, isoacteoside, naringin, and glycyrrhizic acid in XBG were determined as 3.350 ± 0.0502%, 2.641 ± 0.0499%, 2.459 ± 0.0714%, 1.299 ± 0.0661%, 0.958 ± 0.0343%, 26.598 ± 0.609%, and 1.819 ± 0.0542%, respectively.

Table 2. Contents of 7 observed constituents in 15 batches of XBG.

No.	Content (%)
	Hastatoside	Verbenalin	Polydatin	Acteoside	Isoacteoside	Naringin	Glycyrrhizic acid
1	3.331	2.772	2.525	1.231	0.929	27.463	1.792
2	3.318	2.514	2.744	0.982	1.150	28.308	2.180
3	3.399	2.555	2.489	1.575	0.835	26.453	1.839
4	3.549	2.834	2.191	1.538	0.946	26.734	1.778
5	3.309	2.534	2.203	1.407	0.857	26.717	1.646
6	3.240	2.549	2.375	0.812	1.162	26.742	1.894
7	3.501	2.919	2.440	1.720	0.815	26.454	1.893
8	3.203	2.459	2.157	1.354	0.817	26.151	1.518
9	3.529	2.790	2.535	1.687	0.897	25.340	1.918
10	2.790	2.302	1.829	1.069	0.730	19.961	1.282
11	3.408	2.607	2.457	1.253	1.061	29.891	1.903
12	3.466	2.817	2.843	1.209	1.089	29.269	1.959
13	3.555	2.829	2.826	1.307	1.033	28.616	1.940
14	3.431	2.789	2.667	1.219	1.071	26.758	1.928
15	3.220	2.344	2.601	1.125	0.980	24.111	1.817

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Figure 5.

UHPLC chromatograms of (a) mixed standard substances and (b) XBG. (1) Hastatoside, (2) verbenalin, (3) polydatin, (4) acteoside, (5) isoacteoside, (6) naringin, and (8) glycyrrhizic acid.

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The therapeutic efficacy of TCM compounds is the result of multi-component, multi-target, and multi-channel interaction, and clarifying the specific mechanisms of key Q-markers is critical for interpreting their clinical values. For XBG, the seven selected Q-markers (hastatoside, naringin, verbenalin, polydatin, acteoside, glycyrrhizic acid, and isoacteoside) may exhibit distinct yet complementary roles in anti-inflammation, antioxidation, and other activities, collectively forming a robust therapeutic network.

However, in this study, we failed to clarify the synergistic or antagonistic effects between components of XBG, as well as their active or functional mechanisms in the overall effect of this TCM compound. Moreover, due to the small number and representativeness of XBG samples, further in-depth study is needed for the inter-batch consistency evaluation of the dominant pharmacodynamic components. Our study suggests that future studies on XBG should focus on both the small molecular and the polysaccharide components, covering the extraction and transfer optimization of these components from the twelve TCM components to the final XBG preparation, the active and functional positioning of the components, and further verification and application of the in vivo real pharmacodynamic substances of Q-markers based on the dominant components. Modern systematic, pharmacological, molecular biological, and multi-omics techniques, as well as chemoinformatics, machine learning, and computational methods, will be involved in future studies of the complex TCM compounds.