Abstract Background: Yupingfeng San (YPFS) is a classic formula for treating allergic rhinitis (AR), which is composed of Astragalus mongholicus Bunge (AST), Atractylodes macrocephala Koidz (AMR), and Saposhni-kovia divaricata (Turcz.) Schischk (SR) at a ratio of 3:1:1. However, the potential bioactive components of YPFS relevant to AR treatment are currently unknown. Methods: This study combined in vivo chemical profiling, network pharmacology, and experimental validation to identify the substances in YPFS that are active against AR. Results: Firstly, 98 compounds in YPFS were identified using high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) with the assistance of Global Natural Products Social (GNPS) molecular networking. Then, 42 prototype components and 57 metabolites were detected in the plasma, urine, and feces of mice with AR. A network pharmacological analysis based on 42 in vivo prototypical components was also conducted to screen 15 key components and 10 core targets, and 6 key components were further selected through molecular docking. Finally, the four key active components (cimifugin, wogonin, formononetin, and atractylenolide I) were revealed to be the main ingredients of YPFS through validation (in vitro and in vivo). Conclusions: This is the first systematic study of the components of YPFS in AR mice, laying the foundation for elucidating the overall material basis of this formulation. This study provides rich basic data for further pharmacological and mechanistic studies on YPFS. Keywords: Yupingfeng San, allergic rhinitis, prototype component, network pharmacology, molecular docking 1. Introduction Allergic rhinitis (AR), a common allergic condition, is triggered by immunoglobulin E (IgE) in response to allergens such as pollen [[28]1,[29]2]. As per the 2020 publication of the White Book by the World Allergy Organization, the worldwide occurrence of allergic disorders varies, ranging from 10 to 40% of the population, with 400 million cases of AR, urticaria, and anaphylactic shock [[30]3,[31]4]. In China, epidemiological studies showed a high prevalence of AR, affecting approximately 250 million people (17.6%) with a continuous upward trend [[32]2]. Therefore, developing new medications for treating AR is of great practical importance. Yupingfeng San (YPFS) has demonstrated remarkable effectiveness in treating AR, authored Danxixinfa by Zhu Zhenheng during the Yuan Dynasty, and has demonstrated remarkable effectiveness in treating AR. It is composed of three herbs—Astragalus mongholicus Bunge (AST), Atractylodes macrocephala Koidz (AMR), and Saposhnikovia divaricata (Turcz.) Schischk (SR)—at a ratio of 3:1:1 [[33]5]. The chemical components of YPFS are diverse, encompassing flavonoids, saponins, coumarins, chromones, and lactones [[34]5]. However, the material basis on which YPFS treats AR remains unclear, and its complex metabolism in the body complicates the issue [[35]6]. Therefore, it is imperative to employ modern scientific methods to elucidate the material basis and mechanism by which YPFS treats AR. Most prior studies have primarily focused on validating the effectiveness of YPFS in mitigating inflammatory factors in AR [[36]7,[37]8]. Some reports have also identified the absorbed components of YPFS in the blood of rats [[38]9]. However, the prototype components of YPFS in AR models have not been reported. Numerous studies have demonstrated that the absorption and metabolism of traditional Chinese medicine (TCM) differ significantly in pathological conditions compared to natural and healthy states [[39]10,[40]11]. Hence, it is important to identify the essential components of YPFS for managing AR. This study introduced a systematic approach integrating high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC–ESI-Q-TOF-MS/MS) and Global Natural Product Social Molecular Networking (GNPS) to comprehensively identify the chemical components of YPFS. In vivo, we identified the prototype components and metabolites in the plasma, urine, and feces of AR mice after the oral intake of YPFS using mass spectrometry. Then, we constructed a network of the interactions between the prototype components of YPFS and AR-related targets to identify the key components and core targets and predict the potential pathways involved in the regulation of AR. The relationships between the core components and targets were further evaluated through molecular docking, and the components’s anti-AR effects were validated in RBL-2H3 cells and AR mice. This study serves as a foundation for understanding the therapeutic potential of YPFS in AR and provides a scientific basis for screening active ingredients in other medications. 2. Results 2.1. Optimization of Chromatography Conditions The parameters were optimized to increase the separation of YPFS and improve the peak shape and number of peaks. As depicted in [41]Figure S1, the findings revealed that a sample size of 5 mg, flow rate of 1 mL/min, column temperature of 30 °C, and wavelength of 254 nm were the most conducive to the effective separation and identification of the compounds within YPFS. The RSD values of all components were less than 3%, indicating that the method exhibits good repeatability, precision, and stability ([42]Table S1–S3). 2.2. Chemical Profile of YPFS The chemical profile of YPFS was systematically analyzed using manual identification and GNPS methods. A total of 98 compounds ([43]Figure 1A and [44]Table S4) were identified, comprising 20 flavonoids, 8 saponins, 11 lactones, 4 sesquiterpenoids, 10 chromones, 13 coumarins, 12 amino acids, 3 organic acids, and 17 other compounds ([45]Figure 1B). Figure 1. [46]Figure 1 [47]Open in a new tab A total of 98 chemical components of YPFS identified through manual identification and GNPS in positive ion mode. (A) Base peak chromatograms of YPFS obtained using HPLC–Q-TOF-MS in positive modes. (B) Types of chemical compositions in YPFS. The molecular network of YPFS was established using GNPS for the analysis of natural products [[48]12]. As depicted in [49]Figure S2, the generated molecular network comprised 39 clusters (each cluster containing ≥ 2 nodes), totaling 388 nodes, with 89 feature components predicted. The data were subject to the rigorous deduplication and deletion of the endogenous components based on comparisons with literature, chemical compound databases, and MS data [[50]5,[51]13]. In total, 36 potential chemical components of YPFS were obtained. Their chemical structures are shown in [52]Figure 2, including six flavonoids, seven amino acids, two saponins, three organic acids, one chromone, six coumarins, four lactones, and seven other compounds. Notably, 18 components were consistent with previous manual identification predictions, further validating the methodology. This analytical method expedited the identification of chemical constituents within intricate natural product matrices. Figure 2. [53]Figure 2 [54]Open in a new tab The structures of common components identified manually and using GNPS. The node color indicates the type of compound (red: flavonoids; green: saponins; purple: coumarin; blue: lactone; pink: amino acids; orange: others; pale yellow: organic acids). Using the degree of similarity in the MS/MS spectra of individual compounds, which ranges from 0 to 1 in cosine similarity, compounds could be grouped into clusters within a molecular network. This clustering method enabled the rapid classification of components [[55]14]. As illustrated in [56]Figure 2 and [57]Figure S2, the node associated with the ion at m/z 431.134 corresponded to the molecular formula C[22]H[22]O[9]. Analysis of the MS/MS spectrum revealed fragment ions with mass-to-charge ratios of 269, 254, and 137, which are indicative of glycoside ligands, methyl groups, and compounds formed through the retro-Diels–Alder reaction (RDA). By cross-referencing the retention time and MS data with existing databases and scientific literature, the compound was conclusively identified as ononin [[58]15]. The node of the ion at m/z 447.129 was adjacent to the node of ononin, suggesting a high degree of MS/MS spectral similarity with an excimer ion difference of 16 Da. Based on literature reports [[59]16,[60]17], this node had been identified as sissotrin. Similarly, other nodes were identified through this process. 2.3. Characterization of Compounds Compounds with similar structures exhibited comparable cleavage patterns. Therefore, it is imperative to analyze the cleavage rules of the prototype components in order to accurately identify the metabolic components of YPFS in vivo. In this study, we analyzed several typical compounds present in YPFS. 2.3.1. Flavonoids Initially, 19 flavonoids were identified in YPFS, primarily originating from AST. These flavonoids were identified based on characteristic neutral losses of CO (28 Da), CO[2] (44 Da), and CH[3] (15 Da). Calycosin ([61]Figure 3A) and ononin ([62]Figure S3A) exhibited the same cleavage pathway [[63]18,[64]19]. The cleavage rules of flavonoids were represented by the example of calycosin. Calycosin exhibited an [M + H]^+ ion at m/z 285.0753, which was formed by the loss of glucose (C[6]H[10]O[5], 162 Da) from calycosin-7-O-glucoside. The fragment ion at m/z 285.0753 [M + H]^+ further generated the fragment ions at m/z 270.0509, m/z 213.0528 and m/z 137.0221 through losing CH[3] (15 Da), C[3]H[4]O[2] (72 Da) and RDA. Additionally, fragment ions at m/z 225.1409 and 197.0717 were produced through the sequential loss of CO (28 Da) from the fragment ion at m/z 270.0509. Figure 3. [65]Figure 3 [66]Open in a new tab Chemical fragmentation pathways of chemical components in YPFS. (A) calycosin; (B) atractylenolide III. 2.3.2. Lactones Analogously, 12 lactones were identified in YPFS, primarily originating from AMR. The cleavage rules of lactones were represented by the example of atractylenolide III ([67]Figure 3B). Atractylenolide III exhibited an [M + H]^+ ion at m/z 249.1480. Fragment ions at m/z 231.1374 and m/z 213.1277 were generated through the sequential loss of H[2]O (18 Da) from atractylenolide III. The fragment ion at m/z 213.1277 underwent further loss of CO (28 Da) to form the fragment ion at m/z 185.1294. Additionally, fragment ions at m/z 203.1437 and m/z 175.0745 were formed by the sequential removal of CO (28 Da) from the fragment ion at m/z 231.1374. Lastly, the fragment ions at m/z 163.0746 and m/z 189.0927 were generated by the loss of C[5]H[8] (68 Da) and C[3]H[6] (42 Da) groups from the fragment ion at m/z 231.1374. 2.3.3. Coumarins A total of 12 coumarins were identified in YPFS, primarily originating from SR. These coumarins could be classified into two distinct categories, furanocoumarins and coumestrol, based on the structure of their parent nuclei. The characteristic fragment ions of coumarins, specifically those at m/z 131, 143, and 159, were detected in positive mode [[68]20]. Psoralen ([69]Figure S3B) and imperatorin ([70]Figure S4A) shared the same cleavage pathway. Taking imperatorin as an example, the cleavage rule of coumarins is described below. Imperatorin exhibited an [M + H]^+ ion at m/z 271.0956, whose cleavage rule involved the continuous loss of CO (28 Da). The fragment ion at m/z 203.0330 was obtained through the loss of the C[5]H[8] (68 Da) group. There were three possible fragmentation pathways: Firstly, fragment ions at m/z 175.0361, 147.0458, and 119.0856 were produced by the continuous loss of CO (28 Da) from the fragment ion at m/z 203.0330. Alternatively, the fragment ion at m/z 203.0330 could lose CO[2] (44 Da) to form the fragment ion at m/z 159.0965, which subsequently lost CO (28 Da) to form the fragment ion at m/z 131.1069. Additionally, the fragment ion at m/z 203.0330 could lose H[2]O (18 Da) to obtain the fragment ion at m/z 185.0900. Fragment ions at m/z 157.0509 and 129.0552 were generated by the continuous lost of CO (28 Da) from the fragment ion at m/z 185.0900 [[71]21]. 2.3.4. Chromones Currently, the chemical constituents characterized by SR primarily comprise two distinct classes: dihydrofuran chromogen ketones and dihydropyran chromogen ketones. Dihydrofuran chromogen ketones are more abundant, including cimifugin and 5-O-methylvisamminoside, among others. Taking the compound cimifugin ([72]Figure S4B) as an example, the cleavage rule of coumarins was introduced below. Cimifugin exhibited [M + H]^+ at m/z 307.1177. The parent ion at m/z 307.1177 lost H[2]O (18 Da) to form the fragment ion at m/z 289.0778. The parent ion at m/z 307.1177 further lost H[2]O (18 Da) and 2 CH[3] (30 Da), resulting in the fragment ion at m/z 259.0596. The fragment ion at m/z 221.0412 was generated through the loss of the C[3]H[2] (38 Da) group from the fragment ion at m/z 259.0596. The parent ion at m/z 307.1177 lost C[4]H[8]O (72 Da) to generate the fragment ion at m/z 235.1186. The fragment ion at m/z 205.0884 was formed through the loss of CH[2]O (30 Da) from the fragment ion at m/z 235.1186. Lastly, the fragment ion at m/z 177.0554 was obtained through the loss of CO (28 Da) from the fragment ion at m/z 205.0884. 2.4. Identification of YPFS Prototype Components In Vivo To differentiate prototype components from metabolites, we designated compounds detected in drug-associated biological fluids and YPFS extracts as prototype components. Conversely, compounds exclusively detected in the drug-associated biological fluid but absent in the YPFS extract were classified as metabolites. Under the same analytical conditions, mass spectrometry was utilized to analyze plasma, urine, and feces obtained from AR mice treated with YPFS. To screen for absorbed components, total ion chromatogram (TIC) and extraction ion chromatogram (EIC) curves were employed. Combining the results with those obtained from the in vitro analysis of YPFS, a total of 42 prototype components were identified ([73]Table S5), with 11, 28, and 29 prototype components found in the plasma, urine, and feces, respectively. The base peak chromatograms of YPFS are presented in [74]Figure 4A–F, and the structures of the compounds are shown in [75]Figure S5. These compounds include 12 flavonoids, 11 chromones, 6 saponins, 5 lactones, 3 sesquiterpenoids, 2 coumarins, and 3 other compounds. [76]Figure S6 shows the presence of 42 prototype components across the three biological fluids examined. Six prototype components were commonly detected in all three body fluids: blood, urine, and feces. These components were prim-O-glucosylcimifugin, atractylenolide I, formononetin, astramembrannin II, calycosin-7-O-glucoside, and cimifugin. Figure 4. [77]Figure 4 [78]Open in a new tab The total ion chromatography (TIC) of YPFS in biological and blank samples in positive ion mode and prototypical components of YPFS in vivo in AR mice. (A) YPFS plasma sample; (B) blank plasma sample; (C) YPFS urine sample; (D) blank urine sample; (E) YPFS feces sample; (F) blank feces sample. 2.5. Identification of YPFS Metabolites in Vivo Through the in vitro chemical composition analysis of YPFS, the metabolic pathways of its components were elucidated by cross-referencing data from public databases and literature with the cleavage outcomes for YPFS component precursors. A total of 57 metabolites of YPFS were provisionally identified, including 39 metabolites in plasma, 46 in urine, and 28 in feces. All the identified metabolites are listed in [79]Table S6. 2.5.1. Flavonoid Metabolites As depicted in [80]Figure 5 and [81]Figure S7A, 37 metabolites (M1–M37) were identified as flavonoid-related metabolites in plasma, urine, and feces. These metabolites were primarily associated with formononetin, calycosin, calycosin-7-O-glucoside, ononin, and methylnissolin. The pathway of calycosin’s metabolism was used as an example for clarification. Figure 5. [82]Figure 5 [83]Open in a new tab The possible metabolic pathways of formononetin, calycosin, and calycosin-7-O-glucoside from YPFS in AR mice. As depicted in [84]Figure 5, the metabolites of calycosin were identified as M12-M24. Initially, the phase I metabolite-reaction products included M12, M13, M16, and M24. Calycosin exhibited an [M + H]^+ ion at m/z 285.0753, and M12 exhibited an [M + H]^+ ion peak at m/z 271.0619, which was formed by losing 14 Da reduction compared to calycosin. The fragment ion with m/z 137.0244 corresponded to the peak of calycosin, suggesting that M12 could be a demethylated derivative of calycosin. M13 displayed an [M + H]^+ ion peak at m/z 269.0830, revealing a 16 Da decrease compared to calycosin. Its fragment ion at m/z 213.0933 aligned with calycosin, leading to the hypothesis that M13 (formononetin) might be a dehydroxylated product of calycosin. M16 presented an [M + H]^+ ion peak at m/z 255.0676, demonstrating a 30 Da decrement from calycosin. The fragment ions observed at m/z 227.0723 and 199.0764 matched those of calycosin, indicating the possibility that M16 was a demethylated and dehydroxylated variant of calycosin. M24 displayed an [M + H]^+ ion peak at m/z 301.0734, indicating a 16 Da increase over calycosin. Based on its molecular formula and fragmentation pattern, it was inferred that M24 could be a hydroxylated derivative of calycosin. Phase II metabolite-reaction products were categorized into methylation, glucuronidation, and sulfation metabolites. M18 showed an [M + H]^+ ion peak at m/z 299.0936, indicating a 14 Da increase over calycosin, and the fragment ion of m/z 197.0655 corresponded to calycosin, suggesting that M18 was a calycosin methylation product. M14 exhibited an [M + H]^+ ion peak at m/z 447.0955, indicating a mass increase of 176 Da over M12. The fragment ions observed at m/z 225.0702 closely resembled those of calycosin, suggesting that M14 may be the glucuronidation product of M12. Similarly, M17 displayed an [M + H]^+ ion peak at m/z 431.1015, demonstrating a mass increase of 176 Da over M16. It was proposed that M17 was the glucuronidation product of M16. M19 showed an [M + H]^+ ion peak at m/z 475.123, indicating a mass increase of 176 Da over M18. Combined with fragment ions that were identical to the methylation product of calycosin, M19 was determined to be the glucuronidation product of M18. M22 showed an [M + H]^+ ion peak at m/z 461.1082, indicating a mass increase of 176 Da over calycosin. The fragment ions at m/z 225.0580 and 285.0783 matched those of calycosin, suggesting that M22 could be the glucuronidation product of calycosin. M15 displayed an [M + H]^+ ion peak at m/z 351.018, exhibiting a mass variance of 80 Da from M12. Consequently, it was conceivable that M15 could be a sulfated form of M12. Similarly, M20 displayed an [M + H]^+ ion peak at m/z 365.0333, showing a comparable mass increment of 80 Da compared to calycosin. The fragment ions with m/z values of 253.0498 and 225.0544 coincided with those of calycosin, indicating that M20 might also be a sulfation product of calycosin. M23, with the molecular formula C[15]H[10]O[7]S, showed an [M + H]^+ ion peak at m/z 335.0248, displaying a mass increase of 50 Da compared to calycosin. Notably, the fragment ion at m/z 137.0239 was identical to calycosin, leading to the identification of M23 as a sulfated derivative resulting from the demethylation and dihydroxylation of calycosin. Meanwhile, M21 presented an [M + H]^+ ion peak at m/z 541.0647, reflecting a substantial mass augmentation of 256 Da over calycosin. Furthermore, the fragment ion at m/z 270.0515 aligned with calycosin. Taking into account its molecular formula C[22]H[20]O[14]S, it was hypothesized that M21 could be a sulfated and glucuronidation product of calycosin. These results were consistent with literature indicating that phase II conjugation of calycosin in vivo was very common [[85]22]. 2.5.2. Saponins Metabolites As depicted in [86]Figure S7B, three metabolites (M38-M40) were identified as metabolites of astragaloside IV in plasma, urine, and feces. M38 exhibited an [M + Na]^+ ion at m/z 513.3775, indicating a mass 294 Da smaller than astragaloside IV. This observation suggested a molecular formula of C[30]H[50]O[5] for M38. M38 was determined to be the desugaring product of astragaloside IV, specifically resulting from the removal of glucose (C[6]H[10]O[5]) and xylose (C[5]H[8]O[4]), and was identified as cycloastragenol based on a combination of references