Potential anti-ulcerative colitis compounds in Shaoyao decoction explored by bio-affinity ultrafiltration with multiple targets

Potential anti-ulcerative colitis compounds in Shaoyao decoction explored by bio-affinity ultrafiltration with multiple targets | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 23 December 2025 V1 Latest version Share on Potential anti-ulcerative colitis compounds in Shaoyao decoction explored by bio-affinity ultrafiltration with multiple targets Authors : Shujun Wang , Xiaowen Hua , Dan Liu , Satyajit Sarker , Lutfun Nahar , Yingying Zhang , and Mingquan Guo [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176649325.58413573/v1 139 views 84 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose : Shaoyao Decoction (SYD) has shown clinical efficacy in treating ulcerative colitis, yet its precise bioactive constituents remain uncharacterized. This study aimed to systematically screen and identify the key compounds in SYD that exert therapeutic effects against UC by targeting critical inflammatory mediators, including NLRP3, IL-6, and COX-2. Experimental Approach : A multi-target bio-affinity ultrafiltration (UF) screening strategy coupled with UPLC-Q-TOF-MS/MS analysis was developed and employed. This approach aimed to identify compounds in SYD displaying affinity for the three anti-inflammatory drug targets selected, i.e, NLRP3, IL-6, and COX-2. Molecular docking studies were subsequently utilized to validate the potential binding interactions between the identified key ligands and the targets. Key Results : UPLC-Q-TOF-MS/MS analysis revealed the complex composition of SYD, with 42 compounds identified in the positive ion mode, including flavonoids (such as baicalin, wogonin 7-O-glucuronide, wogonin) and alkaloids (such as Jatrorrhizine), and 26 compounds in the negative ion mode, primarily organic acids (such as Gallic acid, 4,5-Dicaffeoylquinic acid) and glycosides (such as Liquiritin). Bio-affinity UF screening revealed that flavonoids, such as baicalin, wogonin 7-O-glucuronide, and wogonin, exhibit strong multi-target binding affinity towards NLRP3, IL-6, and COX-2, suggesting their role as core anti-inflammatory components of SYD. In addition, 4,5-dicaffeoylquinic acid also demonstrated significant multi-target interactions. Molecular docking simulations corroborated these binding findings, further supporting the potential synergistic mechanisms by which SYD components act on the inflammatory targets. Conclusion and Implications : This study establishes a rapid screening strategy for bioactive components in traditional Chinese medicine (TCM) formulations. It identifies core components, including gallic acid, wogonin 7-O-glucuronide, wogonin, baicalin, and 4,5-Dicaffeoylquinic acid, and elucidates their mechanism in alleviating ulcerative colitis by targeting NLRP3, IL-6, and COX-2, providing a basis for clinical application and development. 1. INTRODUCTION Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease (IBD) that primarily affects the colonic mucosa. A rising global incidence of UC has been reported, particularly in industrialized regions, posing a growing challenge to healthcare systems (Ng t et al., 2017; Kaplan & Ng, 2017). The pathogenesis of UC involves a multifactorial interplay of genetic susceptibility, environmental triggers, and immune dysregulation (de Souza & Fiocchi, 2016). Disruption of the intestinal epithelial barrier, alterations in gut microbiota, and persistent activation of inflammatory pathways contribute to disease progression (de Souza & Fiocchi, 2016; Fritsch et al., 2020). Epidemiological studies have shown that the highest incidence of UC in Europe reaches approximately 505 cases per 100,000 individuals (Ng et al., 2017). Environmental factors, dietary changes, and microbial dysbiosis have been implicated in the increasing prevalence of UC (de Souza & Fiocchi, 2016; Kaplan & Ng, 2017; Fritsch et al., 2020). Current pharmacological treatments, including aminosalicylates, corticosteroids, and biologics, often provide only partial remission and are associated with adverse effects, drug resistance, and high relapse rates (Creed & Probert, 2007; Cholapranee et al., 2017). These limitations highlight the need for novel therapeutic strategies that can target multiple inflammatory pathways with improved safety and efficacy. Shaoyao Decoction (SYD) is a classical Chinese medicine formula that originated from the ”Suwen Bingji Qiyi Baoming Ji” by Jin Dynasty physician Liu Wanshu (c. 1186 AD). According to historical Chinese medical records, it is commonly applied to damp-heat dysentery, presenting with symptoms such as abdominal pain, bloody and purulent stools, tenesmus (urgent yet difficult defecation), burning sensation in the anus, and scanty, dark urine. SYD is composed 9 herbs, including Coptis chinensis Franch., Scutellaria baicalensis Georgi, Paeonia lactiflora Pall., Angelica sinensis (Oliv.) Diels, Aucklandia lappa Decne., Areca catechu L., Rheum palmatum L., Cinnamomum cassia Siebold, and Glycyrrhiza uralensis Fisch. ex DC. Clinically, SYD is widely used for treating damp-heat dysentery and is recommended in TCM guidelines for managing colitis. A meta-analysis comprising 23 randomized controlled trials (2,068 patients) demonstrated that SYD, whether used alone or in combination with conventional Western medicine or other Chinese medicine therapies, significantly enhances clinical efficacy and symptom remission rates in UC (Zhu & Liu, 2025). Pharmacological studies have demonstrated that SYD can restore the intestinal mucus layer and reduce inflammation in experimental colitis models (Wang et al., 2020; Wei et al., 2021; Fang et al., 2023). However, the precise bioactive constituents and molecular mechanisms underlying its therapeutic effects remain unclear. The complexity of multi-component herbal formulations presents a major challenge in identifying active compounds and elucidating their mechanisms. Conventional methods such as column chromatography are typically costly, complex to operate, and time-consuming (Wang et al., 2024). Bio-affinity ultrafiltration (UF) combined with multiple target proteins has emerged as a rapid and effective strategy for screening bioactive compounds from complex mixtures (Zhang et al., 2022). This technique exploits the specific binding affinity between target proteins and active molecules, enabling selective enrichment and identification of pharmacologically relevant constituents (Chen et al., 2019; Zhang et al., 2022; Feng et al., 2023). Application of this approach to SYD allows for the identification of compounds that bind to key UC-related targets such as NOD-like receptor family pyrin domain containing 3 (NLRP3), Interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2). These targets are central to the inflammatory processes in UC and represent promising points for therapeutic intervention (Li et al., 2018; Parisinos et al., 2018; Zhen et al., 2019). Characterization of these compounds using UPLC-Q-TOF-MS/MS provides both structural and functional insights, facilitating the development of targeted therapies derived from TCM. Individual components of SYD, such as baicalin and wogonin, have shown anti-inflammatory and antioxidant effects in various disease models. Baicalin has been reported to inhibit oxidative stress and modulate inflammatory signaling pathways (Kang et al., 2018; Shah et al., 2019; Gao et al., 2022). Wogonin has demonstrated the ability to suppress inflammation-associated colorectal carcinogenesis through Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor erythroid 2-related factor (Nrf2) signaling (Yao et al., 2014). Other constituents, including protocatechuic acid and quercetin, have been shown to attenuate inflammation by inhibiting necroptosis and pyroptosis pathways (Xiao et al., 2023). These findings suggest that SYD may exert its effects through a synergistic combination of multi-target compounds with complementary pharmacological actions. 2 METHODS 2.1 Plant materials SYD was prepared according to the traditional Chinese medicine formula, consisting of coptis chinensis (15.0 g), scutellaria baicalensis (15.0 g), paeonia lactiflora (30.0 g), angelica sinensis (15.0 g), aucklandia lappa (6.0 g), areca catechu (6.0 g), rheum palmatum (9.0 g), cinnamomum cassia (5.0 g), and glycyrrhiza uralensis (6.0 g). All plant materials complied with the standards of the Chinese Pharmacopoeia (2020 edition). 2.2 Chemicals and reagents Acetonitrile and formic acid (HPLC grade), p -nitrophenyl-D-glucopyranoside ( p -NPG, ≥99%), and p -nitrophenyl butyrate (p-NPB, ≥98%) were purchased from Aladdin Biochemical Technology Co., Ltd. (China). Recombinant human NLRP3, IL-6, and COX-2 enzymes were obtained from Sigma-Aldrich (USA). Ultrafiltration membranes with molecular weight cut-offs of 30 kDa (YM-30) and 10 kDa (YM-10) were supplied by Millipore Co. Ltd. (Bedford, MA, USA). Carbinol and phosphate-buffered saline (PBS) were obtained from Macklin Biochemical Technology Co., Ltd. (China). Ultra-pure water was prepared using a Milli-Q purification system (USA). 2.3 Preparation of sample extracts The herbal components of SYD, including coptis chinensis (15.0 g), scutellaria baicalensis (15.0 g), paeonia lactiflora (30.0 g), angelica sinensis (15.0 g), aucklandia lappa (6.0 g), areca catechu (6.0 g), rheum palmatum (9.0 g), cinnamomum cassia (5.0 g), and glycyrrhiza uralensis (6.0 g), were accurately weighed. The mixture was soaked in ten times its weight (v/w) of ultrapure water for 30 min, followed by decoction under gentle boiling for 1 h. After high-speed centrifugation, the supernatant was collected. The residue was re-extracted with eight times its weight (v/w) of ultrapure water under the same conditions. The combined supernatants were concentrated using a rotary evaporator and subsequently lyophilized to obtain the SYD extract. 2.4 Identification of bioactive compounds from SYD by UPLC-QTOF-MS/MS A precisely weighed aliquot of the SYD (1.0 g) extract was dissolved in methanol (15 mL). The solution was sonicated for 30 min to ensure complete dissolution, yielding a final concentration of 40 mg/mL. It was stored at 4°C for subsequent analysis. To minimize interference from mirabilite (Mang Xiao), desalting was performed using a C18 solid-phase extraction column prior to LC-MS analysis. The desalted sample was diluted to 4 mg/mL. Chromatographic separation was performed using an Agilent 1290 UPLC system coupled with a 6545 Q-TOF mass spectrometer (Agilent Technologies, USA). An EC-C18 column (3.0 × 150 mm, 2.7 μm) was used and maintained at 25°C. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile), delivered at a flow rate of 0.3 mL/min. The injection volume was 5 μL. The Q-TOF-MS system operated in both positive and negative electrospray ionization (ESI) modes. The parameters were as follows: capillary voltage, 3500 V; capillary temperature, 350 °C; drying gas flow, 8 L/min; and nebulizer pressure, 35 psi. Data acquisition was performed using three collision energies (10, 20, and 40 V) across a mass range of m/z 150-1500. For positive ion mode, the gradient program was: 0–15 min (10% B), 15-22 min (50% B), 22-28 min (80% B), 28-40 min (100% B), maintained until 45 min. For negative ion mode: 0-15 min (50% B), 15-30 min (75% B), 30-40 min (95% B), maintained until 45 min, followed by re-equilibration to 5% B by 50 min. Putative identification of SYD constituents was achieved by comparing precursor ions, characteristic fragment patterns, and retention times with established databases and literature references. 2.5 Bio-affinity ultrafiltration screening A stock solution of SYD extract (1 g/mL) was prepared in Tris-HCl buffer. The extract (100 μL) was then mixed with NLRP3 (10 μL, 4 U), IL-6 (20 μL, 1 μg), and COX-2 (10 μL, 4 U), followed by incubation at 37 °C for 40 minutes. For the negative control, the enzymes were denatured by boiling for 15 minutes prior to incubation, while all other steps were identical to those of the active enzyme group. After incubation, the reaction mixture was transferred to ultrafiltration tubes with molecular weight cut-offs of 30, 30, and 10 kDa, respectively, and centrifuged at 10,000 rpm for 10 minutes. The retained fraction was washed three times with PBS buffer (200 μL, pH 6.8), followed by centrifugation at 10,000 rpm for 10 minutes each time to remove unbound compounds. Subsequently, 200 μL of 90% methanol was added to dissociate potential bioactive compounds specifically bound to NLRP3, IL-6, and COX-2. After standing for 10 minutes, the solution was centrifuged at 10,000 rpm for 10 minutes to collect the eluate. The dissociation procedure was repeated three times. All resulting eluates were combined, evaporated to dryness under a nitrogen stream, and reconstituted in methanol (100 μL) for subsequent UPLC-Q-TOF-MS/MS analysis. The bioactive compounds isolated through affinity ultrafiltration screening underwent UPLC-Q-TOF-MS/MS characterization. An integrated Agilent 1290 Infinity II UHPLC system coupled with a 6530 Q-TOF mass spectrometer was used for analysis. All operational parameters were maintained consistent with the specifications outlined in Section 2.4. 2.6 Molecular docking validation Molecular docking simulations were performed to investigate the interactions between potential bioactive ligands from SYD and key inflammatory targets (NLRP3, IL-6, and COX-2) using AutoDock Vina 1.5.6 and Discovery Studio 4.5 software packages. The three-dimensional crystal structures of NLRP3, IL-6, and COX-2 were retrieved from the RCSB Protein Data Bank (www.rcsb.org). Potential active ligands derived from SYD were energy-minimized and converted into their optimal 3D conformations using ChemBio3D Ultra 14.0. Prior to docking, protein structures were prepared by removing water molecules, adding hydrogen atoms, and calculating partial charges with AutoDockTools. The active binding sites of NLRP3, IL-6, and COX-2 were identified using Discovery Studio 4.5. Molecular docking was carried out using the Genetic Algorithm with 50 independent runs per ligand-receptor pair, while maintaining default parameters in AutoDockTools. For comparative analysis, MCC950, Madindoline A, and meloxicam were included as NLRP3, IL-6, and COX-2 positive control drugs in the docking simulations. 2.8 Statistical analysis The SYD chemical composition, and binding affinity of potential ligands with COX-2, NLRP3, and IL-6, is documented, systematized, and calculated through the implementation of WPS Excel. The molecular docking data were processed using the Genetic Algorithm, which conducted 30 iterations for the interaction between the target and the active ligand. 3 RESULTS 3.1 Identification of chemical components in Shaoyao Decoction In the positive ion mode, UPLC-Q-TOF-MS/MS analysis successfully identified 42 characteristic compounds (Table 1). These compounds can be classified into flavonoids (e.g., wogonin, wogonin 7-O-glucuronide, baicalin, puerarin, oroxin A), alkaloids (e.g., berberrubine, jatrorrhizine, epiberberine, coptisine chloride, palmatine), monoterpene glycosides (e.g., albiflorin, benzoylpaeoniflorin, paeoniflorigenone), and triterpenoid saponins (e.g., glycyrrhizic acid). UPLC-Q-TOF-MS/MS analysis in negative ion mode characterized the chemical profile of the SYD extract, identifying 26 compounds (Table 2). The identified constituents were primarily categorized into organic acids and phenolic acids (e.g., gallic acid, protocatechuic acid, chlorogenic acid, and 4,5-dicaffeoylquinic acid), flavonoids and their glycosides (e.g., baicalein, wogonin, liquiritin, and liquiritin apioside), and triterpenoid saponins (e.g., glycyrrhizic acid). Additionally, other components such as emodin and galloyl paeoniflorin were detected, further illustrating the extract’s chemical diversity. 3.2 Affinity Ultrafiltration Screening for Bioactive Components Affinity ultrafiltration coupled with mass spectrometry was used to screen for potential anti-ulcerative colitis compounds in SYD. The screening was conducted under both positive and negative ionization modes. Figure 1 and 2 show the extracted ion chromatograms, while Tables 3 and 4 summarize the binding degrees of identified compounds with three key inflammatory targets, NLRP3, IL-6, and COX-2. A comprehensive analysis of data from both ion modes revealed that multiple constituents in SYD exhibit distinct binding properties to NLRP3, COX-2, and IL-6 enzymes, suggesting a potential anti-inflammatory mechanism. In positive ion mode, 12 compounds with high binding affinity are presented in Table 3. Specifically, most of the tested compounds showed considerable binding to NLRP3 and IL-6, while binding to COX-2 was less frequent and weaker. Among them, benzoylpaeoniflorin exhibited the highest binding affinity towards NLRP3 (BD% = 26.71%), while glycyrrhizic acid and moslosooflavone also showed strong binding to NLRP3 and IL-6 (BD% of 15.37% and 16.06%, respectively). It is particularly noteworthy that jatrorrhizine was the only compound that demonstrated binding activity against all three targets (NLRP3, COX-2, IL-6), indicating its potential for multi-target action. Figure 3 delineates the compound-target interaction network of SYD, where lines denote affinity interactions between bioactive components and therapeutic targets, and the thickness of the line indicates the degree of connection. It also revealed 12 characteristic constituents exhibiting differential binding capacities with NLRP3, IL-6, and COX-2. This network pharmacology visualization graphically demonstrates SYD’s multi-compound/multi-target therapeutic paradigm through coordinated regulation of inflammasome signaling (via NLRP3), cytokine modulation (via IL-6), and prostaglandin biosynthesis (via COX-2) in UC management. In negative ion mode, 12 compounds were identified. Table 4 presents their binding degrees with the target enzymes. Corroborating the findings from Table 3, Table 4 further delineates its spectrum of activity: the vast majority of components exhibit the strongest and most ubiquitous inhibitory potential against COX-2 (e.g., 4,5-dicaffeoylquinic acid, isoliquiritigenin, and glycyrrhizic acid), constituting the core mechanism for its analgesic and anti-inflammatory effects; concurrently, most components also effectively bind to NLRP3, enabling broad regulation of the upstream inflammasome; whereas Gallic acid acts as a key specific component, uniquely demonstrating exceptionally high affinity for IL-6 (BD% = 54.68%). This indicates that SYD constructs a multi-layered, comprehensive anti-inflammatory network by synergistically inhibiting COX-2 and NLRP3 to control the inflammatory process, while utilizing Gallic acid for precise intervention in the cytokine cascade. As illustrated in Figure 4, the negative ion mode analysis revealed 12 active constituents in SYD exhibiting differential binding affinities with three target proteins. The core target COX-2 is surrounded by the largest number of compounds, and several connections to it (e.g., from Peak 10, 22) are notably thick. This indicates that COX-2 is the primary target strongly and collectively attacked by multiple highly active components in SYD, suggesting that potent COX-2 inhibition is the foremost mechanism for its rapid anti-inflammatory and analgesic effects. For the NLRP3 target, while it is also connected to many compounds, most lines are relatively thinner, indicating generally weaker binding affinity compared to COX-2. This reflects a broad, moderate inhibitory mode on the upstream inflammasome, achieved through the collective action of multiple components. Most critically, the IL-6 target is connected to only a few compounds, but its link to gallic acid is exceptionally thick. This clearly identifies gallic acid as the key, specific, and irreplaceable component within the formula responsible for directly and potently antagonizing IL-6 activity. 3.3 Molecular docking analysis Molecular docking simulations were conducted to further clarify the interaction mechanisms between putative bioactive compounds identified through affinity ultrafiltration and the multi-target enzymes (Supplementary material). The compound demonstrating the highest binding degree (BD) value in affinity ultrafiltration, along with established positive controls for each target enzyme, were subjected to molecular docking analysis. The computational results, encompassing binding energy (BE), inhibition constant (Ki), and hydrogen-bond interactions, are systematically presented in Table 5. Generally, lower BE values indicate stronger binding affinity, while corresponding lower Ki values suggest greater inhibitory potency. Notably, 4,5-dicaffeoylquinic acid demonstrated the most favorable interaction with COX-2, exhibiting the lowest BE (-8.9 kcal/mol) and optimal Ki value (301.28 nM), surpassing the positive control meloxicam (BE: -7.6 kcal/mol; Ki: 2.71 μM). This indicates its potential as a potent COX-2 inhibitor. Similarly, baicalein showed superior IL-6 inhibition with BE of -9.61 kcal/mol and Ki value of 90.76 nM, marginally outperforming the positive control madindoline A (BE: -9.6 kcal/mol; Ki: 91.84 nM), suggesting its strong inhibitory potential. Regarding target selectivity, wogonin 7-O-glucuronide exhibited distinct binding preferences, showing moderate affinity for NLRP3 (BE: -7.74 kcal/mol; Ki: 2.12 μM) but significantly reduced affinity for IL-6 (Ki: 3.09 μM). These findings collectively provide molecular-level insights into the selective targeting and interaction patterns of bioactive components. 4 DISCUSSION Previous studies have developed a multi-target affinity ultrafiltration screening technique, which has been successfully applied to the screening of bioactive components in traditional herbal medicinal plants (Chen et al., 2018; Feng et al., 2023). The technique is particularly suitable for complex formulations such as SYD, which contains a diverse array of phytochemicals with potential therapeutic effects. Pharmacological studies have demonstrated the efficacy of SYD in the prevention and treatment of UC (Wei et al., 2021; Fang et al., 2023). This study successfully established a multi-target bio-affinity ultrafiltration screening model targeting NLRP3, IL-6, and COX-2, enabling rapid identification of potential anti-ulcerative colitis constituents from the complex matrix of SYD, including baicalin, wogonin 7-O-glucuronide, wogonin, 4,5-dicaffeoylquinic acid, and gallic acid et al. This strategy highlights the efficiency and advantage of systematic bioactive compound screening in the context of multi-component and multi-target traditional Chinese medicine. SYD chemical diversity directly reflects the multi-herb composition of SYD, with flavonoids likely originating from radix scutellariae , alkaloids from coptis chinensis and phellodendri chinensis cortex , monoterpene glycosides from paeoniae radix alba , and triterpenoid saponins from glycyrrhizae radix . The coexistence of aglycones (e.g., baicalein) and their corresponding glycosides (e.g., wogonin 7-O-glucuronide) provides valuable insights into the metabolic pathways within the formula. In flavonoids, wogonin 7-O-glucuronide (C 22 H 20 O 11 ) with a mass of 461.1073, coexist. The mass difference between the wogonin and wogonin 7-O-glucuronide is 156.0329 Da, which deviates from the theoretical addition value of glucuronic acid (C 6 H 8 O 6 ), 176.0321 Da. This discrepancy suggests a potential loss of carbon dioxide (decarboxylation) or a neutral fragment during the conjugation process. Baicalin (C 15 H 10 O 5 ) with a mass of 271.0599 exhibits characteristic MS/MS fragments at m/z 123.0071 (decarboxylation product) and m/z 169.0126 (flavone A-ring cleavage), confirming the position of the glucuronic acid group in its structure. Among isoflavone c-glycosides, puerarin (C 21 H 20 O 9 ) with a mass of 417.1180 and oroxin A (C 21 H 20 O 10 ) with a mass of 433.1123 differ by only one oxygen atom in their molecular formulas. However, their MS/MS fragmentation patterns show significant variations: Puerarin has an aglycone fragment at m/z 297.0750, while oroxin A exhibits a dehydroxylated aglycone fragment at m/z 271.0600. These differences suggest distinct glycosylation sites or oxidation states between the two compounds. Flavonoid glycosides typically exist in glycosidic forms, with sugar moieties (e.g., glucuronic acid) linked via glycosidic bonds, which are closely associated with their biological activities in TCM formulations. Pharmacologically, these flavonoids exhibit significant anti-inflammatory and antioxidant properties (Kang et al., 2018; Gao et al., 2022). For instance, baicalin and wogonin exert anti-inflammatory effects by inhibiting the NF-κB pathway (Yao et al., 2014; Shah et al., 2019). These findings not only elucidate the major active components of SYD but also provide a critical foundation for further investigation into its pharmacological mechanisms. Paeonol and peoniflorin derivatives (e.g., galloyl paeoniflorin) originate from paeonia, exhibiting anti-inflammatory and analgesic activities; liquiritin and glycyrrhizic acid come from glycyrrhiza , possessing antioxidant and anti-ulcer effects; baicalein and wogonin are derived from scutellaria , with antibacterial and antiviral properties. Additionally, various phenolic acids (e.g., gallic acid, chlorogenic acid) and flavonoids indicate that SYD has a rich polyphenol content, which may relate to its antioxidant and anti-inflammatory efficacy. Among them, 4,5-dicaffeoylquinic acid (C₂₅H₂₄O₁₂, [M-H]- 515.1199) yielded fragments at m/z 355.0967 and 179.0347, verifying its diacylated configuration and linking it to antioxidant efficacy. Liquiritin apioside (C₂₆H₃₀O₁₃, [M-H]- 549.1609) represented O-glycoside, with their distinct glycosylation patterns potentially affecting metabolic stability and bioavailability. Additionally, protocatechuic acid was tentatively identified based on fragments at m/z 135.0450 and 109.0294. Among the phenolic compounds, catechol despite its simple structures, possess potential antioxidant activity. The organic acids included protocatechuic acid and 4,5-dicaffeoylquinic acid, both of which are associated with anti-inflammatory and free radical-scavenging effects (Jang et al., 2022; Xiao et al., 2023). Notably, wogonin, a flavonoid, was detected with a mass (283.0611) highly consistent with its theoretical value (284.0685), further confirming its structural identity. Overall, the negative ion mode analysis complemented the results obtained in the positive ion mode, revealing a more diverse chemical profile in SYD, particularly with respect to phenolic acids and tannins. This comprehensive characterization of SYD’s chemical constituents provides valuable insights into its bioactive components and supports further exploration of its therapeutic mechanisms. This study selected NLRP3, IL-6, and COX-2 as target molecules based on their interrelated roles in the pathogenesis of UC (Li et al., 2018; Parisinos et al., 2018; Zhen & Zhang, 2019). Activation of the NLRP3 inflammasome triggers pyroptosis and the release of IL-1β, initiating excessive innate immune responses and epithelial barrier disruption (Kelley et al., 2019; Wang et al., 2023). IL-6 drives Th17 cell differentiation and chronic inflammation by activating STAT3 signaling, mediating autoimmune diseases and metabolic disorders, making it a key therapeutic target in inflammation-related pathologies (Hunter & Jones, 2015). COX-2 is a pro-inflammatory enzyme that is highly expressed at inflammatory sites and is commonly used as a therapeutic target for inflammatory diseases (Wang & Dubois, 2010; Lin et al., 2023). Affinity ultrafiltration was employed to screen SYD components capable of binding to multiple targets simultaneously, reflecting the multi-target nature of traditional Chinese medicine. Single-target inhibition often fails to alleviate UC due to compensatory crosstalk among the NLRP3, IL-6, and COX-2 pathways. The screening results in positive ion mode revealed that the primary anti-inflammatory constituents are flavonoids. Previous studies have confirmed that flavonoids exert multi-target anti-inflammatory effects by modulating key signaling pathways such as NF-κB and mitogen-activated protein kinase (MAPK), regulating the production of pro-inflammatory cytokines and mediators, and scavenging reactive oxygen species (ROS) (Wen et al., 2023). A representative example is baicalin, which targets core pathways including NF-κB, MAPK, and NLRP3 inflammasome, thereby suppressing the release of pro-inflammatory factors, attenuating the activation of inflammatory cells (macrophages, neutrophils), and restoring redox homeostasis through ROS elimination and immune regulation. In vivo studies, such as in DSS-induced murine colitis models, have demonstrated that baicalin alleviates intestinal mucosal damage by reducing myeloperoxidase (MPO) activity and tumor necrosis factor-α (TNF-α) levels (Zhang et al., 2017). The screening results in negative ion mode indicate that the primary anti-inflammatory constituents are predominantly composed of flavonoids and their glycosides, including liquiritin, scutellarin methylester, baicalein, wogonin and wogonin 7-O-glucuronide, followed by phenolic acids and their derivatives (e.g., gallic acid and 4,5-dicaffeoylquinic acid). The representative compounds identified in both positive and negative ion modes are baicalein and wogonin 7-O-glucuronide. They sourced from scutellaria baicalensis (a traditional Chinese herb used for clearing heat and detoxifying, anti-inflammatory, and antiviral purposes), exhibited notable binding affinity to the key enzymes. Studies have confirmed its multifaceted pharmacological activities, including anticancer, antiviral, anti-inflammatory, and immunomodulatory effects. Its primary mechanism involves suppressing cell proliferation and modulating the cell cycle through regulation of the PI3K/AKT signaling pathway (Wang et al., 2024). Additionally, other compounds such as liquiritin apioside (Xia et al., 2023), protocatechuic acid (Tsai & Yang, 2012), and 4,5-Dicaffeoylquinic acid (Jang et al., 2021) have demonstrated anti-inflammatory properties. These agents synergistically target multiple pathways, highlighting the multi-target therapeutic strategy of SYD in treating UC. The advantage of SYD in treating UC lies in the synergistic effect likely produced by the combined action of its multiple active compounds. Figure 5 illustrates the potential multi-targeted, multi-pathway mechanisms of action against ulcerative colitis by several bioactive compounds derived from SYD. The majority of active ingredients comprise baicalein, wogonin, wogonin 7-O-glucuronide, gallic acid and 4,5-Dicaffeoylquinic acid appear to target the NLRP3 inflammasome. This complex is crucial for activating cysteinyl aspartate specific proteinase-1 (Caspase-1), which then cleaves pro-interleukin-1β (Pro-IL-1β) into its active, pro-inflammatory form, interleukin-1β (IL-1β). By inhibiting NLRP3, these compounds can potentially suppress the maturation and release of IL-1β, a key driver of inflammation in UC (Xue et al., 2019; Guan et al., 2022). Baicalein, wogonin 7-O-glucuronide and 4,5-Dicaffeoylquinic acid are shown targeting COX-2. Cyclooxygenase-2 is an enzyme induced during inflammation and responsible for producing PEG2 (Jeong et al., 2019), which promotes pain, fever, and further inflammation. Inhibiting COX-2 can reduce PEG2 levels, alleviating inflammatory symptoms. Gallic acid and wogonin 7-O-glucuronide are associated with modulating IL-6. Interleukin-6 is a major cytokine that amplifies inflammatory signals and is involved in the differentiation of pathogenic Th17 cells (Allen et al., 2014). Reducing IL-6 activity can help control the overall inflammatory cascade. The combined effect of these compounds likely works in synergy. By simultaneously inhibiting multiple interconnected pro-inflammatory pathways (NLRP3/Caspase-1/IL-1β; COX-2/PEG2; IL-6 signaling), they create a powerful anti-inflammatory network. This multi-pronged approach not only suppresses active inflammation but may also contribute to mucosal healing and stabilization of the intestinal epithelium, addressing both the symptoms and the underlying damage in UC. In conclusion, this study successfully developed and applied an efficient bio-affinity ultrafiltration combined with UPLC-Q-TOF-MS/MS approach for the rapid screening of multi-target bioactive components from the complex traditional Chinese medicine formula, SYD. A total of 50 compounds were identified, and several key constituents, notably gallic acid, wogonin derivatives (e.g., wogonin 7-O-glucuronide), baicalin, and 4,5-dicaffeoylquinic acid, were pinpointed as the primary multi-target ligands demonstrating significant binding affinity towards the pivotal inflammatory targets NLRP3, IL-6, and COX-2. Molecular docking analysis revealed specific interactions between core components and therapeutic targets, notably showing that 4,5-dicaffeoylquinic acid exhibited superior binding affinity compared with the positive control drug. The presence of flavonoid glycosides and phenolic acids highlights the importance of structural features such as glycosylation in enhancing pharmacological activity. The findings support the existence of synergistic mechanisms within SYD, where multiple compounds act on interconnected targets to produce a coordinated anti-inflammatory response. This study provides a mechanistic foundation for the traditional use of SYD in UC and supports its potential as a source of multi-target therapeutic agents. The results offer a basis for future in vivo validation and clinical development of SYD-based treatments. Natural products with defined multi-target activity may represent a promising direction for the treatment of chronic inflammatory diseases such as UC. ACKNOWLEDGEMENTS This work is partly supported by Ningbo Yongjiang Talent Programme (2024A-427-G), and project of Cixi Leading Medical & Health Discipline, China (Project No.: 2023-ZDFZ03). Lutfun Nahar gratefully acknowledges the support from the European Regional Development Fund (Project ENOCH #CZ.02.1.01/0.0/0.0/ 16_019/0000868), and the Czech Science Foundation (Project #23-05474S), and the Chinese Academy of Sciences (PIFI Project #2025PVA0074). CONFLICT OF INTEREST STATEMENT The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. DATA AVAILABILITY STATEMENT Data are available from the authors on reasonable request. 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Identification of compounds in Shaoyao Decoction extract (positive ion mode) Peak Identification Molecular formula [M+H]+ Characteristic fragment (m/z) 1 Licoricone C 22 H 22 O 6 382.0820 364.0812,172.0390,320.0544,264.0643,200.0334 2 Unidentified - 262.1912 190.1454,217.1691,262.1908 3 Unidentified - 506.2024 344.1483,190.0856,151.0745,326.1376,277.0847 4 N-(2,5-Dihydroxyphenyl)pyridinium(1+) C 11 H 10 NO 2 188.071 118.0655,146.0599,170.0597 5 Kumatakenin C 17 H 14 O 6 314.0660 270.0759,255.0520,224.0703,296.0544,224.0703,212.0700 6 Albiflorin C 23 H 28 O 11 481.1703 105.03335,133.0647,179.0699 7 Avenanthramide 1 C 18 H 17 NO 6 308.0921 280.0964,265.0726,250.0860 8 Unidentified - 566.4276 114.0915,209.1646,228.1589,322.2482,435.3323 9 Unidentified - 486.1763 324.1229,280.0960 10 Luteolin-7-O-β-D-glucuronide C 21 H 18 O 12 463.0873 287.0548,336.1224 11 Berberrubine C 19 H 17 CINO 4 322.1070 307.0837,279.0883,294.0754,266.0804,250.0859 12 Isolicoflavonol C 15 H 10 O 6 354.1698 338.1384,323.1147,308.1271,291.1010,294.1476,308.1271, 13 Puerarin C 21 H 20 O 9 417.1180 297.0750,267.0644,381.0957,243.0283,105.0334, 14 Jatrorrhizine C 20 H 19 NO 4 338.1385 323.1150,294.1123,308.0914,280.0959,265.0726 15 Epiberberine C 20 H 18 NO 4 336.1231 320.0912,321.0997,292.0973,306.0760,278.0810 16 Coptisine chloride C 19 H 14 ClNO 4 320.0915 292.0968,307.0838,277.0729,262.0858,249.0776,234.0905 17 Oroxin A C 21 H 20 O 10 433.1123 271.0600,336.1219,127.0390 18 Palmatine C 21 H 21 NO 4 352.1538 337.1301,308.4837,322.2032 19 Formononetin glucoside C 22 H 22 O 9 431.1332 269.0801,213.0906,336.1223 20 Hispidulin C 16 H 12 O 6 301.0691 286.0471,183.999,156.0051 21 Baicalein C 15 H 10 O 5 271.0599 123.0071,253.0491,169.0130,225.0538 22 Kumatakenin B C 15 H 10 O 4 285.0741 270.0520,168.0048,242.0568 23 Unidentified - 334.1073 319.0829,304.0599,290.0807,276.0649 24 Chrysophanol C 15 H 1 0 O 4 255.0646 103.0541,153.0178,211.0752,255.0646 25 Unidentified 447.0820 271.0594,153.0176,336.1218 26 Wogonin 7-O-glucuronide C 22 H 20 O 11 461.1073 285.0760,168.0049,334.1065 27 Benzoylpaeoniflorin C 30 H 32 O 12 602.2234 105.0335,151.0750,249.0754 28 Unidentified - 295.2265 151.1114,133.1007,145.1003,165.1265,277.2153,249.2185,283.0576 29 Glycyrrhizic acid C 42 H 62 O 16 823.4103 453.3348,353.0710,611.3559,647.3785 30 Paeoniflorigenone C 17 H 18 O 6 318.3000 256.2629,300.2884,212.2363,150.1119,132.1014 31 Tectochrysin C 16 H 12 O 4 269.0802 213.0910,254.0566,237.0540,226.0609,197.0583,165.0170,136.0148 32 Unidentified - 375.1073 345.0602,327.1494,227.0543,197.0075,149.0590 33 Pectolinarigenin C 17 H 14 O 6 315.0846 285.0389,300.0619,257.0434,182.9918,198.0663,154.9973 34 Unidentified - 330.3360 312.3251,141.1129,286.3106,155.1301 35 Unidentified - 281.2949 281.1915,110.7250 36 Moslosooflavone C 17 H 14 O 5 299.0906 284.0669,255.0642,238.0617,267.0633,180.0015 37 Xenognosin B C 16 H 12 O 5 284.3309 105.0331,243.0978,141.1131,183.1847,283.9710 38 Unidentified – 358.3677 106.0860,340.3568 39 Unidentified - 309.3264 309.3260,110.7199 40 Unidentified - 272.2216 109.1007,135.0802,160.0280,248.1626 41 Unidentified - 365.1357 105.0338,141.1131,183.1856,243.0989 42 Unidentified - 670.5066 134.0810,261.2205,116.0704,191.1426,303.2316,233.1530,410.2888,600.4243 Table 2. Identification of compounds in Shaoyao Decoction extract (negative ion mode) Table 3 Binding affinity of active components in Shaoyao Decoction extract with NLRP3, COX-2, and IL-6 enzymes (positive ion mode) Peak Identification BD (%) NLRP3 COX-2 IL-6 6 Albiflorin 19.75 - 2.75 10 Luteolin-7-O-β-D-glucuronide 17.33 - 1.46 11 Berberrubine 7.79 3.3 6.64 14 Jatrorrhizine 5.45 18.92 31.54 16 Coptisine chloride 6.66 - 2.52 17 Oroxin A 6.9 - 2.18 20 Hispidulin 17.55 - 13.47 21 Baicalein 10.23 - 3.23 26 Wogonin 7-O-glucuronide 8.42 - 1.39 27 Benzoylpaeoniflorin 26.71 - 6.9 29 Glycyrrhizic acid 15.37 - 6.45 36 Moslosooflavone 10.58 - 16.06 Table 4 Binding affinity of active components in Shaoyao Decoction extract with NLRP3, COX-2, and IL-6 enzymes (negative ion mode) Peak The chemical constituents Binding Degree (%) NLRP3 COX-2 IL-6 2 Gallic acid 21.35 - 54.68 10 4,5-Dicaffeoylquinic acid 35.17 35.54 - 13 Liquiritin 14.28 32.87 - 14 Galloyl paeoniflorin 20.33 25.42 - 16 Wogonin 7.19 20.16 - 17 Scutellarin methylester 16.49 19.18 - 18 Wogonin 7-O-glucuronide 9.33 50.06 - 19 Baicalein 22.16 40.13 - 20 Glycyrrhizic acid 24.86 36.96 - 21 Formononetin 11 40.08 - 24 Glycycoumarin 16.31 33.6 - 26 Emodin 35.09 - Table 5 . Docking ability and affinity of potential ligands with COX-2, NLRP3, and IL-6. Peaks BE(kcal/mol) Ki Hydrogen Bonds Wogonin 7- O -glucuronide cox-2 -7.37 3.96 μM CYS47 Wogonin cox-2 -7.05 6.82 μM GLU46, LYS137 4,5-Dicaffeoylquinic acid cox-2 -8.9 301.28 nM ARG44 Wogonin 7- O -glucuronide NLRP3 -7.74 2.12 μM ILE482, GLN308, IEU483, IEU266 Wogonin NLRP3 -6.63 13.77 μM ILE370, ALA227 4,5-Dicaffeoylquinic acid NLRP3 -7.46 3.39 μM LYS620, GLN622 Wogonin 7- O -glucuronide IL-6 -7.52 3.09 μM ASN63, LYS66 Wogonin IL-6 -8.09 1.17 μM THR43, LYS46, GLU42 Baicalein IL-6 -9.61 90.76 nM MET184, ASP26 Positive control Meloxicam cox-2 -7.6 2.71 μM CYS41 MCC950 NLRP3 -12.04 1.5 nM LYS615 Madindoline A IL-6 -9.6 91.84 nM PRO65, MET67 FIGURE 1 Affinity ultrafiltration results of Shaoyao Decoction in positive ion mode with (A) NLRP3, (B) COX-2, and (C) IL-6. FIGURE 2 Affinity ultrafiltration results of Shaoyao Decoction in negative ion mode with (A) NLRP3, (B) COX-2, and (C) IL-6. FIGURE 3 Pharmacological interaction network of bioactive components and molecular targets in Shaoyao Decoction (positive ion mode). FIGURE 4 Pharmacological interaction network of bioactive components and molecular targets in Shaoyao Decoction (negative ion mode). FIGURE 5 The potential mechanisms underlying the multi-target synergistic effects of multiple active components in Shaoyao Decoction against ulcerative colitis. Supplementary Material File (image1.jpeg) Download 5.88 MB File (image2.jpeg) Download 4.84 MB Information & Authors Information Version history V1 Version 1 23 December 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Shujun Wang Cixi People's Hospital View all articles by this author Xiaowen Hua Zhejiang International Scientific and Technological Cooperative Base of Biomedical Materials and Technology Zhejiang Engineering Research Center for Biomedical Materials Ningbo Cixi Institute of Biomedical Engineering View all articles by this author Dan Liu Nanchang University Jiangxi Medical College View all articles by this author Satyajit Sarker Liverpool John Moores University Centre for Natural Products Discovery View all articles by this author Lutfun Nahar Palacký University and Institute of Experimental Botany View all articles by this author Yingying Zhang Zhejiang International Scientific and Technological Cooperative Base of Biomedical Materials and Technology Zhejiang Engineering Research Center for Biomedical Materials Ningbo Cixi Institute of Biomedical Engineering View all articles by this author Mingquan Guo [email protected] Zhejiang International Scientific and Technological Cooperative Base of Biomedical Materials and Technology Zhejiang Engineering Research Center for Biomedical Materials Ningbo Cixi Institute of Biomedical Engineering View all articles by this author Metrics & Citations Metrics Article Usage 139 views 84 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shujun Wang, Xiaowen Hua, Dan Liu, et al. Potential anti-ulcerative colitis compounds in Shaoyao decoction explored by bio-affinity ultrafiltration with multiple targets. Authorea . 23 December 2025. DOI: https://doi.org/10.22541/au.176649325.58413573/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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