Multi-Target Inhibition of Streptococcus pyogenes Skin Infections by Officinal Magnolia Bark Rhubarb Decoction: Network Pharmacology and Molecular Docking Insights

Multi-Target Inhibition of Streptococcus pyogenes Skin Infections by Officinal Magnolia Bark Rhubarb Decoction: Network Pharmacology and Molecular Docking Insights | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Multi-Target Inhibition of Streptococcus pyogenes Skin Infections by Officinal Magnolia Bark Rhubarb Decoction: Network Pharmacology and Molecular Docking Insights yuanhao wang, Xinrui Wang, Xueying Zhang, Mengyi Pan, Mingyang Sun, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6231082/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Aug, 2025 Read the published version in Bioresources and Bioprocessing → Version 1 posted 4 You are reading this latest preprint version Abstract Streptococcus pyogenes infection still poses a great threat to humanity on a global scale. The emergence of antibiotic resistance endangers health care systems worldwide. Unlike single component antibiotics, traditional Chinese medicine exerts its effects through multiple pathways and targets, thereby reducing the chance of bacteria developing resistance. The main components in Officinal magnolia bark rhubarb Decoction, namely Officinal magnolia bark, Rhubarb and Immature Bitter Orange, have been proven to have antibacterial effects in previous studies. In this study, through network pharmacology and molecular docking, the interactions between its active components and the targets related to Streptococcus pyogenes skin infection were determined, and enrichment analysis was carried out. Molecular docking studies were conducted on 15 screened drug active components and 12 target proteins. Finally, in vitro experiments were used to prove that Officinal magnolia bark rhubarb Decoction can inhibit the growth of Streptococcus pyogenes and can treat Streptococcus pyogenes skin infections. Network pharmacology Drug discovery Molecular docking Streptococcus pyogenes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The skin covers the entire body surface and is the largest organ in the human body. The skin plays a crucial role in maintaining the stability of the internal environment of the body, protecting the body from daily wear and tear, and regulating body temperature and perception(Piipponen, Li and Landén 2020 , Baker et al. 2023 ).A large number of bacteria colonize on human skin. Under normal circumstances, this colonization is harmless. However, when the body's immunity declines, some bacteria may have flora imbalance and other problems and then attack the human body. Such bacteria are called opportunistic pathogens. As a representative of opportunistic pathogens, Streptococcus pyogenes is a common colonizing bacterium in humans and still poses a great threat to humans worldwide(Howden et al. 2023 ).At the end of 2022, the World Health Organization (WHO) reported that scarlet fever and invasive infections had increased significantly in many developed countries, and children were particularly affected(Wang et al. 2023 ). Streptococcus pyogenes (also known as Group A Streptococcus [GAS]) is an obligate human pathogen associated with many disease states. Streptococcus pyogenes can cause human diseases, ranging from superficial pharyngeal infections such as streptococcal pharyngitis and skin infections such as impetigo and cellulitis to life threatening invasive or toxin mediated manifestations such as necrotizing fasciitis, streptococcal toxic shock syndrome, scarlet fever and septic shock(Bae et al. 2022 ). Of particular concern is Streptococcus pyogenes skin infection (SPSI), which ranges from superficial infections of the epidermis (such as impetigo) to severe invasive infections of the dermis and deeper tissues, including cellulitis and necrotizing fasciitis. Although it is sensitive to many different antibiotics (including beta lactams), the treatment of invasive Streptococcus pyogenes infections is complicated by factors such as its ability to form biofilms and secrete a large number of toxins. A biofilm is an attached and structured bacterial community growing in extracellular polymeric substances, which can enhance the community's ability to resist antibiotic killing. The toxins produced by Streptococcus pyogenes play a major role in destroying host tissues, including several membrane disrupting hemolysins, immunomodulatory superantigens, plasminogen activators, host cell adhesins, complement regulatory proteins, specific and non specific proteases, and a variety of other degradative enzymes(Alves-Barroco et al. 2020 , Zou et al. 2024 ). The emergence of antibiotic resistance threatens healthcare systems worldwide and raises the prospect of a post antibiotic era. Several factors, including the overuse and misuse of antibiotics, as well as exposure to environmental reservoirs of antibiotic resistant bacteria, contribute to the rising rate of antibiotic resistance(Ranjbar and Alam 2023 ).The slowdown in the development of antibacterial drugs has led to a greater reliance on existing ones. Coupled with the poor management of antibiotics, this has further aggravated the development of drug resistance(Baker et al. 2018 ).There is an urgent need for drugs capable of resisting the development of drug resistance to combat multi drug resistant pathogens. The wide use of antibiotics has accelerated the development of drug resistance. Antibiotic resistance has made many common infectious diseases more difficult to treat and may even lead to a situation where there are no drugs available. Unlike single - component antibiotics, traditional Chinese medicine exerts its effects through multiple pathways and targets, thereby reducing the chance of bacteria developing drug resistance. These advantages have made traditional Chinese medicine highly regarded in the fight against bacterial infections, especially drug - resistant bacterial infections. Officinal magnolia bark rhubarb Decoction (OMBRD) is a traditional Chinese medicine prescription, which was first recorded in "Jin Kui Yao Lue" and is mainly used to treat symptoms such as fullness in the chest due to retained fluid and abdominal pain. This prescription consists of Officinal magnolia bark (OMB), Rhubarb (RH) and Aurantii Fructus Immaturus (AF). The decoction is made by boiling these ingredients in water and taking the liquid. OMB has rich pharmacological activities, including antibacterial, antiviral, antitumor and other effects. Magnolol and honokiol interfere with the structure and function of bacterial cell membranes, preventing the reproduction and survival of bacteria. These components have inhibitory effects on a variety of Gram - positive bacteria and some Gram - negative bacteria(Sun et al. 2023 , Zheng et al. 2024 ). The main components of RH, anthraquinone compounds, have relatively strong antibacterial effects. Rhein, emodin and aloe - emodin effectively inhibit the growth and reproduction of a variety of harmful bacteria through multiple mechanisms such as destroying bacterial cell membranes, inhibiting nucleic acid and protein synthesis, and interfering with sugar metabolism(Zheng et al. 2021 ). Hesperidin and other substances in AF have certain antibacterial properties. With the development of multi - omics technologies and the rise of artificial intelligence and big - data analysis, the network pharmacology method has been promoted to develop and be widely applied. Meanwhile, network pharmacology is a new discipline in line with the overall characteristics of traditional Chinese medicine (TCM) and is highly original. More and more researchers are using the network pharmacology method to study the active ingredients, targets, and molecular mechanisms of TCM. Exploring the action targets and molecular mechanisms of TCM is the primary research objective in TCM network pharmacology. Through network construction, network analysis, and network verification, TCM network pharmacology reveals the multi - target and multi - pathway action modes of TCM and explains the action targets and molecular mechanisms by which TCM exerts its efficacy. Chinese herbal compound prescriptions have the characteristics of multi - component and multi - target actions. Due to its holistic and systematic research features, network pharmacology plays a unique role in exploring the compatibility mechanism of Chinese herbal compound prescriptions and provides a theoretical basis for clarifying the compatibility rules of Chinese herbal compound prescriptions(Jiashuo et al. 2022 , Zhou et al. 2023 ). Materials and methods Testing of active ingredients in OMBRD and their potential targets Use the TCMSP database ( https://tcmsp - e.com/tcmsp.php) and search for keywords OMB, RH, and AF. With drug - likeness (DL) ≥ 0.18 as the screening criterion, obtain the active pharmaceutical ingredients of OMBRD. Then, use the PubChem database ( https://pubchem.ncbi.nlm.nih.gov/ ) to obtain the SMILES format of the active compounds and import them into the SwissTargetPrediction platform ( http://www.swisstargetprediction.ch/ ). Set the searched species as “Homo sapiens” and the probability greater than 0 for potential targets. Construction of Staphylococcus aureus and SPSI - related target datasets Take “Streptococcus pyogenes skin infection”as the search term, retrieve relevant targets through the Human Gene Database (GeneCards, https://www.genecards.org/ ) and the Online Mendelian Inheritance in Man Database (OMIM, https://mirror.omim.org/ ), combine the retrieval results of the two databases, remove duplicate targets, and establish an SPSI - related target database. Import the obtained targets and drug active ingredient targets into VENNY2.1 ( https://bioinfogp.cnb.csic.es/tools/venny/index.html/ ), and obtain the intersection targets for OMBRD to treat SPSI. Construction of protein - protein interaction networks and screening of core targets Upload the intersection targets to the STRING platform ( https://cn.stringdb.org/ ) to construct a protein protein interaction (PPI) network. Set the protein species as "Homo sapiens", with the lowest interaction threshold, and keep other default parameters unchanged to obtain the PPI network. Then, import the tsv file into Cytoscape 3.9.1 software and use the Centiscape plugin for topological analysis. Set the conditions as Betweenness unDir 71.059, Closeness unDir 0.006, and Degree unDir 32.28 to screen out the core targets. GO and KEGG Enrichment Analysis Import the core targets into the DAVID ( https://davidbioinformatics.nih.gov/ ) database, and perform GO and KEGG pathway enrichment analyses on the intersection targets with P < 0.05 as the screening condition. Then, use the Wei Sheng Xin ( https://www.bioinformatics.com.cn/ ) website to visualize the results of the enrichment analysis. Among them, the GO enrichment analysis includes biological process (BP), cellular component (CC), and molecular function (MF). Molecular docking Calculate the degree value of active ingredients in the active ingredient - target network graph, and perform molecular docking on the top 15 active ingredients ranked by degree value with the 12 core protein molecules ranked by Degree value in the PPI network. Obtain the "PDB" format files of target protein 3D structure patterns and the "MOL2" format files of active ingredient 2D structure patterns from the PDB database ( http://www.rcsb.org/ ), UniProt database ( https://www.uniprot.org/ ), and TCMSP ( https://tcmsp - e.com/). Use Chem3D and PyMOL3 to optimize the structure transformation energy of active ingredients respectively. Use AutoDockTools 1.5.7 for molecular docking. First, perform operations such as water removal and hydrogen addition on receptor proteins and ligand small molecules, and then perform molecular docking on the 3D structures of the above - processed core components and core targets. Auto Dock Vina is used for molecular docking calculation. Select the result with the lowest binding energy as the best conformation according to the receptor - ligand binding energy, use Oringin2021 to draw the binding energy heat map, and use PyMOL3 for visualization. Cell culture Keratinocytes (HaCat) were purchased from Shanghai Jinyuan Biological Co., Ltd. HaCat cells are grown in DMEM medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin - streptomycin solution, and cultured at 37℃ in 5% CO₂. Cultivation of Streptococcus pyogenes Streptococcus pyogenes with ATCC number BAA − 947 was purchased from Fuxiang Biology and grew in THY broth (Todd - Hewitt broth supplemented with 0.2% yeast extract) at a temperature of 37°C. OMBRD preparation Put 15g of OMB, 18g of RH and 9g of AF into 1L of water, boil for 3 hours and then let it stand until it reaches room temperature. After that, freeze - dry for 18 hours. Dissolve the obtained powder in sterile water to a concentration of 80mg/ml for later use. MIC determination Determine the MIC criteria by the broth micro dilution method recommended by CLSI. Prepare serial two fold dilutions of OMBRD broth in a 96 well plate, and add 50 µL of a 1×10⁶ CFU/mL bacterial suspension to 50 µL of the antibiotic in each well, and then incubate at 37°C for 18 hours. Use 20 µL of resazurin test to observe the MIC and record it as the lowest concentration of the antibiotic(Nwabor et al. 2023 ). ELISA Streptococcus pyogenes invaded human skin substitutes for 3h, collected the supernatant, centrifuged at 1000 × g for 10 min to remove particles and polymers, successively added antibody working solution for incubation and washed for 5 times. When washing, the water was patted dry, added chromogenic solution for incubation, added stop solution and mixed well, and then detected the changes of IL-1 α, IL-6, tnf- α and il-36 at OD600. ELISA kit was purchased from MEIMIAN Biological company. Statistics Experimental conclusions are based on the comparison of averages generated from at least three technical replicates or three biological replicates, and statistical analysis and graphing are carried out using GraphPad Prism 9.0 (GraphPad software). P ≤ 0.05 is considered significant. Results Active ingredients and targets of OMBRD A total of 373 effective components of OMBRD were retrieved through the TCMSP database, among which there were 44 of OMB, 313 of RH, and 46 of AF. The SMILES format of the drug effective components was obtained from the PubChem database and imported into the SwissTargetPrediction database, resulting in a total of 4,933 targets corresponding to the active components. After deleting the identical targets, finally 775 action target diagrams (Fig. 1 A) were obtained. Prediction of the action targets of OMBRD in treating SPSI A total of 553 targets of SPSI were obtained from the GeneCards database, and 260 targets of SPSI were obtained from the OMIM database. After summarizing and deleting the duplicate targets, a total of 795 targets of SPSI were finally obtained. A Venn diagram was drawn to obtain 101 potential targets of OMBRD for treating SPSI (Fig. 1 A). Build a component target network graph In order to further explore the treatment mechanisms of OMBRD against Staphylococcus aureus and SPSI, the potential targets of OMBRD in treating SPSI, as well as their corresponding active ingredients and traditional Chinese medicines, were introduced to construct a composite targeting pathway network using Cytoscape 3.9.1 software. Figure 1 B shows a network composed of 195 nodes and 852 edges. The Analyze Network plug in was used to calculate the topological parameters of network nodes and screen key active ingredients and action targets. The top 14 components in terms of Degree value in the network are MOL001803 (degree = 23), MOL013278 (degree = 22), MOL007879 (degree = 22), MOL013279 (degree = 21), MOL005814 (degree = 20), MOL000006 (degree = 20), MOL000008 (degree = 20), MOL002235 (degree = 20), MOL013277 (degree = 19), MOL001729 (degree = 17), MOL002252 (degree = 17), MOL013430 (degree = 16), MOL002233 (degree = 16), MOL002265 (degree = 16), and MOL005980 (degree = 10), the only effective component in OMB (Table 1 ). As can be seen from Fig. 1 , OMBRD exerts its effect on SPSI through multiple components and multiple targets. Table 1 Information on the core active ingredients of drugs. Mol ID Core component name PubChem CID Targets Number MOL001803 Sinensetin 145659 23 MOL013278 4',5,7,8-Tetramethoxyflavone 629964 22 MOL007879 Tetramethoxyluteolin 631170 22 MOL013279 5,7,4'-Trimethylapigenin 79730 21 MOL005814 tangeretin 68077 20 MOL000006 luteolin 5280445 20 MOL000008 apigenin 5280443 20 MOL002235 EUPATIN 5317287 20 MOL013277 Isosinensetin 632135 19 MOL001729 Crysophanol 10208 17 MOL002252 palmidin B 5320385 17 MOL000476 Physcion 10639 16 MOL002233 2-Methyl cardol 5319544 16 MOL002265 Rheidin B 5320958 16 MOL005980 Neohesperidin 442439 10 Construction of PPI network and screening of core targets Using the STRING database, import 101 intersection targets to draw a Protein - Protein Interaction (PPI) network, and use Cytoscape 3.9.1 to visualize the PPI network, obtaining network Fig. 2 A ; perform topological analysis on the network through Centiscape, and screen with the topological parameters of Betweenness unDir > 71.059, Closeness unDir > 0.006, and Degree unDir > 32.28 as criteria to obtain network Fig. 2 B; perform visualization processing on the 22 core targets in network Fig. 2 B to obtain Fig. 2 C. The results show that there are 12 targets with relatively high degrees in the network, and BCL2, TNF, STAT3, CXCL8, EGFR, IL6, IL1B, GUN, GAPDH, TP53, AKT1, and MMP9 are the core targets for OMBRD to treat SPSI. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis In order to identify which metabolic pathways and biological processes might be affected in the treatment of SPSI by OMBRD, GO and KEGG enrichment analyses were carried out. Enrichment analysis was performed using 101 overlapping genes, and a total of 1,723 entries were obtained, including 1,505 for biological processes, 73 for cellular components, and 145 for molecular functions. The top ten in terms of count for each category were plotted, as shown in Fig. 3 A For biological processes, it was mainly involved in positive regulation of cell migration, cell activation, positive regulation of cytokine production, etc. For cellular components, they were mainly distributed in vesicle lumen, cytoplasmic vesicle lumen, membrane rafts, and membrane microdomains. For molecular functions, it was mainly related to kinase binding, cytokine receptor binding, protein kinase binding, and protein phosphatase binding, etc. Through enrichment analysis, 166 KEGG pathways were determined. We selected 10 of them for presentation, as shown in Fig. 3 B The grouping of KEGG mainly includes metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases. The KEGG pathways are mainly enriched in "Pathways in cancer", "Lipid and atherosclerosis", "Coronavirus disease - COVID − 19", "AGE - RAGE signaling pathway in diabetic complications" and "Chagas disease", etc. Molecular docking Select the main core target proteins, namely BCL2 (with the uniprot identifier of 5vau), TNF (uniprot identifier: 2e7a), STAT3 (uniprot identifier: 6njs), CXCL8 (uniprot identifier: 1icw), EGFR (uniprot identifier: 1xkk), IL6 (uniprot identifier: 1alu), IL1B (uniprot identifier: 1l2h), GUN (uniprot identifier: 1jnm), GAPDH (uniprot identifier: 1u8f), TP53 (uniprot identifier: 1aie), AKT1 (uniprot identifier: 1h10), and MMP9 (uniprot identifier: 1jkd). Carry out molecular docking between the active ingredients that rank among the top 14 in terms of the number of associated target points and the unique active ingredients in OMB. The binding energy (in kcal/mol) is obtained through docking analysis, and the results are presented in Fig. 5 The lower the score, the stronger the binding capacity. A binding score less than 5.0 kcal/mol indicates a moderate affinity, and a score less than 7.0 kcal/mol indicates a high affinity. The results indicate that the docking binding energies of all ligands to receptors are less than 0 kcal/mol, and the docking binding energies of 16 ligands to receptors are less than 9 kcal/mol. Among them, the 12 with the strongest binding energy activities are BCL2 MOL002265, BCL2 MOL002252, BCL2 MOL0059807, EGFR MOL002265, EGFR MOL005980, GAPDH MOL000006, GAPDH MOL002252, GAPDH MOL002265, GAPDH MOL005980, JUN MOL005980, STAT3 MOL002252, and TNF MOL005980.。 The PyMOL3 was utilized to visualize the compound - target interaction complexes with the highest free binding energy for each target as well as their binding mechanisms. Figure 5 shows the two - dimensional and three - dimensional diagrams of the molecular docking between key targets and active compounds. In vitro verification of OMBRD in the treatment of SPSI In order to verify the effect of OMBRD in treating SPSI, we cultured human epidermal cells HaCaT and carried out a bacterial invasion experiment. By measuring the expressions of TNF - α, IL − 1α, IL − 6 and IL − 36, the antibacterial effect of OMBRD was reflected. Specifically, we divided the models into four groups. Group 1 was the PBS group, with 100 µl of PBS added and incubated for 3 hours; Group 2 was the Streptococcus pyogenes invasion group, with 1 × 10⁶ CFU of colonies added. After 3 - hour invasion, the cells were washed three times with PBS and then 100 µl of culture medium was added; Group 3 was the post - invasion treatment group. After being invaded by Streptococcus pyogenes for 3 hours, the cells were washed three times with PBS, and then a mixture of 50 µl of culture medium and 50 µl of OMBRD was added; In Group 4, Streptococcus pyogenes and OMBRD were co - added to the human skin substitute and co - incubated for 3 hours. Then, the cells were washed three times with PBS and 100 µl of culture medium was added. The results showed that, compared with the invasion group, the inflammatory factors in the treatment group and the co - incubation group decreased significantly, proving that OMBRD can inhibit the growth of Streptococcus pyogenes in keratinocytes and has a certain therapeutic effect after Streptococcus pyogenes invasion (Fig. 6 ). Discussion Streptococcus pyogenes is a globally well - known human - specific pathogen, causing more than 600 million cases of pharyngeal infections and over 100 million cases of skin infections every year. Streptococcus pyogenes is a Gram - positive coccus, which is spherical or oval - shaped, with a diameter of 0.6–1µm, and most are arranged in chains(Hurst et al. 2022 ).It widely exists in nature, in human and animal feces, and can also be found in the nasopharynx of healthy people. This bacterium has strong adhesion and a variety of pathogenic factors. It can combine with skin cells and colonize and reproduce on them. The hyaluronidase it produces can decompose connective tissue, making the infection more likely to spread. Common symptoms of Streptococcus pyogenes infection include folliculitis, furuncle, carbuncle, acute cellulitis, abscess, erysipelas, and acute lymphangitis. They are manifested as skin redness, swelling, pain, induration, lumps, increased local skin temperature, and may be accompanied by tenderness. In severe cases, fluctuation, necrosis, ulcer and dysfunction may occur. Some patients may have systemic symptoms such as fever, headache, and general malaise, and in severe cases, complications such as bacteremia and septicemia may be accompanied(Siggins et al. 2020 ).Although people are concentrating on studying invasive indications, there are still many unknowns regarding the natural state of this bacterium during its colonization in the nasopharynx and on the skin. Clinically, antibiotics are usually used to treat infections caused by microorganisms such as Staphylococcus aureus and Streptococcus pyogenes. However, with the widespread use of antibiotics, the number of strains resistant to antibiotics such as macrolides is increasing day by day, making the research and development of new drugs extremely urgent. Based on the advantages of traditional Chinese medicine, such as multi - component, multi - target, wide - ranging pharmacological activities, low toxicity, and less likelihood of developing drug resistance, in this study, we used OMB, RH, and AF aqueous solutions for antibacterial experiments. As far as we know, this is the first study on the fact that the active ingredients in OMBRD can inhibit the growth of Streptococcus pyogenes and treat skin infections caused by Streptococcus pyogenes. In China, according to the theory of traditional Chinese medicine, the main function of OMBRD is to promote qi movement, so OMB is the principal drug in this prescription. In the prescription, OMB promotes qi movement and relieves fullness, Zhishi breaks qi and eliminates accumulation, and RH purges heat and unblocks the bowels. The three drugs are used in combination to achieve the effect of promoting qi movement and relieving constipation. It was recorded in "Jin Kun Yao Lue" written by Zhang Zhongjing, the most famous doctor in China in the early 3rd century AD (the Eastern Han Dynasty). This prescription is composed of OMB, RH and AF. Modern medical research shows that the active ingredients of these three substances can all play an antibacterial role(Guo et al. 2021 , Müller-Heupt et al. 2022 , Gao et al. 2023 ). According to "Chinese Pharmacopoeia", OMB is the dried bark of the trunk or root of Magnolia officinalis or Magnolia officinalis subsp. biloba in the Magnoliaceae family. It has a bitter taste and warm nature, and has the effects of drying dampness, resolving phlegm, and descending qi to relieve fullness. It enters the spleen, stomach, and large intestine meridians, and can be used to treat abdominal fullness caused by damp - obstruction, food - retention, and qi - stagnation; when it enters the lung meridian, it can be used to treat cough, panting, and excessive phlegm. Modern pharmacological studies have shown that OMB has anti - inflammatory, analgesic, anti - tumor, antioxidant and other effects. RH is a plant in the genus Rumex of the Polygonaceae family, an annual herb. Its root is thick, large and yellow. There are more than 200 species of Rumex plants in the world, mainly distributed in Europe and North America(Pliszko 2019 ).RH (bitter - cold in nature, being good at clearing heat and breaking up accumulations, and also able to enter the blood aspect, expelling stasis and dredging menstruation. It is said in "Xue Zheng Lun" that "it can remove the old and promote the new, and damage yang and harmonize yin. Wherever there is disharmony in the blood aspect due to qi reversal, the nature of RH can reach there without exception." Modern research shows that RH has pharmacological effects such as promoting blood circulation and stopping bleeding, anticoagulation, anti - inflammation, and antibacterial. AF is rich in synephrine (alkaloids) and flavonoids, and is mainly used to treat internal retention of food stagnation, constipation, phlegm obstruction and qi stagnation, etc. There are 19 prescriptions in the classic prescriptions in "Shang Han Zang Bing Lun" containing AF. Modern pharmacodynamic research shows that the active pharmaceutical ingredients contained in AF can improve intestinal dryness and constipation, promote gastrointestinal motility, accelerate metabolism, and have pharmacological effects such as antibacterial, anti - infection and lipid metabolism. In conclusion, all three active ingredients in OMBRD have antibacterial and anti - inflammatory effects. In order to study the mechanisms of the antibacterial and anti - infection effects of OMBRD, we adopted the method of network pharmacology to study and found that there are 101 related targets between the active ingredients of OMBRD and SPSI, indicating that the active ingredients of OMBRD may interact with SPSI. This research is based on network pharmacology analysis. Through retrieval in the TCMSP database, a total of 373 effective components of OMBRD were obtained, including 44 of OMB, 313 of RH, and 46 of AF. Among them, the top 14 with the highest Dgree values are Sinensetin, 4',5,7,8 - Tetramethoxyflavone, Tetramethoxyluteolin, 5,7,4' - Trimethylapigenin, tangeretin, luteolin, apigenin, EUPATIN, Isosinensetin, Crysophanol, palmidin B, Physcion, 2 - Methyl cardol, Rheidin B, and Neohesperidin, the only active component in OMB. 101 targets of OMBRD for treating SPSI were obtained. Through PPI network analysis, core targets such as BCL2, TNF, STAT3, CXCL8, EGFR, IL6, IL1B, GUN, GAPDH, TP53, AKT1, MMP9, etc. were screened out. Our analysis results show that Neohesperidin is effective for multiple targets of SPSI. Neohesperidin belongs to flavonoids, which are widely distributed in the plant kingdom. In recent years, they have attracted extensive attention due to their effective antioxidant, metal - chelating, anti - inflammatory, antibacterial, antifungal, anticancer, and antiviral properties(Sage et al. 2022 , Neves et al. 2022 , Li et al. 2024 ).Several studies have shown that Neohesperidin has significant antibacterial potential, and many bacteria and fungi have been studied(El-Kadem et al. 2024 , Akhter et al. 2024 ). Neohesperidin (1.5 gm/l) exhibits moderate antifungal activity, with the maximum inhibition zones being 30%, 10% and 10% respectively against Monilinia fructicola, Botrytis cinerea and Alternaria alternata on fruits(Hernández et al. 2021 ).In vitro studies have shown that neohesperidin (at a concentration of 12.5–50mg/ml) has an enhanced antibacterial activity against Escherichia coli by the agar diffusion method, with the maximum inhibition zone ranging from 17.4 to 24.2 mm. This effect may be mediated through the interaction with the cell membrane of Escherichia coli(Du et al. 2018 ).Neohesperidin at concentrations of 50 and 100 g/ml can treat skin - injury diseases. Its mechanism is to significantly inhibit the formation of RNS (reactive nitrogen species) and ROS (reactive oxygen species), the spread of intercellular adhesion molecule − 1 (ICAM − 1), and induce the synthesis of glycosaminoglycan (GAG) in cells under the action of IFN - γ and histamine, without inhibiting the cell proliferation of the normal human keratinocyte cell line NCTC 2544. This indicates that it has significant concentration - dependent antioxidant and anti - inflammatory effects(Graziano et al. 2012 ).Neohesperidin has also been found to have a significant inhibitory effect on pro - inflammatory mediators and IgE - induced markers (such as HA, TPS, CMA1, IL − 8, MCP − 1, MCP − 1) (at a dose of 400 µm in vitro and 20 mg/kg in vivo)(Liu et al. 2019 ). To further explore and understand the potential functions and interactions of the screening targets, we carried out enrichment analysis on 101 intersection targets. The results showed that they were mainly concentrated in aspects such as cell migration and cytokine production. Subsequently, we performed molecular docking to evaluate the binding situations of 15 active ingredients (Sinensetin, 4',5,7,8 - Tetramethoxyflavone, Tetramethoxyluteolin, 5,7,4' - Trimethylapigenin, tangeretin, luteolin, apigenin, EUPATIN, Isosinensetin, Crysophanol, palmidin B, Physcion, 2 - Methyl cardol, Rheidin B, Neohesperidin) and 12 core proteins (BCL2, TNF, STAT3, CXCL8, EGFR, IL6, IL1B, JUN, GAPDH, TP53, AKT1, MMP9). The results indicated that the binding energies of BCL2 - Neohesperidin, BCL2 - palmidin B, BCL2 - Rheidin B, EGFR - Neohesperidin, EGFR - Rheidin B, GAPDH - luteolin, GAPDH - Neohesperidin, GAPDH - palmidin B, GAPDH - Rheidin B, JUN - Neohesperidin, STAT3 - palmidin B, TNF - Neohesperidin, etc. were very low, suggesting that they might be the main active ingredients and targets for OMBRD to treat SPSI. In order to further verify the inhibitory effect of OMBRD on Streptococcus pyogenes and its effect on treating SPSI (Streptococcus pyogenes - skin and soft tissue infection), in - vitro experiments were carried out. Continuous two - fold dilution broth of OMBRD was prepared in a 96 - well plate. Single colonies with similar morphology were selected from the solid medium and placed in the liquid medium, and cultured at 37°C with 220 rpm for 4–6 hours until the logarithmic growth phase of bacteria, so that the final bacterial suspension concentration reached approximately 5 × 10⁵ CFU/mL. The results showed that the MIC (minimum inhibitory concentration) of OMBRD was 20 mg/mL. IL − 1α, IL − 6, IL − 36 and TNF - α are the most common interleukins secreted by keratinocytes during bacterial infection. We infected HaCaT cells with Streptococcus pyogenes and added OMBRD at the same time. It was found that OMBRD had a significant inhibitory effect on Streptococcus pyogenes after being added. Moreover, 3 hours after Streptococcus pyogenes infected HaCaT cells, after treatment with OMBRD, compared with the positive control group without drug addition, the interleukins also decreased significantly. Our in - vitro experiment preliminarily proved that OMBRD has a certain inhibitory effect on Streptococcus pyogenes and has a therapeutic effect on skin infections caused by Streptococcus pyogenes. In this experiment, through network pharmacology analysis combined with experimental verification, the inhibitory effect of OMBRD on SPSI was explored for the first time. It should be noted that when retrieving OMBRD active ingredients and targets related to SPSI from the database, the reliability and accuracy of the analysis and prediction depend on the quality of the data. To overcome this limitation, we initially carried out in - vitro experiments. The results showed that OMBRD has a certain inhibitory effect on Streptococcus pyogenes and has a therapeutic effect on skin infections caused by Streptococcus pyogenes. However, its specific mechanism still requires additional experiments for verification. Conclusion This study pioneers the systematic exploration of the traditional formula OMBRD against Streptococcus pyogenes and its therapeutic mechanisms. Network pharmacology identified 373 bioactive components and 101 targets, with Neohesperidin, flavonoids, and anthraquinones as key constituents targeting BCL2, TNF, and IL6. Molecular docking revealed strong binding between Neohesperidin and critical targets, suggesting multi-pathway regulation of inflammation and apoptosis. In vitro assays demonstrated OMBRD's antibacterial activity (MIC: 20 mg/mL) and suppression of IL-1α, IL-6, IL-36, and TNF-α in infected HaCaT cells, confirming dual antimicrobial/anti-inflammatory effects. The findings underscore OMBRD's multi-component synergy against drug-resistant infections, while further in vivo validation is needed. This work advances the modernization of herbal formulas for anti-infective drug development. Abbreviations AF Aurantii Fructus Immaturus GAS Group A Streptococcus GO Gene Ontology IL-1α Interleukin-1α IL-36 Interleukin-36 IL-6 Interleukin-6 KEGG Kyoto Encyclopedia of Genes and Genomes MIC Minimum Inhibitory Concentration OMB Officinal magnolia bark OMBRD Officinal magnolia bark rhubarb Decoction RH Rhubarb SPSI Streptococcus pyogenes skin infection TCM traditional Chinese medicine TNF-α tumor necrosis factor-α WHO World Health Organization Declarations Acknowledgements The authors acknowledge the funding support. Author contributions Yuanhao Wang: Search the database and build the dataset, Writing the original draft.Xinrui Wang: Writing the original draft. Xueying Zhang, Mengyi Pan and Mingyang Sun：Complete in vitro experiments. Zhiguo Chen: Validation. Yingli Song: Writing, review and editing, Supervision, Funding acquisition. Funding The work was supported by the National Natural Science Foundation of China (82302535) and the Natural Science Foundation of Heilongjiang Province(LH2023H008）. Availability of data and materials The datasets used during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication All authors agreed to publish this article. Competing interests The authors declare no competing interests. Author details 1 School of Basic Medical Sciences, Harbin Medical University, 157th Rd of Bao jian, Nangang Distinct, Harbin 150081, China. 2 The Second Affiliated Hospital of Harbin University, 246th Rd of Xuefu, Nangang District, Harbin 150086, China. References Akhter S, Arman M S I, Tayab M A, Islam M N & Xiao J (2024) Recent advances in the biosynthesis, bioavailability, toxicology, pharmacology, and controlled release of citrus neohesperidin. Crit. Rev. Food Sci. Nutr. 64: 5073-5092.https://doi.org/10.1080/10408398.2022.2149466 Alves-Barroco C, Paquete-Ferreira J, Santos-Silva T & Fernandes A R (2020) Singularities of Pyogenic Streptococcal Biofilms - From Formation to Health Implication. Front Microbiol 11: 584947.https://doi.org/10.3389/fmicb.2020.584947 Bae S, Gu H, Gwon M-G, An H-J, Han S-M, Lee S-J, Leem J & Park K-K (2022) Therapeutic Effect of Bee Venom and Melittin on Skin Infection Caused by Streptococcus pyogenes. Toxins (Basel) 14.https://doi.org/10.3390/toxins14100663 Baker P, Huang C, Radi R, Moll S B, Jules E & Arbiser J L (2023) Skin Barrier Function: The Interplay of Physical, Chemical, and Immunologic Properties. Cells 12.https://doi.org/10.3390/cells12232745 Baker S J, Payne D J, Rappuoli R & Gregorio E D (2018) Technologies to address antimicrobial resistance. Proc. Natl. Acad. Sci. U. S. A. 115: 12887-12895.https://doi.org/10.1073/pnas.1717160115 Du L, Jiang Z, Xu L, Zhou N, Shen J, Dong Z, Shen L, Wang H & Luo X (2018) Microfluidic reactor for lipase-catalyzed regioselective synthesis of neohesperidin ester derivatives and their antimicrobial activity research. Carbohydr. Res. 455: 32-38.https://doi.org/10.1016/j.carres.2017.11.008 El-Kadem A H, Negm W A, Elekhnawy E, Binsuwaidan R, Attallah N G, Moglad E, Sultan A A J J o D D S & Technology (2024) Neohesperidin-loaded microemulsion for improved healing of Acinetobacter baumannii-infected wound. J Drug Deliv Sci Tec 98: 105905.https://doi.org/10.1016/j.jddst.2024.105905 Gao Z, Jiang S, Zhong W, Liu T & Guo J (2023) Linalool controls the viability of Escherichia coli by regulating the synthesis and modification of lipopolysaccharide, the assembly of ribosome, and the expression of substrate transporting proteins. Food Res. Int. 164: 112337.https://doi.org/10.1016/j.foodres.2022.112337 Graziano A C E, Cardile V, Crascì L, Caggia S, Dugo P, Bonina F & PanicoCaggia A (2012) Protective effects of an extract from Citrus bergamia against inflammatory injury in interferon-γ and histamine exposed human keratinocytes. Life Sci. 90: 968-74.https://doi.org/10.1016/j.lfs.2012.04.043 Guo Y, Hou E, Wen T, Yan X, Han M, Bai L-P, Fu X, Liu J & Qin S (2021) Development of Membrane-Active Honokiol/Magnolol Amphiphiles as Potent Antibacterial Agents against Methicillin-Resistant Staphylococcus aureus (MRSA). J. Med. Chem. 64: 12903-12916.https://doi.org/10.1021/acs.jmedchem.1c01073 Hernández A, Ruiz-Moyano S, Galván A I, Merchán A V, Nevado F P, Aranda E, Serradilla M J, Córdoba M d G & Martín A (2021) Anti-fungal activity of phenolic sweet orange peel extract for controlling fungi responsible for post-harvest fruit decay. Fungal Biol 125: 143-152.https://doi.org/10.1016/j.funbio.2020.05.005 Howden B P, Giulieri S G, Lung T W F, Baines S L, Sharkey L K, Lee J Y H, Hachani A, Monk I R & Stinear T P (2023) Staphylococcus aureus host interactions and adaptation. Nat. Rev. Microbiol. 21: 380-395.https://doi.org/10.1038/s41579-023-00852-y Hurst J R, Shannon B A, Craig H C, Rishi A, Tuffs S W & McCormick J K (2022) The Streptococcus pyogenes hyaluronic acid capsule promotes experimental nasal and skin infection by preventing neutrophil-mediated clearance. PLoS Pathog. 18: e1011013.https://doi.org/10.1371/journal.ppat.1011013 Jiashuo W U, Fangqing Z, Zhuangzhuang L I, Weiyi J & Yue S (2022) Integration strategy of network pharmacology in Traditional Chinese Medicine: a narrative review. J. Tradit. Chin. Med. 42: 479-486.https://doi.org/10.19852/j.cnki.jtcm.20220408.003 Li G, Wang X, Long X, Chen S, Han L, Cheng Y, Chen S, Liu W, Feng R & Sun Z J F B (2024) Attenuation by Zhiqiao neohesperidin on dextran sulfate sodium-induced colitis in mice through inhibition of the TLR4/NF-κB pathway. Food Biosci 62: 105328.https://doi.org/10.1016/j.fbio.2024.105328 Liu T, Ma Y, Zhang R, Zhong H, Wang L, Zhao J, Yang L & Fan X (2019) Resveratrol ameliorates estrogen deficiency-induced depression- and anxiety-like behaviors and hippocampal inflammation in mice. Psychopharmacology (Berl.) 236: 1385-1399.https://doi.org/10.1007/s00213-018-5148-5 Müller-Heupt L K, Vierengel N, Groß J, Opatz T, Deschner J & Loewenich F D v (2022) Antimicrobial Activity of Eucalyptus globulus, Azadirachta indica, Glycyrrhiza glabra, Rheum palmatum Extracts and Rhein against Porphyromonas gingivalis. Antibiotics (Basel) 11.https://doi.org/10.3390/antibiotics11020186 Neves J R, Grether-Beck S, Krutmann J, Correia P, Jr J E G, Sant'Anna B & Kerob D (2022) Efficacy of a topical serum containing L-ascorbic acid, neohesperidin, pycnogenol, tocopherol, and hyaluronic acid in relation to skin aging signs. J. Cosmet. Dermatol. 21: 4462-4469.https://doi.org/10.1111/jocd.14837 Nwabor O F, Chukamnerd A, Terbtothakun P, Nwabor L C, Surachat K, Roytrakul S, Voravuthikunchai S P & Chusri S (2023) Synergistic effects of polymyxin and vancomycin combinations on carbapenem- and polymyxin-resistant Klebsiella pneumoniae and their molecular characteristics. Microbiol Spectr 11: e0119923.https://doi.org/10.1128/spectrum.01199-23 Piipponen M, Li D & Landén N X (2020) The Immune Functions of Keratinocytes in Skin Wound Healing. Int. J. Mol. Sci. 21.https://doi.org/10.3390/ijms21228790 Pliszko A (2019) Typification of two natural hybrids in Rumex (Polygonaceae). Kew Bull. 74: 17.https://doi.org/10.1007/s12225-019-9803-8 Ranjbar R & Alam M (2023) Antimicrobial Resistance Collaborators (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Evid. Based Nurs.https://doi.org/10.1136/ebnurs-2022-103540 Sage J, Renault J, Domain R, Bojarski K K, Chazeirat T, Saidi A, Leblanc E, Nizard C, Samsonov A, Kurfurst R, Lalmanach G & 4 F L (2022) Modulation of the expression and activity of cathepsin S in reconstructed human skin by neohesperidin dihydrochalcone. Matrix Biol. 107: 97-112.https://doi.org/10.1016/j.matbio.2022.02.003 Siggins M K, Lynskey N N, Lucy E. Lamb L A J, Kristin K. Huse M P, Banerji S, Turner C E, Woollard K, Jackson D G & Sriskandan S (2020) Extracellular bacterial lymphatic metastasis drives Streptococcus pyogenes systemic infection. Nat. Commun. 11: 4697.https://doi.org/10.1038/s41467-020-18454-0 Sun J, Xie Y, Chen Z, Fan Y, Liu Y, Gao Q, Li J, Bai J & Yang Y (2023) Antimicrobial activity and mechanism of Magnolia officinalis root extract against methicillin-resistant Staphylococcus aureus based on mannose transporter. Ind Crop Prod 201: 116953.https://doi.org/10.1016/j.phymed.2022.154073 Wang J, Ma C, Li M, Gao X, Wu H, Dong W & We L (2023) Streptococcus pyogenes: Pathogenesis and the Current Status of Vaccines. Vaccines (Basel) 11.https://doi.org/10.3390/vaccines11091510 Zheng Q, Li S, Li X & Liu R (2021) Advances in the study of emodin: an update on pharmacological properties and mechanistic basis. Chin. Med. 16: 102.https://doi.org/10.1186/s13020-021-00509-z Zheng S, Deng R, Huang G, Ou Z & Shen Z (2024) Effects of honokiol combined with resveratrol on bacteria responsible for oral malodor and their biofilm. J Oral Microbiol 16: 2361402.https://doi.org/10.1080/20002297.2024.2361402 Zhou W, Hu Z, Wu X, Zhang S, Jiang Y, Tian L, Huang X, Ma Z, Qiu L, Zheng P, Zhang S & Lu Z (2023) Elucidation of the underlying mechanism of Hua-ban decoction in alleviating acute lung injury by an integrative approach of network pharmacology and experimental verification. Mol. Immunol. 156: 85-97.https://doi.org/10.1016/j.molimm.2023.02.013 Zou Z, Singh P, Pinkner J S, Obernuefemann C L P, Wei Xu 1 T M N, Dodson K W, Almqvist F, Hultgren S J & Caparon M G (2024) Dihydrothiazolo ring-fused 2-pyridone antimicrobial compounds treat Streptococcus pyogenes skin and soft tissue infection. Sci Adv 10: eadn7979.https://doi.org/10.1126/sciadv.adn7979 Supplementary Files 1.jpg Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 26 Aug, 2025 Read the published version in Bioresources and Bioprocessing → Version 1 posted Reviewers agreed at journal 25 Mar, 2025 Reviewers invited by journal 21 Mar, 2025 Editor assigned by journal 18 Mar, 2025 First submitted to journal 14 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6231082","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432018982,"identity":"688f3923-961b-4f95-993d-c287853378be","order_by":0,"name":"yuanhao wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACNv7mAwYfKmzq7Y83HyBOC5/EsYTCGWfSEhjOHEsgToscQ47BZ962wwkMN3wMiHQYwwHDjTPY0vIYZ/B8vPGGwU5Ot4GQFuaGZIMPPDbFzNK9my3nMCQbmx0gbMsxwxkSaYxtMme3SfMwHEjcRlhLYvtvHoPDjD0SOc+I1ZLMYMyTcDhxhkQOG5FaJI4xGM44kGZswHPM2HKOARF+ke/v/2Dw8Z+NnAF788Mbbyrs5AhqQQESPERGDbIWUnWMglEwCkbBiAAAKA5E+81O1joAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0002-9071-0112","institution":"Harbin Medical University","correspondingAuthor":true,"prefix":"","firstName":"yuanhao","middleName":"","lastName":"wang","suffix":""},{"id":432018983,"identity":"431e6b93-1de3-4123-b38d-79dd3f0a308e","order_by":1,"name":"Xinrui Wang","email":"","orcid":"","institution":"Harbin Medical University Second Affiliated Hospital Department of Cardiology","correspondingAuthor":false,"prefix":"","firstName":"Xinrui","middleName":"","lastName":"Wang","suffix":""},{"id":432018984,"identity":"03a71bdb-b946-458d-aa62-0a3e1d526f00","order_by":2,"name":"Xueying Zhang","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xueying","middleName":"","lastName":"Zhang","suffix":""},{"id":432018985,"identity":"109df39c-ecf2-437e-b238-855c4f70ff50","order_by":3,"name":"Mengyi Pan","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengyi","middleName":"","lastName":"Pan","suffix":""},{"id":432018986,"identity":"ac4a63a1-9a4b-44bf-a0d3-f26e2ecf786b","order_by":4,"name":"Mingyang Sun","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingyang","middleName":"","lastName":"Sun","suffix":""},{"id":432018987,"identity":"ef7b7ba5-88bc-47e6-889e-0ad00bfad5bc","order_by":5,"name":"Zhiguo Chen","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhiguo","middleName":"","lastName":"Chen","suffix":""},{"id":432018988,"identity":"9a5d874b-b795-4554-8e6c-7fc9de8fd830","order_by":6,"name":"Yingli Song","email":"","orcid":"https://orcid.org/0000-0002-5808-8557","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yingli","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2025-03-15 07:18:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6231082/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6231082/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40643-025-00933-1","type":"published","date":"2025-08-26T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79088052,"identity":"48bfe264-a67b-4ba1-8800-a0e88fcd40d6","added_by":"auto","created_at":"2025-03-24 09:35:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":248784,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of active ingredients in OMBRD \u003cstrong\u003eA\u003c/strong\u003e Venn diagram of the targets of SPSI and the component targets of OMBRD \u003cstrong\u003eB\u003c/strong\u003e Network diagram of active ingredients in OMBRD and disease targets.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6231082/v1/87d3471325b3a97a19505490.png"},{"id":79088060,"identity":"afe49118-6845-41e3-b966-73319af6fbc1","added_by":"auto","created_at":"2025-03-24 09:35:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":368153,"visible":true,"origin":"","legend":"\u003cp\u003eCore target screening for OMBRD in the treatment of SPSI: \u003cstrong\u003eA \u003c/strong\u003ePPI network diagram of intersection components. \u003cstrong\u003eB \u003c/strong\u003eCore targets after screening by Betweenness unDir \u0026gt; 71.059, Closeness unDir \u0026gt; 0.006, and Degree unDir \u0026gt; 32.28. \u003cstrong\u003eC\u003c/strong\u003e Visualization of core targets.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6231082/v1/5ca43ffd04ac5a94b3abbc6b.png"},{"id":79088051,"identity":"b85fbbe3-3c52-4bb7-857e-8da88841ca0e","added_by":"auto","created_at":"2025-03-24 09:35:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":207183,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment analysis and KEGG pathway enrichment analysis diagrams of 101 intersection targets. \u003cstrong\u003eA\u003c/strong\u003e GO enrichment analysis; \u003cstrong\u003eB\u003c/strong\u003e KEGG pathway analysis diagram.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6231082/v1/58107e3b3d1a2a4962ea9295.png"},{"id":79091183,"identity":"3c5c75a5-f5c1-4b07-bf72-b0829bc85208","added_by":"auto","created_at":"2025-03-24 09:59:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":485446,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of the binding energy (kcal/mol) of key targets and active compounds\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6231082/v1/016ec06de89ae17f93dba2ef.png"},{"id":79088053,"identity":"d0e71148-8bf2-4de7-b753-57e4856959d1","added_by":"auto","created_at":"2025-03-24 09:35:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":561370,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking display of core targets and active ingredient: \u003cstrong\u003eA\u003c/strong\u003eBCL2 MOL002265 \u003cstrong\u003eB\u003c/strong\u003e BCL2 MOL002252 \u003cstrong\u003eC \u003c/strong\u003eBCL2 MOL005980 \u003cstrong\u003eD\u003c/strong\u003e EGFR MOL002265 \u003cstrong\u003eE\u003c/strong\u003e EGFR MOL005980 \u003cstrong\u003eF\u003c/strong\u003e GADPH MOL000006 \u003cstrong\u003eG\u003c/strong\u003e GADPH MOL002252 \u003cstrong\u003eH\u003c/strong\u003e GADPH MOL002265\u003cstrong\u003e I \u003c/strong\u003eGADPH MOL005980\u003cstrong\u003e J \u003c/strong\u003eJUN MOL005980 \u003cstrong\u003eK\u003c/strong\u003e STAT3 MOL002252 \u003cstrong\u003eL \u003c/strong\u003eTNF MOL005980\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6231082/v1/0ec7a633214097e4ad50a36d.png"},{"id":79088055,"identity":"ee16ccc5-bb76-45c4-a806-172d0c8ff3ad","added_by":"auto","created_at":"2025-03-24 09:35:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":237472,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro verification of OMBRD in the treatment of SPSI. \u003cstrong\u003eA\u003c/strong\u003eOMBRD Minimum Inhibitory Concentration (MIC )assay \u003cstrong\u003eB - E\u003c/strong\u003e ELISA was used to evaluate the changes in IL - 1α, IL - 6, TNF - α and IL - 36 in HaCaT cells invaded by Streptococcus pyogenes for 3 hours. PBS: control group; GAS: Streptococcus pyogenes invaded for 3 hours, and then washed with PBS and cultured continuously for 21 hours; GAS + OMBRD: Streptococcus pyogenes invaded for 3 hours, washed with PBS, and then OMBRD was added to the medium for continuous culture for 21 hours; GAS \u0026amp; OMBRD: Streptococcus pyogenes and OMBRD co - infected for 3 hours, washed with PBS and then cultured continuously for 21 hours. *, **, and *** indicate the statistical differences between the groups at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively; ns = not significant. n = 6.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6231082/v1/144620a44d70f2157f4a3fdd.png"},{"id":90344931,"identity":"6709552b-90ea-480a-a1a6-7bad560557a3","added_by":"auto","created_at":"2025-09-01 16:08:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3073511,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6231082/v1/f6c07177-5fc9-4495-b2a0-7432ca8489de.pdf"},{"id":79090015,"identity":"e91e3f82-c474-4001-b025-bc990d93a703","added_by":"auto","created_at":"2025-03-24 09:51:01","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":135143,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6231082/v1/381c857143d98c3a07491266.jpg"}],"financialInterests":"","formattedTitle":"Multi-Target Inhibition of Streptococcus pyogenes Skin Infections by Officinal Magnolia Bark Rhubarb Decoction: Network Pharmacology and Molecular Docking Insights","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe skin covers the entire body surface and is the largest organ in the human body. The skin plays a crucial role in maintaining the stability of the internal environment of the body, protecting the body from daily wear and tear, and regulating body temperature and perception(Piipponen, Li and Land\u0026eacute;n \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Baker et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).A large number of bacteria colonize on human skin. Under normal circumstances, this colonization is harmless. However, when the body's immunity declines, some bacteria may have flora imbalance and other problems and then attack the human body. Such bacteria are called opportunistic pathogens. As a representative of opportunistic pathogens, Streptococcus pyogenes is a common colonizing bacterium in humans and still poses a great threat to humans worldwide(Howden et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).At the end of 2022, the World Health Organization (WHO) reported that scarlet fever and invasive infections had increased significantly in many developed countries, and children were particularly affected(Wang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStreptococcus pyogenes (also known as Group A Streptococcus [GAS]) is an obligate human pathogen associated with many disease states. Streptococcus pyogenes can cause human diseases, ranging from superficial pharyngeal infections such as streptococcal pharyngitis and skin infections such as impetigo and cellulitis to life threatening invasive or toxin mediated manifestations such as necrotizing fasciitis, streptococcal toxic shock syndrome, scarlet fever and septic shock(Bae et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOf particular concern is Streptococcus pyogenes skin infection (SPSI), which ranges from superficial infections of the epidermis (such as impetigo) to severe invasive infections of the dermis and deeper tissues, including cellulitis and necrotizing fasciitis. Although it is sensitive to many different antibiotics (including beta lactams), the treatment of invasive Streptococcus pyogenes infections is complicated by factors such as its ability to form biofilms and secrete a large number of toxins. A biofilm is an attached and structured bacterial community growing in extracellular polymeric substances, which can enhance the community's ability to resist antibiotic killing. The toxins produced by Streptococcus pyogenes play a major role in destroying host tissues, including several membrane disrupting hemolysins, immunomodulatory superantigens, plasminogen activators, host cell adhesins, complement regulatory proteins, specific and non specific proteases, and a variety of other degradative enzymes(Alves-Barroco et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zou et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe emergence of antibiotic resistance threatens healthcare systems worldwide and raises the prospect of a post antibiotic era. Several factors, including the overuse and misuse of antibiotics, as well as exposure to environmental reservoirs of antibiotic resistant bacteria, contribute to the rising rate of antibiotic resistance(Ranjbar and Alam \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).The slowdown in the development of antibacterial drugs has led to a greater reliance on existing ones. Coupled with the poor management of antibiotics, this has further aggravated the development of drug resistance(Baker et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).There is an urgent need for drugs capable of resisting the development of drug resistance to combat multi drug resistant pathogens.\u003c/p\u003e \u003cp\u003eThe wide use of antibiotics has accelerated the development of drug resistance. Antibiotic resistance has made many common infectious diseases more difficult to treat and may even lead to a situation where there are no drugs available. Unlike single - component antibiotics, traditional Chinese medicine exerts its effects through multiple pathways and targets, thereby reducing the chance of bacteria developing drug resistance. These advantages have made traditional Chinese medicine highly regarded in the fight against bacterial infections, especially drug - resistant bacterial infections. Officinal magnolia bark rhubarb Decoction (OMBRD) is a traditional Chinese medicine prescription, which was first recorded in \"Jin Kui Yao Lue\" and is mainly used to treat symptoms such as fullness in the chest due to retained fluid and abdominal pain. This prescription consists of Officinal magnolia bark (OMB), Rhubarb (RH) and Aurantii Fructus Immaturus (AF). The decoction is made by boiling these ingredients in water and taking the liquid. OMB has rich pharmacological activities, including antibacterial, antiviral, antitumor and other effects. Magnolol and honokiol interfere with the structure and function of bacterial cell membranes, preventing the reproduction and survival of bacteria. These components have inhibitory effects on a variety of Gram - positive bacteria and some Gram - negative bacteria(Sun et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Zheng et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The main components of RH, anthraquinone compounds, have relatively strong antibacterial effects. Rhein, emodin and aloe - emodin effectively inhibit the growth and reproduction of a variety of harmful bacteria through multiple mechanisms such as destroying bacterial cell membranes, inhibiting nucleic acid and protein synthesis, and interfering with sugar metabolism(Zheng et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hesperidin and other substances in AF have certain antibacterial properties.\u003c/p\u003e \u003cp\u003eWith the development of multi - omics technologies and the rise of artificial intelligence and big - data analysis, the network pharmacology method has been promoted to develop and be widely applied. Meanwhile, network pharmacology is a new discipline in line with the overall characteristics of traditional Chinese medicine (TCM) and is highly original. More and more researchers are using the network pharmacology method to study the active ingredients, targets, and molecular mechanisms of TCM. Exploring the action targets and molecular mechanisms of TCM is the primary research objective in TCM network pharmacology. Through network construction, network analysis, and network verification, TCM network pharmacology reveals the multi - target and multi - pathway action modes of TCM and explains the action targets and molecular mechanisms by which TCM exerts its efficacy. Chinese herbal compound prescriptions have the characteristics of multi - component and multi - target actions. Due to its holistic and systematic research features, network pharmacology plays a unique role in exploring the compatibility mechanism of Chinese herbal compound prescriptions and provides a theoretical basis for clarifying the compatibility rules of Chinese herbal compound prescriptions(Jiashuo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Zhou et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTesting of active ingredients in OMBRD and their potential targets\u003c/h2\u003e \u003cp\u003eUse the TCMSP database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tcmsp\u003c/span\u003e\u003cspan address=\"https://tcmsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e - e.com/tcmsp.php) and search for keywords OMB, RH, and AF. With drug - likeness (DL)\u0026thinsp;\u0026ge;\u0026thinsp;0.18 as the screening criterion, obtain the active pharmaceutical ingredients of OMBRD. Then, use the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to obtain the SMILES format of the active compounds and import them into the SwissTargetPrediction platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Set the searched species as \u0026ldquo;Homo sapiens\u0026rdquo; and the probability greater than 0 for potential targets.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eConstruction of Staphylococcus aureus and SPSI - related target datasets\u003c/h3\u003e\n\u003cp\u003eTake \u0026ldquo;Streptococcus pyogenes skin infection\u0026rdquo;as the search term, retrieve relevant targets through the Human Gene Database (GeneCards, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the Online Mendelian Inheritance in Man Database (OMIM, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mirror.omim.org/\u003c/span\u003e\u003cspan address=\"https://mirror.omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), combine the retrieval results of the two databases, remove duplicate targets, and establish an SPSI - related target database. Import the obtained targets and drug active ingredient targets into VENNY2.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfogp.cnb.csic.es/tools/venny/index.html/\u003c/span\u003e\u003cspan address=\"https://bioinfogp.cnb.csic.es/tools/venny/index.html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and obtain the intersection targets for OMBRD to treat SPSI.\u003c/p\u003e\n\u003ch3\u003eConstruction of protein - protein interaction networks and screening of core targets\u003c/h3\u003e\n\u003cp\u003eUpload the intersection targets to the STRING platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.stringdb.org/\u003c/span\u003e\u003cspan address=\"https://cn.stringdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to construct a protein protein interaction (PPI) network. Set the protein species as \"Homo sapiens\", with the lowest interaction threshold, and keep other default parameters unchanged to obtain the PPI network. Then, import the tsv file into Cytoscape 3.9.1 software and use the Centiscape plugin for topological analysis. Set the conditions as Betweenness unDir 71.059, Closeness unDir 0.006, and Degree unDir 32.28 to screen out the core targets.\u003c/p\u003e\n\u003ch3\u003eGO and KEGG Enrichment Analysis\u003c/h3\u003e\n\u003cp\u003eImport the core targets into the DAVID (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://davidbioinformatics.nih.gov/\u003c/span\u003e\u003cspan address=\"https://davidbioinformatics.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database, and perform GO and KEGG pathway enrichment analyses on the intersection targets with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as the screening condition. Then, use the Wei Sheng Xin (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) website to visualize the results of the enrichment analysis. Among them, the GO enrichment analysis includes biological process (BP), cellular component (CC), and molecular function (MF).\u003c/p\u003e\n\u003ch3\u003eMolecular docking\u003c/h3\u003e\n\u003cp\u003eCalculate the degree value of active ingredients in the active ingredient - target network graph, and perform molecular docking on the top 15 active ingredients ranked by degree value with the 12 core protein molecules ranked by Degree value in the PPI network. Obtain the \"PDB\" format files of target protein 3D structure patterns and the \"MOL2\" format files of active ingredient 2D structure patterns from the PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"http://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and TCMSP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tcmsp\u003c/span\u003e\u003cspan address=\"https://tcmsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e - e.com/). Use Chem3D and PyMOL3 to optimize the structure transformation energy of active ingredients respectively. Use AutoDockTools 1.5.7 for molecular docking. First, perform operations such as water removal and hydrogen addition on receptor proteins and ligand small molecules, and then perform molecular docking on the 3D structures of the above - processed core components and core targets. Auto Dock Vina is used for molecular docking calculation. Select the result with the lowest binding energy as the best conformation according to the receptor - ligand binding energy, use Oringin2021 to draw the binding energy heat map, and use PyMOL3 for visualization.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eKeratinocytes (HaCat) were purchased from Shanghai Jinyuan Biological Co., Ltd. HaCat cells are grown in DMEM medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin - streptomycin solution, and cultured at 37℃ in 5% CO₂.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCultivation of Streptococcus pyogenes\u003c/h3\u003e\n\u003cp\u003eStreptococcus pyogenes with ATCC number BAA \u0026minus;\u0026thinsp;947 was purchased from Fuxiang Biology and grew in THY broth (Todd - Hewitt broth supplemented with 0.2% yeast extract) at a temperature of 37\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eOMBRD preparation\u003c/h3\u003e\n\u003cp\u003ePut 15g of OMB, 18g of RH and 9g of AF into 1L of water, boil for 3 hours and then let it stand until it reaches room temperature. After that, freeze - dry for 18 hours. Dissolve the obtained powder in sterile water to a concentration of 80mg/ml for later use.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMIC determination\u003c/h2\u003e \u003cp\u003eDetermine the MIC criteria by the broth micro dilution method recommended by CLSI. Prepare serial two fold dilutions of OMBRD broth in a 96 well plate, and add 50 \u0026micro;L of a 1\u0026times;10⁶ CFU/mL bacterial suspension to 50 \u0026micro;L of the antibiotic in each well, and then incubate at 37\u0026deg;C for 18 hours. Use 20 \u0026micro;L of resazurin test to observe the MIC and record it as the lowest concentration of the antibiotic(Nwabor et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eStreptococcus pyogenes invaded human skin substitutes for 3h, collected the supernatant, centrifuged at 1000 \u0026times; g for 10 min to remove particles and polymers, successively added antibody working solution for incubation and washed for 5 times. When washing, the water was patted dry, added chromogenic solution for incubation, added stop solution and mixed well, and then detected the changes of IL-1 α, IL-6, tnf- α and il-36 at OD600. ELISA kit was purchased from MEIMIAN Biological company.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eExperimental conclusions are based on the comparison of averages generated from at least three technical replicates or three biological replicates, and statistical analysis and graphing are carried out using GraphPad Prism 9.0 (GraphPad software). P\u0026thinsp;\u0026le;\u0026thinsp;0.05 is considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eActive ingredients and targets of OMBRD\u003c/h2\u003e \u003cp\u003eA total of 373 effective components of OMBRD were retrieved through the TCMSP database, among which there were 44 of OMB, 313 of RH, and 46 of AF. The SMILES format of the drug effective components was obtained from the PubChem database and imported into the SwissTargetPrediction database, resulting in a total of 4,933 targets corresponding to the active components. After deleting the identical targets, finally 775 action target diagrams (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePrediction of the action targets of OMBRD in treating SPSI\u003c/h2\u003e \u003cp\u003eA total of 553 targets of SPSI were obtained from the GeneCards database, and 260 targets of SPSI were obtained from the OMIM database. After summarizing and deleting the duplicate targets, a total of 795 targets of SPSI were finally obtained. A Venn diagram was drawn to obtain 101 potential targets of OMBRD for treating SPSI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBuild a component target network graph\u003c/h2\u003e \u003cp\u003eIn order to further explore the treatment mechanisms of OMBRD against Staphylococcus aureus and SPSI, the potential targets of OMBRD in treating SPSI, as well as their corresponding active ingredients and traditional Chinese medicines, were introduced to construct a composite targeting pathway network using Cytoscape 3.9.1 software. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows a network composed of 195 nodes and 852 edges. The Analyze Network plug in was used to calculate the topological parameters of network nodes and screen key active ingredients and action targets. The top 14 components in terms of Degree value in the network are MOL001803 (degree\u0026thinsp;=\u0026thinsp;23), MOL013278 (degree\u0026thinsp;=\u0026thinsp;22), MOL007879 (degree\u0026thinsp;=\u0026thinsp;22), MOL013279 (degree\u0026thinsp;=\u0026thinsp;21), MOL005814 (degree\u0026thinsp;=\u0026thinsp;20), MOL000006 (degree\u0026thinsp;=\u0026thinsp;20), MOL000008 (degree\u0026thinsp;=\u0026thinsp;20), MOL002235 (degree\u0026thinsp;=\u0026thinsp;20), MOL013277 (degree\u0026thinsp;=\u0026thinsp;19), MOL001729 (degree\u0026thinsp;=\u0026thinsp;17), MOL002252 (degree\u0026thinsp;=\u0026thinsp;17), MOL013430 (degree\u0026thinsp;=\u0026thinsp;16), MOL002233 (degree\u0026thinsp;=\u0026thinsp;16), MOL002265 (degree\u0026thinsp;=\u0026thinsp;16), and MOL005980 (degree\u0026thinsp;=\u0026thinsp;10), the only effective component in OMB (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, OMBRD exerts its effect on SPSI through multiple components and multiple targets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInformation on the core active ingredients of drugs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMol ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCore component name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePubChem CID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTargets Number\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL001803\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSinensetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e145659\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL013278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4',5,7,8-Tetramethoxyflavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e629964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL007879\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTetramethoxyluteolin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e631170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL013279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,7,4'-Trimethylapigenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e79730\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL005814\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etangeretin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL000006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eluteolin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5280445\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL000008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eapigenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5280443\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL002235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEUPATIN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5317287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL013277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsosinensetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e632135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL001729\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCrysophanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL002252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epalmidin B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5320385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL000476\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhyscion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL002233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Methyl cardol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5319544\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL002265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRheidin B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5320958\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOL005980\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNeohesperidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e442439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of PPI network and screening of core targets\u003c/h2\u003e \u003cp\u003eUsing the STRING database, import 101 intersection targets to draw a Protein - Protein Interaction (PPI) network, and use Cytoscape 3.9.1 to visualize the PPI network, obtaining network Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA ; perform topological analysis on the network through Centiscape, and screen with the topological parameters of Betweenness unDir\u0026thinsp;\u0026gt;\u0026thinsp;71.059, Closeness unDir\u0026thinsp;\u0026gt;\u0026thinsp;0.006, and Degree unDir\u0026thinsp;\u0026gt;\u0026thinsp;32.28 as criteria to obtain network Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; perform visualization processing on the 22 core targets in network Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB to obtain Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. The results show that there are 12 targets with relatively high degrees in the network, and BCL2, TNF, STAT3, CXCL8, EGFR, IL6, IL1B, GUN, GAPDH, TP53, AKT1, and MMP9 are the core targets for OMBRD to treat SPSI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eIn order to identify which metabolic pathways and biological processes might be affected in the treatment of SPSI by OMBRD, GO and KEGG enrichment analyses were carried out. Enrichment analysis was performed using 101 overlapping genes, and a total of 1,723 entries were obtained, including 1,505 for biological processes, 73 for cellular components, and 145 for molecular functions. The top ten in terms of count for each category were plotted, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA For biological processes, it was mainly involved in positive regulation of cell migration, cell activation, positive regulation of cytokine production, etc. For cellular components, they were mainly distributed in vesicle lumen, cytoplasmic vesicle lumen, membrane rafts, and membrane microdomains. For molecular functions, it was mainly related to kinase binding, cytokine receptor binding, protein kinase binding, and protein phosphatase binding, etc.\u003c/p\u003e \u003cp\u003eThrough enrichment analysis, 166 KEGG pathways were determined. We selected 10 of them for presentation, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB The grouping of KEGG mainly includes metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases. The KEGG pathways are mainly enriched in \"Pathways in cancer\", \"Lipid and atherosclerosis\", \"Coronavirus disease - COVID \u0026minus;\u0026thinsp;19\", \"AGE - RAGE signaling pathway in diabetic complications\" and \"Chagas disease\", etc.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eSelect the main core target proteins, namely BCL2 (with the uniprot identifier of 5vau), TNF (uniprot identifier: 2e7a), STAT3 (uniprot identifier: 6njs), CXCL8 (uniprot identifier: 1icw), EGFR (uniprot identifier: 1xkk), IL6 (uniprot identifier: 1alu), IL1B (uniprot identifier: 1l2h), GUN (uniprot identifier: 1jnm), GAPDH (uniprot identifier: 1u8f), TP53 (uniprot identifier: 1aie), AKT1 (uniprot identifier: 1h10), and MMP9 (uniprot identifier: 1jkd). Carry out molecular docking between the active ingredients that rank among the top 14 in terms of the number of associated target points and the unique active ingredients in OMB. The binding energy (in kcal/mol) is obtained through docking analysis, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e The lower the score, the stronger the binding capacity. A binding score less than 5.0 kcal/mol indicates a moderate affinity, and a score less than 7.0 kcal/mol indicates a high affinity. The results indicate that the docking binding energies of all ligands to receptors are less than 0 kcal/mol, and the docking binding energies of 16 ligands to receptors are less than 9 kcal/mol. Among them, the 12 with the strongest binding energy activities are BCL2 MOL002265, BCL2 MOL002252, BCL2 MOL0059807, EGFR MOL002265, EGFR MOL005980, GAPDH MOL000006, GAPDH MOL002252, GAPDH MOL002265, GAPDH MOL005980, JUN MOL005980, STAT3 MOL002252, and TNF MOL005980.。\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PyMOL3 was utilized to visualize the compound - target interaction complexes with the highest free binding energy for each target as well as their binding mechanisms. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the two - dimensional and three - dimensional diagrams of the molecular docking between key targets and active compounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro verification of OMBRD in the treatment of SPSI\u003c/h2\u003e \u003cp\u003eIn order to verify the effect of OMBRD in treating SPSI, we cultured human epidermal cells HaCaT and carried out a bacterial invasion experiment. By measuring the expressions of TNF - α, IL \u0026minus;\u0026thinsp;1α, IL \u0026minus;\u0026thinsp;6 and IL \u0026minus;\u0026thinsp;36, the antibacterial effect of OMBRD was reflected. Specifically, we divided the models into four groups. Group 1 was the PBS group, with 100 \u0026micro;l of PBS added and incubated for 3 hours; Group 2 was the Streptococcus pyogenes invasion group, with 1 \u0026times; 10⁶ CFU of colonies added. After 3 - hour invasion, the cells were washed three times with PBS and then 100 \u0026micro;l of culture medium was added; Group 3 was the post - invasion treatment group. After being invaded by Streptococcus pyogenes for 3 hours, the cells were washed three times with PBS, and then a mixture of 50 \u0026micro;l of culture medium and 50 \u0026micro;l of OMBRD was added; In Group 4, Streptococcus pyogenes and OMBRD were co - added to the human skin substitute and co - incubated for 3 hours. Then, the cells were washed three times with PBS and 100 \u0026micro;l of culture medium was added. The results showed that, compared with the invasion group, the inflammatory factors in the treatment group and the co - incubation group decreased significantly, proving that OMBRD can inhibit the growth of Streptococcus pyogenes in keratinocytes and has a certain therapeutic effect after Streptococcus pyogenes invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStreptococcus pyogenes is a globally well - known human - specific pathogen, causing more than 600\u0026nbsp;million cases of pharyngeal infections and over 100\u0026nbsp;million cases of skin infections every year. Streptococcus pyogenes is a Gram - positive coccus, which is spherical or oval - shaped, with a diameter of 0.6\u0026ndash;1\u0026micro;m, and most are arranged in chains(Hurst et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).It widely exists in nature, in human and animal feces, and can also be found in the nasopharynx of healthy people. This bacterium has strong adhesion and a variety of pathogenic factors. It can combine with skin cells and colonize and reproduce on them. The hyaluronidase it produces can decompose connective tissue, making the infection more likely to spread. Common symptoms of Streptococcus pyogenes infection include folliculitis, furuncle, carbuncle, acute cellulitis, abscess, erysipelas, and acute lymphangitis. They are manifested as skin redness, swelling, pain, induration, lumps, increased local skin temperature, and may be accompanied by tenderness. In severe cases, fluctuation, necrosis, ulcer and dysfunction may occur. Some patients may have systemic symptoms such as fever, headache, and general malaise, and in severe cases, complications such as bacteremia and septicemia may be accompanied(Siggins et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).Although people are concentrating on studying invasive indications, there are still many unknowns regarding the natural state of this bacterium during its colonization in the nasopharynx and on the skin. Clinically, antibiotics are usually used to treat infections caused by microorganisms such as Staphylococcus aureus and Streptococcus pyogenes. However, with the widespread use of antibiotics, the number of strains resistant to antibiotics such as macrolides is increasing day by day, making the research and development of new drugs extremely urgent. Based on the advantages of traditional Chinese medicine, such as multi - component, multi - target, wide - ranging pharmacological activities, low toxicity, and less likelihood of developing drug resistance, in this study, we used OMB, RH, and AF aqueous solutions for antibacterial experiments.\u003c/p\u003e \u003cp\u003eAs far as we know, this is the first study on the fact that the active ingredients in OMBRD can inhibit the growth of Streptococcus pyogenes and treat skin infections caused by Streptococcus pyogenes. In China, according to the theory of traditional Chinese medicine, the main function of OMBRD is to promote qi movement, so OMB is the principal drug in this prescription. In the prescription, OMB promotes qi movement and relieves fullness, Zhishi breaks qi and eliminates accumulation, and RH purges heat and unblocks the bowels. The three drugs are used in combination to achieve the effect of promoting qi movement and relieving constipation. It was recorded in \"Jin Kun Yao Lue\" written by Zhang Zhongjing, the most famous doctor in China in the early 3rd century AD (the Eastern Han Dynasty). This prescription is composed of OMB, RH and AF. Modern medical research shows that the active ingredients of these three substances can all play an antibacterial role(Guo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, M\u0026uuml;ller-Heupt et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Gao et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). According to \"Chinese Pharmacopoeia\", OMB is the dried bark of the trunk or root of Magnolia officinalis or Magnolia officinalis subsp. biloba in the Magnoliaceae family. It has a bitter taste and warm nature, and has the effects of drying dampness, resolving phlegm, and descending qi to relieve fullness. It enters the spleen, stomach, and large intestine meridians, and can be used to treat abdominal fullness caused by damp - obstruction, food - retention, and qi - stagnation; when it enters the lung meridian, it can be used to treat cough, panting, and excessive phlegm. Modern pharmacological studies have shown that OMB has anti - inflammatory, analgesic, anti - tumor, antioxidant and other effects. RH is a plant in the genus Rumex of the Polygonaceae family, an annual herb. Its root is thick, large and yellow. There are more than 200 species of Rumex plants in the world, mainly distributed in Europe and North America(Pliszko \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).RH (bitter - cold in nature, being good at clearing heat and breaking up accumulations, and also able to enter the blood aspect, expelling stasis and dredging menstruation. It is said in \"Xue Zheng Lun\" that \"it can remove the old and promote the new, and damage yang and harmonize yin. Wherever there is disharmony in the blood aspect due to qi reversal, the nature of RH can reach there without exception.\" Modern research shows that RH has pharmacological effects such as promoting blood circulation and stopping bleeding, anticoagulation, anti - inflammation, and antibacterial. AF is rich in synephrine (alkaloids) and flavonoids, and is mainly used to treat internal retention of food stagnation, constipation, phlegm obstruction and qi stagnation, etc. There are 19 prescriptions in the classic prescriptions in \"Shang Han Zang Bing Lun\" containing AF. Modern pharmacodynamic research shows that the active pharmaceutical ingredients contained in AF can improve intestinal dryness and constipation, promote gastrointestinal motility, accelerate metabolism, and have pharmacological effects such as antibacterial, anti - infection and lipid metabolism. In conclusion, all three active ingredients in OMBRD have antibacterial and anti - inflammatory effects. In order to study the mechanisms of the antibacterial and anti - infection effects of OMBRD, we adopted the method of network pharmacology to study and found that there are 101 related targets between the active ingredients of OMBRD and SPSI, indicating that the active ingredients of OMBRD may interact with SPSI.\u003c/p\u003e \u003cp\u003eThis research is based on network pharmacology analysis. Through retrieval in the TCMSP database, a total of 373 effective components of OMBRD were obtained, including 44 of OMB, 313 of RH, and 46 of AF. Among them, the top 14 with the highest Dgree values are Sinensetin, 4',5,7,8 - Tetramethoxyflavone, Tetramethoxyluteolin, 5,7,4' - Trimethylapigenin, tangeretin, luteolin, apigenin, EUPATIN, Isosinensetin, Crysophanol, palmidin B, Physcion, 2 - Methyl cardol, Rheidin B, and Neohesperidin, the only active component in OMB. 101 targets of OMBRD for treating SPSI were obtained. Through PPI network analysis, core targets such as BCL2, TNF, STAT3, CXCL8, EGFR, IL6, IL1B, GUN, GAPDH, TP53, AKT1, MMP9, etc. were screened out. Our analysis results show that Neohesperidin is effective for multiple targets of SPSI. Neohesperidin belongs to flavonoids, which are widely distributed in the plant kingdom. In recent years, they have attracted extensive attention due to their effective antioxidant, metal - chelating, anti - inflammatory, antibacterial, antifungal, anticancer, and antiviral properties(Sage et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Neves et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).Several studies have shown that Neohesperidin has significant antibacterial potential, and many bacteria and fungi have been studied(El-Kadem et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Akhter et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Neohesperidin (1.5 gm/l) exhibits moderate antifungal activity, with the maximum inhibition zones being 30%, 10% and 10% respectively against Monilinia fructicola, Botrytis cinerea and Alternaria alternata on fruits(Hern\u0026aacute;ndez et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).In vitro studies have shown that neohesperidin (at a concentration of 12.5\u0026ndash;50mg/ml) has an enhanced antibacterial activity against Escherichia coli by the agar diffusion method, with the maximum inhibition zone ranging from 17.4 to 24.2 mm. This effect may be mediated through the interaction with the cell membrane of Escherichia coli(Du et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).Neohesperidin at concentrations of 50 and 100 g/ml can treat skin - injury diseases. Its mechanism is to significantly inhibit the formation of RNS (reactive nitrogen species) and ROS (reactive oxygen species), the spread of intercellular adhesion molecule \u0026minus;\u0026thinsp;1 (ICAM \u0026minus;\u0026thinsp;1), and induce the synthesis of glycosaminoglycan (GAG) in cells under the action of IFN - γ and histamine, without inhibiting the cell proliferation of the normal human keratinocyte cell line NCTC 2544. This indicates that it has significant concentration - dependent antioxidant and anti - inflammatory effects(Graziano et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).Neohesperidin has also been found to have a significant inhibitory effect on pro - inflammatory mediators and IgE - induced markers (such as HA, TPS, CMA1, IL \u0026minus;\u0026thinsp;8, MCP \u0026minus;\u0026thinsp;1, MCP \u0026minus;\u0026thinsp;1) (at a dose of 400 \u0026micro;m in vitro and 20 mg/kg in vivo)(Liu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo further explore and understand the potential functions and interactions of the screening targets, we carried out enrichment analysis on 101 intersection targets. The results showed that they were mainly concentrated in aspects such as cell migration and cytokine production. Subsequently, we performed molecular docking to evaluate the binding situations of 15 active ingredients (Sinensetin, 4',5,7,8 - Tetramethoxyflavone, Tetramethoxyluteolin, 5,7,4' - Trimethylapigenin, tangeretin, luteolin, apigenin, EUPATIN, Isosinensetin, Crysophanol, palmidin B, Physcion, 2 - Methyl cardol, Rheidin B, Neohesperidin) and 12 core proteins (BCL2, TNF, STAT3, CXCL8, EGFR, IL6, IL1B, JUN, GAPDH, TP53, AKT1, MMP9). The results indicated that the binding energies of BCL2 - Neohesperidin, BCL2 - palmidin B, BCL2 - Rheidin B, EGFR - Neohesperidin, EGFR - Rheidin B, GAPDH - luteolin, GAPDH - Neohesperidin, GAPDH - palmidin B, GAPDH - Rheidin B, JUN - Neohesperidin, STAT3 - palmidin B, TNF - Neohesperidin, etc. were very low, suggesting that they might be the main active ingredients and targets for OMBRD to treat SPSI.\u003c/p\u003e \u003cp\u003eIn order to further verify the inhibitory effect of OMBRD on Streptococcus pyogenes and its effect on treating SPSI (Streptococcus pyogenes - skin and soft tissue infection), in - vitro experiments were carried out. Continuous two - fold dilution broth of OMBRD was prepared in a 96 - well plate. Single colonies with similar morphology were selected from the solid medium and placed in the liquid medium, and cultured at 37\u0026deg;C with 220 rpm for 4\u0026ndash;6 hours until the logarithmic growth phase of bacteria, so that the final bacterial suspension concentration reached approximately 5 \u0026times; 10⁵ CFU/mL. The results showed that the MIC (minimum inhibitory concentration) of OMBRD was 20 mg/mL. IL \u0026minus;\u0026thinsp;1α, IL \u0026minus;\u0026thinsp;6, IL \u0026minus;\u0026thinsp;36 and TNF - α are the most common interleukins secreted by keratinocytes during bacterial infection. We infected HaCaT cells with Streptococcus pyogenes and added OMBRD at the same time. It was found that OMBRD had a significant inhibitory effect on Streptococcus pyogenes after being added. Moreover, 3 hours after Streptococcus pyogenes infected HaCaT cells, after treatment with OMBRD, compared with the positive control group without drug addition, the interleukins also decreased significantly. Our in - vitro experiment preliminarily proved that OMBRD has a certain inhibitory effect on Streptococcus pyogenes and has a therapeutic effect on skin infections caused by Streptococcus pyogenes.\u003c/p\u003e \u003cp\u003eIn this experiment, through network pharmacology analysis combined with experimental verification, the inhibitory effect of OMBRD on SPSI was explored for the first time. It should be noted that when retrieving OMBRD active ingredients and targets related to SPSI from the database, the reliability and accuracy of the analysis and prediction depend on the quality of the data. To overcome this limitation, we initially carried out in - vitro experiments. The results showed that OMBRD has a certain inhibitory effect on Streptococcus pyogenes and has a therapeutic effect on skin infections caused by Streptococcus pyogenes. However, its specific mechanism still requires additional experiments for verification.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study pioneers the systematic exploration of the traditional formula OMBRD against Streptococcus pyogenes and its therapeutic mechanisms. Network pharmacology identified 373 bioactive components and 101 targets, with Neohesperidin, flavonoids, and anthraquinones as key constituents targeting BCL2, TNF, and IL6. Molecular docking revealed strong binding between Neohesperidin and critical targets, suggesting multi-pathway regulation of inflammation and apoptosis. In vitro assays demonstrated OMBRD's antibacterial activity (MIC: 20 mg/mL) and suppression of IL-1α, IL-6, IL-36, and TNF-α in infected HaCaT cells, confirming dual antimicrobial/anti-inflammatory effects. The findings underscore OMBRD's multi-component synergy against drug-resistant infections, while further in vivo validation is needed. This work advances the modernization of herbal formulas for anti-infective drug development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"320\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eAF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eAurantii Fructus Immaturus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eGAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eGroup A Streptococcus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eGO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eGene Ontology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eIL-1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eInterleukin-1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eIL-36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eInterleukin-36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eIL-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eInterleukin-6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eKEGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eMIC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eMinimum Inhibitory Concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eOMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eOfficinal magnolia bark\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eOMBRD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eOfficinal magnolia bark rhubarb Decoction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eRH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eRhubarb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eSPSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eStreptococcus pyogenes skin infection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eTCM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003etraditional Chinese medicine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003etumor necrosis factor-\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eWHO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 250px;\"\u003e\n \u003cp\u003eWorld Health Organization\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the funding support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuanhao Wang:\u0026nbsp;Search the database and build the dataset, Writing the original draft.Xinrui Wang: Writing the original draft. Xueying Zhang, Mengyi Pan and Mingyang Sun：Complete in vitro experiments. Zhiguo Chen: Validation. Yingli Song: Writing, review and editing, Supervision, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the National Natural Science Foundation of China (82302535) and the Natural Science Foundation of Heilongjiang Province(LH2023H008）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed to publish this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eSchool of Basic Medical Sciences, Harbin Medical University, 157th Rd of Bao jian, Nangang Distinct, Harbin 150081, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eThe Second Affiliated Hospital of Harbin University, 246th Rd of Xuefu, Nangang District, Harbin 150086, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkhter S, Arman M S I, Tayab M A, Islam M N \u0026amp; Xiao J (2024) Recent advances in the biosynthesis, bioavailability, toxicology, pharmacology, and controlled release of citrus neohesperidin. Crit. Rev. Food Sci. Nutr.\u003cem\u003e \u003c/em\u003e64: 5073-5092.https://doi.org/10.1080/10408398.2022.2149466\u003c/li\u003e\n\u003cli\u003eAlves-Barroco C, Paquete-Ferreira J, Santos-Silva T \u0026amp; Fernandes A R (2020) Singularities of Pyogenic Streptococcal Biofilms - From Formation to Health Implication. Front Microbiol\u003cem\u003e \u003c/em\u003e11: 584947.https://doi.org/10.3389/fmicb.2020.584947\u003c/li\u003e\n\u003cli\u003eBae S, Gu H, Gwon M-G, An H-J, Han S-M, Lee S-J, Leem J \u0026amp; Park K-K (2022) Therapeutic Effect of Bee Venom and Melittin on Skin Infection Caused by Streptococcus pyogenes. Toxins (Basel)\u003cem\u003e \u003c/em\u003e14.https://doi.org/10.3390/toxins14100663\u003c/li\u003e\n\u003cli\u003eBaker P, Huang C, Radi R, Moll S B, Jules E \u0026amp; Arbiser J L (2023) Skin Barrier Function: The Interplay of Physical, Chemical, and Immunologic Properties. Cells\u003cem\u003e \u003c/em\u003e12.https://doi.org/10.3390/cells12232745\u003c/li\u003e\n\u003cli\u003eBaker S J, Payne D J, Rappuoli R \u0026amp; Gregorio E D (2018) Technologies to address antimicrobial resistance. Proc. Natl. Acad. Sci. U. S. A.\u003cem\u003e \u003c/em\u003e115: 12887-12895.https://doi.org/10.1073/pnas.1717160115\u003c/li\u003e\n\u003cli\u003eDu L, Jiang Z, Xu L, Zhou N, Shen J, Dong Z, Shen L, Wang H \u0026amp; Luo X (2018) Microfluidic reactor for lipase-catalyzed regioselective synthesis of neohesperidin ester derivatives and their antimicrobial activity research. Carbohydr. Res.\u003cem\u003e \u003c/em\u003e455: 32-38.https://doi.org/10.1016/j.carres.2017.11.008\u003c/li\u003e\n\u003cli\u003eEl-Kadem A H, Negm W A, Elekhnawy E, Binsuwaidan R, Attallah N G, Moglad E, Sultan A A J J o D D S \u0026amp; Technology (2024) Neohesperidin-loaded microemulsion for improved healing of Acinetobacter baumannii-infected wound. J Drug Deliv Sci Tec\u003cem\u003e \u003c/em\u003e98: 105905.https://doi.org/10.1016/j.jddst.2024.105905\u003c/li\u003e\n\u003cli\u003eGao Z, Jiang S, Zhong W, Liu T \u0026amp; Guo J (2023) Linalool controls the viability of Escherichia coli by regulating the synthesis and modification of lipopolysaccharide, the assembly of ribosome, and the expression of substrate transporting proteins. Food Res. Int.\u003cem\u003e \u003c/em\u003e164: 112337.https://doi.org/10.1016/j.foodres.2022.112337\u003c/li\u003e\n\u003cli\u003eGraziano A C E, Cardile V, Crasc\u0026igrave; L, Caggia S, Dugo P, Bonina F \u0026amp; PanicoCaggia A (2012) Protective effects of an extract from Citrus bergamia against inflammatory injury in interferon-\u0026gamma; and histamine exposed human keratinocytes. Life Sci.\u003cem\u003e \u003c/em\u003e90: 968-74.https://doi.org/10.1016/j.lfs.2012.04.043\u003c/li\u003e\n\u003cli\u003eGuo Y, Hou E, Wen T, Yan X, Han M, Bai L-P, Fu X, Liu J \u0026amp; Qin S (2021) Development of Membrane-Active Honokiol/Magnolol Amphiphiles as Potent Antibacterial Agents against Methicillin-Resistant Staphylococcus aureus (MRSA). J. Med. Chem.\u003cem\u003e \u003c/em\u003e64: 12903-12916.https://doi.org/10.1021/acs.jmedchem.1c01073\u003c/li\u003e\n\u003cli\u003eHern\u0026aacute;ndez A, Ruiz-Moyano S, Galv\u0026aacute;n A I, Merch\u0026aacute;n A V, Nevado F P, Aranda E, Serradilla M J, C\u0026oacute;rdoba M d G \u0026amp; Mart\u0026iacute;n A (2021) Anti-fungal activity of phenolic sweet orange peel extract for controlling fungi responsible for post-harvest fruit decay. Fungal Biol\u003cem\u003e \u003c/em\u003e125: 143-152.https://doi.org/10.1016/j.funbio.2020.05.005\u003c/li\u003e\n\u003cli\u003eHowden B P, Giulieri S G, Lung T W F, Baines S L, Sharkey L K, Lee J Y H, Hachani A, Monk I R \u0026amp; Stinear T P (2023) Staphylococcus aureus host interactions and adaptation. Nat. Rev. Microbiol.\u003cem\u003e \u003c/em\u003e21: 380-395.https://doi.org/10.1038/s41579-023-00852-y\u003c/li\u003e\n\u003cli\u003eHurst J R, Shannon B A, Craig H C, Rishi A, Tuffs S W \u0026amp; McCormick J K (2022) The Streptococcus pyogenes hyaluronic acid capsule promotes experimental nasal and skin infection by preventing neutrophil-mediated clearance. PLoS Pathog.\u003cem\u003e \u003c/em\u003e18: e1011013.https://doi.org/10.1371/journal.ppat.1011013\u003c/li\u003e\n\u003cli\u003eJiashuo W U, Fangqing Z, Zhuangzhuang L I, Weiyi J \u0026amp; Yue S (2022) Integration strategy of network pharmacology in Traditional Chinese Medicine: a narrative review. J. Tradit. Chin. Med.\u003cem\u003e \u003c/em\u003e42: 479-486.https://doi.org/10.19852/j.cnki.jtcm.20220408.003\u003c/li\u003e\n\u003cli\u003eLi G, Wang X, Long X, Chen S, Han L, Cheng Y, Chen S, Liu W, Feng R \u0026amp; Sun Z J F B (2024) Attenuation by Zhiqiao neohesperidin on dextran sulfate sodium-induced colitis in mice through inhibition of the TLR4/NF-\u0026kappa;B pathway. Food Biosci\u003cem\u003e \u003c/em\u003e62: 105328.https://doi.org/10.1016/j.fbio.2024.105328\u003c/li\u003e\n\u003cli\u003eLiu T, Ma Y, Zhang R, Zhong H, Wang L, Zhao J, Yang L \u0026amp; Fan X (2019) Resveratrol ameliorates estrogen deficiency-induced depression- and anxiety-like behaviors and hippocampal inflammation in mice. Psychopharmacology (Berl.)\u003cem\u003e \u003c/em\u003e236: 1385-1399.https://doi.org/10.1007/s00213-018-5148-5\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller-Heupt L K, Vierengel N, Gro\u0026szlig; J, Opatz T, Deschner J \u0026amp; Loewenich F D v (2022) Antimicrobial Activity of Eucalyptus globulus, Azadirachta indica, Glycyrrhiza glabra, Rheum palmatum Extracts and Rhein against Porphyromonas gingivalis. Antibiotics (Basel)\u003cem\u003e \u003c/em\u003e11.https://doi.org/10.3390/antibiotics11020186\u003c/li\u003e\n\u003cli\u003eNeves J R, Grether-Beck S, Krutmann J, Correia P, Jr J E G, Sant\u0026apos;Anna B \u0026amp; Kerob D (2022) Efficacy of a topical serum containing L-ascorbic acid, neohesperidin, pycnogenol, tocopherol, and hyaluronic acid in relation to skin aging signs. J. Cosmet. Dermatol.\u003cem\u003e \u003c/em\u003e21: 4462-4469.https://doi.org/10.1111/jocd.14837\u003c/li\u003e\n\u003cli\u003eNwabor O F, Chukamnerd A, Terbtothakun P, Nwabor L C, Surachat K, Roytrakul S, Voravuthikunchai S P \u0026amp; Chusri S (2023) Synergistic effects of polymyxin and vancomycin combinations on carbapenem- and polymyxin-resistant Klebsiella pneumoniae and their molecular characteristics. Microbiol Spectr\u003cem\u003e \u003c/em\u003e11: e0119923.https://doi.org/10.1128/spectrum.01199-23\u003c/li\u003e\n\u003cli\u003ePiipponen M, Li D \u0026amp; Land\u0026eacute;n N X (2020) The Immune Functions of Keratinocytes in Skin Wound Healing. Int. J. Mol. Sci.\u003cem\u003e \u003c/em\u003e21.https://doi.org/10.3390/ijms21228790\u003c/li\u003e\n\u003cli\u003ePliszko A (2019) Typification of two natural hybrids in Rumex (Polygonaceae). Kew Bull.\u003cem\u003e \u003c/em\u003e74: 17.https://doi.org/10.1007/s12225-019-9803-8\u003c/li\u003e\n\u003cli\u003eRanjbar R \u0026amp; Alam M (2023) Antimicrobial Resistance Collaborators (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Evid. Based Nurs.https://doi.org/10.1136/ebnurs-2022-103540\u003c/li\u003e\n\u003cli\u003eSage J, Renault J, Domain R, Bojarski K K, Chazeirat T, Saidi A, Leblanc E, Nizard C, Samsonov A, Kurfurst R, Lalmanach G \u0026amp; 4 F L (2022) Modulation of the expression and activity of cathepsin S in reconstructed human skin by neohesperidin dihydrochalcone. Matrix Biol.\u003cem\u003e \u003c/em\u003e107: 97-112.https://doi.org/10.1016/j.matbio.2022.02.003\u003c/li\u003e\n\u003cli\u003eSiggins M K, Lynskey N N, Lucy E. Lamb L A J, Kristin K. Huse M P, Banerji S, Turner C E, Woollard K, Jackson D G \u0026amp; Sriskandan S (2020) Extracellular bacterial lymphatic metastasis drives Streptococcus pyogenes systemic infection. Nat. Commun.\u003cem\u003e \u003c/em\u003e11: 4697.https://doi.org/10.1038/s41467-020-18454-0\u003c/li\u003e\n\u003cli\u003eSun J, Xie Y, Chen Z, Fan Y, Liu Y, Gao Q, Li J, Bai J \u0026amp; Yang Y (2023) Antimicrobial activity and mechanism of Magnolia officinalis root extract against methicillin-resistant Staphylococcus aureus based on mannose transporter. Ind Crop Prod\u003cem\u003e \u003c/em\u003e201: 116953.https://doi.org/10.1016/j.phymed.2022.154073\u003c/li\u003e\n\u003cli\u003eWang J, Ma C, Li M, Gao X, Wu H, Dong W \u0026amp; We L (2023) Streptococcus pyogenes: Pathogenesis and the Current Status of Vaccines. Vaccines (Basel)\u003cem\u003e \u003c/em\u003e11.https://doi.org/10.3390/vaccines11091510\u003c/li\u003e\n\u003cli\u003eZheng Q, Li S, Li X \u0026amp; Liu R (2021) Advances in the study of emodin: an update on pharmacological properties and mechanistic basis. Chin. Med.\u003cem\u003e \u003c/em\u003e16: 102.https://doi.org/10.1186/s13020-021-00509-z\u003c/li\u003e\n\u003cli\u003eZheng S, Deng R, Huang G, Ou Z \u0026amp; Shen Z (2024) Effects of honokiol combined with resveratrol on bacteria responsible for oral malodor and their biofilm. J Oral Microbiol\u003cem\u003e \u003c/em\u003e16: 2361402.https://doi.org/10.1080/20002297.2024.2361402\u003c/li\u003e\n\u003cli\u003eZhou W, Hu Z, Wu X, Zhang S, Jiang Y, Tian L, Huang X, Ma Z, Qiu L, Zheng P, Zhang S \u0026amp; Lu Z (2023) Elucidation of the underlying mechanism of Hua-ban decoction in alleviating acute lung injury by an integrative approach of network pharmacology and experimental verification. Mol. Immunol.\u003cem\u003e \u003c/em\u003e156: 85-97.https://doi.org/10.1016/j.molimm.2023.02.013\u003c/li\u003e\n\u003cli\u003eZou Z, Singh P, Pinkner J S, Obernuefemann C L P, Wei Xu 1 T M N, Dodson K W, Almqvist F, Hultgren S J \u0026amp; Caparon M G (2024) Dihydrothiazolo ring-fused 2-pyridone antimicrobial compounds treat Streptococcus pyogenes skin and soft tissue infection. Sci Adv\u003cem\u003e \u003c/em\u003e10: eadn7979.https://doi.org/10.1126/sciadv.adn7979\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Network pharmacology, Drug discovery, Molecular docking, Streptococcus pyogenes","lastPublishedDoi":"10.21203/rs.3.rs-6231082/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6231082/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStreptococcus pyogenes infection still poses a great threat to humanity on a global scale. The emergence of antibiotic resistance endangers health care systems worldwide. Unlike single component antibiotics, traditional Chinese medicine exerts its effects through multiple pathways and targets, thereby reducing the chance of bacteria developing resistance. The main components in Officinal magnolia bark rhubarb Decoction, namely Officinal magnolia bark, Rhubarb and Immature Bitter Orange, have been proven to have antibacterial effects in previous studies. In this study, through network pharmacology and molecular docking, the interactions between its active components and the targets related to Streptococcus pyogenes skin infection were determined, and enrichment analysis was carried out. Molecular docking studies were conducted on 15 screened drug active components and 12 target proteins. Finally, in vitro experiments were used to prove that Officinal magnolia bark rhubarb Decoction can inhibit the growth of Streptococcus pyogenes and can treat Streptococcus pyogenes skin infections.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Multi-Target Inhibition of Streptococcus pyogenes Skin Infections by Officinal Magnolia Bark Rhubarb Decoction: Network Pharmacology and Molecular Docking Insights","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 09:34:57","doi":"10.21203/rs.3.rs-6231082/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-25T18:41:08+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-21T09:53:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-18T07:52:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2025-03-15T03:18:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a885af79-765f-43ba-9138-2d62da78e8c7","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T16:03:04+00:00","versionOfRecord":{"articleIdentity":"rs-6231082","link":"https://doi.org/10.1186/s40643-025-00933-1","journal":{"identity":"bioresources-and-bioprocessing","isVorOnly":false,"title":"Bioresources and Bioprocessing"},"publishedOn":"2025-08-26 15:57:11","publishedOnDateReadable":"August 26th, 2025"},"versionCreatedAt":"2025-03-24 09:34:57","video":"","vorDoi":"10.1186/s40643-025-00933-1","vorDoiUrl":"https://doi.org/10.1186/s40643-025-00933-1","workflowStages":[]},"version":"v1","identity":"rs-6231082","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6231082","identity":"rs-6231082","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}