Chemical components and mechanism of Jiuwei Huaban pill in treating psoriasis

SUI Yin-hai, HAO Lei, HUO Zhi-peng, WANG Yu,4, HE Yi?

1.Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China

2.Development Center of Modern Chinese Medicine, Research Institute of Tasly Holding Group Co.Ltd.,Tianjin 300410,China

3.State Key Laboratory of Critical Technology in Innovative Chinese Medicine,Tasly Pharmaceutical Group Co.Ltd.,Tianjin 300410,China

4.School of Pharmaceutical Science and Technology,Tianjin University,Tianjin 300072,China

Keywords:

ABSTRACT Objective: To systematically study the key active ingredients of Jiuwei Huaban pill in the treatment of psoriasis and explore its mechanism of action.Methods: The chemical components of Jiuwei Huaban pill were systematically identified by UPLC-QTOF/MSE, and network pharmacology method was used to study the main pharmacodynamic substances of Jiuwei Huaban pill and discuss the mechanism of action.Molecular docking technology was used to verify the binding activity of key components and target proteins.Results: A total of 75 components of Jiuwei Huaban pill were identified, 35 of which were key components for psoriasis treatment, including luteolin, baicalin, baicalin, paeoniflorin and geniposide.Enrichment analysis of 30 core target pathways showed that Jiuwei Huaban pill played its role in the treatment of psoriasis from the IL-17 signaling pathway, TNF signaling pathway,PI3K-Akt signaling pathway, Th17 signaling pathway, and MAPK signaling pathway.The key active components in Jiuwei Huaban pill and targets with the highest DGREE value in the "component target disease" network have strong binding activity.Conclusion: The active ingredients and mechanism of action of Jiuwei Huaban pill in the treatment of psoriasis were preliminarily clarified, which provids reference for quality control.

1.Introduction

Psoriasis is a chronic inflammatory skin disease that troubles approximately 1% to 3% of patients worldwide[1,2].Its pathological characteristics are mainly divided into three aspects, namely abnormal proliferation of keratinocytes, inflammatory cell infiltration, and vascular proliferation[3].Clinical manifestations are mostly erythema, papules, scales, and itching, which affect the patient’s appearance and are difficult to treat and easy to relapse.Therefore, long-term treatment is necessary and costly[4].Not only does it have a significant negative impact on the physical and mental health of patients, but it also brings a heavy economic burden.

Jiuwei Huaban Fang is a traditional Chinese medicine formula based on the ancient famous formula “Rhinoceros Horn Dihuang Tang”, which is made up of modified prescriptions.The monarch medicine is Huangqin, Jinyinhua, and Shuiniujiao, which are used to cool blood and dissipate stasis.The courtiers medicine is Chishao and Mudanpi, which can not only clear heat and cool blood, but also promote blood circulation and enhance the effect of Huaban.The adjuvants are Jiaozhizi, Shengshigao, and Diyu.The three are used together to prevent blood and heat buildup, making the medicine licorice, which is used to harmonize various medicines.The whole recipe has the effects of clearing heat and detoxifying,promoting blood circulation and resolving blood stasis, and cooling blood and resolving spots[5].After years of clinical application,this formula has shown good efficacy and can significantly reduce the area of skin lesions in patients with psoriasis vulgaris and improve its severity[6,7].On the basis of completing the research on the extraction, refinement, and preparation of the effective ingredients of Jiuwei Huaban Formula in the early stage, this study uses UPLC-Q-TOF/MSEtechnology to analyze and identify the chemical components in Jiuwei Huaban Pill.Based on this, network pharmacology technology is used to screen the active ingredients in Jiuwei Huaban Pill and predict their possible therapeutic mechanisms for psoriasis, In order to preliminarily elucidate the pharmacological substance basis of Jiuwei Huaban pill, and provide reference for further pharmacological research and clinical trials of Jiuwei Huaban Wan in the treatment of psoriasis.

2.Materials and methods

2.1 Medicinal materials and drugs

Slices: Chishao (batch number: 1709133) purchased from Gansu Zhongtian Pharmaceutical Co., Ltd., Jiaozhizi (batch number:1812015466), Mudanpi (batch number: 1812021905), Diyu (batch number: 2103011264), Gancao (batch number: 2107021119)purchased from Hebei Linshi Shengtai Pharmaceutical Co., Ltd.,Jinyinhua (batch number: 220301), Huangqin (batch number:220101) purchased from Bozhou Purun Pharmaceutical Co., Ltd,According to the identification of intermediate engineers Huo Zhipeng and Hao Lei from Tianshili Research Institute, they all comply with the relevant provisions of Part One of the Chinese Pharmacopoeia.

Reference materials: citric acid, gallic acid, luteolin, baicalin,wogonin, luteolin, glycyrrhizic acid, catechin, epicatechin (batch numbers 100396-200301, 110831-20803, 111720-200905, 111701-200501, 111514-20403, 111520-200504, 111610-200604, 877-200001, 110878-200102, respectively), chlorogenic acid, ammonium glycyrrhetinic acid, 3,5-O-dicaffeoyl quinic acid Baicalin, 4,5-Odicaffeoyl quinic acid, baicalin, paeoniflorin, rutin (batch numbers 110753-202018, 110731-201619, 111782-201505, 111595-201607,110736-202145, 100080-202012, 111894-202104, 110715-202122,respectively), 4-caffeioyl quinic acid (batch number 20150401,Tianjin Shilan Technology Co., Ltd.).

2.2 Instruments and reagents

Electronic analytical balance (METTLER TOLEDO, Switzerland),Milli-Q ultra pure water system (Millipore, USA), UPLC-Q-TOFMS mass spectrometer, leucine enkephalin (Waters, USA), formic acid mass spectrometry (Teshia Chemical Industry Development Co., Ltd.), chromatography methanol, mass spectrometry acetonitrile(Merck, Germany), purified water (Hangzhou Wahaha Group Co.,Ltd.).

2.3 Database and software

Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), https://old.tcmsp-e.com/tcmsp.php PubChem database(https://pubchem.ncbi.nlm.nih.gov/)SwissADME platform(http://www.swissadme.ch/)SwissTargetPrediction platform(http://www.swisstargetprediction.ch/)DisGeNET, a database of disease related genes and mutation sites, (https://www.disgenet.org/), GeneCards database(https://www.genecards.org/)DrugBank database(https://go.drugbank.com/)Online Human Mendelian Genetic Database (OMIM, https://www.omim.org/oy)Therapeutic Target Database (TTD, http://db.idrblab.net/ttd/)Global Protein Resource Database (UniProt, https://www.uniprot.org/), Venny 2.1.0(https://bioinfogp.cnb.csic.es/tools/venny/)STRING database(https://www.string-db.org/)PyMOL 2.4.0,DAVID database(https://david.ncifcrf.gov/)WeChat Online Mapping(http://www.bioinformatics.com/), Cytoscape 3.9.1, OpenBabel 2.4.1, RCSBPDB Database(https://www.rcsb.org/)AutoDock Vina,Masslynx 4.1.

2.4 Chemical composition analysis of Jiuwei Huaban Wan based on UPLC-Q-TOF/MSE

2.4.1 Jiuwei Huaban Pill test solution

Accurately weigh 2.0 g of Jiuwei Huaban Pill powder into a conical flask with a stopper, add 50 mL of 50% methanol solution, sonicate for 30 min, shake well, take an appropriate amount, centrifuge for 10 min (speed: 12 000 r/min, radius: 9.8 cm), and take the supernatant for 0.22 μm microporous filter membrane, obtained immediately.

2.4.2 Single drug test solution

Weigh an appropriate amount of each single medicinal herb in Jiuwei Huaban Pill, and process it according to the intermediate preparation method of Jiuwei Huaban Pill to obtain each single medicinal herb extract.Accurately weigh 0.2 g of each single medicinal herb extract, and process it according to the method in 1.4.1 to obtain it.

2.4.3 Reference solution

Accurately weigh an appropriate amount of each reference substance under item 1.1, dissolve it in 50% methanol, and bring to volume in a 100 mL volumetric flask.After passing 0.22 μm microporous filter membrane is used to obtain a mixed reference solution with a mass concentration of approximately 80 mg/mL.

2.4.4 Testing conditions

Chromatographic conditions: Chromatographic column: ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm) ; Mobile phase: 0.1%formic acid aqueous solution (A) - acetonitrile (B); Gradient elution:(0～10 min, 5% B; 10～55 min, 5%～20% B; 55～72 min, 20%～38%B; 72～83 min, 38%～90% B; 83～85 min, 90% B; 85～87 min,90%～5% B; 87～90 min, 5% B); Flow rate: 0.3 mL/min; Column temperature: 30 ℃; Injection volume: 3 μL.

Mass spectrum conditions: electrospray ion source (ESI) is used to scan in positive and negative ion modes, with scanning range ofm/z 50～1 200, capillary voltage of 2.3 kV, taper hole voltage of 40 V,high-purity argon (Ar) as collision gas, atomized gas (N2) flow rate of 50 L/h, desolvent gas (N2) flow rate of 800 L/h, desolvent gas temperature of 350 ℃, ion source temperature of 110 ℃, and low energy scanning without collision energy, High energy MSEmode scanning collision energy of 20-50 eV, dry gas temperature of 350℃, dry gas flow rate of 10 L/min, real-time correction using leucine enkephalin (m/z554.2615 [M-H]-,m/z556.2771 [M+H]+)[8].

2.5 Network pharmacological analysis

2.5.1 Active ingredient screening and target prediction

Import the chemical components identified in item 1.4 into the SwissADME platform for activity screening, and include components that comply with the Lipinski rule.Components that do not meet this condition but are included in the 2020 edition of the Chinese Pharmacopoeia, or components with oral bioavailability(OB) 30 and class drug resistance (DL) 0.18 in the TCMSP database, will still be included.Obtain Canonical SMILES of active ingredients through the PubChem database and import them into the Swiss Target Prediction platform for target prediction to obtain relevant targets of active ingredients.

2.5.2 Screening of related targets for the treatment of psoriasis with Jiuwei Huaban Pill

In the five databases of GeneCards, DisGeNET, DrugBank,OMIM, and TTD, relevant targets were collected using the keyword“Psoriasis”.GeneCards and DisGeNET were screened with“Relevance score” (2 rounds of median screening) and “Score gda” ( 1), respectively, and the collected targets were integrated and deduplicated.Intersect the obtained results with the drug target obtained in 1.5.1 to obtain the drug disease intersection target.

2.5.3 Construction of core target protein protein interaction(PPI) network

Import the intersection targets obtained in 1.5.2 into the STRING database, and import the PPI network obtained into the software Cytoscape 3.9.1.Use the degree values calculated by “CytoNCA”to screen the targets (with a median of 2 rounds of degree value screening) to obtain the core targets.

2.5.4 GO analysis and KEGG pathway enrichment analysis

Using the DAVID database, GO and KEGG analyses were conducted on the core targets obtained in 1.5.3.Species were selected as humans, with an error detection rate (FDR) of 0.01 as the enrichment standard.The results were then imported into the microbiological information online mapping platform for visualization.

Fig 1 BPI chromatograms of Jiuwei Huaban pill by UPLC-Q-TOF/MSE

2.5.5 Construction of the “Component Target Pathway”network for Jiuwei Huaban Pill

Integrate the top 20 pathways obtained from KEGG analysis,corresponding targets, and active ingredients corresponding to the reverse of the targets.Use Cytoscape 3.9.1 to construct a “component target pathway” network for Jiuwei Huaban Pill.Use the plugin“Analyze Network” to analyze and calculate, and find the chemical components with the highest degree ranking, which are the key components of Jiuwei Huaban Pill in treating psoriasis.

2.5.6 Molecular docking

Download the SDF file of the candidate key component 3D structure through the PubChem database, select the appropriate crystal structure in the Uniprot database, download the PDB format file of the crystal structure through RCSBPDB protein data, use OpenBabel 2.4.1 and AutoDockTools 1.5.6 software for preliminary processing, use Autodockvina to run molecular docking and score the docking results.It is generally believed that the binding energy between the receptor and ligand is -4.25 kcal/mol, It indicates that it has corresponding binding ability, -5.0 kcal/mol, indicating good binding activity, and -7.0 kcal/mol has strong binding activity[9-12].Visualize the results of molecular docking using PyMOL 2.4.0 software.

3.Results

3.1 Chemical composition analysis of Jiuwei Huaban Pill

The base peak ion chromatography (BPI diagram) of UPLC-QTOF/MSEanalysis is shown in Figure 1.Combined with the mass spectrometry information of the Jiuwei Huaban Pill test solution, a total of 75 chemical components were identified through comparison of reference materials and reference analysis.The main components are flavonoids and phenolic acids, and the specific results are shown in Table 1.According to the mass spectrometry information of a single herb, the chemical components were classified and classified.Among them, 6 were from Jinyinhua, 23 were from Huangqin, 1 was from Chishao, 3 were from Mudanpi, 5 were from Chishao and Mudanpi, 17 were from Diyu, 3 were from Diyu, Chishao and Mudanpi, 4 were from Jiaozhizi, , 4 were from Gancao, 8 were from Jinyinhua and Jiaozhizi, and 1 was from Chishao, Mudanpi,Huangqin, Jiaozhizi and Jinyinhua have common ingredients.

Tab 1 Identification and attribution of chemical components in Jiuwei Huaban pill

No tR/min Identity Formula Measured mass(m/z) Error(×10-6) MSE Mark 23 23.61 Paeoniflorin* C23H28O11 525.161[M+HCOO]- -0.8 479.156 0,449.143 9,327.106 2,165.054 8,121.028 3 CM2 503.1529[M+Na]+ 0.0 ——24 25.86 3-O-galloyl proanthocyanidin B3[16] C37H30O16 729.1454[M-H]- -1.1 577.134 8,559.122 0,425.086 2,407.076 3,289.071 6,269.045 4 D9 25 26.69 Galloylpaeoniflorin[22] C30H32O16 647.1597[M-H]- 2.8 313.051 1,271.045 9,211.020 4,169.013 7 C1 26 27.06 Tetragalloylglucose[15] C34H28O22 787.1002[M-H]- 1.1 447.057 0,295.045 8,169.013 2 CM3 27 27.26 Secoxyloganin[20] C17H24O11 403.1231[M-H]- -2.2 371.094 4,223.060 8 J3 427.1208[M+Na]+ -1.9 265.068 3,255.084 1,233.039 9 28 28.98 Brevifolincarboxylicacid[16] C13H8O8 291.0173[M-H]- -2.0 211.059 4,196.037 1,181.013 1,153.018 7 D10 29 31.29 Ellagic acid[16] C14H6O8 300.9982[M-H]- -1.3 283.994 1,257.009 8,229.012 8,201.019 2,185.051 8 DCM2 417.1178[M-H]- -1.9 255.064 5,135.006 5,119.048 4 G1 441.1162[M+Na]+ 0.0 257.080 7 31 33.69 2,3,4,6-O-tetragalloyl-β-30 31.87 Liquiritin* C21H22O9 D-glucoside[16] C34H28O22 787.0997[M-H]- -1.1 635.086 5,617.080 3,617.080 3,483.077 3,465.062 8,447.055 8,295.045 6 D11 32 34.55 Rutin * C27H30O16 609.1452[M-H]- 0.5 300.026 2,271.024 4,255.028 5 JZ5 33 34.9 Chrysin 6-C-glucoside 8-Carabinoside or isomers[23] C26H28O13 547.1451[M-H]- 0.2 457.101 6,427.102 4,367.080 9,337.070 5 H2 34 35.98 galloylpaeoniflorin[15,18] C30H32O15 631.1648[M-H]- -2.4 399.092 2,313.055 2,271.045 2,169.012 9 CM4 35 35.98 Cynaroside* C21H20O11 447.0925[M-H]- -0.7 285.038 9 J4 36 36.06 Chrysin 6-C-glucoside 8-Carabinoside or isomers[23] C26H28O13 547.1465[M-H]- 0.9 427.101 1,367.081 0,337.070 2 H3 37 37.48 Chrysin 6-C-glucoside 8-Carabinoside or isomers[23] C26H28O13 547.1464[M-H]- 0.5 457.111 3,427.100 5,367.081 2,337.070 5 H4 38 38.39 Chrysin 6-C-glucoside 8-Carabinoside or isomers[23] C26H28O13 547.1452[M-H]- -0.7 457.111 6,427.101 9,367.080 1,337.070 8,309.075 3,281.078 3 H5 39 40.34 Chrysin 6-C-glucoside 8-Carabinoside or isomers[23] C26H28O13 547.1446[M-H]- -2.4 457.112 0,427.103 1,367.079 5,337.070 2,309.073 4,281.078 9 H6 40 40.8 3,4-Dicaffeoylquinic acid[24] C25H24O12 515.1193[M-H]- 0.6 537.100 9,375.068 3,353.086 7,191.055 6,179.034 2,135.044 2 JZ6 539.1155[M+Na]+ -1.9 377.083 2,163.038 6 41 41.09 3,5-Dicaffeoylquinic acid* C25H24O12 515.1185[M-H]- -0.2 537.100 1,375.068 4,353.085 5,191.054 8,179.034 0,135.044 0 JZ7 539.1148[M+Na]+ -3.2 377.082 6,163.039 1 42 42.25 galloyl-ellagic acid[16] C21H10O13 469.0517[M-H]- 1.2 939.111 3,393.045 9,169.013 3 DCM3 471.0540[M+H]+ 2.7 963.105 7 43 45.1 viscidulin III[25] C17H14O8 345.0601[M-H]- -2.3 330.037 1,315.013 5 H7 44 46.26 4, 5- Dicaffeoylquinic acid * C25H24O12 515.1182[M-H]- 1.0 537.101 4,375.068 9,353.086 7,191.055 9,179.034 1,173.045 2 JZ8 45 47.1 Centauroside[20] C34H46O19 757.2558[M-H]- -0.8 595.203 4,577.193 7,525.160 3,493.171 8 J5 46 48.5 6″-O-trans-p-coumaroylgenipin gentiobioside[26] C32H40O17 695.2185[M-H]- -0.3 663.188 0,469.133 0,225.077 0,145.028 3 Z5 445.0768[M-H]- -0.2 891.161 9,269.044 7 H8 447.0924[M+H]+ -0.7 271.060 9 48 51.40 mudanpioside C[18] C30H32O13 599.1759[M-H]- -2.5 551.156 1,477.136 0,447.127 7,431.133 3 M2 49 52.35 5,6,7-trihydroxyflavanone-7-O-Dglucuronide[27] C21H20O11 447.0917[M-H]- -1.8 271.060 2,243.064 6 H9 50 52.77 3,4’-Di-O-methylellagic acid [16] C16H10O8 329.0293[M-H]- 0.3 314.004 6,298.982 5,285.003 1 D12 51 53.32 Luteolin* C15H10O6 285.0398[M-H]- -2.8 241.047 2 J6 52 54.44 3,4 ‘- O-dimethylellagic acid 4-Oβ- D-xyloside[16] C21H18O12 461.0717[M-H]- -1.7 329.029 4,314.004 9,298.981 8,285.002 0 D13 53 54.77 Wogonin-5-O-D-glucoside[28] C21H18O11 445.0769[M-H]- -0.4 467.057 7,269.044 2 H10 54 55.28 Benzoyloxypeoniflorin[18] C30H32O13 599.1753[M-H]- -2.2 569.176 1,477.138 6,137.022 8 M3 55 56.48 Apigenin-7-glucuronide[29] C21H18O11 445.0769[M-H]- -0.2 497.058 1,299.054 7,269.044 3 H11 57 57.66 Chrysin 7-O-D-glucopyranuronoside[23] C21H18O11 445.1116[M-H]- -1.1 497.058 1,299.054 7 H13 47 50.15 Baicalin* C21H18O11 459.0824[M-H]- -1.3 283.060 5,168.037 0,175.023 1 H14 461.1093[M+H]+ 2.0 483.089 9,285.076 4 59 58.56 3,3 ‘, 4’ - O-trimethyl ellagic acid 4-O-sulfate[16] C17H12O11S 423.0056[M-H]- 1.7 343.044 9,328.022 7,312.998 6 D14 60 59.58 baicalin 6-O-D-glucuronic acid glycoside[27] C21H18O11 445.0765[M-H]- -1.6 467.058 1,269.044 0 H15 58 58.19 Oroxindin[14] C22H20O11 61 60.15 Oroxylin A-7-O-glucuronide[23] C22H20O11 459.0919[M-H]- -1.7 919.192 1,283.060 3,268.036 7 H16 461.1091[M+H]+ 1.5 943.189 8,483.091 0,285.076 4

Note:H:Huangqin;J:Jinyinhua;C:Chishao;M:Mudanpi;Z:Jiaozhizi;D:Diyu;G:Gancao.

3.2 Network Pharmacological Analysis

3.2.1 Active ingredient screening and target prediction

According to the screening conditions set in 1.5.1, 35 active ingredients were selected from the 75 identified components,including 11 from Huangqin, 4 from Jinyinhua, 2 from Jiaozhizi, 5 from Diyu, 2 from Gancao , 1 common ingredient between Chishao and Mudanpi, 7 common ingredients between Jinhua and Jiaozhizi, 2 common ingredients between Chishao and Mudanpi and 1 common ingredient between Huangqin, Jinyinhua , Chishao , Mudanpi Jiaozhizi.388 drug targets were predicted.

Fig 2 Venn diagram of psoriasistargets and Jiuwei Huaban pill

3.2.2 Screening of relevant targets for the treatment of psoriasis with Jiuwei Huaban Pill

According to the method under section 1.5.2, a total of 1274 targets related to psoriasis were collected and screened, and 114 common targets were obtained by intersecting with the drug targets in section 2.2.1.

3.2.3 Construction of core target PPI network

According to the method under section 1.5.3, a core target PPI network consisting of 30 nodes and 305 edges was obtained.The visualization analysis results are shown in Figure 3.The larger the node in the graph, the stronger its interaction with other proteins in the entire network and the closer its relationship.This suggests that targets such as vascular endothelial growth factor A (VEGFA),epidermal growth factor receptor (EGFR), tumor necrosis factor(TNF), signal transduction and transcription activating factor 3(STAT3), prostaglandin endoperoxide synthase 2 (PTGS2), and transcription factor AP-1 (JUN) may play an important role in the treatment of psoriasis with Jiuwei Huaban Pill.

Fig 3 PPI network diagram of core target

Fig 4 GO analysis and KEGG pathway analysis of biological process

3.2.4 GO Function and KEGG Pathway Enrichment Analysis

GO functional enrichment analysis revealed 213 biological processes (BP), mainly related to the forward regulation of protein phosphorylation, angiogenesis, and the forward regulation of phosphatidylinositol 3-kinase signal transduction.It mainly involved 27 molecular functions (MF), including the same protein binding,endopeptidase activity, and transcription factor binding.40 cell compositions (CC) were obtained, indicating that the components of JWHBW mainly act on the extracellular domain and extracellular space The membrane raft and other parts play a role, and the top 10 of BP, MF, and CC are selected for visualization using the microbiome platform, as shown in Figure 4 (A).A total of 85 entries were obtained through KEGG pathway enrichment analysis, and the top 20 were selected for visualization using the microbiome platform, as shown in Figure 4 (B).The core targets are mainly enriched in signaling pathways such as interleukin-17 (IL-17), TNF,phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt), helper T cells (Th17), and mitogen activated protein kinase (MAPK).

3.2.5 Component target pathway network construction

The “active ingredient target pathway” network constructed through Cytoscape is shown in Figure 5.The components with high degree rankings, such as luteolin, baicalin II, baicalin, paeoniflorin,and geniposide, may be key components that work in JWHBW.The top ranked metalloproteinase-2 (MMP2) and MMP9 are the two most well-known members of the matrix metalloproteinase family.They can hydrolyze type IV collagen in the basement membrane,thereby disrupting the tissue barrier and facilitating tumor cell invasion and metastasis.In addition, MMP9 can also promote angiogenesis by releasing VEGF[33].In addition, EGFR, PTGS2,JUN, TNF, and insulin like growth factor 1 receptor (IGF1R) are all important targets.

3.2.6 Molecular docking

Select active ingredients with a moderate value greater than the median (>4) in the “component target pathway” network(supplemented by baicalin and chlorogenic acid, the indicator components of the monarch medicine Huangqin and honeysuckle),and connect them with the core targets EGFR, MMP2, MMP9,PTGS2, JUN, TNF (combined with PPI networks, VEGFA, STAT3)in the top 6 of the network’s moderate value.A total of 120 docking results were obtained, with binding energies -5.9 kcal/mol, Among them, the binding energy of 108 docking results is -7.0 kcal/mol.The specific results are shown in Figure 6.In summary, the key components screened from Jiuwei Huaban Wan have a good combination with core targets.The visualization of some molecular docking results is shown in Figure 7.

Fig 5 Network for active components-targets-pathways of Jiuwei Huaban pill

Fig 6 Results of molecular docking

4.Discussion

This study utilized UPLC-Q-TOF/MSEtechnology to analyze a total of 75 chemical components from Jiuwei Huaban Pill, mostly derived from three medicinal herbs: honeysuckle, scutellaria baicalensis, and Sanguisorba officinalis, with flavonoids and phenolic acids as the main components.After screening, 35 potential active ingredients were identified and 30 core targets were predicted.The results of network pharmacology indicate that the main active components of JWHBW, including luteolin, baicalin II, baicalin, paeoniflorin, and geniposide, act on core targets such as EGFR, MMP2, MMP9, and VEGFA, thereby regulating the signaling pathways of IL-17, PI3KAkt, and MAPK to play a therapeutic role in psoriasis

Fig 7 Results of AutoDock Vina molecular docking

The pathological features of psoriasis are mainly keratinocyte proliferation, inflammation, and angiogenesis[34].In reports on the treatment of psoriasis with various active substances, luteolin can inhibit epidermal cell proliferation and promote apoptosis, as well as inhibit inflammatory factors IL-6 and TNF-α, IL-17, IL-23, and IL-1β The expression of psoriasis can alleviate psoriasis like symptoms[35-37].Paeoniflorin can be expressed through nuclear factors κ B(NF-κB) The pathway inhibits the proliferation of keratinocytes(KC) [38, 39], while reducing the associated inflammatory cytokines(IL-17, INF-γ, IL-6, TNF-α) The expression of[39] and anti angiogenesis[40] can be used to treat psoriasis.The experiment by Lu Chuanjian et al.[41] showed that geniposide has the same anti psoriasis effect as methotrexate, but its safety is better than the latter.As an indicator component of JWHBW, literature has shown that baicalin and chlorogenic acid have therapeutic effects on psoriasis.Baicalin may significantly inhibit the activity of keratinocytes and induce apoptosis by regulating the Notch signaling pathway, tyrosine kinase signaling pathway, and transcription activating protein (JAKSTAT) signaling pathway[42,43], as well as by inhibiting γδ.The activation of T cells and the release of related inflammatory factors(IL-17A, IL-22, and IL-23) exert anti-inflammatory effects[44].Chlorogenic acid inhibits skin inflammatory response to alleviate pathological changes in psoriasis like mouse skin lesions[45].The molecular docking results of this study also showed that the selected active ingredients, such as luteolin, baicalin, paeoniflorin,geniposide, baicalin, and chlorogenic acid, have strong affinity with some core targets.It is speculated that JWHBW mainly exerts therapeutic effects on psoriasis through chemical components such as luteolin, baicalin, paeoniflorin, geniposide, baicalin, and chlorogenic acid.

EGFR is a tyrosine kinase involved in the regulation of the MAPK pathway, PI3K-Akt pathway, and JAK/STAT pathway[46].In psoriasis, high levels of EGFR can lead to excessive proliferation and abnormal differentiation of KC, while also upregulating Decoy Receptor 3 (decoy receptor 3) to promote inflammatory response and angiogenesis[47].VEGF can be secreted by KC cells, and VEGFA is a member of the VEGF family that promotes neovascularization and increases vascular permeability, participating in the pathogenesis of psoriasis[48].High levels of activated STAT3 can be found in the skin of psoriasis patients[49].STAT3 can mediate the secretion of inflammatory factors such as IL-17 and IL-22 by Th17 cells, and can directly activate IL-17A and IL-17F to produce inflammatory reactions.IL-17A and various other inflammatory factors can lead to the proliferation of KC[50].

The KEGG pathway enrichment analysis of JWHBW in the treatment of psoriasis mainly involves signaling pathways such as IL-17, TNF, PI3K-Akt, Th17, and MAPK.The MAPK pathway is composed of four sub pathways: P38 mitogen activated protein kinase (P38 MAPK), extracellular signal-regulated kinase 1/2(ERK1/2), c-Jun amino terminal kinase (JNK), and ERK5[51].P38 MAPK and JNK induce differentiation of Th1 and Th2, respectively,thereby jointly mediating KC cell proliferation.P38 MAPK and ERK can regulate angiogenesis, while P38 MAPK, ERK1/2,and JNK can regulate downstream inflammatory factors, thereby exacerbating the inflammatory response in psoriasis[52].The PI3K/AKT signaling pathway is an important pathway for cell survival,involved in multiple processes such as cell growth, proliferation,energy metabolism, and also involved in angiogenesis [53].The PI3KAkt signaling pathway can affect the proliferation of keratinocytes through phosphorylation and induction of forkhead transcription factor (FOXO) downregulation, which may be a potential target for the treatment of psoriasis[54].The IL-17 pathway, Th17 pathway, and TNF pathway play important roles in the pathogenesis of psoriasis.Mature Th17 cells secrete IL-23 and IL-17, which in turn promote the differentiation and maturation of Th17 cells.IL-17 binds to corresponding receptors and activates P38 MAPK.Additionally,IL-17 can upregulate IL-8 to cause excessive keratinization of the epidermis in psoriatic lesions[55].In addition, IL-17 and TNF- α Can synergistically induce KC cells to produce multiple inflammatory mediators and block TNF-α IL-23 can be downregulated to inhibit Th17 mediated immune response [56].

In summary, this study utilizes UPLC-Q-TOF-MSEtechnology combined with network pharmacology research to speculate that the effective components of JWHBW in the treatment of psoriasis are mainly chemical components such as baicalin, luteolin, geniposide,paeoniflorin, baicalin, chlorogenic acid,etc.By acting on targets such as EGFR, MMP2, MMP9, VEGFA, TNF, STAT3, etc., it regulates the release of inflammatory factors through signaling pathways such as IL-17, TNF, PI3K-Akt, Th17, MAPK, etc.Inhibiting excessive proliferation of keratinocytes and inhibiting angiogenesis in skin lesions.This study preliminarily clarified the pharmacological substance basis of JWHBW in the treatment of psoriasis, providing a basis for its quality standard research, and also providing ideas and references for further in-depth research on the mechanism of action combined with in vitro experiments.

Author’s contribution:

Sui Yinhai: Design and execute research plans, collect and analyze data, write papers, and revise the final version; Hao Lei: Propose research ideas to guide the writing and revision of papers; Huo Zhipeng: Guide experimental operations and participate in paper revision; Wang Yu and He Yi: Financial support and technical guidance for all experimental reagents.

All authors declare no conflict of interest.