Molecular Mechanism of the Therapeutic Effect of Peach Blossom against Constipation: An Exploratory Study Based on Network Pharmacology Analysis and Molecular Docking Verification

Objective The aim of the study is to verify the active ingredients of peach blossom and to explore the molecular mechanisms of their therapeutic effects against constipation through network pharmacology and molecular docking analysis. Methods The potential active ingredients of peach blossom were identified from published literature and the BAT-TCM database, and their potential targets were predicted using the SwissTargetPrediction and PharmMapper platforms. In addition, targets related to constipation were retrieved using OMIM, DrugBank, GeneCards, TTD, and DisGeNET databases. The intersection of drug targets and disease targets was considered as the potential targets of peach blossom in the treatment of constipation. The STRING platform was used to construct a protein interaction network. Gene ontology (GO) functional analysis and KEGG pathway enrichment analysis were performed on key targets using the DAVID database. Molecular docking verification between the active ingredients of peach blossom and the targets was conducted using AutoDock software. Results A total of 33 active ingredients of peach blossom and 185 corresponding targets were identified, and 88 intersection targets were obtained after Venny mapping. These 33 active ingredients (including naringenin, aromadendrin, and cordycepin) in peach blossom may play a role in the treatment of constipation by regulating signaling pathways through targets such as EGFR, VEGFA, ESR1, GSTP1, and PTGS2. Conclusion A variety of active ingredients of peach blossom regulate multiple signaling pathways by acting on targets, which reflects the characteristic of “multiple ingredients-multiple targets-multiple pathways,” thereby playing a role in the treatment of constipation.


Introduction
Constipation is a common gastrointestinal disease characterized by a prolonged or shortened defecation cycle, diffcult defecation, and dry stool. In some conditions, the stool is not hard, but the process of defecation is not smooth though there is a desire to defecate [1]. Constipation can be divided into three subtypes: colonic slow transit constipation, outlet obstructive constipation, and functional defecation disorders [2]. According to the "Guidelines for the diagnosis and treatment of chronic constipation in dextrin [5]), osmotic laxatives (e.g., Polyethylene glycol, sorbitol, and glycerol [6]), and irritant laxatives (e.g., senna and bisacodly [7]). Although these drugs are efective in the short term, there are many adverse reactions, and the constipation is liable to relapse or even aggravate after drug withdrawal. Traditional Chinese medicine (TCM) ofers unique advantages in the treatment of constipation. Te holistic concept of TCM entails performing dialectic analysis, adjusting the balance of yin and yang in the human body, and treatment of both the symptoms and the root cause of the disease. TCM remedies for constipation are safe and have long-term efcacy [8] and can complement the treatment of western medicine. Te top 10 TCM for constipation clinical treatment [9] are rhubarb, Fructus aurantii, Magnolia ofcinalis, Atractylodes, Fructus fructus, Cistanche, Astragalus, peach kernels, and glycyrrhiza. Both alone and in combination with other TCM applications, obvious efcacy without toxic side efects has been observed. In addition, it has been reported that the therapeutic efect of TCM on antipsychogenic constipation was more efective than that of western medicines, such as phenolphthalein and glycerin enema [10].
Peach blossoms are the fowers of rosaceous plants Rosaceae Prunus persica (L.) Batsch or Prunus davidiana (Carr.) Franch. Te blooming of fowers precedes the growth of leaves, and the fowers are harvested between March and April [11]. Peach blossom is bitter in taste and neutral in nature and belongs to the heart, liver, and large intestine meridians. It promotes diuresis and defecation, improves blood circulation, and alleviates blood stasis. It is mainly used in the treatment of constipation, edema, dysuria, phlegm retention, amenorrhea, and mania [12]. Its use has been recorded in many TCM classics including "Tang Materia Medica," "Compendium of Materia Medica," and "Collected Works of Materia Medica." Weng et al. [13] used "Shengdi Baizhu Taohua Decoction" to treat 116 cases of senile habitual constipation. After taking the medicine, approximately 95.69% of patients had smooth bowel movements, and there were no side efects such as abdominal pain. Peach blossom has the efects of slowing down and eliminating accumulation and catharsis, and promoting water fow. It does not cause irritation of the intestinal wall and does not elicit abdominal pain [14]. In addition, a previous study found that ethyl acetate extract from peach blossom can promote gastric emptying, intestinal motility, and secretion of gastrointestinal hormones in rats [15]. In clinical practice, we have observed good clinical efects of peach blossom decoction when used as an adjuvant treatment for constipation (data not shown). Although peach blossom has been used as a natural medicine to promote gastrointestinal motility for many years, the underlying molecular mechanisms of its therapeutic efect against constipation are yet to be elucidated. Network pharmacology and molecular docking analysis not only provide ideas for the research and development of TCM but also provide a theoretical basis for clinical application for constipation treatment. Te underlying mechanism of TCM for constipation treatment could be that the active ingredients from TCM act on targeted genes such as AKT1, thus playing therapeutic roles by regulating cancer, phosphatidylinositol 3-kinase-protein kinase B and p53 signaling pathways [16].
In this study, an "active ingredient-target-signaling pathway" network was constructed based on the network pharmacology analysis, and the potential molecular mechanisms of the therapeutic efect of peach blossom against constipation were explored by molecular docking analysis and verifcation. Our fndings may provide a theoretical basis for the clinical application of peach blossom in the treatment of constipation, and provide novel insights for further research on the mechanisms of peach blossom.

Screening of Active Ingredients of Peach Blossom and
Target Prediction. Te chemical ingredients of peach blossom were retrieved from the BATMAN-TCM database (https://bionet.ncpsb.org/batman-tcm/) [17], and the relevant literature was searched in the CNKI, Wanfang, and PubMed databases [18][19][20][21][22] to supplement the results of the active ingredients of peach blossoms. Te SDF molecular structures of the active ingredients were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov). Te Swis-sADME database (https://www.swissadme.ch/) [23] was searched, and the ingredients satisfying the Lipinski rule [24] and high gastrointestinal (GI) absorption were screened as active ingredients for subsequent analysis. Te potential targets of active ingredients were predicted using the SwissTargetPrediction [25] and PharmMapper [26] platforms.

Venn Analysis of the Potential Targets of Peach Blossom in the Treatment of Constipation.
Te screened targets of active ingredients and disease were uploaded to the Venny 2.1 online tool website (https://bioinfogp.cnb.csic.es/tools/ venny/index.html). A Venn diagram was constructed to obtain the intersection targets, which were the potential targets of peach blossom in the treatment of constipation.
2.4. Construction of the "Active Ingredient-Target" Network. Cytoscape 3.8.0 software [33] was used to construct and analyze the relationship network between the potential active ingredients and the targets of peach blossom in the treatment of constipation. In the network, the active components and targets of the peach blossom were represented by nodes, and the relationship between active components and targets was represented by edges.

Construction of the Protein-Protein Interaction (PPI)
Network. Te drug-disease intersection targets were uploaded to the STRING (https://string-db.org/) database [34] to construct the PPI network. Te species was limited to "Homo sapiens," and "Medium confdence � 0.400" was set to obtain the PPI network. Te ".tsv" format fle was downloaded and imported into the Cytoscape 3.8.0 software. Te plug-in CytoNCA [35] was used to perform a topology analysis of each node in the intersection network. Te parameters such as degree, betweenness, and closeness between each node were adjusted to obtain the most closely related key targets.

Gene Function Annotation and Pathway Enrichment
Analysis. Using the DAVID database (https://david.ncifcrf. gov/) [36], Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed for the key drug-disease targets. P < 0.05 was set as the threshold for screening. Te GO and KEGG enrichment results were plotted into histograms and bubble charts using the bioinformatics online platform (https://www.bioinformatics.com.cn/).

Construction and Analysis of the "Active Ingredient-
Target-Signaling Pathway" Network. Te potential active ingredients of peach blossom, their molecular targets, and the potential signaling pathways mediating the therapeutic efect of peach blossom against constipation were imported into Cytoscape 3.8.0 software to construct a visual network. Te Network Analyzer plug-in was used for network topology analysis to screen important active ingredients and action targets.

Molecular Docking Verifcation.
Te 3D structures of MAPK1, EGFR, VEGFA, PTGS2, and ESR1 proteins were downloaded from the RCSB PDB database (https://www. rcsb.org/), and the SDF structures of the active ingredients of peach blossom were converted into "mol2" format fles using OpenBabel software. Te AutoDock Tools-1.5.6 software was used to dewater and hydrogenate the proteins, and save it as "PDBQT" format. Ten, the processed protein was combined with its corresponding active ingredient by AutoDock Tools-1.5.6 software to obtain the afnity. Afnity < 0 indicated that the ligand and the receptor can spontaneously bind, and the conformation with the lowest afnity was the optimal conformation. Te PyMOL software was used to conduct a visualization analysis of the molecular docking results.

Active Ingredients and the Targets of Peach Blossom.
According to the results of BATMAN-TCM database analysis and literature reports, a total of 79 ingredients were identifed, and the candidate active ingredients with high gastrointestinal absorption were screened. Eventually, 33 active ingredients were selected (Table 1). Te SDF structure information of the 33 ingredients were downloaded on the PubChem website, and the corresponding targets were predicted through the Swiss Prediction platform. Te top 10 targets in the probability ranking were retained. If the target information of the ingredient was not included in the Swiss Prediction website, the PharmMapper was used for supplementation. After deleting the duplicate targets, a total of 185 potential targets were obtained.

Intersection of Peach Blossom Targets and Constipation
Targets. Te disease-related databases were retrieved and the targets were screened according to score and species. Among them, 22, 59, 2778, 9, and 295 targets were obtained in OMIM, DrugBank, GeneCards, TTD, and DisGeNET databases, respectively. A total of 2841 related targets were obtained after the summation and deletion of the duplicate targets. Te screened targets of drug and disease were uploaded to the Venny 2.1 website, and a Venn diagram was generated. Eventually, 88 intersecting targets were extracted for further analysis ( Figure 1).

Construction and Analysis of the "Active Ingredients-
Targets" Network. Te Cytoscape 3.8.0 was used to draw and analyze the network relationship diagram of the potential active ingredients of peach blossom and intersection targets in the treatment of constipation. As shown in Figure 2, the network consisted of 120 nodes and 167 edges. Te size of a node in the diagram represented the corresponding value of a degree, and the degree value was the number of connecting edges. Te larger the degree value, the stronger the pivotal role of the node in the network, the more biological functions involved, and the greater the biological importance. According to the value of degree, the top 5 compounds were naringenin, kaempferol, multiforin A, kaempferol-3-O-rutinoside nicotiforin, and (-)-catechin, which had 9, 8, 8, 7, and 7 corresponding targets, respectively, with relatively strong activity. Te top 5 targets were CA2, CYP19A1, BCHE, TTR, and CYP1D1.

Construction of the PPI Network and Screening of Key
Targets. Te STRING platform was used to construct the interaction relationship of the intersection target proteins, and the PPI-protein interaction network diagram was obtained. As shown in Figure 3, a total of 88 interaction nodes and 446 edges were obtained in the network. Te ".TSV" format fle was downloaded and imported into Cytoscape 3.8.0 software to obtain a visualized network diagram. Te targets that were not connected to other targets were deleted ( Figure 4). Network topology analysis was conducted using the CytoNCA, a Cytoscape 3.8.0 software plug-in. Te degree, betweenness, and closeness parameters were adjusted to screen the targets. Tose with a value larger than the median value were retained. After screening using twice the median value as a threshold, 11 key targets were obtained Evidence-Based Complementary and Alternative Medicine  Evidence-Based Complementary and Alternative Medicine ( Figure 5). According to the value of a degree in the PPI network, the top 5 proteins were ALB (52), VEGFA (37), EGFR (35), PTGS2 (33), and ESR1 (30).

GO and KEGG Enrichment
Analysis. Te David platform was used for enrichment analysis of the above 11 key targets, including the biological process (BP), cellular component (CC), and molecular function (MF) of GO, as well as the KEGG pathway. With P < 0.05 as the screening criterion, the top 10 entries in BP, CC, and MF were selected to draw a histogram ( Figure 6). A bubble chart was drawn for the KEGG pathways with the top 10 ranking of P value (Figure 7). Te color of the bubbles from purple to red represented the P value from small to large. Te smaller the P value, the stronger was the significance. Te size of the bubble represented the count of genes in the pathway, and the horizontal axis represented the ratio of the pathway genes to the total input genes. Te main biological processes involved in the treatment of constipation by peach blossom include negative regulation of the apoptotic process, response to estradiol, longchain fatty acid biosynthetic process, response to immobilization stress, xenobiotic metabolic process, and cellular response to hypoxia. Te molecular functions involved were mainly enzyme binding, identical protein binding, heme binding, nitric-oxide synthase regulator activity, and estrogen 2 -hydroxylase activity.
Te KEGG signaling pathway was mainly enriched in tumor-related pathways, including chemical carcinogenesisreceptor activation, chemical carcinogenesis-DNA adducts, and chemical carcinogenesis-activity oxygen species. It also afected the metabolic pathways, such as the metabolism of xenobiotics by cytochrome P450 and tryptophan metabolism.

Construction and Analysis of the "Active Ingredient-
Target-Signaling Pathway" Network. Cytoscape 3.8.0 was used to draw a network diagram for the top 10 pathways in KEGG analysis and their corresponding active ingredients and targets, and visualization analysis was performed (Figure 8). Te network diagram had a total of 40 nodes and 57 edges. Te size of the node represented the corresponding value of a degree, and the degree value indicated the number of connected edges.
In the diagram, the main active ingredients of peach blossom, such as cordycepin, benzyl-β-D-glucopyranoside, naringenin, aromadendrin, and (-)-catechin, were found to act on the important signaling pathways mediating the therapeutic efect of peach blossom against constipation by binding to targets such as EGFR, VEGFA, and ESR1. Among these, the top 5 targets with a degree ≥2 times of the median were EGFR, VEGFA, ESR1, GSTP1, and PTGS2. Tese fndings suggested that these proteins are potential core targets of peach blossom in the treatment of constipation. Te active ingredients of peach blossom act on multiple targets and diferent pathways interact with each other through multiple common targets, thereby playing a synergistic role in the treatment of constipation.

Molecular Docking Analysis.
Molecular docking was performed for the fve core targets (including EGFR, VEGFA, ESR1, GSTP1, and PTGS2) and their corresponding compounds. It is generally believed that the lower the afnity, the greater is the possibility of the target protein binding to the compound, and the more stable is the binding conformation. Te results of molecular docking between the active ingredients of peach blossom and the core targets are shown in Table 2. Te targets and compounds with a docking afnity less than −5 kcal/ mol are shown in Figure 9.

Discussion
Peach blossom can not only be used as a laxative, as recorded by Shizhen Li in "Compendium of Materia Medica (本草纲目)," but also for dietary therapy to treat dry feces and intestinal obstruction ever since [37]. As recorded in "Qian Jin Fang (千 金方)," consuming a spoonful of peach blossom with water could treat constipation. According to "Taiping Shenghui Fang (太平圣惠方)," ravioli made of peach blossom could treat dry feces, stufy intestines, and abdominal distension and pain peach blossom has a great laxative efect. However, its active ingredients, efect target, and potential mechanisms are still not clear. Molecular docking can be used to virtually screen out active compounds in drugs by scoring the combination of active ingredients and efect target, and analyze by predicting their binding ways and afnities [38]. Tis study was based on network pharmacology and molecular docking analysis, exploring the potential mechanisms of peach blossom treatment of constipation.
A search conducted on the BATMAN-TCM database identifed 79 potential active ingredients of peach blossom.
After the literature review and excluding the ingredients with low gastrointestinal absorption rate, a total of 33 active ingredients were included in the study; most of these were favonoids and alkaloids, including naringenin, hesperetin, aromadendrin, and persicogenin. A previous study [39] found that both naringenin and hesperetin can signifcantly promote small bowel movements in normal mice, and the efect was stronger when the two agents were used together. Kaempferol has been shown to play a regulatory role in the intervention of diarrheal irritable bowel syndrome (IBS-D) and is one of the main compounds of "large-headed atractylodes decoction (参苓白术散)" for IBS-D treatment [40]. "Active ingredient-target-signaling pathway" network analysis showed that peach blossom has multiingredient, multitarget, and multipathway signaling characteristics in the treatment of constipation. EGFR, VEGFA, ESR1, GSTP1, and PTGS2 were identifed as the core targets of peach blossom; of these, EGFR, VEGFA, ESR1, and PTGS2 were the targets predicted by both PPI network analysis and "active ingredient-target-signaling pathway" analysis. During the molecular docking analysis, naringenin  Figure 4: PPI-protein interaction topology analysis network. Te size and color of the node represents the value of the degree. Te node changes from small to large, the color changes from blue to orange, and the corresponding value of the degree changes from small to large. Te thickness and color of the edge represents the value of the overall score. Te edge changes from thin to thick, the color changes from blue to orange, and the corresponding overall score value changes from small to large.  Evidence-Based Complementary and Alternative Medicine and benzyl beta-D-glucopyranoside showed the best binding efects to the ESR1 target and VEGFA target, respectively. Te ESR1 gene encodes the estrogen receptor ERα. Studies have demonstrated upregulation of the expression of ERα in the intestinal mucosa of patients with irritable bowel syndrome, resulting in the dysregulation of estrogen-mediated local immune responses, which may be related to the pathogenesis of irritable bowel syndrome [41]. Vascular endothelial growth factor (VEGF) specifcally promotes vascular endothelial cell proliferation, migration, and angiogenesis, inhibits cell apoptosis, and induces increased vascular permeability [42]. VEGFA is the most important subtype exerting biological functions in the VEGF family, which has been widely studied in infammation and cancers [43]. VEGFA is overexpressed in colitis tissues [44].
In this study, GO enrichment analysis found that the main molecular functions regulated by peach blossom in the treatment of constipation were nitric-oxide synthase (NOS) regulatory activity, heme binding, and efux transmembrane transporter activity. NO plays an important role in regulating gastrointestinal motility and is the main inhibitory neurotransmitter afecting intestinal motility. NOS is a key enzyme for the production of endogenous NO and exists in the gastrointestinal tissues from the esophagus to the internal anal sphincter [45]. Fan et al. [46] found that highdose Zhizhu Tongbian Decoction afected intestinal motility by reducing the expression of NOS mRNA in the colon of rats, thereby signifcantly improving the intestinal transmission and enteric neurotransmission system of slowtransit constipation rats. Chen et al. [47] found that the decrease in distribution and expression of heme oxygenase-2 in the colon tissues of rat may be one of the causes of colonic movement disorder in diabetes. In recent years, studies have demonstrated the expression of a large number of aquaporins (AQPs) in the intestine, which can maintain the homeostasis of the internal and external environment of cells. In the study by Qian et al. [48], Tongbian granules were found to signifcantly improve the defecation function of rats with slow transit constipation, which may be achieved by regulating the absorption and secretion of water by downregulating the expression of AQP3 and AQP8.
KEGG pathway enrichment analysis revealed the potential involvement of several pathways in mediating the therapeutic efect of peach blossom against constipation   Figure 8: "Active ingredient-target-signaling pathway" network diagram of peach blossom in the treatment of constipation. Te orangeyellow rectangular nodes represent targets, the green circular nodes represent active ingredients, and the blue arrows represent pathways.
including cancer signaling pathways, metabolism of cytochrome P450 to exogenous substances, and tryptophan metabolism. Cytochrome P450 (CYP450) is a superfamily of enzymes that exists widely in the body. CYP3A is a subenzyme with the highest content in the CYP450 enzyme system, which mainly exists in the intestine and liver of animals. Approximately, 60% of drugs are catalyzed by the CYP3A enzyme system to complete the metabolic process [49]. Wang et al. [50] found that modulating the tryptophan pathway to regulate the L-tryptophan and serotonin levels can enhance the gastrointestinal motility, suggesting a direct relationship between the tryptophan metabolic pathway and gastrointestinal motility. Studies have shown that Chaihu Shugan powder can signifcantly inhibit the apoptosis of gastric Cajal interstitial cells and efectively improve the gastrointestinal function [51]. Similarly, the efects of peach blossom in the treatment of constipation may be achieved by regulating the biological processes including cell apoptosis and body metabolism, by afecting the functions of molecules such as heme, NOS, and transmembrane transporters, and by participating in the metabolism of exogenous substances by cytochrome P450, tryptophan metabolism, and other pathways, thereby regulating gastrointestinal motility. According to the KEGG analysis of core targets, the processes involve chemical carcinogenesis-receptor activation, chemical carcinogenesis-DNA adducts, bladder cancer, and other cancer-related pathways.

Conclusion
In this study, we performed network pharmacology analysis to systematically analyze the potential active ingredients, efect targets, and signaling pathways mediating the therapeutic efect of peach blossom against constipation. A total of 33 active ingredients (including naringenin, aromadendrin, and cordycepin) in peach blossoms were identifed, which might play a role in the treatment of constipation by regulating signaling pathways through targeted genes such as EGFR, VEGFA, ESR1, GSTP1 and PTGS2, which refects the characteristic of "multi-ingredient-multi-target-multipathway" therapy. Tis study lays a foundation and provides a theoretical basis for further research on the mechanism of the laxative efect of peach blossom.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.  Figure 9: Molecular docking of core targets and active ingredients of peach blossom. Te cyan stick-like structure is the compound, the purple ribbon structure is the protein, the yellow part is the stick-like structure of amino acid residues connected to the compounds, and the green part is the schematic illustration of the structure of amino acid residues connected to the compound. Te number indicates the length of the hydrogen bond of the compound.