Abstract Background: Gastrointestinal motility disorder (GMD) is a common condition characterized by dysfunction or degeneration of the myenteric plexus in specific segments of the gastrointestinal tract. Liupao tea (LPT) is a post-fermented tea that is rich in various secondary metabolites and has demonstrated a range of pharmacological effects, including lipid-lowering properties, antioxidant activity, and modulation of the gut microbiota. However, the underlying mechanisms by which LPT improves GMD remain poorly understood. Methods: Blood was collected after gavage of LPT extract in SD rats. The active components in the aqueous extract of LPT and its serum were analyzed using ultra-high-performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS). The targets of LPT in the treatment of GMD were predicted by network pharmacology and molecular docking. Results: 65 compounds were identified in the water extract of LPT, including flavonoids, phenolic acids, alkaloids, and amino acids. In rats treated with LPT, 14 prototype compounds and 6 metabolites were detected in serum. Network pharmacology and molecular docking analyses revealed 298 common targets between LPT and GMD, including IL-6, AKT1, and TP53. Functional enrichment analysis suggested that LPT may improve GMD through the regulation of immune, inflammatory, and cytokine signaling pathways. Molecular docking further indicated that the primary bioactive components of LPT exhibit a strong affinity for IL-6, AKT1, and TP53. Conclusions: These findings provide new insights into the bioactive components, molecular targets, and mechanisms of LPT, suggesting its potential as a therapeutic strategy for gastrointestinal motility disorders. Keywords: Liupao tea, UPLC-Q-TOF/MS, network pharmacology, molecular docking, gastrointestinal motility disorder 1. Introduction Gastrointestinal motility disorder (GMD) is a common condition characterized by dysfunction or absence of the enteric nervous system in specific segments of the gastrointestinal tract [[38]1]. When the intestine loses its ability to coordinate muscle contractions, motility disturbances arise, leading to various clinical symptoms. These symptoms primarily include bloating, abdominal pain, nausea, poor appetite, and fatigue [[39]2]. Treatment strategies typically involve dietary modifications, vitamin and nutritional supplementation, anti-nausea medications, and promotility agents such as metoclopramide, domperidone, and macrolide antibiotics to enhance gastric emptying [[40]3]. However, prolonged use can produce adverse drug reactions. Tea is traditionally categorized into five types based on its level of fermentation: unfermented tea (green tea), lightly fermented tea (white and yellow teas), semi-fermented tea (oolong tea), fully fermented tea (black tea), and post-fermented tea (dark tea). LPT, a variety of dark tea, is named after its place of origin—Liubao Town in Cangwu County, Guangxi. With a history spanning over 1000 years, LPT was recognized as one of China’s 24 famous teas during the Qing Dynasty [[41]4]. Recent studies have highlighted the various pharmacological properties of LPT, including its ability to lower blood lipids and glucose, provide antioxidant effects, modulate the gut microbiota, and inhibit tumor growth [[42]5,[43]6,[44]7,[45]8,[46]9,[47]10]. Chemical analyses reveal [[48]8] that LPT contains several bioactive compounds, particularly tea polyphenols, which possess antioxidant, anti-inflammatory, and hepatoprotective properties, as well as caffeine, known for its mild stimulating effects that promote gastrointestinal motility, particularly by aiding gastric emptying. Based on these findings, we propose that the polyphenol-rich extract of LPT may enhance gastrointestinal function by exerting anti-inflammatory effects and stimulating smooth muscle activity in the intestines, potentially benefiting individuals with GMD. Therefore, this study employs ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) to conduct a qualitative analysis of the chemical composition of the aqueous extract of LPT, as well as the serum components absorbed into the bloodstream. Additionally, network pharmacology and molecular docking techniques are applied to investigate the mechanisms by which LPT may improve GMD. The findings are expected to demonstrate the prokinetic effects of LPT on the gastrointestinal system, thereby providing a scientific basis for its pharmacological mechanisms. 2. Results 2.1. Total Ion Chromatogram Collection Based on UPLC-Q-TOF-MS/MS Using the established method for analyzing the blood components of LPT, data were collected from both blank serum and LPT-administered serum in positive and negative ion modes. The total ion chromatograms were obtained, as shown in [49]Figure 1. Figure 1. [50]Figure 1 [51]Open in a new tab Total ion chromatograms (TIC) of serum samples based on UPLC-Q-TOF/MS. TIC of blank serum samples in ESI- mode (A), TIC of blank serum samples in ESI+ mode (B), TIC of LPT drug-containing serum samples in ESI- mode (C), and TIC of LPT drug-containing serum samples in ESI+ mode (D). 2.2. Active Components of LPT Based on UPLC-Q-TOF-MS/MS Structural identification of compounds with available reference standards was performed by comparing retention times and secondary mass spectra. The fragmentation patterns of these compounds were further validated by their fragment ions. Using this method, 65 compounds were identified in the aqueous extract of LPT ([52]Supplementary Material S1), including flavonoids, phenolic acids, alkaloids, amino acids, and other compounds. Mass spectrometric data from the LPT aqueous extract, serum containing LPT, and control serum samples were compared to identify blood-absorbed components. A compound was classified as a prototype of LPT absorbed into the bloodstream if it was present in both the LPT aqueous extract and the serum containing LPT, but absent in the control serum. A compound was identified as a metabolite of LPT if it was found exclusively in the serum containing LPT and not in the aqueous extract or control serum [[53]11]. In this study, 20 blood-absorbed components were detected in the serum samples, including 14 prototype compounds and 6 metabolites, as summarized in [54]Table 1. Table 1. Identification results of active components of drug-containing plasma samples. NO. Rt/min Component Name Formula Adduct Precursor Mass Found at Mass Mass Error (ppm) Category 1 1.12 7-methylxanthine C[6]H[6]N[4]O[2] [M-H]^− 165.0418 165.0420 1.3 prototype 2 2.08 esculin hydrate C[7]H[8]N[4]O[2] [M-H]^− 339.0722 339.0722 0.1 metabolite 3 2.16 theobromine C[15]H[16]O[9] [M-H]^− 179.0574 179.0576 0.8 prototype 4 3.34 protocatechuic acid C[7]H[6]O[4] [M-H]^− 153.0193 153.0195 1.0 metabolite 5 4.39 paeonol C[9]H[10]O[3] [M-H]^− 165.0557 165.0560 1.6 prototype 6 5.10 wogonoside C[22]H[20]O[11] [M-H]^− 459.0933 459.0932 −0.3 metabolite 7 5.64 apigenin 7-glucoside C[21]H[20]O[10] [M-H]^− 431.0984 431.0982 −0.4 prototype 8 5.82 3,4-Dihydroxybenzaldehyde C[7]H[6]O[3] [M-H]^− 137.0244 137.0245 0.6 prototype 9 5.89 schaftoside C[26]H[28]O[14] [M-H]^− 563.1406 563.1405 −0.2 prototype 10 6.58 astragalin C[21]H[20]O[11] [M-H]^− 447.0933 447.0935 0.6 prototype 11 7.15 caffeic acid C[9]H[8]O[4] [M-H]^− 179.035 179.0351 0.7 metabolite 12 11.03 catechin C[15]H[14]O[6] [M-H]^− 289.0718 289.0746 9.9 prototype 13 11.03 epicatechin C[15]H[14]O[6] [M-H]^− 289.0718 289.0746 9.9 prototype 14 20.61 ganoderic acid C6 C[30]H[42]O[8] [M-H]^− 529.2807 529.2798 −1.7 metabolite 15 22.40 6-shogaol C[17]H[24]O[3] [M-H]^− 275.1653 275.165 −0.9 prototype 16 3.03 caffeine C[8]H[10]N[4]O[2] [M+H]^+ 195.0877 195.0873 −2.0 prototype 17 21.85 arbutin C[12]H[16]O[7] [M+H]^+ 273.0969 273.0971 0.6 metabolite 18 22.92 palmitic acid C[16]H[33]NO [M+H]^+ 256.2635 256.2635 0.1 prototype 19 23.58 oleamide C[18]H[35]NO [M+H]^+ 282.2791 282.2794 0.9 prototype 20 26.01 stearic acid amide C[18]H[37]NO [M+H]^+ 284.2948 284.2948 0.1 prototype [55]Open in a new tab 2.3. Network Pharmacology and Molecular Docking Experimental Results 2.3.1. Core Targets of LPT’s Active Compounds The Traditional Chinese Medicine System Pharmacology (TCMSP; [56]https://old.tcmsp-e.com/tcmsp.php, accessed on 24 March 2024) and TargetNet ([57]http://targetnet.scbdd.com/calcnet/index/, accessed on 24 March 2024) databases were searched for the targets of active ingredients in LPT. After screening and refinement, 803 potential targets associated with these active ingredients in LPT were ultimately identified. 2.3.2. Targets of Gastrointestinal Motility Disorder Genes associated with gastrointestinal motility dysfunction were retrieved from the OMIM, GeneCards, and TTD databases. Targets from the GeneCards database with relevance scores equal to or greater than the average score were selected. The gastrointestinal motility disorder targets from all three databases were then merged and de-duplicated, yielding a total of 3093 disease-related targets. 2.3.3. Venn Diagram and PPI Network The active compound targets of LPT and the targets associated with GMD were imported into Venny 2.1 ([58]https://bioinfogp.cnb.csic.es/, accessed on 15 April 2024) to generate a Venn diagram ([59]Figure 2) and identify the intersecting targets. A total of 298 intersecting targets were identified, which represent potential pathways through which LPT may ameliorate gastrointestinal dysfunction. These 298 targets were subsequently imported into the STRING online database to construct a protein–protein interaction (PPI) network, as shown in [60]Figure 3A. Topological analysis was then conducted using Cytoscape 3.9.1 software. The core targets were selected based on their degree values. The degree of each gene target was visualized, with darker colors and larger node sizes indicating higher degree values, as shown in [61]Figure 3B. The top six targets, ranked by degree, for LPT’s potential effects on improving gastrointestinal motility disorder were IL-6, TP53, AKT1, EGFR, IL-1β, and TNF. Figure 2. [62]Figure 2 [63]Open in a new tab Venn diagram of active ingredients of LPT and targets related to GMD. Figure 3. [64]Figure 3 [65]Open in a new tab Network diagram of PPI proteins (A), and interaction network of key target (B). A ‘compound–target’ network was constructed using Cytoscape 3.9.1 to elucidate the interactions between the active compounds of LPT and the 298 intersection targets associated with gastrointestinal motility disorder ([66]Figure 4). In this network, orange circular nodes represent the active compounds of LPT, pink triangular nodes indicate LPT itself, and blue triangular nodes denote disease-related intersection targets. The network reveals that LPT exerts its therapeutic effects through multiple components acting on various targets. Among these compounds, quercetin, caffeine, and ellagic acid interact with the greatest number of targets, suggesting that they may play a key role in the therapeutic effects of LPT. Figure 4. [67]Figure 4 [68]Open in a new tab The component–targe network. 2.3.4. GO Functional and Module Analysis, KEGG Enrichment Pathway Analysis A total of 298 potential targets were imported into the DAVID and Metascape databases for functional enrichment analysis based on the Gene Ontology (GO) database ([69]http://geneontology.org/, accessed on 16 April 2024). A significance threshold of p ≤ 0.01 was applied for the selection of 374 biological process (BP) entries, 52 cellular component (CC) entries, and 74 molecular function (MF) entries. The BP, CC, and MF entries are presented in descending order of count values ([70]Figure 5). The results indicate that the primary mechanisms through which LPT affects GMD include responses to xenobiotic stimuli, positive regulation of gene expression, and negative regulation of the apoptotic process. Figure 5. [71]Figure 5 [72]Open in a new tab Analyzing the GO functional enrichment in LPT (top 10 for each BP/CC/MF). In the KEGG pathway enrichment analysis, a significance threshold of p < 0.01 was applied, identifying 137 signaling pathways. The top 20 pathways were visualized in a scatter plot ([73]Figure 6). The results reveal that the potential target pathways are involved in several biologically relevant processes, with pathways related to malignancy and inflammation showing the strongest associations. Among the inflammation-related pathways, the “TNF signaling pathway”, “PI3K-Akt signaling pathway” and “Interleukin-17 signaling pathway” were the most enriched. Figure 6. [74]Figure 6 [75]Open in a new tab Enrichment analysis of KEGG pathway (top 20). 2.3.5. Verification with Molecular Docking The key active compounds of LPT, including quercetin, caffeine, and ellagic acid, were selected as ligands for docking with the key targets IL-6, AKT1, and TP53, which were identified as the top-ranked targets according to their degree values. The binding energy was used to evaluate the affinity between the active compounds and their respective targets. A lower binding energy indicates a greater release of energy, enhancing the likelihood of a successful docking interaction [[76]11]. As detailed in [77]Table 2, all binding energies are negative, indicating thermodynamically favorable binding between the drug molecules and the proteins. Among these, ellagic acid exhibits the strongest binding with IL-6, with a binding energy of −7.1 kcal/mol, suggesting it may be a promising candidate molecule with high target activity. The molecular docking models of key active ingredients and core targets, with binding energies lower than −5 kcal/mol, were generated using PyMOL, as shown in [78]Figure 7. Table 2. Interaction details of molecular docking. Compound Targets Combination of Energy (kcal/mol) Ellagic acid TP53 −6.8 Quercetin TP53 −6.4 Caffeine TP53 −5.1 Ellagic acid IL-6 −7.1 Quercetin IL-6 −6.8 Caffeine IL-6 −5.1 Ellagic acid AKT1 −6.5 Quercetin AKT1 −6.1 Caffeine AKT1 −4.9 [79]Open in a new tab Figure 7. [80]Figure 7 [81]Open in a new tab Visual analysis of molecular docking. AKT1-ellagic acid (A); AKT1-quercetin (B); TP53-caffeine (C); TP53-ellagic acid (D); IL-6-caffeine (E); IL-6-ellagic acid (F); IL-6-quercetin (G); TP53-quercetin (H). 3. Discussion Gastrointestinal dysfunction is strongly influenced by lifestyle, genetic factors, and environmental conditions [[82]12]. Studies have shown that gastrointestinal dysfunction is a common postoperative complication during the perioperative period, which directly affects patients’ postoperative recovery. The rapid recovery of gastrointestinal function is crucial. The longer gastrointestinal suppression persists, the more likely gastrointestinal motility becomes impaired or even absent, leading to increased gas and fluid accumulation in the gut lumen. This can result in intestinal dilation and may lead to severe complications such as adhesive intestinal obstruction, nutritional disorders, poor wound healing, dysbiosis, and multi-organ failure [[83]13]. Treatment strategies typically involve dietary modifications, vitamin and nutritional supplementation, anti-nausea medications, and promotility agents such as metoclopramide, domperidone, and macrolide antibiotics to enhance gastric emptying. In traditional Chinese medicine (TCM), treatment for gastrointestinal dysfunction primarily involves oral herbal therapies, acupuncture, acupoint application, and herbal enemas [[84]2]. China has a long history of using tea for therapeutic purposes. The Compendium of Materia Medica Supplement (1765) [[85]4] records that “Pu-erh tea… relieves greasiness, detoxifies the effects of beef and mutton, and should be avoided by those with deficiency. It is bitter and astringent, promotes phlegm expulsion, and clears the intestines”. Similarly, the Compendium of Materia Medica (1851) states that “Pu-erh tea aids digestion, relieves stagnation, and alleviates dysentery”. In addition, Anhua tea is recognized for its milder digestive benefits. The Compendium of Materia Medica Supplement further notes that “Anhua tea... has a dark color, a bitter taste with a hint of sweetness. It clears the mind, harmonizes the stomach, and warms the body”. It is also known to relieve belching, promote digestion, and dispel cold. LPT, like Pu-erh and Anhua tea, belongs to the category of black tea. Based on these historical references, it is reasonable to speculate