**Background:** Irritable bowel syndrome with constipation (IBS-C) is a common gastrointestinal disorder with limited treatment options. Mao Jian Green Tea (MJGT), a traditional Chinese herbal tea from Dracocephalum rupestre Hance, has been used to aid digestion. Previous work showed MJGT hydro extract promotes gastrointestinal motility in normal rats. This study evaluated the ethanol extract (MJGT_EE) in an IBS-C rat model, focusing on the serotonin (5-HT) signaling pathway and gut microbiota.
**Methods:** IBS-C was induced in 24 offspring rats via maternal separation (3 h/day for 14 days from age 7 days) followed by daily ice water (0–4°C, 1.5 mL/rat) gavage for 14 days. Eight rats without maternal separation served as negative controls. Rats were divided into four groups (n=8): negative control (NC, saline), model (MG, saline), positive drug (MSP, mosapride 1 mg/kg), and MJGT_EE (17 mg/mL, 70% ethanol extract). Treatments were administered by gavage for 30 days. Outcomes included fecal water content (FWC), smallest colorectal distension (CRD) volume, gastric emptying and small intestinal propulsion rates, colonic histology (H&E), 5-HT immunohistochemistry, western blot for TPH1, TPH2, SERT, 5-HT3R, 5-HT4R, CaM, and MLCK, and 16S rDNA sequencing of cecal microbiota. HPLC identified four flavonoid components in MJGT_EE.
**Key Results:** MJGT_EE significantly increased FWC (p < 0.01) and smallest CRD volume (p < 0.05) compared to MG, indicating reduced constipation and visceral hypersensitivity. Gastric emptying and small intestinal propulsion rates were significantly restored (p < 0.01) to near NC levels. H&E staining showed no organic lesions in any group. Immunohistochemistry revealed significantly elevated colonic 5-HT in MG (p < 0.01), which was significantly reduced by MJGT_EE (p < 0.01). Western blot showed MJGT_EE significantly decreased TPH1 and TPH2 expression (p < 0.05) and significantly increased SERT expression (p < 0.05) compared to MG. MJGT_EE significantly upregulated 5-HT4R expression (p < 0.05) but did not affect 5-HT3R. CaM and MLCK expression were significantly lower in MG (p < 0.05 and p < 0.01, respectively) and restored by MJGT_EE. 16S rDNA analysis showed MJGT_EE increased OTU number from 834 (MG) to 926, exceeding NC (882). At phylum level, the Bacteroidetes/Firmicutes ratio was 0.20 (NC), 0.10 (MG), 0.14 (MJGT_EE), and 0.20 (MSP). MJGT_EE increased Lactobacillus abundance from 1.11% (MG) to 6.19%, and enriched Clostridia_UCG-014 (LDA > 4). MJGT_EE reduced Corynebacteriaceae from 2.16% (MG) to 0.92% and Lachnospiraceae from 28.94% (MG) to 14.94%. HPLC identified luteolin-7-O-glucoside, luteolin, eriodictyol-7-O-glucoside, and eriodictyol as major components.
**Clinical Implications:** MJGT_EE demonstrates potential as a therapeutic agent for IBS-C by modulating the 5-HT signaling pathway—reducing 5-HT synthesis via TPH downregulation, enhancing 5-HT reuptake via SERT upregulation, and activating 5-HT4R/CaM/MLCK-mediated smooth muscle contraction. Its effects on gut microbiota, including increasing diversity and beneficial bacteria (Lactobacillus, Ruminococcaceae) while reducing 5-HT-promoting bacteria (Corynebacteriaceae, Lachnospiraceae), suggest a multi-target mechanism. The flavonoid components likely contribute to these effects. However, these findings are from a rat model and require validation in human clinical trials.