Inhibition on XBP1s-driven lipogenesis by Qushi Huayu Decoction contributes to amelioration of hepatic steatosis induced by fructose.

ETHNOPHARMACOLOGICAL RELEVANCE: Qushi Huayu Decoction (QHD) is a traditional Chinese medicine formula consisting of five herbs, which has been used for non-alcoholic fatty liver disease (NAFLD) treatment in clinic for decades in China and validated in several NAFLD animal models. The hepatic de novo lipogenesis (DNL) is enhanced greatly to contribute to steatosis in NAFLD. The spliced form of X-box binding protein 1 (XBP1s) initiates DNL independently of sterol regulatory element-binding protein (SREBP) and carbohydrate-responsive element-binding protein (ChREBP). AIM OF THE STUDY: To disclose the mechanism of inhibition on hepatic DNL by QHD and the responsible compounds. METHODS: The effects of QHD on hepatic DNL were evaluated in mice induced by high-fructose diet (HFru). The effects of the serum-absorbed compounds of QHD on XBP1s were evaluated in HepG2 cells induced by tunicamycin. Hepatic histology, triglyceride (TG) and nonesterified fatty acids were observed. Hepatic apolipoprotein B100 and very low-density lipoprotein were measured to reflect lipid out-transport. The mRNA expression of XBP1s and its target genes were detected by real-time polymerase chain reaction. The protein expression of TG synthetases and DNL enzymes, and inositol requirement enzyme 1 alpha (IRE1α), phosphorylated IRE1α and XBP1s were detected in liver tissue and HepG2 cells by western-blot. The binding activity of SREBP1, protein expression of ChREBP and XBP1s were detected in the nuclear extracts of liver tissue. RESULTS: Dynamical observing suggested feeding with HFru for 2 weeks was sufficient to induce hepatic lipogenesis and XBP1s. QHD ameliorated liver steatosis without enhancing out-transport of lipids, accompanied with more inhibitory effects on DNL enzymes than TG synthetases. QHD inhibits the nuclear XBP1s without affecting ChREBP and SREBP1. In QHD, chlorogenic acid, geniposide and polydatin inhibit lipogenesis initiated by XPB1s. CONCLUSION: QHD probably decreases hepatic DNL by inhibiting XBP1s independent of SREBP1 and ChREBP. Chlorogenic acid, geniposide and polydatin are the potential responsible compounds.

Amelioration of Non-Alcoholic Steatohepatitis by Atractylodes macrocephala Polysaccharide, Chlorogenic Acid, and Geniposide Combination Is Associated With Reducing Endotoxin Gut Leakage.

Gut-derived lipopolysaccharide (LPS) leaking through the dysfunctional intestinal barrier contributes to the onset of non-alcoholic steatohepatitis (NASH) by triggering inflammation in the liver. In the present study, a combination consisting of Atractylodes macrocephala polysaccharide (A), chlorogenic acid (C), and geniposide (G) (together, ACG), was shown to ameliorate NASH in mice and reduce hepatic LPS signaling and endotoxemia without decreasing the abundance of identified Gram-negative bacteria through restoring the intestinal tight junctions. Our data indicated that inhibition of LPS gut leakage by the ACG combination contributed to its amelioration of NASH.

Geniposide and Chlorogenic Acid Combination Improves Non-Alcoholic Fatty Liver Disease Involving the Potent Suppression of Elevated Hepatic SCD-1.

Background: Non-alcoholic fatty liver disease (NAFLD), characterized by the excessive accumulation of hepatic triglycerides (TGs), has become a worldwide chronic liver disease. But efficient therapy keeps unsettled. Our previous works show that geniposide and chlorogenic acid combination (namely the GC combination), two active chemical components combined with a unique ratio (67.16:1), presents beneficial effects on high-fat diet-induced NAFLD rodent models. Notably, microarray highlighted the more than 5-fold down-regulated SCD-1 gene in the GC combination group. SCD-1 is an essential lipogenic protein for monounsaturated fatty acids' biosynthesis and serves as a key regulatory enzyme in the last stage of hepatic de novo lipogenesis (DNL). Methods: NAFLD mice model was fed with 16 weeks high-fat diet (HFD). The pharmacological effects, primarily on hepatic TG, TC, FFA, and liver enzymes, et al. of the GC combination and two individual components were evaluated. Furthermore, hepatic SCD-1 expression was quantified with qRT-PCR, immunoblotting, and immunohistochemistry. Finally, the lentivirus-mediated over-expressed cell was carried out to confirm the GC combination's influence on SCD-1. Results: The GC combination could significantly reduce hepatic TG, TC, and FFA in NAFLD rodents. Notably, the GC combination presented synergetic therapeutic effects, compared with two components, on normalizing murine hepatic lipid deposition and disordered liver enzymes (ALT and AST). Meanwhile, the robust SCD-1 induction induced by HFD and FFA in rodents and ALM-12 cells was profoundly blunted, and this potent suppression was recapitulated in lentivirus-mediated SCD-1 over-expressed cells. Conclusion: Taken together, our data prove that the GC combination shows a substantial and synergetic anti-lipogenesis effect in treating NAFLD, and these amelioration effects are highly associated with the potent suppressed hepatic SCD-1 and a blunted DNL process.

LMR-101, a novel derivative of propofol, exhibits potent anticonvulsant effects and possibly interacts with a novel target on γ-aminobutyric acid type A receptors.

OBJECTIVE: LMR-101 is a bisphenol derivative of propofol, a short-acting general anesthetic, which is also used to manage status epilepticus (SE). We evaluated the sedative and anticonvulsant effects of LMR-101 to discover its potential to manage epilepsy and SE in the clinic. METHODS: Comparative studies between LMR-101 and propofol were performed in mice to elucidate an appropriate dose range for LMR-101 that produced anticonvulsant effects without significant sedation. Then, the anticonvulsive efficacy for LMR-101 was evaluated using seizure models induced by pentylenetetrazol and (+)-bicuculline. The ability of LMR-101 to inhibit SE was assessed using a rat model of SE induced by pilocarpine. Radioligand binding assay profiles for LMR-101 were performed to evaluate the potential mechanisms of action underlying its anticonvulsant properties. RESULTS: In the mouse study, LMR-101 exhibited greater anticonvulsant and lesser sedative effect compared with propofol. LMR-101 completely inhibited pentylenetetrazol-induced seizures at a dose of 50 mg/kg and exhibited heavy sedation at 300 mg/kg. Propofol anesthetized all mice and only decreased the seizure rate at 25 mg/kg. LMR-101 also suppressed seizure behaviors evoked by (+)-bicuculline in mice in a dose-dependent manner. In the pilocarpine-induced SE model, LMR-101 significantly decreased the maximum seizure score and seizure duration in a dose-dependent manner. The median effective dose for LMR-101 was 14.30 mg/kg and 121.87 mg/kg to prevent and inhibit sustained SE, respectively. In binding assays, LMR-101 primarily inhibited tert-[35 S] butylbicyclophosphorothionate binding to γ-aminobutyric acid type A (GABAA ) receptors (half-maximal inhibitory concentration = 2.06 µmol·L-1 ), but it did not affect [3 H] flunitrazepam or [3 H] muscimol binding. SIGNIFICANCE: It is anticipated that LMR-101 might play an essential role in the clinical management of epilepsy and SE. LMR-101 also might bind to a novel target site on the GABAA receptor that is different from existing antiepileptic drugs. Further study of the mechanisms of action of LMR-101 would be of considerable value in the search for new active drug sites on GABAA receptors.

High-trans fatty acid and high-sugar diets can cause mice with non-alcoholic steatohepatitis with liver fibrosis and potential pathogenesis.

BACKGROUND AND AIMS: Even Non-alcoholic steatohepatitis (NASH) has been becoming the key role in process of liver fibrosis or cirrhosis, no any NASH involving liver fibrosis mice model which consistent with the mechanisms of fatty acid and glucose metabolism disorder was widely accepted. Here, we established a mouse model of nonalcoholic steatohepatitis (NASH) with liver fibrosis using a high-fat, high-carbohydrate diet (HFHC) and analyzed the potential pathogenesis using a transcriptome microarray. METHODS: Fifty mice were stratified by weight and randomly divided into the HFHC model and control (Con) groups. Ten mice were sacrificed at the beginning of the experiments, 10 mice of HFHC and Con group were euthanized at the end of 20 and 30 weeks. The following analyses were performed: biochemical analysis; histological assessment; evaluation of hepatic type I collagen (Col-I), α-smooth muscle actin (α-SMA) and transforming growth factor-ß1 (TGF-ß1) protein and mRNA expression levels; and transcriptomic gene chip analysis. RESULTS: Compared with the Con group at each time point, the body weight and liver wet weight of the HFHC model group of mice were significantly higher. At 30th weeks, alanine aminotransferase (ALT), aspartate aminotransferase (AST), fasting blood glucose (FBG) and fasting insulin (FINS) levels or activities and the triglyceride (TG) and hydroxyproline (HYP) content in the HFHC model group were significantly elevated. Severe steatosis was present in the liver tissues contributed from the HFHC group of mice. Typically, substantial perisinusoidal fibrosis with a cage-like structure and bridging formations were observed in the mice liver in HFHC group. Col-I, α-SMA and TGF-ß1 protein and mRNA expression levels in liver tissues of HFHC mice dramatically increased over time. Compared with the Con group, the HFHC group had 151 differentially expressed genes that were involved in 41 signaling pathways. CONCLUSIONS: After keeping 30 weeks HFHC diet treatment, the mice exhibited substantial liver fibrosis, hepatic steatosis, ballooning degeneration and inflammation. Basing on the transcriptome microarray assays, the experimental NASH involving liver fibrosis potentially related to dramatically changed ECM-receptor interaction, Toll-like receptor signaling and other signaling pathways.

Gypenosides regulate farnesoid X receptor-mediated bile acid and lipid metabolism in a mouse model of non-alcoholic steatohepatitis.

BACKGROUND: Gypenosides (Gyp) are the main ingredient of the Chinese medicine, Gynostemma pentaphyllum. They are widely used in Asia as a hepatoprotective agent. Here, we elucidated the mechanism of Gyp in non-alcoholic steatohepatitis (NASH) with a focus on farnesoid X receptor (FXR)-mediated bile acid and lipid metabolic pathways. METHODS: NASH was induced in mice by high-fat diet (HFD) feeding, while mice in the control group were given a normal diet. At the end of week 10, HFD-fed mice were randomly divided into HFD, HFD plus Gyp, and HFD plus obeticholic acid (OCA, FXR agonist) groups and were given the corresponding treatments for 4 weeks. Next, we analyzed the histopathological changes as well as the liver triglyceride (TG) level and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), fasting blood glucose (FBG), fasting insulin (FINS), TG, total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels as well as the bile acid profile. We carried out RT-PCR and western blotting to detect HFD-induced alterations in gene/protein expression related to bile acid and lipid metabolism. RESULTS: The HFD group had histopathological signs of hepatic steatosis and vacuolar degeneration. The liver TG and serum ALT, AST, FBG, FINS, TC, and LDL-C levels as well as the total bile acid level were significantly higher in the HFD group than in the control group (P < 0.01). In addition, we observed significant changes in the expression of proteins involved in bile acid or lipid metabolism (P < 0.05). Upon treatment with Gyp or OCA, signs of hepatic steatosis and alterations in different biochemical parameters were significantly improved (P < 0.05). Further, HFD-induced alterations in the expression genes involved in bile acid and lipid metabolism, such as CYP7A1, BSEP, SREBP1, and FASN, were significantly alleviated. CONCLUSIONS: Gyp can improve liver lipid and bile acid metabolism in a mouse model of NASH, and these effects may be related to activation of the FXR signaling pathway.

Salidroside improves high-fat diet-induced non-alcoholic steatohepatitis by regulating the gut microbiota-bile acid-farnesoid X receptor axis.

BACKGROUND: Our previous studies found that salidroside can effectively treat non-alcoholic steatohepatitis (NASH). Here, we discuss the mechanism of salidroside in the treatment of NASH with a focus on the gut microbiota-bile acid-farnesoid X receptor axis. METHODS: A NASH mouse model was created by providing mice with a high-fat diet (HFD) for 14 weeks. Mice were randomly divided into the HFD group, HFD + salidroside treatment group, and HFD + obeticholic acid treatment group (n = 8 in each group) and were intragastrically administered corresponding drugs for 4 weeks. Hematoxylin-eosin staining was performed to evaluate the histopathological changes associated with the various treatments. In addition, liver triglyceride (TG) content, serum alanine aminotransferase (ALT) activity, serum inflammatory factors, gut microbiota diversity, and the bile acid profile were evaluated. Western blotting and RT-PCR were performed to detect the expressions of FXR and fibroblast growth factor 15 (FGF15). RESULTS: The HFD group displayed obvious signs of hepatic steatosis. The liver TG, serum ALT, and IL-1a, IL-12, MCP-1, KC, MIP-1a, and MIP-1ß were significantly higher in the HFD group than the control group (P < 0.01). Intestinal bacteria and bile acid profiles changed significantly in the HFD group (P < 0.05). Further, the expressions of FXR and FGF15 decreased significantly in the HFD group (P < 0.05). After treatment with salidroside, liver steatosis, TG content, and serum inflammatory factors significantly improved and HFD-induced intestinal bacteria, bile acid disorder, and FXR deficiency were significantly alleviated (P < 0.05). CONCLUSION: Salidroside can improve NASH via the gut microbiota-bile acid-FXR axis.

Amelioration of non-alcoholic steatohepatitis by Qushi Huayu decoction is associated with inhibition of the intestinal mitogen-activated protein kinase pathway.

BACKGROUND: Gut microbiota is increasingly recognized as the key participant in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) by translocation of its products, such as lipopolysaccharide (LPS), via the dysfunctional intestinal barrier. Qushi Huayu decoction (QHD), a traditional Chinese medicine, is developed specially for NAFLD and used in clinic in China for more than a decade and previously found to ameliorate non-alcoholic steatohepatitis (NASH) induced by high-fat diet (HFD) in mice accompanied with inhibited metabolic endotoxemia and hepatic LPS signalling. PURPOSE: To investigate the mechanism of LPS gut-leakage inhibition by QHD in NASH. METHODS: Effects of QHD on gut microbioa and intestinal barrier were evaluated in NASH induced by HFD in mice. 16S rRNA sequencing is employed to analyse the gut microbiota composition. To identify the potential signalling pathway responsible for tight junction regulation, the colonic phosphoprotein profile is screened via the Phospho Explorer Antibody Array and verified in NASH, intestinal barrier dysfunctional mouse and Caco-2 cells. RESULTS: QHD ameliorates NASH accompanied with regulating the gut microbiota composition, protecting intestinal tight junctions and inhibiting LPS gut-leakage without decreasing the abundance of identified Gram-negative bacteria. The validated data of phosphorylated proteins suggested that mitogen-activated protein kinase (MAPK) pathway is predominantly responsible for the colonic tight junction regulation by QHD. CONCLUSION: QHD inhibits LPS gut-leakage in NASH, which is associated with downregulation of intestinal MAPK pathway.

Chinese medicine CGA formula ameliorates liver fibrosis induced by carbon tetrachloride involving inhibition of hepatic apoptosis in rats.

ETHNOPHARMACOLOGICAL REVELVANCE: CGA consisting of Cordyceps sinensis mycelia polysaccharide, gypenosides and amygdalin, was demonstrated to be the effective components formula in Fuzheng Huayu (FZHY) capsule, a traditional Chinese medicine approved by China food and drug administration for treatment of liver fibrosis and to inhibit transforming growth factor-ß1 (TGF-ß1) signaling, previously. AIM OF THE STUDY: To evaluate the effects of CGA on hepatic apoptosis in liver fibrosis induced by carbon tetrachloride (CCl4). MATERIALS AND METHODS: The hepatic injury and histology was detected by serum biomarker assay and hematoxylin-eosin staining. The hepatic collagen was illustrated by Sirius red staining and hydroxyproline (Hyp) concentration. The hepatic stellate cells (HSCs) activation and hepatic apoptosis was visualized by immunohistochemical analysis of α-smooth muscle actin (α-SMA) and terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling (TUNEL) assay respectively. The protein expression of collagen type I (Col-I), α-SMA, TGF-ß1, Fas, tumor necrosis factor receptor 1 (TNF-R1), cleaved-caspase-8, cleaved-caspase-10, cleaved-caspase-9, cleaved-caspase-3, mitochondrial Bcl-2, Bcl-2 associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak), cytochrome C and cytoplasmic cytochrome C was detected by western-blot. RESULTS: CGA or FZHY ameliorated liver histological changes, decreasing serum alanine aminotransferase, aspartate aminotransferase, hepatic Hyp, TUNEL positive-stained area, and down-regulated the protein expression of α-SMA, TGF-ß1, Col-I, Fas, TNF-R1, cleaved-caspase-8, cleaved-caspase-10, cleaved-caspase-9, and cleaved-caspase-3, mitochondrial Bax, Bak, and cytoplasmic cytochrome C, while restored the expression of mitochondrial Bcl-2 and cytochrome C. CONCLUSION: CGA formula ameliorates liver fibrosis induced by CCl4, which is correlated to its inhibition on hepatic apoptosis.

Geniposide and Chlorogenic Acid Combination Ameliorates Non-alcoholic Steatohepatitis Involving the Protection on the Gut Barrier Function in Mouse Induced by High-Fat Diet.

Gut-liver axis is increasingly recognized to be involved in the pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD). The gut microbiota and intestinal permeability have been demonstrated to be the key players in the gut-liver cross talk in NAFLD. Geniposide and chlorogenic acid (GC) combination is derived from a traditional Chinese medicine, Qushi Huayu Decoction (QHD), which has been used in clinic for NAFLD treatment for decades in China and validated in multiple animal models of NAFLD. GC combination previously has been demonstrated to treat NAFLD via modulation on the gut microbiota composition. In the present study, the effects of GC combination on gut barrier function in NAFLD were evaluated, and QHD and sodium butyrate (NaB), the intestinal mucosa protectant, were used as positive control. The therapeutic effect of GC combination on NAFLD were confirmed by amelioration on non-alcoholic steatohepatitis (NASH) induced by high-fat diet (HFD) in mouse, which was comparable to that of QHD. Simultaneously, GC combination was found to reduce the signaling of gut-derived lipopolysaccharide (LPS) including hepatic LPS binding protein, Toll like receptor 4, interleukin-1ß, tumor necrosis factor -α, and Kupffer cells infiltration. Furthermore, GC combination reduced LPS and D-lactate in plasma, restoring the colonic tight junction (TJ) expression and inhibited colonic TJs disassembly by down-regulation on RhoA/ROCK signaling in NASH induced by HFD. On the other hand, NASH was also alleviated in NaB group. The results of the present study suggested the important role of protection on gut barrier function in NAFLD treatment, which contributed to the therapeutic effects of GC combination on NASH.

Yupingfeng Powder relieves the immune suppression induced by dexamethasone in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Yupingfeng Powder (YPF), a Chinese medical formula, is used traditionally for allergic diseases and characterized by reducing allergy relapse. In the present study, we attempted to investigate the effect of YPF on the immunity of mice and the possible mechanisms. MATERIALS AND METHODS: An immunosuppressive mice model induced by Dexamethasone (Dex) was used. Blood samples, spleen and thymus were collected. Then, hematology parameters and organ weight were measured; Phenotypic analyses (CD4+/CD8+) of lymphocytes were performed using a flow cytometer; Phagocytosis of peritoneal macrophages were evaluated by particle tracers; Spleen lymphocytes were isolated, whose proliferation and apoptosis were assessed. NK cells' cytotoxicity was determined using the LDH release assay. RESULTS: YPF could ameliorate weight loss and improve low thymus and spleen coefficients caused by Dex. Treatment with YPF made decreased lymphocytic activity of Dex-treated mice back to normal and inhibited Dex-induced apoptosis of lymphocytes. YPF increased the Dex caused low proportion of CD4+/CD8+, and upregulated Dex-reduced NK cells' activity. CONCLUSION: The series of experiments demonstrated that YPF could exert immune regulation and enhance immunity of immunosuppressive mice through adjusting nonspecific and cellular immunity. The results would provide a basis for clinical application of YPF.

Chinese medicine CGA formula ameliorates DMN-induced liver fibrosis in rats via inhibiting MMP2/9, TIMP1/2 and the TGF-ß/Smad signaling pathways.

AIM: Chinese medicine CGA formula consists of polysaccharide from Cordyceps sinensis mycelia (CS-PS), gypenosides and amygdalin, which is derived from Fuzheng Huayu (FZHY) capsule for treating liver fibrosis. In this study we attempted to confirm the therapeutic effects of CGA formula in dimethylnitrosamine (DMN)-induced liver fibrosis in rats, and to identify the mechanisms of anti-fibrotic actions. METHODS: Rats were injected with DMN (10 mg·kg(-1)·d(-1), ip) for 3 consecutive days per week over a 4-week period. The rats then were orally administered with CGA formula (CS-PS 60 mg·kg(-1)·d(-1), gypenosides 50 mg·kg(-1)·d(-1) and amygdalin 80 mg·kg(-1)·d(-1)) daily in the next 2 weeks. CS-PS, gypenosides or amygdalin alone were administered as individual component controls, whereas colchicine and FZHY were used as positive controls. Serum biomarkers were measured. Hepatic injury, collagen deposition and stellate cell activation were examined. The MMP activities, expression of TIMP protein and proteins involved in the TGF-ß1/Smad signaling pathways in liver tissues were assayed. RESULTS: In DMN-treated rats, administration of CGA formula significantly decreased serum ALT, AST and total bilirubin and hepatic hydroxyproline levels, increased serum albumin level, and attenuated liver fibrosis as shown by histological examination. Furthermore, these effects were comparable to those caused by administration of FZHY, and superior to those caused by administration of colchicine or the individual components of CGA formula. Moreover, administration of CGA formula significantly decreased the protein levels of α-SMA, TGF-ß1, TGF-ß1 receptor (TßR-I), p-TßR-I, p-TßR-II, p-Smad2, p-Smad3, TIMP1 and TIMP2, as well as MMP2 and MMP9 activities in liver tissues of DMN-treated rats. CONCLUSION: Chinese medicine CGA formula ameliorates DMN-induced liver fibrosis in rats, and this effect was likely associated with the down-regulation of MMP2/9 activities, TIMP1/2 protein expression and the TGF-ß1/Smad signaling pathways in the liver.

A Recipe Composed of Chinese Herbal Active Components Regulates Hepatic Lipid Metabolism of NAFLD In Vivo and In Vitro.

This study is to investigate the therapeutic effects of the recipe composed of Atractylodes macrocephala polysaccharide, chlorogenic acid, and geniposide (named ACG) on experimental nonalcoholic fatty liver (NAFL). The research was divided into two parts as screening experiment and verification experiment. In the screening experiment, we used high-fat diet (HFD) induced NAFL rat model and uniform design to get the recipe from five Chinese herbal active components. In the verification experiment, HFD induced fatty liver rat and mouse NAFL models and free fatty acid (FFA) induced HepG2 cell model were used to verify the effects of ACG. According to the multiple regression equation of the hepatic triglyceride (TG) contents of each group in the screening experiment, the recipe ACG was obtained and the doses of Atractylodes macrocephala polysaccharide, chlorogenic acid, and geniposide for rats were 266.67, 3.33, and 45 mg/kg, respectively. The results of verification experiment verified that ACG could significantly reduce hepatic TG contents of NAFL rats and mice, as well as the cellular TG content of FFA-induced HepG2 cells. ACG could also improve HOMA-IR and hepatic mitochondrial ultrastructure of NAFL mice. Our study verified that ACG recipe could regulate lipid metabolism of NAFL in vivo and in vitro.

Antidiarrheal effect of Alpinia oxyphylla Miq. (Zingiberaceae) in experimental mice and its possible mechanism of action.

ETHNOPHARMACOLOGICAL RELEVANCE: The fructus Alpinia oxyphylla Miq. (AOM) has been used for treating diarrhea with spleen deficiency and gastralgia for thousands of years. A number of traditional Chinese medicine formulae provide AOM as an alternative herbal treatment for diarrhea, but the scientific basis for this usage has not been well defined. AIM OF THE STUDY: In this study, we tried to investigate the antidiarrheal activity and possible mechanisms of Fructus AOM, aiming to enrich our understanding to the scientific meanings and theoretical significance of Fructus AOM in clinical practice. MATERIALS AND METHODS: The fructus of AOM collected from Hainan province in China were macerated in the 95% ethanol to obtain the crude 95% ethanol extract, followed by subjected to chromatographic separation over a Diaion HP20 column to obtain 90% and 50% ethanol eluted fractions. The activities of the crude extract and fractions on castor oil induced acute diarrhea, rhubarb induced chronic diarrhea, gastrointestinal transit (GIT) in mice, and contractions of isolated guinea-pig ileum were evaluated. Additionally, nitric oxide (NO), gastrointestinal peptides gastrin (GAS), motilin (MTL) and somatostatin (SS) levels that related to gastrointestinal motilities were detected to demonstrate the potential mechanisms. Ultimately, LC-MS/MS method was utilized to ensure the chemical consistency. RESULTS: The 95% ethanol extract and 90% ethanol eluted fraction significantly delayed the onset time and decreased the wet faeces proportion compared with control group in the castor oil induced acute diarrhea mice. In terms of further evaluation of antidiarrheal activity, the 95% ethanol extract and 90% ethanol elution displayed significant inhibition of the intestinal propulsion at the two highest oral doses of 20 g crude drug/kg and 1g/kg. Moreover the 95% ethanol extract (10 and 20 g crude drug/kg) and 90% ethanol elution (0.5 and 1g/kg) could significantly inhibit the GIT, which was partially attributed to the increase in NO and SS levels, and the decreased MTL. In vitro spontaneous contractions of the isolated guinea pig ileum induced by carbachol, neostigmine and histamine were attenuated by both the extract and elution. Phytochemical analysis of 95% ethanol extract and its fractions identified the presence of diphenylheptanes, sesquiterpenes, and flavones as the major components. CONCLUSIONS: Our in vivo and in vitro data could partly support and justify the traditional usage of Fructus AOM on the treatment of diarrhea in traditional medicine.