[Research on mechanism of hypolipidemic effect of Massa Medicata Fermentata based on metabolomics].

This paper aims to study the therapeutic effect of Massa Medicata Fermentata on hyperlipidemia model rats and investigate its mechanism of hypolipidemic effect with the help of non-targeted metabolomics. The mixed hyperlipidemia model rats were constructed by giving high-fat chow. After successful modeling, the rats were divided into the model group, pravastatin sodium group(4.4 mg·kg~(-1)), lipotropic group(0.1 g·kg~(-1)), high-dose group(2.4 g·kg~(-1)), medium-dose group(1.2 g·kg~(-1)), and low-dose group(0.6 g·kg~(-1)) of Massa Medicata Fermentata, and they were administered for four weeks once daily. An equal volume of ultrapure water was given to the blank group and model group. Serum lipid level and liver hematoxylin-eosin(HE) staining were used as indicators to estimate the intervention effect of Massa Medicata Fermentata on mixed hyperlipidemia, and the changes in metabolites in plasma of mixed hyperlipidemia model rats were analyzed by non-targeted metabolomics. The mechanism of the hypolipidemic effect of Massa Medicata Fermentata was analyzed through metabolite pathway enrichment. The results showed that compared with the model group, the Massa Medicata Fermentata administration group, especially the high-dose group, could significantly reduce the content of total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c)(P<0.05 or P<0.01), and liver HE staining revealed that the number of adipocytes in the high-dose group was reduced to some extent. The potential biomarkers obtained by non-targeted metabolomics screening included glycerol 3-phosphate, sphingomyelin, sphingosine 1-phosphate, and deoxyuridine, which were mainly involved in the sphingolipid metabolism process, glycerophospholipid metabolism process, glycerol ester metabolism pathway, and pyrimidine metabolism pathway, totaling four possible metabolic pathways related to lipid metabolism. This study provides a reference for an in-depth investigation of the hypolipidemic mechanism of Massa Medicata Fermentata, which is of great significance for further promoting the clinical application of Massa Medicata Fermentata and increasing the indications.

The effects of Massa Medicata Fermentata on the digestive function and intestinal flora of mice with functional dyspepsia.

Introduction: The purpose of this study was to identify the chemical components of Massa Medicata Fermentata (MMF) in different fermentation methods, analyze its regulatory effects on gastrointestinal propulsion and intestinal flora in mice with food accumulation, and further explore its mechanism of action in the treatment of dyspepsia. Methods: The chemical compositions of three kinds of MMF were identified using the UPLC-Q- Exactive Orbitrap mass spectrometer. A model of spleen deficiency and food accumulation in mice was established. The gastric emptying rate and intestinal propulsion rate were calculated, serum gastrin concentration and cholinesterase activity were measured, and 16S rRNA microbial detection was performed in different groups of mouse feces. Results: The results showed that a total of 95 chemical components were identified from the three MMF extracts, 62 of which were the same, but there were differences in flavonoids and their glycosides, organic acids, and esters. MMF, PFMMF, and commercial MMF could all significantly improve the gastric emptying rate, intestinal propulsion rate, and GAS concentration in the serum of model mice; PFMMF has a better effect, while there was no significant difference in cholinesterase activity among the groups (p > 0.05). The 16S rRNA sequencing results showed that the MMF and PFMMF could increase the content of beneficial bacteria Bacteroidetes and decrease the pathogenic bacteria Verrucomicrobia in the intestines of model mice, while the commercial MMF could not. Discussion: Studies suggest that MMF has a variety of possible mechanisms for improving food accumulation and treating gastrointestinal dyspepsia, which provides reference value for the quality evaluation and clinical application of MMF.

Exploring the Potential Role of Massa Medicata Fermentata in Alcoholic Liver Injury Disease Based on Network Pharmacology and Animal Experiments.

BACKGROUND: ALD is a chronic liver disease caused by chronic excessive alcohol consumption, for which there are no drugs with better efficacy. Ancient literature and modern studies have shown that Massa Medicata Fermentata (MMF) has a hangover effect and ameliorates hepatic inflammation, so we believe that MMF has a potential role in the treatment of alcoholic liver disease. METHODS: UPLC-Q-Orbitrap HRMS was used to characterize the chemical constituents in MMF. The database was utilized to collect targets for the components and diseases, and cross-targeting analysis of the targets was performed. PPI, KEGG, GO enrichment analysis and molecular docking were performed using the core cross-targeting information to preliminarily validate the mechanism of action of MMF on disease. Finally, animal validation was carried out using male KM mice of the alcoholic liver injury model. RESULTS: MMF could play a role in the therapeutic prevention of alcoholic liver disease through the core targets AKT1, TNF, TP53, IL6 and CASP3 to regulate cancer pathways, lipid, and atherosclerosis, targeting IL-17 signaling, TNF signaling pathway, and hepatitis C, which was confirmed by animal pharmacodynamic experiments. CONCLUSION: This study serves as a rationale to support MMF in the treatment of ALD and meets the urgent need for clinical treatment of ALD. At the same time, it broadens the scope of clinical application of MMF.

Structure and properties of acidic polysaccharides isolated from Massa Medicata Fermentata: Neuroprotective and antioxidant activity.

Massa Medicata Fermentata (MMF) is a fermented food with therapeutic effects. Previous studies suggested that after stir-frying, the uronic acid content in MMF crude polysaccharides increases, and the pH value decreases, which is caused by the change in acidic polysaccharides. However, the detailed physicochemical properties and structure-activity correlation of the acidic polysaccharides in MMF have not been fully explored. In this study, two acidic polysaccharides (SMMFAP and CMMFAP) were isolated from the MMF and its stir-fried product, respectively. Their structural characteristics and bioactivities were comparatively studied, and the structure-activity correlation was examined. Our findings revealed that the SMMFAP had a higher average Mw and higher Gal and Man content than the CMMFAP. Both the SMMFAP and CMMFAP were mainly composed of Xyl, Man, and Gal residues, whereas the CMMFAP had fewer linkage types. Additionally, the CMMFAP exhibited stronger neuroprotective activity than the SMMFAP owing to its higher content of 1,6-linked-Galp, while the SMMFAP exhibited better antioxidant activity, which might be related to its higher average Mw. Our findings suggest that acidic polysaccharides may be the active substances that cause differences in effectiveness between the sheng and chao MMF. Furthermore, the research qualified the SMMFAP and CMMFAP with different potential applications.

Isolation, characterization, trypsin inhibition, liver protective and antioxidant activities of arabinoxylan from Massa Medicata Fermentata and its processed products.

Massa Medicata Fermentata (MMF) is a traditional Chinese medicine widely used in feed additives and human medicine. In this study, two neutral polysaccharides (SMMFP-1 and CMMFP-1) were isolated from two forms of MMF (sheng and chao MMF), and their structural characteristics and bioactivities were studied. The results showed that CMMFP-1 had higher average Mw compared with that of SMMFP-1. SMMFP-1 had a lower proportion of Ara, Xyl, GalA, and GlcA, but higher levels of Fuc, Gal, Man, and GulA. Compared with CMMFP-1, SMMFP-1 had a triple helix structure. SMMFP-1 had a layered structure, whereas CMMFP-1 had a curly layered structure. More glycosidic linkage types were found in SMMFP-1 than in CMMFP-1, and SMMFP-1 had a greater number of side chains. More importantly, SMMFP-1 showed better trypsin inhibition activity in vitro, liver-protective activity in vivo, and stronger antioxidant activity in vivo than CMMFP-1. Thus, arabinoxylans may be one of the active substances for different efficacies between MMF and its processed product. The results of this study facilitate the exploration of the correlation between the structural characteristics and biological functionalities of MMF arabinoxylans. Moreover, a theoretical basis is established for further study of the unique properties of arabinoxylans and their applications.

Processing Mechanism of Massa Medicata Fermentata Based on the Correlation Analysis of Strains, Chemical Compositions and Anti-Inflammatory Activity.

The traditional Chinese medicine of fermented medicine may be under the involvement of multiple strains and the interaction between these microorganisms. Liu Shenqu (Massa Medicata Fermentata, MMF) is one of the most widely used fermented medicines, whose potential processing mechanism is still unclear. In this work, UPLC/MS and GNPS methods were employed to rapidly predict chemical compositions in MMF. Moreover, the dynamic changes of strains, chemical compositions and anti-inflammatory activity of MMF during fermentation process were investigated, and subsequently strains-chemical compositions-efficacy interactions were revealed by Pearson correlation analysis and partial least squares regression (PLSR) analysis. As a result, 24 components were identified, and the potential strains including Bacillus, Burkholderia_Caballeronia_Paraburkholderia, Enterobacter, Aspergillus heterocaryoticus, Rhizopus arrhizus, Kazachstania bulderi, which related to the production of anti-inflammatory active ingredients were exposed. These results demonstrated chemical compositions-strains-efficacy interactions during fermentation of MMF, and provide reference for the exploration of the processing mechanism of MMF.

[Variations of glucose content in Massa Medicata Fermentata during processing based on quantitative proton nuclear magnetic resonance].

A quantitative proton nuclear magnetic resonance(qHNMR) method was established to determine the glucose content in commercially available Massa Medicata Fermentata(MMF) products and explore the variations of glucose content in MMF products during processing. The qHNMR spectrum of MMF in deuterium oxide was obtained with 2,2,3,3-d_4-3-(trimethylsilyl) propionate sodium salt as the internal standard substance. With the doublet peaks of terminal hydrogen of glucose with chemical shift at δ 4.65 and δ 5.24 as quantitative peaks, the content of glucose in MMF samples was determined. The glucose content showed a good linear relationship within the range of 0.10-6.44 mg·mL~(-1). The relative standard deviations(RSDs) of precision, stability, repeatability, and recovery for determination were all less than 2.3%. The glucose content varied in different commercially available MMF samples, which were associated with the different fermentation days, wheat bran-to-flour ratios, and processing methods. The glucose content in MMF first increased and then decreased over the fermentation time. Compared with the MMF products fermented with wheat bran or flour alone, the products fermented with both wheat bran and flour had increased glucose. The glucose content of bran-fried MMF was slightly lower than that of raw MMF, while the glucose content in charred MMF was extremely low. In conclusion, the qHNMR method established in this study is simple, fast, and accurate, serving as a new method for determining the glucose content in MMF. Furthermore, this study clarifies the variations of glucose content in MMF during processing, which can not only indicate the processing degree but also provide a scientific basis for revealing the fermentation mechanism and improving the quality control of MMF.

Variations of glucose content in Massa Medicata Fermentata during processing based on quantitative proton nuclear magnetic resonance / 中国中药杂志

A quantitative proton nuclear magnetic resonance(qHNMR) method was established to determine the glucose content in commercially available Massa Medicata Fermentata(MMF) products and explore the variations of glucose content in MMF products during processing. The qHNMR spectrum of MMF in deuterium oxide was obtained with 2,2,3,3-d_4-3-(trimethylsilyl) propionate sodium salt as the internal standard substance. With the doublet peaks of terminal hydrogen of glucose with chemical shift at δ 4.65 and δ 5.24 as quantitative peaks, the content of glucose in MMF samples was determined. The glucose content showed a good linear relationship within the range of 0.10-6.44 mg·mL~(-1). The relative standard deviations(RSDs) of precision, stability, repeatability, and recovery for determination were all less than 2.3%. The glucose content varied in different commercially available MMF samples, which were associated with the different fermentation days, wheat bran-to-flour ratios, and processing methods. The glucose content in MMF first increased and then decreased over the fermentation time. Compared with the MMF products fermented with wheat bran or flour alone, the products fermented with both wheat bran and flour had increased glucose. The glucose content of bran-fried MMF was slightly lower than that of raw MMF, while the glucose content in charred MMF was extremely low. In conclusion, the qHNMR method established in this study is simple, fast, and accurate, serving as a new method for determining the glucose content in MMF. Furthermore, this study clarifies the variations of glucose content in MMF during processing, which can not only indicate the processing degree but also provide a scientific basis for revealing the fermentation mechanism and improving the quality control of MMF.

[Investigation of microbial community involved in fermentation of Massa Medicata Fermentata].

This study aimed to clarify the microbial diversity, dominant species and the change of community structures in the fermentation of Liushenqu(Massa Medicata Fermentata), and explore the material foundation of its pharmacodynamics effect. On the basis of standardizing the fermentation process, Massa Medicata Fermentata was prepared by screening and optimizing the recipes and the standard formula issued by the Ministry. The community structure and growth process of fungi and bacteria in the samples at five time points(0, 17, 41, 48, 65 h) in the fermentation process of Massa Medicata Fermentata were analyzed by using isolation and culture of eight different media and high-throughput DNA sequencing technology. The results indicated that the samples of the two recipes pre-sented high microbial diversity at the initial fermentation stage, with Aspergillus spp. as the dominant species. As the fermentation process goes forward, Saccharomycopsis fibuligera and Rhizopus oryzae soon became dominant species from 17 h after fermentation commencement point to the fermentation end, while the other species were inhibited at a lower level from 17 h. The species diversity of bacteria in the initial fermentation samples was also high, and Enterobacter was the dominant species. Enterobacter cloacae, Pediococcus pentosaceus and Cronobacter sakazakii became dominant bacterial species 17 h after fermentation commencement, while the species diversity was decreased. Our results will be a scientific basis for promoting the fermentation process of Massa Medicata Fermentata by using pure microbial cultures.

Investigation of microbial community involved in fermentation of Massa Medicata Fermentata / 中国中药杂志

This study aimed to clarify the microbial diversity, dominant species and the change of community structures in the fermentation of Liushenqu(Massa Medicata Fermentata), and explore the material foundation of its pharmacodynamics effect. On the basis of standardizing the fermentation process, Massa Medicata Fermentata was prepared by screening and optimizing the recipes and the standard formula issued by the Ministry. The community structure and growth process of fungi and bacteria in the samples at five time points(0, 17, 41, 48, 65 h) in the fermentation process of Massa Medicata Fermentata were analyzed by using isolation and culture of eight different media and high-throughput DNA sequencing technology. The results indicated that the samples of the two recipes pre-sented high microbial diversity at the initial fermentation stage, with Aspergillus spp. as the dominant species. As the fermentation process goes forward, Saccharomycopsis fibuligera and Rhizopus oryzae soon became dominant species from 17 h after fermentation commencement point to the fermentation end, while the other species were inhibited at a lower level from 17 h. The species diversity of bacteria in the initial fermentation samples was also high, and Enterobacter was the dominant species. Enterobacter cloacae, Pediococcus pentosaceus and Cronobacter sakazakii became dominant bacterial species 17 h after fermentation commencement, while the species diversity was decreased. Our results will be a scientific basis for promoting the fermentation process of Massa Medicata Fermentata by using pure microbial cultures.

NMR-based study of chemical constituents of Massa Medicata Fermentata / 中草药

Objective: To study the chemical constituents from Medicated Leaven (namely Massa Medicata Fermentata in Chinese). Methods: Compounds were isolated and purified from chloroform extract of Medicated Leaven by column chromatograph of silica gel, ODS, Sephadex LH-20, HPLC, and their structure were elucidated on the basis of spectral data and physicochemical property. Results: Eleven chemical compounds were identified as 5-pentacosyl resorcinol (1), 5-heptadecyl resorcinol (2), 5-heneicosyl resorcinol (3), 5-tricosyl resorcinol (4), (9S,10E,12Z)-9-hydroxy-10,12-octadeca-dienoic acid (5), trans-4-hydroxy-2-nonenoic acid (6), 9,10,11-trihydroxy-11(E)-octadecenoic acid (7), (9S,12R,13S)-9,12,13-trihydroxy-10(E)-octadecenoic acid (8), (9S,12S,13S)- 9,12,13-trihydroxy-10(E)-octadecenoic acid (9), (9S,12S,13S)-9,12,13-trihydroxy-10(E)-octadecenoic acid methyl ester (10), and 9,12(Z)-octadecadienoic acid methyl ester (11). Conclusion: All above compounds are all isolated from Massa Medicata Fermentata for the first time, and the chloroform extract of Massa Medicata Fermentata and compound 8 showed inhibitory effect against TNF-α generation of RAW 264.7 induced by LPS in a dose-dependent manner.

Probiotics-fermented Massa Medicata Fermentata ameliorates weaning stress in piglets related to improving intestinal homeostasis.

Weaning stress has serious negative effects on piglets' health and the swine industry. Probiotics-fermented Chinese herbal medicines are potential feed additives to ameliorate weaning stress. In this study, the effects of probiotics-fermented Massa Medicata Fermentata (MMFP) on intestinal homeostasis were evaluated in weaning piglets. Dietary supplementation with MMFP promoted the development of the intestinal structure and elevated the concentrations of lactic acid and short-chain fatty acids (SCFAs) in the intestinal contents and antioxidant capacities in serum. MMFP reduced the levels of inflammatory factors in the intestinal mucosa. Microbial community analysis demonstrated that MMFP led to the selective and progressive enrichment of lactic acid- and SCFA-producing bacteria along the gastrointestinal tract, in particular, OTUs corresponding to Lactobacillus, Streptococcus, Acetitomaculum, Roseburia, and Eubacterium xylanophilum group, while MMFP reduced the relative abundance of pathogenic bacteria. On the contrary, antibiotics had negative effects on intestinal histology and increased the relative abundance of pro-inflammatory bacterium, such as Marvinbryantia, Peptococcus, Turicibacter, and Blautia. Correlation analysis reflected that the bacteria enriched in MMFP group were positively correlated with enhanced intestinal homeostasis, which suggested that dietary supplementation with MMFP enhanced host intestinal homeostasis by modulating the composition of gut microbiota and the levels of beneficial SCFAs, thus ameliorating weaning stress in piglets.

Isolation and Identification of Benzochroman and Acylglycerols from Massa Medicata Fermentata and Their Inhibitory Effects on LPS-Stimulated Cytokine Production in Bone Marrow-Derived Dendritic Cells.

Massa Medicata Fermentata (MMF), known as Shenqu, is an important traditional Chinese medicine widely used to treat indigestion, vomiting, and diarrhea. In this study, a new benzochroman, 3(S)-3,4-dihydro-5,10-di-ß-d-glucopyranoside-2,2-dimethyl-2H-naphtho(2,3-b)pyran-3-ol (1), and five known galactosyl acylglycerols (2⁻6) were isolated from a methanol extract from MMF. In addition, their chemical structures were determined by chemical and spectroscopic methods, which were compared with the previously reported data. Furthermore, the effects of isolated compounds on lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells were investigated. Compounds 1⁻3 exhibited significant inhibitory effects on the LPS-induced production of IL-6 and IL-12 p40, with IC50 values ranging from 1.6 to 10.2 µM. Compounds 2 and 3 also exhibited strong inhibitory effects on the LPS-stimulated production of TNF-α with IC50 values of 12.0 and 11.2 µM, respectively. The results might provide a scientific basis for the development of the active components in MMF, as well as for novel anti-inflammatory agents.

The effects of herbal composition Gambigyeongsinhwan (4) on hepatic steatosis and inflammation in Otsuka Long-Evans Tokushima fatty rats and HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatic steatosis has risen rapidly in parallel with a dramatic increase in obesity. The aim of this study was to determine whether the herbal composition Gambigyeongsinhwan (4) (GGH(4)), composed of Curcuma longa L. (Zingiberaceae), Alnus japonica (Thunb.) Steud. (Betulaceae), and the fermented traditional Korean medicine Massa Medicata Fermentata, regulates hepatic steatosis and inflammation. MATERIALS AND METHODS: The effects of GGH(4) on hepatic steatosis and inflammation in Otsuka Long-Evans Tokushima fatty (OLETF) rats and HepG2 cells were examined using Oil red O, hematoxylin and eosin, and toluidine blue staining, immunohistochemistry, quantitative real-time polymerase chain reaction, and peroxisome proliferator-activated receptor α (PPARα) transactivation assay. RESULTS: Administration of GGH(4) to OLETF rats improved hepatic steatosis and lowered serum levels of alanine transaminase, total cholesterol, triglycerides, and free fatty acids. GGH(4) increased mRNA levels of fatty acid oxidation enzymes (ACOX, HD, CPT-1, and MCAD) and decreased mRNA levels of lipogenesis genes (FAS, ACC1, C/EBPα, and SREBP-1c) in the liver of OLETF rats. In addition, infiltration of inflammatory cells and expression of inflammatory cytokines (CD68, TNFα, and MCP-1) in liver tissue were reduced by GGH(4). Treatment of HepG2 cells with a mixture of oleic acid and palmitoleic acid induced significant lipid accumulation, but GGH(4) inhibited lipid accumulation by regulating the expression of hepatic fatty acid oxidation and lipogenic genes. GGH(4) also increased PPARα reporter gene expression. These effects of GGH(4) were similar to those of the PPARα activator fenofibrate, whereas the PPARα antagonist GW6471 reversed the inhibitory effects of GGH(4) on lipid accumulation in HepG2 cells. CONCLUSIONS: These results suggest that GGH(4) inhibits obesity-induced hepatic steatosis and that this process may be mediated by regulation of the expression of PPARα target genes and lipogenic genes. GGH(4) also suppressed obesity-related hepatic inflammation. Thus, GGH(4) may be a promising drug for the treatment of obesity-related liver diseases.

Herbal composition Gambigyeongsinhwan (4) from Curcuma longa, Alnus japonica, and Massa Medicata Fermentata inhibits lipid accumulation in 3T3-L1 cells and regulates obesity in Otsuka Long-Evans Tokushima Fatty rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Adipocyte lipid accumulation due to impaired fatty acid oxidation causes adipocyte hypertrophy and adipose tissue increment, leading to obesity. The aim of this study was to determine the antiobesity effects of the herbal composition Gambigyeongsinhwan (4) (GGH(4)) composed of Curcuma longa L. (Zingiberaceae), Alnus japonica (Thunb.) Steud. (Betulaceae), and the fermented traditional Korean medicine Massa Medicata Fermentata. MATERIALS AND METHODS: The effects of GGH(4) and the individual components on lipid accumulation in 3T3-L1 adipocytes and body weight gain in Otsuka Long-Evans Tokushima Fatty (OLETF) rats were examined using Oil red O staining, hematoxylin and eosin staining, quantitative real-time PCR, and peroxisome proliferator-activated receptor α (PPARα) transactivation assay. RESULTS: GGH(4), individual components, and an active principle of Curcuma longa curcumin inhibited lipid accumulation and mRNA levels of adipocyte-specific genes (PPARγ, aP2, and C/EBPα) in 3T3-L1 adipocytes compared with control cells. Treatment with GGH(4), the individual components or curcmumin increased mRNA levels of mitochondrial (CPT-1, MCAD, and VLCAD) and peroxisomal (ACOX and thiolase) PPARα target genes. GGH(4) and the individual components also increased PPARα reporter gene expression compared with control cells. These effects were most prominent in GGH(4)-treated cells. However, the PPARα antagonist GW6471 reversed the inhibitory effects of GGH(4) on adipogenesis. An in vivo study showed that GGH(4) decreased body weight gain, adipose tissue mass, and visceral adipocyte size with increasing mRNA levels of adipose tissue PPARα target genes in OLETF rats. CONCLUSIONS: These results demonstrate that GGH(4) has an antiobesity effects through the inhibition of adipocyte lipid accumulation, and this process may be mediated in part through adipose PPARα activation.