阅读说明：本技术 泽泻根茎提取物在改善nafld及调节肠道菌群失调的应用 (Application of rhizoma alismatis rhizome extract in improvement of NAFLD (NAFLD) and regulation of intestinal flora imbalance ) 是由 夏凡 周本杰 乡世健 陈芝娟 邓丽君 纪秋凤 于 2021-10-29 设计创作，主要内容包括：本发明公开了泽泻根茎提取物在改善NAFLD及调节肠道菌群失调的应用,所述泽泻根茎提取物是指从泽泻根茎中提取分离出的AlisolB23-acetate,所述AlisolB23-acetate为一种三萜类化合物,所述泽泻根茎提取物可用于改善NAFLD病程及相关的肠道菌群失调,本发明提供了AB23A的一种新应用,即应用于改善NAFLD及调节相关的肠道菌群失调,所述NAFLD主要指因高脂饮食诱导的非酒精性脂肪肝,包括单纯的肝脏脂质堆积到合并炎症的脂肪性肝炎,所述AB23A不仅对NAFLD有显著的改善作用,还可部分恢复高脂饮食导致的菌群的丰度和多样性的降低,逆转高脂饮食导致的NAFLD中不同菌群分类水平的结构失调并调节其介导的支链氨基酸和脂多糖的过多产生。(The invention discloses an application of rhizoma alismatis rhizome extract in improving NAFLD and adjusting intestinal dysbacteriosis, the rhizoma Alismatis extract is Alisol B23-acetate extracted from rhizoma Alismatis, the AlisolB23-acetate is a triterpenoid, the rhizoma alismatis rhizome extract can be used for improving the disease course of NAFLD and related intestinal dysbacteriosis, the invention provides a new application of AB23A, namely, the application of the compound preparation in improving NAFLD and regulating related intestinal flora imbalance, the NAFLD mainly refers to non-alcoholic fatty liver induced by high fat diet, including simple liver lipid accumulation to steatohepatitis with inflammation, the AB23A has obvious improvement effect on NAFLD, and can partially recover the reduction of abundance and diversity of flora caused by high fat diet, reverse the structural disorder of different flora classification levels in NAFLD caused by high fat diet and regulate the mediated overproduction of branched chain amino acid and lipopolysaccharide.)

1. The application of the rhizoma alismatis rhizome extract in improving NAFLD and regulating intestinal dysbacteriosis is characterized in that the rhizoma alismatis rhizome extract is Alisol B23-acetate extracted and separated from rhizoma alismatis rhizome, the Alisol B23-acetate is a triterpenoid, and the rhizoma alismatis rhizome extract can be used for improving the NAFLD course and the related intestinal dysbacteriosis.

2. The use of an alisma rhizome extract in improving NAFLD and modulating gut flora imbalance according to claim 1, wherein NAFLD comprises mainly non-alcoholic fatty liver induced by high fat diet, including simple liver lipid accumulation to steatohepatitis with associated inflammation.

3. The use of a rhizome extract of alisma orientale in improving NAFLD and modulating gut dysbacteriosis according to claim 1, wherein the gut dysbiosis is caused by NAFLD.

Technical Field

The invention relates to application of rhizoma alismatis rhizome extract, in particular to application of the rhizoma alismatis rhizome extract in improving NAFLD and regulating intestinal flora imbalance.

Background

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease of people, is related to obesity, is the liver manifestation of metabolic syndrome, and as a clinical pathological syndrome, the non-alcoholic fatty liver disease comprises a series of liver diseases, ranging from simple liver lipid accumulation (steatosis) to steatohepatitis (NASH) with inflammation, can cause cirrhosis, hepatocellular carcinoma and death. The intestinal microbiota of non-alcoholic fatty liver is characterized by a reduced diversity of microorganisms and a higher Firmicutes/bacterioidaeota (F/B) ratio, and further studies have shown that gut dysbiosis directly or indirectly regulates the levels of pro-inflammatory cytokines and fat oxidation, which may have important downstream effects on the liver, and that changes in bacterial metabolites may play an important role in this process, such as the levels of Lipopolysaccharide (LPS) and Branched Chain Amino Acids (BCAA), and therefore, in the era of precision medicine, the determination of microbiome structure and mechanism becomes increasingly important;

AlisolB23-acetate (AB23A) is a natural triterpenoid extracted and separated from an extract of rhizome of alisma orientale, which is a medicinal plant and is long-term thought to be widely used as a traditional Chinese medicine, and the biological characteristics and various pharmacological activities of AB23A have been proved in vitro and in vivo, including anti-hepatitis, anti-bacterial, anti-hyperlipidemia, anti-tumor, liver-protecting, anti-inflammatory and anti-oxidation effects, but the application of AB23A in improving NAFLD and regulating the intestinal flora imbalance caused by NAFLD is worthy of further exploration.

Disclosure of Invention

The invention aims to overcome the defects of the technology and provides application of rhizoma alismatis rhizome extract in improving NAFLD and regulating intestinal flora imbalance.

In order to solve the technical problems, the technical scheme provided by the invention is the application of the rhizoma alismatis rhizome extract in improving NAFLD and regulating intestinal dysbacteriosis, the rhizoma alismatis rhizome extract is Alisol B23-acetate extracted and separated from rhizoma alismatis rhizome, the Alisol B23-acetate is a triterpenoid, and the rhizoma alismatis rhizome extract can be used for improving the disease course of NAFLD and the related intestinal dysbacteriosis.

As an improvement, the NAFLD mainly includes non-alcoholic fatty liver induced by high fat diet, including simple liver lipid deposition to steatohepatitis with associated inflammation.

As an improvement, the intestinal dysbacteriosis is the intestinal dysbacteriosis caused by NAFLD.

Compared with the prior art, the invention has the advantages that: the invention provides a new application of AB23A, namely, the application is applied to improve NAFLD and regulate the imbalance of intestinal flora, the NAFLD mainly refers to non-alcoholic fatty liver induced by high-fat diet, including simple liver lipid accumulation to fatty liver with inflammation, the AB23A can inhibit BCAAs (branched chain amino acid) and LPS (lipopolysaccharide) overproduction mediated by intestinal flora, reduce the concentration of LPS and BCAA in vivo, and improve the symptom of NAFLD induced by high-fat diet; the AB23A has good regulating and balancing effects on intestinal flora, and also has inhibiting effect on weight gain of NAFLD induced by high fat diet.

Drawings

FIG. 1 is a schematic view of the structural formula of the rhizome extract AB23A of Alisma orientale of the present invention.

FIG. 2 is the HPLC identification map of the rhizome extract AB23A of Alisma orientale of the present invention.

Fig. 3 shows the effect of alisma rhizome extract AB23A on mouse model body weight NAFLD caused by high fat diet.

Fig. 4 shows the result of histopathological examination of the alisma rhizome extract AB23A on NAFLD mouse model caused by high fat diet.

Where a is a representative liver section stained with hematoxylin and eosin of the SCD, HFD, and HAH groups (magnification × 20, scale bar 100 μm), B is an oil red O stain of a representative liver section of the SCD, HFD, and HAH groups (magnification × 20, scale bar 100 μm), and C and D are analysis charts of oil red O stain data of a representative liver section and a liver section of the SCD, HFD, and HAH groups, respectively.

FIG. 5 shows the effect of Alismatis rhizoma rhizome extract AB23A on the abundance and diversity of microbial flora in NAFLD mouse model caused by high fat diet,

wherein A is Sobs index and B is Shannon index.

FIG. 6 shows the effect of the rhizome extract AB23A of Alisma orientale in improving the intestinal dysbacteriosis caused by NAFLD.

Wherein A is the principal coordinate analysis (PCoA) based on Bray-Curtis, microbial population composition of OTU level in three groups is analyzed, B and C are the ratio of the Bacteromyces backwardiformis, the Proteobacteria and the Bacteroides respectively in the SCD, HFD and HAH three groups, and D is the abundance level distribution of the genus of top 10 in the total abundance ranking of the sequencing in the SCD, HFD and HAH groups.

Fig. 7 shows the results of the prediction and validation of the abundance of gut flora gene function in branched-chain amino acid and lipopolysaccharide synthesis.

Wherein A and B are respectively the results of predicting the abundance of intestinal flora gene function in Lipopolysaccharide (LPS) and Branched Chain Amino Acid (BCAAs) synthesis, and C and D are the results of verifying plasma BCAAs and LPS levels in SCD, HFD and HAH groups.

Figure 8 is a heatmap of spearman (spearman) correlation analysis of bacterial abundance to NAFLD metabolic parameters.

Detailed Description

The following examples are provided to further illustrate the application of the rhizoma alismatis rhizome extract of the present invention in improving NAFLD and regulating the intestinal dysbacteriosis.

Example 1

1. Preparation of the experiment

1) Experimental animals: male C57BL/6J mice (8-9 weeks old) provided by the pharmaceutical animal testing center of the university of traditional Chinese medicine, Guangzhou;

2) experiment feed: standard feed (SCD, 10% fat calories) and high density feed (60% fat calories) provided by the pharmaceutical animal testing center, university of traditional chinese medicine, guangzhou.

2. Procedure of experiment

1) Grouping and process of experimental animals

Mice were housed at 22 ℃ ± 1 ℃ for a 12 hour dark period with adequate food and water supply, all animal maintenance and treatment protocols followed the guidelines for laboratory animal care and use adopted and promulgated by the national institutes of health, and approved by the animal ethics committee of the university of medicine, guangzhou, with 2 weeks of acclimation, and 24 mice were randomly divided into three groups, namely, SCD group (standard diet, n ═ 8), HFD group (high fat diet, 60% of calories from fat, n ═ 8), HFD + high dose AB23A group (HAH ═ 60mg/kg, n ═ 8 per group);

wherein AB32A is administered by intragastric administration to mice once a day;

after 12 weeks of treatment, fecal samples (from 8:00 am to 4:00 pm) were collected using a metabolic cage filled with ice-filled Eppendorf tubes and immediately stored at-80 ℃ until analysis. At the end of the experiment, all animals were sacrificed and serum samples were collected from the orbital plexus, and feces (SCD, HFD and HAH), liver samples were collected for further analysis.

3) Experimental analysis and evaluation

Firstly, evaluating the weight and metabolic parameters of the mouse

The body weight and metabolic parameters of each group of mice were tested and evaluated, wherein the metabolic parameters included Malondialdehyde (MDA), superoxide dismutase (SOD), alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), triglyceride (Serum TG), total cholesterol (SerumTc), Hepatic triglyceride (hepatotic TG), and Hepatic total cholesterol (hepatotic TC).

Histopathological and clinical biochemical assessment

Each group of specimens was randomly selected, fixed with 4% paraformaldehyde solution, histological examination of liver and colon was performed, and then the tissues were embedded with paraffin, stained with hematoxylin-eosin (H & E), frozen liver sections (6 mm thick) were prepared and stained with oil red O-hematoxylin, the stained sections were photographed using a microscope, and the morphology and pathological results of hematoxylin-eosin staining were analyzed and evaluated by a histopathologist of the pathology department (secondary seventh hospital of zhongshan university) according to NAFLD Activity Score (NAS). Quantifying oil red O staining by using Olympus Image-Pro Plus 6.0 software, and calculating staining area;

clinical biochemical analyses including serum Total Cholesterol (TC), Triglycerides (TG), alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) were all detected using a Beckman CX5 automated biochemical analyzer (Beckman Coulter, inc., USA) at the clinical laboratory of the animal laboratory at the university of zhongshan and were performed according to standard routine procedures;

the plasma levels of Interleukin (IL) -6/-1 β and tumor necrosis factor- α (TNF- α) were determined by a specific ELISA kit;

levels of hepatic Malondialdehyde (MDA) and superoxide dismutase (SOD) were monitored using a commercial kit according to instructions for use.

(iii) detection of plasma Lipopolysaccharide (LPS) and Branched Chain Amino Acid (BCAAs) levels

Quantification of plasma LPS and BCAAs was performed according to the instructions of the Pierce Chromogenic endo toxin Quant Kit (A39552, Thermo Scientific) and Branch-ChainAminoAcidAssay Kit (ab83374, Abcam), respectively.

DNA extraction and polymerase chain reaction amplification

Three groups (SCD, HFD and HAH) of bacterial DNA were extracted from fecal samples using the QIAamp FastDNA pool Mini Kit, and the integrity and quality of the DNA was assessed by agarose gel electrophoresis (concentration of agarose gel: 1%; voltage: 5V/cm; electrophoresis time: 20 minutes), and aoDrop 2000(Thermo Fisher Scientific, Wilmington, DE, USA) was used to determine the concentration and purity of the DNA sample, using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). the procedure for the PCR reaction was as follows:

3 minutes at 95 ℃, 30 cycles at 95 ℃, 30 seconds at 60 ℃, 75 seconds at 72 ℃, 10 minutes at 72 ℃;

triplicate PCR reaction mixtures (20. mu.L) contained 4. mu.L of 5 XFastPfu buffer, 2. mu.L of 2.5mM dNTPs, 0.8. mu.L of each primer (5. mu.M), 0.4. mu.L of FastPfu polymerase, and 10ng of template DNA, PCR reactions were performed using appropriate volumes of double-vapor H2O as needed, PCR products were purified using AxyPrep DNA gel extraction kit (Axygen, Biosciences, USA) to remove non-specific products, and DNA concentrations were quantified using QuantiFluor TM-ST (Promega, USA).

Sequencing 16S rRNA gene amplicon and microbiome analysis

The purified amplicons were pooled at equimolar concentrations and paired-end sequencing (2X 300) was performed on the Illumina MiSeq platform (Illumina, San Diego, Calif., USA). (Shanghai) standard protocol;

and demultiplexing and quality filtering the original fastq file by using Trimmomatic, and further combining the original fastq file by using FLASH software v1.2.11. Then inputting the qualified fastq file into QIIME v1.91, referring to a Silva database (138 edition), and clustering sequencing data into an operation classification unit (OTU);

OTUs reaching 97% nucleotide similarity were subjected to α -diversity and β -diversity analysis using mothur software v1.30.2 and QIIME v1.91, respectively, differences in the compositional abundance of intestinal flora of three groups (SCD, HFD and HAH) were verified by principal coordinate analysis (PCoA) based on OTU abundance and distance, bio-correlation and statistical significance were calculated using Spearman scale correlation coefficients, the classification levels between the three groups (SCD, HFD and HAH) were represented to different degrees, microbial function was predicted by the picrus 2 method, and then function was classified according to the KEGG orthography.

4. Results of the experiment

1) Statistical analysis

Differences between groups were analyzed using GraphPadPrism software (version 8.3.0) for one-way analysis of variance (ANOVA) and Tukey multiple comparison tests, and data are presented as mean ± Standard Deviation (SD), differences considered statistically significant at P < 0.05;

intestinal bacterial data were analyzed on the Majorbio cloud platform (www.majorbio.com) with differences between groups by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test with GraphPadPrism software (version 8.3.0), data expressed as mean ± Standard Deviation (SD), differences considered statistically significant at P < 0.05.

2) Conclusion of the experiment

(ii) AB23A had inhibitory effect on the body weight gain of NAFLD mice induced by high fat diet (fig. 3) and regulated metabolic parameters. Metabolic parameters of mice between groups were tested and tabulated as follows:

TABLE 1 comparison of metabolic parameters of mice between different groups

Note: p <0.01 compared to SCD group; # p <0.05 vs HFD group; # p <0.01 compared to the HFD group. P <0.01 compared to SCD group; # p <0.05 vs HFD group; # p <0.01 compared to the HFD group. MDA: malondialdehyde; SOD (superoxide dismutase): superoxide dismutase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; IL-6/-1 β: interleukin-6/-1 β; TNF- α: tumor necrosis factor-alpha; SerumTG: serum triglycerides; a SerumTc: total cholesterol in serum; hepatotic TG: liver tissue triglycerides; hepatotic TC: liver tissue total cholesterol.

② AB23A protective action on liver of high fat diet-induced NAFLD mouse

Staining of liver sections with hematoxylin-eosin (H & E) showed significant balloon-like changes in hepatocytes in HFD-induced mouse livers with severe steatosis, AB23A significantly reduced the extent of hepatocellular steatosis (fig. 4-a and 4-C);

the effect of AB23A treatment on serum and liver lipids was further examined. Triglyceride (TG) and Total Cholesterol (TC) levels were also reduced in serum and liver following AB23A treatment compared to HFD group (table 1);

the results of oil red O stained sections also showed that AB23A caused a significant reduction in red stained areas (FIGS. 4-B and 4-D);

taken together, AB23A not only improved liver steatosis but also reduced pro-inflammatory cytokine production, indicating the protective effect of AB23A on HFD-induced NAFLD mouse liver.

③ AB23A rebalance the composition of the intestinal flora of NAFLD mice induced by high fat diet.

Three groups (SCD, HFD and HAH; n ═ 8) were analyzed for microbial diversity characteristics, and after removing the failing reads, a total of 1250510 raw reads were obtained, with an average of 48396 sequences per sample, in which the Sobs and Shannon indices of the HFD and HAH groups were significantly lower than those of the SCD group, indicating that HFD treatment significantly reduced the abundance and diversity of the flora, and AB23A may restore in small part the reduction in the abundance and diversity of the flora resulting from a high fat diet (fig. 5A and 5B);

analysis of microbiota composition at OTU level based on PCoA of Bray-Curtis showed significant clustering in each group: PC1 and PC2 axis score plots were 46.82% and 12.13%, respectively, indicating a significant difference in bacterial colony distribution characteristics before and after induction with or without AB23A or high fat diet (fig. 6A), and the results of analysis of colony composition at the phylum level showed that AB23A significantly reversed the rise in firmicutes/bacteroidetes (F/B) ratio (fig. 6B) and actinomycetes/bacteroidetes (a/B) ratio (fig. 6C) caused by high fat diet; subsequent analysis results further showed that AB23A dry prognosis could significantly reverse the distribution of abundance levels of the top 10 genera in this sequencing total abundance induced by high fat diet, as evidenced by AB23A reversing high fat diet resulting in significant decrease in abundances of muriciceae (Muri), Lactobacillus (Lactobacillus), while Turicibacter (diriginal), faecalibacillus (faecalibacillus) and Ileibacterium (Ilei) showed significant increase in abundance, etc., and also showed similar regulation for three genera belonging to the family Lachnospiraceae (Lachnospiraceae — NK4a136_ group, unclassified _ f __ Lachnospiraceae and norrank _ f __ Lachnospiraceae) (fig. 6D). Therefore, AB23A could improve NAFLD-related gut flora imbalance.

AB23A reduces intestinal flora-mediated overproduction of BCAAs and LPS.

Picrast 2 analysis based on 16S rRNA gene sequence data was used to predict metabolic changes in NAFLD in these gut microbiomes. The results show that the metabolic function of microorganisms is more involved in several metabolic pathways. Among them, the biosynthesis of LPS and the synthesis of valine, leucine and isoleucine belonging to BCAAs showed a significant difference in each group, indicating that AB23A might reduce the production of BCAAs and LPS (fig. 7A and 7B). To verify that the effect of AB23A on BCAA production and LPS biosynthesis was caused by gut dysbiosis, we further tested the levels of BCAA and LPS in serum. The results show that: HFD-fed mice produced more BCAA than SCD group (fig. 7C and 7D). AB23A treatment partially reversed HFD-induced overproduction of BCAAs. Furthermore, a significant increase in LPS levels was also detected in HFD-fed mice, which was partially reversed in AB23A treatment (fig. 7D). These results indicate that AB23A treatment can ameliorate metabolic disorders caused by dysbacteriosis in HFD.

From the results of the Spearman correlation analysis, it was found that there was a significant positive correlation between the levels of BCAA and LPS in both the Turicibacter (urbanium) and the Ileibacterium (Ilei), and we speculated that the Turicibacter (urbanium) and the Ileibacterium (Ilei) might be closely related to the production of BCAA and LPS, respectively, in this process (fig. 8).

In conclusion, the AB23A has a remarkable improvement effect on NAFLD, and also has a regulation effect on intestinal flora structural disorder in NAFLD and mediated overproduction of branched chain amino acid and lipopolysaccharide, so that the pharmacodynamic effect of the AB23A on treating NAFLD is closely related to the potential prebiotic effect of the AB23A, and the AB23A possibly has certain application and commercial development values.

The invention and its embodiments have been described above, without this being limitative. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.