阅读说明：本技术 一种蒙药肋柱花提取物、制备方法及用途 (Mongolian medicine extract of costaria flowers, preparation method and application ) 是由 付明海 阿丽沙 巴根那 于 2021-10-21 设计创作，主要内容包括：本发明提供了一种蒙药肋柱花提取物、制备方法及用途；将肋柱花全草经二氯甲烷溶液浸泡,用乙醇提取,干燥,得到肋柱花提取物。本发明还涉及蒙药肋柱花提取物的制备方法及用途。本发明所涉及的蒙药肋柱花提取物给药改变了小鼠肠道微生物群的组成,使高脂模型小鼠肠道菌群Firmicute、Proteobacteria相对丰度和Firmicute/Bacteroidetes比值下降,Bacteroidetes相对丰度增加,最终使得本发明所涉及的肋柱花提取物给药改变了小鼠肠道微生物群的组成；本发明通过调节高脂饮食所导致的肠道菌群紊乱,达到减肥作用。(The invention provides a Mongolian medicine extract of costaria, a preparation method and application; soaking the whole plant of the lomatogonia costata in a dichloromethane solution, extracting with ethanol, and drying to obtain the lomatogonia costata extract. The invention also relates to a preparation method and application of the Mongolian medicine ribwort extract. The application of the Mongolian medicine ribbing flower extract changes the composition of mouse intestinal microbiota, so that the relative abundance of high-fat model mouse intestinal flora Firmicute and Proteobacteria and the Firmicute/Bacteroides ratio are reduced, the relative abundance of Bacteroides is increased, and finally the application of the ribbing flower extract changes the composition of mouse intestinal microbiota; the invention achieves the effect of losing weight by adjusting the disturbance of intestinal flora caused by high fat diet.)

1. A Mongolian medicine extract of lomatogonium costatum is characterized in that the whole plant of lomatogonium costatum is soaked in dichloromethane solution, extracted by ethanol and dried to obtain the lomatogonium costatum extract.

2. A method of preparing the extract of the Mongolian medicine, ribwort, as claimed in claim 1, comprising the steps of:

step 1, airing and crushing the whole plant of the Mongolian medicinal material ribbing flower in a shade;

step 2, soaking the mixture in ten times of dichloromethane solution for one night, and extracting the mixture once every 2 hours for 2 times;

and step 3, soaking the medicine residues in 95% ethanol for one night, extracting for 2 times every 3 hours, recovering ethanol, and preparing the medicine residues into powder by using a freeze dryer.

3. Use of the extract of the Mongolian medicine, Rizhilia, as claimed in claim 1, for the preparation of a composition ratio regulator of intestinal flora.

Technical Field

The invention belongs to the field of national medicine; in particular to a Mongolian medicine extract of costaria flowers, a preparation method and application.

Background

Obesity is one of ten chronic metabolic diseases determined by WHO to be caused by the combined action of gene, environment and motion factors, particularly fat accumulation in vivo caused by excessive intake of food nutrients. Obesity not only can bring about changes of body morphology, but also more importantly metabolic syndromes caused by overweight and obesity, namely hypertension, hyperlipidemia, type 2 diabetes mellitus, coronary heart disease, malignant tumor, polycystic ovary syndrome, sleep apnea and other diseases, become healthy invisible killers in our lives. Excess storage of triglycerides in the body of obesity can lead to significant accumulation of macrophages in visceral adipose tissue, resulting in modified release of adipokines and pro-inflammatory cytokines, such as TNF-and IL-6, leading to metabolic abnormalities. Thus, obesity is characterized by a sustained systemic low level inflammatory response. The current treatment methods mainly comprise Roux-en-Y gastric bypass surgery, adjustable gastric band and sleeve gastrectomy, behavior weight-reducing treatment after cognitive remediation, nanoparticle anti-angiogenesis therapy treatment, hypolactamic broad-derived neurotrophic factor (BDNF) or Fibroblast growth factor 21(FGF21) [11] gene therapy, intestinal flora and the like. The intestinal flora is the largest microecosystem of the human body and has great influence on the metabolism of substances and energy of the body. At present, the gut microbiota is considered to be an important factor in the development of obesity.

With the intensive research on the intestinal flora in recent years, it is gradually found that the intestinal flora is closely related to the development and development of obesity. The intestinal flora inhabits the human intestinal tract and is a microbial flora composed of bacteria which depend on the human body to live. The intestinal tract of a human body contains 100-1500 bacteria of nearly hundreds of trillion, the number of the bacteria is close to the total number of cells of the human body, and the ratio is about 1: 1. The intestinal flora has great influence and effect on human health and maintaining human physiological functions, for example, the intestinal flora can regulate the immune system of the body and abnormal metabolic diseases, and can also control the absorption of energy of the human body through the influence on intestinal epithelial cells. Eating high-fat and high-sugar diet for a long time can cause the disturbance of intestinal flora, reduce the proportion of beneficial bacteria and increase the proportion of pathogenic bacteria. Imbalance of the intestinal flora also leads to increased permeability of the intestinal mucosa and the development of chronic inflammation.

Mongolian medicine costal flower (Lomatogeniumrotatum (L.) Fries ex Nym.), Mongolian name Harbiri root-Geddard, which is loaded into the drug Standard Mongolian medicine booklet of Ministry of public health of the people's republic of China in 1998, has the functions of calming ' harmonizing the sun ', clearing heat, invigorating stomach, healing wound and the like. Recent research shows that the components of the xanthone compound, swertiamarin, luteolin, oleanolic acid and the like in the Mongolian costcolumn flower have different degrees of liver protection and cholagogic activity and are one of the main active components of the costcolumn flower. Researchers find that Mongolian medicine lomatogonium carinthiacum has the effects of protecting liver and benefiting gallbladder, and the flavonoids compounds 1-hydroxy-3, 5, 8-trimethoxyflavone, l-hydroxy-3, 7, 8-trimethoxyflavone, swertiamarin and 6, 8-dihydroxy-1, 2-dimethoxyflavone can treat obesity-related diseases, such as hypercholesterolemia and hypertriglyceridemia caused by high-sugar food intake, and improve the lipid and leptin metabolism of insulin resistant rats. However, whether costal Styloides has the potential effect of regulating intestinal microbiota to prevent obesity has not been reported. Therefore, the Mongolian medicine of costaria flowers is selected as the raw material, and the influence of the Mongolian medicine of costaria flowers on intestinal flora is deeply researched.

Disclosure of Invention

The invention aims to provide a Mongolian medicine extract of lomatogonium carinthiacum, a preparation method and application thereof.

The invention is realized by the following technical scheme:

KM male mice were given high-fat diet to establish an obesity model. After 8W, the obese model mice (with the ND group as the experimental control group and the body weight more than 20% of the mice in the ND group) were randomly divided into a high fat diet group (HFD), a high dose group (LR1.8g/kg), a medium dose group (LR0.9g/kg), a low dose group (LR0.18g/kg) and a positive control group (Orlistat, 0.048g/kg) except for the normal diet group (ND). Mouse body weight and food intake were observed and recorded weekly. After 9 weeks of continuous administration, relevant weight loss indexes were examined. Fresh mouse feces were collected and the flora status was detected using 16S rDNA Amplicon sequencing technology.

The invention relates to a Mongolian medicine extract of lomatogonium carinthiacum, which is prepared by soaking whole plant of lomatogonium carinthiacum in dichloromethane solution, extracting with ethanol, and drying.

The invention also relates to a preparation method of the extract of the lomatogonium carinthiacum, which comprises the following steps:

step 1, airing and crushing the whole plant of the Mongolian medicinal material ribbing flower in a shade;

step 2, soaking the mixture in a dichloromethane solution for one night, and extracting the mixture for 2 times every 2 hours;

and step 3, soaking the medicine residues in 95% ethanol for one night, extracting for 2 times every 3 hours, recovering ethanol, and preparing the medicine residues into powder by using a freeze dryer.

The invention also relates to application of the Mongolian medicine ribcage flower extract in preparation of the intestinal flora composition ratio regulator.

The Mongolian medicine ribbing flower extract is used as an intestinal flora composition ratio regulator, and is administered to a high-fat model mouse, so that the composition of intestinal microbiota of the mouse is finally changed, the relative abundance of Firmicute and Proteobacteria of the intestinal flora of the high-fat model mouse is reduced, the Firmicute/Bacteroides ratio is reduced, the relative abundance of Bacteroides is increased, and the composition of the intestinal microbiota of the mouse is finally changed by the administration of the ribbing flower extract; the invention achieves the effect of losing weight by adjusting the disturbance of intestinal flora caused by high fat diet.

The invention has the following advantages:

the application of the Mongolian medicine ribbing flower extract changes the composition of mouse intestinal microflora, so that the relative abundance of high-fat model mouse intestinal flora Firmicute and Proteobacteria and the Firmicute/bacteriodes ratio are reduced, and the relative abundance of bacteriodes is increased, therefore, the ribbing flower extract achieves the weight-reducing effect by regulating the intestinal flora disorder caused by high-fat diet.

Drawings

FIG. 1 is a graph of the effect of extract of the Mongolian agent Rizhilia on Alpha diversity of intestinal microbiota in mice with obese HFD diets;

FIG. 2 is a graph of the effect of extract of the Mongolian agent Ribis on the diversity of intestinal microbiota in mice with obese HFD diets;

FIG. 3 is a graph of the effect of extract of the Mongolian medicine, ribwort on the composition of the intestinal microbiota structure of mice obese on HFD diet.

Detailed Description

The present invention will be described in detail with reference to specific examples. It should be noted that the following examples are only illustrative of the present invention, but the scope of the present invention is not limited to the following examples.

Examples

The embodiment relates to a lomatogonium carinthiacum extract, a preparation method and application.

First, laboratory glassware

Varioskan^TMLUX multifunctional microplate reader (Thermo Fisher Scientific-CN),

sigma 3-18KS high-speed refrigerated centrifuge,

FD-1A-50 freeze dryer (Beijing Bo Yi kang laboratory instruments Co., Ltd.), full-automatic biochemical instrument electronic balance (Saedolis scientific instruments (Beijing) Co., Ltd.).

Second, experimental medicine and reagent

The flower of Ribes nigrum is collected from American flag of Nemongolian Siller, West, and identified as dry whole grass of Lomatogoniumcarinthiacaum (Wulf) Reichb.

High fat diet (Shandong Hengrong Biotech Co., Ltd.),

common feed (Liaoning Biotechnology GmbH, Ltd.).

Third, experimental animal

Male and female SPS-grade KM mice are selected for the experiment, the mice are 5 weeks old, the body mass (35 +/-2) g and 80 mice are purchased from Liaoning Changsheng biotechnology GmbH, the animal license number is as follows: SCXK (Liao) 2015-0001.

Fourth, the method

4.1 preparation of extract of Crotalaria costata

Drying Mongolian medicinal material, air drying and pulverizing in the shade, soaking in dichloromethane solution overnight, extracting for 4h for 2 times, soaking the residue in 95% ethanol overnight, extracting for 6h for 2 times, recovering ethanol, and making into powder with freeze dryer.

4.2 animal grouping, modeling, dosing and sample Collection

After 80 SPS-grade KM mice are adaptively fed for 1 week, the mice are randomly divided into 2 groups, 10 mice are given common feeds to be common diet groups (ND), the rest mice are given high-fat feeds to establish an obesity model, water is freely drunk and food is taken, the animals are in 12h day-night circulation in the room, the room temperature is (22 +/-2) DEG C, and the relative humidity is 40-50%. After 8 weeks, the ordinary diet group was used as an experimental control group, anti-obesity mice were removed, and an average body weight of more than 20% was used as an obesity standard to determine an obesity model. After successful modeling, the obesity model is randomly divided into a high fat diet group (HFD), a high, medium and low dose group (1.8g/kg, 0.9g/kg and 0.18g/kg) and a positive control group (orlistat, 0.048g/kg) on the basis of high fat feed feeding, wherein each group contains 10 n. ND and HFD were given equal volumes of saline 1 time a day for 9 weeks with continuous gavage. Mice body mass, food intake were observed and recorded weekly during this period. After the last administration of the experiment, fasting was performed for 12 hours without water deprivation, the body length, obesity index (Lee's index) were measured after anesthesia by intraperitoneal injection of 10% chloral hydrate 3ml/kg after measurement of body weight, serum was collected, perirenal, peritesticular, periintestinal fat and liver tissue were weighed, stool samples were collected in the colon part immediately after freezing in liquid nitrogen, and stored at-80 ℃.

4.3 intestinal microbiota analysis

Immediately after the mice were sacrificed, fresh stool specimens were taken from the colon, snap frozen in liquid nitrogen and stored at-80 ℃ for total DNA quality testing using Thermo NanoDrop 2000 uv microspectrophotometer and 1% agarose gel electrophoresis. The 16S rDNA amplification selection region was V3-V4 region, and the universal primers used were 341F and 806R (F: CCTACGGGRSGCAGCAG; R: GGACTACVVGGGTATCTAATC). And (3) carrying out PCR by using the diluted genome DNA as a template and using a KAPA HiFi Hotstart ReadyMix PCR kit high-fidelity enzyme to ensure the amplification accuracy and high efficiency. The PCR product was detected by 2% agarose gel electrophoresis, and recovered by cutting with AxyPrep DNA gel recovery kit (AXYGEN).

After recovery, library quality testing was performed using a Thermo NanoDrop 2000 ultraviolet microspectrophotometer and 2% agarose gel electrophoresis. And after the quality of the library is qualified, quantifying the library by using the Qubit, and mixing according to the corresponding proportion according to the data volume requirement of each sample. In-silico sequencing was performed using Illumina Miseq PE 250. 16S specific primers are designed to amplify specific regions, and amplified fragments of about 425bp are obtained. Adding a joint, sequencing by adopting an Illumina platform to obtain Paired-End data of PE250, and splicing to obtain a longer sequence so as to perform 16S analysis.

Performing QC on original data, performing chimera removal and clustering analysis on the data by using Usearch software, sequencing Reads from large to small according to abundance during Usearch clustering, and clustering by 97% similarity to obtain OTUs (operational Taxonomic units), wherein each OTU is considered to represent one species. Counting the number of Reads matched with the OTU of each sample, and in order to avoid analysis deviation caused by different sizes of sample sequencing data, carrying out random flattening processing according to the minimum sequence number matched with the OTU under the condition of enough sequencing depth, and carrying out Alpha Diversity analysis. And extracting a Read from each OTU as a representative sequence, comparing the representative sequence with an RDP (ribosomal database project) database, and carrying out species classification on each OTU to obtain a species abundance table.

Alpha Diversity analyzes the complexity of sample species Diversity by means of the underserved specs index, the Chao1 index, the Shannon index, the Simpson index, and the PD _ whole _ tree5 indices. The Beta Diversity analysis was used to compare the differences in species Diversity between the samples. Both Alpha and Beta diversities were analyzed using QIIME software. To further demonstrate the differences in species diversity between samples and the degree of difference in microbial evolution in different environmental samples, primary coordinate Analysis (PCoA) and arithmetic mean Unweighted Pairing Group Method (UPGMA) were used for clustering. LEfSe uses Linear Discriminant Analysis (LDA) to estimate the magnitude of the effect of abundance of each component on the difference, and to find out the colonies or species that produce significant differential effects on sample partitioning. Significant difference analysis was performed between different groups using the method of Kruskal-Wallis rank sum test and Wilcoxon rank sum test to find species that have significant differential effects on the inter-group partition.

The experimental data were first pre-processed using Excel software and statistical plots were all plotted using Graphpad Prism 8.0.2. Measure data toThe values between groups are compared by using one-way anova, and the comparison between two groups is performed by using t test. The comparison among groups adopts ANOVA variance analysis, and the difference with P less than 0.05 has statistical significance.

Fifth, experimental results

5.1 Effect of extract of Rizhua on the intestinal flora Structure of obese mice

The effect of HFD and LR0.18g/kg on mouse intestinal flora was investigated by sequencing 16S rRNA (V3+ V4 region) using the Illumina MiSeq platform. According to the 97% identity level, the mean OTU values of the ND, HFD and HFD + LR0.18g/kg groups (n ═ 6 per group) were 516, 548 and 506, respectively, the OTU of the HFD group was higher than that of the ND group, while the intercostal administration significantly reduced the number of OTUs. FIG. 1 is a graph showing the effect of costaphania on the diversity of intestinal microbiota Alpha in HFD obese mice, wherein A-B are Shannon and bserved _ species plots and C-D are Shannon and bserved _ species diversity indices, each line represents a sample, and the lines gradually flatten as the sample size increases, as seen from the Shannon plot, and the depth of sequencing has satisfied all the species in the test sample. As shown in FIG. 1, C, D, there was a significant difference between HFD group and ND group in bserved _ species and Shannon index, the diversity of HFD group flora was significantly higher than ND group, and this changed after costal Style treatment (A. crispus)^*P＜0.05)。

FIG. 2 is a graph showing the effect of intercostal flowers on the diversity of intestinal microbiota Beta in HFD diet obese mice, wherein A is a PCoA graph of intestinal microbes based on OTU abundance, and B is a hierarchical cluster analysis graph based on non-weighted group mean method of OTU; PCoA and upma hierarchical clustering analyses were used to show the degree of difference in gut microbiota composition among different environmental samples, and the results are shown in panel a of fig. 2, showing that the HFD group clustered separately from the ND group, and that the ND group and the HFD + lr0.18g/kg group had a more similar gut microbiota composition (P < 0.05) than the HFD group, indicating that the extract of ribbonflower had a substantial effect on the gut microbiota composition of HFD-fed mice. In addition, PC1 accounted for 23.74% of the total variance, which was higher than PC2 for 13.44% of the total variance, indicating that the extract of ribwort could restore the HFD-affected gut microbiome composition to a normal state towards the ND group. The results are shown in graph B in FIG. 2, and as expected, significant separation was also observed in the results of the sample evolutionary trees for the ND, HFD and HFD + LR0.18g/kg groups, consistent with the results for PCoA.

FIG. 3 is a graph showing the effect of intercostal flowers on the composition of intestinal microbiota of HFD diet obese mice, wherein the A-B graph is a composition analysis graph of samples at the level of intestinal microbiota, and the C graph is a composition analysis graph of samples at the level of intestinal microbiota; as can be seen from the A, B chart in FIG. 3, the phylogenetic histogram shows that the mouse gut microflora structure is mainly composed of mycoderm-level Firmicutes and Bacteroides. Obesity on high-fat diet significantly altered the intestinal microflora structure, and thus, in addition to individual sample individual differences, Firmicutes and Proteobacteria (Proteobacteria) were relatively increased and Bacteroidetes (Bacteroidetes) were relatively decreased in the HFD group compared to the ND group. LR1.8g/kg administration decreased Firmicutes and Proteobacteria, and increased Bacteroides and Firmicutes/Bacteroides ratios. Therefore, intercostal flower can significantly attenuate the bacteria increased by high fat diet to a level similar to that of ND group. As shown in the genus-level difference species analysis chart of C in FIG. 3, the groups of HFD and ND have different genera including Clostridium XlVa, Roseburia, Desufovibrio, Acetatofaci, Pseudofularovorifer, Flavobacterium (Flavonivor), and the like 6. After the stigmata is administrated, the genus level of Clostridium XlVa, Roseburia, Acetation factor and Pseudolavavoriferor is remarkably reduced, and the genus level of Desulfovibrio is increased. These results indicate that costal Style may ameliorate the disorders of intestinal flora caused by HFD.

The amount of food consumed by humans or animals varies in many ways, e.g. mood, exercise, food smell or gastrointestinal hormones. Since many toxic substances have a bitter taste, when an animal is exposed to a bitter substance, a strong aversion to taste is generated. Bitter taste is an important defense mechanism, and taste is generated in the brain through interaction of bitter substances with bitter taste receptors (T2Rs) and transmission by neurotransmitters and the like. Bitter substances are also closely related to gastrointestinal hormones. It has been found that bitter substances such as quinine can be removed byCombined with the bitter receptor T2Rs, stimulates L cells to secrete GLP-1 hormone, and GLP-1 has hypoglycemic, lipid metabolism and obesity regulating, food intake regulating and gastric emptying also affecting effects. It was found that T2Rs is also selectively expressed in endocrine cells in the intestinal tract, and when glucagon-like polypeptide 1(STC-1) cells are stimulated to secrete using bitter agonists such as Benzdenatonium (BD), intracellular Ca is rapidly increased²⁺And (4) concentration. Ca²⁺An increase in concentration promotes the release of cholecystokinin (CCK), which in turn regulates gastrointestinal motility and gastric emptying.

The main reason why the host and the microbial flora are in microecological balance when the organism is in a normal state is that the microbial flora in the intestinal tract keeps an interdependent and interdependent relationship. When a human body is in a healthy state, bacteroides and Firmicute in the intestinal tract are dominant and account for more than 90% of the flora. Studies have shown that the relative proportion of bacteroidides is decreased in the obese group and that the relative proportion of Firmicute is increased in the obese group compared to the lean group. And as body weight loss bacteroidides increased, while Firmicute decreased. It has also been shown that long-term feeding of a high fat diet increases the body F/B ratio, and the present invention demonstrates that the extract of Selaginella costata decreases the F/B ratio increase caused by a high fat diet.

The obesity occurs because the outer wall of the cell wall of some gram-negative bacteria such as Proteobacteria and other pathogenic bacteria can cause the generation of a large amount of lipopolysaccharide in the intestinal tract, and the lipopolysaccharide can damage the intestinal barrier, increase the intestinal permeability and increase the fat accumulation. Secondly, the structural characteristics of gram-negative bacteria cause flagella of the gram-negative bacteria to absorb lipid components more easily, so that the accumulation of fat is increased continuously, and obesity is caused and developed. Also, the present invention successfully demonstrates that the extract of ribcage roseum can reduce the relative abundance of intestinal Proteobacteria and reduce obesity.

Bacteroidides and Firmicutes are closely related to weight loss in the body and studies have shown that the gut microbiota plays a role in adipogenesis by promoting the digestion and absorption of nutrients in obese organisms. Therefore, modulation of the relative abundance of Proteobacteria, bacterioides and Firmicutes may be one of the reasons why costal flower has a weight-reducing effect.

In a word, the intercostal flower extract can inhibit weight gain and fat accumulation caused by high-fat diet, regulate serum blood lipid index and has the effect of losing weight. In addition, the extract of the lomatogonium carinthiacum can regulate the disturbance of the mouse intestinal flora caused by high-fat diet, such as reducing relative abundance of Firmicutes, Proteobacteria and F/B ratio, and increasing relative abundance of Bacteroides. These results indicate that the use of the extract of ribwort for regulating the disturbance of the intestinal flora allows effective weight loss.

The 16S rRNA analysis shows that the composition of mouse intestinal microbiota is changed by the administration of the extract of the ribbing flower, so that the relative abundance of high-fat model mouse intestinal microbiota Firmicute and Proteobacteria and the ratio of Firmicute/bacterioides are reduced, and the relative abundance of bacterioides is increased. Conclusion the extract of the lomatogonium carinthiacum achieves the effect of losing weight by regulating the disturbance of intestinal flora caused by high-fat diet.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.