阅读说明：本技术 一种两色金鸡菊的新用途及分析方法 (New application and analysis method of coreopsis bicolor ) 是由 陈晓鹏 刘静 李春霞 何巧玉 于 2021-09-27 设计创作，主要内容包括：本发明提供了两色金鸡菊在制备肠道菌群调节药物方面的新用途,分析方法通过建立动物糖尿病模型,引入高通量测序技术,为后期研究两色金鸡菊治疗糖尿病的作用机制拓展新的方向和思路。(The invention provides a new application of coreopsis bicolor in preparing intestinal flora regulating drugs, and an analysis method develops a new direction and thought for later-stage research on an action mechanism of coreopsis bicolor for treating diabetes by establishing an animal diabetes model and introducing a high-throughput sequencing technology.)

1. New use of coreopsis tinctoria in preparing medicine for regulating intestinal flora is provided.

2. The new use of coreopsis tinctoria according to claim 1, characterized in that: the enteric bacteria are Lactobacillus, Vibrio succinogenes, Treponema and/or undefined ruminobacterium.

3. The new use of coreopsis tinctoria according to claim 1, characterized in that: the coreopsis bicolor is prepared by the following method: precisely weighing 50-100 g of bicolor golden pheasant sample in a 2L beaker, pouring 1-2L of boiling water, covering with tin foil paper, brewing for 25-35 min, filtering out tea soup, continuing to add 1-2L of boiling water for brewing for 25-35 min, simulating a tea brewing process, filtering out the tea soup, combining the two tea soups, shaking up, recovering pure water from the filtrate under reduced pressure, and concentrating to obtain a thick paste, thus obtaining the bicolor golden pheasant extract.

4. The new use of coreopsis tinctoria according to claim 3, characterized in that: the decompression condition is that the temperature is 50-55 ℃, and the vacuum degree is 0.001 Mpa-0.01 Mpa.

5. An analytical method for determining the influence of coreopsis bicolor on intestinal flora is characterized by comprising the following steps: the method comprises the following steps:

(1) administering medicine in an animal model by a gastric lavage way, collecting animal excrement samples, extracting DNA, carrying out PCR amplification by using a 16S rDNA variable region primer, mixing and purifying PCR products, then constructing a library, and carrying out on-machine detection;

(2) and processing sequencing data, performing OUT clustering and species annotation, and comparing and analyzing the alpha and beta diversity of the intestinal flora.

6. The assay for determining the effect of coreopsis tinctoria on the gut flora of claim 5, wherein: the step (1) adopts a CTAB or SDS method to extract the genomic DNA of the sample.

7. The assay for determining the effect of coreopsis tinctoria on the gut flora of claim 5, wherein: in the step (1), PCR is carried out by using a specific primer with Barcode.

8. The assay for determining the effect of coreopsis tinctoria on the gut flora of claim 5, wherein: and (2) carrying out electrophoresis detection on the PCR product in the step (1) by using 2% agarose gel, carrying out magnetic bead purification on the qualified PCR product, carrying out enzyme labeling quantification, carrying out equivalent sample mixing according to the concentration of the PCR product, and detecting the PCR product by using 2% agarose gel electrophoresis after fully and uniformly mixing.

9. The assay for determining the effect of coreopsis tinctoria on the gut flora of claim 5, wherein: and (2) processing the off-line data obtained in the step (1), splitting sample data according to a Barcode sequence and a PCR amplification primer sequence, cutting off the Barcode and the primer sequence, splicing reads of each sample by using FLASH to obtain original data, and then strictly filtering to obtain high-quality data.

10. The assay for determining the effect of coreopsis tinctoria on the gut flora of claim 5, wherein: and (3) clustering the high-quality data of all samples by OUT clustering in the step (2) by using a Upearse algorithm, and clustering the sequences into OTUs by using 97% of consistency as default.

Technical Field

The invention relates to the technical field of biology, in particular to a new application and an analysis method of coreopsis bicolor.

Background

Coreopsis tinctoria Nutt, also known as Kunlun snow chrysanthemum, is a dry capitate inflorescence of a Coreopsis plant of Compositae, is recorded in Xinhua Bencao compendium, has sweet taste, mild nature and large intestine meridian tropism, has the effects of clearing heat and removing toxicity, promoting blood circulation and removing blood stasis and the like, and can treat symptoms such as dryness-heat polydipsia, hypertension, hyperglycemia, palpitation, gastrointestinal discomfort and the like. The medicinal value of the bicolor golden pheasant as a Uyghur national medicine which is widely applied in Xinjiang area is gradually discovered.

Disclosure of Invention

The invention aims to solve the technical problem of providing a new application of coreopsis bicolor.

Another object of the present invention is to provide an analytical method for determining the effect of coreopsis bicolor on the intestinal flora.

The technical scheme adopted by the invention is as follows:

new use of coreopsis tinctoria in preparing medicine for regulating intestinal flora is provided.

Preferably, the new use of the coreopsis tinctoria is the new use of the coreopsis tinctoria, the enterobacteria are Lactobacillus (Lactobacillus), vibrio Succinivibrio (Succinivibrio), Treponema (Treponema) and/or undefined rumen (unidentified-Ruminococcaceae).

Preferably, the new application of the coreopsis tinctoria is prepared by the following method: precisely weighing 50-100 g of bicolor golden pheasant sample in a 2L beaker, pouring 1-2L of boiling water, covering with tin foil paper, brewing for 25-35 min, filtering out tea soup, continuing to add 1-2L of boiling water for brewing for 25-35 min, simulating a tea brewing process, filtering out the tea soup, combining the two tea soups, shaking up, recovering pure water from the filtrate under reduced pressure, and concentrating to obtain a thick paste, thus obtaining the bicolor golden pheasant extract.

Preferably, the new application of the coreopsis tinctoria is prepared by the following method: precisely weighing 60-100 g of bicolor golden pheasant sample in a 2L beaker, pouring 1.5-2L of boiling water, covering tin foil paper, brewing for 30-35 min, filtering out tea soup, continuing to add 1.5-2L of boiling water for brewing for 30-35 min, simulating the tea brewing process, filtering out tea soup, combining the two tea soups, shaking up, recovering pure water from the filtrate under reduced pressure, and concentrating to obtain the bicolor golden pheasant extract.

Preferably, the new application of the coreopsis tinctoria is realized, and the decompression condition is that the temperature is 50-55 ℃ and the vacuum degree is 0.001 Mpa-0.01 Mpa.

An analysis method for determining the influence of coreopsis bicolor on intestinal flora comprises the following steps:

(1) administering medicine in an animal model by a gastric lavage way, collecting animal excrement samples, extracting DNA, carrying out PCR amplification by using a 16S rDNA variable region primer, mixing and purifying PCR products, then constructing a library, and carrying out on-machine detection;

(2) and processing sequencing data, performing OUT clustering and species annotation, and comparing and analyzing the alpha and beta diversity of the intestinal flora.

Preferably, in the analysis method for determining the influence of coreopsis tinctoria on intestinal flora, the step (1) is to extract genomic DNA of the sample by using CTAB or SDS method.

Preferably, in the analysis method for determining the influence of coreopsis tinctoria on intestinal flora, PCR is performed in step (1) by using specific primers with Barcode.

Preferably, in the analysis method for determining the influence of coreopsis tinctoria on intestinal flora, in the step (1), agarose gel with the concentration of 2% is used for electrophoresis detection of a PCR product, magnetic bead purification is performed on the PCR product which is qualified in detection, enzyme labeling quantification is adopted, equivalent sample mixing is performed according to the concentration of the PCR product, and the agarose gel with the concentration of 2% is used for electrophoresis detection of the PCR product after the PCR product is fully and uniformly mixed.

Preferably, the above analysis method for determining the effect of coreopsis tinctoria on intestinal flora, the library constructed in step (1) is usedThe DNA PCR-Free Sample Preparation Kit library construction Kit, the constructed library is subjected to the Qubition and Q-PCR quantification, library qualification, use NovaSeq6000 machine sequencing.

Preferably, in the analysis method for determining the influence of coreopsis bicolor on intestinal flora, in the step (2), off-machine data obtained in the step (1) is processed, sample data is split according to a Barcode sequence and a PCR amplification primer sequence, the Barcode and the primer sequence are cut off, reads of each sample are spliced by using FLASH, original data are obtained, and then high-quality data are obtained through strict filtration processing.

Preferably, in the analysis method for determining the influence of coreopsis tinctoria on intestinal flora, in the step (2), the OUT clustering is performed on high-quality data of all samples by using a Upearse algorithm, and the sequences are clustered into OTUs with 97% of consistency as a default.

Preferably, in the analysis method for determining the influence of coreopsis tinctoria on intestinal flora, species annotation is performed on OTUs sequences in the step (2), and species annotation analysis is performed by using a mortur method and a SSUrRNA database of SILVA138, so that taxonomic information is obtained to count the colony composition of each sample.

Preferably, in the analysis method for determining the influence of coreopsis tinctoria on intestinal flora, the step (2) is used for comparatively analyzing the alpha and beta diversity of the intestinal flora, wherein the relative abundance of Lactobacillus (Lactobacillus), vibrio Succinivibrio (succiivibrio), Treponema (Treponema) and undefined rumenia (unidentified-Ruminococcaceae) is remarkably changed.

The invention has the beneficial effects that:

the new application of the coreopsis bicolor provides a new idea for preparing the intestinal flora regulating medicine. The analysis method aims to explore the regulation effect of the coreopsis bicolor on host intestinal flora, evaluate the influence of the coreopsis bicolor on the composition structure and metabolic products of the intestinal flora and the relation between a diabetes pathogenesis and intestinal microorganisms by establishing an animal (rat) diabetes model and introducing a high-throughput sequencing technology, preliminarily explores the possible mechanism of the antidiabetic effect of the coreopsis bicolor, and develops a new direction and thought for later research on the action mechanism of the coreopsis bicolor for treating diabetes.

Drawings

FIG. 1 is a flow chart of an analysis technique for exploring the influence of coreopsis bicolor on intestinal flora of diabetic rats.

FIG. 2 is a plot of species abundance at the Genus taxonomic level for the samples of example 1. NC-Normal group; a set of Mod-models; the Met-metformin group; JH-two-color coreopsis high dose group; JL-two-color low dose group of golden pheasant.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description is made with reference to the accompanying drawings and the detailed description.

Example 1

The method for researching the influence of coreopsis bicolor on intestinal flora of diabetic rats is shown in figure 1:

(1) preparation of coreopsis tinctoria extract

Precisely weighing 80g of a bicolor golden pheasant sample in a 2L beaker, pouring 2L of boiling water, covering with tin foil paper, brewing for 30min, filtering out tea soup, continuously adding 2L of boiling water to brew for 30min, simulating the tea brewing process, filtering out the tea soup, combining the two tea soups, shaking up, recovering pure water from the filtrate under the reduced pressure of 0.005Mpa at the temperature of 50 ℃, and concentrating to obtain a thick paste, thus obtaining the bicolor golden pheasant extract.

(2) Establishment of diabetic rat model

SD male rats weighing 180-.

(3) Animal grouping, dose setting and administration method

Randomly dividing the diabetic rats successfully molded in the step (2) into the following 4 groups: model group, positive control group (250mg/kg), low-dose group (1000mg/kg) of coreopsis tinctoria, high-dose group (2000mg/kg) of coreopsis tinctoria, and normal control group for 5 groups. The coreopsis tinctoria group and the positive control group are subjected to intragastric perfusion by respectively adopting aqueous solution of coreopsis tinctoria extract and metformin aqueous solution, and rats of the control group and the model group are fed with physiological saline with the same volume in an intragastric perfusion mode for continuous intervention for 8 weeks.

(4) Blood glucose, blood lipid and cholesterol determination

Measuring blood sugar 1 time per week before and during administration, monitoring blood sugar value, blood lipid value and total cholesterol value of each group, fasting (without water supply) for 12h for each group of rats on the day of blood collection, measuring blood sugar of the rats by using a glucometer, and measuring total cholesterol and triglyceride by using a Mirey BS-240VET animal-dedicated full-automatic biochemical analyzer. The total cholesterol level was increased in the model group compared to the normal group, and showed a decrease in the total cholesterol level in the low-dose group of crossostephium bicolor compared to the model group, and its therapeutic effect showed significance (p < 0.05).

(5)16s high throughput sequencing

Collecting animal waste samples of each group in the step (3), extracting genomic DNA of the samples by a CTAB or SDS method, carrying out PCR by using a specific primer (515F: 5'-ACTCCTACGGGAGGCAGCA-3'/806R: 5 '-GGACTACHVGGGTWTCTAAT-3') with Barcode and a 16S rDNA variable region primer, carrying out electrophoresis detection by using 2% agarose gel, carrying out magnetic bead purification on qualified PCR products, carrying out enzyme labeling quantification, carrying out equal sample mixing according to the concentration of the PCR products, fully mixing uniformly, detecting the PCR products by using 2% agarose gel electrophoresis, and then usingThe DNA PCR-Free Sample Preparation Kit library construction Kit is characterized in that a constructed library is subjected to Qubit and Q-PCR quantification, and after the library is qualified, on-machine sequencing is carried out by using NovaSeq 6000.

(6) Microbial diversity analysis

Splitting the sequencing data obtained in the step (5) into sample data according to a Barcode sequence and a PCR amplification primer sequence, splicing reads of each sample by using FLASH after the Barcode sequence and the primer sequence are cut off to obtain original data, obtaining high-quality data through strict filtering treatment, clustering the high-quality data of all samples by using a Uprease algorithm, clustering the sequences into OTUs with 97% consistency by default to perform species annotation on the OTUs sequences, performing species annotation analysis on the OTUs sequences by using a Mothur method and an SSUrRNA database of SILVA138, and obtaining taxonomic information to count community composition of each sample. As shown in FIG. 2, the alpha and beta diversity of the intestinal flora was comparatively analyzed, in which the relative abundance of Lactobacillus (Lactobacillus), Vibrio succinicicus (Succinivibrio), Treponema (Treponema) and undefined rumen (unidentified-Ruminococcaceae) was found to vary significantly.

Of these, the pathogenic (type 2 diabetes) causes significant reduction in the relative abundance of the beneficial bacteria Lactobacillus (Lactobacillus) in the flora, while the harmful bacteria vibrio Succinivibrio (Succinivibrio), Treponema (Treponema) and undefined ruminococcus (unidentified-Ruminococcaceae) are significantly increased. And the florae can be adjusted back to be normal by giving the extract obtained by the calliopsis bicolor simulated tea drink.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.