阅读说明：本技术 一种筛选对肠道微生态具有促进作用的提取物方法及将其用于靶向筛选益生菌的应用 (Method for screening extract with effect of promoting intestinal micro-ecology and application of extract in targeted screening of probiotics ) 是由 李亚峰 董丽娜 张洵 孙龙 于 2021-07-07 设计创作，主要内容包括：本发明涉及一种调控肠道微生态的益生元组合物及其制备方法和应用。组合物中包含白头翁提取物。本发明首次证实了白头翁提取物可降低肠道微生物群的多样性,并且具有促进人体双歧杆菌生长的作用。并且,本发明首次建立了体外发酵体系,能更好的模拟人体内真实环境,从整体丰度,到定量再到菌种水平检测和人粪便来源菌株验证,精准筛选出中药靶向促进生长的益生菌种类,解决了当前的盲目配伍和非人体来源的问题。(The invention relates to a prebiotic composition for regulating and controlling intestinal microecology and a preparation method and application thereof. The composition comprises Pulsatillae radix extract. The invention proves that the pulsatilla chinensis extract can reduce the diversity of intestinal microbiota and has the effect of promoting the growth of human bifidobacteria for the first time. In addition, the invention establishes an in-vitro fermentation system for the first time, can better simulate the real environment in a human body, accurately screen out the probiotic species for promoting the growth of the traditional Chinese medicine in a targeted way from the whole abundance, to the quantification, to the strain level detection and the verification of the human excrement source strain, and solves the problems of blind compatibility and non-human body source at present.)

1. Application of radix Pulsatillae extract in regulating intestinal microecology or in preparing preparation for regulating intestinal microecology is provided.

2. Use according to claim 1, characterized in that the regulation of the intestinal microbiota is in particular a reduction of the diversity of the intestinal microbiota, or a promotion of the growth of bifidobacteria; preferably, the bifidobacterium is bifidobacterium longum, bifidobacterium adolescentis or bifidobacterium pseudocatenulatum; more preferably, the bifidobacterium is bifidobacterium longum.

3. An in vitro fermentation method for screening targeted probiotics, which is characterized by comprising the following steps: collecting fresh excrement; diluting feces, and adding the diluted feces into a culture medium placed in an anaerobic culture system in advance; adding the Chinese medicinal sample to be tested into the culture medium, fermenting for a certain time (preferably 0h, 8h and 24h), respectively sucking liquid, centrifuging, precipitating (at-80 deg.C), and screening out the probiotic species with growth promoting effect by analyzing flora change in the precipitate.

4. The method of claim 3, wherein the analysis of the change in flora in the precipitate is performed by: extracting DNA, PCR amplifying 16S rRNA gene area with the extracted DNA as template, separating and purifying the amplified segment and sequencing, statistical analysis of the sequencing result and screening out the probiotics variety with growth promoting effect.

5. The method of claim 4, wherein the statistical analysis is performed using Dix-seq pipeline for raw 16S sequencing analysis, using principal coordinate analysis (PCoA) and non-metric multi-dimensional scaling (NMDS) based on weighted _ uniform, unweighted _ uniform, and break _ curves distances to evaluate differences between samples, raw count tables for differential abundance analysis at different classification levels (gate, order, class, family, genus, ZOTU).

6. The method of claims 3-5, wherein the Chinese medicinal sample is Pulsatillae radix extract.

7. The use according to any one of claims 1-2 or the method according to claim 6, wherein the extract of pulsatilla chinensis is an aqueous or alcoholic extract of pulsatilla chinensis; preferably, the alcoholic extract is selected from methanol extract, ethanol extract; the ethanol extract is further preferably 70% -95% ethanol extract.

8. The use or method according to claim 7, wherein the aqueous extract of pulsatillae radix is prepared by the following steps: weighing a certain weight of dried Chinese pulsatilla root medicinal material, heating and refluxing for 2 times under the condition that the material-liquid ratio is 1:6, extracting for 1 hour each time, filtering with gauze, collecting and combining two filtrates, concentrating the extracting solution under reduced pressure to 1/4 volumes, and freeze-drying.

9. Use or method according to claim 7, wherein the Pulsatillae alcohol extract is prepared by the following steps: weighing a certain weight of dried Chinese pulsatilla root medicinal material, heating and refluxing for 2 times under the condition that the material-liquid ratio is 1:4, extracting for 1 hour each time, filtering with gauze, collecting and combining two filtrates, concentrating the extracting solution under reduced pressure, evaporating alcohol to dryness, adding distilled water for dissolving, and freeze-drying.

10. The use or method according to claim 9, wherein the pulsatillae radix alcohol extract is preferably pulsatillae radix ethanol extract, wherein ethanol is 95% ethanol.

Technical Field

The invention relates to the field of biological extraction, in particular to a method for screening an extract with a promotion effect on intestinal microecology and application of the extract in targeted screening of probiotics.

Background

The intestinal microecology is closely related to the health and diseases of human beings, and the normal flora of the intestinal tract plays an important role in the aspects of preventing infection, regulating immunity, absorbing nutrient substances, delaying aging and the like of the human body. In recent years, more and more researches show that the traditional Chinese medicine can regulate intestinal microecology and can be used for treating diseases caused by intestinal microecology imbalance. On one hand, the traditional Chinese medicine can be used as a potential prebiotic to promote the growth of beneficial bacteria in the intestinal tract; on the other hand, intestinal microbiota may induce biotransformation of active ingredients of traditional Chinese medicines.

Pulsatilla Radix (PR) is a traditional Chinese medicine with the effects of clearing away heat and toxic materials, cooling blood and stopping dysentery, and is usually used together with other traditional Chinese medicines. Research shows that PR contains various components such as triterpene saponin, triterpenic acid, lignan, pulsatilla chinensis, anemonin, daucosterol and glycoprotein, and PR extract has various pharmacological activities such as antibiosis and antitumor. At present, the association between pulsatillae radix and intestinal microecological flora is rarely reported.

The invention is provided in view of the above.

Disclosure of Invention

The invention proves that the pulsatilla chinensis extract can change the diversity of intestinal microbiota by establishing an in-vitro fermentation system, and the pulsatilla chinensis extract can remarkably promote the growth of bifidobacteria. The invention proves the association between the pulsatilla chinensis and the intestinal microecological flora for the first time, and the established in-vitro fermentation system is beneficial to the targeted screening of the types of probiotics.

In view of this, the present invention claims, in one aspect, an in vitro fermentation method for screening targeted probiotics, comprising the steps of: collecting fresh excrement; diluting feces, and adding the diluted feces into a culture medium placed in an anaerobic culture system in advance; adding the Chinese medicinal sample to be tested into the culture medium, fermenting for a certain time, preferably 0h, 8h and 24h, respectively sucking liquid, centrifuging, precipitating at-80 deg.C, and screening out the probiotic species for promoting growth by analyzing the flora change in the precipitate.

Preferably, the analysis of the change in flora in the precipitate is performed by: extracting DNA, PCR amplifying 16S rRNA gene area with the extracted DNA as template, separating and purifying the amplified segment and sequencing, statistical analysis of the sequencing result and screening out the probiotics variety with growth promoting effect.

Preferably, the traditional Chinese medicine sample to be detected is a pulsatilla chinensis extract; more preferably, the extract is an aqueous or alcoholic extract.

The second aspect of the invention requests to protect the application of the pulsatilla chinensis extract in regulating and controlling the intestinal microecology; or in preparing intestinal microecological preparation.

Preferably, said modulating the intestinal microbiota is in particular reducing the diversity of the intestinal microbiota, or promoting the growth of bifidobacteria; further preferably, the bifidobacterium is bifidobacterium longum, bifidobacterium adolescentis or bifidobacterium pseudocatenulatum. Preferably, the pulsatilla chinensis extract is a pulsatilla chinensis water extract or a pulsatilla chinensis alcohol extract; further preferably, the alcoholic extract is selected from methanol extract, ethanol extract; the ethanol extract is further preferably 70% -95% ethanol extract.

The water extract of the Chinese pulsatilla root is prepared by the following steps: weighing a certain weight of dried Chinese pulsatilla root medicinal material, heating and refluxing for extraction for 2 times under the condition that the material-liquid ratio is 1:4-10, each time for 1-2h, filtering with gauze, collecting and combining two filtrates, concentrating the extracting solution under reduced pressure to 1/4 volumes, and freeze-drying. Preferably, the feed-liquid ratio is 1:4, and the heating reflux time is 1 h.

The pulverata chinensis alcohol extract is prepared by the following steps: weighing a certain weight of dried Chinese pulsatilla root medicinal material, heating and refluxing for extraction for 2 times under the condition that the material-liquid ratio is 1:4-10, each time for 1-2h, filtering with gauze, collecting and combining two filtrates, concentrating the extract under reduced pressure, evaporating to dryness, adding distilled water for dissolution, and freeze-drying. Preferably, the feed-liquid ratio is 1:4, and the heating reflux time is 1 h. A

The invention provides a prebiotic composition capable of regulating intestinal microecological flora, which at least comprises pulsatilla chinensis extract.

Preferably, the composition only contains the pulsatilla chinensis extract, or the composition can further comprise a pharmaceutically acceptable carrier or auxiliary material.

The invention has the following outstanding effects:

at the present stage, many products are based on animal research, because the difference between animal and human flora is very large, and because the human body evaluation in vivo cannot avoid the interference of endogenous factors due to the complexity of the human body. Therefore, the invention establishes an in-vitro fermentation system for the first time, and can better simulate the real environment in a human body and accurately screen out the probiotic species for promoting the growth of the traditional Chinese medicine in a targeted way from the whole abundance, the quantification to the strain level detection and the verification of the human excrement derived strain. Solves the problems of blind compatibility and non-human body source at present.

In addition, the in-vitro fermentation system constructed by the method proves that the pulsatilla chinensis extract can reduce the diversity of intestinal microbiota and has the effect of promoting the growth of human bifidobacteria for the first time. The findings of the research can provide a certain research basis for treating related diseases by bifidobacteria in the future.

Drawings

Figure 1 effect of PRE on gut microbiota diversity. Fig. 1A and 1B show Shannon index differences of α -diversity between 8-hour and 24-hour control groups and PRE, fig. 1C and 1D show Simpson index differences of α -diversity between 8-hour and 24-hour control groups and PRE, fig. 1E and 1F show Anonim index differences of β -diversity between 8-hour and 24-hour control groups and PRE, and fig. 1G shows PCoA analysis results.

FIG. 2 Effect of PRW and PRE on relative abundance of phylum, genus level microflora;

FIG. 3 is a box diagram of Pulsatillae radix extract for promoting Bifidobacterium;

FIG. 4 primer system for qPCR detection;

FIG. 5 shows the growth promoting effect of Pulsatillae radix extract on Bifidobacterium;

FIG. 6 shows the growth promoting effect of Pulsatillae alcohol extract on Bifidobacterium longum.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The following terms or definitions are provided only to aid in understanding the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.

Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The terms "about" and "substantially" in the present invention denote an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The following are specific embodiments.

Example 1 preparation of Pulsatilla extract

The pulsatilla chinensis is purchased from Tongrentang, and the extract is extracted by water and alcohol.

The preparation method of the pulsatilla chinensis water extract (PRW) comprises the following specific steps: weighing 500g of dried Chinese pulsatilla root medicinal material, heating and refluxing for extraction for 2 times at a material-liquid ratio of 1:6, each time for 1h, filtering with gauze, collecting and combining filtrates of the two times. The extract was concentrated under reduced pressure to 1/4 volumes and then freeze-dried.

The preparation method of the Pulsatillae alcohol extract (PRE) comprises the following steps: PRE was extracted with 95% ethanol. Weighing 500g of dried Chinese pulsatilla root medicinal material, heating and refluxing for extraction for 2 times at a material-liquid ratio of 1:4, each time for 1h, filtering with gauze, collecting and combining filtrates of the two times. Concentrating the extractive solution under reduced pressure, evaporating ethanol, dissolving with distilled water, and freeze drying.

Example 2 influence of Pulsatilla extract on intestinal micro-ecology

1. Inoculating and fermenting excrement

Collecting fresh excrement: three healthy adults, without any metabolic and gastrointestinal disease, did not take antibiotics, probiotics or prebiotics three months prior to the donation of the stool sample. All subjects gave written informed consent during the study. The study was approved by the ethical committee of the Shanxi province Hospital study.

One 180ml portion of the medium (composition of the medium is shown in the following table) was taken and placed in the anaerobic culture system overnight one day in advance. Within 15 minutes after fecal excretion, dilutions were made at a ratio of 0.1g fresh feces/1 ml anaerobic PBS (i.e., 10%) and homogenized for 2 minutes. 20ml of the diluted sample was added to the medium previously placed in the anaerobic culture system. PRW or PRE was added to the medium at a ratio of 1g/100 ml. Sucking 3ml of liquid for 0h, 8h and 24h respectively, centrifuging at 4000rpm for 10min, and immediately placing the precipitate at-80 ℃ for analysis.

2. DNA extraction, PCR amplification, 16S rDNA sequencing

The genomic DNA in the sample to be analyzed was extracted using the DNeasy Power Soil Pro Kit (Qiagen, Hiden, Germany). The V4 region of the 16S rRNA gene was PCR-amplified using the genomic DNA as a template. The amplified PCR fragment was purified and quantified. A16S rDNA amplicon library was constructed using the KAPA Hyper Prep Kit (Roche) Kit. The library was double-ended sequenced using the Illumina NovaSeq 600 system.

3. Biological information and statistical analysis

Dix-seq pipeline (version 0.0.1) was used for the original 16S sequencing analysis. Differences between samples were evaluated using principal coordinate analysis (PCoA) and non-metric multi-dimensional scaling (NMDS) based on weighted _ uniform, unweighted _ uniform, and break _ curves distances. The raw count table was used for differential abundance analysis for different classification levels (phylum, order, class, family, genus, ZOTU).

4. Analysis of results

PRW and PRE reduce the diversity of the intestinal microbiota

The shannon index (Simpson index), which reflects the abundance of the species, indicates that there are significant differences between the groups (F ═ 6.708, p ═ 0.004). The analysis results showed that after 24 hours of fermentation, the bacterial species of PRW and PRE were significantly lower than the control group (P0.037 <0.05, P0.000 <0.01), and at 8 hours of fermentation, the bacterial species of the PRW group were also significantly lower than the control group. The extent of species abundance reduction of PRE depends on fermentation time, with 8 hours significantly below 0 hours and 24 hours significantly below 8 hours. The Simpson index also indicates that there are significant differences between the three groups at 24 hours of fermentation. Further analysis showed significant differences in beta diversity between groups. The principal component analysis (PCoA) plot shows that the samples were isolated on a PRE basis, the PRW supplemented the data from the control group, and the two supplemented groups clustered together. FIG. 1 shows the difference between the control group and the PRE group at 8 hours and 24 hours of fermentation.

Effect of PRW and PRE on the relative abundance of phylum and genus level microflora

Differences in relative abundance between groups can be seen at the level of the gates (fig. 2A). PRW and PRE, especially PRE-induced actinomycetemcomita (actinobacillus phylum) increased at 8h and 24h, while Firmicutes and Fusobacteria (fusobacterium) decreased significantly and were time-dependent. Differences in relative abundance between groups for some genera can be seen at the genus level (fig. 2B). Bifidobacterium (bifidobacterialenus) of the PRE group was significantly increased at 8 hours compared to the control group and the PRW group. Both Eggerta (Eggerthella) and Clostridium (Clostridia _ XIVb) were reduced (FIG. 3).

Actinomycetes are gram-positive bacteria with extensive secondary metabolic activity, about two-thirds of the currently clinically used naturally derived antibiotics are produced by actinomycetes, which can also be used for the production of anticancer, anthelmintic and antifungal compounds. Bifidobacteria, belonging to the phylum actinomycetes, are widely used as probiotics, bifidobacteria and their metabolites, in particular short chain fatty acids, are essential for providing energy and effective antibacterial activity for epithelial cell transformation, and show beneficial effects in many pathological conditions. Clostridia are present in many mixed infections and many potential pathogenic bacteria belong to the species of eglata and clostridia. Therefore, it is presumed that the pulsatillae radix extract may exert a disease therapeutic effect by affecting the above-mentioned intestinal flora.

Example 3 Effect of Pulsatillae radix extract on Bifidobacterium

Effect of PRW and PRE on Bifidobacterium abundance and growth

Fresh feces of healthy people are collected, and the influence of the pulsatilla chinensis bunge extract PRW or PRE on flora change is researched according to the feces inoculation fermentation method. The dynamic changes of the flora at 0h, 8h and 24h of fermentation were compared by 16s high throughput sequencing method.

The feces of tested healthy people are subjected to in vitro culture omics detection in vitro, and the separated bifidobacteria mainly comprise bifidobacterium adolescentis, bifidobacterium pseudocatenulatum and bifidobacterium longum. Further detection using qPCR, see fig. 4, it can be seen that pulsatillae radix alcohol extract can produce significant effect on bifidobacterium abundance. As can be seen from fig. 5 and 6, the pulsatillae radix alcohol extract has the most significant growth promoting effect on bifidobacterium longum.

Further, a human feces-derived ATCC15707 Bifidobacterium longum standard strain was purchased, cultured in BBL liquid medium with PRE in place of glucose, and verified by adding 5mg/ml PRE. The results show that Bifidobacterium longum can indeed grow using PRE as a carbon source.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.