CN107753547B

CN107753547B - Saponin compound for improving intestinal flora, preparation method and application thereof

Info

Publication number: CN107753547B
Application number: CN201611018050.8A
Authority: CN
Inventors: 刘洁
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2016-11-17
Filing date: 2016-11-17
Publication date: 2020-11-20
Anticipated expiration: 2036-11-17
Also published as: WO2018090983A9; CN107753547A; WO2018090983A1

Abstract

The invention provides a saponin compound for improving intestinal flora, a preparation method and application thereof. The saponins compound is obtained by extracting the overground part of gynostemma pentaphylla by a solvent, concentrating into an extract, dissolving the extract in water to prepare a dispersion liquid, extracting to obtain an n-butyl alcohol part extract, and finally separating by a chromatographic column and separating and purifying by preparative reverse phase high performance liquid chromatography. The gynostemma total saponin compound can be used for preparing a composition for improving intestinal flora and a composition for promoting characteristic microbial colonization.

Description

Saponin compound for improving intestinal flora, preparation method and application thereof

Technical Field

The invention belongs to the technical field of saponin compounds and preparation and application thereof, and particularly relates to a triterpenoid saponin compound extracted and separated from gynostemma pentaphylla and a preparation method and application thereof.

Background

The human intestinal tract is a complex, active, relatively balanced system that colonizes a large number of structurally complex microbial communities. Human gastrointestinal tract inhabits about 10⁴The normal flora is of the order of magnitude, and 30-40 dominant bacteria in the normal flora constitute 99% of the total intestinal bacteria in the human body. Intestinal microorganisms contain a wide variety of enzyme systems and can participate in a series of physiological processes such as host energy, substance and genetic information transfer. Many researches prove that a dense and inseparable mutual-benefiting symbiotic relationship exists between intestinal flora and a host, intestinal microorganisms can form a biological barrier in the intestinal tract, and play an important role in the aspects of digestion, absorption, immune reaction and metabolic activity of the host through space occupying effect, nutrition competition, various secreted metabolites, bacteriocin and the like. At the phylum level, Firmicutes and bacteroidetes are the two dominant phyla; on the genus level, bacteroides, clostridium tender, lactobacillus and bifidobacterium are four kinds of dominant bacteria closely related to human health and are also the current stage of peopleThe research on intestinal microorganisms is focused, and the occurrence and development of chronic diseases such as obesity, hyperlipidemia and the like have great relation with the structural disorder of intestinal flora.

Gynostemma pentaphylla (Gynostemma pentahyllum (Thunb.) Makino) is a perennial herbaceous vine plant of Gynostemma of Cucurbitaceae (Cucurbitaceae), and its main bioactive component is triterpene saponin compound.

In view of the high medicinal value of triterpenoid saponins, the field needs to research deeply.

Disclosure of Invention

The invention aims to provide a saponin compound and a preparation method and application thereof.

In a first aspect of the invention, the invention provides an application of a gynostemma total saponin compound,

(1) for preparing a composition for improving intestinal flora; and/or

(2) For the preparation of a composition for promoting the colonization of characteristic microorganisms.

In another preferred embodiment, the composition is a pharmaceutical composition or a food composition.

In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is an oral dosage form.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: tablets, capsules or granules.

In another preferred embodiment, said intestinal flora belongs to the phylum firmicutes or bacteroidetes.

In another preferred embodiment, said intestinal flora belongs to the phylum verrucomicrobia or proteobacteria.

In another preferred embodiment, the intestinal flora is selected from the group consisting of: lactic acid bacteria, bifidobacteria or clostridium tender.

In another preferred embodiment, the intestinal flora is Akkermansia muciniphila.

In another preferred embodiment, the food composition is a diet food.

In another preferred embodiment, the pharmaceutically acceptable salt of the total saponin compound is selected from the group consisting of: sodium salt, potassium salt, or a combination thereof.

In another preferred embodiment, the gynostemma pentaphylla is diploid or tetraploid, preferably tetraploid.

In another preferred embodiment, the total saponin compound (designated YJ) includes one or more compounds selected from the group consisting of:

in a second aspect of the invention, there is provided a method for non-therapeutically improving the intestinal flora in vitro comprising the steps of: administering a compound according to the first aspect of the invention or a pharmaceutically acceptable salt thereof at a site in need of treatment.

In another preferred embodiment, the compound or pharmaceutically acceptable salt thereof is administered in a concentration of 0.001-0.02. mu.g/ml, preferably 0.01-0.15. mu.g/ml, more preferably 0.05-0.1. mu.g/ml.

In a third aspect of the present invention, there is provided a probiotic composition, comprising:

(1) a first drug: saponins compound, and

(2) a second drug: one or more probiotics selected from the group consisting of: lactic acid bacteria, bifidobacteria, clostridium tender, Akkermansia muciniphila, or combinations thereof;

wherein the first and second medicaments are located in the same or different containers.

In another preferred embodiment, the mass ratio (by active ingredient) of the first drug to the second drug in the probiotic composition is 1:100-100:1, preferably 1:10-10:1, and more preferably 1:5-5: 1.

In another preferred embodiment, the probiotic composition is a pharmaceutical composition or a food composition.

In a fourth aspect of the present invention, there is provided a method for improving intestinal flora, comprising the steps of: administering to a subject in need thereof a compound according to the first aspect of the invention or a pharmaceutically acceptable salt thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows the prevention effect of different doses of total saponin compound YJ on obesity caused by high fat diet,

a represents a low fat feed; b represents high fat feed feeding; c represents high fat group + low dose YJ (100 mg/Kg); d represents high fat group + high dose YJ (300mg/Kg), 8 mice per group, indicating significant body weight in the low fat group compared to the high fat group, and # indicates very significant P <0.05 level in the total saponin compound-affected group compared to the high fat group.

FIG. 2 shows the effect of total saponin compounds YJ on the community diversity of enterobacteria at the phylum level,

a represents a low fat feed; b represents high fat feed feeding; c represents the high-fat group + the low-dose YJ (100mg/Kg), and D represents the high-fat group + the high-dose YJ (300 mg/Kg).

Figure 3 shows the effect of total saponin compounds YJ on community diversity at the genus level of enterobacteria, a representing low-fat feed feeding; b represents high fat feed feeding; c represents the high-fat group + the low-dose YJ (100mg/Kg), and D represents the high-fat group + the high-dose YJ (300 mg/Kg).

Figure 4 shows PCOA analysis of the effect of total saponin compound YJ on gut flora-genus, a represents low-fat feed feeding; b represents high fat feed feeding; c represents the high-fat group + the low-dose YJ (100mg/Kg), and D represents the high-fat group + the high-dose YJ (300 mg/Kg).

Figure 5 shows the effect of total saponin compounds YJ on gut flora-phylum. HF represents a high-fat diet obesity model group; HF + YJ represents high fat diet +300mg/kg dose YJ.

FIG. 6 shows the effect of total saponin compounds YJ on the population diversity at the phylum level of intestinal bacteria in obese mice, HF representing the high fat diet obesity model group (mice numbered HF 1-8); HF + YJ represents high fat diet +300mg/kg dose YJ (mouse number HF _ YJ 1-8).

FIG. 7 shows the effect of total saponin compounds YJ on the flora diversity at the genus level of intestinal bacteria in obese mice, HF representing the high fat diet obesity model group (mice numbered HF 1-8); HF + YJ represents high fat diet +300mg/kg dose YJ (mouse number HF _ YJ 1-8).

FIG. 8 shows the effect of PCA analysis of total saponin compound YJ on the diversity of intestinal flora in obese mice, HF representing the high fat diet obesity model group (mice numbered HF 1-8); HF + YJ represents high fat diet +300mg/kg dose YJ (mouse number HF _ YJ 1-8).

Detailed Description

The inventor of the application extensively and deeply researches the chemical components and the biological activity of the gynostemma pentaphylla, successfully separates a plurality of novel triterpene saponin compounds from the gynostemma pentaphylla, and discovers for the first time that the triterpene total saponin compound YJ and one or more monomer compounds thereof have the function of preventing and treating obesity by improving the intestinal flora of animals. On the basis of this, the present invention has been completed.

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Gynostemma pentaphylla

Gynostemma pentaphylla is dried whole herb of Gynostemma pentaphylla (Thunb.) Makino) belonging to family Cucurbitaceae, and is a perennial climbing herb. Gynostemma pentaphylla has a long history of eating, and is taken as a wild vegetable from Jiu ban Ben Cao of Ming dynasty. It is called "sweet tea vine" in Japan, and is used as sweet tea substitute or sweetener for diabetic. Gynostemma pentaphylla is also one of the plants with ginsenoside resources in the Araliaceae, has multiple functions of nourishing and health care, resisting cancer and aging, reducing blood fat, reducing blood sugar and the like, and is known as 'southern ginseng'. Gynostemma pentaphylla is one of plants which are published by Ministry of health and can be used for health care products, is suitable for long-term taking due to obvious health care effect and high safety, and is often drunk as lipid-lowering health care tea in folks.

Total saponin compound YJ

The total saponin compounds YJ of the present invention include, but are not limited to, saponin compounds extracted from Gynostemma pentaphyllum and the like, i.e., a series of compounds having saponin structure.

In another preferred embodiment, the total saponin compounds of the present invention include one or more compounds selected from the group consisting of:

intestinal bacteria and obesity

The composition of intestinal flora is closely related to obesity, and research on the animal level shows that compared with a normal mouse, ob/ob obese mice have the advantages that in the intestinal bacteria biological diversity analysis, the number of firmicutes bacteria is increased, and the number of bacteroidetes is reduced by 50%; at the human level, 12 obese patients were treated on a fat-restricted diet and a carbohydrate-restricted diet, respectively, by follow-up studies. After one year, the weight of the former is reduced by 2 percent, the weight of the latter is reduced by 6 percent, and 16s rRNA gene research shows that under the premise that the total energy of ingested food is not changed, the number of firmicutes in intestinal tracts is reduced and the number of bacteroidetes is increased after weight loss through two diets. The data indicate that an increase in the bacteroidetes/firmicutes ratio, which is a major proportion of the intestinal tract, is helpful in the control of obesity.

Akkermansia muciniphila

Akkermansia muciniphila belongs to the family of Verrucomicrobiaceae (Verrucomicrobiaceae), 5.2013, and researchers in Belgium published an article on PNAS, and the health condition of obese mice can be greatly changed by discovering a weight-reducing bacterium through researching an intestinal bacterium and a liquid culture medium containing the bacterium. Researchers at the university of the walwenw astronomical university of belgium fed two to three times more fat than normal rats with a bacterium called Akkermansia muciniphila, which reduced excess body weight by nearly half without altering their diet, and also reduced insulin antibody levels, the main symptom of type II diabetes in vivo. Studies have shown that Akkermansia muciniphila increases intestinal levels of endocannabinoids that control glucose and intestinal homeostasis, and hinders diet-induced reduction of mucosal barrier thickness. In the same year, researchers have published a paper on Gut that metformin significantly increases the amount of Akkermansia Muciniphila to improve the glycemic profile in high fat fed mice. The metformin-treated mice in the high-fat-fed group had more mucin-degrading bacteria Akkermansia than in the group with only high-fat feeding. In addition, the number of mucin-producing goblet cells was also significantly increased in the metformin-treated group. Oral administration of Akkermansia musciniphila to high fat-fed mice significantly improved glucose tolerance even without metformin, which reduced adipose tissue inflammation by inducing Foxp3 regulatory T cells (Tregs) in visceral adipose tissue. Therefore, the use of such bacteria for the prevention or treatment of obesity and type II diabetes has become a potential strategy for the prevention and treatment of obesity.

Preparation method

In another preferred embodiment, the preparation method of the triterpenoid saponin compound comprises the following steps:

(1) extracting the overground part of tetraploid gynostemma pentaphylla with a solvent, and concentrating an extracting solution to obtain an extract;

(2) dissolving the extract in water to obtain dispersion, sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain n-butanol extractive solution, and recovering solvent under reduced pressure to obtain n-butanol extract;

(3) dissolving the n-butanol part extract in water, loading onto D101 macroporous resin column, eluting with pure water to remove sugar and water-soluble impurities, gradient eluting with ethanol-water, collecting different eluted parts, tracking, detecting, and rotary evaporating to concentrate to obtain crude saponin dry powder. Weighing a dissolved pure methanol solution, loading the dissolved pure methanol solution on a Sephadex LH-20 separation column, performing isocratic elution, recovering the solvent under reduced pressure, performing Sephadex LH-20 Sephadex chromatography, performing isocratic elution with the methanol solution, collecting fractions containing the compound, and directly performing separation and purification on the fractions by using a semi-preparative reverse phase high performance liquid chromatography to obtain the compound;

in another preferred example, in the step (1), the overground part of the tetraploid gynostemma pentaphylla is extracted by a solvent for 2-5 times, each time lasts for 1-3 hours, and the extracting solution is concentrated to obtain an extract;

in another preferred example, the solvent in step (1) is selected from one or more of methanol, ethanol, acetone or water;

in another preferred example, the mass ratio of the extract to the water in the step (2) is 1:2-1: 15.

In another preferred example, the volume ratio of the addition amount of the petroleum ether to the dispersion liquid in the step (2) is 1:2-5: 1;

in another preferred embodiment, the volume ratio of the added ethyl acetate to the dispersion liquid in the step (2) is 1:2-5: 1;

in another preferred example, the volume ratio of the adding amount of the n-butanol to the dispersion liquid in the step (2) is 1:2-5: 1.

In another preferred example, in the step (3), the ethanol-water solution is subjected to gradient elution in a volume ratio of 20:1-100: 1; separating by Sephadex LH-20 column chromatography, eluting with pure methanol solvent in sequence, and collecting fraction containing the compound; separating and purifying one flow by semi-preparative reverse phase high performance liquid chromatography with solvent system of 30% acetonitrile-15% methanol-55% water (vol) to obtain saponin compounds 1-9 in turn.

Use of

Through investigating the influence of the total saponin compound on the ratio of intestinal flora bacteroidetes/firmicutes, particularly the weight-reducing bacteria (Akkermansia muciniphila) in the wart microbe family, and researching the action mechanism of the total saponin compound, the compound and the optical isomer, the medicinal salt and the solvate thereof can remarkably adjust the intestinal flora caused by high-fat diet to prevent and treat obesity.

In addition, the compound can be used as a food auxiliary material or a food additive, and is added into food for improving the intestinal flora of animals and promoting the colonization of beneficial microorganisms.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising a safe and effective amount of the active ingredient, and a pharmaceutically acceptable carrier.

The active ingredient refers to the gynostemma total saponin compound.

The active ingredients and the pharmaceutical composition can be used for improving the composition of intestinal flora and promoting the colonization of beneficial microorganisms.

In another preferred embodiment, the composition is used for preparing a medicament for improving the intestinal flora of animals.

"safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of active ingredient per dose, more preferably, 10-200mg of active ingredient per dose. Preferably, said "dose" is a tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of being combined with the active ingredients of the present invention and with each other without significantly diminishing the efficacy of the active ingredient.

Typically, the carrier includes one or more of diluents, fillers, disintegrants, lubricants, colorants, flavoring agents, or other conventional additives.

Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the active ingredient or pharmaceutical composition of the present invention includes oral administration and the like.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

The solid dosage forms may also be prepared using coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be delayed in a certain portion of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, especially cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like. In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active ingredients, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these materials, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

The compounds of the present invention may be administered alone or in combination with other therapeutic agents.

In the case of pharmaceutical compositions, a safe and effective amount of a compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the administration is a pharmaceutically acceptable and effective dose, and the daily dose for a human of 60kg body weight is usually 1 to 2000mg, preferably 20 to 500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

The main advantages of the invention are:

(1) the triterpenoid saponin compound with a novel structure is extracted and separated from tetraploid gynostemma pentaphylla, and the compound, optical isomers, medicinal salts and solvates thereof have the purposes of improving animal intestinal flora, promoting beneficial microorganism colonization and preventing and treating obesity, can be applied in the form of the compound, and can also be applied by forming a medicinal composition with a pharmaceutically acceptable carrier;

(2) the preparation method provided by the invention is simple and feasible and can be completed in general equipment;

(3) the prepared compound has high purity and good economic benefit and application prospect.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1

1.1 preparation of tetraploid Gynostemma pentaphylla Total Saponin YJ

Weighing 18kg tetraploid Gynostemma pentaphyllum leaves, pulverizing, extracting with 20L 95% ethanol under reflux for 3 times (material-liquid ratio of 1:2.6) for 3 hr, 2 hr, and 1 hr, and removing ethanol by rotary evaporation to obtain crude extract

And (6) making paste.

Dispersing the above extract in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether, ethyl acetate, n-butanol and water extract parts. Rotary evaporating to obtain n-butanol fraction of herba Gynostemmatis crude extract

1509g of paste.

Preparing 6mg/mL n-butanol part sample solution, totaling 20mL volume, loading D101 macroporous resin, gradient eluting with 20%, 40% and 60% ethanol, collecting 60% eluate part, rotary evaporating and concentrating to obtain crude saponin dry powder. Then weighing 15g of crude saponin to dissolve in 55mL of methanol solution to sample a Sephadex LH-20 separation column, further purifying by isocratic elution, monitoring in real time, and collecting a sample to obtain a refined saponin total saponin compound YJ.

1.2 animal experiments

SPF grade 5 week old male C57/BL 6 mice (purchased from Peking Wintolite center) were bred in an SPF environment.

1.2.1 prevention experiments

After one week of acclimation, 32 mice were divided into 4 groups of 8 mice each, and were raised in a single cage.

Respectively, group A (low fat group, 10% energy derived from fat), group B (high fat group, 45% energy derived from fat), group C (high fat group +100mg/Kg/D YJ), and group D (high fat group +300mg/Kg/D YJ).

The temperature of the breeding environment is 22 +/-2 ℃, the humidity is 30-70%, and the mice can freely eat and drink water; the experiment lasts for 12 weeks, the weight is recorded every day, fresh food and sterile water are changed every Tuesday and Friday, the weighing record is made, and the food intake is recorded. The padding of the mice is changed every week, and the rearing cage is changed every month. After 12 weeks, feces volume was continuously collected for 3 days, 4 mice were randomly selected per group, DNA was extracted, Illumina Miseq PE250/PE300 sequencing was performed, and fecal sample flora diversity was analyzed.

1.2.2 animal therapy experiments

The mice fed with the high-fat feed for 4 months have the weight of about 40g, and after the obesity resistant mice are removed, the mice are randomly divided into 2 groups of 8 mice each and fed in a single cage.

The model group (HFD) was continuously fed with high fat diet, and the intervention group (HFD + YJ) was fed with high fat diet, and the hose gavage was performed with a 300mg/Kg/d dose of total saponin compound for 2 months, and the weight was recorded every day, and the food and sterile water were renewed every tuesday and friday, and the weight was recorded and the food intake was recorded. The padding of the mice is changed every week, and the rearing cage is changed every month. After 2 months, the fecal contents of all mice were continuously collected for 3 days, DNA was extracted, Illumina Miseq PE250/PE300 sequencing was performed, and the fecal sample flora diversity was analyzed.

1.3 analysis of results

As can be seen from fig. 1, the weight of the mice gradually increased to a significant level after 56 days when the mice were fed the high-fat diet for a long time as compared with the low-fat diet, and the weight increase was strongly inhibited after the YJ intervention, and reached a significant level after 63 days and exhibited a concentration-dose relationship as compared with the high-fat model group.

As can be seen from fig. 2, when the high-fat diet is taken for a long time, the ratio of enterobacteroidetes/firmicutes is significantly decreased, and after the intervention of YJ, the ratio of bacteroides/firmicutes starts to increase, and the significant increase is achieved under the high-dose condition. Also, the high dose intervention group induced Verrucomicrobiaceae (Verrucomicrobiaceae) and increased Proteobacteria (Proteobacteria) numbers. Further classifying enterobacter-genus, high dose total saponin compounds were able to significantly induce a significant increase in Akkermansia muciniphila, a weight-reducing bacterium in the wart family (fig. 3). By analyzing PCOA statistically, the total saponin compound intervention can significantly improve the intestinal bacteria composition change of mice caused by high-fat diet (fig. 4).

As can be seen from fig. 3, YJ has a large influence on the diversity of the following strains: bacteroides, Akkermansia muciniphila, lactic acid bacteria, and the like.

As can be seen from FIG. 5, after the addition of 300mg/kg YJ to intervene for 28 days of obesity, a significant decrease in body weight occurs, demonstrating that YJ has a significant effect of treating obesity.

The results of animal treatment experiments are shown in fig. 6, and it can be seen that using YJ to intervene in mice, Verrucomicrobiaceae (blue marker) was significantly induced to express, and further at genus level, the weight loss of total saponin compounds and its inducing effect on Akkermansia muciniphila were further demonstrated.

FIG. 7 shows the effect of YJ on the flora diversity of obese mouse gut flora at genus level, and similarly, Akkermansia muciniphila was significantly improved, and with the help of PCA analysis, the two groups could be completely separated, demonstrating the significant effect of YJ in regulating the gut flora composition and preventing obesity in obese mice (FIG. 8).

In combination with the above-mentioned prevention experiments, we can judge that the effect of total saponin compounds in preventing and treating obesity is due to the action of directionally inducing "lean bacteria" Akkermansia muciniphila.

Example 2: food product

The weight-reducing food is prepared according to the following formula:

2-5 parts of oatmeal, 1-5 parts of Chinese yam, 10-15 parts of dietary fiber, 1-4 parts of pearl barley, 2-5 parts of inositol, 10-15 parts of maltose and 0.1-0.5% of YJ

The food is taken 3 times a day for 7-15 days.

Example 3: pharmaceutical composition

Mixing the above components, and directly tabletting to obtain tablet composition, which is orally administered 1 tablet each time for 2-3 times a day.

Example 4: pharmaceutical composition

The above mixture is uniformly filled into 1000 capsules, and 1 granule is orally taken every time, 2-3 times a day.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. An application of a gynostemma pentaphylla total saponin compound is characterized in that,

(1) for preparing a composition for improving intestinal flora; and/or

(2) For preparing a composition for promoting the colonization of characteristic microorganisms;

the total saponin compounds include one or more compounds selected from the group consisting of:

2. use according to claim 1, wherein the intestinal flora belongs to the phylum firmicutes or bacteroidetes.

3. The use according to claim 1, wherein the intestinal flora belongs to the phylum verrucomicrobia or proteobacteria.

4. The use according to claim 1, wherein the bacteria of the intestinal flora are lactic acid bacteria.

5. Use according to claim 1, wherein the intestinal flora is Akkermansia muciniphila.

6. The use according to claim 1, wherein the composition is a pharmaceutical composition or a food composition.

7. The use according to claim 1, wherein the gynostemma pentaphyllum is diploid or tetraploid gynostemma pentaphyllum.

8. The use according to claim 1, wherein the gut flora is selected from the group consisting of: bifidobacteria (bifidobacteria) or Clostridium flexibilizatum (Clostridium leptum).

9. The use according to claim 6, wherein the food composition is a diet food.

10. A method for non-therapeutically improving the intestinal flora in vitro comprising the steps of: administering a compound of claim 1 or a pharmaceutically acceptable salt thereof at a site in need of treatment.

11. A probiotic composition, comprising:

(1) a first drug: saponins compound, and

(2) a second drug: one or more probiotics selected from the group consisting of: lactobacillus species Clostridium mollicium (Clostridium leptum), Akkermansia muciniphila, or combinations thereof;

wherein the first and second medicaments are located in the same or different containers;

the saponin compound comprises one or more compounds selected from the group consisting of:

12. the probiotic composition according to claim 11, wherein the lactic acid bacteria is bifidobacteria (bifidobacteria).