WO2018090983A1 - 改善肠道菌群的皂苷类化合物、制备方法及其应用 - Google Patents

改善肠道菌群的皂苷类化合物、制备方法及其应用 Download PDF

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WO2018090983A1
WO2018090983A1 PCT/CN2017/111690 CN2017111690W WO2018090983A1 WO 2018090983 A1 WO2018090983 A1 WO 2018090983A1 CN 2017111690 W CN2017111690 W CN 2017111690W WO 2018090983 A1 WO2018090983 A1 WO 2018090983A1
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intestinal flora
group
composition
use according
saponin compound
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WO2018090983A9 (zh
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刘洁
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北京工商大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)
    • A61K36/424—Gynostemma
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the technical field of saponin compounds and preparation and application thereof. Specifically, the present invention relates to a triterpenoid saponin compound extracted and isolated from Gynostemma pentaphyllum, and a preparation method and use thereof.
  • the human intestine is a complex, active, and relatively balanced system that colonizes a large number of complex microbial communities.
  • the human gastrointestinal tract is home to about 10 4 normal bacteria, of which 30-40 dominant bacteria constitute 99% of the total intestinal bacteria.
  • Intestinal microbes contain a wide variety of enzyme systems that can participate in a range of physiological processes such as host energy, material and genetic information transfer. Many studies have shown that there is an inseparable mutual symbiotic relationship between the intestinal flora and the host.
  • the intestinal microbes can form a biological barrier in the intestinal tract, through mass occupying, nutrient competition and various metabolites secreted by them. And bacteriocins play an important role in the digestion, absorption, immune response and metabolic activity of the host.
  • Firmicutes and Bacteroides are two dominant species; at the genus level, Bacteroides, Clostridium, Clostridium, and Bifidobacteria are four types closely related to human health. Related dominant bacteria are also the focus of research on intestinal micro-organisms at this stage. The occurrence and development of chronic diseases such as obesity and hyperlipidemia are closely related to the structural imbalance of intestinal flora.
  • Gynostemma pentahyllum (Thunb.) Makino is a perennial herbaceous genus of the genus Cucurbitaceae, and its main bioactive component is a triterpenoid saponin.
  • composition for preparing an improved intestinal flora (1) a composition for preparing an improved intestinal flora; and/or
  • the composition is a pharmaceutical composition or a food composition.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is an oral agent.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of a tablet, a capsule or a granule.
  • the intestinal flora belongs to the phylum of the genus Bacteroides or the bacillus.
  • the intestinal flora belongs to the sputum microphylaxis or the proteobacteria.
  • the intestinal flora is selected from the group consisting of lactic acid bacteria, bifidobacteria, or Clostridium natto.
  • the intestinal flora is Akkermansia muciniphila.
  • the food composition is a diet food.
  • the pharmaceutically acceptable salt of the total saponin compound is selected from the group consisting of sodium salts, potassium salts, or combinations thereof.
  • the Gynostemma pentaphyllum is a diploid Gynostemma pentaphyllum or a tetraploid Gynostemma pentaphyllum, preferably a tetraploid Gynostemma pentaphyllum.
  • the total saponin compound (designated YJ) comprises one or more compounds selected from the group consisting of:
  • a method for non-therapeutic improvement of intestinal flora in vitro comprising the step of administering a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, at a locus to be treated.
  • the compound or a pharmaceutically acceptable salt thereof is administered at a concentration of 0.001 to 0.02 ⁇ g/ml, preferably 0.01 to 0.15 ⁇ g/ml, more preferably 0.05. -0.1 ⁇ g/ml.
  • a probiotic composition comprising:
  • a first drug a saponin compound
  • a second drug one or more probiotics selected from the group consisting of lactic acid bacteria, bifidobacteria, Clostridium genus, Akkermansia muciniphila, or a combination thereof;
  • first drug and the second drug are located in the same or different containers.
  • the mass ratio of the first drug to the second drug (in terms of active ingredient) in the probiotic composition is 1:100-100:1, preferably 1: 10-10:1, more preferably 1:5-5:1.
  • the probiotic composition is a pharmaceutical composition or a food composition.
  • a method of improving intestinal flora comprising the step of administering a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.
  • the compound or a pharmaceutically acceptable salt thereof is administered at a concentration of 0.001 to 0.02 ⁇ g/ml, preferably 0.01 to 0.15 ⁇ g/ml, more preferably 0.05. -0.1 ⁇ g/ml.
  • Figure 1 shows the preventive effects of different doses of total saponin compound YJ on obesity caused by a high-fat diet.
  • Figure 2 shows the effect of total saponin compound YJ on community diversity of intestinal bacteria at the gate level.
  • A represents low-fat diet feeding
  • B represents high-fat diet feeding
  • C stands for high-fat group + low-dose YJ (100 mg/Kg)
  • D stands for high-fat group + high-dose YJ (300 mg/Kg).
  • Figure 3 shows the effect of total saponin compound YJ on community diversity of gut bacteria at the genus level, A for low fat feed; B for high fat diet; C for high fat + low dose YJ (100 mg/Kg) D represents a high fat group + a high dose YJ (300 mg/Kg).
  • Figure 4 shows the effect of PCOA on total intestinal saponin compound YJ on intestinal flora-genus, A for low-fat diet feeding; B for high-fat diet; C for high-fat group + low-dose YJ (100 mg/kg), D Represents high fat group + high dose YJ (300mg/Kg).
  • Figure 5 shows the effect of total saponin compound YJ on the intestinal flora-gate.
  • HF stands for high fat diet obesity model group
  • HF+YJ stands for high fat diet +300 mg/kg dose YJ.
  • Figure 6 shows the effect of total saponin compound YJ on the diversity of intestinal bacteria in obese mice at the gate level, HF for the high fat diet obesity model group (mouse number HF 1-8); HF+YJ stands for high Fat feed + 300 mg/kg dose YJ (mouse number HF_YJ 1-8).
  • Figure 7 shows the effect of total saponin compound YJ on the microbial diversity of intestinal bacteria in obese mice
  • HF represents the high fat diet obesity model group (mouse number HF 1-8);
  • HF+YJ stands high Fat feed + 300 mg/kg dose YJ (mouse number HF_YJ 1-8).
  • Figure 8 shows the effect of PCA analysis of total saponin compound YJ on intestinal flora diversity in obese mice, HF for high fat diet obesity model group (mouse number HF 1-8); HF+YJ for high fat diet +300 mg/kg dose YJ (mouse number HF_YJ 1-8).
  • the inventors of the present application have extensively and intensively studied the chemical constituents and biological activities of Gynostemma pentaphyllum, and successfully isolated a plurality of new triterpenoid saponins from Gynostemma pentaphyllum, and first discovered the triterpene total saponin compound YJ and One or more monomeric compounds have a function of preventing obesity by improving the intestinal flora of the animal. On the basis of this, the present invention has been completed.
  • the term “about” means that the value can vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including” may be open, semi-closed, and closed. In other words, the terms also include “consisting essentially of,” or “consisting of.”
  • Gynostemma pentaphyllum is a dry whole grass of Gynostemma pentaphyllum (Thunb. Makino), which is a perennial climbing herb. Gynostemma pentaphyllum has a long history of eating and was first used in the Ming Dynasty's "Rescue Materia Medica” for wild vegetables. The Japanese folks call it "Gancha Man", a sweet tea substitute or a sweetener for diabetics. Gynostemma pentaphyllum is also one of the plants with ginseng saponin resources outside Wujiake. It has many functions such as nourishing health, anti-cancer, anti-aging, blood fat reduction and blood sugar lowering. It is known as “Southern Ginseng”.
  • Gynostemma pentaphyllum is one of the plants that can be used for health care products announced by the Ministry of Health. Because of its remarkable health care effect and high safety, it is suitable for long-term use. It is often used as a lipid-lowering health tea in the folk.
  • the total saponin compound YJ of the present invention includes, but is not limited to, a saponin compound extracted from Gynostemma pentaphyllum and the like, that is, a series of compounds having a saponin structure.
  • the total saponin compound of the present invention comprises one or more compounds selected from the group consisting of:
  • the composition of intestinal flora is closely related to obesity. Studies at the animal level indicate that compared with normal mice, ob/ob obese mice have increased the number of bacteria in the intestinal flora, and the number of Bacteroides Reduced by 50%; at the human level, 12 fat patients were followed up for treatment with a fat-restricted diet and a carbohydrate-restricted diet. After one year, the weight of the former decreased by 2%, and the weight of the latter decreased by 6%. The 16s rRNA gene study showed that the total amount of energy in the food did not change, and the number of thick-walled bacteria in the intestine after losing weight through two diets. Decreased, while the number of Bacteroides gates increased. The data suggest that an increase in the proportion of Bacteroides/Thick Wall bacteria in the gut contributes to the prevention and treatment of obesity.
  • Akkermansia muciniphila belongs to the family Verrucomicrobiaceae. In May 2013, Belgian researchers published an article on PNAS to discover a "slimming bacteria" by studying a gut bacteria. The liquid medium containing this bacteria can be greatly Change the health of obese mice.
  • the method for preparing a triterpenoid saponin compound of the present invention comprises the following steps:
  • the aerial part of the tetraploid Gynostemma pentaphyllum is extracted with a solvent for 2-5 times, each time 1-3 hours, and the extract is concentrated to obtain an extract;
  • the solvent in the step (1) is selected from the mixture of one or more of methanol, ethanol, acetone or water;
  • the mass ratio of the extract to the water in the step (2) is 1:2-1:15.
  • the ratio of the amount of petroleum ether added to the dispersion in the step (2) is 1:2-5:1;
  • the ratio of the amount of ethyl acetate added to the dispersion in the step (2) is 1:2-5:1;
  • the volume ratio of n-butanol to the dispersion in the step (2) is 1:2 to 5:1.
  • the ethanol-water solution is subjected to a gradient elution at a volume ratio of 20:1 to 100:1; and then separated by Sephadex LH-20 column chromatography, and eluted sequentially with a pure methanol solvent. Collecting fractions containing the compound; one fraction is separated and purified by semi-preparative reversed-phase high performance liquid chromatography in a solvent system of 30% acetonitrile-15% methanol-55% water (vol) to obtain saponin compound 1 in sequence -9.
  • the total saponin compounds of the present invention By investigating the effects of the total saponin compounds of the present invention on the proportion of the intestinal flora of the genus Bacteroides and the thick-walled bacteria, especially the slimming bacteria (Akkermansia muciniphila) in the genus Microsporaceae can be significantly improved, and the mechanism of action thereof is studied. It was found that the compounds of the present invention and their optical isomers, pharmaceutically acceptable salts and solvates can be significantly adjusted high. The intestinal flora caused by the fat diet to prevent obesity.
  • the compounds of the present invention can also be used as food adjuvants or food additives, added to foods for improving the intestinal flora of animals and promoting the colonization of beneficial microorganisms.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an active ingredient in a safe and effective amount, together with a pharmaceutically acceptable carrier.
  • the "active ingredient” as used in the present invention means the Gynostemma total saponin compound of the present invention.
  • the "active ingredients" and pharmaceutical compositions of the present invention are useful for improving intestinal flora composition and promoting beneficial microbial colonization.
  • it is used to prepare a medicament which improves the intestinal flora of an animal.
  • the pharmaceutical compositions contain from 1 to 2000 mg of active ingredient per dose, more preferably from 10 to 200 mg of active ingredient per dose.
  • the "one dose” is a tablet.
  • “Pharmaceutically acceptable carrier” means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity.
  • “compatibility” it is meant herein that the components of the composition are capable of intermingling with the active ingredients of the present invention and with respect to each other without significantly reducing the efficacy of the active ingredients.
  • the carrier includes one or more of a diluent, a filler, a disintegrant, a lubricant, a colorant, a flavoring agent, or other conventional additives.
  • Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid). , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as ), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.
  • cellulose and its derivatives such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate,
  • 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.
  • the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerin; (d) a disintegrant such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent such as paraffin; (f) Absorbing accelerators, for example, quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example,
  • the solid dosage forms can also be prepared with coatings and shell materials, such as casings and other materials known in the art. They can An opacifying agent is included and the release of the active ingredient in such a composition can be released in a portion of the digestive tract in a delayed manner.
  • coatings and shell materials such as casings and other materials known in the art.
  • An opacifying agent is included and the release of the active ingredient in such a composition can be released in a portion of the digestive tract in a delayed manner.
  • embedding components that can be employed are polymeric and waxy materials.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.
  • the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances.
  • the compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
  • compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion.
  • Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.
  • the compounds of the invention may be administered alone or in combination with other therapeutic agents.
  • a safe and effective amount of a compound of the invention is administered to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective effective dosage, for a 60 kg body weight
  • the dose to be administered is usually from 1 to 2000 mg, preferably from 20 to 500 mg.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the present invention obtains a novel triterpenoid saponin compound by extracting and separating from tetraploid Gynostemma pentaphyllum, and the compound and their optical isomers, pharmaceutically acceptable salts and solvates thereof have been used for improving intestinal flora of animals.
  • a novel triterpenoid saponin compound by extracting and separating from tetraploid Gynostemma pentaphyllum, and the compound and their optical isomers, pharmaceutically acceptable salts and solvates thereof have been used for improving intestinal flora of animals.
  • beneficial microbial colonization and to prevent and treat obesity which may be administered in its own form or in combination with a pharmaceutically acceptable carrier;
  • the present invention provides a simple and easy preparation method, which can be completed in a general-purpose device
  • the prepared compound has high purity, good economic benefit and application prospect.
  • the 18kg tetraploid Gynostemma pentaphyllum leaves were weighed by a pulverizer and then extracted by 20L 95% ethanol reflux method (feed ratio: 1:2.6), extracted 3 times for 3h, 2h, 1h, and removed by rotary evaporation. Ethanol to obtain crude extract of Gynostemma pentaphyllum paste.
  • the above extract was dispersed in water, and successively extracted with an equal volume of petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether, ethyl acetate, n-butanol and water extracting sites, respectively.
  • mice After 32 mice were acclimated for one week, they were divided into 4 groups, 8 in each group, and fed in a single cage.
  • 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 +100 mg/Kg/d YJ
  • group D High fat group +300mg/Kg/d YJ
  • the breeding environment temperature is 22 ⁇ 2°C, the humidity is 30-70%, the mice are free to eat and drink water; the experiment lasts for 12 weeks, the weight is recorded every day, and the food and sterile water are renewed every Tuesday and Friday. Record the amount and record the amount of food consumed. Change the mouse padding weekly and change the cage every month. After 12 weeks, 3 days of fecal volume was collected continuously. Four mice were randomly selected from each group, DNA was extracted, and Illumina Miseq PE250/PE300 was sequenced to analyze the bacterial diversity of the fecal samples.
  • mice were fed with high-fat diet for 4 months and weighed about 40 g. After obesity-resistant mice were removed, they were randomly divided into 2 groups, 8 in each group, and fed in single cages.
  • the model group (HFD) continued to feed high-fat diet.
  • the tube was given a total dose of 300 mg/Kg/d total saponin compound for 2 months, and the body weight was recorded every day. New food and sterile water will be renewed on Tuesdays and Fridays, and a weighing record will be made to record the food intake. Change the mouse padding weekly and change the cage every month. After 2 months, all mice were continuously collected for 3 days of fecal volume, and DNA was extracted for Illumina Miseq PE250/PE300. Sequencing, analysis of the diversity of fecal sample flora.
  • YJ has a great influence on the diversity of the following strains: Bacteroides, Akkermansia muciniphila, lactic acid bacteria and the like.
  • YJ intervention mouse Verrucomicrobiaceae (blue mark) was significantly induced to express, further at the genus level, further proved the total saponin compound weight loss And its induction to Akkermansia muciniphila. Therefore, YJ is a prebiotic substance that specifically promotes the proliferation of Akkermansia muciniphila. Administration of 300 mg/kg YJ significantly increased the content of Akkermansia muciniphila compared to the control group.
  • Figure 7 shows the effect of YJ on the diversity of intestinal bacteria in obese mice at the genus level. Similarly, Akkermansia muciniphila was significantly improved. By PCA analysis, the two groups could be completely separated, demonstrating that YJ is regulating obese mice.
  • the composition of the intestinal flora constitutes a significant effect on the prevention and treatment of obesity (Fig. 8).
  • the above components are uniformly mixed and directly compressed, that is, a tablet composition is obtained, one tablet per oral administration, 2-3 times a day.
  • the above component mixture was uniformly filled into 1000 capsules, one tablet per oral administration, 2-3 times a day.

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Abstract

一种改善肠道菌群的皂苷类化合物、制备方法及其应用，该皂苷类化合物为绞股蓝总皂苷。所述组合物可改善肠道菌群、促进特征微生物定居。

Description

改善肠道菌群的皂苷类化合物、制备方法及其应用 技术领域
本发明属于皂苷类化合物及其制备和应用技术领域，具体地说，本发明涉及从绞股蓝中提取分离得到的三萜皂苷类化合物及其制备方法和用途。
背景技术
人的肠道是一个复杂、活跃、同时维持着相对平衡的***，定植着数目庞大、结构复杂的微生物群落。人体胃肠道内栖息着大约104数量级的正常菌群，其中的30-40种优势细菌构成了人体肠道细菌总量的99％。肠道微生物含有种类繁多的酶***，可以参与宿主能量、物质及遗传信息转运等一系列生理过程。已有许多研究证明，肠道菌群和宿主之间存在着密不可分的互利共生关系，肠道微生物可以在肠道形成生物屏障，通过占位效应、营养竞争及其所分泌的各种代谢产物和细菌素等在宿主的消化吸收、免疫反应、代谢活性方面都发挥着重要的作用。在门水平上，厚壁菌门(Firmicutes)和拟杆菌门(Bacteroides)为两个优势菌门；在属水平上，拟杆菌、柔嫩梭菌、乳酸菌、双歧杆菌是四类与人类健康密切相关的优势菌，也是现阶段人们对肠道微生物研究的重点，肥胖、高血脂等慢性病的发生、发展与肠道菌群结构失调有很大关系。
绞股蓝(Gynostemma pentahyllum(Thunb.)Makino)是葫芦科(Cucurbitaceae)绞股蓝属多年生草质藤本植物，其主要生物活性成分为三萜皂苷类化合物。
鉴于三萜皂苷类化合物具有较高的药用价值，本领域尚需对其进行深入研究。
发明内容
本发明的目的在于提供一种皂苷类化合物及其制备方法和应用。
本发明第一方面，提供一种绞股蓝总皂苷化合物的用途，
(1)用于制备改善肠道菌群的组合物；和/或
(2)用于制备促进特征微生物定居的组合物。
在另一优选例中，所述组合物为药物组合物或食品组合物。
在另一优选例中，所述药物组合物还包括药学上可接受的载体。
在另一优选例中，所述药物组合物为口服剂。
在另一优选例中，所述药物组合物的剂型选自下组：片剂、胶囊或颗粒。
在另一优选例中，所述肠道菌群属于厚壁菌门或拟杆菌门。
在另一优选例中，所述肠道菌群属于疣微菌门或变形菌门。
在另一优选例中，所述肠道菌群选自下组菌株：乳酸菌、双歧杆菌或柔嫩梭菌。
在另一优选例中，所述肠道菌群为Akkermansia muciniphila。
在另一优选例中，所述食品组合物为减肥食品。
在另一优选例中，所述总皂苷化合物的药学上可接受的盐选自下组：钠盐、钾盐、或其组合。
在另一优选例中，所述绞股蓝为二倍体绞股蓝或四倍体绞股蓝，较佳地为四倍体绞股蓝。
在另一优选例中，所述总皂苷化合物(命名为YJ)包括一种或多种选自下组的化合物：
Figure PCTCN2017111690-appb-000001
Figure PCTCN2017111690-appb-000002
Figure PCTCN2017111690-appb-000003
本发明第二方面，提供一种体外非治疗性地改善肠道菌群的方法，包括步骤：在需要处理的场所施用如本发明第一方面所述的化合物或其药学上可接受的盐。
在另一优选例中，所述方法中，所述化合物或其药学上可接受的盐的施用浓度为0.001-0.02μg/ml，较佳地为0.01-0.15μg/ml，更佳地为0.05-0.1μg/ml。
本发明第三方面，提供一种益生菌组合物，所述益生菌组合物包括：
(1)第一药物：皂苷类化合物，和
(2)第二药物：一种或多种选自下组的益生菌：乳酸菌、双歧杆菌、柔嫩梭菌、Akkermansia muciniphila、或其组合；
其中，所述第一药物和第二药物位于相同或不同的容器中。
在另一优选例中，所述益生菌组合物中，所述第一药物与所述第二药物的质量比(以活性成分计)为1:100-100:1，较佳地为1:10-10:1，更佳地为1:5-5:1。
在另一优选例中，所述益生菌组合物为药物组合物或食品组合物。
本发明第四方面，提供一种改善肠道菌群的方法，包括步骤：对需要的对象施用如本发明第一方面所述的化合物或其药学上可接受的盐。
在另一优选例中，所述方法中，所述化合物或其药学上可接受的盐的施用浓度为0.001-0.02μg/ml，较佳地为0.01-0.15μg/ml，更佳地为0.05-0.1μg/ml。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了不同剂量总皂苷化合物YJ对于高脂饮食导致肥胖的预防作用，
A代表低脂饲料喂食；B代表高脂饲料喂食；C代表高脂组+低剂量YJ(100mg/Kg)；D代表高脂组+高剂量YJ(300mg/Kg)，每组8只老鼠，*表示低脂组同高脂组相比较体重显著，#表示总皂苷化合物干预组同高脂组比较在P<0.05水平下极显著。
图2显示了总皂苷化合物YJ对于肠道菌在门水平下群落多样性的影响，
A代表低脂饲料喂食；B代表高脂饲料喂食；C代表高脂组+低剂量YJ(100mg/Kg)，D代表高脂组+高剂量YJ(300mg/Kg)。
图3显示了总皂苷化合物YJ对于肠道菌在属水平下群落多样性的影响，A代表低脂饲料喂食；B代表高脂饲料喂食；C代表高脂组+低剂量YJ(100mg/Kg)，D代表高脂组+高剂量YJ(300mg/Kg)。
图4显示了PCOA分析总皂苷化合物YJ对于肠道菌群-属的影响,A代表低脂饲料喂食；B代表高脂饲料喂食；C代表高脂组+低剂量YJ(100mg/Kg)，D代表高脂组+高剂量YJ(300mg/Kg)。
图5显示了总皂苷化合物YJ对于肠道菌群-门的影响。HF代表高脂饲料肥胖模型组；HF+YJ代表高脂饲料+300mg/kg剂量YJ。
图6显示了总皂苷化合物YJ对于肥胖小鼠肠道菌在门水平下菌群多样性的影响，HF代表高脂饲料肥胖模型组(小鼠编号为HF 1-8)；HF+YJ代表高脂饲料+300mg/kg剂量YJ(小鼠编号为HF_YJ 1-8)。
图7显示了总皂苷化合物YJ对于肥胖小鼠肠道菌在属水平下菌群多样性的影响，HF代表高脂饲料肥胖模型组(小鼠编号为HF 1-8)；HF+YJ代表高脂饲料+300mg/kg剂量YJ(小鼠编号为HF_YJ 1-8)。
图8显示了PCA分析总皂苷化合物YJ对于肥胖小鼠肠道菌菌群多样性的影响，HF代表高脂饲料肥胖模型组(小鼠编号为HF 1-8)；HF+YJ代表高脂饲料+300mg/kg剂量YJ(小鼠编号为HF_YJ 1-8)。
具体实施方式
本申请的发明人经过对绞股蓝的化学成分及其生物活性进行广泛而深入地研究，从绞股蓝中成功分离得到多个新的三萜皂苷类化合物，并首次发现该三萜总皂苷化合物YJ及其一种或多种单体化合物具有通过改善动物肠道菌群防治肥胖的功能。在此基础上，完成了本发明。
术语说明
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
绞股蓝
绞股蓝为葫芦科植物绞股蓝(Gynostemma pentaphyllum(Thunb.)Makino)的干燥全草，为多年生攀缘草本。绞股蓝具有悠久的食用历史，始载于明代的《救荒本草》作野菜食用。日本民间则称其为“甘茶蔓”，作甜茶代用品或糖尿病人的甜味剂。绞股蓝也是五加科外具有人参皂苷资源的植物之一，具有滋补保健、抗癌防衰、降血脂和降血糖等多种功能，被誉为“南方人参”。绞股蓝作为***公布的可用于保健品的植物之一，因其保健功效显著，且安全性很高，适于长期服用，在民间常被当做降脂保健茶饮用。
总皂苷化合物YJ
本发明的总皂苷化合物YJ，包括但不限于提取自绞股蓝及其类似药物的皂苷化合物，即一系列具有皂苷结构的化合物。
在另一优选例中，本发明所述的总皂苷化合物包括一种或多种选自下组的化合物：
Figure PCTCN2017111690-appb-000004
Figure PCTCN2017111690-appb-000005
Figure PCTCN2017111690-appb-000006
Figure PCTCN2017111690-appb-000007
肠道菌与肥胖
肠道菌群组成同肥胖密切相关，在动物水平上研究表明ob/ob肥胖小鼠同正常小鼠相比较，肠道菌生物多样性分析中，厚壁菌门细菌增多，拟杆菌门的数量减少50％；在人体水平上，通过对12名肥胖患者进行跟踪研究，分别给予脂肪限制的饮食和碳水化合物限制的饮食进行治疗。一年后前者体重下降2％，后者体重下降6％，进行16s rRNA基因研究表明，摄入食物的总能量不变的前提下，通过两种饮食减肥后，肠道内的厚壁菌门数量下降，而拟杆菌门数量上升。数据表明肠道内占主要比例的拟杆菌门/厚壁菌门比值上升有助于防治肥胖。
Akkermansia muciniphila
Akkermansia muciniphila属于疣微菌科(Verrucomicrobiaceae)，2013年5月，比利时研究人员在PNAS上发表文章，通过研究一种肠道细菌发现一种“减肥细菌”，含有这种细菌的液体培养基能大大改变肥胖老鼠的健康状况。比利时鲁汶天主教大学的研究人员给比正常老鼠的脂肪多两至三倍的老鼠喂了一种被称为Akkermansia muciniphila的细菌，在不改变其饮食的情况下，它们的多余体重减少了近一半，而且其体内II型糖尿病的主要症状胰岛素抗体水平也降低了。研究表明Akkermansia muciniphila增加了控制葡萄糖和肠道平衡的内源性***素的肠道水平，并且阻碍了饮食诱导的粘膜屏障厚度的减少。同年，研究人员在Gut上发表文章，二甲双胍可明显提高Akkermansia  Muciniphila数量来改善高脂喂养小鼠的血糖谱。与单纯高脂喂养组相比，二甲双胍处理的高脂喂养组小鼠体内有较多的黏蛋白降解细菌Akkermansia。此外，二甲双胍治疗组中产生黏蛋白的杯状细胞数目亦显著增加。给高脂喂养的小鼠口服Akkermansia Muciniphila，即使不服用二甲双胍也可明显改善糖耐量，其可通过诱导内脏脂肪组织中的Foxp3调节T细胞(Tregs)而减轻脂肪组织炎症。因此，利用这种细菌来预防或治疗肥胖症和II型糖尿病成为潜在的防治肥胖的策略。
制备方法
在另一优选例中，本发明的三萜皂苷类化合物的制备方法，包括以下步骤：
(1)将四倍体绞股蓝地上部分用溶剂提取，浓缩提取液得浸膏；
(2)将浸膏加水溶解得分散液，依次用石油醚、乙酸乙酯、正丁醇进行萃取，萃取后得到正丁醇萃取液，减压回收溶剂，得正丁醇部位浸膏；
(3)将正丁醇部位浸膏水溶解后，上样于D101大孔树脂柱上，先纯水洗脱清除糖及水溶性杂质，然后采用乙醇-水梯度洗脱，收集不同洗脱部位，跟踪检测，旋转蒸发浓缩体积，得到粗皂苷干粉。然后称溶解纯甲醇溶液上样Sephadex LH-20分离柱，等梯度洗脱，减压回收溶剂后进行Sephadex LH-20葡聚糖凝胶色谱，以甲醇溶液进行等度洗脱，收集含所述化合物的流份，将流份直接以半制备型反相高效液相色谱进行分离纯化，得到所述化合物；
在另一优选例中，所述步骤(1)中为将四倍体绞股蓝的地上部分用溶剂提取2-5次，每次1-3小时，浓缩提取液得浸膏；
在另一优选例中，所述步骤(1)中的溶剂选自甲醇、乙醇、丙酮或水中的一种或多种的混合物；
在另一优选例中，所述步骤(2)中的浸膏与水的质量比为1:2-1:15。
在另一优选例中，所述步骤(2)中石油醚的加入量与分散液的体积比为1:2-5:1；
在另一优选例中，所述步骤(2)中乙酸乙酯的加入量与分散液的体积比为1:2-5:1；
在另一优选例中，所述步骤(2)中正丁醇的加入量与分散液的体积比为1:2-5:1。
在另一优选例中，所述步骤(3)中，乙醇-水溶液以体积比20:1-100:1进行梯度洗脱；再通过Sephadex LH-20柱色谱进行分离，以纯甲醇溶剂依次洗脱，收集含所述化合物的流份；一个流份通过半制备型反相高效液相色谱以30％乙腈-15％甲醇-55％水(vol)的溶剂***进行分离纯化，依次得到皂苷化合物1-9。
用途
通过考察本发明总皂苷化合物对肠道菌群拟杆菌门/厚壁菌门比例的影响，尤其是能够显著提高疣微菌科中的减肥细菌(Akkermansia muciniphila)，并对其作用机制研究，研究发现本发明化合物及它们的光学异构体、药用盐、溶剂合物均能显著调节高 脂饮食引起的肠道菌组来防治肥胖。
此外，本发明的化合物还可用作食品辅料或食品添加剂，添加到食品中，用于改善动物肠道菌群，促进有益微生物定居的作用。
药物组合物
本发明还提供了一种药物组合物，它包含安全有效量范围内的活性成分，以及药学上可接受的载体。
本发明所述的“活性成分”是指本发明所述的绞股蓝总皂苷化合物。
本发明所述的“活性成分”和药物组合物可用于改善肠道菌群组成，促进有益微生物定居。
在另一优选例中，用于制备可改善动物肠道菌群的药物。
“安全有效量”指的是：活性成分的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg活性成分/剂，更佳地，含有10-200mg活性成分/剂。较佳地，所述的“一剂”为一个药片。
“药学上可接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺和，而不明显降低活性成分的药效。
通常的，载体包括稀释剂、填充剂、崩解剂、润滑剂、着色剂、调味剂或其它常规添加剂中的一种或多种。
药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2017111690-appb-000008
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明的活性成分或药物组合物的施用方式包括口服等。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。
在这些固体剂型中，活性成分与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和***胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
所述的固体剂型还可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可 包含不透明剂，并且，这种组合物中活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明化合物可以单独给药，或者与其他治疗药物联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1-2000mg，优选20-500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明提到的上述特征，或实施例提到的特征可以任意组合。本案说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
本发明的主要优点在于：
(1)本发明通过从四倍体绞股蓝中提取分离得到结构新颖的三萜皂苷类化合物，该化合物及它们的光学异构体、药用盐、溶剂合物具有用于改善动物肠道菌群，促进有益微生物定居及防治肥胖的用途，它们可以以其自身的形式施用，也可以和药学上可以接受的载体组成药物组合物而应用；
(2)本发明提供制备方法简单易行，可在通用设备中完成；
(3)制备所得化合物纯度高，具有良好经济效益和应用前景。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量 百分比和重量份数。
以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1
1.1制备四倍体绞股蓝总皂苷YJ
称取18kg四倍体绞股蓝叶片经粉碎机粉碎后，采用20L的95％乙醇回流法提取(料液比为1:2.6)，提取3次，时间分别为3h、2h、1h，采用旋转蒸发去除乙醇得到绞股蓝粗提物
Figure PCTCN2017111690-appb-000009
膏。
将上述浸膏加水分散，依次用等体积的石油醚、乙酸乙酯、正丁醇萃取，分别得到石油醚、乙酸乙酯、正丁醇和水提取部位。旋转蒸发最终得绞股蓝粗提物正丁醇部位
Figure PCTCN2017111690-appb-000010
膏1509g。
配制6mg/mL正丁醇部位样品溶液，共计20mL体积，上样D101大孔树脂，采用20％、40％、60％的乙醇梯度洗脱，收集60％洗脱部位，旋转蒸发浓缩体积，得到粗皂苷干粉。然后称量15g粗皂苷溶解于55mL甲醇溶液上样Sephadex LH-20分离柱，等梯度洗脱进一步纯化，实时监测，收集样品得到精制皂苷总皂苷化合物YJ。
1.2动物实验
SPF级的5周龄雄性C57/BL 6小鼠(购于北京维通利华实验动物中心)，在SPF环境下饲养。
1.2.1预防实验
32只小鼠适应一周后，分为4组，每组8只，单笼饲养。
分别为A组(低脂组，10％能量来源于脂肪)、B组(高脂组，45％能量来源于脂肪)、C组(高脂组+100mg/Kg/d YJ)、和D组(高脂组+300mg/Kg/d YJ)。
饲养环境温度为22±2℃，湿度为30-70％，小鼠自由进食和饮水；实验历时12周，每天记录体重，每周二、周五重新换新食物和无菌水，并做好称量记录，记录进食量。每周换老鼠垫料，每月换饲养笼具。12周后，连续收集3天粪便量，每组随机选择4只小鼠，提取DNA，进行Illumina Miseq PE250/PE300测序，对粪便样本菌群多样性进行分析。
1.2.2动物治疗实验
高脂饲料喂食小鼠4个月，体重40g左右，剔除肥胖抵抗型小鼠后，随机分为2组，每组8只，单笼饲养。
模型组(HFD)继续喂食高脂饲料，干预组(HFD+YJ)除喂食高脂饲料外，软管灌胃300mg/Kg/d剂量总皂苷化合物，持续时间2个月，每天记录体重，每周二、周五重新换新食物和无菌水，并做好称量记录，记录进食量。每周换老鼠垫料，每月换饲养笼具。2个月后，连续收集所有小鼠3天粪便量，提取DNA，进行Illumina Miseq PE250/PE300 测序,对粪便样本菌群多样性进行分析。
1.3结果分析
从图1中可以看出，长时间食用高脂饲料同低脂饲料相比，小鼠体重逐渐增加，在56天后达到显著性水平，添加YJ干预后，同高脂模型组相比，体重的增加受到较强抑制，在63天后达到显著性水平并呈现出浓度剂量关系。
从图2中可以看出，同低脂饲料相比，长时间食用高脂饲料会导致肠道菌群拟杆菌门/厚壁菌门比例显著下降，添加YJ干预后，拟杆菌门/厚壁菌门比例开始上升，在高剂量条件下达到显著性提高。并且，高剂量干预组诱导疣微菌科(Verrucomicrobiaceae)和提高变形菌门(Proteobacteria)数量。进一步对肠道菌-属进行归类，高剂量总皂苷化合物能够显著诱导疣微菌科中Akkermansia muciniphila这一减肥细菌的显著提高(图3)。采用统计学分析PCOA，总皂苷化合物干预能够显著改善高脂饮食引起的小鼠肠道菌组成改变(图4)。
由图3可以看出，YJ对以下菌株的多样性影响较大：拟杆菌、Akkermansia muciniphila、乳酸菌等。
由图5可知，添加了300mg/kg YJ干预肥胖28天后，体重出现显著性的降低，证明YJ具有显著性的治疗肥胖作用。
动物治疗实验结果如图6所示，可以看出，使用YJ干预小鼠，疣微菌科(Verrucomicrobiaceae，蓝色标志)得到显著诱导表达，进一步在属水平下，进一步证明了总皂苷化合物的减肥以及其对于Akkermansia muciniphila的诱导作用。所以YJ为特异性促进Akkermansia muciniphila增殖的益生元物质。给予300mg/kg YJ能够显著增加Akkermansia muciniphila的含量同对照组相比较。
图7显示了YJ对于肥胖小鼠肠道菌在属水平下菌群多样性的影响，同样地，Akkermansia muciniphila得到显著提高，借助PCA分析，能够将两组完整分开，证明YJ在调节肥胖小鼠肠道菌群组成，防治肥胖的明显作用(图8)。
结合前述的预防实验，我们可以判断总皂苷化合物防治肥胖的作用是由于定向诱导“瘦细菌”Akkermansia muciniphila起作用的。
实施例2：食品
按以下配方配制减肥食品：
燕麦片2-5重量份、山药1-5重量份、膳食纤维10-15重量份、薏米1-4重量份、肌醇2-5重量份、麦芽糖10-15重量份、YJ 0.1％-0.5％重量份
一日3餐，连续7-15天。
实施例3：药物组合物
Figure PCTCN2017111690-appb-000011
将上述组分混合均匀，直接压片，即得片剂组合物，每次口服1片，一日2-3次。
实施例4：药物组合物
Figure PCTCN2017111690-appb-000012
将上述组分混合物均匀填充成1000个胶囊，每次口服1粒，一日2-3次。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种绞股蓝总皂苷化合物的用途，其特征在于，
    (1)用于制备改善肠道菌群的组合物；和/或
    (2)用于制备促进特征微生物定居的组合物。
  2. 如权利要求1所述的用途，其特征在于，所述肠道菌群属于厚壁菌门或拟杆菌门。
  3. 如权利要求1所述的用途，其特征在于，所述肠道菌群属于疣微菌门或变形菌门。
  4. 如权利要求1所述的用途，其特征在于，所述肠道菌群选自下组菌株：乳酸菌、双歧杆菌或柔嫩梭菌。
  5. 如权利要求1所述的用途，其特征在于，所述肠道菌群为Akkermansia muciniphila。
  6. 如权利要求1所述的用途，其特征在于，所述组合物为药物组合物或食品组合物。
  7. 如权利要求1所述的用途，其特征在于，所述绞股蓝为二倍体绞股蓝或四倍体绞股蓝，较佳地为四倍体绞股蓝。
  8. 如权利要求1所述的用途，其特征在于，所述总皂苷化合物包括一种或多种选自下组的化合物：
    Figure PCTCN2017111690-appb-100001
    Figure PCTCN2017111690-appb-100002
    Figure PCTCN2017111690-appb-100003
  9. 一种体外非治疗性地改善肠道菌群的方法，包括步骤：在需要处理的场所施用如权利要求1所述的化合物或其药学上可接受的盐。
  10. 一种益生菌组合物，所述益生菌组合物包括：
    (1)第一药物：皂苷类化合物，和
    (2)第二药物：一种或多种选自下组的益生菌：乳酸菌、双歧杆菌、柔嫩梭菌、Akkermansia muciniphila、或其组合；
    其中，所述第一药物和第二药物位于相同或不同的容器中。
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