Methods of making and uses of compositions targeting gut microbiota-ucp1 axis

Abstract

The application belongs to the technical field of medicine, and discloses the use of a composition targeting the intestinal flora-UCP1 axis in preventing and / or treating metabolic syndrome and complications thereof. Specifically, the application relates to the use of tea extract, panax notoginseng extract and dendrobium extract in combination as active ingredients in the treatment of metabolic syndrome and complications thereof, and the use of any two or more of tea extract, panax notoginseng extract and dendrobium extract as active ingredients in combination with other active ingredients and excipients in the preparation of a compound drug preparation for clinical use in a daily dosage of 0.001-2000 mg / kg body weight. The application has a synergistic effect, is used for treating metabolic syndrome and complications thereof, reduces the dosage of a single agent, improves the prevention and treatment effect, reduces side effects, lowers the cost, and meets the basic requirements of health economics.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and relates to an application of a composition of scientific proportioning of tea extract, notoginseng extract and dendrobium extract in preparing a medicine for preventing and / or treating metabolic syndrome and complications thereof by targeting an intestinal flora-UCP1 axis. The application mainly relates to an application of the composition of the tea extract, the notoginseng extract and the dendrobium extract in preparing the medicine for preventing and / or treating metabolic syndrome and complications thereof. BACKGROUND

[0002] Intestinal flora often changes with the change of the physiological state of the host, and vice versa. The intestinal flora can affect the metabolic function of the host by affecting the digestion and absorption of food, the synthesis and release of inflammatory factors and other pathways. Intestinal microorganisms can affect the host gene expression through their metabolites and regulate the metabolism of the body. For example, Akkermansia muciniphila can directly improve the metabolic health of the host. It can produce various fermentation products, including SCFA, by degrading mucin, combine with Toll-like receptors on the intestinal epithelial cells, promote the proliferation of epithelial cells, the secretion of IgA into the intestinal cavity and the expression of antibacterial peptides, thereby improving metabolic disorders. The characteristics of intestinal flora include the diversity of the overall flora and the relative abundance of specific flora and strains. Unhealthy living habits represented by a high-fat diet can lead to a decrease in the species diversity of the flora, and such unhealthy characteristics can directly act on the body by affecting intestinal digestion, increasing immune stimulation, damaging mutualistic relationships and other ways, leading to unbalanced blood glucose and blood lipid regulation, blocked thermogenesis, hormone metabolism disorders and other adverse consequences. The abundance ratio of Firmicutes and Bacteroidetes has been proved by multiple studies to be negatively correlated with obesity and other metabolic-related diseases. In addition, studies have shown that the diversity of the intestinal flora of patients with metabolic syndrome is significantly reduced, and the relative abundance of some specific species, including Firmicutes, Bacteroidetes and Akkermansia, is obviously changed. The change in the relative abundance of these flora is closely related to metabolic disorders, and by changing the abundance of these flora, the metabolic state of the host can be significantly improved. In addition, Bacteroidetes and Verrucomicrobia, as producers of short-chain fatty acids, can produce propionate and acetate through interorgan interaction, and systemically promote the thermogenesis of the body to resist metabolic syndrome.

[0003] Recent studies have shown that brown adipose tissue has non-shivering thermogenesis function, which is derived from its rich mitochondria and specific high expression of mitochondrial inner membrane uncoupling protein 1 (UCP1). UCP1 promotes the leakage of protons from the intermembrane space of mitochondria into the mitochondria, thereby making the mitochondrial electron transport chain produce electrochemical potential energy and dissipate in the form of heat energy. In addition, under certain conditions, white fat can also exhibit the characteristics of brown fat, a process known as "browning". Brown fat activation and white fat browning can promote energy consumption in the body and improve glucose and lipid metabolism, and may be a new way to treat metabolic syndrome. Professor Kajimura of UCSF in the United States believes that as long as a drug that increases UCP1 in adipose tissue is found, the purpose of reducing body fat and resisting obesity and its metabolic syndrome will be achieved. However, the development of drugs to promote brown fat activation and white fat browning has not made substantial progress. Therefore, we have constructed a stable transgenic brown adipocyte cell line with Luciferase and tdTomato inserted into the UCP1 promoter region, optimized the high-throughput screening model for UCP1 agonists, and have applied for a national invention patent "Establishment and application of genetically engineered cell lines and high-throughput drug screening models for anti-metabolic syndrome drug target UCP1" (Application No. CN201811248715.3) for finding new UCP1 agonists that play an anti-metabolic syndrome role through thermogenic effect.

[0004] Metabolic syndrome (MetS) refers to a pathological state of metabolic disorder of substances such as protein, fat, and carbohydrate in the human body, which is a complex metabolic disorder syndrome and a risk factor for diabetes and cardiovascular and cerebrovascular diseases. It has the following characteristics: ① multiple metabolic disorders in one body: including obesity, hyperglycemia, hypertension, dyslipidemia, hyperuricemia, fatty liver, and hyperinsulinemia, which are the pathological basis of cardiovascular and cerebrovascular diseases and diabetes. ② Common pathological basis: the common cause is currently believed to be insulin resistance and hyperinsulinemia caused by obesity, especially central obesity. ③ Can increase multiple diseases: such as hypertension, coronary heart disease, stroke, and even certain cancers, including sex hormone-related breast cancer, endometrial cancer, prostate cancer, and digestive system cancers such as pancreatic cancer, hepatobiliary cancer, and colon cancer. ④ Common prevention and treatment measures: preventing and treating one metabolic disorder is also beneficial to the prevention and treatment of other metabolic disorders.

[0005] Metabolic syndrome has become a worldwide public health problem with increasing incidence. The prevalence of metabolic syndrome worldwide ranges from 10% to 84%, and approximately 20% to 25% of adults have metabolic syndrome. According to the National Health and Nutrition Examination Survey data, in the United States, 35% of adults and 50% of people over 60 years old (30.3% of men and 35.6% of women) have been diagnosed with metabolic syndrome; among them, Mexican American women have the highest incidence. According to the International Diabetes Federation diagnostic criteria, in Europe, the prevalence of metabolic syndrome in men is about 41%, and in women, about 38%. Statistics in China show that the prevalence of metabolic syndrome in people aged 15 and above is 24.2%. Another study shows that the prevalence of metabolic syndrome in people aged 60 and above in China is about 32.4%. Metabolic syndrome is prone to cause many complications, such as cardiovascular and cerebrovascular diseases, cancer, liver cirrhosis, polycystic ovary syndrome, and kidney and pancreatic dysfunction. However, there is currently no effective treatment for metabolic syndrome, and the current recommended regimen is basically based on single prevention and treatment of each risk factor, such as lipid-lowering, glucose-lowering, and improvement of fatty liver, etc. Therefore, from the perspective of promoting peripheral heat production and increasing energy consumption, it is of great significance to find a safe and effective comprehensive anti-metabolic syndrome drug with Chinese independent intellectual property rights and based on the comprehensive and overall health concept.

[0006] Tea extract has been found to have good effects on reducing blood sugar and blood lipids, and by improving intestinal metabolites, inhibiting fat absorption and generation, improving oxidative stress, and inhibiting lipase, it can reduce body weight. It is also used to prepare compositions for reducing blood sugar, regulating blood lipids, and improving digestion, as described in the following Chinese patents (Publication Nos. CN105709082A, CN104012712B, CN201911359529, CN104982586A). Panax notoginseng is a medicinal plant of the genus Panax in the family Araliaceae, and is the main component of Yunnan Baiyao. Since ancient times, there has been the saying that "Panax ginseng is the first to supplement qi, and Panax notoginseng is the first to invigorate blood." Traditional Chinese medicine believes that Panax notoginseng is warm in nature, sweet and slightly bitter in taste, and belongs to the liver and stomach meridians, and has the effects of dispersing blood stasis, stopping bleeding, and relieving swelling and pain. Modern pharmacological studies have found that Panax notoginseng extract has the effects of antithrombosis, antibacterial, anticancer, antidepressant, and anti-diabetic retinopathy, and has been used to prepare traditional Chinese medicine compositions for treating fatty liver, as described in the following Chinese patents (Publication Nos. CN103599427A, C1099254544A, CN113663010A, etc.). Studies have shown that 20 mg / kg of Panax notoginseng saponins and Panax notoginseng extract have no effect on reducing the body weight of diet-induced obese mice (Chin Med. 2022; 17(1): 75.). However, no reports have been found on metabolic syndrome. Dendrobium extract has been proven to reduce high-fat diet-induced kidney damage in mice by regulating lipid-induced oxidative stress, and to improve liver glucose metabolism in type II diabetic mice through a glucagon-mediated signaling pathway. In addition, studies have shown that Dendrobium polysaccharides have a certain therapeutic effect on polycystic ovary syndrome in rat models, and have been used to treat metabolic diseases (Publication Nos. CN111773324A, CN111729037A, CN110604789A). They have also been used to prepare traditional Chinese medicine compositions with the effects of reducing blood sugar, improving insulin resistance, or regulating blood lipid metabolism (Publication Nos. CN112457423A, CN112426488A). CN111773324A and CN111729037A use Dendrobium extract to treat metabolic diseases, but only describe the mechanism at the cellular level, including the inhibitory effect of sterol regulatory element binding protein 1 and insulin-induced gene expression. The data in CN110604789A are less than the size of the indicated population, and the improvement effect on diabetes and metabolic syndrome is not very obvious. The examples lack mechanism-related evidence. CN112457423A only focuses on the improvement of insulin resistance, and the pharmacological effect is attributed to the insulin PI3K / AKT signal transduction pathway. CN112426488A lacks mechanism-related research and contains dozens of ingredients in the composition, which is complex and some of the described effects cannot be supported by the published data. Furthermore, there is a lack of mechanism research.

[0007] In summary, the prior art does not disclose the use of the combination of the above three in the prevention and / or treatment of metabolic syndrome and its complications by targeting the intestinal flora-UCP1 axis. The present application aims to prepare a composition targeting the intestinal flora-UCP1 axis by scientifically proportioning tea extract, panax notoginseng extract and dendrobium extract, so as to achieve synergistic pharmacological activity, and ultimately achieve the effect of resisting metabolic syndrome. SUMMARY

[0008] The present application aims to solve the problems of the prior art, and provides a composition for preventing and / or treating metabolic syndrome and its complications by targeting the intestinal flora-UCP1 axis, which has scientific and reasonable proportion, definite curative effect, safety in clinical use and low adverse reactions. Another object of the present application is to provide a preparation method of the composition and the use of the composition in the preparation of a drug for preventing and / or treating metabolic syndrome and its complications.

[0009] In order to achieve the above objects, the technical solutions adopted by the present application are as follows:

[0010] The composition provided by the present application is made of the following raw materials: tea extract, panax notoginseng extract and dendrobium extract. The mixing ratio of each component can be: tea extract 1% to 98%, panax notoginseng extract 1% to 98%, and dendrobium extract 1% to 98%. Preferably, the mixing ratio of tea extract, panax notoginseng extract and dendrobium extract is 60% to 84%, 8% to 28% and 5% to 15% respectively. More preferably, the mixing ratio of tea extract, panax notoginseng extract and dendrobium extract is 70-74%, 16-21% and 8-12% respectively.

[0011] The second composition provided by the present application is made of the following raw materials: tea extract and panax notoginseng extract. The mixing ratio of each component can be: tea extract 1% to 99%, and panax notoginseng extract 1% to 99%. Preferably, the mixing ratio of tea extract and panax notoginseng extract is 60% to 84% and 16% to 40% respectively. More preferably, the mixing ratio of tea extract and panax notoginseng extract is 65-75% and 25-35% respectively.

[0012] The third composition provided by the present application is made of the following raw materials: panax notoginseng extract and dendrobium extract. The mixing ratio of each component can be: panax notoginseng extract 1% to 99%, and dendrobium extract 1% to 99%. Preferably, the mixing ratio of panax notoginseng extract and dendrobium extract is 60% to 84% and 16% to 40% respectively. More preferably, the mixing ratio of panax notoginseng extract and dendrobium extract is 65-75% and 25-35% respectively.

[0013] The fourth composition provided by the present application is made of the following raw materials: tea extract and dendrobium extract. The mixing ratio of each component can be: tea extract 1% to 99%, and dendrobium extract 1% to 99%. Preferably, the mixing ratio of tea extract and dendrobium extract is 60% to 84% and 16% to 40% respectively. More preferably, the mixing ratio of tea extract and dendrobium extract is 65-75% and 25-35% respectively.

[0014] The total daily dose of the above composition is in the range of 0.001-2000 mg / kg body weight, preferably 0.01-1000 mg / kg, more preferably 0.1-500 mg / kg, and most preferably 1-300 mg / kg.

[0015] The present application shows that the composition of tea extract, Panax notoginseng extract and Dendrobium extract has the effect of improving metabolic syndrome and its complications, and is suitable for, but not limited to, the treatment of obesity, abnormal glucose metabolism, abnormal lipid metabolism, fatty liver, hypertension, high blood viscosity, hyperuricemia or gout, and the curative effect is better than that of positive drug metformin.

[0016] The metabolic syndrome is selected from obesity, insulin resistance or hyperinsulinemia, abnormal glucose metabolism, abnormal lipid metabolism, fatty liver, hypertension, high blood viscosity, hyperuricemia or gout. The complications caused by the metabolic syndrome include cardiovascular and cerebrovascular diseases, cancer, liver cirrhosis, polycystic ovary syndrome, and renal and pancreatic dysfunction.

[0017] As a preferred solution, the above-mentioned composition, the tea extract is prepared by using Camellia sinensis (L.) O. Ktze. or Camellia sinensis var. assamica tea raw materials, and according to the requirements of GB / T30766-2014, it is prepared into green tea, white tea, yellow tea, oolong tea, black tea or black tea. After impurity removal, crushing, extraction, centrifugation, filtration, concentration and other steps, finally freeze-drying is carried out to obtain an extract with a particle size of 0.2-0.5 μm.

[0018] As a preferred solution, the above-mentioned composition, the Panax notoginseng extract is prepared by taking 5 kg of roots of Panax notoginseng (Burkill) F. H. Chen ex C. Chow & W. G. Huang of Araliaceae genus Panax, removing impurities, crushing to a particle size of 1-2 mm, adding 25 liters of distilled water, extracting at a temperature of 80℃ for 3 hours, continuously extracting for 3 times, combining the extract, performing vacuum concentration at 50℃ and 3.0 KPa to obtain a vacuum concentrated liquid, and finally freeze-drying the vacuum concentrated liquid at-40℃ and 1.0 KPa to obtain a Panax notoginseng extract with a particle size of 0.1-0.3 μm, wherein the total saponin content of Panax notoginseng is greater than 20%.

[0019] As a preferred solution, the composition described above is prepared by the following method: 10 kg of stems of Dendrobium officinale Kimura et Migo are taken, impurities are removed, and the stems are crushed to a particle size of 1-2 mm, 50 liters of distilled water are added, and the mixture is heated to extract, the extraction temperature is 65°C, and the extraction time is 3 hours, the extraction is repeated 3 times in succession, the extract is combined, and the combined extract is concentrated under reduced pressure at 55°C and 5.0 KPa to obtain a reduced-pressure concentrated extract, the reduced-pressure concentrated extract is finally freeze-dried at -45°C and 609 Pa, and the freeze-dried product is crushed and sieved to obtain a Dendrobium officinale extract with a particle size of 0.5-1.0 μm, wherein the content of Dendrobium officinale polysaccharide is greater than 15%.

[0020] The present application also relates to a pharmaceutical compound preparation for preventing and / or treating metabolic syndrome and complications thereof, characterized in that the active ingredients of the compound preparation comprise at least two of tea extract, notoginseng extract and Dendrobium extract, and further comprise a pharmaceutically acceptable carrier. Therefore, the pharmaceutical compound preparation of the present application comprises, in addition to tea extract, notoginseng extract and Dendrobium extract as the active ingredients, other active ingredients and pharmaceutically acceptable excipients.

[0021] The pharmaceutical compound preparation comprises tablets, capsules, pills, injections, sustained-release pharmaceutical preparations and controlled-release pharmaceutical preparations.

[0022] The pharmaceutically acceptable carrier comprises starch, dextrin, sodium polymethylcellulose, magnesium stearate and talc.

[0023] The present application therefore also relates to a pharmaceutical compound preparation comprising the composition of the present application as the active ingredient. The pharmaceutical compound preparation can be prepared according to methods known in the art. The composition of the present application can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants to form any dosage form suitable for human or animal use.

[0024] The composition of the present application or the pharmaceutical compound preparation comprising the same can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina and rectum.

[0025] The administration form can be a liquid form, a solid form or a semi-solid form. The liquid form can be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type and multiple emulsion), a suspension, an injection (including water injection, powder injection and infusion), eye drops, nose drops, lotion and liniment, etc.; the solid form can be a tablet (including ordinary tablet, enteric-coated tablet, chewable tablet, dispersible tablet, effervescent tablet, oral disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric-coated capsule), a granule, a powder, a pellet, a drop, a suppository, a film, a patch, an aerosol (powder) spray, a spray, etc.; the semi-solid form can be an ointment, a gel, a paste, etc.

[0026] The composition of the present application can be prepared into a common preparation, a sustained-release preparation, a controlled-release preparation, a targeted preparation and various micro-particle administration systems.

[0027] In order to prepare the composition of the present application into a tablet, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropyl alcohol, etc.; the binders can be starch paste, dextrin, sugar syrup, honey, glucose solution, microcrystalline cellulose, acacia paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0028] The tablet can be further prepared into a coated tablet, such as a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, or a double-layer tablet and a multi-layer tablet.

[0029] In order to prepare the administration unit into a capsule, the effective component of the composition of the present application can be mixed with diluents and glidants, and the mixture can be directly placed into a hard capsule or a soft capsule. Alternatively, the effective component of the composition of the present application can be first mixed with diluents, binders and disintegrants to prepare granules or pellets, and then placed into a hard capsule or a soft capsule. The various diluents, binders, wetting agents, disintegrants and glidants used for preparing the tablet of the composition of the present application can also be used for preparing the capsule of the composition of the present application.

[0030] To prepare the injection of the composition of the present application, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as the solvent, and an appropriate amount of solubilizers, co-solvents, pH adjusting agents, osmotic pressure adjusting agents, solubilizers or co-solvents commonly used in the art can be added. The solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjusting agents can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure adjusting agents can be sodium chloride, mannitol, glucose, phosphate, acetate, etc.; if a lyophilized powder injection is prepared, mannitol, glucose, etc. can also be added as a supporting agent.

[0031] In addition, if necessary, coloring agents, preservatives, flavors, taste correctors or other additives can also be added to the pharmaceutical preparation.

[0032] To achieve the purpose of medication and enhance the therapeutic effect, the composition or pharmaceutical combination preparation of the present application can be administered by any known administration method.

[0033] The administration dose of the composition of the present application can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the administration route and dosage form, etc. Generally, the suitable dose of the composition of the present application per day ranges from 0.001 to 2000 mg / kg of body weight, preferably from 0.01 to 1000 mg / kg of body weight, more preferably from 0.1 to 500 mg / kg of body weight, and most preferably from 1 to 300 mg / kg of body weight. The composition of the present application is shown to have an effect of improving metabolic syndrome, and is suitable for, but not limited to, the treatment of obesity, abnormal glucose metabolism, abnormal lipid metabolism, fatty liver, hypertension, hyperviscosity, hyperuricemia or gout. The above-mentioned dose can be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the doctor and the administration regimen including the use of other therapeutic methods. The composition or pharmaceutical combination preparation of the present application can be taken alone or in combination with other therapeutic drugs or symptomatic treatment. When the composition of the present application has a synergistic effect with other therapeutic drugs, the dose thereof should be adjusted according to the actual situation.

[0034] The technical solution of the present application provides the use of the composition in the preparation of a drug for preventing or treating metabolic syndrome and its complications. The metabolic syndrome includes, but is not limited to, obesity, abnormal glucose metabolism, abnormal lipid metabolism, fatty liver, hypertension, hyperviscosity, hyperuricemia or gout. The obesity refers to the body mass index (BMI) of a person being greater than or equal to 28 kg / m 2; the insulin resistance refers to the decrease of the efficiency of insulin in promoting glucose uptake and utilization, and the body compensates for it by hyperinsulinemia; the abnormal glucose metabolism refers to the blood glucose value being higher than the normal value; the abnormal glucose metabolism includes insulin resistance or hyperinsulinemia, abnormal glucose tolerance, prediabetes, diabetes, diabetes complications, and the diabetes complications include diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic liver disease, etc.; the abnormal lipid metabolism refers to the concentration of at least one blood lipid index being increased (decreased) or the abnormal deposition of fat in any tissue of the body; the blood lipid index is selected from triglyceride, total cholesterol, low-density lipoprotein, high-density lipoprotein, free fatty acid, including hypertriglyceridemia, hypercholesterolemia, high low-density lipoprotein, low high-density lipoprotein, and hyperlipidemia; the fatty liver refers to the pathological change of excessive accumulation of fat in liver cells, including non-alcoholic fatty liver disease and non-alcoholic fatty liver hepatitis; the hypertension refers to the increase of arterial blood pressure in the systemic circulation, and the systolic pressure is greater than or equal to 140 mmHg, and the diastolic pressure is greater than or equal to 90 mmHg; the hypercoagulability is the increase of one or more blood viscosity factors, which causes excessive blood viscosity and slow blood flow, and is manifested as abnormal blood rheology parameters; the blood rheology parameters are selected from whole blood viscosity, plasma viscosity, hematocrit, whole blood reduced viscosity, red blood cell aggregation index, red blood cell deformation index, red blood cell rigidity index, and erythrocyte sedimentation rate K value; the hyperuricemia refers to the abnormal increase of uric acid level in blood, which is higher than the upper limit of the normal value; the gout refers to the deposition of urate crystals in the joint caused by the high uric acid content in blood, i.e., hyperuricemia. The complications caused by the metabolic syndrome include cardiovascular and cerebrovascular diseases, cancer, cirrhosis, polycystic ovary syndrome, and renal and pancreatic dysfunction.

[0035] Beneficial technical effects:

[0036] Compared with the prior art, the present application is found through pharmacological research that:

[0037] The composition of tea extract, panax notoginseng extract and dendrobium extract has the value of in-depth research and development. The preparation method and the pharmacological activity of the composition have not been reported so far. Specifically, the beneficial technical effects are as follows:

[0038] (1) The tea extract, panax notoginseng extract and dendrobium extract composition of the present application is based on scientific special proportion, novel thinking and good safety;

[0039] (2) The preparation method is simple, the raw materials are easy to obtain, the preparation process is simple, and the standardized production is easy;

[0040] (3) The tea extract, the panax notoginseng extract and the dendrobium extract are combined in a scientific ratio in the present application, and the synergistic effect is obvious, the use amount of single agent can be reduced, the pharmacological activity is improved, the side effect is reduced, the cost is reduced, and the effect of preventing and treating metabolic syndrome and its complications is finally achieved;

[0041] (4) The mechanisms of tea, panax notoginseng and dendrobium are different, and after being combined in a scientific ratio, the three can extremely effectively activate the mitochondria-specific heat production marker uncoupling protein 1 (UCP1) in vitro, and can obviously promote the brown fat heat production and the white fat browning in vivo, and as the UCP1 activator, can be further developed into a natural heat production activator for promoting heat production, and has great potential in resisting metabolic syndrome and its complications;

[0042] (5) After the three extracts are combined in a scientific ratio, the intestinal flora can be obviously improved, specifically, the species richness of the flora can be restored, the Firmicutes and Proteobacteria can be reduced, the Bacteroidetes and Verrucomicrobia can be increased, the flora structure can be affected, and the whole body can be affected, so that the effect of finally losing weight, resisting metabolic syndrome and its complications is achieved. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Effect of the composition on the liver histopathology of the metabolic syndrome mice induced by high-fat feed after 13 weeks of administration

[0044] Figure 2 Effect of the composition on the heat dissipation of the metabolic syndrome mice induced by high-fat feed after 12 weeks of administration

[0045] Figure 3 Effect of the composition on the subcutaneous fat histopathology of the metabolic syndrome mice induced by high-fat feed after 13 weeks of administration

[0046] Figure 4 Effect of the composition on the visceral fat histopathology of the metabolic syndrome mice induced by high-fat feed after 13 weeks of administration

[0047] Figure 5 Effect of the composition on the brown fat histopathology of the metabolic syndrome mice induced by high-fat feed after 13 weeks of administration

[0048] Figure 6 Effect of the composition on the fecal flora alpha diversity analysis of the metabolic syndrome mice induced by high-fat feed after 12 weeks of administration

[0049] Figure 7 Effect of the composition on the fecal flora species distribution proportion of the metabolic syndrome mice induced by high-fat feed after 12 weeks of administration

[0050] Figure 8Effect of the composition administration for 12 weeks on the abundance of Akkermansia muciniphila strain in the fecal microbiota of high-fat diet-induced metabolic syndrome mice

[0051] Figure 9 Results of pathway enrichment analysis of the fecal microbiota of high-fat diet-induced metabolic syndrome mice administered with the composition for 12 weeks (model control group and normal control group)

[0052] Figure 10 Results of pathway enrichment analysis of the fecal microbiota of high-fat diet-induced metabolic syndrome mice administered with the composition for 12 weeks (model control group and composition 3 administration group)

[0053] Figure 11 Phylum level correlation network diagram of the fecal microbiota of high-fat diet-induced metabolic syndrome mice administered with the composition for 12 weeks

[0054] Figure 12 Phylum level correlation network diagram of the fecal microbiota of high-fat diet-induced metabolic syndrome mice administered with the composition for 12 weeks DETAILED DESCRIPTION

[0055] (I) Preparation experiment

[0056] Example 1, preparation method of black tea extract

[0057] Fresh leaves of Camellia sinensis var. Assamica in Yunnan were taken, and subjected to withering, rolling, fermentation, drying and other processes to produce black tea in strip shape. After impurity removal, the tea was crushed to a particle size of 0.5-2 mm, and then extracted at 70°C for 40 min. After 4 times of extraction, the extracts were combined, filtered through a 0.3 μm ceramic membrane, concentrated by reverse osmosis at 6 MPa, and finally freeze-dried at -35°C and 609 Pa. The crushed tea was sieved to obtain black tea extract with a particle size of 0.2-0.5 μm. The content of each component was as follows: tea polyphenols > 40%; theaflavins > 0.8%; thearubigins 8-15%; theabrownins 10-20%; and caffeine 8-15%.

[0058] Example 2, preparation method of Panax notoginseng extract

[0059] The roots of Panax notoginseng (Burkill) F. H. Chen ex C. Chow & W. G. Huang of Araliaceae were taken, and subjected to impurity removal and crushing to a particle size of 1-2 mm. Then 25 liters of distilled water was added, and the extraction was carried out at 80°C for 3 hours. The extraction was repeated for 3 times, and the extracts were combined. The combined extract was concentrated under reduced pressure at 50°C and 3.0 KPa to obtain a reduced pressure concentrated extract. Finally, the reduced pressure concentrated extract was freeze-dried at -40°C and 1.0 KPa. The crushed tea was sieved to obtain Panax notoginseng extract with a particle size of 0.1-0.3 μm. The content of total saponins in the Panax notoginseng extract was greater than 20%.

[0060] Example 3, Preparation method of Dendrobium officinale Kimura et Migo extract

[0061] Take 10 kg of Dendrobium officinale Kimura et Migo stems, remove impurities, crush to a particle size of 1-2 mm, add 50 liters of distilled water and heat to extract, the extraction temperature is 65°C, the extraction time is 3 hours, after continuous extraction for 3 times, combine the extract, perform vacuum concentration at 55°C and 5.0 KPa to obtain a vacuum concentrated extract, finally perform freeze drying of the vacuum concentrated extract at -45°C and 609 Pa, crush and sieve to obtain a Dendrobium officinale Kimura et Migo extract with a particle size of 0.5-1.0 μm, wherein the content of Dendrobium officinale Kimura et Migo polysaccharide is greater than 15%.

[0062] Example 4, Preparation method of green tea extract

[0063] Take fresh leaves of Camellia sinensis (L.) O. Ktze. or Camellia sinensis var. Assamica as raw materials, and prepare green tea through processes such as fixation, rolling, and drying. Remove impurities from the tea leaves, crush to a particle size of 0.5-2 mm, and extract at 80-90°C for 10-20 minutes, then sieve out tea residue using a 32-60 mesh sieve, and then perform centrifugal filtration using a 300 mesh filter (pore size <10 μm) and 4000 r / min. Concentrate using evaporation, freezing, membrane, and other technologies. Finally, perform spray drying or freeze drying, crush and sieve to obtain a green tea extract with a particle size of 0.2-0.5 μm. The content of each component is: tea polyphenols >25%, catechin >12%, and caffeine 3-12%.

[0064] Example 5, Preparation method of white tea extract

[0065] Take fresh leaves of specific tea tree varieties of Camellia sinensis (L.) O. Ktze. or Camellia sinensis var. Assamica as raw materials, and prepare white tea through processes such as fixation, rolling, yellowing, and drying. Remove impurities from the tea leaves, crush to a particle size of 0.5-2 mm, and extract at 80-90°C for 10-20 minutes, then sieve out tea residue using a 32-60 mesh sieve, and then perform centrifugal filtration using a 300 mesh filter (pore size <10 μm) and 4000 r / min. Concentrate using evaporation, freezing, membrane, and other technologies. Finally, perform spray drying or freeze drying, crush and sieve to obtain a white tea extract with a particle size of 0.2-0.5 μm. The content of each component is: tea polyphenols >25%, catechin >12%, theabrownin >0.8%, and caffeine 5-12%.

[0066] Example 6, Preparation method of yellow tea extract

[0067] Take fresh leaves of Camellia sinensis (L.) O. Ktze. or Camellia sinensis var. Assamica as raw materials, and prepare yellow tea through withering, rolling, fermentation, drying and other processes. The tea leaves are removed of impurities, crushed to a particle size of 0.5-2 mm, and extracted at 80-90°C for 10-20 minutes. The tea residue is removed through a 32-60 mesh sieve, and then filtered through a 300 mesh filter (pore size <10 μm) at 4000 r / min. The extract is concentrated through evaporation, freezing, membrane and other technologies. Finally, spray drying or freeze drying is performed, and the yellow tea extract with a particle size of 0.2-0.5 μm is obtained. The content of each component is: tea polyphenols >25%; theaflavins >0.8%; caffeine 5-12%.

[0068] Example 7, Preparation method of oolong tea extract

[0069] Take fresh leaves of specific tea tree varieties in Fujian as raw materials, and prepare oolong tea through withering, fixing, killing, rolling, drying and other specific processes. The tea leaves are removed of impurities, crushed to a particle size of 0.5-2 mm, and extracted at 90-100°C for 40 minutes. After 4 times of extraction, the extracts are combined, the tea residue is removed through a 32-60 mesh sieve, and then filtered through a 300 mesh filter (pore size <10 μm). The extract is concentrated through evaporation, freezing, membrane and other technologies. Finally, spray drying or freeze drying is performed, and the oolong tea extract with a particle size of 0.2-0.5 μm is obtained. The content of each component is: tea polyphenols >30%; theaflavins >0.8%; caffeine 8-15%.

[0070] Example 8, Preparation method of black tea extract

[0071] Take fresh leaves of Camellia sinensis var. Assamica as raw materials, and prepare black tea through killing, rolling, pile fermentation, drying and other processes. The tea leaves are removed of impurities, crushed to a particle size of 0.5-2 mm, and extracted at 100°C for 40 minutes. After 2 times of extraction, the extracts are combined, the tea residue is removed through a 32-60 mesh sieve, and then filtered through a 300 mesh filter (pore size <10 μm). The extract is concentrated through evaporation, freezing, membrane and other technologies. Finally, spray drying or freeze drying is performed, and the black tea extract with a particle size of 0.2-0.5 μm is obtained. The content of each component is: tea polyphenols 20-30%, tea polysaccharides >5%, theabrownins >20%, caffeine >6%.

[0072] Example 9, Preparation method of Dendrobium nobile extract

[0073] Take Dendrobium nobile Lindl (also known as Dendrobium) stems 20 kg, after the impurities, crushed to a particle size of 1-2 mm, add 100 liters of distilled water heated extraction, extraction temperature 65 °C, extraction time 3 hours, after continuous extraction 3 times combined extract, at 55 °C, 5.0 KPa under reduced pressure concentration, get reduced pressure concentration, finally reduced pressure concentration at -45 °C, 609 Pa under freeze drying, crushing sieve to get a particle size of 0.5-1.0 μm Dendrobium nobile extract, wherein the content of Dendrobium polysaccharide is greater than 12%.

[0074] Example 10, the preparation method of Dendrobium loddigesii extract

[0075] Take Dendrobium loddigesii Rolfe (also known as Dendrobium) stems 5 kg, after the impurities, crushed to a particle size of 1-2 mm, add 25 liters of distilled water heated extraction, extraction temperature 65 °C, extraction time 3 hours, after continuous extraction 3 times combined extract, at 55 °C, 5.0 KPa under reduced pressure concentration, get reduced pressure concentration, finally reduced pressure concentration at -45 °C, 609 Pa under freeze drying, crushing sieve to get a particle size of 0.5-1.0 μm Dendrobium loddigesii extract, wherein the content of Dendrobium polysaccharide is greater than 14.5%.

[0076] Example 11, the preparation method of Dendrobium chrysanthum extract

[0077] Take Dendrobium chrysanthum Lindl (also known as Dendrobium) stems 20 kg, after the impurities, crushed to a particle size of 1-2 mm, add 100 liters of distilled water heated extraction, extraction temperature 65 °C, extraction time 3 hours, after continuous extraction 3 times combined extract, at 55 °C, 5.0 KPa under reduced pressure concentration, get reduced pressure concentration, finally reduced pressure concentration at -45 °C, 609 Pa under freeze drying, crushing sieve to get a particle size of 0.5-1.0 μm Dendrobium chrysanthum extract, wherein the content of Dendrobium polysaccharide is greater than 12.5%.

[0078] Example 12, the preparation method of Dendrobium loddigesii extract

[0079] Take Dendrobium fimbriatum Hook (also known as Dendrobium fimbriatum) stems 15 kg, remove impurities, crush to a particle size of 1-2 mm, add 75 liters of distilled water to heat extraction, extraction temperature 65°C, extraction time 3 hours, continuous extraction 3 times, then combine the extract, reduce pressure concentration at 55°C, 5.0 KPa, to obtain a reduced pressure concentrate, and finally freeze-dry the reduced pressure concentrate at -45°C, 609 Pa, and crush and sieve to obtain Dendrobium fimbriatum extract with a particle size of 0.5-1.0 μm, wherein the Dendrobium polysaccharide content is greater than 10%.

[0080] The above compositions are all provided by the Jitengfei team of the Institute of Materia Medica, Chinese Academy of Medical Sciences.

[0081] (B) Screening experiments

[0082] Experimental Example 1, composition agonizing the upstream promoter activity of uncoupling protein 1 (UCP1)

[0083] Brown adipose tissue has non-shivering thermogenesis function and can dissipate energy by raising body temperature. Its thermogenic function is derived from its rich mitochondria and specific high expression of mitochondrial inner membrane uncoupling protein 1 (UCP1). UCP1 promotes protons to leak from the intermembrane space of mitochondria into the mitochondria, thereby making the mitochondrial electron transport chain produce electrochemical potential energy and dissipate in the form of heat energy. UCP1 agonists that promote brown adipose thermogenesis and white adipose browning, i.e. thermogenic activators, have become a new direction for the development of anti-metabolic syndrome drugs. Therefore, using the high-throughput screening model of UCP1 agonists that we have successfully constructed (application number: CN201811248715.3), the agonizing effect of the composition on the upstream promoter activity of UCP1 in adipocytes was detected.

[0084] Experimental method: insert the UCP1 promoter region into the WT1 stable transfection adipocyte cell strain of Luciferase and tdTomato, seed in a 96-well plate, start fat induction differentiation after 100% fusion, add 10 μmol / L of positive control drug β3-adrenergic receptor agonist CL316,243 (CAS: 138908-40-4) and composition to the cells, and detect the activity of Luciferase using the reporter gene method after 24 h to observe whether the composition has an agonizing effect on the UCP1 promoter activity.

[0085] Experimental method: UCP1 promoter region was inserted into Luciferase and tdTomato stable transgenic adipocyte cell line, which was seeded in 96-well plates, and induced differentiation into adipocytes. On Day 6, 10 μmol / L of positive control drug β3-adrenergic receptor agonist CL316,243 (CAS: 138908-40-4) was added, and the compositions in Experimental Example 1, 2, and 3 were prepared into three different compositions (ratio of black tea extract: notoginseng extract: dendrobium officinale extract: combination 1: 4.5:4.5:1; combination 2: 6:3:1; combination 3: 7.2:1.8:1; 100 μg / ml), black tea extract (100 μg / ml), and notoginseng extract (100 μg / ml) were used to treat the cells, respectively. After 24 h, the activity of Luciferase was detected by the reporter gene method to observe the regulatory effect of the three different combinations, black tea extract, and notoginseng extract on the activity of UCP1 promoter.

[0086] Experimental results: The positive control drug CL316,243 can increase the promoter activity of UCP1 in adipocytes (P<0.05), and similarly, the composition can also enhance the activity of UCP1 promoter, among which combination 3 shows statistical difference (P<0.05) (Table 1). The use of black tea extract alone can also promote the activity of UCP1 promoter (P<0.05), but the luminescence intensity is weaker than that of combination 3, while notoginseng extract alone cannot promote the activity of UCP1 promoter. It is confirmed that combination 3 has a potential thermogenic effect and is better than black tea extract and notoginseng extract alone.

[0087] Table 1 Agonistic effect of composition on UCP1 upstream promoter

[0088]

[0089] Note: n=4, mean±SEM; #P<0.05 (compared with the normal control group)

[0090] Experimental Example 2, composition up-regulates UCP1 expression in mitochondria of adipocytes

[0091] Experimental method: 3T3-L1 adipocyte cell line was seeded in 24-well plates, and adipogenic induction differentiation was performed by the following steps: cells reached confluence, then changed to DMEM medium containing 10% fetal bovine serum, two days later (Day 0) changed to induction medium (10% fetal bovine serum containing 50 nM insulin, 100 nM T3, 0.125 mM indinavir, 2 μg / ml dexamethasone and 0.5 mM IBMX), two days later (Day 2) changed to insulin / T3 medium (10% fetal bovine serum containing 50 nM insulin and 1 nM T3), two days later (Day 4) changed to fresh insulin / T3 medium, and 10, 30, 100 μg / L of combination 3 was added respectively on Day 6, and the cells were collected 24 h after drug addition, and the expression of UCP1 gene was detected by real-time fluorescent quantitative PCR method to observe whether the composition had a regulating effect on Ucp1 mRNA expression.

[0092] Experimental results: the composition can up-regulate the expression of Ucp1 mRNA in adipocytes, and combination 3 is the most significant (P<0.05) (Table 2).

[0093] Table 2 Regulating effect of composition on Ucp1 mRNA expression in mitochondrial inner membrane of adipocytes

[0094]

[0095] Note: n=3, mean±SEM; #P<0.05 (compared with normal control group).

[0096] (Three) Pharmacological animal experiment of Panax notoginseng extract

[0097] Example 3, Effect of Panax notoginseng extract on body weight of mice with metabolic syndrome induced by high-fat feed

[0098] Experimental mice: 24 5-week-old C57BL / 6J male mice, after adaptive feeding for 1 week, 6 of them were randomly selected as normal control group, and the rest were high-fat diet-induced metabolic syndrome group. The normal control group was fed with standard mouse feed, and the high-fat diet-induced metabolic syndrome group was fed with high-fat feed (60% calories from fat, American Research Diets Company). The body weight of the mice was recorded once a week, and the high-fat diet-induced metabolic syndrome group mice were randomly divided into 3 groups, 6 in each group, namely: model control group, metformin hydrochloride positive control group (200 mg / kg), and notoginseng extract treatment group (300 mg / kg). High-fat diet modeling was started at the same time, and gavage was performed (0.5% carboxymethylcellulose sodium (0.5% CMC-Na) was used to prepare the drug), and the normal control group and the model control group were given 0.5% carboxymethylcellulose sodium, 0.1 mL / 10g once a day, and the drug was given continuously for 51 days. The notoginseng extract was provided by the Jitengfei group of the Institute of Materia Medica, Chinese Academy of Medical Sciences.

[0099] Experimental method: The body weight of the mice was recorded every week.

[0100] Experimental results: After feeding with high-fat feed, the body weight of the mice gradually increased, and after 7 days of feeding, the body weight of the high-fat diet-induced metabolic syndrome group mice increased significantly compared with the normal control group (P<0.05). The body weight of the metformin hydrochloride group mice was significantly lower than that of the model control group (P<0.05), and the body weight of the notoginseng extract treatment group mice did not change significantly compared with the model control group. By the end of the 51-day experiment, the body weight of the notoginseng extract group mice decreased by 10.48% compared with the model control group (Table 3). By the end of the experiment, the body weight of the model control group increased by 70.35%, the metformin hydrochloride positive control group by 48.23%, and the notoginseng extract by 48.42% (Table 4). It can be seen that the administration of 300 mg / kg notoginseng extract can inhibit the weight gain of metabolic syndrome mice, and the effect is comparable to that of metformin hydrochloride.

[0101] Table 3 Effect of notoginseng extract on body weight of high-fat diet-induced metabolic syndrome mice

[0102]

[0103]

[0104] Note: n=6, mean±SEM; #P<0.05, ##P<0.01, ###P<0.001 (compared with the model control group).

[0105] Table 4 Effect of notoginseng extract on body weight gain rate of high-fat diet-induced metabolic syndrome mice

[0106]

[0107] Note: n = 6, mean ± SEM; #P < 0.05, ###P < 0.001 (compared with model control group)

[0108] (IV) Pharmacological animal experiments of the composition

[0109] Experimental mice: 48 5-week-old C57BL / 6J male mice, after 1 week of adaptive feeding, 6 of them were randomly selected as the normal control group, and the rest were randomly divided into 6 groups as the high-fat diet-induced metabolic syndrome group. The normal control group was fed with standard mouse feed, and the high-fat diet-induced metabolic syndrome group was fed with high-fat feed (60% calories from fat, American Research Diets company). The 6 groups of the high-fat diet-induced metabolic syndrome group were: model control group (12), metformin hydrochloride positive control group (6, 200 mg / kg), black tea extract group (6, 300 mg / kg), combination of extracts in experimental examples 1, 2 and 3 in proportion, divided into combination 1 group (6, 300 mg / kg: black tea extract 135 mg / kg, panax notoginseng extract 135 mg / kg, dendrobium officinale extract 30 mg / kg), combination 2 group (6, 300 mg / kg: black tea extract 180 mg / kg, panax notoginseng extract 90 mg / kg, dendrobium officinale extract 30 mg / kg), combination 3 group (6, 300 mg / kg: black tea extract 216 mg / kg, panax notoginseng extract 54 mg / kg, dendrobium officinale extract 30 mg / kg). The body weight and food and water intake of the mice were recorded every week. Gavage was performed at a fixed time, once a day, for 13 consecutive weeks.

[0110] Black tea extract, panax notoginseng extract and dendrobium officinale extract were provided by the Jitengfei group of the Institute of Materia Medica, Chinese Academy of Medical Sciences.

[0111] Experimental example 4, effect of the composition on the body weight of high-fat diet-induced metabolic syndrome mice

[0112] Experimental method: The body weight of the mice was recorded every week.

[0113] The experimental results are as follows: the body weight of the mice gradually increased after being fed with high-fat feed, and there was a statistical difference (P<0.05) compared with the normal control group after being fed for 1 week. The continuous administration of the compositions with different ratios gradually reduced the body weight increase of the mice with metabolic syndrome induced by high-fat feed, and inhibited the excessive body weight increase caused by high-fat feed. After the continuous administration of combination 3 for 2 weeks, combination 2 for 7 weeks, combination 1 for 12 weeks, the red tea extract for 5 weeks, and metformin hydrochloride for 11 weeks, the body weight of the mice began to have a statistical difference (P<0.05) compared with the model control group (Table 5). At the end of administration at the 13th week, the body weight increase rate of the model control group was 88.00%, the body weight increase rate of the metformin hydrochloride positive control group was 61.98% (P<0.05), the body weight increase rate of the red tea extract control group was 56.28% (P<0.01), the body weight increase rates of combinations 1, 2 and 3 were 63.64%, 57.20% and 45.28% (P<0.05) respectively. It can be seen that the anti-obesity effects of combinations 2 and 3 are obviously better than that of the positive drug metformin hydrochloride; and the anti-obesity effect of combination 3 containing 216 mg / kg of red tea extract, 54 mg / kg of Panax notoginseng extract and 30 mg / kg of Dendrobium officinale extract is obviously better than that of the red tea extract alone, which shows the synergistic effect of the combination (Table 6).

[0114] Table 5 Effect of the compositions on the body weight of the mice with metabolic syndrome induced by high-fat feed

[0115]

[0116] Note: n=6-12, mean±SEM; #P<0.05, ##P<0.01 (compared with the model control group).

[0117] Table 6 Effect of the compositions administered for 13 weeks on the body weight increase rate of the mice with metabolic syndrome induced by high-fat feed

[0118]

[0119] Note: n=6-12, mean±SEM; #P<0.05, ##P<0.01, ###P<0.001 (compared with the model control group).

[0120] Example 5, Effect of the Compositions on Lipid Synthesis in the Mice with Metabolic Syndrome Induced by High-fat Feed

[0121] 5.1 Effect of the Compositions Administered for 13 Weeks on the Fat Coefficient of the Mice with Metabolic Syndrome Induced by High-fat Feed

[0122] Experimental method: At the end of the experiment, the mice were weighed and then anesthetized with isoflurane. The three types of adipose tissue, including subcutaneous white adipose tissue (SAT), visceral adipose tissue (VAT), and scapular brown adipose tissue (BAT), were quickly removed and weighed. The fat coefficient was calculated.

[0123] Experimental results: Compared with the normal control group, the SAT and VAT fat coefficients of the model control group increased significantly (P < 0.001). After 13 weeks of continuous administration, compared with the model control group, the metformin hydrochloride positive control group, the black tea extract control group, and combinations 1, 2, and 3 could all reduce the fat coefficients of the two types of white adipose tissue in high-fat diet-induced metabolic syndrome mice. Among them, combination 2, black tea extract, and metformin hydrochloride had similar effects, and combination 3 had the most significant effect (P < 0.001), which was significantly better than metformin hydrochloride and black tea extract alone. For brown adipose tissue, there was no significant difference between the high-fat diet model group and the administration groups and the normal control group. Therefore, administration of the combination can reduce fat accumulation in the body, especially the reduction of white adipose tissue, including SAT and VAT, which is closely related to metabolic syndrome, and this reflects the therapeutic effect of the combination (Table 7).

[0124] Table 7 Effect of 13-week administration of the combination on the fat coefficient of high-fat diet-induced metabolic syndrome mice

[0125]

[0126] Note: n = 6-12, mean ± SEM; #P < 0.05, ##P < 0.01, ###P < 0.001 (compared with the model control group).

[0127] 5.2 Effect of 12-week administration of the combination on the fat-water ratio of high-fat diet-induced metabolic syndrome mice

[0128] Experimental method: At the end of the experiment, the fat-water ratio of the mice was detected by nuclear magnetic resonance method. The higher the fat-water ratio, the more severe the obesity of the mice. A lower fat-water ratio indicates that the mice have less fat accumulation and their obesity has improved.

[0129] Results: Compared with the normal control group, the lipid water ratio of the model control group increased significantly (P<0.001). After 12 weeks of continuous administration, compared with the model control group, metformin hydrochloride positive control group, black tea extract control group and combinations 1, 2 and 3 can significantly reduce the lipid water ratio of high-fat diet induced obese mice, combination 2, black tea extract and metformin hydrochloride are close to each other, the lipid water ratio of combination 3 is 0.27 (P<0.001), which is significantly better than metformin hydrochloride and black tea extract alone, so the combination can reduce fat accumulation and fat content in the body, and confirm its anti-obesity effect (Table 8).

[0130] Table 8 Effect of combination on lipid water ratio of high-fat diet induced metabolic syndrome mice

[0131]

[0132] Note: n = 6-12, mean ± SEM; ##P<0.01, ###P<0.001 (compared with the model control group).

[0133] Example 6, Effect of combination on glucose metabolism of high-fat diet induced metabolic syndrome mice

[0134] 6.1 Effect of combination on fasting blood glucose of high-fat diet induced metabolic syndrome mice after 12 weeks of administration

[0135] Experimental method: After 12 weeks of continuous administration of three different combinations, the mice were taken blood from the tail tip after fasting for four hours in the morning, and the fasting blood glucose value of the mice was determined using the auxiliary blood glucose meter and auxiliary blood glucose test paper.

[0136] Results: Compared with the normal control group, the fasting blood glucose of the model control group mice increased significantly (P<0.001), indicating that the model control group mice had hyperglycemia. After 12 weeks of continuous administration of the three combinations, compared with the model control group, they all showed a decrease in fasting blood glucose in mice, and there was a significant statistical significance; and the hypoglycemic effect of combination 3 was better than that of metformin hydrochloride and black tea extract alone (Table 9).

[0137] Table 9 Effect of combination on fasting blood glucose of high-fat diet induced metabolic syndrome mice after 12 weeks of administration

[0138]

[0139] Note: n = 6-12, mean ± SEM; ##P<0.01, ###P<0.001 (compared with the model control group).

[0140] 6.2 Effect of combination on glucose tolerance of high-fat diet induced metabolic syndrome mice after 12 weeks of administration

[0141] Experimental method: After 12 weeks of continuous administration of the three different combinations, the mice were fasted from 8:00 am and administered by gavage, and blood was taken from the tail tip at 12:00. The blood glucose value of the high-fat diet-induced obese mice was measured using the Accu-Check® blood glucose meter and Accu-Check® blood glucose test paper, and recorded as the 0 min blood glucose value. Then 20% glucose was injected intraperitoneally at 2 g / kg. Blood was taken from the tail tip at 30 min, 60 min, and 120 min, and the blood glucose value of the mice was measured using the Accu-Check® blood glucose meter and Accu-Check® blood glucose test paper. The area under the curve was calculated by the formula AUC = 0.25 x fasting blood glucose + 0.5 x 30 min blood glucose + 0.75 x 60 min blood glucose + 0.5 x 120 min blood glucose, and the effects of modeling and administration on glucose tolerance were compared.

[0142] Experimental results: Compared with the normal control group of mice, the blood glucose level of the model control group of mice increased more obviously after intraperitoneal injection of glucose, indicating that the model control group of mice had abnormal glucose tolerance. After 12 weeks of continuous administration of the three combinations, compared with the model control group, the three combinations all showed a decrease in the area under the curve, i.e. an improvement in glucose tolerance, and the effects of combination 2 and combination 3 were statistically different, with combination 3 being the most obvious (P < 0.01), and combination 2 and combination 3 were both better than the red tea extract alone administration group (Table 10, Table 11).

[0143] Table 10 Effect of administration of the combination for 12 weeks on glucose tolerance of high-fat diet-induced metabolic syndrome mice

[0144]

[0145] Note: n = 6-12, mean ± SEM; #P < 0.05, ##P < 0.01, ###P < 0.001 (compared with the model control group).

[0146] Table 11 Area under the curve of glucose tolerance of high-fat diet-induced metabolic syndrome mice after administration of the combination for 12 weeks

[0147]

[0148] Note: n = 6-12, mean ± SEM; #P < 0.05, ##P < 0.01, ###P < 0.001 (compared with the model control group).

[0149] In summary, the red tea extract combination has a lowering effect on the high blood sugar of high-fat diet-induced metabolic syndrome mice, corrects impaired glucose tolerance, and thus improves the abnormal glucose metabolism caused by obesity.

[0150] Experimental example 7, effect of the combination on insulin resistance of high-fat diet-induced metabolic syndrome mice

[0151] 7.1 Effect of 12-week administration of the composition on insulin tolerance of high-fat diet-induced metabolic syndrome mice

[0152] Experimental method: After 12 weeks of continuous administration of the composition with different proportions, the mice were fasted from 8:00 am and administered intragastrically, and blood was taken from the tail tip at 12:00. The blood glucose meter and blood glucose test paper were used to determine the fasting blood glucose value of high-fat diet-induced metabolic syndrome mice, and the value at 0 min was recorded. Then 0.5 U / kg of insulin (insulin injection (400 U: 10 ml), Jiangsu Wanbang Biochemical Pharmaceutical Co., Ltd.) was injected intraperitoneally. Blood was taken from the tail tip at 15 min, 30 min, 60 min, 90 min, and 120 min, and the blood glucose value was determined using the blood glucose meter and blood glucose test paper. The area under the curve was calculated by the formula AUC = 0.25 x fasting blood glucose + 0.5 x 30 min blood glucose + 0.5 x 60 min blood glucose + 0.5 x 90 min blood glucose + 0.25 x 120 min blood glucose, and the effects of modeling and administration on insulin tolerance were compared.

[0153] Experimental results: Compared with the normal control group, the area under the curve of insulin tolerance of the model control group mice increased significantly (P < 0.001), and insulin resistance was observed. After 12 weeks of continuous administration, the three compositions showed an improvement in insulin tolerance of high-fat diet-induced metabolic syndrome mice (P < 0.01), and the area under the curve of insulin tolerance of combinations 2 and 3 was lower than that of the normal control group. The blood glucose of combination 3 continued to decrease after 90 min of insulin injection, and began to increase at 120 min. The effects of combinations 2 and 3 were significantly better than those of the positive drug metformin hydrochloride and the black tea extract control group, confirming that the composition significantly improved insulin resistance and increased insulin sensitivity, and the combination of the three extracts had a significant synergistic effect (Table 12, Table 13).

[0154] Table 12 Effect of 12-week administration of the composition on insulin tolerance of high-fat diet-induced metabolic syndrome mice

[0155]

[0156] Note: n = 6-12, mean ± SEM; #P < 0.05, ##P < 0.01, ###P < 0.001 (compared with the model control group).

[0157] Table 13 Effect of 12-week administration of the composition on the area under the curve of insulin tolerance of high-fat diet-induced metabolic syndrome mice

[0158]

[0159] Note: n = 6-12, mean ± SEM; ##P<0.01, ###P<0.001 (compared with model control group).

[0160] 7.2 Effects of the composition on plasma insulin resistance of high-fat diet-induced metabolic syndrome mice after 12 weeks of administration

[0161] Experimental method: After 12 weeks of continuous administration of different proportions of the composition, blood was collected in heparinized EP tubes, centrifuged at 5000 rpm for 10 minutes at 4°C. The supernatant was transferred to a new EP tube, and the insulin concentration was detected using a mouse insulin ELISA kit (80-INSMSU-E01, ALPCO, USA), and the insulin resistance index (HOMA-IR) was calculated in combination with fasting blood glucose = (fasting insulin concentration * fasting blood glucose) / 22.5.

[0162] Experimental results: Compared with the normal control group, the insulin level and HOMA-IR of the model control group mice were significantly increased (P<0.05); compared with the model control group, after 12 weeks of continuous administration, the three compositions showed effects of reducing the plasma insulin level and HOMA-IR of high-fat diet-induced metabolic syndrome mice, and the reducing effect of metformin was similar to that of combination 2 but weaker than that of combination 3, and the effect of red tea extract alone was better than that of positive drug metformin and weaker than that of combination 3 (Table 14), and the effect of combination 3 was the best, which was equivalent to that of the normal control group, i.e., completely reversing insulin resistance. In summary, the composition can reduce insulin resistance induced by high-fat diet and increase insulin sensitivity.

[0163] Table 14 Effects of the composition on plasma insulin resistance of high-fat diet-induced metabolic syndrome mice after 12 weeks of administration

[0164]

[0165] Note: n = 6-12, mean ± SEM; #P<0.05 (compared with model control group).

[0166] Example 8, Effects of the composition on blood lipid metabolism of high-fat diet-induced metabolic syndrome mice

[0167] Experimental method: After 12 weeks of continuous administration of different proportions of the composition, blood was collected in heparinized EP tubes, centrifuged at 5000 rpm for 10 minutes at 4°C. The supernatant was transferred to a new EP tube, and 12 μL of the supernatant was diluted 5 times with normal saline to 60 μL, and the concentrations of triglycerides (triglyceride assay kit, Beijing Zhensheng Beikong Biotechnology Co., Ltd.) and total cholesterol (total cholesterol assay kit, Beijing Zhensheng Beikong Biotechnology Co., Ltd.) were detected in a fully automatic biochemical analyzer (TBA-40FR, Toshiba, Japan).

[0168] Results: Compared with the normal control group, the model control group showed a rising trend in triglyceride levels. After 12 weeks of continuous administration, all three combinations showed a decrease in plasma triglycerides in mice with high-fat diet-induced metabolic syndrome. The effect of black tea extract alone on triglyceride reduction was similar to that of combination 2 and weaker than that of combination 3. Compared with the normal control group, the model control group showed a significant increase in total cholesterol (P < 0.001). After 12 weeks of administration of the three combinations, there was a statistically significant decrease in total cholesterol. Combination 1, 2, and 3 were more effective than black tea extract alone in reducing total cholesterol. Combination 1 was similar to metformin in reducing total cholesterol, but weaker than combinations 2 and 3 (Table 15).

[0169] Table 15 Effect of combination administration for 12 weeks on blood lipids in mice with high-fat diet-induced metabolic syndrome

[0170]

[0171] Note: n = 6-12, mean ± SEM; #P < 0.05, ##P < 0.01, ###P < 0.001 (compared with the model control group).

[0172] Example 9: Effect of combination on fatty liver in mice with high-fat diet-induced metabolic syndrome

[0173] 9.1 Effect of combination administration for 12 weeks on liver function in mice with high-fat diet-induced metabolic syndrome

[0174] Method: After 12 weeks of continuous administration of different combinations, blood was collected in heparinized EP tubes. Centrifuge at 4°C, 5000 rpm for 10 minutes. Transfer the supernatant to a new EP tube. Dilute 12 μL of supernatant 5 times with normal saline to 60 μL. Detect the concentrations of glutamic transaminase (glutamic transaminase assay kit, Beijing Zhongsheng Beikong Biotechnology Co., Ltd.) and glutamic transaminase (glutamic transaminase assay kit, Beijing Zhongsheng Beikong Biotechnology Co., Ltd.) in the automatic biochemical analyzer (TBA-40FR, Toshiba, Japan).

[0175] Results: Compared with the normal control group, the model control group showed significant liver damage, with significantly increased glutamic transaminase and glutamic transaminase levels. After 12 weeks of continuous administration, all three combinations showed a decrease in plasma glutamic transaminase and glutamic transaminase in mice with high-fat diet-induced metabolic syndrome. The effect of black tea extract alone on liver function was similar to that of combination 2 and weaker than that of combination 3 (Table 16). All three combinations and black tea extract alone were significantly better than metformin.

[0176] Table 16 Effect of the composition on plasma liver function in high-fat diet-induced metabolic syndrome mice after 12 weeks of administration

[0177]

[0178] Note: n = 6-12, mean ± SEM; #P < 0.05, ##P < 0.01 (compared with the model control group).

[0179] 9.2 Effect of the composition on liver coefficient in high-fat diet-induced metabolic syndrome mice after 13 weeks of administration

[0180] Experimental method: At the end of the experiment, the liver was quickly removed after the mice were weighed under isoflurane anesthesia, and the tissue weight was weighed to calculate the liver coefficient.

[0181] Experimental results: Compared with the normal control group, the model control group had fat deposition in the liver, and thus the liver coefficient decreased significantly (P < 0.001). After 13 weeks of continuous administration, compared with the model control group, the metformin hydrochloride positive control group and the combination 3 could significantly increase the liver coefficient of high-fat diet-induced metabolic syndrome mice (Table 17).

[0182] Table 17 Effect of the composition on liver coefficient in high-fat diet-induced metabolic syndrome mice after 13 weeks of administration

[0183]

[0184] Note: n = 6-12, mean ± SEM; #P < 0.05, ###P < 0.001 (compared with the model control group).

[0185] 9.3 Effect of the composition on liver pathology in high-fat diet-induced metabolic syndrome mice after 13 weeks of administration

[0186] Experimental method: After 13 weeks of continuous administration, the mice were sampled, the liver was removed and quickly placed in 10% neutral formalin for fixation, paraffin-embedded, 4 μm section deparaffinization and hydration, hematoxylin staining for 5 min, 1% hydrochloric acid alcohol differentiation for 30 s, eosin staining for 3 min, dehydration, xylene transparency, neutral resin mounting, and observed under an optical microscope.

[0187] Experimental results: Compared with the normal control group, the model control group had hepatosteatosis. After 13 weeks of continuous administration, the three combinations showed a role in reducing fat accumulation in high-fat diet-induced obese mice. The positive drug and combination administration groups could significantly reduce the formation of liver oil droplets and prevent the progression of liver steatosis caused by high-fat diet ( Figure 1 ).

[0188] Experimental Example 10, thermogenic activation of the composition on brown fat thermogenesis and white fat browning

[0189] 10.1 The effect of 12-week administration of the composition on heat dissipation of high-fat diet-induced metabolic syndrome mice

[0190] Experimental method: After 12 weeks of continuous administration of the three compositions, the body temperature of the mice was detected using a handheld infrared thermal imager (E8 Wifi, FLIR, USA).

[0191] Experimental results: Compared with the normal control group, the body temperature of the mice in the model control group increased significantly. After 12 weeks of continuous administration of the three compositions, the body temperature of the high-fat diet-induced metabolic syndrome mice was significantly increased, and the effect of combination 2 and combination 3 was the most significant (P<0.01). The effect of the three compositions on heat dissipation was better than that of metformin and black tea extract alone (P<0.01) (Table 18). Figure 2

[0192] Table 18 The effect of 12-week administration of the composition on the body temperature of high-fat diet-induced metabolic syndrome mice

[0193]

[0194] Note: n=6-12, mean±SEM; #P<0.05, ##P<0.01 (compared with the model control group).

[0195] 10.2 The effect of 12-week administration of the composition on promoting the browning of white adipose tissue and the thermogenesis of brown adipose tissue in high-fat diet-induced metabolic syndrome mice

[0196] Experimental method: After 13 weeks of continuous administration of the composition with different proportions, the mice were taken, and the inguinal subcutaneous fat, epididymal visceral fat, and scapular brown adipose tissue were removed and quickly placed in 10% neutral formalin for fixation, paraffin embedding, 4μm section deparaffinization and hydration, hematoxylin staining for 5min, 1% hydrochloric acid alcohol differentiation for 30s, eosin staining for 3min, dehydration, xylene transparency, neutral resin mounting, and observation under an optical microscope.

[0197] Experimental results: Unilocular cells are the characteristic lipid droplets of white adipose tissue, while multilocular cells are the characteristic lipid droplets of brown adipose tissue. Compared with the normal control group, the adipocytes of white adipose tissue, including visceral fat and subcutaneous fat, in the model control group were significantly hypertrophic, the unilocular cell lipid droplets were enlarged, the multilocular cells of brown adipose tissue were significantly reduced, and unilocular cells with enlarged lipid droplets appeared. Compared with the model control group, after 13 weeks of continuous administration, the administration of the composition can significantly reduce the size of the lipid droplets in the three types of fat. In the subcutaneous fat, the administration of the three combinations and the positive drug can increase the number of multilocular cells and significantly reduce the size of the unilocular cell lipid droplets. Among them, the effect of metformin is weaker than that of black tea extract and the administration of the combination, and the effect of the administration of the combination is the strongest (P<0.01) (Table 19). Figure 3 ​). In the visceral fat, more multilocular cells appeared after the administration of the three combinations, and the monolocular cells were significantly reduced. The positive drug metformin had similar effects to combination 1, but was weaker than combinations 2 and 3, and the black tea extract was weaker than the three combination groups Figure 4 ). It is suggested that the combination can promote the browning of white fat, including the browning of subcutaneous fat and visceral fat. In the brown fat, the multilocular cells significantly increased after the administration of combinations 1, 2 and 3, and the monolocular cells decreased, which is better than the black tea extract and the positive drug metformin, suggesting that the combination can promote the thermogenesis of brown fat Figure 5 ). In summary, the combination has the thermogenic activation effect of promoting the thermogenesis of brown fat and the browning of white fat.

[0198] In summary, the combination promotes the heat dissipation of the back of the high-fat diet-induced metabolic syndrome mice, promotes the browning of white fat and the thermogenesis of brown fat, and can also up-regulate the expression of UCP1 in adipocytes, which has a significant effect of promoting thermogenesis, thereby playing a role as a thermogenic activator to resist metabolic syndrome and complications.

[0199] Experimental Example 11: Improving effect of combination 3 on intestinal flora of high-fat diet-induced metabolic syndrome mice

[0200] Experimental method: After 12 weeks of continuous administration of combination 3, the mice were fasted from 8:00 am and administered orally, and the feces of the mice were taken in a clean bench four hours after fasting and quickly placed in sterile cryogenic tubes and put into liquid nitrogen. The samples were stored in a -80 refrigerator before being sent to Hangzhou Lianchuan Biotechnology Co., Ltd. for 16S rDNA sequencing and corresponding data analysis.

[0201] Experimental results: (1) Alpha diversity analysis showed that combination 3 helped to restore the species richness of the flora and reduce the decrease in the richness of the flora caused by high-fat diet Figure 6 ). It has been proved in the literature that the decrease in the species richness of the intestinal flora and the imbalance of the flora can cause the obstruction of the thermogenic function of the body relying on UCP1, so the restoration of the species of the intestinal flora is very important for the normal thermogenesis of the body. Therefore, the administration of combination 3 can significantly restore the thermogenic dysfunction caused by high-fat diet and prevent the occurrence and development of metabolic syndrome. (2) The species analysis showed that combination 3 helped to regulate the species distribution ratio of the intestinal flora of high-fat fed mice. Compared with the normal control group of mice, the regulation trend was good. It has been proved in the literature that the key indicators related to obesity and metabolic diseases, Firmicutes / Bacteroidetes, have an improving trend after the administration of combination 3 Figure 7 ); Akkermansia muciniphila (Verrucomicrobia), which has been proved to have a negative correlation with metabolic diseases, was significantly up-regulated after administration (p = 0.0036) Figure 8). Meanwhile, Bacteroidetes and Verrucomicrobia are also producers of short-chain fatty acids, which can promote systemic thermogenesis and help fight metabolic syndrome through inter-tissue interactions.(3) Through pairwise comparison of the results of the three groups of pathway enrichment analysis, it can be found that combination 3 helps to promote glycogen synthesis and metabolism in the gut, reduce bacterial metabolism, and reduce bacterial transmembrane transport. The regulation of these pathways (P < 0.05) may be the key to the effect of combination 3 on the bacterial community Figure 9 , Figure 10 ). The improvement of these pathways helps the health of the intestinal flora, among which glycogen synthesis metabolism provides the basis for the mutual symbiosis and diversity of the flora; while the reduction of bacterial metabolism and transmembrane transport may be related to the inhibition of its disorder or the control of inflammatory immunity. The correlation network diagram and the correlation analysis further confirm that combination 3 may affect the structure of the bacterial community and the whole body by reducing Firmicutes and Proteobacteria and increasing the abundance of Bacteroidetes and Verrucomicrobia, so as to achieve the final effect of resisting metabolic syndrome Figure 11 , Figure 12 ).

Claims

1. A composition, characterized in that, The composition is made from the following raw materials in the following weight proportions: 70-74% black tea water extract, 16-21% Panax notoginseng water extract, and 8-12% Dendrobium officinale water extract.

2. The composition according to claim 1, characterized in that, The black tea water extract was prepared by the following method: using Camellia sinensis (L.) O. Ktze. or Camellia sinensis The tea leaves of var. assamica were prepared into black tea according to the requirements of GB / T 30766-2014. After impurity removal, crushing, extraction, centrifugation, filtration and concentration, the tea was finally freeze-dried to obtain a black tea water extract with a particle size of 0.2-0.5μm.

3. The composition according to claim 1, characterized in that, The Panax notoginseng water extract was prepared by the following method: 5 kg of Panax notoginseng (from the Araliaceae family) was crushed into coarse powder, and 25 liters of distilled water was added for extraction. The extraction was carried out by heating and reflux three times, each time for 3 hours. The extracts were combined, concentrated under reduced pressure, and freeze-dried to obtain the Panax notoginseng water extract.

4. The composition according to claim 1, characterized in that, The water extract of Dendrobium officinale was prepared by the following method: 10 kg of Dendrobium officinale stems were taken, crushed into coarse powder, and extracted with 50 liters of distilled water. The extraction was carried out by heating and reflux three times, each time for 3 hours. The extracts were combined, concentrated under reduced pressure, and freeze-dried to obtain the water extract of Dendrobium officinale.

5. A pharmaceutical composition, characterized in that, The composition comprises an effective dose of the composition of claim 1 and a pharmaceutically acceptable carrier or excipient.

6. The composition according to claim 5, characterized in that, The composition is selected from tablets, capsules, pills, and injections.

7. The composition according to claim 5, characterized in that, The composition is selected from sustained-release formulations, controlled-release formulations, and various microparticle delivery systems.

8. Use of the composition of claim 1 in the preparation of a medicament for the treatment and / or prevention of metabolic syndrome.

9. The application according to claim 8, characterized in that, The metabolic syndrome mentioned is selected from obesity, insulin resistance, abnormal glucose metabolism, abnormal lipid metabolism, and fatty liver.

Images