CN111450173A

CN111450173A - Formula preparation method and application of gynostemma pentaphylla and white tea for preventing type 2 diabetes

Info

Publication number: CN111450173A
Application number: CN202010276830.2A
Authority: CN
Inventors: 赵余庆; 夏晓艳; 林振传; 邵克平; 王�华; 黄鸿
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2020-04-09
Filing date: 2020-04-09
Publication date: 2020-07-28

Abstract

The invention belongs to the field of health-care tea, and particularly relates to a formula preparation method and application of gynostemma pentaphylla and white tea for preventing type 2 diabetes. The formula of the white tea comprises Fujian Fuding white tea and gynostemma pentaphylla. The preparation process of the formula mainly comprises the steps of extracting and purifying white tea polyphenol, white tea polysaccharide and white tea total polypeptide, extracting and purifying gynostemma pentaphylla total saponin, preparing different dosage forms of semisolid extract and solid medicinal granules, sterilizing and subpackaging. The formula plays a role in improving the type 2 diabetes through the synergistic effect of the formula and the white tea, and the synergistic effect of the formula on the aspects of reducing fat and regulating sugar and the synergistic effect of intervening in the type 2 diabetes are verified through in-vivo and in-vitro experiments. The invention develops and develops the health care product which can regulate the phenomena of blood sugar imbalance and insulin resistance of the diabetic by activating the AMPK/PI3K pathway, further achieves the effects of preventing and improving type 2 diabetes and the like, and provides health care food with the white tea formula which is beneficial to body health for people.

Description

Formula preparation method and application of gynostemma pentaphylla and white tea for preventing type 2 diabetes

Technical Field

The invention belongs to the field of health-care tea, and particularly relates to a formula preparation method and application of gynostemma pentaphylla and white tea for preventing type 2 diabetes.

Background

As early as 2400 years ago, he bock writing: let food be your medicine and let medicine be your food, and put forward the concept of "health-promoting foods". However, the concept of homology of medicine and food, dietetic invigoration and medicinal diet is existed in China from ancient times, and the traditional method of using natural plants and food to mix in a certain proportion to treat diseases has existed for over 5000 years. The concept of modern functional food is firstly proposed by Japanese researchers, so that the concept of homology of medicine and food is emphasized by people again. China also refers to functional food as health food, which is intended to protect health. The functional food is a kind of food which can promote the improvement of human physiological condition and human health in addition to the properties of common food, and most of the current functional foods are produced by food production enterprises or medicine production enterprises by combining food and health care functions. Tea, one of three kinds of nonalcoholic beverages (coffee, tea and cocoa) in the world at present, has become a plant planted in the world and is more used as a main agricultural product in China. Tea has become a popular functional beverage in the world due to its antioxidant content.

According to the report issued by the WHO, diabetes has become one of three killers threatening human life. In recent years, the incidence of diabetes, particularly type 2 diabetes, has been increasing. The international diabetes consortium (IDF) released recent data that in 2000, globally diabetic adult patients were 1.51 million, which in 2017 reached 4.25 million, with an increase of nearly 2-fold over 17 years, and in the expected 2045 year, diabetic patients may reach 6.29 million. At present, the number of people with diabetes in China is up to 1.14 hundred million, the prevalence rate of diabetes of adults over 18 years old is 10.9%, and the number of diabetes in China is about 1/3 of the total number of diabetes patients all over the world, so that the diabetes mellitus is the first major country of diabetes mellitus in the world. The diabetes mellitus people in China mainly take type 2 diabetes mellitus, and account for more than 90 percent of the total number of diabetes mellitus patients. In western countries, hypoglycemic health foods have developed into an important emerging industry. In the United states, the sale amount of the hypoglycemic health food in the market is up to 25-30 hundred million dollars every year, and the variety is more than one hundred. The incidence rate of Japanese diabetes is close to 4%, the corresponding blood sugar reducing health food industry develops rapidly, the total sale amount breaks through 1 trillion yen, and the blood sugar reducing/weight reducing health food accounts for about 1/2.

White tea is a tea species with the earliest history of processing in our country, the origin of which is at least two thousand years earlier than that of green tea, and is a special treasure in tea species in our country, white tea is a tea species undergoing the least processing, and is considered the oldest tea species, compared to other tea species, a large number of green tea and black tea producers have turned to white tea production due to its organoleptic properties, because it provides a mild, aromatic taste and floral and fruity taste, white tea has been shown to have a lower caffeine content than green tea and to have a stronger antioxidant activity, white tea contains abundant polyphenols, caffeine, gallic acid and other ingredients, EGCG is the compound with the highest content in white tea, secondly caffeine, a modern research finding that white tea has multiple activities, including antioxidant, antibacterial, anticancer, etc., hajiagaiapipr and the like, which are shown to have effects on glucose metabolism reduction and glucose metabolism reduction, and glucose metabolism reduction by the physiological glucose metabolism reduction of white tea, glucose metabolism reduction, and sperm reduction by rat glucose metabolism reduction, and glucose metabolism reduction.

Gynostemma pentaphylla (thostemma pentaphylum (Thunb.) Makino) has been widely used in asian countries, including korea, china, japan and vietnam, as a traditional herbal medicine or tea. The growth environment is mainly distributed in Gansu, Shaanxi and south China, and the growth environment is distributed in the elevation of 600-3200 m. Gynostemma pentaphylla is widely cultivated in southern China and has a long history as a tonic drug among people. Is a unique non-ginseng plant, and is rich in dammarane type triterpene sapogenin. Gynostemma pentaphylla is used as a traditional Chinese medicine (homology of medicine and food) in China, is widely eaten as a dietary supplement, herbal tea, vegetables and the like since the time of the Ming dynasty. The traditional Chinese medicine considers that the gynostemma pentaphylla has multiple effects, such as anti-inflammatory effect, cholesterol reducing effect, immunity enhancing effect and blood pressure reducing effect. Modern researches find that the main bioactive component of gynostemma pentaphylla is gynostemma pentaphylla total saponin, and the gynostemma pentaphylla has various bioactivities such as blood sugar and blood fat reduction, liver protection, cardiovascular protection, oxidation resistance and the like.

At present, the lipid-lowering and sugar-regulating effects of white tea and gynostemma pentaphylla are rarely reported in the prior art, and the research on the simultaneous application of tea polyphenol in the white tea and gypenoside extract in the gynostemma pentaphylla is not reported.

Disclosure of Invention

The purpose of the invention is as follows:

the invention aims to provide a preparation method and application of a formula for preventing type 2 diabetes by using gynostemma pentaphylla and white tea in cooperation, the invention carries out function evaluation on developed white tea series health-care foods according to health-care food inspection and evaluation technical specifications issued by the State food and drug administration, and researches the effect of the formula of the invention on intervening type 2 diabetes through in-vivo experiments of animals so as to develop and research white tea health-care products with the effects of reducing fat, regulating sugar and preventing type 2 diabetes, and provide white tea health-care foods beneficial to body health for people.

The invention is implemented by the following technical scheme:

a formula of gynostemma pentaphylla and white tea for preventing type 2 diabetes mellitus comprises white tea and gynostemma pentaphylla.

Further, the white tea is one or more of pekoe silver needle, white peony, Gongmei and shoumei white tea of Fujian fuding, and the white tea in the formula is one or more of white tea total polyphenol, total polysaccharide or total polypeptide obtained by extraction and purification.

Further, the gynostemma pentaphylla refers to the whole herb, leaf or flower of the plant of the genus gynostemma of the family cucurbitaceae, or the total saponins of gynostemma pentaphylla obtained by extracting and purifying the whole herb of gynostemma pentaphylla.

Further, the formula comprises the following components in percentage by mass: is prepared from one or more of white tea total polyphenol 10 parts, total polysaccharide 10 parts and total polypeptide 10 parts, and herba Gynostemmatis total saponin 1-7.65 parts.

Further, the final form of the white tea formula is a solid preparation of instant micropowder, granule or syrup.

Further, the method for extracting and purifying the white tea total polyphenol, total polysaccharide and total saponin comprises the following steps:

(1) the extraction and purification method of the total polyphenol of the white tea comprises the steps of using 65% ethanol in volume fraction as an extraction solvent, heating and refluxing for 3 times at the material-liquid ratio of 1:20w/v and the extraction temperature of 80 ℃, each time for 40min, combining extracting solutions, standing, filtering to remove precipitates, carrying out rotary evaporation and reduced pressure concentration on a filtrate, cooling, extracting pigment by using petroleum ether, extracting by using chloroform to remove impurities such as caffeine and the like, finally extracting by using ethyl acetate, carrying out reduced pressure vacuum concentration to obtain a concentrated solution, carrying out freeze drying to obtain a crude tea polyphenol extract, filling resin into a column, firstly soaking the resin in 2% NaOH for 2h, then eluting at the flow rate of 2m L/min for 2BV totally, washing the resin to neutrality by using pure water, then eluting by using 4BV ethanol, and then washing by using pure water until no alcohol smell exists, preparing 1.1g/m L crude tea polyphenol concentrate, adsorbing the crude tea polyphenol extract concentrate according to the ratio of the resin amount of 4:2, wherein the adsorption time is 30min, and the total polyphenol extract is obtained by gradient elution according to 10%, 30%, 45-80% ethanol, 60%, 80% water and 95% water is collected;

(2) the extraction and purification method of the white tea total polysaccharide comprises the following steps: extracting white tea powder with distilled water in a boiling water bath twice according to the proportion of 1:40 and 1:20w/v respectively, extracting for 2h and 1.5h respectively, filtering, combining filtrates, concentrating under reduced pressure to 1/10 of the total volume, adding equal volume of 95% ethanol for precipitation, standing overnight, filtering to obtain filtrate, concentrating ethanol for precipitation, filtering, recovering ethanol to obtain crude polysaccharide, adsorbing with weak base resin cellulose to remove pigment, removing macromolecular protein with trichlorotrifluoroethane, and removing excessive water by adopting a freeze drying method to obtain white tea total polysaccharide;

(3) the extraction and purification method of white tea total polypeptide comprises adding white tea powder into aqueous solution containing 2.5% pectinase at a ratio of 1:20w/v, extracting at 45 deg.C for 3h, centrifuging for 10min to remove supernatant, dissolving the precipitate in 1:45v/v ratio in 1.5 mol/L NaOH, further extracting at 90 deg.C for 1.5h, cooling the mixture at room temperature, centrifuging to remove residue, adding HCl (hydrochloric acid) at pH 2.5, standing for 0.5h, precipitating protein in the supernatant, centrifuging, collecting precipitate in dialysis bag 500-550Da, dialyzing with deionized water for 24h, centrifuging again to remove supernatant, dissolving the obtained protein precipitate in 75% acetone at a liquid-solid ratio of (6-8):1, shaking for 10min in a shaker, centrifuging again, repeating the above steps once, washing with pure water, removing acetone-containing supernatant, obtaining solid matter, centrifuging with acidic protease at pH 6332 min, 0.32-0.2 m, and concentrating the supernatant after centrifugation, and concentrating the obtained white tea total polypeptide solution at pH 632-0.5 h and pressure of 0.2 h.

Further, the method for extracting and purifying the gynostemma pentaphylla total saponin comprises the following steps:

(1) extracting the gynostemma total saponin: heating and reflux-extracting herba Gynostemmatis powder with distilled water at material-to-liquid ratio of 1:20w/v at 80 deg.C for 3 times to obtain water extractive solution, evaporating to remove water, concentrating to 1/5 of initial volume, extracting with 60% ethanol for 3 times, and reflux-concentrating under reduced pressure to obtain extract;

(2) and (3) purifying the gynostemma pentaphylla total saponins: recovering ethanol in the step (1), namely crude extract of the gypenosides, eluting with water, 30%, 70% and 90% ethanol through macroporous resin D101/MCI resin respectively to obtain components with different polarities, collecting 70% eluent, performing reflux concentration under reduced pressure to obtain extract, and freeze-drying to obtain the gypenosides total extract.

A formula preparation method of gynostemma pentaphylla and white tea for preventing type 2 diabetes comprises the following steps:

(1) screening raw materials: manually selecting white tea and gynostemma pentaphylla, and respectively sieving the white tea and the gynostemma pentaphylla through a fourth sieve to remove dust and granular impurities in the raw materials;

(2) preparing tea fine powder: respectively crushing the raw materials screened in the step (1) by using a crusher, and sieving to prepare 100-200-mesh medicinal material fine powder;

(3) preparing white tea polyphenol: extracting and purifying the white tea fine powder obtained in the step (2) according to the method of claim 6, and respectively preparing white tea total polyphenol, total polysaccharide and total polypeptide;

(4) preparing gynostemma pentaphylla total saponin: extracting and purifying the fine powder of gynostemma pentaphylla obtained in the step (2) according to the method of claim 7 to prepare total saponins of gynostemma pentaphylla;

(5) and (3) forming a formula: taking the white tea total polyphenol, the total polysaccharide or the total polypeptide and the gynostemma pentaphylla total saponin obtained in the step (3) and the step (4) respectively, and forming an extract formula according to the proportion of (1-1.5), (10), (4-5) and (1-1.15) respectively, wherein the obtained extract formula can be matched pairwise according to the proportion of 1:1 to obtain a formula of white tea polyphenol, white tea polysaccharide and gynostemma pentaphylla total saponin, and a formula of white tea polyphenol, white tea polypeptide, white tea polysaccharide and gynostemma pentaphylla total saponin;

(6) preparation of syrup semisolid tea: mixing the tea extract formula obtained in the step (5) with sucrose according to a ratio of 1:2, adding 0.5 times of water and 0.1% of tartaric acid, heating for dissolving, heating and concentrating at a constant temperature of 80-90 ℃ until the color of sugar liquor is golden, and obtaining syrup semisolid tea;

(7) preparing instant micropowder tea: further grinding the tea extract formula obtained in the step (5) into micro powder, and performing split charging, drying and sterilization processes to prepare the composite instant micro powder tea;

(8) preparing granule: and (3) preparing the tea extract formula obtained in the step (5) into granules by a further spray granulation method, and performing sub-packaging, drying and sterilization to obtain the granules.

Further, the constant-temperature heating concentration in the step (6) means that the moisture content of the final semi-solid extract syrup is 22-25%, the drying steps in the steps (7) and (8) means that the prepared granules or instant micro powder are dried at the constant temperature of 80 ℃ and the moisture content of the granules or instant micro powder is controlled to be 4.5-5.5%, the sterilization steps (7) and (8) are carried out, the packaged finished product is sterilized through ultraviolet lamp irradiation, the minimum package in the step (6) is 50-100m L, and the minimum package in the steps (7) and (8) is 1.5-2.5 g/bag.

The formula of the gynostemma pentaphylla and white tea for preventing type 2 diabetes mellitus is applied to health-care food or health-care medicine for reducing fat and regulating sugar.

The advantages and effects are as follows:

compared with other methods, the method has the advantages that:

(1) the invention provides gynostemma pentaphylla compatible white tea, which can regulate blood sugar and has the effects of reducing fat, losing weight and health care through the synergistic effect of the white tea and the gynostemma pentaphylla.

(2) The white tea disclosed by the invention is cold in nature, can reduce the blood sugar concentration, and has a prevention effect on the occurrence of diabetic complications. Modern pharmacological research shows that polyphenol and lipid in white tea can regulate glycometabolism and promote insulin synthesis, and catechin can remove excessive sugar in blood, lower blood sugar level and prevent and treat diabetes effectively.

(3) The gynostemma pentaphylla is a unique non-ginseng plant and is a plant rich in dammarane type triterpene sapogenin. Gynostemma pentaphylla is widely eaten as a traditional Chinese medicine (homology of medicine and food) in China, a dietary supplement, herbal tea, vegetables and the like. Is used for treating diabetes, namely modern diabetes.

(4) The main bioactive component of the gynostemma pentaphylla in the invention is the total saponins of the gynostemma pentaphylla, which is reported to have various biological activities, such as hyperglycemia, anti-inflammation, anti-blood fat, liver protection, cardiovascular and anti-oxidation effects.

(5) The invention enlarges the drinking form of the white tea, and makes the white tea into the forms of granules, instant micro powder and tea bags, and the forms are various, thereby not only enlarging the application range and the applicable people, but also enriching the taking form of the white tea, leading the white tea to be more convenient and quicker and more convenient to take, and further having wider economic value and benefit.

(6) According to the invention, the white tea and the gynostemma pentaphylla are independently compatible, and the optimal formula is obtained through proportion optimal selection and screening, the effects of reducing fat, regulating sugar and preventing and intervening diabetes mellitus are achieved through verification of the compatibility effect, the formula is simple, the effect is obvious, and scientific combination and proportion for exerting the efficacy of the white tea to the greatest extent are realized.

The invention evaluates and verifies the activity of the enzyme by an in-vitro glucose-reducing and lipid-lowering related enzyme activity determination and evaluation method, and deeply researches a lipid-reducing and glucose-regulating action mechanism and related targets by combining an in-vivo efficacy evaluation method, and the principle lies in jointly regulating AMPK and PI3K pathways. Has wide application prospect in preparing health-care food or health-care medicine for reducing fat and regulating sugar.

Drawings

FIG. 1 is a flow chart of the preparation process of the present invention;

FIG. 2 shows the inhibition of α -glucosidase by white tea polyphenols, gypenosides and formula;

FIG. 3 shows the inhibitory effect of white tea polyphenols, gypenosides and formula on PTP 1B;

FIG. 4 is the effect of white tea polyphenols, gypenosides and formula on the body weight of type 2 diabetic mice;

FIG. 5 shows the effect of white tea polyphenols, gypenosides and formula on water and diet of type 2 diabetic mice;

FIG. 6 shows the effect of white tea polyphenols, gypenosides and formula on blood glucose levels in type 2 diabetic mice;

FIG. 7 is a graph of the effect of white tea polyphenols, gypenosides and formula on OGTT in type 2 diabetic mice;

FIG. 8 shows the effect of white tea polyphenols, gypenosides and formula on the levels of HOMA-IR and HOMA- β in type 2 diabetic mice;

FIG. 9 shows the effect of white tea polyphenols, gypenosides and formulation on pancreatic tissue inflammatory factor expression in type 2 diabetic mice;

FIG. 10 is a graph of the effect of white tea polyphenols, gypenosides and formulation on the expression of antioxidase in pancreatic tissue of type 2 diabetic mice;

FIG. 11 shows the effect of white tea polyphenols, gypenosides and formula on pathological liver injury in type 2 diabetic mice;

FIG. 12 is a graph of the effect of white tea polyphenols, gypenosides and formula on liver lipid accumulation in type 2 diabetic mice;

FIG. 13 shows the effect of white tea polyphenols, gypenosides and formula on liver lipid expression levels in type 2 diabetic mice;

FIG. 14 shows the effect of white tea polyphenols, gypenosides and formula on SREBP pathway in liver tissue of type 2 diabetic mice;

FIG. 15 shows the effect of white tea polyphenols, gypenosides and formula on liver tissue inflammatory factors in type 2 diabetic mice;

FIG. 16 shows the effect of white tea polyphenols, gypenosides and formula on NF κ B pathway in liver tissue of type 2 diabetic mice;

FIG. 17 shows the effect of white tea polyphenols, gypenosides and formula on PPAR pathways in type 2 diabetic mice liver tissues;

FIG. 18 shows the effect of white tea polyphenols, gypenosides and formula on the IRS/PI3K/AKT pathway in liver tissue of type 2 diabetic mice;

FIG. 19 shows the effect of white tea polyphenols, gypenosides and formula on AMPK pathway in liver tissue of type 2 diabetic mice.

Detailed Description

A formula of herba Gynostemmatis and herba Gynostemmatis tea for preventing type 2 diabetes comprises white tea and herba Gynostemmatis or their extracts including white tea polyphenols and herba Gynostemmatis total saponin.

In folk, the white tea is not only popular as a beverage, but also has the efficacy of reducing blood pressure, blood fat, blood sugar, bacteria, viruses, inflammation and the like, namely three-reducing three-resisting, and is used as a good fire-reducing medicine in folk. Modern pharmacological research shows that polyphenols in the white tea can regulate glycometabolism and promote insulin synthesis, and catechin can remove excessive sugar in blood, reduce blood sugar level, and effectively prevent and treat diabetes. The white tea can reduce blood sugar concentration, and has effects of preventing diabetic complication,

gynostemma pentaphyllum, a unique non-ginseng plant, is a plant rich in dammarane-type triterpene sapogenins. Gynostemma pentaphylla is widely eaten as a traditional Chinese medicine (homology of medicine and food) in China, a dietary supplement, herbal tea, vegetables and the like. The traditional Chinese medicine considers that the gynostemma pentaphylla has multiple effects, such as hyperglycemia resisting, inflammation resisting and cholesterol reducing effects, and has the effects of enhancing immunity and reducing blood pressure. The gypenoside is a main bioactive component of the gynostemma pentaphylla, and has various bioactivities, such as biological activities of reducing blood fat, protecting liver, protecting cardiovascular and resisting oxidation.

The invention utilizes the functions of reducing blood sugar and blood fat of the white tea, and combines the gynostemma pentaphylla or the gynostemma pentaphylla total saponin compound with the function of reducing blood fat to achieve the function of regulating the blood fat disorder of the type 2 diabetes.

The white tea is one or more selected from pekoe, white peony, GONGWEI and SHOUWEI white tea, and the herba Gynostemmatis is whole plant of herba Gynostemmatis.

A formula (I) for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in a synergistic mode is as follows: the tea is prepared from 10 parts of white tea total polyphenol and 1-1.5 parts of gypenosides by mass, wherein the white tea is selected from pekoe silver needle, white peony, Gongmei or shoumei of Fujian fuding white tea, and the gynostemma pentaphylla is whole plant of the gynostemma pentaphylla.

A formula (II) for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in a synergistic mode is as follows: the tea is prepared from (by mass) white tea total polysaccharide 10 parts and herba Gynostemmatis total saponin 4-5 parts, wherein the white tea is selected from pekoe, white peony, GONGWEI or SHOUWEI of Fujian Fuding white tea, and herba Gynostemmatis is herba Gynostemmatis whole plant.

A formula (III) for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in a synergistic manner is as follows: the tea is prepared from 10 parts of white tea total polypeptide and 1-1.15 parts of gypenosides by mass, wherein the white tea is white pekoe, white peony, Gongmei or shoumei of Fujian Fuding white tea, and the gynostemma pentaphylla is whole plant of the gypeng.

A formula (IV) for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in a synergistic mode is as follows: the tea is prepared from 10 parts of white tea total polyphenol, 10 parts of white tea total polypeptide and 2-2.65 parts of gypenosides by mass, the white tea is white pekoe, white peony, tribute eyebrow or shoume of Fujian Fuding white tea, and the gynostemma pentaphylla is whole herb of the gynostemma pentaphylla.

A formula (V) for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in a synergistic mode is as follows: the tea is prepared from 10 parts of white tea total polysaccharide, 10 parts of white tea total polypeptide and 5-6.15 parts of gypenosides by mass, the white tea is white pekoe, white peony, tribute eyebrow or shoume of Fujian Fuding white tea, and the gynostemma pentaphylla is whole plant of the gynostemma pentaphylla.

A formula (VI) for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in a synergistic manner is as follows: the tea is prepared from 10 parts of white tea total polyphenol, 10 parts of white tea total polysaccharide and 5-6.5 parts of gypenosides by mass, the white tea is white pekoe, white peony, tribute eyebrow or shoume of Fujian Fuding white tea, and the gynostemma pentaphylla is whole plant of the gynostemma pentaphylla.

The white tea is in the form of instant micropowder, granule, syrup, solid or semisolid granule.

A formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in cooperation, wherein the instant fine tea powder consists of white tea polyphenol and gynostemma pentaphylla total saponin, and consists of 10 parts of white tea polyphenol and 2-3 parts of gynostemma pentaphylla total saponin by mass fraction;

as shown in fig. 1, a preparation method of a formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea, the preparation process mainly comprises the steps of extracting and purifying white tea polyphenol, white tea polysaccharide and white tea total polypeptide, extracting and purifying gynostemma pentaphylla total saponin, preparing different dosage forms of semisolid extract and solid granules, sterilizing and subpackaging.

The preparation method comprises the following steps:

(1) selecting high-quality Fujian Fuding white tea, and selecting one of pekoe silver needle, white peony, Gong eyebrow or shoume;

(2) selecting raw material gynostemma pentaphylla tea for manual selection, and respectively sieving the gynostemma pentaphylla tea by a fourth sieve to remove dust and granular impurities in the raw material;

(3) respectively crushing the white tea and the screened compatible raw materials by adopting a crushing and crushing machine, sieving, and respectively sieving according to the particle size to prepare medicinal material coarse powder and medicinal material fine powder of 10-20 meshes and 100-200 meshes;

(4) mixing the obtained coarse powder of white tea and herba Gynostemmatis according to the ratio of (5-6) to 1 to obtain coarse powder formula of tea, and further subpackaging, drying and sterilizing to obtain compound tea bag;

(5) preparing total polyphenol of white tea, namely taking 65 percent ethanol by volume fraction as an extraction solvent, heating and refluxing for 3 times at the extraction temperature of 80 ℃ for 40min each time, combining extracting solutions, standing, filtering to remove precipitates, carrying out rotary evaporation and reduced pressure concentration on filtrate, cooling, extracting pigment by using petroleum ether, extracting by using chloroform to remove impurities such as caffeine, finally extracting by using ethyl acetate, carrying out reduced pressure vacuum concentration to obtain a concentrated solution, carrying out freeze drying to obtain a crude polyphenol extract, further purifying the obtained crude polyphenol extract by using resin, namely filling the resin into a column, soaking for 2h by using 2 percent NaOH, eluting at the flow rate of 2m L/min, washing with pure water to be neutral, then eluting with ethanol, washing with pure water to be free of alcohol smell, preparing a crude polyphenol concentrated solution of 1.1g/m L, adsorbing by using ethanol at the ratio of 4:2 (the resin amount: the sampling volume), adsorbing for 30min, adsorbing by using 10 percent ethanol, 45 percent, 60 percent water, 95 percent water, carrying out gradient elution, collecting the water, carrying out gradient drying to obtain an extract of the tea, and obtaining tea extract powder by freezing and drying;

(6) preparing white tea total polysaccharide: the white tea powder is extracted twice by distilled water in a boiling water bath according to the proportion of 1:40 and 1:20(w/v), the extraction is carried out for 2h and 1.5h respectively, the filtrate is combined after filtration, reduced pressure concentration is carried out until the total volume is 1/10, equal volume of 95 percent ethanol is added for precipitation, standing overnight is carried out, the filtrate is obtained after filtration, the ethanol precipitation is concentrated and filtered, ethanol is recovered to obtain crude polysaccharide, weak alkaline resin cellulose is used for adsorption to remove pigment, trichlorotrifluoroethane is used for removing macromolecular protein, and a freeze drying method is adopted to remove redundant water to obtain the white tea total polysaccharide.

(7) Preparing the total polypeptide of white tea, taking white tea powder, adding an aqueous solution (pH 3.4) containing 2.5% of pectinase according to the ratio of 1:20(w/v), extracting for 3h at 45 ℃, centrifuging for 10min, removing supernatant, dissolving the obtained precipitate in NaOH (1.5 mol/L) according to the ratio of 1:45(v/v), further extracting for 1.5h at 90 ℃, cooling the mixture at room temperature, centrifuging, removing residues, adding HCl (pH 2.5) dropwise, standing for 0.5h, precipitating the protein in the supernatant, centrifuging, collecting the precipitate in a dialysis bag (500 and 550Da), dialyzing for 24h with deionized water, centrifuging again to remove supernatant, re-dissolving the obtained protein precipitate in 75% acetone according to the ratio of the liquid-solid ratio (6-8):1, shaking for 10min in a shaker, re-centrifuging, repeating the steps once, removing the supernatant containing acetone after final washing with pure water, obtaining a supernatant, centrifuging the obtained by using acid protease at 634 min, obtaining a supernatant after centrifugation for 0.2 m, obtaining a total polypeptide solution, and concentrating the obtained by a drying method after centrifugation at 632-8 m, and obtaining a total polypeptide solution under pressure, and a drying method after centrifugation, wherein the pH of 0.2 m is obtained.

(8) Preparing gypenosides: taking the fine powder of the gynostemma pentaphylla, heating and refluxing the fine powder for 3 times at 80 ℃ by using distilled water with a material-liquid ratio of 1:20(w/v), obtaining water extract, evaporating the water extract to dryness, and concentrating the water extract to 1/5 of the initial volume. Further extracting with 60% ethanol for 3 times, vacuum reflux concentrating to obtain extract, i.e. crude extract of herba Gynostemmatis total saponin, further eluting with macroporous resin D101/MCI resin with water, 30%, 70%, and 90% ethanol respectively to obtain components with different polarities, collecting 70% eluate, vacuum reflux concentrating to obtain extract, and freeze drying to obtain herba Gynostemmatis total saponin total extract;

(9) and (3) forming a formula: taking the white tea total polyphenol, the total polysaccharide or the total polypeptide obtained in the step (5-8) and the gynostemma pentaphylla total saponin respectively according to the proportion of (1-1.5), 10 (4-5) and 10 (1-1.15) to form an extract formula, and matching the obtained extract formulas pairwise according to the proportion of 1:1 to obtain the formulas of the white tea polyphenol, the white tea polysaccharide and the gynostemma pentaphylla total saponin, the white tea polyphenol, the white tea polypeptide and the gynostemma pentaphylla total saponin, and the white tea polypeptide, the white tea polysaccharide and the gynostemma pentaphylla total saponin.

(10) Preparation of syrup semisolid tea: mixing the tea extract formula obtained in the step (9) with sucrose according to a ratio of 1:2, adding 0.5 times of water and 0.1% tartaric acid, heating for dissolving, heating and concentrating at constant temperature of 80-90 ℃ until sugar liquor is golden yellow, and obtaining syrup semisolid tea.

(11) Preparing instant micro-powder tea: and (4) further grinding the tea extract formula obtained in the step (9) into micro powder with the particle size of less than 200 meshes, drying at the constant temperature of 80 ℃, controlling the water content of the particles or the instant micro powder to be 4.5-5.5%, subpackaging to 1.5-2.0 g/bag, irradiating by using an ultraviolet lamp for 40min, and sterilizing to obtain the composite instant micro powder tea.

(12) Preparing granules: and (3) preparing the tea extract formula obtained in the step (9) into granules by a further spray granulation method, drying at a constant temperature of 80 ℃, controlling the water content of the granules or instant micro powder to be 4.5-5.5%, subpackaging into 1.5-2.0 g/bag, and irradiating by using an ultraviolet lamp for 40min for sterilization to obtain the composite granule preparation.

An application of a formula of gynostemma pentaphylla and white tea in preventing type 2 diabetes in preparing a health food or a health medicine for reducing blood fat and regulating sugar.

The invention is further described below with reference to the accompanying drawings:

example 1

A formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in cooperation comprises pekoe polyphenol and gynostemma pentaphylla total saponin in a mass ratio of 10: 1.2. Wherein, the pekoe is taken as tea fine powder with the granularity of 100-200 meshes after being crushed and sieved. Weighing appropriate amount of white tea powder, extracting with 65% ethanol as extraction solvent at a material-to-liquid ratio of 1:20(w/v) and at 80 deg.C under reflux for 3 times, each for 40min, mixing extractive solutions, standing, and filtering to remove precipitate; concentrating the filtrate by rotary evaporation under reduced pressure, cooling, extracting with petroleum ether to remove pigment, extracting with chloroform to remove caffeine and other impurities, extracting with ethyl acetate, vacuum concentrating under reduced pressure to obtain concentrated solution, and freeze drying to obtain crude extract of tea polyphenols. The obtained crude extract of tea polyphenol is further purified by resin, and is eluted according to the gradient of 10%, 30%, 45%, 60%, 80% and 95% ethanol water. Collecting 45-80% ethanol water eluate, concentrating under reduced pressure to obtain extract, and freeze drying to obtain dried tea polyphenols powder. Taking an appropriate amount of herba Gynostemmatis fine powder, heating and refluxing with distilled water at a material-to-liquid ratio of 1:20(w/v) at 80 deg.C for 3 times to obtain water extractive solution, evaporating to remove water, and concentrating to 1/5 of initial volume. Further extracting with 60% ethanol for 3 times, vacuum reflux concentrating to obtain extract, i.e. crude extract of herba Gynostemmatis total saponin, further eluting with macroporous resin D101/MCI resin with water, 30%, 70%, and 90% ethanol respectively to obtain components with different polarities, collecting 70% eluate, vacuum reflux concentrating to obtain extract, and freeze drying to obtain herba Gynostemmatis total saponin total extract.

Mixing the above total polyphenol extract of Ardisia crenata and herba Gynostemmatis total saponin extract at a ratio of 10:1.2, further spray granulating to obtain granule, oven drying at 80 deg.C under constant temperature to control water content of the granule to 4.5-5.5%, irradiating with ultraviolet lamp for 40min, and sterilizing to obtain compound granule preparation with a dose of 1.5 g/bag.

Example 2

A formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in cooperation comprises white peony total polysaccharide and gynostemma pentaphylla total saponin in a mass ratio of 10: 4.4. Wherein, the white peony is crushed and sieved, and then the tea fine powder with the granularity of 100-200 meshes is taken. Extracting the obtained white peony white tea powder twice with distilled water in a boiling water bath according to the proportion of 1:40 and 1:20(w/v), extracting for 2h and 1.5h respectively, filtering, combining filtrates, concentrating under reduced pressure to 1/10 of the total volume, adding equal volume of 95% ethanol for precipitation, standing overnight, filtering to obtain filtrate, concentrating the ethanol precipitate, filtering, recovering ethanol to obtain crude polysaccharide, adsorbing with weak base resin cellulose to remove pigment, removing macromolecular protein with trichlorotrifluoroethane, and removing excessive water by adopting a freeze drying method to obtain white tea total polysaccharide. The white peony total polysaccharide extract and the gynostemma total saponin extract obtained by the method in the embodiment 1 are mixed according to the proportion of 10:4.4, a micro-powder tea formula with the particle size of less than 200 meshes is obtained through further micro-powder preparation, the mixture is dried at the constant temperature of 80 ℃, the water content is controlled to be not higher than 5.0%, and the mixture is irradiated by an ultraviolet lamp for 40min for sterilization to obtain a composite particle preparation, so that a formula instant micro-powder finished product with the weight of 1.5 g/bag is obtained.

Example 3

A formula of gynostemma pentaphylla and white tea for preventing type 2 diabetes mellitus comprises total polypeptides of Gongmei and total saponins of gynostemma pentaphylla, the mass ratio of the total polypeptides to the total saponins is 10:1.12, wherein the Gongmei white tea is crushed and sieved, tea fine powder with the particle size of 100 meshes and 200 meshes is taken, Gongmei white tea powder is taken, aqueous solution (pH 3.4) containing 2.5% of pectinase is added according to the proportion of 1:20(w/v), after extraction for 3h at 45 ℃, centrifugation is carried out for 10min, supernatant is removed, precipitate is obtained and dissolved in NaOH (1.5 mol/L) according to the proportion of 1:45(v/v), the mixture is further extracted for 1.5h at 90 ℃, the mixture is cooled at room temperature and centrifuged, residues are removed, HCl (pH 2.5) is dripped for 0.5h, protein in the supernatant is precipitated, centrifugation is carried out, the precipitate is collected in a dialysis bag, after dialysis is carried out for 24h, the precipitate is centrifuged again according to remove supernatant, the supernatant is obtained, after centrifugation is carried out again according to the proportion of 6: 8: 1.5, the supernatant is added in a syrup, the aqueous solution of the mixture is stirred and the supernatant is added with pure water, the supernatant is added, the supernatant is stirred, the supernatant is added, the mixture is stirred, the temperature is carried out, the mixture is added, the mixture is carried out, the temperature is carried out, the centrifugation is carried out.

Example 4

A formula of gynostemma pentaphylla and white tea for preventing type 2 diabetes is prepared according to example 1, and the formula consists of pekoe polyphenol and gynostemma pentaphylla total saponin, wherein the in vitro lipid-lowering activity of the gynostemma pentaphylla and white tea is evaluated by evaluating the inhibition effect of the gynostemma pentaphylla on lipase.

The specific implementation method comprises the following steps:

a lipase inhibition test is carried out by taking a 96-well plate, arranging a sample hole and a blank hole, adding pancrelipase 30 mu L and Tris-HCl buffer solution 25 mu L into the sample hole and the blank hole, mixing uniformly, incubating for 10min at 37 ℃ in a constant temperature box, adding 5 mu L sample and 80 mu L PNPP into the sample hole, adding 5 mu L buffer solution and 80 mu L PNPP into the blank hole, reacting for 40min at 37 ℃, stopping the reaction by boiling water, measuring absorbance OD value at 405nm by using a microplate reader, measuring lipase activity by measuring PNP absorption at 405nm, and calculating lipase inhibition rate (OD) (% inhibition rate) (OD)_{Blank space}-(OD_{Sample (I)}-OD_Background))/OD_{Blank space}) × 100% in this case, three duplicate wells were set up, three replicates were run, and the IC was further calculated using SPSS₅₀And finally obtaining the evaluation result of the inhibition effect of the formula on the lipase according to the results of three parallel tests, wherein the evaluation result is as follows:

TABLE 1 inhibition of lipase (IC) by pekoe polyphenol, gynostemma total saponin and their formulations₅₀Values) are as follows:

and (4) analyzing results:

the inhibition effect of white tea polyphenol and gynostemma pentaphylla total saponin on lipase is enhanced.

Example 5

A formulation of gynostemma pentaphylla cooperating with white tea for preventing type 2 diabetes, its preparation method refers to example 2, it is made up of total polysaccharides of white peony and total saponin of gynostemma pentaphylla, its activity of reducing blood sugar in vitro is evaluated through evaluating its inhibition to α -glucoside, its method lies in:

α -hydrolysis of glucosidase, in brief, a sample well and a blank well are provided, the sample well is 30 μ L enzyme solution +20 μ L0 sample solution → 37 ℃ water bath 5min → substrate 150 μ L1 +800 μ L buffer solution → 37 ℃ water bath incubation 30min → stop solution 2m L, the blank well is mixed by centrifugation, the blank well is 30 μ L enzyme solution +20 μ L buffer solution → 37 ℃ water bath 5min → substrate 150 μ L +800 μ L buffer solution → 37 ℃ water bath incubation 30min → stop solution 2m L, the mixture is mixed by centrifugation, 150 μ L of the above reaction solution is respectively sucked into a 96-well plate, the absorbance is measured by an enzyme labeling instrument under 405nm, the inhibition ratio is calculated:

inhibition ratio (%) ═ a_{Blank space}-(A_{Sample (I)}-A_Background)/A_{Blank space}]×100％。

The obtained results are shown in fig. 2, according to the results, the pekoe white tea polyphenol and the gynostemma total saponin have different inhibition effects on α -glucosidase, and the inhibition effect of the formula of the pekoe white tea polyphenol and the formula of the gynostemma pentaphyllum total saponin on α -glucosidase is stronger than that of the pekoe white tea polyphenol and the formula of the gynostemma pentaphyllum total saponin when the pekoe white tea polyphenol and the formula of the gynostemma pentaphyllum total saponin are used alone, which shows that the in vitro.

Example 6

A formula of gynostemma pentaphylla and white tea for preventing type 2 diabetes is disclosed, wherein the preparation method of the formula refers to example 3, and the formula comprises tribute eyebrow total polypeptides and gynostemma pentaphylla total saponins, and the in vitro blood glucose reduction activity of the formula is evaluated by evaluating the inhibition effect of PTP 1B. The method comprises the following steps:

assay for the inhibition of PTP1B, which consists in the hydrolysis of PTP1B, briefly, 83 μ L enzyme solution (PTP1B enzyme solution) +10 μ L sample solution +4 μ L substrate → incubation at 37 ℃ for 30min → addition of 5 μ L2 mol/m L NaOH solution to stop the reaction and measuring the absorbance at 405 nm.

Inhibition (% ((OD blank-OD sample)/OD blank) × 100%;

the obtained results are shown in fig. 3, according to the results, the white peony white tea total polypeptide and the gynostemma pentaphylla total saponin have different inhibition effects on PTP1B, and the inhibition effect of the formulas of the white peony white tea total polypeptide and the gynostemma pentaphylla total saponin on PTP1B is stronger than that of the white peony white tea total polypeptide and the gynostemma pentaphylla total saponin when the white peony white tea total polypeptide and the gynostemma pentaphylla total saponin are used alone, which shows that the in vitro hypoglycemic activity.

Example 7

A formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in cooperation, the preparation method of the formula refers to example 1, the formula comprises Gong Mei white tea and gynostemma pentaphylla, the glycolipid metabolism regulation effect of the gynostemma pentaphylla on type 2 diabetes mellitus is achieved by the following evaluation method:

SPF grade C57B L/6 mice (18-20g) were housed in standard polypropylene cages and maintained in a Specific Pathogen Free (SPF) environment for a 12h/12h light/dark cycle with ambient noise of 40 + -10 db, room temperature of 21-23 deg.C, humidity of 50-60%, and allowed free access to water and food for the mice after one week of acclimation above, animals were randomly divided into normal control groups and high fat diet groups for 6 weeks, and after 6 weeks of high fat diet, mice were injected intraperitoneally with STZ solution at a dose of 50mg/kg for 4 consecutive days.

After 72 hours from the completion of the last STZ injection, Fasting Blood Glucose (FBG) values were determined by collecting blood from the tail vein of the mice, the mice with FBG higher than 11.3 mmol/L were considered successful in diabetes modeling, and the mice with FBG lower than 11.3 mmol/L were given STZ (60mg/kg) by further intraperitoneal injection, and the mice that were successful in modeling were further randomly divided into 4 groups (n-8-10) and fed with HFD until the end, including the diabetes model group, white tea group, gynostemma pentaphyllum group and formula group.

After the intervention of the white tea and/or the gynostemma pentaphylla sample for 6 weeks, the animals are fasted for 12 hours overnight after the administration 2 days before the death, and the blood glucose value of the last fasting blood is measured. On the end of the experiment, fasting was carried out for 8h after the last gavage, and blood was taken from the eyeball after the mice were anesthetized with ether, and serum was collected and stored in a refrigerator at-80 ℃ for testing.

The levels of triglyceride, total cholesterol, high density lipoprotein, low density lipoprotein, alanine aminotransferase and aspartate aminotransferase in the mouse serum samples were determined by colorimetry.

During the experiment, the body weight of the mice was measured weekly, and the water intake and food intake of the mice were recorded.

Fasting blood glucose values FBG measured at the same time weekly: after the mice were fasted overnight for 12 hours, blood was collected from the tail vein of the mice using a blood collection needle, and the blood glucose value was measured and recorded using a glucometer.

Steady-state model assessment of insulin resistance (HOMA-IR) calculation by enzyme-linked immunosorbent assay (E L ISA) kit HOMA-IR ═ FBG (mg/dl) x FIN (μ IU/ml)/405; HOMA- β ═ 20 × FIN (mIU/L)/(FBG (mmol/L) -3.5)

OGTT test: after fasting overnight for 12 hours, the mice of each group were gavaged with a glucose solution at a dose of 2g/kg body mass. Blood glucose values were measured by collecting blood from the tail tips at 0, 0.5, 1, 1.5, and 2h after the administration of sugar, respectively, a blood glucose change curve was plotted, and the area under the curve (AUC) was calculated. OGTT_AUC＝1/4(BG_0h)+1/2(BG_0.5h)+3/4(BG_1h)+1/2(BG_2h)。

HE staining: liver tissues of the isolated mice were fixed with a tissue fixing solution, and subjected to embedding, deparaffinization, dehydration, and the like, and the sections were stained with hematoxylin and eosin for morphological analysis, and histopathological analysis was performed.

Dyeing with oil red O: fixing the liver tissue of the isolated mouse by tissue fixing liquid, and completing the oil red O staining process after embedding, slicing and other processes.

Evaluation results were analyzed as follows:

the evaluation results of the weight effect of type 2 diabetic mice were analyzed as follows:

as shown in fig. 4, the white tea and the gynostemma pentaphylla, especially the formula group, slowed down the weight loss of the mice to some extent, suggesting that the white tea and the gynostemma pentaphylla have enhanced effect of improving the weight loss of the mice with type 2 diabetes after being combined.

The evaluation results of the effect on drinking water and diet of type 2 diabetic mice were analyzed as follows:

as shown in fig. 5, the amount of diet and water intake was significantly reduced compared to the model group mice 6 weeks after the administration of white tea. Meanwhile, the intervention of the gynostemma pentaphylla also reverses the food and water intake of the diabetic mice to a certain extent. Meanwhile, the intervention of the combination of the white tea and the gynostemma pentaphylla on the T2DM mouse also obviously regulates the phenomenon of polyphagia and polydipsia of the diabetic mouse, and the effect is even stronger, which indicates that the white tea and the gynostemma pentaphylla are combined to probably have the effect of enhancing and improving the symptoms of diabetes.

The results of the evaluation of the effect on blood glucose levels in type 2 diabetic mice were analyzed as follows:

as shown in fig. 6, while white tea and gynostemma pentaphylla improved the abdominal blood glucose level in diabetic mice, a relatively stronger hypoglycemic effect was observed when white tea was used in combination with gynostemma pentaphylla, indicating that the combined use of the two had an enhanced blood glucose regulating effect.

The results of the evaluation of the effect on OGTT levels in type 2 diabetic mice were analyzed as follows:

as shown in FIG. 7, OGTT is an important index for testing the body's response to acute hyperglycemia, and is generally used for evaluating the glucose metabolism level and the function of pancreas β cells, our research results show that white tea can regulate the glucose metabolism level of diabetic mice to some extent, and the action of gynostemma pentaphylla is relatively weak (p >0.05), but the action is enhanced after the white tea is combined with the gynostemma pentaphylla.

The evaluation results of the effect of insulin resistance in type 2 diabetic mice were analyzed as follows:

as shown in FIG. 8, the white tea and the gynostemma pentaphylla can lower the HOMA-IR level of the diabetes mouse induced by HFD/STZ to different degrees and raise the HOMA- β level to different degrees, which means that the white tea and the gynostemma pentaphylla have different improving effects on the insulin resistance and the insulin function of the diabetes mouse, and the effect of the white tea and the gynostemma pentaphylla is strengthened to a certain degree after the white tea and the gynostemma pentaphylla are combined.

Evaluation results of the effect on lipid metabolism in type 2 diabetic mice were analyzed as follows:

TABLE 2 influence of white tea, Gynostemma pentaphyllum and formulation on dyslipidemia of diabetic mice

Data are expressed as mean ± SEM (n-8), compared to T2DM,^*p<0.05,^**p<0.01; compared with Comb group, ^ p<0.05; in contrast to the Control group,^##p<0.01.

however, compared with two kinds of white tea, the combined T-CHO, TG and L D L are respectively reduced by 21.32 percent, 34.89 percent and 25.99 percent, and the HD L level is increased by 49.63 percent, which shows that the white tea and the gynostemma pentaphylla are combined to enhance the blood fat regulation effect of diabetic mice to a certain extent.

The evaluation results of the ameliorating effect on pancreatic tissue inflammatory injury in type 2 diabetic mice were analyzed as follows:

according to the results shown in fig. 9, the intervention of the white tea and the gynostemma pentaphylla remarkably reduces the expression levels of the I L-6 and TNF- α proteins of the inflammatory factor in pancreatic tissues of the diabetic mice, and the combined use of the white tea and the gynostemma pentaphylla remarkably enhances the inhibition effect of the inflammatory factor compared with the effect of the white tea and the gynostemma pentaphylla when the white tea and the gynostemma pentaphylla are used independently, so that the regulation effect of the white tea and the gynostemma pentaphylla on the inflammatory injury of the pancreatic tissues of the diabetic mice is enhanced.

The evaluation results of the evaluation on the effect of the pancreatic tissue on the expression of the antioxidant enzyme in the type 2 diabetes mouse are analyzed as follows:

according to the results shown in fig. 10, the formula of the white tea and the gynostemma pentaphylla can significantly enhance the expression of HO-1 and GPx in pancreatic tissues of diabetic mice, and according to the experimental results, it is presumed that the effect of improving pancreatic tissue damage by the combination of the white tea and the gynostemma pentaphylla is related to the enhancement of the activity of antioxidant enzymes.

The evaluation results of the improvement effect on the liver tissue damage of the type 2 diabetic mouse were analyzed as follows:

the results of H & E staining analysis of liver tissue are shown in fig. 11: based on the pathological analysis results, the intervention of the white tea and the gynostemma pentaphylla can slow down or block the liver injury of the diabetic mice caused by HFD/STZ to different degrees, including the injuries of fatty lesion, cell necrosis and the like of the liver, and after the white tea and the gynostemma pentaphylla are combined, the protective effect on the liver is enhanced to a certain degree.

The evaluation results of the improvement effect on the lipid accumulation in liver tissue of type 2 diabetic mice were analyzed as follows:

the study performed analysis of mouse livers using oil red O staining, as shown in figure 12. The results show that the livers of the mice in the T2DM model group have a large number of fat drops stained red, and the gynostemma pentaphylla and the white tea especially have the formulas to reduce the fat drops stained red by the oil red O in the livers to different degrees.

The evaluation results of the effect on the expression of the protein associated with lipid accumulation in liver tissue of type 2 diabetic mice were analyzed as follows:

as shown in FIG. 13, ACC activity expression is expressed as p-ACC/ACC ratio, and the ACC protein expression level in liver of mice in T2DM group is significantly increased (p < 0.01) compared to the control group, indicating the overexpression of ACC in liver of diabetic mice. In contrast to the model group, ACC expression was significantly down-regulated in the liver of gynostemma pentaphyllum mice (p < 0.01), whereas intervention with white tea showed a weak effect on ACC expression in the liver of diabetic mice and did not reach statistical significance (p > 0.05). However, the liver ACC expression level of mice in the formula group is remarkably reduced (p < 0.01) and is remarkably enhanced (p < 0.05) compared with that in the white tea group after being combined with gynostemma pentaphylla. Meanwhile, the expression level of FAS in the liver of diabetic mice is determined in the research, and the expression of FAS in each group is based on the similar rule of ACC protein activity.

The evaluation results of the effect of SREBPs protein expression in liver tissue of type 2 diabetic mice were analyzed as follows:

as shown in the results of FIG. 14, the combination of white tea and Gynostemma pentaphyllum can significantly reduce the levels of SREBP-1c and SREBP-2 in the liver of the diabetic mouse, and is stronger than either effect when the white tea and Gynostemma pentaphyllum are used alone, but achieves statistical difference compared with the white tea group. Therefore, the formula of the white tea and the gynostemma pentaphylla can improve the liver steatosis of diabetic mice by regulating the SREBP pathway.

The evaluation results of the effect on the liver tissue inflammatory injury of type 2 diabetic mice were analyzed as follows:

as shown in the result of figure 15, the formula of the white tea and the gynostemma pentaphylla can remarkably reduce the levels of liver I L-6, I L-1 β and TNF- α of the type 2 diabetes mouse, and has stronger effect than that of the formula used alone, so that the formula of the white tea and the gynostemma pentaphylla can improve the liver inflammation injury of the diabetes mouse by inhibiting the expression of inflammatory factors.

The evaluation results of the effect of NF- κ B pathway in liver tissue of type 2 diabetic mice were analyzed as follows:

according to the results shown in fig. 16, the formulation of the white tea and the gynostemma pentaphylla remarkably down-regulates the expression of NF-kappa B p65, so that the formulation of the white tea and the gynostemma pentaphylla can regulate the NF-kappa B signaling pathway and relieve the inflammatory injury of the liver of the type 2 diabetic mouse induced by HFD/STZ.

Evaluation results of effects on PPAR pathway in liver tissue of type 2 diabetic mice were analyzed as follows:

according to the results shown in fig. 17, the formula of the white tea and the gynostemma pentaphylla remarkably enhances the expression activities of damaged PPAR α and PPAR γ in the liver tissues of the type 2 diabetic mice, which indicates that the formula can improve the liver inflammation injury and the steatosis of the diabetic mice through a PPAR pathway.

The evaluation results of the effect of the PI3K pathway on liver tissue of type 2 diabetic mice were analyzed as follows:

according to the results shown in fig. 18, the formula of the white tea and the gynostemma pentaphylla can regulate the related proteins of the liver tissue IRS/PI3K/Akt pathway of type 2 diabetic mice, including IRS, PI3K (p110), PI3K (p85), p-Akt and G L UT2, which indicates that the formula of the white tea and the gynostemma pentaphylla can enhance the expression of G L UT2 through forward regulation of the PI3K pathway, thereby promoting glucose metabolism and further reducing blood sugar, therefore, the formula can regulate the sugar metabolism of type 2 diabetic mice through forward regulation of the PI3K/Akt pathway.

according to the results shown in fig. 19, the formula of the white tea and the gynostemma pentaphylla can regulate the expression levels of proteins related to the AMPK pathway of liver tissues of type 2 diabetic mice, including p-AMPK and G6Pase, so that the formula of the white tea and the gynostemma pentaphylla can regulate gluconeogenesis, reduce blood sugar and inhibit fat synthesis and accumulation by activating the AMPK pathway. Therefore, the formula can regulate the glycolipid metabolism of the type 2 diabetes mouse by regulating the AMPK pathway.

Example 8

A formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in cooperation comprises tea polyphenol of shoumei and gypenosides, wherein the mass ratio of the tea polyphenol to the gypenoside is 10:1. Wherein, the shoumei white tea is taken as tea fine powder with the granularity of 100 plus 200 meshes after being crushed and sieved. Weighing appropriate amount of white tea powder, extracting with 65% ethanol as extraction solvent at a material-to-liquid ratio of 1:20(w/v) and at 80 deg.C under reflux for 3 times, each for 40min, mixing extractive solutions, standing, and filtering to remove precipitate; concentrating the filtrate by rotary evaporation under reduced pressure, cooling, extracting with petroleum ether to remove pigment, extracting with chloroform to remove caffeine and other impurities, extracting with ethyl acetate, vacuum concentrating under reduced pressure to obtain concentrated solution, and freeze drying to obtain crude extract of tea polyphenols. The obtained crude extract of tea polyphenol is further purified by resin, and is eluted according to the gradient of 10%, 30%, 45%, 60%, 80% and 95% ethanol water. Collecting 45-80% ethanol water eluate, concentrating under reduced pressure to obtain extract, and freeze drying to obtain dried tea polyphenols powder. Taking an appropriate amount of herba Gynostemmatis fine powder, heating and refluxing with distilled water at a material-to-liquid ratio of 1:20(w/v) at 80 deg.C for 3 times to obtain water extractive solution, evaporating to remove water, and concentrating to 1/5 of initial volume. Further extracting with 60% ethanol for 3 times, vacuum reflux concentrating to obtain extract, i.e. crude extract of herba Gynostemmatis total saponin, further eluting with macroporous resin D101/MCI resin with water, 30%, 70%, and 90% ethanol respectively to obtain components with different polarities, collecting 70% eluate, vacuum reflux concentrating to obtain extract, and freeze drying to obtain herba Gynostemmatis total saponin. The SHOUJI total polyphenols and herba Gynostemmatis total saponin are mixed at a ratio of 10:1 to form a formula.

The preparation form of the formula comprises: instant micropowder white tea blood sugar reducing tea, granule blood sugar reducing tea and white tea solid blood sugar reducing preparation, and semisolid syrup extract white tea blood sugar reducing preparation.

Example 9

A formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in cooperation comprises tea polyphenol of shoumei and gypenosides, wherein the mass ratio of the tea polyphenol to the gypenoside is 10: 1.5. The preparation method is the same as that of example 8. The formulation form of the formula comprises: instant micropowder white tea blood sugar reducing tea, granule blood sugar reducing tea and white tea solid blood sugar reducing preparation, and semisolid syrup extract white tea blood sugar reducing preparation.

Example 10

A formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in cooperation comprises pekoe total polyphenol, white peony total polysaccharide and gynostemma pentaphylla total saponin in a mass ratio of 10:10: 5.15. Wherein the preparation method of the total polyphenol of the pekoe silver needles is as the embodiment example 1, the preparation method of the total polysaccharide of the white peony is as the embodiment example 2, and the preparation method of the total saponin of the gynostemma pentaphylla is as the embodiment example 1. The obtained total polyphenol of the pekoe, the total polysaccharide of the white peony and the total saponin of the gynostemma pentaphylla are mixed according to the proportion of 10:10:5.15 to form a formula.

The preparation form of the formula comprises the following components: instant micropowder white tea blood sugar reducing tea, granule blood sugar reducing tea and white tea solid blood sugar reducing preparation, and semisolid syrup extract white tea blood sugar reducing preparation.

Example 11

A formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in cooperation is characterized in that pekoe total polyphenols, white peony total polysaccharides, shoumei total polypeptides and gynostemma pentaphylla total saponins are adopted, and the mass ratio is 10:10:10: 3. Wherein the preparation method of the total polyphenol of the pekoe silver needles is as the embodiment example 1, the preparation method of the total polysaccharide of the white peony is as the embodiment example 2, the preparation method of the shoumei total polypeptide is as the embodiment example 3, and the preparation method of the total saponin of the gynostemma pentaphylla is as the embodiment example 1. The obtained total polyphenol of the pekoe, the total polysaccharide of the white peony and the total saponin of the gynostemma pentaphylla are mixed according to the proportion of 10:10:10:3 to form a formula.

Example 12

A formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in cooperation is characterized in that pekoe total polyphenols, white peony total polysaccharides, shoumei total polypeptides and gynostemma pentaphylla total saponins are adopted, and the mass ratio is 10:10:10: 7.65. Wherein the preparation method of the total polyphenol of the pekoe silver needles is as the embodiment example 1, the preparation method of the total polysaccharide of the white peony is as the embodiment example 2, the preparation method of the shoumei total polypeptide is as the embodiment example 3, and the preparation method of the total saponin of the gynostemma pentaphylla is as the embodiment example 1. The obtained total polyphenol of the pekoe, the total polysaccharide of the white peony and the total saponin of the gynostemma pentaphylla are mixed according to the proportion of 10:10:10:7.65 to form a formula.

Claims

1. The formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea in cooperation is characterized in that: the formula consists of white tea and gynostemma pentaphylla.

2. The formula for preventing type 2 diabetes mellitus comprising gynostemma pentaphylla and white tea according to claim 1, wherein the formula comprises: the white tea is one or more of pekoe, white peony, Gongmei and shoumei white tea of Fujian fuding, and the white tea in the formula is one or more of white tea total polyphenol, total polysaccharide or total polypeptide obtained by extraction and purification.

3. The formula for preventing type 2 diabetes mellitus comprising gynostemma pentaphylla and white tea according to claim 1, wherein the formula comprises: herba Gynostemmatis refers to whole herb, leaf or flower of plant of Gynostemma of Cucurbitaceae, or total saponin of herba Gynostemmatis obtained by extracting and purifying whole herb of herba Gynostemmatis.

4. The formula for preventing type 2 diabetes mellitus comprising gynostemma pentaphylla and white tea according to claim 1, wherein the formula comprises: the formula comprises the following components in percentage by mass: is prepared from one or more of white tea total polyphenol 10 parts, total polysaccharide 10 parts and total polypeptide 10 parts, and herba Gynostemmatis total saponin 1-7.65 parts.

5. The formula for preventing type 2 diabetes mellitus comprising gynostemma pentaphylla and white tea according to claim 1, wherein the formula comprises: the final form of the white tea formula is instant micropowder, solid preparation of granule or semi-solid preparation of syrup.

6. The formula for preventing type 2 diabetes mellitus comprising gynostemma pentaphylla and white tea according to claim 2, wherein the formula comprises: the method for extracting and purifying the total polyphenol, the total polysaccharide and the total saponin of the white tea comprises the following steps:

(3) the extraction and purification method of white tea total polypeptide comprises the steps of taking white tea powder, adding an aqueous solution containing 2.5% of pectinase according to the ratio of 1:20w/v, carrying out extraction at 45 ℃ for 3h, centrifuging for 10 minutes, removing supernatant, dissolving the obtained precipitate in 1:45v/v NaOH according to the ratio of 1:45v/v, further extracting at 90 ℃ for 1.5h, cooling the mixture at room temperature, centrifuging, removing residues, dropwise adding HCl, carrying out pH 2.5, standing for 0.5h, precipitating protein in the supernatant, centrifuging, collecting the precipitate in a dialysis bag 500-550Da, dialyzing with deionized water for 24h, centrifuging again to remove supernatant, re-dissolving the obtained protein precipitate in 75% acetone according to the ratio of liquid-solid ratio (6-8):1, shaking for 10min in a shaker, re-centrifuging again, repeating the steps once, finally washing with pure water, removing acetone-containing supernatant, obtaining solid matter, centrifuging the obtained by using acid protease 50m, 0.32 min, obtaining a supernatant, concentrating the obtained white tea powder by a drying method after centrifugation method, and carrying out centrifugation for 2h, and carrying out centrifugation for 0.2-0.2 h, and carrying out pressure drying on the obtained white tea total polypeptide after centrifugation.

7. The formula for preventing type 2 diabetes mellitus comprising gynostemma pentaphylla and white tea according to claim 3, wherein the formula comprises: the extraction and purification method of the gynostemma total saponin comprises the following steps:

8. The preparation method of the formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea as claimed in claim 1, wherein the formula comprises the following components in parts by weight: the method comprises the following steps:

9. The preparation method of the formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea as claimed in claim 8, wherein the constant-temperature heating concentration in step (6) means that the moisture content of the final semi-solid extract syrup is 22-25%, the drying step in steps (7) and (8) means that the prepared granules or instant micro powder are dried at a constant temperature at 80 ℃ to control the moisture content of the granules or instant micro powder to be 4.5-5.5%, the sterilization in steps (7) and (8) means that the packaged finished product is sterilized by irradiation of an ultraviolet lamp, the minimum packaging in step (6) is 50-100m L, and the minimum packaging in steps (7) and (8) is 1.5-2.5 g/bag.

10. The application of the formula for preventing type 2 diabetes mellitus by using gynostemma pentaphylla and white tea as claimed in claims 1-7 in health-care food or health-care medicine for reducing fat and regulating sugar.