A method for promoting the transformation of rare ginsenosides in gynostemma pentaphyllum and therapeutic effect of preparation products thereof on metabolic syndrome

Abstract

The application discloses a method for promoting transformation of rare ginsenoside in gynostemma pentaphyllum, and comprises the following steps: (1) taking a proper amount of dried gynostemma pentaphyllum medicinal material, crushing and then adding water for soaking; (2) soaking for a period of time under a certain water bath temperature; (3) after steaming and drying water, adding a certain concentration of ethanol or methanol, heating and refluxing extraction or ultrasonic extraction, filtering after cooling the extract, reducing pressure concentration, and obtaining a concentrate; and (4) loading the concentrate on a macroporous resin, adsorbing, eluting, collecting 60-80% ethanol elution part, and obtaining dried total saponin. The method has the advantages of mild reaction condition, high efficiency, strong specificity, no pollution, high content of prepared product rare ginsenoside, and can be used for preventing and / or treating metabolic diseases such as non-alcoholic fatty liver disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a method for promoting the conversion of rare ginsenosides in Gynostemma pentaphyllum and the therapeutic effect of the preparation product on metabolic syndrome. BACKGROUND

[0002] Metabolic diseases are metabolic disorders caused by disorders of one or more links of metabolism. Metabolic disorders of glucose, protein and lipid in the body can cause metabolic diseases such as obesity, diabetes, non-alcoholic fatty liver disease, etc. With the improvement of modern living standards, the incidence of metabolic diseases is increasing year by year, which threatens human health. Not only can it cause secondary pathophysiological changes in multiple systems of the patient, but also it brings heavy burden to the patient and the entire medical and health system of the society.

[0003] Gynostemma pentaphyllum (Thumb.) Makino is a perennial vine of Cucurbitaceae. It is called "Southern Ginseng" and "Second Ginseng" because it contains the same components as ginsenosides. Gypenoside is one of the main active ingredients, which has the effects of nourishing the heart and spleen, benefiting qi and blood, removing phlegm and removing blood stasis, etc., and has good market prospects.

[0004] Gynostemma pentaphyllum is rich in tetracyclic triterpene saponins. So far, 327 saponins with clear structure have been isolated and identified from Gynostemma pentaphyllum plants. Six of them, such as gypenoside III, IV, VIII, etc., are homonyms of ginsenosides Rb1, Rb3, Rd, F2, Rg3 and ginsenoside K. The other 77 monomer saponins are all isomers of ginsenosides. Finding a new drug source rich in ginsenosides in plants outside the Araliaceae family not only has important academic significance, but also has great economic and social value. Modern pharmacological activity studies have shown that gypenosides have significant effects on protecting the cardiovascular and cerebrovascular system, anti-tumor, enhancing immunity, and protecting the liver.

[0005] Ginsenosides are one of the main active components of ginseng, which have various important pharmacological activities. Rare ginsenosides such as F1, F2, Rg3, Rh1, Rh2, CK, CY and CMc have very low content in ginseng. However, these rare saponins (with 1-2 sugars in the mother nucleus) have better activity. Therefore, how to prepare rare ginsenosides is a problem of concern to researchers. There are 77 saponins in Gynostemma pentaphyllum which are isomers of ginsenosides. Finding and preparing rare ginsenosides from Gynostemma pentaphyllum has good application prospects. The rare saponins in Gynostemma pentaphyllum have the same parent structure as the high-content saponins, only the number of sugar groups is different. Therefore, the rare ginsenosides can be prepared by selectively hydrolyzing the sugar groups of high-content saponins. Enzymatic method has the advantages of mild reaction conditions, high efficiency, strong specificity and no pollution, so it is the best method for preparing rare saponins. SUMMARY

[0006] Based on this, the present application provides a method for promoting the transformation of rare ginsenosides in Gynostemma pentaphyllum, which comprises the following steps:

[0007] (1) taking a proper amount of dried Gynostemma pentaphyllum medicinal material, crushing it and then adding water to soak it;

[0008] (2) heating it at a certain water bath temperature for a period of time;

[0009] (3) after evaporating the water, adding a certain concentration of ethanol or methanol, heating and refluxing for extraction or ultrasonic extraction, filtering the extract after cooling, and concentrating under reduced pressure to obtain a concentrate; and

[0010] (4) loading the concentrate onto a macroporous resin, adsorbing, eluting, collecting the 60-80% ethanol elution fraction, and obtaining dried total saponins.

[0011] Further, in step (1), the mass-volume ratio (g / L) of the dried Gynostemma pentaphyllum medicinal material to the water is 20:1-100:1.

[0012] Further, the mass-volume ratio (g / L) of the dried Gynostemma pentaphyllum medicinal material to the water is about 50:1.

[0013] Further, the water is deionized water.

[0014] Further, the water is distilled water.

[0015] Further, in step (2), the temperature of the water bath is 30-70°C.

[0016] Further, the temperature of the water bath is 35-50°C.

[0017] Further, the temperature of the water bath is 35-45°C.

[0018] Further, the temperature of the water bath is about 40°C.

[0019] Further, the time of the water bath is 1-12 hours.

[0020] Further, the time of the water bath is 2-4 hours.

[0021] Further, the time of the water bath is about 3 hours.

[0022] Further, in step (4), the concentration of the ethanol or the methanol is 60-80%.

[0023] Further, the concentration of the ethanol or the methanol is about 70%.

[0024] Further, in step (4), the volume ratio of the concentrate to the extract is about 0.06:1.

[0025] Further, in step (4), the volume ratio of the concentrate to the extract is about 0.06:1.

[0026] Further, in step (4), the number of times of the heating reflux extraction or the ultrasonic extraction is 1-4.

[0027] Further, when the number of times of the heating reflux extraction or the ultrasonic extraction is 2-4, the step (4) further comprises a step of combining the filtered extract.

[0028] Further, the number of times of the heating reflux extraction or the ultrasonic extraction is 2.

[0029] Further, the time of the heating reflux extraction or the ultrasonic extraction is 0.5-2 hours.

[0030] Further, the time of the heating reflux extraction or the ultrasonic extraction is about 1 hour.

[0031] Further, in step (4), the mass-volume ratio (g / L) of the sun-dried Gynostemma pentaphyllum medicinal material to the methanol or the ethanol is 20:1-100:1.

[0032] Further, in step (4), the mass-volume ratio (g / L) of the sun-dried Gynostemma pentaphyllum medicinal material to the methanol or the ethanol is about 40:1.

[0033] Further, in step (5), the macroporous resin is D101 resin.

[0034] Further, the adsorption time of the concentrate on the macroporous resin is 20-28 hours.

[0035] Further, the adsorption time of the concentrate on the macroporous resin is about 24 hours.

[0036] Further, in step (5), the elution comprises elution impurity removal using water and 20%-40% ethanol, and elution using 60%-80% ethanol.

[0037] Further, the elution comprises elution impurity removal using water and about 30% ethanol, and elution using about 70% ethanol.

[0038] According to another aspect of the present application, there is provided a preparation product obtained by the above method.

[0039] Further, the saponin component in the preparation product comprises 0%-10% of 3-4 glycosyl-substituted gypenosides.

[0040] Further, the saponin component in the preparation product comprises 90% to 100% of 1 to 2 glycosyl-substituted secondary saponins.

[0041] Further, the 1 to 2 glycosyl-substituted secondary saponins are gypenoside LXXVII, gypenoside TN-1, gypenoside XIII, and / or gypenoside C-K or isomers thereof.

[0042] Further, the 1 to 2 glycosyl-substituted secondary saponins are gypenoside LXXVII, gypenoside TN-1, gypenoside XIII, and / or gypenoside C-K.

[0043] According to another aspect of the present application, a composition comprising the above preparation product is provided, which further comprises one or more substances for preventing and / or treating metabolic diseases, and / or pharmaceutically acceptable excipients.

[0044] According to another aspect of the present application, the above preparation product or the above composition is used in a pharmaceutical product, a food product and / or a health product for preventing and / or treating metabolic diseases.

[0045] Further, the metabolic disease is metabolic syndrome.

[0046] Further, the metabolic disease is obesity, diabetes and / or non-alcoholic fatty liver disease.

[0047] Advantages of the present application:

[0048] The method of the present application has mild reaction conditions, high efficiency, strong specificity, no pollution, and the obtained preparation product has high content of rare ginsenosides, and can be used for preventing and / or treating metabolic diseases such as non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained according to these drawings without exceeding the scope of the present application.

[0050] Figure 1 Schematic diagram of component change results before and after re-enzymolysis of gypenoside medicine materials with different drying methods.

[0051] Figure 2 Schematic diagram of liquid chromatogram for re-enzymolysis time investigation results of sun-dried medicine materials.

[0052] Figure 3The results of the re-enzymolysis time investigation of the dried gynostemma pentaphyllum are shown in the schematic line graph.

[0053] Figure 4 The results of the re-enzymolysis temperature investigation of the dried gynostemma pentaphyllum are shown in the schematic liquid chromatography-mass spectrometry (LC-MS) graph. A-E represent the reaction temperatures of 30°C, 40°C, 50°C, 60°C, and 70°C, respectively.

[0054] Figure 5 The results of the conversion degree of saponins under different re-enzymolysis temperatures are shown in the schematic column graph.

[0055] Figure 6 The preparation process flow chart of the total saponins of gynostemma pentaphyllum dried by the roller dryer is shown.

[0056] Figure 7 The preparation process flow chart of the total saponins of gynostemma pentaphyllum dried by the roller dryer is shown.

[0057] Figure 8 The results of the influence of gynostemma pentaphyllum processed in different ways on the body weight of NAFLD mice (n=8) are shown in the schematic graph. # represents the comparison between C and M, and * represents the comparison among the roller, the dried, and M, * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001.

[0058] Figure 9 The results of the influence of gynostemma pentaphyllum processed in different ways on the excretion of TC and TG in the feces of NAFLD mice for three and six weeks are shown in the schematic graph. # represents the comparison between C and M, and * represents the comparison among the roller, the dried, and M, * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001. A and B represent the excretion of TC and TG in the feces for three weeks, and C and D represent the excretion of TC and TG in the feces for six weeks.

[0059] Figure 10 The results of the influence of gynostemma pentaphyllum processed in different ways on the glucose metabolism of NAFLD mice (n=8) are shown in the schematic graph. A represents the area under the curve of the glucose tolerance test (GTT), B represents the area under the curve of the insulin tolerance test (ITT), and C represents the fasting blood glucose. # represents the comparison between C and M, and * represents the comparison among the roller, the dried, and M, * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001.

[0060] Figure 11 The results of HE staining and oil red O staining of liver of NAFLD mice by different processing methods of Gynostemma pentaphyllum (magnification: 200 times) are shown in the following table.

[0061] Figure 12 The results of the influence of different processing methods of Gynostemma pentaphyllum on adipose tissue of NAFLD mice are shown in the following table. # represents the comparison between C and M, and * represents the comparison between the drum, air-drying and M, * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001.

[0062] Figure 13 The results of the influence of different processing methods of Gynostemma pentaphyllum on FGF15 in serum of NAFLD mice are shown in the following table. # represents the comparison between C and M, and * represents the comparison between the drum, air-drying and M, * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001.

[0063] Figure 14 The results of the influence of different processing methods of Gynostemma pentaphyllum on lipid decomposition of NAFLD mice are shown in the following table. # represents the comparison between C and M, and * represents the comparison between the drum, air-drying and M, * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001. DETAILED DESCRIPTION

[0064] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0065] Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application or the field to which the term applies. Although any methods, conditions, materials, or articles similar or equivalent to those described herein can be used in the practice of the present application, the preferred methods, conditions, materials, or articles are described herein.

[0066] The present invention is intended to embrace all choices, alternatives and equivalents that can fall within the present inventive scope as defined by the claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The invention is not intended to be limited to the embodiments described.

[0067] As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0068] In this disclosure and the accompanying claims, the term "comprising" is synonymous with "including," "containing," or "characterized by." As used herein, the term "comprising" or "comprises" or "including" or "includes" or "containing" or "contains" or "characterized by" means "including, but not limited to," and is intended to be equivalent to the term "including" as used in 35 U.S.C. § 112 or 21 U.S.C. § 321.

[0069] As described in the background section, the current preparation process of Gynostemma pentaphylla has the problem of being unable to prepare rare ginsenosides. In order to solve the above problem, the present invention provides a method for promoting the conversion of rare ginsenosides in Gynostemma pentaphylla, which comprises the following steps:

[0070] (1) Take an appropriate amount of dried Gynostemma pentaphylla medicinal material, crush it, and then soak it in water;

[0071] (2) Heat it at a certain water bath temperature for a period of time;

[0072] (3) After evaporating the water, add a certain concentration of ethanol or methanol, heat and reflux extraction or ultrasonic extraction, filter the cooled extraction liquid, and concentrate it under reduced pressure to obtain a concentrate; and

[0073] (4) Load the concentrate onto a macroporous resin, adsorb it, elute it, collect the 60-80% ethanol elution fraction, and obtain the total saponin of dried Gynostemma pentaphylla.

[0074] In the present application, mass volume ratio, time, temperature, number, pressure, proportion, equivalent, concentration, or other values or parameters are expressed in ranges, preferred ranges, or ranges defined by a series of upper preferred values and lower preferred values, when such ranges are expressed, it should be understood that all ranges formed by any pairing of an upper range limit or preferred value with any lower range limit or preferred value are specifically disclosed, regardless of whether the range is disclosed individually. For example, when the range "60-80" is disclosed, the described range should be interpreted to include the ranges "60-80", "61-80", "62-80", "63-80", "64-80", "65-80", "66-80", "67-80", "68-80", "69-80", "70-80", "71-80", "72-80", "73-80", "74-80", "75-80", "76-80", "77-80", "78-80", "79-80", and the like. When numerical ranges are described herein, unless otherwise stated, the range is intended to include the end values and all integers and fractions within the range.

[0075] In a preferred embodiment, in step (1), the mass volume ratio (g / L) of the dried Gynostemma pentaphyllum medicinal material to the water is 20:1-100:1. In a preferred embodiment, the mass volume ratio (g / L) of the dried Gynostemma pentaphyllum medicinal material to the water is about 50:1.

[0076] In the present application, "about" means a value within ±5% of a particular value. For example, "about 50:1" includes ±5% of 50:1, or from 47.5:1 to 52.5:1.

[0077] In a preferred embodiment, the water is deionized water. In a preferred embodiment, the water is distilled water. In a preferred embodiment, in step (2), the temperature of the water bath is 30°C-70°C. In a preferred embodiment, the temperature of the water bath is 35°C-50°C. In a preferred embodiment, the temperature of the water bath is 35°C-45°C. In a preferred embodiment, the temperature of the water bath is about 40°C.

[0078] In the present application, "about" means a value within ±5% of a particular value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0079] In a preferred embodiment, the time of the water bath is 1-12 hours. In a preferred embodiment, the time of the water bath is 2-4 hours. In a preferred embodiment, the time of the water bath is about 3 hours.

[0080] In the present application, "about" means a value within ±5% of a specified value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0081] In a preferred embodiment, the concentration of the ethanol or the methanol in step (4) is 60% to 80%. In a preferred embodiment, the concentration of the ethanol or the methanol in step (4) is about 70%.

[0082] In the present application, "about" means a value within ±5% of a specified value. For example, "about 70" includes ±5% of 70, or from 66.5 to 73.5.

[0083] In a preferred embodiment, the volume ratio of the concentrate to the extraction liquid in step (4) is 0.02:1 to 0.1:1. In a preferred embodiment, the volume ratio of the concentrate to the extraction liquid in step (4) is about 0.06:1.

[0084] In the present application, "about" means a value within ±5% of a specified value. For example, "about 0.06:1" includes ±5% of 0.06:1, or from 0.057:1 to 0.063:1.

[0085] In a preferred embodiment, the number of times of the heating reflux extraction or the ultrasonic extraction in step (4) is 1 to 4. In a preferred embodiment, when the number of times of the heating reflux extraction or the ultrasonic extraction is 2 to 4, the step (4) further comprises a step of combining the filtered extraction liquids. In a preferred embodiment, the number of times of the heating reflux extraction or the ultrasonic extraction is 2. In a preferred embodiment, the time of the heating reflux extraction or the ultrasonic extraction is 0.5 to 2 hours. In a preferred embodiment, the time of the heating reflux extraction or the ultrasonic extraction is about 1 hour.

[0086] In the present application, "about" means a value within ±5% of a specified value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0087] In a preferred embodiment, the mass to volume ratio (g / L) of the dried gynostemma pentaphyllum medicinal material to the methanol or the ethanol in step (4) is 20:1 to 100:1. In a preferred embodiment, the mass to volume ratio (g / L) of the dried gynostemma pentaphyllum medicinal material to the methanol or the ethanol in step (4) is about 40:1.

[0088] In the present application, "about" means a value within ±5% of a specified value. For example, "about 40:1" includes ±5% of 40:1, or from 38:1 to 42:1.

[0089] In a preferred embodiment, in step (5), the macroporous resin is D101 resin. In a preferred embodiment, the adsorption time of the concentrate on the macroporous resin is 20-28 hours. In a preferred embodiment, the adsorption time of the concentrate on the macroporous resin is about 24 hours.

[0090] In the present application, "about" means a value within ±5% of a specified value. For example, "about 24" includes ±5% of 24, or from 22.8 to 25.2.

[0091] In a preferred embodiment, in step (5), the elution includes elution with water and 20%-40% ethanol for impurity removal, and elution with 60%-80% ethanol. In a preferred embodiment, the elution includes elution with water and about 30% ethanol for impurity removal, and elution with about 70% ethanol.

[0092] In the present application, "about" means a value within ±5% of a specified value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5; "about 70" includes ±5% of 70, or from 66.5 to 73.5.

[0093] According to another aspect of the present application, there is provided a preparation product obtained by the above method.

[0094] In a preferred embodiment, the saponin component in the preparation product comprises 0%-10% of 3-4 glycosyl-substituted gypenosides.

[0095] In a preferred embodiment, the saponin component in the preparation product comprises 90%-100% of 1-2 glycosyl-substituted secondary saponins.

[0096] In a preferred embodiment, the 1-2 glycosyl-substituted secondary saponins are gypenoside LXXVII, gypenoside TN-1, gypenoside XIII, and / or gypenoside C-K or an isomer thereof.

[0097] In a preferred embodiment, the 1-2 glycosyl-substituted secondary saponins are gypenoside LXXVII, gypenoside TN-1, gypenoside XIII, and / or gypenoside C-K.

[0098] According to another aspect of the present application, there is provided a composition comprising the above preparation product, the composition further comprising one or more substances for preventing and / or treating metabolic diseases, and / or a pharmaceutically acceptable excipient.

[0099] In a preferred embodiment, the composition of the present application contains at least one pharmaceutically acceptable excipient in a total amount of 0.00001 to 50 wt.%, or 0.0001 to 10 wt.%, or 0.0001 to 5 wt.%, or 0.005 to 1 wt.%, or 0.1 to 20 wt.%, or 0.5 to 15 wt.%, or 1 to 5 wt.%, calculated on the weight of the composition.

[0100] In the present application, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and which is relatively non-toxic, i.e., the material is not biologically or otherwise undesirable, i.e., the material is not deleterious to the individual when administered in the amounts necessary to achieve the desired therapeutic effect.

[0101] In the present application, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is compatible with the subject and active ingredient in pharmacological and / or physiological terms, i.e., that does not abrogate the biological activity or properties of the active ingredient, and that is relatively non-toxic, i.e., the material is not biologically or otherwise undesirable, i.e., the material is not deleterious to the individual when administered in the amounts necessary to achieve the desired therapeutic effect, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0102] In a preferred embodiment, the excipient is selected from one or more of the following: diluents, disintegrants, dispersants, plasticizers, inclusion agents, wetting agents, sustained release agents, retention agents, lubricants, binders, flavoring agents, opacifiers, and antioxidants.

[0103] These excipients are preferably pharmaceutically inert, or can have a synergistic or additive effect to enhance the therapeutic activity of the pharmaceutical composition, and the above-mentioned excipients are only illustrative, and the types of excipients actually used in the present application are not limited to the above-mentioned types, and can be adjusted according to the actual situation, and all can achieve the effect of the present application.

[0104] According to another aspect of the present application, there is provided a use of the above-prepared product or the above composition in a pharmaceutical product, a food product, and / or a health product for preventing and / or treating metabolic diseases.

[0105] In the present application, the term "treatment" also includes "prevention", unless there is a specific description to the contrary. The terms "therapeutic" and "therapeutically" should be construed accordingly.

[0106] In the present application, the term "treatment" includes alleviating, inhibiting or ameliorating the symptoms or conditions of a disease; inhibiting the development of complications; ameliorating or preventing underlying metabolic syndrome; inhibiting the development of a disease or symptom, such as controlling the progression of a disease or condition; reducing a disease or symptom; causing regression of a disease or symptom; reducing complications resulting from a disease or symptom, or preventing or treating signs resulting from a disease or symptom. As used herein, a disease, symptom, or condition can be improved by a preparation or a composition, as used herein, upon administration, particularly in terms of severity, delay of onset, slowing of progression, or reduction in duration. The condition can be attributed to or associated with the administration, whether fixed or contingent, continuous or intermittent.

[0107] In a preferred embodiment, the metabolic disease is metabolic syndrome.

[0108] In a preferred embodiment, the metabolic disease is obesity, diabetes and / or nonalcoholic fatty liver disease.

[0109] The above-mentioned metabolic diseases of the present application are only listed, and the above-mentioned preparation or the above-mentioned composition of the present application can also be used for preventing and / or treating other metabolic diseases.

[0110] The present application also provides the above-mentioned preparation or the above-mentioned composition for preventing and / or treating a metabolic disease, such as nonalcoholic fatty liver disease, in a subject.

[0111] The present application also provides a method for preventing and / or treating a metabolic disease, such as nonalcoholic fatty liver disease, in a subject, comprising administering to the subject an effective amount of the above-mentioned preparation or the above-mentioned composition.

[0112] In the present application, the term "subject" is a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans can be advantageously used as a subject representing a model of a metabolic disease, such as nonalcoholic fatty liver disease. Preferably, the subject is a human.

[0113] The "effective amount" of the above-mentioned preparation or the above-mentioned composition used in the present application can achieve the desired therapeutic and / or prophylactic effect. The amount effective for this use will depend on, for example, the pharmaceutical composition, the mode of administration, the stage and severity of the disease being treated, the individual's body weight and overall health status, and the judgment of the prescribing physician. The administration of the dose can be once a week, or two days or once a day, or even several times a day. The dose unit can be administered for a short period (e.g., weeks to months) or for a longer period (months to years).

[0114] The application will be further described in connection with the following detailed description of specific embodiments. It should be understood that these embodiments are intended to illustrate the application and are not intended to limit its scope. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred materials and methods are described herein.

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred materials and methods are described herein.

[0116] The above features mentioned in the application, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in the patent specification can be used in any combination, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0117] Experimental instruments (manufacturers)

[0118] EYELA water bath SB-1100 (Shanghai Ailang Instrument Co., Ltd.), digital display electric heating jacket (Shanghai Lichengbang Instrument Technology Co., Ltd.), constant temperature water bath (Zhengzhou Zhuocheng Instrument Technology Co., Ltd.), ACQUITY UPLC system, XevoG2-S QTof mass spectrometer (Waters Corporation, USA), chromatographic column, ACQUITY UPLC BEH C18 (2.1mm×100mm, 1.7μm).

[0119] Blood glucose meter (Roche), high-throughput tissue grinder (Shanghai Bixing), optical microscope (Olympus), analytical balance (Shanghai Jinghai Instrument), multifunctional enzyme label instrument (TECAN), vortex shaker (Hangzhou Aosheng Instrument), microplate constant temperature oscillator (Hangzhou Aosheng Instrument), Multifunctional enzyme label instrument (PE), microcentrifuge (Hangzhou Meiyou), vertical electrophoresis instrument, semi-dry transfer instrument (ATTO), shaking bed (Haimen Qishenbel Instrument), AI600 protein imaging instrument (GE, USA).

[0120] Experimental reagents and materials (manufacturers)

[0121] Gynostemma pentaphyllum medicinal materials were collected from dry leaves in Fengzhou village, Zhangzhou city, Fujian province, acetonitrile (Fisher Company, USA), D-101 macroporous resin (Cangzhou Baonen Absorbing Material Technology Co., Ltd.), ethanol (Beijing Chemical Plant), formic acid (Beijing Chemical Plant), and distilled water from Watsons.

[0122] Triglyceride (TG) test kit, total cholesterol (TC) test kit (Nanjing Jiancheng), biosynthetic human insulin injection (Novo Nordisk), glucose (Sigma), blood glucose test paper (Roche), isoflurane (Rivard Life), general tissue fixative, hematoxylin, eosin, oil red dye (Wuhan Google), chloroform, methanol, isopropanol, physiological saline (National Medicine), Triton X-100 (Biofroxx), Tissue-Tek OCT embedding agent (Sakara), mouse FGF15 enzyme-linked immunoassay kit (Wuhan Huamei), sodium dodecyl sulfate, TEMED, glycine, ammonium persulfate, tris-hydroxymethyl aminomethane, bovine serum albumin (Shanghai Yisheng), Tween 20 (Biofroxx), RIPA protein lysate, protein marker (Thermo), protease inhibitor, phosphatase inhibitor (Roche), developing solution, PVDF membrane (Millipore), Tris-HCl / SDS (1.5 mol / L), pH 8.8, Tris-HCl / SDS (0.5 mol / L), pH 6.8, 30% acrylamide mixture, 10x PBS solution (Dalian Meilun), HSL antibody (#18381), p-HSL antibody (#4137), HSP90 antibody (#4877), FGFR1 antibody (#9740) (Abeam), horseradish peroxidase-labeled goat anti-rabbit IgG (Jackson ImmunoResearch, USA).

[0123] Example one re-enzymolysis process of dried medicinal materials

[0124] 1.1 Comparison of re-enzymolysis reactions of gynostemma pentaphyllum medicinal materials with different drying methods

[0125] In the previous experiments, it was found that the glucose glycoside hydrolase activity still remained in the dried gynostemma pentaphyllum medicinal materials, and the medicinal materials could continue to undergo enzymatic reaction after being rehydrated in a water bath. In order to investigate the rehydration and re-enzymolysis ability of dried medicinal materials, further experiments were carried out. The dried gynostemma pentaphyllum medicinal materials were taken, crushed, and mixed with distilled water at a mass to volume ratio (50:1) (mg:ml) at 40°C for 3 hours in a water bath. Then, methanol was added to the reaction liquid at a volume ratio of 1:4, and ultrasonic extraction was carried out for 1 hour. The supernatant was filtered and subjected to mass spectrometry. The results are shown in Table 1. Figure 1

[0126] ​Chromatographic conditions: column, ACQUITY UPLC BEH C18 (2.1 mm x 100 mm, 1.7 μm); mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid water; gradient elution: 0-3 min 30%-35% A, 3-8 min 35%-45% A, 8-12 min 45%-60% A, 12-15 min 60%-80% A; flow rate 0.4 mL·min-1; injection volume 2 μL; column temperature 35 °C, autosampler temperature 20 °C, PDA detector scan range 200-400 nm.

[0127] Mass spectrometry conditions: ESI ion source, negative ion mode scan, capillary voltage 2 kV, cone hole voltage 40 V, ion source temperature 120 °C, desolvation gas flow rate 600 L / hr, desolvation gas temperature 400 °C, cone hole gas flow rate 50 L / hr, parent ion collision energy 6 eV, fragment ion collision energy 20-50 eV, mass scan range 100-2000 Da, scan time 0.2 s.

[0128] The medicinal materials can continue to undergo enzymatic hydrolysis through rehydration water bath reaction, confirming that the glucose glycoside hydrolase activity is still present in the gynostemma pentaphyllum dried medicinal materials. After 3 hours of re-enzymatic hydrolysis, the gypenoside with 3-4 glycosyl substitutions in the dried medicinal materials is basically (about 90%) completely converted into the secondary saponin with 1-2 glycosyl substitutions, while the tumble-dried sample cannot undergo the above reaction. In order to further utilize the re-enzymatic hydrolysis ability of the dried medicinal materials, the re-enzymatic hydrolysis process optimization is carried out.

[0129] 1.2 Optimal time for re-enzymatic hydrolysis of dried medicinal materials

[0130] The above dried gynostemma pentaphyllum medicinal material powder is taken, mixed with water at a mass to volume ratio of 50:1 (mg:mL), and placed at 40 °C for 12 hours. At 0 h, 1 h, 3 h, and 6 h, the reaction solution is taken, added with methanol at a volume ratio of 1:4, ultrasonically extracted for 1 hour, centrifuged, and the supernatant is taken to pass through a filter membrane and enter mass spectrometry. Figure 2 and Figure 3 It can be seen that after 3 hours of water bath enzymatic hydrolysis, the gypenoside with 3-4 glycosyl substitutions has been basically converted into the secondary saponin with 1-2 glycosyl substitutions. After hydrolysis, the main 4 1-2 sugars generated by mass spectrometry are Gypenoside LXXVII, Gynosaponin TN-1, Gypenoside XIII, Ginsenoside C-K, and isomers thereof. In order to ensure sufficient water bath, 3 hours of water bath is determined as the optimal reaction time.

[0131] Four groups are four different gypenoside compounds from 3-4 sugar substrate enzyme hydrolysis conversion into the corresponding 1-2 sugar product. The numbers are the molecular ion mass in mass spectrometry. Group 1: 1139 is Gypenoside LVI, 1179 is its corresponding malonyl saponin, namely M-Gypenoside LVI, 1181 is its corresponding acetyl saponin. 815 is Gypenoside LXXVII. Group 2: 1007 is Gypenoside XLVI, 1047 is its corresponding malonyl saponin, namely M-Gypenoside XLVI, 1049 is its corresponding acetyl saponin. 683 is Gynosaponin TN-1. Group 3: 1123 is Gypenoside LXIII, 1163 is its corresponding malonyl saponin, namely M-Gypenoside LXIII, 1165 is its corresponding acetyl saponin. 799 is Gypenoside XIII. Group 4: 991 is Ginsenoside Rd, 1031 is its corresponding malonyl saponin, namely M-Ginsenoside Rd, 1033 is its corresponding acetyl saponin. 667 is Ginsenoside C-K.

[0132] Wherein, by the peak area value obtained directly on the mass spectrometry software, the sum of the peak area of the substrate, the intermediate product and the product is calculated. Figure 3 The sum of the peak area of the four groups of substrates and the sum of the peak area of the four groups of products are calculated by the peak area value obtained directly on the mass spectrometry software, wherein the substrate and / (substrate and+product and)×100%=1.8%, the product and / (substrate and+product and)×100%=98.2%.

[0133] 1.3 Optimal temperature investigation of re-enzymolysis of dried medicinal materials

[0134] The above dried gynostemma herb powder is taken, and water is taken in a mass volume ratio of 50:1 (mg:mL), and is placed in a 30°C, 40°C, 50°C, 60°C, 70°C water bath for 1 hour. Methanol is added to the reaction liquid in a volume ratio of 1:4, ultrasonic extraction is carried out for 1 hour, centrifugation is carried out, the supernatant is taken and filtered into mass spectrometry, and the saponin conversion is detected. The peak area sum of the hydrolysis substrate, the intermediate product and the hydrolysis product is calculated. Figure 4 It can be seen that after 1h of reaction, in the 30°C group, a large amount of 3-4 glycosyl-substituted saponins can be detected in the retention time of 2-6 minutes, and the conversion efficiency is low. The conversion of 3-4 glycosyl-substituted saponins is the most complete at a reaction temperature of 40°C, followed by 50°C, and the conversion efficiency is not high at 60°C and 70°C. Figure 5 The peak area sum of the substrate, the intermediate product and the product is calculated by the peak area value obtained directly on the mass spectrometry software. The results show that the optimal reaction temperature is 40°C-50°C, and the most products are obtained.

[0135] Preparation of saponin extract by different processing methods

[0136] 2.1 Preparation of total saponin of Jiaoguleng dried by roller

[0137] Take 50 g of Jiaoguleng dried by roller in Fujian, powder it, add 70% ethanol with the ratio of 1:25, extract it twice for 1 hour each time, combine the extract, cool it, filter it, recover it under reduced pressure to about 150 mL, pass it through 1.0 kg D101 macroporous resin, adsorb it for 24 hours, elute it with water, 30% ethanol, and then 70% ethanol, and collect the elution part of 70% ethanol to obtain 19.53 g of total saponin of Jiaoguleng dried by roller. The flow chart of the preparation of total saponin of Jiaoguleng dried by roller is shown in Figure 6 .

[0138] 2.2 Re-enzymolysis of Jiaoguleng dried in the shade and preparation process of total saponin of Jiaoguleng

[0139] Take 50 g of Jiaoguleng dried in the shade in Fujian, powder it, add 1 L of deionized water, soak it completely, and then put it in a water bath at 40°C for 3 hours, and then evaporate the water. Add 1.25 L of 70% ethanol, extract it twice for 1 hour each time, cool the extract, filter it, recover it under reduced pressure to about 150 mL, pass it through 1 kg of D101 macroporous resin, adsorb it for 24 hours, elute it with water, 30% ethanol, and then 70% ethanol, and collect the elution part of 70% ethanol to obtain the total saponin of Jiaoguleng dried in the shade. The flow chart of the preparation process of total saponin of Jiaoguleng dried in the shade is shown in Figure 7 .

[0140] 2.3 Detection of saponin content

[0141] The chromatographic conditions and mass spectrometric conditions of Example 2 are the same as those of Example 1.

[0142] Take an appropriate amount of the above two sample extract solutions, pass them through a 0.22 μm filter membrane, and then inject them for mass spectrometric detection. It is found that the saponin components contained in the high-temperature roller-dried sample are mainly concentrated in 4-8 min, and the parent nucleus mainly contains 3-4 sugars. The Jiaoguleng dried in the shade is mostly concentrated in 8-13 min, and the parent nucleus mainly contains 1-2 sugars. This part of saponin is similar in structure to the rare ginsenosides reported at present, and has better activity.

[0143] From the above results, it can be seen that there is a chemical transformation during the drying of fresh Jiaoguleng in the shade. There may be glucosidase in fresh Jiaoguleng, and under the action of glucosidase, hydrolysis reaction may occur during the drying of Jiaoguleng in the shade, and a large amount of rare ginsenosides are generated by desugarification. Under high-temperature conditions, the enzyme is inactivated during roller drying, which easily leads to a decrease in the activity of glucosidase in vivo, so the hydrolysis reaction of saponin in Jiaoguleng in vivo is slow, and the original saponin mainly exists in the form of saponin.

[0144] The above research results suggest that the gynostemma pentaphyllum drying process is more conducive to the generation of rare saponins than drum drying, and therefore the preparation method of the high-activity rare ginsenoside has important significance.

[0145] Example Three: Comparison of the glycolipid metabolic activity of gynostemma pentaphylla saponins prepared by different processing methods

[0146] 3.1 Experimental method

[0147] 3.1.1 Preparation of drug feed

[0148] The drug (200mg·kg -1 The drum gynostemma pentaphyllum saponin, 200mg·kg -1 The gynostemma pentaphyllum saponin was added to the crushed high-fat high-sugar high-cholesterol feed, and the mixture was shaped and sterilized under ultraviolet light to prepare the feed for the mice in the drug administration group.

[0149] 3.1.2 Animal grouping, modeling, and drug administration method

[0150] After the C57BL / 6J mice were adaptively fed for one week, the normal control group (C, n = 6) was randomly selected and fed with ordinary feed, and the remaining mice were modeled using high-fat high-sugar high-cholesterol feed for 16 weeks. The NAFLD model group (M, n = 6), the drum gynostemma pentaphyllum saponin (200mg·kg -1 ) group (drum, n = 8), and the sun-dried gynostemma pentaphyllum saponin (200mg·kg -1 ) group (sun-dried, n = 8) were prepared. The C group continued to be fed with ordinary feed for 6 weeks, the M group continued to be fed with high-fat high-sugar high-cholesterol feed for 6 weeks, and the drum group and the sun-dried group were fed with the respective drug feed for 6 weeks. The body weight and food intake of the mice in each group were recorded every week.

[0151] 3.1.3 Glucose tolerance test

[0152] The mice in each group were fasted for 12h, and the fasting blood glucose value was measured after intraperitoneal injection of glucose solution (2g / kg). Blood samples were taken from the tail tip at 15, 30, 60, and 120min to measure the blood glucose value.

[0153] 3.1.4 Insulin tolerance test

[0154] The mice in each group were fasted for 5h, and the fasting blood glucose value was measured after intraperitoneal injection of insulin solution (0.75U / kg). Blood samples were taken from the tail tip at 15, 30, 60, and 120min to measure the blood glucose value.

[0155] 3.1.5 Collection of animal tissue samples and serum

[0156] After 6 weeks of administration, the mice were weighed, anesthetized with isoflurane, and whole blood was taken by enucleation. The liver, epididymal fat, inguinal fat, and brown fat of the mice were carefully removed and weighed, and then stored in a -80°C refrigerator. The liver was weighed, and the liver-to-body weight ratio (liver weight / body weight

[0157] × 100%) was calculated. The whole blood was allowed to stand at room temperature for 2 h, and then centrifuged (4°C) at 3500 rpm for 15 min. The supernatant was collected and stored in a -80°C refrigerator.

[0158] 3.1.6 Extraction and determination of TC and TG in mouse feces

[0159] The feces of the mice were collected at 3 and 6 weeks, dried, and ground. About 50 mg of the feces of the mice was weighed, homogenized with 0.5 mL of PBS solution, and then mixed with 0.5 mL of a chloroform-methanol mixture (chloroform:methanol

[0160] = 2:1) by vortexing. After centrifugation at 3000 rpm for 15 min, the chloroform layer was collected, dried, and then redissolved with 10% Triton X-100 solution. The content of TC and TG in the feces was calculated according to the test kit instructions.

[0161] 3.1.7 HE staining of liver and adipose tissue

[0162] The same parts of the liver lobe tissue and adipose tissue were fixed in a general tissue fixative, and then paraffin-embedded, sectioned, and placed on a glass slide. After deparaffination, the tissue was stained with hematoxylin for about 3 min, and then washed with running water for 1 min. The tissue was immersed in an eosin solution for 30 s, and then washed with running water for 1 min. After dehydration with ethanol, the tissue was baked, and then sealed with a 50% neutral gum xylene solution, and dried. The morphological changes of the liver tissue and adipose tissue were observed under a microscope.

[0163] 3.1.8 Oil red O staining of liver tissue

[0164] The same parts of the liver lobe tissue were fixed in a general tissue fixative, and then embedded with Tissue-Tek OCT embedding medium on dry ice, and stored in a -80°C refrigerator. After sectioning with a freezing microtome, the tissue was infiltrated with 60% isopropanol, and then slowly dripped with oil red O working solution for staining, for about 15 min. After staining, the tissue was washed with 60% isopropanol three times, and then washed with running water for 30 s. The tissue was stained with a hematoxylin solution for about 5 min, washed with running water for 30 s, and then sealed with glycerol gelatin. The lipid accumulation in the liver tissue was observed under a microscope.

[0165] 3.1.9 Western Blot protein blotting analysis

[0166] 3.1.9.1 Extraction of tissue proteins

[0167] White adipose tissue about 100 mg was cut on dry ice, and 400 μL of RIPA solution containing phosphatase and protease inhibitors was added, and homogenized for 1 min. It was placed on ice for 30 min, and vortexed gently once every 10 min, and the protein was fully lysed. After lysis, it was placed in a 4°C centrifuge, centrifuged at 12000 rpm for 15 min, and the supernatant solution was the extracted protein solution.

[0168] 3.1.9.2 Protein concentration determination

[0169] The protein standard solution was diluted in RIPA solution according to Table 3-1 to prepare a protein standard curve. The sample solution was diluted 40 times, and 20 μL of the standard solution and the sample solution were added to a 96-well plate, and 200 μL of working solution was added using a gun. Incubate at 37°C for 30 min on a constant temperature shaker at 300 rpm. Use a microplate reader to detect the absorbance value at 562 nm wavelength, and calculate the concentration of each sample.

[0170] Table 1 Standard curve preparation system

[0171]

[0172] 3.1.9.3 Protein sample preparation

[0173] The sample concentration was quantified as 10 μg / 10 μL, and the sample was diluted using RIPA lysis solution containing phosphatase and protease inhibitors, and an appropriate amount of 5x loading buffer solution was added and vortexed. Heat at 95°C for 10 min, and cool the sample to room temperature, and store in a -20°C refrigerator.

[0174] 3.1.9.4 Western Blot experiment

[0175] (1) Preparation of reagents

[0176] ① 10x SDS electrophoresis buffer was prepared according to Table 3-2.

[0177] Table 2 Preparation of 10x SDS electrophoresis buffer

[0178] Reagent name Volume / weight Tris 112.12g Glycine 576.56g SDS 40g <![CDATA[dd H2O]]> Up to 4L

[0179] ② 10x transfer solution was prepared according to Table 3-3.

[0180] Table 3 Preparation of 10x transfer solution

[0181]

[0182]

[0183] iii. 10X PBST solution was prepared as per Table 3-4.

[0184] Table 4 10X PBST solution preparation

[0185] Reagent name Volume / weight 10 x PBS 500 mL Tween 20 5g

[0186] iv. 1X SDS running buffer: 10X SDS running buffer was diluted with ddH20 to 1X. v. 1X transfer buffer was prepared as per Table 3-5.

[0187] Table 5 1X transfer buffer preparation

[0188] Reagent name Volume 10 x transfer buffer 10 mL ddH2O 70 mL Methanol 20 mL

[0189] vi. 1X PBST solution: 10X PBST solution was diluted with ddH20 to 1X.

[0190] vii. 5% BSA solution was prepared as per Table 3-6.

[0191] Table 6 5% BSA preparation

[0192] Reagent name Volume / weight BSA 2g 1 x PBST 40 mL

[0193] viii. 10% APS solution was prepared as per Table 3-7.

[0194] Table 7 10% APS preparation

[0195] Reagent name Volume / weight APS 1g ddH2O 10 mL

[0196] (2) WB experimental procedure

[0197] i. Gel preparation: ddH20 was used to clean the glass plates, dry them, place the short plate on the experimental table (convex side up), stick the adhesive tape, cover the long glass plate on the short glass plate, keep the bottom end flush, use the clamp to clamp the two glass plates, and place them on the table. According to the molecular weight of the target protein, the concentration of the separation gel was selected, and the preparation method of the separation gel is shown in Table 3-8 and Table 3-9.

[0198] Table 8 7.5% separation gel preparation

[0199] Reagent name Volume required for two gels 4 x Tris pH 8.8 4 mL 30% AA 10 mL 10% APS 160 μL TEMED 16 μL ddH2O 8 mL

[0200] Table 9 10% separation gel preparation

[0201] Reagent name Volume required for two gels 4 x Tris pH 8.8 4 mL 30% AA 5.33 mL 10% APS 160 μL TEMED 16 μL ddH2O 6.67 mL

[0202] Slowly pour the separating gel between the two plates to 80% of the height of the short plate, then add an appropriate amount of anhydrous ethanol to the level of the separating gel to remove the bubbles and flatten the liquid surface. After the separating gel between the two plates solidifies, use thick filter paper to wipe off the alcohol between the two plates. Pour the concentrated gel on top of the separating gel, and the preparation method of the concentrated gel is shown in Table 3-10.

[0203] Table 10 Preparation of concentrated gel

[0204] Reagent name Volume required for two gels 4 x Tris pH 6.8 1.5 mL 30% AA 0.66 mL 10% APS 46.6 μL TEMED 6.6 μL ddH2O 3.83 mL

[0205] Vortex the concentrated gel in a 50 mL centrifuge tube, mix thoroughly, and pour it on top of the separating gel between the two plates. Insert the comb at an angle to prevent air bubbles from entering. Note that the gel and the comb should be kept horizontal, and the concentrated gel should be allowed to solidify.

[0206] ② Loading: Place the protein sample in a 75°C constant temperature heater and heat for 10 min. Vortex and centrifuge the sample. Pour 1x SDS electrophoresis solution to 1 / 3 of the height of the electrophoresis tank. Remove the clamp and gel strip of the glass plate, and slowly tilt the glass plate into the loading tank to remove the air bubbles at the bottom. Fill the space between the two plates with 1x SDS electrophoresis solution, and horizontally pull out the comb to blow out the broken gel in the hole. From right to left, sequentially add 5 μL protein marker and 10 μL sample by full suction and half-punch.

[0207] ③ Electrophoresis: Insert the electrodes (red to red and black to black), 300 V, 65 mA (two blocks), and run the gel for 60 min. When the bromophenol blue reaches the bottom of the gel, stop the electrophoresis and flush the 1x SDS electrophoresis solution in the loading tank with running water.

[0208] ④ Membrane transfer: Cut a 6x9 cm filter paper and a PVDF membrane. Activate the PVDF membrane in methanol solution for 1 min, and then soak the filter paper and PVDF membrane in 1x membrane transfer solution. Remove the glass plate, place the short plate at the bottom, and use a gel cutter to pry open the two plates to remove the concentrated gel. Place the PVDF membrane on the separating gel and align it. Use tweezers to clamp the PVDF membrane and the gel together, and place them on the membrane transfer instrument on which a layer of wet filter paper has been placed. Note that air bubbles should be removed between the gel and the PVDF membrane. Cover another layer of wet filter paper, and finally cover the membrane transfer instrument cover. Perform membrane transfer at 30 V and 250 mA (two blocks) for 90 min.

[0209] ⑤ Blocking: After the membrane transfer is completed, discard the gel and filter paper, and place the PVDF membrane in a 5% BSA solution for 2-3 h of room temperature blocking.

[0210] ⑥ Membrane washing: After the blocking is completed, discard the BSA solution, and place the PVDF membrane in 1x PBST on a shaker for 10 min of washing, and repeat the washing three times.

[0211] ⑦Primary antibody incubation: The PVDF membrane was placed in plastic wrap, and the target protein bands were cut according to the protein marker. The primary antibody was diluted with 1xPBST, and the cut bands were placed in different antibody solutions. The bands should not have bubbles near them. Incubate overnight at 4°C with slow shaking on a shaker.

[0212] ⑧Washing the membrane: Recover the primary antibody solution, and place the bands in 1xPBST. Wash once for 10 minutes on a shaker, and repeat three times.

[0213] ⑨Secondary antibody incubation: Choose the corresponding secondary antibody for the species, dilute it with 1xPBST, and place the bands in the secondary antibody solution. Incubate for 1 hour at room temperature with slow shaking on a shaker.

[0214] ⑩Washing the membrane and developing: Recover the secondary antibody solution, and place the bands in 1xPBST. Wash once for 10 minutes on a shaker, and repeat three times. Prepare the developing solution according to the instructions, and immerse the bands in the developing solution for a few moments before developing. Use β-ACTIN, GAPDH, or HSP90 as the internal reference protein to analyze the expression of the target protein.

[0215] 3.1.10 Statistical processing

[0216] The experimental results were analyzed using Graphpad Prism 9.3 software, and the mean ± standard error (Mean ± SEM) was used to represent the experimental results. One-way ANOVA was used for multiple group data comparison, and student's t test was used for two group data comparison. If p<0.05, it was considered statistically significant, *p<0.05, **p<0.01, ***p<0.001.

[0217] 3.2 Experimental results

[0218] Effect of different processing methods of Gynostemma pentaphyllum on glucose and lipid metabolism in non-alcoholic fatty liver disease (NAFLD) mice

[0219] NAFLD mouse models were established by high-fat high-sugar high-cholesterol diet to evaluate the effect of different processing methods of Gynostemma pentaphyllum saponins on improving the metabolic phenotype of NAFLD mice.

[0220] 3.2.1 Effect of different processing methods of Gynostemma pentaphyllum on body weight of NAFLD mice

[0221] The experimental results are shown in Figure 8 The body weight of the successfully modeled NAFLD mice was significantly increased compared to the C group mice. After treatment with dried Gynostemma pentaphyllum saponins, the body weight of the NAFLD mice was stably reduced from the second week. The roller-dried saponin group showed a trend of reducing the body weight of the NAFLD mice, but there was no statistically significant difference.

[0222] 3.2.2 The effect of different processing methods of Gynostemma pentaphyllum on the excretion of fecal TC and TG in NAFLD mice for three and six weeks

[0223] The experimental results are shown in Figure 9 The dried Gynostemma pentaphyllum saponins can significantly increase the excretion of total cholesterol (TC) and triglyceride (TG) in mouse feces.

[0224] 3.2.3 The effect of different processing methods of Gynostemma pentaphyllum on the glucose metabolism of NAFLD mice

[0225] The experimental results are shown in Figure 10 After intraperitoneal injection of glucose solution, the blood glucose levels of mice in each group increased, and the blood glucose level of mice in group M increased significantly compared with that in group C, and decreased slowly. After treatment with different processing methods of Gynostemma pentaphyllum total saponins, the glucose tolerance of NAFLD mice was effectively regulated. By calculating the area under the curve (AUC) value of glucose tolerance test (GTT), it was found that the AUC value of the dried group was significantly reduced ( Figure 10 -A), and the fasting blood glucose value of the mice was significantly reduced ( Figure 10 -C). The saponins of the drum-dried group showed a downward trend in the relevant indicators, but the difference was not significant.

[0226] After intraperitoneal injection of insulin solution, the blood glucose levels of mice in each group decreased, and the blood glucose level of mice in group C decreased slowly compared with that in group M, and could recover to the blood glucose level before insulin injection at 120 min. After treatment with different processing methods of Gynostemma pentaphyllum total saponins, the insulin resistance of NAFLD mice was effectively improved. By calculating the AUC value of insulin tolerance test (ITT), it was found that the AUC value of the dried group was significantly reduced ( Figure 10 -B). It was shown that the dried group could restore the insulin sensitivity of NAFLD mice and improve the glucose metabolism capacity. The saponins of the drum-dried group showed a downward trend in the relevant indicators, but the difference was not significant.

[0227] 3.2.4 The effect of different processing methods of Gynostemma pentaphyllum on the lipid accumulation in the liver of NAFLD mice

[0228] The experimental results are shown in Figure 11 It was found by HE and oil red O staining of adipose tissue that the hepatocytes in the liver of the model group of mice were fatty degenerative, and a large number of fat vacuoles appeared. The total saponins of the dried group could significantly reduce the lipid infiltration in the liver, reduce the number of lipid droplets, and reduce the fat vacuoles in the liver. Compared with the model group, the size of the hepatocytes was uniform, and the arrangement was more orderly, while the improvement of the total saponins of the drum-dried group was weaker.

[0229] 3.2.5 The effect of different processing methods of Gynostemma pentaphyllum on the adipose tissue of NAFLD mice

[0230] The experimental results are shown in Figure 12 As shown in Table 6, the gynohepatitis group of NAFLD mice was significantly reduced in the weight of epididymal fat and inguinal fat by gynohepatitis, while the drum had no significant effect on the weight of adipose tissue in NAFLD mice, and both had no significant effect on the weight of brown adipose tissue.

[0231] 3.2.6 Effect of different processing methods of Gynostemma pentaphyllum on FGF15 in serum of NAFLD mice

[0232] The experimental results are shown in Figure 13 As shown in Table 6, the gynohepatitis group of NAFLD mice was significantly reduced in the weight of epididymal fat and inguinal fat by gynohepatitis, while the drum had no significant effect on the weight of adipose tissue in NAFLD mice, and both had no significant effect on the weight of brown adipose tissue.

[0233] 3.2.7 Effect of different processing methods of Gynostemma pentaphyllum on lipid decomposition in NAFLD mice

[0234] The experimental results are shown in Figure 14 As shown in Table 6, the gynohepatitis group of NAFLD mice was significantly reduced in the weight of epididymal fat and inguinal fat by gynohepatitis, while the drum had no significant effect on the weight of adipose tissue in NAFLD mice, and both had no significant effect on the weight of brown adipose tissue.

[0235] The above describes the embodiments of the present application in detail, and the principles and implementation methods of the present application are described by applying specific examples. The above description of the embodiments is only used to help understand the method of the present application and its core idea. Meanwhile, changes or deformations made by those skilled in the art according to the principles of the present application, based on the specific implementation methods and application scope of the present application, all belong to the scope of protection of the present application. In summary, the content of the specification should not be understood as a limitation of the present application.

Claims

1. Use of shade-dried total saponins in the preparation of a medicament for preventing and / or treating non-alcoholic fatty liver disease, characterized in that: The shade-dried total saponins are obtained by a method consisting of the following steps: (1) Take an appropriate amount of shade-dried Gynostemma pentaphyllum, crush it, and then add water to soak it; (2) heating in a water bath at about 40°C for about 3 hours; (3) after evaporating the water, adding a certain concentration of ethanol or methanol, heating and refluxing extraction or ultrasonic extraction, cooling the extract, filtering, and concentrating under reduced pressure to obtain a concentrate; and (4) loading the concentrate onto a macroporous resin for adsorption, eluting with water and about 30% ethanol to remove impurities, and eluting with about 70% ethanol, collecting the portion eluted with about 70% ethanol to obtain shade-dried total saponins; The saponin components in the shade-dried total saponins are composed of 0% to 10% of gypenosides substituted with 3 to 4 glycosyl groups and 90% to 100% of secondary saponins substituted with 1 to 2 glycosyl groups; Wherein, the secondary saponins substituted with 1 to 2 sugar groups are gypenosides LXXVII, gypenosides TN-1, gypenosides XIII and gypenosides CK.

2. The use according to claim 1, characterized in that In step (1), the mass volume ratio of the shade-dried Gynostemma pentaphyllum medicinal material to the water is 20:1 to 100:1, and the unit is g / L.

3. The use according to claim 2, characterized in that The mass volume ratio of the shade-dried Gynostemma pentaphyllum medicinal material to the water is about 50:1, and the unit is g / L.

4. The use according to claim 3, characterized in that The water is deionized water.

5. The use according to claim 3, characterized in that The water is distilled water.

6. The use according to claim 1, characterized in that In step (3), the concentration of the ethanol or the methanol is 60% to 80%.

7. The use according to claim 6, characterized in that The concentration of the ethanol or the methanol is about 70%.

8. The use according to claim 1, characterized in that In step (3), the volume ratio of the concentrate to the extract is 0.02:1 to 0.1:

1.

9. The use according to claim 8, characterized in that In step (3), the volume ratio of the concentrate to the extract is about 0.06:

1.

10. The use according to claim 1, characterized in that In step (3), the heating reflux extraction or the ultrasonic extraction is performed 1 to 4 times.

11. The use according to claim 1, characterized in that When the heating reflux extraction or the ultrasonic extraction is performed 2 to 4 times, the step (3) further comprises the step of combining the filtered extracts.

12. The use according to claim 11, characterized in that The heating reflux extraction or the ultrasonic extraction is performed twice.

13. The use according to claim 1, characterized in that The time for the heating reflux extraction or the ultrasonic extraction is 0.5 to 2 hours.

14. The use according to claim 13, characterized in that The time for the heating reflux extraction or the ultrasonic extraction is about 1 hour.

15. The use according to claim 1, characterized in that In step (3), the mass volume ratio of the shade-dried Gynostemma pentaphyllum medicinal material to the methanol or the ethanol is 20:1 to 100:1, and the unit is g / L.

16. The use according to claim 15, characterized in that The mass volume ratio of the shade-dried Gynostemma pentaphyllum medicinal material to the methanol or the ethanol is about 40:1, and the unit is g / L.

17. The use according to claim 1, characterized in that In step (4), the macroporous resin is D101 resin.

18. The use according to claim 1, characterized in that The adsorption time of the concentrate on the macroporous resin is 20 to 28 hours.

19. The use according to claim 18, characterized in that The adsorption time of the concentrate on the macroporous resin was about 24 hours.

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