Method for promoting conversion of rare ginsenoside in gynostemma pentaphylla and treatment effect of prepared product on metabolic syndrome

Abstract

The invention discloses a method for promoting conversion of rare ginsenoside in gynostemma pentaphylla and a therapeutic effect of a prepared product on metabolic syndrome. Specifically, the invention discloses a method for promoting conversion of rare ginsenoside in gynostemma pentaphylla, and the method comprises the following steps: (1) taking a proper amount of gynostemma pentaphylla medicinal material dried in the shade, crushing, and adding water for infiltration; (2) soaking for a period of time at a certain water bath temperature; (3) evaporating to dryness, adding ethanol or methanol with a certain concentration, carrying out heating reflux extraction or ultrasonic extraction, cooling an extracting solution, filtering, and carrying out vacuum concentration to obtain a concentrate; and (4) loading the concentrate on macroporous resin, adsorbing, eluting, and collecting the 60-80% ethanol elution part to obtain the total saponins dried in the shade. The method is mild in reaction condition, high in efficiency, high in specificity and free of pollution, and the prepared product is high in rare ginsenoside content and can be used for preventing and / or treating metabolic diseases such as non-alcoholic fatty liver diseases.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202310364731.3, filed on April 7, 2023, entitled "A method for promoting the conversion of rare ginsenosides in Gynostemma pentaphyllum and the therapeutic effect of the prepared product on metabolic syndrome". Technical Field

[0002] This invention relates to the field of pharmaceutical technology, specifically to a method for promoting the conversion of rare ginsenosides in Gynostemma pentaphyllum and the therapeutic effect of the prepared product on metabolic syndrome. Background Technology

[0003] Metabolic diseases are metabolic disorders caused by disturbances in one or more aspects of metabolism. Disorders in the metabolism of glucose, protein, and lipids can lead to metabolic diseases such as obesity, diabetes, and non-alcoholic fatty liver disease. With the improvement of modern living standards, the incidence of metabolic diseases is increasing year by year, threatening human health. These diseases not only cause secondary pathophysiological changes in multiple systems throughout the body but also place a heavy burden on individual patients and the entire social healthcare system.

[0004] Gynostemma pentaphyllum (Thumb.) Makino is a perennial creeping herbaceous plant belonging to the Cucurbitaceae family. Because it contains components similar to ginsenosides, it is known as "Southern Ginseng" or "Second Ginseng." Gynostemma pentaphyllum saponins are one of its main active ingredients, possessing functions such as nourishing the heart and spleen, replenishing qi and blood, and resolving phlegm and removing blood stasis, thus showing promising market prospects.

[0005] Gynostemma pentaphyllum is rich in tetracyclic triterpenoid saponins. To date, 327 structurally distinct saponins have been isolated and identified from the plant. Six of these, such as gypenosides III, IV, and VIII, are synonyms of ginsenosides Rb1, Rb3, Rd, F2, Rg3, and ginsenoside K. An additional 77 monomeric saponins are isomers of ginsenosides. The discovery of a new medicinal source rich in ginsenosides from plants outside the Araliaceae family is not only of significant academic importance but also has substantial economic and social value. Modern pharmacological activity studies have shown that gypenosides have significant effects in protecting the cardiovascular system, anti-tumor activity, enhancing immunity, and protecting the liver.

[0006] Ginsenosides are one of the main active components of ginseng, possessing a variety of important pharmacological activities. Rare ginsenosides, such as F1, F2, Rg3, Rh1, Rh2, CK, CY, and CMC, are present in very small amounts in ginseng; however, these rare saponins (whose parent nucleus typically contains 1-2 sugars) exhibit better activity. Therefore, the preparation of rare ginsenosides is a research focus. Gynostemma pentaphyllum contains 77 saponins that are isomers of ginsenosides. Discovering and preparing rare ginsenosides from Gynostemma pentaphyllum has promising application prospects. Rare saponins in Gynostemma pentaphyllum share the same parent structure as high-content saponins, differing only in the number of sugar groups. Rare ginsenosides can be prepared by selectively hydrolyzing the sugar groups of high-content saponins. Enzymatic methods, with their advantages of mild reaction conditions, high efficiency, strong specificity, and no pollution, have become the optimal method for preparing rare saponins. Summary of the Invention

[0007] Based on this, the present invention provides a method for promoting the conversion of rare ginsenosides in Gynostemma pentaphyllum, the method comprising the following steps:

[0008] (1) Take an appropriate amount of dried Gynostemma pentaphyllum, crush it, and then soak it in water;

[0009] (2) Heat at a certain water bath temperature for a period of time;

[0010] (3) After evaporating the water, add a certain concentration of ethanol or methanol, heat and reflux for extraction or ultrasonic extraction, cool the extract, filter, and concentrate under reduced pressure to obtain the concentrate; and

[0011] (4) The concentrate was loaded onto a macroporous resin for adsorption and elution. The eluted fraction was collected with 60-80% ethanol to obtain total saponins that were dried in the shade.

[0012] Further, in step (1), the mass-volume ratio (g / L) of the dried Gynostemma pentaphyllum medicinal material to the water is 20:1 to 100:1.

[0013] Furthermore, the mass-to-volume ratio (g / L) of the dried Gynostemma pentaphyllum to the water is approximately 50:1.

[0014] Furthermore, the water is deionized water.

[0015] Furthermore, the water is distilled water.

[0016] Furthermore, in step (2), the temperature of the water bath is 30°C to 70°C.

[0017] Furthermore, the temperature of the water bath is 35℃~50℃.

[0018] Furthermore, the temperature of the water bath is 40℃~50℃.

[0019] Furthermore, the temperature of the water bath is 35℃~45℃.

[0020] Furthermore, the temperature of the water bath is approximately 40°C.

[0021] Furthermore, the water bath lasts for 1 to 12 hours.

[0022] Furthermore, the water bath lasts for 2 to 4 hours.

[0023] Furthermore, the water bath lasts for approximately 3 hours.

[0024] Furthermore, in step (3), the concentration of the ethanol or the methanol is 60% to 80%.

[0025] Furthermore, the concentration of the ethanol or methanol is approximately 70%.

[0026] Further, in step (3), the volume ratio of the concentrate to the extract is 0.02:1 to 0.1:1.

[0027] Furthermore, in step (3), the volume ratio of the concentrate to the extract is approximately 0.06:1.

[0028] Furthermore, in step (3), the number of times the heating reflux extraction or the ultrasonic extraction is performed is 1 to 4.

[0029] Furthermore, when the number of heating reflux extractions or ultrasonic extractions is 2 to 4, step (3) further includes the step of merging the filtered extracts.

[0030] Furthermore, the heating reflux extraction or the ultrasonic extraction is performed twice.

[0031] Furthermore, the extraction time for heating and reflux or ultrasonic extraction is 0.5 to 2 hours.

[0032] Furthermore, the heating reflux extraction or the ultrasonic extraction takes approximately 1 hour.

[0033] Further, in step (3), the mass-volume ratio (g / L) of the dried Gynostemma pentaphyllum medicinal material to the methanol or the ethanol is 20:1 to 100:1.

[0034] Furthermore, in step (3), the mass-to-volume ratio (g / L) of the dried Gynostemma pentaphyllum to the methanol or ethanol is approximately 40:1.

[0035] Furthermore, in step (4), the macroporous resin is D101 resin.

[0036] Furthermore, the adsorption time of the concentrate on the macroporous resin is 20-28 hours.

[0037] Furthermore, the concentration is adsorbed onto the macroporous resin for approximately 24 hours.

[0038] Furthermore, the volume-to-mass ratio (mL / g) of the concentrate to the macroporous resin is 0.15:1.

[0039] Further, in step (4), the elution includes elution with water and 20% to 40% ethanol to remove impurities, and elution with 60% to 80% ethanol.

[0040] Furthermore, the elution process includes eluting with water and approximately 30% ethanol to remove impurities, and eluting with approximately 70% ethanol.

[0041] According to another aspect of the present invention, a preparation product obtained by the above method is provided.

[0042] Furthermore, the saponin components in the prepared product include 0% to 10% of 3 to 4 glycosyl-substituted Gynostemma pentaphyllum saponins.

[0043] Furthermore, the saponin components in the prepared product contain 90% to 100% secondary saponins with 1 to 2 glycosyl substitutions.

[0044] Furthermore, the 1-2 glycosyl-substituted secondary saponins are gypenosides LXXVII, gypenosides TN-1, gypenosides XIII, and / or gypenosides CK or their isomers.

[0045] Furthermore, the 1-2 glycosyl-substituted secondary saponins are gypenosides LXXVII, gypenosides TN-1, gypenosides XIII and / or gypenosides CK.

[0046] According to another aspect of the invention, a composition comprising the above-described prepared product is provided, the composition further comprising one or more substances for the prevention and / or treatment of metabolic diseases, and / or pharmaceutically acceptable excipients.

[0047] According to another aspect of the present invention, the use of the above-prepared product or the above-prepared composition in pharmaceuticals, foods and / or health products for the prevention and / or treatment of metabolic diseases is provided.

[0048] Furthermore, this metabolic disorder is metabolic syndrome.

[0049] Furthermore, this metabolic disorder is glucose and lipid metabolism syndrome.

[0050] Furthermore, the effects of the prepared product or the composition on the prevention and / or treatment of metabolic diseases are achieved through one or more of the following mechanisms: reducing body weight, increasing total cholesterol excretion, increasing triglyceride excretion, improving glucose metabolism, reducing hepatic lipid accumulation, reducing adipose tissue weight, increasing serum FGF15 levels, and increasing the expression of lipid breakdown-related proteins.

[0051] Furthermore, this improved glucose metabolism includes regulating glucose tolerance, reducing fasting blood glucose, and / or improving insulin resistance.

[0052] Furthermore, this reduction in hepatic lipid accumulation includes a decrease in the number of hepatic lipid droplets and / or a reduction in hepatic fat vacuoles.

[0053] Furthermore, the adipose tissue includes epididymal adipose tissue and / or inguinal adipose tissue.

[0054] Furthermore, the lipidolysis-related proteins include hormone-sensitive lipase and / or fibroblast growth factor receptor 1.

[0055] Furthermore, the metabolic disease is obesity, diabetes, and / or non-alcoholic fatty liver disease.

[0056] The beneficial effects of this invention are:

[0057] The method of this invention has mild reaction conditions, high efficiency, strong specificity, and no pollution. The obtained product has a high content of rare ginsenosides and can be used to prevent and / or treat metabolic diseases such as non-alcoholic fatty liver disease. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0059] Figure 1 This diagram illustrates the changes in the composition of Gynostemma pentaphyllum before and after enzymatic hydrolysis of the herb prepared using different drying methods.

[0060] Figure 2 The liquid chromatography-mass spectrometry diagram shows the results of the enzymatic hydrolysis time study of air-dried medicinal materials.

[0061] Figure 3 This is a schematic line graph showing the results of the investigation on the re-enzymatic hydrolysis time of air-dried medicinal materials.

[0062] Figure 4 The liquid chromatography-mass spectrometry (LC-MS) plots show the results of the enzymatic hydrolysis of air-dried medicinal materials. The A and E reaction temperatures were 30℃, 40℃, 50℃, 60℃, and 70℃.

[0063] Figure 5 A bar chart illustrating the degree of saponin conversion at different re-enzymatic hydrolysis temperatures.

[0064] Figure 6 Flowchart of the process for preparing total saponins from Gynostemma pentaphyllum by drum drying.

[0065] Figure 7 This is a process flow diagram for the enzymatic hydrolysis of air-dried Gynostemma pentaphyllum and the preparation of total saponins from Gynostemma pentaphyllum.

[0066] Figure 8 This is a schematic diagram showing the effect of different processing methods of Gynostemma pentaphyllum on the body weight of NAFLD mice (n=8). # represents the comparison between C and M, and * represents the comparison between drum drying, air drying, and M. * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001.

[0067] Figure 9 This diagram illustrates the effects of different processing methods of Gynostemma pentaphyllum on fecal TC and TG excretion in NAFLD mice at three and six weeks. # represents the comparison between C and M, and * represents the comparison between drum drying, air drying, and M. * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001. Where A and B are the excretion of TC and TG in feces over three weeks; C and D are the excretion of TC and TG in feces over six weeks.

[0068] Figure 10 This diagram illustrates the effects of different processing methods of Gynostemma pentaphyllum on glucose metabolism in NAFLD mice (n=8). A: Area under the curve for GTT; B: Area under the curve for ITT; C: Fasting blood glucose. # represents the comparison between C and M, and * represents the comparison between drum drying, air drying, and M. * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001.

[0069] Figure 11 Schematic diagram of HE staining and Oil Red O staining results of Gynostemma pentaphyllum processed with different methods on the liver of NAFLD mice (magnification: 200x).

[0070] Figure 12This diagram illustrates the effects of different processing methods of Gynostemma pentaphyllum on the adipose tissue of NAFLD mice. # represents the comparison between C and M, and * represents the comparison between drum drying, air drying, and M. * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001.

[0071] Figure 13 This diagram illustrates the effects of different processing methods of Gynostemma pentaphyllum on serum FGF15 in NAFLD mice. # represents the comparison between C and M, and * represents the comparison between drum drying, air drying, and M. * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001.

[0072] Figure 14 This diagram illustrates the effects of different processing methods of Gynostemma pentaphyllum on lipid metabolism in NAFLD mice. # represents the comparison between C and M, and * represents the comparison between drum drying, air drying, and M. * p<0.05, ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001. Detailed Implementation

[0073] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0074] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0075] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0076] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0077] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0078] As described in the background section, current Gynostemma pentaphyllum preparation processes have the problem of failing to prepare rare ginsenosides. To address this problem, the present invention provides a method for promoting the conversion of rare ginsenosides in Gynostemma pentaphyllum, the method comprising the following steps:

[0079] (1) Take an appropriate amount of dried Gynostemma pentaphyllum, crush it, and then soak it in water;

[0080] (2) Heat at a certain water bath temperature for a period of time;

[0081] (3) After evaporating the water, add a certain concentration of ethanol or methanol, heat and reflux for extraction or ultrasonic extraction, cool the extract, filter, and concentrate under reduced pressure to obtain the concentrate; and

[0082] (4) The concentrate was loaded onto a macroporous resin for adsorption and elution. The eluted fraction was collected with 60-80% ethanol to obtain total saponins that were dried in the shade.

[0083] In this invention, when mass-volume ratio, time, temperature, number of times, pressure, proportion, equivalent, concentration, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range "60-80" is disclosed, the described range should be interpreted as including 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", etc. When a range of values ​​is described in this document, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0084] In a preferred embodiment, in step (1), the mass-to-volume ratio (g / L) of the dried Gynostemma pentaphyllum to the water is 20:1 to 100:1. In another preferred embodiment, the mass-to-volume ratio (g / L) of the dried Gynostemma pentaphyllum to the water is approximately 50:1.

[0085] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50:1" includes ±5% of 50:1, or from 47.5:1 to 52.5:1.

[0086] 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 to 70°C. In a preferred embodiment, the temperature of the water bath is 35°C to 50°C. In a preferred embodiment, the temperature of the water bath is 40°C to 50°C. In a preferred embodiment, the temperature of the water bath is 35°C to 45°C. In a preferred embodiment, the temperature of the water bath is approximately 40°C.

[0087] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0088] In one preferred embodiment, the water bath lasts for 1 to 12 hours. In another preferred embodiment, the water bath lasts for 2 to 4 hours. In yet another preferred embodiment, the water bath lasts for approximately 3 hours.

[0089] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes 3 ± 5%, or from 2.85 to 3.15.

[0090] In a preferred embodiment, in step (3), the concentration of the ethanol or the methanol is 60% to 80%. In a preferred embodiment, the concentration of the ethanol or the methanol is about 70%.

[0091] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 70" includes ±5% of 70, or from 66.5 to 73.5.

[0092] In a preferred embodiment, in step (3), the volume ratio of the concentrate to the extract is 0.02:1 to 0.1:1. In a preferred embodiment, in step (3), the volume ratio of the concentrate to the extract is approximately 0.06:1.

[0093] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.06:1" includes ±5% of 0.06:1, or from 0.057:1 to 0.063:1.

[0094] In a preferred embodiment, in step (3), the number of times the heating reflux extraction or the ultrasonic extraction is performed is 1 to 4. In a preferred embodiment, when the number of times the heating reflux extraction or the ultrasonic extraction is performed is 2 to 4, step (3) further includes a step of combining the filtered extracts. In a preferred embodiment, the number of times the heating reflux extraction or the ultrasonic extraction is performed is 2. In a preferred embodiment, the time for the heating reflux extraction or the ultrasonic extraction is 0.5 to 2 hours. In a preferred embodiment, the time for the heating reflux extraction or the ultrasonic extraction is about 1 hour.

[0095] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0096] In a preferred embodiment, in step (3), the mass-to-volume ratio (g / L) of the dried Gynostemma pentaphyllum to the methanol or ethanol is 20:1 to 100:1. In a preferred embodiment, in step (3), the mass-to-volume ratio (g / L) of the dried Gynostemma pentaphyllum to the methanol or ethanol is approximately 40:1.

[0097] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40:1" includes ±5% of 40:1, or from 38:1 to 42:1.

[0098] In a preferred embodiment, in step (4), 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 approximately 24 hours.

[0099] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 24" includes ±5% of 24, or from 22.8 to 25.2.

[0100] In a preferred embodiment, the volume-to-mass ratio (mL / g) of the concentrate to the macroporous resin is 0.15:1.

[0101] In a preferred embodiment, in step (4), the elution includes eluting with water and 20% to 40% ethanol to remove impurities, and eluting with 60% to 80% ethanol. In a preferred embodiment, the elution includes eluting with water and about 30% ethanol to remove impurities, and eluting with about 70% ethanol.

[0102] In this invention, "about" refers to a value within a range of ±5% of a specific 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.

[0103] According to another aspect of the present invention, a preparation product obtained by the above method is provided.

[0104] In a preferred embodiment, the saponin component in the prepared product comprises 0% to 10% of 3 to 4 glycosyl-substituted Gynostemma pentaphyllum saponins.

[0105] In a preferred embodiment, the saponin component in the prepared product comprises 90% to 100% of secondary saponins with 1 to 2 glycosyl substitutions.

[0106] In a preferred embodiment, the 1-2 glycosyl-substituted secondary saponins are gypenosides LXXVII, gypenosides TN-1, gypenosides XIII, and / or gypenosides CK or their isomers.

[0107] In a preferred embodiment, the 1-2 glycosyl-substituted secondary saponins are gypenosides LXXVII, gypenosides TN-1, gypenosides XIII and / or gypenosides CK.

[0108] According to another aspect of the invention, a composition comprising the above-described prepared product is provided, the composition further comprising one or more substances for the prevention and / or treatment of metabolic diseases, and / or pharmaceutically acceptable excipients.

[0109] In a preferred embodiment, the composition of the present invention contains at least one pharmaceutically acceptable excipient in an 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.% relative to the total weight of the composition.

[0110] In this invention, the term "pharmaceutically acceptable" means a substance, such as a carrier or diluent, that does not destroy the biological activity or properties of a compound and is relatively non-toxic, such that, when administered to an individual, it will not cause unwanted biological effects or interact with any of its constituent components in a harmful manner.

[0111] In this invention, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0112] In a preferred embodiment, the excipient is selected from one or more of the following: diluents, disintegrants, dispersants, plasticizers, encapsulating agents, wetting agents, sustained-release agents, retention aids, lubricants, adhesives, flavoring agents, light-blocking agents, and antioxidants.

[0113] These excipients are preferably drug-inert, or may have synergistic or enhancing effects to enhance the therapeutic activity of the drug composition. The types of excipients listed above are merely examples, and the types of excipients actually used in this invention are not limited to those listed above. They can be adjusted according to the actual situation to achieve the effects of this invention.

[0114] According to another aspect of the present invention, the use of the above-prepared product or the above-prepared composition in pharmaceuticals, foods and / or health products for the prevention and / or treatment of metabolic diseases is provided.

[0115] In this invention, the term "treatment" also includes "prevention," unless specifically stated otherwise. The terms "therapeutic" and "therapeutically" should be understood accordingly.

[0116] In this invention, the term "treatment" includes alleviating, suppressing, or improving symptoms or conditions of a disease; suppressing the development of complications; improving or preventing underlying metabolic syndromes; suppressing the development of a disease or symptom, such as controlling the progression of a disease or condition; reducing a disease or symptom; alleviating a disease or symptom; reducing complications arising from a disease or symptom; or preventing or treating signs arising from a disease or symptom. As used herein, a preparation or composition, when administered, can improve a disease, symptom, or condition, particularly by improving its severity, delaying onset, slowing disease progression, or reducing the duration of the condition. Whether the administration is fixed or intermittent, continuous or intermittent, it can be attributed to or related to the administration.

[0117] In a preferred embodiment, the metabolic disease is metabolic syndrome.

[0118] In a preferred embodiment, the metabolic disorder is glucose and lipid metabolism syndrome.

[0119] In a preferred embodiment, the prepared product or the composition is used to prevent and / or treat metabolic diseases through one or more of the following mechanisms: reducing body weight, increasing total cholesterol excretion, increasing triglyceride excretion, improving glucose metabolism, reducing hepatic lipid accumulation, reducing adipose tissue weight, increasing serum FGF15 levels, and increasing the expression of lipid breakdown-related proteins.

[0120] In a preferred embodiment, the improved glucose metabolism includes regulating glucose tolerance, reducing fasting blood glucose, and / or improving insulin resistance.

[0121] In a preferred embodiment, the reduction of hepatic lipid accumulation includes a decrease in the number of hepatic lipid droplets and / or a reduction in hepatic fat vacuoles.

[0122] In a preferred embodiment, the adipose tissue includes epididymal adipose tissue and / or inguinal adipose tissue.

[0123] In a preferred embodiment, the lipidolysis-related protein includes hormone-sensitive lipase and / or fibroblast growth factor receptor 1.

[0124] In a preferred embodiment, the metabolic disease is obesity, diabetes, and / or non-alcoholic fatty liver disease.

[0125] The metabolic diseases described above in this invention are merely examples. The prepared products or compositions of this invention may also be used to prevent and / or treat other metabolic diseases.

[0126] The present invention also provides the above-prepared product or the above-described composition for the prevention and / or treatment of metabolic diseases such as non-alcoholic fatty liver disease in subjects.

[0127] The present invention also provides a method for preventing and / or treating metabolic diseases such as non-alcoholic fatty liver disease in a subject, comprising administering an effective amount of the above-described prepared product or the above-described composition to the subject.

[0128] In this invention, the term "subject" refers to a mammal. A 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 subjects representing models of metabolic diseases such as non-alcoholic fatty liver disease. Preferably, the subject is a human.

[0129] The "effective amount" of the above-described prepared product or composition used in this invention can achieve the desired therapeutic and / or preventative effects. The effective amount for this purpose will depend on factors such as the pharmaceutical composition, the route of administration, the stage and severity of the disease being treated, the individual's weight and overall health status, and the judgment of the prescribing physician. Dosage can be administered once a week, every two days, once daily, or even several times daily. Dosage units can be administered over a short period (e.g., weeks to months) or a longer period (months to years).

[0130] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0131] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0132] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0133] Laboratory instruments (manufacturer)

[0134] SB-1100 EYELA water bath (Shanghai Ailang Instrument Co., Ltd.), digital display heating mantle (Shanghai Lichen Bangxi 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 (2.1 mm × 100 mm, 1.7 μm).

[0135] Blood glucose meter (Roche), high-throughput tissue homogenizer (Shanghai Biheng), optical microscope (Olympus), analytical balance (Shanghai Jinghai Instruments), multi-functional microplate reader (TECAN), vortex mixer (Hangzhou Aosheng Instruments), microplate constant temperature shaker (Hangzhou Aosheng Instruments), EnVision® multi-functional microplate reader (PE), microcentrifuge (Hangzhou Mio), vertical electrophoresis apparatus, semi-dry transfer apparatus (ATTO), shaker (Haimen Qilinbei Instruments), AI600 protein imager (GE, USA).

[0136] Experimental reagents and materials (manufacturer)

[0137] The medicinal material of Gynostemma pentaphyllum was obtained from dried leaves of Yingfu Village, Zhangzhou City, Fujian Province, along with chromatographic acetonitrile (Fisher Corporation, USA), D-101 macroporous resin (Cangzhou Baoen Adsorption Materials Technology Co., Ltd.), ethanol (Beijing Chemical Plant), formic acid (Beijing Chemical Plant), and Watson's distilled water.

[0138] Triglyceride (TG) test kit, total cholesterol (TC) test kit (Nanjing Jiancheng), biosynthetic human insulin injection (Novo Nordisk), glucose (Sigma), blood glucose test strips (Roche), isoflurane (Reward Life), universal tissue fixative, hematoxylin, eosin, oil red dye (Wuhan Google), chloroform, methanol, isopropanol, physiological saline (Sinopharm), Triton X-100 (Biofroxx), Tissue-Tek OCT embedding medium (Sakara), mouse FGF15 ELISA kit (Wuhan Huamei), sodium dodecyl sulfate, TEMED, glycine, ammonium persulfate, tris(hydroxymethyl)aminomethane, bovine serum albumin (Shanghai Yisheng), Tween 20 (Biofroxx), RIPA protein lysis buffer, 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) mol / L), pH 6.8, 30% acrylamide mixture, 10×PBS solution (Dalian Meilun), HSL antibody (#18381), p-HSL antibody (#4137), HSP90 antibody (#4877), FGFR1 antibody (#9740) (Abcam), horseradish peroxidase-labeled goat anti-rabbit IgG (Jackson ImmunoResearch, USA).

[0139] Example 1: Investigation of the process of rehydration and enzymatic hydrolysis of air-dried medicinal materials

[0140] 1.1 Comparison of re-enzymatic hydrolysis reactions of Gynostemma pentaphyllum medicinal materials under different drying methods

[0141] Previous experiments revealed that glucosinolate hydrolase activity remained in the shade-dried Gynostemma pentaphyllum. The herb could continue enzymatic hydrolysis after rehydration in a water bath. To investigate the rehydration and re-enzymatic hydrolysis capability of the dried herb, further experiments were conducted. Shade-dried Gynostemma pentaphyllum was pulverized and mixed with distilled water at a mass-to-volume ratio of (50:1) (mg:ml). The mixture was incubated in a 40°C water bath for 3 hours. Then, methanol was added to the reaction solution at a volume ratio of 1:4, and the mixture was ultrasonically extracted for 1 hour. The supernatant was centrifuged, filtered through a membrane, and subjected to mass spectrometry. The results are as follows: Figure 1 As shown.

[0142] Chromatographic conditions: Column: ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm); Mobile phase A: acetonitrile, Mobile phase B: 0.1% formic acid in 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℃, Autosampler temperature: 20℃, PDA detector scan range: 200–400 nm.

[0143] Mass spectrometry conditions: ESI ion source, negative ion mode scanning, capillary voltage 2 kV, cone voltage 40 V, ion source temperature 120℃, desolvation gas flow rate 600 L / hr, desolvation gas temperature 400℃, cone gas flow rate 50 L / hr, precursor ion collision energy 6 eV, fragment ion collision energy 20-50 eV, mass scan range 100-2000 Da, scan time 0.2 s.

[0144] The medicinal material underwent further enzymatic hydrolysis via a rehydration bath, confirming that glucosinolate hydrolase activity remained in the shade-dried Gynostemma pentaphyllum. After 3 hours of further enzymatic hydrolysis, approximately 90% of the 3-4 glycosyl-substituted Gynostemma pentaphyllum saponins in the shade-dried material were essentially converted to 1-2 glycosyl-substituted secondary saponins, while the drum-dried sample did not exhibit this reaction. To further utilize the re-enzymatic hydrolysis capability of the shade-dried material, the re-enzymatic hydrolysis process was optimized.

[0145] 1.2 Investigation on the optimal time for re-enzymatic hydrolysis of air-dried medicinal materials

[0146] Take the above-mentioned dried Gynostemma pentaphyllum powder, mix it with water at a mass-to-volume ratio of 50:1 (mg:mL), and place it at 40℃ for 12 hours. At 0h, 1h, 3h, and 6h, respectively, add methanol to the reaction solution at a volume ratio of 1:4, and extract by ultrasonication for 1 hour. Centrifuge, collect the supernatant, filter it through a membrane, and insert it into a mass spectrometer. Figure 2 and Figure 3 It was found that after 3 hours of enzymatic hydrolysis in a water bath, the 3-4 glycosyl-substituted Gynostemma pentaphyllum saponins were basically converted into 1-2 glycosyl-substituted secondary saponins. Mass spectrometry analysis after hydrolysis indicated that the four main 1-2 sugars generated were Gypenoside LXXVII, Gynosaponin TN-1, Gypenoside XIII, Ginsenoside CK, and their isomers. To ensure sufficient water bath time, 3 hours was determined to be the optimal reaction time.

[0147] The four groups represent the products of four different Gynostemma pentaphyllum saponin compounds converted from 3-4 sugar substrates by enzyme hydrolysis to the corresponding 1-2 sugars. All numbers represent molecular ion masses in mass spectrometry. Group 1: 1139 is Gypenoside LVI, 1179 is its corresponding malonyl saponin (M-Gypenoside LVI), and 1181 is its corresponding acetyl saponin. 815 is Gypenoside LXXVII. Group 2: 1007 is Gypenoside XLVI, 1047 is its corresponding malonyl saponin (M-Gypenoside XLVI), and 1049 is its corresponding acetyl saponin. 683 is Gynosaponin TN-1. Group 3: 1123 is Gypenoside LXIII, 1163 is its corresponding malonyl saponin (M-Gypenoside LXIII), and 1165 is its corresponding acetyl saponin. 799 is Gypenoside XIII. Group 4: 991 is Ginsenoside Rd, 1031 is its corresponding malonyl saponin, i.e., M-Ginsenoside Rd, and 1033 is its corresponding acetyl saponin. 667 is Ginsenoside CK.

[0148] Among them, the peak area values ​​obtained directly from the mass spectrometry analysis software are used to calculate... Figure 3 The sum of the peak areas of the four substrates and the sum of the peak areas of the four products after 3 hours of reaction were statistically analyzed. The sum of the peak areas of the four substrates and the sum of the peak areas of the four products were 1.8% (substrate sum / (substrate sum + product sum) × 100%) and 98.2% (product sum / (substrate sum + product sum) × 100%).

[0149] 1.3 Investigation on the optimal temperature for re-enzymatic hydrolysis of air-dried medicinal materials

[0150] The above-mentioned dried Gynostemma pentaphyllum powder was mixed with water at a mass-to-volume ratio of 50:1 (mg:mL), and reacted in water baths at 30℃, 40℃, 50℃, 60℃, and 70℃ for 1 hour each. Methanol was added to the reaction solution at a volume ratio of 1:4, and the mixture was ultrasonically extracted for 1 hour. The supernatant was centrifuged, filtered through a membrane, and subjected to mass spectrometry to detect saponin conversion. The peak areas of the hydrolysate, intermediate, and hydrolysate were statistically analyzed. Figure 4 It can be seen that after 1 hour of reaction, in the 30℃ group, with a retention time of 2-6 minutes, a large number of saponins with 3-4 glycosyl substitutions can be detected, indicating a low conversion efficiency. At a reaction temperature of 40℃, the conversion of saponins with 3-4 glycosyl substitutions is the most complete, followed by 50℃. The conversion efficiency is not high at 60℃ and 70℃. Figure 5 The peak areas of the substrate, intermediate product, and product were statistically analyzed by directly obtaining peak area values ​​from the mass spectrometry analysis software. The results showed that 40℃-50℃ was the optimal reaction temperature, yielding the most product.

[0151] Example 2: Preparation of saponin extracts by different processing methods

[0152] 2.1 Preparation of total saponins from Gynostemma pentaphyllum by drum drying

[0153] 50g of drum-dried Gynostemma pentaphyllum from Fujian was ground into powder. At a material-to-liquid ratio of 1:25, 70% ethanol was added, and the mixture was heated and refluxed twice for 1 hour each time. The extracts were combined, cooled, and filtered. The solution was collected under reduced pressure to approximately 150 mL, which was then passed through 1.0 kg of D101 macroporous resin for 24 hours. Impurities were removed by elution with water and 30% ethanol, followed by elution with 70% ethanol. 19.53g of total saponins from the drum-dried Gynostemma pentaphyllum were collected. The flowchart for the preparation of total saponins from drum-dried Gynostemma pentaphyllum is shown below. Figure 6 As shown.

[0154] 2.2 Process for re-enzymatic hydrolysis of air-dried Gynostemma pentaphyllum and preparation of total saponins from Gynostemma pentaphyllum

[0155] Take 50g of dried Gynostemma pentaphyllum from Fujian, grind it into powder, add 1L of deionized water, completely soak it, and heat it in a 40℃ water bath for 3 hours, then evaporate it to dryness. Add 1.25L of 70% ethanol, heat and reflux twice, 1 hour each time. After cooling, filter the extract and recover approximately 150mL under reduced pressure. Pass the extract through 1kg of D101 macroporous resin for 24 hours. Elute with water and 30% ethanol to remove impurities, then elute with 70% ethanol. Collect the 70% ethanol eluent to obtain the total saponins from the dried Gynostemma pentaphyllum. The process flow for the enzymatic hydrolysis of the dried Gynostemma pentaphyllum and the preparation of total saponins from Gynostemma pentaphyllum is as follows: Figure 7 As shown.

[0156] 2.3 Saponin Content Detection

[0157] The chromatographic and mass spectrometric conditions for Example 2 were the same as those for Example 1.

[0158] Appropriate amounts of the extracts from the two samples were filtered through a 0.22 μm filter membrane and then injected for mass spectrometry analysis. The results showed that the saponin components in the high-temperature drum-dried samples were mainly concentrated in the 4-8 min range, with the core containing 3-4 sugars, while those in the air-dried samples were mostly concentrated in the 8-13 min range, with the core containing 1-2 sugars. These saponins are structurally similar to the rare ginsenosides currently reported and have better activity.

[0159] The results above show that chemical transformation occurs during the air-drying process of fresh Gynostemma pentaphyllum. Fresh Gynostemma pentaphyllum may contain glucosidase. During the air-drying process, under the action of glucosidase, hydrolysis may occur, resulting in the desugaring and generation of corresponding secondary glycosides, producing a large amount of rare ginsenosides. However, drum drying at high temperatures inactivates enzymes, easily causing a decrease in the activity of glucosidase in the body. Therefore, the hydrolysis rate of saponins in Gynostemma pentaphyllum is slow, and they mainly exist in the form of protopanaxadiones.

[0160] The above research results suggest that the shade-drying process of Gynostemma pentaphyllum is more conducive to the formation of rare saponins than drum drying. Therefore, the preparation method of highly active rare ginsenosides of the present invention is of great significance.

[0161] Example 3: Comparison of the glycolipid metabolism activities of Gynostemma pentaphyllum saponins processed using different methods

[0162] 3.1 Experimental Methods

[0163] 3.1.1 Preparation of feed for drug administration

[0164] The drug (200 mg / kg) -1 Gynostemma pentaphyllum saponins, 200 mg·kg -1 The dried Gynostemma pentaphyllum saponins were added to the pulverized high-fat, high-sugar, and high-cholesterol feed, mixed and shaped again, and sterilized under ultraviolet light to prepare the feed for the mice in the drug administration group.

[0165] 3.1.2 Animal grouping, model establishment, and drug administration methods

[0166] After one week of acclimatization, C57BL / 6J mice were randomly divided into a normal control group (C, n=6) fed a standard diet, and the remaining mice were fed a high-fat, high-sugar, and high-cholesterol diet to induce NAFLD for 16 weeks. These groups were designated as the NAFLD model group (M, n=6) and the control group (Gynostemma pentaphyllum saponin 200 mg / kg). -1 Group (drum, n=8), shade-dried Gynostemma pentaphyllum saponins (200 mg·kg) -1 Group C (air-dried, n=8) was divided into two groups. Group C continued to be fed a normal diet for 6 weeks, Group M continued to be fed a high-fat, high-sugar, and high-cholesterol diet for 6 weeks, and the roller-dried and air-dried groups were fed their respective drug-treated diets for 6 weeks. The body weight and food intake of mice in each group were recorded weekly.

[0167] 3.1.3 Glucose tolerance test

[0168] Mice in each group were fasted for 12 hours. After measuring fasting blood glucose levels, they were injected intraperitoneally with glucose solution (2 g / kg). Blood glucose levels were measured by collecting blood from the tail tip at 15, 30, 60 and 120 minutes.

[0169] 3.1.4 Insulin Tolerance Test

[0170] Mice in each group were fasted for 5 hours. After measuring fasting blood glucose levels, insulin solution (0.75 U / kg) was injected intraperitoneally. Blood glucose levels were measured by collecting blood from the tail tip at 15, 30, 60 and 120 minutes.

[0171] 3.1.5 Collection of animal tissue samples and serum

[0172] Six weeks after drug administration, the mice were weighed, anesthetized with isoflurane, and whole blood was collected by enucleation. The liver, epididymal fat, inguinal fat, and brown fat were carefully removed, weighed, and stored at -80 °C. The liver weight-to-body weight ratio (liver weight / body weight × 100%) was calculated. The whole blood was allowed to stand at room temperature for 2 h, then centrifuged at 3500 rpm for 15 min at 4 °C. The supernatant was collected and stored at -80 °C.

[0173] 3.1.6 Extraction and determination of TC and TG in mouse feces

[0174] Feces were collected from mice at three and six weeks of age, dried, and pulverized. Approximately 50 mg of feces was weighed and homogenized in 0.5 mL of PBS solution. Then, 0.5 mL of a chloroform-methanol mixture (chloroform:methanol = 2:1) was added and vortexed. After centrifugation at 3000 rpm for 15 min, the chloroform layer was collected, evaporated to dryness, and reconstituted with 10% Triton X-100 solution. The experimental procedures were performed according to the test kit instructions, and the contents of TC and TG in the feces were calculated.

[0175] 3.1.7 HE staining of liver and adipose tissue

[0176] Hepatic lobe tissue and adipose tissue from the same location were harvested, fixed in a general-purpose tissue fixative, routinely embedded in paraffin, sectioned, and placed on glass slides. After dewaxing, hematoxylin was stained for approximately 3 minutes, followed by rinsing with running water for 1 minute. The slides were then immersed in eosin solution for 30 seconds, rinsed with running water for 1 minute, dehydrated with ethanol, dried, mounted with 50% neutral resin xylene solution, and air-dried. Morphological changes in the liver and adipose tissues were observed under a microscope.

[0177] 3.1.8 Oil Red O staining of liver tissue

[0178] Hepatic lobe tissues from the same location were harvested, fixed in a general-purpose tissue fixative, embedded on dry ice using Tissue-Tek OCT embedding medium, and frozen at -80 °C. After sectioning using a cryostat, the tissue was infiltrated with 60% isopropanol, followed by slow dripping of Oil Red O working solution for staining for approximately 15 min. After staining, the tissue was washed three times with 60% isopropanol and rinsed with running water for 30 s. Hematoxylin solution was then stained for approximately 5 min, rinsed with running water for 30 s, and mounted with glycerol gelatin. Lipid accumulation in the liver tissue was observed under a microscope.

[0179] 3.1.9 Western Blot Protein Imprinting Analysis

[0180] 3.1.9.1 Extraction of tissue protein

[0181] Approximately 100 mg of white adipose tissue was cut off on dry ice and added to 400 µL of RIPA solution containing phosphatase inhibitors and protease inhibitors. The mixture was homogenized for 1 min. The mixture was then placed on ice for lysis for 30 min, gently vortexing every 10 min to ensure complete protein lysis. After lysis, the mixture was centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected as the extracted protein solution.

[0182] 3.1.9.2 Protein concentration determination

[0183] Dilute the protein standard solutions with RIPA solution according to Table 3-1 to prepare a protein standard curve. Dilute the sample solutions to be tested 40-fold, add 20 µL of standard solution and sample solution to each well of a 96-well plate, and then add 200 µL of working solution using a multipipeline. Incubate at 37 °C and 300 rpm for 30 min using a constant temperature shaker. Measure the absorbance at 562 nm using a microplate reader and calculate the concentration of each sample.

[0184] Table 1 Standard Curve Preparation System

[0185]

[0186] 3.1.9.3 Protein Sample Preparation

[0187] The sample concentration was quantified to 10 µg / 10 µL. The sample was diluted with RIPA lysis buffer containing phosphatase and protease inhibitors, and then an appropriate amount of 5× loading buffer solution was added and vortexed to mix. The sample was heated at 95°C for 10 min, and after cooling to room temperature, it was stored in a -20°C refrigerator.

[0188] 3.1.9.4 Western Blot Experiment

[0189] (1) Preparation of reagents

[0190] ① Prepare 10×SDS electrophoresis buffer according to Table 3-2.

[0191] Table 2 Preparation of 10×SDS Electrophoresis Buffer

[0192]

[0193] ② Prepare the 10× transfer solution according to Table 3-3.

[0194] Table 3 Preparation of 10× Transfer Buffer

[0195]

[0196] ③ Prepare 10×PBST solution according to Table 3-4.

[0197] Table 4 Preparation of 10×PBST solution

[0198]

[0199] ④ 1×SDS electrophoresis buffer: Dilute 10×SDS electrophoresis buffer to 1× with ddH2O.

[0200] ⑤ Prepare 1× transfer solution according to Table 3-5.

[0201] Table 5. Preparation of 1× Transfer Buffer

[0202]

[0203] ⑥ 1×PBST solution: Dilute 10×PBST solution with ddH2O to 1×.

[0204] ⑦ Prepare the 5% BSA solution according to Table 3-6.

[0205] Table 6 5% BSA Preparation

[0206]

[0207] ⑧ Prepare the 10% APS solution according to Table 3-7.

[0208] Table 7 10% APS Preparation

[0209]

[0210] (2) WB experimental procedure

[0211] ① Gel preparation: Clean and dry the glass plates using ddH2O. Place the shorter glass plate on the lab bench (convex side up), attach the adhesive strip, and cover the shorter glass plate with the longer glass plate, keeping the bottom edges flush. Clamp the two glass plates together and place them flat on the table. Select the separating gel concentration according to the molecular weight of the target protein. The preparation methods for the separating gel are shown in Tables 3-8 and 3-9.

[0212] Table 8. Preparation of 7.5% separating gel

[0213]

[0214] Table 9. Preparation of 10% separating gel

[0215]

[0216] Slowly pour the separating gel between the two plates until it reaches 80% of the height of the shorter plate. Then add an appropriate amount of anhydrous ethanol to the surface of the separating gel, remove air bubbles, and flatten the liquid surface. After the separating gel between the two plates has solidified, wipe the alcohol off the plates with thick filter paper. Pour in the stacking gel; the preparation method for the stacking gel is shown in Table 3-10.

[0217] Table 10 Preparation of Concentrated Gel

[0218]

[0219] Vortex the concentrated gel in a 50 mL centrifuge tube to mix thoroughly. Pour the mixture onto the separating gel in both plates, tilting the tube and inserting a comb to prevent air bubbles from entering. Ensure the gel and comb are level and allow the concentrated gel to set.

[0220] ② Sample loading: Place the protein sample in a 75 ℃ constant temperature heater and heat for 10 min, then vortex centrifuge. Pour 1×SDS electrophoresis buffer to 1 / 3 of the height of the electrophoresis tank. Remove the clamps and gel strips from the glass plate, and slowly tilt the glass plate into the sample loading tank, removing air bubbles from the bottom, and fix it with a stopper plate. Fill the space between the two plates with 1×SDS electrophoresis buffer, pull out the comb horizontally, and blow out any broken gel fragments from the wells. From right to left, add 5 µL of protein marker and 10 µL of sample in half a dozen batches.

[0221] ③ Electrophoresis: Insert the electrodes (red to red, black to black), set the voltage to 300 V, 65 mA (two electrodes), and run the gel electrophoresis for 60 min. When the bromophenol blue reaches the bottom of the gel, stop the electrophoresis and rinse the sample tank with running water to remove the 1×SDS electrophoresis solution.

[0222] ④ Transfer: Cut 6×9 cm filter paper and PVDF membrane. Activate the PVDF membrane by immersing it in methanol solution for 1 min. Then, wet the filter paper and PVDF membrane with 1× transfer buffer. Remove the glass plate, placing the shorter plate underneath. Use a gel cutter to pry the two plates apart and remove the concentrating gel. Place the PVDF membrane on top of the separating gel and align it. Use tweezers to pick up the PVDF membrane and gel together and place it on the transfer apparatus with a layer of moistened filter paper already placed on it, being careful to remove any air bubbles between the gel and the PVDF membrane. Cover with another layer of moistened filter paper, and finally close the transfer apparatus lid. Perform the transfer at 30 V, 250 mA (two plates), for 90 min.

[0223] ⑤ Sealing: After the membrane transfer is complete, discard the gel and filter paper, and place the PVDF membrane in a 5% BSA solution for sealing at room temperature for 2-3 hours.

[0224] ⑥ Washing the membrane: After blocking, discard the BSA solution, place the PVDF membrane in 1×PBST, place it on a shaker, and wash it once every 10 minutes, for a total of three washes.

[0225] ⑦ Primary antibody incubation: Place the PVDF membrane between plastic wrap and cut bands according to the protein marker instructions, based on the molecular weight of the target protein. Dilute the primary antibody with 1×PBST, and place the cut bands into different antibody solutions, ensuring there are no air bubbles near the bands. Incubate overnight at 4 ℃ with gentle shaking on a shaker.

[0226] ⑧ Washing the membrane: Recover the primary antibody solution, place the band in 1×PBST, and wash once every 10 minutes on a shaker, for a total of three washes.

[0227] ⑨ Secondary antibody incubation: Select the secondary antibody corresponding to the species, dilute it with 1×PBST, put the band into the secondary antibody solution, and incubate it slowly on a shaker at room temperature for 1 h.

[0228] ⑩ Washing and Development: Recover the secondary antibody solution, place the band in 1×PBST, and wash three times in a shaker for 10 minutes each time. Prepare the developing solution according to the manufacturer's instructions, immerse the band in the developing solution for a short time, and then develop. Use β-ACTIN, GAPDH, or HSP90 as internal control proteins to analyze the expression of the target protein.

[0229] 3.1.10 Statistical Analysis

[0230] Graphpad Prism 9.3 software was used for analysis. Experimental results are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used for comparisons of multiple groups, and student's t-test was used for comparisons of two groups. A p < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0231] 3.2 Experimental Results

[0232] Effects of different processing methods on glucose and lipid metabolism in mice with non-alcoholic fatty liver disease (NAFLD)

[0233] A NAFLD mouse model was established by setting up a high-fat, high-sugar, and high-cholesterol diet to evaluate the effects of Gynostemma pentaphyllum saponins processed in different ways on improving the metabolic phenotype of NAFLD mice.

[0234] 3.2.1 Effects of different processing methods on the body weight of Gynostemma pentaphyllum mice with DNAFLD

[0235] Experimental results are as follows Figure 8As shown, the NAFLD mice that successfully developed the model had a significantly higher body weight than the mice in group C. Treatment with air-dried Gynostemma pentaphyllum saponins steadily reduced the body weight of NAFLD mice from the second week onwards. The drum-dried saponin group showed a trend towards reducing the body weight of NAFLD mice compared to the model group, but the difference was not statistically significant.

[0236] 3.2.2 Effects of different processing methods on fecal TC and TG excretion in NAFLD mice at three and six weeks of age.

[0237] Experimental results are as follows Figure 9 As shown, shade-dried Gynostemma pentaphyllum saponins can significantly increase the excretion of total cholesterol (TC) and triglycerides (TG) in mouse feces.

[0238] 3.2.3 Effects of different processing methods on blood glucose metabolism in NAFLD mice

[0239] Experimental results are as follows Figure 10 As shown, after intraperitoneal injection of glucose solution, blood glucose levels increased in all groups of mice. Group M mice showed a significantly higher blood glucose level than group C mice, and the decrease was slower. Treatment with total saponins from Gynostemma pentaphyllum processed in different ways effectively regulated glucose tolerance in NAFLD mice. Statistical analysis of the area under the curve (AUC) of the glucose tolerance test (GTT) revealed that the shade-dried group significantly reduced the AUC value. Figure 10 -A), and significantly reduced fasting blood glucose levels in mice ( Figure 10 -C), the saponins in the drum-dried group showed a decreasing trend in related indicators, but the differences were not significant.

[0240] After intraperitoneal injection of insulin solution, blood glucose levels decreased in all groups of mice. Group C mice showed a slower decline in blood glucose compared to group M mice, and their blood glucose levels recovered to pre-insulin injection levels within 120 minutes. Treatment with total saponins from Gynostemma pentaphyllum processed using different methods effectively improved insulin resistance in NAFLD mice. Statistical analysis of the AUC values ​​in the insulin tolerance test (ITT) revealed that air-drying significantly reduced the AUC value (…). Figure 10 -B). This indicates that air drying can restore insulin sensitivity and improve glucose metabolism in NAFLD mice. In the drum-dried group, saponins and related indicators showed a decreasing trend, but the differences were not significant.

[0241] 3.2.4 Effects of different processing methods on lipid accumulation in the liver of Gynostemma pentaphyllum mice

[0242] Experimental results are as follows Figure 11As shown, HE and Oil Red O staining results of adipose tissue revealed that hepatocytes in the model group mice exhibited steatosis and numerous fat vacuoles. Total saponins in the air-dried group significantly reduced lipid infiltration in the liver, decreased the number of lipid droplets, and reduced the size of fat vacuoles. Compared with the model group, hepatocytes were more uniform in size and more neatly arranged, while the improvement effect of total saponins in the roller-dried group was weaker.

[0243] 3.2.5 Effects of Gynostemma pentaphyllum processed in different ways on adipose tissue of NAFLD mice

[0244] Experimental results are as follows Figure 12 As shown, the saponins in the air-dried group significantly reduced the weight of epididymal and inguinal fat in NAFLD mice, while the roller had no significant effect on the weight of adipose tissue in NAFLD mice, and neither had a significant effect on the weight of brown fat.

[0245] 3.2.6 Effects of different processing methods on FGF15 levels in the serum of Gynostemma pentaphyllum mice

[0246] Experimental results are as follows Figure 13 As shown, by measuring the serum FGF15 content, it was found that the serum FGF15 level of NAFLD mice was significantly increased after intervention with air-dried Gynostemma pentaphyllum saponins.

[0247] 3.2.7 Effects of different processing methods on lipid metabolism in Gynostemma pentaphyllum mice

[0248] Experimental results are as follows Figure 14 As shown, the saponins of Gynostemma pentaphyllum in the shade-dried group increased the expression of hormone-sensitive lipase (HSL), a key protein in lipolysis, and increased the expression of fibroblast growth factor receptor FGFR1, the receptor protein of FGF15 in adipose tissue.

[0249] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for promoting the conversion of rare ginsenosides in Gynostemma pentaphyllum, characterized in that, The method includes the following steps: (1) Take an appropriate amount of dried Gynostemma pentaphyllum, crush it, and then soak it in water; (2) Heat at a certain water bath temperature for a period of time; (3) After evaporating the water, add a certain concentration of ethanol or methanol, heat and reflux for extraction or ultrasonic extraction, cool the extract, filter, and concentrate under reduced pressure to obtain the concentrate; and (4) The concentrate is loaded onto a macroporous resin for adsorption and elution. The eluted fraction with 60-80% ethanol is collected to obtain total saponins that are dried in the shade.

2. The method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio (g / L) of the dried Gynostemma pentaphyllum to the water is 20:1 to 100:1; Preferably, the mass-to-volume ratio (g / L) of the dried Gynostemma pentaphyllum to the water is (47.5~52.5):1; More preferably, the water is deionized water; Preferably, the water is distilled water.

3. The method according to claim 1, characterized in that, In step (2), the temperature of the water bath is 30℃~70℃; Preferably, the temperature of the water bath is 35℃~50℃; Preferably, the temperature of the water bath is 40℃~50℃; More preferably, the temperature of the water bath is 35°C to 45°C; Preferably, the temperature of the water bath is 37.5℃~42.5℃; Particularly preferably, the water bath time is 1 to 12 hours; Particularly preferably, the water bath time is 2 to 4 hours; Most preferably, the water bath time is 2.85 to 3.15 hours.

4. The method according to claim 1, characterized in that, In step (3), the concentration of the ethanol or the methanol is 60% to 80%; Preferably, the concentration of the ethanol or the methanol is 66.5% to 73.5%; More preferably, in step (3), the volume ratio of the concentrate to the extract is 0.02:1 to 0.1:1; Preferably, in step (3), the volume ratio of the concentrate to the extract is (0.057~0.063):

1.

5. The method according to claim 1, characterized in that, In step (3), the number of times the heating reflux extraction or the ultrasonic extraction is performed is 1 to 4; Preferably, when the number of heating reflux extraction or ultrasonic extraction is 2 to 4 times, step (3) further includes the step of merging the filtered extracts; Preferably, the heating reflux extraction or the ultrasonic extraction is performed twice; More preferably, the time for the heating reflux extraction or the ultrasonic extraction is 0.5 to 2 hours; Particularly preferred is that the heating reflux extraction or the ultrasonic extraction time is 0.95~1.05 hours; Particularly preferably, in step (3), the mass-to-volume ratio (g / L) of the dried Gynostemma pentaphyllum to the methanol or the ethanol is 20:1 to 100:1; Most preferably, in step (3), the mass-to-volume ratio (g / L) of the dried Gynostemma pentaphyllum to the methanol or the ethanol is (38~42):

1.

6. The method according to claim 1, characterized in that, In step (4), the macroporous resin is D101 resin; Preferably, the adsorption time of the concentrate on the macroporous resin is 20-28 hours; More preferably, the adsorption time of the concentrate on the macroporous resin is 22.8 to 25.2 hours; More preferably, the volume-to-mass ratio (mL / g) of the concentrate to the macroporous resin is 0.15:

1.

7. The method according to claim 1, characterized in that, In step (4), the elution includes elution with water and 20% to 40% ethanol to remove impurities, and elution with 60% to 80% ethanol; Preferably, the elution includes eluting with water and 28.5%~31.5% ethanol to remove impurities, and eluting with 66.5%~73.5% ethanol.

8. A preparation product obtained by the method of any one of claims 1 to 7; Preferably, the saponin components in the prepared product include 0% to 10% of 3 to 4 glycosyl-substituted gypenosides; More preferably, the saponin components in the prepared product comprise 90% to 100% of secondary saponins with 1 to 2 glycosyl substitutions; Preferably, the secondary saponins with 1-2 glycosyl substitutions are gypenosides LXXVII, gypenosides TN-1, gypenosides XIII, and / or gypenosides CK or their isomers. Particularly preferred, the 1-2 glycosyl-substituted secondary saponins are gypenosides LXXVII, gypenosides TN-1, gypenosides XIII and / or gypenosides CK.

9. A composition comprising the preparation product of claim 8, characterized in that, The composition further comprises one or more substances for the prevention and / or treatment of metabolic diseases, and / or pharmaceutically acceptable excipients.

10. Use of the product prepared according to claim 8 or the composition according to claim 9 in pharmaceuticals, foods and / or health products for the prevention and / or treatment of metabolic diseases; Preferably, the metabolic disease is metabolic syndrome; Preferably, the metabolic disease is glucose and lipid metabolism syndrome; Preferably, the prepared product or the composition has the effect of preventing and / or treating metabolic diseases through one or more of the following mechanisms: reducing body weight, increasing total cholesterol excretion, increasing triglyceride excretion, improving glucose metabolism, reducing hepatic lipid accumulation, reducing adipose tissue weight, increasing serum FGF15 levels, and increasing the expression of lipid breakdown-related proteins. More preferably, the improvement in glucose metabolism includes regulating glucose tolerance, reducing fasting blood glucose, and / or improving insulin resistance; More preferably, the reduction of hepatic lipid accumulation includes a decrease in the number of hepatic lipid droplets and / or a reduction in hepatic fat vacuoles; More preferably, the adipose tissue includes epididymal adipose tissue and / or inguinal adipose tissue; More preferably, the lipidolysis-related protein includes hormone-sensitive lipase and / or fibroblast growth factor receptor 1; More preferably, the metabolic disease is obesity, diabetes, and / or non-alcoholic fatty liver disease.

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