Preparation method of gynostemma pentaphyllum secondary saponin with hypoglycemic activity

Abstract

The invention discloses a preparation method of gynostemma pentaphyllum secondary saponins with hypoglycemic activity, and relates to the technical field of preparation of gynostemma pentaphyllum secondary saponins, the preparation method comprises the following steps: mixing gynostemma pentaphyllum total saponins with an ethanol aqueous solution, and preparing a secondary saponin product with hypoglycemic activity by controlling hydrolysis conditions, according to the preparation method of the gynostemma pentaphyllum secondary saponin with the hypoglycemic activity, the hypoglycemic activity of the gynostemma pentaphyllum secondary saponin is remarkably improved by accurately controlling key conditions such as the concentration of the ethanol aqueous solution, the solid-liquid ratio, the hydrolysis temperature and time, the preparation process is simple, convenient, efficient and environmentally friendly, the product purity is high, and the stability is good; meanwhile, the effective components of the product and the good combining capacity of the effective components and diabetes treatment targets are verified through component analysis and molecular docking, scientific basis and technical support are provided for developing novel hypoglycemic drugs or health care products, and the application prospect and market potential are wide.

Description

Technical Field

[0001] This invention relates to the field of preparation technology of Gynostemma pentaphyllum secondary saponins, specifically a method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity. Background Art

[0002] Gynostemma pentaphyllum (Thunb.) Makino. is a perennial herbaceous vine belonging to the genus Gynostemma of the Cucurbitaceae family. It is an important plant resource in my country, with its history dating back to the Spring and Autumn and Warring States periods. In 1986, it was listed as one of the precious Chinese medicinal materials to be developed, and in 2002, it was included in the list of health products. Because it is rich in ginsenosides, it is also known as "Southern Ginseng". Gynostemma pentaphyllum prefers a warm, humid, and shady ecological environment, and its natural communities are mainly distributed in a belt along the East Asian monsoon belt.

[0003] Extraction methods for Gynostemma pentaphyllum saponins include distilled water extraction, organic solvent extraction, enzymatic extraction, ultrasonic-assisted extraction, and microwave-assisted extraction. Gynostemma pentaphyllum saponins are easily degraded by acids, alkalis, and enzymes. The main hydrolysis methods include acid hydrolysis, alkali hydrolysis, and enzymatic hydrolysis. After the glycosidic bonds of saponins are hydrolyzed, not only are sugars removed to generate secondary glycosides, but the structure of the aglycone also changes. Ultimately, they are metabolized to generate secondary saponins and aglycones that are less polar, easier to absorb, and have stronger biological activity.

[0004] Acid hydrolysis using strong acids may damage the aglycone structure of saponins, leading to loss of active ingredients, numerous side reactions, and complex product impurities. Alkaline hydrolysis is effective for saponins containing ester bonds or specific glycosidic bonds, but less effective for most Gynostemma pentaphyllum saponins (mainly oxyglycosidic bonds). Strong alkalis may also cause isomerization or degradation of the aglycone (such as saponin ring cleavage). Enzymatic hydrolysis uses expensive enzyme preparations and requires strict control of reaction conditions (such as temperature, pH, ionic strength, etc.). In contrast, the hot ethanol hydrolysis method for total Gynostemma pentaphyllum saponins uses ethanol as a solvent to dissolve saponins while the high temperature promotes the hydrolysis of glycosidic bonds. Compared with traditional acid hydrolysis, hot ethanol hydrolysis can shorten the reaction time and improve efficiency. Ethanol is also more efficient than other methods. The reagents are safer and can be recycled and reused through distillation, reducing production costs and waste discharge, which aligns with green chemistry principles. Ethanol is slightly acidified at high temperatures, providing weakly acidic conditions for hydrolyzing glycosidic bonds. Compared to strong acids, it is less likely to damage the aglycone structure (e.g., oxidation, dehydration). The weakly acidic conditions inhibit caramelization of sugars and saponin ring cleavage, resulting in fewer product impurities and less pressure for subsequent purification. Ethanol (moderately polar) has high solubility for saponins and their hydrolysis products, while inhibiting the dissolution of large molecular impurities such as polysaccharides and proteins, thus improving product purity. The reaction vessel provides a stable heating environment, avoiding local overheating that could lead to saponin degradation, while also reducing solvent evaporation losses, preventing external contamination, and ensuring product purity.

[0005] To expand the sources of hypoglycemic active substances from Gynostemma pentaphyllum, the total saponins of Gynostemma pentaphyllum were hydrolyzed using hot water. Hydrolysis time, hydrolysis temperature, ethanol concentration, and solid-liquid ratio were investigated as factors, and α-glucosidase inhibition rate was used as the hypoglycemic activity index. Based on single-factor experiments, response surface methodology was employed to optimize the hot water hydrolysis process of total saponins from Gynostemma pentaphyllum. Experimental results showed that the optimal hot water hydrolysis conditions were a hydrolysis time of 8.1 h, a hydrolysis temperature of 179.5 °C, a solid-liquid ratio of 1.0 mg / mL, and an ethanol concentration of 20%. Under these conditions, the hydrolysate of total saponins from Gynostemma pentaphyllum exhibited an α-glucosidase inhibition rate of 83.02%. This process is simple to operate and can provide a reference for the screening and further development and utilization of hypoglycemic active substances from Gynostemma pentaphyllum. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing secondary saponins of Gynostemma pentaphyllum with hypoglycemic activity, the preparation method comprising mixing total saponins of Gynostemma pentaphyllum with an aqueous ethanol solution, and preparing secondary saponin products with hypoglycemic activity by controlling hydrolysis conditions.

[0008] Preferably, the concentration of the ethanol-water solution is precisely controlled at 20% (v / v) as a solvent for the hydrolysis reaction to promote the conversion of total saponins from Gynostemma pentaphyllum.

[0009] Preferably, the ratio of total saponins from Gynostemma pentaphyllum to the aqueous ethanol solution is set to 1.0 mg / mL to ensure sufficient contact between the reactants and high hydrolysis efficiency.

[0010] Preferably, the hydrolysis reaction is carried out in a hot water reactor, and the reaction temperature is controlled by an electrically heated drying oven to ensure the stability of temperature and pressure during the reaction.

[0011] Preferably, the temperature of the hydrolysis reaction is precisely controlled at 179.5°C to accelerate the hydrolysis reaction and optimize the generation of secondary saponins.

[0012] Preferably, the hydrolysis reaction time is set to 8.1 hours to ensure that the total saponins of Gynostemma pentaphyllum are fully hydrolyzed and converted into secondary saponins.

[0013] Preferably, after the hydrolysis reaction is completed, the reaction solution is cooled, and then most of the solvent is removed by a concentration step to obtain a concentrated product.

[0014] Preferably, the concentrated product is diluted to a final volume using 70% ethanol.

[0015] Preferably, the preparation method further includes drying the obtained Gynostemma pentaphyllum secondary saponins to obtain the product in solid powder form.

[0016] Preferably, the total saponin content of the Gynostemma pentaphyllum is 65.13%.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention significantly improves the hypoglycemic activity of Gynostemma pentaphyllum secondary saponins by precisely controlling key conditions such as the concentration of ethanol-water solution, the material-to-liquid ratio, hydrolysis temperature, and time. The preparation process is simple, efficient, and environmentally friendly, and the product has high purity and good stability. Furthermore, component analysis and molecular docking have verified the effective components of the product and their good binding ability with diabetes treatment targets. This provides a scientific basis and technical support for the development of novel hypoglycemic drugs or health products, and has broad application prospects and market potential. Attached Figure Description

[0019] Figure 1 This is a linear graph showing the effect of ethanol concentration on the inhibition rate of α-glucosidase according to the present invention.

[0020] Figure 2 This is a linear graph showing the effect of the feed-to-liquid ratio on the α-glucosidase inhibition rate of the present invention.

[0021] Figure 3 This is a linear graph showing the effect of hydrolysis temperature on the inhibition rate of α-glucosidase in this invention.

[0022] Figure 4 This is a linear graph showing the effect of hydrolysis time on the inhibition rate of α-glucosidase in this invention.

[0023] Figure 5 This is the base peak chromatogram of the chemical components (positive ions) of Gynostemma pentaphyllum saponins obtained by hydrolysis in this invention;

[0024] Figure 6 This is the base peak chromatogram of the chemical components (negative ions) of Gynostemma pentaphyllum saponins obtained by hydrolysis in this invention. Detailed Implementation

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] Example 1: Please refer to Figures 1 to 6The present invention provides a technical solution: a method for preparing secondary saponins of Gynostemma pentaphyllum with hypoglycemic activity, the preparation method comprising mixing total saponins of Gynostemma pentaphyllum with an aqueous ethanol solution, and preparing secondary saponin products with hypoglycemic activity by controlling hydrolysis conditions.

[0027] The concentration of the ethanol-water solution was precisely controlled at 20% (v / v) as a solvent for the hydrolysis reaction to promote the conversion of total saponins from Gynostemma pentaphyllum.

[0028] The ratio of total saponins from Gynostemma pentaphyllum to ethanol aqueous solution was set at 1.0 mg / mL to ensure sufficient contact between reactants and high hydrolysis efficiency.

[0029] The hydrolysis reaction is carried out in a hot water reactor, and the reaction temperature is controlled by an electrically heated drying oven to ensure the stability of temperature and pressure during the reaction.

[0030] The hydrolysis reaction temperature was precisely controlled at 179.5℃ to accelerate the hydrolysis reaction and optimize the formation of secondary saponins.

[0031] The hydrolysis reaction time was set at 8.1 hours to ensure that the total saponins of Gynostemma pentaphyllum were fully hydrolyzed and converted into secondary saponins.

[0032] After the hydrolysis reaction is completed, the reaction solution is cooled, and then most of the solvent is removed by a concentration step to obtain the concentrated product.

[0033] The concentrated product was brought to a final volume using 70% ethanol.

[0034] The preparation method also includes drying the obtained Gynostemma pentaphyllum secondary saponins to obtain the product in solid powder form.

[0035] The total saponin content of Gynostemma pentaphyllum is 65.13%.

[0036] The specific implementation method of this embodiment is as follows:

[0037] 40.0 mg of total saponins from Gynostemma pentaphyllum were weighed into a hydrothermal reactor. A certain volume and concentration of ethanol-water solution were added, and the mixture was hydrolyzed in an electric heating drying oven at a certain temperature for a certain time. The hydrolysis product was concentrated and diluted to 50 mL with 70% ethanol as a sample solution. 50 μL of the sample solution was taken, and 50 μL of α-glucosidase solution (0.2 U / mL) was added. The mixture was kept at 37℃ for 10 min, and then 50 μL of 1.0 mmol / mL 4-nitrophenyl-β-D-glucopyranoside (PNPG) solution was added. The mixture was kept at 37℃ for 30 min, and then 50 μL of 0.5 mol / L Na2CO3 solution was added to terminate the reaction. The absorbance (As) was measured at 405 nm. 50 μL of phosphate buffer (0.0125 mol / L) was used as the sample control (Ab) instead of α-glucosidase, and 70% ethanol solution was used as the blank control (Ac) instead of the sample solution. The formula for calculating the inhibition rate of the sample against α-glucosidase is as follows:

[0038]

[0039] Single-factor experiment:

[0040] (1) Effect of ethanol concentration:

[0041] With fixed material-to-liquid ratio, hydrolysis temperature, and hydrolysis time, hot water hydrolysis experiments of total saponins from Gynostemma pentaphyllum were conducted by adjusting the ethanol concentration. After treatment according to the above experimental method, the inhibition rate of α-glucosidase by the hot water hydrolysis products of total saponins from Gynostemma pentaphyllum at different ethanol concentrations was measured using an enzyme-linked immunosorbent assay (ELISA) analyzer. The effect of ethanol concentration on the inhibition rate was investigated, as shown in the table below:

[0042] Single-factor (ethanol concentration) experimental level table

[0043]

[0044] (2) Effect of feed-to-liquid ratio:

[0045] With fixed ethanol concentration, hydrolysis temperature, and hydrolysis time, hot water hydrolysis experiments of total saponins from Gynostemma pentaphyllum were conducted by adjusting the solid-liquid ratio. After treatment according to the above experimental method, the inhibition rate of α-glucosidase by the hot water hydrolysis products of total saponins from Gynostemma pentaphyllum with different solid-liquid ratios was measured using an enzyme-linked immunosorbent assay (ELISA) analyzer. The effect of solid-liquid ratio on the inhibition rate was investigated, as shown in the table below:

[0046] Single-factor (solid-to-liquid ratio) experimental level table

[0047]

[0048]

[0049] (3) Effect of hydrolysis temperature:

[0050] With fixed material-to-liquid ratio, ethanol concentration, and hydrolysis time, hot hydrolysis experiments of total saponins from Gynostemma pentaphyllum were conducted by adjusting the hydrolysis temperature. After treatment according to the above experimental method, the inhibition rate of α-glucosidase by the hot hydrolysis products of total saponins from Gynostemma pentaphyllum at different hydrolysis temperatures was measured using an enzyme-linked immunosorbent assay (ELISA) analyzer. The effect of hydrolysis temperature on the inhibition rate was investigated, as shown in the table below:

[0051] Single-factor (hydrolysis temperature) experimental level table

[0052]

[0053] (4) Effect of hydrolysis time:

[0054] With fixed material-to-liquid ratio, ethanol concentration, and hydrolysis temperature, hot water hydrolysis experiments were conducted on total saponins from Gynostemma pentaphyllum by adjusting the hydrolysis time. After treatment according to the above experimental method, the inhibition rate of α-glucosidase by the hydrolysates of total saponins from Gynostemma pentaphyllum at different hydrolysis times was determined using an enzyme-linked immunosorbent assay (ELISA) analyzer. The effect of hydrolysis time on the inhibition rate was investigated, as shown in the table below:

[0055] Single-factor (hydrolysis time) experimental level table

[0056]

[0057] The experimental scheme was optimized using response surface methodology software (Design-Expert Version 8.0.6). Based on the single-factor experiments, response surface experiments were designed to optimize each factor, as shown in the table below. Finally, the optimal hot water hydrolysis conditions for total saponins from Gynostemma pentaphyllum were determined.

[0058] Response Surface Design Level Table

[0059]

[0060] Results of the single-factor experiment:

[0061] like Figure 1 As shown, when the ethanol concentration is in the range of 20% to 40%, the inhibition rate gradually increases. The higher the ethanol concentration, the greater the hydrolytic ability of the total saponins of Gynostemma pentaphyllum. When the ethanol concentration is 40%, the inhibition rate reaches its maximum value. When the ethanol concentration is greater than 40%, the inhibition rate shows a decreasing trend. Although the ethanol concentration is positively correlated with the hydrolytic ability, under higher temperature conditions, the solvent has a greater destructive force on the aglycone structure of the total saponins of Gynostemma pentaphyllum, resulting in the loss of active ingredients. Therefore, ethanol concentrations of 20%, 40%, and 60% were selected as the factor levels for response surface methodology optimization.

[0062] like Figure 2As shown, the inhibition rate gradually increases when the solid-liquid ratio is in the range of 0.6–0.8 mg / mL. The inhibition rate reaches its maximum when the solid-liquid ratio is 0.8 mg / mL. When the solid-liquid ratio is greater than 0.8 mg / mL, the inhibition rate shows a decreasing trend. Increasing the solid-liquid ratio is equivalent to increasing the contact area between the total saponins of Gynostemma pentaphyllum and the solvent, thus increasing the amount of active ingredients in the hydrolysate of total saponins of Gynostemma pentaphyllum. Therefore, solid-liquid ratios of 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL were selected as the factor levels for optimization in the response surface methodology experiment.

[0063] like Figure 3 As shown, when the hydrolysis temperature is in the range of 120 to 180℃, the inhibition rate gradually increases with the increase of the hydrolysis temperature. When the hydrolysis temperature is 180℃, the inhibition rate reaches its maximum value. When the hydrolysis temperature is greater than 180℃, the inhibition rate shows a decreasing trend. Ethanol provides weak acidic conditions at high temperature to hydrolyze glycosidic bonds, has high hydrolysis ability, and inhibits the dissolution of large molecular impurities such as polysaccharides and proteins. The hydrolysis products have high purity. Therefore, hydrolysis temperatures of 160℃, 180℃, and 200℃ were selected as the factor level values ​​for response surface methodology optimization.

[0064] like Figure 4 As shown, when the hydrolysis time is in the range of 5 to 8 hours, the inhibition rate gradually increases with the increase of hydrolysis time. When the hydrolysis time is 8 hours, the inhibition rate reaches the maximum value. When the hydrolysis time is in the range of 8 to 11 hours, the inhibition rate first decreases and then increases with the increase of hydrolysis time. This may be because after the hydrolysis time exceeds 9 hours, the total saponins of Gynostemma pentaphyllum are hydrolyzed into secondary glycosides and then further hydrolyzed into highly active aglycones. Therefore, hydrolysis times of 7 hours, 8 hours, and 9 hours were selected as the factor level values ​​for response surface methodology optimization.

[0065] The experimental data were processed and analyzed using Design-Expert Version 8.0.6 software. Ethanol concentration, solid-liquid ratio, hydrolysis time, and hydrolysis temperature were selected as the main influencing factors. Three levels were chosen for each of these four factors. The response surface methodology and results for the hot water hydrolysis of total saponins from Gynostemma pentaphyllum are shown in the table below, using α-glucosidase inhibition rate as the indicator.

[0066] Response surface experimental design and results

[0067]

[0068]

[0069] Based on the results of single-factor experiments, a Box-Behnken Design experiment was conducted with hydrolysis time (7h, 8h, 9h), hydrolysis temperature (160℃, 180℃, 200℃), ethanol concentration (20%, 40%, 60%), and solid-liquid ratio (0.6mg / mL, 0.8mg / mL, 1.0mg / mL) as levels. The quadratic polynomial regression equation for the α-glucosidase inhibition rate Y against A (solid-liquid ratio), B (ethanol concentration), C (hydrolysis time), and D (hydrolysis temperature) was obtained.

[0070] Y=76.44+2.04*A-2.46*B+0.64*C+1.36*D-2.90*A*B+1.05*A*C+2.28*A* D+0.60*B*C+4.43*B*D+1.08*C*D-1.23*A2-2.38*B2-3.95*C2-13.48*D2

[0071] The optimal process conditions were finally obtained: hydrolysis time 8.1 h, hydrolysis temperature 179.5 °C, feed-to-liquid ratio 1.0 mg / mL, and ethanol concentration 20.0%. Under these conditions, the inhibition rate reached 83.02% in three repeated experiments.

[0072] Composition analysis:

[0073] (1) Sample preparation

[0074] Accurately weigh 40 mg of gypenosides into a 100 mL reactor, add 40 mL of 20% ethanol aqueous solution, and hydrolyze at 179.5 °C for 8.1 h. Concentrate the solution and freeze-dry to obtain the hydrolyzed product of gypenosides. Take 2 mg of the product and add 500 μL of anhydrous methanol, sonicate to dissolve, centrifuge at 13000 rpm for 10 min, and collect the supernatant for analysis (LCMS).

[0075] (2) Chromatographic conditions

[0076] The UltiMate 3000U HPLC was used as the liquid chromatography instrument, and a Thremo Hypersil gold C18 (1.9 μm, 2.1 mm × 100 mm) column was used as the chromatographic column. The flow rate was set to 0.3 mL / min, the injection volume to 15 μL, and the mobile phase to be 0.1% formic acid / acetonitrile (B) – 0.1% formic acid / water (A). The gradient elution program is shown in the table below.

[0077] mobile phase gradient elution program

[0078]

[0079]

[0080] (3) Mass spectrometry conditions

[0081] Take the hydrolysate of Gynostemma pentaphyllum saponins as a test sample and use a super-resolution liquid chromatography-mass spectrometry (QExactiveHF-X) instrument to collect the primary and secondary spectra of the test sample;

[0082] The mass spectrometer used a Q-Exactive (ThermoFisher Scientific, CA, USA) HESI source with an ion source temperature of 310℃ and a capillary temperature of 320℃. The sheath gas flow rate was 30 units, and the auxiliary gas flow rate was 10 units. The spray voltages for positive and negative ion modes were 3 kV and 2.8 kV, respectively. Data-dependent scan analysis (DDA) was used, with a loop count set to 10 and HCD energies set to step-wise normalized collision energies of 10, 28, and 35 eV. The primary mass spectrometer had a scan range of 100–1500 m / z, with resolution, AGC target, and injection time set to 70,000, 3E6, and 200 ms, respectively. The secondary mass spectrometer had resolution, AGC target, and injection time set to 17,500, 1E5, and 50 ms, respectively.

[0083] (4) Data processing

[0084] The above LCMS data were automatically searched using Compounddiscover software (V3.2, ThermoFisherScientific, CA, USA) to identify potential chemical components, including the Chemspider plant database (CHEBI, CHEMBL), natural product databases, OTC databases, and mzcloud database. Components with high matching scores or high content were retained.

[0085] (5) Identification and preparation of treatment targets for diabetes

[0086] Alpha-glucosidase (AG) is mainly located in the pancreas of the small intestine in the human body. It can hydrolyze carbohydrates such as starch and sucrose into monosaccharides such as glucose, which are then absorbed by the intestine and raise blood sugar. It is a key target enzyme for the treatment of diabetes and obesity.

[0087] Dipeptidyl peptidase-4 (DPP-4) is a serine protease widely expressed in various tissues (such as the pancreas, liver, intestines, and immune system). It is an important enzyme in glucose metabolism and is widely expressed in most tissues and cells. DPP-IV rapidly degrades glucagon-like peptide-1 (GLP-1) in vivo, thereby affecting insulin secretion and glucose metabolism.

[0088] Glycogen synthase kinase-3 (GSK-3) is a serine / threonine protein kinase. The level of GSK-3β in patients with diabetic nephropathy can affect their urinary microalbumin, blood glucose, lipid metabolism, etc. Therefore, serum GSK-3β level can be used as an important and sensitive indicator in the clinical treatment of diabetic nephropathy.

[0089] Sodium-glucose cotransporter 2 (SGLT2) is a transmembrane protein located on the brush border of epithelial cells in the S1-S2 segment of the proximal tubule of the kidney. It reduces glucose reabsorption by inhibiting sodium-glucose cotransporter (SGLT) in the S1 and S2 segments of the proximal tubule, thereby lowering blood glucose levels.

[0090] Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and belong to ligand-activated transcription factors. After PPARγ activation, it promotes adipocyte differentiation, increases the storage capacity of adipose tissue, reduces the lipotoxicity of free fatty acids to muscle and liver, and improves insulin resistance.

[0091] Angiotensin-converting enzyme (ACE) is a zinc-containing membrane-bound glycoprotein. Under pathological conditions, ACE / Ang II (angiotensin II) can induce oxidative stress, protein glycosylation, inflammatory response, vasoconstriction, and increased insulin resistance in diabetes.

[0092] Glucokinase (GK) is one of the key enzymes regulating blood glucose in the human body. It is mainly distributed in tissues such as the liver, pancreatic β cells, intestinal endocrine cells and hypothalamus. It plays a core role in the regulation of blood glucose homeostasis. In the pancreas, GK can directly and indirectly promote the secretion of insulin by β cells by promoting Ca2+ influx, repairing Ca2+ response defects, reducing β cell oxygen consumption and promoting proliferation.

[0093] Therefore, these seven targets were selected as the research objects for this molecular docking. The crystals of the seven targets were imported into Sybyl software for processing to determine the binding sites, which served as the basis for subsequent molecular docking.

[0094] (6) Molecular docking

[0095] Molecular docking was performed on the core target of the hydrolyzed Gynostemma pentaphyllum saponins obtained by UHPLC-QExactiveOrbitrapHRMS technology. The 3D structure of the core target protein in PDB format was obtained from the PDB database (https: / / www.rcsb.org / ), and the active component in mol2 format was obtained from the PubChem database. Preprocessing of the protein target receptor was performed using PyMOL software, including removing the proligand, removing water of crystallization, removing lone pairs, and re-adding all polar / nonpolar hydrogen atoms. AutoDock version 4.2.6 was used for molecular docking. Generally, lower affinity indicates a more stable binding conformation, while higher affinity indicates a less stable binding conformation. Finally, PyMOL software was used for visualization to clearly demonstrate the effect of molecular docking.

[0096] (7) Molecular docking results

[0097] Sixteen key active ingredients of Gynostemma pentaphyllum saponins were selected and molecularly docked with seven key targets for the treatment of diabetes: alphaglucosidaseare (PDBID: 1LF9), GSK-3 (PDBID: 1O6L), SGLT2 (PDBID: 3DH4), PPARγ (PDBID: 3U9Q), DPP-IV (PDBID: 4A5S), GK (PDBID: 4BB9), and ACE (PDBID: 4BZR). The docking results are shown in the table below:

[0098] docking results

[0099]

[0100]

[0101] Generally, the results of molecular docking are mainly judged based on the docking binding energy. When the docking binding energy is less than -1.2 kcal / mol, it proves that there is an affinity between the two molecules; when the docking binding energy is less than -5 kcal / mol, it proves that the affinity between the two molecules is relatively good. In summary, when the docking binding energy is less than -5 kcal / mol, the docking result is good.

[0102] Among the seven diabetes targets that docked with Gynostemma pentaphyllum saponin A, the best docking results were observed for sodium-glucose cotransporter 2, peroxidase proliferation factor activating receptor γ, and dipeptidyl peptidase-IV, with binding energies of -7.08 kcal / mol, -5.02 kcal / mol, and -6.78 kcal / mol, respectively.

[0103] Among the seven diabetes targets that docked with ginsenoside compound K, α-glucosidase showed the best docking result, with a binding energy of -5.4 kcal / mol.

[0104] Among the seven diabetes targets that were docked with ginsenoside Rg6, dipeptidyl peptidase-IV showed the best docking result, with a docking result of -5.06 kcal / mol.

[0105] Among the seven diabetes targets that were docked with ginsenoside Rg5, dipeptidyl peptidase-IV showed the best docking result, with a docking result of -5.61 kcal / mol.

[0106] Among the seven diabetes targets that were docked with ginsenoside Rh1, the docking results were better for glycogen synthase kinase-3, sodium-glucose cotransporter 2, peroxidase proliferation factor activating receptor γ, and dipeptidyl peptidase-IV, with docking results of -6.95 kcal / mol, -6.01 kcal / mol, -6.1 kcal / mol, and -5.39 kcal / mol, respectively.

[0107] Among the 16 Gynostemma pentaphyllum saponins listed above, Gynostemma pentaphyllum saponin A and ginsenoside Rh1 have binding energies of less than -5 kcal / mol to three or more diabetes targets, indicating good binding results and suggesting that they may exert their hypoglycemic effects through these targets.

[0108] (8) Analysis Results

[0109] The prepared test solution was analyzed using UHPLC-Q Exactive Orbitrap HRMS under the chromatographic conditions described in 2.1.2 and the mass spectrometric conditions described in 2.1.3. Qualitative analysis of the hydrolyzed chemical components of Gynostemma pentaphyllum saponins was performed. Base peak chromatograms of the test sample were acquired in both positive ion (POS) and negative ion (NEG) modes. Figure 5 and Figure 6 As shown;

[0110] A total of 158 chemical components were obtained from the hydrolysis of Gynostemma pentaphyllum saponins, including 61 positive ions and 97 negative ions. The main components were saponins, organic acids, flavonoids, alkaloids, etc. 14 saponin components were identified, including 11 ginsenosides, 1 Gynostemma pentaphyllum saponin, and 2 bupleurum saponins. The saponins with a peak area percentage of more than 1% were ginsenoside Rg5, ginsenoside Rh1, ginsenoside compound K, ginsenoside F2, ginsenoside F1, and ginsenoside Rg3. The hydrolysis products of Gynostemma pentaphyllum saponins contained a variety of trace ginsenosides, such as ginsenoside Rk1, ginsenoside Rg5 and ginsenoside Rg6

[37] . Although the content of trace saponins was low, they played an important role in pharmacological activity. Ginsenoside F2 had a hypoglycemic effect, and ginsenoside Rg1 could reduce glucose output to alleviate insulin resistance. At the same time, it could also play a therapeutic role in hyperphosphorylation-induced diabetic retinopathy.

[0111] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0112] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity, characterized in that: The preparation method includes mixing total saponins from Gynostemma pentaphyllum with an aqueous ethanol solution, and preparing secondary saponin products with hypoglycemic activity by controlling the hydrolysis conditions.

2. The method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity according to claim 1, characterized in that: The concentration of the ethanol-water solution is precisely controlled at 20% (v / v) as a solvent for the hydrolysis reaction to promote the conversion of total saponins from Gynostemma pentaphyllum.

3. The method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity according to claim 1, characterized in that: The ratio of total saponins from Gynostemma pentaphyllum to the aqueous ethanol solution was set at 1.0 mg / mL to ensure sufficient contact between the reactants and high hydrolysis efficiency.

4. The method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity according to claim 1, characterized in that: The hydrolysis reaction is carried out in a hot water reactor, and the reaction temperature is controlled by an electrically heated drying oven to ensure the stability of temperature and pressure during the reaction.

5. The method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity according to claim 1, characterized in that: The temperature of the hydrolysis reaction is precisely controlled at 179.5℃ to accelerate the hydrolysis reaction and optimize the generation of secondary saponins.

6. The method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity according to claim 1, characterized in that: The hydrolysis reaction time was set at 8.1 hours to ensure that the total saponins of Gynostemma pentaphyllum were fully hydrolyzed and converted into secondary saponins.

7. The method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity according to claim 1, characterized in that: After the hydrolysis reaction is completed, the reaction solution is cooled, and then most of the solvent is removed by a concentration step to obtain a concentrated product.

8. The method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity according to claim 7, characterized in that: The concentrated product was diluted to a final volume using 70% ethanol.

9. The method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity according to claim 1, characterized in that: The preparation method further includes drying the obtained Gynostemma pentaphyllum secondary saponins to obtain the product in solid powder form.

10. The method for preparing Gynostemma pentaphyllum secondary saponins with hypoglycemic activity according to claim 1, characterized in that: The total saponin content of the Gynostemma pentaphyllum is 65.13%.

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