Microwave-assisted method for preparing rare ginsenosides F4 and Rk1, composition and application

Abstract

The invention discloses a microwave-assisted method for preparing rare ginsenosides F4 and Rk1, a composition and application, the method utilizes the synergistic effect of microwave reaction and cooling crystallization, adopts a one-pot method to prepare high-purity ginsenosides, saves energy, reduces the use of chemical reagents, is green and environment-friendly, adopts simple and easy-to-control equipment, is beneficial to industrialization, and is suitable for industrial production. The purity of the prepared rare ginsenosides F4 and Rk1 is greater than or equal to 95% through HPLC (High Performance Liquid Chromatography) detection. The rare ginsenoside F4 / Rk1 composition can obviously reduce lipid deposition, inflammatory cell infiltration and formation of fat vacuoles in liver tissue, so that the pathological state of the liver tissue is obviously improved, and the composition has the effect of effectively improving and / or preventing and treating the non-alcoholic fatty liver disease and has obvious clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a method for preparing rare ginsenoside F4 and Rk1 assisted by microwave, a composition and an application thereof. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease characterized by abnormal accumulation of fat in the liver without significant alcohol intake. The incidence of NAFLD is on the rise globally, especially among patients with obesity, diabetes and metabolic syndrome. The pathological process of NAFLD includes simple fatty liver, non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis and liver cancer. Although the incidence of NAFLD is high, there is currently no specific drug available for the treatment of NAFLD, and the existing treatment methods mainly focus on lifestyle intervention and management of metabolic syndrome.

[0003] Ginsenosides are the main bioactive components of plants in the genus Panax of the Araliaceae family (such as Panax ginseng, Panax quinquefolius, Panax notoginseng, etc.), and their chemical structure belongs to tetracyclic triterpenoid compounds. According to the difference in the number of hydroxyl groups on the aglycone nucleus, ginsenosides can be divided into two major categories: the diol group (such as Rb1, Rb2, Rc, etc.) and the triol group (such as Rg1, Re, Rf, etc.). Modern pharmacological studies have confirmed that ginsenosides have a wide range of biological activities, including anti-tumor, lipid-lowering, anti-inflammatory, antioxidant, neuroprotection (such as enhancing memory and preventing Alzheimer's disease) and other pharmacological effects. In recent years, it has been found that specific types of ginsenosides (such as CK, Rh4) and their compositions show potential therapeutic value in improving non-alcoholic hepatic fibrosis and insulin resistance.

[0004] The preparation of ginsenosides mainly uses pyrolysis method, metal ion catalytic conversion method and degradation method. However, the pyrolysis method requires high temperature and long reaction time, with high energy consumption. High temperature is likely to cause excessive decomposition of the target product or side reactions, affecting purity and yield. When preparing Rk1, complex degradation products will be generated, increasing the separation difficulty. Although the metal ion catalytic conversion method reduces the reaction temperature and time, metal ions are likely to introduce impurities, and subsequent impurity removal increases the cost and process complexity, and it is difficult to recycle and is not environmentally friendly. The degradation method for preparing ginsenosides such as F4 requires a large amount of chemical reagents, harsh reaction conditions, pollutes the environment, and has limited reaction selectivity, easily generating impurities such as isomers, reducing the product purity. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method, composition and application for microwave-assisted preparation of rare ginsenosides F4 and Rk1 in view of the deficiencies of the above-mentioned prior art. This method uses microwave assistance to prepare rare ginsenosides F4 and Rk1. Under microwave radiation, the reaction system rapidly heats up, and the acidic environment provided by vitamin C promotes the hydrolysis reaction of glycosidic bonds in saponin molecules. The synergistic effect of the thermal effect and non-thermal effect of microwaves is as follows: the thermal effect accelerates molecular movement, enabling the reaction system to quickly reach the required temperature, greatly shortening the reaction time and improving efficiency; the non-thermal effect may change the electron cloud distribution and activity of molecules, reducing the reaction activation energy, thereby accelerating the hydrolysis reaction rate, enhancing the reaction selectivity, increasing purity and yield. Under these conditions, the sugar groups at specific positions of ginsenosides are gradually hydrolyzed and detached, and through a series of intermediate transition states, the target products, rare ginsenosides, are finally formed.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: A method for microwave-assisted preparation of rare ginsenosides F4 and Rk1, characterized by including the following steps:

[0007] Step 1: Place the starting material in a reaction system containing vitamin C, and react at a temperature of 150°C to 200°C with a microwave power of 1000W for 20 min to 40 min; when preparing ginsenoside F4, the starting material is ginsenoside Re of the panaxatriol group, and when preparing ginsenoside Rk1, the starting material is ginsenoside Rb1 of the panaxatriol group;

[0008] Step 2: Add an ethanol solution to the reaction solution after the reaction in Step 1, stir evenly, and then transfer it to a programmed cooling device. First, cool it at a rate of 5°C / min to 100°C, then cool it at a rate of 2°C / min to 60°C and keep it at a constant temperature for 10 min, and then cool it to room temperature at a rate of 1°C / min to obtain ginsenoside F4 or ginsenoside Rk1 accordingly.

[0009] In the above-mentioned method for microwave-assisted preparation of rare ginsenosides F4 and Rk1, the concentration of vitamin C in the reaction system containing vitamin C in Step 1 is 10 mmol / L to 15 mmol / L, and the concentration of the starting material is 5 g / L to 100 g / L.

[0010] In the above-mentioned method for microwave-assisted preparation of rare ginsenosides F4 and Rk1, the solvent of the reaction system in Step 1 is an ethanol aqueous solution with a mass concentration of 30% to 60%.

[0011] In the above-mentioned method for microwave-assisted preparation of rare ginsenosides F4 and Rk1, the mass concentration of the ethanol solution in Step 2 is 30% to 60%, and the added volume of the ethanol solution is 1 to 3 times the volume of the reaction solution.

[0012] Furthermore, the present invention provides a composition of rare ginsenoside F4 and Rk1, which is characterized by comprising ginsenoside F4, ginsenoside Rk1, and a pharmaceutically acceptable carrier, and the mass ratio of ginsenoside F4 to ginsenoside Rk1 is 1:(1-3).

[0013] Furthermore, the present invention provides an application of the above composition in the preparation of drugs and / or health products for improving and / or preventing and treating non-alcoholic fatty liver.

[0014] The present invention has the following advantages compared with the prior art:

[0015] 1. The present invention uses microwave-assisted preparation of rare ginsenoside F4 and Rk1. Under microwave radiation, the reaction system rapidly heats up. The acidic environment provided by vitamin C promotes the hydrolysis reaction of the glycosidic bond in the saponin molecule. The thermal effect and non-thermal effect of microwave act synergistically. The thermal effect accelerates the molecular movement, enabling the reaction system to quickly reach the required temperature, greatly shortening the reaction time and improving the efficiency. The non-thermal effect may change the electron cloud distribution and activity of the molecule, reduce the reaction activation energy, thereby accelerating the hydrolysis reaction rate, enhancing the reaction selectivity, increasing the purity and yield. Under this condition, the sugar groups at specific positions of ginsenoside are gradually hydrolyzed and detached, and after a series of intermediate transition states, the target product, rare ginsenoside, is finally formed.

[0016] 2. By adding an ethanol solution to the reaction solution after the reaction in the present invention, it helps the precipitation of the target product and the removal of impurities. Then, a cooling procedure is adopted. First, cool down to 100 °C at a rate of 5 °C / min, then cool down to 60 °C at a rate of 2 °C / min and keep it constant for 10 min, and then cool down to room temperature at a rate of 1 °C / min. This can make the target product crystallize out more fully, and at the same time avoid the co-precipitation of impurities caused by too rapid cooling.

[0017] 3. By utilizing the synergistic effect of microwave reaction and cooling crystallization, the present invention prepares high-purity ginsenoside by a one-pot method, which is energy-saving, reduces the use of chemical reagents, is green and environmentally friendly, and the equipment is also simple and easy to control, facilitating industrialization. The prepared rare ginsenoside F4 and Rk1, detected by HPLC high-performance liquid chromatography, have a purity ≥ 95%.

[0018] 4. The composition of rare ginsenosides F4 and Rk1 of the present invention can significantly reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are liver function indicators in serum. At the same time, this composition can effectively regulate lipid metabolism and reduce the contents of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL) in serum and liver. Histopathological observations show that the ginsenoside F4 / Rk1 composition can significantly reduce lipid deposition, inflammatory cell infiltration, and the formation of lipid vacuoles in liver tissue, thus significantly improving the pathological state of liver tissue, having the effect of effectively improving and / or preventing and treating non-alcoholic fatty liver, and possessing significant clinical application value.

[0019] The following further describes the technical solutions of the present invention in detail with reference to the accompanying drawings and embodiments. Brief Description of the Drawings

[0020] Figure 1 is the chemical structural formula of ginsenoside F4;

[0021] Figure 2 is the high-performance liquid chromatography (HPLC) chromatogram of the ginsenoside F4 standard and the HPLC chromatogram of the ginsenoside F4 prepared in Example 1 of the present invention;

[0022] Figure 3 is the chemical structural formula of ginsenoside Rk1;

[0023] Figure 4 is the high-performance liquid chromatography (HPLC) chromatogram of the ginsenoside Rk1 standard and the HPLC chromatogram of the ginsenoside Rk1 prepared in Example 1 of the present invention;

[0024] Figure 5 is the Oil Red O staining image of lipid droplets in HpeG2 cells after the treatment in Example 5;

[0025] Figure 6 is the measurement image of the contents of TG and TC in HpeG2 cells after the treatment in Example 6;

[0026] Figure 7 is the weight gain trend graph of each group of mice within 16 weeks after the treatment in Example 7;

[0027] Figure 8 is the Oil Red O staining image of lipid droplets in the liver of mice and the HE staining image of the liver tissue of the group of mice after the treatment in Example 8;

[0028] Figure 9 is the measurement image of the contents of TG, TC, and LDL-C in the serum of mice after the treatment in Example 9;

[0029] Figure 10 is the measurement image of the contents of ALT and AST in the serum of mice after the treatment in Example 10;

[0030] Figure 11 It is a measurement chart of the contents of SOD and MDA in the liver of mice after the treatment of Example 11. Detailed implementation manners

[0031] The present invention will be specifically described below through examples, which are only used for further illustration of the present invention and cannot be construed as limiting the protection scope of the present invention. For the experimental methods without specific conditions noted in the examples, they are usually carried out according to the conventional conditions and the conditions described in the manuals, or according to the conditions recommended by the manufacturers; for the equipment, materials, reagents, etc., unless otherwise specified, they can be obtained from commercial channels.

[0032] Example 1: Microwave-assisted preparation of rare ginsenoside F4 and Rk1

[0033] Step 1: Place the starting material in a reaction system containing vitamin C, and react at a temperature of 200°C and a microwave power of 1000W for 30 min; when preparing ginsenoside F4, the starting material is ginsenoside Re of the panaxatriol group, and when preparing ginsenoside Rk1, the starting material is ginsenoside Rb1 of the panaxatriol group; the concentration of vitamin C in the reaction system is 12 mmol / L, and the concentration of the starting material is 50 g / L; the solvent of the reaction system is an ethanol aqueous solution with a mass concentration of 50%.

[0034] Step 2: Add an ethanol solution with a mass concentration of 50% to the reaction solution after the reaction in Step 1. The added volume of the ethanol solution is twice the volume of the reaction solution. Stir evenly, and then transfer it to a programmed cooling device. First, cool it at a rate of 5°C / min to 100°C, then cool it at a rate of 2°C / min to 60°C and keep it at a constant temperature for 10 min, and then cool it to room temperature at a rate of 1°C / min to obtain ginsenoside F4 or ginsenoside Rk1 accordingly.

[0035] The composition of rare ginsenoside F4 and Rk1 in this example includes ginsenoside F4 and ginsenoside RK1, and a pharmaceutically acceptable carrier. The mass ratio of ginsenoside F4 to ginsenoside RK1 is 1:1.

[0036] Example 2: Microwave-assisted preparation of rare ginsenoside F4 and Rk1

[0037] Step 1: Place the starting material in a reaction system containing vitamin C, and react at a temperature of 150°C and a microwave power of 1000 W for 40 min; when preparing ginsenoside F4, the starting material is ginsenoside Re of the panaxatriol group, and when preparing ginsenoside Rk1, the starting material is ginsenoside Rb1 of the panaxatriol group; the concentration of vitamin C in the reaction system is 10 mmol / L, and the concentration of the starting material is 100 g / L; the solvent of the reaction system is an ethanol aqueous solution with a mass concentration of 30%.

[0038] Step 2: Add an ethanol solution with a mass concentration of 30% to the reaction solution after the reaction in Step 1. The added volume of the ethanol solution is 3 times the volume of the reaction solution. Stir evenly, and then transfer it to a programmed cooling device. First, cool it at a rate of 5°C / min to 100°C, then cool it at a rate of 2°C / min to 60°C and keep it constant for 10 min, and then cool it to room temperature at a rate of 1°C / min to obtain ginsenoside F4 or ginsenoside Rk1 accordingly.

[0039] The composition of rare ginsenoside F4 and Rk1 in this example includes ginsenoside F4 and ginsenoside RK1, as well as a pharmaceutically acceptable carrier. The mass ratio of ginsenoside F4 to ginsenoside RK1 is 1:2.

[0040] Example 3: Microwave-assisted preparation of rare ginsenoside F4 and Rk1

[0041] Step 1: Place the starting material in a reaction system containing vitamin C, and react at a temperature of 180°C and a microwave power of 1000 W for 20 min; when preparing ginsenoside F4, the starting material is ginsenoside Re of the panaxatriol group, and when preparing ginsenoside Rk1, the starting material is ginsenoside Rb1 of the panaxatriol group; the concentration of vitamin C in the reaction system is 15 mmol / L, and the concentration of the starting material is 5 g / L; the solvent of the reaction system is an ethanol aqueous solution with a mass concentration of 60%.

[0042] Step 2: Add an ethanol solution with a mass concentration of 60% to the reaction solution after the reaction in Step 1. The added volume of the ethanol solution is 1 time the volume of the reaction solution. Stir evenly, and then transfer it to a programmed cooling device. First, cool it at a rate of 5°C / min to 100°C, then cool it at a rate of 2°C / min to 60°C and keep it constant for 10 min, and then cool it to room temperature at a rate of 1°C / min to obtain ginsenoside F4 or ginsenoside Rk1 accordingly.

[0043] The composition of rare ginsenoside F4 and Rk1 in this example includes ginsenoside F4 and ginsenoside RK1, as well as a pharmaceutically acceptable carrier. The mass ratio of ginsenoside F4 to ginsenoside RK1 is 1:3.

[0044] Example 4: HepG2 cell culture

[0045] (1) MEM culture medium: Add 10% fetal bovine serum and 1% penicillin / streptomycin mixture to 500 mL of MEM basal medium, mix well and store in a refrigerator at 4°C. HepG2 cells are cultured adherently using MEM medium in a biochemical incubator with 5% CO2, 37°C and 95% humidity. Passage culture or replacement of cell culture medium is carried out according to the cell growth conditions, and the cells are cryopreserved using serum-free cell cryopreservation solution.

[0046] (2) Weigh PA (palmitic acid) and OA (oleic acid) respectively and dissolve them in 1 mL of absolute ethanol. Use MEM containing only 1% double antibody to dissolve BSA into a 20% solution. Mix the three to obtain a lipid medium with a final concentration of 200 μM for PA and 400 μM for OA.

[0047] (3) Experimental grouping: The cells are divided into a blank control group, a model group and a drug administration group. The blank group is cultured with blank medium for 24 h, and the model group is cultured with the above-mentioned culture medium for 24 h to obtain a HepG2 cell fatty liver model. The drug administration concentrations of HepG2 cells in the drug administration group are F4 (40 μM), Rk1 (40 μM), and F4 / Rk1 (1:1, 30 μM).

[0048] Example 5: Oil Red O staining of HepG2 cells

[0049] Disperse HepG2 cells in a 6-well plate, with 3×10 5 cells per well, and set up 3 replicates for each group;

[0050] Remove the cell culture medium, wash twice with PBS, and add ORO Fixative to fix for 20 - 30 min;

[0051] Discard the fixative and wash twice with distilled water;

[0052] Add 60% isopropanol and soak for 5 min;

[0053] After discarding 60% isopropanol, add freshly prepared ORO Stain and stain for 10 - 20 min;

[0054] Discard the staining solution, wash 2 - 5 times with water until there is no excess staining solution. Add Mayer hematoxylin staining solution to counterstain the nucleus for 1 - 2 min, and then wash 2 - 5 times with water after discarding the staining solution;

[0055] Add ORO Buffer for 1 min and then discard it; Add distilled water to cover the cells, observe and take pictures under a microscope, and the results are shown in Figure 5, As can be seen from the figure, the edges of HepG2 cells in the control group were clear, no red lipid droplets were seen around the cells, and the nuclear membrane was intact. After 24 hours of treatment with a lipid medium, the morphology of some cells in the Model group changed, and a large number of orange-red and red small lipid droplets aggregated around the cells. In the groups treated with ginsenoside F4 and ginsenoside Rk1, we observed that the number of lipid droplets aggregated around the cells decreased significantly.

[0056] Example 6: Determination of the contents of TG and TC in HepG2 cells

[0057] Collect the cells, centrifuge at 1000 rpm for 10 min, discard the supernatant, wash the cell pellet with PBS, centrifuge again at 1000 rpm for 10 min, discard the supernatant, and keep the cell pellet; add PBS and use liquid nitrogen to quickly freeze and thaw the cells repeatedly 6 times to break the cells; measure according to the kit manufacturer's instructions, and use an enzyme-linked immunosorbent assay colorimetry method to measure the absorbance. The results are shown in Figure 6 , compared with the normal group, the contents of TG and TC in the cells of the Model group were higher, while after treatment with ginsenosides, the contents of TG and TC in the cells decreased significantly. The above data indicate that ginsenoside F4 and ginsenoside Rk1 have a certain ability to resist lipid accumulation.

[0058] Example 7: Establishment of a NAFLD mouse model

[0059] The C57BL / 6 mice (male, 8 weeks old) used in this experiment generally weighed 18 - 20 g. All the mice were randomly grouped and placed in a pathogen-free breeding box at a room temperature of 25 ± 2 °C, a relative humidity of 50% ± 5%, and a light / dark cycle of 12 h. After one week of adaptive feeding of the mice, 50 mice were randomly divided into 6 groups, including 10 mice in the normal group (Normal Group). The mice in the normal group were fed tap water and laboratory mouse food, and the remaining 8 mice in each group were fed a D12492 high-fat diet and 30% sugar water for 12 weeks. Starting from the 13th week, the number of mice in the 5 groups was equal to 8 and there was no significant difference in body weight among the groups. According to the corresponding doses of F4 (60 mg / kg), Rk1 (60 mg / kg), and F4 / Rk1 (50 mg / kg), the mice were intragastrically administered drugs every day. The normal group and the model group were given a single solvent by gavage for four weeks. The mice in each experimental group were weighed weekly, and the body weight of the mice was recorded. The results are shown in Figure 7 , as shown in the figure, the body weight of the mice in the high-fat and high-sugar diet group increased steadily in the first 12 weeks before modeling, showing a significant difference from the body weight of the mice in the normal group. During the last four weeks of drug administration, except for the normal group, the body weight of the mice in the Model group continued to increase, while the body weight of the mice in the drug administration group showed a downward trend, indicating that ginsenoside F4 and ginsenoside Rk1 have a good effect on reducing the body weight of NAFLD mice. After 16 weeks of the experiment, the mice in each experimental group were sacrificed by cervical dislocation, and blood and liver tissues were collected.

[0060] Example 8: HE staining and Oil Red O staining of mouse liver tissue

[0061] Deparaffinize the paraffin sections to water: sequentially place the sections into environment-friendly deparaffinizing solution I for 20 min - environment-friendly deparaffinizing solution II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and wash with tap water.

[0062] Restore the temperature and fix the frozen sections: take out the frozen sections from the -20°C refrigerator and restore them to room temperature, fix them with tissue fixative for 15 min, and then rinse with running water;

[0063] Pretreatment: immerse the sections in a high-definition constant staining pretreatment solution for 1 min;

[0064] Hematoxylin staining: immerse the sections in hematoxylin staining solution for 3 - 5 min, wash with tap water, differentiate with differentiating solution, wash with tap water, blue with bluing solution, and rinse with running water;

[0065] Eosin staining: dehydrate the sections in 95% alcohol for 1 min, and immerse them in eosin staining solution for 15 s;

[0066] Dehydration and mounting: sequentially place the sections into absolute ethanol I for 2 min - absolute ethanol II for 2 min - absolute ethanol III for 2 min - n-butanol I for 2 min - n-butanol II for 2 min - xylene I for 2 min - xylene II for 2 min for clearing, and mount with neutral balsam;

[0067] Microscopic examination, image acquisition and analysis, the results are shown in Figure 8 , it can be seen from the figure that in the Model group, dilation of the central vein, widening of the hepatic cord, disordered arrangement, congestion of some hepatic sinusoids, unclear cell boundaries accompanied by extensive fatty degeneration, and edema degeneration of some hepatocytes can be observed, which are in line with the histopathological characteristics of NAFLD. After treatment with ginsenoside F4, ginsenoside Rk1 and their combination, the phenomena of unclear cell boundaries, fatty degeneration and edema degeneration were significantly improved.

[0068] Example 9: Determination of TG, TC, and LDL-C contents in mouse serum

[0069] Obtain mouse blood by the method of eye blood collection; let it stand at room temperature, and after the serum separates out, centrifuge at 3000 rpm / min for 5 min, and aspirate the upper serum; according to the instructions, use TG, TC, and LDL-C test kits to detect the contents in mouse serum, and measure the absorbance value of each well with an enzyme-linked immunosorbent assay reader, the results are shown in Figure 9, Long-term high-fat diet led to serum metabolic disorders in mice, with the influx of blood lipids in the body exceeding the excretion. Therefore, the contents of TG, TC, and LDL-C in the serum of Model mice all increased significantly, which was consistent with the disease characteristics of NAFLD, manifested as a significant increase in serum lipid content. Consistent with the expected results, after treatment with ginsenoside F4 and ginsenoside Rk1, the contents of TG, TC, and LDL-C in the serum decreased significantly.

[0070] Example 10: Determination of the contents of ALT and AST in mouse serum

[0071] Using the method of eye blood collection to obtain mouse blood; standing at room temperature, after serum precipitation, centrifuging at 3000 rpm / min for 5 min, and sucking the upper-layer serum; according to the instruction manual, using the ALT and AST test kits to detect the contents of each index in mouse serum, and using an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value of each well. The results are shown in Figure 10 , Compared with the normal group, the levels of ALT and AST in the serum of Model group mice were higher, while after treatment with ginsenoside F4 and ginsenoside Rk1, the contents of ALT and AST in the serum of mice decreased significantly.

[0072] Example 11: Determination of the contents of SOD and MDA in mouse liver

[0073] According to the kit instruction manual, accurately weigh the weight of animal tissues, add homogenization medium in a certain proportion, mechanically homogenize under ice-water bath conditions, and centrifuge, then suck the supernatant into a new EP tube. The MDA and SOD indexes of the livers of each group were measured by ELISA kits (Nanjing Jiancheng). To measure the SOD activity, 30 μg of the extracted protein was incubated with the analysis reagent at room temperature for 10 min, and then its OD value was measured at the absorbance of 550 nm. To quantify MDA, 30 μg of the extracted protein was incubated with the analysis reagent at 95 °C for 40 min, and then quickly cooled to room temperature. After centrifugation, the OD value of the supernatant was measured at 532 nm.

[0074] One-way analysis of variance was used for multiple comparisons and statistical significance between different groups. Statistical analysis was performed using GraphPad Prism 10. The results are shown in Figure 11 , Through long-term high-fat and high-sugar diet, severe liver oxidative stress reaction occurred in the livers of Model group mice, manifested as a significant increase in MDA content and a significant decrease in the corresponding SOD activity. After intervention with ginsenoside F4 and ginsenoside Rk1, the MDA content in the liver decreased, the SOD activity and content increased, and the liver oxidative stress reaction was significantly improved.

[0075] The composition of rare ginsenosides F4 and Rk1 of the present invention can significantly reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are liver function indicators in serum. At the same time, this composition can effectively regulate lipid metabolism and reduce the contents of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) in serum and liver. Histopathological observation shows that the ginsenoside F4 / Rk1 composition can significantly reduce lipid deposition, inflammatory cell infiltration and the formation of fat vacuoles in liver tissue, thus significantly improving the pathological state of liver tissue, having the effect of effectively improving and / or preventing and treating non-alcoholic fatty liver, and having significant clinical application value.

[0076] As mentioned above, it is only a preferred embodiment of the present invention and does not impose any limitation on the present invention. Any simple modification, change and equivalent structural change made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing rare ginsenosides F4 and Rk1 by microwave-assisted method, characterized in that: The following steps are involved: Step 1, placing the starting material in a reaction system containing vitamin C, reacting at a temperature of 150° C. to 200° C. and a microwave power of 1000 W for 20 min to 40 min; when preparing ginsenoside F4, the starting material is ginsenoside Re of the ginsenoside group, and when preparing ginsenoside Rk1, the starting material is ginsenoside Rb1 of the ginsenoside group; Step 2: Add ethanol solution to the reaction solution after the reaction in step 1, stir evenly, and then transfer to a programmed cooling device, first cool to 100°C at a rate of 5°C / min, then cool to 60°C at a rate of 2°C / min and keep the constant temperature for 10 minutes, and then cool to room temperature at a rate of 1°C / min to obtain ginsenoside F4 or ginsenoside Rk1 accordingly.

2. The method for preparing rare ginsenosides F4 and Rk1 by microwave-assisted method according to claim 1, characterized in that: The concentration of vitamin C in the reaction system containing vitamin C in step 1 is 10 mmol / L to 15 mmol / L, and the concentration of the starting material is 5 g / L to 100 g / L.

3. The method for preparing rare ginsenosides F4 and Rk1 by microwave-assisted method according to claim 1, characterized in that: The solvent of the reaction system in step 1 is an ethanol aqueous solution with a mass concentration of 30% to 60%.

4. The method for preparing rare ginsenosides F4 and Rk1 by microwave-assisted method according to claim 1, characterized in that: The mass concentration of the ethanol solution in step 2 is 30% to 60%, and the added volume of the ethanol solution is 1 to 3 times the volume of the reaction solution.

5. A rare ginsenoside F4 and Rk1 composition, characterized in that: The invention comprises ginsenoside F4 and ginsenoside Rk1, and a pharmaceutically acceptable carrier, wherein the mass ratio of ginsenoside F4 to ginsenoside Rk1 is 1:(1-3).

6. Use of the composition according to claim 5 in the preparation of medicines and / or health products for improving and / or preventing and treating non-alcoholic fatty liver disease.

Images