Application of oleanolic acid in tumor resistance

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an application of oleanolic acid in tumor resistance, and the application of oleanolic acid in tumor resistance specifically comprises the following steps: inhibiting tumor cell proliferation and inducing apoptosis; tumor angiogenesis is inhibited; the drug resistance of tumor cells is reversed; the immune function is regulated, and the anti-tumor immunity is enhanced; tumor invasion and metastasis are inhibited; according to the method, supercritical CO2 extraction is adopted, recoverable CO2 is used as an extraction medium, and no toxic organic solvent such as chloroform is used in the whole process; a small amount of ethanol is used only in the links of resin activation and elution, and is recovered through vacuum concentration, so that the use amount is extremely small, the treatment is easy, and the environmental pollution caused by solvent leakage or residue is avoided from the source.

Description

TECHNICAL FIELD

[0001] The application relates to the field of medicine, in particular to application of oleanolic acid in antitumor. BACKGROUND

[0002] Oleanolic acid is a widely distributed pentacyclic triterpenoid compound in nature, has various important biological activities, such as antitumor, anti-HIV virus, liver protection, anti-inflammatory and the like, but the activity is weak, and the bioavailability is low, so that the application in the clinic is limited.

[0003] Researches show that oleanolic acid has different degrees of inhibitory activity on various tumor cell strains, has remarkable prevention and treatment effects on cancer in various animal models, and shows clear anticancer properties after activity evaluation. Oleanolic acid has an apoptosis-inducing activity on leukemia cells HL-60, and the action path is to activate apoptosis protein-3 and apoptosis protein-9, and meanwhile, DNA repair enzyme (PARP) is cleaved. Oleanolic acid can also induce differentiation of mouse mammary epithelial cells, and induce differentiation of mouse granulocytic leukemia (M1) cells and human acute promyelocytic leukemia (HL-60) cells.

[0004] However, the following technical problems still exist in the preparation process of oleanolic acid: Currently, organic solvents such as ethanol and chloroform are used in the extraction process of oleanolic acid, and if not properly handled, environmental pollution is easy to be caused; in addition, the impurity components in plants are complex, and repeated impurity removal is needed in the subsequent purification link, which not only increases the operation difficulty, but also is easy to cause the problem of substandard purity, and affects the product quality; Therefore, the application of oleanolic acid in antitumor is proposed to solve the above problems. SUMMARY

[0005] The application aims to provide the application of oleanolic acid in antitumor to solve the problems in the background.

[0006] To achieve the above-mentioned purpose, the application provides the following technical scheme: the application of oleanolic acid in antitumor, the application of oleanolic acid in antitumor specifically includes: (1) inhibiting tumor cell proliferation and inducing apoptosis; (2) inhibiting tumor angiogenesis; (3) reversing tumor cell drug resistance; (4) regulating immune function and enhancing antitumor immunity; (5) inhibiting tumor invasion and metastasis; The preparation steps of the oleanolic acid are as follows: Step one, raw material pretreatment: remove impurities from ligustrum lucidum, crush and pass through a 50-80 mesh sieve to obtain powder; Step two, supercritical CO2 extraction: 200g powder into supercritical CO2 extraction kettle, 35-55℃, 15-35MPa, with 18-24kg / h flow extraction 60-120min, separated by separation kettle, get the crude extract, CO2 recovery; Step three, preliminary enrichment: the crude extract is added to 5 times distilled water, stirred in water bath at 50℃ for 30min, and the filter residue is taken out by suction filtration; Step four, macroporous resin purification: AB-8 macroporous resin is activated and columned, the filter residue is dissolved with a small amount of ethanol, diluted to 1-2mg / mL sample solution, and then columned, washed with distilled water and 30% ethanol in turn, and eluted with 70-80% ethanol, and the eluate is collected; Step five, drying: the eluate is concentrated under reduced pressure to remove ethanol, and distilled water is added to refrigerate and crystallize, then the crystals are filtered and washed, and vacuum dried at 60℃ for 4-6h to obtain the product with a purity of more than 90%, which uses less ethanol and no harmful solvent in the whole process.

[0007] Preferably, the mechanism of oleanolic acid in inhibiting tumor cell proliferation and inducing apoptosis is as follows: by regulating cell cycle-related proteins, the tumor cells are blocked in G0 / G1 or S phase, their division and proliferation are inhibited, the apoptosis signal pathway is activated, and the programmed death of tumor cells is promoted, which has inhibitory effect on liver cancer, lung cancer, gastric cancer and other tumor cells.

[0008] Preferably, the mechanism of oleanolic acid in inhibiting tumor angiogenesis is as follows: by down-regulating the expression of vascular endothelial growth factor and basic fibroblast growth factor angiogenesis-related factors, the formation of tumor neovasculature is blocked, the tumor nutrition supply and metastasis channel are reduced, and the tumor growth and distant metastasis are inhibited.

[0009] Preferably, the mechanism of oleanolic acid in reversing tumor cell drug resistance is as follows: by regulating the expression of drug resistance-related proteins, the efflux capacity of tumor cells to chemotherapeutic drugs is reduced, the accumulation of chemotherapeutic drugs in tumor cells is enhanced, and the chemotherapeutic effect is improved, especially for drug-resistant liver cancer and breast cancer cells.

[0010] Preferably, the mechanism of oleanolic acid in regulating immune function and enhancing anti-tumor immunity is as follows: promoting the activation of macrophages, the proliferation of lymphocytes and the secretion of cytokines, improving the immune suppression state in the tumor microenvironment, activating the body's immune surveillance system, and indirectly killing tumor cells Preferably, the mechanism of oleanolic acid in inhibiting tumor invasion and metastasis is as follows: down-regulating the activity of matrix metalloproteinase, reducing the invasion and destruction of tumor cells to the surrounding tissues, and inhibiting the epithelial-mesenchymal transition process to reduce the migration ability of tumor cells.

[0011] Preferably, the specific preparation steps of oleanolic acid are as follows: Step one, raw material pretreatment: select the raw material of Ligustrum lucidum, remove the stones, dry branches, avoid impurities affecting the subsequent extraction effect, put the purified Ligustrum lucidum into the pulverizer, crush, sieve after crushing, get uniform Ligustrum lucidum powder; Step two, supercritical CO2 extraction: take 200g of pretreated Ligustrum lucidum powder, evenly put it into the extraction kettle of supercritical CO2 extraction device, ensure that the material is flat, avoid local accumulation leading to uneven extraction, set the extraction parameters, the extraction kettle temperature is controlled at 35-55℃, the pressure is adjusted to 15-35MPa, the flow of carbon dioxide is stabilized at 18-24kg / h, the extraction time is set to 60-120min; start the extraction device, CO2 enters the extraction kettle under high pressure and fully contacts with the raw material, dissolves the active ingredients such as oleanolic acid, then the CO2 fluid containing the target components enters the separation kettle in turn, the temperature of separation kettle I and II is controlled at 5-45℃, through gradually reducing the pressure, CO2 and oleanolic acid are separated, finally the extract in the separation kettle is collected, oleanolic acid crude extract is obtained, and CO2 can be recycled, no waste gas and waste liquid pollution; Step three, preliminary impurity removal and enrichment: add 5 times the volume of distilled water to the obtained crude extract, stir in a 50℃ water bath for 30min, dissolve a small amount of water-soluble impurities in the crude extract, hot filtration, remove water-soluble impurities and a small amount of incomplete pulverization solid particles, collect the filter residue; Step four, precise purification of macroporous resin: AB-8 type macroporous resin is selected, first soaked with 95% ethanol for 24h, then washed with distilled water until no ethanol residue, loaded into the chromatography column, ensure uniform resin loading, no bubbles, dissolve the filter residue in step three with a small amount of ethanol, dilute with distilled water to a concentration of 1-2mg / mL of sample solution, slowly inject into the chromatography column, control the sample flow rate at 1-2mL / min, let oleanolic acid be fully adsorbed by the resin, first wash the chromatography column with distilled water at a flow rate of 2mL / min, wash for 2-3 column volumes, remove the residual water-soluble impurities in the column; then wash with 30% ethanol, elute part of the weakly adsorbed neutral impurities, this process does not need to be repeated several times, finally use 70-80% ethanol as eluent, elute at a flow rate of 1mL / min, collect the eluate.

[0012] Compared with the prior art, the beneficial effects of the present application are: 1) The present application uses supercritical CO2 extraction, uses recyclable CO2 as the extraction medium, and does not use toxic organic solvents such as chloroform throughout the process; only a small amount of ethanol is used in the activation and elution of the resin, and it is recovered by vacuum concentration, the amount is very small and easy to handle, which avoids environmental pollution caused by solvent leakage or residue from the source; 2) In the pretreatment stage of this application, the impurities of the raw materials are reduced by crushing and sieving. The initial impurity removal utilizes the difference in water solubility. A large number of water-soluble impurities are removed by hot water stirring and filtration. The key is to use AB-8 macroporous resin for precise purification. Its strong specific adsorption of eparginal acid can efficiently separate neutral impurities. Different types of impurities are removed stepwise by gradient elution without repeated operation. Finally, the materials are further purified by crystallization rinsing and vacuum drying to ensure stable quality. Attached Figure Description

[0013] Fig. 1 A detailed diagram illustrating the application of oleanolic acid in anti-tumor therapy; Fig. 2 This is a flowchart of the preparation steps for oleanolic acid. Detailed Implementation

[0014] 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 embodiments of the present invention, and not all embodiments. 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.

[0015] Example: Please see Figs. 1-2 The present invention provides a technical solution: An application of oleanolic acid in antitumor activity, specifically including: (1) Inhibits tumor cell proliferation and induces apoptosis; (2) Inhibits tumor angiogenesis; (3) Reversing drug resistance in tumor cells; (4) Regulates immune function and enhances anti-tumor immunity; (5) Inhibits tumor invasion and metastasis; Application scenarios of oleanolic acid: Adjuvant therapy for malignant tumors: Currently, it is mostly used as an adjuvant drug for chemotherapy and radiotherapy to improve the clinical symptoms of patients with malignant tumors such as liver cancer, lung cancer, stomach cancer, and breast cancer, reduce adverse reactions such as nausea, vomiting, and bone marrow suppression caused by radiotherapy and chemotherapy, and improve the quality of life of patients. Synergistic effect of combination therapy: When used in combination with traditional chemotherapy drugs (such as 5-fluorouracil and paclitaxel) and targeted drugs, the anti-tumor effect can be enhanced through synergistic effects, the dosage of chemotherapy drugs can be reduced, and the toxic side effects can be reduced. Cancer prevention: Based on its antioxidant, anti-inflammatory and cell metabolism-regulating effects, it can be used for cancer prevention in high-risk groups (such as preventing liver cancer in patients with chronic hepatitis and preventing gastric cancer in patients with atrophic gastritis), reducing the risk of cancer by inhibiting the progression of precancerous lesions; Experimental research and drug development: As a natural active compound, oleanolic acid is an important lead compound for the development of anti-tumor drugs. Through structural modification (such as esterification and glycosylation), its water solubility and bioavailability can be improved, and novel anti-tumor derivatives with high efficiency and low toxicity can be developed.

[0016] The preparation steps of this oleanolic acid are as follows: Step 1: Raw material pretreatment: Remove impurities from privet fruit, crush it and pass it through a 50-80 mesh sieve to obtain powder; Step 2, Supercritical CO2 extraction: 200g of powder is added to a supercritical CO2 extraction vessel and extracted at 35-55℃ and 15-35MPa with a flow rate of 18-24kg / h for 60-120min. After separation by depressurization in a separation vessel, crude extract is obtained and CO2 is recovered. Step 3: Preliminary impurity removal and enrichment: Add 5 times the amount of distilled water to the crude extract, stir in a 50°C water bath for 30 minutes, and filter to collect the residue; Step 4: Precise purification of macroporous resin: After activation, AB-8 macroporous resin is packed into a column. The filter residue is dissolved in a small amount of ethanol and diluted to a 1-2 mg / mL loading solution. The solution is passed through the column and washed successively with distilled water and 30% ethanol, and then eluted with 70-80% ethanol. The eluent is collected. Step 5, Refining and Drying: The eluent is concentrated under reduced pressure to remove ethanol, distilled water is added and the mixture is refrigerated to crystallize. After filtration and rinsing, the crystals are dried under vacuum at 60°C for 4-6 hours to obtain a high-quality product with a purity of over 90%. Ethanol is used sparingly throughout the process, and no harmful solvents are used.

[0017] The specific mechanism by which oleanolic acid inhibits tumor cell proliferation and induces apoptosis is as follows: by regulating cell cycle-related proteins, it arrests tumor cells in the G0 / G1 phase or S phase, inhibiting their division and proliferation, while activating apoptosis signaling pathways to promote programmed cell death in tumor cells. It has an inhibitory effect on various tumor cells, including liver cancer, lung cancer, and gastric cancer.

[0018] The specific mechanism by which oleanolic acid inhibits tumor angiogenesis is as follows: it downregulates the expression of angiogenesis-related factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), blocks tumor angiogenesis, reduces tumor nutrient supply and metastasis pathways, and inhibits tumor growth and distant metastasis.

[0019] The specific mechanism by which oleanolic acid reverses drug resistance in tumor cells is as follows: by regulating the expression of drug resistance-related proteins (such as P-glycoprotein), it reduces the efflux capacity of tumor cells to chemotherapeutic drugs (such as cisplatin and doxorubicin), enhances the accumulation of chemotherapeutic drugs in tumor cells, and improves the efficacy of chemotherapy, especially for drug-resistant liver cancer and breast cancer cells.

[0020] The specific mechanisms by which oleanolic acid regulates immune function and enhances anti-tumor immunity are as follows: it promotes macrophage activation, lymphocyte proliferation, and cytokine (such as TNF-α and IL-2) secretion, improves the immunosuppressive state in the tumor microenvironment, activates the body's own immune surveillance system, and indirectly kills tumor cells. The specific mechanism by which oleanolic acid inhibits tumor invasion and metastasis is as follows: it downregulates the activity of matrix metalloproteinases (MMP-2, MMP-9), reduces the invasion and damage of tumor cells to surrounding tissues, and inhibits the epithelial-mesenchymal transition (EMT) process, thereby reducing the migration ability of tumor cells.

[0021] The specific preparation steps for this oleanolic acid are as follows: Step 1: Raw material pretreatment: Select privet fruit as raw material, remove stones and dead branches to avoid impurities affecting the subsequent extraction effect, put the purified privet fruit into a pulverizer to crush it, and then pass it through a 50-80 mesh sieve to obtain uniform privet fruit powder. Powdered raw material can increase the contact area with the extraction medium, improve the extraction efficiency, and at the same time reduce material loss during subsequent separation. By removing impurities, crushing and sieving, mechanical impurities such as stones and dead branches are removed, and the privet fruit is made into a uniform powder. This increases the contact area with the extraction medium and improves the subsequent extraction efficiency. At the same time, it reduces the interference of impurities in the raw material on the extraction process and reduces the difficulty of subsequent separation. Step 2, Supercritical CO2 Extraction: Weigh approximately 200g of pretreated privet fruit powder and evenly place it into the extraction vessel of the supercritical CO2 extraction device, ensuring the material is evenly spread to avoid uneven extraction due to local accumulation. Set the extraction parameters: control the extraction vessel temperature at 35-55℃, adjust the pressure to 15-35MPa, stabilize the carbon dioxide flow rate at 18-24kg / h, and set the extraction time to 60-120min. This parameter range ensures that CO2 is in a supercritical state with good dissolving capacity, efficiently extracts oleanolic acid, and does not damage its active structure. Start the extraction device. CO2 enters the extraction vessel under high pressure and fully contacts the raw material, dissolving oleanolic acid and other active ingredients. Subsequently, the CO2 fluid containing the target components enters the fractionation separation vessel sequentially. The temperature of separation vessels I and II is controlled at 5-45℃. By gradually reducing the pressure, CO2 and oleanolic acid are separated. Finally, collect the extract in the separation vessel to obtain crude oleanolic acid extract. The CO2 can be recycled and reused, with no waste gas or waste liquid pollution. Using recyclable supercritical CO2 as the extraction medium, oleanolic acid is efficiently dissolved and separated under specific temperature and pressure, replacing traditional organic solvents and avoiding pollution at the source. The separation of CO2 and crude extract is achieved through a staged separation vessel. CO2 can be recycled, which is both environmentally friendly and cost-effective, while preserving the active structure of oleanolic acid. Step 3: Preliminary impurity removal and enrichment: Add 5 times the volume of distilled water to the obtained crude extract, place it in a 50℃ water bath and stir for 30 minutes to dissolve a small amount of water-soluble impurities in the crude extract. Filter while hot to remove water-soluble impurities and a small amount of incompletely crushed solid particles. Collect the filter residue. This step can initially remove impurities with large differences in solubility from oleanolic acid, reducing the pressure of subsequent purification. By taking advantage of differences in water solubility, water-soluble impurities in the crude extract are dissolved by stirring with hot water. After filtration, these impurities and incompletely crushed particles are removed, and oleanolic acid is initially purified and enriched, reducing the pressure of subsequent purification and laying the foundation for precise purification. Step 4: Precise Purification with Macroporous Resin: AB-8 macroporous resin is selected. It is first activated by soaking in 95% ethanol for 24 hours, then rinsed with distilled water until no ethanol residue remains. The resin is then packed into the chromatography column, ensuring uniform packing and no air bubbles. AB-8 macroporous resin has a strong specific adsorption capacity for eparginal acid, effectively separating impurities. The filter residue from Step 3 is dissolved in a small amount of ethanol, then diluted with an appropriate amount of distilled water to a loading solution concentration of 1-2 mg / mL. This solution is slowly injected into the chromatography column, controlling the loading flow rate to 1... To allow oleanolic acid to be fully adsorbed by the resin, first rinse the column with distilled water at a flow rate of 2 mL / min for 2-3 column volumes to remove residual water-soluble impurities. Then rinse with 30% ethanol to elute some weakly adsorbed neutral impurities. This process does not require repeated impurity removal. Finally, use 70-80% ethanol as the eluent at a flow rate of 1 mL / min and collect the eluent. This concentration of ethanol can efficiently desorb oleanolic acid and is easy to remove subsequently.

[0022] AB-8 macroporous resin exhibits strong and specific adsorption of oleanolic acid, achieving efficient separation of the target component from impurities. Water-soluble impurities and weakly adsorbed neutral impurities are removed stepwise through gradient rinsing with distilled water and low-concentration ethanol, eliminating the need for repeated operations. High-concentration ethanol elution allows for the targeted recovery of oleanolic acid, significantly improving product purity and resolving the issue of substandard purity caused by complex impurities.

[0023] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

[0024] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An application of oleanolic acid in antitumor activity, characterized in that, The specific applications of oleanolic acid in anti-tumor treatment include: (1) Inhibits tumor cell proliferation and induces apoptosis; (2) Inhibits tumor angiogenesis; (3) Reversing drug resistance in tumor cells; (4) Regulates immune function and enhances anti-tumor immunity; (5) Inhibits tumor invasion and metastasis; The preparation steps of this oleanolic acid are as follows: Step 1: Raw material pretreatment: Remove impurities from privet fruit, crush it and pass it through a 50-80 mesh sieve to obtain powder; Step 2: Take 200g of supercritical CO2 powder into a supercritical CO2 extraction vessel, extract at 35-55℃ and 15-35MPa with a flow rate of 18-24kg / h for 60-120min, separate by depressurization in a separation vessel to obtain crude extract, and recover CO2. Step 3: Preliminary impurity removal and enrichment: Add 5 times the amount of distilled water to the crude extract, stir in a 50°C water bath for 30 minutes, and filter to collect the residue; Step 4: Precise purification of macroporous resin: After activation, AB-8 macroporous resin is packed into a column. The filter residue is dissolved in a small amount of ethanol and diluted to a 1-2 mg / mL loading solution. The solution is passed through the column and washed successively with distilled water and 30% ethanol, and then eluted with 70-80% ethanol. The eluent is collected. Step 5, Refining and Drying: The eluent is concentrated under reduced pressure to remove ethanol, distilled water is added and the mixture is refrigerated to crystallize. After filtration and rinsing, the crystals are dried under vacuum at 60°C for 4-6 hours to obtain a high-quality product with a purity of over 90%. Ethanol is used sparingly throughout the process, and no harmful solvents are used.

2. The application of oleanolic acid in antitumor activity according to claim 1, characterized in that: The specific mechanism by which oleanolic acid inhibits tumor cell proliferation and induces apoptosis is as follows: by regulating cell cycle-related proteins, it arrests tumor cells in the G0 / G1 phase or S phase, inhibiting their division and proliferation, while activating apoptosis signaling pathways to promote programmed cell death in tumor cells. It has an inhibitory effect on various tumor cells, including liver cancer, lung cancer, and gastric cancer.

3. The application of oleanolic acid in antitumor activity according to claim 1, characterized in that: The specific mechanism by which oleanolic acid inhibits tumor angiogenesis is as follows: it downregulates the expression of vascular endothelial growth factor, basic fibroblast growth factor and other angiogenesis-related factors, blocks tumor angiogenesis, reduces tumor nutrient supply and metastasis pathways, and inhibits tumor growth and distant metastasis.

4. The application of oleanolic acid in antitumor activity according to claim 1, characterized in that: The specific mechanism by which oleanolic acid reverses drug resistance in tumor cells is as follows: by regulating the expression of drug resistance-related proteins, it reduces the efflux capacity of tumor cells to chemotherapeutic drugs, enhances the accumulation of chemotherapeutic drugs in tumor cells, and improves the efficacy of chemotherapy, especially for drug-resistant liver cancer and breast cancer cells.

5. The application of oleanolic acid in antitumor activity according to claim 1, characterized in that: The specific mechanisms by which oleanolic acid regulates immune function and enhances anti-tumor immunity are as follows: it promotes macrophage activation, lymphocyte proliferation and cytokine secretion, improves the immunosuppressive state in the tumor microenvironment, activates the body's own immune surveillance system, and indirectly kills tumor cells.

6. The application of oleanolic acid in antitumor activity according to claim 1, characterized in that: The specific mechanism by which oleanolic acid inhibits tumor invasion and metastasis is as follows: it downregulates the activity of matrix metalloproteinases, reduces the invasion and damage of tumor cells to surrounding tissues, and inhibits the epithelial-mesenchymal transition process, thereby reducing the migration ability of tumor cells.

7. The application of oleanolic acid in antitumor activity according to claim 1, characterized in that: The specific preparation steps for this oleanolic acid are as follows: Step 1: Raw material pretreatment: Select privet fruit as raw material, remove stones and dead branches to avoid impurities affecting the subsequent extraction effect, put the purified privet fruit into a pulverizer to crush it, and then pass it through a 50-80 mesh sieve to obtain uniform privet fruit powder. Step 2, Supercritical CO2 Extraction: Weigh approximately 200g of pretreated privet fruit powder and evenly place it into the extraction vessel of the supercritical CO2 extraction device, ensuring the material is evenly spread to avoid uneven extraction due to local accumulation. Set the extraction parameters: control the extraction vessel temperature at 35-55℃, adjust the pressure to 15-35MPa, stabilize the carbon dioxide flow rate at 18-24kg / h, and set the extraction time to 60-120min. Start the extraction device; CO2 enters the extraction vessel under high pressure and fully contacts the raw material, dissolving active ingredients such as oleanolic acid. Subsequently, the CO2 fluid containing the target components enters the fractionation separation vessel sequentially. The temperatures of separation vessels I and II are both controlled at 5-45℃. By gradually reducing the pressure, CO2 is separated from oleanolic acid. Finally, collect the extract in the separation vessel to obtain crude oleanolic acid extract. The CO2 can be recycled and reused, with no waste gas or waste liquid pollution. Step 3: Preliminary impurity removal and enrichment: Add 5 times the volume of distilled water to the obtained crude extract, place it in a 50℃ water bath and stir for 30 minutes to dissolve a small amount of water-soluble impurities in the crude extract, filter while hot to remove water-soluble impurities and a small amount of incompletely crushed solid particles, and collect the filter residue. Step 4: Precise purification with macroporous resin: Select AB-8 type macroporous resin, first soak it in 95% ethanol for 24 hours to activate it, then rinse it with distilled water until there is no ethanol residue, pack it into the chromatography column, ensuring that the resin is packed evenly and without air bubbles, dissolve the filter residue from step 3 with a small amount of ethanol, add an appropriate amount of distilled water to dilute it to a concentration of 1-2 mg / mL, slowly inject it into the chromatography column, control the sample loading rate at 1-2 mL / min, so that oleanolic acid is fully adsorbed by the resin, first rinse the chromatography column with distilled water at a flow rate of 2 mL / min for 2-3 column volumes to remove residual water-soluble impurities in the column; Then rinse with 30% ethanol to wash away some weakly adsorbed neutral impurities. This process does not require repeated impurity removal. Finally, use 70-80% ethanol as the eluent and elute at a flow rate of 1 mL / min. Collect the eluent. Step 5, Refining and Drying: Place the collected eluent in a vacuum concentration apparatus and concentrate it at 50℃ and a vacuum degree of 0.08-0.09 MPa to remove a small amount of ethanol, obtaining a concentrated oleanolic acid solution. Add a small amount of distilled water to the concentrate, stir well, and refrigerate overnight to allow oleanolic acid to crystallize. Filter and collect the crystals, wash them 2-3 times with a small amount of cold distilled water to further remove residual trace impurities, and place the crystals in a vacuum drying oven at 60℃ for 4-6 hours to obtain a white, high-quality oleanolic acid solution.

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