Method for extracting high-content oleanolic acid from olive leaves

Abstract

The invention relates to the technical field of natural product extraction, in particular to a method for extracting high-content oleanolic acid from olive leaves, which comprises the following steps: drying fresh olive leaves, crushing, extracting raw materials by using an ethanol solution through a hot reflux method, and filtering to obtain an oleanolic acid extract; concentrating the filtrate into extract, performing alkaline dissolution and acidification precipitation to obtain a crude product, performing macroporous resin adsorption purification, silica gel column chromatography and recrystallization purification treatment on the crude product in sequence, and performing vacuum drying to obtain the high-content oleanolic acid. Through the combination of the steps of macroporous resin purification, silica gel column chromatography, recrystallization and the like, the purity of the oleanolic acid can reach 95% or above, and the high-purity requirements in the fields of medicine, food and the like are met. And through mechanical application of the solvent and optimized process steps, the use amount of the solvent and production procedures are reduced, and the production cost is reduced. In addition, ethanol is adopted as a main extraction solvent, and the ethanol is volatile and environment-friendly, so that the pollution to the environment is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of natural product extraction, in particular to a method for extracting high-content oleanolic acid from olive leaves. Background Art

[0002] Oleanolic acid is a pentacyclic triterpenoid compound isolated and extracted from the whole herb of Swertia serrata or the fruit of Ligustrum lucidum (Gentianaceae). It exists in various plants as a free form and glycosides. Oleanolic acid is a broad-spectrum antibacterial agent clinically used to protect the liver and lower enzyme levels, and to treat bronchitis, pneumonia, acute tonsillitis, periodontitis, bacillary dysentery, acute gastroenteritis, and urinary tract infections.

[0003] Currently, the methods for extracting oleanolic acid from plants mainly include solvent extraction, ultrasonic-assisted extraction, and microwave-assisted extraction. However, these traditional methods have some shortcomings in practical applications. For example, the traditional solvent extraction method has a low extraction efficiency, and the purity of the oleanolic acid obtained is not high. It usually requires multiple purifications to reach a higher purity, which not only increases the production process and time, but also increases production costs. Moreover, during the purification process, the reagents used in some methods may be environmentally unfriendly, or the purification effect is unsatisfactory, resulting in unstable quality of the final product.

[0004] Therefore, there is an urgent need to develop an efficient, environmentally friendly method for extracting high-content oleanolic acid to meet the market demand for high-quality oleanolic acid. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a method for extracting high-content oleanolic acid from olive leaves, which can improve the extraction efficiency and purity of oleanolic acid, reduce production costs, and has the advantages of simple operation and environmental protection.

[0006] To achieve the above objectives, the present invention is implemented by the following technical solution: a method for extracting high-content oleanolic acid from olive leaves, comprising the following steps: S1. Raw material pretreatment Take fresh olive leaves and put them into an oven to dry at 40-50℃, and crush them into 40-60 mesh after drying. Drying fresh olive leaves can remove moisture from the leaves, making it easier to crush them in the subsequent crushing operation, while also preventing excessive moisture from causing the raw materials to deteriorate during storage. Controlling the drying temperature at 40-50℃ is to prevent excessive temperature from destroying the structure of oleanolic acid, affecting its activity and extraction effect. Crushing into 40-60 mesh can increase the surface area of ​​the raw material, increase the contact area between the solvent and the raw material in the subsequent extraction process, and thus improve the extraction efficiency; S2. Solvent Extraction The crushed raw material is extracted using a hot reflux method using an 85%-90% ethanol solution by volume. The extract is filtered and the filtrate is retained. The 85%-90% ethanol solution is chosen as the extraction solvent because oleanolic acid is a moderately polar triterpenic acid, and this concentration of ethanol has good solubility for it, enabling efficient extraction. The hot reflux method allows for continuous solvent circulation under heating conditions, improving extraction efficiency.

[0007] The process parameters for the hot reflux method are: a solid-liquid ratio of 1:8-12, an extraction temperature of 65-75°C, a reflux extraction time of 1.5-2.5 hours per extraction, and two extractions. The solvent from the second extraction is then used for the first extraction of the next batch of raw materials. The choice of solid-liquid ratio affects the solvent's ability to wet the raw materials and the dissolution of oleanolic acid. A solid-liquid ratio of 1:8-12 ensures efficient extraction while conserving solvent. Controlling the extraction temperature between 65-75°C accelerates the dissolution of oleanolic acid while preventing excessive ethanol volatilization and decomposition of oleanolic acid due to high temperatures. Each extraction time is 1.5-2.5 hours, ensuring that oleanolic acid is fully dissolved in the solvent. Performing two extractions and reusing the solvent from the second extraction for the first extraction of the next batch of raw materials reduces solvent usage, thereby lowering costs.

[0008] The filtration process uses centrifugal filtration or plate and frame filtration, and the filtration temperature is controlled at 50-60°C. Filtration at 50-60°C can avoid the precipitation of oleanolic acid due to low temperature, which increases the cost of repeated operations, and ensure the content of oleanolic acid in the filtrate; S3. Concentration and preliminary purification The filtrate is concentrated under reduced pressure to an extract form, and the crude product is obtained by sequentially performing the steps of dissolving impurities with cold water, dissolving with alkaline solution, and acidifying and precipitating. Concentration under reduced pressure can be performed at a lower temperature to reduce the loss of oleanolic acid.

[0009] Cold water dissolves impurities by stirring with purified water at 2-5 times the volume and a temperature of ≤20°C. Adjust the pH value to 10-12 for alkaline dissolution and to 2-3 for acidification precipitation. Dissolving impurities with low-temperature purified water can fully dissolve the impurities, while oleanolic acid has a low solubility at low temperatures, thereby achieving a preliminary separation of impurities and oleanolic acid. Adjusting the pH value to 10-12 can convert oleanolic acid into sodium salt and dissolve it, making it easier to separate it from insoluble impurities; adjusting the pH value to 2-3 again will convert sodium salt into oleanolic acid and precipitate, thus obtaining a crude product; S4. Deep Purification The crude product was purified by macroporous resin adsorption, silica gel column chromatography and recrystallization in sequence.

[0010] The specific steps for macroporous resin purification are as follows: Use D101, AB-8, or HPD-100 macroporous resins; after eluting impurities with warm water at 30-40°C, use a gradient elution of 30%-70% ethanol to elute the target component. These macroporous resins have excellent adsorption and separation properties for oleanolic acid. Elution with warm water at 30-40°C removes some water-soluble impurities. Then, a gradient elution of 30%-70% ethanol allows for gradual elution of oleanolic acid based on its solubility in different ethanol concentrations, improving purification efficiency.

[0011] The eluent for silica gel column chromatography is a petroleum ether-ethyl acetate mixed solvent, with the gradient elution ratio gradually adjusted from 15:1 to 3:1. Recrystallization uses a methanol-water or ethanol-water mixed solvent system, with 2 or more crystallizations. The petroleum ether-ethyl acetate mixed solvent gradient elution can further separate oleanolic acid from other impurities, improving its purity. Recrystallization can form purer crystals of oleanolic acid, and 2 or more crystallizations can ensure that the purity meets the requirements. S5. Drying and identification The purified product is vacuum dried at a temperature of 45-65°C, with a purity of ≥95%. Vacuum drying can be performed at a lower temperature to avoid decomposition of oleanolic acid due to high temperatures and ensure product quality. Purity is tested by HPLC, and at least one of the following optimization steps may also be included: Adding activated carbon decolorization treatment before acidification and precipitation can remove impurities such as pigments from the extract and improve the color and purity of the product. When using activated carbon decolorization, add 1-5g of activated carbon per liter of extract, stir at 50-70℃ for 30-60 minutes, and then filter to remove the activated carbon.

[0012] Adding preparative HPLC purification after silica gel column chromatography can further improve the purity of oleanolic acid. The preparative HPLC conditions can be adjusted according to actual conditions, such as using a C18 column, a column temperature of 30°C, a flow rate of 1.0-5.0 mL / min, a mobile phase of methanol-water, and a detection wavelength of 210 nm.

[0013] Combining mass spectrometry or nuclear magnetic resonance for structural confirmation during the drying and identification steps can more accurately determine the structure of the product and ensure the correctness of the product.

[0014] The present invention provides a method for extracting high-content oleanolic acid from olive leaves. The method has the following beneficial effects: 1. The present invention, through a combination of steps such as macroporous resin purification, silica gel column chromatography, and recrystallization, can achieve a purity of oleanolic acid of more than 95%, meeting the high-purity requirements in the fields of medicine and food. Furthermore, through the application of solvents and optimized process steps, the amount of solvent used and the production process are reduced, thereby lowering production costs. In addition, ethanol is used as the main extraction solvent, which is volatile and environmentally friendly, thereby reducing environmental pollution. DETAILED DESCRIPTION

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all 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.

[0016] Example 1: The embodiment of the present invention provides a method for extracting high-content oleanolic acid from olive leaves, comprising the following steps: S1. Raw material pretreatment Take 500g of fresh olive leaves, put them in an oven, and dry them at 45°C to constant weight. Put the dried olive leaves into a grinder and grind them into 50 mesh size for later use.

[0017] S2. Solvent Extraction Place the crushed olive leaf powder in a round-bottom flask and add 5000 mL of 88% ethanol (1:10 solid-liquid ratio). Extraction is performed using a hot reflux method. The extraction temperature is controlled at 70°C, and each reflux extraction is repeated for 2 hours. The solvent from the second extraction is collected and used for the first extraction of the next batch of raw materials. After the extraction is complete, the extract is filtered hot through a plate and frame filter at 55°C, and the filtrate is retained.

[0018] S3. Concentration and preliminary purification Transfer the filtrate to a rotary evaporator and concentrate under reduced pressure at -0.07 MPa until the extract is concentrated to an extract-like consistency (relative density approximately 1.15). Add 2500 mL of purified water (5 times the extract mass) at ≤20°C to the concentrated extract and stir for 2 hours to fully dissolve the impurities. Then, filter using a plate and frame filter to collect the solids. Add the solids to an appropriate amount of water and adjust the pH to 11 with NaOH solution to convert the oleanolic acid to sodium oleanolic acid, which dissolves the solids. Adjust the pH to 2.5 with HCl solution, acidify the precipitate, and collect the crude product by centrifugation.

[0019] S4. Deep Purification A D101 macroporous resin was pretreated and loaded into a chromatography column. The crude product was dissolved in an appropriate amount of water and loaded onto the column. Impurities were first eluted with warm water at 35°C until the elution became colorless. Gradient elution was then performed using 30%, 50%, and 70% ethanol solutions, and the eluate with the highest ethanol concentration was collected. The eluate was concentrated under reduced pressure to an appropriate volume and then subjected to silica gel column chromatography. The silica gel was 200-300 mesh, and the eluent was petroleum ether:ethyl acetate, with the gradient elution ratio gradually adjusted from 10:1 to 5:1. TLC monitoring was performed, and the eluate containing oleanolic acid was collected. After the eluate was concentrated under reduced pressure, a methanol:water (9:1 by volume) mixed solvent system was added, heated to dissolve, and then cooled to crystallize. This recrystallization process was repeated twice.

[0020] S5. Drying and identification The recrystallized product was placed in a vacuum drying oven and dried at 55°C to constant weight, yielding a white crystalline powder. Its purity was determined by HPLC using the following chromatographic conditions: a C18 column (250 mm × 4.6 mm, 5 μm), a column temperature of 30°C, a flow rate of 1.0 mL / min, a mobile phase of methanol-water (volume ratio 85:15), and a detection wavelength of 210 nm. The product purity was 97.5%.

[0021] Example 2: The embodiment of the present invention provides a method for extracting high-content oleanolic acid from olive leaves, comprising the following steps: S1. Raw material pretreatment Take 1000 g of fresh olive leaves, put them in an oven and dry them at 40°C to constant weight. Grind the dried olive leaves into 40 mesh and set aside.

[0022] S2. Solvent Extraction Place the crushed olive leaf powder in a round-bottom flask and add 8000 mL of 85% ethanol (1:8 solid-liquid ratio). Extraction is performed using reflux. The extraction temperature is controlled at 65°C, and each reflux extraction is repeated for 2.5 hours. The solvent from the second extraction is reused for the first extraction of the next batch of raw materials. After the extraction, the extract is centrifuged and filtered at 50°C. The filtrate is retained.

[0023] S3. Concentration and preliminary purification The filtrate was concentrated under reduced pressure at -0.06 MPa to an extract (relative density of approximately 1.1). 2000 mL of purified water (4 times the mass of the extract) at a temperature ≤ 20°C was added to the extract and stirred for 2 hours. The solids were then collected using a plate and frame filter press. The solids were dissolved in water and the pH was adjusted to 10 with NaOH solution and then to 3 with HCl solution. The precipitate was acidified and the crude product was collected by centrifugation.

[0024] S4. Deep Purification Select AB-8 macroporous resin and load it into a chromatography column. Dissolve the crude product and apply it to the sample. Elute impurities with 30°C warm water, followed by a gradient elution using 30%-70% ethanol. Collect and concentrate the eluate, then perform silica gel column chromatography using petroleum ether:ethyl acetate in a gradient elution ratio gradually adjusted from 15:1 to 3:1. Collect the desired eluate, concentrate it, and recrystallize it using a mixed solvent of ethanol:water (8:2 by volume). Repeat three times.

[0025] S5. Drying and identification The product was dried under vacuum at 45°C to constant weight to obtain a slightly yellow crystalline powder with a purity of 95.8% as determined by HPLC.

[0026] Example 3: The embodiment of the present invention provides a method for extracting high-content oleanolic acid from olive leaves, comprising the following steps: S1. Raw material pretreatment Take 800g of fresh olive leaves, put them in an oven, and dry them at 50°C to constant weight. Put the dried olive leaves into a grinder and grind them into 60 mesh size for later use.

[0027] S2. Solvent Extraction Place the crushed olive leaf powder in a round-bottom flask and add 9600 mL of 90% ethanol (1:12) to extract the leaves using reflux. The extraction temperature was controlled at 75°C, and each reflux extraction lasted 1.5 hours. This was repeated twice. The solvent from the second extraction was collected and used for the first extraction of the next batch of raw materials. After the extraction, the extract was filtered hot through a plate and frame filter at 55°C, and the filtrate was retained.

[0028] S3. Concentration and preliminary purification Transfer the filtrate to a rotary evaporator and concentrate under reduced pressure at -0.07 MPa until the extract is concentrated to an extract-like consistency (relative density approximately 1.2). Add 4000 mL of purified water (5 times the extract mass) at ≤20°C to the concentrated extract and stir for 2 hours to fully dissolve the impurities. Then, filter using a plate and frame filter to collect the solids. Add the solids to an appropriate amount of water and adjust the pH to 12 with NaOH solution to convert the oleanolic acid to sodium oleanolic acid, which dissolves the solids. Adjust the pH to 2 with HCl solution, acidify the precipitate, and collect the crude product by centrifugation.

[0029] S4. Deep Purification HPD-100 macroporous resin was pretreated and loaded into a chromatography column. The crude product was dissolved in an appropriate amount of water and loaded onto the column. Impurities were first eluted with warm water at 40°C until the elution became colorless. Gradient elution was then performed using 30%, 50%, and 70% ethanol solutions, and the eluate with the highest ethanol concentration was collected. The eluate was concentrated under reduced pressure to an appropriate volume and then subjected to silica gel column chromatography. The eluent used for column chromatography was petroleum ether:ethyl acetate, with the gradient elution ratio gradually adjusted from 10:1 to 5:1. TLC monitoring was performed, and the eluate containing oleanolic acid was collected. After the eluate was concentrated under reduced pressure, a methanol:water (8:2 by volume) mixed solvent system was added, heated to dissolve, and then cooled to crystallize. Recrystallization was repeated twice.

[0030] S5. Drying and identification The recrystallized product was placed in a vacuum drying oven and dried at 65°C to constant weight, yielding a white crystalline powder. HPLC analysis revealed a purity of 98.2%. Furthermore, prior to acidification and precipitation, activated carbon decolorization was performed by adding 3g of activated carbon per liter of extract, stirring at 60°C for 45 minutes, and filtering before subsequent operations. This resulted in a whiter product.

[0031] Comparative Example: A conventional solvent extraction method was used, with the same raw material pretreatment as in Example 1. Solvent extraction employed a 70% ethanol solution by volume, a solid-liquid ratio of 1:10, an extraction temperature of 60°C, and a single extraction without solvent application. Subsequent purification steps involved simple filtration and concentration, without macroporous resin purification, silica gel column chromatography, or recrystallization. After drying, the oleanolic acid purity was 78.3%.

[0032] By comparing Examples 1 to 3 with Comparative Example 1, it can be seen that the method provided by the present invention can significantly improve the extraction purity of oleanolic acid, and has obvious advantages.

[0033] 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 extracting high-content oleanolic acid from olive leaves, characterized in that: The following steps are involved: S1. Raw material pretreatment Take fresh olive leaves and put them into an oven to dry at 40-50℃, then crush them into 40-60 mesh size. S2. Solvent Extraction The crushed raw material is extracted by a hot reflux method using an ethanol solution with a volume fraction of 85% to 90%, and the extract is filtered and the filtrate is retained; S3. Concentration and preliminary purification The filtrate is concentrated under reduced pressure to an extract form, and the crude product is obtained by sequentially performing the steps of dissolving impurities with cold water, dissolving with alkaline solution, and precipitating by acidification; S4. Deep Purification The crude product was purified by macroporous resin adsorption, silica gel column chromatography and recrystallization in sequence; S5. Drying and identification The purified product was dried under vacuum and its purity was checked by HPLC.

2. A method for extracting high-content oleanolic acid from olive leaves according to claim 1, characterized in that: In the step S2, the process parameters of the thermal reflux method are: The material-liquid ratio is 1:(8-12), the extraction temperature is 65-75°C, each reflux extraction takes 1.5-2.5 hours, the extraction times are 2 times, and the solvent after the second extraction is used for the first extraction of the next batch of raw materials.

3. A method for extracting high-content oleanolic acid from olive leaves according to claim 1, characterized in that: In the step S2, the filtration process adopts centrifugal filtration or plate and frame filtration, and the filtration temperature is controlled at 50-60°C.

4. A method for extracting high-content oleanolic acid from olive leaves according to claim 1, characterized in that: In the step S3, the impurities are dissolved in cold water by stirring with purified water of 2-5 times the volume and a temperature of ≤20°C. The pH value is adjusted to 10-12 during alkaline dissolution and to 2-3 during acidification precipitation.

5. A method for extracting high-content oleanolic acid from olive leaves according to claim 1, characterized in that: In the S4 step, the specific steps of macroporous resin purification are as follows: Use D101, AB-8 or HPD-100 macroporous resin; After eluting impurities with warm water at 30-40°C, the target component was eluted with a gradient of 30%-70% ethanol.

6. A method for extracting high-content oleanolic acid from olive leaves according to claim 1, characterized in that: In the step S4, the eluent for silica gel column chromatography is a petroleum ether-ethyl acetate mixed solvent, and the gradient elution ratio is gradually adjusted from 15:1 to 3:1; the recrystallization adopts a methanol-water or ethanol-water mixed solvent system, and the number of crystallizations is ≥2 times.

7. A method for extracting high-content oleanolic acid from olive leaves according to claim 1, characterized in that: In the step S5, the vacuum drying temperature is 45-65° C., and the purity of the product after drying is ≥95%.

8. A method for extracting high-content oleanolic acid from olive leaves according to claim 1, characterized in that: Also includes at least one of the following optimization steps: Add activated carbon decolorization treatment before acidification and precipitation in step S3; In step S4, preparative HPLC purification was added after silica gel column chromatography; In step S5, the structure is confirmed by combining mass spectrometry or nuclear magnetic resonance.