Method for efficiently preparing oleanolic acid

Abstract

The invention discloses a method for preparing high-content oleanolic acid from chaenomeles speciosa, and belongs to the technical field of natural pharmaceutical chemistry. The method comprises the following specific steps: firstly, extracting a crude extract from chaenomeles speciosa by using a mixed solvent extraction method, and then carrying out multi-stage silica gel column chromatography separation, activated carbon decoloration and recrystallization to obtain high-content oleanolic acid. According to the invention, multiple mixed solvent extraction conditions are studied to obtain an optimal method, and it is found that the yield of oleanolic acid is up to 0.3060% and the content of oleanolic acid is up to 95.5% after the mixed solvent with the volume ratio of alcohol to ester being 1: 6 is used for extracting chaenomeles speciosa through proper treatment. The preparation process is fine, the conditions are mild, the extraction and separation efficiency is high, the loss is small, the content of the obtained oleanolic acid is high, the method is suitable for large-scale production, and the chaenomeles speciosa resources are fully utilized.

Description

Technical fields:

[0001] This invention belongs to the field of natural medicinal chemistry technology, specifically relating to a method for extracting the active ingredient oleanolic acid from Chaenomeles speciosa. Background technology:

[0002] Chinese quince, the fruit of the flowering quince (Chaenomeles speciosa) in the Rosaceae family, is produced in Xuancheng, Anhui Province. Li Shizhen, in his "Compendium of Materia Medica" during the Ming Dynasty, mentioned that "quinces are found everywhere, but those from Xuancheng are the best." Compared to quinces from other regions, the flesh of the Xuancheng quince is "thick, glutinous, purple, and oily," exhibiting better biological activity and higher medicinal value. It can relieve muscle tension, soothe the liver and relieve pain, strengthen the spleen and stomach, and dispel wind and dampness.

[0003] The main bioactive component of Chaenomeles speciosa is oleanolic acid, and it also contains ursolic acid, β-sitosterol, β-sitosterol glucoside, and hyperoside, among other effective components. Oleanolic acid is a pentacyclic triterpenoid compound with the molecular formula C2. 30 H 48 O3, with a molecular weight of 456, has the chemical formula shown in the figure below. Oleanolic acid appears as white, clustered needle-like crystals, colorless, tasteless, and odorless. It is insoluble in water but soluble in solvents such as methanol, ethanol, and acetone. It possesses anti-aging, anti-mutagenic, hydroxyl free radical scavenging, and abnormal protein metabolism corrective effects.

[0004]

[0005] Chinese patent CN102229638B discloses a method for separating and purifying oleanolic acid from Chaenomeles speciosa. This patent involves soaking Chaenomeles speciosa powder in an alcohol-water solution and using ultrasound-assisted extraction. The resulting extract is then extracted with a saturated aqueous solution of n-butanol. After further concentration, the extract is eluted using a macroporous resin with 20%, 50%, and 95% ethanol solutions as the mobile phase at a flow rate of 1.5 mL / min. The eluent is concentrated to obtain a crude product, which is then purified by column chromatography using 95% methanol as the eluent. The eluent is concentrated and evaporated to dryness to obtain high-purity oleanolic acid, with a purity of up to 97.5%, but the yield is low, only 0.123 g / 100 g. Chinese patent CN1919862A reports a method for preparing oleanolic acid from wrinkled papaya. This patent involves immersing dried wrinkled papaya powder in an alcohol-water solution and extracting it using percolation. The extract is then vortexed into a paste, dissolved in ethanol, and decolorized with an appropriate amount of activated carbon. The decolorized solution is then mixed with an appropriate amount of silica gel powder, evaporated, packed into a silica gel column, and eluted with a mixed solvent of petroleum ether and ethyl acetate to obtain oleanolic acid, with a yield of only 62 mg / 100 g. The literature "Study on Extraction and Separation of Major Physiologically Active Substances from Chaenomeles speciosa Fruit [D]. Zhang Guimin, Anhui Agricultural University, 2010" involves a different separation and purification method. This method involves ultrasonic-assisted extraction with 95% ethanol, followed by decolorization with activated carbon and rotary evaporation to obtain an extract. After dissolving in water, the extract is extracted three times with diethyl ether. The ether layer is then rotary evaporated to obtain the extract, followed by washing with water and chloroform layers. The washings are combined, the aqueous layer is discarded, and the chloroform layer is washed six times with water. The aqueous layer is discarded, and the chloroform layer is then distilled to obtain a solution containing oleanolic acid. The content is determined using semi-preparative high-performance liquid chromatography (HPLC) with a Phenomenex C18 column. The mobile phase is V(methanol):V(phosphoric acid aqueous solution, pH=2.0)=85:15, and the flow rate is 0.8 mL / min. The yield is 0.2%, and the purity is 57.08%. All of the aforementioned literature reports the preparation of oleanolic acid, but they generally use ethanol extraction as the sole solvent, resulting in incomplete extraction of oleanolic acid. Furthermore, the separation methods were not refined enough, leading to the failure to effectively separate oleanolic acid from other active ingredients, resulting in low yields reported in these studies. While the literature "Study on Extraction and Separation of Major Physiologically Active Substances from Chaenomeles speciosa Fruit" achieved a yield of 0.2%, its purity was only 57.08%, indicating that the separation and purification method needs further improvement. As a major bioactive component of Chaenomeles speciosa, oleanolic acid is present in low concentrations. Improving its yield and purity will play a crucial role in the development of Chaenomeles speciosa medicinal materials.

[0006] This application utilizes the principle of "like dissolves like" to efficiently extract the effective components from Chaenomeles speciosa using a mixed solvent. Subsequently, a multi-stage separation process is employed to efficiently separate oleanolic acid. This method yields oleanolic acid with high purity. Oleanolic acid has various isomers, such as ursolic acid and betulinic acid, all belonging to the pentacyclic triterpenoid class of compounds. In the extraction and separation of oleanolic acid from Chaenomeles speciosa, oleanolic acid often appears mixed with ursolic acid, resulting in lower purity of the prepared oleanolic acid. Currently, semi-preparative high-performance liquid chromatography (HPLC) is commonly used to separate the two substances, typically achieving a purity of 95%–98%. However, this separation method involves high equipment costs, complex operation, and demanding maintenance, making it unsuitable for industrial production. This method employs multi-stage extraction and separation to effectively separate the two substances. This method first utilizes the difference in solubility of the two substances in chloroform for primary separation. The chloroform layer contains only a small amount of ursolic acid, resulting in extremely high purity oleanolic acid (approximately 98%) after column chromatography purification. During extraction, a small amount of oleanolic acid may not be completely extracted. Since oleanolic acid has good solubility in ethyl acetate, a second extraction with ethyl acetate is used. Acetone-petroleum ether is used as the eluent to significantly separate oleanolic acid and ursolic acid, maximizing the yield of oleanolic acid from *Chaenomeles speciosa* and simultaneously obtaining high-purity ursolic acid. This method utilizes the principle of "like dissolves like" and multi-stage separation techniques to greatly improve the purity of oleanolic acid while ensuring high yield. Furthermore, experimental data shows that the yield of oleanolic acid prepared using this method is significantly higher than that reported in existing literature. The separation method is simple and easy to operate, with mild conditions and low cost, and can yield high-content oleanolic acid. This method provides important technical support for the preparation of high-content, high-purity oleanolic acid from Chaenomeles speciosa. Currently, there are no related literature or patent reports. Summary of the Invention:

[0007] The purpose of this invention is to provide a method for preparing oleanolic acid with high yield and high purity. A mixed solvent is used to extract the active ingredient from Chaenomeles speciosa, resulting in a high yield. Multi-stage separation is employed to separate and purify the crude extract, yielding oleanolic acid with high purity. The specific technical solution of this invention is as follows:

[0008] A method for efficiently preparing oleanolic acid, the specific steps of which are as follows:

[0009] (1) Dry the papaya, pulverize it, and pass it through a 60-mesh sieve to obtain papaya powder; weigh the papaya powder, put it into a flask, add mixed solvent A, keep the ratio of the mass of the papaya powder to the volume of the total extraction solvent within a certain range, heat to a certain temperature, reflux and extract 3 times, 1 hour each time; combine the extracts, then rotary evaporate the extract to recover the organic solvent, and the resulting extract is the crude extract of papaya.

[0010] (2) Dissolve the crude extract of Chaenomeles speciosa in water until it is completely dissolved, then add chloroform of the same volume as water for extraction. Take the extracted chloroform phase and add a certain amount of 100-300 mesh silica gel powder to rotary evaporation and mixing to obtain a mixture of chloroform layer extract and silica gel powder. Pack the mixture into a silica gel column, and the amount of silica gel used in the column is a certain multiple of the mixture.

[0011] (3) Take the aqueous layer extracted in step (2) and add an equal amount of ethyl acetate for extraction again. Take the extracted ethyl acetate phase and add a certain amount of 100-300 mesh silica gel powder. Stir and mix by rotary evaporation to obtain a mixture of ethyl acetate layer extract and silica gel powder. Pack the mixture into a silica gel column. The amount of silica gel used in the column is a certain multiple of the mixture.

[0012] (4) Use mixed solvent B as the eluent to elute the silica column in step (2), and after a large amount of pigment, β-sitosterol and other substances are eluted, continue to elute the sample with mixed solvent C to obtain eluent C. Rotary evaporate eluent C to obtain a green powder containing oleanolic acid.

[0013] (5) Use mixed solvent D as the eluent to elute the silica column in step (3). After eluting out ursolic acid and other substances, continue to elute the sample with mixed solvent E to obtain eluent E. Rotary evaporate eluent E to obtain a green powder containing oleanolic acid.

[0014] (6) Dissolve the green oleanolic acid powder containing the oleanolic acid from steps (4) and (5) completely with anhydrous ethanol, add an appropriate amount of activated carbon, heat and stir at a certain temperature for 1 hour, filter to obtain an ethanol solution of oleanolic acid, evaporate to dryness and recrystallize with 95% ethanol to obtain oleanolic acid.

[0015] Furthermore, the mixed solvent A mentioned in step (1) above is a mixed solvent in which the volume ratio of ethanol to ethyl acetate is 1:6;

[0016] In step (1) above, the ratio of the mass of papaya powder to the total mass-volume of the extraction solvent is in the range of 1:(18~20)(g:mL);

[0017] In step (1) above, the extraction temperature of Chaenomeles speciosa is 80℃~85℃;

[0018] The amount of silica powder added in steps (2) and (3) above is twice the amount of crude extract from the papaya extract layer, and the amount of silica gel used for column packing is 2 to 4 times the amount of silica powder mixed powder.

[0019] In steps (4) and (5) above, the mixed solvent B is a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 8:1, the mixed solvent C is a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 1:1, the mixed solvent D is a mixed solvent with a volume ratio of petroleum ether to acetone of 5:1, and the mixed solvent E is a mixed solvent with a volume ratio of petroleum ether to acetone of 3:1.

[0020] In step (6) above, the activated carbon is heated and stirred at 50°C, and the appropriate mass ratio of activated carbon to green oleanolic acid powder is 1:4.

[0021] This invention provides an efficient method for preparing oleanolic acid, which improves the extraction rate of oleanolic acid and results in high purity oleanolic acid after separation by using a relatively simple extraction and purification method. Attached image description:

[0022] Appendix Figure 1 Photo of an oleanolic acid sample.

[0023] Appendix Figure 2 This is the ESI-MS (positive) mass spectrum of oleanolic acid.

[0024] Appendix Figure 3 For oleanolic acid 1 H-NMR spectrum.

[0025] Appendix Figure 4 This is the UV absorption spectrum of oleanolic acid.

[0026] Appendix Figure 5 This is a microscopic image of oleanolic acid crystals.

[0027] Among them, by appendix Figure 1 It can be seen that the prepared oleanolic acid is a white powder. Figure 2 The ESI-MS (positive) m / z value is 455 [MH]. + Therefore, the molecular weight of this substance is 456. Figure 3 of 1 The H-NMR spectrum determined the molecular formula of the substance to be C. 30 H 48 O3. Figure 4 The UV absorption spectrum of the solution is obtained by dissolving green oleanolic acid powder in ethanol, with the absorption peak located at 211 nm. Figure 5 It can be seen that oleanolic acid is in the form of needle-like crystals. In summary, this method can be used to prepare high-purity oleanolic acid. Detailed implementation method:

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0029] Example 1: Method for preparing ethanol using a single solvent

[0030] (1) The papaya was dried in an oven at 60℃ for 8 hours, pulverized, and passed through a 60-mesh sieve to obtain papaya powder. 100g of the powder was placed in a 2L round-bottom flask, 800mL of 95% ethanol was added, and the mixture was heated and extracted for 1 hour. The extract was then separated. Subsequently, 600mL of 95% ethanol was added to the round-bottom flask, and the mixture was heated and extracted for 1 hour. The extract was then separated for the second time. 600mL of 95% ethanol was added again, and the mixture was heated and extracted for 1 hour. The extract was then separated for the third time. The three extracts were combined, and the solvent was recovered by rotary evaporation at 55℃ to obtain 25.6g of papaya extract, which is the crude extract of papaya.

[0031] (2) Dissolve the crude extract of Chaenomeles speciosa in 250 mL of water, add 250 mL of chloroform for extraction, take the chloroform layer and add 10 g of 100 mesh silica powder and stir to mix, remove the solvent by rotary evaporation at 55 °C, and obtain a mixed powder of chloroform layer extract and silica powder. Pack the mixed powder into a silica column.

[0032] (3) Take the aqueous phase remaining after extraction in the above steps, add 250 mL of ethyl acetate and extract again. Then take the ethyl acetate layer, add 12 g of 100 mesh silica powder and stir to mix. Remove the solvent by rotary evaporation at 55°C to obtain a mixed powder of ethyl acetate layer extract and silica powder. Pack the mixed powder into a silica column.

[0033] (4) Take 1L of mixed solvent B to elute the silica gel column with chloroform layer, remove pigment and β-sitosterol, and then use 600mL of mixed solvent C to elute oleanolic acid to obtain eluent. Rotary evaporate the C eluent to obtain green powder containing oleanolic acid.

[0034] (5) Take 800 mL of mixed solvent D to elute the silica column containing ethyl acetate, remove ursolic acid and other substances, and then use 600 mL of mixed solvent E to elute oleanolic acid to obtain the eluent. Rotary evaporate the eluent E to obtain a green powder containing oleanolic acid.

[0035] (6) Dissolve the green oleanolic acid powder containing a total of 0.32 g from steps (4) and (5) completely with anhydrous ethanol, add 0.064 g of activated carbon, heat and stir at 50 °C for 1 h, filter to obtain an ethanol solution of oleanolic acid, evaporate to dryness and recrystallize with 95% ethanol to obtain 0.2849 g of oleanolic acid needle crystals with a content of 91.1% and a yield of 0.2849%.

[0036] Example 2: Preparation method of ethyl acetate using a single solvent

[0037] (1) The papaya was dried in an oven at 60℃ for 8 hours, pulverized, and passed through a 60-mesh sieve to obtain papaya powder. 100g of the powder was placed in a 2L round-bottom flask, 800mL of 95% ethanol was added, and the mixture was heated and extracted for 1 hour. The extract was then separated. Subsequently, 600mL of 95% ethanol was added to the round-bottom flask, and the mixture was heated and extracted for 1 hour. The extract was then separated for the second time. 600mL of 95% ethanol was added again, and the mixture was heated and extracted for 1 hour. The extract was then separated for the third time. The three extracts were combined, and the solvent was recovered by rotary evaporation at 55℃ to obtain 23.5g of papaya extract, which is the crude extract of papaya.

[0038] (2) Dissolve the crude extract of Chaenomeles speciosa in 250 mL of water, add 250 mL of chloroform for extraction, take the chloroform layer and add 9.2 g of 100 mesh silica powder and stir to mix, remove the solvent by rotary evaporation at 55 °C, and obtain a mixed powder of chloroform layer extract and silica powder. Pack the mixed powder into a silica column.

[0039] (3) Take the aqueous phase remaining after extraction in the above steps, add 250 mL of ethyl acetate and extract again. Then take the ethyl acetate layer and add 11.3 g of 100 mesh silica powder and stir to mix. Remove the solvent by rotary evaporation at 55°C to obtain a mixed powder of ethyl acetate layer extract and silica powder. Pack the mixed powder into a silica column.

[0040] (4) Take 1L of mixed solvent B to elute the silica gel column with chloroform layer, remove pigment and β-sitosterol, and then use 600mL of mixed solvent C to elute oleanolic acid to obtain eluent. Rotary evaporate the C eluent to obtain green powder containing oleanolic acid.

[0041] (5) Take 800 mL of mixed solvent D to elute the silica column containing ethyl acetate, remove ursolic acid and other substances, and then use 600 mL of mixed solvent E to elute oleanolic acid to obtain the eluent. Rotary evaporate the eluent E to obtain a green powder containing oleanolic acid.

[0042] (6) Dissolve the green oleanolic acid powder containing a total of 0.3025 g from steps (4) and (5) completely with anhydrous ethanol, add 0.0605 g of activated carbon, heat and stir at 50 °C for 1 h, filter to obtain an ethanol solution of oleanolic acid, evaporate to dryness and recrystallize with 95% ethanol to obtain 0.2692 g of needle-like oleanolic acid crystals with a content of 93.7% and a yield of 0.2692%.

[0043] Example 3: Preparation method of mixed solvent with an alcohol-ester volume ratio of 1:3

[0044] (1) The papaya was dried in an oven at 60℃ for 8 hours, pulverized, and passed through a 60-mesh sieve to obtain papaya powder. 100g of the powder was placed in a 2L round-bottom flask, 800mL of 95% ethanol was added, and the mixture was heated and extracted for 1 hour. The extract was then separated. Subsequently, 600mL of 95% ethanol was added to the round-bottom flask, and the mixture was heated and extracted for 1 hour. The extract was then separated for the second time. 600mL of 95% ethanol was added again, and the mixture was heated and extracted for 1 hour. The extract was then separated for the third time. The three extracts were combined, and the solvent was recovered by rotary evaporation at 55℃ to obtain 24.3g of papaya extract, which is the crude extract of papaya.

[0045] (2) Dissolve the crude extract of Chaenomeles speciosa in 250 mL of water, add 250 mL of chloroform for extraction, take the chloroform layer and add 9.6 g of 100 mesh silica powder and stir to mix, remove the solvent by rotary evaporation at 55 °C, and obtain a mixed powder of chloroform layer extract and silica powder. Pack the mixed powder into a silica column.

[0046] (3) Take the aqueous phase remaining after extraction in the above steps, add 250 mL of ethyl acetate and extract again. Then take the ethyl acetate layer and add 11.8 g of 100 mesh silica powder and stir to mix. Remove the solvent by rotary evaporation at 55°C to obtain silica powder adsorbing the sample. A mixed powder of ethyl acetate layer extract and silica powder is obtained. The mixed powder is loaded into a silica column.

[0047] (4) Take 1L of mixed solvent B to elute the silica gel column with chloroform layer, remove pigment and β-sitosterol, and then use 600mL of mixed solvent C to elute oleanolic acid to obtain eluent. Rotary evaporate the C eluent to obtain green powder containing oleanolic acid.

[0048] (5) Take 800 mL of mixed solvent D to elute the silica column containing ethyl acetate, remove ursolic acid and other substances, and then use 600 mL of mixed solvent E to elute oleanolic acid to obtain the eluent. Rotary evaporate the eluent E to obtain a green powder containing oleanolic acid.

[0049] (6) Dissolve the green oleanolic acid powder containing a total of 0.3089 g from steps (4) and (5) in anhydrous ethanol. Add 0.0618 g of activated carbon, heat and stir at 50 °C for 1 h, filter to obtain an ethanol solution of oleanolic acid, evaporate to dryness, and recrystallize with 95% ethanol to obtain 0.2749 g of needle-like oleanolic acid crystals with a content of 94.2% and a yield of 0.2749%.

[0050] Example 4: Preparation method of mixed solvent with an alcohol-ester volume ratio of 1:5

[0051] (1) The papaya was dried in an oven at 60℃ for 8 hours, pulverized, and passed through a 60-mesh sieve to obtain papaya powder. 100g of the powder was placed in a 2L round-bottom flask, 800mL of 95% ethanol was added, and the mixture was heated for 1 hour. The extract was then separated. Subsequently, 600mL of 95% ethanol was added to the round-bottom flask, and the mixture was heated for 1 hour. The extract was then separated for the second time. 600mL of 95% ethanol was added again, and the mixture was heated for 1 hour. The extract was then separated for the third time. The three extracts were combined, and the solvent was recovered by rotary evaporation at 55℃ to obtain 26g of papaya extract, which is the crude extract of papaya.

[0052] (2) Dissolve the crude extract of Chaenomeles speciosa in 250 mL of water, add 250 mL of chloroform for extraction, take the chloroform layer and add 10.6 g of 100 mesh silica powder and stir to mix, remove the solvent by rotary evaporation at 55 °C, and obtain a mixed powder of chloroform layer extract and silica powder. Pack the mixed powder into a silica column.

[0053] (3) Take the aqueous phase remaining after extraction in the above steps, add 250 mL of ethyl acetate and extract again. Then take the ethyl acetate layer and add 12.5 g of 100 mesh silica powder and stir to mix. Remove the solvent by rotary evaporation at 55°C to obtain a mixed powder of ethyl acetate layer extract and silica powder. Pack the mixed powder into a silica column.

[0054] (4) Take 1L of mixed solvent B to elute the silica gel column with chloroform layer, remove pigment and β-sitosterol, and then use 600mL of mixed solvent C to elute oleanolic acid to obtain eluent. Rotary evaporate the C eluent to obtain green powder containing oleanolic acid.

[0055] (5) Take 800 mL of mixed solvent D to elute the silica column containing ethyl acetate, remove ursolic acid and other substances, and then use 600 mL of mixed solvent E to elute oleanolic acid to obtain the eluent. Rotary evaporate the eluent E to obtain a green powder containing oleanolic acid.

[0056] (6) Dissolve the green oleanolic acid powder containing a total of 0.325 g from steps (4) and (5) in anhydrous ethanol. Add 0.065 g of activated carbon, heat and stir at 50 °C for 1 h, filter to obtain an ethanol solution of oleanolic acid, evaporate to dryness, and recrystallize with 95% ethanol to obtain 0.2922 g of oleanolic acid needle crystals with a content of 94.7% and a yield of 0.2922%.

[0057] Example 5: Preparation method of a mixed solvent with an alcohol-ester volume ratio of 1:6 (preferred method)

[0058] (1) The papaya was dried in an oven at 60℃ for 8 hours, pulverized, and passed through a 60-mesh sieve to obtain papaya powder. 100g of the powder was placed in a 2L round-bottom flask, 800mL of 95% ethanol was added, and the mixture was heated and extracted for 1 hour. The extract was then separated. Subsequently, 600mL of 95% ethanol was added to the round-bottom flask, and the mixture was heated and extracted for 1 hour. The extract was then separated for the second time. 600mL of 95% ethanol was added again, and the mixture was heated and extracted for 1 hour. The extract was then separated for the third time. The three extracts were combined, and the solvent was recovered by rotary evaporation at 55℃ to obtain 30.3g of papaya extract, which is the crude extract of papaya.

[0059] (2) Dissolve the crude extract of Chaenomeles speciosa in 250 mL of water, add 250 mL of chloroform for extraction, take the chloroform layer and add 12.2 g of 100 mesh silica powder and stir to mix, remove the solvent by rotary evaporation at 55 °C, and obtain a mixed powder of chloroform layer extract and silica powder. Pack the mixed powder into a silica column.

[0060] (3) Take the aqueous phase remaining after extraction in the above steps, add 250 mL of ethyl acetate and extract again. Then take the ethyl acetate layer and add 13.9 g of 100 mesh silica powder and stir to mix. Remove the solvent by rotary evaporation at 55°C to obtain a mixed powder of ethyl acetate layer extract and silica powder. Pack the mixed powder into a silica column.

[0061] (4) Take 1L of mixed solvent B to elute the silica gel column with chloroform layer, remove pigment and β-sitosterol, and then use 600mL of mixed solvent C to elute oleanolic acid to obtain eluent. Rotary evaporate the C eluent to obtain green powder containing oleanolic acid.

[0062] (5) Take 800 mL of mixed solvent D to elute the silica column containing ethyl acetate, remove ursolic acid and other substances, and then use 600 mL of mixed solvent E to elute oleanolic acid to obtain the eluent. Rotary evaporate the eluent E to obtain a green powder containing oleanolic acid.

[0063] (6) Dissolve the green oleanolic acid powder containing a total of 0.34 g from steps (4) and (5) completely with anhydrous ethanol, add 0.068 g of activated carbon, heat and stir at 50 °C for 1 h, filter to obtain an ethanol solution of oleanolic acid, evaporate to dryness and recrystallize with 95% ethanol to obtain 0.3060 g of needle-like crystals of oleanolic acid with a content of 95.5% and a yield of 0.3060%.

[0064] Example 6: Preparation method of mixed solvent with an alcohol-ester volume ratio of 1:8

[0065] (1) The papaya was dried in an oven at 60℃ for 8 hours, pulverized, and passed through a 60-mesh sieve to obtain papaya powder. 100g of the powder was placed in a 2L round-bottom flask, 800mL of 95% ethanol was added, and the mixture was heated and extracted for 1 hour. The extract was then separated. Subsequently, 600mL of 95% ethanol was added to the round-bottom flask, and the mixture was heated and extracted for 1 hour. The extract was then separated for the second time. 600mL of 95% ethanol was added again, and the mixture was heated and extracted for 1 hour. The extract was then separated for the third time. The three extracts were combined, and the solvent was recovered by rotary evaporation at 55℃ to obtain 25.8g of papaya extract, which is the crude extract of papaya.

[0066] (2) Dissolve the crude extract of Chaenomeles speciosa in 250 mL of water, add 250 mL of chloroform for extraction, take the chloroform layer and add 10.3 g of 100 mesh silica powder to stir and mix, remove the solvent by rotary evaporation at 55 °C, and obtain a mixed powder of chloroform layer extract and silica powder. Pack the mixed powder into a silica column.

[0067] (3) Take the aqueous phase remaining after extraction in the above steps, add 250 mL of ethyl acetate and extract again. Then take the ethyl acetate layer and add 12.2 g of 100 mesh silica powder and stir to mix. Remove the solvent by rotary evaporation at 55°C to obtain a mixed powder of ethyl acetate layer extract and silica powder. Pack the mixed powder into a silica column.

[0068] (4) Take 1L of mixed solvent B to elute the silica gel column with chloroform layer, remove pigment and β-sitosterol, and then use 600mL of mixed solvent C to elute oleanolic acid to obtain eluent. Rotary evaporate the C eluent to obtain green powder containing oleanolic acid.

[0069] (5) Take 800 mL of mixed solvent D to elute the silica column containing ethyl acetate, remove ursolic acid and other substances, and then use 600 mL of mixed solvent E to elute oleanolic acid to obtain the eluent. Rotary evaporate the eluent E to obtain a green powder containing oleanolic acid.

[0070] (6) Dissolve the green oleanolic acid powder containing a total of 0.322 g from steps (4) and (5) completely with anhydrous ethanol, add 0.065 g of activated carbon, heat and stir at 50 °C for 1 h, filter to obtain an ethanol solution of oleanolic acid, evaporate to dryness, and then recrystallize with 95% ethanol to obtain 0.2866 g of needle-like oleanolic acid crystals with a content of 94.5% and a yield of 0.2866%.

[0071] Table 1. Yield and content of oleanolic acid in Examples 1 to 6

[0072]

[0073] In summary, this invention utilizes a mixed solvent extraction method to extract crude extract from Chaenomeles speciosa, followed by multi-stage silica gel column chromatography, activated carbon decolorization, and recrystallization to obtain a high-purity oleanolic acid. Mass spectrometry, nuclear magnetic resonance (NMR), and ultraviolet spectrophotometry confirmed that the prepared product is high-purity oleanolic acid. As shown in Table 1, compared to single solvent extraction, Example 5, using a mixed solvent with an ethanol to ethyl acetate volume ratio of 1:6, yielded the highest oleanolic acid yield and total mass, reaching 0.3066% and 0.2922 g, respectively. In terms of content, Example 5 also showed that the ethanol to ethyl acetate volume ratio of 1:6 was the optimal choice, achieving a content as high as 95.5%. Compared to the oleanolic acid obtained from Chaenomeles speciosa in patent CN102229638B, the yield of this method is approximately 2.5 times higher. Compared with the literature "Study on Extraction and Separation of Major Physiologically Active Substances from Chaenomeles speciosa Fruit [D]. Zhang Guimin, Anhui Agricultural University, 2010," the optimal method of this invention nearly doubles the yield of oleanolic acid, and the purity is much higher than the reported value of 57.08%. The content of oleanolic acid in Chaenomeles speciosa is low. The extraction method of this invention greatly improves the extraction rate of oleanolic acid, and the separation method is simple, highly pure, and superior to the methods reported in the current literature.

[0074] Therefore, this invention provides a method for efficiently preparing oleanolic acid from Chaenomeles speciosa. The preparation process of this invention is relatively refined, the conditions are mild, it does not require large-scale equipment, it has high separation efficiency, and it ensures high purity while achieving high yield, making full use of Chaenomeles speciosa resources.

[0075] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for efficiently preparing oleanolic acid, characterized in that, The specific steps are as follows: (1) Dry the papaya, pulverize it, and pass it through a 60-mesh sieve to obtain papaya powder; weigh the papaya powder, put it into a flask, add mixed solvent A, keep the ratio of the mass of the papaya powder to the volume of the total extraction solvent within a certain range, heat to a certain temperature, reflux and extract 3 times, 1 hour each time; combine the extracts, then rotary evaporate the extract to recover the organic solvent, and the resulting extract is the crude extract of papaya; (2) Dissolve the crude extract of Chaenomeles speciosa in water until it is completely dissolved, then add chloroform of the same volume as water for extraction. Take the extracted chloroform phase and add a certain amount of 100-300 mesh silica gel powder to rotary evaporation and mix well to obtain a mixture of chloroform layer extract and silica gel powder. Pack it into a silica gel column, and the amount of silica gel used in the column is a certain multiple of the mixture. (3) Take the water layer extracted in step (2) and add an equal amount of ethyl acetate for extraction again. Take the ethyl acetate phase after extraction and add a certain amount of 100-300 mesh silica gel powder. Stir and mix by rotary evaporation to obtain a mixture of ethyl acetate layer extract and silica gel powder. Pack the mixture into a silica gel column. The amount of silica gel used in the column is a certain multiple of the mixture. (4) Use mixed solvent B as the eluent to elute the silica column in step (2), and after a large amount of pigment, β-sitosterol and other substances are eluted, continue to elute the sample with mixed solvent C to obtain eluent C. Rotary evaporate eluent C to obtain green powder containing oleanolic acid. (5) Use mixed solvent D as the eluent to elute the silica column in step (3). After eluting out ursolic acid and other substances, continue to elute the sample with mixed solvent E to obtain eluent E. Rotary evaporate eluent E to obtain green powder containing oleanolic acid. (6) Dissolve the green powder containing oleanolic acid from steps (4) and (5) completely with anhydrous ethanol, add an appropriate amount of activated carbon, heat and stir at a certain temperature for 1 hour, filter to obtain an ethanol solution of oleanolic acid, evaporate to dryness and recrystallize with 95% ethanol to obtain oleanolic acid. The mixed solvent A mentioned in step (1) above is a mixed solvent in which the volume ratio of ethanol to ethyl acetate is 1:6; In step (1) above, the ratio of the mass of papaya powder to the total mass-volume of the extraction solvent is in the range of 1:(18~20)(g:mL); In step (1) above, the extraction temperature of Chaenomeles speciosa is 80℃~85℃; The amount of silica powder added in steps (2) and (3) above is twice the amount of crude extract from the papaya extract layer, and the amount of silica gel used for column packing is 2 to 4 times the amount of silica powder mixed powder. In steps (4) and (5) above, the mixed solvent B is a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 8:1, the mixed solvent C is a mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 1:1, the mixed solvent D is a mixed solvent with a volume ratio of petroleum ether to acetone of 5:1, and the mixed solvent E is a mixed solvent with a volume ratio of petroleum ether to acetone of 3:

1. In step (6) above, the activated carbon is heated and stirred at 50°C, and the appropriate mass ratio of activated carbon to green oleanolic acid powder is 1:

4.

2. As described in claim 1, a method for efficiently preparing oleanolic acid, wherein the preferred method is characterized in that the yield of oleanolic acid reaches 0.3060% and the content reaches 95.5%.

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