A green method for the extraction of ursolic acid from hawthorn

Abstract

The present application relates to ursolic acid extraction technical field, propose a kind of green method for extracting ursolic acid from hawthorn, comprising the following steps: S1, hawthorn powder is added to hydrophobic low eutectic solvent, ultrasonic extraction, filtration, obtain extract;S2, extract is purified by fractional elution by chromatographic separation column, obtain primary eluent and secondary eluent, primary eluent is concentrated, centrifuged, obtain recovered hydrophobic low eutectic solvent;Secondary eluent is concentrated, dried, obtain ursolic acid product;Hydrophobic low eutectic solvent includes hydrogen bond donor and hydrogen bond acceptor;Hydrogen bond donor is thymol;Hydrogen bond acceptor includes pyruvic acid.Through the above technical scheme, the problem of low ursolic acid extraction rate and insufficient purity in the prior art is solved.

Description

Technical Field

[0001] This invention relates to the field of ursolic acid extraction technology, specifically to a green method for extracting ursolic acid from hawthorn. Background Technology

[0002] Ursolic acid is a natural active ingredient with a pentacyclic triterpenoid structure, widely found in plants such as hawthorn, privet, and bearberry. It possesses various pharmacological activities, including anti-inflammatory, antioxidant, lipid-regulating, liver-protective, and anti-tumor effects, making it highly valuable for applications. Hawthorn, a traditional food and medicine source, is abundant and inexpensive, making it an ideal raw material for extracting ursolic acid. Therefore, developing efficient and green ursolic acid extraction technology from hawthorn has significant practical importance and market prospects.

[0003] Eutectic solvents, as a novel type of green solvent, are increasingly being used in the extraction of natural products. Currently, most eutectic solvents used for ursolic acid extraction are hydrophilic systems (such as combinations based on choline chloride-ethylene glycol, choline chloride-lactic acid, etc.). These hydrophilic eutectic solvents are easily miscible with water in hawthorn, leading to decreased solvent system stability and affecting the smooth progress of the extraction process. Furthermore, while hydrophilic solvents have a strong ability to dissolve hydrophilic impurities, they have poor solubility for ursolic acid, resulting in a low extraction rate. Therefore, a green method for extracting ursolic acid from hawthorn with a high extraction rate is urgently needed. Summary of the Invention

[0004] This invention proposes a green method for extracting ursolic acid from hawthorn, which solves the problems of low extraction rate and insufficient purity of ursolic acid in related technologies.

[0005] The technical solution of the present invention is as follows: This invention proposes a green method for extracting ursolic acid from hawthorn, comprising the following steps: S1. Add hawthorn powder to a hydrophobic eutectic solvent, extract by ultrasonication, filter, and obtain the extract; S2. The extract is purified by fractional elution using a chromatographic column to obtain a primary eluent and a secondary eluent. The primary eluent is concentrated and centrifuged to obtain the recovered hydrophobic eutectic solvent. The secondary eluent is concentrated and dried to obtain the ursolic acid product. The hydrophobic eutectic solvent includes hydrogen bond donors and hydrogen bond acceptors; The hydrogen bond donor is thymol; the hydrogen bond acceptor includes pyruvate.

[0006] As a further technical solution, the preparation method of the hawthorn powder includes the following steps: drying fresh hawthorn, pulverizing and sieving, and drying again to obtain the hawthorn powder.

[0007] As a further technical solution, the preparation method of the hydrophobic eutectic solvent includes the following steps: mixing thymol and the hydrogen bond acceptor, stirring, to obtain the hydrophobic eutectic solvent.

[0008] As a further technical solution, when the hydrogen bond acceptor is pyruvic acid, the molar ratio of thymol to pyruvic acid is 1:1~2.

[0009] As a further technical solution, the stirring temperature is 70~80℃, and the stirring time is 30~40min.

[0010] As a further technical solution, the mass-to-volume ratio of the hawthorn powder and the hydrophobic eutectic solvent is 1g:5~10mL.

[0011] As a further technical solution, the temperature of the ultrasonic extraction is 50~60℃, and the ultrasonic extraction time is 30~40min.

[0012] As a further technical solution, the hydrogen bond acceptor also includes undecenoic acid.

[0013] In this invention, the extraction rate of ursolic acid is further improved by adding undecenoic acid and pyruvate together as hydrogen bond acceptors. The addition of undecenoic acid improves the hydrophobicity of the eutectic solvent, enhances its solubility for ursolic acid, and enables more precise and selective extraction of ursolic acid. At the same time, the long-chain structure of undecenoic acid can reduce the viscosity of the solvent to a certain extent, resulting in higher mass transfer efficiency during extraction and better flowability when subsequently loaded onto macroporous resin, reducing problems such as clogging or excessively slow flow rates.

[0014] As a further technical solution, the molar ratio of thymol to undecenoic acid is 1:0.5~1.

[0015] As a further technical solution, the chromatographic separation column is a reversed-phase chromatographic separation column.

[0016] As a further technical solution, the eluent used in the elution and purification process is an aqueous ethanol solution.

[0017] The working principle and beneficial effects of this invention are as follows: In this invention, thymol and pyruvate are used as hydrophobic eutectic solvents to extract ursolic acid from hawthorn, thereby improving the extraction rate of ursolic acid. Ursolic acid is a lipophilic, weakly polar, weakly acidic pentacyclic triterpenoid compound. Therefore, hydrophobic eutectic solvents exhibit higher selectivity for extracting lipophilic ursolic acid compared to hydrophilic eutectic solvents, reducing the dissolution of water-soluble impurities. In the hydrophobic eutectic solvent of this invention, thymol provides a hydrophobic framework, while pyruvate introduces polar carbonyl and carboxyl groups, making the overall polarity similar to ursolic acid, thus improving the solubility of ursolic acid in the eutectic solvent. Furthermore, pyruvate can form intermolecular hydrogen bonds with ursolic acid, further enhancing the solvent's ability to dissolve ursolic acid. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] In the following examples and comparative examples, the mass percentage of ursolic acid in the hawthorn powder was 0.4%. The extraction rate of ursolic acid (%) = (mass of ursolic acid product × purity of ursolic acid product) / mass of ursolic acid in hawthorn powder × 100%.

[0020] Example 1 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix thymol and pyruvic acid in a molar ratio of 1:1 and stir at 70°C for 40 min to obtain a hydrophobic eutectic solvent. S3. Add 300g of hawthorn powder to 1500mL of hydrophobic eutectic solvent, extract by ultrasonication at 50℃ for 40min, filter, and obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 0.967 g of ursolic acid product.

[0021] Example 2 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix thymol and pyruvic acid in a molar ratio of 1:2 and stir at 80°C for 30 min to obtain a hydrophobic eutectic solvent. S3. Take 300g of hawthorn powder and add it to 3000mL of hydrophobic eutectic solvent. Extract by ultrasonication at 60℃ for 30min. Filter to obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 0.965 g of ursolic acid product.

[0022] Example 3 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix thymol and pyruvic acid in a molar ratio of 1:1.2 and stir at 75°C for 35 min to obtain a hydrophobic eutectic solvent. S3. Add 300g of hawthorn powder to 1500mL of hydrophobic eutectic solvent, extract by ultrasonication at 55℃ for 35min, filter, and obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 0.969 g of ursolic acid product.

[0023] Example 4 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix thymol, pyruvic acid and undecenoic acid in a molar ratio of 1:1.2:0.5 and stir at 80°C for 30 min to obtain a hydrophobic eutectic solvent. S3. Add 300g of hawthorn powder to 1500mL of hydrophobic eutectic solvent, extract by ultrasonication at 60℃ for 30min, filter, and obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 1.072 g of ursolic acid product.

[0024] Example 5 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix thymol, pyruvic acid and undecenoic acid in a molar ratio of 1:1.2:1 and stir at 80°C for 30 min to obtain a hydrophobic eutectic solvent. S3. Add 300g of hawthorn powder to 1500mL of hydrophobic eutectic solvent, extract by ultrasonication at 60℃ for 30min, filter, and obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 1.081 g of ursolic acid product.

[0025] Example 6 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix thymol, pyruvic acid and undecenoic acid in a molar ratio of 1:1.2:0.2 and stir at 80°C for 30 min to obtain a hydrophobic eutectic solvent. S3. Add 300g of hawthorn powder to 1500mL of hydrophobic eutectic solvent, extract by ultrasonication at 60℃ for 30min, filter, and obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 1.025 g of ursolic acid product.

[0026] Example 7 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix thymol, pyruvic acid and undecenoic acid in a molar ratio of 1:1.2:1.5 and stir at 80°C for 30 min to obtain a hydrophobic eutectic solvent. S3. Add 300g of hawthorn powder to 1500mL of hydrophobic eutectic solvent, extract by ultrasonication at 60℃ for 30min, filter, and obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 1.024 g of ursolic acid product.

[0027] Comparative Example 1 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix thymol and linalool in a molar ratio of 1:1.2 and stir at 80°C for 30 min to obtain a hydrophobic eutectic solvent. S3. Add 300g of hawthorn powder to 1500mL of hydrophobic eutectic solvent, extract by ultrasonication at 60℃ for 30min, filter, and obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 0.881 g of ursolic acid product.

[0028] Comparative Example 2 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix thymol and hexanoic acid in a molar ratio of 1:1.2 and stir at 80°C for 30 min to obtain a hydrophobic eutectic solvent. S3. Add 300g of hawthorn powder to 1500mL of hydrophobic eutectic solvent, extract by ultrasonication at 60℃ for 30min, filter, and obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 0.893 g of ursolic acid product.

[0029] Comparative Example 3 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix menthol and pyruvic acid in a molar ratio of 1:1.2 and stir at 80°C for 30 min to obtain a hydrophobic eutectic solvent. S3. Add 300g of hawthorn powder to 1500mL of hydrophobic eutectic solvent, extract by ultrasonication at 60℃ for 30min, filter, and obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 0.891 g of ursolic acid product.

[0030] Comparative Example 4 A green method for extracting ursolic acid from hawthorn includes the following steps: S1. Dry fresh hawthorn, pulverize it, pass it through a 60-mesh sieve, and dry it again to obtain hawthorn powder; S2. Mix thymol and undecenoic acid in a molar ratio of 1:0.5 and stir at 80°C for 30 min to obtain a hydrophobic eutectic solvent. S3. Add 300g of hawthorn powder to 1500mL of hydrophobic eutectic solvent, extract by ultrasonication at 60℃ for 30min, filter, and obtain the extract. S4. The extract was diluted with an equal volume of 65% ethanol aqueous solution and then purified by reversed-phase chromatography. First, a primary elution was performed using 65% ethanol aqueous solution as the eluent. After 5 BV, the primary eluent was obtained. The primary eluent was concentrated under reduced pressure and centrifuged, and the supernatant was collected as the recovered hydrophobic eutectic solvent. Then, a secondary elution was performed using 85% ethanol aqueous solution as the eluent. After 5 BV, the secondary eluent was obtained. The secondary eluent was concentrated and dried to obtain 0.925 g of ursolic acid product.

[0031] Experimental Example 1 The purity of the ursolic acid products obtained in Examples 1-8 and Comparative Examples 1-3 was determined by HPLC, and the extraction rate of ursolic acid was calculated. The results are shown in Table 1. Table 1. Purity and Extraction Rate Results

[0032] By comparing the data of Examples 1-3 and Comparative Examples 1-3, Examples 1-3 showed that the extraction rate of ursolic acid from hawthorn was higher than that of Comparative Examples 1-3 when a hydrophobic eutectic solvent composed of thymol and pyruvic acid was used. This indicates that the extraction rate of ursolic acid from hawthorn can be improved by using a hydrophobic eutectic solvent composed of thymol and pyruvic acid.

[0033] By comparing the data from Examples 3, 4-7, and Comparative Example 4, it was found that Examples 4-7, using pyruvic acid and undecenoic acid together as hydrogen bond acceptors, resulted in a higher ursolic acid extraction rate than Examples 3 and Comparative Example 4. This indicates that using a hydrophobic eutectic solvent composed of thymol, pyruvic acid, and undecenoic acid to extract ursolic acid from hawthorn can improve the extraction rate. By comparing the data from Examples 4-7, it was found that when the hydrogen bond acceptor also contains undecenoic acid, optimizing the molar ratio of thymol to undecenoic acid to 1:0.5-1 can further improve the extraction rate of ursolic acid from hawthorn.

[0034] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A green method for extracting ursolic acid from hawthorn, characterized in that, Includes the following steps: S1. Add hawthorn powder to a hydrophobic eutectic solvent, extract by ultrasonication, filter, and obtain the extract; S2. The extract is purified by fractional elution using a chromatographic column to obtain a primary eluent and a secondary eluent. The primary eluent is concentrated and centrifuged to obtain the recovered hydrophobic eutectic solvent. The secondary eluent is concentrated and dried to obtain the ursolic acid product. The hydrophobic eutectic solvent includes hydrogen bond donors and hydrogen bond acceptors; The hydrogen bond donor is thymol; the hydrogen bond acceptor includes pyruvate.

2. The green method for extracting ursolic acid from hawthorn according to claim 1, characterized in that, The method for preparing the hydrophobic eutectic solvent includes the following steps: mixing the hydrogen bond donor and the hydrogen bond acceptor, stirring, to obtain the hydrophobic eutectic solvent.

3. The green method for extracting ursolic acid from hawthorn according to claim 1, characterized in that, The molar ratio of thymol to pyruvic acid is 1:1~2.

4. A green method for extracting ursolic acid from hawthorn according to claim 2, characterized in that, The stirring temperature is 70~80℃, and the stirring time is 30~40min.

5. A green method for extracting ursolic acid from hawthorn according to claim 1, characterized in that, The mass-to-volume ratio of the hawthorn powder and the hydrophobic eutectic solvent is 1g:5~10mL.

6. A green method for extracting ursolic acid from hawthorn according to claim 1, characterized in that, The ultrasonic extraction temperature is 50~60℃, and the ultrasonic extraction time is 30~40min.

7. A green method for extracting ursolic acid from hawthorn according to claim 1, characterized in that, The hydrogen bond acceptor also includes undecenoic acid.

8. A green method for extracting ursolic acid from hawthorn according to claim 7, characterized in that, The molar ratio of thymol to undecenoic acid is 1:0.5~1.

9. A green method for extracting ursolic acid from hawthorn according to claim 1, characterized in that, The chromatographic separation column is a reversed-phase chromatographic separation column.

10. A green method for extracting ursolic acid from hawthorn according to claim 1, characterized in that, During the elution and purification process, the eluent is an aqueous ethanol solution.

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