Method for recovering ginkgo phenolic acid from ginkgo biloba leaf extract residue

Abstract

The application discloses a method for recovering ginkgolic acid from ginkgo leaf extract residue, and adopts DES solvent ultrasonic extraction. The DES solvent is formed by mixing a hydrogen bond acceptor and a hydrogen bond donor with water. The hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is urea. The ginkgo leaf is changed from waste to treasure, economic value is provided, and the environment is protected. The method is simple in operation and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of ginkgo leaf extract waste residue extraction and recycling technology, specifically to a method for recovering ginkgolic acid from ginkgo leaf extract waste residue. Background Technology

[0002] Ginkgo leaves are the dried leaves of Ginkgo biloba L., a plant in the Ginkgoaceae family. They are harvested in autumn while the leaves are still green and dried promptly. They have the effects of promoting blood circulation and removing blood stasis, clearing the meridians and relieving pain, astringing the lungs and relieving asthma, and resolving turbidity and lowering lipids. They are used for blood stasis obstructing the meridians, chest pain, hemiplegia due to stroke, cough and asthma due to lung deficiency, and hyperlipidemia.

[0003] Ginkgo leaf extract is a light yellow-brown powder obtained from the dried leaves of Ginkgo biloba (a plant in the Ginkgoaceae family) through reflux extraction with dilute ethanol and resin purification. It contains ≥24.0% total flavonol glycosides and ≥6.0% terpene lactones, and has effects such as improving blood circulation, anti-oxidation, and neuroprotection. Its active components, flavonol glycosides and terpene lactones, can dilate cerebral blood vessels and inhibit platelet aggregation, making it useful as an adjunct treatment for diseases such as cerebral ischemia and diabetic retinopathy. It is also used as a food additive in the dairy and fruit juice industries.

[0004] During the extraction of ginkgo leaves, the residue still contains valuable components. Improper disposal of this residue not only pollutes the environment but also wastes resources. For example, ginkgolic acid, an alkylphenol and alkylphenolic acid compound found in ginkgo, belongs to the long-chain phenol class of compounds. It includes ginkgolic acid, hydroginkgolic acid, bilobol, and ginkgol, among other components. These components may be associated with allergies and mutagenesis and are considered toxic side effects of ginkgo leaf extract. The discarded residue contains some ginkgolic acid. Improper disposal or handling of this residue can further harm the environment.

[0005] Currently, there are no reports on the recovery and separation of ginkgolic acid from the residue of ginkgo leaf extract. Summary of the Invention

[0006] To address the above problems, the present invention aims to provide a method for recovering ginkgolic acid from the waste residue of ginkgo leaf extract. This process achieves effective secondary recycling of the waste residue of ginkgo leaf extract, is stable, produces high purity ginkgolic acid monomers, and is simple to operate, making it suitable for industrial production.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a method for recovering ginkgolic acid from waste residue of ginkgo leaf extract, characterized by comprising the following steps: (1) Dry the ginkgo leaves under vacuum at 45-55℃ for 1-2 hours. Accurately weigh the dried ginkgo leaves, grind them into coarse powder, add methanol and reflux extract at 40-60℃ 1-3 times. The ratio of ginkgo leaves to methanol is 1g:20-30mL. Each extraction lasts 50-70min. After extraction, filter the solution. The filtrate is the ginkgo leaf alcohol extract. Collect the filter residue as ginkgo leaf extract waste residue A. Then concentrate the ginkgo leaf alcohol extract at 45-55℃ to remove alcohol. After standing overnight, filter to separate the precipitate and the concentrated ginkgo leaf alcohol extract. Combine the precipitate and ginkgo leaf extract waste residue A to obtain ginkgo leaf extract waste residue B. (2) Take the waste residue B of Ginkgo biloba extract, add DES solvent, sonicate at 30-40℃, centrifuge after sonication, take the supernatant, filter, and obtain the Ginkgo biloba extract.

[0008] In step (1) above, the ginkgo leaves were vacuum dried at 50°C for 1.5 hours. The dried ginkgo leaves were accurately weighed, ground into coarse powder, and extracted twice by reflux with methanol at 50°C. The ratio of ginkgo leaves to methanol was 1 g: 25 mL. Each extraction lasted 60 minutes. After extraction, the mixture was filtered, and the filtrate was the ginkgo leaf alcohol extract. The filter residue was collected as ginkgo leaf extract waste residue A. The ginkgo leaf alcohol extract was then concentrated at 50°C to remove the alcohol. After standing overnight, the precipitate and the concentrated ginkgo leaf alcohol extract were separated by filtration. The precipitate and ginkgo leaf extract waste residue A were combined to obtain ginkgo leaf extract waste residue B.

[0009] In step (2) above, the DES solvent is made by mixing hydrogen bond acceptor and hydrogen bond donor with water. The hydrogen bond acceptor is choline chloride and the hydrogen bond donor is urea. The volume ratio of choline chloride, urea and water is 1:1:1-3.

[0010] Specifically, in step (2) above, the DES solvent is made by mixing hydrogen bond acceptor and hydrogen bond donor with water. The hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is urea. The volume ratio of choline chloride, urea and water is 1:1:2.

[0011] In step (2) above, the solid-liquid ratio of Ginkgo biloba extract residue B to DES solvent is 1g:45-55mL.

[0012] Specifically, in step (2) above, the solid-liquid ratio of Ginkgo biloba extract residue B to DES solvent is 1g:50mL.

[0013] In step (2) above, the ultrasonic power is 150-300W and the ultrasonic time is 10-30min.

[0014] Specifically, in step (2) above, the ultrasonic power is 200W and the ultrasonic time is 20min.

[0015] Compared with the prior art, the present invention has the following advantages: 1. The waste residue from the production of ginkgo leaf extract, which is used as a raw material, is produced at no cost. By extracting ginkgo phenolic acid, it can be comprehensively utilized, turning waste into benefit; 2. Extraction and separation are performed using eutectic solvents, which is green and pollution-free, simple to operate, and has high extraction efficiency; 3. The recycling of ginkgo biloba phenolic acid has a promising future in the preparation of biological fungicides, turning waste into treasure and possessing certain economic value; at the same time, it reduces the environmental pollution of ginkgo processing and lays the foundation for the development of green plant-based biological pesticides.

[0016] 4. This invention has significant economic, environmental and social benefits, as it promotes the rapid development of the entire ginkgo planting and processing industry, increases the economic income of local people, and lays the foundation for adding a new type of plant-derived biological pesticide to the domestic market. Detailed Implementation

[0017] The present invention will now be described in detail with reference to specific embodiments. The embodiments are merely supplementary explanations and illustrations of the present invention, and not intended to limit the invention.

[0018] Example 1: Method for recovering ginkgolic acid from waste residue of ginkgo leaf extract (1) Dry the ginkgo leaves under vacuum at 50℃ for 1.5h. Accurately weigh the dried ginkgo leaves, grind them into coarse powder, add methanol and reflux extract twice at 50℃. The ratio of ginkgo leaves to methanol is 1g:25mL. Each extraction is 60min. After extraction, filter the solution. The filtrate is the ginkgo leaf alcohol extract. Collect the filter residue as ginkgo leaf extract waste residue A. Then concentrate the ginkgo leaf alcohol extract at 50℃ to remove alcohol. After standing overnight, filter to separate the precipitate and the concentrated ginkgo leaf alcohol extract. Combine the precipitate and ginkgo leaf extract waste residue A to obtain ginkgo leaf extract waste residue B. (2) Take the waste residue B of Ginkgo biloba extract and add it to DES solvent. DES solvent is a mixture of hydrogen bond acceptor and hydrogen bond donor with water. The hydrogen bond acceptor is choline chloride and the hydrogen bond donor is urea. The volume ratio of choline chloride, urea and water is 1:1:2. The solid-liquid ratio of Ginkgo biloba extract waste residue B to DES solvent is 1g:50mL. Sonicate at 30-40℃ for 20min with an ultrasonic power of 200W. After centrifugation, take the supernatant and filter to obtain the Ginkgo biloba extract.

[0019] Example 2: Method for recovering ginkgolic acid from waste residue of ginkgo leaf extract (1) Dry the ginkgo leaves under vacuum at 55℃ for 1 hour. Accurately weigh the dried ginkgo leaves, grind them into coarse powder, add methanol and reflux extract at 60℃ 3 times. The ratio of ginkgo leaves to methanol is 1g:30mL. Each extraction is 70min. After extraction, filter the solution. The filtrate is the ginkgo leaf alcohol extract. Collect the filter residue as ginkgo leaf extract waste residue A. Then concentrate the ginkgo leaf alcohol extract at 55℃ to remove alcohol. After standing overnight, filter to separate the precipitate and the concentrated ginkgo leaf alcohol extract. Combine the precipitate and ginkgo leaf extract waste residue A to obtain ginkgo leaf extract waste residue B. (2) Take the waste residue B of Ginkgo biloba extract and add it to DES solvent. DES solvent is a mixture of hydrogen bond acceptor and hydrogen bond donor with water. The hydrogen bond acceptor is choline chloride and the hydrogen bond donor is urea. The volume ratio of choline chloride, urea and water is 1:1:1. The solid-liquid ratio of waste residue B of Ginkgo biloba extract to DES solvent is 1g:45mL. Sonicate at 30℃ for 30min with a sonication power of 300W. After sonication, centrifuge and take the supernatant. Filter to obtain the Ginkgo biloba extract.

[0020] Example 3: Method for recovering ginkgolic acid from waste residue of ginkgo leaf extract (1) Dry the ginkgo leaves under vacuum at 45℃ for 2 hours. Accurately weigh the dried ginkgo leaves, grind them into coarse powder, add methanol and reflux extract once at 40℃. The ratio of ginkgo leaves to methanol is 1g:20mL. Each extraction lasts for 50min. After extraction, filter the solution. The filtrate is the ginkgo leaf alcohol extract. Collect the filter residue as ginkgo leaf extract waste residue A. Then concentrate the ginkgo leaf alcohol extract at 45℃ to remove alcohol. After standing overnight, filter to separate the precipitate and the concentrated ginkgo leaf alcohol extract. Combine the precipitate and ginkgo leaf extract waste residue A to obtain ginkgo leaf extract waste residue B. (2) Take the waste residue B of Ginkgo biloba extract and add it to DES solvent. DES solvent is a mixture of hydrogen bond acceptor and hydrogen bond donor with water. The hydrogen bond acceptor is choline chloride and the hydrogen bond donor is urea. The volume ratio of choline chloride, urea and water is 1:1:3. The solid-liquid ratio of Ginkgo biloba extract waste residue B to DES solvent is 1g:55mL. Sonicate at 30-40℃ for 10min with a sonication power of 150W. After sonication, centrifuge and collect the supernatant. Filter to obtain the Ginkgo biloba extract.

[0021] Example 4: Method for recovering ginkgolic acid from waste residue of ginkgo leaf extract (1) Dry the ginkgo leaves under vacuum at 45℃ for 1 hour. Accurately weigh the dried ginkgo leaves, grind them into coarse powder, add methanol and reflux extract at 45℃ 1-3 times. The ratio of ginkgo leaves to methanol is 1g:20mL. Each extraction is 65min. After extraction, filter the solution. The filtrate is the ginkgo leaf alcohol extract. Collect the filter residue as ginkgo leaf extract waste residue A. Then concentrate the ginkgo leaf alcohol extract at 55℃ to remove alcohol. After standing overnight, filter to separate the precipitate and the concentrated ginkgo leaf alcohol extract. Combine the precipitate and ginkgo leaf extract waste residue A to obtain ginkgo leaf extract waste residue B. (2) Take the waste residue B of Ginkgo biloba extract and add it to DES solvent. DES solvent is a mixture of hydrogen bond acceptor and hydrogen bond donor with water. The hydrogen bond acceptor is choline chloride and the hydrogen bond donor is urea. The volume ratio of choline chloride, urea and water is 1:1:1.5. The solid-liquid ratio of Ginkgo biloba extract waste residue B to DES solvent is 1g:55mL. Sonicate at 35℃ for 20min with a sonication power of 250W. After sonication, centrifuge and collect the supernatant. Filter to obtain the Ginkgo biloba extract.

[0022] To verify the feasibility and effectiveness of the separation and purification method of this invention, the inventors conducted numerous experiments. The following are some of the experimental findings. 1. Optimization of the extraction process of ginkgolic acid from ginkgo leaves 1.1 Experimental reagents Raw material: Ginkgo leaves were collected from the West Campus of Guizhou University, vacuum dried at 50℃ for 1-2 hours, and then sealed and stored in a cool place for later use.

[0023] Reference standards and reagents: see Table 1 and Table 2. All other reagents were of analytical grade.

[0024] Table 1. Reference Standards Table 2 Experimental Reagents 1.2 Content determination methods and results 1.2.1 Sample solution preparation Accurately weigh 0.01 g of the ginkgo biloba extract obtained in Example 1 and place it in a 10 mL volumetric flask. Add methanol to dissolve the extract, make up to volume, and shake well to obtain the test sample.

[0025] 1.2.2 Instrument: Preparative high-performance liquid chromatography (LC-20AR) (Shimadzu Corporation, Japan) 2.3.2.1 Chromatographic conditions Preparative HPLC conditions: SHIMADZU C18 (20 mm × 250 mm, 5 µm) preparative column; mobile phase: methanol (B) - 0.1% trifluoroacetic acid water (A) 95:5; detection wavelength: 310 nm; injection volume: 100 µL; flow rate: 8 mL / min; analysis time: 60 min.

[0026] Analytical HPLC conditions: Column: Supersil-U C18 (4.6 mm × 150 mm, 5 µm); Mobile phase: Acetonitrile-0.1% trifluoroacetic acid water (0 min 75:25→30 min 90:10→35 min; 90:10→35.5 min 75:25→38 min 75:25); Detection wavelength: 310 nm; Injection volume: 10 µL; Column temperature: 45 ℃; Flow rate: 1 mL / min; Analysis time: 40 min.

[0027] The results are shown in Table 3: Table 3. Validation of Ginkgo Biloba Extraction Process (n=3) The results showed that the content of ginkgo biloba acid in the waste residue from the production of ginkgo biloba extract was 15.64%, and the content of ginkgo biloba acid after extraction could reach more than 53%, with a recovery rate of 90%.

Claims

1. A method for recovering ginkgolic acid from waste residue of ginkgo leaf extract, characterized in that: Includes the following steps: (1) Dry the ginkgo leaves under vacuum at 45-55℃ for 1-2 hours. Accurately weigh the dried ginkgo leaves, grind them into coarse powder, add methanol and reflux extract at 40-60℃ 1-3 times. The ratio of ginkgo leaves to methanol is 1g:20-30mL. Each extraction lasts 50-70min. After extraction, filter the solution. The filtrate is the ginkgo leaf alcohol extract. Collect the filter residue as ginkgo leaf extract waste residue A. Then concentrate the ginkgo leaf alcohol extract at 45-55℃ to remove alcohol. After standing overnight, filter to separate the precipitate and the concentrated ginkgo leaf alcohol extract. Combine the precipitate and ginkgo leaf extract waste residue A to obtain ginkgo leaf extract waste residue B. (2) Take the waste residue B of Ginkgo biloba extract, add DES solvent, sonicate at 30-40℃, centrifuge after sonication, take the supernatant, filter, and obtain the Ginkgo biloba extract.

2. The method for recovering ginkgolic acid from the waste residue of ginkgo leaf extract according to claim 1, characterized in that: In step (1), the ginkgo leaves are vacuum dried at 50°C for 1.5 hours. The dried ginkgo leaves are accurately weighed, ground into coarse powder, and extracted twice by reflux with methanol at 50°C. The ratio of ginkgo leaves to methanol is 1g:25mL. Each extraction lasts for 60 minutes. After extraction, the mixture is filtered, and the filtrate is the ginkgo leaf alcohol extract. The filter residue is collected as ginkgo leaf extract waste residue A. The ginkgo leaf alcohol extract is then concentrated at 50°C to remove alcohol. After standing overnight, the precipitate and the concentrated ginkgo leaf alcohol extract are separated by filtration. The precipitate and ginkgo leaf extract waste residue A are combined to obtain ginkgo leaf extract waste residue B.

3. The method for recovering ginkgolic acid from the waste residue of ginkgo leaf extract according to claim 1, characterized in that: In step (2), the DES solvent is a mixture of hydrogen bond acceptor and hydrogen bond donor with water. The hydrogen bond acceptor is choline chloride and the hydrogen bond donor is urea. The volume ratio of choline chloride, urea and water is 1:1:1-3.

4. The method for recovering ginkgolic acid from the waste residue of ginkgo leaf extract according to claim 3, characterized in that: In step (2), the DES solvent is a mixture of hydrogen bond acceptor and hydrogen bond donor with water. The hydrogen bond acceptor is choline chloride and the hydrogen bond donor is urea. The volume ratio of choline chloride, urea and water is 1:1:

2.

5. The method for recovering ginkgolic acid from the waste residue of ginkgo leaf extract according to claim 1, characterized in that: In step (2), the solid-liquid ratio of Ginkgo biloba extract waste residue B to DES solvent is 1g:45-55mL.

6. The method for recovering ginkgolic acid from the waste residue of ginkgo leaf extract according to claim 5, characterized in that: In step (2), the solid-liquid ratio of Ginkgo biloba extract waste residue B to DES solvent is 1g:50mL.

7. The method for recovering ginkgolic acid from the waste residue of ginkgo leaf extract according to claim 1, characterized in that: In step (2), the ultrasonic power is 150-300W and the ultrasonic time is 10-30min.

8. The method for recovering ginkgolic acid from the waste residue of ginkgo leaf extract according to claim 7, characterized in that: In step (2), the ultrasonic power is 200W and the ultrasonic time is 20min.

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