Method for separating and preparing natural 6-hydroxykynurenic acid from ginkgo leaves

Abstract

The invention relates to a method for separating and preparing natural 6-hydroxykynurenic acid from ginkgo leaves, and belongs to the technical field of extraction and separation of natural plant products. The method comprises the following steps: firstly extracting 6-HKA from ginkgo leaf powder by using a low-alcohol solution, then adsorbing and separating by using a polyamide column, and finally obtaining the high-purity 6-HKA by using a high-speed counter-current chromatography. According to the method, low-concentration ethanol is adopted as a solvent and an elution reagent, the purification steps are few, the purity of the 6-HKA product is high, and compared with the prior art, the extraction preparation method is easy and convenient to operate, high in safety, low in requirement for instruments and equipment, low in cost and suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and separation of natural plants, and specifically relates to a method for separating and preparing natural 6-hydroxykynurenic acid from ginkgo leaves. Background Art

[0002] Ginkgo biloba leaves ( Ginkgo biloba ) The unique medicinal value of ginkgo has been recorded in medical literature both in ancient and modern times, especially in improving blood circulation and enhancing memory. Ginkgo biloba contains rich flavonoids, terpenes and organic acids, which have strong antioxidant effects. As we age, free radicals in the human body will continue to accumulate, causing damage to cells, which may lead to serious diseases such as heart disease, cancer and Alzheimer's disease. The antioxidants in ginkgo biloba can effectively remove these free radicals and protect cells from damage. In addition, ginkgo biloba can significantly improve blood circulation. It effectively prevents diseases such as varicose veins and intermittent claudication caused by poor blood circulation by dilating blood vessels and reducing blood viscosity. More importantly, ginkgo biloba has a significant effect on improving memory and preventing memory loss. Although ginkgo biloba extracts have been developed as cardiovascular and cerebrovascular disease drug preparations in many countries, such as Ginaduo (Schwabe, Germany), Tianbaoning (Kangenbei, China), Danakang Tablets (Ipsen, France), etc., their bioactive components have not yet been fully elucidated. 6-Hydroxykynurenic acid (6-HKA) in Ginkgo biloba leaves is a nitrogen-containing phenolic acid with peroxynitrite scavenging activity. Similar to kynurenic acid, it is a glutamate receptor antagonist that can improve neurological dysfunction, reduce infarct area, and lower superoxide dismutase and malondialdehyde concentrations, indicating that 6-HKA can repair neurons and has a protective effect on brain tissue.

[0003] With the vigorous development of the ginkgo industry and the rapid development of science and technology, the separation, purification, pharmacology and efficacy of the components in ginkgo leaves and their preparations have become more and more in-depth. At present, the efficient separation and extraction of active ingredients in ginkgo leaf extracts has become the core focus of the research and development field, especially the separation and extraction of key components such as ginkgo flavonoids, ginkgo lactones and ginkgo phenolic acid, which has received extensive attention and in-depth research. However, the functional components in ginkgo leaves are very complex. In addition to ginkgo flavonoids (such as quercetin, kaempferol, isorhamnetin), ginkgo lactones (A, B, C) and other components, it also contains organic acids (shikimic acid, 6-HKA, protocatechuic acid, quinic acid, chlorogenic acid), proanthocyanidins, polyprenols, polysaccharides, proteins and other components. How to efficiently purify 6-HKA from complex compounds has become an important problem.

[0004] Patent CN 103784448 B discloses a method for preparing 6-HKA using a polyamide resin and a crystallization method. The macroporous resin water wash solution is directly adsorbed and separated by a polyamide resin, and the pH of the eluted concentrate is adjusted to obtain a 6-HKA extract. The purity of 6-HKA is 50% to 90%, and the 6-HKA can be used to prepare foods, health foods or medicines for preventing or treating nervous system diseases, cardiovascular and cerebrovascular diseases or depression.

[0005] Patent CN 115006435 B discloses a method for preparing an organic acid extract of ginkgo biloba leaves. By controlling the contents of protocatechuic acid, 6-HKA, gallic acid and p-hydroxybenzoic acid, and controlling the content ratio of protocatechuic acid to the other three organic acids, the obtained organic acid extract of ginkgo biloba leaves has a significant effect in treating chronic ischemic stroke, achieving synergistic synergy. The present invention also provides a preparation process for the extract, including dissolution of ginkgo biloba extract, polyamide column chromatography, macroporous resin column chromatography and concentration and drying. However, this method does not purify 6-HKA, and the purity of 6-HKA is 3% to 8%.

[0006] High-speed countercurrent chromatography (HSCCC) technology is a continuous liquid-liquid partition chromatography technology that does not require a solid carrier. Its uniqueness lies in that the stationary phase is stably retained in the separation column through the synergistic effect of the gravity field and centrifugal force, thereby avoiding denaturation and irreversible adsorption caused by chemical reactions that may occur between the solid carrier and the sample. This feature makes HSCCC excellent in sample recovery and can efficiently prepare high-purity monomer compounds from crude samples. Therefore, HSCCC technology has been widely used in biochemical engineering, medicine, natural product extraction, food science and other fields. The HSCCC separation solvent system in the present invention successfully improves the yield and purity of 6-HKA, providing a new and effective method for the separation and purification of organic phenolic acids. Summary of the invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for separating and preparing natural 6-hydroxykynurenic acid from ginkgo leaves. The method overcomes the problems of low extraction rate and more impurities in the extract existing in the prior preparation method, and has the advantages of high efficiency, simplicity, low cost and suitability for a certain scale of production. The method is specifically achieved through the following technical solutions: A method for separating and preparing natural 6-hydroxykynurenic acid from ginkgo leaves comprises the following steps: 1) Preparation of crude extract: adding crushed ginkgo leaf powder to ethanol aqueous solution, performing ultrasonic-assisted extraction to obtain an extract, filtering and collecting the filtrate, and concentrating the filtrate under reduced pressure to obtain a crude extract; 2) Preparation of organic acid extract from ginkgo leaves: the crude extract is loaded onto a polyamide resin column, eluted with hydrochloric acid solution and ethanol solution in sequence, the ethanol eluate is collected, the eluate is concentrated and dried to obtain the organic acid extract from ginkgo leaves; 3) Solvent preparation: petroleum ether, methanol, ethyl acetate and water were mixed in a volume ratio of 9:6:3:1, shaken vigorously and allowed to stand until the two phases were in equilibrium, and then separated into upper and lower phases. The upper phase was used as the stationary phase and the lower phase as the mobile phase. 4) Sample preparation: dissolving the ginkgo leaf organic acid extract obtained in step 2) in a mobile phase to obtain a sample solution; 5) Isolation and purification: The sample solution obtained in step 4) is subjected to high-speed countercurrent chromatography, and the fractions with a peak time of 115 to 125 min are collected, concentrated and dried to obtain the compound 6-hydroxykynurenic acid.

[0008] The structural formula of 6-hydroxykynurenic acid (6-HKA) is as follows:

[0009] The above method for preparing 6-HKA adopts an unconventional preparation route design, creatively combines polyamide column adsorption and high-speed countercurrent chromatography to efficiently purify 6-HKA. At the same time, the preparation process achieves the goals of environmental protection, energy saving, safety and high efficiency.

[0010] Furthermore, in step 1), the concentration of the ethanol aqueous solution is 0-10%, and the solid-liquid ratio of the ginkgo leaf powder to the ethanol aqueous solution is 1:10-100 (g:mL).

[0011] Furthermore, in step 1), the conditions for ultrasonic-assisted extraction are: ultrasonic extraction time: 30 to 90 minutes, ultrasonic power 300 to 500W.

[0012] Furthermore, in step 2), the concentration of the hydrochloric acid solution is 0-0.01%; and the ethanol solution is 20-70% ethanol containing 0-0.1% hydrochloric acid.

[0013] Furthermore, in step 2), the ethanol solution is eluted at 5 BV, the flow rate is 1 BV / h, and the concentration temperature is 60°C.

[0014] Furthermore, in step 5), the separation conditions are: flow rate 2 mL / min, main engine speed 900 rpm, temperature 25° C., and detection wavelength 350 nm.

[0015] The method of the invention uses ginkgo leaves as raw materials, and through low-alcohol extraction, polyamide column adsorption, and high-speed countercurrent chromatography purification, high-purity 6-HKA can be obtained through only two purification steps. The steps are simple and the purity is high. The method obtains an unexpected purification effect, and the requirements for instruments and equipment in the preparation process are low, the cost is low, and it is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the chromatogram of 6-HKA reference substance; Figure 2 HSCCC separation diagram of 6-HKA from Ginkgo biloba leaves; Figure 3 This is the HPLC chart of Ginkgo biloba 6-HKA. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution. Example

[0018] The ginkgo leaves used in this example were purchased from a Chinese herbal medicine market, and milli-Q (18 MΩ) system pure water, analytical grade petroleum ether, methanol, and ethyl acetate were used for HSCCC separation.

[0019] The specific method for separating 6-HKA from Ginkgo biloba leaves is as follows: Step 1, preparation of crude extract: 500 g of ginkgo leaf powder was added to 20 L of 10% ethanol solution, and ultrasonic extraction was performed at an ultrasonic power of 400 W for 50 min to obtain an extract, which was filtered through filter paper to obtain a filtrate, and concentrated under reduced pressure and 45° C. to obtain a crude extract.

[0020] Step 2, preparation of ginkgo leaf organic acid extract: the crude extract was loaded onto a polyamide resin column, first eluted with 0-0.01% hydrochloric acid, then eluted with 20-70% (v / v) ethanol solution containing 0-0.1% hydrochloric acid for 5 BV, at an elution rate of 1 BV / h, the ethanol eluate was collected, concentrated and dried at 60°C to obtain a ginkgo leaf organic acid extract sample.

[0021] Step 3, HSCCC separation preparation: Add a two-phase solvent system containing petroleum ether-methanol-ethyl acetate-water (9:6:3:1, v / v) into a separatory funnel and shake it vigorously. Let it stand for 15 min until the two phases are in equilibrium. The upper and lower phases serve as the stationary phase and the mobile phase, respectively.

[0022] Step 4: Add 500 mg of ginkgo leaf extract to a 10 mL centrifuge tube, add 5 mL of the upper phase and 5 mL of the lower phase of the petroleum ether-methanol-ethyl acetate-water (9:6:3:1, v / v) solvent system, respectively, to completely dissolve the sample, and perform high-speed countercurrent chromatography separation at a flow rate of 2 mL / min, a main engine speed of 900 rpm, a temperature of 25°C, and a detection wavelength of 350 nm. Collect the fractions within the peak time of 115 to 125 min, concentrate and dry, and obtain compound 6-HKA.

[0023] Detection method of 6-HKA content in Ginkgo biloba extract: Instrument: Agilent HPLC1260; Stationary phase: SB-C18 column (4.6x250mm, 5μm); The liquid chromatography conditions for analyzing 6-HKA in Ginkgo biloba extract were as follows: SB-C18 column (4.6×250mm, 5μm) with diisobutylsilane bonded silica as filler, mobile phase A was 0.1% phosphoric acid aqueous solution (pH 2.5), mobile phase B was acetonitrile, and mobile phase C was methanol. 0-14min: 94%A, 5%B, 1%C; 16-25min: 40%A, 50%B, 10%C; 28-30min: 94%A, 5%B, 1C. UV detector, detection wavelength 350nm, column temperature 25℃; injection volume 10μL; flow rate 1.0mL / min.

[0024] The analysis results are as follows: (1) The content of 6-HKA in Ginkgo biloba leaves is 0.049%; (2) The yield of ginkgo leaf extract was 3.14%, and the 6-HKA content was 0.259%.

[0025] (3) The HPLC results of 6-HKA standard are shown in Figure 1 , HSCCC separation spectrum see Figure 2 , the HPLC results of 6-HKA purification are shown in Figure 3 .

[0026] The present invention establishes a method for separating and preparing 6-HKA from ginkgo leaves by low-concentration ethanol extraction and elution and high-speed countercurrent chromatography purification. Liquid phase results show that the purity of the obtained 6-HKA is 92%. The method is simple, rapid, and reproducible, suitable for large-scale preparation, and lays a material foundation for further activity research of 6-HKA.

Claims

1. A method for separating and preparing natural 6-hydroxykynurenic acid from ginkgo leaves, characterized in that: The method comprises the following steps: 1) Preparation of crude extract: adding crushed ginkgo leaf powder to ethanol aqueous solution, performing ultrasonic-assisted extraction to obtain an extract, filtering and collecting the filtrate, and concentrating the filtrate under reduced pressure to obtain a crude extract; 2) Preparation of organic acid extract from ginkgo leaves: the crude extract is loaded onto a polyamide resin column, eluted with hydrochloric acid solution and ethanol solution in sequence, the ethanol eluate is collected, the eluate is concentrated and dried to obtain the organic acid extract from ginkgo leaves; 3) Solvent preparation: petroleum ether, methanol, ethyl acetate and water were mixed in a volume ratio of 9:6:3:1, shaken vigorously and allowed to stand until the two phases were in equilibrium, and then separated into upper and lower phases. The upper phase was used as the stationary phase and the lower phase as the mobile phase. 4) Sample preparation: dissolving the ginkgo leaf organic acid extract obtained in step 2) in a mobile phase to obtain a sample solution; 5) Isolation and purification: The sample solution obtained in step 4) is subjected to high-speed countercurrent chromatography, and the fractions with a peak time of 115 to 125 min are collected, concentrated and dried to obtain the compound 6-hydroxykynurenic acid.

2. A method for separating and preparing natural 6-hydroxykynurenic acid from ginkgo leaves as claimed in claim 1, characterized in that In step 1), the concentration of the ethanol aqueous solution is 0-10%, and the solid-liquid ratio of the ginkgo leaf powder to the ethanol aqueous solution is 1:10-100.

3. A method for separating and preparing natural 6-hydroxykynurenic acid from ginkgo leaves as claimed in claim 1, characterized in that In step 1), the conditions for ultrasonic-assisted extraction are: ultrasonic extraction time: 30 to 90 minutes, ultrasonic power 300 to 500W.

4. A method for separating and preparing natural 6-hydroxykynurenic acid from Ginkgo biloba leaves as claimed in claim 1, characterized in that In step 2), the concentration of the hydrochloric acid solution is 0-0.01%; the ethanol solution is 20-70% ethanol containing 0-0.1% hydrochloric acid.

5. A method for separating and preparing natural 6-hydroxykynurenic acid from ginkgo leaves as claimed in claim 1, characterized in that In step 2), 5 BV of ethanol solution was eluted at a flow rate of 1 BV / h and a concentration temperature of 60°C.

6. A method for separating and preparing natural 6-hydroxykynurenic acid from ginkgo leaves as claimed in claim 1, characterized in that In step 5), the separation conditions are: flow rate 2 mL / min, main engine speed 900 rpm, temperature 25°C, and detection wavelength 350 nm.

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