A method for extracting a ketone ester from ginkgo leaves, and products and applications thereof

Abstract

The application discloses a method for extracting ketone ester from ginkgo leaves, products and applications thereof, and relates to the technical field of plant extraction. The method comprises the following steps: uniformly mixing ginkgo leaf dry powder with lime water suspension, then performing hydrothermal extraction, then separating to obtain filtrate, and adjusting the pH value of the filtrate to neutral to obtain pH-neutral filtrate; using a macroporous resin to perform adsorption and elution treatment on the pH-neutral filtrate to obtain eluate, and performing drying treatment on the eluate to obtain ginkgo leaf ketone ester; the elution treatment uses ethanol aqueous solutions with increasing concentrations in sequence. The method provided by the application can effectively improve the extraction yield of ginkgo leaf ketone ester, and the antioxidant effect of ginkgo leaf ketone ester obtained by using the method is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extraction, in particular to a method for extracting ketone ester from ginkgo leaves, products thereof and application. BACKGROUND

[0002] Studies have shown that free radicals in the blood can attack normal cells indiscriminately, causing excessive lipid particles in the blood and accelerating arteriosclerosis; at the same time, free radicals can also attack nucleic acid molecules in cells, becoming an important factor in inducing cancer; in addition, excessive oxidation caused by free radicals can accelerate cell and even human aging. How to remove free radicals and excess cholesterol and other substances in the blood vessels of the human body, prevent cardiovascular and cerebrovascular diseases, and delay aging is a major problem facing people.

[0003] In view of the above problems, people have begun to explore. Ginkgo is a unique ancient tree species in China, and Chinese people have been in love with it for thousands of years. Ginkgo has been used as medicine in China for a long time. Li Shizhen recorded in "Compendium of Materia Medica" during the Ming Dynasty that ginkgo "cooked food can warm the lungs, benefit qi, stop cough, shrink the small intestine, and stop leukorrhea. Raw food can reduce phlegm and kill insects". "Chinese Pharmacopoeia" clearly points out that "ginkgo leaf is sweet, bitter, astringent and flat, and belongs to heart and lung channels, and its functions are to astringe the lung, relieve asthma, activate blood and resolve stasis, and relieve pain, and it is used for lung deficiency cough and asthma, coronary heart disease, angina pectoris, and high blood lipids". Ginkgo leaf is a traditional heart-clearing and depression-relieving folk medicine. It can not only improve memory and cognitive ability, increase brain blood circulation, and improve nerve conduction speed, thereby promoting brain function, but also protect the nervous system and prevent and treat nervous system diseases. Ginkgo leaf extract also has the effects of lowering blood pressure, blood lipids and blood sugar. Ginkgo leaf extract is rich in flavonoids and phenolic acids, which have strong antioxidant effects and can scavenge free radicals and reduce oxidative stress, thereby protecting cell health. Ginkgo leaf extract has anti-inflammatory effects and can inhibit inflammatory reactions, reduce pain and swelling, etc. Ginkgo leaf extract can inhibit the growth and spread of tumor cells and has an anti-tumor effect.

[0004] Modern scientific research has proved that ginkgo biloba extract contains various active substances such as flavonoids and terpene lactone compounds, has the effects of improving cardiovascular and cerebrovascular circulation, resisting viruses, resisting cancer, preventing aging and reducing cholesterol, and has little toxic and side effects, and is safe to eat. Flavonoids and lactone compounds are two kinds of natural active ingredients, widely exist in the plant kingdom, and are effective ingredients of many Chinese herbal medicines. In recent years, due to the progress of free radical life science, ketone ester with strong antioxidant and free radical scavenging effect has been unprecedentedly valued. Ketone ester participates in the metabolism of phosphoric acid and arachidonic acid, protein phosphorylation, calcium ion transfer, free radical scavenging, antioxidant activity enhancement, oxidation and reduction, chelation and gene expression. Naturally derived bioflavonoids and lactone have small molecular weight, can be rapidly absorbed by the human body, can pass through the blood-brain barrier, and can enter fat tissue, thereby exhibiting the following functions: eliminating fatigue, protecting blood vessels, preventing arteriosclerosis, expanding capillaries, dredging microcirculation, activating brain and other organ cell functions, resisting fat oxidation, and resisting aging.

[0005] At present, the extraction yield of ginkgo biloba ketone ester is low, and the application of ginkgo biloba ketone ester generally adopts traditional experience and quality standards. The main features of the current ginkgo biloba ketone ester quality standards include: (1) the total ginkgo acid mass fraction is less than 5 mg / kg; (2) the total flavonoid glycoside mass fraction is 24.0-35.0%; and the total terpene lactone mass fraction is 6.0-12.0%. How to break the fixed limit, improve the extraction yield of ginkgo biloba ketone ester and the antioxidant effect has become a technical problem to be solved in the field. SUMMARY

[0006] The purpose of the present application is to provide a method for extracting ketone ester from ginkgo biloba leaves and its products and applications, so as to solve the problems existing in the prior art. The method can effectively improve the extraction yield of ginkgo biloba ketone ester, and the antioxidant effect of ginkgo biloba ketone ester obtained by the method is greatly improved.

[0007] To achieve the above purpose, the present application provides the following scheme:

[0008] The present application provides a method for extracting ketone ester from ginkgo biloba leaves, comprising the following steps:

[0009] After the ginkgo biloba leaf dry powder is uniformly mixed with lime water suspension, hydrothermal extraction is carried out, then the filtrate is separated, and the pH value of the filtrate is adjusted to neutral, so as to obtain a pH neutral filtrate;

[0010] The pH neutral filtrate is subjected to adsorption and elution treatment by using macroporous resin, so as to obtain an eluate, and the eluate is subjected to drying treatment, so as to obtain ginkgo biloba ketone ester;

[0011] The elution treatment comprises the steps of sequentially using ethanol aqueous solutions with increasing concentrations for elution.

[0012] Further, the content of Ca(OH)2 in the lime water suspension is 1.8-3 g / L.

[0013] Further, the mass-volume ratio of the dry ginkgo leaf powder to the lime water suspension is 1000 g:(1000-2000) mL.

[0014] Further, the temperature of the hydrothermal extraction is 80-100℃.

[0015] Further, the time of the hydrothermal extraction is 1 h.

[0016] Further, the macroporous resin is D101 resin.

[0017] The application also provides a ginkgo leaf ketone ester prepared according to the above method.

[0018] The application also provides the use of the above ginkgo leaf ketone ester in the preparation of an antioxidant product.

[0019] The application also provides an antioxidant product, and the active ingredient includes the above ginkgo leaf ketone ester.

[0020] Further, the product is a medicine or a health product.

[0021] The application discloses the following technical effects:

[0022] (1) The method for extracting ketone ester from ginkgo leaves provided by the application can reduce the loss of ketone ester in ginkgo leaves during the extraction process, thereby effectively increasing the total amount of ginkgo leaf ketone ester and further reducing the production cost of ginkgo leaf ketone ester.

[0023] (2) The ginkgo leaf ketone ester extracted by the method has a significantly increased proportion of terpene lactones and an effectively improved antioxidant function. The method can obtain ginkgo leaf ketone ester products with high antioxidant effect through one extraction, breaks the fixed limitation, realizes the synchronous improvement of the extraction yield and the antioxidant effect of ginkgo leaf ketone ester, and has important market application prospects.

[0024] (3) The ginkgo leaf ketone ester extracted by the application can be prepared into an antioxidant product, such as an effervescent tablet or an effervescent granule, and the effervescent tablet or the effervescent granule has the advantages of good water solubility, easy carrying, good taste and the like. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0026] Figure 1 Statistical diagram of inhibition rates of vitamin C and different ginkgo biloba leaf ketosterols on O 2- Statistical diagram of inhibition rates of vitamin C and different ginkgo biloba leaf ketosterols on O

[0027] Figure 2 Statistical diagram of inhibition rates of vitamin C and different ginkgo biloba leaf ketosterols on O DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0029] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range, and any other stated value or intermediate value in the stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0031] Various modifications and changes can be made to the specific implementation of the present application described in the specification without departing from the scope or spirit of the application. Other implementations of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples are illustrative only.

[0032] As used herein, the terms "comprise", "comprising", "including", "include", "contain", "containing", "have", "having" or variants thereof are open-ended, and mean including, but not limited to.

[0033] The ginkgo leaf dry powder used in the following examples was obtained by cleaning fresh ginkgo leaves, drying them in a dryer, crushing them, and passing them through an 80-mesh sieve. The total flavonoid glycoside content was 0.7 wt%, and the total terpene lactone content was 0.3 wt%.

[0034] The detection method for the content of total flavonoid glycosides and total terpene lactones is described in the reference "Zhang Guoyong, Liang Xiaomei, Fang Lei. Quality comparison and drug cost analysis of ginkgo biloba leaves and ginkgo biloba ketone ester tablets. China Modern Applied Pharmacy. 2014 31(9): 1136-1140".

[0035] The calculation formula for the extraction rate of ginkgo biloba ketone esters is:

[0036] Extraction rate = total amount of ketone esters in ginkgo biloba extract / total amount of ketone esters in ginkgo biloba dry powder x 100%.

[0037] Example 1

[0038] (1) Take 1 kg of ginkgo leaf dry powder, add 1000 mL of lime water suspension containing 1.8 g of Ca(OH)2, stir uniformly, heat to 90°C, heat for 1 h, then cool to room temperature, filter the filtrate; adjust the pH of the filtrate to neutral with hydrochloric acid with a pH of 2.

[0039] (2) Pack the D101 resin into the adsorption column, activate the resin in the adsorption column with 30% ethanol aqueous solution, and activate for 24 h.

[0040] (3) Pass 4 BV of ethanol aqueous solution (30% ethanol aqueous solution) through the column at a flow rate of 2 BV / h, then pass distilled water through the column at a flow rate of 2 BV / h until there is no alcohol smell; then pass 4 BV of hydrochloric acid (2 wt.% hydrochloric acid) through the column at a flow rate of 4 BV / h, then pass distilled water through the column at a flow rate of 4 BV / h until the eluate is neutral; finally, pass 4 BV of NaOH solution (2 wt.% NaOH solution) through the column at a flow rate of 4 BV / h, then pass distilled water through the column at a flow rate of 4 BV / h until the eluate is neutral, to obtain the adsorption resin column.

[0041] (4) First, pass the filtrate obtained in step (1) with a neutral pH through the column at a flow rate of 2 BV / h, and there should be no air bubbles in the resin layer; then sequentially elute with 30% ethanol aqueous solution (3 BV), 40% ethanol aqueous solution (4 BV), and 50% ethanol aqueous solution (5 BV) at a flow rate of 3 BV / h, collect the eluate, and dry to obtain 13.5 g of ginkgo biloba ketone ester product. The total ginkgo acid content of the ginkgo biloba ketone ester product was less than 5 mg / kg, the total flavonoid glycoside content was 29 wt%, the total terpene lactone content was 41 wt%, and the extraction rate of the ketone ester was calculated to be 94.5%.

[0042] Example 2

[0043] (1) Take 1 kg of dry powder of Ginkgo biloba leaves, add lime water suspension containing 4.5 g of Ca(OH)2, 1500 mL, stir uniformly, heat to 80°C, heat for 1 h, then reduce to room temperature, filter out the filtrate; adjust the pH of the filtrate to neutral with hydrochloric acid with pH 3.

[0044] (2) Pack the adsorption column with D101 resin, activate the resin in the adsorption column with 50% ethanol aqueous solution by volume for 24 h.

[0045] (3) Pass 4 BV of ethanol aqueous solution (30% ethanol aqueous solution by volume) through the column at a flow rate of 2 BV / h, then pass distilled water through the column at a flow rate of 2 BV / h until there is no alcohol smell; then pass 4 BV of hydrochloric acid (2 wt.% hydrochloric acid) through the column at a flow rate of 4 BV / h, then pass distilled water through the column at a flow rate of 4 BV / h until the eluate is neutral; finally, pass 4 BV of NaOH solution (2 wt.% NaOH solution) through the column at a flow rate of 4 BV / h, then pass distilled water through the column at a flow rate of 4 BV / h until the eluate is neutral, to obtain the adsorption resin column.

[0046] (4) First, pass the filtrate obtained in step (1) with neutral pH through the column at a flow rate of 2 BV / h, and there should be no air bubbles in the resin layer; then elute with 30% ethanol aqueous solution (4 BV), 50% ethanol aqueous solution (4 BV) and 60% ethanol aqueous solution (4 BV) at a flow rate of 3 BV / h in sequence, collect the eluate, and dry to obtain 13.4 g of Ginkgo biloba leaf ketone ester product, which is determined to have a total ginkgo acid content of less than 5 mg / kg, a total flavonoid glycoside content of 31 wt%, and a total terpene lactone content of 37 wt%, and the extraction rate of the ketone ester is calculated to be 91.1%.

[0047] Example 3

[0048] (1) Take 1 kg of dry powder of Ginkgo biloba leaves, add lime water suspension containing 4.5 g of Ca(OH)2, 1500 mL, stir uniformly, heat to 80°C, heat for 1 h, then reduce to room temperature, filter out the filtrate; adjust the pH of the filtrate to neutral with hydrochloric acid with pH 3.

[0049] (2) Pack the adsorption column with D101 resin, activate the resin in the adsorption column with 50% ethanol aqueous solution by volume for 24 h.

[0050] (3) The column was washed with 4 BV of 30% ethanol aqueous solution at a flow rate of 2 BV / h, and then with distilled water at a flow rate of 2 BV / h until the eluate was alcohol-free. Then the column was washed with 4 BV of 2% hydrochloric acid at a flow rate of 4 BV / h, and then with distilled water at a flow rate of 4 BV / h until the eluate was neutral. Finally, the column was washed with 4 BV of 2% NaOH solution at a flow rate of 4 BV / h, and then with distilled water at a flow rate of 4 BV / h until the eluate was neutral, to obtain the adsorption resin column.

[0051] (4) The filtrate obtained in step (1) was first passed through the column at a flow rate of 2 BV / h, and no air bubbles were generated in the resin layer. Then the column was eluted with 20% ethanol aqueous solution (3 BV), 30% ethanol aqueous solution (4 BV) and 40% ethanol aqueous solution (5 BV) at a flow rate of 3 BV / h, respectively, and the eluate was collected. After drying, ginkgo leaf ketone ester product 14.1 g was obtained. The total ginkgo acid content of the ginkgo leaf ketone ester product was less than 5 mg / kg, the total flavonoid glycoside content was 30 wt%, and the total terpene lactone content was 35 wt%. The extraction rate of the ketone ester was calculated to be 91.7%.

[0052] Comparative Example 1

[0053] The same as Example 1, except that in step (1), the lime water suspension containing 20 g of Ca(OH)2was replaced by a NaOH aqueous solution with a pH of 12.

[0054] The ginkgo leaf ketone ester product obtained in this comparative example was 13.8 g, and the total ginkgo acid content was less than 5 mg / kg, the total flavonoid glycoside content was 28 wt%, and the total terpene lactone content was 22 wt%. The extraction rate of the ketone ester was calculated to be 69%.

[0055] Comparative Example 2

[0056] The same as Example 1, except that in step (1), the lime water suspension containing 20 g of Ca(OH)2was replaced by water.

[0057] The ginkgo leaf ketone ester product obtained in this comparative example was 14.0 g, and the total ginkgo acid content was less than 5 mg / kg, the total flavonoid glycoside content was 34 wt%, and the total terpene lactone content was 12 wt%. The extraction rate of the ketone ester was calculated to be 64%.

[0058] Example 4

[0059] Weigh 10 g of the ginkgo biloba ester product prepared in Example 3 and 1 g of citric acid (up to 3 g), pour them into a granulator, add 10 mL of water, and mix them to form granules; weigh 10 g of the ginkgo biloba ester product prepared in Example 3 and 1 g of sodium bicarbonate (up to 3 g), pour them into a granulator, add 10 mL of water, and mix them to form granules.

[0060] The above two granules were mixed in a mass ratio of 1:1, dried at 60°C and then pressed into effervescent tablets.

[0061] The effervescent tablet has a light yellow appearance and tastes sweet and sour when dissolved in water. The solution is clear and transparent, and there is no insoluble matter. This shows that it has good water solubility. The effervescent tablets prepared in this embodiment were tested for solubility, friability and foaming amount. The test results are as follows: the solubility experiment shows that a large amount of carbon dioxide gas is generated within 5 minutes, showing an effervescent state, a friability of <0.8%, and it quickly disintegrates and produces bubbles within 5 minutes, all of which meet the requirements of the Chinese Pharmacopoeia (2020) edition.

[0062] Effect verification

[0063] The commercially available Ginkgo biloba ketone ester used in the following effect verification experiment was tested to have a total ginkgolic acid mass fraction of less than 5 mg / kg, a total flavonol glycoside content of 32 wt %, and a total terpenoid lactone content of 11 wt %.

[0064] 1. Ginkgo biloba ketone esters 2- The clearing effect

[0065] 1) Determination of the auto-oxidation rate of pyrogallol: 9 mL of Tris-HCl-EDTA buffer was added to each of two test tubes. 40 mL of distilled water was added to the control tube and mixed. The absorbance was adjusted to zero at 420 nm using a 1 cm cuvette. 40 mL of pyrogallol was added to the measurement tube and, after mixing, a 1 cm cuvette was immediately added. The absorbance A0 at 420 nm was read every minute. This was repeated three times. The auto-oxidation curve of pyrogallol was plotted with time as the abscissa and absorbance as the ordinate.

[0066] 2) Ginkgo biloba ketone esters 2- The clearance rate was determined as follows: different concentrations of the ginkgo biloba esters obtained in Example 1 and Comparative Examples 1-2, commercially available ginkgo biloba esters, and vitamin C (Vc) were added to the above-mentioned Tris-HCl-EDTA buffer, and compared with a blank control group containing distilled water. After 10 minutes of reaction, the absorbance of the solution at 420 nm was measured. 2- The clearance rate D=(A 空白 -A 样品 ) / A 空白 × 100%. The results are shown in Table 1 and Figure 1 .

[0067] Table IVc and the inhibition rate of different ginkgo leaf ketone esters on O 2-

[0068] 0.1 mg / mL 0.2 mg / mL 0.3 mg / mL 0.4 mg / mL 0.5 mg / mL Example 1 56％ 89％ 92％ 93％ 95％ Comparative Example 1 40％ 47％ 59％ 64％ 75％ Comparative Example 2 32％ 35％ 50％ 58％ 69％ Commercially available ginkgo biloba leaf ketone ester 31％ 38％ 45％ 57％ 62％ Vc 20％ 32％ 39％ 46％ 55％

[0069] As can be seen from Table 1, the ability of the ginkgo leaf ketone ester prepared in Example 1 to scavenge superoxide anions is obviously higher than that of the ginkgo leaf ketone esters of Comparative Examples 1-2, commercially available ginkgo leaf ketone ester and Vc. According to the extraction method of the present application, the proportion of total terpene lactones in ginkgo leaf ketone ester can be effectively improved. In combination with the scavenging effect of the ginkgo leaf ketone ester of Example 1 and commercially available ginkgo leaf ketone ester, it can be seen that after the proportion of total terpene lactones is improved, the effect of ginkgo leaf ketone ester on scavenging superoxide anions can be effectively improved.

[0070] 2. Scavenging effect of ginkgo leaf ketone ester on hydroxyl radicals (·OH)

[0071] 1) Bromocresol purple-hydrochloric acid-Fe 2+ Solution blank experiment

[0072] In a 10 mL colorimetric tube, 0.4 mL of bromocresol purple solution, 0.5 mL of 0.1 mol / L hydrochloric acid, 1.0 mL of 1 mmol / L ferric sulfate solution and 0.5 mL of 0.1 mol / L hydrogen peroxide solution were added, diluted with water to the mark, shaken well, placed in a 30°C constant temperature water bath for reaction for 8 min, then 0.2 mL of 0.05 mol / L NaF solution was quickly added after taking out, mixed well to terminate the reaction. With 1 cm cuvette, water as reference, the absorbance of the solution at 420 nm was determined, and the absorbance A0 of the bromocresol purple-hydrochloric acid solution blank test solution was determined by the same method.

[0073] 2. Determination of scavenging rate of Vc and ginkgo leaf ketone ester on hydroxyl radicals (·OH)

[0074] In the above bromocresol purple-hydrochloric acid-Fe 2+ Solution, different concentrations of ginkgo leaf ketone esters prepared in Example 1 and Comparative Examples 1-2, commercially available ginkgo leaf ketone ester and vitamin C were added, and a blank control with distilled water was compared, and the absorbance A of the solution at 420 nm was determined.

[0075] The scavenging rate of hydroxyl radicals (·OH) D = (A 空白 -A 样品 ) / A 空白 ×100% was calculated to obtain the antioxidant capacity of ginkgo leaf ketone ester, and the results are shown in Table 2 and Figure 2 .

[0076] ​As can be seen from Table 2, the ability of the ginkgo leaf ketone ester prepared in Example 1 to scavenge hydroxyl radicals is obviously higher than that of the ginkgo leaf ketone esters of Comparative Examples 1-2, the commercially available ginkgo leaf ketone ester and Vc. In combination with the scavenging effect of the ginkgo leaf ketone ester of Example 1 and the commercially available ginkgo leaf ketone ester, it can be seen that after the proportion of total terpene lactones is increased, the effect of the ginkgo leaf ketone ester on scavenging hydroxyl radicals can be effectively improved.

[0077] Table 2 Inhibition rate of Vc and different ginkgo leaf ketone esters on hydroxyl radicals (·OH)

[0078]

[0079] The above-described examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope defined by the claims of the present application.

Claims

1. A method for extracting ketone esters from Ginkgo biloba leaves, characterized in that: The following steps are involved: After uniformly mixing the ginkgo leaf dry powder and the lime water suspension, performing hydrothermal extraction, separating to obtain a filtrate, and adjusting the pH value of the filtrate to neutral to obtain a pH-neutral filtrate; Adsorbing and eluting the pH-neutral filtrate with a macroporous resin to obtain an eluate, and drying the eluate to obtain ginkgo biloba ketone ester; The elution treatment comprises the steps of sequentially eluting with ethanol aqueous solutions of increasing concentrations; The content of Ca(OH)2 in the limewater suspension is 1.8-3g / L; The mass volume ratio of the ginkgo leaf dry powder to the lime water suspension is 1000 g: (1000-2000) mL; The temperature of the hydrothermal extraction is 80-100°C; The hydrothermal extraction time is 1h; The macroporous resin is D101 resin.

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