Method for extracting ketones and esters from ginkgo leaves, and product and use thereof

Abstract

A method for extracting ketones and esters from ginkgo leaves, and a product and use thereof. The method comprises: uniformly mixing a ginkgo leaf dry powder and a lime water suspension, performing hot water extraction, separating to give a filtrate, and adjusting the filtrate to a neutral pH to give a neutral filtrate; performing adsorption and elution processing on the neutral filtrate using a macroporous resin to give an eluent, and performing drying processing on the eluent to give the ginkgo leaf ketones and esters, wherein the elution processing is performed sequentially using ethanol aqueous solutions with ascending concentrations. The ginkgo leaf ketones and esters have anti-oxidation effects.

Description

A method for extracting ketone esters from ginkgo leaves and its products and applications Technical Field

[0001] The present invention relates to the technical field of plant extraction, and in particular to a method for extracting ketone esters from ginkgo leaves, and a product and application thereof. Background Art

[0002] Nowadays, life is stressful, and people at all stages of life experience varying degrees of stress, which can easily lead to restlessness, chest tightness, and heartache. At the same time, many unhealthy lifestyle habits, such as smoking, alcoholism, frequent consumption of high-fat, high-protein, and high-calorie foods, and lack of exercise, can easily lead to elevated levels of free radicals and cholesterol in the blood. Studies have shown that free radicals in the blood indiscriminately attack normal cells, causing excessive lipids in the blood and accelerating arteriosclerosis. Free radicals also attack nucleic acid molecules in cells, becoming a significant factor in inducing cancer. Furthermore, the excessive oxidation produced by free radicals can easily accelerate cellular and even human aging. How to remove free radicals and excess cholesterol from human blood vessels, prevent cardiovascular and cerebrovascular diseases, and delay aging is a major issue facing people.

[0003] In response to these questions, people have begun to explore the potential of ginkgo trees. Ginkgo biloba is an ancient tree species unique to China, and the Chinese have had a special fondness for it for thousands of years. Ginkgo biloba has a long history of medicinal use in my country. Li Shizhen of the Ming Dynasty recorded in his Compendium of Materia Medica that ginkgo biloba, when cooked, warms the lungs and replenishes qi, calms asthma and coughs, reduces urination, and stops leukorrhea. When eaten raw, it reduces phlegm and disinfects and kills insects. The Chinese Pharmacopoeia clearly states: "Ginkgo biloba leaves are sweet, bitter, astringent, and neutral in nature, and enter the heart and lung meridians. They function to astringe the lungs, calm asthma, promote blood circulation and remove blood stasis, and relieve pain. They are used for cough and asthma caused by lung deficiency, coronary heart disease, angina pectoris, and hyperlipidemia." Ginkgo biloba is a traditional folk remedy for clearing the heart and relieving depression. It not only improves memory and cognition, increases blood circulation in the brain, and increases nerve conduction speed, thereby promoting brain function, but also protects the nervous system and prevents and treats neurological diseases. Ginkgo biloba extract also has the effects of lowering blood pressure, blood lipids, and blood sugar. Ginkgo biloba extract is rich in flavonoids and phenolic acids, which have strong antioxidant properties. These compounds can scavenge free radicals and reduce oxidative stress, thereby protecting cellular health. Ginkgo biloba extract also has anti-inflammatory properties, inhibiting inflammation and alleviating symptoms such as pain and swelling. Ginkgo biloba extract can also inhibit the growth and spread of tumor cells, exerting an anti-tumor effect.

[0004] Modern scientific research has shown that ginkgo biloba extract contains a variety of active substances, such as flavonoids and terpene lactones, which have the potential to improve cardiovascular circulation, provide antiviral and anticancer effects, promote anti-aging, and lower cholesterol. Furthermore, ginkgo biloba extract has minimal toxicity and side effects and is safe to consume. Flavonoids and lactones are two natural active ingredients widely found in the plant kingdom and are active ingredients in many Chinese herbal medicines. In recent years, advances in free radical life sciences have led to unprecedented attention for ketoesters, which possess potent antioxidant and free radical scavenging properties. Ketoesters are involved in the metabolism of phosphate and arachidonic acid, protein phosphorylation, calcium ion transfer, free radical scavenging, antioxidant activity enhancement, redox reactions, chelation, and gene expression. Naturally derived bioflavonoids and lactones have a low molecular weight and are rapidly absorbed by the human body. They can cross the blood-brain barrier and enter adipose tissue, where they exhibit the following benefits: fatigue relief, vascular protection, arteriosclerosis prevention, capillary dilation, microcirculation dredging, activation of brain and other organ cell functions, anti-fat oxidation, and anti-aging.

[0005] Currently, the extraction yield of ginkgo biloba esters is low, and the application of ginkgo biloba esters generally relies on traditional experience and quality standards. The main characteristics of the current ginkgo biloba ester quality standards include: (1) a total ginkgolic acid mass fraction of less than 5 mg / kg; (2) a total flavonol glycoside mass fraction of 24.0-35.0%; and a total terpenoid lactone mass fraction of 6.0-12.0%. How to overcome these inherent limitations and improve the extraction yield and antioxidant effect of ginkgo biloba esters has become a technical problem that needs to be solved in this field. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for extracting ketone esters from ginkgo leaves, its products and applications, in order to solve the problems existing in the above-mentioned prior art. This method can effectively improve the extraction yield of ginkgo leaf ketone esters, and the antioxidant effect of the ginkgo leaf ketone esters obtained by this method is greatly improved.

[0007] To achieve the above object, the present invention provides the following solutions:

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

[0009] 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;

[0010] 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;

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

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

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

[0014] Furthermore, the temperature of the hydrothermal extraction is 80-100°C.

[0015] Furthermore, the hydrothermal extraction time is 1 hour.

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

[0017] The present invention also provides a ginkgo biloba ketone ester prepared according to the above method.

[0018] The present invention also provides the use of the above-mentioned ginkgo biloba ketone ester in the preparation of antioxidant products.

[0019] The present invention also provides an antioxidant product, the active ingredient of which includes the above-mentioned ginkgo biloba ketone ester.

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

[0021] The present invention discloses the following technical effects:

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

[0023] (2) The ginkgo biloba ketone esters extracted using the method of the present invention have a significantly increased proportion of terpenoid lactones and an effectively enhanced antioxidant function. The present invention can obtain a ginkgo biloba ketone ester product with a high antioxidant effect through a single extraction. This method breaks through inherent limitations and achieves a simultaneous increase in the extraction yield and antioxidant effect of ginkgo biloba ketone esters, which has important market application prospects.

[0024] (3) The ginkgo biloba ketone esters extracted by the present invention can be prepared into antioxidant products, such as effervescent tablets or effervescent granules, and the effervescent tablets or effervescent granules have the advantages of good water solubility, easy portability, and good taste. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 shows the effect of vitamin C and different ginkgo biloba ketone esters on O 2- Statistical graph of the inhibition rate;

[0027] FIG2 is a statistical graph showing the inhibition rates of vitamin C and different ginkgo biloba ketone esters on hydroxyl radicals (·OH). DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

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

[0034] The method for determining the content of total flavonol glycosides and total terpenoid lactones can be found in the reference “Zhang Guoyong, Liang Xiaomei, Fang Lei. Quality comparison and cost analysis of Ginkgo biloba leaves and Ginkgo biloba ester tablets. Chinese Journal of Modern Applied Pharmacy. 201431(9):1136-1140”.

[0035] The calculation formula of the extraction rate of Ginkgo biloba ketone ester is:

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

[0037] Example 1

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

[0039] (2) The adsorption column was filled with D101 resin, and the resin in the adsorption column was activated with a 30% by volume ethanol aqueous solution for 24 h.

[0040] (3) Use 4BV of ethanol aqueous solution (the volume fraction of ethanol aqueous solution is 30%) to pass through the column at a flow rate of 2BV / h, and then use distilled water to pass through the column at a flow rate of 2BV / h to wash until there is no alcohol smell; then use 4BV of hydrochloric acid (the concentration of hydrochloric acid is 2wt.%) to pass through the column at a flow rate of 4BV / h, and then use distilled water to pass through the column at a flow rate of 4BV / h until the eluent is neutral; finally, use 4BV of NaOH solution (the concentration of NaOH solution is 2wt.%) to pass through the column at a flow rate of 4BV / h, and then use distilled water to pass through the column at a flow rate of 4BV / h until the eluent is neutral, thereby obtaining an adsorption resin column.

[0041] (4) First, the pH-neutral filtrate obtained in step (1) was passed through the column at a flow rate of 2 BV / h, and no bubbles were generated in the resin layer; then, elution was performed 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, and the eluate was collected and dried to obtain 13.5 g of ginkgo biloba ketone ester product. The total ginkgo acid content of the ginkgo biloba ketone ester product was determined to be less than 5 mg / kg, the total flavonol glycoside content was 29 wt%, and the total terpenoid lactone content was 41 wt%. The extraction rate of the ketone ester was calculated to be 94.5%.

[0042] Example 2

[0043] (1) Weigh 1 kg of dry ginkgo leaf powder, add 1500 mL of lime water suspension containing 4.5 g of Ca(OH)2, stir evenly, heat to 80°C for 1 hour, then cool to room temperature and filter the filtrate; adjust the pH of the filtrate to neutral using hydrochloric acid with a pH of 3.

[0044] (2) The adsorption column was filled with D101 resin, and the resin in the adsorption column was activated with a 50% by volume ethanol aqueous solution for 24 h.

[0045] (3) Use 4BV of ethanol aqueous solution (the volume fraction of ethanol aqueous solution is 30%) to pass through the column at a flow rate of 2BV / h, and then use distilled water to pass through the column at a flow rate of 2BV / h to wash until there is no alcohol smell; then use 4BV of hydrochloric acid (the concentration of hydrochloric acid is 2wt.%) to pass through the column at a flow rate of 4BV / h, and then use distilled water to pass through the column at a flow rate of 4BV / h until the eluent is neutral; finally, use 4BV of NaOH solution (the concentration of NaOH solution is 2wt.%) to pass through the column at a flow rate of 4BV / h, and then use distilled water to pass through the column at a flow rate of 4BV / h until the eluent is neutral, thereby obtaining an adsorption resin column.

[0046] (4) First, the pH-neutral filtrate obtained in step (1) was passed through the column at a flow rate of 2 BV / h, and no bubbles were generated in the resin layer; then, elution was performed 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, and the eluate was collected and dried to obtain 13.4 g of ginkgo biloba ketone ester product. The total ginkgo acid content of the ginkgo biloba ketone ester product was determined to be less than 5 mg / kg, the total flavonol glycoside content was 31 wt%, and the total terpenoid lactone content was 37 wt%. The extraction rate of the ketone ester was calculated to be 91.1%.

[0047] Example 3

[0048] (1) Weigh 1 kg of dry ginkgo leaf powder, add 2000 mL of lime water suspension containing 4.0 g of Ca(OH)2, stir evenly, heat to 100°C for 1.5 h, then cool to room temperature and filter the filtrate; adjust the pH of the filtrate to neutral using hydrochloric acid (pH 5).

[0049] (2) The adsorption column was filled with D101 resin, and the resin in the adsorption column was activated with an ethanol aqueous solution with a volume fraction of 75% for 24 hours.

[0050] (3) Use 4BV of ethanol aqueous solution (the volume fraction of ethanol aqueous solution is 30%) to pass through the column at a flow rate of 2BV / h, and then use distilled water to pass through the column at a flow rate of 2BV / h to wash until there is no alcohol smell; then use 4BV of hydrochloric acid (the concentration of hydrochloric acid is 2wt.%) to pass through the column at a flow rate of 4BV / h, and then use distilled water to pass through the column at a flow rate of 4BV / h until the eluent is neutral; finally, use 4BV of NaOH solution (the concentration of NaOH solution is 2wt.%) to pass through the column at a flow rate of 4BV / h, and then use distilled water to pass through the column at a flow rate of 4BV / h until the eluent is neutral, thereby obtaining an adsorption resin column.

[0051] (4) First, the pH-neutral filtrate obtained in step (1) was passed through the column at a flow rate of 2 BV / h, and no bubbles were generated in the resin layer; then, elution was performed 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, and the eluate was collected and dried to obtain 14.1 g of ginkgo biloba ketone ester product. The total ginkgo acid content of the ginkgo biloba ketone ester product was determined to be less than 5 mg / kg, the total flavonol glycoside content was 30 wt%, and the total terpenoid 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)2 is replaced by a NaOH aqueous solution with a pH of 12.

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

[0055] Comparative Example 2

[0056] Same as Example 1, except that in step (1), the limewater suspension containing 20 g of Ca(OH)2 is replaced by water.

[0057] The obtained ginkgo biloba ketone ester product in this comparative example is 14.0 g, whose total ginkgolic acid content is less than 5 mg / kg, total flavonol glycoside content is 34 wt %, total terpenoid lactone content is 12 wt %, and the extraction rate of the ketone ester is 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 1 Effects of Vc and different Ginkgo biloba ketone esters on O 2 - The inhibition rate

[0068] As can be seen from Table 1, the ability of the ginkgo biloba ester prepared in Example 1 to scavenge superoxide anions is significantly higher than that of the ginkgo biloba esters of Comparative Examples 1-2, commercially available ginkgo biloba esters, and Vc. According to the extraction method of the present invention, the proportion of total terpenoid lactones in the ginkgo biloba ester can be effectively increased. Combining the scavenging effects of the ginkgo biloba ester of Example 1 and commercially available ginkgo biloba esters, it can be seen that increasing the proportion of total terpenoid lactones can effectively improve the superoxide anion scavenging effect of the ginkgo biloba ester.

[0069] 2. Scavenging effect of Ginkgo biloba ketone esters on hydroxyl radicals (·OH)

[0070] 1) Bromocresol purple-hydrochloride-Fe 2+ Solution blank test

[0071] In a 10mL colorimetric tube, add 0.4mL of bromocresol purple solution, 0.5mL of 0.1mol / L hydrochloric acid, 1.0mL of 1mmol / L ferric sulfate solution, and 0.5mL of 0.1mol / L hydrogen peroxide solution, respectively. Dilute to the mark with water, shake well, and place in a 30°C constant temperature water bath to react for 8 minutes. Remove from the tube and quickly add 0.2mL of 0.05mol / L NaF solution, mixing well to terminate the reaction. Using a 1cm cuvette, measure the absorbance of the solution at 420nm with water as the reference. Determine the absorbance A0 of a blank test solution of bromocresol purple-hydrochloric acid solution in the same manner.

[0072] 2) Determination of the scavenging rate of hydroxyl radicals (·OH) by Vc and Ginkgo biloba ketone esters

[0073] In the above bromocresol purple-hydrochloride-Fe 2+ Different concentrations of the ginkgo biloba ketone esters prepared in Example 1 and Comparative Examples 1-2, commercially available ginkgo biloba ketone esters, and vitamin C were added to the solution, and compared with a blank control in which distilled water was added. The absorbance A of the solution at 420 nm was measured.

[0074] Calculate the scavenging rate of hydroxyl radical (·OH) D=(A 空白 -A 样品 ) / A 空白 × 100% to obtain the antioxidant capacity of Ginkgo biloba ketone esters. The results are shown in Table 2 and Figure 2.

[0075] As can be seen from Table 2, the ability of the ginkgo biloba ester prepared in Example 1 to scavenge hydroxyl radicals is significantly higher than that of the ginkgo biloba ester prepared in Comparative Examples 1-2, commercially available ginkgo biloba ester, and Vc. Combining the scavenging effects of the ginkgo biloba ester prepared in Example 1 and commercially available ginkgo biloba ester, it can be seen that increasing the proportion of total terpenoid lactones can effectively improve the scavenging effect of ginkgo biloba esters on hydroxyl radicals.

[0076] Table 2 Inhibition rate of hydroxyl radical (·OH) by Vc and different ginkgo biloba ketone esters

[0077] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

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 process comprises the steps of sequentially using ethanol aqueous solutions with increasing concentrations for elution.

2. The method according to claim 1, characterized in that The content of Ca(OH)2 in the limewater suspension is 1.8-3g / L.

3. The method according to claim 1, characterized in that The mass volume ratio of the ginkgo leaf dry powder to the lime water suspension is 1000g:(1000-2000)mL.

4. The method according to claim 1, wherein The temperature of the hydrothermal extraction is 80-100°C.

5. The method according to claim 1, wherein The hydrothermal extraction time is 1 hour.

6. The method according to claim 1, characterized in that The macroporous resin is D101 resin.

7. A ginkgo biloba ketone ester prepared according to the method according to any one of claims 1 to 6.

8. Use of the ginkgo biloba ketone ester according to claim 7 in the preparation of antioxidant products.

9. An antioxidant product, characterized in that The active ingredient comprises the ginkgo biloba ketone ester according to claim 7.

10. The product according to claim 9, characterized in that The product is a medicine or a health product.

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