CN107158048A - A kind of high efficiency from ginkgo leaf extracts flavones and the method for being converted into glucoside type flavone - Google Patents

Abstract

A method for efficiently extracting flavonoids from ginkgo leaves and converting them into aglycon-type flavonoids. Ginkgo leaves are cleaned, crushed, mixed with water and put into a fermenter, sterilized at 121°C, and then steam exploded; then Aspergillus niger is added Fermentation; then add mixed enzyme solution, the amount added is 2%-5% of the reaction substrate, and the reaction time is 3-6 hours at 25-30°C; then add β-glucosidase, the amount added is the reaction substrate 1%, the reaction conditions are pH 4.3 at 42°C, and the reaction time is 5 hours; then 1% protease is added, and the reaction time is 3-4 hours at 25-30°C; the temperature is raised to 95°C for 10 minutes, and then the extraction extract flavonoids, centrifuge at 12000g for 20min, take the supernatant and concentrate it, extract it by ethanol leaching, collect the ethanol eluate, concentrate it, and use freeze-drying to obtain the product.

Description

A method for efficiently extracting flavonoids from ginkgo leaves and converting them into aglycon-type flavones

technical field

The invention relates to the technical field of extraction of ginkgo flavonoids, in particular to a method for efficiently extracting flavonoids from ginkgo leaves and converting them into aglycon-type flavones.

Background technique

Ginkgo flavonoids are Ginkgo biloba extracts, which can increase cerebral blood flow, improve cerebral vascular circulation, protect brain cells, expand coronary arteries, prevent angina pectoris and myocardial infarction, prevent thrombosis, and improve the body's immunity. It is very beneficial to patients with coronary heart disease, angina pectoris, cerebral arteriosclerosis, senile dementia, and hypertension. However, ginkgolic acid is often adulterated in ginkgo biloba extract. Ginkgolic acid has potential sensitization and mutagenic effects, and strong cytotoxicity, which can cause severe allergic reactions, gene mutations, nerve damage, nausea and heartburn, anaphylactic shock, allergic purpura, exfoliative dermatitis, gastrointestinal Adverse reactions such as mucous membrane allergy, spasm and nerve paralysis. Therefore, it is necessary to remove these impurities in the extraction method. There are many production techniques for Ginkgo biloba extract, and organic solvent method is often used abroad. The representative techniques are a series of patents DE2117419, DE1767098, DE3940092 of German Schwabe company; and the patented technique EP0360556 of Italian Indena company. A large amount of organic solvents are used in the production process, such as acetone, ethanol, carbon tetrachloride, n-hexane, methyl ethyl ketone, n-butanol, etc. The advantage of the process is that the product quality is good and stable, and the disadvantage is that the production cost is extremely high and the production process is safe. Poor performance, great impact on the environment.

At present, ginkgo flavonoids are mainly extracted from ginkgo leaves as raw materials at home and abroad. There are many methods for separating and purifying ginkgo flavonoids, including solvent extraction, supercritical method, ultrasonic method, microwave-assisted extraction, microwave method, high-speed countercurrent method, macroporous resin adsorption method, polyamide column chromatography, and enzymatic method. Among them, the most mature and commonly used extraction process is solvent extraction.

For example, CN 102805755 A relates to a preparation method for extracting high-quality ginkgo flavonoids from ginkgo leaves. The crude extract is extracted from Ginkgo biloba leaves with deionized water, and after filtration, alcohol precipitation removes impurities, clarifying agent removes impurities, enriches ginkgo flavonoids and lactones, and uses polar and weak polar macroporous resins for primary separation. Resin removal of ginkgolic acid for final Ginkgo biloba extract product. The technical advantages of the invention are as follows: a) Macroporous resins with different properties are used in combination to enrich ginkgo flavonoids and lactones and extract ginkgolic acid, so that the product quality is obviously improved and the product is guaranteed. b) Simplify the production process, shorten the production cycle, and reduce the production cost. c) The use of non-toxic and harmless organic solvents and natural clarifiers greatly reduces the residual amount of toxic and harmful substances in the product and improves the quality of the product. However, in the separation process, alcohol precipitation and impurity removal steps are complicated.

CN 101463051 A discloses a preparation process for extracting ginkgo flavonoids with calcium carbonate, including: (1) taking ginkgo leaves as raw materials and pulverizing them; adding water to 6-8 times the weight of ginkgo leaves, and adding 0.5 ～1.5% calcium carbonate, after boiling for 1～1.5 hours, filter to obtain a water extract, which is a primary extraction; in the same way, perform a secondary extraction to obtain a secondary water extract; combine the primary and secondary water extracts, (2) Concentrate the combined solution in vacuum at a temperature of 60-70°C to 0.5-0.6 times the volume of the combined solution, add ethanol until the ethanol degree is 40-50%, fully stir, and let stand for 3-4 Filter after hours; add water to dilute the filtrate so that the ethanol degree is 15 to 20%, and adjust the pH value to 3.0 with dilute sulfuric acid to obtain a diluted filtrate, and the diluted filtrate is adsorbed with a DM131 resin column; (3) the ethanol degree is 20% The ethanol aqueous solution is adjusted to a pH value of 3.0 with dilute sulfuric acid to elute the resin column impurities, and then rinsed with water to neutrality; (4) finally, the active ingredient is eluted with an ethanol aqueous solution with an ethanol degree of 70%, and then the eluent Concentrate and dry under vacuum at a temperature of 60-70° C. to obtain ginkgo flavonoids extract. The method requires the use of a large amount of acid solution, and the yield is not very high, and the process is complex and unfavorable for industrial scale production.

Disclosed in CN 1166675 A is a process for producing high-activity ginkgo leaf extract, which is characterized in that the process comprises the following steps: (1) adding extractant sodium sulfite or sodium bisulfite to the ginkgo leaf to be treated, adding The amount is 0.001-0.5 according to the w/w ratio, and water is added to 100; (2) according to w/w, the clean ginkgo leaves are fed and extracted according to the ratio of raw materials and extraction agent to 1:5-20, and the Raise the temperature at 30-100°C, process for 30 minutes-20 hours, stir the raw materials intermittently during the extraction process, control the cooling rate at 1-10°C/hour, then keep the material liquid, and then add 5-20 times the extractant of ginkgo leaves Carry out the second extraction and combine the two feed liquids; (3) filter the feed liquid obtained in step (2) to remove impurities below 200 mesh, and cool the feed liquid to below 30°C; (4) obtain from step (3) The feed liquid flows through the resin adsorption column for refining and purification, after washing with water to remove impurities, then rinse the resin column with 30%-95% lower alcohol to obtain an eluent; (5) the eluent described in step (4) Concentrate in vacuum at a temperature below 50° C., reclaim the solvent, and obtain a concentrated solution; (6) dry the concentrated solution obtained in step (5), and (7) quantify the product obtained in step (6) by reverse-phase high-performance liquid chromatography Analysis of ginkgo flavonoids, terpene lactones, proanthocyanidins and ginkgolic acids. The yield of this method is also not high, and the preparation process is relatively complicated, so there is huge room for improvement.

In addition, in the prior art, the extractant usually has relatively high toxicity, is also relatively harmful to the human body, and is also potentially dangerous during the production process.

Finally, the metabolic pathways of flavonoids in animals are different. Only aglycon-type flavonoids can be directly absorbed into the blood of animals, while most glycoside-type flavonoids cannot enter the blood through the small intestine wall in humans, but only A small part of flavonoids can be hydrolyzed into aglycones under the action of colonic microorganisms before they can enter the blood. Therefore, the bioavailability of flavone glycosides in animals is much lower than that of aglycone flavones. Therefore, improving the configuration of flavonoids and increasing their absorption rate in blood is an important way to increase the bioavailability of flavonoid products. At present, there are mainly chemical and biological methods for the configuration transformation of flavone glycosides. Many researchers at home and abroad use chemical methods to hydrolyze flavone glycosides, as well as chemical derivatization reactions such as etherification, esterification, and acylation of flavones. Can change the properties of flavonoids. However, these methods often shield the main functional group of flavonoids—the phenolic hydroxyl group—in the reaction process, which has adverse effects on antioxidants. Simultaneously, these methods have many by-products, which easily cause environmental pollution. Therefore, in order to improve the absorption effect of flavonoids in the human body, it is also urgent to develop a high-efficiency conversion of flavones into aglycone flavones.

Contents of the invention

The invention provides a method for efficiently extracting flavonoids from ginkgo leaves and converting them into aglycon-type flavones, which mainly includes crushing, sterilization, steam explosion, enzymolysis, extraction, ethanol treatment, concentration, drying and the like.

Specifically, the present invention provides a method for efficiently extracting flavonoids from ginkgo leaves and converting them into aglycon-type flavonoids. The ginkgo leaves are cleaned and crushed into 20-100 meshes. The ginkgo leaves are mixed with water, and the amount of water added is 40%-60 %; the mixture was put into a fermenter, sterilized at 121°C, and then steam exploded; inoculated with Aspergillus niger, 10 ⁸ /mL seed liquid inoculum was 100mL/100kg, and the fermentation condition was 30°C for 3 days; then added mixed enzyme liquid, added The amount is 2%-5% of the reaction substrate, and the reaction time is 3-6 hours at 25-30°C; then add β-glucosidase, the addition amount is 1% of the reaction substrate, and the reaction condition is 42°C Under the conditions, the pH is 4.3, and the reaction time is 5 hours; then add 1% protease, and the reaction time is 3-4 hours at 25-30°C; raise the temperature to 95°C for 10 minutes, then add the extractant to extract flavonoids, centrifuge at 12000g for 20 minutes , after the supernatant was taken and concentrated, extracted by ethanol leaching, the ethanol eluate was collected, concentrated, and freeze-dried to obtain the product.

In another aspect of the present invention, the steam explosion uses saturated water vapor as the working medium, the pressure is 0.73 MPa, and the pressure holding time is 70 seconds.

In another aspect of the present invention, the mixed enzyme liquid is a combination of the three enzyme liquids of cellulase, hemicellulase, and pectinase; the specific composition ratio is cellulase: hemicellulase: pectinase = 2:1:5.

In another aspect of the present invention, the extractant is 4% sodium citrate with a pH of 8.0. The extraction conditions are as follows: the ratio of the weight of ginkgo biloba leaves to the extractant is 1:2-4, feeding and extracting, the extraction temperature is 30-100°C, the treatment is 1-5 hours, and the raw materials are stirred intermittently for 10 minutes during the extraction process.

In another aspect of the present invention, the raw material for production can be fresh ginkgo leaves or dried ginkgo leaves, and the leaves do not need to be pulverized.

In another aspect of the invention, the ethanol eluate is concentrated under vacuum.

In another aspect of the present invention, the Aspergillus niger has the preservation number CCTCCM2012515.

In another aspect of the present invention, the production process adopts freeze drying, spray drying or vacuum drying to dry the concentrate.

In another aspect of the present invention, the method for detecting ginkgo flavonoids, ginkgolides and ginkgolic acid content in the extract with high performance liquid chromatography (HPLC) is respectively: the detection method of ginkgo flavones is: mobile phase: methanol: phosphoric acid: water =57:0.2:42.8, flow rate is 1.0ml/min, detection wavelength is 370nm, chromatographic column temperature: 25 ℃, injection volume: 20 μ l; The detection method of ginkgolide is: mobile phase is methanol:water=25:75 , flow rate 1.0mL/min, detector is ELSD; proanthocyanidin detection method is detection wavelength: 280nm; column temperature 30 ℃; mobile phase flow rate is 1.0ml/min; mobile phase: A, 20.0% acetic acid; B: 80% acetonitrile; Gradient elution: 0min-85min, 100%-20%A; 6min-85min, 4%-80%B, injection volume: 10μL; ginkgolic acid detection method is methanol:water=90:10, flow rate is 1.0 mL/min, the detection wavelength is 310nm.

Compared with the prior art, the method for extracting flavonoids of the present invention has the following advantages:

1. The present invention uses Aspergillus niger fermentation and compound enzyme solution to pre-treat the ginkgo leaf tissue, which improves the mass transfer characteristics of the intracellular components of the material; the plant cell wall plays a protective role to prevent the random entry and exit of intracellular and extracellular substances, mainly composed of cellulose Composed of pectin and cellulase, cellulase and pectinase are used to dissolve the cell wall, which can effectively promote the dissolution of active ingredients in plant cells. After fermentation and enzyme liquid mixed treatment, it has better treatment effect.

2. The present invention has explored a large number of conditions in the early stage, and uses β-glucosidase to convert the aglycon-type flavonoids for the products of the previous extraction steps. The conversion efficiency is very high and has a good effect.

3. Steam explosion is used for specific treatment, especially the conditions for blasting are optimized for Ginkgo biloba tissue, which can quickly release flavonoids in the tissue.

4. Through a large number of experiments, sodium citrate was finally selected as the extractant, which has a non-toxic effect than the prior art such as sodium bisulfite, is more efficient to use, and has a better extraction effect. The most important thing is to effectively remove ginkgolic acid.

5. The extraction process of the present invention does not require the use of macroporous resins, which saves costs. At the same time, ethanol is used as extraction, the extraction rate is higher, and the extraction temperature is lower, and there is no organic solvent residue, which reduces the dissolving ability of impurities, so that flavonoids can be extracted The primary processing purity of the extract reaches more than 98.6%, therefore, the present invention has the advantages of high extract purity, good extraction rate and high safety;

6. Freeze-drying is used to keep the active ingredients of flavonoids from being destroyed, and the biological activity is high. No toxic and harmful organic solvents are ever used in the process, and the final product obtained has no organic solvent residues; it is suitable for human use with confidence.

detailed description

Below in conjunction with embodiment the present invention is described in further detail. The specific embodiment only shows the best implementation, but cannot limit the protection scope of the present invention.

Example 1 Efficient extraction of flavonoids from Ginkgo biloba

Take 100kg of clean and dry ginkgo leaves, crush them into 20 meshes, add water to the ginkgo leaves, and add 50% of the water; put the mixture into a fermenter, sterilize at 121°C, and then carry out steam explosion, which uses saturated water vapor as the working medium , the pressure is 0.73MPa, and the holding time is 70 seconds; inoculate Aspergillus niger, the inoculation amount of 108/mL seed solution is 100mL/100kg, and the fermentation condition is 30°C for 3 days; then add mixed enzyme solution: cellulase: hemicellulose Enzyme: pectinase=2:1:5, the amount added is 2% of the reaction substrate, the reaction time is 6 hours at 30°C, then 1% protease is added, and the reaction time is 4 hours at 30°C; Raise the temperature to 95°C for 10 minutes, then add sodium citrate with a pH of 8.0 and 4%. The extraction conditions are as follows: 1:3 according to the weight of the ginkgo leaf and the extraction agent. Stir the raw materials intermittently for 10min50r/min during the extraction process. 12000g centrifugal 20min, take supernatant and adopt concentrating tower to carry out concentrating and concentrating ratio is 1:10, concentrated solution is leached with absolute ethanol, and the final concentration of ethanol is 90%, receives ethanol extracting liquid, concentrating tower carries out concentrating and concentrating ratio is 1:15, using a spray freeze dryer to freeze-dry to obtain 4.36kg light yellow ginkgo leaf extract product.

The specific components were analyzed by high-performance liquid chromatography, and the test results were: total flavonoid glycosides 38.0%, total terpene lactones 10.6%, proanthocyanidins 12.3%, ginkgolic acid 0.1ppm, solubility in water 11.8%, of which ginkgolic acid content 0.01ppm Almost undetectable.

Example 2 Comparative Example 1

The composition and proportion of the extractant (calculated by weight): sodium bisulfite 10kg; tap water 8000kg; pH5.0.

Clean 300kg of dried ginkgo leaves, filter the water, put them into the extraction tank, add 5000kg of extractant at 94°C, cover and seal, raise the temperature in the extraction tank to 97°C, and keep 1 atmospheric pressure, stirring every half an hour For 1 minute, the temperature in the tank was controlled to drop by 2.5°C per hour. After leaching for 16 hours, 4400kg of the feed liquid was filtered out. Then add 3000kg of extractant solution to the extraction tank for the second leaching, filter out 3100kg of feed liquid after 10 hours, combine the two feed liquids with a centrifugal filter to coarsely filter out impurities below 20 mesh, and sand filter to fine filter out Impurities below 200 mesh were removed, and the feed liquid was cooled to 30°C.

The feed liquid flows through the NKA-9 resin adsorption column at a flow rate of 1500kg/hour, and then washes with water to remove impurities that are not adsorbed or adsorbed, and washes with deionized water twice the column volume, and then rinses the resin with 70% ethanol for adsorption 1500kg of ethanol eluate was collected, and the ethanol eluate was concentrated 20 times in a three-effect concentration tower at a temperature of 43°C, and the concentrated solution was freeze-dried to obtain 9.75kg of product. Test results: total flavonoid glycosides 28.1%, total terpene lactones 9.2%, proanthocyanidins 10.3%, ginkgolic acid 3.8ppm, water solubility 7.7%.

Example 3 Comparative Example 2

(1) Pulverize 100 kilograms of ginkgo leaves with a pulverizer, pour them into an extraction tank, add 700 kilograms of water, and add 1.5 kilograms of calcium carbonate at the same time, heat, filter after boiling for 1.2 hours, and obtain a water extract; the same method Extract again, combine the first and second water extracts to obtain the combined solution;

(2) Concentrate the combined solution to 50L in a vacuum concentration tank at 65°C, add ethanol until the ethanol degree is 45%, stir fully, filter after standing for 3.5 hours, discard the alcohol sediment; dilute the filtrate with water, so that The ethanol degree is 18%, is adjusted to pH value 3.0 with dilute sulfuric acid, obtains dilute filtrate; The dilute filtrate is adsorbed with DM131 resin column, and resin column diameter height ratio is 1:5, flow velocity 1Bv/h, resin consumption is resin consumption is 50 kg (volume 50.1L);

(3) Adjust 100L of ethanol aqueous solution with 20% alcohol concentration to pH 3.0 with dilute sulfuric acid, elute impurities for 2 hours, and the elution flow rate is 1BV/h, and then wash with water until neutral;

(4) 70% ethanol aqueous solution was used to elute the active ingredient for 3 hours with 400L ethanol degree, flow velocity 2BV/h, then the eluent was vacuum concentrated and dried at 60°C to obtain 2.5 kilograms of ginkgo flavonoids extract, wherein The total flavonoid glycosides are 25.3%, the total terpene lactones are 7.2%, the proanthocyanidins are 9.0%, and the ginkgolic acid is 3.4ppm.

Example 4 A method for efficiently extracting flavonoids from ginkgo leaves and converting them into aglycon-type flavones

Take 100kg of clean and dry ginkgo leaves, crush them into 20 meshes, add water to the ginkgo leaves, and add 50% of the water; put the mixture into a fermenter, sterilize at 121°C, and then carry out steam explosion, which uses saturated water vapor as the working medium , the pressure is ^0.73MPa , and the holding time is 70 seconds; inoculate Aspergillus niger, the inoculation amount of 108/mL seed solution is 100mL/100kg, and the fermentation condition is 30°C for 3 days; then add mixed enzyme solution: cellulase: hemicellulose Sulfase: pectinase=2:1:5, the addition is 2% of the reaction substrate, and the reaction time is 6 hours at 30°C; then add β-glucosidase, the addition is 1% of the reaction substrate %, the reaction conditions are pH 4.3 at 42°C, and the reaction time is 5 hours; then 1% protease is added, and the reaction time is 4 hours at 30°C; the temperature is raised to 95°C for 10 minutes, and then the pH is 8.0, 4% The sodium citrate, the extraction condition is according to the ratio of ginkgo leaf weight and extractant is 1: 3, feeds and extracts and extracts, extracts temperature 60 ℃, handles 3 hours, and leaching process intermittently 10min 50r/min stirs raw material and gets final product. 12000g centrifugal 20min, take supernatant and adopt concentrating tower to carry out concentrating and concentrating ratio is 1:10, concentrated solution is leached with absolute ethanol, and the final concentration of ethanol is 90%, receives ethanol extracting liquid, concentrating tower carries out concentrating and concentrating ratio is 1:15, using a spray freeze dryer to freeze-dry to obtain 4.37kg light yellow ginkgo leaf extract product.

The specific components were analyzed by high-performance liquid chromatography, and the test results were: total flavonoid glycosides 38.2%, total terpene lactones 10.5%, proanthocyanidins 12.4%, ginkgolic acid 0.1ppm, solubility in water 11.7%, of which ginkgolic acid content 0.01ppm Almost undetectable. Among the total flavonoid glycosides, the aglycon type flavones accounted for 92.3%.

As can be seen from the above examples, the extract of the present invention has a better extraction effect, especially ginkgolic acid has a significant downward trend, and the proportion of aglycon-type flavones in the total flavonoid glycosides is relatively high, which also shows that the glycosides of the present invention The conversion efficiency of aglycon-type flavones is also higher, which shows that the enzymes of the present invention have a better synergistic effect, and at the same time, the loss of aglycone-type flavones is also lower during the extraction process, which has a better extraction effect. Moreover, the overall extraction steps of the present invention are also simple, and are suitable for industrialized production. The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (7)

1. A method for efficiently extracting flavonoids from Ginkgo biloba and converting them into aglycon-type flavones, mainly comprising steps such as crushing, sterilization, steam explosion, fermentation, enzymolysis, extraction, ethanol treatment, concentration, and drying. 2. A method for efficiently extracting flavonoids from ginkgo leaves and converting them into aglycon-type flavonoids. The ginkgo leaves are cleaned and crushed into 20-100 meshes. The ginkgo leaves are mixed with water, and the amount of water added is 40%-60%; the mixture is packed into Fermentation tank, sterilized at 121°C, followed by steam explosion; inoculated with Aspergillus niger, ¹⁰⁸ /mL seed liquid inoculum amount was 100mL/100kg, and the fermentation condition was 30°C for 3 days; then added mixed enzyme liquid, the amount added was the reaction substrate The reaction time is 3-6 hours at 25-30°C, then add β-glucosidase in an amount of 1% of the reaction substrate, and the reaction conditions are pH 4.3 at 42°C , the reaction time is 5 hours; then add protease 1%, at 25-30°C, the reaction time is 3-4 hours; heat up to 95°C, 10min, then add extractant to extract flavonoids, centrifuge at 12000g for 20min, take the supernatant After concentration, ethanol extraction is used to extract, and the ethanol extract is collected, concentrated, and freeze-dried to obtain the product; Wherein, the steam explosion uses saturated water vapor as the working medium, the pressure is 0.73MPa, and the pressure holding time is 70 seconds; The enzyme liquid is a combination of the three enzyme liquids of cellulase, hemicellulase and pectinase; the specific composition ratio is cellulase: hemicellulase: pectinase=2:1:5; The extractant is 4% sodium citrate, the pH is 8.0; the extraction condition is 1:2-4 according to the ratio of ginkgo leaf weight to the extractant, feeding and extracting, the extraction temperature is 30-100°C, and the treatment is 1-2. 5 hours, stirring the raw materials intermittently for 10 minutes during the leaching process. 3. according to the method for claim 1 or 2, described production raw material is ginkgo fresh leaf or through the dried ginkgo leaf. 4. The method according to claim 3, the ethanol extract is concentrated under vacuum conditions. 5. The method according to claim 1, 2 or 4, wherein said freeze-drying is drying the concentrate under vacuum conditions. 6. the ginkgo flavonoid extract that the method for preparing according to claim 2 or 4 obtains. 7. The flavonoid extract prepared according to claim 6 is used in the preparation of health products or medicines.