阅读说明：本技术 一种银杏酚酸溶液剂及制备方法 (Ginkgolic acid solution and preparation method thereof ) 是由 汤先伟 汤海江 于 2021-08-15 设计创作，主要内容包括：本发明公开了一种银杏酚酸溶液剂,其中银杏酚酸是制剂中的唯一活性成分,银杏酚酸溶液剂中含有以重量百分比计30%～70%的银杏酚酸提取物,与余量的溶剂定容至100%混合均匀后,用G-(3)纯熔玻璃漏斗过滤,分装制成；其中银杏酚酸提取物中的银杏酚酸的含量超过60重量%；其中所述的溶剂为甘油缩甲醛、DMF、DMA、DMSO中的任意一种或两种任意比例的混合物,或甘油缩甲醛、DMF、DMA、DMSO重量30%～60%的水溶液或乙醇溶液；本发明银杏酚酸溶液剂作为防治农业作物病原菌和病虫害中的应用。(The invention discloses a ginkgolic acid solution, wherein ginkgolic acid is the only active component in the preparation, the ginkgolic acid solution contains 30-70% of ginkgolic acid extract by weight percentage, the ginkgolic acid extract and the rest solvent are added to the volume of 100% and mixed uniformly, and G is used for diluting the solution to the desired volume 3 Filtering with a pure molten glass funnel, and subpackaging to obtain the final product; wherein the content of ginkgolic acid in the ginkgolic acid extract exceeds 60 percent by weight; wherein the solvent is any one of glycerol formal, DMF, DMA and DMSO or a mixture of two of glycerol formal, DMF, DMA and DMSO in any proportion, or an aqueous solution or an ethanol solution with the weight of 30-60% of glycerol formal, DMF, DMA and DMSO; the ginkgolic acid solution is applied to preventing and treating pathogenic bacteria and plant diseases and insect pests of agricultural crops.)

1. A ginkgolic acid solution is characterized in that the ginkgolic acid solution is prepared from 30-70 percent of ginkgolic acid extract by weight percentage and the balance of solvent; the preparation method comprises mixing 30-70 wt% of ginkgolic acid extract with the rest solvent to 100% of constant volume, and mixing with G₃Filtering with a pure molten glass funnel, and packaging; the ginkgolic acid extract is obtained by extracting the following steps:

(1) drying and crushing ginkgo biloba testa or ginkgo leaf waste residue, sieving with a 200-300 mesh sieve to obtain ginkgo biloba testa powder or ginkgo leaf waste residue powder, placing the ginkgo biloba testa powder or ginkgo leaf waste residue powder in an ethanol solution with the weight 10 times of the content and the concentration of 85% V/V, leaching for 24-48 h under the condition of low temperature, stirring for 20min every 2h during leaching, rough filtering after leaching, fine filtering and recovering ethanol to obtain concentrated thick paste; adding water with the weight being 30-50 times of that of the thick paste, heating to 95-98 ℃ to a near-boiling state, continuously stirring and preserving heat for 30min, stopping heating, standing and cooling, standing and precipitating at the temperature of 2-4 ℃ for 24h, discharging supernatant by a siphon method, and centrifuging the precipitate to obtain a paste extract;

(2) adding the paste obtained in the step (1) into a methanol solution with 10 times of weight and 90% V/V concentration, leaching for 5-7 h at room temperature, stirring for 20min every 1h during leaching, performing rough filtration after leaching, recovering methanol after fine filtration to obtain a concentrated thick paste, adding water with 20-30 times of weight into the thick paste, heating to 70-80 ℃, continuously stirring and preserving heat for 30min, stopping heating, standing and cooling, standing and precipitating at 2-4 ℃ for 24h, discharging a supernatant by using a siphon method, and centrifuging the precipitate to obtain the paste;

(3) adding the paste obtained in the step (2) into petroleum ether with the weight of 10 times, stirring and leaching for 3-4 hours at the temperature of 15-20 ℃ and the rotating speed of 20r/min, after leaching, performing rough filtration, then recovering the petroleum ether through fine filtration to obtain concentrated thick paste, then adding the thick paste into water with the weight of 20-30 times, heating to 70-80 ℃, keeping the temperature and stirring for 30 minutes, stopping heating, standing and cooling, standing and precipitating for 24 hours at the temperature of 2-4 ℃, discharging supernatant by using a siphon method, and then performing centrifugal treatment on the precipitate to obtain the paste; the paste is ginkgolic acid extract; the content of the ginkgolic acid in the ginkgolic acid extract exceeds 60 percent by weight;

the solvent is any one or a mixture of two of glycerol formal, N-dimethylformamide, N-dimethylacetamide and dimethyl sulfoxide in any proportion; or water solution or ethanol solution with 30 to 60 percent of the weight of glycerol formal, N-dimethylformamide, N-dimethylacetamide and dimethyl sulfoxide; the ethanol solution is ethanol with the concentration of 60 percent V/V; the water is aqueous solution.

2. The ginkgolic acid solution of claim 1, wherein the mild condition is 40-50 ℃.

3. The ginkgolic acid solution of claim 1, wherein the conditions of rough filtration and fine filtration are that the leaching solution is filtered for three times of 100 meshes, 300 meshes and 500 meshes, and then filtered by using a 0.2 μm microporous filter membrane.

4. The solution of ginkgolic acid according to claim 1, wherein said petroleum ether is petroleum ether with a boiling range of 60-90 degrees.

5. The ginkgolic acid solution of claim 1, wherein the centrifugation treatment is performed by any one of a three-leg centrifuge or a tubular high-speed centrifuge; the centrifugal conditions are that the rotating speed is 3000-5000 r/min and the centrifugal time is 20 min.

6. The method for preparing the ginkgolic acid solution of claim 1, which comprises the following steps:

(1) drying and crushing ginkgo biloba testa or ginkgo leaf waste residue, sieving with a 200-300 mesh sieve to obtain ginkgo biloba testa powder or ginkgo leaf waste residue powder, placing the ginkgo biloba testa powder or ginkgo leaf waste residue powder in an ethanol solution with the weight 10 times of the content and the concentration of 85% V/V, leaching for 24-48 h under the condition of low temperature, stirring for 20min every 2h during leaching, rough filtering after leaching, fine filtering and recovering ethanol to obtain concentrated thick paste; adding water with the weight being 30-50 times of that of the thick paste, heating to 95-98 ℃ to a near-boiling state, continuously stirring and preserving heat for 30min, stopping heating, standing and cooling, standing and precipitating at the temperature of 2-4 ℃ for 24h, discharging supernatant by a siphon method, and centrifuging the precipitate to obtain a paste extract;

(2) adding the paste obtained in the step (1) into a methanol solution with 10 times of weight and 90% V/V concentration, leaching for 5-7 h at room temperature, stirring for 20min every 1h during leaching, performing rough filtration after leaching, recovering methanol after fine filtration to obtain a concentrated thick paste, adding water with 20-30 times of weight into the thick paste, heating to 70-80 ℃, continuously stirring and preserving heat for 30min, stopping heating, standing and cooling, standing and precipitating at 2-4 ℃ for 24h, discharging a supernatant by using a siphon method, and centrifuging the precipitate to obtain the paste;

(3) adding the paste obtained in the step (2) into petroleum ether with the weight of 10 times, stirring and leaching for 3-4 hours at the temperature of 15-20 ℃ and the rotating speed of 20r/min, after leaching, performing rough filtration, then recovering the petroleum ether through fine filtration to obtain concentrated thick paste, then adding the thick paste into water with the weight of 20-30 times, heating to 70-80 ℃, keeping the temperature and stirring for 30 minutes, stopping heating, standing and cooling, standing and precipitating for 24 hours at the temperature of 2-4 ℃, discharging supernatant by using a siphon method, and then performing centrifugal treatment on the precipitate to obtain the paste; the paste is ginkgolic acid extract;

(4) 30-70% of the ginkgolic acid extract obtained in the step (3) in percentage by weight is mixed with the rest solvent until the volume is 100%, and G is used for uniformly mixing₃Filtering with a pure molten glass funnel, and packaging.

7. The method according to claim 6, wherein the solvent is any one of glycerol formal, N-dimethylformamide, N-dimethylacetamide and dimethyl sulfoxide or a mixture of two of glycerol formal, N-dimethylformamide, N-dimethylacetamide and dimethyl sulfoxide in any proportion; or water solution or ethanol solution with 30 to 60 percent of the weight of glycerol formal, N-dimethylformamide, N-dimethylacetamide and dimethyl sulfoxide; the ethanol solution is 60% V/V ethanol water solution.

8. The method of claim 6, wherein said mild conditions are between 40 ℃ and 50 ℃.

9. The method as claimed in claim 6, wherein the conditions of rough filtration and fine filtration are that the leaching solution is filtered for three times of 100 meshes, 300 meshes and 500 meshes, and then filtered by using a 0.2 μm microporous membrane.

10. The method according to claim 6, wherein the centrifugal treatment is performed by using any one of a three-leg type centrifuge and a tubular type high-speed centrifuge; the centrifugal conditions are that the rotating speed is 3000-5000 r/min and the centrifugal time is 20 min.

Technical Field

The invention belongs to the technical field of biopesticide processing, also relates to the technical field of medical treatment of preparations for treating skin diseases, and particularly relates to a preparation method of a ginkgolic acid solution.

Background

Ginkgolic acids (Ginkgolic acids) are organic components with important biological activity in ginkgo leaves, fruits, testa and rhizomes, and are derivatives of 6-alkyl or 6-alkenyl phenolic acids. At present, the sources of ginkgolic acid are mostly obtained from waste residues and waste liquids of ginkgo biloba extracts, ginkgo nuts, ginkgo biloba leaves, ginkgo biloba testa and ginkgo rhizomes, wherein the content of dry ginkgo biloba leaves is about 1-2%, the content of ginkgo biloba testa (dry) is about 3-5%, the content of waste liquids after processing the ginkgo biloba extracts is also rich, and in addition, chemical synthetics exist.

The existing research shows that the ginkgolic acid has good inhibition effect on a plurality of bacteria in vitro and can inhibit the growth of part of agricultural pathogenic bacteria; the extraction of ginkgolic acid and the in vitro antibacterial activity, the anti-agricultural pathogenic bacteria and pathogenic protozoa and the anti-tumor activity have been reported in documents. For years, Chinese scholars have long studied the antibacterial and insecticidal effects of ginkgolic acid, and the results show that ginkgolic acid can replace pesticides to control agricultural pests such as aphids, python wax, cabbage caterpillars and red spiders and various plant pathogenic fungi (CN 109287665A); is used for preventing and treating cotton aphid, rice borer and killing bacillus (CN 101326921A); the application of the compound as a medicament for preventing and treating rice sheath blight (CN 108207954A); also has application in killing ampullaria gigas by ginkgolic acid (CN 101911938A); the application of ginkgolic acid in killing blue algae (microcystis aeruginosa) (CN 103299998A); there are reports related to prevention and treatment of schistosomiasis. In the patent documents published by the state, the ginkgolic acid is used for preparing external medicines, cosmetics or disinfectants for preventing and treating skin diseases such as cutaneous pruritus, herpes or corn callus and the like (CN 102319272A), external preparations for preventing and treating bromidrosis, hyperhidrosis and chromhidrosis (CN 102349919A) and application of ginkgolic acid nano liposome in treating gynecology and venereal diseases.

The inventor is dedicated to research and development of preparation and application of ginkgolic acid and ginkgolic acid contained in ginkgo biloba and ginkgo biloba exocarp for many years, and the existing published literature reports that the ginkgolic acid preparation has the beneficial effects of preventing and treating crop aphid, cabbage caterpillar, thrips, moth larva and other insect pests, downy mildew, powdery mildew, gray mold, epidemic disease, pear scab, rice blast, rice sheath blight, tomato leaf blight and the like.

A large number of experiments show that the ginkgolic acid preparation has a prevention rate of more than 80 percent on insect pests such as aphids, cabbage caterpillars, thrips and the like; has a control rate of more than 90 percent on fungal diseases such as cucumber downy mildew, strawberry gray mold, tomato leaf blight, rice sheath blight and the like (CN 110973164A).

Aim at the existing published literatureThe preparation method of the Chinese patent application number 201510415427.2, which is used for extracting the ginkgolic acid from the ginkgo episperm as a pesticide preparation, comprises the following steps: (1) the preparation method of the mother medicine comprises extracting with ethanol over 80% or methanol over 90% to obtain extract such as ginkgol. Removing impurities by water extraction method, and purifying ginkgol as mother medicine. (2) The preparation method comprises adding dispersing agent and solubilizer into the parent drug, stirring and heating to mix well to obtain the soluble ginkgol solution. The method of application No. 201811193998.6 is: extracting ginkgolic acid substances in the ginkgo episperm with 80% ethanol or methanol as solvent, removing impurities by water extraction method, and purifying the extract, wherein the extract contains about 31% ginkgolic acid as the mother medicine containing effective components. The preparation method of the preparation comprises the following steps: with G₁₈And O_SHeating and mixing the mother medicine and the solubilizer as solubilizer to prepare the preparation containing about 23% of ginkgolic acid serving as an effective component. The ginkgolic acid soluble solution is not only an insecticide, but also a bactericide. The pesticide has a control rate of more than 80% on pests such as aphids, cabbage caterpillars, thrips and the like, and has a control rate of more than 90% on agricultural fungal diseases such as cucumber downy mildew, strawberry gray mold, tomato leaf blight, rice sheath blight and the like.

How to improve the prior art to improve the extraction efficiency, reduce the mixing of excessive impurities and the stability of products in the extraction process, and avoid or reduce the solubilizer, thereby not only bringing convenience to the dilution operation in use, but also reducing the manufacturing cost; and the research on improving the extraction content and realizing the quantification of the ginkgolic acid product is needed.

Disclosure of Invention

Aiming at overcoming the defects of low extraction rate of active components of ginkgolic acid, high impurity content, unstable preparation and the like in the prior art, the ginkgolic acid solution and the preparation method thereof are provided, and the quality of a product is improved on the basis of the existing ginkgolic acid solution and the preparation method thereof. In order to realize the purpose, the technical scheme adopted by the invention is as follows:

a ginkgolic acid solution, wherein ginkgolic acid is the only active ingredient in the preparation, 30-70% of ginkgolic acid extract is contained in the ginkgolic acid solution by weight percentage, and the balance is solvent;

the ginkgolic acid extract is obtained by extracting the following steps:

(1) drying and crushing ginkgo biloba testa or ginkgo leaf waste residue, sieving with a 200-300 mesh sieve to obtain ginkgo biloba testa powder or ginkgo leaf waste residue powder, placing the ginkgo biloba testa powder or ginkgo leaf waste residue powder in an ethanol solution with the weight 10 times of the content and the concentration of 85% (V/V), leaching for 24-48 h under the condition of low temperature, stirring for 20min every 2h during leaching, rough filtering after leaching, fine filtering and recovering ethanol to obtain concentrated thick paste; adding water with the weight being 30-50 times of that of the thick paste, heating to 95-98 ℃ to a near-boiling state, continuously stirring and preserving heat for 30min, stopping heating, standing and cooling, standing and precipitating at the temperature of 2-4 ℃ for 24h, discharging supernatant by a siphon method, and centrifuging the precipitate to obtain a paste extract;

(2) adding the paste obtained in the step (1) into a methanol solution with 10 times of weight and 90% (V/V) of content concentration, leaching for 5-7 h at room temperature, stirring for 20min every 1h during leaching, performing rough filtration after leaching, recovering methanol after fine filtration to obtain concentrated thick paste, adding water with 20-30 times of weight into the thick paste, heating to 70-80 ℃, continuously stirring and preserving heat for 30min, stopping heating, standing and cooling, standing and precipitating at 2-4 ℃ for 24h, discharging supernatant by using a siphon method, and centrifuging the precipitate to obtain the paste;

(3) adding the paste obtained in the step (2) into petroleum ether with the weight of 10 times, stirring and leaching for 3-4 hours at the temperature of 15-20 ℃ and the rotating speed of 20r/min, after leaching, performing rough filtration, then recovering the petroleum ether through fine filtration to obtain concentrated thick paste, then adding the thick paste into water with the weight of 20-30 times, heating to 70-80 ℃, keeping the temperature and stirring for 30 minutes, stopping heating, standing and cooling, standing and precipitating for 24 hours at the temperature of 2-4 ℃, discharging supernatant by using a siphon method, and then performing centrifugal treatment on the precipitate to obtain the paste; the paste is ginkgolic acid extract;

the weight of the ginkgolic acid extract is taken as a reference; high Performance Liquid Chromatography (HPLC) analysis shows that the content of ginkgolic acid in the paste extract exceeds 60 wt%.

The pulverization is ultramicro pulverization which is a known and mature common method, and 200-300-mesh powder is not difficult to obtain by adopting a ball mill pulverization, a vibration mill pulverization or a jet mill pulverization according to the requirements of the invention;

the micro-temperature condition is 40-50 ℃;

the water is any one of distilled water, ion exchange water, reverse osmosis water, electrodialysis water or purified water;

the conditions of coarse filtration and fine filtration are that the leaching liquor is filtered for three times of 100 meshes, 300 meshes and 500 meshes and then filtered by a 0.2 mu m microporous filter membrane;

the centrifugal treatment conditions are that the rotating speed is 3000-5000 r/min, the centrifugation is 20min, and any one of a three-leg centrifuge or a tubular high-speed centrifuge is adopted.

(4) 30-70% of the ginkgolic acid extract obtained in the step (3) in percentage by weight is mixed with the rest solvent until the volume is 100%, and G is used for uniformly mixing₃Filtering with a pure molten glass funnel, and packaging.

The solvent is any one or a mixture of two of glycerol formal (1, 3 dioxane-5-alcohol), N-Dimethylformamide (DMF), N-Dimethylacetamide (DMA) and dimethyl sulfoxide (DMSO) in any proportion;

wherein the glycerol formal, the dimethylformamide, the dimethylacetamide and the dimethyl sulfoxide can be directly used as a solvent, and can also be diluted by water to form an aqueous solution or an ethanol solution with the weight of 30-60% according to the requirement; the ethanol solution is 60% V/V ethanol water solution.

The petroleum ether is petroleum ether with a boiling range of 60-90 degrees. In practical application, the use concentration of the formula raw materials is determined after dissolution test and stability test are carried out on the formula raw materials.

The glycerol formal is a solvent which is obtained by the reaction of glycerol and formaldehyde and is used as pesticide and pharmaceutical preparation;

glycerol formal is capable of dissolving many drugs, especially organic drugs and herbal extracts, in any ratio in propylene glycol, water, glycerol, ether, acetone, refined oils, ethanol, etc.

Rat LD₁₀₀3g/kg is injected into the abdominal cavity.

Oral administration of LD to rats₅₀It was 8 ml.kg.

Oral administration of LD to mice₅₀It was 8.6 ml/kg.

N, N-Dimethylformamide (DMF) is colorless or yellowish clear liquid, and can be mixed with water, ethanol, chloroform, acetone, diethyl ether, etc. as common organic solvent.

DMF is of low toxicity type.

Mouse LD₅₀Injecting 1.122g/kg into the abdominal cavity; intravenous injection is 100 mg/kg.

Rat LD₅₀It is administered orally at a dose of 7 g/kg.

Rabbit LD₅₀5g/kg was injected subcutaneously.

The maximum concentration allowed in air is 10ppm.

DMF is a non-associative polar solvent with very stable properties, which can dissolve many insoluble organic substances, especially some high polymers, and is called "universal solvent". Can be used as solvent for various medicines.

N, N-Dimethylacetamide (DMA) is colorless clear neutral liquid, and can be mixed with water and ethanol. Is very soluble in organic solvent or mineral oil.

DMA belongs to the low toxicity class.

Mouse LD₅₀3.236g/kg is injected intraperitoneally.

Mouse LD₁₀₀5.02g/kg is injected into the abdominal cavity.

The maximum concentration in air was allowed to be 10ppm.

Dimethyl sulfoxide (DMSO) is a viscous liquid which is almost odorless, colorless, transparent and oily, and has strong water absorption property, and the water absorption property can exceed 70% of the weight of the DMSO. Can be optionally mixed with water, ethanol, acetone, etc.

Toxicity:

mouse LD₅₀The injection is carried out in the abdominal cavity at 22.4 g/kg.

Rat LD₅₀The injection is carried out in the abdominal cavity at 22.4 g/kg.

The product is a highly polar solvent, has a wide dissolution range, can be dissolved in water-soluble and fat-soluble medicines which are insoluble in water, glycerol, ethanol, propylene glycol and fatty oil, and is also called as a universal solvent because many inorganic salts can be dissolved in the product.

The invention has the advantages of

Aiming at the defects in the prior art, the inventor overcomes the technical defects through a large number of repeated tests, so that the content of active ingredients in the ginkgolic acid extract is increased, the excessive impurities in the preparation process are reduced, the prepared biopesticide product is easy to precipitate or turbidity, the instability of the inherent quality caused by the instability of the storage period of the product is influenced, and simultaneously, a large amount of ethanol, polyalcohol ethylene oxide polymer and other nonionic surfactants used in the preparation process are avoided as a dispersing agent and a solubilizer.

The invention adopts glycerol formal, DMF, DMA and DMSO as solvents of the ginkgolic acid solution, and can be used as single solvents in practical application to achieve better dissolving effect, for example, the glycerol formal, DMF, DMA and DMSO are prepared into 30-60% aqueous solution or ethanol solution, so that the ginkgolic acid extract prepared in the invention has better dissolving effect, the product liquid is clear and transparent, and the appearance of the product is unchanged after being placed at normal temperature for 12 months, which is enough to indicate that the existing methods of adding solubilizer and the like are not needed, and the solubility and stability of the product can be improved.

In the invention, the ginkgo episperm or ginkgo biloba waste residue is subjected to micro-pulverization to obtain 200-300 mesh powder, the surface area is increased, and the cells overflow and are easy to dissolve out during leaching; the stirring during the leaching is beneficial to the replacement of the substances and the solvent through osmotic pressure so as to achieve more sufficient leaching effect.

According to the method, aiming at the property that the ginkgolic acid is easily dissolved in ethanol, methanol and petroleum ether but not dissolved in water, the ethanol, the methanol and the petroleum ether are respectively adopted for gradient replacement extraction, the extracted paste is diluted by water every 1 time of extraction, and simultaneously, water-insoluble floating or suspended impurities, foreign matters and the like in the paste are fully filtered by filtering processes with different meshes, so that the influence on the content of the ginkgolic acid in a final product due to excessive impurities mixed in the precipitation process is reduced. Because the invention adopts the continuous process of ethanol extraction → recovered ethanol → water precipitation, methanol extraction → recovered methanol → water precipitation, petroleum ether extraction → recovered petroleum ether → water precipitation, the content of the ginkgolic acid in the finally obtained ginkgolic acid extract exceeds 60 percent by weight. Compared with the method adopted by the disclosed invention document, the method not only greatly improves the content of the ginkgolic acid in the extract and has less impurity content, but also fully recycles the leaching solvent in the process, thereby saving the production cost.

The ginkgolic acid solution is applied to the prevention and treatment of agricultural pests and diseases. Wherein the agricultural pests are aphids, cabbage caterpillars, thrips or leek root maggots; the diseases are agricultural fungal diseases such as cucumber downy mildew, strawberry gray mold, tomato leaf blight, rice sheath blight or ginkgo leaf blight and the like.

Detailed Description

Example 1

1. Preparing the ginkgolic acid extract:

(1) drying ginkgo biloba exocarp, weighing 10kg, ball milling and crushing by a ball mill crusher to prepare 250-mesh powder, adding 100kg of ethanol with the concentration of 85% V/V into the ginkgo biloba exocarp powder, leaching for 36h at 45 ℃, stirring for 20min every 2h during leaching, filtering through a 100-mesh sieve to remove filter residue after leaching is finished, filtering through a 300-mesh sieve, filtering the filtrate through a 500-mesh sieve, collecting the filtrate, filtering through a 0.2 mu m microporous filter membrane once, recovering ethanol from the filtrate to obtain 3860g of concentrated thick paste, adding 154.4kg of ion exchange water into the thick paste, heating to 96 ℃, continuously stirring and preserving heat for 30min, stopping heating, standing and cooling, standing and precipitating for 24h at the temperature of 2-4 ℃, discharging supernatant by a siphon method, centrifuging the precipitate for 20min by a tripodia centrifuge at 4000r/min, obtaining 3088g of pasty extract;

(2) adding 3088g of the paste extract into 30.88kg of 90% methanol solution, leaching for 6 hours at 25 ℃, stirring for 20 minutes every 1 hour during leaching, filtering with a 100-mesh sieve to remove insoluble impurities after leaching is finished, filtering with a 300-mesh sieve, filtering with a 500-mesh sieve, collecting filtrate, fine-filtering with a 0.2-micron microporous filter membrane, and recovering methanol from the filtrate to obtain 2162g of concentrated extract; adding 54.05kg of ion exchange water into the concentrated thick paste, heating to 75 ℃, continuously stirring and preserving heat for 30min, stopping heating, standing and cooling, standing and precipitating for 24h at the temperature of 2-4 ℃, discharging supernatant by using a siphon method, and centrifuging the precipitate for 20min at 4000r/min by using a three-foot centrifuge to obtain 1517g of paste extract;

(3) adding 1517g of the paste extract into 15.17kg of petroleum ether, stirring and leaching for 3.5h at the temperature of 18 ℃ and the rotation speed of 20r/min, filtering by a 100-mesh sieve after leaching is finished, filtering the filtrate by 300-mesh and 500-mesh sieves respectively, collecting the filtrate, filtering once by a 0.2-micron microporous filter membrane, recovering the petroleum ether from the filtrate to obtain 1238g of concentrated thick paste, adding 30.95kg of ion exchange water into the concentrated thick paste, heating to 75 ℃ and keeping the temperature and stirring for 30min, stopping heating, standing and cooling, standing and precipitating at the temperature of 2-4 ℃ for 24h, discharging supernatant by a siphon method, centrifuging the precipitate by a tubular high-speed centrifuge for 20min at 5000r/min to obtain 1062g of paste extract, namely the ginkgolic acid extract;

the content of ginkgolic acid in the obtained ginkgolic acid extract is 64.08 wt% by High Performance Liquid Chromatography (HPLC) method.

The process for preparing ginkgolic acid in this example illustrates that the method of the present invention comprises ethanol leaching, water precipitation, centrifugation of the precipitate, methanol leaching, water precipitation, centrifugation of the precipitate, further petroleum ether leaching, and centrifugation of the precipitate to obtain a ginkgolic acid extract, wherein ethanol, methanol, petroleum ether insoluble components and water soluble components are removed by multiple filtration, so that the obtained ginkgolic acid has a high content, and the relative content of inactive components is low, which has a good beneficial effect on ensuring the internal quality and stability of the further preparation of the ginkgolic acid solution.

2. Preparing a ginkgolic acid solution agent:

(4) weighing 650G of the ginkgolic acid extract prepared in the step (3), adding glycerol formal, stirring to dissolve, fixing the volume to 1000G of the total volume, mixing uniformly, and adding G₃Filtering with a pure melting glass funnel, and subpackaging to obtain the ginkgolic acid solution product. The prepared ginkgolic acid solution product is measured by an HPLC method, and the content of ginkgolic acid is 41.65%.

Example 2

1. Preparing the ginkgolic acid extract:

(1) drying ginkgo biloba exocarp, weighing 10kg, ball milling and crushing by a ball milling crusher to prepare 200-mesh powder, adding 100kg of ethanol with the concentration of 85% V/V into the ginkgo biloba exocarp powder, leaching for 48h at 40 ℃, stirring for 20min every 2h during leaching, filtering the ginkgo exocarp powder by a 100-mesh sieve to remove filter residue after leaching, filtering the ginkgo exocarp powder by a 300-mesh sieve, filtering the filtrate by a 500-mesh sieve, collecting the filtrate, filtering the filtrate by a 0.2 mu m microporous filter membrane, recovering the ethanol from the filtrate to obtain 3942g of concentrated thick paste, adding 118.3kg of ion exchange water into the thick paste, heating to 95 ℃, continuously stirring and preserving the temperature for 30min, stopping heating, standing and cooling, standing and precipitating for 24h at the temperature of 2-4 ℃, discharging supernatant by a siphon method, centrifuging the precipitate by a tripodia centrifuge for 20min at 3000r/min, obtaining 3127g of paste extract;

(2) adding 3127g of the paste extract into 31.27kg of 90% V/V methanol solution, leaching at 20 deg.C for 5 hr, stirring every 1 hr for 20min during leaching, filtering with 100 mesh sieve to remove insoluble impurities, filtering with 300 mesh sieve, filtering with 500 mesh sieve, collecting filtrate, fine filtering with 0.2 μm microporous membrane, and recovering methanol from the filtrate to obtain 2301g of concentrated soft extract; adding 46.02kg of ion exchange water into the concentrated thick paste, heating to 70 ℃, continuously stirring and preserving heat for 30min, stopping heating, standing and cooling, standing and precipitating for 24h at the temperature of 2-4 ℃, discharging supernatant by using a siphon method, and centrifuging the precipitate for 20min at 3000r/min by using a three-foot centrifuge to obtain 1721g of paste extract;

(3) adding 1721g of the paste extract into 17.21kg of petroleum ether, stirring and leaching for 3h at the temperature of 15 ℃ and the rotation speed of 20r/min, filtering the mixture through a 100-mesh sieve after leaching is finished, filtering the filtrate through a 300-mesh sieve and a 500-mesh sieve respectively, collecting the filtrate, filtering the filtrate once through a 0.2-micron microporous filter membrane, recovering the petroleum ether from the filtrate to obtain 1399g of concentrated thick paste, adding 27.98kg of ion exchange water into the concentrated thick paste, heating to 70 ℃, keeping the temperature and stirring for 30min, stopping heating, standing and cooling, standing and precipitating at the temperature of 2-4 ℃ for 24h, discharging supernatant liquid by a siphon method, centrifuging the precipitate by a tubular high-speed centrifuge for 20min at 4000r/min to obtain 1327g of paste extract, namely the ginkgolic acid extract;

the content of ginkgolic acid in the obtained ginkgolic acid extract is 60.16 wt% by High Performance Liquid Chromatography (HPLC) method.

2. Preparing a ginkgolic acid solution agent:

(4) weighing 500G of the ginkgolic acid extract prepared in the step (3), adding glycerol formal, stirring to dissolve, fixing the volume to 1000G of the total volume, uniformly mixing, and adding G₃Filtering with a pure melting glass funnel, and subpackaging to obtain the ginkgolic acid solution product. The prepared ginkgolic acid solution product is determined by an HPLC method, and the content of ginkgolic acid is 30.08%.

Example 3

(1) Drying the ginkgo biloba exocarp, weighing 10kg, crushing by a vibration mill crusher to prepare 300-mesh powder, adding 100kg of ethanol with the concentration of 85% V/V into the ginkgo biloba exocarp powder, leaching for 24h at 50 ℃, stirring for 20min every 2h during leaching, after leaching, filtering through a 100-mesh sieve to remove filter residue, filtering through a 300-mesh sieve, filtering the filtrate through a 500-mesh sieve, collecting the filtrate, filtering through a 0.2 mu m microporous filter membrane once, recovering the ethanol from the filtrate to obtain 3172g of concentrated thick paste, adding 185.6kg of ion exchange water into the thick paste, heating to 98 ℃, continuously stirring and preserving heat for 30min, stopping heating, standing and cooling, standing and precipitating for 24h at the temperature of 2-4 ℃, discharging the supernatant by a siphon method, centrifuging the precipitate by a centrifuge at a high speed of a tubular type, centrifuging for 20min at 3500r/min, 2920g of pasty extract is obtained;

(2) adding 2920g of the paste extract into 29.2kg of 90% V/V methanol solution, leaching for 7 hours at 30 ℃, stirring for 20 minutes every 1 hour during leaching, filtering through a 100-mesh sieve to remove insoluble impurities after leaching is finished, filtering through a 300-mesh sieve, filtering the filtrate through a 500-mesh sieve, collecting the filtrate, finely filtering through a 0.2-micron microporous filter membrane, and recovering methanol from the filtrate to obtain concentrated thick paste 2014 g; adding 60.42kg of ion exchange water into the concentrated thick paste, heating to 80 ℃, keeping the temperature for 30min under continuous stirring, stopping heating, standing for cooling, standing and precipitating for 24h at the temperature of 2-4 ℃, discharging the supernatant by a siphon method, and centrifuging the precipitate for 20min at 4000r/min by using a tubular high-speed centrifuge to obtain 1362g of paste extract;

(3) adding 1362g of the paste extract into 13.62kg of petroleum ether, stirring and leaching for 4h at the temperature of 20 ℃ and the rotation speed of 20r/min, filtering the extract by using a 100-mesh sieve after leaching is finished, filtering the filtrate by using a 300-mesh sieve and a 500-mesh sieve respectively, collecting the filtrate, filtering the filtrate by using a 0.2-micron microporous filter membrane once, recovering the petroleum ether from the filtrate to obtain 1104g of concentrated thick paste, adding 33.12kg of ion exchange water into the concentrated thick paste, heating to 70 ℃, keeping the temperature and stirring for 30min, stopping heating, standing and cooling, standing and precipitating for 24h at the temperature of 2-4 ℃, discharging supernatant by using a siphon method, centrifuging the precipitate by using a tubular high-speed centrifuge for 20min at 5000r/min to obtain 986g of paste extract, wherein the paste extract is the ginkgolic acid extract obtained by the invention;

the content of ginkgolic acid in the obtained ginkgolic acid extract is measured by a High Performance Liquid Chromatography (HPLC), and the content of ginkgolic acid is 65.23 wt%.

2. Preparing a ginkgolic acid solution agent:

(4) weighing 500g of the ginkgolic acid extract prepared in the step (3), adding SMF, stirring to dissolve, and fixing the volume to be full1000G, mixing well, adding G₃Filtering with a pure melting glass funnel, and subpackaging to obtain the ginkgolic acid solution product. The prepared ginkgolic acid solution product is determined by an HPLC method, and the content of ginkgolic acid is 32.62%.

The present invention has been tested on the stability of the solvent (solvent) of the ginkgolic acid extract prepared by the present invention using glycerol formal, DMF, DMA and DMSO respectively, and the test examples are as follows:

test example 1

The formulations of steps (2) to (4) of the foregoing example 1 were used as test examples.

Test example 2

Taking 500G of ginkgolic acid extract prepared in the steps (1) to (3) of the embodiment 1 of the invention, adding DMF to dissolve the ginkgolic acid extract, fixing the volume to 1000G, stirring the mixture evenly, and using G₃Filtering with sintered glass funnel, and packaging;

the content of ginkgolic acid is 32.04 percent by HPLC detection.

Test example 3

Taking 550G of ginkgolic acid extract prepared in the steps (1) to (3) of the embodiment 1 of the invention, adding DMA to dissolve the ginkgolic acid extract, fixing the volume to 1000G, stirring the mixture evenly, and using G₃Filtering with sintered glass funnel, and packaging;

the content of ginkgolic acid is 35.24 percent by HPLC detection.

Test example 4

Taking 600G of ginkgolic acid extract prepared in the steps (1) to (3) of the embodiment 1 of the invention, adding DMSO for dissolving, fixing the volume to 1000G, and using G₃Filtering with sintered glass funnel, and packaging;

the content of ginkgolic acid is 38.45 percent by HPLC detection.

For the test examples 1 to 4, which were stored at room temperature for 12 months, the appearance and the active ingredient content were periodically compared, and the results are shown in the following table:

table 1:

as can be seen from the observation of the sample storage in Table 1, the appearance color and clarity of the product are not changed basically when the product is stored at room temperature, the content of the active ingredient is not changed greatly, only the active ingredient of test example 1 is reduced by 0.01% after 6 months of storage, the active ingredient is reduced by 0.02% after 12 months of storage, the active ingredient of example 3 is reduced by 0.02% and the active ingredient of example 4 is reduced by 0.02% after 12 months of storage, and the content changes of the examples are all changes after decimal points, and the solvent used in the test examples is reliable to the quality stability of the product of the invention after the observation and analysis of the results of the test examples after 12 months of storage.

The invention also carries out stability tests by taking water solutions of glycerol formal, DMF, DMA and DMSO with different weight percentages as the solvents in the invention, and the test examples are as follows:

test example 5

Taking 600G of the ginkgolic acid extract prepared in the steps (1) to (3) of the embodiment 1 of the invention, adding an aqueous solution containing 60 percent of glycerol formal by weight for dissolving, fixing the volume to 1000G, stirring uniformly, and using G₃Filtering with sintered glass funnel, and packaging;

the content of ginkgolic acid is 38.45 percent by HPLC detection.

Test example 6

Taking 400G of ginkgolic acid extract prepared in the steps (1) to (3) of the embodiment 1, adding an aqueous solution containing 50% of DMF (dimethyl formamide) by weight for dissolving, fixing the volume to 1000G, stirring uniformly, and using G₃Filtering with sintered glass funnel, and packaging;

the content of ginkgolic acid is 25.63 percent by HPLC detection.

Test example 7

350G of the ginkgolic acid extract prepared in the steps (1) to (3) of the embodiment 1 is taken, an aqueous solution containing 50 percent of DMA (direct memory access) is added to dissolve the ginkgolic acid extract, the volume is determined to be 1000G, the mixture is uniformly stirred, and G is used₃Filtering with sintered glass funnel, and packaging;

the content of ginkgolic acid is 22.43 percent by HPLC detection.

Test example 8

The ginkgolic acid extract prepared in the steps (1) to (3) of the embodiment 1 is taken320G, adding aqueous solution containing 30% DMSO by weight for dissolving, fixing volume to 1000G, stirring well, and adding G₃Filtering with sintered glass funnel, and packaging;

the content of ginkgolic acid is 20.51 percent by HPLC detection.

The results of comparing the appearance and the active ingredient content regularly when the test examples 5 to 8 were stored at room temperature for 12 months, are shown in Table 2:

TABLE 2

As can be seen from the observation results of the sample storage in Table 2, the product has basically no change in appearance color and clarity after being stored for 12 months at room temperature, the active ingredients are stable and have no big change, and the contents of experimental examples 5, 6, 7 and 8 are respectively reduced by 0.11%, 0.08%, 0.02% and 0.09%; as can be seen from the observation and analysis of the results of 12-month storage at room temperature in the test examples, the use of aqueous solutions of glycerol formal, DMF, DMA and DMSO as solvents ensures the stability of the quality of the product of the present invention.