A composition of ginkgo biloba leaf extract and a method for preparing the same

Abstract

This invention provides a composition of Ginkgo biloba extract and its preparation method. The composition consists of Ginkgo biloba extract (calculated as total flavonol glycosides) and dipyridamole in a dosage ratio of 10:4. The preparation method can significantly improve product yield, reduce production costs, and significantly increase the content of the main components of the produced Ginkgo biloba extract, namely total flavonol glycosides and terpene lactones. It can also reduce the content of poorly water-soluble ginkgolides and harmful impurities such as ginkgolic acid, control its microbial level, and improve the stability and safety of the Ginkgo biloba extract preparation product of this invention.

Description

[0001] This application is a divisional application of the invention patent application filed by Tonghua Guhong Pharmaceutical Co., Ltd. on November 5, 2025, with application number 202511606949.0 and titled "A Composition of Ginkgo Leaf Extract and its Preparation Method". Technical Field

[0002] This invention belongs to the field of extraction, purification and separation of effective components of traditional Chinese medicine, and particularly relates to a composition of ginkgo leaf extract and its preparation method. Background Technology

[0003] The composition of this invention mainly comprises total flavonoids from ginkgo biloba and dipyridamole, derived from the applicant's secondary development technology of its already marketed product, Ginkgo biloba extract injection. The efficacy and function of this drug are applicable to the prevention and treatment of coronary heart disease and thromboembolic diseases. Ginkgo biloba total flavonoids dilate coronary and cerebral blood vessels, improving symptoms and memory function caused by cerebral ischemia; dipyridamole inhibits platelet aggregation, and at high concentrations, it can inhibit platelet release. The combination of these two components allows the drug to have a systemic vascular unblocking effect, and it can also treat vascular blockages in other parts of the body, such as cerebral embolism, pulmonary embolism, and splenic artery embolism. This drug can be used to adjust any condition belonging to the qi stagnation and blood stasis type, and its clinical application effect is quite significant.

[0004] A systematic review of existing technical documents for this product reveals the following related technology disclosed in patent document CN1939357A: Ginkgo leaves are pulverized, 10 times the amount of 70% ethanol is added, and the mixture is refluxed at 50°C twice, for 3 hours each time. The extracts are combined, filtered, and the filtrate is concentrated under reduced pressure to 1 / 8 of the extract volume. 1.5 times the volume of the concentrate is added with distilled water, and 1.5% of the concentrate volume of co-solvent is added for filtration. The filtrate is adsorbed by macroporous resin and desorbed with 70% ethanol solution. The desorbed solution is added to 60% of the prepared volume of fresh water for injection, ultrasonically treated for 30 minutes, refrigerated at 4°C for 24 hours, and ultrafiltered using an ultrafiltration membrane with a cutoff molecular weight of 10,000 to obtain the ginkgo leaf extract. The patent text CN102626427A describes the following: 10 kg of cleaned coarse ginkgo leaf fibers are placed in an extraction tank and soaked in 10 times and 8 times the amount of 70-80% ethanol for 1-2 hours. Then, the mixture is heated and refluxed for 2 hours and 1 hour respectively. The extracts are combined, filtered, and the ethanol is recovered from the filtrate. An appropriate amount of purified water is added, and the mixture is allowed to stand and refrigerate for 12 hours. The filtrate is then filtered through filter paper. The filtrate is added to a pre-treated macroporous resin column and washed sequentially with purified water, 20% ethanol, and 60% ethanol. The eluent containing 60% ethanol is collected, and the ethanol is recovered from the eluent until there is no alcohol odor. The eluent is then concentrated, and the collected extract is dried, pulverized, and sieved. The formulation of the Ginkgo biloba extract drug composition is characterized in that each 1 ml of the injection contains 0.8-1.2 mg of total Ginkgo flavonoid glycosides, 0.35-0.5 mg of dipyridamole, and 0.3-0.6 mg of total Ginkgo lactones; the pH value is 4.5-5.5; wherein the total Ginkgo lactones are composed of ginkgolides, ginkgolides A, ginkgolides B, ginkgolides C and ginkgolides M in a weight ratio of 22-30:15-18:5-8:8-22:4-8. CN102626383A discloses a method for preparing ginkgolide injection. The effective fraction content of the ginkgolide extract prepared by this method is ≥95%, wherein the content of ginkgolide is 25.0%~50.0%, the content of ginkgolide A is 20.0%~45.0%, the content of ginkgolide B is 10.0%~30.0%, the content of ginkgolide C is 5.0%~15.0%, and the total ginkgolic acid content is less than 5ppm. This method solves the problem of residual macromolecules, polymers and proteins in the production process of traditional Chinese medicine injections and reduces the occurrence of adverse reactions.

[0005] There are many traditional Chinese medicine injection products using Ginkgo biloba extract as raw material. For example, the main components and contents of Ginkgo biloba extract injection are strictly regulated (e.g., containing 24% total flavonol glycosides and 6% terpene lactones). It can be used to improve blood circulation disorders in the brain and peripheral blood. Other products include Shuxuening injection and Ginkgo lactone injection. Due to the special nature of traditional Chinese medicine injections, the requirements for raw materials of the above products are very strict, and the following problems exist: (1) The extraction process of the raw material is difficult to stabilize, which may lead to the inability to guarantee the clinical effect of the preparation product; (2) Ginkgo biloba extract is extracted with ethanol and contains components with poor water solubility. Since the final product of the extract is a high-temperature sterilized injection, it is easy to cause problems such as poor stability and increased visible foreign matter, which leads to high drug safety risks; (3) The existing technology has low yield, low content of effective ingredients, many impurities, and the microbial level cannot meet the requirements of raw materials for injection. In view of this, the present invention develops an extract composition with high content of effective ingredients and fewer impurities, and develops a more advanced preparation process to overcome the defects of the above traditional Chinese medicine injections. Summary of the Invention

[0006] This invention provides a ginkgo leaf extract composition and its preparation method. The preparation method can significantly improve the yield of the product (Ginkgo biloba extract injection), reduce production costs, and significantly increase the content of the main components of the ginkgo leaf extract, namely total flavonol glycosides and terpene lactones. It can also reduce the content of poorly water-soluble ginkgolides and harmful impurities such as ginkgolic acid, control the microbial level, and improve the stability and safety of the ginkgo leaf extract preparation product of this invention.

[0007] The technical problem this invention aims to solve is as follows: Ginkgo biloba extract is extracted with ethanol and contains components with poor water solubility. Because the final product of the extract is a high-temperature sterilized injection, it is prone to poor stability and may exhibit increased visible foreign matter, thus posing a safety risk to the use of Ginkgo biloba extract injections. Furthermore, existing technologies also suffer from low yields of Ginkgo biloba extract, low content of effective active ingredients, high impurity levels, and the inability to meet the microbial requirements for injectable raw materials. (The 2020 edition of the Chinese Pharmacopoeia specifies the following requirements for Ginkgo biloba extract in Ginkgo biloba injection products: total flavonol glycosides ≥ 25.5%, ginkgolides not less than 6.2%, and total ginkgolic acid ≤ 5 ppm).

[0008] The technical solution of this invention patent application is as follows: A composition of Ginkgo biloba extract, wherein the composition comprises Ginkgo biloba extract (calculated as total flavonol glycosides): dipyridamole in a dosage ratio of 10:4, wherein the Ginkgo biloba extract comprises the following molar percentages of active pharmaceutical ingredients: 27-35% total flavonol glycosides of Ginkgo biloba, 7.0%-8.6% ginkgolides, of which ginkgolides A 3.5%-4.5%, ginkgolides B 2%-2.7%, ginkgolides C 1.5%-1.8%, ginkgolides ≤0.01%, total ginkgolic acid ≤0.01%, flavonoid aglycones ≤1.3%-1.5%, free quercetin ≤0.05%, free kaempferol ≤0.05%, free isorhamnetin ≤0.01%, and the remainder being other components of the Ginkgo biloba extract.

[0009] Preferably, the ginkgo leaf extract comprises the following raw material composition in molar percentages: 29% total ginkgo flavonol glycosides and 7.8% ginkgo terpene lactones, of which ginkgolide A is 3.9%, ginkgolide B is 2.2%, ginkgolide C is 1.6%, ginkgolide ≤0.01%, flavonoid aglycones ≤1.3%, free quercetin ≤0.05%, free kaempferol ≤0.05%, and free isorhamnetin ≤0.01%, with the remainder being other components of the ginkgo extract.

[0010] More preferably, the ginkgo leaf extract comprises the following raw material composition in molar percentages: 33% total ginkgo flavonol glycosides, 8.5% ginkgo terpene lactones, of which ginkgolide A 4.3%, ginkgolide B 2.5%, ginkgolide C 1.8%, ginkgolide ≤0.01%, flavonoid aglycones ≤1.5%, free quercetin ≤0.03%, free kaempferol ≤0.04%, free isorhamnetin ≤0.01%, and the remainder is other components of the ginkgo extract.

[0011] The preparation method of Ginkgo biloba extract of the present invention includes the following steps: (1) Extraction of Ginkgo biloba leaves Take ginkgo leaves and add 6-8 times the amount of 70-80% ethanol, and heat and reflux at 75-80℃ for 1-2 hours; for the second extraction: add 4-6 times the amount of 70-80% ethanol, and reflux for 0.5-1.5 hours; concentrate at 50-80℃, vacuum degree -0.050 to -0.090 MPa, concentrate to a relative density of 1.05-1.15, and concentrate under reduced pressure to obtain ginkgo extract; (2) Water sedimentation of Ginkgo biloba extract Take the ginkgo extract from step (1) and add 3-5 times the amount of purified water. Stir for 20-40 minutes, centrifuge, cool the centrifuged liquid to 0-8℃, let it settle for 12-48 hours, and then filter to obtain the water-precipitated ginkgo leaf filtrate. (3) Purification of Ginkgo Leaf Water Settling Filtrate Take the water-precipitated filtrate of Ginkgo biloba leaves from step (2) and add it to macroporous adsorption resin column A. Discard the effluent. Elute with purified water and discard the eluent. Elute with 1-3 column volumes of 10-20% ethanol and discard the eluent. Connect resin column B in series with column A and elute with 60-80% ethanol for 2-5 column volumes. Collect the eluent. (4) Concentration of Ginkgo Eluent Take the eluent from step (3), adjust the pH to 5.0-8.5, concentrate under reduced pressure to about 1 / 10 of the volume, replace the ethanol with purified water, slowly cool to 0-8℃, let stand for 12-48 hours for cold precipitation, filter, and obtain the filtered drug solution. (5) Resin purification treatment The filtered solution from step (4) is fed into a resin C and D tandem column, and the effluent is discarded; the solution is eluted with purified water, and the eluent is discarded; the solution is eluted with 5-8 column volumes of 30-50% ethanol, and the eluent is collected; the solution is concentrated under reduced pressure and dried to obtain crude Ginkgo biloba extract. ⑹ Refined Add the crude Ginkgo biloba extract from step (5) to ethanol, add activated carbon, heat to boiling, then cool, filter, concentrate under reduced pressure; vacuum dry, pulverize and mix to obtain Ginkgo biloba extract.

[0012] As a further preferred embodiment of the present invention, the composition of the ginkgo leaf extract is composed of ginkgo leaf extract (calculated as total flavonol glycosides): dipyridamole in a dosage ratio of 10:4, characterized in that the ginkgo leaf extract comprises the following molar percentages of raw materials: total ginkgo flavonol glycosides 27-35%, ginkgo terpene lactones 7.0%-8.6%, ginkgolide A 3.5%-4.5%, ginkgolide B 2%-2.7%, ginkgolide C 1.5%-1.8%, ginkgolide ≤0.01%, total ginkgolic acid ≤0.01%, flavonoid aglycones ≤1.3%-1.5%, free quercetin ≤0.05%, free kaempferol ≤0.05%, free isorhamnetin ≤0.01%, and the remainder being other components of the ginkgo extract; The preparation method includes the following steps: (1) Extraction of Ginkgo biloba leaves Take ginkgo leaves and add 6-8 times the amount of 70-80% ethanol, and heat and reflux at 75-80℃ for 1-2 hours; for the second extraction: add 4-6 times the amount of 70-80% ethanol, and reflux for 0.5-1.5 hours; concentrate at 50-80℃, vacuum degree -0.050 to -0.090 MPa, concentrate to a relative density of 1.05-1.15, and concentrate under reduced pressure to obtain ginkgo extract; (2) Water sedimentation of Ginkgo biloba extract Take the ginkgo extract from step (1) and add 3-5 times the amount of purified water. Stir for 20-40 minutes, centrifuge, cool the centrifuged liquid to 0-8℃, let it settle for 12-48 hours, and then filter to obtain the water-precipitated ginkgo leaf filtrate. (3) Purification of Ginkgo Leaf Water Settling Filtrate Take the water-precipitated filtrate of Ginkgo biloba leaves from step (2) and add it to macroporous adsorption resin column A. Discard the effluent. Elute with purified water and discard the eluent. Elute with 1-3 column volumes of 10-20% ethanol and discard the eluent. Connect resin column B in series with column A and elute with 60-80% ethanol for 2-5 column volumes. Collect the eluent. (4) Concentration of Ginkgo Eluent Take the eluent from step (3), adjust the pH to 5.0-8.5, concentrate under reduced pressure to about 1 / 10 of the volume, replace the ethanol with purified water, slowly cool to 0-8℃, let stand for 12-48 hours for cold precipitation, filter, and obtain the filtered drug solution. (5) Resin purification treatment The filtered solution from step (4) is fed into a resin C and D tandem column, and the effluent is discarded; the solution is eluted with purified water, and the eluent is discarded; the solution is eluted with 5-8 column volumes of 30-50% ethanol, and the eluent is collected; the solution is concentrated under reduced pressure and dried to obtain crude Ginkgo biloba extract. ⑹ Refined Add the crude Ginkgo biloba extract from step (5) to ethanol, add activated carbon, heat to boiling, then cool, filter, concentrate under reduced pressure; vacuum dry, pulverize and mix to obtain the Ginkgo biloba extract.

[0013] Preferably, the dosage form of the ginkgo leaf extract composition is an injection.

[0014] Preferably, in step (2) of the preparation method, the water settling time of the ginkgo extract is 12-24 hours, and the amount of water added is 5 times.

[0015] As a preferred embodiment of the present invention, in step (3) of the preparation method for purifying the water-precipitated filtrate of Ginkgo biloba leaves, the macroporous adsorption resin column A is of type DM130 or HPD-450, and the column B is one of type LX-158 or ADS-17.

[0016] As a preferred embodiment of the present invention, in step (5) of the preparation method for purifying the water-precipitated filtrate of Ginkgo biloba leaves, the macroporous adsorption resin column C is of type DM130 or HPD-450, and the column D is one of LX-158 or ADS-17.

[0017] Preferably, in step (6) of the preparation method, the amount of ethanol used in the refining process is 15-30 times that of the crude Ginkgo biloba extract.

[0018] Preferably, the amount of activated carbon added in step (6) of the preparation method is 0.1-0.3%.

[0019] Preferably, the composition of the Ginkgo biloba extract is used in the preparation of drugs for treating coronary heart disease, thromboembolic diseases, and cardiovascular and cerebrovascular diseases.

[0020] To better demonstrate the innovativeness of the preparation process technology of this invention, we have summarized the preferred process steps of the process parameters of this invention as follows.

[0021] 1. Extraction Parameter Selection Ginkgo leaves require extraction using an ethanol-water mixture. This study investigates the effects of ethanol dosage and concentration, as well as the number of extractions and extraction times, on the extraction efficiency. An orthogonal experimental design was used to test different factors and levels to examine relevant extract indicators. Based on the results, the process parameters were determined through analysis.

[0022] 1.1 Operating Procedures 300 g of ginkgo leaves were placed in a flask. According to the experimental design, different volumes and concentrations of ethanol were added and extracted for a corresponding time while maintaining a gentle boil. The extracts were combined, concentrated to about 300 ml, dried, and the total flavonoid content and lactone content were tested. Foreign matter was visible in the finished product after preparing ginkgo biloba extract injection.

[0023] 1.2 Influencing Factors and Levels Table 1. Factors and levels affecting the extraction process

[0024] 1.3 Experimental Design and Results Table 2 Extraction test plan and results

[0025] 1.4 Summary (1) The extraction rate is highest when the amount of alcohol added is 6-8 times; (2) 70-80% ethanol is beneficial for the extraction of flavonoids and lactones; (3) The more extraction times and the longer extraction time, the higher the extraction yield of total flavonoids and lactones. From the perspective of cost control, it is advisable to choose to extract twice. The content is reduced in the second extraction, and 1 hour is appropriate.

[0026] The final extraction process was determined as follows: First extraction of ginkgo leaf slices: add 6-8 times the amount of 70-80% ethanol, reflux at 75-80℃ for 2 hours; Second extraction: add 4-6 times the amount of 70-80% ethanol, reflux for 1 hour.

[0027] 2. Selection of process conditions for water settling step 2.1 Influencing Factors and Levels Table 3. Factors and Levels Affecting the Water Submersion Process

[0028] 2.2 Experimental Design and Results Table 4. Test Scheme and Results of Water Submersion Process

[0029] 2.3 Results (1) The rate of visible foreign matter after 24 hours of water immersion is relatively high, but the improvement compared to 12 hours of water immersion is not significant; (2) The amount of water added to the water-sinking solution has little impact on each of the test items, with 3 times and 5 times the amount of water added having slightly better effects; (3) Better sedimentation effect can be obtained under the conditions of water settling temperature of 0-8℃ and water addition temperature of 0-5℃; The water settling time is 12-24 hours, the water volume is 5 times, the water settling temperature is 0-8℃, and the water addition temperature is 0-5℃.

[0030] 3. Verification of secondary water settling parameters The changes in total flavonoids, lactones, insoluble microparticles, total solids, and precipitate weight during the second water sedimentation process were examined to determine whether the water sedimentation process could achieve the expected results.

[0031] 3.1 Experimental Methods Take 1000ml of the concentrated Ginkgo biloba leaf extract after water replacement in process step (4), and cool it rapidly and slowly to 0-8℃ for 10, 12, 16, 24 and 48 hours respectively. Filter the water-precipitated liquid through a 0.45μm filter membrane and measure the total precipitate weight. Then take samples to measure the total flavonoids, lactones, number of insoluble particles and total solids of each filtrate. The results are shown in Tables 5 and 6.

[0032] Table 5. Process investigation of water settling time - rapid cooling (≈3℃ / min)

[0033] Table 6. Process investigation of water settling time - slow cooling (≈0.5℃ / min)

[0034] 3.2 Results The results showed that the content of total flavonoids and lactones decreased slightly with the extension of water settling time during the water settling process; after 12 hours, the insoluble microparticles met the quality standard requirements, indicating that the water settling effect can basically achieve the quality control purpose when the water settling time is 12 hours. The water settling time limit can be adjusted according to actual production, but it should not be less than 12 hours.

[0035] Slow cooling can form finer precipitates, reducing the inclusion degree of active ingredients and thus increasing product yield.

[0036] Pilot-scale testing and process verification have demonstrated that a water-settling process with a water-settling time of 12-48 hours, a water-settling temperature of 0-8℃, and slow cooling can produce products that meet the quality standards of Ginkgo biloba extract and Ginkgo biloba extract injection. This process can be implemented in production.

[0037] 4. Resin separation and purification process conditions 4.1 Resin Selection Macroporous resins are increasingly widely used in the purification of ginkgo flavonoids and ginkgolides. Numerous manufacturers produce macroporous resins with varying models, specifications, and performance. Based on a review of relevant literature and application data from related manufacturers, we further screened DM130, HPD-450, SP850, LX-158, ADS-17 macroporous resins and polyamide resins.

[0038] 4.2 Investigation of Adsorption Capacity Weigh out 10g each of the following wet resins: DM130, SP850, HPD-450, LX-158, and ADS-17. Concentrate 2000mL of Ginkgo biloba extract under vacuum to 200mL, pour the concentrated extract into 400mL of cold water while still hot, stir, allow to stand for separation, filter, and obtain the extract. Measure the volume and retain a sample to determine the content of lactones and flavonoids. Add 59mL of extract to every 10g of wet resin, perform static adsorption on a shaker overnight, filter, and obtain the adsorbed solution. Return the filter residue to the bottle and add 100mL of 70% ethanol, perform static desorption on a shaker overnight, filter, discard the filter residue, and obtain the desorbed solution.

[0039] Table 7. Adsorption capacity of five macroporous resins for ginkgo flavonoids and ginkgo lactones.

[0040] 4.3 Examination of desorption rate Following the method in 4.2, after sufficient adsorption, 30 ml of 70%, 80%, and 90% ethanol were precisely added to the resin, respectively, and the resin was soaked for 10 h. After filtration, the concentrations of flavonoids and lactones in the filtrate were measured, and the desorption rate was calculated based on the adsorption amount. The results are shown in Table 8.

[0041] Table 8. Desorption rates of Ginkgo biloba leaf components by different resins

[0042] The experimental results show that, through adsorption and desorption tests, DM130 and HPD-450 have high adsorption and desorption rates for flavonoids, while LX-158 and ADS-17 have high adsorption and desorption rates for lactones. Flavonoids and lactones adsorbed on the resin can be eluted using 60%-80% ethanol.

[0043] 4.4 Selection of Elution Solvent Amount (1) Column saturation test method Based on the relative content of the two main components in Ginkgo biloba leaves and the relative ratio of the adsorption amounts of the two components by DM130 macroporous resin, the amount of flavonoids loaded onto the column can be determined by the saturation adsorption capacity of the flavonoids.

[0044] Take 2 drops of the post-column supply solution and place them on a white porcelain plate. Add 1 drop of glacial acetic acid and 1 drop of 1% ferrous sulfate solution. If the solution turns dark blue or black, the column is saturated. If the solution does not change color, the column is not saturated.

[0045] (2) Investigation on the amount of eluent used Collect the filtered extract of Ginkgo biloba leaves after water precipitation and add it to a pre-treated DM130 macroporous resin column (dosage: 100g of dried resin). The column flow rate should be controlled at 1-3 drops per second. Perform a saturation test on the eluent every 2 minutes, stopping the loading once the column is saturated. Record the volume of the loaded solution. Then, pass the eluent through the column with distilled water until the eluent is clear, recording the volume of distilled water used. Next, pass the eluent through the column with 70% ethanol, collecting the ethanol solution at a rate of 50-60 ml / min. Perform a flavonoid test on the eluent every 2 minutes until no flavonoid reaction occurs, stopping the loading once the reaction stops, recording the volume of ethanol used.

[0046] 4.5 Results The results showed that the amount of distilled water used was about 2.5 times that of the concentrate, and the amount of elution ethanol used was about 2 times that of the concentrate. To ensure the elution of flavonoids, the amount of ethanol used should be 2-5 times that of the concentrate.

[0047] 4.6 Verification of purification effect Ginkgo leaf aqueous extract was added to a pre-treated resin column (DM130 1kg), and the eluent was discarded. A saturation test was performed using 1% ferrous sulfate. After ginkgo flavonoids and ginkgolides were adsorbed onto the purification column, the column was washed with purified water. When the eluent became clearer, it was eluted with 3 times the volume of the concentrated drug solution in 70% ethanol at a rate of 100 ml / min. The ethanol eluent was collected, diluted to volume, and samples were taken to determine the total solids, total flavonol glycosides, and total lactone content. The experiment was repeated 3 times, and the results are shown in Table 9. Table 9 Purification effect of macroporous resin

[0048] As shown in the table above, after the drug solution was passed through the DM130 macroporous resin column, the total solids decreased by 75.3%, while the transfer rate of total flavonol glycosides reached 94.6%, accounting for 27.6% of the total solids. The transfer rate of ginkgolide was 85.5%, accounting for 8.6% of the total solids. The purification effect was obvious, the loss of effective components was small, and the process was stable.

[0049] 4.7 pH test for removing visible foreign matter from the solution The existing technology for producing Ginkgo biloba leaf extract has low water solubility, while its terpenoid lactones are fat-soluble components, including lactone A, lactone B, lactone C, and ginkgolide. Ginkgoolide is unstable at high temperatures and has a high content, resulting in a high visible foreign matter rate in the prepared injection formulation after sterilization. Furthermore, ginkgolide has limited pharmacological activity; therefore, this component can be removed. Ginkgoolide, unlike lactones A, B, and C, is a sesquiterpene lactone, which is degradable under heating and alkaline conditions. Therefore, a series of experiments were designed, and the results showed that the degradation rate of ginkgolide increased with increasing pH. After the first resin treatment, adjusting the pH of the solution, followed by concentration and ethanol replacement, effectively degraded most of the ginkgolide.

[0050] Table 10 Degradation experiments of ginkgolide

[0051] As can be seen from the experimental results in Table 10 above, the content of ginkgolides in the ginkgo extract decreased significantly from pH 3.5 before addition to pH 2.98 after heating, which was 6.56%, to pH 5.2 after heating, which was 0.28%.

[0052] The beneficial effects of the patented technical solution of this invention are as follows: (1) Compared with existing technologies, the optimized extraction process of this invention ensures the complete and thorough extraction of effective components from ginkgo leaves (total ginkgo flavonol glycosides, ginkgo terpene lactones, ginkgolide A, ginkgolide B%, ginkgolide C, ginkgolides, flavonoid aglycones, etc.). This invention reduces ethanol usage, shortens extraction time, simplifies extraction steps, effectively reduces costs, and improves production efficiency. Through extensive experimentation, the optimal preparation process parameters have finally been found.

[0053] The final extraction process for ginkgo leaves was determined as follows: First extraction of ginkgo leaf slices: add 6-8 times the amount of 70-80% ethanol and reflux at 75-80℃ for 2 hours; Second extraction: add 4-6 times the amount of 70-80% ethanol and reflux for 1 hour.

[0054] (2) Water settling process: Based on the existing technology, the water settling process is refined. By using different temperature control methods, impurities are fully removed, such as controlling the temperature difference between the drug solution and water, the cooling rate, and adjusting the rate and morphology of precipitation in different water settling steps, ensuring that impurities are fully removed and the effective components are retained. Through the centrifugation step, most of the precipitate is removed, reducing the inclusion of effective components, increasing the product yield by nearly 60%, and significantly shortening the filtration time, reducing a large amount of precipitate discharged with the water, alleviating the pressure on wastewater treatment, and reducing environmental protection costs. Resin treatment: Compared with the existing technology 1 (CN1939357A), this invention uses more types of macroporous resins and optimizes the elution method of the existing technology 2 (CN102626427A), which can effectively remove harmful components such as ginkgolic acid, far below the Chinese Pharmacopoeia standard, and retain the total flavonoids and lactones to the maximum extent. Exploration and optimization experiments revealed that the qualified rate of visible foreign matter was relatively high after 24 hours of water immersion, but the improvement was not significant compared to 12 hours. The amount of water added had little impact on each test item, with 3 times and 5 times the amount of water added showing slightly better results. Among these conditions, a better sedimentation effect could be obtained under the conditions of water immersion temperature of 0-8℃ and water addition temperature of 0-5℃. Finally, the optimal process for water immersion of Ginkgo leaves was determined to be: water immersion time of 12-24 hours, water addition of 5 times the amount of water, water immersion temperature of 0-8℃, and water addition temperature of 0-5℃.

[0055] (3) Ginkgolide is unstable at high temperatures and has a high content, resulting in a high visible foreign matter rate in the prepared injection formulation after sterilization. A higher pH environment can remove ginkgolide. During the second vacuum concentration, the pH of the extract was increased, reducing the ginkgolide content. This resulted in a decrease in the ginkgolide content in the Ginkgo extract from 6.56% to 0.28%, further improving the water solubility and formulation stability of the Ginkgo leaf extract.

[0056] (4) The preparation process of this invention also examines the effects of different resins on Ginkgo biloba extract from three aspects: adsorption capacity, desorption rate, and elution solvent volume. Using Ginkgo biloba flavonol glycosides and Ginkgo biloba lactones as indicator components, appropriate elution process parameters are selected. Through adsorption and desorption experiments, the results show that DM130 and HPD-450 have high adsorption and desorption rates for flavonoids, while LX-158 and ADS-17 have high adsorption and desorption rates for lactones. Using 60%-80% ethanol can elute the flavonoids and lactones adsorbed on the resin. The amount of elution ethanol is approximately twice that of the concentrate; to ensure the elution of flavonoid components, the amount of ethanol should be 2-5 times the concentrate volume. After passing the drug solution through a DM130 macroporous resin column, the total solids decreased by 75.3%, while the transfer rate of total flavonol glycosides reached 94.6%, accounting for 27.6% of the total solids. The transfer rate of ginkgolides was 85.5%, accounting for 8.6% of the total solids. The optimized resin purification method showed significant results, with minimal loss of active ingredients and stable process.

[0057] This invention removes harmful ginkgolic acid through resin treatment while maximizing the retention of total flavonoids and lactones. Microbial limits are controlled by adsorbing the ginkgo leaf extract with activated carbon and then subjecting it to heat treatment, which meets the quality requirements for injectable raw materials.

[0058] (5) Compared with the preparation processes described in prior art 1 (CN1939357A) and prior art 2 (CN102626427A), the content of each component in the ginkgo extract of the present invention is more reasonable, the content of total ginkgo flavonol glycosides can be increased by 3-7%, and the content of ginkgo lactones is reduced by 95%. The water solubility of the above products is significantly improved, the microbial limit meets the standard that the total number of aerobic bacteria in 1g of ginkgo leaf extract does not exceed 100cfu, and the ginkgolic acid content is less than 0.5ppm.

[0059] (6) The content of the Ginkgo extract prepared according to the optimal preparation process parameters of this invention is as follows: total Ginkgo flavonol glycosides 27-35%, Ginkgo terpene lactones 7.0%-8.6%, Ginkgolide A 3.5%-4.5%, Ginkgolide B 2%-2.7%, Ginkgolide C 1.5%-1.8%, Ginkgolide ≤0.01%, total Ginkgolic acid ≤0.01%, flavonoid aglycones ≤1.3%-1.5%, free quercetin ≤0.05%, free kaempferol ≤0.05%, free isorhamnetin ≤0.01%, and the remainder is other components of the Ginkgo extract. The above preparation process is stable and reliable, and has been used as a key factor in stabilizing the content of effective components in Ginkgo leaf injections. It has also been applied in the production of raw materials for Ginkgo biloba extract injections, and has a very large market application prospect. Detailed Implementation

[0060] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0061] Example 1 The present invention optimizes the extraction, water precipitation, resin treatment, and refining steps, and the process is as follows: (1) Extraction of Ginkgo biloba leaves Take ginkgo leaves, add 7 times the amount of 75% ethanol, and heat and reflux at 78℃ for 2 hours; for the second extraction, add 5 times the amount of 75% ethanol and reflux for 1 hour; concentrate at 70℃, vacuum degree -0.050~-0.090MPa, concentrate to a relative density of 1.05-1.15, and concentrate under reduced pressure to obtain ginkgo extract. (2) Water sedimentation of Ginkgo biloba extract Take the ginkgo extract from step (1) and add 4 times the amount of purified water. Stir for 30 minutes, centrifuge, cool the centrifuged liquid to 5°C, let it settle for 24 hours, and then filter to obtain the water-precipitated ginkgo leaf filtrate. (3) Purification of Ginkgo Leaf Water Settling Filtrate Take the water-precipitated filtrate of Ginkgo biloba leaves from step (2), add it to a macroporous adsorption resin column DM130, and discard the effluent; then elute with purified water and discard the eluent; elute with 2 column volumes of 15% ethanol and discard the eluent; connect a macroporous adsorption resin column LX-158 in series with the macroporous adsorption resin column DM130, and elute with 70% ethanol for 4 column volumes, and collect the eluent; (4) Concentration of Ginkgo Eluent Take the eluent from step (3), adjust the pH to 6.0, concentrate under reduced pressure, replace the ethanol with purified water, slowly cool to 3°C, let stand for 24 hours to allow to settle, and then filter to obtain the filtered solution. (5) Resin purification treatment The filtered solution from step (4) was fed into a series of HPD-450 and ADS-17 macroporous adsorption resin columns, and the effluent was discarded. The solution was eluted with purified water, and the eluent was discarded. The solution was then eluted with 7 column volumes of 40% ethanol, and the eluent was collected. The solution was concentrated under reduced pressure and dried to obtain crude Ginkgo biloba extract. ⑹ Refined Add the crude ginkgo leaf extract from step (5) to 20 times the volume of ethanol, add 0.02% activated carbon, heat to boiling, then cool, filter, concentrate under reduced pressure; vacuum dry, pulverize and mix to obtain ginkgo leaf extract.

[0062] Table 11 Results of determination of effective components in Ginkgo biloba extract of the present invention

[0063] Example 2 (1) Extraction of Ginkgo biloba leaves Take ginkgo leaves and add 8 times the amount of 70% ethanol, and heat and reflux at 80℃ for 1 hour; second extraction: add 6 times the amount of 70% ethanol, reflux for 1.5 hours; concentration temperature 80℃, vacuum degree -0.050~-0.090MPa, concentrate to a relative density of 1.05-1.15, and concentrate under reduced pressure to obtain ginkgo extract. (2) Water sedimentation of Ginkgo biloba extract Take the ginkgo extract from step (1) and add 5 times the amount of purified water. Stir for 40 minutes, centrifuge, cool the centrifuged liquid to 0℃, let it settle for 48 hours, and then filter to obtain the water-precipitated ginkgo leaf filtrate. (3) Purification of Ginkgo Leaf Water Settling Filtrate Take the water-precipitated filtrate of Ginkgo biloba leaves from step (2), add it to a macroporous adsorption resin column DM130, and discard the effluent; then elute with purified water and discard the eluent; elute with 3 column volumes of 20% ethanol and discard the eluent; connect a macroporous adsorption resin column ADS-17 in series with the macroporous adsorption resin column DM130, and elute with 80% ethanol for 5 column volumes, and collect the eluent; (4) Concentration of Ginkgo Eluent Take the eluent from step (3), adjust the pH to 8.5, concentrate under reduced pressure, replace the ethanol with purified water, slowly cool to 8°C, let stand for 12 hours to allow to settle, and then filter to obtain the filtered solution. (5) Resin purification treatment The filtered solution from step (4) was fed into a tandem column of macroporous resin DM130 and macroporous resin ADS-17, and the eluent was discarded. The solution was eluted with purified water, and the eluent was discarded. The solution was then eluted with 8 column volumes of 50% ethanol, and the eluent was collected. The solution was concentrated under reduced pressure and dried to obtain crude Ginkgo biloba extract. ⑹ Refined Add the crude Ginkgo biloba extract from step (5) to 15 times the volume of ethanol, add 0.01% activated carbon, heat to boiling, then cool, filter, concentrate under reduced pressure; vacuum dry, pulverize and mix to obtain the Ginkgo biloba extract.

[0064] Example 3 (1) Extraction of Ginkgo biloba leaves Take ginkgo leaves, add 6 times the amount of 80% ethanol, and heat and reflux at 75℃ for 2 hours; for the second extraction, add 4 times the amount of 80% ethanol and reflux for 0.5 hours; concentrate at 50℃, vacuum degree -0.050~-0.090MPa, concentrate to a relative density of 1.05-1.15, and concentrate under reduced pressure to obtain ginkgo extract. (2) Water sedimentation of Ginkgo biloba extract Take the ginkgo extract from step (1) and add 3 times the amount of purified water. Stir for 20 minutes, centrifuge, cool the centrifuged liquid to 8°C, let it settle for 12 hours, and then filter to obtain the water-precipitated ginkgo leaf filtrate. (3) Purification of Ginkgo Leaf Water Settling Filtrate Take the water-precipitated filtrate of Ginkgo biloba leaves from step (2), add it to the HPD-450 macroporous adsorption resin column, and discard the effluent; then elute with purified water and discard the eluent; elute with 1 column volume of 10% ethanol and discard the eluent; connect LX-158 macroporous adsorption resin in series to the HPD-450 macroporous adsorption resin column, and elute with 60% ethanol for 2 times the total column volume, and collect the eluent; (4) Concentration of Ginkgo Eluent Take the eluent from step (3), adjust the pH to 5.0, concentrate under reduced pressure, replace the ethanol with purified water, slowly cool to 0°C, let stand for 48 hours to allow to settle, and then filter to obtain the filtered solution. (5) Resin purification treatment The filtered solution from step (4) was fed into a tandem column of resin HPD-450 and LX-158, and the eluent was discarded. The solution was eluted with purified water, and the eluent was discarded. The solution was then eluted with 5 column volumes of 30% ethanol, and the eluent was collected. The solution was concentrated under reduced pressure and dried to obtain crude Ginkgo biloba extract. ⑹ Refined Add the crude Ginkgo biloba extract from step (5) to 30 times the volume of ethanol, add 0.01% activated carbon, heat to boiling, then cool, filter, concentrate under reduced pressure; vacuum dry, pulverize and mix to obtain the Ginkgo biloba extract.

[0065] Example 4 (1) Extraction of Ginkgo biloba leaves Take ginkgo leaves, add 7 times the amount of 75% ethanol, and heat and reflux at 76℃ for 1.5 hours; for the second extraction, add 6 times the amount of 80% ethanol and reflux for 0.5 hours; concentrate at 60℃, vacuum degree -0.050~-0.090MPa, concentrate to a relative density of 1.05-1.15, and concentrate under reduced pressure to obtain ginkgo extract. (2) Water sedimentation of Ginkgo biloba extract Take the ginkgo extract from step (1) and add 5 times the amount of purified water. Stir for 40 minutes, centrifuge, cool the centrifuged liquid to 3°C, let it settle for 24 hours, and then filter to obtain the water-precipitated ginkgo leaf filtrate. (3) Purification of Ginkgo Leaf Water Settling Filtrate Take the water-precipitated filtrate of Ginkgo biloba leaves from step (2), add it to a DM130 macroporous adsorption resin column, and discard the effluent; then elute with purified water and discard the eluent; elute with 2 column volumes of 15% ethanol and discard the eluent; connect ADS-17 macroporous adsorption resin to the DM130 macroporous adsorption resin column, and elute with 75% ethanol for 3 total column volumes, and collect the eluent; (4) Concentration of Ginkgo Eluent Take the eluent from step (3), adjust the pH to 6.5, concentrate under reduced pressure, replace the ethanol with purified water, slowly cool to 5°C, let stand for 16 hours to allow to settle, and then filter to obtain the filtered solution. (5) Resin purification treatment The filtered solution from step (4) was fed into a tandem column of macroporous adsorption resin DM130 and macroporous adsorption resin ADS-17, and the effluent was discarded. The solution was eluted with purified water, and the eluent was discarded. The solution was then eluted with 6 column volumes of 40% ethanol, and the eluent was collected. The solution was concentrated under reduced pressure and dried to obtain crude Ginkgo biloba extract. ⑹ Refined Add the crude Ginkgo biloba extract from step (5) to 20 times the volume of ethanol, add 0.02% activated carbon, heat to boiling, then cool, filter, concentrate under reduced pressure; vacuum dry, pulverize and mix to obtain the Ginkgo biloba extract.

[0066] Example 5 (1) Extraction of Ginkgo biloba leaves Take ginkgo leaves and add 8 times the amount of 80% ethanol, and heat and reflux at 80℃ for 2 hours; second extraction: add 6 times the amount of 80% ethanol, reflux for 0.5 hours; concentration temperature 60℃, vacuum degree -0.050~-0.090MPa, concentrate to a relative density of 1.05-1.15, and concentrate under reduced pressure to obtain ginkgo extract. (2) Water sedimentation of Ginkgo biloba extract Take the ginkgo extract from step (1) and add 4 times the amount of purified water. Stir for 30 minutes, centrifuge, cool the centrifuged liquid to 7°C, let it settle for 36 hours, and then filter to obtain the water-precipitated ginkgo leaf filtrate. (3) Purification of Ginkgo Leaf Water Settling Filtrate Take the water-precipitated filtrate of Ginkgo biloba leaves from step (2), add it to the HPD-450 macroporous adsorption resin column, and discard the effluent; then elute with purified water and discard the eluent; elute with 3 column volumes of 20% ethanol and discard the eluent; connect LX-158 macroporous adsorption resin in series to the HPD-450 macroporous adsorption resin column, and elute with 70% ethanol for 2-5 column volumes, and collect the eluent; (4) Concentration of Ginkgo Eluent Take the eluent from step (3), adjust the pH to 6.5, concentrate under reduced pressure, replace the ethanol with purified water, slowly cool to 6°C, let stand for 12 hours to allow to settle, and then filter to obtain the filtered solution. (5) Resin purification treatment The filtered solution from step (4) was fed into a tandem column of resin DM130 and macroporous adsorption resin ADS-17, and the eluent was discarded. The solution was eluted with purified water, and the eluent was discarded. The solution was then eluted with 8 column volumes of 40% ethanol, and the eluent was collected. The solution was concentrated under reduced pressure and dried to obtain crude Ginkgo biloba extract. ⑹ Refined Add the crude Ginkgo biloba extract from step (5) to 20 times the volume of ethanol, add 0.03% activated carbon, heat to boiling, then cool, filter, concentrate under reduced pressure; vacuum dry, pulverize and mix to obtain the Ginkgo biloba extract.

[0067] After optimizing the preparation process according to this invention, the ginkgolides in Ginkgo biloba extract were not detected, and the water solubility of Ginkgo biloba extract was significantly improved. The visible foreign matter rate of Ginkgo biloba extract injection and Shuxuening injection both decreased, with an improvement of 2-5%. Microbial limit control: By reconstituted the crude Ginkgo biloba extract and treating it with activated carbon, the quality requirements of the injectable raw material can be met. Compared with the existing technology, the two methods can achieve the same effect, but the activated carbon treatment is simpler and cheaper than the ultrafiltration process.

[0068] Compared with the processes described in existing technologies 1 (CN1939357A) and 2 (CN102626427A), the optimized process has a more reasonable content of each component, with a 3-7% increase in total ginkgo flavonol glycosides and a 95% reduction in ginkgo lactones. The water solubility of the above products is significantly improved, and the microbial limit meets the standard that the total number of aerobic bacteria in 1g of ginkgo leaf extract does not exceed 100cfu, and the ginkgolic acid content is less than 0.5ppm.

Claims

1. A composition of Ginkgo biloba extract, said composition comprising Ginkgo biloba extract (calculated as total flavonol glycosides): dipyridamole in a dosage ratio of 10:4, characterized in that, The Ginkgo biloba extract comprises the following raw material components in the following molar percentages: total flavonol glycosides of Ginkgo biloba 27-35%, ginkgolides 7.0%-8.6%, ginkgolides A 3.5%-4.5%, ginkgolides B 2%-2.7%, ginkgolides C 1.5%-1.8%, ginkgolide ≤0.01%, total ginkgolic acid ≤0.01%, flavonoid aglycones ≤1.3%-1.5%, free quercetin ≤0.05%, free kaempferol ≤0.05%, free isorhamnetin ≤0.01%, and the remainder being other components of the Ginkgo biloba extract; The preparation method includes the following steps: (1) Extraction of Ginkgo biloba leaves Take ginkgo leaves and add 6-8 times the amount of 70-80% ethanol, and heat and reflux at 75-80℃ for 1-2 hours; for the second extraction: add 4-6 times the amount of 70-80% ethanol, and reflux for 0.5-1.5 hours; concentrate at 50-80℃, vacuum degree -0.050 to -0.090 MPa, concentrate to a relative density of 1.05-1.15, and concentrate under reduced pressure to obtain ginkgo extract; (2) Water sedimentation of Ginkgo biloba extract Take the ginkgo extract from step (1) and add 3-5 times the amount of purified water. Stir for 20-40 minutes, centrifuge, cool the centrifuged liquid to 0-8℃, let it settle for 12-48 hours, and then filter to obtain the water-precipitated ginkgo leaf filtrate. (3) Purification of Ginkgo Leaf Water Settling Filtrate Take the water-precipitated filtrate of Ginkgo biloba leaves from step (2) and add it to macroporous adsorption resin column A. Discard the effluent. Elute with purified water and discard the eluent. Elute with 1-3 column volumes of 10-20% ethanol and discard the eluent. Connect resin column B in series with column A and elute with 60-80% ethanol for 2-5 column volumes. Collect the eluent. (4) Concentration of Ginkgo Eluent Take the eluent from step (3), adjust the pH to 5.0-8.5, concentrate under reduced pressure to about 1 / 10 of the volume, replace the ethanol with purified water, slowly cool to 0-8℃, let stand for 12-48 hours for cold precipitation, filter, and obtain the filtered drug solution. (5) Resin purification treatment The filtered solution from step (4) is fed into a resin C and D tandem column, and the effluent is discarded; the solution is eluted with purified water, and the eluent is discarded; the solution is eluted with 5-8 column volumes of 30-50% ethanol, and the eluent is collected; the solution is concentrated under reduced pressure and dried to obtain crude Ginkgo biloba extract. ⑹ Refined Add the crude Ginkgo biloba extract from step (5) to ethanol, add activated carbon, heat to boiling, then cool, filter, concentrate under reduced pressure; vacuum dry, pulverize and mix to obtain the Ginkgo biloba extract.

2. The application of the Ginkgo biloba extract composition as described in claim 1, characterized in that, The dosage form of the Ginkgo biloba extract composition is an injection.

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