CN1944626A - Microbial bacteria for preparing ginkalide by microbial fermenting and method for preparing ginkalide - Google Patents

Abstract

The invention discloses a fungus isolated and screened from the inner side of ginkgo bark. The invention is used to produce and prepare ginkgolide A, ginkgolide B and bilobalide. What used was the three-line Fusarium, and the preservation number was CGMCC No1677. Its process is as follows: (1) the nutrition in the culture medium adopts carbon source and nitrogen source, and wherein carbon source can be glucose, starch, dextrin, maltose, and nitrogen source can be peptone, peanut cake powder, soybean cake powder, beef extract, In addition to fishmeal, inorganic salts need to be added. (2) Fermentation process: using this production strain, its metabolite - ginkgolide can be obtained through the method of shake flask secondary fermentation. The cultivation method includes: activation of the original strain, Shake flask seed preparation, shake flask fermentation, post-treatment of fermentation broth, extraction and purification to prepare ginkgolide. The invention overcomes the restriction of ginkgo tree resources that the original ginkgolide is extracted from ginkgo leaves or bark, changes the production mode of ginkgolide, greatly improves the output of the product, and has stable quality.

Description

Microbial bacteria for preparing ginkgolide by microbial fermentation and method for preparing ginkgolide

Technical field:

The invention relates to the technical field of biopharmaceuticals, in particular to a method for preparing ginkgolide, a precious traditional Chinese medicine component, by microbial fermentation, a microbial bacterium (Fusarium oxysporum) for preparing ginkgolide by microbial fermentation, and a method for preparing ginkgolide.

Background technique:

Ginkgo biloba L., also known as Gongsun tree, is one of the ancient tree species and is a special plant of one family and one genus. Ginkgo trees can survive more than two million years of geological changes and vicissitudes, and they can still survive, so there must be something unique about them. Therefore, since the 1960s, domestic and foreign plant chemists have done a lot of research work on the chemical components and pharmacological effects of ginkgo tree tissues, and now there are about 70 components with clear structures. Table 1 lists some biologically active substances in Ginkgo biloba. At present, Ginkgo Biloba extract (GBE) is widely used in medicines, food supplements, cosmetics, etc. Although ginkgo is now planted in Europe and North America, China is still the main producing area. In 2004, China provided 24,000 tons of ginkgo leaves to the world, with a sales volume of about 600 million yuan, while the total sales of ginkgo preparations worldwide reached 5 billion US dollars, 98% of which were produced by foreign companies.

Table 1 The main components with biological activity in Ginkgo biloba type name molecular formula source of organization biological activity flavonoid aglycone Kaempferol C15H10O6 Ginkgo biloba Dilates coronary vessels and inhibits the activity of angiotensin-converting enzyme (ACEZ). Kaempferol-3-O-glucoside C21H20O11 Kaempferol-3-O-rutinoside C27H30O15 and flavonoid glycosides Kaempferol-7-O-glucoside C21H20O11 Kaempferol-3-glucose-2,6-dirhamnoside C33H40O19 Quercetin C15H10O7 quercetin-3-O-glucoside C21H20O12 quercetin-3-O-rhamnoside C21H20O11 quercetin-3-O-rutinoside C27H30O16 quercetin-3-glucose-2,6-dirhamnoside C33H40O20 isorhamnetin C16H12O7 isorhamnetin-3-O-glucoside C22H22O12 isorhamnetin-3-O-rutinoside C28H32O16 Biflavones Ginkgo biloba C32H22O10 Ginkgo biloba Ginkgo exocarp Cytotoxicity-mediated inhibition of NO synthesis Isoflavones C32H22O10 Desmethyl Ginkgo biloba Flavonoids C31H20O10 Biflavone C30H18O10 Qiansong Biflavone C33H24O10 5-Methoxynor Ginkgo biloba Flavone C32H22O11 Ginkgolides bilobalide C15H18O8 Ginkgo biloba root bark tissue The effect on the central nervous system, the effect of anti-platelet activating factor (PAF) Ginkgolide A C20H24O9 Ginkgolide B C20H24O10 Ginkgolide C C20H24O11 Ginkgolide J C20H24O10 Ginkgolide M C20H24O10 only in the root bark organic acid 6-Hydroxykynurenic acid C10H7O4N Ginkgo biloba N-methyl-D-aspartate receptor antagonists Alkylphenolic acid 4-methoxypyridoxic acid C9H13O3N ginkgo fruit Cytotoxin Ginkgool C21H34O ginkgo fruit Antitumor effect ginkgodiol C21H34O2 ginkgo fruit Ginkolic acid C22H34O3 ginkgo fruit Antitumor effect, antibacterial effect Hydrogenated Ginolic Acid C22H36O3 ginkgo fruit Anti-inflammatory, anti-allergic, antibacterial Hydrogenated Binolic Acid C21H34O3 ginkgo fruit

As can be seen from the above table, ginkgolides are specific platelet activating factor (PAF) antagonists. Platelet activating factor is an endogenous physiologically active substance in mammals, which is mainly related to the onset of allergies, ulcers, asthma, thrombosis, certain inflammations, allogeneic rejection of organ transplantation, and myocardial injury caused by hypoxia in coronary heart disease patients. Closely related, so ginkgolides are also mainly used for the treatment of the above diseases, clinically mainly used for senile dementia, cardiovascular and cerebrovascular diseases, bronchial asthma and allogeneic rejection of organ transplantation.

At present, the active ingredients in ginkgo tissue mainly come from leaves, bark and fruit of ginkgo. Because ginkgo trees are limited by climate, region, and harvest season, and more importantly, large-scale felling of ginkgo trees will destroy the ecological balance and is not conducive to environmental protection. Therefore, it is necessary to develop other resources to replace the extraction of ginkgo active ingredients from ginkgo trees.

Invention content:

The purpose of the present invention is to provide a kind of microbial strain (preservation date: on April 17th, 2006) that can produce ginkgolide by fermentation, depositor name: China Microorganism Culture Preservation Management Committee General Microorganism Center (CGMCC), depositor address: No. 13, North Yiyi, Zhongguancun, Haidian District, Beijing, preservation number: CGMCC No1677, strain classification name: Fusarium oxysporum). The strain is isolated from the inner side of ginkgo bark and obtained through screening. GB-1-3 is Fusarium tricinctum (three-line Fusarium), which belongs to Deuteromycotina, Hyphomycetes, Tumor Tuberculares, Tuberculariaceae, Fusarium fungi. .

The invention also provides a method for preparing ginkgolide by microbial fermentation, including various steps of activation of original strains, seed culture in shaking flasks, fermentation culture, and post-extraction.

The skimmed milk freeze-dried tube bacterial strain of the present invention refers to a storage method for producing the bacterial strain. It is to add the required proportion of sterile skim milk solution into the cultivated inclined test tube, use the inoculation ring to gently scrape off the mycelium and break it up as much as possible to make it evenly dispersed into a suspension. Use a sterile long dropper to dispense the suspension into the bottom of sterile clean ampoules (0.9 ml/each). Then pre-freeze the divided ampoule tubes in dry ice alcohol between -25 and -40°C for 1 hour, put the ampoule tubes into a vacuum, start the vacuum pump to dry, and finally seal the tubes.

The slant-plane activation of the strains in the present invention refers to opening the preserved freeze-dried tube, picking the strains and evenly spreading them on the slant medium, and culturing them at 28° C. for 7 days.

The shake flask seed culture of the present invention refers to adopting the carbon source, nitrogen source, inorganic salt etc. needed for bacterial strain growth, after being sterilized, excavate appropriate amount of thalline inclined plane, transfer in the seed shake flask, in automatic rotation shake Shake culture on a bottle machine, 200rpm, 28°C, and cultivate for 22-26 hours to obtain shake flask seeds.

Fermentation of the present invention refers to the carbon source, nitrogen source, inorganic salt, antifoaming agent etc. needed for the growth of bacterial strains, after sterilization, according to a certain inoculum ratio (10-15%), transfer to the shake flask The seeds are shaken and cultured on an automatic rotary shaker at 220-240 rpm, 28° C., and cultured for 140-150 hours to obtain fermentation metabolites.

The post-processing method of the fermented liquid of the present invention refers to that after the fermented liquid is placed in a bottle, centrifuge at room temperature and normal pressure with a centrifuge at 4000 rpm for 30 minutes, discard the fermentation supernatant, collect mycelium, and dry at 55°C Until the weight is halved, suspend the mycelium with 10 times the amount of water, then add 2 times the amount of petroleum ether, stir evenly and layer thickly, collect the water phase, repeat the above petroleum ether extraction operation once, and the purpose is to remove polarity. Substances such as small fatty acids have little effect on the extraction of lactones. Add 2N CaOH2 to the water phase, adjust the pH to 8.0, and make the free fatty acid salt precipitate (because the free fatty acid calcium salt is a hydrophobic substance, the solubility in water is very small), after the precipitation is sufficient, centrifuge to obtain the supernatant 1 and the precipitate 1. Precipitate 1 was shaken and washed with twice the amount of water, centrifuged at 3000 rpm for 20 minutes, and repeated twice. Supernatant 2 was collected and combined with supernatant 1. Add the ethyl acetate of equivolume amount then, after extraction 1 time, obtain ester phase 1 and water phase 1, wherein water phase 1 kind adds 2N HCl and adjusts pH to 4.7 (experimental result of the present invention shows, the range of pH4.7-6.1 It is beneficial to improve the yield of ginkgolides), then add 3 times the amount of ethyl acetate and extract fully for 3 times, and the obtained ester phase 2 is combined with the ester phase 1. Ethyl acetate was evaporated to obtain a dried extract. The mixed sample was applied to a silica gel column, and chloroform-methanol was used for gradient elution, and the fractions were collected to obtain ginkgolides A, B, and bilobalide.

The advantages of the present invention are:

1. Alternative to plant sources, providing new sources of ginkgo active ingredients

The present invention is the first use of microbial fermentation to prepare ginkgolides, completely replacing plant-derived ginkgo trees, so it is more environmentally friendly, does not destroy the ecological balance, and can control its output and quality during the fermentation process without being affected by changes in regions and seasons The impact is the greatest advantage of the present invention.

2. Does not contain harmful substances in ginkgo biloba extract - ginkgol and ginkgolic acid

Ginkgols and ginkgolic acids can cause severe allergic reactions. Such reactions are attributed to alkanols of the urshiol type, some of which are considered among the most potent contact allergens in nature. Several cases of circulatory disturbances including anaphylactic shock have been reported in patients treated with Ginkgo biloba. Therefore, in August 1999, the Veterinary Drugs Committee of the European Community Drug Review Committee issued a summary report on Ginkgo biloba extract (EMEA/MRL/668/99 final draft), which stipulated ginkgolic acid (less than 5mg/kg). The EMEA document emphasizes that the standard is also used for humans, and the standard has also been adopted by North American countries. No ginkgolic acid is found in the metabolites prepared by the microbial fermentation of the present invention.

3. Low production cost and no pollution

The raw material used in the invention has low cost, advanced production technology, and is suitable for large-scale industrial production, and the production equipment can be used in common with other microbial products. In addition, there is no emission of harmful substances in the production process of this product, no pollution to the surrounding environment, no pollution, and meets environmental protection requirements.

Strain preservation in the present invention:

Date of preservation: April 17, 2006.

Name of depository unit: General Microorganism Center (CGMCC) of China Committee for Culture Collection of Microorganisms.

Deposit number: CGMCC No1677.

Bacteria taxonomic name: Fusarium oxysporum.

Description of drawings:

Figure 1 is a flow chart of the production process

Figure 2 is a flow chart of the fermentation broth post-treatment process

Detailed ways:

Embodiment 1: The characteristics of the microbial strain GB-1-3 used for fermenting and preparing ginkgolide according to the present invention.

After two days of cultivation on the PDA medium, white cotton-like mycelium grows, and the edge of the colony is ciliated; on the third day of cultivation, the mycelium in the base is red; the aerial mycelium is white, grows luxuriantly, and covers the whole dish. There are no chlamydospores on the hyphae observed under an optical microscope. The conidia are divided into two types of spores. The megaspores are sickle-shaped, colorless, with 3-5 septa, and the size is 25-48×3-4.5 μm; the microspores are long Oval, colorless, 1-2 compartments, 12-15×2.4-3μm in size. Use shake flask seed medium to obtain mycelia, extract its chromosome, use ITS primers, use the chromosome of the thalline as a template, carry out PCR amplification, and recover, clone and sequence the PCR product, and the obtained sequence is in Compare and analyze in Genbank. At the same time, combined with the investigation of culture characteristics and mycelial morphology, it was determined that the strain was Fusarium tricinctum (Fusarium tricinctus), which belonged to the subphylum Deuteromycotina, Hyphomycetes, Tuberculariales, and Tumors. Tuberculariaceae, Fusarium fungi.

The production technology of ginkgolide prepared by microbial fermentation of the present invention

1. Culture medium

1. Inclined culture medium:

Glucose 10g/L, potato (peeled) 200g/L, agar 15g/L, prepared with distilled water, pH7.2-7.4, 121℃ for 30min.

Preparation of potato dipping juice: Take 200g of peeled potatoes, cut into small pieces, boil in 1000ml of water for an hour, filter, and fill the filtrate with cold boiled water to 1000ml.

2. Shake flask seed medium

Glucose 20g/L, peanut powder 3.0g/L, NH ₄ Cl 3.0g/L, KH ₂ PO ₄ 3.0g/L, MgSO ₄ 7H2O 3.0g/L, CaCO ₃ 4.0g/L,

NaAC·3H ₂ O 1.0g/L, prepared in normal water, pH 6.0 before sterilization.

Divide into 40ml/250ml Erlenmeyer flasks, 121°C for 30min.

3. Fermentation tank culture medium

Glucose 10g/L, peanut cake powder 10g/L, soybean cake powder 10g/L,

Corn starch 40g/L, NH4Cl 3g/L, KH ₂ PO4 3g/L,

NaAC.3H ₂ O 3g/L, CaCO ₃ 4g/L, MgSO ₄ .7H ₂ O 3g/L.

Prepared in normal water, pH 6.0 before sterilization, divided into 50ml/250ml Erlenmeyer flasks, 121°C for 30min.

Peanut cake powder, soybean cake powder and cornstarch in the above composition are required to be fresh and not moldy and pass through a 160-mesh sieve.

2. Fermentation production

1. Slope activation of the bacteria: Open the freeze-dried tube of the skim milk of the bacteria, pick a small amount of powder and spread it evenly on the sterilized slope, and cultivate it at 28°C for 5-7 days, until the lawn grows to white The aerial mycelium is dense, covered with slopes, free of bacteria, and stored in the refrigerator for 4 hours.

2. Shake flask seed culture: Inoculate 1*1cm2 of the activated slant bacteria into each seed culture bottle, culture at a constant temperature of 28°C, 200rpm, 22-26 hours, and check the growth and stretch of mycelia under the microscope , deep staining, and no bacteria.

3. Fermentation: The formula of the fermentation medium is as above. After sterilization, the cultivated seeds are transferred to the fermentation bottle according to the transfer amount of 10%, 180-240rpm, 25-28°C, and the cultivation time is 140-150 hours .

4. After terminating the fermentation, centrifuge the fermentation broth at room temperature and normal pressure with a centrifuge at a speed of 4000rpm for 30 minutes, discard the fermentation supernatant, collect the mycelia, dry at 55°C until the weight is reduced by half, and use 10 times amount of water-suspended mycelium, then add petroleum ether with 2 times the amount of water, stir evenly and layer thickly, collect the water phase, repeat the above-mentioned petroleum ether extraction operation once, and the purpose is to remove substances such as fatty acids with less polarity. The extraction of esters has little effect. Add 2N CaOH ₂ to the water phase, adjust the pH to 8.0, and make the free fatty acid salt precipitate (because the free fatty acid calcium salt is a hydrophobic substance, the solubility in water is very small), and after the precipitation is sufficient, centrifuge to obtain the supernatant 1 and Precipitation 1. Precipitate 1 was shaken and washed with twice the amount of water, centrifuged at 3000 rpm for 20 minutes, and repeated twice. Supernatant 2 was collected and combined with supernatant 1. Then add the ethyl acetate of equivolume amount, after extracting 1 time, obtain ester phase 1 and water phase 1, wherein water phase 1 kind adds 2N HCl and adjusts pH to 4.7 (experimental result of the present invention shows, the range of pH4.7-6.1 It is beneficial to improve the yield of ginkgolides), then add 3 times the amount of ethyl acetate and extract fully for 3 times, and the obtained ester phase 2 is combined with the ester phase 1. Ethyl acetate was evaporated to obtain a dried extract. The mixed sample was applied to a silica gel column, and chloroform-methanol was used for gradient elution, and the fractions were collected to obtain ginkgolides A, B, and bilobalide.

Example 2 The detection of ginkgolide prepared by microbial fermentation of the present invention

The fermentation broth of strain GD3-1 was centrifuged to obtain mycelia, and after being treated according to the extraction method described in Example 1, the dry sample of the crude extract obtained after volatilizing the ethyl acetate phase was dissolved in methanol, and the configuration was 7.6mg/ml Solution: After dissolving ginkgolides A, B, C and bilobalide respectively, configure a solution containing the above-mentioned various standard substances at a concentration of 0.2 mg/ml. Using the above-mentioned standard solution as a control, the samples were analyzed by a combination of external standard method and internal addition method. . The comparison of the retention time and peak area of each adjacent peak position between the standard and the sample is shown in Table 2. As can be seen from Table 2, there are indeed chromatographic peaks overlapping with the standards GA, GB, GC and BB in the sample.

Table 2 Statistics of chromatographic data of substances with retention times similar to those of the standard in the sample Standard (0.2mg/ml) GA GB GC BB Retention time t`R(min) 16.375 18.492 8.748 7.937 Peak area Ar 50708 45875 58342 34699 Sample (7.6mg crude extract/ml) GA' GB' GC' BB' Retention time tR(min) 16.668 18.15 8.542 8.005 Peak area Ar 3426 30169 11906 66403 Sample (7.6mg crude extract/ml) + standard (0.2mg/ml) Retention time tR(min) 16.59 18.775 8.757 8.173 Peak area Ar 53995 86291 65164 96603

Example 3 Primary Mass Spectrometry Identification of Ginkgolide Components in Microbial Fermentation Products of the Present Invention

Taking GD 3-1 as the starting strain for shake flask culture, the ethyl acetate extract obtained after the mycelium was treated according to the extraction process, and the standard products (gingkolides A, B, C and bilobalide) were used as controls. Perform HPLC analysis under the same conditions, collect chromatographic peaks with a retention time close to that of the standard, concentrate and then use the standard as a control for mass spectrometry analysis. The comparison and analysis of the mass spectrometry results of standard products BB, GA and GB are shown in Table 4, the comparison of BB standard product and sample mass spectrometry results is shown in Table 5, the comparison of GA standard product and sample mass spectrometry results is shown in Table 6, and the comparison of GB standard product and sample mass spectrometry results is shown in Table 6. The comparison of the mass spectrometry results of the samples is shown in Table 7.

Table 4 Comparison and analysis of standard mass spectrometry analysis results Graph properties BB GA GB analyze Standard Molecular Weight 326 408 424 m Full spectrum of positive ions (m/z) ⁺ 426.0833.4838.7855.5 442.0463.0865.7886.8 M+NH ₄ ⁺ M+K ⁺ 2M+NH ₄ ⁺ 2M+Na ⁺ 2M+K ⁺ Positive ion secondary spectrum (m/z) ⁺ 440.5425.7 456.6442.5378.6 M+CH ₃ OH+H ⁺ M+NH ₄ ⁺ M-CO ₂ +H ⁺ Full spectrum of negative ions (m/z) ^- 325.0370.6650.9 406.9452.8814.9 422.9458.9468.6847.0 M-1M+Cl ^- M+HCOO ^- 2M-1 Negative ion secondary spectrum (m/z) ^- 325.0306.5250.9206.8163.3 406.8378.6351.0 423.0394.7367.0 M-1[MH ₃ O ⁺ ][M-CO-H ⁺ ] ^- [M-2CO-H ⁺ ] ^- 250.9-CO ₂ 206.8-CO ₂

Table 5 Comparison of the results of bilobalide and its samples Graph properties BB standard BB' sample analyze full spectrum of negative ions 325.0650.9 325.0 M-12M-1 325 negative ion secondary spectrum 325.8325.0306.5250.9206.8163.3 326.3325.2306.1250.9206.9163.2 M-1[MH ₃ O ⁺ ] ^- [M-CO-H ⁺ ] ^- 250.9-CO ₂ 206.9-CO ₂

Table 6 Comparison of the results of GA standard and its samples Graph properties GA standard GA' sample analyze full spectrum of positive ions 426.0833.4839.7 425.9 M+NH ₄ ⁺ 2M+NH ₄ ⁺ 2M+Na ⁺ 426 Positive Ion Secondary Spectrum 441.7440.5426.5425.7 441.8440.6426.4425.0 M+CH ₃ OH+H ⁺ M+NH ₄ ⁺ full spectrum of negative ions 406.9452.8811.0 452.9810.9 M-1M+HCOO ^- 2M-1 453 negative ion secondary spectrum 452.5406.8378.6351.0 453.1406.9378.6350.9 M+HCOO ^- M-1M-CO-H ⁺ M-2CO-H ⁺

Table 7 Comparison of the results of GB standard and its samples Graph properties GB standard GB' sample analyze full spectrum of positive ions 442.0865.7886.7 442.0463.4 M+NH ₄ ⁺ M+K ⁺ 2M+NH ₄ ⁺ 2M+K ⁺ 442.0 positive ion secondary spectrum 457.7456.6442.5378.6 457.6456.6442.2378.7 M+CH ₃ OH+H ⁺ M+NH ₄ ⁺ M-CO ₂ +H ⁺ full spectrum of negative ions 422.9468.6847.0 423.0458.9 M-1M+Cl ^- M+HCOO ^- 2M-1 423.0 negative ion secondary spectrum 424.0423.0394.7367.0 424.4423.0394.8366.9 M-1M-CO-H ⁺ M-2CO-H ⁺

The above data show that there are substances with similar retention time and the same quasi-molecular ions as the standard products BB, GA, and GB in the fermentation broth extraction sample.

Example 4 High performance liquid phase/electrospray mass spectrometry (HPLC/ESI-MS) analysis and identification of ginkgolide B components in microbial fermentation products of the present invention

The ethyl acetate extract of mycelia after GD3-1 fermentation was subjected to silica gel column chromatography and chloroform-methanol gradient elution, and it was found that the effluent of chloroform-methanol 96:4 contained Consistent substance; cut off this section fraction, obtain about 50 μ g sample (GB'), dissolve with 4mL methanol, use GB standard as contrast, carry out high-performance liquid phase/electrospray mass spectrometry (HPLC/ESI under the same chromatographic and mass spectrometry conditions -MS) analysis, it was found that the two were consistent, further confirming the existence of ginkgolide B.

Claims (5)

1. A microbial bacterium for preparing ginkgolide by microbial fermentation, characterized in that: Fusarium oxysporum is used, the preservation number is CGMCC No1677, and the classification name of the strain is Fusariumoxysporum. 2. A method for cultivating strains of Fusarium tritina as claimed in claim 1, characterized in that: the preparation of shake flask seeds: culture medium Ph6.0 before sterilization, digging blocks and inoculating 1* ^1cm slant of the production strain For bacterial lawn, the rotation speed is 200rpm, the temperature is 25-28°C, and the cultivation time is 30-36 hours. 3. The cultivation method of the three-line Fusarium according to claim 2, characterized in that: the shaking flask fermentation process is as follows: pre-sterilization culture medium Ph6.0, inoculation according to 10-15% inoculum size, stirring rotation number 200- 240rpm, the temperature is 25-28°C, and the incubation time is 140-148 hours. 4. A method for preparing ginkgolides by microbial fermentation, characterized in that: the used production strain GB-1-3 is a three-line Fusarium, belonging to Deuteromycotina, Hyphospora, Tumoria, Tumoraceae, Fusarium fungus, its technology is as follows: (1) the nutrition in culture medium seed and fermentation medium adopts carbon source and nitrogen source, and wherein carbon source can be glucose, starch, dextrin, maltose, nitrogen The source can be peptone, peanut cake powder, soybean cake powder, beef extract, fish meal, and also need to add inorganic salts, NH ₄ Cl, KH ₂ PO ₄ , MgSO ₄ .7H ₂ O, CH ₃ COONa, CaCO ₃ , (2 ) fermentation process using the production strain, the metabolite - ginkgolide can be obtained through the shake flask secondary fermentation method. The cultivation method includes: activation of the original strain, preparation of shake flask seeds, shake flask fermentation, fermentation broth post-treatment, extraction and purification to prepare ginkgolides. 5. A method for preparing ginkgolides by microbial fermentation according to claim 4, characterized in that: its fermentation broth post-treatment process is as follows: Suspend the crude cell extract with 10 times the amount of water, add 2 times the amount of petroleum ether to the obtained feed solution, extract twice, separate the ether phase and the water phase; add 2N CaOH ₂ to the water phase, adjust the pH to 8.0, Salt precipitation of free fatty acids, after the precipitation is sufficient, centrifuge at 3000rpm for 20 minutes to obtain supernatant 1 and precipitation 1; precipitation 1 is washed with twice the amount of water, centrifuged at 3000rpm for 20 minutes, and repeat twice to obtain precipitation and supernatant 2. Collect the supernatant 2 and combine it with the supernatant 1, then add an equal volume of ethyl acetate, and extract once to obtain the ester phase 1 and the water phase 1, wherein 2N HCl is added to the water phase 1 to adjust the pH to 4.7 , and then add 3 times the amount of ethyl acetate to fully extract 3 times, and the obtained ester phase 2 is combined with the ester phase 1; the ethyl acetate is evaporated to obtain a dry product of the extract, and the sample is mixed on a silica gel column and carried out using chloroform-methanol. Gradient elution, each fraction was collected to obtain ginkgolides A, B and bilobalide.

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