WO2019113861A1 - 一种银杏提取物药用原料及其制备方法 - Google Patents

一种银杏提取物药用原料及其制备方法 Download PDF

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Publication number
WO2019113861A1
WO2019113861A1 PCT/CN2017/116018 CN2017116018W WO2019113861A1 WO 2019113861 A1 WO2019113861 A1 WO 2019113861A1 CN 2017116018 W CN2017116018 W CN 2017116018W WO 2019113861 A1 WO2019113861 A1 WO 2019113861A1
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total
lactone
flavonoid
purified product
content
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PCT/CN2017/116018
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English (en)
French (fr)
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巴卫松
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巴卫松
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Application filed by 巴卫松 filed Critical 巴卫松
Priority to CN201780003764.8A priority Critical patent/CN110198713A/zh
Priority to PCT/CN2017/116018 priority patent/WO2019113861A1/zh
Priority to DE112017006865.7T priority patent/DE112017006865T5/de
Publication of WO2019113861A1 publication Critical patent/WO2019113861A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/16—Ginkgophyta, e.g. Ginkgoaceae (Ginkgo family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps

Definitions

  • the invention relates to the field of medicine, in particular to a medicinal raw material of ginkgo biloba extract and a preparation method thereof.
  • Ginkgo biloba extract has a wide range of pharmacological effects, and its rich ginkgo biloba and ginkgo total lactone are the most important material basis for exerting such pharmacological effects.
  • the scavenging of oxygen free radicals (superoxide anion O-2) and the anti-platelet aggregation of platelet-activating factor (PAF) are the two most prominent pharmacological effects in many biological activities.
  • Ginkgo flavonoids have clear ability to scavenge oxygen free radicals and express different activities according to the structure of flavonoids. Studies have shown that flavonoids also have certain anti-platelet aggregation effects.
  • Ginkgolides can competitively antagonize platelet-activating factor (PAF) to exert strong anti-platelet aggregation, and its activity has a structure-activity relationship.
  • PAF platelet-activating factor
  • ginkgolide B is the most active in ginkgo diterpene lactone.
  • Ginkgolides also have a relatively weak activity of scavenging oxygen free radicals, that is, there are extremely complicated relationships between the two types of substances that cross each other and complement each other.
  • German scientists have conducted systematic and in-depth research on Ginkgo biloba extract (from ginkgo planting, supply of bottom nutrient, dwarfing, timing of collection, determination of active ingredient enrichment, etc.) and Ginkgo biloba extract International medicinal standards (total flavonoids ⁇ 24%, total lactone ⁇ 6%, ginkgolic acid ⁇ 5ppm).
  • this standard was established based on the current extraction preparation process and was not based on the evaluation of the pros and cons of the drug activity.
  • In order to better play the pharmacological activities of the two active ingredients reduce the clinical dose, and further reduce the insecurity and interference caused by impurities, it is necessary to explore a better proportional relationship between the two active ingredients! At present, such research has not been discovered.
  • One of the objects of the present invention is to provide a medicinal raw material for ginkgo biloba extract which has strong ability to scavenge oxygen free radicals and has strong anti-platelet aggregation activity.
  • Another object of the present invention is to provide a preparation method of a medicinal raw material of Ginkgo biloba extract, which is simple in preparation method and easy to operate, and extracts total flavonoids and total flavonoids from total lactone purified product and total flavonoid purified product.
  • the content of lactone is reasonable and it is more conducive to compounding.
  • a ginkgo biloba extract medicinal raw material comprising a mixture of a total lactone purified product extracted from a crude extract of Ginkgo biloba leaves and a purified total flavonoid, wherein the total lactone purified product comprises a first total lactone and The first total flavonoid, the total flavonoid purified product comprises a second total lactone and a second total flavonoid, and in the mixture, the content of the first total lactone and the second total lactone in the mixture is the same as the first total flavonoid and the second total
  • the ratio of the content of the flavonoids in the mixture is 1-2: 1-2, preferably 1-2:1, more preferably 1:1.
  • the total total lactone content in the above total lactone purified product is 36%-46%, the first total flavonoid content is 13%-19%, and the sum of the first total lactone content and the first total flavonoid content More than 50%; the total total lactone content in the total flavonoid purified product is 11%-14%, the second total flavonoid content is 42%-47%, and the sum of the second total lactone content and the second total flavonoid content is greater than 50 %.
  • a preparation method of medicinal raw material of ginkgo biloba extract extracting total lactone purified product and total flavonoid purified product from crude ginkgo leaf extract, and determining content of first total lactone and first total flavonoid in total lactone purified product , determining the content of the second total lactone and the second total flavonoid in the purified total flavonoid, and then compounding the total lactone purified product and the total flavonoid purified product to satisfy the first total lactone and the second total lactone
  • the ratio of the content in the mixture to the content of the first total flavonoids and the second total flavonoids in the mixture is 1-2: 1-2, preferably 1-2:1, more preferably 1:1.
  • the extracting the total lactone purified product and the total flavonoid purified product from the crude extract of Ginkgo biloba comprises: adding the Ginkgo biloba extract powder to water for the first ultrasonic dissolution, and centrifuging to obtain the first filtrate and the first filter residue.
  • the first extracting agent is subjected to extraction; the first ester phase and the first aqueous phase are respectively taken, the total lactone purified product is extracted in the first ester phase, and the total flavonoid purified product is extracted in the first aqueous phase.
  • the pH of the total filtrate is first adjusted to 4.5-5.0 prior to the addition of an equal volume of the first extractant to the total filtrate for extraction.
  • the first extracting agent comprises first ethyl acetate and n-heptane
  • the volume ratio of the first ethyl acetate to n-heptane is from 6 to 10:1, preferably from 7 to 9:1, more preferably from 7 to 8:1.
  • the ultrasonication time of the first sonication described above is 1-3 min, preferably 1-2 min, more preferably 1-1.5 min.
  • the ultrasonication time of the second sonication described above is 10-20 min, preferably 12-18 min, more preferably 14-16 min.
  • extracting the total lactone purified product in the first ester phase comprises: distilling the first ester phase under reduced pressure, recovering the solvent, and then adding the first ethanol to the concentrate to evaporate to dryness, recovering the first solid, and freeze-drying To obtain a total lactone purified product.
  • extracting the total flavonoid purified product in the first aqueous phase comprises: adjusting the pH of the first aqueous phase to 7-8, extracting with an equal volume of the second extractant multiple times, and discarding the plurality of extracted extracts
  • the aqueous second phase is combined with a plurality of second ester phases, and the solvent is distilled off under reduced pressure. Then, the second ethanol is evaporated to dryness in the concentrate, and the second solid is recovered and freeze-dried to obtain a purified flavonoid.
  • the second extracting agent comprises a second ethyl acetate and n-butanol
  • the second ethyl acetate and n-butanol have a volume ratio of from 4 to 8:1, preferably from 5 to 7:1; more preferably 6:1.
  • the Ginkgo biloba extract medicinal raw material provided in the present embodiment is compounded by purifying the total lactone purified product and the total flavonoid extract extracted from the crude extract of Ginkgo biloba, and by measuring the total total amount in the total lactone purified product.
  • the content of the ester and the first total flavonoids and the content of the second total lactone and the second total flavonoids in the purified total flavonoids are adjusted by adjusting the ratio of the total lactone purified product and the total flavonoid purified product.
  • the ratio of the total content of the first total lactone and the second total lactone in the mixture to the total content of the first total flavonoid and the second total flavonoid in the mixture is in the range of 1-2: 1-2
  • the obtained medicinal raw material of Ginkgo biloba extract has better free radical scavenging ability and excellent anti-platelet aggregation activity.
  • the conventional ginseng extract contains ⁇ 24% total flavonoids and total lactone ⁇ 6%.
  • the quality standard of Ginkgo biloba extract is more refined and the biological activity is better. .
  • the preparation method of the ginkgo biloba extract medicinal raw material provided in this embodiment is obtained by extracting the total lactone purified product and the total flavonoid purified product, determining the content, calculating the reasonable ratio, and configuring the first total lactone and the second in the formed mixture.
  • the ratio of the total content of the total lactone in the mixture to the total content of the first total flavonoids and the second total flavonoids in the mixture is in the range of 1-2: 1-2, and the obtained medicinal raw materials of the ginkgo biloba extract are compared.
  • the preparation method is simple in operation, easy to implement, and suitable for mass production.
  • ginkgo biloba extract medicinal raw material and the preparation method thereof according to the examples of the present invention are specifically described below.
  • a ginkgo biloba extract medicinal raw material comprising a mixture of a total lactone purified product extracted from a crude extract of Ginkgo biloba leaves and a purified total flavonoid, the total lactone purified product comprising a first total lactone and The first total flavonoid, the total flavonoid purified product comprises a second total lactone and a second total flavonoid, in the mixture, the total content of the first total lactone and the second total lactone in the mixture is combined with the first total flavonoid and the second
  • the ratio of the total content of total flavonoids in the mixture is from 1-2:1 to 2, preferably from 1 to 2:1, more preferably 1:1.
  • the total lactone purified product and the total flavonoid purified product contain flavonoids. And lactone two components, but the content is different, wherein the total lactone purified product contains more total lactone content, less total flavonoid content, and the total lactone content in the total flavonoid purified product Less, total flavonoids are more.
  • a single total lactone purified product and a total flavonoid purified product have a poor ratio of total flavonoids and total lactones, resulting in a poor effect of a single total lactone purified product and a total flavonoid purified product.
  • the treatment results are not ideal.
  • the content of the first total lactone and the first total flavonoid in the total lactone purified product is determined. Simultaneously determining the content of the second total lactone and the second total flavonoid in the purified total flavonoids, thereby forming a mixture by compounding the total lactone purified product and the total flavonoid purified product, so that the first total lactone and the second total in the mixture.
  • the ratio of the content of the lactone in the mixture to the content of the first total flavonoids and the second total flavonoids in the mixture is 1-2:1 to 2, preferably 1-2:1, more preferably 1:1.
  • the ratio of total lactone (the sum of the first total lactone and the second total lactone) to the total flavonoid (the sum of the first total flavonoid and the second total flavonoid) in the mixture formed by the combination is found.
  • the pharmacological activity of the total lactone and total flavonoids can be better exerted at this time, the free radical scavenging and the anti-platelet aggregation effect are good, and the clinical dosing is also reduced, and the impurities are further reduced. Safety and interference.
  • the content of the first total lactone and the first total flavonoid in the total lactone purified product is also defined, and the second total lactone and the second total in the total flavonoid purified product are also defined.
  • the content of flavonoids is limited, thereby ensuring the superiority of the total flavonoid purified product and the total lactone purified product.
  • the total total lactone content in the total lactone purified product is 36%-46%
  • the first total flavonoid content is 13%-19%
  • the sum of the first total lactone content and the first total flavonoid content More than 50%
  • the total total lactone content in the total flavonoid purified product is 11%-14%
  • the second total flavonoid content is 42%-47%
  • the sum of the second total lactone content and the second total flavonoid content is greater than 50 %.
  • the content of the first total lactone may be any one of 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46% or both
  • the range value between the first total flavonoids may be any one of 13%, 14%, 15%, 16%, 17%, 18%, 19% or a range between any two.
  • the content of the second total lactone may be any one of 11%, 12%, 13%, 14% or a range between any two; the content of the second total flavonoid may be 42%, 43%, 44 A range value between any one of %, 45%, 46%, 47% or any two.
  • the total lactone purified product and the total flavonoid purified product satisfy the above content requirements, the total lactone purified product and the total flavonoid purified product have good quality, and can better exert the pharmacological activity of total lactone and total flavonoids. It has a good effect of scavenging free radicals and anti-platelet aggregation.
  • This embodiment also provides a preparation method of a medicinal raw material of Ginkgo biloba extract, which specifically comprises the following steps:
  • the crude extract of Ginkgo biloba leaves is dissolved in water, and the first ultrasonic solution is dissolved, followed by centrifugation to obtain the first filtrate and the first filter residue, and the first filter residue is added with disodium hydrogen phosphate for the second ultrasonic dissolution, and the mixture is centrifuged to obtain the first
  • the second filtrate and the second filter residue combine the first filtrate and the second filtrate to form a total solution, and add an equal volume of the first extractant to the total filtrate for extraction; respectively, the first ester phase and the first aqueous phase are taken.
  • the pH of the total filtrate is first adjusted to 4.5-5.0 before the addition of an equal volume of the first extractant to the total filtrate for extraction. Adjusting the pH of the total filtrate to 4.5 to 5.0 is advantageous for better dispersion of the total filtrate and better extraction.
  • the first extracting agent comprises a first ethyl acetate and n-heptane; preferably, the first ethyl acetate and n-heptane are in a volume ratio of from 6 to 10:1, preferably from 7 to 9:1, more preferably from 7 to 8: 1. It has been found by the inventors that the use of a specific volume ratio of the first ethyl acetate and n-heptane as the first extractant is advantageous for enhancing the extraction effect.
  • the sonication time for the first sonication is 1-3 min, preferably 1-2 min, more preferably 1-1.5 min.
  • the sonication time for the second sonication is 10-20 min, preferably 12-18 min, more preferably 14-16 min. According to the study by the inventors, after two different times of ultrasonic dissolution, the total flavonoids and total lactones of Ginkgo biloba extract powder can be fully extracted, and the extraction effect is better.
  • Extracting the total lactone purified product in the first ester phase heating the first ester phase under reduced pressure, recovering the solvent, and then adding the first ethanol to the concentrate to evaporate to dryness, recovering the first solid, and freeze-drying to obtain total lactone Purified product.
  • the organic solvent can be recovered, and the organic solvent remaining in the concentrate can be easily taken out by mixing the first ethanol with the organic solvent, thereby further reducing the amount of the organic solvent remaining in the purified product. Residual organic solvent.
  • Extracting the total flavonoids in the first aqueous phase first adjusting the pH of the first aqueous phase to 7-8, then extracting it multiple times with an equal volume of the second extractant, discarding the first aqueous phase after multiple extractions And combining the second ester phase multiple times is advantageous for increasing the yield of the purified total flavonoids, and further, recovering the solvent by the operation of concentration distillation under reduced pressure. Thereafter, a second ethanol was added to the concentrate to evaporate to dryness, and the second solid was recovered and lyophilized to obtain a purified flavonoid.
  • the second extractant for multiple extractions, the total flavonoid purified product in the first aqueous phase can be further extracted, and the extraction effect is better, and the solvent is recovered by the operation of concentration distillation under reduced pressure, which can not only be realized.
  • the solvent can be recycled, and the purity of the purified flavonoids can be further improved when the solvent is recovered.
  • the second ethanol is evaporated to dryness in the concentrated solution, and the organic solvent remaining in the concentrated solution can be taken out together. Reducing the residual of the organic solvent in the purified total flavonoids.
  • the second extracting agent comprises a second ethyl acetate and n-butanol; preferably, the second ethyl acetate and n-butanol have a volume ratio of 4-8:1, preferably 5-7:1; more preferably 6: 1.
  • a second mixture of ethyl acetate and n-butanol is selected as the second extractant, which can effectively extract the purified flavonoids in the aqueous phase, and the extraction effect is good.
  • the volume ratio of the second ethyl acetate to n-butanol is within the above ratio, the extraction effect is better.
  • the total total lactone content in the total lactone purified product is determined to be 36% to 46%, the first total flavonoid content is 13% to 19%, and the sum of the first total lactone content and the first total flavonoid content is greater than 50%. .
  • the second total lactone content in the purified total flavonoids is 11%-14%, the second total flavonoid content is 42%-47%, and the sum of the second total lactone content and the second total flavonoid content is greater than 50%. .
  • the total lactone purified product and the total flavonoid purified product are compounded and satisfy the total content of the first total lactone and the second total lactone in the mixture and the total content of the first total flavonoid and the second total flavonoid in the mixture.
  • the ratio is 1-2:1-2, preferably 1-2:1, more preferably 1:1.
  • the total product lactone purified product and the total flavonoid purified product are compounded so that the final product satisfies the requirements of the above content ratio.
  • the ginkgo biloba extract medicinal raw material satisfying the above content ratio has better radical scavenging ability and excellent anti-platelet aggregation activity.
  • the method for flavonoid determination and lactone determination in the International Pharmaceutical Standard is used to determine the total flavonoid purified product and the total lactone by using the total flavonoid purified product and the total lactone purified product in the present embodiment as samples. The content of each flavonoid and lactone in the purified product.
  • the ethyl acetate extract and the washing liquid were combined, washed twice with water, 20 mL each time, and the aqueous liquid was separated, washed with ethyl acetate 10 mL, and the ethyl acetate solution was combined, and the obtained extract was concentrated to dryness under reduced pressure and transferred with methanol.
  • the Ginkgo biloba extract medicinal raw material provided in the present embodiment is compounded by purifying the total lactone purified product and the total flavonoid extract extracted from the crude extract of Ginkgo biloba, and determining the total lactone purified product.
  • the content of the first total lactone and the first total flavonoid in the total flavonoid purified product and the content of the second total lactone and the second total flavonoid in the total flavonoid purified product are adjusted by adjusting the ratio of the total lactone purified product and the total flavonoid purified product.
  • the ratio of the content of the first total lactone and the second total lactone in the mixture to the content of the first total flavonoid and the second total flavonoid in the mixture is 1-2: 1-2.
  • the obtained medicinal raw material of Ginkgo biloba extract has better free radical scavenging ability and excellent anti-platelet aggregation activity.
  • the preparation method of the ginkgo biloba extract medicinal raw material provided in this embodiment is obtained by extracting the total lactone purified product and the total flavonoid purified product, determining the content, calculating the reasonable ratio, and configuring the first total lactone and the second in the formed mixture.
  • the ratio of the total content of the total lactone in the mixture to the total content of the first total flavonoids and the second total flavonoids in the mixture is in the range of 1-2: 1-2, and the obtained medicinal raw materials of the ginkgo biloba extract are compared.
  • the preparation method is simple in operation, easy to implement, and suitable for mass production.
  • ginkgo biloba extract medicinal raw material of the present invention and a preparation method thereof are further described below in conjunction with the examples.
  • This embodiment provides a medicinal raw material of ginkgo biloba extract and a preparation method thereof.
  • the Ginkgo biloba extract medicinal raw material is a mixture of a total lactone purified product and a total flavonoid purified product, wherein the total lactone purified product comprises a first total lactone and a first total flavonoid, and a total flavonoid purified product.
  • the second total lactone and the second total flavonoid are included, and the ratio of the content of the first total lactone and the second total lactone in the mixture to the content of the first total flavonoid and the second total flavonoid in the mixture is 1:2.
  • the medicinal raw material of the ginkgo extract is prepared by the following method:
  • the first ester phase was distilled under reduced pressure, and the solvent was recovered. Then, the first ethanol was added to the concentrate to evaporate to dryness, and the first solid was recovered and freeze-dried to obtain a total lactone purified product.
  • the second ester phase is combined and the solvent is distilled off under reduced pressure, and then the second ethanol is evaporated to dryness in the concentrate to recover the second solid, which is freeze-dried to obtain total flavonoids. Purified product.
  • the content of the first total lactone and the first total flavonoid in the total lactone purified product was determined, and the total total lactone content in the total lactone purified product was 36%, the first total flavonoid content was 19%, and the first total lactone was used. The sum of the content and the first total flavonoid content is 55%;
  • the content of the second total lactone and the second total flavonoid in the purified total flavonoids was determined, and the total total flavonoid content in the total flavonoid purified product was 11%, the second total flavonoid content was 47%, and the second total lactone content was The sum of the second total flavonoid content was 58%.
  • the total lactone purified product and the total flavonoid purified product are then compounded and satisfy the content of the first total lactone and the second total lactone in the mixture and the content of the first total flavonoid and the second total flavonoid in the mixture.
  • the ratio is 1:2.
  • This embodiment provides a medicinal raw material of ginkgo biloba extract and a preparation method thereof.
  • the Ginkgo biloba extract medicinal raw material is a mixture of a total lactone purified product and a total flavonoid purified product, wherein the total lactone purified product comprises a first total lactone and a first total flavonoid, and a total flavonoid purified product.
  • the second total lactone and the second total flavonoid are included, and the ratio of the content of the first total lactone and the second total lactone in the mixture to the content of the first total flavonoid and the second total flavonoid in the mixture is 2:1.
  • the medicinal raw material of the ginkgo extract is prepared by the following method:
  • the first ester phase was distilled under reduced pressure, and the solvent was recovered, followed by evaporation of the first ethanol in the concentrate to dryness, and the first solid was recovered and freeze-dried to obtain a total lactone purified product.
  • the second ester phase is combined multiple times, and the solvent is distilled off under reduced pressure, and then the second ethanol is evaporated to dryness in the concentrate to recover the second solid, which is freeze-dried to obtain total flavonoids. Purified product.
  • the total total lactone content in the total lactone purified product was determined to be 40%, the first total flavonoid content was 15%, and the sum of the first total lactone content and the first total flavonoid content was 55%.
  • the total total lactone content in the purified total flavonoids was determined to be 13%, the second total flavonoid content was 45%, and the sum of the second total lactone content and the second total flavonoid content was 58%.
  • the total lactone purified product and the total flavonoid purified product are then compounded and satisfy the content of the first total lactone and the second total lactone in the mixture and the content of the first total flavonoid and the second total flavonoid in the mixture.
  • the ratio is 2:1.
  • This embodiment provides a medicinal raw material of ginkgo biloba extract and a preparation method thereof.
  • the Ginkgo biloba extract medicinal raw material is a mixture of a total lactone purified product and a total flavonoid purified product, wherein the total lactone purified product comprises a first total lactone and a first total flavonoid, and a total flavonoid purified product.
  • the second total lactone and the second total flavonoid are included, and the ratio of the content of the first total lactone and the second total lactone in the mixture to the content of the first total flavonoid and the second total flavonoid in the mixture is 1:1.
  • the medicinal raw material of the ginkgo extract is prepared by the following method:
  • the first ester phase was distilled under reduced pressure, and the solvent was recovered. Then, the first ethanol was added to the concentrate to evaporate to dryness, and the first solid was recovered and freeze-dried to obtain a total lactone purified product.
  • the second ester phase is combined and the solvent is distilled off under reduced pressure, and then the second ethanol is evaporated to dryness in the concentrate to recover the second solid, which is freeze-dried to obtain total flavonoids. Purified product.
  • This embodiment provides a medicinal raw material of ginkgo biloba extract and a preparation method thereof.
  • the Ginkgo biloba extract medicinal raw material is a mixture of a total lactone purified product and a total flavonoid purified product, wherein the total lactone purified product comprises a first total lactone and a first total flavonoid, and a total flavonoid purified product.
  • the second total lactone and the second total flavonoid are included, and the ratio of the content of the first total lactone and the second total lactone in the mixture to the content of the first total flavonoid and the second total flavonoid in the mixture is 1:1.
  • the medicinal raw material of the ginkgo extract is prepared by the following method:
  • the first ester phase was distilled under reduced pressure, and the solvent was recovered. Then, the first ethanol was added to the concentrate to evaporate to dryness, and the first solid was recovered and freeze-dried to obtain a total lactone purified product.
  • the second ester phase is combined and the solvent is distilled off under reduced pressure, and then the second ethanol is evaporated to dryness in the concentrate to recover the second solid, which is freeze-dried to obtain total flavonoids.
  • the total total lactone content in the total lactone purified product was determined to be 37%, the first total flavonoid content was 14%, and the sum of the first total lactone content and the first total flavonoid content was 51%.
  • the total total lactone content in the purified flavonoids was determined to be 12%, the second total flavonoid content was 46%, and the sum of the second total lactone content and the second total flavonoid content was 58%.
  • the total lactone purified product and the total flavonoid purified product are then compounded and satisfy the content of the first total lactone and the second total lactone in the mixture and the content of the first total flavonoid and the second total flavonoid in the mixture.
  • the ratio is 1:1.
  • This embodiment provides a medicinal raw material of ginkgo biloba extract and a preparation method thereof.
  • the Ginkgo biloba extract medicinal raw material is a mixture of a total lactone purified product and a total flavonoid purified product, wherein the total lactone purified product comprises a first total lactone and a first total flavonoid, and a total flavonoid purified product.
  • the second total lactone and the second total flavonoid are included, and the ratio of the content of the first total lactone and the second total lactone in the mixture to the content of the first total flavonoid and the second total flavonoid in the mixture is 1:1.
  • the medicinal raw material of the ginkgo extract is prepared by the following method:
  • the first ethyl acetate and n-heptane have a volume ratio of 10:1, and the first ester phase and the first aqueous phase are respectively taken.
  • the first ester phase was distilled under reduced pressure, and the solvent was recovered.
  • the first ethanol was added to the concentrate to evaporate to dryness, and the first solid was recovered and freeze-dried to obtain a total lactone purified product.
  • the second ester phase is combined and the solvent is distilled off under reduced pressure, and then the second ethanol is evaporated to dryness in the concentrate to recover the second solid, which is freeze-dried to obtain total flavonoids. Purified product.
  • the total total lactone content in the total lactone purified product was determined to be 45%, the first total flavonoid content was 17%, and the sum of the first total lactone content and the first total flavonoid content was 62%.
  • the total total lactone content in the purified total flavonoids was 11%
  • the second total flavonoid content was 46%
  • the sum of the second total lactone content and the second total flavonoid content was 57%.
  • the total lactone purified product and the total flavonoid purified product are then compounded and satisfy the content of the first total lactone and the second total lactone in the mixture and the content of the first total flavonoid and the second total flavonoid in the mixture.
  • the ratio is 1:1.
  • the ginkgo biloba extract medicinal raw material and the preparation method thereof are prepared according to the preparation method of the ginkgo biloba extract, and the total lactone purified product and the total flavonoid are adjusted when the total lactone purified product and the total flavonoid purified product are compounded.
  • the experiments were carried out one by one with the two pharmacodynamic indicators of scavenging oxygen free radicals and anti-platelet aggregation, and negative and positive control groups were set up. From the seven groups, the group with the strongest activity of scavenging free radicals and anti-platelet aggregation was selected.
  • vitamin C sodium ascorbate
  • Three reference control groups edaravone injection Ginkgo biloba extract injection And ginkgo total lactone injection
  • the experimental method is the same as the test sample, and the DPPH free radical scavenging ability of the test sample and the reference reference product is compared.
  • Data processing and analysis were performed using EXCEL and ORIGIN 8.0, and curve fitting was performed to determine the EC50 concentration.
  • the concentration of the total extract corresponding to the ratio was calculated and labeled as: total flavonoid glycoside concentration/total extract concentration.
  • the EC 50 concentration of DPPH free radicals in Comparative Example 1-4 was 138.99 ⁇ g.mL -1 /396.23 ⁇ g.mL -1 , 89.64 ⁇ g.mL -1 /337.58 ⁇ g.mL -1 , 67.11 gg.mL -1 /377.27 ⁇ g.mL -1 , 186.75 ⁇ g.mL -1 /443.63 ⁇ g.mL -1 ,167.11 ⁇ g.mL -1 /425.45 ⁇ g.mL -1 , 50.02 ⁇ g.mL -1 /410.74 ⁇ g .mL -1 , 42.53 ⁇ g.mL -1 /376.87 ⁇ g.mL -1 .
  • the EC 50 concentrations of the reference control edaravone and Ginaton were 86.88 ⁇ g.mL -1 and 157.10 ⁇ g.mL -1 /654.58 ⁇ g.mL - 1.
  • the reference substance Ginkgo biloba total lactone injection (Golden Lye) solution (total lactone concentration of 5000 ⁇ g.mL -1 ) had a DPPH removal capacity of only 30.08%, and no EC 50 concentration was obtained.
  • the drug was administered by single tail vein injection, and the test samples 1 to 3 were given to the test cases 1-3, the test samples 4-7 were given the comparative examples 1-4, and the reference control group 1 was given the Jinnadu.
  • mice were subjected to intraocular blood sampling with a broken capillary (about 1.5 cm long). After removing the first drop of blood, one drop of blood was dropped on the slide, and the stopwatch was immediately used every 30 seconds. Use a pin to pick light from the edge of the blood drop to the middle once, stop timing when there is bloodshot, and record the time, which is the in vitro clotting time.
  • results were expressed as mean ⁇ standard deviation (x ⁇ s).
  • Statistical analysis was performed using EXCEL software. The difference in clotting time between each group and the negative control group was compared by paired T test. The test P value was 0.05.
  • Free radical scavenging ability Under the premise of achieving the same EC50 concentration for scavenging DPPH free radicals, the concentrations of the total extracts required in Test Examples 1-3, Comparative Examples 1-4 are different, and the concentration is smaller. The stronger the ability to clear. The results of the scavenging free radical experiment showed that the test examples 1-3, the effects of the comparative examples 1-4 were significantly better than the Ginaton and ginkgo total lactone injections.
  • the obtained medicinal raw material of Ginkgo biloba extract has better free radical scavenging ability and excellent anti-platelet aggregation activity.
  • the medicinal raw material of Ginkgo biloba extract obtained by the preparation method of the medicinal raw material of Ginkgo biloba extract has better free radical scavenging ability and excellent anti-platelet aggregation activity.
  • the preparation method is simple in operation, easy to implement, and suitable for mass production.

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Abstract

一种银杏提取物药用原料及其制备方法，银杏提取物药用原料包括将从银杏叶粗提物中提取的总内酯纯化品和总黄酮纯化品复配形成的混合物，总内酯纯化品内包括第一总内酯和第一总黄酮，总黄酮纯化品包括第二总内酯和第二总黄酮，在混合物中，第一总内酯和第二总内酯的总含量与第一总黄酮和第二总黄酮的总含量的比例为1-2:1-2。制备方法为：通过测定第一总内酯、第一总黄酮、第二总内酯和第二总黄酮的含量，然后按照上述比例对总内酯纯化品和总黄酮纯化品进行复配形混合物。

Description

一种银杏提取物药用原料及其制备方法 技术领域
本发明涉及医药领域，具体而言，涉及一种银杏提取物药用原料及其制备方法。
背景技术
银杏叶提取物具有广泛的药理作用，其所富含的银杏总黄酮和银杏总内酯是其发挥此类药理作用最主要的物质基础。而清除氧自由基(超氧阴离子O-·2)抗氧化和拮抗血小板活化因子(PAF)抗血小板凝聚又是其众多生物活性中最为突出的两个药理作用。银杏黄酮类化合物具有明确的清除氧自由基能力，并根据黄酮类化合物结构差异表达不同的活性，研究表明黄酮类化合物也具有一定的抗血小板凝聚作用。银杏内酯类物质可以竞争性拮抗血小板活化因子(PAF)从而发挥强大的抗血小板凝聚作用，其活性强弱存在构效关系，其中以银杏二萜内酯中的银杏内酯B活性最强。银杏内酯类物质也具有相对比较弱的清除氧自由基的活性，即两类物质之间存在着相互交叉、相辅相成等极其复杂的关系。德国科学家曾对银杏叶提取物进行过比较系统、深入的研究(从银杏树的种植、底肥营养的供给、矮化、采集时机把握、活性成分富集量的测定等)并制定了银杏提取物国际药用标准(总黄酮≥24％、总内酯≥6％、银杏酸＜5ppm)。但这个标准是根据当时的提取制备工艺而确立，不是根据药物活性的优劣评估产生的。为了更好的发挥两类有效成分的药理活性，减少临床给药剂量，进一步降低杂质带来的不安全性及干扰，探究寻找出两类有效成分更好的比例关系就显得很有必要！目前此类研究尚无被发现。
发明内容
本发明的目的之一在于提供一种银杏提取物药用原料，其清除氧自由基的能力强，抗血小板凝聚的活性强。
本发明的目的中的另一目的在于提供一种银杏提取物药用原料的制备方法，其制备方法简单，操作容易，提取获得的总内酯纯化品和总黄酮纯化品中的总黄酮和总内酯的含量分布合理，更有利于复配。
本发明的实施例是这样实现的：
一种银杏提取物药用原料，其包括将从银杏叶粗提物中提取的总内酯纯化品和总黄酮纯化品复配形成的混合物，总内酯纯化品内包括第一总内酯和第一总黄酮，总黄酮纯化品包括第二总内酯和第二总黄酮，在混合物中，第一总内酯和第二总内酯在混合物中的含量 与第一总黄酮和第二总黄酮在混合物中的含量的比例为1-2:1-2，优选为1-2:1，更优选为1:1。
可选地，上述总内酯纯化品中第一总内酯含量为36％-46％、第一总黄酮含量为13％-19％，第一总内酯含量与第一总黄酮含量之和大于50％；总黄酮纯化品中第二总内酯含量为11％-14％，第二总黄酮含量为42％-47％，第二总内酯含量与第二总黄酮含量之和大于50％。
一种银杏提取物药用原料的制备方法，从银杏叶粗提物中提取总内酯纯化品和总黄酮纯化品，测定总内酯纯化品中第一总内酯和第一总黄酮的含量，测定总黄酮纯化品中第二总内酯和第二总黄酮的含量，然后将总内酯纯化品和总黄酮纯化品复配形混合物并满足第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为1-2:1-2，优选为1-2:1，更优选为1:1。
可选地，上述从银杏叶粗提物中提取总内酯纯化品和总黄酮纯化品包括：将银杏叶提取物粉加水进行第一次超声溶解，离心分离，获得第一滤液和第一滤渣，向第一滤渣中加入磷酸氢二钠溶液进行第二次超声溶解，离心分离，获得第二滤液和第二滤渣，合并第一滤液和第二滤液形成总滤液；向总滤液中加入等体积的第一萃取剂进行萃取；分别取第一酯相和第一水相，在第一酯相内提取总内酯纯化品，在第一水相内提取总黄酮纯化品。
可选地，上述在向总滤液中加入等体积的第一萃取剂进行萃取之前，先将总滤液的pH值调至4.5-5.0。
可选地，上述第一萃取剂包括第一乙酸乙酯和正庚烷；
优选地，第一乙酸乙酯和正庚烷的体积比为6-10:1，优选为7-9:1，更优选为7-8:1。
可选地，上述第一次超声溶解的超声时间为1-3min，优选为1-2min，更优选为1-1.5min。
可选地，上述第二次超声溶解的超声时间为10-20min，优选为12-18min，更优选为14-16min。
可选地，上述在第一酯相内提取总内酯纯化品包括：减压蒸馏第一酯相，回收溶剂，之后在浓缩液中加入第一乙醇蒸发至干，回收第一固体，冷冻干燥，得到总内酯纯化品。
可选地，上述在第一水相内提取总黄酮纯化品包括：调节第一水相的pH至7-8，用等体积的第二萃取剂萃取多次，弃除多次萃取后的第二水相，合并多次第二酯相，减压蒸馏浓缩回收溶剂，之后在浓缩液中加第二乙醇蒸发至干，回收第二固体，冷冻干燥，得到总黄酮纯化品。
可选地，上述第二萃取剂包括第二乙酸乙酯和正丁醇；
优选地，第二乙酸乙酯和正丁醇的体积比为4-8:1，优选为5-7:1；更优选为6:1。
本发明实施例的有益效果例如包括：
本实施例中提供的银杏提取物药用原料通过将从银杏叶粗提物中提取的总内酯纯化品和总黄酮纯化品进行复配，并且通过测定总内酯纯化品中第一总内酯和第一总黄酮的含量以及总黄酮纯化品中第二总内酯和第二总黄酮的含量，通过调节总内酯纯化品和总黄酮纯化品的复配比例，使得复配后形成的混合物中，第一总内酯和第二总内酯在混合物中的总含量与第一总黄酮和第二总黄酮在混合物中的总含量的比例在1-2:1-2这个范围内时，获得的银杏提取物药用原料的具有较佳的自由基清除能力和优良的抗血小板凝聚的活性。本实施例中，改变并优化常规的控制银杏提取物含总黄酮≥24％、总内酯≥6％这一国际药用标准，对银杏提取物的质量标准更加精细化，使生物活性更加优良。
本实施例提供的银杏提取物药用原料的制备方法通过提取总内酯纯化品和总黄酮纯化品，测定含量，通过计算合理进行配比，配置形成的混合物中第一总内酯和第二总内酯在混合物中的总含量与第一总黄酮和第二总黄酮在混合物中的总含量的比例在1-2:1-2这个范围内，获得的银杏提取物药用原料的具有较佳的自由基清除能力和优良的抗血小板凝聚的活性。并且该制备方法操作简单，容易实现，适合大批量生产。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本发明实施例的银杏提取物药用原料及其制备方法进行具体说明。
一种银杏提取物药用原料，其包括将从银杏叶粗提物中提取的总内酯纯化品和总黄酮纯化品复配形成的混合物，该总内酯纯化品包括第一总内酯和第一总黄酮，总黄酮纯化品包括第二总内酯和第二总黄酮，在混合物中，第一总内酯和第二总内酯在混合物中的总含量与第一总黄酮和第二总黄酮在混合物中的总含量的比例为1-2:1-2，优选为1-2:1，更优选为1:1。
也即是，本实施例中，通过复配银杏叶提取物中的两类成分，具体为总内酯纯化品和总黄酮纯化品，由于总内酯纯化品和总黄酮纯化品内均含有黄酮和内酯这两类组分，但其含量不同，其中，总内酯纯化品内含有的总内酯含量较多，总黄酮含量较少，而总黄酮纯化品内含有的总内酯含量较少，总黄酮含量较多。经发明人研究发现，单一的总内酯纯化品和总黄酮纯化品，由于其总黄酮和总内酯的含量比例欠佳，导致单一的总内酯纯化品和总黄酮纯化品效果欠佳，治疗结果不理想。
本实施例中，通过将不同含量的黄酮和内酯的总内酯纯化品和总黄酮纯化品进行复配， 通过测定总内酯纯化品中第一总内酯和第一总黄酮的含量，同时测定总黄酮纯化品中第二总内酯和第二总黄酮的含量，从而通过复配总内酯纯化品和总黄酮纯化品形成混合物，使得混合物中，第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为1-2:1-2，优选为1-2:1，更优选为1:1。经发明人研究发现，当复配形成的混合物中的总内酯(第一总内酯和第二总内酯之和)与总黄酮(第一总黄酮和第二总黄酮之和)的比例满足上述条件时，此时能够更好的发挥总内酯和总黄酮的药理活性，清除自由基和抗血小板凝聚效果好，同时还有利于减少临床给药计量，同时进一步降低杂质带来的不安全性和干扰。
进一步地，本实施例中，还对总内酯纯化品中的第一总内酯和第一总黄酮的含量进行了限定，同时对总黄酮纯化品中的第二总内酯和第二总黄酮的含量进行限定，进而保证了总黄酮纯化品和总内酯纯化品的优异性。
具体来说，总内酯纯化品中第一总内酯的含量为36％-46％、第一总黄酮含量为13％-19％，第一总内酯含量与第一总黄酮含量之和大于50％；总黄酮纯化品中第二总内酯含量为11％-14％，第二总黄酮含量为42％-47％，第二总内酯含量与第二总黄酮含量之和大于50％。
第一总内酯的含量可以为36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％中的任意一者或者任意两者之间的范围值；第一总黄酮的含量可以为13％、14％、15％、16％、17％、18％、19％中的任意一者或者任意两者之间的范围值。
第二总内酯的含量可以为11％、12％、13％、14％中的任意一者或者任意两者之间的范围值；第二总黄酮的含量可以为42％、43％、44％、45％、46％、47％中的任意一者或者任意两者之间的范围值。
经发明人研究，在总内酯纯化品和总黄酮纯化品满足上述含量要求时，总内酯纯化品和总黄酮纯化品质量佳，能够更好的发挥总内酯和总黄酮的药理活性，清除自由基和抗血小板凝聚效果好。
本实施例还提供了一种银杏提取物药用原料的制备方法，其具体包括一下步骤：
S1：银杏叶粗提物的预处理。
将银杏叶粗提物加水溶解，进行第一次超声溶解，接着离心分离，获得第一滤液和第一滤渣，向第一滤渣加入磷酸氢二钠进行第二次超声溶解，离心分离，获得第二滤液和第二滤渣，合并第一滤液和第二滤液形成总溶液，向总滤液中加入等体积的第一萃取剂进行萃取；分别取第一酯相和第一水相。
具体地，在向总滤液中加入等体积的第一萃取剂进行萃取之前，先将总滤液的pH值调至4.5-5.0。调节总滤液的pH值至4.5～5.0有利于总滤液的分散度更佳，萃取效果更好。
第一萃取剂包括第一乙酸乙酯和正庚烷；优选地，第一乙酸乙酯和正庚烷的体积比为6-10:1，优选为7-9:1，更优选为7-8:1。经发明人研究发现，选用特定的体积比的第一乙酸乙酯和正庚烷作为第一萃取剂，有利于提升萃取效果。
第一次超声溶解的超声时间为1-3min，优选为1-2min，更优选为1-1.5min。第二次超声溶解的超声时间为10-20min，优选为12-18min，更优选为14-16min。经发明人研究发现，经过两次不同时间的超声溶解，能够将银杏叶提取物粉的总黄酮和总内酯充分提取，提取效果更佳。
S2：从银杏叶中提取总内酯纯化品。
在第一酯相内提取总内酯纯化品：加热减压蒸馏第一酯相，回收溶剂，之后在浓缩液中加入第一乙醇蒸发至干，回收第一固体，冷冻干燥，得到总内酯纯化品。本实施例中，通过加热蒸馏第一酯相，能够将有机溶剂予以回收，利用第一乙醇与有机溶剂混溶容易将残留在浓缩液中的有机溶剂一并带出，进一步降低纯化品中的有机溶剂残留。
S3：从银杏叶中提取总黄酮纯化品。
在第一水相内提取总黄酮纯化品：先调节第一水相的pH值至7-8，然后用等体积的第二萃取剂萃取多次，弃除多次萃取后的第一水相，并且合并多次第二酯相，有利于提升总黄酮纯化品的收率，进一步地，通过减压蒸馏浓缩的操作回收溶剂。之后在浓缩液中加入第二乙醇蒸发至干，回收第二固体，冷冻干燥，得到总黄酮纯化品。本实施例中，利用第二萃取剂进行多次萃取，能够将第一水相内的总黄酮纯化品进一步提取，提取效果更好，同时利用减压蒸馏浓缩的操作回收溶剂，不仅仅能够实现溶剂的回收利用，同时还能在回收溶剂时进一步提升总黄酮纯化品的纯度，此外，在浓缩液中加入第二乙醇挥发至干，能够将残留在浓缩液中的有机溶剂一并带出，降低有机溶剂在总黄酮纯化品中的残留。
其中，第二萃取剂包括第二乙酸乙酯和正丁醇；优选地，第二乙酸乙酯和正丁醇的体积比为4-8:1，优选为5-7:1；更优选为6:1。本实施例中，选择第二乙酸乙酯和正丁醇的混合液作为第二萃取剂，能够有效萃取水相内的总黄酮纯化品，萃取效果佳。并且经发明人研究发现，第二乙酸乙酯和正丁醇的体积比在上述比例范围内时，其萃取效果更佳。
S4：测定总内酯纯化品中第一总内酯和第一总黄酮的含量。
测得总内酯纯化品中第一总内酯含量为36％—46％、第一总黄酮含量为13％—19％，第一总内酯含量与第一总黄酮含量之和大于50％。
S5：测定总黄酮纯化品中第二总内酯和第二总黄酮的含量。
测得总黄酮纯化品中的第二总内酯含量为11％—14％，第二总黄酮含量为42％—47％，第二总内酯含量与第二总黄酮含量之和大于50％。
S6：复配总内酯纯化品和总黄酮纯化品。
将总内酯纯化品和总黄酮纯化品复配形混合物并满足第一总内酯和第二总内酯在混合物中的总含量与第一总黄酮和第二总黄酮在混合物中的总含量的比例为1-2:1-2，优选为1-2:1，更优选为1:1。通过计算复配总内酯纯化品和总黄酮纯化品，使得最终产物满足上述含量比的要求。满足上述含量比的要求的银杏提取物药用原料具有较佳的自由基清除能力和优良的抗血小板凝聚的活性。
此外，本实施例中利用国际药用标准中关于黄酮测定和内酯测定的方法分别以本实施例中的总黄酮纯化品和总内酯纯化品作为样品，测定总黄酮纯化品和总内酯纯化品中各自的黄酮和内酯的含量。
1.黄酮测定
1.1样品前处理 取纯化品约35mg，加甲醇-25％盐酸(4:1，临用新配)混合溶液25mL，于80℃水浴回流30min，迅速冷却至室温，转移至50mL容量瓶中，用甲醇稀释至刻度，摇匀，过0.45微米滤膜备用。
1.2色谱分析 高效液相色谱仪为安捷伦公司1200型高效液相色谱，配阵列二极管检测器，条件流动相：甲醇:0.1％磷酸水溶液＝48:52，流速0.8mL/min，波长360nm，柱温30℃。
1.3测定结果 以槲皮素、山奈酚、异鼠李素为对照品按1.2方法进样分析，测得纯化品中黄酮含量。
2.内酯测定
2.1样品前处理 取纯化品约0.05g，加蒸馏水10mL，置水浴中温热溶解，加2％盐酸两滴，用乙酸乙酯振摇提取4次(15mL、10mL、10mL、10mL)，合并提取液，用5％乙酸钠溶液20mL洗涤，分取乙酸钠溶液，再用乙酸乙酯10mL洗涤。合并乙酸乙酯提取液及洗液，用水洗涤2次，每次20mL，分取水液，用乙酸乙酯10mL洗涤，合并乙酸乙酯液，将得到的萃取液减压浓缩至干，用甲醇转移至5mL容量瓶中，稀释至刻度，摇匀，过0.45微米滤膜备用。
2.2色谱分析 高效液相色谱仪为日本岛津公司LC-20AT型。色谱条件：流动相：甲醇:水＝75:25，流速0.8mL/min，柱温30℃。检测器：蒸发光散射检测器，漂移管温度105℃，氮气流速3mL/min。
2.3测定结果 以银杏内酯A、B、C和白果内酯为对照品按2.2方法进样分析，测得纯化品中4种内酯的总含量。
综上所述，本实施例中提供的银杏提取物药用原料通过将从银杏叶粗提物中提取的总内酯纯化品和总黄酮纯化品进行复配，并且通过测定总内酯纯化品中第一总内酯和第一总黄酮的含量以及总黄酮纯化品中第二总内酯和第二总黄酮的含量，通过调节总内酯纯化品 和总黄酮纯化品的复配比例，使得复配后形成的混合物中，第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例在1-2:1-2这个范围内时，获得的银杏提取物药用原料的具有较佳的自由基清除能力和优良的抗血小板凝聚的活性。
本实施例提供的银杏提取物药用原料的制备方法通过提取总内酯纯化品和总黄酮纯化品，测定含量，通过计算合理进行配比，配置形成的混合物中第一总内酯和第二总内酯在混合物中的总含量与第一总黄酮和第二总黄酮在混合物中的总含量的比例在1-2:1-2这个范围内，获得的银杏提取物药用原料的具有较佳的自由基清除能力和优良的抗血小板凝聚的活性。并且该制备方法操作简单，容易实现，适合大批量生产。
以下结合实施例对本发明的银杏提取物药用原料及其制备方法进一步进行阐述。
实施例1
本实施例提供了一种银杏提取物药用原料及其制备方法。
该银杏提取物药用原料是由总内酯纯化品和总黄酮纯化品复配而成的混合物，其中，总内酯纯化品内包括第一总内酯和第一总黄酮，总黄酮纯化品包括第二总内酯和第二总黄酮，在混合物中，第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为1:2。
该银杏提取物药用原料是按照以下方法制备而得：
称取银杏叶粗提物1g加水20ml，在60℃下进行第一次超声溶解1min，沉降式离心分离，获得第一滤液和第一滤渣，向第一滤渣中加入磷酸氢二钠溶液进行第二次超声溶解10min，离心分离，获得第二滤液和第二滤渣，合并第一滤液和第二滤液形成总滤液；向总滤液中加入等体积的第一乙酸乙酯和正庚烷作为第一萃取剂进行萃取，其中，第一乙酸乙酯和正庚烷的体积比为6:1，分别取第一酯相和第一水相。
加热减压蒸馏第一酯相，回收溶剂，之后在浓缩液中加入第一乙醇蒸发至干，回收第一固体，冷冻干燥，得到总内酯纯化品。
调节第一水相的pH至7，用等体积的第二乙酸乙酯和正丁醇作为第二萃取剂萃取多次，其中，第二乙酸乙酯和正丁醇的体积比为4:1，弃除多次萃取后的第一水相，合并多次第二酯相，减压蒸馏浓缩回收溶剂，之后在浓缩液中加第二乙醇蒸发至干，回收第二固体，冷冻干燥，得到总黄酮纯化品。
测定总内酯纯化品中第一总内酯和第一总黄酮的含量，总内酯纯化品中第一总内酯含量为36％、第一总黄酮含量为19％，第一总内酯含量与第一总黄酮含量之和为55％；
测定总黄酮纯化品中第二总内酯和第二总黄酮的含量，总黄酮纯化品中第二总内酯含量为11％，第二总黄酮含量为47％，第二总内酯含量与第二总黄酮含量之和为58％。
然后将总内酯纯化品和总黄酮纯化品复配形混合物并满足第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为1:2。
实施例2
本实施例提供了一种银杏提取物药用原料及其制备方法。
该银杏提取物药用原料是由总内酯纯化品和总黄酮纯化品复配而成的混合物，其中，总内酯纯化品内包括第一总内酯和第一总黄酮，总黄酮纯化品包括第二总内酯和第二总黄酮，在混合物中，第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为2:1。
该银杏提取物药用原料是按照以下方法制备而得：
将银杏叶粗提物1g加水25ml，在55℃下进行第一次超声溶解3min，离心分离，获得第一滤液和第一滤渣，向第一滤渣中加入磷酸氢二钠溶液进行第二次超声溶解10min，离心分离，获得第二滤液和第二滤渣，合并第一滤液和第二滤液形成总滤液；调节总滤液的pH值至5。接着向总滤液中加入等体积的第一乙酸乙酯和正庚烷作为第一萃取剂进行萃取，其中，第一乙酸乙酯和正庚烷的体积比为7:1，分别取第一酯相和第一水相。
减压蒸馏第一酯相，回收溶剂，之后在浓缩液中接着加入第一乙醇蒸发至干，回收第一固体，冷冻干燥，得到总内酯纯化品。
调节第一水相的pH至8，用等体积的第二乙酸乙酯和正丁醇作为第二萃取剂萃取多次，其中，第二乙酸乙酯和正丁醇的体积比为8:1，弃除多次萃取后的第一水相，合并多次第二酯相，减压蒸馏浓缩回收溶剂，之后在浓缩液中加第二乙醇挥发至干，回收第二固体，冷冻干燥，得到总黄酮纯化品。
测得总内酯纯化品中第一总内酯含量为40％、第一总黄酮含量为15％，第一总内酯含量与第一总黄酮含量之和为55％。
测得总黄酮纯化品中第二总内酯含量为13％，第二总黄酮含量为45％，第二总内酯含量与第二总黄酮含量之和为58％。
然后将总内酯纯化品和总黄酮纯化品复配形混合物并满足第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为2:1。
实施例3
本实施例提供了一种银杏提取物药用原料及其制备方法。
该银杏提取物药用原料是由总内酯纯化品和总黄酮纯化品复配而成的混合物，其中，总内酯纯化品内包括第一总内酯和第一总黄酮，总黄酮纯化品包括第二总内酯和第二总黄 酮，在混合物中，第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为1:1。
该银杏提取物药用原料是按照以下方法制备而得：
将银杏叶粗提物1g加水20ml，进行第一次超声溶解2min，离心分离，获得第一滤液和第一滤渣，向第一滤渣中加入磷酸氢二钠溶液进行第二次超声溶解15min，离心分离，获得第二滤液和第二滤渣，合并第一滤液和第二滤液形成总滤液；向总滤液中加入等体积的第一乙酸乙酯和正庚烷作为第一萃取剂进行萃取，其中，第一乙酸乙酯和正庚烷的体积比为8:1，分别取第一酯相和第一水相。
加热减压蒸馏第一酯相，回收溶剂，之后在浓缩液中加入第一乙醇蒸发至干，回收第一固体，冷冻干燥，得到总内酯纯化品。
调节第一水相的pH至7.5，用等体积的第二乙酸乙酯和正丁醇作为第二萃取剂萃取多次，其中，第二乙酸乙酯和正丁醇的体积比为6:1，弃除多次萃取后的第一水相，合并多次第二酯相，减压蒸馏浓缩回收溶剂，之后在浓缩液中加第二乙醇蒸发至干，回收第二固体，冷冻干燥，得到总黄酮纯化品。
实施例4
本实施例提供了一种银杏提取物药用原料及其制备方法。
该银杏提取物药用原料是由总内酯纯化品和总黄酮纯化品复配而成的混合物，其中，总内酯纯化品内包括第一总内酯和第一总黄酮，总黄酮纯化品包括第二总内酯和第二总黄酮，在混合物中，第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为1:1。
该银杏提取物药用原料是按照以下方法制备而得：
将银杏叶粗提物1g加水22ml，在57℃下进行第一次超声溶解3min，离心分离，获得第一滤液和第一滤渣，向第一滤渣中加入磷酸氢二钠溶液进行第二次超声溶解19min，离心分离，获得第二滤液和第二滤渣，合并第一滤液和第二滤液形成总滤液；向总滤液中加入等体积的第一乙酸乙酯和正庚烷作为第一萃取剂进行萃取，其中，第一乙酸乙酯和正庚烷的体积比为9:1，分别取第一酯相和第一水相。
加热减压蒸馏第一酯相，回收溶剂，之后在浓缩液中加入第一乙醇蒸发至干，回收第一固体，冷冻干燥，得到总内酯纯化品。
调节第一水相的pH至7.3，用等体积的第二乙酸乙酯和正丁醇作为第二萃取剂萃取多次，其中，第二乙酸乙酯和正丁醇的体积比为7:1，弃除多次萃取后的第一水相，合并多次第二酯相，减压蒸馏浓缩回收溶剂，之后在浓缩液中加第二乙醇蒸发至干，回收第 二固体，冷冻干燥，得到总黄酮纯化品。测得总内酯纯化品中第一总内酯含量为37％、第一总黄酮含量为14％，第一总内酯含量与第一总黄酮含量之和为51％。
测得总黄酮纯化品中第二总内酯含量为12％，第二总黄酮含量为46％，第二总内酯含量与第二总黄酮含量之和为58％。
然后将总内酯纯化品和总黄酮纯化品复配形混合物并满足第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为1:1。
实施例5
本实施例提供了一种银杏提取物药用原料及其制备方法。
该银杏提取物药用原料是由总内酯纯化品和总黄酮纯化品复配而成的混合物，其中，总内酯纯化品内包括第一总内酯和第一总黄酮，总黄酮纯化品包括第二总内酯和第二总黄酮，在混合物中，第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为1:1。
该银杏提取物药用原料是按照以下方法制备而得：
将银杏叶粗提物1g加水27ml，在54℃下进行第一次超声溶解3min，离心分离，获得第一滤液和第一滤渣，向第一滤渣中加入磷酸氢二钠溶液进行第二次超声溶解20min，离心分离，获得第二滤液和第二滤渣，合并第一滤液和第二滤液形成总滤液；向总滤液中加入等体积的第一乙酸乙酯和正庚烷作为第一萃取剂进行萃取，其中，第一乙酸乙酯和正庚烷的体积比为10:1，分别取第一酯相和第一水相。加热减压蒸馏第一酯相，回收溶剂，之后在浓缩液中加入第一乙醇蒸发至干，回收第一固体，冷冻干燥，得到总内酯纯化品。
调节第一水相的pH至8，用等体积的第二乙酸乙酯和正丁醇作为第二萃取剂萃取多次，其中，第二乙酸乙酯和正丁醇的体积比为8:1，弃除多次萃取后的第一水相，合并多次第二酯相，减压蒸馏浓缩回收溶剂，之后在浓缩液中加第二乙醇蒸发至干，回收第二固体，冷冻干燥，得到总黄酮纯化品。
测得总内酯纯化品中第一总内酯含量为45％、第一总黄酮含量为17％，第一总内酯含量与第一总黄酮含量之和为62％。
测得总黄酮纯化品中第二总内酯含量为11％，第二总黄酮含量为46％，第二总内酯含量与第二总黄酮含量之和为57％。
然后将总内酯纯化品和总黄酮纯化品复配形混合物并满足第一总内酯和第二总内酯在混合物中的含量与第一总黄酮和第二总黄酮在混合物中的含量的比例为1:1。
对比例
按照实施例2提供的银杏提取物药用原料及其制备方法对银杏提取物药用原料进行制备，在复配总内酯纯化品和总黄酮纯化品时，调节总内酯纯化品和总黄酮纯化品和量，从而使得对照例中的总内酯(第一总内酯和第二总内酯的含量之和)与总黄酮(第一总黄酮和第二总黄酮的含量之和)具有不同的比例。具体地，
试验例1中，总黄酮：总内酯＝2:1；
试验例2中，总黄酮：总内酯＝1:1；
试验例3中，总黄酮：总内酯＝1:2；
对比例1中，总黄酮：总内酯＝4:1；
对比例2中，总黄酮：总内酯＝3:1；
对比例3中，总黄酮：总内酯＝1:3；
对比例4中，总黄酮：总内酯＝1:4。
以清除氧自由基和抗血小板凝聚两个药效指标逐个进行实验，并设阴性和阳性对照组。从七组中筛选出兼备清除自由基活性和抗血小板凝聚活性最强组。
(一)清除氧自由基活性筛选：
采用已知强还原剂维生素C(抗坏血酸钠)作为阳性对照。试验设三个参比对照组，依达拉奉注射液
Figure PCTCN2017116018-appb-000001
银杏叶提取物注射液
Figure PCTCN2017116018-appb-000002
和银杏总内酯注射液
Figure PCTCN2017116018-appb-000003
Figure PCTCN2017116018-appb-000004
实验方法同供试品，比较供试品与参比对照品的清除DPPH自由基能力。采用EXCEL和ORIGIN 8.0进行数据处理和分析，并进行曲线拟合，求得EC50浓度。
结果：以总黄酮苷浓度为基准并换算出该比例所对应的总提取物供试品浓度，标记为：总黄酮苷浓度/总提取物供试品浓度。试验例1-3，对比例1-4清除DPPH自由基的EC 50浓度依次为138.99μg.mL -1/396.23μg.mL -1、89.64μg.mL -1/337.58μg.mL -1、67.11μg.mL -1/377.27μg.mL -1、186.75μg.mL -1/443.63μg.mL -1、167.11μg.mL -1/425.45μg.mL -1、50.02μg.mL -1/410.74μg.mL -1、42.53μg.mL -1/376.87μg.mL -1。参比对照品依达拉奉和金纳多(以总黄酮苷浓度/总提取物浓度计)的EC 50浓度分别为86.88μg.mL -1和157.10μg.mL -1/654.58μg.mL -1，参比对照品银杏总内酯注射液(金阁莱)原液(总内酯浓度为5000μg.mL -1)对DPPH的清除能力仅为30.08％，未得到EC 50浓度。
结论：在本试验条件下，试验例1-3，对比例1-4对DPPH自由基的清除的有效性成立。试验例1-3，对比例1-4对DPPH自由基清除能力均明显强于银杏总内酯注射液
Figure PCTCN2017116018-appb-000005
试验例1-3，对比例1-4对DPPH自由基清除EC 50总提取物供试品浓度都低于银杏叶提取物注射液
Figure PCTCN2017116018-appb-000006
试验例1-3，对比例1-4对DPPH自由基清除EC 50总提取物供试品浓度都高于依达拉奉
Figure PCTCN2017116018-appb-000007
结论表明：随着总黄酮：总内酯比例的改变，提取物清除自由基能力也随之改变，即 所需要的总提取物供试品浓度值不同。其实试验例1-3，对比例1-4以及依达拉奉注射液
Figure PCTCN2017116018-appb-000008
Figure PCTCN2017116018-appb-000009
明显优于银杏叶提取物注射液
Figure PCTCN2017116018-appb-000010
和银杏总内酯注射液
Figure PCTCN2017116018-appb-000011
其中尤以总黄酮：总内酯＝1:1组对DPPH自由基清除EC 50总提取物供试品浓度值在试验例1-3，对比例1-4中是最低的(是金纳多总提取物浓度的337.58/654.58≈1/1.94、是银杏总内酯注射液
Figure PCTCN2017116018-appb-000012
总浓度的337.58/5000≈1/14.8)，即该组清除自由基能力是最强的。
(二)抗血小板凝聚活性实验筛选：
以肝素钠作为阳性对照组，以银杏叶提取物注射液
Figure PCTCN2017116018-appb-000013
和银杏总内酯注射液
Figure PCTCN2017116018-appb-000014
作为参比对照品，与总提取物供试品进行比较。
方法：取检疫合格的KM小鼠66只，4～6周龄，雄雌各半，按性别随机分为11组(即阴性对照组、阳性对照组、供试品1组、供试品2组、供试品3组、供试品4组、供试品5组、供试品6组、供试品7组、参比对照品1组、参比对照品2组)，每组6只。采用单次尾静脉注射方式给药，供试品1～3组依次给予试验例1-3，供试品4-7组分别给予对比例1-4，参比对照品1组给予金纳多注射液，参比对照品2组给予银杏总内酯注射液
Figure PCTCN2017116018-appb-000015
Figure PCTCN2017116018-appb-000016
给药剂量均为2.45mg/kg，给药容积均为0.07mL/10g，阴性对照组给予同体积的生理盐水，阳性对照组给予同体积肝素钠注射液(给药浓度为1250单位/mL)。给药后10分钟，用折断的毛细管(约1.5cm长)对小鼠进行眼內眦取血，去掉第1滴血后，在载玻片上滴1滴血，立即用秒表计时，每隔30s用大头针自血滴边缘向中间轻挑1次，当有血丝挑起时停止计时，并记录时间，即为体外凝血时间。
试验结果以均数±标准差(x±s)表示，采用EXCEL软件进行统计学处理，各组与阴性对照组间的凝血时间差异采用配对T检验比较，检验水准P值为0.05。
结果：预试验及两次正式试验中，阳性对照品(肝素钠)组所有小鼠凝血时间均＞600s，统计学显著长于阴性对照组，认为本试验方法可靠。结果汇总请参阅表1：
表1.试验结果
Figure PCTCN2017116018-appb-000017
Figure PCTCN2017116018-appb-000018
备注：1：试验例1-3，对比例1-4及金纳多组给药剂量以银杏叶提取物计，银杏总内酯注射液
Figure PCTCN2017116018-appb-000019
组给药剂量以萜烯内酯计；2：肝素钠预试验给药剂量为12500单位/kg，正式试验剂量为8750单位/kg；*：P＜0.05；**：P＜0.01。
结论：在本试验条件下，系列总提取物供试品(试验例1-3，对比例1-4)及参比对照品延长小鼠凝血时间的有效性成立。与阴性对照品相比，试验例1-3，对比例1-4及参比对照品在近似金纳多临床剂量下均可延长小鼠凝血时间，且试验例1-3及银杏总内酯注射液
Figure PCTCN2017116018-appb-000020
效果强于金纳多。试验例1-3，对比例1-4中，试验例1-3效果较优，尤其是试验例2(即总黄酮：总内酯＝1：1)效果最优。
接下来，对试验例1-3，对比例1-4中、金纳多、金阁莱品注射液和依达拉奉进行药效学筛选试验，结果请参阅表2：
表2.药效学筛选试验结果
Figure PCTCN2017116018-appb-000021
Figure PCTCN2017116018-appb-000022
自由基清除能力：在达到同样的对清除DPPH自由基的EC50浓度前提下，试验例1-3，对比例1-4中所需要的总提取物供试品浓度是不同的，浓度越小则清除能力越强。清除自由基实验结果显示试验例1-3，对比例1-4的效果明显优于金纳多和银杏总内酯注射液
Figure PCTCN2017116018-appb-000023
Figure PCTCN2017116018-appb-000024
抗血小板凝聚活性：在相同的给药浓度条件下，试验例1-3、对比例1-4及金纳多、银杏总内酯注射液
Figure PCTCN2017116018-appb-000025
谁的体外凝血时间越长则抗凝活性越强。从预试验结果、第一次试验结果、第二次实验结果都显示出1:1的抗凝聚活性最强。综合两项药效试验结果分析认定：黄酮：内酯＝1:1组是兼备清除自由基和抗血小板凝聚最佳的比例配比。同时优于金纳多和银杏总内酯注射液
Figure PCTCN2017116018-appb-000026
以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
工业实用性
获得的银杏提取物药用原料的具有较佳的自由基清除能力和优良的抗血小板凝聚的活性。通过银杏提取物药用原料的制备方法而获得的银杏提取物药用原料的具有较佳的自由基清除能力和优良的抗血小板凝聚的活性。并且该制备方法操作简单，容易实现，适合大 批量生产。

Claims (11)

  1. 一种银杏提取物药用原料，其特征在于，其包括将从银杏叶粗提物中提取的总内酯纯化品和总黄酮纯化品复配形成的混合物，所述总内酯纯化品内包括第一总内酯和第一总黄酮，所述总黄酮纯化品包括第二总内酯和第二总黄酮，在所述混合物中，所述第一总内酯和所述第二总内酯在所述混合物中的含量与所述第一总黄酮和所述第二总黄酮在所述混合物中的含量的比例为1-2:1-2，优选为1-2:1，更优选为1:1。
  2. 根据权利要求1所述的银杏提取物药用原料，其特征在于，所述总内酯纯化品中所述第一总内酯含量为36％-46％、所述第一总黄酮含量为13％-19％，所述第一总内酯含量与所述第一总黄酮含量之和大于50％；所述总黄酮纯化品中所述第二总内酯含量为11％-14％，所述第二总黄酮含量为42％-47％，所述第二总内酯含量与所述第二总黄酮含量之和大于50％。
  3. 一种银杏提取物药用原料的制备方法，其特征在于，从银杏叶粗提物中提取总内酯纯化品和总黄酮纯化品，测定所述总内酯纯化品中第一总内酯和第一总黄酮的含量，测定总黄酮纯化品中第二总内酯和第二总黄酮的含量，然后将所述总内酯纯化品和所述总黄酮纯化品复配形混合物并满足所述第一总内酯和所述第二总内酯在所述混合物中的含量与所述第一总黄酮和所述第二总黄酮在所述混合物中的含量的比例为1-2:1-2，优选为1-2:1，更优选为1:1。
  4. 根据权利要求3所述的银杏提取物药用原料的制备方法，其特征在于，从银杏叶中提取总内酯纯化品和总黄酮纯化品包括：将银杏叶粗提物粉加水进行第一次超声溶解，离心分离，获得第一滤液和第一滤渣，向所述第一滤渣中加入磷酸氢二钠溶液进行第二次超声溶解，离心分离，获得第二滤液和第二滤渣，合并所述第一滤液和所述第二滤液形成总滤液；向所述总滤液中加入等体积的第一萃取剂进行萃取；分别取第一酯相和第一水相，在所述第一酯相内提取所述总内酯纯化品，在所述第一水相内提取所述总黄酮纯化品。
  5. 根据权利要求4所述的银杏提取物药用原料的制备方法，其特征在于，在向所述总滤液中加入等体积的所述第一萃取剂进行萃取之前，先将所述总滤液的pH值调至4.5-5.0。
  6. 根据权利要求4所述的银杏提取物药用原料的制备方法，其特征在于，所述第一萃取剂包括第一乙酸乙酯和正庚烷；
    优选地，所述第一乙酸乙酯和所述正庚烷的体积比为6-10:1，优选为7-9:1，更优选为7-8:1。
  7. 根据权利要求4所述的银杏提取物药用原料的制备方法，其特征在于，所述第一 次超声溶解的超声时间为1-3min，优选为1-2min，更优选为1-1.5min。
  8. 根据权利要求4所述的银杏提取物药用原料的制备方法，其特征在于，所述第二次超声溶解的超声时间为10-20min，优选为12-18min，更优选为14-16min。
  9. 根据权利要求4-8任一项所述的银杏提取物药用原料的制备方法，其特征在于，在所述第一酯相内提取所述总内酯纯化品包括：加热减压蒸馏所述第一酯相，回收溶剂，之后在浓缩液中加入第一乙醇蒸发至干，回收第一固体，冷冻干燥，得到所述总内酯纯化品。
  10. 根据权利要求4-8任一项所述的银杏提取物药用原料的制备方法，其特征在于，在所述第一水相内提取所述总黄酮纯化品包括：调节所述第一水相的pH至7-8，用等体积的第二萃取剂萃取多次，弃除多次萃取后的所述第一水相，合并多次第二酯相，减压蒸馏浓缩回收溶剂，之后在浓缩液中加第二乙醇蒸发至干，回收第二固体，冷冻干燥，得到所述总黄酮纯化品。
  11. 根据权利要求10所述的银杏提取物药用原料的制备方法，其特征在于，所述第二萃取剂包括第二乙酸乙酯和正丁醇；
    优选地，所述第二乙酸乙酯和所述正丁醇的体积比为4-8:1，优选为5-7:1；更优选为6:1。
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CN115594654A (zh) * 2022-10-28 2023-01-13 河北荃美生物科技有限公司（Cn） 7-去甲基银杏双黄酮和银杏酮酯的提取方法及其应用

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