WO2019128498A1 - 一种银杏二萜内酯组合物 - Google Patents

一种银杏二萜内酯组合物 Download PDF

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WO2019128498A1
WO2019128498A1 PCT/CN2018/115166 CN2018115166W WO2019128498A1 WO 2019128498 A1 WO2019128498 A1 WO 2019128498A1 CN 2018115166 W CN2018115166 W CN 2018115166W WO 2019128498 A1 WO2019128498 A1 WO 2019128498A1
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composition
ginkgolides
ginkgo
diterpene lactone
lactone
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PCT/CN2018/115166
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English (en)
French (fr)
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萧伟
常秀娟
周恩丽
康小东
王永香
胡晗绯
吴云
王振中
章晨峰
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江苏康缘药业股份有限公司
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Priority to JP2020536623A priority Critical patent/JP7090164B2/ja
Priority to KR1020207019560A priority patent/KR102515713B1/ko
Priority to EP18897379.6A priority patent/EP3708160A4/en
Priority to US16/771,677 priority patent/US11564904B2/en
Publication of WO2019128498A1 publication Critical patent/WO2019128498A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the invention belongs to the field of medical technology, in particular to a ginkgo diterpene lactone composition and application thereof.
  • Ginkgo biloba leaves have a wide range of biological activities, including a variety of chemical components, including flavonoids, terpenoids, polysaccharides, phenols, organic acids, alkaloids, amino acids, steroids, trace elements.
  • flavonoids include flavonoids, terpenoids, polysaccharides, phenols, organic acids, alkaloids, amino acids, steroids, trace elements.
  • vitamin C, vitamin E, carotene and calcium, phosphorus, boron, selenium and other mineral elements are also very rich, and its most important medicinal value components are flavonoids and anthraquinones. Flavonoids and anthraquinones have many functions such as vasodilation and anti-oxidation.
  • Ginkgolide is one of the main active ingredients in Ginkgo biloba leaves. It is a rare natural compound that has not been found in other plants. It has anti-oxidation, anti-inflammatory, anti-platelet aggregation, anti-apoptosis and death, and expansion. Pharmacological effects such as blood vessels, protection of central nervous system and ischemic tissue. Ginkgolide has a strong specific inhibitory activity on receptors and is a specific platelet activating factor (PAF) antagonist, which inhibits PAF-induced platelet aggregation and thromboxane increase in healthy people, and its anti-inflammatory activity is closely related to its chemical structure. Related. PAF is a soluble phospholipid substance that can cause platelet aggregation.
  • PAF platelet activating factor
  • Ginkgo biloba lactone is currently considered to be the most natural PAF receptor antagonist in clinical application.
  • Ginkgo biloba lactone mainly includes ginkgolides A (GA), ginkgolides B (GB), ginkgolides C (GC), ginkgolides L (GL), ginkgolides M belonging to the diterpene lactones.
  • Ginkgolide K is a novel monomer whose structure is similar to that of ginkgolides B.
  • CN1424031A reports a ginkgolide preparation in which the content of ginkgolides A, B and K is 30-40% of ginkgolides A, 50-55% of ginkgolides B, and 0.5-5% of ginkgolides K. This patent does not conduct an in-depth study of the proportional relationship between various diterpene lactones. .
  • the present invention has been further studied on the basis of the prior art, and the weight percentage of ginkgolide A, B, and K which is superior in ratio is obtained by using an ischemic stroke disease model and an evaluation index for inhibiting platelet aggregation.
  • the inventors were surprised to find that the ratio between the ginkgolide B and K is within a certain range, and the ginkgo diterpene lactone composition has better anti-stroke effect.
  • the present invention relates to a ginkgo diterpene lactone composition, characterized in that the composition comprises: 32-36% by weight of ginkgolides A, 55-60% of ginkgolides B, 2.2-3.6 % ginkgolides K, the weight ratio of the ginkgolides B to ginkgolides K is 18-22:1.
  • ginkgolides A Preferably, 33.5-35.2% of ginkgolides A, 56.1-60.1% of ginkgolides B, 2.6-3.0% of ginkgolides K, the weight ratio of ginkgolides B and ginkgolides K is 20.0- 21.5:1.
  • the content of the ginkgolides A is 32-34%, and/or the content of the ginkgolides B is 56-58%, and/or the content of the ginkgolides K is 2.4-3.4%. .
  • the ratio of ginkgolides B/K is from 19 to 21:1, more preferably 20:1.
  • the composition may be in the form of an extract or a form of a compound
  • the present invention also provides a ginkgo lactone formulation comprising the above composition (including an extract), characterized in that the formulation further comprises a pharmaceutically acceptable adjuvant.
  • the preparation is a ginkgo bilobalide injection.
  • the injection is a 1 ml or 5 ml or 10 ml dose containing 5 ⁇ 0.5 mg or 25 ⁇ 0.5 mg or 50 ⁇ 0.5 mg of the ginkgo diterpene lactone composition.
  • the invention also proposes the use of the above composition in the preparation of a medicament for preventing and treating stroke.
  • the present invention also provides the use of the above composition for the preparation of a medicament for inhibiting platelet aggregation.
  • the invention also proposes the use of the above composition for the preparation of a neuroprotective drug.
  • the "application” refers to a subject in which the above composition (including an extract) is administered to a subject having a corresponding disease or a disease for the purpose of imparting a therapeutic effect, such as curing, alleviating, altering, influencing, ameliorating or preventing the above.
  • a therapeutic effect such as curing, alleviating, altering, influencing, ameliorating or preventing the above.
  • the disease, its symptoms, or its propensity One skilled in the art will be able to readily determine a particular effective dosage depending on the type of disease being treated, the route of administration, and the use of excipients, which may vary depending on the concurrent use of other drugs.
  • the invention adopts a rat model of focal cerebral ischemia-reperfusion injury and finds that the ginkgo diterpene lactone composition can reduce the neurological function score, reduce the area of cerebral ischemia and reduce the brain tissue edema after cerebral ischemia reperfusion.
  • the water content, increasing the SOD activity reduced the MDA content, and found that the effect of the ginkgo diterpene lactone monomer at a certain ratio was significantly improved.
  • the present invention also demonstrates that the ginkgo diterpene lactone composition inhibits platelet aggregation induced by PAF and ADP inducers at different time points and reduces the maximum aggregation rate. Similarly, the ginkgo diterpene lactone monomer has a certain ratio. Better antithrombotic effects.
  • the present invention is directed to providing a ginkgo diterpene lactone composition.
  • the details will be specifically described below in conjunction with the contents of the embodiments.
  • Example 1 Protective effect of ginkgo diterpene lactone composition on ischemic brain disease model
  • 1.1 Ginkgo biloba lactone composition can be prepared by the following methods: taking ginkgo biloba leaves by ethanol extraction, concentration, adsorbent removal, organic solvent elution, washing purification, recrystallization, etc., and extraction parameters, adsorbents Selection, eluent selection, washing solution selection, and recrystallization solvent selection for adjustment; or as prepared by the method disclosed in CN1424031A, and prepared by adjusting the preparation parameters; the composition may also be compounded by compound And get it.
  • the ginkgo diterpene lactone composition of Table 1 ratio was obtained in the above manner.
  • composition or extract with different weight percentages was obtained according to the above methods, and the percentage content of the ginkgo diterpene lactones A, B, K was determined as follows:
  • Nimodipine produced by Shandong Xinhua Pharmaceutical Co., Ltd., batch number 1609215; 2,3,5-triphenyltetrazolium chloride, produced by sigma; superoxide dismutase (SOD), malondialdehyde (MDA)
  • SOD superoxide dismutase
  • MDA malondialdehyde
  • TSQ-280 constant temperature culture oscillator (Shanghai Jinghong Experimental Equipment Co., Ltd.), 5804R type refrigerated centrifuge (Eppendorf), BD224S type electronic balance (Sedolis Scientific Instrument Co., Ltd.).
  • Rats with qualified weight were randomly divided into 14 groups, namely sham operation group, model group, nimodipine group (10.8 mg/kg) and ginkgo diterpene lactone composition group (Table 1) 2.4 g ⁇ kg -1 vein. Injection administration. All animals were given the corresponding drugs at 1 d before the model establishment and 30 min after the reperfusion (the sham operation group and the model group were injected with the same volume of normal saline at the same time limit).
  • the middle cerebral artery occlusion MCAO model was prepared by internal carotid artery suture method.
  • the right external carotid artery was isolated, ligated and severed.
  • the stump of the external carotid artery was slowly inserted into the head along the common carotid artery and internal carotid artery.
  • the enlarged nylon fish line is about 18mm, which blocks the ischemia of the middle cerebral artery, and is reperfused for 3 hours after ischemia. After 24 hours of reperfusion, the brain is decapitated.
  • the sham operation group only separated the common carotid artery, the external carotid artery, and the internal carotid artery but did not insert the wire.
  • the neurological function score was based on the Longa score. The postoperative evaluation of 1 to 2 points was the success of the operation; 0 points: no obvious neurological symptoms; 1 point: the left forelimb could not be fully extended; 2 points: rotated to the left; 3 points: dump to the left while walking; 4 points: can not walk on their own.
  • the ginkgo diterpene lactone composition group and the nimodipine group can significantly reduce the neurobehavioral score of rats (P ⁇ 0.05, P ⁇ 0.01), significantly improving the rats.
  • the behavioral disorder after ischemia-reperfusion significantly reduced the infarct size (P ⁇ 0.05, P ⁇ 0.01).
  • the results showed that the ginkgo diterpene lactone composition can reduce the nerve damage and reduce the cerebral infarction rate in rats, as shown in Table 2.
  • the water content of brain tissue in the model group was significantly increased, which was significantly different from that in the sham operation group (p ⁇ 0.01).
  • the nimodipine and ginkgo diterpene lactone composition groups were significantly different from the model group (P ⁇ 0.05, P ⁇ 0.01). ), indicating that the ginkgo diterpene lactone composition can significantly reduce the water content after brain tissue edema, see Table 2.
  • the MDA content of the model group increased (p ⁇ 0.01) and the SOD activity decreased (p ⁇ 0.01).
  • the SOD activity of the rats in the nimodipine group increased, and the MDA content decreased significantly, and the difference was significant ( p ⁇ 0.01)
  • the SOD activity of the ginkgo biloba lactone composition group was significantly increased (P ⁇ 0.05, P ⁇ 0.01)
  • the MDA content was significantly decreased (P ⁇ 0.05, P ⁇ 0.01), as shown in Table 2.
  • Example 1-3 group of ginkgo diterpene lactone composition the content of ginkgolide A is in the range of 32-36%, the content of ginkgolide B is in the range of 55-60%, and the content of ginkgolide K is in Within the range of 2.2-3.6%, and the weight ratio of ginkgolides B to ginkgolides K is in the range of 18-22:1.
  • the Ginkgo biloba lactone compositions of Examples 1-3 reduced the neurological function score, reduced the area of cerebral ischemia, and decreased the cerebral ischemia-reperfusion rats.
  • the water content after brain tissue edema increase SOD activity and reduce the protective effect of MDA on focal cerebral ischemia-reperfusion injury in rats, which has better effect on focal cerebral ischemia-reperfusion injury in rats. Good protection.
  • the invention also provides a preparation method of an extract capable of obtaining a relatively stable component ratio: specifically, a ginkgo leaf (dry leaf of Ginkgo biloba Ginkgo biloba L., purchased from Wuzhou, Jiangsu province) is 50 kg, plus 8 times the amount of 10% ethanol was heated and refluxed for 2 times for 1.5 hours, and the extracts were combined, concentrated, and filtered to obtain a concentrate. To the concentrate, a polyamide having a weight of 15% of Ginkgo biloba leaves was added, and the mixture was stirred for 36 hours, left to stand overnight, and the supernatant was discarded to collect the adsorbent.
  • a ginkgo leaf dry leaf of Ginkgo biloba Ginkgo biloba L., purchased from Wuzhou, Jiangsu province
  • a polyamide having a weight of 15% of Ginkgo biloba leaves was added, and the mixture was stirred for 36 hours, left to stand overnight, and the supernatant was discarded to collect the adsorb
  • the crude ginkgo total lactone was washed 3 times with 1 time (weight ratio) of water, the water solution was discarded, and the precipitate was added to 3 times (weight ratio) ethanol and repeatedly recrystallized 3 times, and dried to obtain a ginkgo diterpene lactone raw material.
  • the ginkgo diterpene lactone A content was 35%
  • the ginkgo diterpene lactone B was 58%
  • the ginkgo diterpene lactone K was 3.1%.
  • the content of ginkgo dibenzo lactone A, ginkgo diterpene lactone B, and ginkgo diterpene lactone K is small, all within 5%.
  • the specific proportion of the drug group is the same as in Table 1.
  • Nimodipine produced by Shandong Xinhua Pharmaceutical Co., Ltd., batch number 1609215; adenosine diphosphate (ADP), Shanghai Boao Biotechnology Co., Ltd. (imported packaging); platelet activating factor (PAF), Provided by sigma company; trisodium citrate, Shanghai Lingfeng Chemical Reagent Co., Ltd.
  • New Zealand white rabbit provided by Lefu Farm in Pukou District, Nanjing, laboratory animal production license SCXK (Su) 2014-0004, license: SYXK (Su) 2013-0021.
  • Rabbits underwent local anesthesia with procaine, carotid intubation and bloodletting, anticoagulation with 3.8% sodium citrate solution, centrifugation at 1000 r/min for 10 min, platelet-rich plasma (PRP), and the remainder was centrifuged at 3000 r/min. That is, platelet-poor plasma (PPP) was taken, and the aggregation inducing agent was ADP (final concentration 5.4 ⁇ g/ml) and PAF (final concentration 0.37 ⁇ g/ml), respectively.
  • PPP platelet-poor plasma
  • Aggregation inhibition rate (%) (maximum aggregation rate of blank control group - maximum aggregation rate of administration group) / maximum aggregation rate of blank control group ⁇ 100%.
  • Example 1-3 group of ginkgo diterpene lactone composition the content of ginkgolide A is in the range of 32-36%, the content of ginkgolide B is in the range of 55-60%, and the content of ginkgolide K is in Within the range of 2.2-3.6%, and the weight ratio of ginkgolides B to ginkgolides K is in the range of 18-22:1.
  • the Ginkgo biloba lactone compositions of Examples 1-3 inhibited platelet aggregation of rabbits induced by PAF and ADP inducers at different time points, and decreased the maximum. The aggregation rate achieved better results, indicating that it has a better antithrombotic effect, which is closely related to its efficient and extensive inhibition of platelet aggregation.

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Abstract

本发明涉及一种银杏二萜内酯组合物，该提取物可降低脑缺血再灌注大鼠神经功能评分，减少脑缺血面积，降低脑组织水肿后的含水量，提高SOD活性降低MDA含量，且该组合物能抑制不同时间点由PAF和ADP诱导剂诱导的家兔血小板聚集，降低最大聚集率，此外，通过实验表明，银杏二萜内酯组合物中各单体化合物在一定比例下其效果显著提高。

Description

一种银杏二萜内酯组合物 技术领域
本发明属于涉及医药技术领域，特别是涉及一种固定组分的银杏二萜内酯组合物及应用。
背景技术
银杏叶有广泛生物活性，含有多种化学成分，主要包括黄酮类、萜类、多糖类、酚类、有机酸、生物碱、氨基酸、甾体化合物、微量元素等。其中，维生素C、维生素E、胡萝卜素以及钙、磷、硼、硒等矿物元素含量也十分丰富，其最主要药用价值成分是黄酮类和萜类。黄酮类和萜类具有扩血管、抗氧化等多方面的作用。
银杏内酯作为银杏叶中主要的活性成分之一，是一类罕见的天然化合物，迄今尚未发现存在于其他植物中，具有抗氧化、抗炎、抗血小板聚集、抗细胞凋亡及死亡、扩张血管、保护中枢神经和缺血组织等药理作用。银杏内酯对受体有强大的特异性抑制活性，是特异的血小板活化因子(PAF)拈抗剂，能抑制PAF引起健康人的血小板聚集和血栓素增加，其拈抗作用活性与化学结构密切相关。PAF是一种可引起血小板聚集的可溶性磷脂类物质，同时也是目前为止发现的最强的脂质介质之一，在多种病理、生理过程中有关键作用。PAF发挥其生物学作用主要是通过PAF受体，PAF受体拈抗剂主要通过抑制PAF受体，拈抗PAF受体的生物学效应。银杏二萜内酯是目前被认为最具有临床应用前景的天然PAF受体拈抗剂。银杏二萜内酯主要包括属于二萜内酯类的银杏内酯A(GA)、银杏内酯B(GB)、银杏内酯C(GC)、银杏内酯L(GL)、银杏内酯M(GM)、银杏内酯J(GJ)、银杏内酯K(GK)和属于倍半萜的白果内酯，其中银杏内酯B对PAF具有较强的拈抗作用。银杏内酯K为一种新单体，其结构与银杏内酯B相似。
CN1424031A报道了一种银杏内酯制剂，银杏内酯A、B、K的含量为银杏内酯A 30-40％，银杏内酯B 50-65％，银杏内酯K 0.5-5％。该专利并未对各种二萜内酯之间的比例关系进行深入的研究。。
发明内容
本发明在现有技术的基础上进行更深入地研究，利用缺血性脑中风疾病模型以及抑制血小板聚集等评价指标，获得了配比更优的银杏内酯A、B、K的重量百分比。在此基础上，发明人惊讶地发现，银杏内酯B和K之间的比例在一定范围内，银杏二萜内酯组合物的抗脑卒中效果更优。
本发明涉及一种银杏二萜内酯组合物，其特征在于，所述组合物包含：以重量计，32-36％的银杏内酯A、55-60％的银杏内酯B、2.2-3.6％的银杏内酯K，所述银杏内酯B和银杏内酯K的重量比为18-22∶1。
优选地，33.5-35.2％的银杏内酯A、56.1-60.1％的银杏内酯B、2.6-3.0％的银杏内酯K，所述银杏内酯B和银杏内酯K的重量比为20.0-21.5∶1。
优选地，所述银杏内酯A的含量为32-34％，和/或所述银杏内酯B的含量为56-58％，和/或所述银杏内酯K的含量为2.4-3.4％。
优选地，银杏内酯B/K的比例为19-21∶1，更优选为20∶1。
优选地，所述组合物可为提取物形式或复配物形式；
本发明还提出一种包含上述组合物(包括提取物)的银杏二萜内酯制剂，其特征在于，该制剂还包含药学上可接受的辅料。
进一步地，所述制剂为银杏二萜内酯注射剂。
具体地，该注射剂为1ml或5ml或10ml剂量，其含有5±0.5mg或25±0.5mg或50±0.5mg所述银杏二萜内酯组合物。
进一步地，该注射剂还包含葡甲胺和氯化钠，各组分的重量比为：银杏二萜内酯组合物∶葡甲胺∶氯化钠＝(2-8)∶(2∶-8)∶(4-12)。
本发明还提出了上述组合物在制备防治脑卒中药物中的应用。
本发明还提出了上述的组合物在制备抑制血小板聚集药物中的应用。
本发明还提出了上述的组合物在制备神经保护药物中的应用。
所述“应用”是指将上述组合物(包括提取物)给予具有相应疾病或该疾病的倾向的受试者，其目的为赋予治疗效果，例如治愈、缓解、改变、影响、改善或预防上述疾病、其症状或其倾向。本领域技术人员，能根据所治疗的疾病的类型、给药途径、赋形剂的使用容易确定具体的有效剂量，该剂量可能会因为同时使用其它药物而有所差异。
本发明通过大鼠局灶性脑缺血再灌注损伤实验，发现银杏二萜内酯组合物均可降低脑缺血再灌注大鼠神经功能评分，减少脑缺血面积，降低脑组织水肿后的含水量，提高SOD活性降低MDA含量，并且发现银杏二萜内酯单体在一定比例下的效果显著提高。本发明还证实了银杏二萜内酯组合物可抑制不同时间点由PAF和ADP诱导剂诱导的家兔血小板聚集，降低最大聚集率，同样地，银杏二萜内酯单体在一定比例下有更好的抗血栓形成作用。
具体实施方式
如前所述，本发明旨在提供一种银杏二萜内酯组合物。以下将结合实施例的内容进行具体描述。
特别需要指出的是，针对本发明所做出的类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
需要注意的是，如未注明具体条件者，均按照常规条件或制造商建议的条件进行，所用原料药或辅料，以及所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。除非另外说明，否则所有的百分数、比率、比例或份数按重量计。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用与本发明。
实施例1银杏二萜内酯组合物对缺血性脑疾病模型的保护作用
1.材料
1.1银杏二萜内酯组合物可以通过以下方法制备：取银杏叶经乙醇提取，浓缩，吸附剂除杂，有机溶剂洗脱，洗涤纯化，重结晶等步骤，并对其中的提取参数、吸附剂选择、洗脱剂选择、洗涤溶液选择及重结晶溶剂选择进行调整而获得；或如CN1424031A所公开的方法进行制备，并通过调整制备参数来进行制备；所述组合物也可以通过化合物的复配而得到。通过上述方式获得表1配比的银杏二萜内酯组合物。
按以上各方法得到重量百分比不同的组合物或提取物，并对其银杏二萜内酯A、B、K的百分比含量进行测定，如下：
表1银杏二萜内酯组合物中各组分配比
Figure PCTCN2018115166-appb-000001
尼莫地平，山东新华制药股份有限公司生产，批号1609215；2，3，5- 氯化三苯基四氮唑，sigma公司生产；超氧化物岐化酶(SOD)、丙二醛(MDA)试剂盒均购自南京建成生物工程研究所。
1.2动物
SD大鼠150只，雄性，体重220～250g，清洁级，上海西普而-必凯实验动物有限公司，合格证号：SCXK(沪)2013-0016。
1.3仪器
TSQ-280型恒温培养振荡器(上海精宏实验设备有限公司)，5804R型冷冻离心机(Eppendorf公司)，BD224S型电子天平(赛多利斯科学仪器有限公司)。
2.方法
2.1分组及模型制作
选取体重合格大鼠随机分为14组，即假手术组、模型组、尼莫地平组(10.8mg/kg)、银杏二萜内酯组合物组(如表1)2.4g·kg -1静脉注射给药。所有动物于造模前1d及再灌注后30min尾静脉注射给予相应药物(假手术组和模型组于相同时限注射等容量生理盐水)。
根据Zea Longa等方法，采用颈内动脉线栓法制备大鼠右侧大脑中动脉栓塞(middle cerebral artery occlusion MCAO)模型。主要步骤：10％水合氯醛(350mg·kg -1)腹腔注射麻醉大鼠，分离、结扎并切断右侧颈外动脉，由颈外动脉残端沿颈总动脉和颈内动脉缓慢插入头端膨大的尼龙鱼线约18mm，阻塞大脑中动脉入口造成缺血，缺血3h再灌注，再灌24h后断头取脑。假手术组仅分离颈总动脉、颈外动脉和颈内动脉但不插线。
2.2检测指标
2.2.1神经功能评分参照Longa评分标准，术后评1～2分者为手术成功标志；0分：无明显神经病学症状；1分：不能完全伸展左前肢；2分：向左侧旋转；3分：行走时向左侧倾倒；4分：不能自行行走。
2.2.2脑组织含水量大鼠再灌24h后断头取脑，分别测定其湿重及在电热恒温干燥箱中100℃干燥48h至恒重后的干重，以计算含水量。含水量(％)＝(湿重-干重)/湿重×100％。
2.2.3脑梗死面积测定再灌24h后断头取脑，将大脑作连续2mm冠状切片，共5片，将脑片放入2％TTC磷酸盐缓冲液中，37℃恒温孵育30min，正常脑组织染为红色，梗死灶呈白色。滤纸吸去表面液体后用数码相机拍照，通过软件计算每片大脑两面的缺血面积取其平均值。
2.2.4血清活力、MDA含量的检测再灌24h后取血分离血清，按说明书操作测定OD值，计算SOD、MDA含量。
2.3统计学处理
实验数据以
Figure PCTCN2018115166-appb-000002
表示，SPSS 17.0统计软件进行分析，多组间均数比较采用单因素方差分析，两组间样本均数比较采用q检验。P＜0.05为有统计学意义.
3.结果
3.1神经功能评分
脑缺血再灌注后，与模型组比较，银杏二萜内酯组合物组和尼莫地平组均能明显降低大鼠的神经行为学评分(P＜0.05，P＜0.01)，显著改善大鼠缺血再灌注后的行为障碍，明显减少梗死面积(P＜0.05，P＜0.01)，结果表明，银杏二萜内酯组合物可降低大鼠神经损伤，降低脑梗死率，见表2。
3.2脑组织含水量
模型组脑组织含水量明显增加，与假手术组比较有显著差异(p＜0.01)，尼莫地平、银杏二萜内酯组合物组与模型组比较有显著差异(P＜0.05，P＜0.01)，表明银杏二萜内酯组合物能显著降低脑组织水肿后的含水量，见表2。
3.3脑梗死面积
与假手术相比，模型组MDA含量增加(p＜0.01)，SOD活性减弱(p＜0.01)；与模型组比较，尼莫地平组大鼠的SOD活性增强，MDA含量显著降低，差异显著(p＜0.01)，银杏二萜内酯组合物组大鼠的SOD活性显著提高(P＜0.05，P＜0.01)，MDA的含量明显减少(P＜0.05，P＜0.01)，见表2。
表2银杏二萜内酯组合物对大鼠神经功能评分、脑组织含水量、SOD活力及MDA的影响(
Figure PCTCN2018115166-appb-000003
n＝10)
Figure PCTCN2018115166-appb-000004
与假手术组比较：*p＜0.05，**p＜0.01；与实施例1比较： ▲p＜0.05， ▲▲p＜0.01。
4结论
由表2可以看出，实施例2-3组与实施例1组比较，无显著性差异，即，实施例1组与对比例1-8组相比有显著性差异。
实施例1-3组银杏二萜内酯组合物，银杏内酯A的含量在32-36％范围内、银杏内酯B的含量在55-60％的范围内、银杏内酯K的含量在2.2-3.6％的范围内，且银杏内酯B和银杏内酯K的重量比在18-22∶1的范围内。实施例1-3组银杏二萜内酯组合物与对比例1-8组银杏二萜内酯组合物相比，在降低脑缺血再灌注大鼠神经功能评分，减少脑缺血面积，降低脑组织水肿后的含水量，提高SOD活性降低MDA含量对大鼠局灶性脑缺血再灌注损伤的保护作用取得更好的效果，即对大鼠局灶性脑缺血再灌注损伤具有更好的保护作用。
本发明还给出了一种能制得相对稳定组分比例的提取物制备方法： 具体为，取银杏叶(银杏科植物银杏Ginkgo biloba L.的干燥叶，购于江苏邳州)药材50kg，加8倍量10％乙醇加热回流提取2次，每次1.5小时，合并提取液，浓缩，滤过，得浓缩液。向浓缩液中加入银杏叶药材重量15％的聚酰胺，搅拌吸附36小时，静置过夜，弃去上清液，收集吸附剂。向吸附剂中加入30L 95％乙醇，加热至沸1.5小时后，放冷，取上清液，提取后的吸附剂再加入30L乙醇重复提取3次，合并以上4次提取液，减压浓缩，得浓缩液(d＝1.40)冷藏5天。取冷藏后的浓缩液离心，收集沉淀，沉淀加入水洗涤3次，弃去水液，沉淀加2倍量乙醇回流提取3次，每次1小时，提取液滤过，收集提取液，回收溶剂至干，得银杏总内酯粗品。银杏总内酯粗品加入1倍(重量比)的水洗涤3次，弃去水液，沉淀加入3倍量(重量比)乙醇反复重结晶3次，干燥，得银杏二萜内酯原料。经检测，该原料中，银杏二萜内酯A含量为35％，银杏二萜内酯B为58％，银杏二萜内酯K为3.1％。
按上述步骤重复三次，银杏二萜内酯A，银杏二萜内酯B，银杏二萜内酯K的含量SD值小，均在5％以内。
实施例2银杏二萜内酯组合物对家兔血小板聚集的影响
1.实验材料
1.1仪器
STEELIEX血小板聚集凝血因子分析仪，北京世帝科学仪器公司；LDZ5-2离心机，北京医用离心机厂。
1.2试剂和药物
药物组的具体比例同表1。
尼莫地平，山东新华制药股份有限公司生产，批号1609215；二磷酸腺苷(adenosin diphosphate，ADP)，上海伯奥生物科技有限公司(进口分装)；血小板活化因子(platelet activating factor，PAF)，由sigma公司提供；柠檬酸三钠，上海凌峰化学试剂有限公司。
1.3实验动物
新西兰白兔，由南京市浦口区莱芙养殖场提供，实验动物生产许可 证SCXK(苏)2014-0004，使用许可证：SYXK(苏)2013-0021。
2.实验方法
2.1家兔体外血小板聚集率测定
家兔采用普鲁卡因局部麻醉，颈动脉插管放血，3.8％的枸橼酸钠溶液抗凝，以1000r/min离心10min，取富血小板血浆(PRP)，剩余部分以3000r/min离心，即取贫血小板血浆(PPP)，聚集诱导剂分别采用ADP(终浓度5.4μg/ml)和PAF(终浓度0.37μg/ml)。每管250μl PRP中加入银杏二萜内酯组合物各药液10μl(终浓度为9.26μg/ml)，尼莫地平10μl(终浓度为0.05μg/ml)，对照组PRP中加入生理盐水10μl，温育5min，然后依次加入146μg/ml的ADP10μl、10μg/ml的PAF10μl，分别检测两种诱导剂诱导的1min、5min时的血小板聚集率和最大聚集率，并按公式计算血小板聚集抑制率。
聚集抑制率(％)＝(空白对照组最大聚集率-给药组最大聚集率)/空白对照组最大聚集率×100％。
2.2统计学处理
实验数据以
Figure PCTCN2018115166-appb-000005
表示，SPSS17.0统计软件进行分析，多组间均数比较采用单因素方差分析，两组间样本均数比较采用q检验。P＜0.05为有统计学意义.
3.结果
3.1银杏二萜内酯组合物体外给药对PAF诱导家兔血小板聚集率的影响
结果表明，银杏二萜内酯组合物体外给药终浓度为9.26μg/ml对PAF诱导的家兔血小板最大聚集率有明显的抑制作用，并能够显著抑制血小板1min和5min的聚集率，与空白对照组比较，差异有显著性(p＜0.01，P＜0.05)。结果见表3。
表3.银杏二萜内酯组合物对PAF诱导家兔血小板聚集率的影响(
Figure PCTCN2018115166-appb-000006
n＝10)
Figure PCTCN2018115166-appb-000007
Figure PCTCN2018115166-appb-000008
与对照组比较**p＜0.01，*p＜0.05；与实施例1比较 ▲p＜0.05， ▲▲p＜0.01
3.2银杏二萜内酯组合物体外给药对ADP诱导家兔血小板聚集率的影响
结果表明，银杏二萜内酯组合物体外给药在终浓度为9.26μg/ml对ADP诱导的家兔血小板1min、5min聚集率有一定的抑制作用，与空白对照组比较，差异有显著性(p＜0.05，0.01)。结果见表4。
表4.银杏二萜内酯组合物对ADP诱导家兔血小板聚集率的影响(
Figure PCTCN2018115166-appb-000009
n＝10)
Figure PCTCN2018115166-appb-000010
Figure PCTCN2018115166-appb-000011
与对照组比较**p＜0.01，*p＜0.05；与实施例1比较 ▲p＜0.05， ▲▲p＜0.01
4.结论
由表3可以看出，实施例2-3组与实施例1组比较，无显著性差异，即，实施例1组与对比例1-8组相比有显著性差异。
实施例1-3组银杏二萜内酯组合物，银杏内酯A的含量在32-36％范围内、银杏内酯B的含量在55-60％的范围内、银杏内酯K的含量在2.2-3.6％的范围内，且银杏内酯B和银杏内酯K的重量比在18-22∶1的范围内。实施例1-3组银杏二萜内酯组合物与对比例1-8组银杏二萜内酯组合物相比，在抑制不同时间点由PAF和ADP诱导剂诱导的家兔血小板聚集，降低最大聚集率取得更好的效果，表明其有更好的抗血栓形成作用，该作用与其高效广泛地抑制血小板聚集密切相关。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种银杏二萜内酯组合物，其特征在于，所述组合物包含：以重量计，32-36％的银杏内酯A、55-60％的银杏内酯B、2.2-3.6％的银杏内酯K，所述银杏内酯B和银杏内酯K的重量比为18-22∶1。
  2. 根据权利要求1所述的组合物，其特征在于，所述银杏内酯A的含量为32-34％，和/或所述银杏内酯B的含量为56-58％，和/或所述银杏内酯K的含量为2.4-3.4％。
  3. 根据权利要求1所述的组合物，其特征在于，33.5-35.2％的银杏内酯A、56.1-60.1％的银杏内酯B、2.6-3.0％的银杏内酯K，所述银杏内酯B和银杏内酯K的重量比为20.0-21.5∶1。
  4. 一种包含权利要求1-3任一所述组合物的银杏二萜内酯制剂，其特征在于，该制剂还包含药学上可接受的辅料。
  5. 包含权利要求1-3任一所述组合物的银杏二萜内酯注射剂。
  6. 根据权利要求5所述的注射剂，其特征在于，该注射剂为1ml或5ml或10ml剂量，其含有5±0.5mg或25±0.5mg或50±0.5mg所述银杏二萜内酯组合物。。
  7. 根据权利要求5或6所述的注射剂，其特征在于，该注射剂还包含葡甲胺和氯化钠，各组分的重量比为：银杏二萜内酯组合物∶葡甲胺∶氯化钠＝(2-8)∶(2：-8)∶(4-12)。
  8. 权利要求1-3任一项所述的组合物物在制备防治脑卒中药物中的应用。
  9. 权利要求1-3任一项所述的组合物在制备抑制血小板聚集药物中的应用。
  10. 权利要求1-3任一项所述的组合物在制备神经保护药物中的应用。
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CN107898782B (zh) * 2017-12-29 2019-03-26 江苏康缘药业股份有限公司 一种银杏二萜内酯组合物
CN110876746B (zh) * 2018-09-05 2022-04-22 江苏康缘药业股份有限公司 银杏二萜内酯眼用制剂及其制备方法和应用
WO2020207398A1 (zh) * 2019-04-10 2020-10-15 成都百裕制药股份有限公司 银杏萜内酯在制备预防和/或治疗吉兰-巴雷综合征药物中的用途
CN111803487B (zh) * 2019-04-10 2022-12-02 成都百裕制药股份有限公司 银杏萜内酯在制备预防和/或治疗吉兰-巴雷综合征药物中的用途
CN110051664A (zh) * 2019-04-16 2019-07-26 江苏康缘药业股份有限公司 一种银杏内酯组合物在制备用于急性冠状动脉综合征的药物中的应用

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KR20200097303A (ko) 2020-08-18
CN107898782B (zh) 2019-03-26
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