CN105801600B - A kind of Bilobalide, ginkalide A, the preparation method of ginkolide B and ginkalide C - Google Patents
A kind of Bilobalide, ginkalide A, the preparation method of ginkolide B and ginkalide C Download PDFInfo
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Abstract
本发明提供了一种白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的制备方法，包括：将银杏叶进行处理，得到银杏总内酯粗品；将所述银杏总内酯粗品和硅胶混合，得到样品硅胶；将样品硅胶在真空液相色谱分离装置中进行梯度洗脱，所述梯度洗脱过程中弱极性溶剂A和强极性溶剂B的洗脱比例为(8.2～7.8)：(1.8～2.2)，得到白果内酯；洗脱比例为(7.2～6.8)：(3.2～2.8)，得到银杏内酯C；洗脱比例为(6.2～5.8)：(4.2～3.8)，得到银杏内酯A；洗脱比例为1：(0.5～1.5)，得到银杏内酯B。本发明提供的方法制备得到的白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的收率和纯度均较高。
The invention provides a preparation method of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, comprising: processing ginkgo leaves to obtain crude ginkgolides; The crude product is mixed with silica gel to obtain sample silica gel; the sample silica gel is carried out gradient elution in a vacuum liquid chromatography separation device, and the elution ratio of weak polar solvent A and strong polar solvent B in the gradient elution process is (8.2 ～7.8): (1.8～2.2), to obtain bilobalide; the elution ratio is (7.2～6.8): (3.2～2.8), to obtain ginkgolide C; the elution ratio is (6.2～5.8): (4.2～ 3.8), to obtain ginkgolide A; the elution ratio is 1: (0.5-1.5), to obtain ginkgolide B. The yield and purity of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C prepared by the method provided by the invention are relatively high.
Description
技术领域technical field
本发明涉及银杏技术领域，尤其涉及一种白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的制备方法。The invention relates to the technical field of ginkgo, in particular to a preparation method of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C.
背景技术Background technique
银杏内酯(英文名：ginkgolide)化合物属于萜类化合物，由倍半萜内酯和二萜内酯组成，是银杏叶中一类重要的活性成分。白果内酯(bilobalide；BB)属倍半萜内酯，是目前从银杏叶中发现的唯一的一个倍半萜内酯化合物。银杏内酯A(ginkgolide A；GA)、银杏内酯B(ginkgolide B；GB)、银杏内酯C(ginkgolide C；GC)、银杏内脂M(ginkgolide M；GM)、银杏内脂J(ginkgolide J；GJ)为二萜类化合物，其差别在于含有的羟基数目和羟基连接的位置不同。银杏内酯分子具有独特的十二碳骨架结构，嵌有一个叔丁基和六个五元环，包括一个螺壬烷、一个四氢呋喃环和三个内酯环。银杏内酯对血小板活化因子(PAF)受体有强大的特异性抑制作用，其中银杏内酯的抗PAF活性最高。PAF是血小板多种炎症组织分泌产生的一种内源性磷脂，是迄今发现的最有效的血小板聚集诱导剂，它与许多疾病的产生、发展密切相关。而银杏内酯目前被认为是最有临床应用前景的天然PAF受体拮抗剂。Ginkgolide (English name: ginkgolide) compounds belong to terpenoids, composed of sesquiterpene lactones and diterpene lactones, and are an important class of active ingredients in Ginkgo biloba leaves. Bilobalide (BB) is a sesquiterpene lactone, which is the only sesquiterpene lactone compound found in Ginkgo biloba. Ginkgolide A (ginkgolide A; GA), Ginkgolide B (ginkgolide B; GB), Ginkgolide C (ginkgolide C; GC), Ginkgolide M (ginkgolide M; GM), Ginkgolide J (ginkgolide J; GJ) are diterpenoids, the difference lies in the number of hydroxyl groups contained and the position of hydroxyl connection. Ginkgolide molecule has a unique twelve-carbon skeleton structure, embedded with a tert-butyl group and six five-membered rings, including a spirononane, a tetrahydrofuran ring and three lactone rings. Ginkgolides have a strong and specific inhibitory effect on platelet-activating factor (PAF) receptors, and ginkgolides have the highest anti-PAF activity. PAF is an endogenous phospholipid secreted by platelets in various inflammatory tissues. It is the most effective inducer of platelet aggregation found so far, and it is closely related to the occurrence and development of many diseases. Ginkgolides are currently considered to be the most promising natural PAF receptor antagonists for clinical application.
申请号为201310624971.9的中国专利公开了一种银杏内酯A与银杏内酯B的精制方法，包括：取银杏叶提取物浸膏1000g，用水溶液稀释至0.5g生药/mL，加入1000mL乙酸乙酯进行萃取，共萃取3次，合并乙酸乙酯萃取液，于45℃减压浓缩至无溶剂，加入10％的乙醇使浓缩液浓度为0.5g/mL，湿法装柱，使药液通过MCI树脂，先用30％的乙醇洗脱树脂除杂，以除去水溶性和大极性杂质，共洗脱3BV，洗脱速度为1BV/h，再用40％的乙醇以同样的流速洗脱树脂，分别收集前2BV洗脱液和后2BV洗脱液，分别于45℃减压浓缩至1g/mL浸膏，加入500mL 80％乙醇，70℃加热至溶解，4℃冰箱静置24小时析晶，析出晶体后抽滤，分别得到银杏内酯A和银杏内酯B的粗品，将得到的银杏内酯A和银杏内酯B的粗品分别同50mL 80％的乙醇重结晶1次，温度4℃，静置时间24h。析出晶体，抽滤，母液合并，结晶分别于80℃烘干，得到银杏内酯A 0.76g、银杏内酯B 1.07g，银杏内酯A的纯度为95.6％，银杏内酯B的纯度为96.2％。The Chinese patent with application number 201310624971.9 discloses a refining method of ginkgolide A and ginkgolide B, including: take 1000g of ginkgo biloba extract extract, dilute it to 0.5g crude drug/mL with aqueous solution, add 1000mL of ethyl acetate Extraction was carried out for a total of 3 times, the ethyl acetate extracts were combined, concentrated under reduced pressure at 45°C until there was no solvent, and 10% ethanol was added to make the concentration of the concentrated solution 0.5g/mL. Resin, first use 30% ethanol to elute the resin to remove impurities, to remove water-soluble and large polar impurities, elute 3BV in total, the elution speed is 1BV/h, then use 40% ethanol to elute the resin at the same flow rate , respectively collect the first 2BV eluate and the last 2BV eluate, concentrate under reduced pressure at 45°C to 1g/mL extract, add 500mL 80% ethanol, heat to dissolve at 70°C, and stand in the refrigerator at 4°C for 24 hours to crystallize , after the crystals were precipitated, the crude products of ginkgolide A and ginkgolide B were obtained respectively, and the obtained crude products of ginkgolide A and ginkgolide B were recrystallized once with 50mL of 80% ethanol respectively at a temperature of 4°C , standing time 24h. Crystals were precipitated, filtered by suction, the mother liquors were combined, and the crystals were dried at 80°C to obtain 0.76 g of ginkgolide A and 1.07 g of ginkgolide B. The purity of ginkgolide A was 95.6%, and the purity of ginkgolide B was 96.2%. %.
现有技术提供的这种银杏内酯A和银杏内酯B的精制方法虽然工艺简单、损失较少，能够同时分离纯化银杏内酯A和银杏内酯B，但是这种方法制备银杏内酯A和银杏内酯B的纯度和收率较低。Although the refining method of this ginkgolide A and ginkgolide B provided by the prior art has simple process and less loss, it can simultaneously separate and purify ginkgolide A and ginkgolide B, but this method prepares ginkgolide A And the purity and yield of ginkgolide B are low.
发明内容Contents of the invention
有鉴于此，本发明的目的在于提供一种白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的制备方法，本发明提供的方法制备白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的纯度和收率均较高。In view of this, the object of the present invention is to provide a kind of preparation method of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, the method provided by the invention prepares bilobalide, ginkgolide A, ginkgolide The purity and yield of lactone B and ginkgolide C are high.
本发明提供了一种白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的制备方法，包括：The invention provides a preparation method of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, comprising:
将银杏叶采用醇类化合物回流提取后经过滤过、浓缩、分散、再次滤过，得到滤液；Ginkgo biloba is refluxed and extracted with an alcohol compound, filtered, concentrated, dispersed, and filtered again to obtain a filtrate;
将所述滤液萃取后依次进浓缩和干燥，得到银杏总内酯粗品；After the filtrate is extracted, it is concentrated and dried successively to obtain the crude product of total ginkgo lactones;
将所述银杏总内酯粗品在溶剂中和硅胶混合后粉碎，得到样品硅胶；The crude ginkgo lactones are mixed with silica gel in a solvent and pulverized to obtain sample silica gel;
将所述样品硅胶在真空液相色谱分离装置中进行梯度洗脱，按照所述梯度洗脱过程中的洗脱液包括弱极性溶剂A和强极性溶剂B，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(8.2～7.8)：(1.8～2.2)，得到白果内酯；所述弱极性溶剂A和强极性溶剂B的洗脱比例为(7.2～6.8)：(3.2～2.8)，得到银杏内酯C；所述弱极性溶剂A和强极性溶剂B的洗脱比例为(6.2～5.8)：(4.2～3.8)，得到银杏内酯A；所述弱极性溶剂A和强极性溶剂B的洗脱比例为1：(0.5～1.5)，得到银杏内酯B；The sample silica gel is subjected to gradient elution in a vacuum liquid chromatography separation device, according to which the eluent in the gradient elution process includes a weak polar solvent A and a strong polar solvent B, and the weak polar solvent A And the elution ratio of strong polar solvent B is (8.2～7.8): (1.8～2.2), obtains bilobalide; The elution ratio of described weak polar solvent A and strong polar solvent B is (7.2～6.8 ): (3.2～2.8), ginkgolide C is obtained; the elution ratio of the weak polar solvent A and strong polar solvent B is (6.2～5.8): (4.2～3.8), ginkgolide A is obtained; The elution ratio of the weak polar solvent A and the strong polar solvent B is 1: (0.5-1.5), and ginkgolide B is obtained;
所述弱极性溶剂A包括石油醚或环己烷；The weakly polar solvent A includes sherwood oil or cyclohexane;
所述强极性溶剂B包括乙酸乙酯或丙酮。The strong polar solvent B includes ethyl acetate or acetone.
优选的，所述醇类化合物为碳原子数为1～5的醇类化合物。Preferably, the alcohol compound is an alcohol compound with 1-5 carbon atoms.
优选的，所述醇类化合物为甲醇或乙醇。Preferably, the alcohol compound is methanol or ethanol.
优选的，所述萃取的试剂为乙酸乙酯。Preferably, the extraction reagent is ethyl acetate.
优选的，所述溶剂包括甲醇、乙醇、丙酮或乙酸乙酯。Preferably, the solvent includes methanol, ethanol, acetone or ethyl acetate.
优选的，所述硅胶的粒度为80目～100目。Preferably, the particle size of the silica gel is 80 mesh to 100 mesh.
优选的，所述银杏总内酯粗品和硅胶的质量比为1：(0.8～1.2)。Preferably, the mass ratio of the crude total ginkgo lactones to silica gel is 1: (0.8-1.2).
优选的，所述弱极性溶剂A和强极性溶剂B的洗脱比例为8：2，得到白果内酯；Preferably, the elution ratio of the weak polar solvent A and the strong polar solvent B is 8:2 to obtain bilobalide;
所述弱极性溶剂A和强极性溶剂B的洗脱比例为7：3，得到银杏内酯C；The elution ratio of the weak polar solvent A and the strong polar solvent B is 7:3, and ginkgolide C is obtained;
所述弱极性溶剂A和强极性溶剂B的洗脱比例为6：4，得到银杏内酯A；The elution ratio of the weak polar solvent A and the strong polar solvent B is 6:4 to obtain ginkgolide A;
所述弱极性溶剂A和强极性溶剂B的洗脱比例为1：1，得到银杏内酯B。The elution ratio of the weak polar solvent A and the strong polar solvent B is 1:1 to obtain ginkgolide B.
优选的，所述洗脱液为石油醚和乙酸乙酯。Preferably, the eluent is petroleum ether and ethyl acetate.
优选的，所述梯度洗脱过程中洗脱液的用量为6BV～10BV。Preferably, the amount of eluent used in the gradient elution process is 6BV-10BV.
本发明提供的方法通过对银杏叶进行合适的处理，制备样品硅胶，采用真空液相色谱分离装置同时控制梯度洗脱的工艺条件制备银杏内酯，这种方法制备得到的白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的收率和纯度均较高。此外，本发明提供的方法能够同时制备得到白果内酯、银杏内酯A、银杏内酯B和银杏内酯C，制备过程简单、快速、制备量大。The method provided by the invention prepares sample silica gel by properly treating ginkgo leaves, and uses a vacuum liquid chromatography separation device to simultaneously control the process conditions of gradient elution to prepare ginkgolides. The bilobalide and ginkgolides prepared by this method are The yield and purity of ester A, ginkgolide B and ginkgolide C were high. In addition, the method provided by the present invention can simultaneously prepare bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, and the preparation process is simple, fast and has a large amount of preparation.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only It is an embodiment of the present invention, and those skilled in the art can also obtain other drawings according to the provided drawings without creative work.
图1为本发明实施例提供的制备白果内酯、银杏内酯A、银杏内酯B和银杏内酯C方法的流程图；Fig. 1 is the flow chart of the preparation bilobalide, ginkgolide A, ginkgolide B and ginkgolide C method that the embodiment of the present invention provides;
图2为本发明实施例1制备得到的银杏总内酯粗品的蒸发光散射检测图；Fig. 2 is the evaporative light scattering detection figure of the total ginkgo lactone crude product that the embodiment of the present invention 1 prepares;
图3为银杏内酯混合对照品的蒸发光散射检测图；Fig. 3 is the evaporative light scattering detection figure of ginkgolide mixed reference substance;
图4为本发明实施例1制备得到的白果内酯的蒸发光散射检测图；Fig. 4 is the evaporative light scattering detection diagram of the bilobalide prepared in Example 1 of the present invention;
图5为本发明实施例1制备得到的银杏内酯A的蒸发光散射检测图；Fig. 5 is the evaporative light scattering detection figure of the ginkgolide A prepared by the embodiment of the present invention 1;
图6为本发明实施例1制备得到的银杏内酯B的蒸发光散射检测图；Fig. 6 is the evaporative light scattering detection figure of the ginkgolide B prepared by the embodiment of the present invention 1;
图7为本发明实施例1制备得到的银杏内酯C的蒸发光散射检测图。Fig. 7 is an evaporative light scattering detection diagram of ginkgolide C prepared in Example 1 of the present invention.
具体实施方式Detailed ways
下面将对本发明实施例中的技术方案进行清楚、完整地描述，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。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
本发明提供了一种白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的制备方法，包括：The invention provides a preparation method of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, comprising:
将银杏叶采用醇类化合物回流提取后经过滤过、浓缩、分散、再次滤过，得到滤液；Ginkgo biloba is refluxed and extracted with an alcohol compound, filtered, concentrated, dispersed, and filtered again to obtain a filtrate;
将所述滤液萃取后依次进浓缩和干燥，得到银杏总内酯粗品；After the filtrate is extracted, it is concentrated and dried successively to obtain the crude product of total ginkgo lactones;
将所述银杏总内酯粗品在溶剂中和硅胶混合后粉碎，得到样品硅胶；The crude ginkgo lactones are mixed with silica gel in a solvent and pulverized to obtain sample silica gel;
将所述样品硅胶在真空液相色谱分离装置中进行梯度洗脱，所述梯度洗脱过程中的洗脱液包括弱极性溶剂A和强极性溶剂B，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(8.2～7.8)：(1.8～2.2)，得到白果内酯；所述弱极性溶剂A和强极性溶剂B的洗脱比例为(7.2～6.8)：(3.2～2.8)，得到银杏内酯C；所述弱极性溶剂A和强极性溶剂B的洗脱比例为(6.2～5.8)：(4.2～3.8)，得到银杏内酯A；所述弱极性溶剂A和强极性溶剂B的洗脱比例为1：(0.5～1.5)，得到银杏内酯B；The sample silica gel is subjected to gradient elution in a vacuum liquid chromatography separation device, and the eluent in the gradient elution process includes a weak polar solvent A and a strong polar solvent B, and the weak polar solvent A and The elution ratio of strong polar solvent B is (8.2～7.8): (1.8～2.2), and bilobalide is obtained; the elution ratio of described weak polar solvent A and strong polar solvent B is (7.2～6.8) : (3.2～2.8), ginkgolide C is obtained; the elution ratio of the weak polar solvent A and strong polar solvent B is (6.2～5.8): (4.2～3.8), ginkgolide A is obtained; The elution ratio of the weak polar solvent A and the strong polar solvent B is 1: (0.5～1.5), and ginkgolide B is obtained;
所述弱极性溶剂A包括石油醚或环己烷；The weakly polar solvent A includes sherwood oil or cyclohexane;
所述强极性溶剂B包括乙酸乙酯或丙酮。The strong polar solvent B includes ethyl acetate or acetone.
本发明将银杏叶采用醇类化合物进行回流提取。本发明对所述银杏叶没有特殊的限制，采用本领域技术人员熟知的银杏叶药材即可，可由市场购买获得，如可采用徐州市淳康食品有限公司提供的产品。In the present invention, the ginkgo leaves are reflux extracted with alcohol compounds. The present invention has no special restrictions on the ginkgo leaves, and the ginkgo leaves known to those skilled in the art can be used, which can be purchased from the market, such as the products provided by Xuzhou Chunkang Food Co., Ltd.
在本发明的实施例中，所述醇类化合物为碳原子数为1～5的醇类化合物；在其他的实施例中，所述醇类化合物为甲醇或乙醇。在本发明的实施例中，所述醇类化合物以醇类化合物溶液的形式使用。在本发明的实施例中，所述醇类化合物溶液为醇类化合物的水溶液。在本发明的实施例中，所述醇类化合物溶液的质量浓度为70％～80％；在其他的实施例中，所述醇类化合物溶液的质量浓度为72％～78％；在另外的实施例中，所述醇类化合物溶液的质量浓度为74％～76％。在本发明的实施例中，所述醇类化合物溶液为乙醇溶液；在其他的实施例中，所述醇类化合物溶液为质量浓度为75％的乙醇水溶液。In an embodiment of the present invention, the alcohol compound is an alcohol compound with 1-5 carbon atoms; in other embodiments, the alcohol compound is methanol or ethanol. In an embodiment of the present invention, the alcohol compound is used in the form of an alcohol compound solution. In an embodiment of the present invention, the alcoholic compound solution is an aqueous solution of the alcoholic compound. In an embodiment of the present invention, the mass concentration of the alcohol compound solution is 70% to 80%; in other embodiments, the mass concentration of the alcohol compound solution is 72% to 78%; in another In an embodiment, the mass concentration of the alcohol compound solution is 74%-76%. In an embodiment of the present invention, the alcoholic compound solution is an ethanol solution; in other embodiments, the alcoholic compound solution is an aqueous ethanol solution with a mass concentration of 75%.
在本发明的实施例中，所述银杏叶和醇类化合物的质量比为1：(8～12)；在其他的实施例中，所述银杏叶和醇类化合物的质量比为1：(9～11)；在另外的实施例中，所述银杏叶和醇类化合物的质量比为1:10。In an embodiment of the present invention, the mass ratio of the ginkgo leaf and the alcohol compound is 1: (8-12); in other embodiments, the mass ratio of the ginkgo leaf and the alcohol compound is 1: ( 9～11); In another embodiment, the mass ratio of described Ginkgo biloba and alcohol compound is 1:10.
在本发明的实施例中，将银杏叶采用醇类化合物回流提取1～3次；在其他的实施例中，将银杏叶采用醇类化合物回流提取2次。在本发明的实施例中，所述回流提取的时间为1.5h/次～2.5h/次；在其他的实施例中，所述回流提取的时间为1.8h/次～2.2h/次；在另外的实施例中，所述回流提取的时间为2h/次。In the embodiments of the present invention, the ginkgo leaves are extracted 1 to 3 times with alcohol compounds; in other embodiments, the ginkgo leaves are extracted twice with alcohol compounds. In an embodiment of the present invention, the time of the reflux extraction is 1.5h/time to 2.5h/time; in other embodiments, the time of the reflux extraction is 1.8h/time to 2.2h/time; In another embodiment, the time of the reflux extraction is 2h/time.
将银杏叶采用醇类化合物回流提取后，本发明将回流提取后的产物进行滤过。在本发明的实施例中，所述滤过的设备为布氏漏斗。在本发明的实施例中，所述滤过介质的孔径为80μm～120μm；在其他的实施例中，所述滤过介质的孔径为90μm～110μm；在另外的实施例中，所述滤过介质的孔径为100μm。在本发明的实施例中，所述滤过的次数为1次～3次；在其他的实施例中，所述滤过的次数为2次。在本发明的实施例中，当滤过的次数为多次时将多次滤过后产生的滤液合并。在本发明的实施例中，将回流提取后的产物经过2次滤过后产生的滤液合并。After the ginkgo leaves are reflux extracted with alcohol compounds, the product after reflux extraction is filtered in the present invention. In an embodiment of the present invention, the filtering device is a Buchner funnel. In an embodiment of the present invention, the filter medium has a pore size of 80 μm to 120 μm; in other embodiments, the filter medium has a pore size of 90 μm to 110 μm; in another embodiment, the filter The pore size of the medium is 100 μm. In an embodiment of the present invention, the number of times of filtering is 1 to 3 times; in other embodiments, the number of times of filtering is 2 times. In an embodiment of the present invention, when the number of times of filtration is several times, the filtrates produced after multiple times of filtration are combined. In an embodiment of the present invention, the filtrates obtained after the reflux-extracted product is filtered twice are combined.
将回流提取后的产物滤过后，本发明将滤过后得到的滤液进行浓缩。在本发明的实施例中，所述浓缩的方法为减压浓缩。在本发明的实施例中，所述浓缩使得到的浓缩液无醇味即可。After filtering the product after reflux extraction, the present invention concentrates the filtrate obtained after filtering. In an embodiment of the present invention, the method of concentration is concentration under reduced pressure. In an embodiment of the present invention, the concentration only needs to make the obtained concentrated solution have no alcohol smell.
将滤过后产生的滤液浓缩后，本发明将浓缩后的浓缩液进行分散。在本发明的实施例中，将浓缩后的浓缩液在水中分散。在本发明的实施例中，所述浓缩液和水的质量比为1：(0.8～2.5)；在其他的实施例中，所述浓缩液和水的质量比为1：(1～2)；在另外的实施例中，所述浓缩液和水的质量比为1：(1.2～1.8)。After the filtrate produced after filtration is concentrated, the present invention disperses the concentrated liquid. In an embodiment of the present invention, the concentrated liquid is dispersed in water. In an embodiment of the present invention, the mass ratio of the concentrate to water is 1: (0.8-2.5); in other embodiments, the mass ratio of the concentrate to water is 1: (1-2) ; In another embodiment, the mass ratio of the concentrate to water is 1: (1.2-1.8).
将浓缩后得到的浓缩液分散后，本发明将分散后的分散液再次滤过，除去其中的不溶物，得到澄清的滤液。在本发明的实施例中，可以将分散后的分散液在布氏漏斗中进行滤过。在本发明的实施例中，将分散液进行滤过的介质孔径为80μm～120μm。After dispersing the concentrated liquid obtained after concentration, the present invention filters the dispersed dispersion liquid again to remove insoluble matter therein to obtain a clear filtrate. In an embodiment of the present invention, the dispersed dispersion liquid may be filtered in a Buchner funnel. In an embodiment of the present invention, the pore size of the medium through which the dispersion is filtered is 80 μm-120 μm.
将分散后的分散液再次滤过后，本发明将得到的澄清的滤液进行萃取。在本发明的实施例中，所述萃取的试剂为乙酸乙酯。在本发明的实施例中，所述澄清的滤液和萃取试剂的体积比为1：(0.8～1.2)；在其他的实施例中，所澄清的滤液和萃取试剂的体积比为1：(0.9～1.1)；在另外的实施例中，所述澄清的滤液和萃取试剂的体积比为1:1。在本发明的实施例中，所述萃取的次数为2次～4次；在其他的实施例中，所述萃取的次数为3次。在本发明的实施例中，将萃取后得到的萃取产物进行合并。After the dispersed dispersion liquid is filtered again, the present invention extracts the obtained clear filtrate. In an embodiment of the present invention, the extraction reagent is ethyl acetate. In an embodiment of the present invention, the volume ratio of the clarified filtrate and extraction reagent is 1:(0.8～1.2); in other embodiments, the volume ratio of the clarified filtrate and extraction reagent is 1:(0.9 ~1.1); In another embodiment, the volume ratio of the clarified filtrate and the extraction reagent is 1:1. In an embodiment of the present invention, the number of extractions is 2 to 4 times; in other embodiments, the number of extractions is 3 times. In an embodiment of the present invention, the extracted products obtained after extraction are combined.
将澄清的滤液萃取后，本发明将得到的萃取产物进行浓缩。在本发明的实施例中，将得到的萃取产物进行浓缩的方法为减压浓缩。After extracting the clarified filtrate, the present invention concentrates the obtained extraction product. In the embodiment of the present invention, the method of concentrating the obtained extracted product is concentrating under reduced pressure.
将得到的萃取产物进行浓缩后，本发明将得到的浓缩产物进行干燥，得到银杏总内酯粗品。在本发明的实施例中，所述干燥的方法为减压真空干燥。在本发明的实施例中，所述干燥的温度为45℃～60℃；在其他的实施例中，所述干燥的温度为50℃～55℃。在本发明的实施例中，所述干燥的时间为2h～4h；在其他的实施例中，所述干燥的时间为2.5h～3.5h。After the obtained extraction product is concentrated, the present invention drys the obtained concentrated product to obtain a crude product of total ginkgo lactones. In an embodiment of the present invention, the drying method is vacuum drying under reduced pressure. In an embodiment of the present invention, the drying temperature is 45°C-60°C; in other embodiments, the drying temperature is 50°C-55°C. In an embodiment of the present invention, the drying time is 2h-4h; in other embodiments, the drying time is 2.5h-3.5h.
得到银杏总内酯粗品后，本发明将所述银杏总内酯粗品在溶剂中和硅胶混合。在本发明的实施例中，将所述银杏总内酯粗品溶解在溶剂中，向得到的溶解液中加入硅胶混合。在本发明的实施例中，在搅拌的条件下进行混合。本发明对所述搅拌的方法没有特殊的限制，将银杏总内酯粗品、溶剂和硅胶搅拌均匀即可。After the crude total ginkgo lactones are obtained, the present invention mixes the crude total ginkgo lactones with silica gel in a solvent. In an embodiment of the present invention, the crude total ginkgolides are dissolved in a solvent, and silica gel is added to the obtained solution for mixing. In an embodiment of the present invention, mixing is performed under agitation. The present invention has no special limitation on the stirring method, and the crude total ginkgo lactone, solvent and silica gel can be uniformly stirred.
在本发明的实施例中，所述溶剂为甲醇、乙醇、丙酮或乙酸乙酯。本发明对所述溶剂的用量没有特殊的限制，所述溶剂的用量能够使银杏总内酯粗品完全溶解即可。In an embodiment of the present invention, the solvent is methanol, ethanol, acetone or ethyl acetate. The present invention has no special limitation on the amount of the solvent, as long as the amount of the solvent can completely dissolve the crude product of total ginkgo lactones.
在本发明的实施例中，所述硅胶为工业柱层析硅胶。在本发明的实施例中，所述硅胶的粒度为80目～100目；在其他的实施例中，所述硅胶的粒度为85目～95目；在另外的实施例中，所述硅胶的粒度为90目。在本发明的实施例中，所述硅胶的比表面积为≥550m2/g。在本发明的实施例中，所述硅胶的孔容为≤0.70mL/g。在本发明的实施例中，所述硅胶的孔径为200μm～500μm；在其他的实施例中，所述硅胶的孔径为250μm～450μm；在另外的实施例中，所述硅胶的孔径为300μm～400μm。在本发明的实施例中，所述硅胶的pH值为6.0～7.0；在其他的实施例中，所述硅胶的pH值为6.2～6.8；在另外的实施例中，所述硅胶的pH值为6.4～6.6。本发明对所述硅胶的来源没有特殊的限制，可由市场购买获得，如可采用青岛海洋化工有限公司提供的ZCX-1型号的产品。In an embodiment of the present invention, the silica gel is industrial column chromatography silica gel. In an embodiment of the present invention, the particle size of the silica gel is 80 mesh to 100 mesh; in other embodiments, the particle size of the silica gel is 85 mesh to 95 mesh; in another embodiment, the silica gel particle size The particle size is 90 mesh. In an embodiment of the present invention, the specific surface area of the silica gel is ≥550 m 2 /g. In an embodiment of the present invention, the pore volume of the silica gel is ≤0.70 mL/g. In an embodiment of the present invention, the pore diameter of the silica gel is 200 μm to 500 μm; in other embodiments, the pore diameter of the silica gel is 250 μm to 450 μm; in another embodiment, the pore diameter of the silica gel is 300 μm to 400 μm. In an embodiment of the present invention, the pH value of the silica gel is 6.0 to 7.0; in other embodiments, the pH value of the silica gel is 6.2 to 6.8; in another embodiment, the pH value of the silica gel is 6.4 to 6.6. The present invention has no special limitation on the source of the silica gel, which can be purchased from the market, such as the ZCX-1 product provided by Qingdao Ocean Chemical Co., Ltd.
在本发明的实施例中，所述银杏总内酯粗品和硅胶的质量比为1：(0.8～1.2)；在其他的实施例中，所述银杏总内酯粗品和硅胶的质量比为1：(0.9～1.1)；在另外的实施例中，所述银杏总内酯粗品和硅胶的质量比为1:1。In an embodiment of the present invention, the mass ratio of the crude total ginkgo lactones to silica gel is 1: (0.8-1.2); in other embodiments, the mass ratio of the crude total ginkgo lactones to silica gel is 1 : (0.9～1.1); In another embodiment, the mass ratio of the total ginkgo lactone crude product and silica gel is 1:1.
将银杏总内酯粗品在溶剂中和硅胶混合后，本发明将得到的混合物进行粉碎，得到样品硅胶。在本发明的实施例中，将得到的混合物干燥后粉碎。在本发明的实施例中，将混合物干燥的方法为烘干。在本发明的实施例中，所述烘干的温度为50℃～60℃；在其他的实施例中，所述烘干的温度为55℃。在本发明的实施例中，所述烘干的时间为2h～3h；在其他的实施例中，所述烘干的时间为2.5h。在本发明的实施例中，所述粉碎的方法为研磨。在本发明的实施例中，所述研磨的设备为研磨粉碎机。在本发明的实施例中，所述研磨的时间为4min～6min；在其他的实施例中，所述研磨的时间为5min。After mixing crude ginkgo lactones with silica gel in a solvent, the present invention grinds the obtained mixture to obtain sample silica gel. In an example of the present invention, the obtained mixture was dried and pulverized. In an embodiment of the present invention, the method of drying the mixture is drying. In an embodiment of the present invention, the drying temperature is 50°C-60°C; in other embodiments, the drying temperature is 55°C. In an embodiment of the present invention, the drying time is 2h-3h; in other embodiments, the drying time is 2.5h. In an embodiment of the present invention, the crushing method is grinding. In an embodiment of the present invention, the grinding equipment is a grinding mill. In an embodiment of the present invention, the grinding time is 4 minutes to 6 minutes; in other embodiments, the grinding time is 5 minutes.
得到样品硅胶后，本发明将所述样品硅胶在真空液相色谱分离装置中进行梯度洗脱。在本发明中，所述真空液相色谱分离装置可分离多种组分，设备简单，溶剂消耗少、回收率高、分离容量大、分离效果好，本发明采用真空液相色谱装置进行白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的制备，制备过程简单、快速、制备量大。本发明对所述真空液相色谱分离装置没有特殊的限制，可提供图纸由厂家制作，如可由济南博纳生物技术有限公司代为制作。After the sample silica gel is obtained, the present invention performs gradient elution on the sample silica gel in a vacuum liquid chromatography separation device. In the present invention, the vacuum liquid chromatography separation device can separate various components, the equipment is simple, the solvent consumption is small, the recovery rate is high, the separation capacity is large, and the separation effect is good. The preparation of ester, ginkgolide A, ginkgolide B and ginkgolide C has a simple, fast and large preparation process. The present invention has no special limitation on the vacuum liquid chromatography separation device, and the drawings can be provided to be made by the manufacturer, for example, Jinan Bona Biotechnology Co., Ltd. can make it on behalf.
本发明将银杏内酯粗品用TLC法筛选，确定洗脱剂及洗脱比例。在本发明中，所述梯度洗脱过程中的洗脱液包括弱极性溶剂A和强极性溶剂B。在本发明中，所述弱极性溶剂A包括石油醚或环己烷。在本发明中，所述强极性溶剂B包括乙酸乙酯或丙酮。在本发明的实施例中，所述梯度洗脱过程中的洗脱液包括石油醚-乙酸乙酯、石油醚-丙酮、环己烷-乙酸乙酯或环己烷-丙酮；在其他的实施例中，所述梯度洗脱过程中的洗脱液为石油醚-乙酸乙酯。In the present invention, the crude ginkgolide is screened by the TLC method, and the eluent and the elution ratio are determined. In the present invention, the eluent in the gradient elution process includes weak polar solvent A and strong polar solvent B. In the present invention, the weak polar solvent A includes petroleum ether or cyclohexane. In the present invention, the strong polar solvent B includes ethyl acetate or acetone. In an embodiment of the present invention, the eluent in the gradient elution process includes petroleum ether-ethyl acetate, petroleum ether-acetone, cyclohexane-ethyl acetate or cyclohexane-acetone; in other implementations In an example, the eluent in the gradient elution process is petroleum ether-ethyl acetate.
在本发明中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(8.2～7.8)：(1.8～2.2)，得到白果内酯。在本发明的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(8.1～7.9)：(1.9～2.1)，得到白果内酯；在其他的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为8：2，得到白果内酯。在本发明的实施例中，所述制备白果内酯过程中洗脱液的用量为6BV～10BV；在其他的实施例中，所述制备白果内酯过程中洗脱液的用量为7BV～9BV；在另外的实施例中，所述制备白果内酯过程中洗脱液的用量为8BV。In the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is (8.2-7.8): (1.8-2.2), and bilobalide is obtained. In an embodiment of the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is (8.1-7.9): (1.9-2.1), to obtain bilobalide; in other embodiments, The elution ratio of the weak polar solvent A and the strong polar solvent B is 8:2 to obtain bilobalide. In an embodiment of the present invention, the amount of eluent used in the process of preparing bilobalide is 6BV-10BV; in other embodiments, the amount of eluent used in the process of preparing bilobalide is 7BV-9BV ; In another embodiment, the amount of eluent in the process of preparing bilobalide is 8BV.
在本发明的实施例中，弱极性溶剂A和强极性溶剂B的洗脱比例为(8.2～7.8)：(1.8～2.2)洗脱后，将得到的白果内酯粗品依次进行馏分收集、浓缩、干燥、溶解、过滤和重结晶，得到白果内酯。在本发明的实施例中，可以将得到的白果内酯粗品进行馏分收集。本发明对所述馏分收集的方法没有特殊的限制，采用本领域技术人员熟知的馏分收集器将真空液相色谱分离装置分离出的成分进行收集即可。在本发明的实施例中，将馏分收集后的物质进行浓缩。在本发明的实施例中，将馏分收集后的物质进行浓缩的方法为减压浓缩。在本发明的实施例中，将浓缩后的物质进行过干燥。在本发明的实施例中，将浓缩后的物质进行干燥的方法为减压真空干燥。在本发明的实施例中，将浓缩后的物质干燥的时间为1h～2h。在本发明的实施例中，将浓缩后的物质干燥的温度为50℃～60℃。In the embodiment of the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is (8.2-7.8): (1.8-2.2) After elution, the obtained crude bilobalide is sequentially collected by fractions , concentrated, dried, dissolved, filtered and recrystallized to obtain bilobalide. In the embodiment of the present invention, the obtained crude product of bilobalide can be collected for fractionation. The method of the present invention has no special limitation on the fraction collection method, and the components separated by the vacuum liquid chromatography separation device can be collected by using a fraction collector well known to those skilled in the art. In an embodiment of the present invention, the collected fractions are concentrated. In an embodiment of the present invention, the method of concentrating the collected fractions is concentrating under reduced pressure. In an example of the present invention, the concentrated material was over-dried. In an embodiment of the present invention, the method for drying the concentrated substance is vacuum drying under reduced pressure. In an embodiment of the present invention, the time for drying the concentrated substance is 1 h to 2 h. In an embodiment of the present invention, the temperature for drying the concentrated substance is 50°C-60°C.
在本发明的实施例中，将干燥的后产物进行溶解。在本发明的实施例中，将干燥后的产物加热溶解。在本发明的实施例中，所述加热的温度为70℃～85℃。在本发明的实施例中，所述加热的时间为10min～15min。In an embodiment of the present invention, the dried product is dissolved. In an embodiment of the present invention, the dried product is heated and dissolved. In an embodiment of the present invention, the heating temperature is 70°C-85°C. In an embodiment of the present invention, the heating time is 10 minutes to 15 minutes.
在本发明的实施例中，将干燥后的产物溶解的试剂为甲醇、乙醇、丙酮或乙酸乙酯；在其他的实施例中，将干燥后的产物溶解的试剂为乙醇。在本发明的实施例中，将干燥后的产物溶解的试剂为乙醇溶液；在其他的实施例中，将干燥后的产物溶解的试剂为乙醇的水溶液。在本发明的实施例中，所述乙醇溶液的质量浓度为10％～70％；在其他的实施例中，所述乙醇溶液的质量浓度为20％～60％；在另外的实施例中，所述乙醇溶液的质量浓度为30％～50％。In an embodiment of the present invention, the reagent for dissolving the dried product is methanol, ethanol, acetone or ethyl acetate; in other embodiments, the reagent for dissolving the dried product is ethanol. In an embodiment of the present invention, the reagent for dissolving the dried product is an ethanol solution; in other embodiments, the reagent for dissolving the dried product is an aqueous solution of ethanol. In an embodiment of the present invention, the mass concentration of the ethanol solution is 10% to 70%; in other embodiments, the mass concentration of the ethanol solution is 20% to 60%; in another embodiment, The mass concentration of the ethanol solution is 30%-50%.
在本发明的实施例中，所述干燥后的产物和溶解的试剂的质量比为1：(15～20)；在其他的实施例中，所述干燥后的产物和溶解的试剂的质量比为1：(16～19)；在另外的实施例中，所述干燥后的产物和溶解的试剂的质量比为1：(17～18)。In an embodiment of the present invention, the mass ratio of the dried product to the dissolved reagent is 1: (15-20); in other embodiments, the mass ratio of the dried product to the dissolved reagent is 1:(16-19); in another embodiment, the mass ratio of the dried product to the dissolved reagent is 1:(17-18).
在本发明的实施例中，将干燥后的产物溶解后，将得到的溶解液过滤。在本发明的实施例中，所述过滤的设备为布氏漏斗。在本发明的实施例中，所述过滤介质的孔径为80μm～120μm。在本发明的实施例中，所述过滤的温度为25℃。In an embodiment of the present invention, after dissolving the dried product, the obtained solution is filtered. In an embodiment of the present invention, the filtering device is a Buchner funnel. In an embodiment of the present invention, the filter medium has a pore size of 80 μm˜120 μm. In an embodiment of the present invention, the filtration temperature is 25°C.
在本发明的实施例中，将溶解液过滤后，将得到的过滤产物重结晶，得到白果内酯。在本发明的实施例中，所述重结晶的温度为2℃～6℃；在其他的实施例中，所述重结晶的温度为3℃～5℃；在另外的实施例中，所述重结晶的温度为4℃。在本发明的实施例中，所述重结晶的时间为8小时～12小时；在其他的实施例中，所述重结晶的时间为9小时～11小时；在另外的实施例中，所述重结晶的温度为10小时。In the embodiment of the present invention, after the solution is filtered, the obtained filtered product is recrystallized to obtain bilobalide. In an embodiment of the present invention, the recrystallization temperature is 2°C to 6°C; in other embodiments, the recrystallization temperature is 3°C to 5°C; in another embodiment, the The recrystallization temperature was 4°C. In an embodiment of the present invention, the recrystallization time is 8 hours to 12 hours; in other embodiments, the recrystallization time is 9 hours to 11 hours; in another embodiment, the The recrystallization temperature was 10 hours.
在本发明中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(7.2～6.8)：(3.2～2.8)，得到银杏内酯C。在本发明的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(7.1～6.9)：(2.9～3.1)，得到银杏内酯C；在其他的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为7：3，得到银杏内酯C。在本发明的实施例中，所述制备银杏内酯C过程中洗脱液的用量为6BV～10BV；在其他的实施例中，所述制备银杏内酯C过程中洗脱液的用量为7BV～9BV；在另外的实施例中，所述制备银杏内酯C过程中洗脱液的用量为8BV。In the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is (7.2-6.8): (3.2-2.8), and ginkgolide C is obtained. In an embodiment of the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is (7.1-6.9): (2.9-3.1), and ginkgolide C is obtained; in other embodiments , the elution ratio of the weak polar solvent A and the strong polar solvent B is 7:3, and ginkgolide C is obtained. In an embodiment of the present invention, the amount of eluent used in the preparation of ginkgolide C is 6BV to 10BV; in other embodiments, the amount of eluent used in the preparation of ginkgolide C is 7BV ~9BV; In another embodiment, the amount of eluent used in the preparation of ginkgolide C is 8BV.
在本发明的实施例中，弱极性溶剂A和强极性溶剂B的洗脱比例为(7.2～6.8)：(3.2～2.8)洗脱后，将得到的银杏内酯C粗品依次进行馏分收集、浓缩、干燥、溶解、过滤和重结晶，得到银杏内酯C。在本发明中，将银杏内酯C粗品依次进行馏分收集、浓缩、干燥、溶解、过滤和重结晶的方法与上述将白果内酯粗品依次进行馏分收集、浓缩、干燥、溶解、过滤和重结晶的方法一致，在此不再赘述。在本发明的实施例中，将银杏内酯C粗品进行溶解采用的乙醇溶液的质量浓度为50％～80％；在其他的实施例中，将银杏内酯C粗品进行溶解采用的乙醇溶液的质量浓度为60％～70％。In the embodiment of the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is (7.2～6.8): (3.2～2.8) After elution, the obtained ginkgolide C crude product is sequentially fractionated Collect, concentrate, dry, dissolve, filter and recrystallize to obtain ginkgolide C. In the present invention, the method of sequentially collecting fractions, concentrating, drying, dissolving, filtering and recrystallizing the crude product of ginkgolide C is the same as the method of sequentially collecting fractions, concentrating, drying, dissolving, filtering and recrystallizing the crude product of bilobalide C The method is the same and will not be repeated here. In an embodiment of the present invention, the mass concentration of the ethanol solution used for dissolving the crude ginkgolide C is 50% to 80%; in other embodiments, the mass concentration of the ethanol solution used for dissolving the crude ginkgolide C is The mass concentration is 60% to 70%.
在本发明中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(6.2～5.8)：(4.2～3.8)，得到银杏内酯A。在本发明的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(6.1～5.9)：(3.9～4.1)，得到银杏内酯A；在其他的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为6：4，得到银杏内酯A。在本发明的实施例中，所述制备银杏内酯A过程中洗脱液的用量为6BV～10BV；在其他的实施例中，所述制备银杏内酯A过程中洗脱液的用量为7BV～9BV；在另外的实施例中，所述制备银杏内酯A过程中洗脱液的用量为8BV。In the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is (6.2-5.8): (4.2-3.8), and ginkgolide A is obtained. In an embodiment of the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is (6.1-5.9): (3.9-4.1), and ginkgolide A is obtained; in other embodiments , the elution ratio of the weak polar solvent A and the strong polar solvent B is 6:4, and ginkgolide A is obtained. In an embodiment of the present invention, the amount of eluent used in the preparation of ginkgolide A is 6BV to 10BV; in other embodiments, the amount of eluent used in the preparation of ginkgolide A is 7BV ~9BV; In another embodiment, the amount of eluent used in the preparation of ginkgolide A is 8BV.
在本发明的实施例中，弱极性溶剂A和强极性溶剂B的洗脱比例为(6.2～5.8)：(4.2～3.8)洗脱后，将得到的银杏内酯A粗品依次进行馏分收集、浓缩、干燥、溶解、过滤和重结晶，得到银杏内酯A。在本发明中，将银杏内酯A粗品依次进行馏分收集、浓缩、干燥、溶解、过滤和重结晶的方法与上述将白果内酯粗品依次进行馏分收集、浓缩、干燥、溶解、过滤和重结晶的方法一致，在此不再赘述。在本发明的实施例中，将银杏内酯A粗品进行溶解采用的乙醇溶液的质量浓度为60％～95％；在其他的实施例中，将银杏内酯A粗品进行溶解采用的乙醇溶液的质量浓度为70％～85％；在另外的实施例中，将银杏内酯A粗品进行溶解采用的乙醇溶液的质量浓度为75％～80％。在本发明的实施例中，将银杏内酯A粗品进行重结晶的次数的为1次～3次；在其他的实施例中，将银杏内酯A粗品进行重结晶的次数为2次。In the embodiment of the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is (6.2～5.8): (4.2～3.8) After elution, the obtained ginkgolide A crude product is sequentially fractionated Collect, concentrate, dry, dissolve, filter and recrystallize to obtain ginkgolide A. In the present invention, the method of sequentially collecting fractions, concentrating, drying, dissolving, filtering and recrystallizing the crude product of ginkgolide A is the same as the above method of sequentially collecting fractions, concentrating, drying, dissolving, filtering and recrystallizing the crude product of bilobalide The method is the same and will not be repeated here. In an embodiment of the present invention, the mass concentration of the ethanol solution used for dissolving the crude ginkgolide A is 60% to 95%; in other embodiments, the mass concentration of the ethanol solution used for dissolving the crude ginkgolide A is The mass concentration is 70%-85%; in another embodiment, the mass concentration of the ethanol solution used for dissolving the crude ginkgolide A is 75%-80%. In the embodiments of the present invention, the number of recrystallization of the crude ginkgolide A is 1 to 3 times; in other embodiments, the number of recrystallization of the crude ginkgolide A is 2 times.
在本发明中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为1：(0.5～1.5)，得到银杏内酯B。在本发明的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为1：(0.8～1.2)，得到银杏内酯B；在其他的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为1：1，得到银杏内酯B。在本发明的实施例中，所述制备银杏内酯B过程中洗脱液的用量为6BV～10BV；在其他的是实施例中，所述制备银杏内酯B过程中洗脱液的用量为7BV～9BV；在另外的实施例中，所述制备银杏内酯B过程中洗脱液的用量为8BV。In the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is 1: (0.5-1.5), and ginkgolide B is obtained. In an embodiment of the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is 1: (0.8-1.2), and ginkgolide B is obtained; in other embodiments, the weak polar solvent The elution ratio of polar solvent A and strong polar solvent B is 1:1, and ginkgolide B is obtained. In an embodiment of the present invention, the amount of eluent in the process of preparing ginkgolide B is 6BV～10BV; in other embodiments, the amount of eluent in the process of preparing ginkgolide B is 7BV～9BV; in another embodiment, the amount of eluent used in the process of preparing ginkgolide B is 8BV.
在本发明的实施例中，弱极性溶剂A和强极性溶剂B的洗脱比例为1：(0.5～1.5)洗脱后，将得到的银杏内酯B粗品依次进行馏分收集、浓缩、干燥、溶解、过滤和重结晶，得到银杏内酯B。在本发明中，将银杏内酯B粗品依次进行馏分收集、浓缩、干燥、溶解、过滤和重结晶的方法与上述将白果内酯粗品依次进行馏分收集、浓缩、干燥、溶解、过滤和重结晶的方法一致，在此不再赘述。在本发明的实施例中，将银杏内酯B粗品进行溶解采用的乙醇溶液的质量浓度为70％～100％(当乙醇溶液的质量浓度为100％时即为乙醇纯品)；在其他的实施例中，将银杏内酯B粗品进行溶解采用的乙醇溶液的质量浓度为75％～95％；在另外的实施例中，将银杏内酯B粗品进行溶解采用的乙醇溶液的质量浓度为80％～90％。在本发明的实施例中，将银杏内酯B粗品进行重结晶的次数的为1次～3次；在其他的实施例中，将银杏内酯B粗品进行重结晶的次数为2次。In the embodiment of the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is 1: (0.5-1.5) After the elution, the obtained crude ginkgolide B is sequentially collected, concentrated, Ginkgolide B is obtained by drying, dissolving, filtering and recrystallizing. In the present invention, the method of sequentially collecting fractions, concentrating, drying, dissolving, filtering and recrystallizing the crude product of ginkgolide B is the same as the above method of sequentially collecting fractions, concentrating, drying, dissolving, filtering and recrystallizing the crude product of bilobalide The method is the same and will not be repeated here. In an embodiment of the present invention, the mass concentration of the ethanol solution used for dissolving the ginkgolide B crude product is 70% to 100% (when the mass concentration of the ethanol solution is 100%, it is pure ethanol); In an embodiment, the mass concentration of the ethanol solution used for dissolving the ginkgolide B crude product is 75% to 95%; in another embodiment, the mass concentration of the ethanol solution used for dissolving the ginkgolide B crude product is 80% %~90%. In the embodiments of the present invention, the number of recrystallization of the crude ginkgolide B is 1 to 3 times; in other embodiments, the number of recrystallization of the crude ginkgolide B is 2 times.
在本发明的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(9.2～8.8)：1，能够去除银杏总内酯粗品中脂肪酸类杂质；在其他的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(9.1～8.9)：1；在另外的实施例中，所述弱极性溶剂A和强极性溶剂B的洗脱比例为9：1。在本发明的实施例中，去除银杏总内酯粗品中脂肪酸类杂质过程中洗脱液的用量为6BV～10BV；在其他的实施例中，去除银杏总内酯粗品中脂肪酸类杂质过程中洗脱液的用量为7BV～9BV；在另外的实施例中，去除银杏总内酯粗品中脂肪酸类杂质过程中洗脱液的用量为8BV。In an embodiment of the present invention, the elution ratio of the weak polar solvent A and the strong polar solvent B is (9.2～8.8):1, which can remove fatty acid impurities in the crude product of total ginkgo lactones; in other implementations In the example, the elution ratio of the weak polar solvent A and the strong polar solvent B is (9.1～8.9):1; in another embodiment, the elution ratio of the weak polar solvent A and the strong polar solvent B The elution ratio is 9:1. In an embodiment of the present invention, the amount of eluent used in the process of removing fatty acid impurities in the crude product of total ginkgo lactones is 6BV～10BV; The amount of dehydration is 7BV-9BV; in another embodiment, the amount of eluent used in the process of removing fatty acid impurities in the crude total ginkgo lactones is 8BV.
图1为本发明实施例提供的制备白果内酯、银杏内酯A、银杏内酯B和银杏内酯C方法的流程图，如图1所示：Fig. 1 is the flow chart of the preparation bilobalide, ginkgolide A, ginkgolide B and ginkgolide C method that the embodiment of the present invention provides, as shown in Fig. 1:
将银杏叶采用质量浓度为75％的乙醇溶液进行回流提取，得到提取液；Ginkgo leaves are reflux extracted with an ethanol solution with a mass concentration of 75%, to obtain an extract;
将所述提取液进行减压浓缩，得到流浸膏；Concentrating the extract under reduced pressure to obtain a liquid extract;
将所述流浸膏用1～2倍的水进行分散，得到分散液；Disperse the liquid extract with 1 to 2 times of water to obtain a dispersion;
将所述分散液用乙酸乙酯萃取3次后进行浓缩和干燥，得到银杏总内酯粗品；The dispersion was extracted 3 times with ethyl acetate and then concentrated and dried to obtain the crude product of total ginkgo lactones;
将所述银杏总内酯粗品用甲醇溶解，将得到的溶解液和粒度为80目～100目的硅胶混合后研磨，得到样品硅胶；Dissolving the crude ginkgo lactones in methanol, mixing the obtained solution with silica gel with a particle size of 80 mesh to 100 mesh, and grinding to obtain a sample silica gel;
将所述样品硅胶在真空液相色谱分离装置中进行梯度洗脱，所述梯度洗脱过程中的洗脱液为石油醚和乙酸乙酯，所述石油醚和乙酸乙酯的洗脱比例为9:1，去除银杏内酯粗品中的脂肪酸类杂质；所述石油醚和乙酸乙酯的洗脱比例为8:2，得到白果内酯粗品，将所述白果内酯粗品进行重结晶，得到白果内酯(BB)；所述石油醚和乙酸乙酯的洗脱比例为7:3，得到银杏内酯C粗品，将所述银杏内酯C粗品进行重结晶，得到银杏内酯C(GC)；所述石油醚和乙酸乙酯的洗脱比例为6:4，得到银杏内酯A粗品，将所述银杏内酯A粗品进行重结晶，得到银杏内酯A(GA)；所述石油醚和乙酸乙酯的洗脱比例为1:1，得到银杏内酯B粗品，将所述银杏内酯B粗品进行重结晶，得到银杏内酯B(GB)；所述石油醚和乙酸乙酯的洗脱比例为1:0，除去脂肪酸类等杂质。The sample silica gel is subjected to gradient elution in a vacuum liquid chromatography separation device, the eluent in the gradient elution process is petroleum ether and ethyl acetate, and the elution ratio of the petroleum ether and ethyl acetate is 9:1, remove the fatty acid impurities in the crude ginkgolide; the elution ratio of the petroleum ether and ethyl acetate is 8:2, the crude bilobalide is obtained, and the crude bilobalide is recrystallized to obtain Bilobalide (BB); The elution ratio of the petroleum ether and ethyl acetate is 7:3, and the crude product of Ginkgolide C is obtained, and the crude product of Ginkgolide C is recrystallized to obtain Ginkgolide C (GC ); the elution ratio of the petroleum ether and ethyl acetate is 6:4, and the crude product of ginkgolide A is obtained, and the crude product of ginkgolide A is recrystallized to obtain ginkgolide A (GA); the petroleum The elution ratio of ether and ethyl acetate is 1:1, and the crude product of ginkgolide B is obtained, and the crude product of ginkgolide B is recrystallized to obtain ginkgolide B (GB); the petroleum ether and ethyl acetate The elution ratio is 1:0 to remove impurities such as fatty acids.
真空液相色谱(Vacuum Liquid Chromatography，VLC)于1979年由Targett命名，它是利用柱后减压，使洗脱剂迅速通过固定相，从而很好地分离样品。具有设备简单、溶剂消耗少、回收率高、分离容量大、分离效果好，节省劳动力等优点。本发明以中药银杏叶为研究对象，对银杏内酯的系统色谱分离条件进行探索，通过TLC多次展开筛选流动相，建立银杏内酯的分离条件，实现了银杏内酯的快速分离，提高了分离效率。Vacuum Liquid Chromatography (VLC) was named by Targett in 1979. It uses decompression after the column to make the eluent pass through the stationary phase quickly, so as to separate the samples well. It has the advantages of simple equipment, less solvent consumption, high recovery rate, large separation capacity, good separation effect and labor saving. The present invention takes Chinese medicine Ginkgo biloba as the research object, explores the system chromatographic separation conditions of ginkgolides, and establishes the separation conditions of ginkgolides through TLC multiple expansion and screening mobile phases, realizes the rapid separation of ginkgolides, and improves the separation efficiency.
将本发明提供的方法制备得到的白果内酯、银杏内酯A、银杏内酯B和银杏内酯C采用蒸发光散射检测器(ELSD)进行纯度检测，检测条件为：Acuity C18(250mm×4.6mm,5μm)色谱柱，流动相为甲醇、四氢呋喃和水的混合液，流动相流速为1.0mL/min，洗脱比例为甲醇：四氢呋喃：水为25:10:65，洗脱时间为0～30min，进样体积为10μL，柱温为30℃。The bilobalide, ginkgolide A, ginkgolide B and ginkgolide C prepared by the method provided by the invention are tested for purity using an evaporative light scattering detector (ELSD), and the detection conditions are: Acuity C18 (250mm×4.6 mm, 5μm) chromatographic column, the mobile phase is a mixture of methanol, tetrahydrofuran and water, the flow rate of the mobile phase is 1.0mL/min, the elution ratio is methanol: tetrahydrofuran: water 25:10:65, and the elution time is 0～ 30min, the injection volume was 10μL, and the column temperature was 30°C.
检测结果为，本发明提供的方法制备得到的白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的纯度均＞99％。The detection result is that the purity of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C prepared by the method provided by the invention is all >99%.
检测本发明提供的方法制备白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的收率，检测结果为，本发明提供的方法制备得到的白果内酯的收率≥85％，银杏内酯A的收率＞81％，银杏内酯B的收率＞82％，银杏内酯C的收率＞82％，各种产品的收率均较高。Detect the yield of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C prepared by the method provided by the invention, and the test result is that the yield of bilobalide prepared by the method provided by the invention is ≥85% , the yield of ginkgolide A > 81%, the yield of ginkgolide B > 82%, the yield of ginkgolide C > 82%, and the yields of various products are higher.
本发明以下实施例所用的原料均为市售商品，所用的银杏叶药材为徐州市淳康食品有限公司提供的产品；所用的硅胶为青岛海洋化工公司提供的产品；所用的真空液相色谱分离装置由济南博纳生物技术有限公司代为制作。The raw materials used in the following examples of the present invention are commercially available, and the ginkgo leaf medicinal material used is the product provided by Xuzhou Chunkang Food Co., Ltd.; the silica gel used is the product provided by Qingdao Ocean Chemical Company; the used vacuum liquid chromatography The device was produced by Jinan Bona Biotechnology Co., Ltd.
实施例1Example 1
取20Kg银杏叶药材采用质量浓度为75％乙醇溶液回流提取两次(2h/次)，所述乙醇溶液的质量是银杏叶药材制备的10倍；将得到的提取液滤过，合并两次滤液；将合并后的滤液减压浓缩至无醇味；将得到的浓缩液加入到水分散，所述水的用量和浓缩液的用量相同；将得到的分散液滤过，除去不溶物，得澄清的滤液；将得到的澄清的滤液加入到等体积的乙酸乙酯中萃取3次，合并萃取后的产物进行浓缩、干燥，得银杏总内酯粗品245g。Get 20Kg Ginkgo biloba medical material and adopt mass concentration to be that 75% ethanol solution reflux extracts twice (2h/time), the quality of described ethanol solution is 10 times that Ginkgo biloba medical material is prepared; The extract that obtains is filtered, merge twice filtrate Concentrate the combined filtrate under reduced pressure until there is no alcohol smell; add the obtained concentrated solution to water for dispersion, and the amount of the water is the same as that of the concentrated solution; filter the obtained dispersion to remove insolubles, and obtain clarification The filtrate; The clarified filtrate obtained is added to an equal volume of ethyl acetate to extract 3 times, and the product after the merging and extraction is concentrated and dried to obtain 245 g of ginkgo lactone crude product.
将200g的银杏总内酯粗品用甲醇溶解，向得到的溶解液中加入与银杏总内酯粗品等质量的90目的硅胶拌样，搅拌均匀，烘干后研磨，得到样品硅胶。Dissolve 200 g of crude ginkgo lactones in methanol, add 90-mesh silica gel with the same quality as the crude total ginkgo lactones to the obtained solution, mix the sample, stir evenly, dry and grind to obtain sample silica gel.
将样品硅胶加入到真空液相色谱分离装置中进行梯度洗脱，采用的洗脱液为石油醚-乙酸乙酯。The sample silica gel was added to a vacuum liquid chromatography separation device for gradient elution, and the eluent used was petroleum ether-ethyl acetate.
石油醚：乙酸乙酯的洗脱比例为9:1用量为8BV洗脱，除去银杏总内酯粗品中脂肪酸类杂质；Petroleum ether: The elution ratio of ethyl acetate is 9:1, and the dosage is 8BV elution to remove fatty acid impurities in the crude product of total ginkgo lactone;
石油醚：乙酸乙酯的洗脱比例为8:2用量为8BV洗脱，收集洗脱后的馏分，将得到馏分浓缩干燥得到淡黄色粉末，用质量浓度为50％的乙醇溶液进行加热溶解，所述乙醇溶液的用量为干燥后的产物的16倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏10h进行重结晶，得到48.6g的白果内酯；The elution ratio of petroleum ether: ethyl acetate is 8:2, and the amount is 8BV for elution. Collect the eluted fractions, concentrate and dry the obtained fractions to obtain a light yellow powder, and heat and dissolve them with an ethanol solution with a mass concentration of 50%. The amount of the ethanol solution was 16 times that of the dried product, the dissolved solution was filtered, and the obtained filtrate was refrigerated at 4° C. for 10 h for recrystallization to obtain 48.6 g of bilobalide;
石油醚：乙酸乙酯的洗脱比例为7:3用量为8BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到淡黄色粉末，用质量浓度为70％的乙醇溶液进行加热溶解，所述乙醇溶液的用量为干燥后的产物的17倍，将溶解后的溶液过滤，将得到的滤液4℃冷藏11h进行重结晶，得到19.5g的银杏内酯C；The elution ratio of petroleum ether: ethyl acetate is 7:3, and the dosage is 8BV. Collect the eluted fractions, concentrate and dry the obtained fractions to obtain a light yellow powder, and heat and dissolve with an ethanol solution with a mass concentration of 70%. , the amount of the ethanol solution is 17 times that of the dried product, the dissolved solution is filtered, and the obtained filtrate is refrigerated at 4° C. for 11 h for recrystallization to obtain 19.5 g of ginkgolide C;
石油醚：乙酸乙酯的洗脱比例为6:4用量为8BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到黄色粉末，用质量浓度为90％的乙醇溶液加热溶解，所述乙醇溶液的用量为干燥后的产物的18倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏9h进行2次重结晶，得到17.6g的银杏内酯A；The elution ratio of petroleum ether: ethyl acetate is 6:4, and the amount is 8BV for elution. The eluted fractions are collected, concentrated and dried to obtain a yellow powder, which is heated and dissolved with an ethanol solution with a mass concentration of 90%. The amount of the ethanol solution is 18 times that of the dried product, the dissolved solution is filtered, and the obtained filtrate is refrigerated at 4° C. for 9 h for 2 recrystallizations to obtain 17.6 g of ginkgolide A;
石油醚：乙酸乙酯的洗脱比例为1:1用量为8BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到黄色粉末，用质量浓度为95％的乙醇溶液进行加热溶解，所述乙醇溶液的用量为干燥后的产物的18倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏9h进行2次重结晶，得到14.8g的银杏内酯B。Petroleum ether: The elution ratio of ethyl acetate is 1:1, and the amount is 8BV for elution. Collect the eluted fractions, concentrate and dry the obtained fractions to obtain a yellow powder, and heat and dissolve with an ethanol solution with a mass concentration of 95%. The amount of the ethanol solution was 18 times that of the dried product. The dissolved solution was filtered, and the obtained filtrate was refrigerated at 4° C. for 9 h for 2 recrystallizations to obtain 14.8 g of ginkgolide B.
按照上述技术方案所述的方法，对银杏总内酯粗品、银杏内酯混合对照品(银杏内酯A(批号110862-201310)；银杏内酯B(批号110863-201209)；银杏内酯C(批号110864-201307)；白果内酯(批号110861-201305)均购自中国食品药品检定研究院)、白果内酯、银杏内酯A、银杏内酯B和银杏内酯C采用蒸发光散射检测器进行检测，检测结果如图2～图7所示，图2为本发明实施例1制备得到的银杏总内酯粗品的蒸发光散射检测(ELSD)图，图3为银杏内酯混合对照品的蒸发光散射检测图，图4为本发明实施例1制备得到的白果内酯的蒸发光散射检测图，图5为本发明实施例1制备得到的银杏内酯A的蒸发光散射检测图，图6为本发明实施例1制备得到的银杏内酯B的蒸发光散射检测图，图7为本发明实施例1制备得到的银杏内酯C的蒸发光散射检测图；由图2～图7可知，本发明实施例1制备得到的白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的纯度均大于99.9％。According to the method described in the above-mentioned technical scheme, to total ginkgolide crude product, ginkgolide mixed reference substance (Ginkgolide A (lot number 110862-201310); Ginkgolide B (lot number 110863-201209); Ginkgolide C ( Batch No. 110864-201307); bilobalide (batch No. 110861-201305) were purchased from China National Institutes for Food and Drug Control), bilobalide, ginkgolide A, ginkgolide B and ginkgolide C with an evaporative light scattering detector Detect, and test result is as shown in Figure 2～Fig. 7, and Fig. 2 is the evaporative light scattering detection (ELSD) figure of the total ginkgolide crude product that the embodiment of the present invention 1 prepares, and Fig. 3 is the mixed reference substance of ginkgolide Evaporative light scattering detection figure, Fig. 4 is the evaporative light scattering detection figure of the bilobalide prepared in the embodiment of the present invention 1, Fig. 5 is the evaporative light scattering detection figure of the ginkgolide A prepared in the embodiment of the present invention 1, Fig. 6 is the evaporative light scattering detection diagram of ginkgolide B prepared in Example 1 of the present invention, and FIG. 7 is the evaporative light scattering detection diagram of ginkgolide C prepared in Example 1 of the present invention; , the purity of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C prepared in Example 1 of the present invention is greater than 99.9%.
本发明实施例1制备得到的白果内酯的收率为86.8％、银杏内酯A的收率为81.5％、银杏内酯B的收率为83.2％、银杏内酯C的收率为82.8％，各种产品的收率均较高。The yield of bilobalide prepared in Example 1 of the present invention is 86.8%, the yield of ginkgolide A is 81.5%, the yield of ginkgolide B is 83.2%, and the yield of ginkgolide C is 82.8% , the yields of various products are higher.
实施例2Example 2
取20Kg银杏叶药材采用质量浓度为75％甲醇溶液回流提取两次(2h/次)，所述乙醇溶液的质量是银杏叶药材制备的10倍；将得到的提取液滤过，合并两次滤液；将合并后的滤液减压浓缩至无醇味；将得到的浓缩液加入到水分散，所述水的用量和浓缩液的用量相同；将得到的分散液滤过，除去不溶物，得澄清的滤液；将得到的澄清的滤液加入到等体积的乙酸乙酯中萃取3次，合并萃取后的产物进行浓缩、干燥，得银杏总内酯粗品262g。Get 20Kg Ginkgo biloba medical material and adopt mass concentration and be that 75% methanol solution reflux extracts twice (2h/time), the quality of described ethanol solution is 10 times that Ginkgo biloba medical material is prepared; The extract that obtains is filtered, merge twice filtrate Concentrate the combined filtrate under reduced pressure until there is no alcohol smell; add the obtained concentrated solution to water for dispersion, and the amount of the water is the same as that of the concentrated solution; filter the obtained dispersion to remove insolubles, and obtain clarification The filtrate; The clarified filtrate obtained is added to an equal volume of ethyl acetate to extract 3 times, and the product after the merging and extraction is concentrated and dried to obtain 262 g of ginkgo lactone crude product.
将200g的银杏总内酯粗品用乙醇溶解，向得到的溶解液中加入与银杏总内酯粗品等质量的80目的硅胶拌样，搅拌均匀，烘干后研磨，得到样品硅胶。Dissolve 200 g of crude ginkgo lactones in ethanol, add 80-mesh silica gel with the same quality as the crude total ginkgo lactones to the obtained solution, stir evenly, dry and grind to obtain sample silica gel.
将样品硅胶加入到真空液相色谱分离装置中进行梯度洗脱，采用的洗脱液为环己烷-丙酮。The sample silica gel was added to a vacuum liquid chromatography separation device for gradient elution, and the eluent used was cyclohexane-acetone.
环己烷：丙酮的洗脱比例为9.2:1用量为6BV洗脱，除去银杏总内酯粗品中脂肪酸类杂质；The elution ratio of cyclohexane: acetone is 9.2:1 and the dosage is 6BV elution to remove fatty acid impurities in the crude product of total ginkgo lactones;
环己烷：丙酮的洗脱比例为8.2:2用量为6BV洗脱，收集洗脱后的馏分，将得到馏分浓缩干燥得到淡黄色粉末，用质量浓度为10％的乙醇溶液进行加热溶解，所述乙醇溶液的用量为干燥后的产物的15倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏8h进行重结晶，得到49.5g的白果内酯；The elution ratio of cyclohexane: acetone is 8.2:2 and the dosage is 6BV for elution. The eluted fractions are collected, concentrated and dried to obtain a light yellow powder, which is heated and dissolved with an ethanol solution with a mass concentration of 10%. The amount of the ethanol solution is 15 times that of the dried product, the dissolved solution is filtered, and the obtained filtrate is refrigerated at 4°C for 8 hours for recrystallization to obtain 49.5 g of bilobalide;
环己烷：丙酮的洗脱比例为7.2:3用量为6BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到淡黄色粉末，用质量浓度为50％的乙醇溶液进行加热溶解，所述乙醇溶液的用量为干燥后的产物的15倍，将溶解后的溶液过滤，将得到的滤液4℃冷藏8h进行重结晶，得到21.2g的银杏内酯C；The elution ratio of cyclohexane: acetone is 7.2:3, and the dosage is 6BV. Collect the eluted fractions, concentrate and dry the obtained fractions to obtain a light yellow powder, and heat and dissolve them with an ethanol solution with a mass concentration of 50%. The amount of the ethanol solution was 15 times that of the dried product, the dissolved solution was filtered, and the obtained filtrate was refrigerated at 4° C. for 8 hours for recrystallization to obtain 21.2 g of ginkgolide C;
环己烷：丙酮的洗脱比例为6.2:4用量为6BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到黄色粉末，用质量浓度为60％的乙醇溶液加热溶解，所述乙醇溶液的用量为干燥后的产物的15倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏8h进行重结晶，得到19.5g的银杏内酯A；The elution ratio of cyclohexane: acetone is 6.2:4, and the dosage is 6BV for elution. The eluted cuts are collected, and the obtained cuts are concentrated and dried to obtain a yellow powder, which is dissolved by heating with an ethanol solution with a mass concentration of 60%. The amount of the ethanol solution was 15 times that of the dried product, the dissolved solution was filtered, and the obtained filtrate was refrigerated at 4° C. for 8 hours for recrystallization to obtain 19.5 g of ginkgolide A;
环己烷：丙酮的洗脱比例为1:0.5用量为6BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到黄色粉末，用质量浓度为70％的乙醇溶液进行加热溶解，所述乙醇溶液的用量为干燥后的产物的15倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏8h进行重结晶，得到16.5g的银杏内酯B。The elution ratio of cyclohexane: acetone is 1:0.5 and the dosage is 6BV for elution. The eluted fractions are collected, concentrated and dried to obtain a yellow powder, which is heated and dissolved with an ethanol solution with a mass concentration of 70%. The amount of the ethanol solution was 15 times that of the dried product, the dissolved solution was filtered, and the obtained filtrate was refrigerated at 4° C. for 8 hours for recrystallization to obtain 16.5 g of ginkgolide B.
按照实施例1所述的方法，对银杏总内酯粗品、银杏内酯混合对照品、白果内酯、银杏内酯A、银杏内酯B和银杏内酯C采用蒸发光散射检测器进行检测，检测结果为，本发明实施例2制备得到的白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的纯度为均大于99.9％。According to the method described in Example 1, the total ginkgolide crude product, ginkgolide mixed reference substance, bilobalide, ginkgolide A, ginkgolide B and ginkgolide C are detected by an evaporative light scattering detector, The test result shows that the purity of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C prepared in Example 2 of the present invention is greater than 99.9%.
本发明实施例2制备得到的白果内酯的收率为85.9％、银杏内酯A的收率为82.2％、银杏内酯B的收率为83.7％、银杏内酯C的收率为83.2％，各种产品的收率均较高。The yield of bilobalide prepared in Example 2 of the present invention is 85.9%, the yield of ginkgolide A is 82.2%, the yield of ginkgolide B is 83.7%, and the yield of ginkgolide C is 83.2%. , the yields of various products are higher.
实施例3Example 3
取20Kg银杏叶药材采用质量浓度为75％乙醇溶液回流提取两次(2h/次)，所述乙醇溶液的质量是银杏叶药材制备的10倍；将得到的提取液滤过，合并两次滤液；将合并后的滤液减压浓缩至无醇味；将得到的浓缩液加入到水分散，所述水的用量和浓缩液的用量相同；将得到的分散液滤过，除去不溶物，得澄清的滤液；将得到的澄清的滤液加入到等体积的乙酸乙酯中萃取3次，合并萃取后的产物进行浓缩、干燥，得银杏总内酯粗品253g。Get 20Kg Ginkgo biloba medical material and adopt mass concentration to be that 75% ethanol solution reflux extracts twice (2h/time), the quality of described ethanol solution is 10 times that Ginkgo biloba medical material is prepared; The extract that obtains is filtered, merge twice filtrate Concentrate the combined filtrate under reduced pressure until there is no alcohol smell; add the obtained concentrated solution to water for dispersion, and the amount of the water is the same as that of the concentrated solution; filter the obtained dispersion to remove insolubles, and obtain clarification The filtrate; The clarified filtrate obtained is added to an equal volume of ethyl acetate to extract 3 times, and the product after the merging and extraction is concentrated and dried to obtain 253 g of ginkgo lactone crude product.
将200g的银杏总内酯粗品用丙酮溶解，向得到的溶解液中加入与银杏总内酯粗品等质量的100目的硅胶拌样，搅拌均匀，烘干后研磨，得到样品硅胶。Dissolve 200 g of crude ginkgo lactones in acetone, add 100-mesh silica gel with the same quality as the crude total ginkgo lactones to the obtained solution, stir evenly, dry and grind to obtain sample silica gel.
将样品硅胶加入到真空液相色谱分离装置中进行梯度洗脱，采用的洗脱液为石油醚-丙酮。The sample silica gel was added to a vacuum liquid chromatography separation device for gradient elution, and the eluent used was petroleum ether-acetone.
石油醚：丙酮的洗脱比例为8.8:1用量为10BV洗脱，除去银杏总内酯粗品中脂肪酸类杂质；The elution ratio of petroleum ether: acetone is 8.8:1, and the amount is 10BV for elution to remove fatty acid impurities in the crude product of total ginkgo lactones;
石油醚：丙酮的洗脱比例为7.8:2用量为10BV洗脱，收集洗脱后的馏分，将得到馏分浓缩干燥得到淡黄色粉末，用质量浓度为70％的乙醇溶液进行加热溶解，所述乙醇溶液的用量为干燥后的产物的20倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏12h进行重结晶，得到48.9g的白果内酯；The elution ratio of petroleum ether: acetone is 7.8:2, and the amount is 10BV for elution. The eluted fractions are collected, and the obtained fractions are concentrated and dried to obtain a light yellow powder, which is heated and dissolved with an ethanol solution with a mass concentration of 70%. The amount of the ethanol solution was 20 times that of the dried product, the dissolved solution was filtered, and the obtained filtrate was refrigerated at 4°C for 12 hours for recrystallization to obtain 48.9 g of bilobalide;
石油醚：丙酮的洗脱比例为6.8:3用量为10BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到淡黄色粉末，用质量浓度为80％的乙醇溶液进行加热溶解，所述乙醇溶液的用量为干燥后的产物的20倍，将溶解后的溶液过滤，将得到的滤液4℃冷藏12h进行重结晶，得到20.2g的银杏内酯C；The elution ratio of petroleum ether: acetone is 6.8:3, and the amount is 10BV for elution. The eluted fractions are collected, concentrated and dried to obtain a light yellow powder, which is heated and dissolved with an ethanol solution with a mass concentration of 80%. The amount of the ethanol solution is 20 times that of the dried product, the dissolved solution is filtered, and the obtained filtrate is refrigerated at 4° C. for 12 hours for recrystallization to obtain 20.2 g of ginkgolide C;
石油醚：丙酮的洗脱比例为5.8:4用量为10BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到黄色粉末，用质量浓度为95％的乙醇溶液加热溶解，所述乙醇溶液的用量为干燥后的产物的20倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏12h进行重结晶，得到18.3g的银杏内酯A；The elution ratio of petroleum ether: acetone is 5.8:4, and the amount is 10BV for elution. Collect the eluted cuts, concentrate and dry the obtained cuts to obtain a yellow powder, and heat and dissolve the ethanol solution with a mass concentration of 95%. The ethanol The amount of the solution was 20 times that of the dried product, and the dissolved solution was filtered, and the obtained filtrate was refrigerated at 4° C. for 12 hours for recrystallization to obtain 18.3 g of ginkgolide A;
石油醚：丙酮的洗脱比例为1：1.5用量为10BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到黄色粉末，用乙醇纯品进行加热溶解，所述乙醇溶液的用量为干燥后的产物的20倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏12h进行重结晶，得到14.2g的银杏内酯B。The elution ratio of petroleum ether: acetone is 1:1.5, and the dosage is 10BV for elution. The eluted fractions are collected, concentrated and dried to obtain a yellow powder, which is heated and dissolved with pure ethanol, and the amount of the ethanol solution is The dried product was 20 times larger, and the dissolved solution was filtered, and the obtained filtrate was refrigerated at 4° C. for 12 hours for recrystallization to obtain 14.2 g of ginkgolide B.
按照实施例1所述的方法，对银杏总内酯粗品、银杏内酯混合对照品、白果内酯、银杏内酯A、银杏内酯B和银杏内酯C采用蒸发光散射检测器进行检测，检测结果为，本发明实施例3制备的到的白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的纯度为大于99.9％。According to the method described in Example 1, the total ginkgolide crude product, ginkgolide mixed reference substance, bilobalide, ginkgolide A, ginkgolide B and ginkgolide C are detected by an evaporative light scattering detector, The test result shows that the purity of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C prepared in Example 3 of the present invention is greater than 99.9%.
本发明实施例3制备得到的白果内酯的收率为84.9％、银杏内酯A的收率为81.5％、银杏内酯B的收率为82.1％、银杏内酯C的收率为82.8％，各种产品的收率均较高。The yield of bilobalide prepared in Example 3 of the present invention is 84.9%, the yield of ginkgolide A is 81.5%, the yield of ginkgolide B is 82.1%, and the yield of ginkgolide C is 82.8%. , the yields of various products are higher.
实施例4Example 4
取20Kg银杏叶药材采用质量浓度为75％乙醇溶液回流提取两次(2h/次)，所述乙醇溶液的质量是银杏叶药材制备的10倍；将得到的提取液滤过，合并两次滤液；将合并后的滤液减压浓缩至无醇味；将得到的浓缩液加入到水分散，所述水的用量和浓缩液的用量相同；将得到的分散液滤过，除去不溶物，得澄清的滤液；将得到的澄清的滤液加入到等体积的乙酸乙酯中萃取3次，合并萃取后的产物进行浓缩、干燥，得银杏总内酯粗品258g。Get 20Kg Ginkgo biloba medical material and adopt mass concentration to be that 75% ethanol solution reflux extracts twice (2h/time), the quality of described ethanol solution is 10 times that Ginkgo biloba medical material is prepared; The extract that obtains is filtered, merge twice filtrate Concentrate the combined filtrate under reduced pressure until there is no alcohol smell; add the obtained concentrated solution to water for dispersion, and the amount of the water is the same as that of the concentrated solution; filter the obtained dispersion to remove insolubles, and obtain clarification The filtrate; The clarified filtrate obtained is added to an equal volume of ethyl acetate to extract 3 times, and the product after the merging and extraction is concentrated and dried to obtain 258 g of ginkgo lactone crude product.
将200g的银杏总内酯粗品用乙酸乙酯溶解，向得到的溶解液中加入与银杏总内酯粗品等质量的95目的硅胶拌样，搅拌均匀，烘干后研磨，得到样品硅胶。Dissolve 200 g of crude ginkgo lactones with ethyl acetate, add 95-mesh silica gel with the same quality as the crude total ginkgo lactones to the obtained solution, mix the sample, stir evenly, dry and grind to obtain sample silica gel.
将样品硅胶加入到真空液相色谱分离装置中进行梯度洗脱，采用的洗脱液为环己烷-乙酸乙酯。The sample silica gel was added to a vacuum liquid chromatography separation device for gradient elution, and the eluent used was cyclohexane-ethyl acetate.
环己烷：乙酸乙酯的洗脱比例为9:1用量为7BV洗脱，除去银杏总内酯粗品中脂肪酸类杂质；The elution ratio of cyclohexane: ethyl acetate is 9:1 and the dosage is 7BV elution to remove fatty acid impurities in the crude product of total ginkgo lactones;
环己烷：乙酸乙酯的洗脱比例为8:2用量为7BV洗脱，收集洗脱后的馏分，将得到馏分浓缩干燥得到淡黄色粉末，用质量浓度为60％的乙醇溶液进行加热溶解，所述乙醇溶液的用量为干燥后的产物的17倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏9h进行重结晶，得到50.6g的白果内酯；The elution ratio of cyclohexane:ethyl acetate is 8:2, and the dosage is 7BV for elution. Collect the eluted fractions, concentrate and dry the obtained fractions to obtain a light yellow powder, and heat and dissolve with an ethanol solution with a mass concentration of 60%. , the amount of the ethanol solution is 17 times that of the dried product, the dissolved solution is filtered, and the obtained filtrate is refrigerated at 4° C. for 9 hours for recrystallization to obtain 50.6 g of bilobalide;
环己烷：乙酸乙酯的洗脱比例为7:3用量为7BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到淡黄色粉末，用质量浓度为60％的乙醇溶液进行加热溶解，所述乙醇溶液的用量为干燥后的产物的16倍，将溶解后的溶液过滤，将得到的滤液4℃冷藏10h进行重结晶，得到22.3g的银杏内酯C；The elution ratio of cyclohexane:ethyl acetate is 7:3, and the dosage is 7BV. Collect the eluted fractions, concentrate and dry the obtained fractions to obtain a light yellow powder, and heat it with an ethanol solution with a mass concentration of 60%. Dissolved, the amount of the ethanol solution was 16 times that of the dried product, the dissolved solution was filtered, and the obtained filtrate was refrigerated at 4°C for 10 hours for recrystallization to obtain 22.3g of ginkgolide C;
环己烷：乙酸乙酯的洗脱比例为6:4用量为9BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到黄色粉末，用质量浓度为85％的乙醇溶液加热溶解，所述乙醇溶液的用量为干燥后的产物的18倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏11h进行重结晶，得到19.6g的银杏内酯A；The elution ratio of cyclohexane:ethyl acetate is 6:4, and the amount is 9BV for elution. Collect the eluted fractions, concentrate and dry the obtained fractions to obtain a yellow powder, and heat and dissolve with an ethanol solution with a mass concentration of 85%. The amount of the ethanol solution was 18 times that of the dried product, the dissolved solution was filtered, and the obtained filtrate was refrigerated at 4° C. for 11 h for recrystallization to obtain 19.6 g of ginkgolide A;
环己烷：乙酸乙酯的洗脱比例为1:1用量为9BV洗脱，收集洗脱后的馏分，将得到的馏分浓缩干燥得到黄色粉末，用乙醇纯品进行加热溶解，所述乙醇溶液的用量为干燥后的产物的19倍，将溶解后的溶液过滤，将得到的滤液在4℃冷藏9h进行重结晶，得到15.3g的银杏内酯B。The elution ratio of cyclohexane:ethyl acetate is 1:1, and the dosage is 9BV for elution. The eluted fractions are collected, and the obtained fractions are concentrated and dried to obtain a yellow powder, which is heated and dissolved with pure ethanol, and the ethanol solution The amount of Ginkgolide is 19 times that of the dried product, the dissolved solution is filtered, and the obtained filtrate is refrigerated at 4° C. for 9 hours for recrystallization to obtain 15.3 g of ginkgolide B.
按照实施例1所述的方法，对银杏总内酯粗品、银杏内酯混合对照品、白果内酯、银杏内酯A、银杏内酯B和银杏内酯C采用蒸发光散射检测器进行检测，检测结果为，本发明实施例4制备得到的白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的纯度为大于99.9％。According to the method described in Example 1, the total ginkgolide crude product, ginkgolide mixed reference substance, bilobalide, ginkgolide A, ginkgolide B and ginkgolide C are detected by an evaporative light scattering detector, The test result shows that the purity of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C prepared in Example 4 of the present invention is greater than 99.9%.
本发明实施例4制备得到的白果内酯的收率为87.8％、银杏内酯A的收率为83.6％、银杏内酯B的收率为83.8％、银杏内酯C的收率为83.5％，各种产品的收率均较高。The yield of bilobalide prepared in Example 4 of the present invention is 87.8%, the yield of ginkgolide A is 83.6%, the yield of ginkgolide B is 83.8%, and the yield of ginkgolide C is 83.5%. , the yields of various products are higher.
由以上实施例可知，本发明提供了一种白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的制备方法，包括：将银杏叶采用醇类化合物回流提取后经过滤过、浓缩、分散、再次滤过，得到滤液；将所述滤液萃取后依次进浓缩和干燥，得到银杏总内酯粗品；将所述银杏总内酯粗品在溶剂中和硅胶混合后粉碎，得到样品硅胶；将所述样品硅胶在真空液相色谱分离装置中进行梯度洗脱，所述梯度洗脱过程中的洗脱液包括弱极性溶剂A和强极性溶剂B，所述弱极性溶剂A和强极性溶剂B的洗脱比例为(8.2～7.8)：(1.8～2.2)，得到白果内酯；所述弱极性溶剂A和强极性溶剂B的洗脱比例为(7.2～6.8)：(3.2～2.8)，得到银杏内酯C；所述弱极性溶剂A和强极性溶剂B的洗脱比例为(6.2～5.8)：(4.2～3.8)，得到银杏内酯A；所述弱极性溶剂A和强极性溶剂B的洗脱比例为1：(0.5～1.5)，得到银杏内酯B；所述弱极性溶剂A包括石油醚或环己烷；所述强极性溶剂B包括乙酸乙酯或丙酮。Known from the above examples, the present invention provides a kind of preparation method of bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, comprising: after Ginkgo biloba adopts alcoholic compound to reflux extract, filter, Concentrate, disperse, and filter again to obtain a filtrate; extract the filtrate and then concentrate and dry it successively to obtain the crude product of total ginkgo lactones; mix the crude product of total ginkgo lactones with silica gel in a solvent and pulverize to obtain a sample silica gel ; The sample silica gel is carried out gradient elution in a vacuum liquid chromatography separation device, and the eluent in the gradient elution process includes a weak polar solvent A and a strong polar solvent B, and the weak polar solvent A And the elution ratio of strong polar solvent B is (8.2～7.8): (1.8～2.2), obtains bilobalide; The elution ratio of described weak polar solvent A and strong polar solvent B is (7.2～6.8 ): (3.2～2.8), ginkgolide C is obtained; the elution ratio of the weak polar solvent A and strong polar solvent B is (6.2～5.8): (4.2～3.8), ginkgolide A is obtained; The elution ratio of the weak polar solvent A and the strong polar solvent B is 1: (0.5～1.5), to obtain ginkgolide B; the weak polar solvent A includes sherwood oil or cyclohexane; the strong polar solvent Polar solvent B includes ethyl acetate or acetone.
本发明提供的方法通过对银杏叶进行合适的处理，制备样品硅胶，采用真空液相色谱分离装置同时控制梯度洗脱的工艺条件制备银杏内酯，这种方法制备得到的白果内酯、银杏内酯A、银杏内酯B和银杏内酯C的收率和纯度均较高。此外，本发明提供的方法能够同时制备得到白果内酯、银杏内酯A、银杏内酯B和银杏内酯C，制备过程简单、快速、制备量大。The method provided by the invention prepares sample silica gel by properly treating ginkgo leaves, and uses a vacuum liquid chromatography separation device to simultaneously control the process conditions of gradient elution to prepare ginkgolides. The bilobalide and ginkgolides prepared by this method are The yield and purity of ester A, ginkgolide B and ginkgolide C were high. In addition, the method provided by the present invention can simultaneously prepare bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, and the preparation process is simple, fast and has a large amount of preparation.
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