CN101926835B - Method for extracting gingko total terpene lactones and injection containing same - Google Patents
Method for extracting gingko total terpene lactones and injection containing same Download PDFInfo
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Abstract
The invention relates a method for extracting gingko total terpene lactones and injection containing the gingko total terpene lactones, belonging to the traditional Chinese medicine technical field. The invention overcomes the defects of low purity, easily damaged active ingredients, unstable injection and the like in the existing extraction separation technology, and provides a method for extracting and separating the gingko total terpene lactones by comprehensively applying multi-technology ways, as well as discloses the injection containing the gingko total terpene lactones prepared by the method. Compared with the prior gingko injection, the injection prepared by the invention has the advantages of high total terpene lactones content and full ingredients; and the ingredients content of the injection reaches above 85% by detection.
Description
Technical field
The present invention relates to a kind of injection that extracts the method for gingko total terpene lactones and contain gingko total terpene lactones, belong to technical field of Chinese medicines.
Background technology
Folium Ginkgo extract has the dual function of treatment and health care, and it can improve cardiovascular and cerebrovascular circulation, the treatment cardiovascular and cerebrovascular disease, and improving memory is removed oxygen-derived free radicals, slow down aging.Main active is flavonoid and terpene lactones in the Semen Ginkgo extrac, terpene lactones mainly comprises bilobalide and ginkalide A, B, C, M, J etc., wherein bilobalide belongs to sesquiterpenoids, and ginkalide A, B, C, M, J belong to diterpene-kind compound.Bilobalide is nervous system, psychotic medicine, and the moronism phenomenon that occurs is with age had unusual curative effect, also is used for the treatment of neuropathy, encephalopathy and myelopathy etc.; Bilobalide has important pharmacological action as the higher platelet antagonism factor of a kind of activity in the pathological processes such as shock that thrombosis, organ transplantation rejection, senile dementia, heart allergy, endotoxin cause, development and application has a extensive future.
Bilobalide and bilobalide are present unique particular matters of finding from Semen Ginkgo, because the space structure complexity, chirality is changeable, and synthetic very trouble can only be extracted from Folium Ginkgo at present.Scientist begins one's study to bilobalide and comprises separation and purification, makes basic research such as structure from the beginning of the sixties in last century.The mid-1970s Semen Ginkgo extrac (GBE) listing, countries in the world food and health food manufacturer all put into the exploitation of Folium Ginkgo with great enthusiasm.Adopt organic solvent multitple extraction and kieselguhr, silica gel adsorption-eluting or activated carbon adsorption-eluting and lead acetate to precipitate to send out bonded extracting method at present abroad, step is numerous and diverse, yield rate is low, purity is also lower.Domestic also have the lixiviate of employing, column purification, extraction, separation, method for crystallising to prepare bilobalide, but purity is still lower.
Solvent extraction method is the representative method for preparing Semen Ginkgo extrac, Schwabe as Germany, the Flaschman pharmaceutical factory, be mainly Semen Ginkgo cured leaf coarse powder, its preparation technology is that 60% acetone extracts, filter pressing, filtrate is with the carbon tetrachloride eluting, the acetone water layer is saturated with ammonium sulfate, extract with butanone, be made into certain density alcoholic solution behind the extracting solution concentrating under reduced pressure, add an amount of PB (OH) 2 and filter, it is saturated at an ammonium sulfate that filtrate concentrates the back, the butanone extraction, the dry back that concentrates of extracting solution is with dry GBE (the Deutsche Bundespatent ED2117429 of getting of the refining reconcentration of ethanol, 1767098).
Bilobalide-like substances content in Folium Ginkgo is very low, generally has only about 0.3%, and changes along with changes of seasons, therefore, the content of gingko total terpene lactones in the Semen Ginkgo thing extract to be improved as far as possible, and on this basis, impurity be removed as much as possible.Because Folium Ginkgo terpene lactones is slightly soluble in water, so according to its physicochemical property, the dissolubility of selecting for use suitable approach to strengthen it also is the key of injection technological forming, and this process can't be destroyed the chiral structure of Ginkgo total lactones.
Extracting method for Folium Ginkgo terpene lactones, domestic existing detailed bibliographical information, for example, people such as Li Xingang have introduced the experiment extracting method of bilobalide (with reference to Li Xingang etc., the laboratory Study on Extraction Method of bilobalide in the Folium Ginkgo, assorted 1998 the 1st phases of Chinese Medicine industry), Chinese patent literature CN1195665A has introduced the extracting method of bilobalide and has contained the preparation of bilobalide, this method is that Folium Ginkgo is extracted with water boil, with adsorbent extraction filtrate is adsorbed the reuse ethanol elution, reclaim ethanol, with the crystallization dissolving of separating out, recrystallization, drying makes Folium Ginkgo terpene lactones.Chinese patent literature CN1313287A also discloses a kind of production technology of bilobalide.The method of separating ginkgolide monomer in the middle of the total extract of bilobalide, bibliographical information is also arranged, for example, people such as You Song have introduced the separation of bilobalide and structure determination method (referring to You Song etc., the separation of bilobalide and structure determination in the Folium Ginkgo, Chinese pharmaceutical chemistry magazine the 4th phase of nineteen ninety-five).Chinese patent literature CN1287121A discloses the method that is prepared ginkalide A, B by Folium Ginkgo or Folium Ginkgo extractum.Bilobalide is made injection, a very important technical problem is how to solve its slightly solubility, up to the present, and in the middle of the bilobalide injector preparation process, selection for cosolvent is also extremely important, and this directly has influence on the stability and the clinical efficacy of product.
According to the lactonic ring structure of bilobalide, generally be easy to expect making its open loop with acid or alkali, perhaps use organic solvent dissolution, but its dissolubility in general organic solvent is also very poor, as at PEG400, and glycerol, ethanol, the dissolubility in the propylene glycol is all very little.So at present the bilobalide method for preparing injection has the dissolubility by using aqueous slkali to increase bilobalide to make, CN200510040497.0 for example, CN02134223.7, CN02134223.7; Under the situation that adds meglumine, the dissolubility of bilobalide also can well be increased, for example CN02128962.X; Adopt the HP-solubilising also can make its aqueous solution to bilobalide, CN200510072466.3 for example, CN200410013937.9, CN200410041120.2; In addition, contain 5-99% Folium Ginkgo extract, 1-95% poloxamer 188 and pH value regulator and other excipient in the open injection of CN0310034.6; The preparation method of a CN03137720.3 injection of ginkgolide B, bilobalide B raw material is dissolved in the water for injection solvent Polyethylene Glycol, Polyethylene Glycol can use separately or be made into mixed solvent with dehydrated alcohol, by volume, and Polyethylene Glycol: ethanol=0.5: 0.5～0.9.
But, add alkali or add meglumine and all belong to and allow lactonic ring open loop salify, under neutrality or acid condition, be difficult to form again the lactone original shape of effective situation, even do not have bilobalide.And in the Folium Ginkgo leaching process, bilobalide is hydrolysis or oxidation very easily.And bilobalide is present chemical constituent of greatest concern; the animal experiment report; bilobalide has the effect that promotes nerve growth; and the effect that prevents brain, spinal nerves demyelination; its neurotrophy, neuroprotective are stronger than bilobalide; be that content of bilobalide is 2.6-3.2% to the requirement of Semen Ginkgo extrac in the world at present.The Folium Ginkgo extract injection of listing mainly contains the Ginaton injection that the big pharmaceutical factory of homemade SHUXUENING ZHUSHEYE and German Weil-McLain Shu Pei produces both at home and abroad.Because need high temperature sterilize in the injection production process, wherein bilobalide and ginkalide B isoreactivity composition major part are destroyed in the bilobalide, content is very low in the preparation.For Folium Ginkgo extract active component terpene lactone class, Ginaton injection does not pass a test because of preparation process, and the bilobalide major part is destroyed, does not detect its total terpene lactones in the end product quality standard; Homemade SHUXUENING ZHUSHEYE only requires to detect the content of ginkalide A.After testing, the content of two kinds of product bilobalide and ginkalide B is all very low more than.
In order to solve purity problem and validity problem, the invention discloses a kind of process for purification of gingko total terpene lactones, and make stable injection, and each component ratio is very approaching in its composition and the Folium Ginkgo with this total terpene lactones.
Summary of the invention
First technical problem to be solved by this invention is to overcome that existing extraction and separation technology moderate purity is not high, effective ingredient is destroyed easily and the deficiency of injection instability etc., provides a kind of integrated use multiple technologies means to extract, separate the method for gingko total terpene lactones.
The method of extraction separation gingko total terpene lactones involved in the present invention comprises the steps:
1) get Folium Ginkgo, add ethanol and citric acid or tartaric acid, reflux, extract, concentrates alcohol extract;
2) macroporous resin column on the step 1) concentrate, gradient elution is collected eluent, concentrates;
3) step 2) silicagel column on the concentrate, eluent ethyl acetate is collected eluent, drying;
4) the dry thing of step 3) adds water through dissolve with ethanol, places time crystallized product of winning; Mother solution reclaims ethanol, and the water liquid cooling is heavy, gets crystallized product for the second time; Merge twice crystallized product and promptly get gingko total terpene lactones.
Preferably, the described concentration of ethanol of step 1) is 60%～80%, and described citric acid or tartaric addition are 0.1%～1%;
Preferably, the step 1) ratio that concentrates alcohol extract for the medical material ratio be 1: 0.8～1;
Preferably, step 2) wash 3～5 times of column volumes during eluting with water, with 3～5 times of column volumes of ethanol elution of 20%, remove impurity discards again, uses the column volume of 3～6 times of 75% ethanol elutions then;
Preferably, on the described sample of step 3) before the silicagel column, with step 2) extract obtainedly add an amount of water, fully mix thoroughly with an amount of silica gel, go up silicagel column behind the vacuum drying;
Second technical problem to be solved by this invention provides a kind of injection that contains above-mentioned gingko total terpene lactones.Because Folium Ginkgo terpene lactones is slightly soluble in water, so how to increase the key that its dissolubility in injection solvent is the preparation injection.
The injection that contains gingko total terpene lactones involved in the present invention, it is grouped into by following one-tenth:
Gingko total terpene lactones 0.8%～1.2%
Excipient 10%～60%
The PH regulator is an amount of
Antioxidant 0.02%～0.04%
Water for injection 40%～89%
Wherein, described excipient comprises ethylene glycol, ethanol, Polyethylene Glycol, 1, in the 2-propylene glycol one or both, described PH regulator is a kind of of following buffer: dibastic sodium phosphate-biphosphate is received, citric acid-sodium citrate or acetic acid-sodium acetate, and the consumption of PH regulator is to make the PH of injection transfer to 3.5～5.0 to get final product; Described antioxidant is a kind of in sodium sulfite, the disodiumedetate.
Preferably, the described PH regulator dibastic sodium phosphate-biphosphate that is a kind of of following buffer: PH3.2～4.8 receive, the citric acid-sodium citrate of PH4.4～4.8, the acetic acid-sodium acetate of PH3.8～5.2.
Compared with the prior art, the inventive method has following obvious improvement:
1) in bilobalide and the bilobalide molecular structure a lot of chiral carbon is arranged, the fracture of lactonic ring or conformational change all may have influence on its pharmacology, drug effect, the present invention creatively uses buffer solution to transfer pH value to 3.5～5.5, can avoid the fracture of lactonic ring effectively, thereby make gained injection curative effect better;
2) adopt two kinds of column chromatography method combinations of macroporous resin and silicagel column to carry out remove impurity, can farthest remove impurity; Macroporous resin column can be adsorbed the relative less material with the flavonoid isopolarity of lactone effectively, can remove saccharide and the bigger impurity of aminoacid isopolarity; Silicagel column can be removed ginkgetin glycoside impurity effectively;
3) adopt the mode of fractional crystallization that the lactone composition is separated out one by one, make that lactone composition purity is higher, impurity still less;
4) compare with existing Semen Ginkgo injection, with the resulting total terpene lactones content height of the present invention, composition is complete, after measured, its total terpene lactones content reaches more than 85%, wherein, not only contain bilobalide, also contain ginkalide A, B, C etc., the most approaching with each component ratio of Folium Ginkgo.
5) because of terpene lactones is insoluble in water, and unstable, the present invention has adopted double solvent to make injection, thereby has improved the stability of the dissolubility and the injection of terpene lactones effectively;
6) before extraction, in ethanol, added an amount of citric acid, tartaric acid, can stop the hydrolysis of bilobalide effectively, make natural ratio basically identical in the ratio of lactone and the leaf.
The specific embodiment
The preparation of embodiment 1 gingko total terpene lactones
1) gets 10 kilograms of dry Folium Ginkgos, add 60% alcohol reflux twice, each reflux, extract, 1 hour, alcohol adding amount is 50 kilograms and (wherein adds citric acid 50 grams for the first time, i.e. 0.1% citric acid), alcohol adding amount is 40 kilograms (wherein adding citric acid 40 gram, i.e. 0.1% citric acid) for the second time, merges alcohol extract twice, filter with 200 purpose filter clothes, decompression recycling ethanol under 0.06～0.08 atmospheric pressure is concentrated into 8 kilograms with extracting solution, and is stand-by.
2) get 10 kilograms of the macroporous resins (AB-8 type) handled well, wet method dress post, last sample, flow velocity is 120ml/min, then, and successively with 30 kilograms of water elutions, flow velocity is 250ml/min, and 30 kilograms of the ethanol elutions with 20%, flow velocity are 280ml/min, discard, with 30 kilograms of 70% ethanol elutions, flow velocity is 260ml/min then, collect eluent, at 80 ℃, reclaim under reduced pressure alcohol eluen under 0.06～0.08 atmospheric pressure, be concentrated into 5 kilograms, stand-by.
3) accurate weighing 200 purpose silica gel Hs are 3 kilograms, and wet method dress post slowly adds above-mentioned 5 kilograms sample, the adjustment flow velocity is 180ml/min, and last sample finishes, with 30 kilograms of eluent ethyl acetates, flow velocity is 250ml/min, collect eluent,, eluent ethyl acetate liquid is concentrated in 0.06 atmospheric pressure, 70 ℃ of reclaim under reduced pressure ethyl acetate, 0.08 individual atmospheric pressure, 95 ℃ of vacuum dryings get dry thing 385 grams, and are stand-by.
4) above-mentioned dry thing is restrained dissolve with ethanols with 1200, add 360 gram water, room temperature was placed 20 hours down, got primary crystallization product (main bilobalide-containing A, B, C); Mother solution is decompression recycling ethanol on rotation volatilization instrument, and water liquid filters 2～4 ℃ of Cryoprecipitation 24 hours, crystallized product (contain bilobalide, and a spot of ginkalide A, B, C) for the second time, merge last twice crystallized product, promptly get exsiccant gingko total terpene lactones 60.0 grams, standby.
Measure (with reference to the mensuration of 2005 editions the 221st page of Folium Ginkgo terpene lactones of Chinese Pharmacopoeia) through high performance liquid chromatogram, its bilobalide, Ginkgo total lactones A, B, the total content of four kinds of terpene lactoness of C are 95.6%.
The preparation of embodiment 2 gingko total terpene lactones
1) gets 10 kilograms of dry Folium Ginkgos, add 80% alcohol reflux twice, each reflux, extract, 2 hours, alcohol adding amount is 90 kilograms and (wherein adds citric acid 900 grams for the first time, i.e. 1% citric acid), alcohol adding amount is 70 kilograms (wherein adding citric acid 700 gram, i.e. 1% citric acid) for the second time, merges alcohol extract twice, filter with 200 purpose filter clothes, decompression recycling ethanol under 0.06～0.08 atmospheric pressure is concentrated into 10 kilograms with extracting solution, and is stand-by.
2) get 10 kilograms of the macroporous resins (D-101 type) handled well, wet method dress post, last sample, flow velocity is 120ml/min, then, and successively with 50 kilograms of water elutions, flow velocity is 250ml/min, and 50 kilograms of the ethanol elutions with 20%, flow velocity are 280ml/min, discard, with 60 kilograms of 70% ethanol elutions, flow velocity is 260ml/min then, collect eluent, at 80 ℃, reclaim under reduced pressure alcohol eluen under 0.06～0.08 atmospheric pressure, be concentrated into 5 kilograms, stand-by.
3) accurate weighing 200 purpose silica gel Hs are 3 kilograms, and wet method dress post slowly adds above-mentioned 5 kilograms sample, the adjustment flow velocity is 180ml/min, and last sample finishes, with 60 kilograms of eluent ethyl acetates, flow velocity is 250ml/min, collect eluent,, eluent ethyl acetate liquid is concentrated in 0.06 atmospheric pressure, 70 ℃ of reclaim under reduced pressure ethyl acetate, 0.08 individual atmospheric pressure, 95 ℃ of vacuum dryings get dry thing 412 grams, and are stand-by.
4) above-mentioned dry thing is restrained dissolve with ethanols with 1200, add 360 gram water, room temperature was placed 20 hours down, got primary crystallization product (main bilobalide-containing A, B, C); Mother solution is decompression recycling ethanol on rotation volatilization instrument, and water liquid filters 2～4 ℃ of Cryoprecipitation 24 hours, crystallized product (contain bilobalide, and a spot of ginkalide A, B, C) for the second time, merge last twice crystallized product, promptly get exsiccant gingko total terpene lactones 58.5 grams, standby.
Measure (with reference to the mensuration of 2005 editions the 221st page of Folium Ginkgo terpene lactones of Chinese Pharmacopoeia) through high performance liquid chromatogram, its bilobalide, Ginkgo total lactones A, B, the total content of four kinds of terpene lactoness of C are 95.1%.
The preparation of embodiment 3 gingko total terpene lactones
1) gets 10 kilograms of dry Folium Ginkgos, add 70% alcohol reflux twice, each reflux, extract, 1.5 hours, alcohol adding amount is 70 kilograms and (wherein adds citric acid 350 grams for the first time, i.e. 0.5% citric acid), alcohol adding amount is 60 kilograms (wherein adding citric acid 300 gram, i.e. 0.5% citric acid) for the second time, merges alcohol extract twice, filter with 200 purpose filter clothes, decompression recycling ethanol under 0.06～0.08 atmospheric pressure is concentrated into 9 kilograms with extracting solution, and is stand-by.
2) get 10 kilograms of the macroporous resins (HPD-100 type) handled well, wet method dress post, last sample, flow velocity is 120ml/min, then, and successively with 40 kilograms of water elutions, flow velocity is 260ml/min, and 40 kilograms of the ethanol elutions with 20%, flow velocity are 280ml/min, discard, with 40 kilograms of 70% ethanol elutions, flow velocity is 260ml/min then, collect eluent, at 80 ℃, reclaim under reduced pressure alcohol eluen under 0.06～0.08 atmospheric pressure, be concentrated into 5.2 kilograms, stand-by.
3) accurate weighing 200 purpose silica gel Hs are 3 kilograms, and wet method dress post slowly adds above-mentioned 5.2 kilograms sample, the adjustment flow velocity is 180ml/min, and last sample finishes, with 40 kilograms of eluent ethyl acetates, flow velocity is 250ml/min, collect eluent,, eluent ethyl acetate liquid is concentrated in 0.06 atmospheric pressure, 70 ℃ of reclaim under reduced pressure ethyl acetate, 0.08 individual atmospheric pressure, 95 ℃ of vacuum dryings get dry thing 395 grams, and are stand-by.
4) above-mentioned dry thing is restrained dissolve with ethanols with 1200, add 365 gram water, room temperature was placed 20 hours down, got primary crystallization product (main bilobalide-containing A, B, C); Mother solution is decompression recycling ethanol on rotation volatilization instrument, and water liquid filters 2～4 ℃ of Cryoprecipitation 24 hours, crystallized product (contain bilobalide, and a spot of ginkalide A, B, C) for the second time, merge last twice crystallized product, promptly get exsiccant gingko total terpene lactones 60.6 grams, standby.
Measure (with reference to the mensuration of 2005 editions the 221st page of Folium Ginkgo terpene lactones of Chinese Pharmacopoeia) through high performance liquid chromatogram, its bilobalide, Ginkgo total lactones A, B, the total content of four kinds of terpene lactoness of C are 96.5%.
The preparation of embodiment 4 gingko total terpene lactones
1) gets 10 kilograms of dry Folium Ginkgos, add 75% alcohol reflux twice, reflux, extract, is 2 hours for the first time, for the second time each reflux, extract, 1.5 hours, for the first time alcohol adding amount is 80 kilograms of (tartarize 480 grams wherein, i.e. 0.6% citric acid), for the second time alcohol adding amount is 60 kilograms and (wherein adds tartaric acid 360 grams, i.e. 0.6% citric acid), merge alcohol extract twice, filter decompression recycling ethanol under 0.06～0.08 atmospheric pressure with 200 purpose filter clothes, extracting solution is concentrated into 8.6 kilograms, stand-by.
2) get 10 kilograms of the macroporous resins (HPD-100 type) handled well, wet method dress post, last sample, flow velocity is 115ml/min, then, and successively with 30 kilograms of water elutions, flow velocity is 260ml/min, and 60 kilograms of the ethanol elutions with 20%, flow velocity are 290ml/min, discard, with 50 kilograms of 70% ethanol elutions, flow velocity is 275ml/min then, collect eluent, at 80 ℃, reclaim under reduced pressure alcohol eluen under 0.06～0.08 atmospheric pressure, be concentrated into 4.9 kilograms, stand-by.
3) accurate weighing 200 purpose silica gel Hs are 3 kilograms, and wet method dress post slowly adds above-mentioned 4.9 kilograms sample, the adjustment flow velocity is 175ml/min, and last sample finishes, with 50 kilograms of eluent ethyl acetates, flow velocity is 250ml/min, collect eluent,, eluent ethyl acetate liquid is concentrated in 0.06 atmospheric pressure, 70 ℃ of reclaim under reduced pressure ethyl acetate, 0.08 individual atmospheric pressure, 95 ℃ of vacuum dryings get dry thing 398 grams, and are stand-by.
4) above-mentioned dry thing is restrained dissolve with ethanols with 1150, add 365 gram water, room temperature was placed 20 hours down, got primary crystallization product (main bilobalide-containing A, B, C); Mother solution is decompression recycling ethanol on rotation volatilization instrument, and water liquid filters 2～4 ℃ of Cryoprecipitation 24 hours, crystallized product (contain bilobalide, and a spot of ginkalide A, B, C) for the second time, merge last twice crystallized product, promptly get exsiccant gingko total terpene lactones 61.6 grams, standby.
Measure (with reference to the mensuration of 2005 editions the 221st page of Folium Ginkgo terpene lactones of Chinese Pharmacopoeia) through high performance liquid chromatogram, its bilobalide, Ginkgo total lactones A, B, the total content of four kinds of terpene lactoness of C are 94.1%.
The preparation of embodiment 5 gingko total terpene lactones
1) gets 10 kilograms of dry Folium Ginkgos, add 85% alcohol reflux twice, reflux, extract, is 1.5 hours for the first time, for the second time each reflux, extract, 2 hours, for the first time alcohol adding amount is 90 kilograms of (tartarize 720 grams wherein, i.e. 0.8% tartaric acid, for the second time alcohol adding amount is 50 kilograms and (wherein adds tartaric acid 400 grams, i.e. 0.8% tartaric acid), merge alcohol extract twice, filter decompression recycling ethanol under 0.06～0.08 atmospheric pressure with 200 purpose filter clothes, extracting solution is concentrated into 9.15 kilograms, stand-by.
2) get 10 kilograms of the macroporous resins (HPD-100 type) handled well, wet method dress post, last sample, flow velocity is 125ml/min, then, and successively with 40 kilograms of water elutions, flow velocity is 260ml/min, and 40 kilograms of the ethanol elutions with 20%, flow velocity are 290ml/min, discard, with 60 kilograms of 70% ethanol elutions, flow velocity is 275ml/min then, collect eluent, at 80 ℃, reclaim under reduced pressure alcohol eluen under 0.06～0.08 atmospheric pressure, be concentrated into 5.05 kilograms, stand-by.
3) accurate weighing 200 purpose silica gel Hs are 3 kilograms, and wet method dress post slowly adds above-mentioned 5.05 kilograms sample, the adjustment flow velocity is 185ml/min, and last sample finishes, with 55 kilograms of eluent ethyl acetates, flow velocity is 2650ml/min, collect eluent,, eluent ethyl acetate liquid is concentrated in 0.06 atmospheric pressure, 70 ℃～80 ℃ reclaim under reduced pressure ethyl acetate, 0.08 individual atmospheric pressure, 95 ℃ of vacuum dryings get dry thing 402.5 grams, and are stand-by.
4) above-mentioned dry thing is restrained dissolve with ethanols with 1180, add 375 gram water, room temperature was placed 20 hours down, got primary crystallization product (main bilobalide-containing A, B, C); Mother solution is decompression recycling ethanol on rotation volatilization instrument, and water liquid filters 2～4 ℃ of Cryoprecipitation 24 hours, crystallized product (contain bilobalide, and a spot of ginkalide A, B, C) for the second time, merge last twice crystallized product, promptly get exsiccant gingko total terpene lactones 60.8 grams, standby.
Measure (with reference to the mensuration of 2005 editions the 221st page of Folium Ginkgo terpene lactones of Chinese Pharmacopoeia) through high performance liquid chromatogram, its bilobalide, Ginkgo total lactones A, B, the total content of four kinds of terpene lactoness of C are 94.7%.
The preparation of embodiment 6 gingko total terpene lactones
1) gets 10 kilograms of dry Folium Ginkgos, add 80% alcohol reflux twice, reflux, extract, is 1.5 hours for the first time, for the second time each reflux, extract, 1 hour, for the first time alcohol adding amount is 80 kilograms of (tartarize 720 grams wherein, i.e. 0.9% tartaric acid, for the second time alcohol adding amount is 80 kilograms and (wherein adds tartaric acid 720 grams, i.e. 0.9% tartaric acid), merge alcohol extract twice, filter decompression recycling ethanol under 0.06～0.08 atmospheric pressure with 200 purpose filter clothes, extracting solution is concentrated into 9.26 kilograms, stand-by.
2) get 10 kilograms of the macroporous resins (AB-8 type) handled well, wet method dress post, last sample, flow velocity is 135ml/min, then, and successively with 50 kilograms of water elutions, flow velocity is 260ml/min, and 50 kilograms of the ethanol elutions with 20%, flow velocity are 290ml/min, discard, with 60 kilograms of 70% ethanol elutions, flow velocity is 275ml/min then, collect eluent, at 80 ℃, reclaim under reduced pressure alcohol eluen under 0.06～0.08 atmospheric pressure, be concentrated into 4.98 kilograms, stand-by.
3) accurate weighing 200 purpose silica gel Hs are 3 kilograms, and wet method dress post slowly adds above-mentioned 4.98 kilograms sample, the adjustment flow velocity is 195ml/min, and last sample finishes, with 60 kilograms of eluent ethyl acetates, flow velocity is 255ml/min, collect eluent,, eluent ethyl acetate liquid is concentrated in 0.06 atmospheric pressure, 70 ℃～80 ℃ reclaim under reduced pressure ethyl acetate, 0.08 individual atmospheric pressure, 95 ℃ of vacuum dryings get dry thing 399.5 grams, and are stand-by.
4) above-mentioned dry thing is restrained dissolve with ethanols with 1180, add 375 gram water, room temperature was placed 20 hours down, got primary crystallization product (main bilobalide-containing A, B, C); Mother solution is decompression recycling ethanol on rotation volatilization instrument, and water liquid filters 2～4 ℃ of Cryoprecipitation 24 hours, crystallized product (contain bilobalide, and a spot of ginkalide A, B, C) for the second time, merge last twice crystallized product, promptly get exsiccant gingko total terpene lactones 60.2 grams, standby.
Measure (with reference to the mensuration of 2005 editions the 221st page of Folium Ginkgo terpene lactones of Chinese Pharmacopoeia) through high performance liquid chromatogram, its bilobalide, Ginkgo total lactones A, B, the total content of four kinds of terpene lactoness of C are 94.1%.
The preparation of embodiment 7 gingko total terpene lactones injection
Prescription: Folium Ginkgo terpene lactones 60.0 grams
1,2500 milliliters of 2-propylene glycol
500 milliliters of 95% ethanol
It is an amount of that dibastic sodium phosphate-biphosphate is received buffer solution
2000 milliliters of waters for injection
Make 500 (10 milliliters /)
Get total terpene lactones extract 60.0 grams of the foregoing description 1 preparation, earlier, slowly add 1 again, the 2-propylene glycol with 95% dissolve with ethanol, add water for injection at last, water for injection, 1 wherein, 2-propylene glycol and consumption of ethanol ratio mix and are water for injection: ethanol (95%): 1,2-propylene glycol=5: 1: 4, the water for injection that promptly in the total terpene lactones extract of 60 grams, adds 2500 milliliters, 500 milliliters of 95% ethanol and 1,2000 milliliters of 2-propylene glycol.Add sodium sulfite 1 gram, i.e. 0.02% ratio stirs, and slowly to add PH be that dibastic sodium phosphate-biphosphate of 3.2 is received buffer solution, transfer PH to 3.5, filter with 0.22 micron cellulose acetate sodium filter membrane, filtering fill with ultrafilter membrane (molecular cut off is 10,000), sterilization (105 ℃ following 30 minutes), promptly get 500 of 10 milliliters of Ginkgo total lactones injection finished products, measure through high-efficient liquid phase technique, every contains about 58.15 milligrams of gingko total terpene lactones.
The preparation of embodiment 8 gingko total terpene lactones injection
Prescription: Folium Ginkgo terpene lactones 58.5 grams
1,2000 milliliters of 2-propylene glycol
500 milliliters of 95% ethanol
Citric acid-sodium citrate buffer solution is an amount of
2500 milliliters of waters for injection
Make 500 (10 milliliters /)
Get total terpene lactones extract 58.5 grams of embodiment 2 preparations, earlier, slowly add 1,2 propylene glycol again with 95% dissolve with ethanol, add at last as water for injection, wherein water for injection, 1,2 propylene glycol and ethanol usage ratio are water for injection: ethanol (95%): 1,2 propylene glycol=6: 1: 3, the water for injection that promptly in the total terpene lactones extract of 60 grams, adds 2500 milliliters, 500 milliliters of 95% ethanol and 1,2000 milliliters of 2-propylene glycol.And adding sodium sulfite 1.75 grams, i.e. 0.025% ratio, stir, and slowly to add PH be citric acid-sodium citrate buffer solution of 4.8, transfer PH to 5.0, filter with 0.22 micron cellulose acetate sodium filter membrane, filtering with ultrafilter membrane (molecular cut off is 10,000), fill, sterilization (105 ℃ following 30 minutes), promptly get 500 of 10 milliliters of Ginkgo total lactones injection finished products, measure (with reference to the mensuration of 2005 editions the 221st page of Folium Ginkgo terpene lactones of Chinese Pharmacopoeia) through high-efficient liquid phase technique, every contains about 56.03 milligrams of gingko total terpene lactones.
The preparation of embodiment 9 gingko total terpene lactones injection
Prescription: Folium Ginkgo terpene lactones 60.6 grams
1,2000 milliliters of 2-propylene glycol
Acetic acid-sodium acetate buffer solution is an amount of
3000 milliliters of waters for injection
Make 500 (10 milliliters /)
Get total terpene lactones extract 60.6 grams of embodiment 3 preparations, earlier, slowly add 1,2 propylene glycol again with 95% dissolve with ethanol, add water for injection at last, wherein water for injection, 1,2 propylene glycol and consumption of ethanol ratio are mixed and are water for injection: ethanol (95%): 1,2 propylene glycol=6: 1: 3, the water for injection that promptly in the total terpene lactones extract of 60 grams, adds 3000 milliliters, 500 milliliters of 95% ethanol and 1,1800 milliliters of 2-propylene glycol.Add sodium sulfite 1.5 grams, i.e. 0.03% ratio stirs, and slowly to add PH be citric acid-sodium citrate buffer solution of 4.4, transfer PH to 4.5, filter with 0.22 micron cellulose acetate sodium filter membrane, filtering fill with ultrafilter membrane (molecular cut off is 10,000), sterilization (105 ℃ following 30 minutes), promptly get 500 of 10 milliliters of Ginkgo total lactones injection finished products, measure through high-efficient liquid phase technique, every contains about 58.12 milligrams of gingko total terpene lactones.
The preparation of embodiment 10 gingko total terpene lactones injection
Prescription: Folium Ginkgo terpene lactones 61.6 grams
The polyethylene glycol 1500 milliliter
Acetic acid-sodium acetate buffer solution is an amount of
3500 milliliters of waters for injection
Make 500 (10 milliliters /)
Get total terpene lactones extract 61.6 grams of embodiment 4 preparations, earlier with 95% dissolve with ethanol, slowly add 1 again, 2 propylene glycol add water for injection at last, and wherein water for injection, ethylene glycol and consumption of ethanol ratio are mixed and be, water for injection: ethanol (95%): ethylene glycol=6: 1: 3, the water for injection that promptly in the total terpene lactones extract of 60 grams, adds 3000 milliliters, 500 milliliters of 95% ethanol and 1,1500 milliliters of 2-propylene glycol.Add EDTA-Na2 (disodiumedetate) 1.5 grams, i.e. 0.03% ratio stirs, and slowly to add PH be 3.8 acetic acid-sodium acetate buffer solution, transfer PH to 4.0, filter with 0.22 micron cellulose acetate sodium filter membrane, filtering fill with ultrafilter membrane (molecular cut off is 10,000), sterilization (105 ℃ following 30 minutes), promptly get 500 of 10 milliliters of Ginkgo total lactones injection finished products, measure through high-efficient liquid phase technique, every contains about 58.35 milligrams of gingko total terpene lactones.
The preparation of embodiment 11 gingko total terpene lactones injection
Prescription: Folium Ginkgo terpene lactones 60.8 grams
500 milliliters of 95% ethanol
Citric acid-sodium citrate buffer solution is an amount of
4500 milliliters of waters for injection
Make 500 (10 milliliters /)
Get total terpene lactones extract 60.8 grams of embodiment 5 preparations, earlier with 95% dissolve with ethanol, slowly add Polyethylene Glycol again, add water for injection at last, wherein water for injection, ethylene glycol and consumption of ethanol ratio are mixed and be water for injection: ethanol (95%): ethylene glycol=5.5: 1: 3.5 promptly restrains the water for injection that adds 2750 milliliters in total terpene lactones extract 60,500 milliliters of 95% ethanol and 1,1750 milliliters of 2-propylene glycol.Add EDTA-Na2 (disodiumedetate) 2.0 grams, i.e. 0.04% ratio stirs, and slowly to add PH be 4.8 citric acid-sodium citrate buffer solution, transfer PH to 5.0, filter with 0.22 micron cellulose acetate sodium filter membrane, filtering fill with ultrafilter membrane (molecular cut off is 10,000), sterilization (105 ℃ following 30 minutes), promptly get 500 of 10 milliliters of Ginkgo total lactones injection finished products, measure through high-efficient liquid phase technique, every contains about 60.80 milligrams of gingko total terpene lactones.
The preparation of embodiment 12 gingko total terpene lactones injection
Prescription: Folium Ginkgo terpene lactones 60.2 grams
1,1500 milliliters of 2-propylene glycol
500 milliliters of 95% ethanol
It is an amount of that dibastic sodium phosphate-biphosphate is received buffer solution
3000 milliliters of waters for injection
Make 500 (10 milliliters /)
Get total terpene lactones extract 60.2 grams of embodiment 6 preparations, earlier with 95% dissolve with ethanol, slowly add Polyethylene Glycol again, add water for injection at last, wherein water for injection, ethylene glycol and consumption of ethanol ratio are mixed and are water for injection: ethanol (95%): 1,2-propylene glycol=5: 1: 4, the water for injection that promptly in the total terpene lactones extract of 60.2 grams, adds 2500 milliliters, 500 milliliters of 95% ethanol and 1,2000 milliliters of 2-propylene glycol.Add EDTA-Na2 (disodiumedetate) 1.75 grams, i.e. 0.035% ratio stirs, and slowly to add PH be 5.2 acetic acid-sodium acetate buffer solution, transfer PH to 5.3, filter with 0.22 micron cellulose acetate sodium filter membrane, filtering fill with ultrafilter membrane (molecular cut off is 10,000), sterilization (105 ℃ following 30 minutes), promptly get 500 of 10 milliliters of Ginkgo total lactones injection finished products, measure through high-efficient liquid phase technique, every contains about 57.93 milligrams of gingko total terpene lactones.
The study on the stability of embodiment 13 gingko total terpene lactones injection
The room temperature investigation that keeps sample: the sample that embedding is good place company's laboratory room temperature keep sample the chamber (25 ℃ ± 2 ℃ of temperature, 60 ± 10%RH), detect respectively at sampling regularly in the 3rd, 6,9,12,18 month, and with 0 month result relatively, see Table 1.By the stability test result as can be known, with the Folium Ginkgo terpene lactones injection that this method is made, when it is 4.5 left and right sides at pH value, have good stability.
0th, 3,6,9,12,18 months sample is respectively on March 20th, 2007,20,, 2007 Decembers of on June 20th, 2007,2007 on JIUYUE on June 20th, 20 days 1 sample time.Sample is the thick general laboratory self-control of Lunan Pharmaceutical Co., Ltd., and prescription, technology are seen embodiment 9.
Table 1, gingko total terpene lactones injection stability test are analyzed (room temperature)
Claims (7)
1. a method of extracting gingko total terpene lactones is characterized in that, comprises the steps:
1) get Folium Ginkgo, add ethanol and citric acid or tartaric acid, reflux, extract, concentrates alcohol extract;
2) macroporous resin column on the step 1) concentrate, gradient elution is collected eluent, concentrates;
3) step 2) silicagel column on the concentrate, eluent ethyl acetate is collected eluent, drying;
4) the dry thing of step 3) adds water through dissolve with ethanol, places, and gets the primary crystallization product; Mother solution reclaims ethanol, and the water liquid cooling is heavy, gets crystallized product for the second time; Merge twice crystallized product and promptly get gingko total terpene lactones.
2. the method for claim 1 is characterized in that, the described concentration of ethanol of step 1) is 60%～80%, and described citric acid or tartaric addition are 0.1%～1% (g/ml).
3. the method for claim 1 is characterized in that, the weight of step 1) alcohol extraction concentrated solution and the weight ratio of medical material are 1: 0.8～1.
4. the method for claim 1 is characterized in that step 2) wash 3～5 times of column volumes with water during eluting, remove impurity discards; With 3～5 times of column volumes of ethanol elution of 20%, remove impurity discards again; Use the column volume of 3～6 times of 75% ethanol elutions then, collect alcohol eluen, concentrate, stand-by.
5. the method for claim 1 is characterized in that, on the described sample of step 3) before the silicagel column, with step 2) extract obtainedly add an amount of water, fully mix thoroughly with an amount of silica gel, go up silicagel column behind the vacuum drying.
6. an injection that contains gingko total terpene lactones is characterized in that, and is composed of the following components:
Wherein, described excipient comprises ethylene glycol, ethanol, Polyethylene Glycol, 1, in the 2-propylene glycol one or both, described PH regulator is a kind of of following buffer: dibastic sodium phosphate-biphosphate is received, citric acid-sodium citrate or acetic acid-sodium acetate, and the consumption of PH regulator is to make the pH of injection transfer to 3.5～5.0 to get final product; Described antioxidant is a kind of in sodium sulfite, the disodiumedetate.
7. injection as claimed in claim 6 is characterized in that, dibastic sodium phosphate-biphosphate that described PH regulator is a kind of of following buffer: pH3.2～4.8 is received, the acetic acid-sodium acetate of the citric acid-sodium citrate of pH4.4～4.8 and pH3.8～5.2.
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| CN2009101494741A CN101926835B (en) | 2009-06-19 | 2009-06-19 | Method for extracting gingko total terpene lactones and injection containing same |
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| CN103664981B (en) * | 2013-12-02 | 2015-12-09 | 沃太能源南通有限公司 | The extracting method of lactone in a kind of gingko |
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| Title |
|---|
| 韩金玉.银杏萜内酯提取与纯化技术.《中草药》.2002,第33卷(第11期),附2-附5. * |
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