CN101182325A

CN101182325A - Intravenous administration bilobalide B and extraction method

Info

Publication number: CN101182325A
Application number: CNA2006100974926A
Authority: CN
Inventors: 张国清; 吴洪君
Original assignee: JIANGSU PENGYAO PHARMACEUTICAL Inc
Current assignee: JIANGSU PENGYAO PHARMACEUTICAL Inc
Priority date: 2006-11-13
Filing date: 2006-11-13
Publication date: 2008-05-21

Abstract

The present invention relates to a ginkgolide B of intravenous administration and an extracting method thereof, which is characterized in that the content of the ginkgolide B is more than or equal to 97 percent; ginkgoic acid is less than or equal to 2ppm; bilobalide is detected to be not found, so the ginkgolide B can be used as the medical materials of mainline or intravenous drip; the extracting is that the ethanol aqueous solution with 8 to 25 times of the quantity of leaf dry weight and the concentration of 5 percent to 30 percent is used for reflux extraction for a plurality of times for the filtration and separation; filtrate is concentrated to the quantity of 0.05 to 0.2 time of the former volume, sorbent is added to absorb completely under the stirring state for filtering, a filter cake is processed for the reflux extraction for a plurality of times by the ethanol with 2 to 10 times of the quantity; extract solution is combined and concentrated to the quantity of 0.01 to 0.2 time of the former volume for standing crystallization to be filtered to obtain the ginkgolide; the ginkgolide is recrystallized for a plurality of times by the methanol with 10 to 50 times of the quantity, the crystallization temperature is subzero 10 DEG C to subzero 25 DEG C, and the ginkgolide B is obtained after the filtration, crystallization and drying. Compared with the prior extracting and separating method, the present invention has the advantages of simple technical, being reliable, good economic character, being especially applicable to the industrialized production, low requirement towards equipments, the high yield of the ginkgolide B of about 0.4 percent, the high purity of the ginkgolide B, no pollution and good safety.

Description

Intravenous administration bilobalide B and extracting method

Technical field

The present invention is to medicine material---the improvement of Ginkgolide B and extracting method, relate in particular to a kind of purity height, Ginkgolide B content 〉=97%, ginkgoic acid content≤2pmm, bilobalide does not detect (evaporative light scattering detection), can be directly used in the Ginkgolide B and the extracting method of intravenously administrable.

Background technology

The medicinal function of bilobalide and value are approved by medical circle, along with the further investigation to bilobalide, it is found that each lactone drug effect has nothing in common with each other, and wherein Ginkgolide B is than other lactone compositions, and pharmacologically active is the strongest, so receive much attention.

The source of Ginkgolide B can be divided into artificial culture extraction, chemosynthesis and culture plant cell.Chemosynthesis exists space structure complexity, chirality changeable, synthetic very tired lock, and complex process, the high deficiency of cost still are confined to learning value at present, still do not have the production meaning; Culture plant cell, because technical difficulty is big, reasons such as production cost height do not have the actual production meaning at present yet.Present medicinal Ginkgolide B mainly extracts from the artificial culture Ginkgo Leaf.But be about 0.2% because Ginkgolide B content in Ginkgo Leaf is low, (Ginkgo biloba Extract also has only 1%～2% in GBE) at Semen Ginkgo extrac.And Ginkgolide A, B, C, J etc. and ginkgoic acid, bilobalide exist jointly, and each monomer structure is very similar again, and polarity difference is very little, and the monomer separation purifying is difficulty comparatively.General or the resin column chromatographic separation of the more employing traditional colour of prior art, not only higher to equipment requirements, cause the cost of investment height, and the effective extraction yield of Ginkgolide B is not high enough, and owing to the extracting method reason, contain a certain amount of virose ginkgoic acid and bilobalide, and separation purity is not high enough, is difficult to directly apply to intravenously administrable.Be difficult to be directly used in the intravenously administrable higher so extract Ginkgolide B usually to purity requirement, can only be as the oral medication raw material.And its pharmacodynamic feature performance of oral administration can not show a candle to the intravenously administrable height, can not give full play to the drug effect of Ginkgolide B.Therefore, seek effective Ginkgolide B and extract and purification process, make it to be directly used in intravenously administrable, become the vital task of ginkgo exploitation.

Chinese patent 01119824.9 has been introduced Folium Ginkgo extract system separation method, carries by adding 1-2% oxidation inhibitor poach, crosses the polyamides adsorption column, crosses macroporous adsorbent resin, uses ethyl acetate extraction three times, gets total lactone; Last silicagel column separates, and uses the chloroform-methanol gradient elution, gets bilobalide and ginkgolectone AB; The bilobalide recrystallization gets colourless bilobalide, and ginkgolectone AB gets Ginkgolide B through recrystallization repeatedly.Ginkgo total flavones content＞50% that this method report can obtain respectively, Ginkgolide B content＞98%, content of bilobalide＞98.5%.But this method, extraction and separation method complexity not only, the Ginkgolide B yield is not high yet, has only 0.015%, and contains higher about 5ppm left and right sides ginkgoic acid.

The disclosed method of extracting Ginkgolide A, B from Ginkgo Leaf or Ginkgo Leaf medicinal extract of Chinese patent 00117758.3, at first adopt the saltout agent leaching of ethanol or methyl alcohol or acetone or butanone or ethyl acetate organic solvent and muriate, to obtaining the enriched material of Ginkgolide A and B, adopt preparative liquid chromatography that the enriched material that contains Ginkgolide A and B is separated, obtain Ginkgolide A and Ginkgolide B.Carry out recrystallization with methyl alcohol and water, can obtain monoesters purity about 96.This method adopts liquid chromatography to separate, the working condition complexity, and manufacturing requirements is higher, causes the production cost height, and ginkgoic acid and bilobalide is not made quantitative target, and report can not be directly used in intravenously administrable; Secondly, this method Ginkgolide B yield is lower, has only about 0.001%.

The simulated moving bed chromatography that Chinese patent 200310104958.7 is introduced is separated purification Ginkgolide B method, at first adopts water-boiling method to obtain the ginkgo biloba extract of total flavones 24%, total lactone 6%; Add the 0.8-1.2L ethyl acetate by every gram extract powder again, carry out the liquid extraction under 30-35 ℃, filter, the filtrate decompression evaporate to dryness gets the bilobalide crude extract; Get mixture through simulation moving-bed purifying again; At last, use ethyl alcohol recrystallization earlier, the freezing placement of refrigerator gets Ginkgolides; Use recrystallizing methanol again, be cooled to subzero 10-18 ℃ gradually, crystallization twice gets Ginkgolide B.It is the 90-95% Ginkgolide B that report can obtain purity, but does not report bilobalide, ginkgoic acid content, and not only purity is still lower, and preparation is complicated, and report can not be used for intravenously administrable.

The Chinese patent 200410061284.1 disclosed methods of from Ginkgo Leaf, extracting Ginkgolide B and bilobalide, comprise: A, extract 2-3 time united extraction liquid, concentrating under reduced pressure respectively with 50-60% ethanol at 65-75 ℃, reclaim ethanol, with concentrated solution 1-2 water doubly, precipitation, centrifugal is got adsorption column on the supernatant liquor, elder generation's water flushing, with 75-80% ethanolic soln wash-out, collect ethanol eluate, concentrate; B, concentrated solution is added ethyl acetate extraction 2-3 time of 1-2 times of water and concentrated solution 30-40%, water liquid concentrates, 65-75 ℃ vacuum-drying, is the ginkgo flavonoid glycoside product, the organic phase that extracting and separating goes out concentrates, dry, be crushed to the 90-100 order, be bilobalide; C, add 85-95% ethanol, last acidic alumina column is collected effluent liquid, when being concentrated into solid substance 25-30%, stop heating, crystallization is separated out, filter, use 95% dissolve with ethanol crystallization again, recrystallization, isolate the crystallization Ginkgolide A, the solution recrystallize is Ginkgolide B, with 95% alcohol flushing of crystalline 20-40%, suction filtration is used 95% dissolve with ethanol again, recrystallization, Ginkgolide B is through three recrystallizations; D, mother liquor is added 1-2 times of water, boil off ethanol, cooling at 75-80 ℃, separate out crystallization, crystallization is a ginkalide C, isolates crystallization, the mother liquor evaporated under reduced pressure, pulverizing the 90-100 order is the yellow powder bilobalide, with crystalline 20-40% dehydrated alcohol flushing bilobalide, suction filtration, use 95% dissolve with ethanol, recrystallization is the bilobalide of white, uses 95% dissolve with ethanol solution again, recrystallization again, bilobalide is through three recrystallizations.Though this method report can obtain purity 95% above Ginkgolide B and bilobalide, but not unexposed yet ginkgoic acid content of bilobalide, also report can not be directly used in intravenously administrable, and the extracting method complexity, separate and adopt separation method one by one, processing step is many and complicated.

Chinese patent 200510023164.7 openly adopts the preparation method of high-speed counter-current look popularize law (HSCCC) preparation high purity ginkgo lactone, though can obtain purity up to 99% various high purity ester monomers, but adopt three individual system, the separation method one by one of secondary separation process, technology is very complicated, and need to be difficult in actual production, use by look general instrument.

The disclosed bilobalides of Chinese patent 200510041266.1 extract and purifying process, add the ethanol or the methanol extraction of several times amount by medicinal material weight, and extracting solution reclaims the degreasing solvent removal of impurities of alcohol back, extraction, and removal of impurities again gets raw product; Use ethyl alcohol recrystallization again, filter, dry bilobalide highly finished product, but be not Ginkgolide B, but total amount 92%, Ginkgolide B more than 40%, ginkgolic acid are less than 2/1000000ths mixing lactone.

All there is certain deficiency in above-mentioned numerous method, is not separate Ginkgolide B separately, can not obtain purity height, the low Ginkgolide B that can be directly used in intravenously administrable of ginkgoic acid content exactly; Can obtain the higher Ginkgolide B of purity, often extraction, separating technology complexity need to adopt the separating for several times method, are difficult to use in actual production; And the Ginkgolide B extraction yield is not high; Especially most of technology is not all reported poisonous ginkgoic acid content, does not therefore all see to be directly used in the intravenously administrable report.

Summary of the invention

The objective of the invention is to overcome the deficiency of above-mentioned prior art, a kind of purity height is provided, Ginkgolide B content 〉=97%, ginkgoic acid≤2ppm does not detect the intravenous administration bilobalide B of bilobalide.

Another object of the present invention is to provide a kind of extracting method that obtains above-mentioned intravenous administration bilobalide B.

Intravenous administration bilobalide B of the present invention is characterized in that Ginkgolide B content 〉=97%, ginkgoic acid≤2ppm, and bilobalide does not detect.

Intravenous administration bilobalide B extraction separation of the present invention, comprise the Ginkgo Leaf lixiviate is separated the acquisition bilobalide, after recrystallizing methanol is purified, it is characterized in that said lixiviate is: with leaf dry weight 8～25 times of amounts, concentration is 5%-30% alcohol reflux several times, filtering separation; Filtrate is concentrated into 0.02～0.2 times of amount of original volume, adds sorbent material fully absorption under whipped state, filter, filter cake is with 3～10 times of amount alcohol reflux several times, and united extraction liquid is concentrated into 0.01～0.2 times of amount of original volume, leave standstill crystallization, filter bilobalide; Bilobalide carries out the recrystallization several times with the methyl alcohol of 10～50 times of amounts, recrystallization temperature-10 ℃～-25 ℃, filtering for crystallizing, drying, Ginkgolide B.

Lixiviate of the present invention, a kind of be preferably aqueous ethanolic solution be leaf dry weight 10-20 doubly; Described alcohol concn, more preferably 10%-25%.

Among the present invention:

Selecting to adopt concentration is the lixiviate of 5%-30% aqueous ethanolic solution, be the experiment gained, be that the present invention innovates, this vat liquor can as much as possiblely obtain Ginkgolide B (Ginkgolide B 〉=45%), other bilobalides are low relatively in the extracting solution simultaneously, improve the yield of Ginkgolide B, seldom bring ginkgoic acid and bilobalide (ginkgoic acid≤5ppm again into, bilobalide fails to detect), more help follow-up removing.Detect through respectively the bilobalide that obtains with 10% extraction using alcohol and recrystallization of 50% extraction using alcohol being carried out chromatogram, collection of illustrative plates 1-4 shows and shows: the bilobalide that adopts lower concentration 10% extraction using alcohol of the present invention, be mainly A and B, ginkalide C content is few, and Ginkgolide B content (〉=50%) is higher than Ginkgolide A (43.3%), does not contain bilobalide; And adopt 50% extraction using alcohol bilobalide, and not only Ginkgolide B content decreases, and is 30.1%, Ginkgolide A higher (54.2%), and ginkalide C content raises to some extent, also contains bilobalide.Show: low-concentration ethanol has better choice to Ginkgolide B, adopts low-concentration ethanol to extract Ginkgolide B and is better than the high density ethanol-extracted.Collection of illustrative plates 5-8 shows and shows: adopt the bilobalide of lower concentration 10% extraction using alcohol of the present invention, ginkgoic acid content lower (4.3ppm); And adopt the bilobalide of 50% extraction using alcohol, and ginkgoic acid higher relatively (15.4ppm) exceeds about 4 times approximately, i.e. and lower concentration alcohol extracting ginkgoic acid content is significantly less than the high density alcohol extracting.Collection of illustrative plates shows, the present invention obtains the Ginkgolide B extracting method and adopts low-concentration ethanol to extract, and has good selectivity, obviously is better than high density alcohol, and more is better than water and carries (the lactone water solubility is lower).

Filtrate concentrates, and is similar to common Chinese medicine such as Ginkgo Leaf lixiviate, and effect mainly is to water in the vat liquor and ethanol decrement.

Sorbent material, main effect is to impel extract one bilobalide enrichment, to improve lactone B content and recovery rate; Whip attachment more helps fully contacting of lactone and sorbent material in the filtrate, improves the adsorption and enrichment rate; Sorbent material can adopt the sorbent material commonly used that meets traditional Chinese medicine extraction, as activated carbon, diatomite, aluminum oxide etc.

Dissolve with methanol low temperature (10 ℃～-25 ℃) frozen recrystallization, be the another innovation of the inventive method, test shows adopts dissolve with methanol cryogenic freezing recrystallization, can improve the purity of Ginkgolide B, reduce other foreign matter contents, can obtain Ginkgolide B content 〉=97%, ginkgoic acid≤2ppm, not detect high-purity Ginkgolide B of bilobalide through this process.Collection of illustrative plates 9-13 shows and shows: methyl alcohol-15 ℃ freezing and crystallizing, and gained Ginkgolide B content is up to 98.5%, and ginkgoic acid does not detect; And 0 ℃ of freezing and crystallizing of methyl alcohol, gained Ginkgolide B content only 94.6%.

Refluxing extraction and recrystallization number of times, theoretically often, extraction yield and purity all can improve, test shows, take into account interest rate, purity and economy, repeat 2-4 time in the technology of the present invention and just can obtain more satisfactory result, increase number of times again and improve effect and not obvious, therefore the present invention takes into account economy and extraction yield and purity, think 2-4 time comparatively economical and practical.

Used ethanol, methyl alcohol in the inventive method, as do not indicate concentration all refer to commercially available technical grade, for example concentration be 95% and more than.

The present invention extracts the Ginkgolide B method from Ginkgo Leaf, gained Ginkgolide B purity height, content 〉=97%, bilobalide and ginkgoic acid content are extremely low, ginkgoic acid≤2ppm, bilobalide does not detect (evaporative light scattering detection), thereby can be a difference greatly of Ginkgolide B of the present invention and additive method gained Ginkgolide B directly as intravenous injection or intravenous drip bulk drug.Adopting concentration is the lixiviate of 5%-30% aqueous ethanolic solution, not only extraction process is simple, be applicable to industrial production, and Ginkgolide B content height, Ginkgolide B 〉=45% can also reduce and extract that other do not need the content of impurity in the ginkgo ester, ginkgoic acid≤5ppm for example, bilobalide fails to detect, and other bilobalides are less, helps obtaining the higher Ginkgolide B of purity; Methyl alcohol-10 ℃～-25 ℃ of low temperature recrystallization purifyings, select, separation and purification is effective, method is simple, practical, not only can obtain the Ginkgolide B of higher degree, and this process can separate simultaneously again and remove ginkgoic acid and other unwanted impurity, and single selective is strong, has improved purity, security and the validity of gained Ginkgolide B greatly.The inventive method is simple, reliable, good economy performance, and Ginkgolide B interest rate height can reach about 0.04%, and cost is low, is particularly suitable for large-scale commercial production, and pollution-free, and is low for equipment requirements, extracts, separation costs is low.

Gained Ginkgolide B of the present invention:

Long term toxicity test (seeing appendix) toxicity symptom do not occur when being equivalent to the clinical maximal dose of people 50 times (dogs) and 75 times (rats) respectively by kg body weight calculating yet, remains safe.

Acute toxicity test (seeing appendix), mouse intravenous injection are equivalent to 1075 times of clinical medicine dose, any tangible toxicity symptom do not occur, and no autonomic activities and other spirit, nervous system abnormality do not have animal dead yet.The huge inspection of necrotomy does not find that histoorgan is unusual, and administration group and control group compare, the weight increase no significant difference.

Mutagenicity test (seeing appendix), three mutagenicity test results show: in this test conditions and dosage (concentration) scope, the injection bilobalide does not have potential mutagenicity.

Showing that Ginkgolide B of the present invention is directly used in intravenously administrable, is safe; And pharmacodynamics test shows: the treatment cardiovascular and cerebrovascular diseases is had unique curative effect.

Below in conjunction with the description of several specific embodiments, in conjunction with pharmacodynamics and toxicity test result, further specify the present invention, following embodiment only is used to illustrate the present invention, and should not be construed as limitation of the present invention.

Description of drawings

Fig. 1 is Ginkgolide A, B mixing contrast solution (sample introduction 5 μ l) standard diagram.

Fig. 2 is Ginkgolide A, B mixing contrast solution (sample introduction 10 μ l) standard diagram.

Fig. 3 is 10% extraction using alcohol bilobalide color atlas.

Fig. 4 is 50% extraction using alcohol bilobalide color atlas.

Fig. 5 is a total ginkgolic acids reference colour spectrogram.

Fig. 6 is total ginkgolic acids reference colour spectrogram (C13:0 location).

Fig. 7 is 10% extraction using alcohol total ginkgolic acids collection of illustrative plates.

Fig. 8 is 50% extraction using alcohol total ginkgolic acids collection of illustrative plates.

Fig. 9 is Ginkgolide B contrast solution (sample introduction 5 a μ l) standard diagram.

Figure 10 is Ginkgolide B contrast solution (sample introduction 10 a μ l) standard diagram.

Figure 11 is frozen recrystallization (15 a ℃) Ginkgolide B collection of illustrative plates.

Figure 12 is recrystallization (0 a ℃) Ginkgolide B collection of illustrative plates.

Figure 13 is frozen recrystallization (15 a ℃) total ginkgolic acids collection of illustrative plates.

Figure 14 respectively organizes cerebral tissue infarct stained brain sheet after the Ginkgolide B pre-treatment is dyeed to TTC.

Figure 15 is that the Ginkgolide B pre-treatment is to cortex and hippocampus CA1 district histopathology colored graph.

Specific implementation method

Embodiment 1: get dry Folium Ginkgo 10kg, add the 30% ethanolic soln refluxing extraction secondary of 150kg, each 2 hours, the extracting solution core filtered; Merging filtrate, filtrate recycling ethanol is concentrated into about 25kg, and concentrated solution added an amount of activated carbon adsorption 24 hours, and constantly stirred; Filter, filter cake merges ethanol extract with 10kg95% ethanol gradation refluxing extraction 3 times, reclaims ethanol and is concentrated into about 1Kg, leaves standstill crystallization, filter crude product 10.5g; Use the 100g95% recrystallizing methanol again 3 times, recrystallization temperature-12 ℃ is filtered, drying, Ginkgolide B 3.6 grams.The sample detection ginkgoic acid is less than 2PPM, and bilobalide does not detect, Ginkgolide B purity 97.8%.

Embodiment 2: get dry Folium Ginkgo 10kg, add the 10% ethanolic soln refluxing extraction secondary of 150kg, each 2 hours, the extracting solution core filtered; Merge and carry filtrate, filtrate recycling ethanol is concentrated into about 24kg, and concentrated solution added an amount of activated carbon adsorption 32 hours, and constantly stirs; Filter, filter cake merges ethanol extract with 20Kg95% ethanol gradation refluxing extraction 3 times, reclaims ethanol and is concentrated into about 1.5kg, leaves standstill crystallization, filter crude product 13.4g; Use crude product 300g95% recrystallizing methanol 3 times again, recrystallization temperature-15 ℃ is filtered, drying, Ginkgolide B 4.2 grams.The sample detection ginkgoic acid is less than 2PPM, and bilobalide does not detect, Ginkgolide B purity 98.5%.

Embodiment 3: get dry Folium Ginkgo 10kg, add the 5% ethanolic soln refluxing extraction secondary of 150kg, each 2 hours, the extracting solution core filtered; United extraction liquid, filtrate recycling ethanol are concentrated into about 30kg and doubly measure, and concentrated solution adds proper amount of active carbon absorption 48 hours, and constantly stir; Filter, filter cake merges ethanol extract with 30kg95% ethanol gradation refluxing extraction 3 times, reclaims ethanol and is concentrated into about 2.0kg and doubly measures, and leaves standstill crystallization, filter crude product 11.3g; Use the 450g95% recrystallizing methanol again 3 times, recrystallization temperature-25 ℃ is filtered, drying, Ginkgolide B 3.8 grams.The sample detection ginkgoic acid is less than 2PPM, and bilobalide does not detect, Ginkgolide B purity 98.4%.

Chromatogram detects explanation:

The bilobalide chromatographic determination, chromatographic instrument: day island proper Tianjin LC-10ATVP of company pump, the U.S. ELSD800 of Ao Tai company detector, chromatographic column: Hypersil ODS 5 μ m Dalian Yi Lite, moving phase: water: tetrahydrochysene furan south: n-propyl alcohol (690: 300: 10), flow velocity: 1.0ml/min, integrative approach: area external standard method.

Ginkgo total acid chromatographic determination, chromatographic instrument: U.S. Lab Alliance company, chromatographic column: LichrospherC18 7C post 4.6*250mm 5 μ m Han Bang scientific ﹠ technical corporation, moving phase: 1% Glacial acetic acid: methyl alcohol (10: 90), wavelength: 310mm, column temperature: 40 ℃, flow velocity: 1.0ml/min, integrative approach: area external standard method.

Appendix: correlation test and result

Intravenous administration bilobalide B pharmacodynamics test of the present invention

One, is subjected to reagent

1, title: the present invention extracts Ginkgolide B (Ginkgolide B purity 97.4%, ginkgoic acid are less than 2PPM, and bilobalide does not detect)

2, be subjected to the reagent preparation method: prepare the Ginkgolide B liquid of various concentration with sodium chloride injection, place refrigerating chamber and keep in Dark Place.

Two, experimental animal

The healthy male SD rat of SPF level, body weight 250～350g, 10～12 ages in week.Raise with the standard particle complete feed, freely drink cleaning water.Testing indoor feeding 3 days, room temperature before the test: 18～22 ℃, relative humidity 60～80%.

Three, reagent

1, TCC (2,3,5-Tripheny-2H-Tetrazolium chloride, TTC), Shanghai chemical reagents corporation, lot number F990930 is mixed with 2% working fluid with 1% phosphoric acid salt PBS solution.

2, mda (MDA) is measured test kit; Superoxide-dismutase (SOD) is measured test kit; Protein quantification test kit (TP) is measured test kit, and bio-engineering research institute is built up in Nanjing.

3, nimodipine (nimodipine, NIM), lot number: 20050218, Jiangsu JumpCan Medicines Co., Ltd..

4, Chloral Hydrate (choral hydrate), analytical pure, lot number: 20040420, Shanghai chemical reagents corporation of Chinese Medicine group is mixed with 10% solution with distilled water.

5, Paraformaldehyde 96 (paraformaldehyde), analytical pure, lot number: 031119, Shanghai Ling Feng chemical reagents corporation.

Four, experimental implementation

1, animal grouping

According at random, double blinding, contrast and parallel principle, rat is divided into following group:

1, pseudo-operation group;

2, ischemia-reperfusion injury model group (solvent control);

3, the positive medicine contrast of nimodipine medicine, 5mg.kg ^-1

4, Ginkgolide B pretreated group: Ginkgolide B 10mg.kg ^-1, 20mg.kg ^-1, 40mg.kg ^-1

2, experimental arrangement

(300mg/kg ip) under the anesthesia, cuts skin of neck to rat at Chloral Hydrate, separate the outer superficial vein of neck, pseudo-operation group and solvent control group vein give physiological saline, and experimental group is irritated preceding 1 hour femoral vein again at ischemic and given Ginkgolide B 10～40mg/kg, administration volume 1ml.kg ^-1

3, focal brain ischemia-reperfusion injury in rats model

Embolism line preparation: nylon wire one end of diameter 2.6mm is carefully polished smooth, do sign at distance top 18mm, 20mm, 22mm place respectively.

Adopt improvement Koizumi legal system to make arteria cerebri media blocking-up (MACO) model, monitoring anus temperature makes it to remain on 37 ± 0.5 ℃ in the surgical procedure.Rat is used chloral hydrate anesthesia (300mg/kg, ip), neck median incision, expose tracheae and right common carotid artery, at internal carotid artery and external carotid artery tapping point,, separate the ligation external carotid artery from bifurcation from proximal part ligation arteria carotis communis, on arteria carotis communis, cut a little otch apart from bifurcation 2mm, after internal carotid artery is placed the about 20mm of embolism line or met obstructions, stop by incision.Sew up the skin of neck otch.The embolism line of intravasation not is retained in outside the cervical incision.Behind the ischemic 2h embolism alignment is extracted about 10mm outward, make the logical again 22h of blood flow.Pseudo-operation group rat is except that internal carotid artery not the plug line, and other is with the ischemia-reperfusion injury model group.Model group is rejected the rat of death or the behavior of impassivity afunction.

4, neural function study of behaviour scoring

After rat ischemia was irritated again, except that dead mouse, most mouse had been in waking state.5 point-scores by Longa carry out the scoring of neural function study of behaviour to rat, and standards of grading are as follows: 0 minute: the impassivity defective symptom; 1 minute: left fore can not stretch fully; 2 minutes: to anticlockwise; 3 minutes: topple over to the left; 4 minutes: can not walk or go into a coma.

5, the infarcted region scope is measured

After pouring into the scoring of last neural function study of behaviour again,, get brain rapidly, brain is placed after (refrigerator) preserves 10min under-20 ℃ of environment the rat sacrificed by decapitation.Full brain is cut the crown brain sheet of the about 2mm of thickness in the past backward continuously, and every brain is cut 5.The brain sheet is put into 2%TTC solution immediately.Because TTC can make normal cerebral tissue take on a red color, the damaged cerebral tissue is white in color.The brain sheet keeps 15min under 37 ℃ of airtight light tight environment, after place 10% formalin to keep in Dark Place the brain sheet, took out the brain sheet and take pictures in 7 days.According to colour developing infarcted region brain district is separated with normal cerebral tissue, weigh respectively, calculate every mouse cerebral infarction per-cent (the infarcted region cerebral tissue accounts for the per-cent of full brain weight) and compare significant difference between administration group (Ginkgolide B and nimodipine) and model control group value.

6, cerebral tissue SOD and MDA measure

The same legal system is equipped with the rat brain focal ischemia and irritates damage model again, and ditto grouping and administration with the rat sacrificed by decapitation, got brain rapidly after multiple filling finishes, and makes 10% brain tissue homogenate.Press the test kit specification sheets and measure MDA content with thiobarbituricacid (TBA) method, measure SOD content with xanthine oxidase, biuret method is measured the protein concentration in the homogenate.

7, encephalopathic reason histological examination

The same legal system is equipped with the cerebral ischemia model.The same grouping, 3 of every group of SD rats are poured into the end again and open the rat thoracic cavity rapidly and expose heart, and the perfusion pin is inserted the rat apex, upwards arrive the rat aorta ascendens.Cut an osculum with scissors in right atrium.Irritate about the about 200ml of cold saline earlier perfusion beginning back, irritates Paraformaldehyde 96 150～200ml of 4% in succession.After perfusion finishes brain placed 4% paraformaldehyde solution to preserve down for 4 ℃.Get and respectively organize the cerebral tissue of rat optic chiasma to the infundibular stalk coronal section, serial section is done in the routine paraffin wax embedding, and slice thickness is 5

μ m.Get

3 and 7 of sections, conventional H E dyeing is observed medicine and ischemic is irritated again the influence that causes cortex and hippocampus (mainly being the CA1 district) pathology damage.

8, statistical procedures

Experimental result is used Two sample t check is relatively adopted in expression between group.There is statistical significance P＜0.05 for difference.

Five, experimental result

1, the Ginkgolide B pre-treatment is to the influence of MACO rat behavior

The result shows: Ginkgolide B 10mg/kg, 20mg/kg and 40mg/kg, iv pre-treatment dosage improves neuroethology function behind the rat MACO with relying on, but only Ginkgolide B 40mg/kg class value and solvent control group comparison difference have statistical significance (P＜0.05, table 1), 10 and 20mg/kg group though improvement trend is arranged, with solvent control class value not statistically significant relatively.Nimodipine 5mg/kg, the injury of brain function (p＜0.05) that the iv pre-treatment also has minimizing MACO to cause.

Table 1 Ginkgolide B is to the reperfusion injury neurological deficit scoring influence of rat brain focal ischemia.

Annotate: ^*For with solvent control group P＜0.05 relatively,

2, the Ginkgolide B pre-treatment is to the influence of rat cerebral infarction volume

Ginkgolide B 10～40mg/kg dosage reduces the cerebral infarction volume after focal cerebral ischemia in rats 2h pours into 22h again with relying on, wherein 20mg/kg and 40mg/kg class value and model control group value are relatively, difference has statistical significance (P＜0.05), Ginkgolide B 10mg seemingly has the effect that reduces the cerebral infarction volume, but compares difference not statistically significant (Figure 14 and table 2) with the solvent control group.

The explanation of Figure 14 TTC stained brain sheet: infarcted region is a white, and non-infarcted region is red: the pseudo-operation group of a; B solvent control group; C Ginkgolide B 10mg/kg group; D Ginkgolide B 20mg/kg group; E Ginkgolide B 40mg/kg group; F nimodipine group.

The pre-treatment of table 2 Ginkgolide B is irritated damage again to rat brain medium sized artery ischemic and is caused cerebral infarction dead band weight (g) influence

Group	Full brain weight	Non-infarcted region	Infarcted region	Infarcted region %
Group	Full brain weight	Non-infarcted region	Infarcted region	Infarcted region %	Solvent control	1.62±0.04	1.08±0.07	0.538±0.05	33±3
Ginkgolide B 10mg/kg	1.583±0.07	1.116±0.065	0.474±0.06	30.55±3	Solvent control	1.62±0.04	1.08±0.07	0.538±0.05	33±3
Ginkgolide B 10mg/kg	1.583±0.07	1.116±0.065	0.474±0.06	30.55±3	Ginkgolide B 20mg/kg	1.60±0.06	1.11±0.07	0.368±0.05 ^**	21.6±4 ^**
Ginkgolide B 40mg/kg	1.59±0.07	1.366±0.06 ^**	0.239±0.04 ^**	13.6±3 ^**	Ginkgolide B 20mg/kg	1.60±0.06	1.11±0.07	0.368±0.05 ^**	21.6±4 ^**
Ginkgolide B 40mg/kg	1.59±0.07	1.366±0.06 ^**	0.239±0.04 ^**	13.6±3 ^**	Nimodipine 5mg/kg	1.62±0.03	1.185±0.02 ^**	0.428±0.03W	26.2±2 ^**

Expressed?as

； ^**p＜0.01?compared?with?vehicle?control

3, the Ginkgolide B pre-treatment is to the influence of brain MDA content and SOD vigor

Ginkgolide B 20mg/kg group and 40mg/kg group can increase the SOD activity (P＜0.05) that focal cerebral ischemia in rats 2h pours into the cerebral tissue behind the 22h again, minimizing MDA content (P＜0.05), and dose-dependence (table 3) is arranged.

Table 3 Ginkgolide B to rat brain focal ischemia reperfusion injury after the influence of cerebral tissue MDA and SOD

Annotate: ^*For comparing P＜0.05 with the solvent control group; # compares P＜0.05 with puppet operation group,

4, the influence of Ginkgolide B pre-treatment pallium and hippocampus CA1 district neurone pathological change

Histopathology result shows: pseudo-operation group rat layer and hippocampus CA1 district neurone marshalling are methodically arranged acellular necrosis, no inflammatory cell infiltration; This brain district neuronal quantity of model control group obviously reduces, arrangement disorder, and a large amount of non-viable non-apoptotic cells, pericaryon swelling, nuclear hyperchromatism, pyknosis, cracked or dissolving disappearance, inflammatory cell (being mainly neutrophil leucocyte) soaks into obviously, and intercellular substance is sparse; Ginkgolide B 10mg/kg group is similar to the solvent control group, and more non-viable non-apoptotic cell is arranged, in the form of sheets or the kitchen range shape distribute, cell is arranged the level disappearance, tissue edema is obvious; Ginkgolide B 20mg/kg and Ginkgolide B 40mg/kg and nimodipine group group pathology are slight, visible spotty necrosis, and Mild edema, neuronal quantity is more than the solvent control group but be less than pseudo-operation group (seeing Figure 15).Respectively organize cerebral tissue CA1 district and cortex HE dyeing by Figure 15 and show, the Ginkgolide B pre-treatment can alleviate pathology damage behind the cerebral tissue ischemia-reperfusion.

Figure 15 explanation: rat layer and hippocampus CA1 district pathological observation, HE dyeing, the 200 times of pseudo-operation group of the visual field: a1 cortexes; The pseudo-operation group of a2 hippocampus; B1 solvent control group cortex; B2 solvent control group hippocampus; C1 Ginkgolide B 10mg organizes hippocampus; C2 Ginkgolide B 10mg organizes cortex; D1 Ginkgolide B 20mg organizes hippocampus; D2 Ginkgolide B 20mg organizes cortex; E1 Ginkgolide B 40mg organizes hippocampus; E2 Ginkgolide B 40mg organizes cortex; F1 nimodipine group hippocampus; F2 nimodipine group cortex.

Six, conclusion

This experiment confirm: the intravenously administrable quiet injecting amount of Ginkgolide B 10-40mg/kg, rely on reduction rat arteria meningea media ischemic and irritate the Infarction volume that causes again, increase the SOD of ischemic region, reduce the MDA level, reduce ischemic and irritate cortex and the damage of hippocampus CA1 district that causes again.Show: intravenously administrable has Chinese People's Anti-Japanese Military and Political College's mouse focal cerebral ischemia injury effect with Ginkgolide B, shows that this product has unique curative effect to the treatment cardiovascular and cerebrovascular diseases.

Intravenous administration bilobalide B long term toxicity test of the present invention

One, research purpose

By beasle dog and the 13 all chronic toxicity tests of rat intravenous injection Ginkgolide B, observe the untoward reaction that excess drug may cause, comprise the character, degree of untoward reaction etc.; Infer that Ginkgolide B repeats the clinical toxicity target organ or the target tissue of intravenously administrable.

Two, be subjected to reagent

1. the Ginkgolide B produced of the present invention;

2. solvent: sodium chloride injection specification: 4.5g/500mL lot number: 0512196, production unit: Yangzhou Zhongbao Pharmaceutical Co., Ltd.;

3. be subjected to the reagent preparation method to prepare injection Ginkgolide B liquid with sodium chloride injection, solution is placed on refrigerating chamber and keeps in Dark Place.

Three, experimental animal

Dog: 12 of 6 monthly age Beagle dogs, female body weight 5-6.5kg, male 7-8.5kg, two groups (high dose group and solvent control group), each 3 of every group of male and female, totally 12.Provide by Nanjing An Limo Science and Technology Ltd..

Rat: 6 age in week the SD rat: 4 groups, each 10 of every group of male and female, every cage is with 5 of sexes.Female mouse body weight 70-90g during the experiment beginning, male mouse 80-110g is provided by animal reproduction field, Green Dragon mountain, Jiangning, Nanjing, animal quality conformity certification SCXK (Soviet Union) 2002-0018.

Four, route of administration intravenous injection (bolus) dog forearm vein, the rat stomach vein

Five, administration volume and injection speed dog 2.0mL/kg/min, rat: 1.0mL/kg/min

Six, 13 weeks of administration time limit

Seven, dosage is selected

Dog: Ginkgolide B 100mg/kg; Rat: 125mg/kg, 50mg/kg, 20mg/kg does solvent control with equal-volume physiological saline, once a day, 6 days weekly.

Eight, observation index

1. general situation is observed

During administration, observe dog and rat outward appearance sign, behavioral activity, glandular secretion, breathing, ight soil shape, food ration, administration local reaction once a day.Body weight is weekly.

2. hematological indices

Red blood cell count(RBC), oxyphorase, red cell volume, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration (MCHC), white blood cell count(WBC) and classification thereof, platelet count, reticulocyte count, prothrombin time etc.

3. blood biochemical is learned index

Aspartic transaminase, alanine aminotransferase, alkaline phosphatase, γ glutamyl transferase, blood urea nitrogen, creatinine, albumin, total protein, total bilirubin, blood sugar, total cholesterol, triglyceride level, Na ion concentration, potassium concentration, chlorine ion concentration, creatine phosphokinase.

4. uroscopy (only dog)

Urine outward appearance, proportion, pH, glucose in urine, urine protein, urine bilirubin, urobilinogen, ketoboidies, occult blood, white corpuscle.

5. system becomes celestial and histopathological examination

5.1 system's necrotomy

Duration of test in time observes the symptoms, and in time finds dying animal, carries out pathological examination.Off-test is lived and is killed all survival dogs, and behind the collection blood specimen, conscientious scrutiny organ is found the timely record of pathological change, measure organ weights by the governing principle requirement, and the sample that should check send Pathological Staff Room to do pathological examination.

5.2 organ coefficient

Internal organs such as the heart, brain, liver,kidney,spleen, suprarenal gland, thymus gland, lung, testis, epididymis, ovary and uterus are weighed, and calculate organ coefficient.

5.3 histopathological examination

Internal organs and the tissue checked comprise: the heart, liver, spleen, lung, kidney, brain, oesophagus, stomach, small intestine, large intestine, hypophysis, spinal cord, marrow, lymphoglandula, bladder, testis, epididymis, uterus, ovary, thymus gland, suprarenal gland, optic nerve (dog), Tiroidina, parathyroid gland, sialisterium, gall-bladder (dog), pancreas, tracheae, aorta, prostate gland, mammary gland, sciatic nerve, eye (dog) and administration local organization etc.

6. the time of observation index and number of times

Clinical symptom is in time found record once a day, and body weight is weekly, and food-intake (rat) is weekly.

Before on-test and carry out the hematological examination of dog after finishing, blood biochemical and routine urianlysis, rat lives in taking a blood sample before killing at the experiment closing day and carries out hematology, blood biochemistry checking.

Carry out necrotomy and pathological examination after the off-test.

Eye examination: before the dog medicine and before living extremely, check the pupil size, light reflex, eyelid and conjunctiva have or not hyperemia, ulcer.

Electrocardiogram(ECG: respectively carry out once before the dog administration and after the administration end.

Nine, result

(1) dog

1) clinical symptom

Dog: after the administration of Ginkgolide B group dog, with the control group ratio, generalized case is good, and no dystropy does not see that respiratory rate and amplitude change weight increase and control group no significant difference, ight soil Huang Cheng shape.Administration is local do not have red and swollen, fester and local vascular irritation such as scleroma.

2) dead control group and administration group dog and rat all do not occur dead in 13 weeks of test

3) body weight dog: do not see relevant change with administration, 0～13 all body weight change see table 4 for details

4) the food consumption dog is raised by animal center is unified, and every day, each once supplied food and water sooner or later, and administration is carried out before the feeding in the morning, and daily inspection finds no leftover.

5) the eye examination viewing duration is not seen pupil, the conjunctivae and selerae variation relevant with administration.

6) ECG; Do not see the variation relevant (heart rate and electrocardiogram(ECG index) with administration, dog electrocardiogram(ECG before the administration and after the administration, administration group and control group comparison, the P ripple, the PR interval, the QRS ripple, the T ripple, the ST section is all no abnormal, irregular pulse all do not occur.

Table 4 dog intravenous injection Ginkgolide B 100mg/kg/d, 13 all body weight change

7) the main hematological indices of hematology solvent control group and Ginkgolide B group dog changes, and sees Table 5-1. table 5-2

With control group relatively, every hematological indices does not occur relevant with administration unusual, the control group male and female respectively have the white blood cell count(WBC) of 2 dogs to be lower than 4 * 10 in the hematological indices ⁹/ L, but the difference of not statistically significant, all the other hematological indices administration groups and solvent control group be no significant difference relatively all.

Table 5-1 dog intravenous injection Ginkgolide B 100mg/kg.13 week is to hematological indices influence (basic value before the medicine)

Table 5-2 dog Ginkgolide B 100mg/kg.iv, the main hematological indices in 13 week backs

8) control group and the every blood parameters of administration group before the clinical chemistry administration, no significant difference.The amino transpeptidase of total serum protein, albumin, gpt, glutamic-oxal(o)acetic transaminase and glutamy seemingly has increase behind the intravenous injection Ginkgolide B male and female dog medicine, but with solvent control class value comparing difference not statistically significant, other change slight and fragmentary, do not think meaningful.See Table 6-1,6-2.

9) variation relevant with administration do not seen in uroscopy

10) organ weight

The female dog heart of Ginkgolide B group, liver,kidney,spleen organ weights and a little higher than contrast class value of organ coefficient (P＜0.05).Male dog organ weights and organ coefficient and control group compare the difference not statistically significant.See table 7 for details

Table 6-1 dog Ginkgolide B 100mg/kg, 13 weeks of intravenous injection are to the influence of blood parameters--blood biochemical basic value before the-medicine

Table 6--2 dog Ginkgolide B 100m/kg 13 week of intravenous injection back is to the influence of blood parameters

^*With the difference of comparing statistical significance

11) variation relevant with administration do not seen in huge inspection, but the organ weights of some organ and coefficient Ginkgolide B group and control group comparing difference have the significance meaning.

12) microscopy is not seen relevant with administration unusually, sees pathological replacement for details.

13) body temperature sees table 8 for details

14) injection site naked eyes and test under microscope are not seen obvious vascular stimulation symptom.

Table 7-1 female dog intravenous injection Ginkgolide B 13 weeks of 100mg/kg are to the influence of organ weights and organ coefficient

^*Compare with the solvent control group P＜0.05

Table 7--2 male dogs intravenous injection Ginkgolide B 100mg/kg is to the influence of organ weights and organ coefficient

(2) long-term intravenous injection bilobalide toxicologic study result of 13 weeks of rat

1. generalized case rat: behind the quiet notes Ginkgolide B, three dosage groups (20,50 and 125mg/kg) and control group are not relatively seen spirit and dystropy, food-intake and body weight change with contrast mouse comparison, no significant difference.

2. during dead this experimental observation all rats comprise the solvent control group and low, in and high dose group male and female rat all survive to the observation period and finish.

3. body weight when body weight male and female rat forward and backward body weight change weekly of continuous quiet notes Ginkgolide B and off-test, with control group comparing difference not statistically significant, the quiet notes of prompting Ginkgolide B do not influence rat body weight to be increased.See Table 9

Table 8 dog intravenous injection Ginkgolide B is to the influence of dog body temperature

Three months intravenous injection body weight change of table 9-1 male rat Ginkgolide B

4. hematological examination

The result shows: male and female rat Ginkgolide B 125mg/kg, and continuous 13 weeks of iv do not have obviously influence to every hematological indices, and each respective value compares between administration group and solvent control group, and difference does not have the significance meaning, promptly finds the change relevant with administration.See Table 10

Three months intravenous injection body weight change of table 9-2 female rats Ginkgolide B

The quiet notes Ginkgolide B of table 10 male and female SD rat 125mg/kg, 13 weeks are to the influence of hematological indices

5. to the influence of blood coagulation

Male and female rat Ginkgolide B 125mg/kg, continuous 13 weeks of iv do not have the obvious influence relevant with administration to coagulation indexes (going out clotting time and prothrombinogen time),

6. the influence of blood parameters

The result shows: male and female rat Ginkgolide B 125mg/kg, and continuous 13 weeks of iv do not have obviously influence to every blood parameters, and each respective value compares between administration group and solvent control group, and difference does not have the significance meaning, promptly finds the change relevant with administration.See Table 11.

The quiet notes Ginkgolide B of table 11 male and female SD rat 125mg/kg, the influence to blood parameters in 3 months

7. organ weight

The result shows: male and female rat Ginkgolide B 125mg/kg, and iv continuous 13 all administrations do not have significantly relevant influence with administration to organ weights, and each respective value compares between administration group and solvent control group, and difference does not have the significance meaning, promptly finds the change relevant with administration.See Table 12

The long-term Ginkgolide B 125mg/kg of table 12 rat intravenous injection is to the influence of organ weights

8. the long-term Ginkgolide B 125mg/kg of rat intravenous injection is to the influence of organ coefficient

The result shows: male and female rat Ginkgolide B 125mg/kg, continuous 3 months of iv do not have obvious influence to organ coefficient, between administration group and solvent control group each respective value relatively, difference does not have the significance meaning, promptly finds the change relevant with administration.See Table 13

9. pathological examination

Ginkgolide B 125mg/kg back male and female rat pathological examination of quiet 13 week of notes result shows the not discovery pathological change relevant with administration.

10. injection site visual control dog injection site does not find that the change of phlebitis sample appears in the administration blood vessel.

The long-term Ginkgolide B 125mg/kg of table 13 rat intravenous injection is to the influence of organ coefficient

Ten, evaluation of result

This test-results shows: beasle dog intravenous injection Ginkgolide B 100mg/kg, rat Ginkgolide B 20mg/kg, 50mg/kg and 125mg/kg, continuous 13 all intravenous injections, dog all growth, behavior, spirit, body weight, food consumption, hematology and the blood clotting relevant with administration, blood biochemical, routine urianlysis etc. occur unusually with rat during the administration, the change of aspects such as internal organ structure organization form is not found in pathology naked eyes and microscopic examination yet, can not determine the toxicity target organ.Organ coefficient etc. and the more every index difference of control group not statistically significant (P＞0.05), because the restriction of medicine dissolution and administration volume, this test is calculated by kg body weight and is not occurred toxicity symptom yet when this dosage is equivalent to the clinical maximal dose of people 50 times (dogs) and 75 times (rats) respectively, remains safe.

Intravenous administration bilobalide B acute toxicity test of the present invention

One, test objective

Carry out the observation of acute toxic reaction by intending clinical administration approach (filling stomach), the Ginkgolide B security is carried out clinical preceding the evaluation.

Two, tried thing

(1) title: the Ginkgolide B that the present invention produces, room temperature preservation;

(2) solvent and/or auxiliary material: tween-80 (Chemical Reagent Co., Ltd., Sinopharm Group), dehydrated alcohol (Shanghai reagent one factory, lot number: 050701).

Three, laboratory animal

Healthy adult mouse SPF level, body weight 18～22g; Male and female half and half, 5 in every cage.The Zong Yuan of Nanjing Military Command provides, conformity certification number: SCXK:2003-0004

Four, test method

4.1 trial test

The design of trial test grouping and dosage: engler Horn method is suddenly taked in this trial test, and dosage is from 10g/kg, 4.64g/kg, 2.15g/kg and 1.0g/kg, each 2 of every group of male and female.Observe animal dead number in 24 hours.

Preliminary experiment is intended using anhydrous alcohol solution, behind the tween-80 hydrotropy Ginkgolide B, dilutes with physiological saline again.Mouse vein administration and mouse stomach volume are respectively 10ml/kg and 20mL/kg.Owing to be subjected to reagent dissolving restriction, this test is with 11.75% Ginkgolide B solution.Vein and gastric infusion volume are 20mL, but administration (morning 8:00 and afternoon 4:00) at twice, the trial test dosage is respectively 2.15g/kg/d, 1.00g/kg/d and 0.64g/kg/d.The result shows and is subjected to all none dead mouses of reagent group and vehicle group after the administration of mouse Ginkgolide B in 7 days.Mouse feed, spirit and nervous activity, ight soil there is no unusually.Because solvability and administration volume restrictions, this test intended is with the evaluation index of maximum dosage as acute toxicity test.

4.2 official test

4.2.1 animal-origin and raising condition are the same, 40 of 18-22g kunming mices, male and female half and half.

4.2.2 dosage 2150mg/kg (iv), 4300mg/kg (ig)

4.2.3 route of administration

Originally being tried thing is injection, thus test take intravenous injection (iv) with irritate stomach (ig) administration.Administration time 8:00 in the morning and afternoon 4:00.Fasting 16 hours during oral administration but can't help water.

4.2.4 administration capacity tail vein injection (bolus): 10ml/kg (0.1ml/10g/ time, 2 times/day);

Irritate stomach: 20mL/kg (0.2mL/10g/ time, 2 times/day)

4.2.5 observe 14 days time limits, slower if toxic reaction occurs, answer proper extension observing time.

4.2.6 observation index

Comprise the weight of animals variation, diet, outward appearance, behavior, secretory product, movement, death condition and toxic reaction (symptom of toxic reaction, severity, time of origin, time length, whether reversible) etc.Should in time carry out gross anatomy to dying and dead animal, other animals carry out gross anatomy after the observation period finishes, and when finding that organ volume, color, quality etc. occur and changes, then the organ that changes are carried out histopathological examination.

4.2.7 result

In 14 days observation periods, mouse mainline Ginkgolide B 2150mg/kg and irritate stomach 4300mg/kg and the overt toxicity reaction all do not occur, animal spontaneous activity reduces after the administration, but recovers normal after 2 hours, and all mouse survive to the observation period and finish.Weight increase is subjected to no significant difference between reagent group and the solvent control group.Observation period finishes back necrotomy visual inspection and does not find that abnormal change appears in important organ.See Table 14

Table 14 Ginkgolide B vein 2150mg/kg or irritate before the stomach 4300mg/kg medicine and body weight change n=20 behind the medicine

Five, interpretation of result

This test-results shows: intravenous injection 2.15g/kg of mouse Ginkgolide B (be equivalent to clinical medicine dose 1075 times) and 4.3g/kg irritated behind the stomach in 14 days, any tangible toxicity symptom does not appear, no autonomic activities and other spirit, nervous system abnormality do not have animal dead yet.Observation period finishes the huge inspection of back necrotomy and does not find that histoorgan is unusual, and administration group and control group compare, the weight increase no significant difference.

Intravenous administration bilobalide B mutagenicity test of the present invention

One, test objective

Estimate the genetoxic that may exist of " intravenous administration bilobalide B " and the possibility of potential carcinogenesis, for clinical study and safe medication provide information.

Two, the Ginkgolide B that produced by reagent the present invention

Three, content of the test

3.1 external salmonella typhimurium reverse mutation test

3.2 Mammals marrow polychromatophilia micronucleus in erythrocytes test in the body

3.3 Chinese hamster pneumonocyte (CHL) chromosomal aberration test

Four, histidine auxotroph ames test

4.1 Ginkgolide B histidine auxotroph ames test (Salmonella reversion test), the result shows: 4 bacterial strains of 5 test dose groups (TA98, TA100, TA102, TA97), S no matter whether ₉Activation system exists, and the Salmonella reversion test of Ginkgolide B is negative findings.

4.2 solvent: DMSO specification 500mL/ bottle lot number 20021109 (Chinese Medicine group) sodium chloride injection specification 4.5g: 500mL production unit: Yangzhou Zhongbao Pharmaceutical Co., Ltd.

4.3 prepared by reagent: with DMSO Ginkgolide B is mixed with 0.5,1.5,5.0,15 and 50mg/ml totally 5 concentration, lucifuge is cold deposits.

4.4 reference substance

4.4.1 positive control drug: Quinacrime, to nitroquinoline, methylmethane sulphonate, 2-aminofluorene, 1,8-dihydroxyanthraquinone.

4.4.2 solvent control: DMSO (chromatographically pure).

4.4.3 blank: do not add and tried thing and solvent.

4.5 bacterial strain

4.5.1 test strain title: histidine auxotroph Salmonella typhimurium (S.Typhimurium), four strain bacterial strains are TA ₉₇, TA ₉₈, TA ₁₀₀, TA ₁₀₂

4.5.2 source and preservation: U.S. University of California department of biochemistry provides liquid nitrogen cryopreservation.

4.5.3 identification of strains: before the official test, carrying out test strain genotype identification (histidine auxotroph is identified, degree of depth rough type is identified, the R factor is identified and Δ uvrB identifies) and spontaneous answer number identifies, test meets the requirements with the bacterial strain biological characteristics, and test is 1-2 * 10 with bacterial concentration ⁹/ ml.

4.6 metabolism activation system

Mammals hepatomicrosome enzyme (S ₉) activation system: Shanghai Disease Prevention and Control Centre provides, and recording protein content by the Lowry method is 35mg/ml.Liquid nitrogen is preserved.

4.7 dosage and selection reason

Dosage is provided with: Ginkgolide B can be dissolved in DMSO, after prerun, establishes 5 of dosage levels, is respectively 0.05,0.15,0.5,1.5, the 5.0mg/ ware.

4.8 test method

Operation steps: the bacterial strain of accreditation is experimentized.Employing standard plate participates in method, establishes 0.05,0.15,0.5,1.5 and 5 serial dosage groups such as 5.0mg/ ware, 4 bacterial strains of each dosage group, and test divides an activation test (+S ₉) and disactivation test (S ₉), establish the corresponding positive and negative control group simultaneously.Each dosage group and control group are all made 3 plates, 48 hours observationss.And repeat once.

4.9 the result judges

Increased 4.9.1 try the recovery mutation colony number that thing brings out, surpass 2 times of solvent control group, dose-response relationship is arranged;

4.9.2 certain test point surpasses 2 times of contrasts, need present repeatability, and statistical significance is arranged.

Meet one of above-mentioned person, can be judged to the Salmonella reversion test positive, otherwise negative.

4.10 result

Ginkgolide B from 0.05,0.15,0.5,1.5, the 5.0mg/ ware, no matter add S ₉Or do not add S ₉To TA ₉₇, TA ₉₈, TA ₁₀₀And TA ₁₀₂The recovery mutation colony number of 4 bacterial strains all surpasses 2 times of spontaneous reverse mutation number; Each positive control compound group all is and surpasses 2 times positive reaction.Revision test is unanimity as a result.The prompting Ginkgolide B does not have the change effect of bringing out back to histidine auxotroph Salmonella typhimurium (S.Typhimurium).See Table 15.

Table 15 Ginkgolide B Salmonella reversion test result (6 ware means standard deviation)

The result is twice repeated experiments result's a average in the table.

^*Positive thing: do not add and use S ₉: TA ₉₇500 μ g/ ware Quinacrime; TA ₉₈200 μ g/ wares are to nitroquinoline; TA ₁₀₀And TA ₁₀₂1 μ l/ ware methylmethane sulphonate; Add and use S ₉: TA ₉₇, TA ₉₈, TA ₁₀₀10 μ g/ ware 2-aminofluorenes, TA ₁₀₂500 μ g/ wares 1, the 8-dihydroxyanthraquinone.

4 bacterial strains of each experimental group add and do not add S ₉Activation system is returned and is become the bacterium colony mean and be above negative control and return 2 times that become colony number, so can think that the Ames of Ginkgolide B tests negative result.

Five, mouse marrow cell micro nuclear test and Chinese hamster pneumonocyte chromosomal aberration test

5.1. being subjected to reagent prepares:, be soluble in DMSO because of Ginkgolide B is insoluble in water.

Accurately the weighing Ginkgolide B is used for the cell chromosome aberration test with the DMSO dissolving.

With 0.5% carboxymethyl cellulose (CM cellulose sodium salt 300-800) chemical pure, lot number F20040419, Chemical Reagent Co., Ltd., Sinopharm Group) make the suspension of desired concn for mouse marrow cell micro nuclear test usefulness.

5.2. reference substance

5.2.1. positive reference substance:

5.2.1.1 micronucleus test: endoxan.

5.2.1.2 Chinese hamster pneumonocyte (CHL) chromosomal aberration test: ametycin, endoxan.

5.2.1.3 blank product: DMSO.

5.3 mouse marrow cell micro nuclear test

5.3.1 animal

The sexual maturity Kunming mouse is provided by Shanghai Slac Experimental Animal Co., Ltd.'s (Chinese Academy of Sciences's Shanghai Experimental Animal Center).Production licence number: SCXK (Shanghai) 2003-0003; Animal occupancy permit number: SYXK (Soviet Union) 2002-0130.

5.3.2 dosage and selection reason

Dosage is provided with: 3 of dosage levels: be respectively 1.08,0.54,0.27g/Kg.bw

5.3.3 the route of administration per os is irritated stomach.

5.3.4 method

5.3.4.1 operation steps:

Through determine after the prerun sampling time be administration after 30 hours.50 of the Kunming mouses of selection body weight 18-24 gram are divided into 5 groups at random, and 10 every group, male and female half and half.Test group is respectively with 1/2,1/4 and 1/8LD ₅₀Three dosage per os gastric infusions, negative control are with isopyknic 0.5% carboxymethyl cellulose, and positive control is with the endoxan abdominal injection of 40mg/Kg.bw, and all animals is administered twice, in putting to death animal behind the 6h after the administration second time.Get the conventional film-making of breastbone, Giemsa dyeing, every animal is observed 1000 polychromatic erythrocytes under the opticmicroscope, calculates the permillage of micronucleated cell.

5.3.4.2. discrimination standard as a result:

The increase of the micronuclear rates that is a. tried thing and brought out has dose-response relationship;

B. certain test point micronucleus increase presents repeatability, and statistical significance is arranged.

Meet one of above-mentioned person, can be judged to the micronucleus test positive, otherwise negative.

5.4 result

Test-results: mouse marrow cell micro nuclear test result.See Table 16

Table 16 Ginkgolide B its mouse oral administration bone marrow cell micronucleus test-results

^*With negative control group than P＜0.001

By table 16 as seen: high, medium and low three the dosage group mouse polychromatic erythrocytes microkernel incidences of the B difference (P＞0.05) of comparing with negative control group that there are no significant in the silver, positive controls and negative control group ratio then have notable difference (P＜0.001).

5.5 conclusion

Ginkgolide B can not cause that under this experiment condition the mouse Bone marrow cells micronucleus rate raises.The prompting Ginkgolide B does not cause that the mouse somatocyte undergos mutation.

Six, CHL cell chromosome aberration test

6.1 cell Chinese hamster pneumonocyte (CHL cell).

6.3 dosage and selection reason

Dosage is provided with: under the maxima solubility (10.00mg/ml) of trial test at medicine, cell survival rate is greater than 90%, so establish 3 dosage levels: be respectively 10.0,5.0,2.5mg/ml.Other establishes positive control and negative control group.Direct positive reaction is ametycin 0.2 a μ g/mL nutrient solution, and the positive is endoxan 20 μ g/mL nutrient solutions indirectly, and directly feminine gender is a DMSO 0.1ml/L nutrient solution, and feminine gender is 50ml hepatomicrosome enzyme/L nutrient solution indirectly.

6.4 method

6.4.1. operation steps:

Cell is put in 37 ℃ of nutrient solutions and is cultivated 3d, adds B effect 48h in the silver of various dose respectively, at maxima solubility (10.00mg/ml) time cell survival rate greater than 90%, so be the maximum dose level of chromosomal aberration test with 10.00mg/ml.

During official test, do not adding liver microsomes enzyme (S ₉) time, harvested cell behind the processing 24h.Adding S ₉₉During mixture, behind the drug treating 6h, flush away S ₉Mixture and medicine are cultivated the 18h harvested cell again, and 2h adds colchicine before harvested cell, and dosage is 0.2 μ g/ml, prepares karyomit(e) according to a conventional method, dyeing then, microscopy.Repeat above-mentioned test, whether observations is consistent.

Each dosage is observed the chromosome aberration number of 100 metacinesis phase cells, and record gap (g), fracture (b), exchange (t), ring-type (r), 5 types of distortion of polyploid (p) (wherein g is not counted in total aberration rate) are represented with percentage.

6.5 discrimination standard as a result:

Aberration rate＜5% feminine gender (-)

Aberration rate＞5% suspicious (±)

Aberration rate＞10% positive (+)

Aberration rate＞20% strong positive (++)

A certain test point presents repeatably and has the increase of statistical significance; Meet above-mentioned one and can be judged to the positive.

6.6 result

CHL cell chromosome aberration test result.See Table 17

Table 17 Ginkgolide B is to the influence of CHL cell dyeing body structure

^*Compare P＜0.01 with negative control group

As known from Table 17: high, medium and low three the dosage groups of Ginkgolide B, no matter add and do not add activation system, CHL cell chromosome aberration rate is all＜5%.And positive controls adds and do not add activation system, and CHL cell chromosome aberration rate is then respectively up to 28% and 29%, 30%.Show that Ginkgolide B does not cause CHL cell chromosome textural anomaly.

6.7 conclusion

Whether Ginkgolide B salmonella typhimurium reverse mutation test is the result show, exist S9 metabolic activation thing all not have mutagenesis.Injection silver Ginkgolide B, mouse marrow cell micro nuclear test and Chinese hamster pneumonocyte (CHL) chromosomal aberration test result show: under this test conditions, the injection Ginkgolide B does not bring out mouse Bone marrow cells micronucleus to be increased; Do not bring out the CHL cell chromosome yet and distortion occurs.The result of three mutagenicity tests shows: in this test conditions and dosage (concentration) scope, the injection bilobalide does not have potential mutagenicity.

Claims

1. intravenous administration bilobalide B is characterized in that Ginkgolide B content 〉=97%, ginkgoic acid≤2ppm, and bilobalide does not detect.

2. according to the described intravenous administration bilobalide B extracting method of claim 1, comprise the Ginkgo Leaf lixiviate is separated the acquisition bilobalide, after recrystallizing methanol is purified, it is characterized in that said lixiviate is: with leaf dry weight 8～25 times of amounts, concentration is 5%-30% alcohol reflux several times, filtering separation; Filtrate is concentrated into 0.02～0.2 times of amount of original volume, adds sorbent material fully absorption under whipped state, filter, filter cake is with 3～10 times of amount alcohol reflux several times, and united extraction liquid is concentrated into 0.01～0.2 times of amount of original volume, leave standstill crystallization, filter bilobalide; Bilobalide carries out the recrystallization several times with the methyl alcohol of 10～50 times of amounts, recrystallization temperature-10 ℃～-25 ℃, filtering for crystallizing, drying, Ginkgolide B.

3. according to the described intravenous administration bilobalide B extracting method of claim 2, it is characterized in that said aqueous ethanolic solution is 10-20 a times of leaf dry weight.

4. according to the described intravenous administration bilobalide B extracting method of claim 2, it is characterized in that said described alcohol concn is 10%-25%.