CN1977868A - Ginkgo biloba leaf total terpene lactone extract, and its preparing method, medicinal composition and use - Google Patents
Ginkgo biloba leaf total terpene lactone extract, and its preparing method, medicinal composition and use Download PDFInfo
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Abstract
The present invention relates to a new-type high-effective safety medicine-ginkgo leaf total lactone for curing depression. It is a terpene lactone extracted from ginkgo biloba. L or other portions of said plant, such as ginkgo nut, ginkgo root bark and ginkgo twing, in which five terpene lactone compounds of bilobalide A, bilobalide B, bilobalide C, bilobalide J and bilobalide are mainly contained.
Description
Technical field
The present invention relates to fields such as phytochemistry and pharmaceutical chemistry, particularly, the present invention relates to Semen Ginkgo plant total lactone extract and its production and use.
Background technology
Dysthymia is listed as the 5th in the whole world ten big diseases, expects the year two thousand twenty and will rise to second, and the numeral of WHO shows: the patients with depression in the current whole world has 8,500 ten thousand crowd, expects 2005, and the sickness rate of depression will reach 10% of total population; And the annual suicidal thought that also has 1,000 ten thousand-2,000 ten thousand people, although depression seriousness is so big, but do not obtain treatment fully among this class patient, only have 53% patient once to heal with medicine, the patient of existing depression obtains appropriate amount of drug therapist less than 10%.Fluorine west fourth, Sertraline, paroxetine etc. are SSRI class medicine, be the first-selected active drugs of many countries as the treatment depression, but the unusual sickness rate of sexual function is up to 43%, main adverse reaction is sexual dysfunction and ahedonia, and part patient is not good to its reaction, and multiple side effect and contraindication are arranged, patient can not be tolerated, be difficult to reach satisfactory effect, the relapse rate height, and also various antidepressants all might bring out manic.Withdraw from rate according to nearest investigation SSRI and be about 11% common patient practitioner.Over nearly 10 years, the goal of the invention of new antidepressants is to increase therapeutic effect, improves toleration, reduces drug interaction, reduces untoward reaction.
It is flavone and ginkgolide compound that Semen Ginkgo (Ginkgo biloba L.) mainly contains effective constituent, is used for the treatment of cerebrovascular circulation and peripheral circulation disorders, as angina pectoris, coronary heart disease, cerebral infarction, apoplexy etc.(Wu Chunfu such as Wu Chunfu, the trip pine, Liu Wen etc., ". bilobalide and Semen Ginkgo extrac are to the influence of striatum and limbic system dopamine and metabolite content thereof ", " Chinese herbal medicine ", 1995,16 (5): 253～254,262) carried out bilobalide and Folium Ginkgo extract to striatum and limbic system dopamine and metabolite content thereof research, prompting bilobalide and Folium Ginkgo extract have certain inhibitory action to rat striatum and limbic system DA metabolism, and infer that at Folium Ginkgo extract to central dopamine in the metabolic influence, bilobalide may play an important role.But bilobalide is not seen the research of anti-zoopery depression of sex so far as yet.We screen and further investigate the depressed effective site of Folium Ginkgo, have confirmed that the bilobalide of Folium Ginkgo extraction, enrichment, purification is the strongest effective site of antidepressant pharmacology effect, are the active substance groups of extract of ginkgo biloba for treating depression; And confirm that further total lactone is more effective than monomer lactone, be very reasonably compatibility of monomer lactone, and have certain proportionate relationship between the monomer lactone that a better ratio is arranged.The technical study of ginkgo biloba leaf total terpene lactone extract of the present invention has filtered out optimum process as index, has finished the present invention thus.
Summary of the invention
Antidepressant drug one ginkgo biloba leaf total terpene lactone extract that the purpose of this invention is to provide a kind of new and effective, safety, non-evident effect;
Another object of the present invention provides the preparation method of ginkgo biloba leaf total terpene lactone extract;
Another object of the present invention provides the purposes that Folium Ginkgo or its total lactone extract are used to prepare the medicine for the treatment of depression, and this purposes preferably uses ginkgo biloba leaf total terpene lactone extract of the present invention to realize;
Another object of the present invention has provided the pharmaceutical composition of the treatment depression that contains ginkgo biloba leaf total terpene lactone extract of the present invention.
The terpene lactones of ginkgo biloba leaf total terpene lactone extract system of the present invention extraction from Folium Ginkgo (Ginkgo biloba L.) or other positions of this plant (as Semen Ginkgo, root bark, stem branch etc.), wherein mainly contain 5 kinds of terpene lactones chemical compounds, ginkalide A (Ginkgolide A; (15291-75-5) BN-52020; C
20H
24O
9), ginkalide B (Ginkgolide B; (15291-77-7) BN-52021; C
20H
24O
10), ginkalide C (GinkgolideC; (15291-76-6) BN-52022; C
20H
24O
11), bilobalide J (Ginkgolide J; (107438-79-9) BN-52024; C
20H
24O
10) and bilobalide (Bilobalide, C
15H
18O
8) (following GA, GB, GC, GJ and the BB of being called for short respectively.The content of each lactone in total lactone, wherein GJ accounts for total lactone 3%-15%, and GC accounts for total lactone 8%-30%, and BB accounts for total lactone 20%-65%, and GA accounts for total lactone 15%-50%, and GB accounts for total lactone 3%-40%.Content 〉=50% of total lactone in the ginkgo biloba leaf total terpene lactone extract, preferred 〉=70%.The inventor discovers that Folium Ginkgo or its total lactone can be used for the treatment of depression, and this purposes preferably uses ginkgo biloba leaf total terpene lactone extract of the present invention to realize.
Ginkgo biloba leaf total terpene lactone extract of the present invention can prepare by following method:
The Folium Ginkgo coarse powder with 50%～80% (v/v) ethanol or use acetone extraction, reclaims solvent, last macroporous adsorptive resins, the washing decontamination is behind 5%～20% (v/v) ethanol elution decontamination, use 50%～80% (v/v) ethanol elution instead, collect alcohol eluen, reclaim ethanol, last polyamide column, water elution, collect water elution liquid, concentrate ethyl acetate extraction.Reclaim solvent, vacuum drying gets ginkgo biloba leaf total terpene lactone extract.
The preferred manufacturing procedure of ginkgo biloba leaf total terpene lactone extract of the present invention is:
The Folium Ginkgo coarse powder, acetone extraction reclaims solvent, last macroporous adsorptive resins, the washing decontamination is behind 5%～20% (v/v) ethanol elution decontamination, use 60%～80% (v/v) ethanol elution instead, collect alcohol eluen, reclaim ethanol, last polyamide column, water elution, collect water elution liquid, concentrate ethyl acetate extraction.Reclaim solvent, vacuum drying gets ginkgo biloba leaf total terpene lactone extract.
The preferred preparation method of ginkgo biloba leaf total terpene lactone extract of the present invention is:
The Folium Ginkgo coarse powder, 70%～80% (v/v) ethanol extraction reclaims solvent, last macroporous adsorptive resins, the washing decontamination is behind 10%～15% (v/v) ethanol elution decontamination, use 60%～70% (v/v) ethanol elution instead, collect alcohol eluen, reclaim ethanol, last polyamide column, water elution, collect water elution liquid, concentrate ethyl acetate extraction.Reclaim solvent, or recrystallization, vacuum drying gets ginkgo biloba leaf total terpene lactone extract.
The present invention from Folium Ginkgo, extract the method for terpene lactones with other and the product that obtains different, its distinguishing feature is: the present invention has adopted macroporous adsorbent resin and polyamide column series connection method to extract and has handled.If single macroporous adsorbent resin of using, except total lactone that can't reach high-load (＞75%), more can't remove the flavone part in the Folium Ginkgo extract, not only and this flavone part does not have the antidepressant activity, on the contrary, also present antagonism and the effect of obvious suppression appetite.The present invention adopts macroporous adsorbent resin and polyamide column series connection method to extract and handles, polyamide column firmly is adsorbed on the chromocor compound that macroporous adsorptive resins elutes on the post, and terpene lactones is easy to be got off by water elution, thereby reach complete isolating purpose, prepare ginkgo biloba leaf total terpene lactone extract of the present invention.Although (Li Xingang such as Li Xingang, Wei Wei, Chen Wei. be rich in the Folium Ginkgo dry extract preparation technology of bilobalide. Chinese Medicine technology magazine, 1998,29 (1): 8) also adopted macroporous adsorbent resin and polyamide column series connection method, but the purpose of connecting with polyamide column fundamental difference, they are intended to remove tannin, so macroporous adsorbent resin 95% ethanol elution effluent, directly go up polyamide column without reclaiming ethanol, this is to adopt the macroporous adsorbent resin key point different with the polyamide column series connection method with the present invention, as a result in the effluent terpene lactones and the flavone component while by eluting, the product flavones content 27.4% that obtains, bilobalide content 10.6%; Two of difference: we adopt water elution by polyamide column, and Li Xingang etc. adopt 95% ethanol elution; Three of difference: we adopt polyamide column water elution liquid to concentrate, and the purification of ethyl acetate extraction and process for refining have only by this step to reach total lactone content＞75% target; And Li Xingang etc. do not design this step.Therefore the product that obtains of employing macroporous adsorbent resin such as Li Xingang and polyamide column series connection method has been said nothing of activity intensity with ginkgo biloba leaf total terpene lactone extract depression.
The ginkgo biloba leaf total terpene lactone extract that preparation in accordance with the present invention prepares, GJ wherein, GC, BB, the content relative fixed of GA and GB, this compatibility is very useful to the antidepressant effect of Folium Ginkgo total lactones of the present invention.
In the ginkgo biloba leaf total terpene lactone extract that the present invention prepares, the weight content of each lactone in total lactone: wherein the content of GJ is 3%-15%, and the content of GC is 8%-30%, and the content of BB is 20%-65%, the content of GA is 15%-50%, and the content of GB is 3%-40%;
The preferred ginkgo biloba leaf total terpene lactone extract of the present invention is, the weight content of each lactone in total lactone: wherein the content of GJ is 3%-10%, and the content of GC is 10%-20%, and the content of BB is 38%-55%, the content of GA is 18%-30%, and the content of GB is 4%-15%;
The preferred ginkgo biloba leaf total terpene lactone extract of the present invention is, the weight content of each lactone in total lactone: wherein the content of GJ is 3%-5%, the content of GC is 12%-20%, the content of BB is 40%-52%, the content of GA is 20%-30%, the content of GB is 5%-15%, total lactone content 〉=70%.
Ginkgo biloba leaf total terpene lactone extract of the present invention has good antidepressant effect, can be used to prepare the medicine for the treatment of depression.
The ginkgo biloba leaf total terpene lactone extract pharmaceutical composition of treatment depression of the present invention is made up of ginkgo biloba leaf total terpene lactone extract of the present invention and pharmaceutically acceptable auxiliaries, and it can prepare by the conventional method of pharmaceutical field.
The present invention discovers, ginkgo biloba leaf total terpene lactone extract, the mixture of GA and GB (purity 〉=98%, GA content is a little more than GB), BB (purity 〉=98%) and high-activity ginkgo leaf extract (wherein total lactone 12%, total flavones 48%) rat and mice all there is antidepressant effect, and ginkgo biloba leaf total terpene lactone extract (gastric infusion dosage: the total lactone 7.2mg/kg of mice, the total lactone 3.6mg/kg of rat) pharmacological action and other 3 kinds more all have significant difference P＜0.01, and with imipramine (gastric infusion dosage: mice 50mg/kg, rat 25mg/kg) more then there is not significant difference (P＞0.05).Their pharmacological action intensity: mixture=high-activity ginkgo leaf extract＞BB of total lactone＞GA and GB.Ginkgo biloba leaf total terpene lactone extract of the present invention is all very effective to multiple depression animal model, can reduce outstanding tail mice dead time, reduce the forced swimming mice dead time, can increase due to shocking by electricity the success of depressed rat the number of times of runing away, act on similar to imipramine.The mixture of GA and GB can increase the weight of the anxiety of rat, and BB then has angst resistance effect.Total lactone is equivalent to the compound preparation of Folium Ginkgo terpene lactones in the ginkgo biloba leaf total terpene lactone extract that the present invention prepares, wherein BB has angst resistance effect, help anxiety-depression patient's treatment, simultaneously antagonism GA and GB mixture increase the weight of the side effect of anxiety again, this shows, in the ginkgo biloba leaf total terpene lactone extract that the present invention prepares, each lactone proportioning is to the antidepressant effect significance, and the terpene lactones prescription is very reasonable.Contain total lactone 12% in the high-activity ginkgo leaf extract, though total lactone amount wherein equates with total lactone dosage in the ginkgo biloba leaf total terpene lactone extract that the present invention prepares, but the former obviously is not so good as ginkgo biloba leaf total terpene lactone extract of the present invention by antidepressant effect, from preparation technology and principal component analysis, high-activity ginkgo leaf extract is than the ginkgo biloba leaf total terpene lactone extract flavone of manying part, infer that thus the flavone in the high-activity ginkgo leaf extract does not partly have the antidepressant activity, on the contrary, as if also present antagonism, in addition, the part of the flavone in the high-activity ginkgo leaf extract still has the effect of obvious suppression appetite.6 months long term toxicity tests of acute toxicity test in mice and rat successive administration and 9 months long term toxicity tests of Beegle dog successive administration show that it is low that ginkgo biloba leaf total terpene lactone extract of the present invention has toxicity, and toxic dose and effective dose distance are bigger.It is all no abnormal to sexual cycle, farrowing rate, filial mice survival rate, the filial mice growth promoter of mating ability, sperm quantity and the vigor of male rat and female rats to observe ginkgo biloba leaf total terpene lactone extract of the present invention simultaneously.Test shows that also ginkgo biloba leaf total terpene lactone extract long-term prescription body of the present invention does not produce physical dependence and drug dependence.
Therefore, the present invention studies have shown that Folium Ginkgo total lactones provided by the invention and ginkgo biloba leaf total terpene lactone extract thereof have clear and definite antidepressant effect, can be used for the treatment of depressive illness.The advantage of ginkgo biloba leaf total terpene lactone extract of the present invention be effective dose low (for imipramine 1/7), safety is big, side effect is little, significantly do not cause the anxiety effect as GA and GB mixture, not significantly as the effect of high-activity ginkgo leaf extract appetite-suppressing, more do not have the appetite effect of imipramine severe inhibition and cause the disequilibrium effect, under the situation that especially is subjected to shocking by electricity repeatedly, the general situation of ginkgo biloba leaf total terpene lactone extract treated animal is better than other each group.Research of the present invention shows that also ginkgo biloba leaf total terpene lactone extract of the present invention is not seen obvious influence to sexual function and reproduction, does not have recessiveness, to the not influence of cardiovascular, motor system, mutual aid balance and muscular strength; The acute toxicity of ginkgo biloba leaf total terpene lactone extract of the present invention and long term toxicity test are not found the overt toxicity reaction.
Ginkgo biloba leaf total terpene lactone extract of the present invention can make up with pharmaceutically acceptable auxiliaries, is made into pharmaceutical composition, is used for the treatment of depression.This pharmaceutical composition can be forms such as tablet, capsule, granule, drop pill, injection, oral cavity rapid release preparation, sustained-release preparation.
The specific embodiment
Further specify the present invention below by embodiment.It should be understood that embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.In addition, in the present invention, (v/v) expression volume by volume concentration or volume ratio; (w/w) expression weight ratio concentration or weight ratio; (w/v) expression weight/volume specific concentration or by weight/volume, its corresponding unit is (grams per milliliter); (rpm) expression per minute revolution (rev/min); (d) expression day, (h) expression hour, (min) expression minute, (s) expression second.Except as otherwise noted, percent of the present invention is percetage by weight, and removing other has regulation, and the ginkgo biloba leaf total terpene lactone extract that the present invention uses embodiment 1 to prepare carries out pharmacological testing and formulation preparation.
Embodiment 1: the preparation of ginkgo biloba leaf total terpene lactone extract of the present invention
Get Folium Ginkgo coarse powder 2kg; add 20 liters of 60% ethanol; reflux, extraction 2 times each 3 hours, are reclaimed ethanol; last DM-130 macroporous adsorbent resin (production of resin subsidiary factory of Lukang Medical Co., Ltd., Shandong) post; 60% ethanol Xian takes off, and collects ethanol elution, reclaims ethanol; last polyamide column (column chromatography; Shanghai chemical reagents corporation produces), water elution is collected water elution liquid; be evaporated to an amount of; use ethyl acetate extraction, reclaim solvent, 80 ℃ of vacuum dryings; get Folium Ginkgo total lactones 3.8g, total lactone content 83.38%.The content of each lactone in total lactone, wherein the content of GJ is 3.62%, and the content of GC is 12.70%, and the content of BB is 49.59%, and the content of GA is 27.27%-, the content of GB is 6.83%.
Embodiment 2: the preparation of ginkgo biloba leaf total terpene lactone extract of the present invention
Get Folium Ginkgo coarse powder 2kg; add 24 liters of reflux of acetone; extract 2 times, each 2 hours, reclaim acetone; last DM-130 macroporous adsorbent resin (production of resin subsidiary factory of Lukang Medical Co., Ltd., Shandong) post; 60% ethanol Xian takes off, and collects ethanol elution, reclaims ethanol; last polyamide column (column chromatography; Shanghai chemical reagents corporation produces), water elution is collected water elution liquid; be evaporated to an amount of; use ethyl acetate extraction, reclaim solvent, 80 ℃ of vacuum dryings; get Folium Ginkgo total lactones 3.95g, total lactone content 85.11%.The content of each lactone in total lactone, wherein the content of GJ is 7.37%, and the content of GC is 14.94%, and the content of BB is 47.84%, and the content of GA is 24.83%, the content of GB is 5.02%.
Embodiment 3: the preparation of ginkgo biloba leaf total terpene lactone extract of the present invention
Get Folium Ginkgo coarse powder 2kg, add 24 liters of reflux of acetone, extract each 2 hours 2 times; reclaim acetone, last D101 macroporous adsorbent resin (production of Tianjin insecticide factory) post, 70% ethanol Xian takes off; collect ethanol elution, reclaim ethanol, last polyamide column (column chromatography; Shanghai chemical reagents corporation produces), water elution is collected water elution liquid; be evaporated in right amount, use ethyl acetate extraction, reclaim solvent; 80 ℃ of vacuum dryings get Folium Ginkgo total lactones 4.87g, total lactone content 71.76%.The content of each lactone in total lactone, wherein the content of GJ is 9.41%, and the content of GC is 20.22%, and the content of BB is 40.58%, and the content of GA is 24.65%, the content of GB is 5.15%.
Embodiment 4: the preparation of ginkgo biloba leaf total terpene lactone extract of the present invention
Get Folium Ginkgo coarse powder 2kg, add 24 liters of 70% ethanol, reflux; extract 2 times; each 3 hours, reclaim ethanol, last DM-130 macroporous adsorbent resin (production of resin subsidiary factory of Lukang Medical Co., Ltd., Shandong) post; behind the 10% ethanol flush away impurity, change 70% ethanol Xian and take off, collect 70% ethanol elution; reclaim ethanol; last polyamide column (column chromatography, Shanghai chemical reagents corporation produces), water elution; collect water elution liquid; be evaporated in right amount, use ethyl acetate extraction, washing ethyl acetate extraction liquid; anhydrous sodium sulfate dehydration; reclaim solvent, recrystallization, 80 ℃ of vacuum dryings; get Folium Ginkgo total lactones 3.10g, total lactone content 93.13%.The content of each lactone in total lactone, wherein the content of GJ is 4.11%, and the content of GC is 15.08%, and the content of BB is 45.64%, and the content of GA is 25.14%, the content of GB is 10.03%.
Embodiment 5: the preparation of high-activity ginkgo leaf extract
Get Folium Ginkgo coarse powder 2kg; add 24 liters of 70% ethanol, reflux, extraction 2 times, each 3 hours; reclaim ethanol; last DM-130 macroporous adsorbent resin (production of resin subsidiary factory of Lukang Medical Co., Ltd., Shandong) post, 70% ethanol Xian takes off, and collects ethanol elution; reclaim ethanol; last polyamide column (column chromatography, Shanghai chemical reagents corporation produces), water elution; collect water elution liquid; be evaporated in right amount, use ethyl acetate extraction, reclaim solvent; 80 ℃ of vacuum dryings; get ginkgo biloba leaf total terpene lactone extract 4.5g, total lactone content 87.13%, standby.Polyamide column behind the water elution, behind 10% ethanol flush away impurity, with rare pure eluting, vacuum concentration, spray drying gets Folium Ginkgo total flavones extract 13.7g, general flavone content 55.87% adds the ginkgo biloba leaf total terpene lactone extract 2.2g of front gained, mix homogeneously, get high-activity ginkgo leaf extract 15.9g, wherein total lactone content 12.05%, the content of each lactone in total lactone, wherein the content of GJ is 3.62%, the content of GC is 14.67%, the content of BB is 45.68%, and the content of GA is 26.04%-, and the content of GB is 9.99%; General flavone content 48.04%.
Embodiment 6: Folium Ginkgo total lactones antidepressant effect research of the present invention
Material
Bilobalide (BB): purity is 〉=99% (being that the inventor adopts Folium Ginkgo total lactones to obtain through the silica gel column chromatography separation)
Ginkgo biloba leaf total terpene lactone extract: embodiment 1 preparation; Hereinafter to be referred as: total lactone
Lactone GA: GB (57: 43) mixture: purity is (being that the inventor adopts Folium Ginkgo total lactones to separate through silica gel column chromatography that to obtain monomer formulated, the slightly high GB of the ratio of GA) for 〉=98%
High-activity ginkgo leaf extract: embodiment 5 preparations; Hereinafter to be referred as: high activity
Distinguish in order with above-mentioned Folium Ginkgo extract melt into 0.675mg.ml with distilled water
-1, 1.15mg.ml
-1, 0.675mg.ml
-1, 8.2mg.ml
-1The medicine liquid irrigation stomach use;
Imipramine hydrochloride 25mg/ sheet is produced by Shanghai nine good fortune Pharma Inc.s, lot number 990409, same melt into 12.5mg.ml
-1And 5mg.ml
-1The medicine liquid irrigation stomach use;
SD rat, Kunming mouse.
Method and result
1.1 mouse tail suspension test
Choose 60 of the male mices of body weight 20～24g, be divided into 6 groups at random, be i.e. normal saline matched group (NS), imipramine hydrochloride group, BB group, total lactone group, GA: GB group and high activity group, 10 every group by body weight.By body weight gastric infusion respectively, dosage sees Table 1, and the normal saline group such as gavages at the capacity normal saline, once a day, and totally 5 days.30 minutes begin to test after the 5th administration.Position with mice tail end 2cm during experiment is attached on the waddy, makes animal become reversal of the natural order of things state, the overhead about 5cm of its head.Both sides separate the animal sight line with plate, the dead time of back 3min in the record animal 6min.Real test is 1 time before the administration, and administration was tested once after 5 days.Calculate the difference of twice dead time of mice self and after 1g (X+86) conversion, carry out statistical analysis, the results are shown in Table 1.
4 kinds of Folium Ginkgos carry that mouse tail suspension dead time and NS group prolong more to some extent after the medication of thing thing, find through the F check analysis, the difference of dead time and NS group relatively has significant difference before and after the medication of 4 medication treated animals, P＜0.01, promptly these 4 kinds of Folium Ginkgo extract have antidepressant effect on this model.
Table 1: four kinds of influences that Folium Ginkgo extract is tested mouse tail suspension
N=10)
| Group | Medicine (mgkg -1) | Dead time (s) | Difference 1g (X+86) | |
| Before the medication | After the medication | |||
| The total lactone GA of NS BB: GB high activity imipramine | 5.0 7.2 * 5.0 60.0 50.0 | 81.4±32.8 81.9±19.1 78.4±25.3 76.5±15.8 86.7±38.4 90.2±32.8 | 65.1±30.8 45.1±15.3 36.9±17.6 60.0±18.7 59.5±28.9 31.7±16.3 | 1.826±0.645 2.082±0.080 2.135±0.058 2.008±0.066 2.026±0.169 F＝1 6.45 P＜0.01 2.149±0.092 |
The ANOV statistical analysis method
*: the ginkgo biloba leaf total terpene lactone extract dosage is in wherein total lactone (as follows).
1.2 mice forced swimming test
Choose 60 of the male mices of body weight 20～24g, grouping and administration are with test 1.1.See Table 2.30 minutes begin to test after the 5th administration.Mice is put into the graduated cylinder (high 20cm, diameter 14cm) of depth of water 10cm, 30 ℃ of water temperatures.Calculate the difference of twice dead time of mice self and after 1g (X+45) conversion, carry out statistical analysis, the results are shown in Table 2.
The result confirms, the dead time when 4 kinds of Folium Ginkgo extract that tried and imipramine all can prolong the mice forced swimming, with the normal saline group relatively, can prolong 18.09～34.14%.Through variance analysis, 6 experimental mice dead time differences are significantly different, and highly significant statistical significance (P＜0.01) is arranged.By through the Newman-Keuls statistical analysis, relatively there is remarkable statistical significance (P all＜0.01) dead time that imipramine and 4 kinds of Folium Ginkgo extract reduce in the mice forced swimming with normal saline respectively.Imipramine, total lactone group drug effect are than GA: GB group strong (P all＜0.01) again.
Table 2: plant the influence of Folium Ginkgo extract to the mice forced swimming test
N=10)
| Group | Medicine (mgkg -1) | Dead time (s) | Difference 1g (X+45) | |
| Before the medication | After the medication | |||
| The total lactone GA of NS BB: GB high activity imipramine | 5.0 7.2 * 5.0 60.0 50.0 | 107.4±16.4 99.6±23.9 99.2±22.3 102.7±16.7 106.7±16.6 99.8±13.1 | 105.1±21.7 87.4±21.7 25.8±16.6 38.3±13.2 29.4±13.7 25.9±17.7 | 1.550±0.376 1.830±0.172 ※※2.081±0.082 ※※2.008±0.066 ※※2.021±0.197 ※※2.079±0.105 ※※F＝13.24 P＜0.01 |
※ ※ANOV and Newman-Keuls statistical analysis method are compared with the NS group in P＜0.01
1.3 the depressed test of rat anti
Observe the antidepressant effect of medicine with acquired helpless electric shock model.Choose 70 of male rats, body weight 180～200g is divided into 7 groups at random by body weight, promptly normal control group, normal saline matched group (model group, NS), BB group, total lactone group, GA: GB group, high activity group and imipramine group, 10 every group.By body weight gastric infusion respectively, dosage sees Table, and the normal saline group such as gavages at the capacity normal saline, once a day, and totally 35 days.Begin experiment after 30 minutes in the 30th administration.Test and carried out " helpless bringing out " in first day, with one 20 * 10 * 10cm
3The bottom be the cage of copper grid, impose unavoidable foots electric shock (every 1min ± 15s gives once for 0.8mA, 15s) at random 60 times to animal, rats in normal control group is put into the identical time of cage but is not given electric shock.Begin to carry out avoidance training behind the 48h.With 60 * 20 * 30cm
3Shuttle box, the base copper grid are spaced apart 1.0cm.Animal is individually put into shuttle box one end, allow it adapt to the avoidance training that carries out 20 times behind the 5min, each 30s at interval.Give a light signal during training earlier, allow animal to reach the other end during this period to escape electric shock, if reactionless generation, then optical signal continues to occur 3s again, a 0.8mA also appears simultaneously, the foot electric shock of 3s, if still reactionless generation, electric shock and optical signal stop immediately and remember and do once to escape failure.Training was carried out 5 days altogether.Write down the escape number of success of every rat when training every day, the 5th training result is carried out statistical analysis.
According to test result analysis, the NS group compares with normal group, and rat successfully escapes number of times and obviously reduces (t=3.99, P＜0.01), illustrates that the rat depression model is successful.Test all has the improvement effect with 4 kinds of Folium Ginkgo extract and imipramine to the depressive state rat that unavoidable electric shock causes, especially remarkable with total lactone group, through variance analysis, 7 experimental group rats escape significantly difference of number of success, and highly significant statistical significance (P＜0.01) is arranged.Further analyze through the Newman-Keuls statistic law again, 4 kinds of Folium Ginkgo extract and imipramine relatively have remarkable statistical significance (P all＜0.01) to effect and the normal saline of depressed rat, and more all there were significant differences for the pharmacological action of total lactone and other 3 kinds of extracts, P all＜0.01 does not more then have marked difference (P＞0.05) with imipramine.A little less than in addition drug effect with BB was between 3 kinds of extracts, drug effect was not then seen marked difference between GA: GB and high activity extract.These 3 kinds of extract drug effects are weak (P all＜0.01) than imipramine all.See Table 3.
Table 3: four kinds of Folium Ginkgo extract are to the influence of rat depressive state
N=10)
| Group | Medicine (mgkg -1) | Number of times (5d) is escaped in success |
| The normal total lactone GA of NS BB: GB high activity imipramine | 5.0 7.2 * 5.0 60.0 50.0 | 18.7±1.3 12.9±4.4 9.2±4.8 ※※△△ 17.7±1.9 ※※ 14.8±2.5 ※※△△ 15.2±4.3 ※※△△ 17.5±1.8 ※※ F＝9.157 P＜0.01 |
※ ※Compare with the NS group P＜0.01,
△ △Compare with total lactone group P＜0.01.ANOV and Newman-Keuls statistical analysis method
Conclusion:
1,4 kinds of extracts of Folium Ginkgo all have antidepressant effect with the dosage that adopted in this test to rat and mice.Their the total lactone=imipramine of pharmacological action intensity＞GA: GB=high activity＞BB.
2, GA, GB mixture can increase the weight of the anxiety of rat and cause stiff carpentery workshop usefulness, and BB then has angst resistance effect.The obvious appetite-suppressing effect of high-activity ginkgo leaf extract.
3, the antidepressant advantage of ginkgo biloba leaf total terpene lactone extract is that side effect is little, do not have tangible GA, GB to cause anxiety and cause stiff carpentery workshop to use, there is not the effect of tangible high-activity ginkgo leaf extract appetite-suppressing, more do not have imipramine severe inhibition appetite and cause the disequilibrium effect, especially under the situation that is subjected to shocking by electricity repeatedly, the general situation of total lactone treated animal is better than other each groups.
4,4 kinds of extracts of Folium Ginkgo all have sedation with the dosage that adopted in this test, but do not influence the activity such as motion, feed of animal.
Embodiment 7: ginkgo biloba leaf total terpene lactone extract is to the influence of depressive state
Material
Be subjected to the reagent thing
Ginkgo biloba leaf total terpene lactone extract: embodiment 1 preparation, irritating the stomach test is 2.5% arabic gum normal saline suspension with medicinal liquid.
Animal: Kunming mouse, SD rat.
Other medicine: imipramine hydrochloride is Shanghai nine good fortune pharmaceutical factory product (lot numbers: 990401).
1.1 influence to the mice dead time
Choose 50 of the male mices of body weight 20～24g, be divided into 5 groups at random by body weight, i.e. normal saline matched group, ginkgo biloba leaf total terpene lactone extract 12.5,25.0,50.0mgkg
-1Group, imipramine hydrochloride group 30.0mgkg
-1, 10 every group.By body weight gastric infusion 1 time respectively, the normal saline group such as gavages at the capacity normal saline with above dosage, during experiment mice is become the reversal of the natural order of things state, and other are with 1.1 experimental technique among the embodiment 6.Experiment is 1 time before the administration, and the 1h experiment once after the administration.Calculate the difference of twice dead time of mice self, do variance analysis.
Other gets 50 of the male mices of body weight 20～24g, and the grouping situation is the same.Divided dose is by body weight ig administration respectively, and capacity normal saline such as normal saline group ig are total to 3d every day 1 time, test once before the same method administration, and test once behind the administration 3d.Calculate the ratio of mice dead time/6min and the difference of self twice test, do variance analysis.
Mouse tail suspension dead time and normal saline group prolong more to some extent after the ginkgo biloba leaf total terpene lactone extract medication, and increase with dosage, mice dead time dose dependent ground prolongs, find through the F check analysis, difference has significant difference before and after the medication of 5 groups of mices, P＜0.01, promptly ginkgo biloba leaf total terpene lactone extract has antidepressant effect on this model.
1.2 make the ED of mouse tail suspension dead time minimizing
50Measure
Choose 100 of the male mices of body weight 20～24g, be divided into 10 groups at random by body weight, i.e. ginkgo biloba leaf total terpene lactone extract 5.12,6.4,8.0,10.0,12.5mgkg
-1Group, imipramine hydrochloride 12.29,15.36,19.2,24.0,30.0mgkg
-1Group, 10 every group.The same method is carried out the mouse tail suspension experiment, and reaching 10% with the front and back difference is that medicine is effective, and the result calculates the ginkgo biloba leaf total terpene lactone extract ED that the mouse tail suspension dead time of sening as an envoy to reduces with the bliss method
50± L
95=7.27 ± 0.91mgkg
-1, imipramine ED
50± L
95=16.90 ± 2.40mgkg
-1
1.3 influence to the mice forced swimming dead time
Choose 50 of the male mices of body weight 20～24g, grouping and administration be with test 1.1, administration 4d, and 1h begins experiment after the 4th administration.Mice is put into the graduated cylinder (high 20cm, diameter 14cm) of depth of water 10cm, 25 ℃ of water temperatures respectively.The dead time of back 4min in the record animal 6min.Calculate the ratio of mice dead time/6min, compare, do variance analysis with matched group.
The result confirms, ginkgo biloba leaf total terpene lactone extract and imipramine 30.0mgkg
-1Dead time in the time of all prolonging the mice forced swimming, compare ginkgo biloba leaf total terpene lactone extract 12.5-50.0mgkg with the normal saline group
-1Can prolong 17.98～37.49%.Through variance analysis, 5 experimental mice dead time differences are significantly different, and significance statistical significance (P＜0.01) is arranged.
1.4 influence to depressed rat
With acquired helpless electric shock model
[4]Observe the antidepressant effect of medicine.Choose 60 of male rats, body weight 180～200g is divided into 6 groups at random by body weight, i.e. normal control group, normal saline matched group (model group), ginkgo biloba leaf total terpene lactone extract 7.5,15.0,30.0mgkg
-1Group and imipramine 20.0mgkg
-1Group, 10 every group.By body weight ig administration respectively, capacity normal saline such as normal saline group ig, every day 1 time, 35d altogether.Behind the 28th administration 1h, begin experiment.Test 1d and carry out " helpless bringing out ", other are with 1.3 experimental technique among the embodiment 6.
Through variance analysis, 6 experimental group rats escape significantly difference of number of success, and remarkable statistical significance (P＜0.01) is arranged according to test.Depressive state rat that unavoidable electric shock causes and rats in normal control group are escaped number of success remarkable significant difference (P＜0.01), the depressive state rat that ginkgo biloba leaf total terpene lactone extract causes unavoidable electric shock successfully escapes electric shock positive role, and dose dependent is arranged.Imipramine also has improvement effect (P＜0.01) to the depressive state rat.
Embodiment 8: to cardiovascular, motor system pharmacological research
Material
Be subjected to the reagent thing
Ginkgo biloba leaf total terpene lactone extract: embodiment 1 preparation, irritating the stomach test is 2.5% arabic gum normal saline suspension with medicinal liquid.
Animal: Kunming mouse, SD rat.
Other medicine and reagent: scopolamine (Scopolamine, Sco) be Guangdong emigrant's industry pharmaceutical Co. Ltd product, (Xylazine Xyl) is provided by Military Medical Science Institute's chemical defence hydrochloric acid thiophene piperazine, and imipramine hydrochloride is Shanghai nine good fortune pharmaceutical factory product (lot numbers: 990401).
Instrument and equipment: CPRS blood pressure cardiac electrophysiology computer monitoring system (Jiangxi Medical College's function center development is by provincial-level appraisal), pressure transducer etc.
Statistical analysis: the result carries out statistical analysis with the NDST software of Wannan Medical College's development.
Method and result
2.1 ginkgo biloba leaf total terpene lactone extract is to cardiovascular influence
Select experimental rat for use, urethane 1.5gkg
-1Ip anesthesia, the common carotid artery intubate connects with the CPRS physiological monitoring system after connecting pressure transducer, electrocardio lead, treat that reaction of animals is stablized after, write down one section administration preceding observed value, i.e. heart rate, the rhythm of the heart, systolic pressure, diastolic pressure, respiratory frequency, joint rate and amplitude of respiration.The duodenum injection gives ginkgo biloba leaf total terpene lactone extract then, and dosage is respectively 7.5,15.0,30.0,50.0,75.0,100.0mgkg
-1, the administration volume is 2.5mlkg
-1, matched group waits the capacity normal saline, respectively at administration 20,30, and above each index of observed and recorded behind the 60min.
The result shows, ginkgo biloba leaf total terpene lactone extract single administration 7.5-50.0mgkg
-1Anesthetized rat systolic pressure, diastolic pressure, the rhythm of the heart, heart rate, respiratory frequency, the rhythm and pace of moving things, the degree of depth are not all made significant difference, and after the administration 20,30, relatively, difference does not have significance (P＞0.05) before each observed value of 60min and the self administration.After the administration in 2 hours rat arrhythmia does not appear.When dosage reaches 75mgkg
-1The time, the about 20mmHg of rat blood pressure decline behind the administration 20min can return to the preceding blood pressure level of medication behind the 40min.But dosage reaches 100.0mgkg
-1When above, blood pressure drops is more, and can not return to the preceding level of administration.Above results suggest rat is lower than 50.0mgkg with Folium Ginkgo total lactones
-1It is safe dose.
2.2 ginkgo biloba leaf total terpene lactone extract is to the influence of coordinated balance motion
2.2.1 mice blance test and rotating stick test
Get mice, body weight 19～22g, ♂ ♀ dual-purpose is divided into 6 groups at random by the sex body weight, 12 every group: 1. 2.-5. ginkgo biloba leaf total terpene lactone extract 12.5,25.0,50.0,100.0mgkg of matched group
-1Organize 6. thiophene piperazine 10.0mgkg
-1Group.Carry out rotating stick test before the administration earlier, mice is placed on the homemade horizontal transfer rod dish, shank diameter is 2.5cm, and 16 times/min of rotating speed is selected in the above mice of excellent upward stop 3min and carries out next step experiment.Each group is by above-mentioned dosage ig administration, and matched group waits the capacity normal saline, administration volume 0.2ml/10g, and every day 1 time, 7d tests behind the last administration 1h continuously.Be recorded in the Mus number of loss of equilibrium on the transfer rod dish during rotating stick test, it is long mice to be placed on a 60cm again, and diameter is on the unsettled horizon bar of 0.8cm, falls in all 3min of animal that the person is disequilibrium more than 3 times, and record falls and falls Mus number more than 3 times in the 3min.
Experiment confirm, alpha-2 receptor blocker Xyl 10.0mgkg
-1Make 8/12 mice bull stick anergy, calm with it relevant with the effect of flesh pine.Ginkgo biloba leaf total terpene lactone extract 12.5-50.0mgkg
-1Mutual aid coordinated balance to mice does not have harmful effect, 100.0mgkg
-1More than influence the mutual aid coordinated balance ability of mice, reduce mice bull stick and holding rod ability, but not statistically significant (P＞0.05).
2.2.2 inclined plane method
Get 40 of rats, body weight 160～200g, ♂ ♀ dual-purpose is divided into 4 groups at random by the sex body weight: 1. 2.-4. ginkgo biloba leaf total terpene lactone extract 7.5,15.0,30.0mgkg of matched group
-1Group, be selected in before the administration on the transfer rod dish (shank diameter is 7.5cm, rotating speed be 8 times/min) stopping the above rat of 3min carries out next step experiment.Each group is by above-mentioned dosed administration, and matched group waits the capacity normal saline, administration volume 1.0ml/100g, and every day 1 time, 7d tests behind the last administration 1h continuously.Be recorded in the Mus number of loss of equilibrium on the transfer rod dish during rotating stick test, rat be placed on the level and smooth plank again, with one downward-sloping 45 ° of planks, record slides number of animals.
Rat is used ginkgo biloba leaf total terpene lactone extract 7.5-30.0mgkg
-1Do not influence the coordinated balance function, compare there was no significant difference, P＞0.05 with control rats.
2.2.3 swimming method
Get the 2.2.2 experimental mouse, other gets 10 of rats, and body weight is close with the 2.2.2 experimental mouse, ig imipramine hydrochloride 30mgkg
-1, once a day, be total to 7d.In administration 1d, 2d, and 7d puts into Morris water labyrinth and swims.
Analysis result finds that imipramine group rat begins loss of equilibrium when 2d swim, and body inclination is to a side in water, but the swimming rate and the direction that do not influence in the water labyrinth discern, and during to 5d, balance-loss state alleviates.Ginkgo biloba leaf total terpene lactone extract is respectively organized rat does not then have the loss of equilibrium phenomenon, and damage is light than imipramine to the prompting ginkgo biloba leaf total terpene lactone extract to equilibrium function.
2.3 influence to muscular strength
Observe the muscular strength of mice and rat with pulling test.Get male mice and male rat, dosage sees Table, every day 1 time, be total to 7d, 1h is placed on (rat 0.2cm, mice 0.1cm) on the horizontal wire with two fore paws of animal after the last administration, as animal in the 3s fall or only a pawl catch tinsel all to belong to failure, record failure number of animals.
The result shows, ginkgo biloba leaf total terpene lactone extract mice dosage 12.5-50.0mgkg
-1, rat dosage 7.5-30.0mgkg
-1, medication 7d is to nibbling the not influence of muscular strength of food class animal.
Conclusion
Mice gives 12.5-50.0mgkg
-1, rat 7.5-30.0mgkg
-1Ginkgo biloba leaf total terpene lactone extract, medication 7 days mutual aid coordinated balance and the not influence of muscular strength to nibbling food class animal is with control animals zero difference relatively.Compare with the imipramine treated animal in swimming test, the damage of equilibrium function is littler.
Embodiment 9: to the influence of sexual function and reproduction
Be subjected to the reagent thing: with embodiment 8, be made into suitable concentration, directly irritate stomach and use to animal with 5% arabic gum normal saline solution.Animal and statistical analysis: with embodiment 8 and the experimental result software NDST of Wannan Medical College analytical calculation.
3.1 influence to the female rats oestrous cycle:
Get 3 months female experimental mouse of administration in the long term toxicity test, 10.0,60.0,120.0mgkg
-1Group and normal saline matched group, 10 every group, body weight 205-266g gets the rat vagina cell behind the normal saline mixing with cotton swab, to low power microscope observation down, every day 1 time, totally 6 times.
Observed result shows, the oestrous cycle of respectively organizing female rats of being tried ginkgo biloba leaf total terpene lactone extract of the present invention is not affected, rutting period and diestrus difference are obviously, the visible superficial cell of later stage of oestrusing, shape is big and irregular, similar to matched group, the estrus cycle is then only seen a spot of leukocyte, no keratinization epithelium.
3.2 influence to male rat mating ability and sperm quantity, vigor
With experiment 3.1, with 3 months male mouse of administration in the long term toxicity test, get 6 for every group, with female: male 1: 1 ratio and adult virgin Mus and cage, with two weeks interior female Mus vaginas sperm occurs and think that male Mus has mating ability, calculate male Mus mating rate (%).Put to death rat then, take off epididymis, at one end cut off, draw the 0.1ml seminal fluid, put in the test tube that fills the 2ml normal saline and shake up by cross with shears, leave standstill 30min after, drip on counting chamber, observe motility of sperm and counting.
Ginkgo biloba leaf total terpene lactone extract long term administration (10.0-120.0mgkg
-1) to the mating ability of male rat, sperm count, the sperm motility influence is not obvious, with matched group relatively, not statistically significant (p＞0.05).
3.3 influence to the rat reproduction
Get 120.0mgkg in 3.2 experiments
-1Become pregnant 2 of female rats of group copulation, the back 21d that becomes pregnant farrow respectively 11 (7 ♂, 4 ♀) and (6 ♂, 6 ♀), with matched group become pregnant the farrowing of female Mus (2,10 of farrowing, 4 ♂, 6 ♀, 11 5 ♂ of a farrowing, 6 ♀) relatively, the filial mice outward appearance does not have deformity, body weight equilibrium between the filial mice, physically well develops none death of filial mice at body weight gain.Can think ginkgo biloba leaf total terpene lactone extract 120.0mgkg
-1Below litter size, filial mice survival rate, filial mice outward appearance, the growth promoter of female rats had no adverse effects.
Embodiment 10: become recessive test
Be subjected to reagent thing, statistical method: with embodiment 8.Other medicines: morphine hydrochloride powder pin is the Shenyang No. 1 Pharmaceutical Factory product; Stable powder pin is Shanghai pharmacy three factory's products.
Method and result
4.1 mice natural withdrawal test
Get 50 of mices, body weight 18-22g, ♂ ♀ half and half.Be divided into 1. 2. 3. ginkgo biloba leaf total terpene lactone extract 12.5mgkg of positive controls of normal control group at random by the sex body weight
-1Organize 4. ginkgo biloba leaf total terpene lactone extract 25.0mgkg
-1Organize 5. ginkgo biloba leaf total terpene lactone extract 50mgkg
-1Group.Morphine hydrochloride is mixed in the mixed feed (every gram is expected hydrochloric morphine 1.1mg) feeds and 2. organizes mice.3.-5. (ig: the ginkgo biloba leaf total terpene lactone extract expression oral administration), normal control group ig normal saline once a day, is total to 7d by various dose ig to organize mice.After the 7d administration, weigh immediately, begin then to give up, promptly 2. organize the mixed feed raising that mice is changed not dusting powder drug, 3.-5. organize mice and stop gastric infusion.Each is organized mice and claims body weight one time every 4h, till recovering normally.To give up preceding body weight is radix, calculates the percentage rate of giving up each time point mice weight loss of back, adds up the difference degree of each group of ig ginkgo biloba leaf total terpene lactone extract and positive controls body weight change respectively.
By experimental result as seen, behind the ginkgo biloba leaf total terpene lactone extract administration 7d after the drug withdrawal suddenly mice withdrawal symptom does not appear, with the withdrawal symptom of morphine group such as body weight change relatively, difference has highly significant (p＜0.01).
4.2 rat substitutes experiment
Get 30 of ♂ rats, body weight 300g ± 65s is divided into 3 groups at random by body weight: 1. positive controls, in feedstuff, stabilize 52-160mgkg with drug-admixed food
-1, 2.-3. ginkgo biloba leaf total terpene lactone extract 30.0mgkg
-1, 60.0mgkg
-1Group, ig after continuous 12 weeks, interrupts the stable and ginkgo biloba leaf total terpene lactone extract ig of feeding suddenly once a day, observes rat and withdrawal symptom whether occurs, and weigh, the stable 10.0mgkg of positive controls ig behind the 36h
-1, 2., 3. organize ig ginkgo biloba leaf total terpene lactone extract 30.0,60.0mgkg
-1, observe the rat symptom and whether disappear, whether body weight is recovered.
Experiment shows that it is stable to feed for a long time, and after the drug withdrawal suddenly, withdrawal symptom appears in rat, show as excited uneasiness, appetite reduces, sialorrhea, and weight loss is obvious behind the 36h, stabilize the back sx immediately, weight recovery is very fast, and ig ginkgo biloba leaf total terpene lactone extract 30-60mgkg
-1And the withdrawal symptom of addictive rats is not had improvement, body weight is not also recovered behind the ig, compares p＞0.05 with positive controls.Can think that ginkgo biloba leaf total terpene lactone extract does not have alternative cancellation effect to the withdrawal symptom of stable addictive rats.
4.3 conditioned place preference
With the square box of two sizes consistent 50 * 25 * 25, the box wall is smooth, makes black and white, and the box floor is respectively matsurface and shiny surface, and there is active clapboard the centre, is used for passage between two boxes during unlatching, and add a cover at the top.
Get 30 of rats, body weight 200g ± 20g, ♂ ♀ dual-purpose, by the sex body weight be divided at random normal saline matched group and ginkgo biloba leaf total terpene lactone extract (30.0,60.0mgkg
-1) group.Test 1d and rat is put into the box of taking out dividing plate, the time that stops respectively in the inherent black/white box of record rat 20min, then by various dose ig ginkgo biloba leaf total terpene lactone extract, seal passage between two boxes, rat is positioned in the white box, take out same time of 2d, capacity such as ig 5% arabic gum normal saline solution behind the 20min, put it in the black box and take out behind the stop 20min, test in 7d after repeating 3 times, promptly under the situation of not administration, extract two box space bars, rat is put into box, the time that stops respectively in the inherent black/white box of record rat 20min, compare with test for the first time in the preference position of observation animal.
By test result analysis, ginkgo biloba leaf total terpene lactone extract can not increase the time of staying of rat in white box, compare with the normal saline group, and p＞0.05, promptly ginkgo biloba leaf total terpene lactone extract does not influence the position preference of rat.
Embodiment 11: acute toxicity test
Ginkgo biloba leaf total terpene lactone extract is to the disposable gastric infusion of mice, male LD
50Be 3030.5 ± 646.6mgkg
-1, female LD
50Be 2828.7 ± 574.3mgkg
-1Intraperitoneal injection male mice LD
50Be 412.2 ± 47.6mgkg
-1, female mice LD
50Be 457.9 ± 67.4mgkg
-1To the disposable gastric infusion of rat, male LD
50Be 1073.7 ± 92.8mgkg
-1, female LD
50Be 1096.2 ± 139.0mgkg
-1Intraperitoneal injection male rat LD
50Be 410.2 ± 44.7mgkg
-1, female rats LD
50Be 410.2 ± 45.1mgkg
-1Asexuality difference.
Embodiment 12: the rat long term toxicity test
Ginkgo biloba leaf total terpene lactone extract was with three dosage 10.0mg/kg/ days, 60.0mg/kg/ day, 120.0mg/kg/ day successive administration 6 months,, body weight gain less except that heavy dose of treated animal rats eating slowed down, rat outward appearance sign, behavioral activity, defecation, hematological indices, blood biochemical learned that index, system become celestial, the histopathologic examination of organ coefficient, whole body major organs tissue does not all find other toxic reaction.
Embodiment 13: the dog long term toxicity test
Ginkgo biloba leaf total terpene lactone extract was with three dosage 81.0mg/kg/ days, 27.0mg/kg/ day, 9.0mg/kg/ day successive administration 9 months.The test initial stage, in, vomiting after eating reaction appears in heavy dose of administration group part animal, continues for 3 weeks approximately, stops vomiting after 3 weeks substantially, other animal activity freely, defecation is normal, body weight increases naturally.The important organ coefficient: notable difference is found at 17 organ coefficient ends such as the brain of each treated animal, heart, liver, lungs, spleen, kidney; Check pathological section: totally 26 internal organs pathological sections such as the brain of each treated animal, spinal cord, thymus, thyroid etc. are not all found obvious pathologic pathological changes; Convalescent period is checked and finds: the every index of hematology and uroscopy is all in normal range; The rarely seen arrhythmia of electrocardiogram is substantially in normal range; Each internal organs of pathological examination do not find that all obvious pathologic changes.
Embodiment 14: tablet
Tablet formulation
Ginkgo biloba leaf total terpene lactone extract 0.3kg
Corn starch 0.6kg
Hydroxypropyl starch 0.06kg
Microcrystalline Cellulose 0.02kg
Aluminium silicate 0.02kg
Low-substituted hydroxypropyl cellulose 0.03kg
Magnesium stearate 0.0003kg
Method for making is with ginkgo biloba leaf total terpene lactone extract (crossing 80 mesh sieves); adding starch (80 ℃ of bakings), hydroxypropyl starch, microcrystalline Cellulose, aluminium silicate, low-substituted hydroxypropyl cellulose puts in the fluidised bed granulator spraying 2%HPMC ethanol and granulates; cross 14 mesh sieve granulate; add magnesium stearate; mixing; press 10000, promptly.
Embodiment 15: capsule
The capsule prescription
Ginkgo biloba leaf total terpene lactone extract 0.3kg
Starch 0.45kg
Microcrystalline Cellulose 0.025kg
Magnesium stearate 0.004kg
Method for making adds starch (80 ℃ of bakings), microcrystalline Cellulose, magnesium stearate with ginkgo biloba leaf total terpene lactone extract (crossing 80 mesh sieves), and mixing is sub-packed in 10000 capsules, promptly.
Embodiment 16: drop pill
Pill prescription
Ginkgo biloba leaf total terpene lactone extract 0.3kg
Macrogol 4000 1.0kg
After the fusion of method for making Polyethylene Glycol, add ginkgo biloba leaf total terpene lactone extract fine powder mix homogeneously, insulation is the 3.mm dropper with internal diameter below 80 ± 2 ℃, splashes in the methyl-silicone oil with 60～70 droplets/minute, collects drop pill, the absorption liquid coolant, promptly.
Embodiment 17: granule
The granule prescription
Ginkgo biloba leaf total terpene lactone extract 0.3kg
Betacyclodextrin 2.4kg
Icing Sugar 0.3kg
Dextrin 0.1kg
Method for making is got betacyclodextrin (Yunan County, Guangdong Province light monosodium glutamate the Industrial Co., Ltd. forever produces) embedding that the ginkgo biloba leaf total terpene lactone extract fine powder adds 8 times of amounts, adds an amount of Icing Sugar and dextrin, 30% ethanol, and granulation, drying, granulate, packing are promptly.
Embodiment 18: injection
Injection formula
Ginkgo biloba leaf total terpene lactone extract 5g
Propylene glycol 30ml
Water for injection adds to 1000ml
Method for making is got the ginkgo biloba leaf total terpene lactone extract micronizing, adds the propylene glycol dissolving, adds an amount of water for injection, transfers pH to 7.0, add active carbon 0.2g, heat 80 ℃ of insulations, No. 4 sintered glass filter pre-flocks add the injection water to 1000ml, 0.2 μ m filtering with microporous membrane, embedding, promptly.
Claims (10)
1. ginkgo biloba leaf total terpene lactone extract, it is characterized in that: wherein the weight content of bilobalide J is 3%-15%, the weight content of ginkalide C is 8%-30%, the weight content of bilobalide is 20%-65%, the weight content of ginkalide A is 15%-50%, and the weight content of ginkalide B is 3%-40%.
2. according to the ginkgo biloba leaf total terpene lactone extract of claim 1, wherein the weight content of bilobalide J is 3%-10%, the weight content of ginkalide C is 10%-20%, the weight content of bilobalide is 38%-55%, the weight content of ginkalide A is 18%-30%, and the weight content of ginkalide B is 4%-15%.
3. according to the ginkgo biloba leaf total terpene lactone extract of claim 2, wherein the weight content of bilobalide J is 3%-5%, the weight content of ginkalide C is 12%-20%, the weight content of bilobalide is 40%-52%, the weight content of ginkalide A is 20%-30%, and the weight content of ginkalide B is 5%-15%.
4. according to the preparation method of the ginkgo biloba leaf total terpene lactone extract of one of claim 1-3, this method comprises: get the Folium Ginkgo coarse powder, with 50%～80% (v/v) ethanol or acetone extraction, reclaim solvent, last macroporous adsorptive resins, washing decontamination, behind 5%～20% (v/v) ethanol elution decontamination, use 50%～80% (v/v) ethanol elution instead, collect alcohol eluen, reclaim ethanol, last polyamide column, water elution is collected water elution liquid, concentrates, ethyl acetate extraction, reclaim solvent, vacuum drying gets ginkgo biloba leaf total terpene lactone extract.
5. according to the preparation method of the ginkgo biloba leaf total terpene lactone extract of claim 4, this method comprises: get the Folium Ginkgo coarse powder, acetone extraction, reclaim solvent, last macroporous adsorptive resins, washing decontamination, behind 5%～20% (v/v) ethanol elution decontamination, use 60%～80% (v/v) ethanol elution instead, collect alcohol eluen, reclaim ethanol, last polyamide column, water elution, collect water elution liquid, concentrate ethyl acetate extraction, reclaim solvent, vacuum drying gets ginkgo biloba leaf total terpene lactone extract.
6. according to the preparation method of the ginkgo biloba leaf total terpene lactone extract of claim 4, this method comprises: get the Folium Ginkgo coarse powder, 70%～80% (v/v) ethanol extraction reclaims solvent, last macroporous adsorptive resins, the washing decontamination, behind 10%～15% (v/v) ethanol elution decontamination, use 60%～70% (v/v) ethanol elution instead, collect alcohol eluen, reclaim ethanol, last polyamide column, water elution is collected water elution liquid, concentrate, ethyl acetate extraction reclaims solvent, or recrystallization, vacuum drying gets ginkgo biloba leaf total terpene lactone extract.
7. Folium Ginkgo or be used to prepare the purposes of the medicine for the treatment of depression according to the ginkgo biloba leaf total terpene lactone extract of one of claim 1-3.
8. according to the purposes of claim 7, wherein Folium Ginkgo total lactones is selected from the total lactone extract of one of claim 1-3.
9. pharmaceutical composition for the treatment of depression, it contains ginkgo biloba leaf total terpene lactone extract and the pharmaceutically acceptable auxiliaries of one of with good grounds claim 1-3.
10. according to the pharmaceutical composition of claim 9, it is tablet, capsule, granule, drop pill, injection, oral cavity rapid release preparation or sustained-release preparation.
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| CN101134758B (en) * | 2007-10-15 | 2010-04-14 | 桂林市振达生物科技有限责任公司 | Method for extracting and separating bilobalide A, B, C, J and bilobalide monomer from ginkgo leaf |
| CN101412722B (en) * | 2007-10-15 | 2010-12-08 | 桂林市振达生物科技有限责任公司 | Method for extracting and separating bilobalide C from ginkgo leaf |
| CN101412725B (en) * | 2007-10-15 | 2010-12-08 | 桂林市振达生物科技有限责任公司 | Method for extracting and separating bilobalide B from ginkgo leaf |
| CN101773528B (en) * | 2010-02-23 | 2011-09-28 | 广州汉方现代中药研究开发有限公司 | Preparation method of total terpene lactones with low phenolic acid |
| CN102250106A (en) * | 2011-05-19 | 2011-11-23 | 华东理工大学 | Extraction method of sesquiterpene lactone compound from Chinese mugwort leaf and application thereof |
| CN102266380A (en) * | 2011-07-26 | 2011-12-07 | 天津泰阳制药有限公司 | Traditional Chinese medicine composition for treating depression |
| CN109793233A (en) * | 2018-07-25 | 2019-05-24 | 上海药辰生物科技有限公司 | A kind of deep working method of ginkgo nut endosperm |
| CN109925310A (en) * | 2013-12-10 | 2019-06-25 | 成都百裕制药股份有限公司 | Blood-pressure drug containing therapeutically effective amount ginkgolides |
| CN109985074A (en) * | 2017-12-29 | 2019-07-09 | 成都百裕制药股份有限公司 | A kind of extraction separation method of Total Terpene Lactones |
| WO2020037736A1 (en) * | 2018-08-20 | 2020-02-27 | 上海上药杏灵科技药业股份有限公司 | Ginkgo biloba leaf extract capsule and preparation method thereof |
| WO2020037735A1 (en) * | 2018-08-20 | 2020-02-27 | 上海上药杏灵科技药业股份有限公司 | Ginkgo biloba leaf extract particle and preparation method thereof |
| CN113491672A (en) * | 2020-04-07 | 2021-10-12 | 成都百裕制药股份有限公司 | A dripping pill containing bilobalide as effective component and its preparation method |
| CN114224922A (en) * | 2021-11-17 | 2022-03-25 | 湖南科技学院 | Preparation method and application of anti-depression ginkgo leaf extract |
| WO2022122040A1 (en) * | 2020-12-11 | 2022-06-16 | 成都百裕制药股份有限公司 | Use of bilobalide or ginkgolide composition in preparation of sedative drug |
| RU2778292C1 (en) * | 2018-08-20 | 2022-08-17 | Спх Син Лин Сай. & Тек. Фармасьютикал Ко., Лтд. | Granular complex ketone ether of ginkgo biloba and method for its preparation |
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| CN1249067C (en) * | 2003-08-30 | 2006-04-05 | 曹明成 | Extracting process for bilobalide A, B, C and its preparation |
| CN1569105A (en) * | 2004-05-12 | 2005-01-26 | 河南大学 | Natural herb made healthy product for resisting depression |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101412722B (en) * | 2007-10-15 | 2010-12-08 | 桂林市振达生物科技有限责任公司 | Method for extracting and separating bilobalide C from ginkgo leaf |
| CN101412725B (en) * | 2007-10-15 | 2010-12-08 | 桂林市振达生物科技有限责任公司 | Method for extracting and separating bilobalide B from ginkgo leaf |
| CN101134758B (en) * | 2007-10-15 | 2010-04-14 | 桂林市振达生物科技有限责任公司 | Method for extracting and separating bilobalide A, B, C, J and bilobalide monomer from ginkgo leaf |
| CN101773528B (en) * | 2010-02-23 | 2011-09-28 | 广州汉方现代中药研究开发有限公司 | Preparation method of total terpene lactones with low phenolic acid |
| CN102250106A (en) * | 2011-05-19 | 2011-11-23 | 华东理工大学 | Extraction method of sesquiterpene lactone compound from Chinese mugwort leaf and application thereof |
| CN102266380A (en) * | 2011-07-26 | 2011-12-07 | 天津泰阳制药有限公司 | Traditional Chinese medicine composition for treating depression |
| CN109925310A (en) * | 2013-12-10 | 2019-06-25 | 成都百裕制药股份有限公司 | Blood-pressure drug containing therapeutically effective amount ginkgolides |
| CN109985074A (en) * | 2017-12-29 | 2019-07-09 | 成都百裕制药股份有限公司 | A kind of extraction separation method of Total Terpene Lactones |
| CN109793233A (en) * | 2018-07-25 | 2019-05-24 | 上海药辰生物科技有限公司 | A kind of deep working method of ginkgo nut endosperm |
| WO2020037736A1 (en) * | 2018-08-20 | 2020-02-27 | 上海上药杏灵科技药业股份有限公司 | Ginkgo biloba leaf extract capsule and preparation method thereof |
| WO2020037735A1 (en) * | 2018-08-20 | 2020-02-27 | 上海上药杏灵科技药业股份有限公司 | Ginkgo biloba leaf extract particle and preparation method thereof |
| RU2778292C1 (en) * | 2018-08-20 | 2022-08-17 | Спх Син Лин Сай. & Тек. Фармасьютикал Ко., Лтд. | Granular complex ketone ether of ginkgo biloba and method for its preparation |
| CN113491672A (en) * | 2020-04-07 | 2021-10-12 | 成都百裕制药股份有限公司 | A dripping pill containing bilobalide as effective component and its preparation method |
| WO2022122040A1 (en) * | 2020-12-11 | 2022-06-16 | 成都百裕制药股份有限公司 | Use of bilobalide or ginkgolide composition in preparation of sedative drug |
| CN114224922A (en) * | 2021-11-17 | 2022-03-25 | 湖南科技学院 | Preparation method and application of anti-depression ginkgo leaf extract |
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