CN1424315A - Ginkgo lactone compound and its preparation and medicinal composition containing it - Google Patents
Ginkgo lactone compound and its preparation and medicinal composition containing it Download PDFInfo
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Abstract
A novel compound, ginkolide K, is separated from the general extract of ginkolide. It can be used to prevent and treat cerebral ischemia. Its preparing process and its medical composition are also disclosed.
Description
Technical field
The present invention relates to a kind of new compound, this compound is a kind of of bilobalide, is that the bilobalide that extracts from the herbal medicine Ginkgo Leaf separates a kind of monomer that obtains.The present invention is with its called after bilobalide K.
Background technology
Bilobalide is to extract the active substance that obtains in the middle of the herbal medicine Ginkgo Leaf, this material can also further be subdivided into Ginkgolide A on structure, B, C etc., extracting method for bilobalide has had detailed bibliographical information, for example, people such as Li Xingang have introduced the laboratory extracting method of bilobalide (referring to Li Xingang etc., the laboratory Study on Extraction Method of bilobalide in the Ginkgo Leaf, 1998 the 1st phases of Chinese Journal of Pharmaceuticals), Chinese patent literature CN1195665A has introduced the extracting method of bilobalide and has contained the preparation of bilobalide, this method is that Ginkgo Leaf is extracted with water boil, with sorbent material extraction filtrate is adsorbed, use the ethanol desorption again, reclaim ethanol, with the crystallization dissolving of separating out, recrystallization, drying makes bilobalide.Chinese patent literature CN1313287A discloses a kind of production technique of bilobalide.The method of separating ginkgolide monomer in the middle of the general extractive of bilobalide, a lot of relevant bibliographical informations are also arranged, for example, people such as You Song have introduced the separation of bilobalide in the Ginkgo Leaf and structure determination method (referring to You Song etc., the separation of bilobalide and structure determination in the Ginkgo Leaf, Chinese pharmaceutical chemistry magazine the 4th phase of nineteen ninety-five).Chinese patent literature CN1287121A discloses the method that is prepared medicine Ginkgolide A, B by Ginkgo Leaf or Ginkgo Leaf medicinal extract.Bilobalide can be prepared into it different clinical medicine formulations as an effective active substance.The method for preparing injection with bilobalide is open in the middle of Chinese patent literature CN1315175A.Use Ginkgo Leaf injections such as Liu Jie treatments acute cerebral infarction obtained certain curative effect [referring to Liu Jie etc., YINXINGYE ZHUSHEYE to acute cerebral infarction after the influence of extremity motor function and TXB 2,6-Keto-PGF (1a)].About the pharmacological action of bilobalide, comparatively detailed bibliographical information (referring to Chen Weijun etc., the chemical structure of bilobalide and Advance on Pharmacological Activities, 1998 the 9th phases of Chinese Pharmaceutical Journal) has been arranged.Although the research for bilobalide and preparation thereof in the middle of the prior art has had a lot of reports,, never stop for the research of bilobalide, particularly, bibliographical information is arranged constantly for the separation of ginkgolide monomer and the research of derivative thereof.Present research for effective monomer in the middle of the bilobalide, structures such as Ginkgolide A, B, C have been reported, because bilobalide is a general name of extracting a class material that obtains in the middle of Ginkgo Leaf, to wherein monomeric further separation, it is the important subject of this area, because the pharmacologically active of bilobalide general extractive is generally approved, therefrom further isolated effective monomer, will be for people provide a kind of new active compound, for the research and development of new drug provides material base.The present invention be exactly to the further extraction of bilobalide effective monomer with separate.
Summary of the invention
The object of the invention provides a kind of new medical compounds, and this compound is a kind of of bilobalide, and has certain pharmacologically active.
Compound of the present invention is that the contriver passes through to extract and separates, and the material of classifying and obtaining in the middle of the bilobalide general extractive with its called after bilobalide K, is a kind of new compound.
The extraction and separation method of this compound is:
Get bilobalide crude product 3g and put in the apparatus,Soxhlet's, add ethyl acetate (or acetone) 2500ml and refluxed 24 hours, reclaim ethyl acetate to the medicinal extract shape, mix sample with diatomite, oven dry, porphyrize, use sherwood oil: ethyl acetate/wet method was loaded on low pressure silicagel column (pressure 0.2-0.3k/cm in 9: 1
2), with sherwood oil: the ethyl acetate gradient increases progressively wash-out, when sherwood oil: during ethyl acetate/6: 4,97～121 flow points, through TCL check (toluene: ethyl acetate: acetone: methyl alcohol/5: 2.5: 2.5: 0.3,4%NaAc silica gel H plate) occur the bilobalide spot (with the bilobalide reference substance relatively), it merged concentrate, place the appearance precipitation, mother liquor is continued to employ.To precipitate with acetone solution,, can get bilobalide K with preparation type high performance liquid phase purifying.
Prepare the raw material bilobalide crude product that bilobalide K of the present invention uses, can extract acquisition through commercially available purchase or according to currently known methods, this crude product is the bilobalide general extractive, and thin-layer chromatography is differentiated should check out bilobalide K.
The bilobalide K that said extracted obtains is white needle (MeoH) through conclusive evidence, and mp＞300 ℃ (decomposition) is soluble in alcohol, acetone, is slightly soluble in water.Show slightly faint yellow (place in the air and fade to white).
Its infrared spectrum feature is as follows:
IRV
max kBr(cm
- 1)：3500，3385，3130，2960，2915，2857，1783，1757，1700，1600，1465，1437，1400，1376，1368，1356，1340，1305，1280，1260，1243，1231，1215，1183，1174，1150，1124，1110，1087，1063，1037，1021，957，750；
Mass spectrum: ECI-MS (m/z): 405[M-H]
-
The hydrocarbon signal ownership and the proton signal of being correlated with (DMSO-d6) thereof of bilobalide K see Table 1.
The hydrocarbon signal ownership and the proton signal of being correlated with (DMSO-d6) position thereof of table 1 bilobalide K
13C-NMR
1H-NMR HMBC1 84.00 4.80 (dd) 5.11 (1-OH), (6) 5.53 (2) 2 80.20 6.53 5.47 (dd) 4.83 (1), 5.28 (1-OH) 3 160.33 2.90 (1 6), 5.53 (2) 4 90.50 6.06 (12) 5 76.39 2.19 (7), 3.83 (1), (10) 6 88.91 6.47 5.00 (d) 2.19 (7), 3.83 (1) 7 35.61 2.208 50.83 1.87 2.87 (8) 9 69.17 10 69.05 6.00 (d) 7.18 (10-OH) 11 74.11 5.00 (10), (12) 12 90.58 6.01 6.06 (S) 1.87 (8) 13 69.18 5.47 (6) 14 25.14 1.91 (16), 5.53 (2) 15 173.43 1.91 (16) 16 18.83 1.91 (d) 17 31.97 1.05 (18-20) 18-20 30.81 1.87 (8)
Annotate:
13C-NMR,
1H-NMR, HMBC refer to carbon-13 nmr spectra, proton nmr spectra, the long-range relevant spectrum of two-dimentional C-H respectively.
By physico-chemical analysis and spectroscopic technique in modern age (C-H is relevant, H-H is relevant, HMQC and HMBC spectrum) the hydrocarbon signal of bilobalide K compound is carried out full ownership, and determines that its chemical structure is as follows:
The chemical name of bilobalide K of the present invention is: 1,10-dihydroxyl-3,1 4-two dehydrogenation bilobalides (1,10-Dihydroxy-3,14-didehydroginkgolide).Molecular formula C
20H
22O
9, molecular weight is 406.
Bilobalide K of the present invention as activeconstituents, can be prepared into the clinical medicine preparation, for example, and tablet, capsule, oral liquid and injection.
Therefore, the present invention also provides the pharmaceutical composition that contains bilobalide K, and said composition contains the bilobalide K for the treatment of significant quantity, and the content of preferred bilobalide K is greater than 0.1%.
Pharmaceutical composition of the present invention preferably contains the bilobalide K of 0.1-90%, further preferably contains the bilobalide K of 0.5-60%, is more preferably the bilobalide K that contains 0.5-30%, preferably contains the bilobalide K of 0.5-5%.
Bilobalide K of the present invention preferably uses meglumine as solubility promoter when being prepared into liquid preparation, and particularly when bilobalide K was prepared into injection, meglumine was preferred solubility promoter.
Bilobalide K of the present invention can be prepared into medicine separately as activeconstituents, also can become medicine with other activeconstituents combined preparation, for example, can make up with other bilobalide, is prepared into medicine.
The pharmaceutical composition that bilobalide K and Ginkgolide A, B are combined to form has treatment and prophylactic effect to ishemic stroke.Said composition composed as follows:
Ginkgolide A 30-40%, Ginkgolide B 50%-65%, bilobalide K 0.5-5%.
The best proportioning of above-mentioned each component is: 35.59% Ginkgolide A, 58.39% Ginkgolide B, 2.07% bilobalide K.
Prove that through activity experiment bilobalide K of the present invention has pharmacologically active.Bilobalide K (GinkgolideK) intravenous injection can significantly reduce the infarction size and the water content of rat cerebral tissue after the Focal Cerebral Ischemia Reperfusion, improves its neurobehavioral.Brain tissue homogenate's biochemical indicator detects finds that this medicine can significantly reduce MDA in the cerebral tissue, LA content, rising GSH, SOD content.The cerebral tissue pathological section shows that this medicine is obvious to the neurocyte protection effect.Show that this medicine has significant provide protection to the rat experiment focal cerebral ischemia.Righting reflex time of recovery and the EEG time of recovery of experimental dispersivity global brain ischemia rat can be obviously shortened in bilobalide K (GinkgolideK) intravenous injection; can also significantly reduce behind the cerebral ischemia re-pouring content of Evans Blue in the cerebral tissue; promptly reduce cerebrovascular permeability, show that this medicine has provide protection to rat experiment dispersivity global brain ischemia.
The injection made from the composition of bilobalide K, Ginkgolide A and Ginkgolide B composition has carried out following a series of pharmacological evaluation, has proved the pharmacological action of said composition.
(1), bilobalide composite injection 7.5,15.0,30.0mg/kg iV can make the cerebral apoplexy scoring of MCAO rat reduce, the MCAO infarction size dwindles, brain water content reduces (P＜0.01), and its action intensity and Ginaton injection liquid be no significant difference relatively.
(2), bilobalide composite injection 7.5,15.0,30.0mg/kg iV can make the interior MDA of cerebral tissue of focal cerebral ischemia rat reperfusion injury, the LA equal size reduces (P＜0.01), shows that cerebral tissue hypoxic-ischemic and peroxidation degree are subjected to obvious inhibition; SOD and GSH content increase (P＜0.01) simultaneously, have reflected that medicine has raising to antioxidant ability of organism and the ability of removing free radical.
(3), bilobalide composite injection 7.5,15.0,30.0mg/kg iV can obviously protect the cerebral tissue structure of focal cerebral ischemia rat reperfusion injury; pyknosis, the karyolysis degree of pallium pyramidal cell and brain essence neurocyte obviously alleviate than ischemic control group, and softening kitchen range reduces.
(4), bilobalide composite injection 7.5,15.0,30.0mg/kg iV compare with model group that the EEG that can make diffusivity global brain ischemia rat recovers normal time and (P＜0.01) is obviously shortened in righting reflex time of recovery, make the Evans Blue permeability significantly reduce (P＜0.01), its effect does not relatively have statistical significant difference with the Ginaton injection liquid.
(5), the bilobalide composite injection can obviously increase the survival number of acute imperfection cerebral ischemia mouse.Press the ED that the Bliss method is calculated bilobalide injection iV
50Be 21.3 (17.6-25.6) mg/kg; The ED of bilobalide ig
50Be 29.2 (16.5-51.7) mg/kg; The ED of Ginaton iV
50Count 12.2 (6.3-23.3) mg/kg, ED with Semen Ginkgo extrac
50Value compares indifference through between t check group.
(6), bilobalide composite injection iV10min promptly begins onset, about peak time 20min, the time length can reach more than the 90min; And this medicine ig onset time 20-30min, about peak time 45min, the time length can reach more than the 120min.The effect of same dosage group iV is better than ig.
(7) but, bilobalide composite injection 12.0,24.0, the survival time (P＜0.01) of 48.0mg/kg iV significant prolongation mouse under anoxic condition.
(8), bilobalide composite injection 2.5,5.0,10.0mg/kg iV all do not have obvious influence (P＞0.05) to anesthetized dog CBF, CVR, SBP, DBP, MBP and HR, shows that three dosage of this medicine do not influence cerebral blood flow (CBF), cerebral vascular resistance, blood pressure and the heart rate of anesthetized dog.
(9), bilobalide composite injection 0.99,1.96,3.80 μ g/ml induce the rabbit extracorporeal platelet aggregation that obvious restraining effect (P＜0.01) is all arranged to PAF, and obvious unzipping (P＜0.01) are arranged.To ADP induced platelet congregation weak (P＜0.05 or P＜0.01), there is not obvious unzipping (P＞0.05).This medicine iV0.75,1.50,3.0mg/kg all have obvious restraining effect (P＜0.01) to platelet aggregation in the PAF inductive rabbit body, and middle and high dosage group also has obvious unzipping (P＜0.01).The rabbit platelet number there is not obvious effect (P＞0.05).
(10), bilobalide composite injection iV1.50,3.0mg/kg can obviously reduce rabbit erythrocyte hematocrit (P＜0.01) and whole blood viscosity (ratio) especially to whole blood viscosity (ratio) effect more obvious (P＜0.01) of high shear rate.
(11), it is moving that bilobalide composite injection iv 0.75,1.50,3.0mg/kg can obviously suppress rabbit---the thrombosis (P＜0.01) of vein bypass.
(12), bilobalide composite injection iv 3,6,12mg/kg are moving to rat---and vein bypass thrombus has obvious thrombolytic effect (P＜0.01).
(13), bilobalide composite injection iv6,12,24mg/kg can obviously prolong clotting time of mice (P＜0.01).
(14), obvious prolong rats clotting time during 10min-20min behind the bilobalide composite injection iv6mg/kg, during the peak about 12min, can make rat cruor time extending about 32%, 45min is effective behind the ig50mg/kg, continue 120mm, during the peak about 69min, rat cruor time extending about 34%.
(15), just can obviously suppress PAF behind the bilobalide composite injection iv 1.5mg/kg medicine immediately induces and exempts from the body platelet aggregation and obvious unzipping (P＜0.01) is arranged, effect continues 30min, during its peak 5min, the anticoagulant rate can reach about 80%, and depolymerization in 1 minute is about 81%.30min can obviously suppress PAF and induces in the rabbit body platelet aggregation and obvious unzipping (P＜0.01) is arranged behind the bilobalide ig25mg/kg medicine, continue 60min, be about 42min during its peak, the maximum gathering of thrombocyte inhibiting rate is about 38%, and the depolymerization rate was about 32% in 1 minute.
(16), bilobalide composite injection iV prolongs the IC of clotting time of mice
50Be 6.42mg/kg, bilobalide ig prolongs the IC of clotting time of mice
50Be 29.41mg/kg.Bilobalide injection iv is to the IC of platelet aggregation in tame rabbit platelet and the PAF inductor
50Be 2.32mg/kg, bilobalide ig is to the IC of platelet aggregation in tame rabbit platelet and the PAF inductor
50Be 87.72mg/kg.
Above-mentioned experiment shows that bilobalide composite injection of the present invention is safe and effective, can be used for the prevention and the clinical treatment of ishemic stroke.
The pharmacological evaluation of bilobalide K is as follows:
Experimental example one bilobalide K intravenous injection is to the influence of focal rats with cerebral ischemia brain function, infraction rate and brain water content
1. be subjected to the reagent thing: bilobalide K is provided by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov; Nimotop injection liquid (nimotop vial) is for German Baeyer is produced lot number: CATEZ1, specification: 50ml:10mg; Ginaton injection, German Shu Pei produces lot number in big pharmaceutical factory: 6580199, specification: 17.5mg; Semen Ginkgo extrac, 5ml/ props up.
2, animal: SD big white mouse, body weight are 300-400g, and male and female half and half are provided by China Medicine University's animal feeding room, conformity certification number: Soviet Union's kinoplaszm 97004.
3, test method and result
Get 80 of male SD rats, body weight 300-400g, be divided into 8 groups at random by body weight, be respectively bilobalide high dose group 2.0mg/kg, middle dosage group 1.0mg/kg, low dose group 0.5mg/kg, the solvent control group, positive control nimotop injection liquid group 0.2mg/kg, positive control Ginaton injection group 1.0ml/kg, sham operated rats and ischemia model control group (all giving equivalent physiological saline 0.6ml/100g).In preceding four days of experiment, every day, tail vein injection was administered once, and the 4th day for the last time in ischemic administration in preceding 30 minutes.With reference to people's such as Longa method, adopt internal carotid artery line bolt legal system to be equipped with intraluminal middle cerebral artery occlusion in rats obturation (MCAO) model.Rat is anaesthetized with 10% chloral hydrate (300mg/kg) ip, the neck median incision on the constant temperature operating table of lying on the back, expose right carotid, outwards draw biventer and nutator, free successively by arteria carotis communis crotch head-end, ligation and the branch of cutting off external carotid artery: artery and superior thyroid artery under the occipital bone, in the ligation of external carotid artery far-end, cutting off external carotid artery makes its trunk free standby, separate internal carotid artery then, make a call to one with silk thread at the external carotid artery root and release, folder closes arteria carotis communis and internal carotid artery.With nylon wire (long 4cm, diameter 0.26mm) through external carotid artery trunk otch, slowly going into the cranium direction to internal carotid artery advances, with the arteria carotis communis crotch is mark, feel resistance when advancing the 20mm left and right sides, promptly reached in the thinner arteria cerebri anterior, all blood of having blocked MCA are tightened the external carotid artery root and are released for the source.After one hour, extract nylon wire, tighten the artery stump.Skin suture is finished MCAO and is caused focal cerebral ischemia-irritate again model.Behind the sham operated rats rat anesthesia, only expose the inside and outside aortic bifurcation of neck, not inaccessible MCA.
Postoperative 24h carries out rank scores by the method for Bederson to the behavioral deficiency of animal, and standard is as follows:
0 grade: do not observe nervous symptoms;
1 grade: carry tail when unsettled, the operation offside forelimb of animal shows as wrist elbow flexing, the shoulder inward turning, and the elbow abduction is close to the wall of the chest;
2 grades: animal is placed on the smooth flat, and pushing hands art side is takeed on to side shifting the time, and resistance reduces;
3 grades: side ring is changeed or turn-take to operation during the animal walking freely;
4 grades; Collapse from physical exhaustion, limbs do not have spontaneous activity.
Behind the MCAO 24h, the sacrificed by decapitation rat is taken out full brain, and left and right sides brain cuts respectively, weighs.At right brain optic chiasma and each 2mm place, front and back thereof, do crown cutting, brain section lucifuge in 1%TTC solution was hatched 25 minutes for 37 ℃, separated pale district (infarct) and non-pale district (normal district) with the ophthalmology tweezer, and it is as follows to calculate infraction per-cent:
Infraction per-cent (%)=pale district weight/(pale district weight+non-pale district weight) * 100%
Cerebral tissue after the dyeing is placed 110 ℃ of oven for drying, and it is as follows that contrast brain weight in wet base is obtained brain water content:
Brain water content (%)=(1-cerebral tissue dry weight/cerebral tissue weight in wet base) * 100%
As shown in Table 2, bilobalide K 0.5,1.0,2.0mg/kg iv can make the cerebral apoplexy scoring of MCAO rat reduce (P＜0.01), the MCAO infarction size dwindles (P＜0.01), and brain water content reduces (P＜0.01), and its height, middle dosage action intensity and Ginaton be no significant difference relatively.
---0.2 ± 0.4 0 77.7 ± 0.6 model contrast---contrast of 3.2 ± 0.8 21.0 ± 2.3 81.0 ± 1.7 solvents---3.0 ± 0.8 that table 2 silver contains lactone Kiv to impact (x ± s, n=10) group dosage (mg/kg) neural behavior scoring cerebral infarction rate (%) brain water content (%) the sham-operation group of Level In Rats With Focal Cerebral Ischemia neurobehavioral, cerebral infarction rate and brain water content* △ △ △ ◇ ◇ ◇20.7 ± 2.0
* △ △ △ ◇ ◇ ◇81.1 ± 1.4
* △ △ ◇ ◇ ◇
2.0 1.5 ± 0.7
* * △ ◇ ◇ ◇13.8 ± 2.0
* * △ ◇ ◇ ◇78.1 ± 0.9
* * △ ◇Ginkgo propyl ester K 1.0 2.1 ± 0.7
* △ ◇ ◇16.4 ± 2.4
* * △ ◇ ◇ ◇79.2 ± 1.2
* △ ◇ ◇ ◇
0.5 2.9 ± 0.7
* △ ◇ ◇ ◇18.4 ± 2.0
* △ △ ◇ ◇ ◇80.0 ± 1.4
* △ △ ◇ ◇ ◇Ginaton group 1.0ml/kg 1.9 ± 0.7
* ◇ ◇ ◇15.6 ± 3.0
* * ◇ ◇ ◇78.0 ± 42.1
* ◇Nimotop group 0.2 1.9 ± 0.7
* ◇ ◇ ◇16.9 ± 1.9
* * ◇ ◇ ◇78.4 ± 1.3
* ◇ *P＞0.05,
*P＜0.05,
* *P＜0.01 vs model contrast
△P＞0.05,
△ △P＜0.05,
△ △ △P＜0.01 vs Ginaton group
◇P＞0.05,
◇ ◇P＜0.05,
◇ ◇ ◇P＜0.01 vs sham operated rats
Experimental example two bilobalide K intravenous injections are to the influence of focal rats with cerebral ischemia brain homogenate biochemical indicator
1. be subjected to the reagent thing: bilobalide K is provided by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov; Nimotop injection liquid (nimotop vial) is for German Baeyer is produced lot number: CATEZ1, specification: 50ml:10mg; Ginaton injection, German Shu Pei produces lot number in big pharmaceutical factory: 6580199, specification: 17.5mg; Semen Ginkgo extrac, 5ml/ props up.
2, animal: SD big white mouse, body weight are 300-400g, and male and female half and half are provided by China Medicine University's animal feeding room, conformity certification number: Soviet Union's kinoplaszm 97004.
3, test method and result
Get 80 of male rats, 300-400g, grouping administration and operation method are with experimental example one.Behind the MCAO 24h, the rat sacrificed by decapitation is got brain, removes cerebellum.Brain is made 10% homogenate with physiological saline, build up the test kit specification sheets that bio-engineering research provides, measure superoxide-dismutase (SOD), gsh (GSH), mda (MDA) and lactic acid (LD) content in the cerebral tissue by Nanjing.The brain homogenate protein content is pressed the Xylene Brilliant Cyanine G method and is measured.
Can be made the MDA in the cerebral tissue of focal cerebral ischemia rat reperfusion injury by the visible bilobalide K of table 3, the LA equal size reduces, and shows that tissue ischemia anoxic and peroxidation degree are subjected to obvious inhibition; SOD and GSH content increase simultaneously, have reflected that medicine has raising (P＜0.01) to antioxidant ability of organism and the ability of removing free radical.
---57.3 ± 17.1 1.9 ± 0.6 935.8 ± 52.9 79.8 ± 20.1 models contrast---contrast of 116.9 ± 24.3 8.8 ± 1.5 363.8 ± 121.1 38.1 ± 15.1 solvents---104.6 ± 23.6 that table 3 bilobalide K iv is to impact (x ± s, n=10) group dosage (mg/kg) MDA (nmol/mgprot) LA (mmol/gprot) SOD (NU/mgprot) GSH (mg/mgprot) the sham-operation group of Level In Rats With Focal Cerebral Ischemia brain homogenate biochemical indicator* △ △ △ ◇ ◇ ◇8.0 ± 1.0
* △ △ △ ◇ ◇ ◇432.7 ± 34.0
* △ △ △ ◇ ◇ ◇44.5 ± 7.8
* △ △ △ ◇ ◇ ◇
2.0 56.2 ± 16.2
* * △3.4 ± 0.4
* * △ ◇ ◇ ◇760.5 ± 53.6
* * △ ◇ ◇ ◇60.5 ± 14.1
* * △ △ ◇Bilobalide 1.0 58.4 ± 7.3
* * △4.3 ± 0.8
* * △ ◇ ◇ ◇757.7 ± 56.9
* * △ ◇ ◇ ◇50.8 ± 11.6
* △ ◇ ◇ ◇K 0.5 78.1 ± 14.5
* * △ △ △ ◇ ◇6.3 ± 1.0
* * △ △ △ ◇ ◇ ◇631.0 ± 117.7
* * △ △ △ ◇ ◇ ◇46.0 ± 15.4
* △ △ △ ◇ ◇ ◇Ginaton group 1.0ml/kg 57.3 ± 10.2
* *4.0 ± 0.6
* * ◇ ◇ ◇720.0 ± 36.3
* * ◇ ◇ ◇53.9 ± 18.9
* * ◇ ◇ ◇Nimotop group 0.2 56.9 ± 22.3
* *3.3 ± 0.7
* * ◇ ◇ ◇799.1 ± 75.4
* * ◇ ◇ ◇61.3 ± 16.7
* * ◇ ◇
*P＞0.05,
*P＜0.05,
* *P＜0.01 vs model contrast
△P＞0.05,
△ △P＜0.05,
△ △ △P＜0.01 vs Ginaton group
◇P＞0.05,
◇ ◇P＜0.05,
◇ ◇ ◇P＜0.01 vs sham operated rats
The influence that experimental example three bilobalide Ks are learned focal rats with cerebral ischemia cerebral tissue
1. be subjected to the reagent thing: bilobalide K is provided by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov; Nimotop injection liquid (nimotop vial) is for German Baeyer is produced lot number: CATEZ1, specification: 50ml:10mg; Ginaton injection, German Shu Pei produces lot number in big pharmaceutical factory: 6580199, specification: 17.5mg; Semen Ginkgo extrac, 5ml/ props up.
2, animal: SD big white mouse, body weight are 300-400g, and male and female half and half are provided by China Medicine University's animal feeding room, conformity certification number: Soviet Union's kinoplaszm 97004.
3, test method and result
Get 40 of male rats, 300-400g is divided into 5 every group at random, and administration and operation method are with experimental example one.24h sacrificed by decapitation rat behind the MCAO, it is fixing in 10% formaldehyde solution to take out full brain.Do H.E. dyeing after sample is cut into slices in cured molding jig, the pallium hippocampus is carried out histopathologic examination.The result is by shown in pathological section report and the photo:
Sham operated rats: each routine cerebral tissue neurone and spongiocyte are normal, and nuclear membrane is clear, and kernel is obvious, do not see obviously pathological change such as neurodegeneration, necrosis and cell infiltration.
The ischemia model group: each routine cerebral tissue sees that all neuronal structure is fuzzy, cell space swelling, and nissl bodies reduces or disappears, and nuclear hyperchromatism in various degree occurs, pyknosis, karyolysis, nucleome is irregular, and sees that obviously softening kitchen range forms.
Three dosage groups of bilobalide K, pyknosis, the karyolysis degree of pallium pyramidal cell and brain essence neurocyte obviously alleviate than ischemic control group, and softening kitchen range reduces.Wherein the high dosage effect is more remarkable, and the DeGrain of low dose group.
The brain tissue impairment that Ginaton injection control group and nimotop injection liquid control group also cause cerebral ischemia has obvious provide protection.
The result shows, bilobalide K 0.5,1.0, but 4 days dosage correlation ground of 2.0mg/kg continuous intravenous injection improves the neural function of focal cerebral ischemia rat due to the medium sized artery obturation, obviously dwindles infarct size, alleviates cerebral edema; But this medicine is remarkable mda (MDA) and lactic acid (LA) content that reduces in the inaccessible rat brain tissue of medium sized artery in dosage correlation ground also, increases superoxide-dismutase (SOD) and Triptide (GSH) content.Pathological section shows that also bilobalide can obviously improve the brain metabolism of rat under the focal cerebral ischemia; Keep the normal morphology and the function of neurocyte under the cerebral ischemic condition, delay, alleviate its necrosis.
Experimental example four bilobalide K intravenous injections are to the provide protection of rat experiment dispersivity thoroughness cerebral ischemia
1. be subjected to the reagent thing: bilobalide K is provided by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov; Nimotop injection liquid (nimotop vial) is for German Baeyer is produced lot number: CATEZ1, specification: 50ml:10mg; Ginaton injection, German Shu Pei produces lot number in big pharmaceutical factory: 6580199, specification: 17.5mg; Semen Ginkgo extrac, 5ml/ props up.
2, animal: SD big white mouse, body weight are 300-400g, and male and female half and half are provided by China Medicine University's animal feeding room, conformity certification number: Soviet Union's kinoplaszm 97004.
3, test method and result
Get 80 of male SD rats, body weight 300-400g is divided into 8 groups at random, 10 every group.Be divided into bilobalide K high dose group 2.0mg/kg, middle dosage group 1.0mg/kg, low dose group 0.5mg/kg, nimotop injection liquid group 0.2mg/kg, Ginaton injection group 1.0ml/kg, solvent control group, model control group and sham operated rats (all giving equivalent physiological saline 0.6ml/100g).In the experiment first three day, every day tail vein injection once.30min after the administration in the 3rd day, with 10% chloral hydrate anesthesia (300mg/kg, ip).Make rat acute thoroughness cerebral ischemic model then.Under occipital bone, make 1cm left and right sides otch, separate both sides latissimus dorsi muscle and trapezius muscle, expose two foramen transversariums about atlas, insert the boiling hot bilateral vertebral artery that closes of foramen transversarium with electric iron in the center.Last administration behind the 24h, the etherization state cuts calvarium skin down, in sagittal suture and crossing temporal bone and the lambdoidal suture intersection difference heeling-in brain electrode of reaching of coronal suture, be connected to the SJ-42 polygraph, with electroencephalogram (EEG) and the righting reflex time of recovery of record rat in process of the test.Rat back of the body position is fixing, separate bilateral common carotid arteries.Arteriole folder folder with the band silicone tube under 30min rat waking state after the administration closes bilateral common carotid arteries, visible rat consciousness and righting reflex loss, and eye bleaches, perpendicular hair, accelerated breathing.Remove bulldog clamp behind the 30min, cause re-perfusion model, irritate vena femoralis injection 60mg/kg Evans Blue simultaneously again.The rat broken end is got brain behind the 60min, use physiological saline and acetone mixed solution (3: 7v/v) make 10% homogenate (W/V) then, after sealing is placed and is spent the night, the centrifugal 15min of 2500rpm, get supernatant liquor, with physiological saline-acetone mixed solution zeroing, survey absorbancy at the 620nm place and represent that [with physiological saline-acetone mixed solution is the typical curve that solvent is set up Evans Blue absorbance and concentration to Evans Blue content in the cerebral tissue, reflects the influence of this medicine to cerebrovascular permeability with this.The result is by shown in the table 4, bilobalide K 0.5,1.0, but 4 days dosage correlation ground of 2.0mg/kg continuous intravenous injection shortens EEG and the righting reflex time of recovery of the full brain dispersivity ischemic rat of four artery ligations-due to irritating again, reduces brain homogenate Evans Blue content (high dose group p＜0.01).Brain function after bilobalide K can be protected ischemic is described, reduces capillary permeability.
Table 4 bilobalide K iv is to the provide protection of rat experiment dispersivity thoroughness cerebral ischemia (n=10, group dosage EEG time of recovery (min) the righting reflex Evans Blue time of recovery content of x ± s)
(mg/kg) (min) (μ g/g brain is heavy) sham operated rats------contrast of 7.51 ± 1.0 models---46.5 ± 7.8 30.1 ± 4.3 17.5 ± 1.6
◇ ◇ ◇Nimotop group 0.2 39.0 ± 5.7
*24.7 ± 4.8
*15.5 ± 2.2
* ◇ ◇ ◇Ginaton group 1.0ml/kg 37.5 ± 6.8
*24.1 ± 5.5
*15.2 ± 1.9
* ◇ ◇ ◇The solvent contrast---47.0 ± 4.8
* △ △ △31.9 ± 2.8
* △ △ △17.9 ± 1.4
* △ △ △ ◇ ◇ ◇
20.0 38.7 ± 3.5
* * △23.9 ± 5.1
* * △15.0 ± 1.5
* * △ ◇ ◇ ◇Bilobalide K 1.0 41.0 ± 2.1
* △26.4 ± 3.3
* △15.3 ± 2.0
* △ ◇ ◇ ◇
0.5 40.5 ± 3.7
* △26.5 ± 3.2
* △15.9 ± 1.6
* △ ◇ ◇ ◇ *P＞0.05,
*P＜0.05,
* *P＜0.01 vs model control group
△P＞0.05,
△ △P＜0.05,
△ △ △P＜0.01 vs Ginaton group
◇P＞0.05,
◇ ◇P＜0.05,
◇ ◇ ◇P＜0.01 vs sham operated rats
Embodiment
Embodiment 1
Bilobalide K 5.0g
Meglumine 5.0g
NaCl 8.0g
10% citric acid solution is an amount of
Get meglumine and add fresh water for injection 1000ml dissolving, in 120 ℃ of autoclaving 30min, take out the back and be heated to 80 ℃ in gradating material chambers 10,000, add porphyrize and cross the bilobalide K of 80 mesh sieves, be heated to dissolving, transfer pH to 8.0 with 10% aqueous citric acid solution after being cooled to room temperature, add NaCl 8.0g, needle-use activated carbon 2.0g, stirring at room absorption 30min, filter carbon removal, add fresh water for injection, serve as a contrast 0.22 μ m filtering with microporous membrane down with aseptic filter plate in 100 grades of sterile workshop to total amount 1000ml, be sub-packed in the 10ml ampoule fusion sealing, quality inspection, lettering, packing.
Embodiment 2
Ginkgolide A 1.78g
Ginkgolide B 2.72g
Bilobalide K 0.1g
Meglumine 5.0g
NaCl 8.0g
10% citric acid solution is an amount of
Get meglumine and add fresh water for injection 1000ml dissolving, in 120 ℃ of autoclaving 30min, take out the back and be heated to 80 ℃ in gradating material chambers 10,000, add porphyrize and cross Ginkgolide A, B, the K of 80 mesh sieves, be heated to dissolving, transfer pH to 8.0 with 10% aqueous citric acid solution after being cooled to room temperature, add NaCl 8.0g, needle-use activated carbon 2.0g, stirring at room absorption 30min, filter carbon removal, add fresh water for injection, serve as a contrast 0.22 μ m filtering with microporous membrane down with aseptic filter plate in 100 grades of sterile workshop to total amount 1000ml, be sub-packed in the 10ml ampoule fusion sealing, quality inspection, lettering, packing.
Claims (10)
1. a compound is characterized in that this compound monomer is to separate the white needle that obtains in the middle of the bilobalide general extractive, and fusing point is soluble in alcohol, acetone, is slightly soluble in water greater than 300 ℃, shows slightly faint yellow, places in the air and fades to white, molecular formula C
20H
22O
9, molecular weight is 406, its infrared spectrum is characterized as:
IRV
max kBr(cm
-1)：
3500，3385，3130，2960，2915，2857，1783，1757，1700，1600，1465，1437，1
400，1376，1368，1356，1340，1305，1280，1260，1243，1231，1215，1183，11
74，1150，1124，1110，1087，1063，1037，1021，957，750；
Mass spectrum: ECI-MS (m/z): 405[M-H]
-
The hydrocarbon signal ownership and the proton signal DMSO-d6 that is correlated with thereof are as follows: the position
13C-NMR
1H-NMR HMBC1 84.00 4.80 (dd) 5.11 (1-OH), (6) 5.53 (2) 2 80.20 6.53 5.47 (dd) 4.83 (1), 5.28 (1-OH) 3 160.33 2.90 (16), 5.53 (2) 4 90.50 6.06 (12) 5 76.39 2.19 (7), 3.83 (1), (10) 6 88.91 6.47 5.00 (d) 2.19 (7), 3.83 (1) 7 35.61 2.208 50.83 1.87 2.87 (8) 9 69.1710 69.05 6.00 (d) 7.18 (10-OH) 11 74.11 5.00 (10), (12) 12 90.58 6.01 6.06 (S) 1.87 (8) 13 69.18 5.47 (6) 14 25.14 1.91 (16), 5.53 (2) 15 173.43 1.91 (16) 16 18.83 1.91 (d) 17 31.97 1.05 (18-20) 18-20 30.81 1.87 (8)
2. the described compound of claim 1 is characterized in that this compound prepares with following method:
Get bilobalide crude product 3g and put in the apparatus,Soxhlet's, add ethyl acetate or acetone 2500ml and refluxed 24 hours, reclaim ethyl acetate to the medicinal extract shape, mix sample with diatomite, oven dry, porphyrize, use sherwood oil: ethyl acetate/wet method was loaded on the low pressure silicagel column in 9: 1, pressure 0.2-0.3k/cm
2, with sherwood oil: the ethyl acetate gradient increases progressively wash-out, when sherwood oil: during ethyl acetate/6: 4,97～121 flow points are checked toluene: ethyl acetate: acetone: methyl alcohol/5: 2.5: 2.5: 0.3 through TCL, 4%NaAc silica gel H plate, the bilobalide spot relatively occurs with the bilobalide reference substance, its merging is concentrated, place the appearance precipitation, mother liquor is continued to employ, to precipitate with acetone solution,, promptly get this compound with preparation type high performance liquid phase purifying.
3. bilobalide compound is characterized in that the chemical name of this compound is: 1, and 10-dihydroxyl-3,14-two dehydrogenation bilobalides, its structural formula is:
4. the preparation method of bilobalide K compound is characterized in that this method comprises:
Get bilobalide crude product 3g and put in the apparatus,Soxhlet's, add ethyl acetate or acetone 2500ml and refluxed 24 hours, reclaim ethyl acetate to the medicinal extract shape, mix sample with diatomite, oven dry, porphyrize, use sherwood oil: ethyl acetate/wet method was loaded on the low pressure silicagel column in 9: 1, pressure 0.2-0.3k/cm
2, with sherwood oil: the ethyl acetate gradient increases progressively wash-out, when sherwood oil: during ethyl acetate/6: 4,97～121 flow points are checked toluene: ethyl acetate: acetone: methyl alcohol/5: 2.5: 2.5: 0.3 through TCL, 4%NaAc silica gel H plate, the bilobalide spot relatively occurs with the bilobalide reference substance, its merging is concentrated, place the appearance precipitation, mother liquor is continued to employ, to precipitate with acetone solution,, promptly get this compound with preparation type high performance liquid phase purifying.
5. a pharmaceutical composition is characterized in that said composition contains the bilobalide K for the treatment of significant quantity.
6. according to the pharmaceutical composition of claim 5, it is characterized in that this pharmaceutical composition contains the bilobalide K of 0.1-90%.
7. according to the pharmaceutical composition of claim 6, it is characterized in that this pharmaceutical composition contains the bilobalide K of 0.5-60%.
8. according to the pharmaceutical composition of claim 7, it is characterized in that this pharmaceutical composition contains the bilobalide K of 0.5-5%.
9. according to the pharmaceutical composition of claim 5, it is characterized in that this pharmaceutical composition contains: Ginkgolide A 30-40%, Ginkgolide B 50%-65%, bilobalide K 0.5-5%.
10. according to the pharmaceutical composition of claim 9, it is characterized in that this pharmaceutical composition contains: 35.59% Ginkgolide A, 58.39% Ginkgolide B, 2.07% bilobalide K.
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| CN105524077B (en) * | 2015-11-18 | 2019-03-01 | 江苏康缘药业股份有限公司 | Bilobalide K crystalline substance K-type and preparation method and its composition and purposes |
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| CN107898782B (en) * | 2017-12-29 | 2019-03-26 | 江苏康缘药业股份有限公司 | A kind of ginkgo diterpenoid-lactone composition |
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| JP7090164B2 (en) | 2017-12-29 | 2022-06-23 | ジアンスー カニョン ファーマシューティカル カンパニー リミテッド | Ginkgo diterpene lactone composition |
| US11564904B2 (en) | 2017-12-29 | 2023-01-31 | Jiangsu Kanion Pharmaceutical Co., Ltd. | Gingko diterpene lactone composition |
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