CN101653528A - Use of Chinese medicinal composition in preparation of medicaments for inhibiting cell apoptosis - Google Patents
Use of Chinese medicinal composition in preparation of medicaments for inhibiting cell apoptosis Download PDFInfo
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Abstract
The invention discloses the use of a Chinese medicinal composition in the preparation of medicaments for inhibiting cell apoptosis. The medicament is mainly prepared from the following main active rawmaterials in percentage by weight: red ginseng 6.05-15.8, panax pseudo-ginseng wall 8.95-10.08, Chinese angelica 5.12-8.34, suberect spatholobus stem 7.76-8.97, salvia miltiorrhiza bunge 8.01-11.13,safflower 5.18-8.84, ginkgo leaf 8.87-10.11, hawthorn 7.76-8.55, chrysanthemum 5.78-6.89, sea-ear shell 5.80-6.53, tuber fleeceflower root 7.77-8.84, grassleaf sweelflag rhizome 5.03-5.74, and kudzu-vine root 7.56-8.65. The Chinese medicinal composition has the function of inhibiting the cell apoptosis, thereby being capable of treating diseases related to cell apoptosis. The Chinese medicinal composition is a novel targeted medicament for treating diseases related to cell apoptosis.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to the application of a kind of known Chinese medicine composition in the preparation medicaments for inhibiting cell apoptosis.
Background technology
Apoptosis is one of dead process of the cell that proposed by people such as Kerr, Wyllie ', Currie or mode (referring to Brit, J.Cancer, 26,239 (1972)).Know now that in fetology it is dead cell to take place in the embryogenetic regular period in certain place, on the dead meaning of the cell that produces along with the program that takes place, also be known as " the program cell is dead ".Therefore, in morphology, be subjected to obstacle and downright bad process is distinguished to some extent with the cell of necessity.As apoptotic morphologic feature, can enumerate with on every side cell and lack contact, cytoplasmic enrichment, relevant with the activity of Cobra venom endonuclease chromatinic condensing and examine the merogenesisization etc. of condensing and nuclear, (blister of cell surface forms: membrane blebbing) etc. can to observe the Microvillares disappearance of cell surface and the smoothing of cell surface in addition.In addition, can also observe the phenomenon of DAN fragmentation by endonuclease activity, cell itself forms the cell fragment that is called apoptotic body, formed apoptotic body rapidly by around cell or decompositions of eating such as macrophage, the generation apoptosis.
Know that now dynamic equilibrium (homeostasis) is regulated, kept to organism automatically by the balance of cell proliferation and cell death.In the past, the Control Study of people's on cell proliferation more, but known to the control of pair cell death then almost has no.As everyone knows, apoptosis is inductive (referring to Cell by shortage, the apoptosis inducing factor (factor of tumor necrosis factor (TNF) and lymphocytotoxin etc. and C-myC, p-53 gene outcome etc.) of biological active substances (nerve growth factor (NGF) or cluster stimulating factor (CSF) etc.), 69,119 (1992); Nature, 362,849 (1993)), on the other hand, apoptosis be subjected to survivin (bCl-2 etc.) inhibition (referring to Nature, 359,552 (1992); Nature, 359,554 (1992)) etc.
Recently, people know that this apoptosis all plays an important role in multiple disease, and carried out various tests, and to attempt to diagnose, prevent and treat these diseases by the apoptosis of inducing or control cell, the concern that causes common people is (referring to Science, 267,1456 (1995)).
In addition, now, in the nerve cell death after ischemia the tardy property of Hippocampus nerve cell death owing to the apoptosis-related attention that is subjected to people.That is, open-minded again after two bilateral common carotid artery short time (～10 minutes) obturation with pallasiomy or rat, the cell of observing Hippocampus CA1 zone, both sides after 2 days (48 hours) is dead, when (96 hours) were above through 4 days, cell dwindles, is dendrite, observes empty bag, eliminates fully after a week.
Brain be whole body tissue in the highest tissue of oxygen consumption, and be the position that consumes big energy.Therefore, be the position of a weakness for ischemia, cause disordered brain function easily because of neurocyte is downright bad.Nerve cell death can be observed in growth course significantly, and cell is constantly dead after producing.According to reports, in people's cerebral cortex, every day nearly 100,000 nerve cell deaths.Neurocyte is unrenewable, therefore, if the excessively death owing to certain obstacle will cause dysfunction.Debatable especially is the nerve cell death that causes owing to ischemia, medicine, stress or virus etc., understands that the treatment of the multiple sacred diseases such as neurological disorder that its mechanism causes for the healing potion of development cerebral ischemia obstacle or because of AIDS and the clue of definite Therapeutic Method and understanding neurocyte long term survival ability all are vital.
In addition, recently, apoptosis with comprise that the relation of the neurodegenerative disease of Alzheimer and parkinson etc. also is subjected to people's attention, understand fully that the mechanism of morbidity and the method and the healing potion of development prevention become the problem that becomes more and more important.
The applicant has applied for that the patent No. is 00131558.7, to be called " a kind of medicine and production method thereof for the treatment of apoplexy ", Granted publication day be that June 11, publication number in 2003 are CN1111054C to name.This disclosure of the Invention a kind of Chinese medicine preparation for the treatment of apoplexy, the component of its main activated feedstock and weight percentage are: Radix Ginseng Rubra 6.05-15.8, Radix Notoginseng 8.95-10.08, Radix Angelicae Sinensis 5.12-8.34; Caulis Spatholobi 7.76-8.97, Radix Salviae Miltiorrhizae 8.01-11.13, Flos Carthami 5.18-8.84; Folium Ginkgo 8.87-10.11, Fructus Crataegi 7.76-8.55, Flos Chrysanthemi 5.78-6.89, Concha Haliotidis 5.80-6.53, Radix Polygoni Multiflori 7.77-8.84, Rhizoma Acori Graminei 5.03-5.74, Radix Puerariae 7.56-8.65.This medicine is to utilize alcohol extraction, water the modern pharmaceutical production technology such as to put forward above-mentioned raw material of Chinese medicine to make capsule or granule, oral liquid, tablet.This medicine has benefiting QI for activating blood circulation, disperse blood stasis and dredge collateral, the effect of the eliminating phlegm that relieves dizziness, high fever, infantile convulsions, epilepsy, etc., can suppress, eliminate atherosclerosis, effects such as inhibition hypercholesterolemia and hyperlipidemia, promoting blood circulation thrombus dissolving, resisting cerebrovascular wear out, cure mainly syndrome of blood stasis due to qi deficiency, the wind-phlegm blood stasis impatency venation card of cerebral infarction (cerebral infarction), convalescent period apoplex involving the channels and collaterals, it is early stage also to can be used for acute cerebral ischemic stroke, evident in efficacy, treat the disease spectrum width, and treating both the principal and secondary aspects of a disease.Its nomenclature of drug is " a brain arteries and veins Thailand ", because its determined curative effect since listing, has won the trust of extensive patients.
In recent years, the applicant continues to excavate other effects of above-mentioned composition, Everything comes to him who waits, through clinical and experimental study in recent years, the applicant finds that above-mentioned Chinese medicine composition suppresses apoptotic effect in addition, and up to the present, do not find that also above-mentioned composition suppresses apoptotic report.Though the report of above-mentioned composition treatment apoplexy is arranged, and the treatment of apoplexy is the result, and unspecified be that effect by which or several links or target spot obtains medical treatment apoplexy, also do not see that above-mentioned composition suppresses apoptotic report.
The applicant finds that described compositions suppresses apoptotic effect in addition, thereby can be used for treating some diseases relevant with apoptosis by concentrating on studies, and becomes the targeted drug of a kind of new treatment disease relevant with apoptosis.
In view of this, special proposition the present invention.
Summary of the invention
The object of the present invention is to provide the application of a kind of Chinese medicine composition in the preparation medicaments for inhibiting cell apoptosis.
For achieving the above object, the present invention adopts following technical scheme:
The application of a kind of Chinese medicine composition in the preparation medicaments for inhibiting cell apoptosis is characterized in that the component and the weight percentage of the main activated feedstock of described medicine are as follows: Radix Ginseng Rubra 6.05～15.8, Radix Notoginseng 8.95～10.08, Radix Angelicae Sinensis 5.12～8.34; Caulis Spatholobi 7.76～8.97, Radix Salviae Miltiorrhizae 8.01～11.13, Flos Carthami 5.18～8.84; Folium Ginkgo 8.87～10.11, Fructus Crataegi 7.76～8.55, Flos Chrysanthemi 5.78～6.89, Concha Haliotidis 5.80～6.53, Radix Polygoni Multiflori 7.77～8.84, Rhizoma Acori Graminei 5.03～5.74, Radix Puerariae 7.56～8.65.
Chinese medicine composition of the present invention suppresses by Akt, bcl-2, Bax or/and the application in the apoptosis medicine that caspase3 causes in preparation.
Discover have many genes to participate in the regulating cell apoptosis, these genes can participate in promoting or suppressing apoptotic generation, development.The gene relevant with cell regulate and control has Akt, bcl-2, Bax, caspase3.Bcl-2 has antioxidant action, thereby suppresses apoptosis; Bcl-2 albumen can suppress the inductive apoptosis of p53; Bcl-2 and Bax are combined into heterodimer, suppress the apoptosis-promoting effect of Bax.Bcl-2 is an anti-apoptotic albumen, pro apoptotic protein Bax then is considered to the important inducing molecule of mitochondrial injury, after dead signal stimulates, activated Bax moves on the mitochondrial adventitia, change the permeability of mitochondrial membrane, Bax can also directly be attached to mitochondrial membrane permeability transhipment hole (MPTP), strengthens the permeability of mitochondrial membrane, promote antiapoptotic factors (AIF) to discharge, thereby start the apoptotic signal approach; Moreover cytochrome C combines with apoptotic proteins enzyme activity factor Apaf-1 after mitochondrion is discharged into cytoplasm, activates Caspase-9, and Caspase-3 is activated in Caspase-9 cutting back, and Caspase-3 decomposes aminoacid sequence, thereby causes apoptosis.
The mitosis of PI3-K/Akt signal path wide participation cell, the differentiation of cell, the survival and the migration of cell, and relevant with rearrangement of cytoskeleton etc.Akt (protein kinase B) is the crucial effector molecule in phosphatidylinositols-3-hydroxyl kinases downstream, and it is the important prerequisite of the short cells survival effect of multiple somatomedin performance that Akt activates.A lot of experiment in vivo and vitro all prove in succession, increase phosphatidylinositols-3 hydroxyl kinases/protein kinase B (PI3-K/Akt) signal path and activate Akt, in close relations with the survival propagation of cell, can promote cell survival by deactivation apoptosis effect molecule such as Caspase 9, glycogen synthase kinase 3 and BAD etc. after the activation, the biologic activity that promptly increases Akt can improve the ability of cerebral tissue ischemia resisting damage.
The inventor has the MCAO of the increasing bcl-2 of rat cerebral tissue, the proteic expression of Akt by the discovery of concentrating on studies by Chinese medicine composition provided by the present invention, reduces the expression of Caspase-3 and Bax, thereby has the apoptotic effect of inhibition.
Another object of the present invention is to provide the application of Chinese medicine composition of the present invention in the medicine of the preparation treatment disease relevant with apoptosis.
Among the present invention, the application of described Chinese medicine composition in preparation treatment myocardial infarction, angina pectoris, blood stasis DHF, arrhythmia, apoplexy, cerebral infarction, cerebral thrombosis, vascular dementia, Alzheimer or the parkinsonian medicine relevant with apoptosis.
The inventor is by the discovery of concentrating on studies, have the MCAO rat cerebral tissue of increasing gene bcl-2, the Akt proteic expression relevant by Chinese medicine composition provided by the present invention with cell regulate and control, reduce gene C aspase-3 relevant and the expression of Bax, thereby have the apoptotic effect of inhibition with cell regulate and control.And apoptosis all plays important effect in multiple disease.Thereby, Chinese medicine composition of the present invention can be used for treating some diseases relevant with apoptosis, especially treat myocardial infarction, angina pectoris, blood stasis DHF, arrhythmia, apoplexy, cerebral infarction, cerebral thrombosis, Alzheimer or the parkinson relevant with apoptosis, clinical data shows, the total effective rate for the treatment of above-mentioned disease reaches 89%, curative effect is apparently higher than other drug, becomes the targeted drug of a kind of new treatment disease relevant with apoptosis.
Among the present invention, the dosage form of described medicine is a said dosage form on any pharmaceutics, preferred capsule, granule, oral liquid or tablet.The preparation method of described medicine can apply for that the patent No. is 00131558.7 referring to the applicant, to be called " a kind of medicine and production method thereof for the treatment of apoplexy ", Granted publication day be that June 11, publication number in 2003 are the patent of invention of CN1111054C to name.
The present invention compared with prior art has following advantage:
(1) the present invention's preferred above-mentioned Chinese medicine under Chinese medical theory instructs, science compatibility in addition, make that each medicine cooperatively interacts, learns from other's strong points to offset one's weaknesses, synergism, all medicines share, the deficiency of vital energy can be mended, and blood stasis can loose, and wind-phlegm can be dispelled, suppressing the hyperactive liver and subsiding YANG is played benefiting QI for activating blood circulation, the effect of the eliminating phlegm that relieves dizziness, high fever, infantile convulsions, epilepsy, etc., disperse blood stasis and dredge collateral, treating both the principal and secondary aspects of a disease altogether;
(2) through verification experimental verification, Chinese medicine composition of the present invention has the apoptotic effect of inhibition, especially suppresses by bcl-2, Bax, Akt or/and the effects such as apoptosis that caspase3 causes;
(3) through clinical verification, Chinese medicine composition of the present invention has the apoptotic effect of inhibition, thereby can be used for treating some diseases relevant with apoptosis, especially treat myocardial infarction, angina pectoris, blood stasis DHF, arrhythmia, apoplexy, cerebral infarction, cerebral thrombosis, vascular dementia, Alzheimer or the parkinson relevant with apoptosis, treat the disease spectrum width, with strong points, evident in efficacy;
(4) product of the present invention is safe by intending with using the daily dose and the course of treatment, no obvious adverse reaction and toxic and side effects.
Description of drawings
Fig. 1-A is the influence of Sham group to MCAO rat model TUNEL protein expression;
Fig. 1-B is the influence of MCAO group to MCAO rat model TUNEL protein expression;
Fig. 1-C is the influence of the safe heavy dose of group of brain arteries and veins to MCAO rat model TUNEL protein expression;
Fig. 1-D be in the brain arteries and veins Thailand dosage group to the influence of MCAO rat model TUNEL protein expression;
Fig. 1-E is the influence of the safe small dose group of brain arteries and veins to MCAO rat model TUNEL protein expression;
Fig. 2-A is the influence of Sham group to ischemia rat bcl-2;
Fig. 2-B is the influence of MCAO group to ischemia rat bcl-2;
Fig. 2-C is the influence of the safe heavy dose of group of brain arteries and veins to ischemia rat bcl-2;
Fig. 2-D be in the brain arteries and veins Thailand dosage group to the influence of ischemia rat bcl-2;
Fig. 2-E is the influence of the safe small dose group of brain arteries and veins to ischemia rat bcl-2;
Fig. 3-A is the influence of Sham group to ischemia rat Bax;
Fig. 3-B is the influence of MCAO group to ischemia rat Bax;
Fig. 3-C is the influence of the safe heavy dose of group of brain arteries and veins to ischemia rat Bax;
Fig. 3-D be in the brain arteries and veins Thailand dosage group to the influence of ischemia rat Bax;
Fig. 3-E is the influence of the safe small dose group of brain arteries and veins to ischemia rat Bax;
Fig. 4-A is the influence of Sham group to ischemia rat caspase 3;
Fig. 4-B is the influence of MCAO group to ischemia rat caspase 3;
Fig. 4-C is the influence of the safe heavy dose of group of brain arteries and veins to ischemia rat caspase 3;
Fig. 4-D be in the brain arteries and veins Thailand dosage group to the influence of ischemia rat caspase 3;
Fig. 4-E is the influence of the safe small dose group of brain arteries and veins to ischemia rat caspase 3;
Fig. 5-A is the influence of Sham group to ischemia rat Akt;
Fig. 5-B is the influence of MCAO group to ischemia rat Akt;
Fig. 5-C is the influence of the safe heavy dose of group of brain arteries and veins to ischemia rat Akt;
Fig. 5-D be in the brain arteries and veins Thailand dosage group to the influence of ischemia rat Akt;
Fig. 5-E is the influence of the safe small dose group of brain arteries and veins to ischemia rat Akt.
The specific embodiment
The present invention is further illustrated below in conjunction with embodiment, and following each embodiment only is used to the present invention is described and is not limitation of the present invention.
Embodiment 1
Take by weighing following Chinese medicine ingredients (unit: g):
Radix Ginseng Rubra 6 Radix Notoginseng 9.0 Radix Angelicae Sinensis 5.5 Caulis Spatholobis 7.8 Radix Salviae Miltiorrhizaes 8.5
Flos Carthami 5.5 Folium Ginkgos 9.0 Fructus Crataegis 7.8 Flos Chrysanthemis 6.0 Concha Haliotidis 5.9
Radix Polygoni Multiflori 7.5 Rhizoma Acori Graminei 5.1 Radix Puerariaes 7.6
Its production stage is:
1, get Radix Ginseng Rubra 4g, Radix Notoginseng 6g pulverizes, and crosses 80 mesh sieves, sterilize intermediate products A;
2, remaining Radix Ginseng Rubra 2g, Radix Notoginseng 3g and Folium Ginkgo 9.0g, Radix Salviae Miltiorrhizae 8.5g were with 4 times of amount alcohol reflux of 70% 2 hours, and alcohol extract reclaims ethanol and gets intermediate products B;
3, the medicinal residues of alcohol extraction part add twice of 5 times of water gagings extraction with nine flavor medical materials such as Radix Angelicae Sinensis 5.5g, Caulis Spatholobi 7.8g, Flos Carthami 5.5g, Fructus Crataegi 7.8g, Flos Chrysanthemi 6.0g, Concha Haliotidis 5.9g, Radix Polygoni Multiflori 7.5g, Rhizoma Acori Graminei 5.1g, Radix Puerariae 7.6g, each 2 hours, get extracting solution, partially mixed with B, concentrate, get fluid extract, spray drying gets the dried cream C of intermediate products;
4, get intermediate products A, C, mixing granulation is used capsule subpackage, and the reuse In Aluminium Foil Packing gets finished product, and name of product is " the safe capsule of three gold medal brain arteries and veins ".
Embodiment 2
Take by weighing following Chinese medicine ingredients (unit: g):
Radix Ginseng Rubra 8 Radix Notoginseng 10 Radix Angelicae Sinensis 6 Caulis Spatholobis 8.2 Radix Salviae Miltiorrhizaes 9.2
Flos Carthami 5.8 Folium Ginkgos 9.5 Fructus Crataegis 8.1 Flos Chrysanthemis 6.6 Concha Haliotidis 6.6
Radix Polygoni Multiflori 8.0 Rhizoma Acori Graminei 5.5 Radix Puerariaes 7.9
Its production stage is:
1, get whole Radix Ginseng Rubra 8g, Radix Notoginseng 10g and Folium Ginkgo 9.5g, Radix Salviae Miltiorrhizae 9.2g were with 4.5 times of amount alcohol reflux of 60% 2 hours, and alcohol extract reclaims ethanol and gets intermediate products B
1
2, the medicinal residues of alcohol extraction part add twice of 8 times of water gagings extraction with nine flavor medical materials such as Radix Angelicae Sinensis 6g, Caulis Spatholobi 8.2g, Flos Carthami 5.8g, Fructus Crataegi 8.1g, Flos Chrysanthemi 6.6g, Concha Haliotidis 6.6g, Radix Polygoni Multiflori 8.0g, Rhizoma Acori Graminei 5.5g, Radix Puerariae 7.9g, each 2 hours, get extracting solution, with B
1Partially mixed, concentrate, relative density is 1.05～1.20/55-60 ℃ fluid extract C
1
3, get intermediate products fluid extract C
1, with appropriate amount of auxiliary materials mixing granulation, packing, pack finished product, name of product is " three gold medal brain arteries and veins safe granule ".
Embodiment 3
Take by weighing following Chinese medicine ingredients (unit: g):
Radix Ginseng Rubra 10 Radix Notoginseng 8 Radix Angelicae Sinensis 7 Caulis Spatholobis 8.5 Radix Salviae Miltiorrhizaes 10.2
Flos Carthami 6.5 Folium Ginkgos 10 Fructus Crataegis 8.2 Flos Chrysanthemis 6.5 Concha Haliotidis 6.2
Radix Polygoni Multiflori 8.5 Rhizoma Acori Graminei 5.3 Radix Puerariaes 8.3
Its production stage is:
1, get whole Radix Ginseng Rubra 10g, Radix Notoginseng 8g and Folium Ginkgo 10g, Radix Salviae Miltiorrhizae 10.2g were with 7 times of amount alcohol reflux of 50% 2～3 hours, and alcohol extract reclaims ethanol and gets intermediate products B
2
2, the medicinal residues of alcohol extraction part add twice of 8 times of water gagings extraction with nine flavor medical materials such as Radix Angelicae Sinensis 7g, Caulis Spatholobi 8.5g, Flos Carthami 6.5g, Fructus Crataegi 8.2g, Flos Chrysanthemi 6.5g, Concha Haliotidis 6.2g, Radix Polygoni Multiflori 8.5g, Rhizoma Acori Graminei 5.3g, Radix Puerariae 8.3g, each 2 hours, get extracting solution, partially mixed with B2, concentrate, relative density is 1.05～1.20/55-60 ℃ fluid extract C
2
3, get intermediate products fluid extract C
2, with an amount of syrup or Mel mixing, sterilization, packing, sterilization again, pack finished product, name of product be " three gold medal brain arteries and veins Thailand oral liquid ".
Embodiment 4
Take by weighing following Chinese medicine ingredients (unit: g):
Radix Ginseng Rubra 8.0 Radix Notoginseng 9.0 Radix Angelicae Sinensis 5.8 Caulis Spatholobis 8.0 Radix Salviae Miltiorrhizaes 8.8
Flos Carthami 5.6 Folium Ginkgos 9.2 Fructus Crataegis 8.1 Flos Chrysanthemis 6.2 Concha Haliotidis 6.3
Radix Polygoni Multiflori 7.8 Rhizoma Acori Graminei 5.5 Radix Puerariaes 7.8
Its production stage is:
1, get Radix Ginseng Rubra 5g, Radix Notoginseng 5g pulverizes, and crosses 80 mesh sieves, sterilize intermediate products A
3
2, remaining Radix Ginseng Rubra 3g, Radix Notoginseng 4g and Folium Ginkgo 9.2g, Radix Salviae Miltiorrhizae 8.8g were with alcohol reflux 2-3 hour of 5 times of amounts 70%, and alcohol extract reclaims ethanol and gets intermediate products B
3
3, the medicinal residues of alcohol extraction part add twice of 5 times of water gagings extraction with nine flavor medical materials such as Radix Angelicae Sinensis 5.8g, Caulis Spatholobi 8.0g, Flos Carthami 5.6g, Fructus Crataegi 8.1g, Flos Chrysanthemi 6.2g, Concha Haliotidis 6.3g, Radix Polygoni Multiflori 7.8g, Rhizoma Acori Graminei 5.5g, Radix Puerariae 7.8g, each 2 hours, get extracting solution, with B
3Partially mixed, concentrate, get fluid extract, spray drying gets the dried cream C of intermediate products
3
4, get intermediate products A
3, C
3, mixing granulation, tabletting, packing gets finished product, and name of product is " the safe sheet of three gold medal brain arteries and veins ".
Following the applicant will have the apoptotic purposes of inhibition to this Chinese medicine composition by concrete pharmacodynamics test and describe.
[test example 1]
1 materials and methods
1.1 material (1) medicine and reagent: the safe capsule of brain arteries and veins is provided by Sanjin Pharmaceutical Co., Ltd., Guilin.(93 defend the accurate word X-81 of medicine number, people pharmaceutical factory of Tianjin aminoacid company purchases in pharmacy nimodipine (nimodipine nimo) injection.The TUNEL test kit is buied by Wuhan doctor's moral company.Akt, bcl-2, Bax, the anti-Akt of caspase3 immunohistochemical staining rabbit, bcl-2, Bax, caspase3 polyclonal antibody (concentration is 1: 150) and biotinylation goat anti-rabbit igg and DAB developer are all purchased the Bioisystech Co., Ltd in Beijing Zhong Shan.(2) 60 rats of animal: SpragueDawley, body weight (300 ± 30) g, the cleaning level is available from the laboratory animal department of the Chinese Academy of Sciences of Central South University.(3) key instrument equipment: Kodak DX.3900 digital camera; Electronic balance (model: PF-300E); (Japan produces high speed low temperature centrifugal machine.Model: SCR20BC).
1.2 method
60 SD rats 1.2.1 divide into groups, be divided into 6 groups (n=10) at random: sham operated rats (sham-operation), cerebral ischemia re-pouring model group (middle cerebral artery occlusion, MCAO), the safe heavy dose of group of brain arteries and veins (the safe 2.24gkg of MCAO+ brain arteries and veins
-1), dosage group (the safe 1.12gkg of MCAO+ brain arteries and veins in the brain arteries and veins Thailand
-1), the safe small dose group of brain arteries and veins (the safe 0.56gkg of MCAO+ brain arteries and veins
-1) and nimodipine group (MCAO+nimo 10mg kg-1).Safe group of brain arteries and veins and nimodipine group begin gastric infusion in MCAO the first five day, every day secondary, continuous five days.
1.2.2 the Focal Cerebral Ischemia Reperfusion modelling is made the focal brain ischemia-reperfusion injury in rats model by the Longa legal system, rat is weighed, lumbar injection volume fraction 10% chloral hydrate 3mlkg
-1Anesthesia, do the cervical region median incision, expose right common carotid artery, external carotid artery and internal carotid artery, do a little otch at external carotid artery apart from the about 2mm in its top place, the 3-0 nylon wire bead end that is roasted into pellet shapes with an end inserts external carotid artery through incision, again through internal carotid artery to middle cerebral artery top, the thromboembolism middle cerebral artery, cause focal cerebral ischemia: rat cerebral ischemia was extracted nylon wire after 90 minutes, recovered the cerebral tissue blood supply.The sham operated rats rat is extracted after the little pommel of nylon wire is inserted into middle cerebral artery top immediately: keep rectal temperature in the experimentation at 38 ± 1 ℃.
1.2.3 organized processing is behind cerebral ischemia re-pouring 24h, with 10% chloral hydrate anesthesia rat, open the thoracic cavity, cut an osculum, insert conduit to aorta from left ventricle in right auricle portion, in aorta, slowly inject the normal saline 200ml of 37 ℃ of heparinizations, become limpid to the right atrium effluent, inject 4% paraformaldehyde phosphate buffer 350ml then, behind the perfusion fixation 30min, broken end is got brain, puts into fixedly 72h of same fixed liquid., waxdip transparent, embedding through gradient alcohol dehydration, dimethylbenzene, with optic chiasma and the crown section in 4mm place thereafter, the thick 4 μ m of sheet.Microscope slide is handled through DEPC.Apoptotic cell detects and adopts TUNEL method original position labeled dna fragment to detect apoptosis, and test kit is buied by Wuhan doctor's moral company, and strict by specification is operated.Akt, Bcl-2, Bax and the anti-Akt of caspase3 immunohistochemical staining rabbit, bcl-2, Bax and caspase3 polyclonal antibody (concentration is 1: 50) and biotinylation goat anti-rabbit igg and DAB developer are all purchased the Bioisystech Co., Ltd in Beijing Zhong Shan.Adopt the dyeing of S-P method, strictness is operated by the test kit description.According to the localization method of rat cerebral ischemia half blanking bar, the homonymy volume cortex of parietal lobe top of middle cerebral artery occlusion is defined as the equivalence district of half blanking bar.The positive is pale brown color or sepia, is graininess, and background is hyacinthine.The TUNEL positive material is positioned at nucleus, bcl-2, and the Bax positive material is positioned at kytoplasm, to examine periphery obviously, adopt the full-automatic image analysis system of Nikon ECLIPSE E600, under the identical visual field, count the total cellular score of contiguous slices respectively, apoptotic cell and Akt, bcl-2, Bax, caspase3 immunoreation positive cell number calculates positive rate of apoptosis cells and Akt, bcl-2, Bax, caspase3 cell positive rate.
1.2.7 all data of statistical procedures represent that with X ± S statistical analysis adopts SPSS11.0 software.Mean relatively with the F check, is relatively checked with q between mean in twos between many groups.
2 results
2.1 brain arteries and veins Thailand is mainly seen in infarction kitchen range striatum and volume top cortex on every side to the apoptosis neuron positive cell that influences of rat half blanking bar neuronal apoptosis, the Sham group has a small amount of apoptotic cell to exist.When MCAO group rat ischemia 1.5h pours into 24h again, visible more apoptotic cell, its positive rate significantly increases, and relatively there were significant differences (P＜0.01) with the Sham group.The safe heavy dose of group of brain arteries and veins, middle dosage group and nimodipine group rat positive cell rate and MCAO group rat comparing difference be (P＜0.01) significantly.See Table 1.
Table 1 brain arteries and veins Thailand is to the influence of cerebral ischemia re-pouring rat half blanking bar apoptotic cell (x ± s)
Group (gkg
-1) positive cell rate
Sham 4.52±1.56
MCAO 47.81±5.33
ΔΔ
MCAO+ brain arteries and veins safe 0.56 39.25 ± 9.22
MCAO+ brain arteries and veins safe 1.12 31.07 ± 3.89
*
MCAO+ brain arteries and veins safe 2.24 27.44 ± 4.15
*
MACO+nimo organizes 10mg 27.09 ± 3.36
*
MCAO VS Sham: the safe vs MCAO:*P＜0.05**P of Δ Δ P＜0.01MCAO+ brain arteries and veins＜0.01
2.2 brain arteries and veins Thailand to cerebral ischemia re-pouring rat half blanking bar bcl-2, Bax, Caspase3, Akt express influence ischemia 1.5h and pour into 24h again the time, the positive rate of MCAO group bcl-2, Bax, Caspase3, Akt and Sham group significantly increase (P＜0.01), the bcl-2 positive cell rate of the safe heavy dose of group of brain arteries and veins, middle dosage group further increases, the positive cell rate of Bax significantly descend (P＜0.05, P＜0.01); The caspase-3 positive cell number obviously reduces, and the Akt positive cell number further increases, and compares with the MCAO group, and significant difference (P＜0.05, P＜0.01) is arranged.See Table 2 and Fig. 2-A-E, Fig. 3-A-E, Fig. 4-A-E, Fig. 5-A-E.
The influence that table 2 brain arteries and veins Thailand expresses cerebral ischemia re-pouring rat half blanking bar bcl-2, Bax, Caspase3, Akt (x ± s)
MCAO VS Sham: the safe vs MCAO:*P＜0.05**P of Δ Δ P＜0.01MCAO+ brain arteries and veins＜0.01
This test shows that the safe increase of the brain arteries and veins MCAO bcl-2 of rat cerebral tissue, the proteic expression of Akt reduce the expression of Caspase-3 and Bax, thereby suppress apoptosis.
In addition, clinical data shows, Chinese medicine composition of the present invention can be used for treating some diseases relevant with apoptosis, especially treat myocardial infarction, angina pectoris, blood stasis DHF, arrhythmia, apoplexy, cerebral infarction, cerebral thrombosis, vascular dementia, Alzheimer or the parkinson relevant with apoptosis, clinical data shows, the total effective rate for the treatment of above-mentioned disease reaches 89%, and curative effect is apparently higher than other drug, and does not have obvious adverse reaction and toxic and side effects.
Claims (6)
1, the application of a kind of Chinese medicine composition in the preparation medicaments for inhibiting cell apoptosis is characterized in that the component and the weight percentage of the main activated feedstock of described medicine are as follows: Radix Ginseng Rubra 6.05～15.8, Radix Notoginseng 8.95～10.08, Radix Angelicae Sinensis 5.12～8.34; Caulis Spatholobi 7.76～8.97, Radix Salviae Miltiorrhizae 8.01～11.13, Flos Carthami 5.18～8.84; Folium Ginkgo 8.87～10.11, Fructus Crataegi 7.76～8.55, Flos Chrysanthemi 5.78～6.89, Concha Haliotidis 5.80～6.53, Radix Polygoni Multiflori 7.77～8.84, Rhizoma Acori Graminei 5.03～5.74, Radix Puerariae 7.56～8.65.
2, the described Chinese medicine composition of claim 1 suppresses by bcl-2, Bax, Akt or/and the application in the apoptosis medicine that caspase3 causes in preparation.
3, claim 1 or the 2 described Chinese medicine compositions application in the medicine of the preparation treatment disease relevant with apoptosis.
4, the application of the described Chinese medicine composition of claim 3 in preparation treatment myocardial infarction, angina pectoris, blood stasis DHF, arrhythmia, apoplexy, cerebral infarction, cerebral thrombosis, vascular dementia, Alzheimer or the parkinsonian medicine relevant with apoptosis.
According to the application of any described Chinese medicine composition of claim 1-4, it is characterized in that 5, the dosage form of described medicine is a said dosage form on any pharmaceutics.
6, the application of Chinese medicine composition according to claim 5 is characterized in that, the dosage form of described medicine is capsule, granule, oral liquid or tablet.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104922611A (en) * | 2015-07-17 | 2015-09-23 | 江苏苏南药业实业有限公司 | Traditional Chinese medicine composition for treating arrhythmia and preparation method thereof |
| CN107338223A (en) * | 2017-05-27 | 2017-11-10 | 温州医科大学 | Method and SCF on a kind of suppression photoperiod sensitivity gene are overexpressed application of the virus on suppression photoperiod sensitivity gene medicine is prepared |
-
2008
- 2008-08-20 CN CN200810147517A patent/CN101653528A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 冯建玉: "大鼠局灶性脑缺血再灌注后Caspase-3、Bcl-2和Bax的表达与神经元凋亡的实验研究", 《中国优秀博硕士论文全文数据库(硕士) 医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104922611A (en) * | 2015-07-17 | 2015-09-23 | 江苏苏南药业实业有限公司 | Traditional Chinese medicine composition for treating arrhythmia and preparation method thereof |
| CN104922611B (en) * | 2015-07-17 | 2018-06-01 | 江苏苏南药业实业有限公司 | A kind of Chinese medicine composition for treating arrhythmia cordis and preparation method thereof |
| CN107338223A (en) * | 2017-05-27 | 2017-11-10 | 温州医科大学 | Method and SCF on a kind of suppression photoperiod sensitivity gene are overexpressed application of the virus on suppression photoperiod sensitivity gene medicine is prepared |
| CN107338223B (en) * | 2017-05-27 | 2021-01-26 | 温州医科大学 | Method for inhibiting apoptosis of photoreceptor cells and application of SCF overexpression virus in preparation of drugs for inhibiting apoptosis of photoreceptor cells |
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