CN101322713A - Applications of gastrodine in preparing cysteine proteinase-3 active inhibitor - Google Patents
Applications of gastrodine in preparing cysteine proteinase-3 active inhibitor Download PDFInfo
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- CN101322713A CN101322713A CNA2007101189006A CN200710118900A CN101322713A CN 101322713 A CN101322713 A CN 101322713A CN A2007101189006 A CNA2007101189006 A CN A2007101189006A CN 200710118900 A CN200710118900 A CN 200710118900A CN 101322713 A CN101322713 A CN 101322713A
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- gastrodine
- active inhibitor
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- cysteine proteinase
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses the application of gastrodine in the preparation of caspase-3 active inhibitor and belongs to the field of pharmacy. The gastrodine is applied to the preparation of caspase-3 active inhibitor, the caspase-3 active inhibitor can be used for treating diseases including neurodegenerative diseases, cardiovascular and cerebrovascular diseases, osteoarthritis, rheumatoid arthritis, and the like, specifically such as Parkinson disease, Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, traumatic brain injury, spinal cord injury, severe hepatitis, liver dysfunction, osteoarthritis, rheumatoid arthritis, acquired immunodeficiency syndrome, myocardial infarction, cerebral ischemia and the like. The application of gastrodine has the advantage of blocking the specific protease needed by apoptosis without any adverse reaction which is caused by the application of the majority of broad spectrum drugs.
Description
Technical field
The present invention relates to the application of gastrodine in preparation cysteine proteinase-3 (Caspase-3) activity inhibitor, belong to pharmaceutical field.
Background technology
Apoptotic is the protease cascade course of reaction of the Caspase family guiding of a complexity, although the Caspase that participates in the apoptotic process that brings out for different cells or unlike signal pathway is different, but Caspase-3 is the only way which must be passed of caspase-3 cascade reaction, also is the key enzyme and the executor of apoptosis.Genetic knock-out experiment and animal model show that blocking-up Caspase activity has promptly blocked external and intravital apoptosis.Therefore, Caspase-3 is expected to become the novel targets of neurodegenerative diseases that the treatment aberrant apoptosis causes, cardiovascular and cerebrovascular disease, rheumatic arthritis etc., and this just needs the research and development of effective Caspase-3 inhibitor.
The Caspase-3 inhibitor will become and stop apoptotic important new drug, and its potential advantage is: can block the required specific proteases of apoptosis and the untoward reaction that can not produce most broad-spectrum medicinals.Viral and endogenous Caspase inhibitor can not be used as medicine, though the research of peptide class Caspase-3 inhibitor has obtained some progress, peptide is unstable in vivo, and this has just limited its application.Therefore, the application of the non-peptide class of micromolecule Caspase-3 inhibitor have most prospect [Ren Sumei waits .Caspase-3 inhibitor progress for Liu Zhelin, Jiang Tao. ACAD J GCP, 2004; 20 (6): 669-670].
The Caspase-3 inhibitor can be used for treating diseases such as neurodegenerative diseases that aberrant apoptosis causes, cardiovascular and cerebrovascular disease, rheumatic arthritis.Concrete as [Chen Yihua such as parkinson disease, Alzheimer, amyotrophic lateral sclerosis, Huntington Chorea, traumatic brain injury, spinal cord injury, hepatitis gravis, liver dysfunction, osteoarthritis, rheumatic arthritis, acquired immune deficiency syndrome (AIDS), myocardial infarction, cerebral ischemias, Huajie Zhang, Nan Fajun, Deng the .Caspases Research development of inhibitors. life sciences, 2006; 18 (3): 247-254; Liu Zhelin, Jiang Tao, Ren Sumei waits .Caspase-3 inhibitor progress. ACAD J GCP, 2004; 20 (6): 669-670; Publication number: CN1662505, denomination of invention: the quinoline and preparation method thereof and the Pharmaceutical composition that can be used as the Caspase-3 inhibitor].
Gastrodine (gastrodine) is the main component of orchid Rhizoma Gastrodiae, molecular formula: C
13H
18O
7, molecular weight: 295.38, structural formula is as follows:
Pharmacological evaluation shows that gastrodine can recover the dysequilibrium between cerebral cortex excitement and process of inhibition, has calmness, sleeps peacefully and central inhibitory action such as analgesia.Expand blood vessel in addition, improve myocardium microcirculation, increase the myocardial nutrition blood flow, improve oxygen delivery capacity; Hypotensive effect.Clinical neurasthenia, angioneurotic headache, trigeminal neuralgia, sciatica, the vertigo of being used for the treatment of.Active influence does not appear in the newspapers but gastrodine ex hoc genus anne chemical compound is to Caspase-3.
Summary of the invention
The technical problem to be solved in the present invention is: the application of gastrodine in preparation Caspase-3 activity inhibitor is provided.
For achieving the above object, the present invention is by the following technical solutions:
The application of gastrodine in the preparation cysteine proteinase-3 active inhibitor.
The disease that described Caspase-3 inhibitor can be used for treating comprises neurodegenerative diseases that aberrant apoptosis causes, cardiovascular and cerebrovascular disease, rheumatic arthritis etc.Concrete as parkinson disease, Alzheimer, amyotrophic lateral sclerosis, Huntington Chorea, traumatic brain injury, spinal cord injury, hepatitis gravis, liver dysfunction, osteoarthritis, rheumatic arthritis, acquired immune deficiency syndrome (AIDS), myocardial infarction, cerebral ischemia etc.
Gastrodine is a kind of known material with pharmaceutically active, and therefore, formulation method is prepared into clinical suitable any medicament, for example oral solid formulations such as tablet, capsule, granule, oral liquid, injection etc. with it routinely.
The usage and dosage of gastrodine when preparation Caspase-3 activity inhibitor medicine: calculate that in conjunction with the gastrodine physicochemical property dosage range is 100-500mg/ people/day according to the in vitro tests result, usage can oral, injection.Because of the inside and outside diversity, actual clinical consumption usage can be adjusted to some extent.
The invention will be further described below in conjunction with the drawings and specific embodiments, is not limitation of the present invention, every any this area of making according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is caspase3 and A β 42 immunofluorescence dyeing results; (a) red fluorescence is shown as caspase3 immunofluorescence dyeing positive cell; (b) green fluorescence is shown as A β 42 immunofluorescence dyeing positive cells; (a+b) blue-fluorescence is the painted DAPI dyeing of nucleus, and orange-yellow fluorescence is shown as caspase3 and A β 42 equal positive cells in the endochylema; (1) cell of transfection PS1 saltant M146L; (2) Chinese hamster ovary celI of transfection wild type PS1; (3) Chinese hamster ovary celI of transfection PS1 saltant M146L behind the 25ug/ml gastrodine effect 24h.
Fig. 2 is the immunoblotting Western blot testing result of caspase3 activated protein: 1) be the untransfected Chinese hamster ovary celI; 2) for the Chinese hamster ovary celI of transfection wild type PS1; 3) Chinese hamster ovary celI of transfection PS1 saltant M146L; 7) Chinese hamster ovary celI of gastrodine effect 24h transfection PS1 saltant M146L; 4,5,6 Chinese hamster ovary celIs that are the transfection PS1 saltant M146L after the effect of other drug monomer.Caspase3 activated protein band approximately is positioned at the 17kDa place.β-actin among the figure (confidential reference items) shows that the albumen applied sample amount of sample keeps homogeneous.
The specific embodiment
Gastrodine, gastrodini, gastrodine capsule, gastrodine injection are all the listing product of granted code, all can obtain by buying commercially available product, as all available from Kunming Medicine Group Stock Co., Ltd.
Embodiment 1: gastrodine is to the active influence of Caspase-3
One. purpose
Utilize Western blot immunoblotting, cellular immunofluorescence dyeing experimental technique and method, observe effective natural drug monomer pair cell apoptosis and carry out the influence of gene caspase3 protein expression, and then inquire into the mechanism of action of natural drug monomer anti-apoptotic from apoptosis gene expression aspect.
Two. materials and methods
1. experiment material and equipment
(1) cell source
Dual-transfected cell line (the PS1M146L/APP of saltant PS1M146L and wild type APP751
751), the dual-transfected Chinese hamster ovary oncocyte system of wild type PS1 and wild type APP751 (TW), Chinese hamster ovary oncocyte system (CHO) make up by Harvard University medical college sacred disease research center, represent through the Liangping professor of basic research institute of the Chinese Academy of Medical Sciences.
(2) reagent and medicine
A β
42Mouse anti human monoclonal antibody, the anti-mouse monoclonal antibody of Caspase3 rabbit, the anti-mouse antibodies of actin multi-clone rabbit are given by Xuan Wu hospital polypeptide chamber.Lowlenthal serum confining liquid (Lot Number:245742) is purchased in middle mountain biological product company.Texas
Goat-anti mouse immune fluorescence two anti-(Lot Number:51216), Cy
TM2 goat-anti rabbit immunofluorescences two anti-(Lot Number:51742), DAPI dye liquor (Lot Number:38495) are American I R company and produce.Horseradish peroxidase (HRP) coupling connection goat-anti mice two anti-and goat-anti rabbits two anti-(LotNumber:73452), ECL luminescent solution (Lot Number:EV052) is Hong Kong Amersham Biosciences Ltd company and produces.Pvdf membrane (Lot Number:2378) U.S. MA company produces.BCA protein quantification test kit is that U.S. Promega (Lot Number:EDM938) company produces.
Cell culture medium: high sugared DMEM (Lot No:12100-061), MEM (Lot No:41500-034), F12 (Lot No:21700-075), Fetal Bovine Serum (Lot No:16000-044), Penicillin-Streptomycin (Lot No:15070-063), Zeocin (Lot No:R25005), G-418 (Lot No:11811-031) are U.S. GIBCO biological product company and produce.Puromycin (LotNo:3025B52) is produced by U.S. company BD.Tissue Culture Plate is produced by U.S. COSTER company.
Gastrodine (gastrodine, Gastro), the crude drug standard substance are all available from Chinese biological goods calibratings institute.
(3) instrument and equipment
The instrument title place of production
Constant temperature CO
2Incubator FORMA SCIENTIFIC, USA
Inverted phase contrast microscope OLYMPUS, Japan
Inversion differs fluorescence microscope Nikon 2000-U, Japan
Desk centrifuge Beckman, Germany
Wuhan Science Instrument Factory, Chinese Academy of Sciences of a complete set of system of transfer printing
SPOT-RT cooled digital micro-analysis camera system Nikon, Japan
2. experimental technique
(1) cell culture and group technology
Chinese hamster ovary celI system cultivates with high sugared DMEM, adds 20%FBS, 2%Penicillin-Streptomycin, and wherein PS1 M146L/APP cell culture medium adds 250ug/ml G418,2.5ug/ml Puromycin.All cells is all at 37 ℃, 5%CO
2, cultivate under the saturated humidity, 0.25% trypsinization is gone down to posterity.Cell is with 5 * 10
5Be inoculated in 6 orifice plates and hatch 48h, abandoning culture medium, to add final concentration be 25ug/ml natural drug monomer serum-free medium, hatches to draw materials after adding 4uM sodium butyrate effect 24h behind the 24h.
(2) cellular immunofluorescence dyeing
With 30 * 10
4Individual cell density 50ul inoculates 24 orifice plates, and 37 ℃ discard culture medium, continuous 4 times of the adherent flushing of cold PBS 5min after hatching 24h.Every hole 500ul 4% paraformaldehyde fixed cell, room temperature is placed 1h.Turnover panel is abandoned liquid, adds PBS and adds the every hole 500ul of lowlenthal serum sealing working solution behind 4 each 15min of flushing continuously, and room temperature is placed 1h.Discard the lowlenthal serum confining liquid, directly add the anti-mouse monoclonal antibody of Caspase3 rabbit (1: 150), the A β that dilute with the sealing working solution in advance
1-42Mouse anti human monoclonal antibody (1: 250), every hole cumulative volume 500ul spends the night for 4 ℃.Discard anti-PBS 4 the each 15min of flushing continuously that add.Add Texas
Goat-anti mouse immune fluorescence two anti-or Cy
TM2 goat-anti rabbits two are anti-, dilute according to 1: 200 with PBS, and every hole 250ul, 37 ℃ of black outs are hatched 2h.Discard two and resist, 4 each 15min of PBS black out flushing.Every hole adds cold PBS diluting cells nuclear dye liquor DAPI (1: 1000) the working solution 250ul for preparing in advance, and the room temperature lucifuge is dyed 20min, and the result is watched under the fluorescence microscope in PBS flushing 2min * 4 time.SPOT-RT cooled digital micro-analysis camera system is collected image, and Image-ProPlus 5.0 image analysis software are carried out graphical analysis.
(3) Western blot immunoblotting
Each cell line is all cultivated with equal densities, washes once with cold PBS, adds the RIPA lysis buffer of pre-cooling, gets cell and at cracking 30min on ice, with lysate with 14,4 ℃ of centrifugal 30min of 000rpm, collection supernatant.It is quantitative with BCA protein quantification test kit to get the 10ul sample.Remaining sample adds 5 * upward sample buffering buffer (100mmol/L Tris, 4%SDS, 0.2% bromophenol blue, 20% glycerol, 200mmol/L dithiothreitol, DTT) and fully mixes.After being applied sample amount 10%SDS-PAGE electrophoretic separation with 25ug/, 350 milliamperes of 3h are transferred to pvdf membrane with albumen.Methanol soaks 15S after drying 1h, 5% defatted milk powder sealing 1h, adding Caspase3 one according to 1: 1000 resists, mouse anti human β-actin monoclonal antibody (1: 500), 4 ℃ of horseradish peroxidases that spend the night (HRP) coupling connection goat-anti mice, two anti-(1: 2000), goat-anti rabbit two anti-(1: 2000) incubated at room 2h are with chemoluminescence method (ECL) testing result.The protein expression band is analyzed by the Bandscan image analysis software.
3. statistical method
One factor analysis of variance (ONE-WAY ANOVA) is added up in the experimental result employing SPSS11.5 statistical analysis software.Wherein optical density (OD) value of immunofluorescence value, immunoblotting image is all compared with mutated genes groups of cells maximum and is carried out standardization of data and handle laggard line data statistical analysis.
Three. the result
1. cellular immunofluorescence dyeing
The cellular immunofluorescence coloration result shows A β
42Expression all is positioned at endochylema with capase3 positive expression position, and both positive expression positions have concordance (Fig. 1).Cell A β on the same group not
42, caspase3 expresses IOD value analysis result and shows that saltant PS1M146L organizes A β
42Expression is many than transfection wild type PS1 (WT group) with capase3 positive cell number average, statistical analysis significant difference (F
A β 42=3.489, F
Caspase3=1.211, P＜0.05).Add β with natural drug monomer gastrodine (Gastro) group A
42Expression obviously reduces than saltant PS1M146L group (matched group control), and statistics shows significant difference P＜0.05.
Tab.1?Comparation?of?effect?of?different?naturally?medical?monomeron?expression?of?Aβ?42?and?caspase3(x±s，n＝8)
Note：#vs?WT?group?P＜0.05；*vs?PS1M146L?group?P＜0.05；
2.caspase3 Western blot immunoblotting
Caspase3 active fragment band (Fig. 2) appears in each group about molecular weight 17kDa, results of statistical analysis shows respectively organizes group difference significantly (F=4.365, P＜0.05).Saltant PS1M146L group caspase3 activated protein expression is than transfection wild type PS1 (WT group) showed increased, and statistics shows significant difference P＜0.05.Add with natural drug monomer gastrodine (Gastro) group and saltant PS1M146 (control) the group caspase3 activated protein expression that does not add with the processing of natural drug monomer minimizing in various degree, statistical analysis significant difference P＜0.05 are arranged.
Tab.2?Comparation?of?effect?of?different?naturally?medicalmonomer?on?expression?of?caspase3?activation(x±s，n＝6)
Note：#vs?WT?group?P＜0.05；*vs?PS1M146L?group?P＜0.05.
Claims (4)
1. the application of gastrodine in the preparation cysteine proteinase-3 active inhibitor.
2. the application of gastrodine according to claim 1 in the preparation cysteine proteinase-3 active inhibitor, it is characterized in that: the disease that described cysteine proteinase-3 active inhibitor can be used for treating comprises neurodegenerative diseases, cardiovascular and cerebrovascular disease, traumatic brain injury, spinal cord injury, hepatitis gravis, liver dysfunction, osteoarthritis, rheumatic arthritis, the acquired immune deficiency syndrome (AIDS) that aberrant apoptosis causes.
3. the application of gastrodine according to claim 2 in the preparation cysteine proteinase-3 active inhibitor, it is characterized in that: described neurodegenerative diseases is parkinson disease, Alzheimer, amyotrophic lateral sclerosis, Huntington Chorea.
4. the application of gastrodine according to claim 2 in the preparation cysteine proteinase-3 active inhibitor, it is characterized in that: described cardiovascular and cerebrovascular disease is myocardial infarction, cerebral ischemia.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188438A (en) * | 2010-03-05 | 2011-09-21 | 昆明制药集团股份有限公司 | Application of acetagastrodine in preparing medicines to prevent and treat vascular dementia (VD) and Alzheimer disease (AD) |
| CN103385884A (en) * | 2013-07-05 | 2013-11-13 | 昆明医科大学 | Application of gastrodin to preparing medicaments for preventing and treating Alzheimer's disease |
| CN115721667A (en) * | 2021-08-25 | 2023-03-03 | 昆药集团股份有限公司 | Application of panax notoginseng saponins and acegastrodine in amyotrophic lateral sclerosis |
| CN115813941A (en) * | 2021-09-17 | 2023-03-21 | 佛教慈济医疗财团法人 | Application of gastrodin in preventing or treating amyotrophic lateral sclerosis |
| TWI837513B (en) | 2021-09-17 | 2024-04-01 | 佛教慈濟醫療財團法人 | Use of gastrodin for prevention or treatment of amyotrophic lateral sclerosis |
| US11963969B2 (en) * | 2021-09-17 | 2024-04-23 | Buddhist Tzu Chi Medical Foundation | Method for prevention or treatment of amyotrophic lateral sclerosis by administering gastrodin |
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2007
- 2007-06-14 CN CNA2007101189006A patent/CN101322713A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188438A (en) * | 2010-03-05 | 2011-09-21 | 昆明制药集团股份有限公司 | Application of acetagastrodine in preparing medicines to prevent and treat vascular dementia (VD) and Alzheimer disease (AD) |
| CN103385884A (en) * | 2013-07-05 | 2013-11-13 | 昆明医科大学 | Application of gastrodin to preparing medicaments for preventing and treating Alzheimer's disease |
| CN115721667A (en) * | 2021-08-25 | 2023-03-03 | 昆药集团股份有限公司 | Application of panax notoginseng saponins and acegastrodine in amyotrophic lateral sclerosis |
| CN115813941A (en) * | 2021-09-17 | 2023-03-21 | 佛教慈济医疗财团法人 | Application of gastrodin in preventing or treating amyotrophic lateral sclerosis |
| TWI837513B (en) | 2021-09-17 | 2024-04-01 | 佛教慈濟醫療財團法人 | Use of gastrodin for prevention or treatment of amyotrophic lateral sclerosis |
| US11963969B2 (en) * | 2021-09-17 | 2024-04-23 | Buddhist Tzu Chi Medical Foundation | Method for prevention or treatment of amyotrophic lateral sclerosis by administering gastrodin |
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