CN103006633A - Application of hydroxysafflor yellow A in preparation of medicament for resisting Alzheimer disease - Google Patents
Application of hydroxysafflor yellow A in preparation of medicament for resisting Alzheimer disease Download PDFInfo
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Abstract
本发明涉及羟基红花黄色素A在制备抗阿尔茨海默病药物中的应用。本发明所述的药物由羟基红花黄色素A和医学上可接受的辅料组成，其中，羟基红花黄色素A在药物中的质量百分含量为1%～66.7%。本发明所述药物中的化合物羟基红花黄色素A可显著提高Aβ(25-35)损伤的PC12细胞的存活率，增加模型细胞内GSH含量、线粒体膜电位以及Bcl-2/Bax的比值，降低MDA含量、细胞内活性氧(ROS)的水平并减少DNA碎片的形成，从而通过保护细胞线粒体功能、抑制细胞异常凋亡、清除体内氧化应激所产生的自由基和抑制脂质过氧化等途径，提高机体抗氧化能力，改善Aβ(25-35)所致的神经细胞损伤，达到预防和/或治疗阿尔茨海默病的目的。The invention relates to the application of hydroxysafflor yellow A in the preparation of anti-Alzheimer's disease medicine. The medicine of the present invention is composed of hydroxysafflower yellow A and medically acceptable auxiliary materials, wherein the mass percentage of hydroxysafflower yellow A in the medicine is 1%-66.7%. The compound hydroxysafflor yellow A in the medicine of the present invention can significantly improve the survival rate of PC12 cells damaged by Aβ (25-35) , increase the GSH content in model cells, the mitochondrial membrane potential and the ratio of Bcl-2/Bax, Reduce the content of MDA, the level of reactive oxygen species (ROS) in cells and reduce the formation of DNA fragments, so as to protect the mitochondrial function of cells, inhibit abnormal apoptosis of cells, remove free radicals produced by oxidative stress in the body, and inhibit lipid peroxidation, etc. Ways to improve the body's antioxidant capacity, improve nerve cell damage caused by Aβ (25-35) , and achieve the purpose of preventing and/or treating Alzheimer's disease.
Description
Technical field
The present invention relates to contain the pharmaceutical product of organic effective ingredient, be specifically related to the new medicine use of S-A Hydroxysafflor yellow A (Hydroxysafflor yellow A, HSYA).
Background technology
S-A Hydroxysafflor yellow A (HSYA) is the main water solublity drug effect monomer component of Chinese traditional herbs Flos Carthami (Carthamus tinctorius L.), it is the chemical compound with single chalcone glycoside structure, shown in the following formula I of structural formula, molecular formula is C
27H
32O
16, molecular weight is 612.53, is yellow or brown color powder, soluble in water and ethanol water.
Modern pharmacological research shows that HSYA has multiple pharmacologically active, and overview gets up to say that the pharmacological action of present published bibliographical information is as follows:
(1) to the protective effect of cerebral ischemia (Yuan Yumei, Qian Xiaodong, Cao Hengbin. S-A Hydroxysafflor yellow A anti-cerebral ischemia damnification effect progress. medical Leader, 2012,31 (8): 1045-1049; Tian Jingwei, Fu Fenghua, Jiang Wanglin, etc. the protective effect of hydroxysafflor yellow A against rat cortex mitochondrial injuries induced by cerebral ischemia. Acta Pharmaceutica Sinica, 2004,39 (10): 774-777.)
(2) protective effect of cardiovascular system (is contained woods, Bi Shaojie, burnt ripple, the inhibitory action of the vascular smooth muscle cell proliferation of OxLDL ELISA being induced etc. S-A Hydroxysafflor yellow A. Chinese J Pharmacol Toxicol, 2012,26 (2): 194-199; Jin Hongguang, Jiang Chen, Tian Yudan. S-A Hydroxysafflor yellow A is on the experimentation of atherosclerotic rabbit impact. journal of shanghai Chinese medicine, 2011,45 (4): 67-68; Female precious rosy clouds, the gold ring, compass in ancient China, etc. S-A Hydroxysafflor yellow A is to the antagonism of platelet activating factor. Acta Pharmaceutica Sinica, 2002,37 (9): 696.)
(3) to neural protective effect (Liu Xingmiao, Sun Li, Liang Hao, etc. the impact that S-A Hydroxysafflor yellow A is expressed glutamate induction injured neuron peroxisome proliferation-activated receptors γ. contemporary Chinese sacred disease magazine, 2012,12 (3): 330-336; Zhu H, Wang Z, Ma C, Tian J, Fu F, Li C, Guo D, Roeder E, Liu K.Neuroprotective effects of hydroxysafflor yellow A:in vivo and invitro studies.Planta Med, 2004,69:429-433.)
(4) antiinflammatory action (Wu Y, Wang L, Jin M, Zang BX.Hydroxysafflor yellow A alleviates early inflammatoryresponse of bleomycin-induced mice lung injury.Biol Pharm Bull, 2012,35:515-22; Chen Tingting, Du Yujuan, Liu Xiaolei, etc. S-A Hydroxysafflor yellow A is to the inhibitory action of Cortex Inflammatory Signal Transduction approach correlation factor. Acta Pharmaceutica Sinica, 2008,43 (6): 570-575.)
(5) antitumor action (Chinese patent-patent No.: 200810226990, denomination of invention: S-A Hydroxysafflor yellow A the preparation antitumor drug new purposes)
(6) antioxidation (gold ring, Li Jinrong, Wu Wei. the research of antioxidative effect of Safflor Yellow. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2012,29 (5): 447-448; Xu Jing, Cai Xiaojun. the experimentation of study on HSYA preventing lens from oxidative stress. Recent Advances in Ophthalmology, 2008,28 (3): 190-194; Lu element prunus mume (sieb.) sieb.et zucc., Liu Luhua, Sun Tao, etc. the protective effect of hydroxysafflor yellow A on oxidative neurotoxicity induced by glutamate. journal of Shandong university (medicine), 2008,46 (3): 232-236.)
More than research prompting, HSYA has protection cardiovascular system and nervous system, the effects such as anti-cerebral ischemia damnification, antioxidation, antitumor, antiinflammatory.At present, HSYA uses clinically as the medicine for the treatment of cardiovascular and cerebrovascular disease, there is not yet the research report that is applied to prevent and treat Alzheimer (Alzheimer ' s Disease, AD).
Senile dementia is a kind of degenerative disease take the infringement of the cognitive disorder of carrying out property and memory as the central nervous system of main clinical manifestation, is the syndrome of intelligence infringement.This disease generally can be divided into Alzheimer (AD), vascular dementia (VD) and Mixed dementia (MD) three classes.Alzheimer and vascular dementia are two large topmost types in the senile dementia, prevalence accounts for more than 90% of all dementias, wherein AD accounts for half, and the vascular dementia common with the another kind of geratic period has essential distinction at aspects such as the cause of disease, pathogeny and prognosis.Foreign data shows that worldwide, spend 1,000 hundred million dollar because of AD every year.Become the 4th cause of death after heart disease, tumor, apoplexy at western countries AD.China's Epidemiological study finds that the prevalence of crowd more than 60 years old is up to 4%-6%, according to China's aging population 1.67 hundred million calculating more than 60 years old, conservative estimation China number of patients surpasses 6,500,000, account for 1/4 of all numbers of patients of the whole world, therefore seeking effectively to treat and improve this sick medicine also becomes an extremely urgent task.
Summary of the invention
The technical problem to be solved in the present invention provides the new purposes of S-A Hydroxysafflor yellow A, i.e. new application in pharmacy.
Above-mentioned new application in pharmacy is the application of S-A Hydroxysafflor yellow A (Hydroxysaffor yellow A, HSYA) in preparation anti-Alzheimer disease medicine.
In the above-mentioned application, described medicine is comprised of S-A Hydroxysafflor yellow A and medically acceptable adjuvant, and wherein, the quality percentage composition of S-A Hydroxysafflor yellow A in medicine is 1%～66.7%.Described medicine can be injection, transfusion or freeze-dried powder, also can be common oral formulations, such as tablet, capsule or oral liquid.
Application of the present invention, wherein said chemical compound S-A Hydroxysafflor yellow A can be that the described method of CN 1475272A application for a patent for invention is extracted acquisition from Chinese herbal medicine Flos Carthami (Carthamus tinctorius L.) by publication number, also can be obtained by synthetic, also can directly buy in market, buy as directly helping Industrial Co., Ltd. from the Shanghai coloured silk.
Alzheimer (Alzheimer's disease, AD) be a kind of central nervous system's constitutional degeneration degenerative disease, mainly clinical is chronic brain syndrome mutually, the lasting comprehensive hypophrenia that namely under the state of Consciousness, occurs, showing as memory, computing power, judgment, attention, abstract thinking ability, language function goes down, emotion and behavior disorder are lived on one's own life and ability to work forfeiture etc.Application of the present invention, wherein said chemical compound S-A Hydroxysafflor yellow A can significantly improve A β
(25-35)The survival rate of the PC12 cell of damage; increase the ratio of GSH content, mitochondrial membrane potential and Bcl-2/Bax in the model cell; reduce the level of MDA content, reactive oxygen species (ROS) and reduce the formation of DNA fragment; thereby by the Cell protection mitochondrial function, suppress the cellular abnormality apoptosis, remove the free radical that vivo oxidation stress produce and suppress the approach such as lipid peroxidation; improve antioxidant ability of organism, improve A β
(25-35)Due to neural cell injury, reach the purpose that prevents and/or treats AD.
Following experimental result has illustrated the new drug effect of S-A Hydroxysafflor yellow A, has shown beneficial effect of the present invention.
S-A Hydroxysafflor yellow A (HSYA) is to A β
(25-35)Experiment material and the method for the protective effect of the PC12 cell injury of inducing:
1. instrument and material
A β
(25-35)(Sigma-Aldrich company, purity is greater than 97%), PC12 cell (available from Chinese Academy of Sciences's Shanghai cell bank), MTT (sigma company), DMEM culture medium (low sugar), hyclone, horse serum, penicillin and streptomycin (Gibco product), DCFH-DA(is available from Invitrogen company), Bcl-2 and Bax antibody (available from Santa Cruz biotech company), Rhodamine 123 (available from Sigma-Aldrich company), malonaldehyde (MDA) test kit, glutathion (GSH) test kit, Cell Death Detection ELISA
PlusKit (building up biological study institute available from Nanjing).
Given the test agent S-A Hydroxysafflor yellow A (HSYA) is that the described method of CN 1475272A application for a patent for invention is extracted acquisition from Chinese herbal medicine Flos Carthami (Carthamus tinctorius L.) by publication number, and purity is greater than 96%.
SW-CJ-1F superclean bench (Suzhou Decontamination Equipment Plant), NU2600E type CO2 gas incubator (U.S. Precision company), 3K18 low-temperature and high-speed centrifuge (U.S. Sigma company product), PM-30 inverted microscope-Computer digital image analysis (Japanese Olympus company), homogenizer (Shanghai experimental apparatus factory), MODEL E 960 type microplate reader (Metertech Inc company), DYCZ-40B transfer printing electrophresis apparatus (Beijing Liuyi Instrument Factory), XS1050 precise electronic balance (Switzerland MettlerToledo company), 631 pure water instrument (German Sartorius company).
2. reagent preparation
A β
(25-35)The preparation of solution: with sterile distilled water preparation 1mM A β
(25-35)Solution, after PBS is diluted to variable concentrations, reference literature (XianYF, Lin ZX, Zhao M, Mao QQ, Ip SP, Che CT.Uncaria rhynchophylla ameliorates cognitive deficits induced byD-galactose in mice.Planta Med, 2011,77:1977-1983; Xian YF, Lin ZX, Mao QQ, Zhao M, Hu Z, Ip SP.Bioassay-guided isolation of neuroprotective compounds from Uncaria rhynchophylla againstbeta-amyloid-induced neurotoxicity in PC12 cells.Evid Based Complement Alternat Med, 2012:802625.) method is for subsequent use after aging 4 days in 37 ℃.The preparation of HSYA solution: with HBSS HSYA is mixed with the mother solution that concentration is 8.16mM, 0.2 μ m membrane filtration degerming, and in-20 ℃ of preservations, be diluted to three concentration of 20 μ M, 40 μ M and 80 μ M during use with serum-free DMEM culture fluid.
3. cell culture
The PC12 cell DMEM culture medium (100U/ml penicillin, 100 μ g/ml streptomycins) that contains 6% Ox blood serum and 6% horse serum, at 37 ℃, 5%CO
2, cultivate in the cell culture incubator of saturated humidity.Changed a subculture, went down to posterity once in 3-4 days in per 2 days.The trophophase cell of taking the logarithm is inoculated in culture plate, is used for different experiments.
4. grouping and administration:
1) normal group: only use DMEM culture medium culturing PC12 cell, do not add HSYA and A β
(25-35)Intervene.
2) model group: with A β
(25-35)Act on the PC12 cell.
3) after HSYA protection group: HSYA is hatched PC12 cell 2h, give again A β
(25-35)Effect 24h, according to Concentraton gradient, the HSYA protection group is divided into again three concentration groups of 20 μ M, 40 μ M, 80 μ M.
5. cell viability detects
Detect cell survival rate with mtt assay.The take the logarithm PC12 cell of trophophase is made into unicellular solution with the DMEM complete medium, with 2 * 10
4The density of individual cells/well is inoculated in 96 orifice plates, every hole 100 μ l, and every group of sample established 6 parallel holes, postvaccinal Tissue Culture Plate put into 37 ℃, 5%CO
2, behind the cultivation 24h, adding respectively final concentration is the HSYA pretreatment 2h of 20 μ M, 40 μ M and 80 μ M in the cell culture incubator of saturated humidity, adding final concentration is the A β of 20 μ M again
(25-35)Behind the damage 24h, add 10 μ l MTT (5mg/ml) and continue to cultivate 4h, suck supernatant, every hole adds the crystallization of DMSO150 μ l dissolve purple, measures each hole absorbance (OD with microplate reader under the 570nm wavelength
570Nm), be calculated as follows cell survival rate:
Cell survival rate=experimental group (OD
570Nm)/blank group (OD
570Nm) * 100%.
6.ROS detect
This experiment is with reference to Hong and Joeph method and improve to some extent, and behind the PC12 cell inoculation 24h, changes liquid, and adding respectively final concentration is the HSYA of 20 μ M, 40 μ M, 80 μ M, blank group and A β
(25-35)The damage group adds respectively serum-free DMEM culture fluid, behind the effect 2h, adds A β
(25-35)(20 μ M) hatches 24h, adds DCFH-DA (10 μ M) again, and 37 ℃ of lucifuges are hatched 30min, shift out the solution that contains DCFH-DA, the PBS washing, and every hole fluorescence intensity detects (excitation wavelength 485nm, emission wavelength 538nm) with microplate reader.
7.MDA, the GSH assay
With the Tissue Culture Dish cultured cell of 100mm, Normal group, A β are established in experiment
(25-35)Five groups of model group and HSYA protection groups (20 μ M, 40 μ M, 80 μ M).Collecting cell when drug effect stops, HBSS rinsing 2 times is collected in the centrifuge tube, the centrifugal 5min of 1000rpm, 500mlHBSS suspension cell in the EP pipe, ultrasonic disruption instrument smudge cells (amplitude 14 μ m, supersound process 5 times, each 8s, interval 10s); The centrifugal 30min of 4000rpm, get supernatant, building up malonaldehyde (MDA), glutathion (GSH) that bio-engineering research provides according to Nanjing respectively measures the test kit description and carries out each step reaction, use 722 type ultraviolet-uisible spectrophotometers, measure and respectively organize MDA and GSH content in the cell.
Wherein, the MDA test kit adopts thiobarbituricacidα-(Thibabituric Acid, TBA) method to measure MDA content, malonaldehyde can with the thiobarbituricacidα-condensation, form red product, at the 532nm place maximum absorption band is arranged.GSH measures when test kit utilizes dithio dinitrobenzoic acid and sulfhydryl compound reaction can produce a kind of yellow compound, at the 412nm place maximum absorption band is arranged, and can carry out colorimetric assay and measure.
8.DNA shive content is measured
With 2 * 10
4The density plasma cell of individual cells/well is inoculated in 96 orifice plates, every hole 100 μ l, and every group of sample established 6 parallel holes, postvaccinal Tissue Culture Plate put into 37 ℃, 5%CO
2, behind the cultivation 24h, adding respectively final concentration is the HSYA pretreatment 2h of 20 μ M, 40 μ M and 80 μ M in the cell culture incubator of saturated humidity, adding final concentration is the A β of 20 μ M again
(25-35)Behind the damage 24h, abandon supernatant, add 200 μ l lysates, cracking 30min under the room temperature in the centrifugal 10min of 200 * g, gets supernatant with this 96 orifice plate, carry out each step reaction according to Elisa test kit description, measure the content of respectively organizing DNA fragment in the cell in the 405nm place with microplate reader.
9. cell membrane potential (MMP) is measured
With 2 * 10
4The density plasma cell of individual cells/well is inoculated in 96 orifice plates, every hole 100 μ l, and every group of sample established 6 parallel holes, postvaccinal Tissue Culture Plate put into 37 ℃, 5%CO
2, behind the cultivation 24h, adding respectively final concentration is the HSYA pretreatment 2h of 20 μ M, 40 μ M and 80 μ M in the cell culture incubator of saturated humidity, adding final concentration is the A β of 20 μ M again
(25-35)Behind the damage 24h, add Rhodamine 123 (5mg/L), 37 ℃ of lucifuges are hatched 30min, shift out the solution that contains Rhodamine 123, HBSS washing 2 times, and every hole fluorescence intensity detects (excitation wavelength 488nm, emission wavelength 510nm) with microplate reader.
10. immunoblotting assay
The PC12 cell of cultivating in the Tissue Culture Dish of results 100mm extracts cell protein with the NonidetP-40 lysate, adopts improvement Lowry method to measure cell protein content.Get protein sample 30-40 μ g, with 5 * sample-loading buffer 4:1 volume mixture, 95 ℃ of heating 5min make albuminous degeneration.Protein sample after processing is slowly added in the well, and a swimming lane adds 5 μ l and dyes in advance molecular weight of albumen Marker therein.80V constant voltage electrophoresis when the extremely concentrated glue of protein sample swimming and separation gel intersection, changes 100V constant voltage electrophoresis into.When treating the swimming of bromophenol blue line extremely apart from glue edge, stop electrophoresis.With reference to molecular weight Marker, downcut the required part of separation gel, it is for subsequent use to immerse transferring film liquid.Take off pvdf membrane, the one side of labelling carrier protein molecule is dipped among the TBST, washes film 3 times, each 5min.Pvdf membrane is dipped in 5% defatted milk powder room temperature sealing 2h.Pvdf membrane is dipped among the TBST, washes film 3 times, each 10min.Take out pvdf membrane, immerse the primary antibodie with the TBST dilution, incubated at room 1h.Pvdf membrane is dipped among the TBST, washes film 3 times, each 10min.Two anti-(Bax with the anti-IgG, β-actin, Bcl-2 goat anti-mouse IgG of exempting from of goat) that adds Radix Cochleariae officinalis enzyme labelling corresponding to each antibody, incubated at room 2h.Pvdf membrane is dipped among the TBST, washes film 3 times, each 10min.Photographic fixing is developed in chemiluminescence.Strip analysis is used Image Pro-Plus software analysis purpose band gray value, relatively each histone differential expression.
Interpretation of result:
1.HSYA to A β
(25-35)The protective effect of the PC12 cell of damage
Cell survival rate adopts mtt assay to detect.Originally studies show that model group PC12 cell adds A β
(25-35)After (20 μ M) hatched 24h, cell survival rate compared with normal matched group significantly reduced, and was 62.2% of Normal group.Compare HSYA(20 μ M, 40 μ M, 80 μ M with model group) protection group, can reduce the PC12 cell injury in dose dependent ground, cell survival rate significantly improves, and is respectively 70.2%, 80.9% and 91.5% of Normal group.Experimental data adopts the SPSS17.0 statistical software to carry out data analysis with Mean ± SEM (n=6) expression, take group as abscissa, take comparative survival rate of cells % as vertical coordinate, draws bar diagram.The anti-A β of variable concentrations HSYA
(25-35)Cause the protective effect of PC12 cellular neural damage to the results are shown in bar diagram 1.
2.HSYA to A β
(25-35)The impact of ROS in the PC12 cell of damage
Active oxygen (ROS) is at A β
(25-35)Important role in the process of cell death of mediation can be estimated HSYA at A β by ROS level in the DCFH-DA fluorescent probe mensuration born of the same parents
(25-35)Inducing cell produces the effect in the oxidative stress.Experiment shows that the PC12 cell is exposed to A β
(25-35)Behind the 24h, the ROS level obviously increases in the cell, is 247.3% of normal group in (20 μ M); Variable concentrations HSYA(20,40,80 μ M) intervene after, the increase of fluorescence intensity in the born of the same parents is alleviated on dose dependent ground, wherein the HSYA effect of 40 and 80 μ M is the most remarkable, the interior ROS level of cell drops to respectively 179.5% and 122.8% of normal group.Experimental data adopts the SPSS17.0 statistical software to carry out data analysis with Mean ± SEM (n=6) expression, take group as abscissa, take ROS relative level % as vertical coordinate, draws bar diagram.Variable concentrations HSYA is to A β
(25-35)The impact of inducing the PC12 cell to produce the ROS level the results are shown in bar diagram 2.
3.HSYA to A β
(25-35)The PC12 cell GSH of damage, the impact of MDA content
GSH is important antioxidant and free radical scavenger in the body, and the height reflection body of its content is to the removing ability of free radical; MDA is that free radical causes the end product that lipid peroxidation decomposes, and also is the major incentive that causes the necrosis of AD neuronal degeneration.This result of study shows that the interior MDA content of model group cell obviously increases, GSH content obviously reduces, and is respectively 186.4% and 61.5% of normal group; HSYA20,40,80 μ M protection groups can significantly reduce MDA content, increase GSH content, and prompting HSYA can effectively remove the free radical that vivo oxidation stress produce, and suppresses lipid peroxidation, improves antioxidant ability of organism, thereby improves A β
(25-35)Oxidative stress damage due to (20 μ M).Experimental data adopts the SPSS17.0 statistical software to carry out data analysis with Mean ± SEM (n=6) expression, respectively take group as abscissa, take MDA relative amount %, GSH relative amount % as vertical coordinate, draws bar diagram.Variable concentrations HSYA is to A β
(25-35)Induce the impact of MDA, GSH content in the PC12 neurocyte to see bar diagram 3 and bar diagram 4.
4.HSYA to A β
(25-35)The impact of DNA shive content in the PC12 cell of damage
An apoptotic key character is the excessive fragmentation of DNA.This result of study shows that the PC12 cell is exposed to A β
(25-35)Behind the 24h, the content of DNA fragment significantly increases in the born of the same parents, is 207.0% of normal group in (20 μ M); Variable concentrations HSYA(20,40,80 μ M) intervene after, its content dose dependent ground reduction, wherein 40 and 80 μ M HSYA effects are the most remarkable, are respectively 139.9% and 136.0% of normal group.Experimental data adopts the SPSS17.0 statistical software to carry out data analysis with Mean ± SEM (n=6) expression, take group as abscissa, take DNA fragment relative amount % as vertical coordinate, draws bar diagram.Variable concentrations HSYA is to A β
(25-35)Induce the impact of DNA shive content in the PC12 neurocyte to see bar diagram 5.
5.HSYA to A β
(25-35)The impact of MMP in the PC12 cell of damage
The decline of mitochondrial membrane potential (MMP) is an early stage significant event of apoptosis.But Rhodamine123 is a kind of cationic fluorescent dyestuff of permeate through cell membranes, can rely in normal cell that mitochondrial transmembrane potential enters mitochondrial matrix and cell is painted, the decline of mitochondrial membrane potential during the cell apoptosis, reduce so that enter Intramitochondrial Rh123 dyestuff, so detected fluorescence dies down, come the variation of detection line mitochondrial membrane potential and the generation of apoptosis by the power of fluorescence signal.This result of study shows that the PC12 cell is exposed to A β
(25-35)In (20 μ M) behind 24h, MMP obviously descend (for normal group 69.0%), cause the cell permeability to increase the trigger cell apoptosis; Variable concentrations HSYA(20,40,80 μ M) intervene after, dose dependent ground rising cell MMP, inhibited apoptosis, wherein 40 and 80 μ M HSYA effects are the most remarkable, and cell is had remarkable protective effect.Experimental data adopts the SPSS17.0 statistical software to carry out data analysis with Mean ± SEM (n=6) expression, take group as abscissa, take MMP relative level % as vertical coordinate, draws bar diagram.Variable concentrations HSYA is to A β
(25-35)Due to the impact that changes of PC12 mitochondrial membrane potential in anoxic see bar diagram 6.
6.HSYA to A β
(25-35)Bcl-2, the Bax of the PC12 cell of damage and the impact of Bax/Bcl-2
Bcl family is the class regulatory factor in the apoptosis process, and whether the balance of anti-apoptotic genes expression Bcl-2 and short apoptogene Bax enters apoptosis pathway to cell and play an important role in the family.This experimental result shows A β
(25-35)Obviously raise the expression of Bax in the cell, reduce simultaneously the Bcl-2 protein level, the ratio of Bcl-2/Bax is reduced (for normal group 48.8%), promote apoptosis; Be the pre-Cell protection 2h of HSYA of 20,40,80 μ M with concentration, all can significantly suppress A β
(25-35)The apoptosis of inducing, reduce Bax expression, raise the Bcl-2 protein level, thereby improve the ratio (be respectively normal group 62.8%, 89.8% and 80.4%) of Bcl-2/Bax in the cell, improve by A β
(25-35)The excessive Apoptosis of the PC12 cell of inducing.Adopt gel imaging system that immunoblotting gained film is taken pictures, get gel electrophoresis Fig. 7; And with the molecular weight of gel images processing system evaluating objects band and clean optical density value, experimental data adopts the SPSS17.0 statistical software to carry out data analysis, take group as abscissa with Mean ± SEM (n=6) expression, take Bax/Bcl-2 relative expression quantity % as vertical coordinate, draw bar diagram 8.Variable concentrations HSYA is to A β
(25-35)Due in the PC12 cell Bcl-2, Bax and β-actin express the impact that changes and see electrophoretogram 7.Variable concentrations HSYA is to A β
(25-35)Due in the PC12 cell Bax/Bcl-2 express the impact that changes and see bar diagram 8.
Description of drawings:
Fig. 1 is for showing the anti-A β of variable concentrations HSYA
(25-35)The bar diagram that causes PC12 cellular neural damage effect.
Fig. 2 suppresses A β for showing variable concentrations HSYA
(25-35)Induce the PC12 cell to produce the bar diagram of ROS effect.
Fig. 3 suppresses A β for showing variable concentrations HSYA
(25-35)Induce the PC12 cell to produce the bar diagram of MDA effect.
Fig. 4 recovers A β for showing variable concentrations HSYA
(25-35)Suppress the bar diagram that the PC12 cell produces the GSH effect.
Fig. 5 suppresses A β for showing variable concentrations HSYA
(25-35)Induce the PC12 cell to produce the bar diagram of DNA fragment effect.
Fig. 6 recovers A β for showing variable concentrations HSYA
(25-35)Reduce the bar diagram of PC12 cell membrane potential effect.
Fig. 7 is for showing that variable concentrations HSYA is to A β
(25-35)Due in the PC12 cell Bcl-2, Bax and β-actin express the electrophoretogram of variable effect.
Fig. 8 is for showing that variable concentrations HSYA is to A β
(25-35)Due in the PC12 cell Bax/Bcl-2 express the bar diagram of variable effect.
Among above-mentioned Fig. 1～6 and Fig. 8, the implication of label symbol is respectively in the relevant bar diagram: ﹟ represents with normal group significant difference P＜0.001 is arranged relatively; * expression relatively has significant difference P＜0.05 with model group; * represents with model group significant differences P＜0.01 is arranged relatively; * * represents with model group utmost point significant difference P＜0.001 is arranged relatively.
The specific embodiment
Example 1 (injection)
Get S-A Hydroxysafflor yellow A 15g, adding sodium chloride 9g adds water to 1000m1, adjusts pH to 4.5-7.0 with the 1mol/ml sodium hydroxide solution, filters, and filtrate is potted in the ampoule bottle of 5ml, 100 ℃ of sterilization 30min, and labeling is packed and be get final product.But this product intravenous injection or intramuscular injection, each one bottle, once a day, 14 days is a course for the treatment of, can use continuously the 1-2 course for the treatment of.
Example 2 (lyophilized injectable powder)
Take by weighing 40g mannitol, place in the appropriate containers, add 200ml water for injection, add pin with charcoal 0.2g (0.1%w/v), be heated to 80 ℃, stir 30min, 0.22um filtering with microporous membrane, filtrate for later use.
Take by weighing the 80g S-A Hydroxysafflor yellow A, inject water to 1000ml, stirring is dissolved S-A Hydroxysafflor yellow A fully.Mix S-A Hydroxysafflor yellow A solution and mannitol solution, add water for injection to 2000ml, use the 0.22um filtering with microporous membrane, packing, loading amount is every hydroxyl carthamin yellow A-containing 80mg, lyophilization.Lid is rolled in the vacuum tamponade, and labeling is packed and be get final product.But this product intravenous injection is got one of above-mentioned injection S-A Hydroxysafflor yellow A and is added in the 250ml injection normal saline, slowly intravenous drip, and every day 1 time, 14 days is a course for the treatment of.
Example 3 (tablet)
Get S-A Hydroxysafflor yellow A 20g, add lactose 48g, starch 110g mix homogeneously, starch slurry 30g with 7% is as binding agent, wet granulation, oven dry, the magnesium stearate 6.24g that adds grain amount 3%, then mixing is pressed into 600 tablets of tablets with decompressor with granule, namely gets the tablet for the treatment of senile dementia.This tablet is oral, each 2-3 sheet, and 3 times on the one, 15-20 days is a course for the treatment of, can use continuously the 2-3 course for the treatment of.
Example 4 (capsule)
Get S-A Hydroxysafflor yellow A 50g, add lactose 48g, starch 110g mix homogeneously, starch slurry 30g with 7% is as binding agent, wet granulation, oven dry, cross 20 mesh sieve granulate, fill in relative humidity is lower than 74% environment in No. 3 capsulae vacuuses, the heavy 123mg of the average content of every capsules namely gets the capsule of control AD.This capsule is oral, and 3 times on the one, each 2-3 grain, 15-20 days is a course for the treatment of, can use continuously the 2-3 course for the treatment of.
Example 5 (oral liquid)
Press the asepsis operation, S-A Hydroxysafflor yellow A 10g is dissolved in (about 30ml) in the suitable quantity of water, be stirred to fully dissolving, pour in the 1000ml beaker, add glycerol 120ml again and mix, adding distil water is to 400ml, as I liquid.The about 0.5g of taking ethylparaben, accurately weighed, pour in the 500ml beaker, add about 200ml distilled water, it is all soluble in water to be heated to ethyl hydroxybenzoate, as II liquid.II liquid is slowly added in the I liquid, and the limit edged stirs, and is settled to 1000ml again, stirs, and filters, and fill is in 10mlA type oral liquid bottle and get final product under the aseptic condition.Every day oral 2 bottles (20ml), divide and take for 1-2 time, serve on for 1 week.
Claims (3)
1. the application of S-A Hydroxysafflor yellow A in preparation anti-Alzheimer disease medicine.
2. application according to claim 1 is characterized in that, described medicine is comprised of S-A Hydroxysafflor yellow A and medically acceptable adjuvant, and wherein, the quality percentage composition of S-A Hydroxysafflor yellow A in medicine is 1%～66.7%.
3. application according to claim 1 and 2 is characterized in that, described medicine is injection, freeze-dried powder, tablet, capsule or oral liquid.
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| CN2012104924508A CN103006633A (en) | 2012-11-27 | 2012-11-27 | Application of hydroxysafflor yellow A in preparation of medicament for resisting Alzheimer disease |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107056857A (en) * | 2017-03-31 | 2017-08-18 | 湖南中医药大学 | A kind of new flavone compound and preparation method and application |
| CN112142806A (en) * | 2020-09-22 | 2020-12-29 | 山西德元堂药业有限公司 | Compound and salt, pharmaceutical composition and application thereof |
| CN113350358A (en) * | 2021-07-08 | 2021-09-07 | 上海市普陀区中心医院 | Application of polygala tenuifolia sapogenin single or combined with hydroxysafflor yellow A in preparation of medicine for improving cognitive impairment after ischemic stroke |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107056857A (en) * | 2017-03-31 | 2017-08-18 | 湖南中医药大学 | A kind of new flavone compound and preparation method and application |
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| CN112142806A (en) * | 2020-09-22 | 2020-12-29 | 山西德元堂药业有限公司 | Compound and salt, pharmaceutical composition and application thereof |
| CN112142806B (en) * | 2020-09-22 | 2023-03-03 | 山西德元堂药业有限公司 | Compound and salt, pharmaceutical composition and application thereof |
| CN113350358A (en) * | 2021-07-08 | 2021-09-07 | 上海市普陀区中心医院 | Application of polygala tenuifolia sapogenin single or combined with hydroxysafflor yellow A in preparation of medicine for improving cognitive impairment after ischemic stroke |
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