CN103432118B - The application of andrographolidume derivative in preparation prevention and treatment nerve degenerative diseases medicine - Google Patents
The application of andrographolidume derivative in preparation prevention and treatment nerve degenerative diseases medicine Download PDFInfo
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Abstract
The invention belongs to technical field of pharmaceuticals, disclose the application of a kind of andrographolidume derivative in preparation prevention and treatment nerve degenerative diseases medicine. Described andrographolidume derivative is the andrographolidume derivative AL-1 preparing with andrographolide and alpha-lipoic acid. In body, show that with experiment in vitro result AL-1 has neuroprotection, can improve the behaviouristics of Parkinson disease mice. The mechanism of AL-1 neuroprotection with remove free radical, anti-oxidant and suppress expression of nuclear factor kappa B (NF-κ B) activate relevant. Therefore, described andrographolidume derivative AL-1 can be used as prevention and the especially Parkinsonian medicine for the treatment of nerve degenerative diseases, and can make various formulations with available pharmaceutical carrier.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to a kind of andrographolidume derivative in preparation prevention andApplication in treatment nerve degenerative diseases medicine.
Background technology
Nerve degenerative diseases is that the caused central functions of nerve cell carrying out property degeneration necrosis goes down and leadsThe disease causing, is accompanied by the aging of population in the world and is and increase the weight of trend, comprises Alzheimer disease(Alzheimer ' sdisease), Parkinson's (Parkinson ' sdisease, PD), ALSIn (Atrophylateralsclerosis, ALS), Huntington's disease (Huntington ' sdisease, HD) and brainWind (Stroke) and cardiovascular and cerebrovascular dementia (Vasculardementia, VD). Its major effect patient's cognitionFunction and motor function, disable, fatal rate is high, brings great psychology and warp to patient family and societyJi burden. Most of nerve degenerative diseases there is no the cure diseases of block nerves cell degeneration process effectivelyMeans, be mainly taking medicine symptomatic treatment as main clinically, can only improve symptom. Fundamentally solve godThe misery of bringing to the mankind through DD and the method for burden are can slow down or the change of block nerves cellProperty process, even prophylactic generation, carry out can the death of block nerves cell degeneration drug researchBecome the focus of various countries' scientific research and pharmaceutical manufacturer.
At present, the pathogenesis of nerve degenerative diseases is still not very clear, thinks and oxidative stress, nerveInflammation, excitatory toxicity and nerve cell apoptosis are relevant. Therefore, there is anti-oxidant, anti-inflammatory, anti-excitabilityToxicity and anti-apoptosis activity compound are likely had a neuroprotection, can delay the disease of nerve degenerative diseasesJourney.
Chinese medicine Andrographis Paniculata has anti-inflammatory, antiviral, antibacterial, strengthens the effects such as immunity and antithrombotic[Clinicalandexperimentalpharmacologyandphysiology.2000,27:358-363.]. PunchingLotus lactone (Andrographolide, Andro) is the main active ingredient of Chinese medicine Herba Andrographitis, diabetes is had goodGood curative effect [JournalofClinicalInvestigation.1990,85:962.]. In addition, andrographolide hasThe antitumaous effect [J.Exp.Ther.Oncol.2003,3,147-158] of wide spectrum. The structural formula of andrographolide asUnder:
Andrographolide can reduce inflammation, the clinical symptoms of heating, bacterium and disease of viral infection[J.Bacteriol.2001,183,7126-7134]. Because it derives from natural traditional Chinese medicine and good effect, this class medicine is commonBe called " green antibiotic ". In recent years, research finds that andrographolide is a kind of strong NF-kB inhibitor,Reduce Neuroinflammation and there is neuroprotective activity.
Chiou etc. [Br.J.Pharmcol.2009,129,1553-1560] research thinks that andrographolide existsIt is synthetic by reducing induction type NO synthase (iNOS) protein expression inhibition NO in RAW264.7 cell,Stop from the beginning synthetic and by accelerated degradation, reduce iNOS protein stability. Shen etc.[Br.J.Pharmacol.2002,135,399-406] illustrated andrographolide at neutrophil cell inflammation-inhibitingThe mechanism of reaction is the generation that the approach by regulating PKC dependence stops or partly stop active oxygen (ROS),And lower Mac-1 high expressed. Herba Andrographitis is found in the research of Xia etc. [J.Immunol.2004,173,4207-4217]Lactone suppresses NF-kB activity. Hidalgo etc. [Br.J.Pharmacol.2005,144,680-686] are about Herba AndrographitisLactone Anti-inflammatory Mechanism research discovery andrographolide is brought into play anti-inflammatory by the combination that suppresses NF-κ B and DNAEffect, and then reduce proinflammatory albumen as the expression of COX-2 etc.
The andrographolide neuroprotection that has also been in the news. Chan etc. are reported in headstroke model, punchingLotus lactone oral administration 0.1mg/kg, maximum can reduce 50% brain infarction area [Br.J.Pharmcol.2010,161:668-679]. In addition, andrographolide can suppress p65 from Chromosome migration to nucleus, thereby presses downHaving made NF-κ B activates. Andrographolide, by suppressing the activity of NF-κ B and microglia cell, reducesTNF (TNF-α), interleukin 1 and inflammatory factor (PGE) reach neural and protectThe effect of protecting. This class finds to show, andrographolide is useful on the potential quality for the treatment of headstroke. In addition, punchingThe functions such as the anti-inflammatory of lotus lactone and raising immunity also play useful effect to neuroprotective. At middle cranial nerveIn unit and spongiocyte co-culture model, andrographolide can reduce inflammation by suppressing microglial activationThe dopamine neuron damage [JPharmacolExpTher.2004,308 (3): 975-83] of reaction mediation.
Alpha-lipoic acid (Alpha-lipoicacid, LA), or claim alpha-lipoic acid, be present in various protokaryons andEukaryotic. Alpha-lipoic acid, as a kind of powerful trace nutrient, has multiple pharmacological activity and antioxygenChange feature. In Germany, alpha-lipoic acid is approved for the nerve symptom pathology that causes for the treatment of diabetes to be hadMore than 20 years historical. Alpha-lipoic acid structure is as follows:
Alpha-lipoic acid, as the typical antioxidant of one, is called as " chondriosome nutrient " [ToxicologyandAppliedpharmacology.2002,182:84-90]. Alpha-lipoic acid is the freedom in scavenger-cell matter directlyBase, generates iron ion chelate and increases intracytoplasmic glutathione. There are some researches show alpha-lipoic acid to godThere is certain protective effect [AnnalsoftheNewYorkAcademyofSciences. through DD2008,1147:395-412]。
Lot of documents has reported that chondriosome nutrient and antioxidant comprise AD and PD at nerve degenerative diseasesOn protective effect [Annalsofneurology2003,53:S39-S48]. Research shows, alpha-lipoic acid can be carriedThe cognitive ability of high aged rats. Alpha-lipoic acid can improve the long-term memory of NMRI mouse in open field the insideAbility, also can reduce deficiency [Pharmacology, the biochemistryand of NMDA path acceptorBehavior1993]. Aspect anti-oxidant, alpha-lipoic acid can reduce products of oxidative stress such as MDA etc.,Increase Sudismase SOD activity, entirety improves anti-oxidation function [JournalofneuroscienceResearch2006,83:1584-1590]. The comparison of alpha-lipoic acid report aspect the sick AD for the treatment of neurologicalMany [Pharmacology&Therapeutics.2007,113:154-164.]. Alpha-lipoic acid is mainly by recoveringWith raising acetylcholinesterase and Na+,K+The activity of-ATPase reaches neuroprotection. Alpha-lipoic acidCan increase the activity [RejuvenationResearch. of acetylcholinesterase at positions such as cortex, midbrain, hippocampus2006,9:198-201]. In senile rat body, Na+,K+The activity of-ATPase significantly reduces, give α-After lipoic acid, the Na in rat body+,K+The activity of-ATPase [the RejuvenationResearch. that increases2006,9:198-201]. Alpha-lipoic acid can be protected the cortical neuron of amyloid-beta and hydrogen peroxide-inducedDamage. In addition, alpha-lipoic acid can increase Akt level. Can find out the god of alpha-lipoic acid from this pointThrough protective effect mainly by modulin kinase b (PKB)/Akt signal path [Neuroscienceletters.2001,312:125-128]。
In addition, other chondriosome nutrient of alpha-lipoic acid coupling, such as Co-Q10, can more effectively improveCognitive disorder, the disorder of the mitochondrial function that minimizing oxidative stress causes. Therefore, alpha-lipoic acid and other line grainThe coupling of body trophic factors may be a kind of well minimizing injury of mitochondria to the patient of neurodegeneration and recognizeKnow the method for ability obstacle. Aspect treatment PD, it is oxygen that levodopa treatment PD exists a large problemChange effect, in order to address this problem, similarly many with levodopa and the synthetic a series of structures of alpha-lipoic acidFunction medicament is considered to have very large potentiality. Studies have shown that, this class medicine is than using separately levodopaMore effectively [Journalofmedicinalchemistry.2006,49:1486-1493] for the treatment of PD.
In sum, the application of natural products andrographolide and alpha-lipoic acid has very long history, hasGood security, can be by unique their drug activity of mechanism performance. Application number 200710029644.3Chinese invention patent the andrographolidume derivative AL-1 preparing with andrographolide and alpha-lipoic acid is disclosedApplication in preparation treatment cancer drug, inflammation medicine, diabetes medicament, bacterium and virus infective medicament.
Summary of the invention
The object of the invention is to openly a kind of new purposes of andrographolidume derivative, andrographolide spreads outBiological application in preparation prevention and treatment nerve degenerative diseases medicine.
Described andrographolidume derivative is that the andrographolide of preparing with andrographolide and alpha-lipoic acid is derivativeThing AL-1, its structural formula as the formula (1):
Formula (1)
Described nerve degenerative diseases be Parkinson's, Alzheimer disease, ALS orHeadstroke; The preferred Parkinson's of described nerve degenerative diseases.
Described andrographolidume derivative can be made various formulations with available pharmaceutical carrier; Described formulation comprises sheetAgent, granule, injection, pulvis, capsule, suspending agent.
The medicine of prevention prepared by described andrographolidume derivative and treatment nerve degenerative diseases can be by variousThe common process preparation of preparation. The pharmaceutically useful excipient and the additive that in medicine preparation, use comprise nontoxicCompatible filler, adhesive, disintegrant, buffer, anticorrisive agent, antioxidant, lubricant, rectifyTaste agent, thickener, colouring agent, emulsifying agent or stabilizing agent.
Principle of the present invention:
Oxidative stress, inflammatory reaction and NF-κ B activate in the generation of nerve degenerative diseases, evolutionPlay key player. Andrographolide can suppress NF-kB activity, and having the inflammatory reaction of minimizing and be oxidized shouldSwash damaging action. Alpha-lipoic acid is a kind of strong antioxidant and mitochondrion protecting agent, has neuroprotective activity.Andrographolidume derivative AL-1 of the present invention is the couplings of andrographolide and alpha-lipoic acid, simultaneouslyThere is the multi-functional that suppresses NF-κ B, anti-inflammatory and anti-oxidant and chondriosome protective, therefore there is prevention and controlTreat the potential of nerve degenerative diseases.
The present invention has following advantage and effect:
(1) the present invention has found the new purposes of andrographolidume derivative AL-1, prevents and controls in preparationTreat the application in nerve degenerative diseases.
(2) coupling that andrographolidume derivative AL-1 provided by the invention is andrographolide and alpha-lipoic acidCompound, has anti-oxidant, to suppress NF-κ B and anti-Neuroinflammation effect concurrently, can suppress to cause simultaneouslySeveral different pathological factors of the chronic sex change of neuron, play neuroprotection; And above paathogenic factor isThe common factor of different nerve degenerative diseases neuronal degenerations, therefore andrographolide provided by the invention spreads outBiological AL-1 is to different nerve degenerative diseases, as Parkinson's, Alzheimer disease, amyotrophic lateral sclerosis sideRope sclerosis or headstroke, have preventive and therapeutic action.
(3) existing treatment nerve degenerative diseases medicine clinically, as the acetyl courage for the treatment of Alzheimer diseaseAlkali esterase inhibitor and the Parkinsonian MAOI class medicine for the treatment of, can only improve symptom, noCan delay and stop generation, the development of disease, andrographolidume derivative AL-1 provided by the invention act asNeuroprotective agent, has the generation of prevention of neurodegenerative diseases and delays and stop effect of advancing of disease.
(4) andrographolidume derivative AL-1 provided by the invention can move back with existing treatment nerve clinicallyRow disease medicament is combined use, improves curative effect, reduces the side effect of existing clinical medicine, has both improved symptom,Can delay even to stop again the course of disease of nerve degenerative diseases, treat both principal and secondary aspect of disease.
Brief description of the drawings
Fig. 1 be AL-1 in vitro respectively to hydroxyl radical free radical (OH), ultra-oxygen anion free radical (O2-), mistakeThe removing of oxygen nitrite ion (ONOO-) and four kinds of free radicals of 1,1-diphenyl-2-phenylhydrazine free radical (DPPH)Effect.
Fig. 2 is that AL-1 is to MPP+The neuroprotection of the SH-SY5Y cellular damage model of induction; WhereinSelegiline (Dep) concentration is 100 μ M, and MPP+ concentration is 2mM. ###p < 0.001 and control group (Ctrl)Relatively;*P < 0.05 and the comparison of MPP+ model group.
Fig. 3 is that AL-1 is to MPP+The neuroprotection of the cerebellar granule neuron cellular damage model of induction,Wherein selegiline (Dep) concentration is 100 μ M, MPP+Concentration is 200 μ M. ###p < 0.001 and control group(Ctrl) relatively;**P < 0.01 He***P < 0.001 and MPP+Model group comparison.
Fig. 4 is that gait and the motor function of the PD mouse of AL-1 on MPTP induction affects result. #p < 0.05Compare with control group (Ctrl);*p<0.05,**P < 0.01 He***P < 0.001 and the comparison of MPTP model group.
Fig. 5 is that AL-1 expresses positive DOPA to PD mouse Substantia Nigra (SNpc) tyrosine hydroxylase (TH)Amine neuron form and quantity affect result. Compare with control group (Ctrl) ###p < 0.001;*p<0.05,**P < 0.01 He***P < 0.001 and the comparison of MPTP model group.
Fig. 6 is the affect result of AL-1 on the PD mouse black substance TH of portion and the expression of NF-kB protein. #p < 0.05And compare with control group (Control) ##p < 0.01;*P < 0.05 He**P < 0.01 and the comparison of MPTP model group.
Detailed description of the invention
Below in conjunction with drawings and Examples, the present invention is described in further detail, and these embodiment sayBright property, therefore should be interpreted as limitation of the scope of the invention.
Embodiment 1. andrographolidume derivative AL-1 are in vitro respectively to hydroxyl radical free radical (OH), super oxygen the moonIon free radical (O2-), peroxynitrite salt ion (ONOO-) and 1,1-diphenyl-2-phenylhydrazine free radical(DPPH) the removing effect of four kinds of free radicals
Hydroxyl radical free radical (OH): adopt Phen-metal ion-H2O2, react (H by Fenton2O2+Fe2+→·OH+H2O+Fe3+) generate hydroxy radical, impel Phen-Fe2+Be oxidized to adjacent phenodiazinePhenanthrene-Fe3+, cause the maximum disappearance at wavelength 440nm place of its aqueous solution, calculate its clearance rate. Concrete stepsBe: the andrographolidume derivative that adds 300 μ L distilled waters (blank) or variable concentrations in 48 orifice platesAL-1(dissolves and is configured to 10mM storage liquid with DMSO, is then diluted to 0.01 μ M, 0.1 with distilled waterμ M, 1 μ M, 10 μ M), (configuration 1.0mM is dissolved in 50mM to add 50 μ Lof1.0mM PhensNaCl solution), then add respectively 125 μ L1.0mMH2O2With 125 μ L2.0mMFe2+Mix, useBioTekSynergyHT ELIASA is measured 100 seconds inherent 440nm wavelength place light absorption values and is reduced percentage. HydroxylBase free radical scavenging activity computing formula is: clearance rate (%)=[1-(A0－A100)/A0]×100%，A0And A100Be respectively the light absorption value in the time of 0 second and 100 seconds.
Ultra-oxygen anion free radical (O2 -): adopt pyrogallol Autoxidation Method, concrete steps are: at 48 orifice platesThe middle 250 μ L50mMTris-HCl buffer solutions (pH8.2) that add respectively, 300 μ L distilled waters (blank group)Or the andrographolidume derivative AL-1(of variable concentrations dissolves and is configured to 10mM storage liquid with DMSO,Then be diluted to 0.01 μ M, 0.1 μ M, 1 μ M, 10 μ M with distilled water). Add 50 μ L2.0mM neighboursBenzenetriol, mixes with eddy mixer. Compare under 320nm wavelength with blank, every 30 seconds records 1Sub-value, 30min measures light absorption value with BioTekSynergyHT ELIASA continuously, under similarity condition, measuresSample light absorption value, oxidation rate refers to the increment of absorbance per minute. Linear regression method, with the time (second)For abscissa, absorbance is ordinate mapping, draws absorbance and the linear relationship between the time, meterThe autoxidation rate result of calculating pyrogallol represents with the increment dA/dt of absorbance per second, i.e. equation of linear regressionMiddle y=ax+b, a value in R2. Clearance rate (%)=(dA/dt-dAs/dt)/(dA/dt). DA/dt is nothingThe autoxidation rate of pyrogallol under sample existence condition, dAs/dt is that pyrogallol under sample existence condition is from oxygenRate.
Peroxynitrite salt ion (ONOOˉ): SIN-1 (PBSpH7.4) under weak basic condition decomposes,Can produce ultra-oxygen anion free radical (O simultaneously2 ˉ) and nitric oxide free radical (NO·), the two momentProduce again peroxynitrite (ONOO-). ONOO-react with luminol, excites its oxidation, sends outPenetrate the light of 425nm wavelength. Experimental implementation concrete steps are: in the opaque ELISA Plate of 96 hole black matrix, useMicropipettor adds 60 μ L0.1mol/LPBS (pH=7.4) solution, the punching of 100 μ L variable concentrations successivelyLotus lactone derivatives AL-1(dissolves and is configured to 10mM storage liquid with DMSO, then dilutes with distilled waterBecome 0.01 μ M, 0.1 μ M, 1 μ M, 10 μ M) or 0.1mol/LPBS (pH=7.4) solution (control group),Use charger under hole pattern, to add 20 μ L1.0mmol/LLuminol solution and 20 μ L3mg/mLSIN-1Hydrochloride solution, with kinetics model total time 00:33:20, the time interval is 00:01:40.Measure luminous intensity just can judge ONOO-generation, the competition of antioxidant (AH) and luminol (L) andONOO-reaction, reduced ONOO-with LH-Reaction, reduced luminous intensity. Its luminous intensity is with anti-The oxidation resistance of oxygen agent is negative correlation, records at set intervals luminous intensity on Chemiluminescence Apparatus, makesWith clearance rate describe antioxidant catch ONOO-ability. Clearance rate (%)=(Actrl－Asample)/Actrl×100%。
1,1-diphenyl-2-phenylhydrazine free radical (DPPH): 1,1-diphenyl-2-phenylhydrazine free radical (DPPH) point flash rangingThe measuring principle of determining method is to have the last one to absorb at 517nm place according to DPPH, and its methanol solution is darkviolet.In the time having free radical scavenger to exist, owing to its absorption being faded away with its single electron pairing, it fadesDegree becomes quantitative relationship with the electron number of its acceptance. Thereby before and after available reaction, the variation of absorbance is carried out quantitativelyAnalyze. Thereby by detecting the each sample of variable concentrations, the absorbance of DPPH is changed, by clearance rateRepresent the power of its removing free radical ability. Specific experiment step is: in 96 orifice plates, add respectively 100 μ LThe sample solution (sample sets) of each concentration or 100 μ L methyl alcohol (blank group), then add rapidlyThe DPPH methanol solution (final concentration is 50 μ M) of 100 μ L100 μ M, each sample concentration 3-5 multiple hole,Under the even rear lucifuge condition of shaking, room temperature is placed one hour, then on ELIASA, under 517nm wavelength, measures extinctionDegree value, clearance rate is calculated with following formula:
Clearance rate (%)=(Actrl－Asample)/Actrl×100%。
Andrographolidume derivative AL-1 is in vitro respectively to hydroxyl radical free radical (OH), and superoxide anion freelyBase (O2 ˉ), peroxynitrite salt ion (ONOOˉ) and four kinds of 1,1-diphenyl-2-phenylhydrazine free radicals (DPPH) fromBy the effect data of the scavenging action of base as shown in Figure 1.
Embodiment 2. andrographolidume derivative AL-1 are external to MPP+The SH-SY5Y cell of induction damagesThe prolection of wound
To after SH-SY5Y cell dissociation, be seeded in 96 orifice plates (2 × 104Individual/hole), every hole 100 μ l. CarefullyBorn of the same parents cultivated after 24 hours in incubator, discarded nutrient solution. The andrographolidume derivative that contains variable concentrationsAL-1(0.01 μ M, 0.1 μ M, 1 μ M, 10 μ M) culture medium (containing 2%FBS) protection after 2 hours,Add again the MPP of 2mM+Hatch 24 hours, then suck after old culture medium and add and contain 5mg/mlMTTNew culture medium solution. In incubator, lucifuge is hatched after 4 hours and is sucked culture medium, and every hole adds 150 μ lDMSO, lucifuge shakes up after precipitation is dissolved completely, measures the absorbance at 570nm place with ELIASA. WithNormal cell survival rate is 100%, calculates the cell survival rate of different disposal group.
Andrographolidume derivative AL-1 is to MPP+The neuroprotective of the SH-SY5Y cellular damage model of inductionEffect experimental data as shown in Figure 2.
Embodiment 3. andrographolidume derivative AL-1 are to MPP+The former culture cerebellar granule neuron of inductionThe protective effect of cell
Primary cerebellar granule cell is inoculated in 96 orifice plates after extracting from 3 days large newborn rat (body weight 20-25g)（2×105Individual/hole), every hole 100 μ l cultivated after 24 hours in incubator, added 10 μ M toward culture medium the insideCytarabine. Cultivate after the 8th day, add variable concentrations andrographolidume derivative AL-1(0.01 μ M,0.1 μ M, 1 μ M, 10 μ M) after protection, after the MPP+ induction 24h with 200 μ M, adopt mtt assay mensurationEach group absorbance.
Andrographolidume derivative AL-1 is to MPP+The god of the cerebellar granule neuron cellular damage model of inductionThrough protective effect experimental data as shown in Figure 3.
PD mice behavior impaired change of embodiment 4. andrographolidume derivative AL-1 to MPTP inductionKind effect
BALB/C57 is MPTP (i.p. for mouse; 30mg/kg; 5d) after modeling, with the Herba Andrographitis of various doseLactone derivatives AL-1 (30,60,120mg/kg) gavage was treated after 2 weeks, adopted the inspection of CatWalk systemSurvey methods experiment and investigate andrographolidume derivative AL-1 to PD mouse movement gait and total momental shadowRing.
In CatWalk experiment, each treated animal administration starts after one week to train every day, allows to pass its every dayGlazing channel [Journalofneurotrauma2005,22 (2): 214-225.]. Last administration is after 24 hours, formalTest. Every mouse freely passes through glazing channel, and every animal system successfully gathers three secondary data backsights for inspectionSurvey successfully. The index detecting comprises Static State Index, dynamic indicator and equilibrium index. System is the number of output automaticallyAccording to a lot, comprise span, across width, movement velocity, cycle and ground connection time.
Gait and the motor function of the PD mouse of andrographolidume derivative AL-1 on MPTP induction affect resultData as shown in Figure 4.
Embodiment 5. andrographolidume derivative AL-1 are to PD mouse SNpc district TH positive neuron quantityImpact
After mouse modeling and administration, carry out fixing in perfusion, take out brain tissue further fixing, dehydration and stoneWax embedding, carries out TH immunohistochemical staining after making section.
Choose the section SABC between arriving-3.20 microns from bregma position-3.08. Process is as follows: 1) by stoneWax section is put into dewaxing to paraffin in 65 DEG C of baking ovens and all melts, and room temperature is cooling. 2) section is put into successivelyIn three bottles of dimethylbenzene, dewax, each 8 minutes, take off and wash 3 times, if paraffin is de-clean, can increase dewaxingTime or the dimethylbenzene renewing continue dewaxing. 3) thoroughly after dewaxing, by section put into successively gradient ethanol (100%,100%, 90%, 80%, 70%) aquation in, each concentration aquation 3 minutes. 4) with deionized water gentlyWash out after grease and (avoid tissue to be washed out), section is put into and contained 3%H2O2In solution, lucifuge is hatchedWithin 10 minutes, eliminate endogenous catalase. 5), after deionized water rinsing, section is put into TBS solutionRinse each 2 minutes 3 times. 6) then section all being immersed to microwave method in citrate buffer solution resistsFormer reparation, micro-wave oven is made as high fire 5 minutes, in low fiery 10 minutes. Attention can not allow chip drying. 7)Naturally cool to after room temperature, hatch 1h after covering tissue with 10% horse serum, non-specific in sealing tissueProperty binding site. 8) sop up serum, add TH(1:1000) primary antibodie, overnight incubation under 4 DEG C of conditions.9) sop up primary antibodie, section is put into TBS solution gently and wash 2 times, each 3 minutes. 10) drip horseradishTwo anti-(providing in DAB kit) of peroxidase labelling cover tissue, incubated at room 30min. 11)TBS develops a film 2 times, and each 2min adds the DAB colour developing working solution of now joining in tissue by kit stepIn section, incubated at room 30-60s, controls the dyeing depth. Then carry out counterstain with haematoxylin and strengthen backgroundContrast. 12) through gradient ethanol (concentration from low to high) aquation, dimethylbenzene is transparent, finally utilizes neutral treeRubber seal sheet.
Every mouse tissue is got the identical section of fine and close position 6 tension positions of black substance, observes and claps with 50 power microscopesAccording to. Positive cell in black substance region is counted, and each picture two people count and average respectively, eachGroup cell number is averaged and is compared.
Andrographolidume derivative AL-1 expresses PD mouse Substantia Nigra (SNpc) tyrosine hydroxylase (TH)Positive dopamine neuron form and quantity affect result as shown in Figure 5.
Embodiment 6. andrographolidume derivative AL-1 are to dopamine, homovanillic acid in PD mouse striaturnAnd the content influence of 3,4-dihydroxyphenyl acetic acid (DOPAC) (HVA)
After mouse modeling and administration, after chloral hydrate anesthesia, de-neck is put to death, and takes out rapidly brain. At ice bathUnder condition, separate and take out bilateral corpus straitum, at once proceed in liquid nitrogen and save backup. Detect the same day, tissue is weighedRear each tissue add 500 μ L0.3M perchloric acid extracts in ice bath, after homogenate, will organize centrifugal, 12000R/min, 4 DEG C of centrifugal 10min, get supernatant for subsequent use. Get supernatant 200ul and carry out electrochemistry HPLC analysis. StreamMoving phase: buffer solution: methyl alcohol=90:10(v/v). Flow: 1.0ml/min, column temperature: 40 DEG C. Coulomb battle array mutuallyRow electrochemical detector is set two channel electrode electromotive forces: passage 1:-150mV; Passage 4:+450mV.
Andrographolidume derivative AL-1 is to dopamine and metabolites homovanillic acid in PD mouse striaturn(HVA) and 3,4-dihydroxyphenyl acetic acid DOPAC content to affect result as shown in table 1. Wherein, ##p < 0.01Compare with control group (Control);*P < 0.05 He**P < 0.01 and the comparison of MPTP model group.
Table 1AL-1 is to dopamine and metabolites homovanillic acid (HVA) in PD mouse striaturn and 3,4-dihydroxyphenyl acetic acid DOPAC contentAffect result
Embodiment 7. andrographolidume derivative AL-1 to NO, CAT in PD mouse striaturn, MDA andThe impact of SOD
After mouse modeling and administration, after taking-up corpus straitum is weighed, each tissue adds 500 μ LPBS solution at iceIn bath, after homogenate, will organize centrifugal, 12000r/min, 4 DEG C of centrifugal 10min, get supernatant.
It is tight that the content of NO builds up bio tech ltd's (article No.: A013-2) description step according to NanjingLattice operation. NO chemical property is active, is metabolized to very soon in vivo NO2 ﹣And NO3 ﹣，NO2 ﹣FinallyFurther change into NO3 ﹣. Utilize nitrate reductase by NO3 ﹣Reductive NO2 ﹣, detect dense by the colour developing depthThe height of degree. NOS catalysis L-Arghe and molecular oxygen reaction generate NO, and NO and nucleophile generate faceColor substance detects absorbance under 530 nano wave lengths, thereby calculates NOS vigor. Organize NOS enzymeUnit of activity definition: every milligram of histone generation per minute 1nmolNO is an enzyme activity unit.
Catalase (CAT) measure according to Nanjing build up Catalase determination kit (article No.:A007-2) description has operated: catalase decomposing H2O2Reaction run into ammonium molybdate and rapidly stop,Remaining H2O2Produce faint yellow complex compound with ammonium molybdate reaction, have absorption maximum at 405nm place, measureAbsorption value can be calculated the vigor of CAT. Activity of catalase definition: every milligram of histone divides each secondSeparate 1 μ molH2O2Amount be a unit of activity.
MDA (MDA) mensuration is built up Mda kit (article No.: A003-1) according to Nanjing and is saidBright book has operated. Under high temperature and acid condition, MDA reacts generation with thiobarbituricacidα-(TBA) redLook condensation product MDA-TBA, detects the absorbance of this product at 532nm place with ELIASA. MDA containsThe calculating of amount: calculate the molar concentration of each group of sample MDA according to calibration curve, then divided by each groupProtein concentration in sample is μ M/mg albumen by the concentration conversion of MDA.
Total superoxide dismutase (SOD) according to Nanjing build up total superoxide dismutase measure box (article No.:A001-1) description step. WST-I method is surveyed SOD vigor in striatum homogenate: principle: fast at HuangUnder the oxidasic catalysis of purine, WST-1 and superoxide anion reaction Sheng is Chenged formazan, and SOD can block thisThe carrying out of reaction. By utilizing ELIASA to carry out colorimetric analysis to the color of product, then below basisFormula calculates the vigor of SOD enzyme: SOD enzyme activity unit=inhibition percentage/(1-inhibition percentage).Inhibition percentage=(ABlank 1－ABlank 2－ASample)/(ABlank 1－ABlank 2)×100%。
Andrographolidume derivative AL-1 is to NO, NOS, CAT, MDA and SOD in PD mouse striaturnTo affect result as shown in table 2. Wherein, compare with control group (Control) #p < 0.05 and ##p < 0.01;*p<0.05With**P < 0.01 and the comparison of MPTP model group.
The affect result of table 2AL-1 on NO, NOS, CAT, MDA and SOD in PD mouse striaturn
Embodiment 8. andrographolidume derivative AL-1 express the PD mouse black substance TH of portion and NF-kB proteinImpact
Every group of 6 mouse, after above method modeling and administration, get black substance albumen 30mg, add for every group200 μ lRIPA lysates (1%ProteaseInhibitorCocktail, 1%PMSF), on ice, machinery splitsSeparate. 12000r/min, 4 DEG C of centrifugal 10min, get supernatant. Get 40 μ l protein quantification concentration for every group, otherPacking immediately after be stored in 80 DEG C of refrigerators of ﹣, use in 2 weeks.
Westernblotting routinely experimental technique carries out, and wherein primary antibodie is pressed 1:1000 dilution proportion, and two is anti-Press 1:2000 dilution.
The impact knot of andrographolidume derivative AL-1 on the PD mouse black substance TH of portion and the expression of NF-kB proteinFruit data as shown in Figure 6.
Result of study shows that andrographolidume derivative AL-1 can remove dissimilar free radical, guarantor in vitroProtect MPP+The neural cell injury of induction can be protected black substance DOPA in the PD mouse model of MPTP inductionAmine neuron, improves the activity of antioxidase in brain and the generation of minimizing MDA, reduces MPTP and luresIn the brain of leading, NF-kB protein p65 subunit phosphorylation raises, and the mice behavior that can improve MPTP induction is subject toDamage, and effect is suitable with clinical treatment PD medicine selegiline (Deprenyl, Dep). Result of study provesAndrographolidume derivative AL-1 is expected to for the preparation of prevention and treatment Parkinson's medicine.
Above-described embodiment is preferably embodiment of the present invention, but embodiments of the present invention are not subject to above-mentioned enforcementThe restriction of example, other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, replacesGeneration, combination, simplification, all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (5)
1. the andrographolidume derivative suc as formula structure shown in (1) moves back in preparation prevention and treatment nerveApplication in row disease medicament:
Described nerve degenerative diseases be Parkinson's, Alzheimer disease, ALS orHeadstroke, wherein said derivative is used for preventing and treating the effect of nerve degenerative diseases in described medicineComprise removing free radical.
2. the application of andrographolidume derivative according to claim 1, is characterized in that: described godBe Parkinson's through DD.
3. the application of andrographolidume derivative according to claim 1, is characterized in that: described in wearHeart lotus lactone derivatives can prevent and treatment nerve degenerative diseases medicine separately or with other drug combination preparation.
4. the application of andrographolidume derivative according to claim 1, is characterized in that: described in wearHeart lotus lactone derivatives can be made various formulations with available pharmaceutical carrier.
5. the application of andrographolidume derivative according to claim 4, is characterized in that: described doseType comprises: tablet, granule, injection, pulvis, capsule, suspending agent.
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