CN104013609A - Purpose of asphonin in preparation of medicine for treating Parkinson's disease - Google Patents
Purpose of asphonin in preparation of medicine for treating Parkinson's disease Download PDFInfo
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- CN104013609A CN104013609A CN201410226395.7A CN201410226395A CN104013609A CN 104013609 A CN104013609 A CN 104013609A CN 201410226395 A CN201410226395 A CN 201410226395A CN 104013609 A CN104013609 A CN 104013609A
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- chinonin
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Abstract
The invention belongs to the technical field of medicine, and particularly relates to purpose of flavonoid asphonin in preparation of medicines for treating Parkinson's disease. The invention establishes a Parkinson's disease animal and cell models, and comprehensively utilizes various behavioral, immunological, biochemical indicator research methods; and experiments confirm that the medicine can significantly improve dyskinesia symptoms of mice in the Parkinson's disease model, improve levels of striatal dopamine and its metabolites and reduce the dopaminergic neuron degeneration of compact part of substantia nigra; and it is found that the protective effects are correlative to antioxidant activity in scavenging free radicals and reduction of oxidative stress effect. Asphonin is rich in raw material sources, and has significant efficacy and multiple effect targets, so asphonin has certain development value and prospects in clinical application.
Description
Technical field
The invention belongs to medical technical field, particularly the purposes of flavone compound Chinonin in preparation treatment parkinson disease medicine.
Background technology
Parkinson disease (parkinson ' s disease, PD) 1817, by James Parkinson, propose first and deliver, a kind of common nervus centralis degenerative disease, often with dyskinesia symptoms such as bradykinesia, muscle rigidity, static tremor, posture shakinesses.Main pathological characters is that black substance compact part (SNpc) dopaminergic neuron is degenerated, and striatal dopamine fiber is lost.Parkinsonian pathogenesis is very complicated, and mitochondria dysfunction, environmental toxin, oxidative stress, excitatory toxicity are participated.Except the significant dyskinesia and the change on pathology that these occur clinically, also have some other delayed ischemic neurological deficits, as sleep disorder, cognitive disorder, and approach 40% people and can suffer from depression, having a strong impact on patient's quality of life.Along with social development, social senilization further develops, and Parkinsonian sickness rate also increases gradually.
The medicine using clinically at present mainly contains anticholinergic agents, Loratadine, dopamine alternative medicine, dopamine-receptor stimulant, inhibition Dopamine Metabolism In The Rat medicine etc.These medicines just improve symptom, can not effect a radical cure, and long-term taking there will be some side effect.As dopamine replaces medicine levodopa all effective to all parkinson disease, can improve patient's ability to work and independent living ability, reduce case fatality rate.But patient is prone to " switch " phenomenon, patient's symptom, alleviating (opening the phase) and increase the weight of fluctuation between (pass phase) suddenly, alternately occurs rapidly repeatedly.A minority there will be nausea and vomiting, involuntary movement, mental symptom (hallucination and illusion), anxious state of mind, drowsiness, the side effect such as cognitive dysfunction.In dopamine agonist, most drug prices are expensive, and individual variation is large, easily produces postural hypotension, nausea and vomiting, constipation, headache, the side effect such as sleepy.Suppress the general and other drug of Dopamine Metabolism In The Rat medicine and share, when having strengthened drug effect, also can expand side effects of pharmaceutical drugs etc.
In recent years, the active substance extracting from natural plants progressively becomes neurologic agent and researches and develops the particularly focus of PD treatment, and the related drugs Chinonin in the present invention belongs to the flavone compound in natural plants.Chinonin (chinonin), chemistry 2-(D-glucopyranosyl-1,3,6,7-hydroxyl-9H-phylloxanthin-9-ketone) by name, molecular formula is C
19h
18o
11, structure is as follows:
Chinonin is a kind of natural polyphenol compound, and its source is abundant, can be from fruit, leaf, the bark of Anacardiaceae plant Fructus Mangifera Indicae, and the rhizome of the liliaceous plant Rhizoma Anemarrhenae, aerial parts, extracts and obtains in the flower of irides, the plant such as dry.Chinonin is a polyphenol compound, four phenolic hydroxyl groups are avtive spots that Chinonin is removed free radical, have stronger Polarography, and phenyl ring has good energy trapping, its strong anti-oxidation ability is proved in different cells, comprises neurocyte.Chinonin can strengthen the anesthetic action of cyclopropylene barbital sodium, can reverse the sedation that reserpine causes, can strengthen rat that DOPA causes and the piloerection of mice, salivation, mobility, aggressivity.Visible Chinonin has obvious central nervous system's excitation, and chlorpromazine can block the central nervous excitation that chimonin causes, infers that its action site is the agonist of mesolimbic system D2 receptor.At MPP
+on the N2A cell Parkinson disease model of induction, research is found, Chinonin can be by regulating the level of GSH, and the formation that suppresses ROS reduces MPP
+toxicity.Chinonin can reduce intracellular Ca
2+concentration, thereby the damage of minimizing neurocyte.
Summary of the invention
The deficiency and the shortcoming that in order to overcome existing treatment parkinson disease medicine, exist, primary and foremost purpose of the present invention is to provide the purposes of a kind of Chinonin in preparation treatment parkinson disease medicine.Chinonin is abundant, the evident in efficacy flavone compounds of a kind of raw material sources; Chinonin itself has phenolic hydroxyl structure feature, removes free radical ability, Cell protection apoptosis; can penetrate smoothly the characteristics such as blood brain barrier, utilize Chinonin to carry out evaluating drug effect to parkinson disease.
The object of the invention is achieved through the following technical solutions:
The purposes of Chinonin in preparation treatment parkinson disease medicine.
Described medicine contains mass percent and is greater than pharmaceutically acceptable carrier or the excipient that 99% Chinonin and mass percent are less than 1%.
The present invention possesses following outstanding advantages and effect with respect to prior art:
The present invention is by setting up C57BL/6J MPTP-induced Parkinson disease mice model, observe Chinonin to parkinsonian mouse dyskinesia symptom, striatal dopamine and metabolite level thereof and black substance compact part dopaminergic neuron, cerebral tissue superoxide dismutase activity, the impact of glutathion, malonaldehyde level.Proving the experimental result of Chinonin in integral level shows, Chinonin can be removed free radical ability by strengthening brain, reduce level of lipid, to reduce oxidative stress degree, significantly improve the bradykinesia symptom of parkinsonian mouse, improve striatal dopamine content and reduce its metabolism, improving distribution and the form of black substance compact part dopaminergic neuron.Thereby proof Chinonin all has good neuroprotective to inside and outside parkinson disease.
Accompanying drawing explanation
The openfield behavioristics experimental result picture of Fig. 1 Chinonin to MPTP induction mouse model of Parkinson's disease.
The CatWalk behavioristics experimental result picture of Fig. 2 Chinonin to MPTP induction mouse model of Parkinson's disease.
The affect figure of Fig. 3 Chinonin on MPTP induction mouse model of Parkinson's disease Substantia Nigra dopaminergic neuron cell.
The affect figure of Fig. 4 Chinonin on MPTP induction mouse model of Parkinson's disease striatal dopamine and metabolite content thereof.
The affect figure of Fig. 5 Chinonin on the super oxygen dismutase activity of MPTP induction mouse model of Parkinson's disease striatum, glutathione level and mda content.
The specific embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
The foundation of animal model and grouping:
By body weight 20-25g, in age in 8-10 week, male C57BL/6 mice, is divided into matched group at random, MPTP model group, positive drug group, the basic, normal, high dosage group of Chinonin, 18 every group.Be placed in 23 ± 2 ℃, lighting hours is 12h/day, and animal starts to give medicine after conforming one week.Except matched group, other respectively organize lumbar injection MPTP every day (30mg/kg) once, positive drug group injection selegiline (10mg/kg) after 30min, Chinonin administration group injection Chinonin (10mg/kg, 20mg/kg, 40mg/kg), model group gives isopyknic normal saline, after the 12h of interval, be administered once again, continue 7 days.Matched group whole process gives normal saline.First carry out subsequently by the method for openfield and catwalk, the behavioristics of rat being marked, then by Immunohistochemical Method, detect the variation of Substantia Nigra dopaminergic neuron, HPLC-ECD detects the content of Dopamine In Striatum and metabolite, test kit detects striatal oxidative damage, and the method for open field (open field) and catwalk is marked to the behavioristics of rat.By above method, evaluate the therapeutical effect of Chinonin to PD rat model.
1. Behavioral assessment
Each is organized mice and before giving medicine, carries out respectively behavioristics's training, as self-blank contrast, carries out behavioristics's detection at the 7th day that gives MPTP.
1.1 experimental technique
1.1.1Openfield
Open field (openfield) is mainly to detect the autonomic activities ability of mice.Mice is put into the lucite box of 50cm * 50cm * 40cm, places same angle, and mouse adapts to after 2min, in quiet, dim environment, detects.With the relevant speed of ethvision xt8.0 system acquisition mouse movable 10min in box, moving displacement, central area motion frequency etc.
1.1.2CatWalk
Adopt the variation of the dynamic and static gait index of CatWalk gait analysis system detection mice, as length and the width of pawl seal.Pawl hang time and speed, the indexs such as left and right foot spacing, step order.Under quiet, non-stimulated condition, every mice is by fluorescent track under faint red light irradiates, and with high frequency camera shooting machine record, every mice runs 3 times continuously, gets its meansigma methods and compares.Parameter in the CatWalk system of choosing in this experiment is described below:
(1) Run duration: mice is from required time of runway end to end (s), the i.e. period of motion.(2) Stance: mice extremity continue the time contacting with floor, i.e. contact time (s).(3) Swing: the time (s) that pawl is unsettled.(4) Swing speed: the speed striding across when pawl is unsettled (cm/s), equals stride length/swing.(5) Regularity index (RI): normally walk the ratio (%) that order accounts for total step order.
1.2 experimental result
1.2.1Openfield
Utilize open field to detect the autonomic activities ability of mice, testing result shows, with the comparison of blank group mice, the displacement of model group mouse movement reduces, and the speed of motion slows down.Pathway figure shows that mice zone of action scope reduces, the only surrounding activity of surrounding wall, and in central area, movable number of times tails off, and result confirms that the mice autonomic activities ability of injection MPTP weakens.Positive drug group mice increases compared with the displacement of model group mice, and speed speeds, and central movable frequency increases, and relatively has significant difference (P<0.05) with model group.Meanwhile, Chinonin dosage group mice all can be improved the moving displacement of mice, movement velocity, and range of activity, wherein middle dosage Chinonin can significantly increase moving displacement and the movement velocity of mice, and the movable number of times in central area increases.Result confirms that Chinonin can effectively improve the autonomic activities ability (as shown in Figure 1) of parkinsonian mouse.
1.2.2CatWalk
Through CatWalk, detect and find, compare with working as parkinsonian mouse, positive drug group mice ran the time shorten of passage, and front and back limb contact time shortens, and normally walked order and improved than recovering.Described Chinonin has good improvement effect (as shown in Figure 2) to the behavior disorder symptom of parkinsonian mouse.
2. Immunohistochemical Method detects the variation of Substantia Nigra dopaminergic neuron
2.1 sample treatment
Get 6 mices for every group, the chloral hydrate anesthesia with mass fraction 10%, breaks right auricle, and from left ventricle inserting needle perfusion normal saline, the percentage by volume that instils after flushing blood is 4% paraformaldehyde 50mL.Open cranium and get brain, immerse after 4 ℃ of Bollinger body liquid 6h fixing.Through gradient alcohol dehydration, dimethylbenzene is transparent, and paraffin embedding is also made pathological section (5 μ m).
2.2 SABC
Get continuously 5 of the crown sections in cerebral tissue black substance position, dimethylbenzene dewaxing, gradient ethanol elution (volume fraction is followed successively by 100%, 100%, 90%, 80%, 70%) eluting, the H that volume fraction is 3%
2o
2(PBS preparation) incubated at room 10min is to eliminate the activity of endogenous peroxydase, PBST embathes, 3min * 3 time, put in microwave oven, heating citrate buffer is repaired antigen, adopt high fiery 5min, in low fiery 10min, cooling rear PBST embathes 3min * 3 time, volume fraction is 0.1% hyclone incubated at room 1h, add 4 ℃ of overnight incubation of primary antibodie (volume ratio is 1:500), add two anti-(1:200) incubated at room 1.5h, the DAB 2min that develops the color, gradient ethanol dehydration, dimethylbenzene is transparent, and mounting dries naturally.
2.3 experimental result
Take normal mouse as benchmark, the neuronal cell of untreated parkinsonian mouse declines 65.93%, positive drug group mice black substance compact part positive neuron number significantly increases, reduce by 21.23%, through the neuronal cell of the sick mice of handkerchief gold of the Chinonin treatment 43.7%-50% that only declines, and it is sparse to improve nerve fiber, dimness, the symptoms such as cell shape variation.Described Chinonin has good improvement effect to the tyrosine hydroxylase positive neurons cell distribution of parkinsonian mouse black substance compact part and form.Result as shown in Figure 3.
3.HPLC-ECD detects the content of Dopamine In Striatum and metabolite
3.1 sample treatment
Get 6 mices for every group, after mass fraction is 10% chloral hydrate anesthesia, normal saline rinses blood from heart perfusion, gets brain and peels off bilateral striatum, and every operation all carries out on ice bath, and weighs.It is 3% perchloric acid homogenate that striatum adds 0.3mL mass fraction, 12000r/min, and 4 ℃ of centrifugal 10min, 0.2 μ m membrane filtration ,-20 ℃ store for future use.Adopt HPLC-ECD method to detect the content of mouse brain striatum DA and metabolite dihydroxyphenyl acetic acid DOPAC and 4-hydroxy-3-methoxy-.alpha.-toluic acid. HVA.
3.2HPLC-ECD detect
3.2.1 chromatographic condition
Chromatographic column: the Develosil ODS-UG-5 of NOMURA CHEMICAL company (4.6mm * 150mm, 5 μ m); Guard column: U.S. Phenomenex company (4.0Lmm * 3.0LD mm); The preparation of mobile phase: take monohydrate potassium 15.0g, trisodium citrate 20.2g, EDTANa
20.036g, eight sodium alkyl sulfonate 0.18g, add pure water (>=18M Ω) 900mL; Add methanol 100mL, standby after 0.2 μ m membrane filtration ultrasonic degas; Column temperature: 38 ℃; Flow velocity: 0.8mLmin
-1; Coulomb array electrochemical detector arranges three channel electrode electromotive forces: passage 1 (reduction potential) :-150mV; Passage 2 (NE, DOPAC, DA, 5-HIAA, 5-HT sense channel) :+350mV; Passage 3 (HVA sense channel) :+450mV.
3.2.2 the preparation of standard curve
The reference substance of DA, DOPAC and HVA is mixed with to the stock solution that concentration is 100 μ g/mL by mobile phase, then by mobile phase, is diluted to 5,10,20,50,100,200,400, the reference substance solution of 600,800 μ g/L, gets 20 μ L injecting chromatographs and analyzes, and obtains reference substance solution chromatogram.The concentration of each reference substance solution of take is abscissa (X), and the peak area of each component is that vertical coordinate (Y) carries out linear regression, obtains standard curve equation.
DA?Y＝10.806X+2.7491，R＝0.9999
DOPAC?Y＝11.067X-2.4808，R＝0.9998
HVA?Y＝15.969X+6.9319，R＝0.9999
3.3 experimental result
With normal mouse comparison, untreated parkinsonian mouse DOPAMINE CONTENT IN RABBIT obviously declines, and the content of its metabolite dihydroxyphenyl acetic acid DOPAC and 4-hydroxy-3-methoxy-.alpha.-toluic acid. HVA significantly reduces.With the comparison of model group mice, the mice dopamine that positive drug group gives selegiline obviously increases.The sick mouse striaturn DOPAMINE CONTENT IN RABBIT of handkerchief gold through Chinonin treatment obviously improves.Result shows that Chinonin has improved the content of dopamine, has suppressed it simultaneously and has decomposed, and this is conducive to the storage of brain dopamine.Described Chinonin has good improvement effect (as shown in Figure 4) to parkinsonian mouse striatal dopamine and metabolite thereof.
4. test kit detects striatum oxidative damage
4.1 sample treatment
In treatment model, get 6 mices for every group, after the chloral hydrate anesthesia that mass fraction is 10%, normal saline rinses blood from heart perfusion, gets brain and peels off bilateral striatum, and every operation all carries out on ice bath, and weighs.Mouse striaturn adds 0.5mL PBS homogenate, 12000r/min, and 4 ℃ of centrifugal 10min, get supernatant, and-20 ℃ store for future use.
4.2 detection method
MDA detects by TBA method, and SOD is by colorimetric determination, and GSH utilizes DTNB circular response to detect, and every operation builds up the description of related kit with reference to Nanjing.
4.3 experimental result
With the comparison of blank group, parkinsonian mouse SOD, GSH are active significantly to decline, and MDA level value increases.Positive drug group SOD level value rises, and GSH level rises, and MDA level value declines, and has significant difference with model group.In the sick mouse striaturn of handkerchief gold of Chinonin treatment, SOD is active improves, and GSH level improves, and therefore organizes MDA level to reduce.Results suggest Chinonin can be removed free radical by increasing GSH level, effectively improve Mice Body inner lipid peroxide level, thereby reduce oxidative stress effect, has good antioxidant effect and reaches the effect of neuroprotective, and result is as Fig. 5.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (2)
1. Chinonin is treated the purposes in parkinson disease medicine in preparation.
2. purposes according to claim 1, is characterized in that: described medicine contains mass percent and is greater than pharmaceutically acceptable carrier or the excipient that 99% Chinonin and mass percent are less than 1%.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101919839A (en) * | 2009-06-12 | 2010-12-22 | 海南德泽药物研究有限公司 | Usage of mangiferin calcium salt taken as AMPK agonist |
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| CN101919839A (en) * | 2009-06-12 | 2010-12-22 | 海南德泽药物研究有限公司 | Usage of mangiferin calcium salt taken as AMPK agonist |
Non-Patent Citations (2)
| Title |
|---|
| LA¨ILA AMAZZAL ,ET AL.: "Mangiferin protects against 1-methyl-4-phenylpyridinium toxicity mediated by oxidative stress in N2A cells", 《NEUROSCIENCE LETTERS》 * |
| 任晓光等: "芒果苷药理活性研究进展", 《中成药》 * |
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Application publication date: 20140903 |