CN105902535A - Application of baicalein to preparation of medicament for preventing and treating Parkinson's disease - Google Patents

Abstract

The invention discloses application of baicalein(chemical name:5,6,7-trihydroxy flavone;English name: baicalein) shown as a general formula (I) to preparation of a medicament for preventing and treating Parkinson's disease. The baicalein can lower the tremorfrequency and amplitude of the Parkinson's disease, and improve and treat Parkinson's disease symptoms; the baicalein can protect dopaminergic neurons from being injured, and can effectively prevent and treat pathological change of the Parkinson's disease and the development of the Parkinson's disease; through the anti-apoptosis, anti-inflammation and anti-oxidation functions of the baicalein, the effect of protecting thedopaminergic neurons and the effect of restraining the symptoms of the Parkinson's disease are achieved. The invention relates to separate use of the baicalein to preparation of the medicament for preventing and treating the Parkinson's disease and preparation of the baicalein into various preparations in combination with other substances. The general formula (I) is show in the description.

Description

Baicalin application in preparation prevention and treatment anti-parkinson drug

Technical field

The present invention relates to baicalin for preparing prevention or treatment parkinson disease or being characterized with parkinson disease related symptoms The medicine of disease.Further relate to baicalin and prevent and treat Parkinsonian medicine with flavone compound and other combinations of substances for preparing Thing preparation, belongs to pharmaceutical field,

Background technology

Parkinson disease (PD) have become as at present in the world after Alzheimer the second largest common age be correlated with Neurodegenerative diseases.The sickness rate of more than the 60 years old old man in the whole world is 1%, and increases and sickness rate increase with the age.At me State, there are about ten thousand Parkinsonian more than 170 in more than 55 years old old people.Understanding to PD major pathologic features is that black substance causes at present Compact part dopaminergic neuron Progressive symmetric erythrokeratodermia disappearance and death, make nigro-striatal path dopamine (DA) release reduce, cause stricture of vagina Shape body DA content significantly reduces.Clinically to tremble, myotonia, bradykinesia, posture gait disorder be characterized.

The most unclear to the pathogenesis of PD, the most also do not have to reverse, stop or slow down patient PD The medicine of substantia nigra dopaminergic neuron degenerative process.Traditional PD medicine mainly has: levodopa and Carbidopa thereof Preparation, dopamine-receptor stimulant, anticholinergic agents, oxidase inhibitor etc..These medicines are sent out by different approaches Wave the effect of suppression parkinson patient symptom, it is possible to be obviously improved Parkinsonian symptom, but the process of disease can not be delayed, no Can the regression of prevention of dopaminergic neurone, and prolonged application has serious toxic and side effects, even results in the deterioration of disease.According to Medicinal application situation analysis at present is visible, either improves symptom or suppression PD, and prevention disease occurs or treatment The newtype drug of parkinson disease disease is all the demand of clinical treatment.

Baicalin (baicalein) is Labiatae (Labiatase) plant Radix Scutellariae (Scutelaria baicalensis Georgi) flavone compound contained in dry root, has the structure of formula (I).

Baicalin has multiple pharmacological effect, and the pharmacological action of existing document report includes: antibacterial, antiviral, antiinflammatory, anti- Allergy, antioxidation, scavenging activated oxygen, antitumor, anticoagulant, antithrombus formation and the liver protecting, cardiovascular and cerebrovascular vessel and nerve The effects such as unit.

" the extraction of baicalin of China Medicine University's invention has been recorded in Chinese patent CN 1556108A (publication number) Technique, Pharmaceutical composition and preparation process thereof "^[1].Wherein, relate to baicalin and pharmaceutical composition thereof and treat various diseases Poison infectious disease such as hepatitis, viral influenza, virus infect the application in the acute respiratory syndrome caused.

In Chinese patent CN 1608619A (publication number), record car celebrate the " medicament composing prescription of baicalin of bright invention ”^[2].Wherein, relate to baicalin and pharmaceutically acceptable salt class thereof with in, western drug coordinate prescription, for preparing medicine New application, specifically includes for preparing antipyretic-antalgic anti-inflammatory agent thing；For preparing antibacterial, antiviral drugs；For preparing double controlling Antipyretic-antalgic antiinflammatory and antibacterial, antiviral drugs.

Chinese patent CN 1606979A (publication number) has been recorded car celebrate bright invention " baicalin is as antipyretic, town Bitterly, antiinflammatory, antibacterial, the effect of antiviral agent "^[3].Wherein, relate to baicalin and pharmaceutically acceptable salt class thereof for making For antipyretic, analgesia, antiinflammatory, antibacterial, antiviral drug.

" the baicalin drop pill of Tianjin changzheng Hospital's invention has been recorded in Chinese patent CN 1939295A (publication number) Agent and preparation method thereof "^[4].Wherein, relate to natural plants Radix Scutellariae by conventional extraction and separation, use new medicinal Adjuvant, is developed into baicalin drop pill, is used for treating anaphylaxis dermatosis.

No.1 People's Hospital, Hangzhou City Zhang Xiping invention has been recorded in Chinese patent CN 1559400A (publication number) " compound method of a kind of baicalin liquid "^[5].Wherein, relate to the compound method of a kind of baicalin liquid, it is removing oxygen freely The aspects such as base, alleviates tissue ischemia reperfusion injury, regulates immunologic function, hepatic cholagogic, infection, antitumor all have certain Effect, is plurality of Chinese injection, oral liquid, capsule, decoction pieces, ball dissipates, cream is red constituent.

" the treatment of the inventions such as Xi'an Communications University Lee Zong Fang has been recorded in Chinese patent CN 1528289A (publication number) Compound rubarb element baicalin injection of acute pancreatitis and preparation method thereof " [6].Wherein, relate to a kind of compound rubarb element The preparation method of baicalin injection, its effect in treatment acute pancreatitis.

Summary of the invention

The present invention to solve the technical problem that the new application in pharmacy of the baicalin shown in the formula of being to provide (I), i.e. Huang The application in preparation prevention and treatment anti-parkinson drug of a kind of reed mentioned in ancient books element.

The present invention prevents, alleviates and/or treats parkinson disease or symptom includes releasing disease symptoms and prevention, delaying disease Feelings development or reverse pathological development process.

The baicalin of invention can effectively alleviate the symptom of parkinson patient；Solution is divided by trembling for principal character Parkinson symptom；Having invented baicalin can protect the pathologic of neurocyte to change, and is conducive to suppressing Parkinsonian development, Promote Parkinsonian reverse or recovery.

Described prevention, alleviate and/or treat parkinson disease or symptom selected from improving Parkinsonian symptom and/or the fortune of trembling Dynamic dysfunction symptom.

Improve frequency that Parkinsonian symptom of trembling mainly trembled by minimizing, reduce the amplitude trembled and realize.

The medicine for preventing, alleviate and/or treat parkinson relevant disease can also be prepared according to baicalin of the present invention.

Described parkinson relevant disease is that neural cell injury, neurodegenerative diseases and/or neurotransmitter metabolism are different The most relevant disease.

Can also prepare for preventing, alleviate and/or treat tremble symptom and relevant disease thereof according to baicalin of the present invention Medicine in application.

Described tremble symptom and relevant disease is frequently characterized with limbs myoelectrical activity.

Present invention also offers a kind of prevention, alleviate and/or treat the pharmaceutical composition of parkinson disease or symptom, containing pre- Anti-or the baicalin of therapeutically effective amount, and optional pharmaceutically acceptable carrier and/or adjuvant.

Baicalin can also prevent and treat parkinson disease with the combination of other compounds for preparation, particularly natural with flavonoid product Prepared by thing combination is used for treating anti-parkinson drug preparation.

Therefore the pharmaceutical composition of the present invention can also contain other chromocor compounds.Preferably chromocor compound is selected from Mongolian oak In Pi Su, rutin, luteolin at least one.

The pharmaceutical composition of the present invention, can be in selected from following dosage form according to pharmaceutical composition described in route of administration: solution, Suspension, Emulsion, pill, tablet, capsule, powder, control release or extended release preparation.

Present invention discover that baicalin has a following pharmacological action:

1. baicalin can prevent or treat parkinson disease.

By baicalin has carried out series of experiments, the present invention proves that it is prevention and treats Parkinsonian effective medicine By setting up the PD rat model of 6-hydroxyl DOPA (6-OHDA) damage Brain Function of Rats, thing, determines that PD animal model is transported by baicalin The improvement result of the behavioral indexes such as dynamic obstacle, in the C57BL mouse Parkinson disease model of MPTP induction, it was demonstrated that baicalin The spontaneous activity in mice caused by MPTP can be significantly improved decline, shorten the mice pole-climbing time lengthening caused by MPTP.

2. baicalin can treat Parkinsonian symptom of trembling.

In the rat model of 6-OHDA damage, baicalin can obviously improve rat muscle caused by 6-OHDA and trembles performance, Reduce the frequency and amplitude trembled, and prove that this protective effect mainly improves the maincenter pathology shape of animal model for parkinsonism The result of state, with peripheral nervous conduction without direct relation, is baicalin to one of important feature for the treatment of of Parkinson disease.

3. baicalin can regulate animal model for parkinsonism brain neurotransmitter.

Baicalin can change the monoamine neurotransmitter in the regions such as Parkinson disease model animal brain striatum, aminoacid The metabolic process of isoreactivity composition and concentration, can improve striatal dopamine, 4-hydroxy-3-methoxy-.alpha.-toluic acid. and the level of 5-hydroxyl color ammonia, increase Substantia nigra dopaminergic neuron number and DAT number, inhibited oxidation with inflammation, thus stress play preventing and treating handkerchief gold The effect of gloomy disease.

4. baicalin can protect neurocyte.

The 6-OHDA that alleviates that the present invention relates to baicalin energy concentration dependent causes the vigor decline of SH-SY5Y cell, alleviates 6-OHDA causes the apoptosis of SH-SY5Y cell, prompting baicalin prevention and treat Parkinsonian effect and its anti-apoptotic, antiinflammatory with Antioxidation is relevant.

Present invention also offers baicalin as active ingredient and customary pharmaceutical excipients or the pharmaceutical composition of adjuvant.

The pharmaceutical composition of the compounds of this invention can be prepared according to method well known in the art, time for this purpose, if Needing, can the compounds of this invention and one or more solids or liquid pharmaceutical excipients and/or adjuvant be combined, making can conduct Suitable administration form that people's medicine or veterinary drug use or dosage form.

The such as preparation of baicalin tablet can use various carrier well known in the art.Example about carrier is, example Such as diluent and absorbent, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, carbamide, carbonic acid Calcium, kaolin, microcrystalline Cellulose, aluminium silicate etc.；Wetting agent and binding agent, such as water, glycerol, Polyethylene Glycol, ethanol, propanol, shallow lake Slurry, dextrin, syrup, Mel, glucose solution, mucialga of arabic gummy, gelatine size, sodium carboxymethyl cellulose, lac, Methyl cellulose Element, potassium phosphate, polyvinylpyrrolidone etc.；Disintegrating agent, such as, be dried starch, alginate, agar powder, laminaran, carbonic acid Hydrogen sodium and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, dodecyl sodium sulfate, methylcellulose, ethyl fibre Dimension element etc.；Disintegrate inhibitor, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil and fat etc.；Absorption enhancer, such as quaternary ammonium Salt, sodium lauryl sulphate etc.；Lubricant, such as Pulvis Talci, silicon dioxide, corn starch, stearate, boric acid, liquid stone Wax, Polyethylene Glycol etc..Tablet can also be made coated tablet, such as sugar coated tablet, thin membrane coated tablet, enteric coating further Sheet, or double-layer tablet and multilayer tablet.

The preparation of baicalin pill can use various carrier well known in the art, and the example about carrier is, the dilutest Release agent and absorbent, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Kaolin, Talcum Powder etc.；Binding agent, such as arabic gum, Tragacanth, gelatin, ethanol, Mel, liquid sugar, rice paste or batter etc.；Disintegrating agent, such as agar Powder, dry starch, alginate, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc..

Baicalin can be mixed by the preparation of baicalin capsule with above-mentioned various carriers, and is put by thus obtained mixture In hard gelatine capsule or soft capsule.Also effective ingredient the compounds of this invention can be made microcapsule, be suspended in aqueous medium Middle formation suspensoid, also can load in hard capsule or make injection application.

The preparation of baicalin injection preparation, such as solution, suspensoid solution, Emulsion, lyophilized injectable powder, this preparation Can be aqueous or non-aqueous, acceptable carrier, diluent, binding agent, lubrication on a kind of and/or multiple pharmacodynamics can be contained Agent, preservative, surfactant or dispersant.As diluent is selected from water, ethanol, Polyethylene Glycol, 1,3-PD, ethyoxyl The isooctadecanol changed, polyoxygenated isooctadecanol, Polyoxyethylene Sorbitol Fatty Acid Esters etc..It addition, in order to prepare isotonic injection Liquid, can add appropriate sodium chloride, glucose or glycerol in injection preparation, further, it is also possible to add conventional hydrotropy Agent, buffer agent, pH adjusting agent etc..These adjuvants are commonly used in the art

Additionally, if desired, can also add in pharmaceutical preparation coloring agent, preservative, spice, correctives, sweeting agent or Other material.

Form of administration can be liquid dosage form, solid dosage forms.If liquid dosage form can be true solution class, colloidal type, microgranule Dosage form, emulsion dosage form, mixed suspension form.Other dosage forms such as tablet, capsule, drop pill, aerosol, pill, powder, solution, mixed Suspension, Emulsion, granule, suppository, lyophilized injectable powder etc..

The compounds of this invention can be made ordinary preparation, can also be slow releasing preparation, controlled release preparation, targeting preparation and various Particulate delivery system.

Present invention also offers by preventing with baicalin or its pharmaceutical composition, alleviate and/or treat parkinson Disease or the method for symptom.

For reaching medication purpose, strengthening therapeutic effect, the medicine of the present invention or pharmaceutical composition can be given known to any Prescription method is administered.

The compounds of this invention or the pharmaceutical composition containing it can be administered in a unit, and route of administration can be intestinal Or non-bowel, such as oral, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritoneum or rectum etc..

The compounds of this invention or the route of administration containing its pharmaceutical composition can be drug administration by injection.Injection includes that vein is noted Penetrate, intramuscular injection, subcutaneous injection, intradermal injection and acupoint injection therapy etc..

The dosage of the compounds of this invention pharmaceutical composition depends on many factors, such as, to prevent or treat disease Character and the sex of the order of severity, patient or animal, age, body weight, personality and individual reaction, route of administration, administration number of times, Therapeutic purposes, therefore the therapeutic dose of the present invention can have change on a large scale.In general, the use of Pharmaceutical Compositions of the present invention Dosage is to well known to a person skilled in the art.Can be according to reality contained in preparation last in the compounds of this invention compositions Border medication amount, in addition suitable adjustment, to reach the requirement of its therapeutically effective amount, complete prevention or the treatment mesh of the present invention 's.

The required accumulated dose of each treatment is divided into repeatedly or is administered by dose.The compound of the present invention or compositions Can individually take, or merge use with other treatment medicine and adjust dosage.

Accompanying drawing explanation

Fig. 1. baicalin causes the impact of parkinsonian mouse striatum monoamine transmitters to MPTP^#P<0.05,^##P<0.01vs MPTP model group, * P < 0.05, * * P < 0.01vs Normal group.

Fig. 2. baicalin causes parkinsonian mouse substantia nigra TH, the impact of DAT, GFAP to MPTP.

^#P<0.05,^##P < 0.01vs MPTP model group, * P < 0.05, * * P < 0.01vs Normal group.

Fig. 3. baicalin side inclined on 6-OHDA injury rats trembles the impact of activity.

^#P<0.05,^##P < 0.01vs 6-OHDA model group, * P < 0.05, * * P < 0.01vs sham operated rats.

Fig. 4. the impact of baicalin side inclined on 6-OHDA injury rats Conduction Velocity of Peripheral Nerves.

^#P < 0.05vs 6-OHDA model group, * P < 0.05vs sham operated rats.

Fig. 5. baicalin side inclined to 6-OHDA injury rats substantia nigra TH, the impact of GFAP.

^#P<0.05,^##P < 0.01vs 6-OHDA model group, * P < 0.05, * * P < 0.01vs sham operated rats.

Fig. 6. baicalin causes the impact of SH-SY5Y cell injury survival rate to 6-OHDA

^#P < 0.05vs 6-OHDA, * P < 0.05vs blank group.

Fig. 7. nuclei dyeing color method detection baicalin causes impact (a: the blank group of SH-SY5Y natural death of cerebral cells to 6-OHDA；b: 6-OHDA group；C: baicalin low dose group；D: dosage group in baicalin；E: baicalin high dose group)

Fig. 8. flow cytomery baicalin causes the impact of SH-SY5Y natural death of cerebral cells to 6-OHDA.

^#P < 0.05vs 6-OHDA, * P < 0.05, * * P < 0.01vs blank group.

Term and abbreviation

MPTP:1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine

6-OHDA:6-hydroxy dopamine

Detailed description of the invention

Following embodiment is particularly shown the application of the present invention.But the present embodiment does not limit the range of the present invention.

Embodiment 1, the baicalin improvement result to MPTP induction PD mice behavior

Model is set up and packet

C57BL/6 mice is randomly divided into 3 groups, respectively Normal group, MPTP model group, baicalin administration group (200mg/kg), often 12 are organized.Matched group and model group give to fill to feed normal saline in advance, and administration group gives the medicine of above-mentioned dosage Thing, continuous gastric infusion is after one week, in the 8th day model group and administration group lumbar injection MPTP (30mg/kg), once a day, even Continuous 5 days, within the 13rd day, observe the behavioral indexes detection of mice.

Behavioristics is detected

1. spontaneous activity experiment uses mice autonomic activities instrument (ZIL-2 type mice autonomic activities instrument, the Chinese Academy of Medical Sciences Institute of materia medica) measure spontaneous activity in mice counting, mice is put in autonomic activities case (high 13cm, diameter 25cm, every time Measure 4 mices simultaneously, in each active box 1), monitor automatically record mice movable, before measuring every mice first from By movable 3 minutes, then measure the movable number of times in every mice 5min.

2. the cork bead of an a diameter of 2.5cm is fixed on the rod top of a root length 50cm diameter 1cm by Grasping clubglass test, Gauze it is wrapped in anti-slip, as experiment pole-climbing on rod.During experiment, mice is guided every day to climb at the bottom of bar from masthead, every day 2 Secondary.During test, tested mice is placed on cork bead, records the time required for mice climbs at the bottom of bar the credit that takes statistics analysis, More than 60s in terms of 60s.The most first measure all mice pole-climbing time, and 1h after last lumbar injection MPTP, again Measuring its pole-climbing time, every mice is surveyed and averages for 3 times.The difference of pole-climbing time before being administered is deducted with the last pole-climbing time, The limbs coordination ability is caused to decline the index of degree as measurement mice because of MPTP.

MPTP can significantly reduce the Assay of spontaneous activity of mice and extend the mice pole-climbing time, and baicalin can significantly change Kind spontaneous activity in mice caused by MPTP declines, and shortens the prolongation of mice pole-climbing time, and this display baicalin can be obviously improved PD The limbs coordination ability of model mice, and increase the motion (table 1,2) of mice.

The impact of the PD spontaneous activity in mice that MPTP is induced by table 1 baicalin

Note:^#P < 0.05vs.MPTP model group, * P < 0.05vs. Normal group N=12. (mean ± S.D)

The impact of the PD mice pole-climbing time that MPTP is induced by table 2 baicalin

Note:^#P < 0.05vs.MPTP model group, * P < 0.05vs. Normal group N=12. (mean ± S.D)

Embodiment 2, baicalin are on the impact of monoamine transmitters in MPTP induction PD mouse brain striatum

Model is set up with packet ibid.Often group takes 6 mices and draws materials, and broken end is opened cranium and taken brain, and ice pan separates both sides Black substance and striatum, frozen in liquid nitrogen.Take side striatum and carry out the mensuration of neurotransmitter.Take striatum weigh, record, ice bath In in 0.2ml homogenate be homogenized 40s, 80% ultrasonic energy, cycle=0.7,4 DEG C of 12000rpm are centrifuged 30min, take supernatant The liquid method of high-efficient liquid phase electrochemical detects dopamine (DA) and metabolite dihydroxyphenyl acetic acid (DOPAC), 4-hydroxy-3-methoxy-.alpha.-toluic acid. (HVA), 5-hydroxy tryptamine (5-HT) and the level of metabolite 5-hydroxyindoleacetic acid (5-HIAA) thereof, each sample introduction 20 μ l.Flowing Phase: 0.1mol/l NaH₂PO₄Aqueous solution contains 0.85mmol/l OSA, 0.5mmol/l EDTANa₂With 11% methanol, use strong phosphoric acid Adjust pH to 3.25,0.45 μm microporous filter membrane sucking filtration degassing；Fixing phase: Hypersil C185 μm fills stainless steel column.Running voltage + 0.75mV, column temperature 35 DEG C, flow velocity 0.8ml/min.

Compared with Normal group, mice continuous lumbar injection MPTP is after 5 days, the DA in striatum and metabolite thereof DOPAC, HVA content is all remarkably decreased.After baicalin pretreatment, DA and HVA content all dramatically increases, but the content of DOPAC Without significant change (Figure 1A).The 5-HT content that MPTP may also lead in striatum significantly declines, but does not has the content of 5-HIAA There is significant impact.After baicalin pretreatment, in striatum, the content of 5-HT can dramatically increase (Figure 1B).

Embodiment 3, baicalin are to MPTP induction PD mouse brain substantia nigra tyrosine hydroxylase (TH), DAT (DAT) impact that, glial cell line-derived fibrin (GFAP) is expressed

Model is set up with packet ibid.Often group takes 6 row SABC detections.Fix by 4% chloral hydrate anesthesia, open Thoracic cavity, fixing with normal saline and the perfusion of 4% paraformaldehyde respectively, open cranium and take brain.1 day is fixed after being placed in 4% paraformaldehyde, Change to again in the 0.01M PBS containing 30% sucrose.20 μ m-thick coronal planes are cut with freezing microtome after cerebral tissue sinks Brain sheet.PBST (PH=7.4) the vibration rinsing 3 times of brain sheet, each 10min；Again with 3% H₂O₂Process 10min, PBST vibration Rinse 3 times, each 10min；1h is hatched with confining liquid；Add anti-(TH, a 1:500；DAT, 1:200；GFAP, 1:400), 4 DEG C Overnight incubation.PBST vibration rinsing 3 times, after each 10min, respectively with biotin labeled two anti-(1:300) incubated at room 1h； PBST vibration rinsing 3 times, after each 10min, with avidin-biotin composite (ABC) incubated at room 2h；PBST vibration again Rinse 3 times, each 10min.DAB develops the color, Microscopic observation, terminates reaction with PBS in good time.SABC matched group is not added with one and resists, Replacing with PBS, remaining step is the same.Paster, dries, Gradient elution using ethanol, and dimethylbenzene is transparent, and neutral gum mounting, under light microscopic Observe and take the photograph sheet.SABC section uses IPP5.1 image analysis system to carry out graphical analysis, and all sections all use same Amplification (× 200).Every mice chooses 3 sections, respectively accumulative left side and the immunoreation positive neurons of right substantia nigra Unit's number also calculates its mean.

After MPTP abdominal cavity injects 5 days continuously, the number of black substance TH immuning positive neuron and DAT immuning positive neuron shows Write and decline, give baicalin in advance and TH positive neuron number and DAT positive neuron number can be made respectively to have a certain degree of Increase, illustrate that baicalin has certain neuroprotective.It addition, MPTP can make GFAP immuno positive star glue in black substance Cell plastid number dramatically increases, and baicalin can significantly reduce the number of GFAP immuno positive astrocyte, and Radix Scutellariae is described Element can suppress inflammatory reaction (Fig. 2) by suppression glial cell reactive hyperplasia.

Embodiment 4, baicalin are to MPTP induction PD mouse brain superoxide dismutase (SOD), glutathione peroxidase Enzyme (GSH-Px) activity and the impact of malonaldehyde (MDA) content

Model is set up with packet ibid.Mus broken end opens cranium, takes cerebral tissue specimen, blots the residual blood in surface with filter paper, and place to go is smelt Ball, brain stem, cerebellum.Cerebral tissue specimen is respectively placed in glass homogenizer, with ice normal saline by the ratio that quality and volume ratio are 1:9 It is prepared as the brain tissue homogenate of 10% under example ice bath, is immediately placed in-40 DEG C of Refrigerator stores to be measured.With Coomassie Brilliant Blue before experiment Measure cerebral tissue protein quantification.Bioengineering Research Institute's test kit description requirement is built up, with Spectra Max M5 by Nanjing Microplate reader measures SOD, GSH-Px activity and MDA content in cerebral tissue respectively.

MPTP lumbar injection may result in MDA level in cerebral tissue for 5 days and significantly raises, and GSH-Px level significantly reduces, and SOD Content has no significant change, and baicalin can significantly reduce MDA level in cerebral tissue, increases SOD activity and GSH-Px level (table 3), obvious antioxidant activity is shown.

PD Mice brain tissues SOD, GSH-Px that MPTP is induced by table 3 baicalin activity and the impact of MDA level

Note:^#P < 0.05vs.MPTP model group,^*P < 0.05vs. Normal group, N=6. (mean ± S.D)

Embodiment 5, baicalin side inclined to 6-OHDA injury rats trembles the improvement result of activity

Prepared by model

Healthy male SD rat is randomly divided into 2 groups, respectively sham operated rats and model group.3% pentobarbital sodium (50mg/ Kg) intraperitoneal injection of anesthesia, after conventional transdermal sterilization, is fixed on rat head on stereotaxic instrument, exposes reference after skull Paxinos and Watson Mus Naoliqing capsule collection of illustrative plates, is equivalent to the skull surface boring in MFB district, diameter about 2.5cm in left side. After cleaning meninges, at the descending MFB of guiding 2 injection 6-OHDA (4mg/ml) of three-dimensional pusher.Coordinate is as follows: (1) TB:- 2.3mm,AP:-4.4mm,ML:1.2mm,V:-7.8mm；(2)TB:+3.4mm,AP:-4.0mm,ML:0.8mm,V:-8.0mm.Two The amount of some injection 6-OHDA is 3 μ l, and injection speed controls at 1 μ l/min, slowly goes out pin after after per injection, let the acupuncture needle remain at a certain point 10min. Postoperative continuous 3d intramuscular injection penicillin 200,000 U/d is in case infecting.The normal saline of rats in sham-operated group injection equivalent.In the postoperative 14th The circling behavior that it test apomorphine (APO, subcutaneous injection, 0.2mg/kg) is induced.After APO injection, animal is to injection 6- The offside of OHDA rotates, often motion 360 °, as once rotating.Choose the rat of rotating speed >=240r/40min as one-tenth Merit PD rat model.Success PD rat model is randomly divided into 3 groups, often group 12, respectively model group, baicalin group (baicalein, 200mg/kg), positive drug madopar group (madopar, 50mg/kg).Additionally take sham operated rats 12 be only used as right According to, give normal saline every day once, within the 3rd week after modeling, carry out rat and tremble the mensuration of activity.

Tremble the detection of activity

Double recording electrode is inserted on rat buttocks muscles, reference electrode ground connection.Use BL-420E biological signal collecting device Activity of trembling Conscious Rat hind leg measures, and evaluates its tremble frequency and tremor amplitude.Stimulus signal selects 1 millivolt (mv), 0.015 millisecond (ms), scanning speed is 0.01 second (s), and 10kHz filters.Respectively record 3 in 1 minute rat tremble Frequency and amplitude, average.

Test result shows, compared with sham operated rats, 6-OHDA damage group tremble frequency and tremor amplitude have have aobvious The increase write；Compared with model group, baicalin and madopar can obviously improve the shake of rat model of Parkinson disease caused by 6-OHDA Quiver activity, reduce frequency of trembling, reduce tremor amplitude (Fig. 3).

The impact of embodiment 6, baicalin side inclined on 6-OHDA injury rats Conduction Velocity of Peripheral Nerves

Model preparation is with packet ibid.Rat is through pentobarbital sodium intraperitoneal anesthesia, prostrate fixing.Stimulating electrode is crosspointer electricity Pole, inserts at sciatic nerve nest near sciatic nerve-trunk, and leading electrode is crosspointer electrode, inserts gastrocnemius belly of muscle, two electrode phases Away from about 2cm.Equipment uses BL-420E biological signal collecting device, and stimulus signal selects 0.6 millivolt (mv), 0.02 millisecond (ms), list Stimulating, scanning speed is 0.01 second (s), and 1kHz filters, and guides signal to amplify 50 times, and stimulation per minute once, stimulates six times altogether. Calculating neuromuscular action potential incubation period, conduction velocity computing formula is:

V=stimulating electrode is to distance (the mm)/incubation period (ms) of leading electrode.

Experimental result shows, 6-OHDA model group rats Conduction Velocity of Peripheral Nerves relatively sham operated rats substantially speeds, Radix Scutellariae Element is to Conduction Velocity of Peripheral Nerves without obvious inhibitory action (Fig. 4), and this prompting baicalin is to 6-OHDA damage rat model Protective effect is conducted without direct relation with peripheral nervous.

Embodiment 7, baicalin side inclined to 6-OHDA injury rats substantia nigra tyrosine hydroxylase (TH), glial cell line-derived fiber The impact that albumen (GFAP) is expressed

Model is set up with packet ibid.Often group takes 6 row SABC detections.Fix by 4% chloral hydrate anesthesia, open Thoracic cavity, fixing with normal saline and the perfusion of 4% paraformaldehyde respectively, open cranium and take brain.1 day is fixed after being placed in 4% paraformaldehyde, Change to again in the 0.01M PBS containing 30% sucrose.20 μ m-thick coronal planes are cut with freezing microtome after cerebral tissue sinks Brain sheet.PBST (PH=7.4) the vibration rinsing 3 times of brain sheet, each 10min；Again with 3% H₂O₂Process 10min, PBST vibration Rinse 3 times, each 10min；1h is hatched with confining liquid；Add anti-(TH, a 1:500；GFAP, 1:400), 4 DEG C of overnight incubation. PBST vibration rinsing 3 times, after each 10min, respectively with biotin labeled two anti-(1:300) incubated at room 1h；PBST vibrates Rinse 3 times, after each 10min, with avidin-biotin composite (ABC) incubated at room 2h；PBST vibration rinsing 3 times again, 10min every time.DAB develops the color, Microscopic observation, terminates reaction with PBS in good time.SABC matched group is not added with one and resists, and uses PBS generation Replacing, remaining step is the same.Paster, dries, Gradient elution using ethanol, and dimethylbenzene is transparent, neutral gum mounting, and light Microscopic observation is also taken the photograph Sheet.SABC section uses IPP5.1 image analysis system to carry out graphical analysis, and all sections all use same amplification (×200).Every rat chooses 3 sections, and the immunoreactive positive neurons number of accumulative left side and right substantia nigra is also respectively Calculate its mean.

After 6-OHDA brain locating injection, black substance TH immuning positive neuron number is remarkably decreased, and giving baicalin in advance can Make TH positive neuron number have a certain degree of increase, illustrate that baicalin has certain neuroprotective.It addition, 6- In OHDA can make black substance, GFAP immuno positive astrocyte number dramatically increases, and baicalin can significantly reduce GFAP immunity The number of astrocyte, illustrates that baicalin can suppress inflammatory reaction by suppression glial cell reactive hyperplasia (Fig. 5).

The impact on 6-OHDA damaging cells vigor of embodiment 8, baicalin

The cultivation of cell and process

SH-SY5Y cell with complete RMPI 1640 culture medium (containing 10% hyclone, 100U/ml penicillin, 100 μ g/ Ml streptomycin, 1% glutamine), in 37 DEG C, cultivate under the conditions of 5%CO2 saturated humidity.Often within 2-3 days, change a not good liquor.Take the logarithm The cell of trophophase, adjusts cell concentration for 1 × 10 with complete medium⁵Individual/ml, is seeded to 96 well culture plates, every hole 200 μ L, cultivates 24h to subconfluence.Use serum-free medium instead, be simultaneously introduced baicalin (final concentration 5,0.5,0.05 μ g/ml) After hatching 1h, add 6-OHDA (final concentration 100 μMs), be measured after 24h.

The mensuration of cell viability

Add MTT 200 μ l, the final concentration of 0.5mg/ml of serum-free medium preparation, after 37 DEG C are continued to cultivate 4h, move Walk MTT, add dimethyl sulfoxide 200 μ l and dissolve the crystal of cell dyeing, at 570nm, read absorption value.Thin with Normal group Born of the same parents' survival rate is 100%, calculates different disposal group cell survival rate.

MTT testing result shows, 100 μMs of 6-OHDA effect SH-SY5Y cell 24h may result in cell viability and are remarkably decreased, And giving the survival ability that baicalin (0.5,5 μ g/ml) is remarkably improved cell in advance, result represents (figure with cell survival rate 6)。

6-OHDA damaging cells is adjusted morphologic impact of dying by embodiment 9, baicalin

The cultivation of cell is with process ibid.After medicine effect terminates, exhaust culture fluid, wash one time with PBS.Add 200 μ l Fixative, fixing 10min.Discard fixative, add the PBS liquid containing Hoechest 33258 (final concentration 10 μ g/ml), be placed in thin Born of the same parents' incubator hatches 5min, in fluorescence microscopy Microscopic observation, with ultraviolet excitation, takes pictures simultaneously.

Hoechest coloration result shows, more consistent sized by the nucleus majority of the normal cellular control unit cultivated, dye The uniform circular kernel of color, accidental pyknosis dense dye core；And process the cell not only cell number minimizing of 12h through 6-OHDA, and occur withering The nucleus died increases, chromatin generation pyknosis, the fine and close dense dye of nucleus, in irregular shape, in granule, little bulk.Give Huang A kind of reed mentioned in ancient books element can significantly improve the change (Fig. 7) of the relevant morphocytology of above-mentioned apoptosis.

6-OHDA damaging cells is adjusted the impact of rate of dying by embodiment 10, baicalin

Medicine effect collects cell after terminating, and washes twice with PBS.The cell precipitation collected adds 70% ethanol and mixes, 4 DEG C Fixing overnight, 1000rpm is centrifuged 5min, discards fixative.3ml PBS resuspended precipitation 5min, filters with 400 mesh steel wires, 4 DEG C, 1500rpm is centrifuged 10min, abandons supernatant.In precipitation, add 1ml PBS, add PI (final concentration 100 μ g/ml).4 DEG C of lucifuges 30min, with 10000 cells of flow cytomery, excitation wavelength is 488nm, a length of 630nm of transmitted wave, and calculates apoptosis Percentage rate.

Flow cytomery apoptosis rate result shows, apoptosis rate is less than 5% under normal circumstances, and 100 μMs 6-OHDA act on SH-SY5Y cell 12h and can substantially increase apoptosis rate, give 0.5, the baicalin of 5 μ g/ml can be notable Inhibited apoptosis (Fig. 8).

Claims (7)

1. the baicalin as shown in logical formula (I) is used for by zoopery 200mg/kg corresponding dosage and cell experiment 0.5 μ in preparation The prevention that uses under g/ml or 5 μ g/ml respective concentration, alleviate and/or treat in the medicine of Parkinsonian symptom of trembling should With

Application the most according to claim 1, it is characterised in that described in the tremble improvement of symptom be to reduce to tremble under given dose Frequency, reduce the amplitude trembled.

Application the most according to claim 1, it is characterised in that described symptom of trembling is frequently to be characterized with limbs myoelectrical activity , improve cell survival rate, inhibited apoptosis.

4. a pharmaceutical composition is in preparing the medicine for preventing, alleviate and/or treat Parkinsonian symptom of trembling Application, it is characterised in that described pharmaceutical composition contains prevention or the baicalin of therapeutically effective amount, and optional pharmacy can connect The carrier being subject to and/or adjuvant.

Application the most according to claim 4, it is characterised in that described pharmaceutical composition is possibly together with other chromocor compounds.

Application the most according to claim 5, it is characterised in that described chromocor compound is selected from Quercetin, rutin, luteolin In at least one.

Application the most according to claim 4, it is characterised in that can be in selected from following according to pharmaceutical composition described in route of administration Dosage form: solution, suspension, Emulsion, pill, tablet, capsule, powder, control release or extended release preparation.