CN101313912A - Application of echinacoside in preparing medicament for treating parkinsonism - Google Patents

Abstract

The invention relates to an application of echinacoside in the preparation of drugs for treating Parkinson's disease. The invention discloses that echinacoside has a remarkable protective effect against the MMP+ mediated cell injury. The echinacoside can be used to prepare drugs for the treating Parkinson's disease and offers a new treatment for people with the Parkinson's disease.

Description

The application of echinacoside in the Parkinsonian medicine of preparation treatment

Technical field

The present invention relates to the purposes of echinacoside, relate in particular to new purposes at pharmaceutical field.

Background technology

Herba Cistanches (Hera CistaneMs) is the kind commonly used in the traditional Chinese medical science tonification medicine, belong to the Orobanchaceae Cistanche Hoffmgg. et Link plants, pharmaceutical products is the chylocaulous of its dry zone scale leaf, have another name called greatly rue, smart Radix Crotalariae szemoensis, vertical Rong, dried meat floss Rong, Herba Stellariae Saxatilis, beginning is stated from Shennong's Herbal, classify " top grade " as, have the effect of the kidney invigorating and essence nourishing, loosening bowel to relieve constipation, cure mainly cards such as impotence in male, infertilitas feminis.

Echinacoside is a kind of chemical compound of separation, purification from Cistanche Tubulosa (Cistanche tubulosa).The echinacoside that studies show that in the past can improve the content of multiple central neurotransmitter, has very strong antioxidation, also can prolong animal life-span and anti-aging effects.Be used for the treatment of defying age or alzheimer disease at present.

Parkinson disease (PD) are middle-aged and elderly people nervous system common diseases.Its characteristic pathological change is that the degeneration of substantia nigra of midbrain dopaminergic neuron reduces, and causes the interior dopamine concentration of striatum obviously to descend, the main dopamine alternative medicine that relies on of treatment at present.Although the neuroscientist takes to comprise the various Therapeutic Method of Dopar, the progress that can't stop the dopaminergic neuron degeneration to reduce is all pointed out in all at present research.

Summary of the invention

The object of the present invention is to provide the application of echinacoside in the Parkinsonian medicine of preparation treatment.

Echinacoside is a kind of chemical compound that extracts from Herba Cistanches, and its known chemical structural formula is:

The invention provides the application of echinacoside in preparation treatment parkinson disease medicine.

Above-mentioned echinacoside can be made oral solutions, tablet, pill, electuary, capsule, injection, drop pill, syrup, unguentum with correctives, solvent or adjuvant in preparation treatment parkinson disease medicine.

Owing to adopted above-mentioned technical solution, echinacoside can be used for preparing the Parkinsonian medicine of treatment, brought new hope for Parkinsonian's treatment.

Description of drawings

Fig. 1 echinacoside is to the protective effect of the parkinson disease cell model of MPP+ mediation;

The expression electrophoretogram of the mRNA of Fig. 2 GADD153 in different groups;

Fig. 3 echinacoside is to the influence of the cell injury model GADD153mRNA expression of MPP+ mediation;

The expression electrophoretogram of the albumen of Fig. 4 GADD153 in different groups;

Fig. 5 echinacoside is to the influence of the SH-SY5Y cell GADD153 protein expression of MPP+ mediation.

The specific embodiment

In order to understand essence of the present invention better, will illustrate that it is being used for preparing the new purposes for the treatment of Parkinsonian medicine with pharmacological testing and result below.

Echinacoside can adopt the record preparation method preparation of present disclosed document.As adopting following method preparation:

After the dry meat stem 10kg of Cistanche Tubulosa (place of production is a hotan) pulverized, be 95% alcohol reflux 3 times, 2h at every turn with volume fraction.Extracting solution is merged, be evaporated to paste, add low amounts of water and make suspension.Extract respectively successively 3 times with petroleum ether, acetic acid ethyl ester, water-saturated n-butanol, extract concentrates standby.Get n-butyl alcohol extract 10g, with about 2 times water dissolution it, annotate people D101 macroporous adsorptive resins (the about 1000ml of bed volume) subsequently, earlier with 3000ml deionized water eluting, discard water elution liquid, be 5% methanol aqueous solution eluting again with the volume fraction of 3 times of bed volumes, promptly about 3000ml discards.Adopting body to know mark at last is that 20% methanol aqueous solution is made eluent, with 500ml is a fraction, knowing with the inspection of poly-phthalein amine thin layer chromatography, is that 5% ferric chloride alcoholic solution is made developer with mass fraction, and the fraction of collecting and be associated with impact point carries out concentrating under reduced pressure.Concentrate is through the sephadex lh-20 column chromatography, is 10% methanol aqueous solution isocratic elution with volume fraction.The fraction that will contain higher degree merges, and is evaporated to driedly, obtains 2g sample (the water-bath temperature of all concentrating under reduced pressure all is lower than 60 ℃).Measure peak area ratio with HPLC, the relative amount of SONGGUOJU former times is 87%.Above-mentioned crude product is mixed with the methanol solution of 0.15kg/L, adopts half preparative high performance liquid chromatography purification.Sample size is 120 μ L, and collecting retention time is the component of 13.6min.Its concentrating under reduced pressure (below 60 ℃) is taken out when small amount of liquid is arranged, lyophilization, at last canescence amorphous powder 220mg, this is the standard substance of echinacoside.The known chemical structural formula of echinacoside is:

1, echinacoside is to 1 methyl-4 phenyl-pyridinium ion (1-methyl-4-phenylpyridiniumion, MPP+) the protective effect test of the SH-SY5Y dopaminergic cell of mediation system damage

Reduce because the Parkinsonian cause of disease is a dopaminergic neuron, the SH-SY5Y dopaminergic cell is the Parkinsonian typical cells model of research.(1-methyl-4-phenylpyridinium ion MPP+) makes SH-SY5Y dopaminergic cell cell survival rate with MPP to the 1 methyl-4 phenyl-pyridinium ion that studies show that in the past behind the treatment S H-SY5Y dopaminergic cell ⁺Concentration increase and reduce.(1-methyl-4-phenylpyridinium ion, MPP+) the SH-SY5Y dopaminergic cell of Jie Dao damage is the Parkinsonian cell model of research with 1 methyl-4 phenyl-pyridinium ion of 1mM in this test.

1.1 cell culture and grouping

Per 2～the 3d of SH-SY5Y cell changes liquid 1 time with the 10%DMEM culture fluid.When treating that cell enlargement to 80% merges, use 0.25% trypsin digestion cell, go down to posterity, place the cell culture incubator of 5%CO2 by 1 * 105 fen bottle.Test is divided into matched group, MPP+ group and echinacoside variable concentrations group with cell.With the DMEM culture fluid MPP+ is become 400mM by 1: 10 gradient dilution, add in 96 orifice plates or 6 orifice plates, make the MPP+ final concentration be respectively 0.25mM, 0.5mM, 1mM, 2mM, 4mM by 1: 100 volume ratio.Echinacoside becomes 1mg/ml and two working concentrations of 10ug/ml with the DMEM culture fluid by 1: 10 gradient dilution.Added in 96 orifice plates or 6 orifice plates by 1: 100, make final concentration be respectively 1 μ g/ml, 10 μ g/ml, 100 μ g/ml.The every hole of six orifice plates cumulative volume is 2ml, and the every hole of 96 orifice plates cumulative volume is 200 μ l.Every identical experiment all repeats 3 times.

1.2 detection cytoactive

MTT (tetrazole indigo plant) is a Dutch Duchefa company product.MPP+ is to the influence of SH-SY5Y cell survival rate: 1 * 104 density SH-SY5Y cell 200ul is inoculated in 96 orifice plates, changes behind the 24h to contain the MPP+ final concentration and be respectively 0.25mM, 0.5mM, 1mM, 2mM, the culture fluid of 4mM, every concentration 3 multiple holes, place the 5%CO2 incubator to hatch for 37 ℃, respectively at 6h, 12h, 24h, each hole adds MTT20 μ l during 48h, continues to hatch 4h and takes out culture plate.Culture fluid is abandoned in suction, and every hole adds DMSO 150 μ l, fully shakes 10min, treats that blue particle dissolves the back fully and measures absorbance with full-automatic microplate reader (Biorad product) 570nm place.Echinacoside is to the influence of the SH-SY5Y cell injury of MPP+ mediation: final concentration 1mMMPP+ and echinacoside are respectively 1 μ g/ml, and 10 μ g/ml, 100 μ g/ml add 96 orifice plates simultaneously and put the 5%CO2 incubator and hatch 48h for 37 ℃.Surplus the same.

1.3 result of the test

Fig. 1: echinacoside is to the cell injury model tool protective effect of MPP+ mediation.The SH-SY5Y dopaminergic cell survival rate that the echinacoside of three kinds of concentration is handled all obviously increases (P＜0.01) than the MPP+ group

48 hours survival rates of SH-SY5Y dopaminergic cell that MPP+1mM handles are 45.3 ± 3.21%, 1 μ g/ml, to correspond to 85.97 ± 3.41%, 10 μ g/ml be that 129.97 ± 4.84%, 100 μ g/ml are 164.1 ± 3.91% and echinacoside is handled the cell survival rate of above-mentioned cell.Three kinds of concentration of echinacoside and MPP+1mM control treatment compare, and cell survival rate has notable difference (P＜0.01).Therefore echinacoside not only has neuroprotective preferably, but also has the promotion cel l proliferation.1 μ g/ml, 10 μ g/ml, the SH-SY5Y dopaminergic cell survival rate of 100 μ g/ml echinacoside all obviously increases (P＜0.01) than MPP+ group, and cell survival rate and echinacoside concentration are dose-dependence.Therefore result of the test shows that echinacoside has significant protective effect to the damage of MPP+ mediation.

2, echinacoside is to the mechanism test of the protective effect of the SH-SY5Y dopaminergic cell of the damage of mediation

GADD153 is the co-channel of er stress and other stress state, simultaneously can cause apoptotic important molecule again, and this exists at present in the research of multiple pathogenetic disease consequence is arranged in parkinson disease for it.

2.1RT-PCR detecting the mRNA of GADD153 expresses

Reverse transcription test kit (RevertAidTM First Strand cDNA Synthesis Kit) is available from MBI company.

Final concentration 1mM MPP+ and echinacoside are respectively 1 μ g/ml, and 10 μ g/ml, 100 μ g/ml add 6 orifice plates simultaneously and put the 5%CO2 incubator and hatch 48h for 37 ℃.Take out 6 well culture plates, the PBS flushing is extracted total RNA by the Trizol method.Get 1 μ gRNA, by reverse transcription test kit explanation carrying out reverse transcription.The polymerase chain reaction cumulative volume is 25ul, primer sequence be GADD153 (go up primer: 5 '-GCACCTCCCAGAGCCCTCACTCTCC-3 ' and following primer: 5 '-GTCTACTCCAAGCCTTCCCCCTGCG-3 '). β-Actin (last primer: 5 '-ATCGTGGGCCGCCCTAGGCAC-3 ', following primer: 5 '-TGGCCTTAGGGTTCAGAGGGGC-3 ').The length of amplification purpose product is respectively 422bp, 244bp.94 ℃ of pre-degeneration 5min, loop parameter is: 95 ℃ of degeneration 0.5min, 60 ℃ of annealing 0.5min, and extends 1min totally 35 circulations for 72 ℃.Get PCR product 4ul, take pictures behind the electrophoresis.

2.2Western Blot detects the GADD153 protein expression

Final concentration 1mM MPP+ and echinacoside are respectively 1 μ g/ml, and 10 μ g/ml, 100 μ g/ml add 6 orifice plates simultaneously and put the 5%CO2 incubator and hatch 48h for 37 ℃.Take out 6 well culture plates, the PBS flushing adds 60 μ L protein lysate cracking, ultrasonic concussion, 100 ℃ of degeneration 10min.With lowry method working sample protein concentration.Electrophoresis: spacer gel 60V 30min, separation gel 120V 90min; Pvdf membrane changes film 110V 100min.Add 1: 50 mouse anti human GADD153 monoclonal antibody, 4 ℃ are spent the night.TBST solution washing 3 times, each 10min adds the anti-mice two anti-incubated at room 2h of the rabbit that contains HRP, chemoluminescence method development X film.The immunoblotting band of each sample is all analysed with the β-actin of the same sample credit that takes statistics after relatively.

Fig. 2: at matched group (Con), MPP+ organizes (M), 1 μ g/ml echinacoside group (E1), 10 μ g/ml echinacoside groups (CE2), the expression of the mRNA of the GADD153 in the 100 μ g/ml echinacoside groups (CE3).

Fig. 3: GADD153 mRNA level is than matched group obviously raise (P＜0.01) behind the MPP+1mM treatment S H-SY5Y cell.And the GADD153 mRNA level that adds the echinacoside group is organized obviously descend (P＜0.01) than MPP+.

Fig. 4: at matched group (Con), MPP+ organizes (M), 1 μ g/ml echinacoside group (E1), 10 μ g/ml echinacoside groups (CE2), the proteic expression of GADD153 in the 100 μ g/ml echinacoside groups (CE3).

The proteic expression of GADD153 of Fig. 5: MPP+ group shows a marked increase than matched group, and the proteic expression of GADD153 of the echinacoside group of variable concentrations is compared obvious reduction with the MPP+ group.(P＜0.01)

By RT-PCR and Western-Blot, find that obviously reduction is compared also in the expression of GADD153 with matched group.Wherein dropped to minimum (P＜0.01), and the result of Western Blot finds that also the protein expression of GADD153 will arrive minimum (P＜0.01) simultaneously in echinacoside maximum concentration group (100mg/L) GADD153 mRNA level.Based on The above results, can think that echinacoside may realize by regulation and control GADD153 the protective effect of the SH-SY5Y cell injury model of MPP+ mediation.

Above-mentioned result of the test shows that echinacoside has significant protective effect to the cell injury of MPP+ mediation.And echinacoside is realized by regulation and control GADD153 the protective effect of the SH-SY5Y cell injury model of MPP+ mediation.Echinacoside can be used for preparing the Parkinsonian medicine of treatment.

Using method:

The dosage of echinacoside 50mg/kg mice body weight is irritated stomach, becomes body weight for humans to press 60kg and calculates, and 20mg is oral for every day.

Embodiment

Above-mentioned echinacoside can with acceptable adjuvant (if necessary) on the pharmaceutics, according to conventional pharmaceutical methods, make oral agents and injection.

Embodiment 1 makes tablet, pill, electuary, syrup, unguentum.

Except that echinacoside, can add correctives as required, or adjuvant is as strengthening vitamin, protein and the mineral of nutrition or immunity, or can treats Parkinsonian medicine by synergism.This tablet, pill, electuary, syrup, unguentum composition comprise:

Echinacoside 5%-90% (weight ratio)

Correctives 0.1-1% (weight ratio)

Adjuvant is an amount of

Above-mentioned correctives can be selected from alpha-cyclodextrin (α-CD), beta-schardinger dextrin-(one or more in β-CD), N-LOK modified starch, sucrose, xylitol, high fructose, cyclamate, stevioside and the protein sugar.

Adjuvant can be selected from vitamin or the aminoacid that human body needs, as vitamin B, and vitamin C, lysine, glutamic acid etc.

Embodiment 2 makes capsule.This capsule composition comprises:

Echinacoside 15%-30% (weight ratio)

Correctives 0.5-10% (weight ratio)

Above-mentioned correctives mainly contains: hydroxypropyl emthylcellulose (HPMC), Polyethylene Glycol (PEG), acrylic resin etc.Also can add adjuvant, adjuvant can be selected from vitamin or the aminoacid that human body needs, as vitamin B, and vitamin C, lysine, glutamic acid etc.

Embodiment 3 makes injection, drop pill.This injection, drop pill composition comprise:

Echinacoside 20%-30% (weight ratio)

Solvent is an amount of

Above embodiment is only for the usefulness that the present invention is described, but not limitation of the present invention, person skilled in the relevant technique, under the situation that does not break away from the spirit and scope of the present invention, can also make various conversion or modification, therefore all technical schemes that are equal to also should belong to category of the present invention, should be limited by each claim.

Claims (2)

1, the application of echinacoside in preparation treatment parkinson disease medicine, wherein, described echinacoside extracts from Herba Cistanches, and its chemical structural formula is:

2, echinacoside as claimed in claim 1 can be made oral solutions, tablet, pill, electuary, capsule, injection, drop pill, syrup, unguentum with correctives, solvent or adjuvant in preparation treatment parkinson disease medicine.

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