CN101434593B - Two crystal substances of baicalin, and preparations, pharmaceutical composition and uses thereof - Google Patents
Two crystal substances of baicalin, and preparations, pharmaceutical composition and uses thereof Download PDFInfo
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Abstract
The invention discloses a Baicalien compound (with the chemical name of 5, 6, 7 - Genistein and the English name of Baicalien), two crystal form substances and a preparation thereof; a medicament combination containing Baicalien Alpha crystal form, Beta crystal form and (Alpha plus Beta) mixed crystal form and a Baicalien crystal form substance are taken as active ingredients of a medicament for preventing and treating nervous system diseases, cardiovascular and cerebrovascular diseases, infectious diseases caused by bacteria and virus, inflammation and diseases of the immune system, metabolic diseases, infirmities of age, especially senile dementia and paralysis agitans. Besides, the invention also relates to a medicament combination which takes the Baicalien Alpha crystal form, the Betacrystal form and (Alpha plus Beta) the mixed crystal form solid substances and flavonoid substances (or other chemical substances) and/or Chinese medicinal herbs as the active ingredients.
Description
Technical field
The invention discloses two kinds of crystal-form substances of scutellarin compound and preparation method thereof, the pharmaceutical composition that contains scutellarin alpha-crystal form, beta crystal and (alpha+beta) mixing crystal formation, and the scutellarin crystal-form substances is being prevented and treated nervous system disorders, is being prevented and treated the application in diseases of cardiovascular and cerebrovascular systems, inflammation and the diseases such as disease of immune system, metabolic disease, geriatric disease and bacterium and virus infection as effective ingredient.
Background technology
Scutellarin (chemical name: 5，6，7-trihydroxyflavone; English name: Baicalien)
The scutellarin molecular formula
In Chinese patent CN 1556108A (publication number), put down in writing " extraction process of scutellarin, medicinal compositions and the preparation process thereof " of China Medicine University's invention
[1]Wherein, related to the application in the acute respiratory syndrome that treatment various diseases of viral infection such as hepatitis, viral cold, virus infection cause of scutellarin and pharmaceutical composition thereof.
In Chinese patent CN 1608619A (publication number), put down in writing " medicament composing prescription of scutellarin " of Che Qingming invention
[2]Wherein, related to scutellarin and pharmacy acceptable salt class thereof with in, western drug cooperates prescription, for the preparation of the new purposes of medicine, specifically comprise for the preparation of the antipyretic-antalgic anti-inflammatory agent thing; For the preparation of antibiotic, antiviral; For the preparation of double antipyretic-antalgic anti-inflammatory and antibiotic, the antiviral controlled.
In Chinese patent CN 1606979A (publication number), put down in writing " scutellarin is as the effect of analgesic, analgesia, anti-inflammatory, antibiotic, antiviral agent " of Che Qingming invention
[3]Wherein, scutellarin and pharmacy acceptable salt class thereof have been related to for the preparation of analgesic, analgesia, anti-inflammatory, antibiotic, antiviral drug.
In Chinese patent CN 1939295A (publication number), put down in writing Tianjin changzheng Hospital's invention " baical skullcap root dropping balls and preparation method thereof "
[4]Wherein, related to the natural phant root of large-flowered skullcap is passed through conventional extraction and separates, adopted new pharmaceutical excipient, be developed into the scutellarin dripping pill, be used for the treatment of anaphylaxis dermatosis.
In Chinese patent CN 1559400A (publication number), put down in writing " a kind of compound method of scutellarin liquid " of the Zhang Xiping of No.1 People's Hospital, Hangzhou City invention
[5]Wherein, the compound method that has related to a kind of scutellarin liquid, it is removing oxyradical, alleviate the tissue ischemia reperfusion injury, regulate immunologic function, hepatic cholagogic, anti-infective, the aspect such as antitumor all has certain effect, and is plurality of Chinese injection liquid, oral liquid, capsule, medicine materical crude slice, loose, the red moiety of cream of ball.
In Chinese patent CN 1528289A (publication number), put down in writing the inventions such as Lee Zong Fang of Xi'an Communications University " compound rubarb element scutellarin injection liquid for the treatment of acute pancreatitis and preparation method thereof "
[6]Wherein, related to a kind of preparation method of compound rubarb element scutellarin injection liquid, its effect in the treatment acute pancreatitis.
Summary of the invention
One of the object of the invention: two kinds of crystal formation solid matter samples that provide scutellarin alpha-crystal form and beta crystal.
Two of the object of the invention: the process of preparing that provides two kinds of crystal formation solid matter samples of scutellarin alpha-crystal form and beta crystal.
Three of the object of the invention: provide the solid pharmaceutical and the composition thereof that contain scutellarin alpha-crystal form sterling, beta crystal sterling or mixed the different crystal forms that forms by (alpha+beta) crystal formation by the different ratios proportioning.
Four of the object of the invention: provide the solid crystal formation medicine that contains scutellarin alpha-crystal form, beta crystal, (alpha+beta) crystal formation in performance pharmacological agent mechanism and produce the impact of specific absorption difference.
Five of the object of the invention: provide the effect of scutellarin crystal-form substances in preventing and treating nervous system disorders, diseases of cardiovascular and cerebrovascular systems or other class disease, and the therapeutic action that in treatment various diseases process, improves Plasma Concentration performance in the organism owing to the crystal formation effect.
Six of the object of the invention: provide and use the scutellarin alpha-crystal form, beta crystal and (alpha+beta) mix the crystal formation solid matter, and the combination that becomes to grade (contains the different chemical composition kind with flavonoid chemical substance (or other class chemical substance), the different content proportioning) jointly as the raw material of effective ingredient, the compound chemical medicine that exploitation is made and its various preparations are at the control diseases of mental and nervous system, diseases of cardiovascular and cerebrovascular systems, inflammation and disease of immune system, metabolic disease (such as diabetes), application in the diseases such as geriatric disease and bacterium and virus infection.Especially the application in control nerve degenerative diseases such as the diseases such as senile dementia, Parkinson's disease.
Seven of the object of the invention: provide and use the scutellarin alpha-crystal form, beta crystal and (alpha+beta) mix the crystal formation solid matter and herbal medicine (contains Chinese medicine compound prescription, Chinese medicinal materials, extract parts etc.) combinations of substances (contains different sorts, the different content proportioning) jointly as the raw material of effective ingredient, the herbal mixture medicine that exploitation is made and its various preparations are at the control diseases of mental and nervous system, diseases of cardiovascular and cerebrovascular systems, inflammation and disease of immune system, metabolic disease (such as diabetes), application in the diseases such as geriatric disease and bacterium and virus infection.Especially the application in control nerve degenerative diseases such as the diseases such as senile dementia, Parkinson's disease.
This patent has been invented two kinds of different crystal forms (alpha-crystal form and beta crystal) solid matter existence of scutellarin compound, and has invented the preparation method of two kinds of crystal form samples; The present invention finds that there is absorptivity difference in vivo in two kinds of crystal formation solid matters of scutellarin, wherein the beta crystal specific absorption is greater than alpha-crystal form, the comparable alpha-crystal form of the specific absorption of beta crystal improves more than 2 times, on because specific absorption is different, and the Plasma Concentration that causes thus changes and causes medicine biological action difference in performance pharmacological agent; In addition, the present invention has found the effect of Huang Qinsu in preventing and treating other class diseases such as nervous system disorders, diseases of cardiovascular and cerebrovascular systems, especially the application in control senile dementia and Parkinson's disease.
Technical characterictic
1. the morphological specificity of alpha-crystal form scutellarin sample:
1.1. scutellarin alpha-crystal form solid matter of the present invention is characterized in that (CuK when using powder x-ray diffraction analysis
αRadiation), show as the diffraction peak position: the 2-Theta value (°) or the d value (
) and diffraction peak relative intensity: crystalline state solid matter when having following characteristic peaks of peak height value (Height%) or peak area value (Area%) (table 1, Fig. 1):
The powder x-ray diffraction characteristic peaks of table 1 alpha-crystal form scutellarin sample
1.2. scutellarin alpha-crystal form solid matter of the present invention when using DSC collection of illustrative plates (Fig. 2) to analyze, is characterized in that having an endotherm(ic)peak in its DSC collection of illustrative plates, transformation value is about 271 ℃.
1.3. scutellarin alpha-crystal form solid matter of the present invention, when using the KBr compressing tablet to analyze, the infrared absorption spectrum of acquisition (Fig. 3) is characterized in that its infrared absorption exists: 3411.1,3090.0,1656.5,1618.3,1586.0,1564.4,1505.3,1492.7,1471.0,1449.2,1412.1,1389.2,1338.8,1297.8,1243.9,1211.8,1182.0,1162.4,1103.2,1085.2,1032.5,1021.7,998.6,966.8,916.1,897.6,853.5,826.7,777.6,748.5,733.1,715.9,705.7,682.7,640.0,619.4,599.9,573.2,522.2,488.0,461.0,425.2cm
-1There are absorption peak, wherein 1564.4,853.5,826.7,715.9 cm in the place
-1The peak is the principal character absorption peak of scutellarin alpha-crystal form solid matter.
1.4. scutellarin alpha-crystal form solid matter of the present invention, its melting point values are about 267 to 269 ℃.
2. the morphological specificity of beta crystal scutellarin sample:
2.1. scutellarin beta crystal solid matter of the present invention is characterized in that (CuK when using powder x-ray diffraction analysis
αRadiation), show as the diffraction peak position: the 2-Theta value (°) or the d value (
) and diffraction peak relative intensity: solid matter when having following characteristic peaks of peak height value (Height%) or peak area value (Area%) (table 2, Fig. 4):
The powder x-ray diffraction characteristic peaks of table 2 beta crystal scutellarin sample
2.2. scutellarin beta crystal solid matter of the present invention when using DSC collection of illustrative plates (Fig. 5) to analyze, is characterized in that having two endotherm(ic)peaks in its DSC collection of illustrative plates, its transformation value is respectively about 111 ℃ and about 146 ℃.
2.3. scutellarin beta crystal solid matter of the present invention, when using the KBr compressing tablet to analyze, the infrared absorption spectrum of acquisition (Fig. 6) is characterized in that its infrared absorption exists: 3411.7,3089.4,1656.7,1619.4,1586.3,1506.0,1492.6,1471.1,1449.2,1390.0,1339.4,1298.5,1243.9,1211.4,1182.1,1103.3,1085.2,1033.7,1021.8,999.0,967.5,916.0,897.9,853.0,826.1,777.3,733.5,717.2,706.4,682.3,640.3,619.5,599.4,573.1,521.8,487.9,461.0,425.7cm
-1There is an absorption peak at the place, wherein 853.0,826.1,717.2cm
-1The peak is the principal character absorption peak of scutellarin beta crystal solid matter.
2.4. scutellarin beta crystal solid matter of the present invention, its melting point values are about 264 to 266 ℃.
3. the bioavailability feature of scutellarin sample:
Scutellarin alpha-crystal form sterling of the present invention and beta crystal sterling all can be passed through oral absorption, but bioavailability there are differences, the bioavailability of oral beta crystal sterling is higher more than 2 times than alpha-crystal form, and the mixed crystal solid matter sample that obtains after two kinds of crystal formations make up in any proportion also has different bioavailability difference.
4. scutellarin is prevented and treated the feature of nervous system disorders and other diseases
The purposes feature of disease preventing and treating of the present invention is the provide protection that comprises neurocyte; improvement effect to symptoms such as the dyskinesia of nerve injury animal model and muscular tremors; to the therapeutic action of the animal movement obstacle of inducing of dopamine system and cholinergic nerve system, and to the restraining effect of related enzyme activity.
In addition, the application in control diseases of mental and nervous system, diseases of cardiovascular and cerebrovascular systems, inflammation and disease of immune system, metabolic disease (as: diabetes), geriatric disease and the diseases such as bacterium and virus infection.
5. the dosage of scutellarin sample and pharmaceutical preparation feature:
Further aspect of the present invention also relates to the pharmaceutical composition of the compounds of this invention as active ingredient.Pharmaceutical preparation feature of the present invention is to contain the mixing crystal formation solid sample that scutellarin alpha-crystal form sterling, beta crystal sterling, the combination of (alpha+beta) crystal formation arbitrary proportion obtain, it is characterized in that every day dosage in the 2000mg scope with interior pharmaceutical composition.Pharmaceutical composition of the present invention, contain effective dose such as claim 1 (alpha-crystal form) and or claim 4 (beta crystal) and claim 7 (alpha+beta mixing crystal formation) described scutellarin solid matter and pharmaceutically acceptable carrier.Pharmaceutical composition also contains the flavonoid chemical substance, or also contains the herbal medicine material.Described herbal medicine material comprises Chinese medicine compound prescription, Chinese medicinal materials, extract part.This pharmaceutical composition includes the different pharmaceutical dosage forms such as tablet, capsule, pill, injection, sustained release preparation, controlled release preparation.
This pharmaceutical composition can be according to method preparation well known in the art.Can be by the pharmaceutically acceptable solid of the compounds of this invention and one or more or liquid excipient and/or assistant agent being combined, making any formulation that is suitable for human or animal's use.The content of the compounds of this invention in its pharmaceutical composition is generally the 0.1-95 % by weight.
The compounds of this invention or contain its pharmaceutical composition can the unit dosage form administration, route of administration can be enteron aisle or non-enteron aisle, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum etc.
Form of administration can be liquid dosage form, solid dosage or semisolid dosage form.Liquid dosage form can be solution (comprising true solution and colloidal solution), emulsion (comprising o/w type, w/o type and emulsion), suspensoid, injection (comprising aqueous injection, powder injection and transfusion), eye drops, nasal drop, lotion and liniment etc.; Solid dosage can be tablet (comprising ordinary tablet, enteric coated tablet, lozenge, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), capsule (comprising hard capsule, soft capsule, enteric coated capsule), granule, powder, micropill, dripping pill, suppository, film, paster, the agent of gas (powder) mist, sprays etc.; Semisolid dosage form can be ointment, gelifying agent, paste etc.
The compounds of this invention can be made ordinary preparation, also make is sustained release preparation, controlled release preparation, targeting preparation and various particulate delivery system.
For the compounds of this invention is made tablet, can be widely used various vehicle well known in the art, comprise thinner, tamanori, wetting agent, disintegrating agent, lubricant, glidant.Thinner can be starch, dextrin, sucrose, glucose, lactose, N.F,USP MANNITOL, sorbyl alcohol, Xylitol, Microcrystalline Cellulose, calcium sulfate, secondary calcium phosphate, calcium carbonate etc.; Wetting agent can be water, ethanol, Virahol etc.; Tackiness agent can be starch slurry, dextrin, syrup, honey, glucose solution, Microcrystalline Cellulose, mucialga of arabic gummy, gelatine size, Xylo-Mucine, methylcellulose gum, Vltra tears, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyoxyethylene glycol etc.; Disintegrating agent can be dry starch, Microcrystalline Cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, croscarmellose sodium, sodium starch glycolate, sodium bicarbonate and Citric Acid, polyoxyethylene sorbitol fatty acid ester, sodium laurylsulfonate etc.; Lubricant and glidant can be talcum powder, silicon-dioxide, stearate, tartrate, whiteruss, polyoxyethylene glycol etc.
Tablet further can also be made coating tablet, for example sugar coated tablet, thin membrane coated tablet, ECT, or double-layer tablets and multilayer tablet.
For capsule is made in the administration unit, the effective constituent the compounds of this invention can be mixed with thinner, glidant, mixture is directly placed hard capsule or soft capsule.Also can with effective constituent the compounds of this invention elder generation and thinner, tamanori, disintegrating agent granulation or micropill, place again hard capsule or soft capsule.The capsule that also can be used for preparing the compounds of this invention for the preparation of each thinner, tamanori, wetting agent, disintegrating agent, the glidant kind of the compounds of this invention tablet.
For the compounds of this invention is made injection, can water, ethanol, Virahol, propylene glycol or their mixture as solvent and add an amount of this area solubilizing agent commonly used, solubility promoter, pH and adjust agent, osmotic pressure regulator.Solubilizing agent or solubility promoter can be poloxamer, Yelkin TTS, hydroxypropyl-beta-cyclodextrin etc.; PH adjustment agent can be phosphoric acid salt, acetate, hydrochloric acid, sodium hydroxide etc.; Osmotic pressure regulator can be sodium-chlor, N.F,USP MANNITOL, glucose, phosphoric acid salt, acetate etc.As prepare lyophilized injectable powder, also can add N.F,USP MANNITOL, glucose etc. as propping agent.
In addition, such as needs, also can in pharmaceutical preparation, add tinting material, sanitas, spices, correctives or other additive.
For reaching the medication purpose, strengthen result for the treatment of, medicine of the present invention or pharmaceutical composition can be with any known medication administrations.
The dosage of the compounds of this invention pharmaceutical composition is according to character and the severity that will prevent or treat disease, the individual instances of patient or animal, and route of administration and formulation etc. can have large-scale variation.In general, the appropriate dose scope of the every day of the compounds of this invention is the 0.001-150mg/Kg body weight, is preferably the 0.1-100mg/Kg body weight, and more preferably the 1-60mg/Kg body weight most preferably is the 2-30mg/Kg body weight.Above-mentioned dosage can a dose unit or is divided into several dose unit administrations, and this depends on doctor's clinical experience and comprises the dosage regimen of using other treatment means.
Compound of the present invention or composition can be taken separately, or merge use with other treatment medicine or symptomatic drugs.When compound of the present invention and other medicine existence synergy, should adjust according to practical situation its dosage.
Description of drawings
The x-ray diffractogram of powder spectrum of Fig. 1 alpha-crystal form scutellarin sample
The DSC collection of illustrative plates of Fig. 2 alpha-crystal form scutellarin sample
The infrared absorption pattern of Fig. 3 alpha-crystal form scutellarin sample
The x-ray diffractogram of powder spectrum of Fig. 4 beta crystal scutellarin sample
The DSC collection of illustrative plates of Fig. 5 beta crystal scutellarin sample
The infrared absorption pattern of Fig. 6 beta crystal scutellarin sample
Fig. 7 scutellarin is on the impact of 6-OHDA damage SH-SY5Y cell viability
Fig. 8 scutellarin is on the impact of 6-OHDA damage SH-SY5Y cell ATP content
Fig. 9 scutellarin is on the impact of SH-SY5Y apoptosis rate due to the 6-OHDA
Different time blood Chinese traditional medicine concentration behind Figure 10 scutellarin Oral Administration in Rats (200mg/kg)
Embodiment
The crystal formation preparation
The preparation method 1 of scutellarin alpha-crystal form sample:
Contain a kind of preparation method of scutellarin alpha-crystal form sample, it is characterized in that using the single solvent systems such as chloroform, acetonitrile, tetrahydrofuran (THF), dioxane, Glacial acetic acid, formic acid, ether, methylene dichloride, toluene, benzene, normal hexane, hexanaphthene, DMF, sherwood oil, ammoniacal liquor, n-propyl alcohol, water.First the scutellarin sample is dissolved fully, is placed on temperature range at 4 ℃ to 250 ℃, the relative humidity scope 90% with interior envrionment conditions under, through recrystallization growth time 1 day to 60 days, the alpha-crystal form solid sample for preparing; Under the complete dissolved state of scutellarin sample, utilize vacuum and control the alpha-crystal form solid sample that temperature range prepares-50 to 100 ℃ (refrigeration or heating).
Embodiment 2
The preparation method 2 of scutellarin alpha-crystal form sample:
The preparation method who contains a kind of scutellarin alpha-crystal form sample, it is characterized in that using methyl alcohol, ethanol, Virahol, 95% ethanol, chloroform, acetone, ethyl acetate, propyl carbinol, acetonitrile, tetrahydrofuran (THF), dioxane, Glacial acetic acid, formic acid, ether, methylene dichloride, toluene, benzene, normal hexane, hexanaphthene, dioxane, DMF, sherwood oil, ammoniacal liquor, n-propyl alcohol, water equal solvent kind, the mixed solvent system of making through various combination (two or more solvent use) and different proportionings.First the scutellarin sample is dissolved fully, is placed on temperature range at 4 ℃ to 250 ℃, the relative humidity scope 90% with interior envrionment conditions under, through recrystallization growth time 1 day to 60 days, the alpha-crystal form solid sample for preparing; Under the complete dissolved state of scutellarin sample, utilize vacuum and control the alpha-crystal form solid sample that temperature range prepares-50 to 100 ℃ (refrigeration or heating).
The preparation method 1 of scutellarin beta crystal sample:
The preparation method of scutellarin beta crystal sample is characterized in that using scutellarin alpha-crystal form solid sample as raw material, turns the beta crystal solid sample that brilliant processing condition and other physics rotating crystal method prepare through solid abrasive, pressurization, heating etc.
Embodiment 4
The preparation method 2 of scutellarin beta crystal sample:
The preparation method of scutellarin beta crystal sample, after it is characterized in that using the single solvents such as chloroform, acetonitrile, tetrahydrofuran (THF), dioxane, Glacial acetic acid, formic acid, ether, methylene dichloride, toluene, benzene, normal hexane, hexanaphthene, dioxane, DMF, sherwood oil, ammoniacal liquor, n-propyl alcohol, water or mixed solvent system with the scutellarin sample dissolution, re-use the cold spray method and obtain fast the beta crystal solid sample.
Preparation
The preparation method 1 (tablet) of combined pharmaceutical formulation:
A kind of preparation method of combined pharmaceutical formulation, it is characterized in that using solid matter that several vehicle mix by the different ratios proportioning with scutellarin alpha-crystal form sterling, beta crystal sterling or by (alpha+beta) crystal formation as the bulk drug of medicinal composition, make every content of dispersion at the tablet of 10～500mg, table 3 provides tablet formulation:
The preparation prescription of table 3 scutellarin medicinal composition tablet
With scutellarin alpha-crystal form sterling, beta crystal sterling or by the method that the bulk drug that (alpha+beta) crystal formation mixes by the different ratios proportioning is prepared into tablet formulation be: several vehicle and bulk drug are mixed, it is an amount of to add 1% sodium cellulose glycolate solution, make soft material, the granulation of sieving, wet grain oven dry, the whole grain that sieves adds Magnesium Stearate and talcum powder and mixes, compressing tablet, and get final product.
Embodiment 6
The preparation method 2 (capsule) of combined pharmaceutical formulation:
A kind of preparation method of combined pharmaceutical formulation, it is characterized in that using solid matter that several vehicle mix by the different ratios proportioning with scutellarin alpha-crystal form sterling, beta crystal sterling or by (alpha+beta) crystal formation as the bulk drug of medicinal composition, make every content of dispersion at the capsule of 10～500mg, table 4 provides capsule formula:
The preparation prescription of table 4 scutellarin medicinal composition capsule
With scutellarin alpha-crystal form sterling, beta crystal sterling or by the method that the bulk drug that (alpha+beta) crystal formation mixes by the different ratios proportioning is prepared into capsule preparations be: several vehicle and bulk drug are mixed, it is an amount of to add 1% sodium cellulose glycolate solution, make wet grain, oven dry, whole grain sieves, add Magnesium Stearate, mix, insert capsule and make.Or do not use granulation step, and and directly that bulk drug and several mixed with excipients is even, after sieving, directly incapsulate and make.
Pharmacological evaluation
Embodiment 7
Scutellarin damages the provide protection of SH-SY5Y cell to 6-OHDA:
7.1 at 37 ℃, 5%CO
2In the constant incubator, cultivate the SH-SY5Y cell with 1640 substratum (containing 10% foetal calf serum, penicillin 100U/ml, Streptomycin sulphate 100ug/ml).After cell is paved into individual layer, absorb former substratum, add the 6-OHDA 100ul of final concentration 100uM, test behind the 24h.Adopt mtt assay to measure cell viability; Adopt CellTiter-Glo
The content of ATP in the Luminescent kit measurement cell.The result shows 10
-5The mol/L scutellarin can obviously suppress the decline (Fig. 7) of the SH-SY5Y cell viability due to the 6-OHDA.
7.2 under 7.1 embodiment experiment conditions, 10
-5Mol/L, 10
-6The mol/L scutellarin can reduce the decline (Fig. 8) of the SH-SY5Y cell ATP content due to the 6-OHDA, and the SH-SY5Y cell injury due to the 6-OHDA is had provide protection.
7.3 culturing cell is inoculated in six well culture plates be paved into individual layer after, absorb former substratum, add the scutellarin (10 of different concns
-5, 10
-6, 10
-7Mol/L) preincubate added 6-OHDA after 1 hour, and making its final concentration is 100uM, tests behind the 24h.Adopt flow cytometer to detect apoptosis rate.The result shows 10
-5Mol/L, 10
-6The mol/L scutellarin can reduce the apoptosis (Fig. 9) of the SH-SY5Y cell due to the 6-OHDA.
Embodiment 8
Scutellarin is to the neuroprotective of parkinsonian mouse model due to the MPTP:
8.1 scutellarin is to parkinsonian mouse model autonomic activities effect due to the MPTP
Male 8 the week age C57BL/6J mouse, be divided at random 3 groups, 10 every group, be respectively control group, model group, scutellarin group (280mg/kg).Continuously gastric infusion is after one week, at the 8th day gavage 1h pneumoretroperitoneum injection MPTP30mg/kg, continuous 5 days, carries out the study of behaviour index in 1 day after last 1 administration and detects.
Use ZIL-2 type mouse autonomic activities instrument to measure spontaneous activity in mice and counting, mouse is put into autonomic activities case (high 13cm, diameter 25cm, measure simultaneously 4 mouse at every turn, in each active box 1), automatically record the mouse activity by registering instrument, measure the movable number of times in every mouse 5min, carry out statistical procedures.The result shows, compares with control group, and MPTP can obviously reduce the spontaneous activity of mouse, gives can obviously increase behind the scutellarin (280mg/kg) autonomic activities (table 5) of mouse.
Table 5. scutellarin on the impact of parkinsonian mouse autonomic activities due to the MPTP (
):
*P＜0.05 (comparing with control group),
#P＜0.05 (comparing with model group)
8.2 the effect that scutellarin is coordinated animal movement---pole-climbing test
Under the condition of above-mentioned animal model preparation, be the rod top that the cork bead of 2.5cm is fixed in a thick 1cm of long 50cm with a diameter, be wrapped with gauze on the rod with anti-slip, then tested mouse is put on the bead, the record mouse climbs to the time value at the bottom of the bar, surpass 60s in 60s.The result shows the pole-climbing time that MPTP can the significant prolongation mouse, and scutellarin (280mg/kg) can significantly improve the increase (table 6) of parkinsonian mouse pole-climbing time due to the MPTP.
Table 6. scutellarin to the effect of parkinsonian mouse pole-climbing ability due to the MPTP (
):
*P＜0.05 (comparing with control group),
#P＜0.05 (comparing with model group)
Embodiment 9
Scutellarin brings out the therapeutic action of hemiparkinsonian disease rat model to the 6-OHDA locating injection:
9.1 scutellarin is on the impact of animal model myoelectricity
Model is set up: 3% vetanarcol (50mg/kg) intraperitoneal injection of anesthesia, take the hind leg rebound reflex disappear and absent corneal reflex as the anesthesia index, can low dose append in case of necessity.Rat head is fixed on the stereotaxic instrument, with reference to Paxinos and Watson mouse brain stereotaxic atlas, is equivalent to the skull surface boring in MFB district in the left side, diameter 2.5cm behind the exposure skull.Behind the cleaning meninx, at 2 injections of the descending MFB of the guiding of three-dimensional pusher 6-OHDA (4mg/ml).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.The amounts of 2 injection 6-OHDA are 3 μ l, and injection speed is controlled at 1 μ l/min, slowly go out pin behind let the acupuncture needle remain at a certain point after the per injection 10min.The physiological saline of sham operated rats locating injection equivalent.Postoperative 7 days, (circling behavior of 0.2mg/kg) inducing is chosen the rat of rotating speed＞240r/40min as successful PD rat model to 14 days test apomorphines for APO, subcutaneous injection.
Successful PD rat model is divided into two groups at random, 10 every group, be respectively model group and scutellarin group (200mg/kg), get in addition 10 of sham operated rats in contrast.In carrying out the rat behavior of trembling, the mensuration of autonomic activities and transfer rod ability the 5th week.
Use BL-420S biological signal collecting device that rat hindlimb is carried out myoelectricity and measure, estimate its tremble frequency and tremor amplitude.The result shows that scutellarin (200mg/kg) can obviously improve the reaction of trembling of rat model of Parkinson disease due to the 6-OHDA, reduces the frequency of trembling, and reduces tremor amplitude.The result shows that scutellarin has certain improvement effect (table 7) of trembling to PD rat model due to the MPTP.
Table 7. scutellarin to the effect of trembling of rat model of Parkinson disease due to the 6-OHDA (
):
*P＜0.05 (comparing with sham operated rats),
#P＜0.05 (comparing with model group)
9.2 scutellarin affects the animal model autonomic activities
Under above-mentioned 9.1 prepared animal model conditions, use the movable number of times in every rat of DZIL-2 type rat autonomic activities instrument record 10 minutes.The result shows that scutellarin group rat autonomic activities number compared significant increase with model group, and scutellarin can improve the spontaneous activity (table 8) of rat model of Parkinson disease.
Table 8. scutellarin on the impact of rat model of Parkinson disease spontaneous activity due to the 6-OHDA (
)
*P＜0.05 (comparing with sham operated rats),
#P＜0.05 (comparing with model group)
The scutellarin solid material medicine of alpha-crystal form and beta crystal Absorption Characteristics and Plasma Concentration feature in vivo:
To giving respectively alpha-crystal form, beta crystal scutellarin solid material medicinal powder end in the cleaning level rat stomach, dosage is 200mg/kg, after administration, extract in the different time points rat artery hematometry scutellarin content (table 9, Figure 10).The result proves: different crystal forms scutellarin bulk drug under the condition of oral same dosage, the drug level in the blood and the asynchronism(-nization) of concentration of peaking, wherein the Plasma Concentration of alpha-crystal form is starkly lower than the Plasma Concentration of beta crystal.
The Plasma Concentration (HPLC detection) of different time behind the table 9. Oral Administration in Rats scutellarin different crystal forms sample
Claims (11)
1. a scutellarin beta crystal is characterized in that, uses powder x-ray diffraction analysis to adopt CuK
αThe radiation experiments condition, the diffraction peak position: the 2-Theta value (°) or the d value
With the diffraction peak relative intensity: peak height value (Height%) or peak area value (Area%) have following feature:
2. two kinds of crystal form samples of beta crystal that contain scutellarin alpha-crystal form and claim 1 make up the mixing crystal formation solid matter that obtains in any proportion after physical mixed; Described scutellarin alpha-crystal form uses powder x-ray diffraction analysis to adopt CuK
αThe radiation experiments condition, the diffraction peak position: the 2-Theta value (°) or the d value
With the diffraction peak relative intensity: peak height value (Height%) or peak area value (Area%) have following feature:
3. the preparation method of scutellarin beta crystal according to claim 1 is characterized in that, uses scutellarin alpha-crystal form solid sample as raw material, the beta crystal solid sample that process solid abrasive, pressurization, heating prepare; Described scutellarin alpha-crystal form uses powder x-ray diffraction analysis to adopt CuK
αThe radiation experiments condition, the diffraction peak position: the 2-Theta value (°) or the d value
With the diffraction peak relative intensity: peak height value (Height%) or peak area value (Area%) have following feature:
4. the preparation method of scutellarin beta crystal according to claim 1, it is characterized in that, use chloroform, acetonitrile, tetrahydrofuran (THF), dioxane, Glacial acetic acid, formic acid, ether, methylene dichloride, toluene, benzene, normal hexane, hexanaphthene, DMF, sherwood oil, ammoniacal liquor, n-propyl alcohol, any single solvent of water or mixed solvent, after the scutellarin sample dissolution, use the cold spray method to obtain fast the beta crystal solid sample.
5. pharmaceutical composition, contain effective dose such as claim 1 scutellarin beta crystal and pharmaceutically acceptable carrier.
6. pharmaceutical composition contains mixing crystal formation solid matter as claimed in claim 2 and the pharmaceutically acceptable carrier of effective dose.
7. each pharmaceutical composition according to claim 5 or in 6 is characterized in that described pharmaceutical composition is selected from tablet, capsule, pill, injection.
8. each pharmaceutical composition according to claim 5 or in 6 is characterized in that described pharmaceutical composition is selected from sustained release preparation, controlled release preparation.
The scutellarin beta crystal of claim 1 preparation prevent and treat nervous system disorders, the control diseases of cardiovascular and cerebrovascular systems, inflammation and disease of immune system, metabolic disease and bacterium and virus infection medicine in application.
The mixing crystal formation solid matter of claim 2 preparation prevent and treat nervous system disorders, the control diseases of cardiovascular and cerebrovascular systems, inflammation and disease of immune system, metabolic disease and bacterium and virus infection medicine in application.
11. each application is characterized in that according to claim 9 or in 10, described nervous system disorders is senile dementia (A Erzi adopts silent sick), Parkinson's disease; Described metabolic disease is diabetes.
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| WO2012040919A1 (en) * | 2010-09-29 | 2012-04-05 | 中国医学科学院药物研究所 | Use of baicalein in manufacturing medicament for preventing and treating parkinson's disease |
| CN104592184B (en) | 2014-12-15 | 2017-09-29 | 云南省药物研究所 | Scutellarein crystal formation and preparation method thereof |
| CN104829577B (en) * | 2015-05-07 | 2017-04-05 | 诸城市浩天药业有限公司 | A kind of baicalin γ crystal formations, its preparation method and its pharmaceutical composition and purposes |
| CN105218500B (en) * | 2015-11-02 | 2018-06-15 | 诸城市浩天药业有限公司 | Baicalein caffeine eutectic, preparation method, pharmaceutical composition and its application |
| CN110314156A (en) * | 2018-03-28 | 2019-10-11 | 中国医学科学院药物研究所 | Hundred can application of the benefit in preparation prevention and treatment central neurotransmitter disorder and the product that trembles |
| CN112007023A (en) * | 2019-05-31 | 2020-12-01 | 中国医学科学院药物研究所 | Application of baicalein in preparing medicine for preventing and treating obesity and its complication |
| CN112274504A (en) * | 2019-07-25 | 2021-01-29 | 中国医学科学院药物研究所 | Application of Baikeli in preparation of medicine for treating diseases caused by intestinal flora disorder |
| CN110483464A (en) * | 2019-09-09 | 2019-11-22 | 南开大学 | Adenosine A1Receptor antagonist and its application |
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