CN101926788A

CN101926788A - Cardiovascular/cerebral and ophthalmological medicines, and preparation and use thereof

Info

Publication number: CN101926788A
Application number: CN2009100627708A
Authority: CN
Inventors: 刘力
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-06-18
Filing date: 2009-06-18
Publication date: 2010-12-29
Anticipated expiration: 2029-06-18
Also published as: CN101926788B

Abstract

The invention relates to cardiovascular/cerebral and ophthalmological medicines-puerarin crystalline hydrate for the treatment of high blood pressure, coronary heart disease, cerebral infarction, cerebral thrombosis and sequela thereof, cerebral circulation improvement, vertebral-basilar insufficiency vertigo, post-deep venous thrombosis syndrome, diabetes, diabetes complication, diabetic nephropathy, diabetic peripheral neuropathy, diabetic retinopathy, retinal artery ostruction, retinal vein obstruction, sudden hearing loss, glaucoma and other diseases, and further relates to preparation and use thereof; the derivative has excellent storage stability and is suitable for the use of preparing the medicines for the prevention and the treatment of cardiovascular/cerebral diseases and ophthalmological diseases.

Description

Cardiovascular and cerebrovascular vessel and opthalmological and preparation thereof and purposes

Technical field

The present invention relates to medical technical field, specifically provide cardiovascular and cerebrovascular vessel and opthalmological and preparation thereof and purposes.

Background technology

Still do not have at present disclosed document only reported puerarin [8-β-D-glucopyanosyl-4 ', 7-dihydroxy isoflavone, molecular formula: C ₂₁H ₂₀O ₉, molecular weight: 416.37, CAS:3681-99-0], up to the present, still do not have disclosed bibliographical information puerarin crystalline hydrate [C both at home and abroad ₂₁H ₂₀O ₉N H ₂O, n=0.8～1.3] and its production and use.

Summary of the invention

Involved in the present invention is that report is used for hypertension, coronary heart disease, pulmonary heart disease, heart failure, angina pectoris, myocardial infarction, cardiogenic shock, arrhythmia, myocarditis, ischemic encephalopathy, cerebral infarction, cerebral thrombosis and sequela thereof, improve cerebral circulation, megrim caused by vertebra-fundus artery blood supply insufficiency, syndrome behind the deep vein thrombosis of lower extremity, diabetes, the diabetic complication diabetic nephropathy, diabetic peripheral neuropathy, retinopathy, retinal artery occlusion, vein obstruction, sudden deafness, reduce intraocular pressure, the control glaucoma, cardiovascular and cerebrovascular vessel and opthalmological and the preparation and the purposes of disease prevention such as blood lipid regulation or treatment.Its chemical name is: its molecular formula is C ₂₁H ₂₀O ₉N H ₂O, n=0.8～1.3, n can be 0.85,1,1.1,1.25 or the numeral between it.

Surprisingly, the puerarin that does not contain water of crystallization draws the moist puerarin that contains water of crystallization that obtains far above the present invention, the hydrate of the puerarin that contains water of crystallization of the present invention than do not contain water of crystallization more can be stable existence, be convenient to store and transportation, and at room temperature have good flowability, be easy to be used for the preparation of preparation.Distinctive, distinctive weightless platform between the heat analysis of hydrate of the present invention (TG-DSC or TG-DTA) collection of illustrative plates step state different as can be seen, level of approximation (approximately between 60-150 ℃) has the endothermic peak of intensive correspondence, and the thermal analysis curue spectrum demonstrates puerarin 1 hydrate (C ₂₁H ₂₀O ₉H ₂O), puerarin 1.25 hydrate (C ₂₁H ₂₀O ₉1.25H ₂O) etc., it is consistent with hot analysis result to measure the moisture result with the karl Fischer method.

Puerarin crystalline hydrate of the present invention can stable storage.With the hydrate of above-mentioned puerarin and puerarin anhydride sample airtight respectively with cillin bottle in carry out accelerated stability test (chromatographic condition: chromatographic column: C ₁₈(150mm * 4.6mm, 5 μ m); Mobile phase; Citric acid soln-methanol of 0.1% (75: 25); Flow velocity: 1ml/min; Temperature: room temperature; Detect wavelength: 250nm), find that unexpectedly the content and the related substance of the hydrate of puerarin of the present invention do not have significant change, puerarin anhydride accelerated test 6 months was compared (40 ℃ with 0 month, RH75%), related substance increases is higher than puerarin 1 hydrate.Draw moist test by the Chinese Pharmacopoeia requirement: get puerarin anhydride and the about 5g of crystalline hydrate of the present invention, place the surface plate of dry constant weight, precision is weighed.25 ℃, relative humidity are 75%, respectively at test 0h and 48h sampling, calculate the percentage rate that draws wet weightening finish, and the result shows that anhydride draws moist more much higher than hydrate of the present invention, illustrates that puerarin crystalline hydrate of the present invention has better storage stability.The results are shown in Table 1～3.

In addition, the deliquescence of anhydride makes wants secluding air to prevent adhesion etc. when handling, and does not have good sliding like hydrate, improves the operability of preparation.

Table 1. puerarin 1 hydrate accelerated stability test result of the present invention

Table 2. puerarin 1 hydrate long-term stable experiment result of the present invention

Table 3. draws the wet test result

Cardiovascular and cerebrovascular vessel and opthalmological preparation method comprise:

Method A. is in reaction vessel, one or more carry out cold extraction with the low mass molecule alcohol of powder of Radix Puerariae water or C1-C6, supersound extraction, microwave extraction or backflow are carried once to merging for several times, filter, microporous filter membrane or ceramic membrane filter, filtrate concentrates, concentrate Radix Puerariae extractum, add water, regulate PH3-5 with acid, reuse alkali or sodium carbonate or sodium bicarbonate a kind of or several to regulate PH be between the 6-9, filter, use the lower ketones of low mass molecule alcohol or the C3-C8 of C3-C6 then, as hexone, or one or more of the lower member ester of C2-C8 extract for solvent, the extract evaporate to dryness gets solid, add water, the low mass molecule alcohol of reuse C3-C6 or the lower ketones of C3-C8 are as hexone, or one or more of the lower member ester of C2-C8 extract for solvent, concentrate, leave standstill, filter, with water, the lower halogenated hydrocarbon of C1-C6, the lower ketones of C3-C8 is as acetone, the lower member ester of C2-C8, the low mass molecule alcohol of C1-C6 such as methanol, ethanol, isopropyl alcohol, the lower fatty acid of C1-C6, oxolane, one or more of acetonitrile are recrystallisation solvent, carry out recrystallization once or for several times, filter washing, with the solid drying of gained, promptly get the puerarin crystalline hydrate;

Perhaps the water of method B. Radix Puerariae extractum or Radix Puerariae total flavones or Radix Puerariae is carried the pure water extraction concentrate of concentrate or Radix Puerariae, add water or C1-C6 low mass molecule alcohol one or more, microporous filter membrane (comprising ceramic membrane) filters, cross the neutral alumina chromatographic column, silica gel column chromatography and macroporous resin, or polyamide column chromatography or glucose gel resin, the low mass molecule alcohol of water or C1-C6, the lower halogenated hydrocarbon of C1-C6, one or more carry out eluting, concentrated or ultrafilter membrane concentrates, leave standstill, filter, with water, the lower halogenated hydrocarbon of C1-C6, the lower ketones of C3-C8, as acetone, the lower member ester of C2-C8, the low mass molecule alcohol of C1-C6 such as methanol, ethanol, isopropyl alcohol, the lower fatty acid of C1-C6, oxolane, one or more of acetonitrile are recrystallisation solvent, carry out recrystallization once or for several times, filter, washing with the solid drying of gained, promptly gets the puerarin crystalline hydrate;

Perhaps the water of method C. Radix Puerariae extractum or Radix Puerariae total flavones or Radix Puerariae is carried the pure water extraction concentrate of concentrate or Radix Puerariae, add water or C1-C6 low mass molecule alcohol one or more, through microporous filter membrane, the film of doughnut, ceramic membrane, ultrafilter membrane, once or for several times the filtering or concentrate of one or more of NF membrane, with water, the lower halogenated hydrocarbon of C1-C6, the lower ketones of C3-C8, as acetone, the lower member ester of C2-C8, the low mass molecule alcohol of C1-C6 such as methanol, ethanol, isopropyl alcohol, the lower fatty acid of C1-C6, oxolane, one or more of acetonitrile are recrystallisation solvent, carry out recrystallization once or for several times, filter, washing, with the solid drying of gained, promptly get the puerarin crystalline hydrate;

Perhaps the water of method D. Radix Puerariae extractum or Radix Puerariae flavone or Radix Puerariae is carried the pure water extraction concentrate of concentrate or Radix Puerariae, film through doughnut, ceramic membrane, ultrafilter membrane, one or more of NF membrane once or multiple times of filtration or concentrate, with water, chloroform, the lower ketones of C3-C6, as acetone, the lower member ester of C2-C8, the low mass molecule alcohol of C1-C6 such as methanol, ethanol, isopropyl alcohol, the lower fatty acid of C1-C6, oxolane, one or more of acetonitrile are solvent, placing magnetic flux is to carry out the one or many crystallization under the magnetic field of 0.1-5 tesla, filter, washing, drying promptly gets the puerarin crystalline hydrate;

Different step in above-mentioned each method can be used alternatingly, so that preparation puerarin crystalline hydrate.

Identical puerarin hydrate or its crystal formation of puerarin 1 hydrate that above-mentioned distinct methods obtains are different, and the performance on X powder diffraction is different.

The purposes of cardiovascular and cerebrovascular vessel and opthalmological is: at preparation injection freeze-dried powder or little water needle injection, great transfusion preparation (comprising the glucose of puerarin crystalline hydrate, the great transfusion preparation of sodium chloride), through the intestinal canal administration preparation, comprise tablet, capsule, granule, the application on dosing eyes preparation (eye drop, gel for eye use).

The purposes of cardiovascular and cerebrovascular vessel and opthalmological is: in preparation hypertension, coronary heart disease, pulmonary heart disease, heart failure, angina pectoris, myocardial infarction, cardiogenic shock, arrhythmia, myocarditis, ischemic encephalopathy, cerebral infarction, cerebral thrombosis and sequela thereof, improve cerebral circulation, megrim caused by vertebra-fundus artery blood supply insufficiency, syndrome behind the deep vein thrombosis of lower extremity, diabetes, the diabetic complication diabetic nephropathy, diabetic peripheral neuropathy, retinopathy, retinal artery occlusion, vein obstruction, sudden deafness, reduce intraocular pressure, the control glaucoma, the application in the medicine of disease prevention such as blood lipid regulation or treatment.

The water miscible clathrate (also being complex) that the puerarin crystalline hydrate can form with cyclodextrin, the preferred 2-hydroxypropyl of cyclodextrin (2-HP-β-CYD), 3-hydroxypropyl (3-HP-β-CYD), sulfobutyl ether-beta-schardinger dextrin-(one or more among SBE-β-CD) etc., and preparing the preparation that can more stablely deposit thus, this was not reporting in open source literature.Puerarin crystalline hydrate and cyclodextrin or cyclodextrin derivative form water miscible clathrate, and wherein the quality of puerarin crystalline hydrate and cyclodextrin or cyclodextrin derivative or weight ratio are 1: 5 to 1: 60.

Be used to prepare tablet through the intestinal canal administration preparation, capsule, granule wherein can contain pharmaceutically acceptable filler, as starch, modified starch, lactose, sucrose, microcrystalline Cellulose, cyclodextrin, sorbitol, mannitol, calcium phosphate, aminoacid etc.; Pharmaceutically acceptable disintegrating agent is as starch, modified starch, microcrystalline Cellulose, carboxymethyl starch sodium, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl starch, sodium carboxymethyl cellulose, melon that natural gum, methylcellulose, surfactant; Pharmaceutically acceptable wetting agent and binding agent are as gelling starch, methylcellulose, sodium carboxymethyl cellulose, ethyl cellulose, polyvinylpyrrolidone, alginic acid and salt thereof; Pharmaceutically acceptable lubricant and fluidizer are as a kind of of stearic acid, magnesium stearate, zinc stearate, micropowder silica gel, Pulvis Talci, Macrogol 2000-8000, Stepanol MG or several; Pharmaceutically acceptable sweeting agent is as cyclamate, aspartame, saccharin sodium, sweetleaf centautin, xylitol, sorbitol, xylose, lactose, glycyrrhizin, sucrose, sucralose etc.; The suspendible stabilizing agent of Jie Shouing pharmaceutically is as your natural gum of xanthan gum, melon, methylcellulose, ethyl cellulose, sodium carboxymethyl cellulose, carboxymethyl starch sodium, Macrogol 2000-8000, surfactant, alginic acid and salt thereof etc.; Binding agent is a kind of in polyvinylpyrrolidone, methylcellulose, ethyl cellulose, hydroxypropyl emthylcellulose, pregelatinized Starch, starch, gelatin, the arabic gum or several, and binding agent can be a solvent by water or ethanol or both mixture.

In the preparation of puerarin crystalline hydrate eye drop, the parts by weight of each component can be puerarin crystalline hydrate 0.2-2, cosolvent 1-10, antioxidant 0.02-0.5, metal chelating agent 0.01-0.2, osmotic pressure regulator 0.5-10, antiseptic 0.002-0.4, water is 10-100 part, and it is an amount of that other adds the PH regulator.

Its preparation method can be: the puerarin hydrate is added in an amount of water for injection, add cosolvent, stir, the examination dissolving, add antioxidant, antiseptic, osmotic pressure regulator, stabilizing agent, PH regulator, water stirs, and becomes the solution shape, make pH=5.0-7.0, filter in modes such as microporous filter membrane or ultrafiltration, detect, aseptic subpackaged in sterilized clean plastics eyedrops bottle promptly.

Puerarin crystalline hydrate gel for eye use, the parts by weight of each component can be puerarin crystalline hydrate 0.2-2, gel-type vehicle 0.05-1, osmotic pressure regulator 1-8, antioxidant 0.01-0.5, metal chelating agent 0.01-0.2, antiseptic 0.002-0.4 in 100 parts of gels, all the other are water, and it is an amount of that other adds the PH regulator.

The preparing gel of puerarin hydrate eye usefulness: puerarin hydrate and 50-95% substrate mixing, substrate can be water, ethanol, glycerol, triethanolamine, glycerin gelatine, methylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, xanthan gum, Macrogol 200-8000 (comprises PEG200, PEG300, PEG400, PEG600, PEG800,1000, PEG1540, PEG4000 etc.), poloxamer series (can be poloxamer-188, poloxamer-237, poloxamer-338, poloxamer-407), polyvinylpyrrolidone, semi-synthetic hard fatty acids fat, the water solublity monoglyceride, carbomer series is (as carbomer 931,934,940,974, AA-1,1342 etc.) a kind of etc. or several, can contain pharmaceutically receivable antibacterial and stabilizing agent, pharmaceutically receivable PH regulator.Can be earlier during preparation with aqueous dispersion such as gel-type vehicle carbomer or glycerin gelatine, methylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, xanthan gum, poloxamer, Polyethylene Glycol, add glycerol, heating in water bath, stirring and evenly mixing, add the puerarin hydrate, osmotic pressure regulator, antioxidant of recipe quantity, pharmaceutically receivable antibacterial and stabilizing agent, stir, with pharmaceutically about receivable PH regulator adjusting pH=5.0-7.0, add water to full dose, be stirred to even, filtration, sterilization, packing, promptly.

Or with puerarin hydrate, osmotic pressure regulator, antioxidant, pharmaceutically receivable antibacterial and stabilizing agent, pharmaceutically receivable PH regulator stirs, make dissolving, filter in modes such as microporous filter membrane or ultrafiltration,, filtrate is joined in the dispersive gel-type vehicle of water for injection, stir, mixing is regulated pH=5.0-7.0, adds water to capacity, stir, sterilization, packing promptly get the gel for eye use of puerarin hydrate.

The injection of puerarin crystalline hydrate, its preparation method is:

The preparation method of freeze-dried powder is: get the puerarin hydrate, adding pharmaceutically acceptable cosolvent, frozen-dried supporting agent or auxiliary shape agent, add injection and blunge and make dissolving, is 4.0～7.5 with pharmaceutically acceptable acid-alkali accommodation pH, add activated carbon 0.005～0.5% (W/V) and stir 15～45min, filter moisturizing, aseptic filtration, by the packing of 50～600mg/ bottle, lyophilization, tamponade gets finished product.

Pharmaceutically acceptable frozen-dried supporting agent or auxiliary shape agent can contain one or more of lactose, glucose, mannitol, sorbitol, xylitol, dextran, ascorbic acid, aminoacid, glycine, taurine, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium deoxycholate etc.

Puerarin hydrate injection with small volume and preparation technology thereof: the puerarin hydrate adds injection water and pharmaceutically acceptable additives, for example: add pharmaceutically acceptable cosolvent, pharmaceutically acceptable pH regulator agent, pharmaceutically acceptable antioxidant, noble gas, the sterilization injection with small volume is made in filtration, degerming, make that its specification is that 50～800mg/ props up, its pH value is between 3.5～7.5.

Transfusion of puerarin hydrate and preparation technology thereof: the puerarin hydrate adds injection water and pharmaceutically acceptable additives, for example: add pharmaceutically acceptable cosolvent, pharmaceutically acceptable isoosmotic adjusting agent, pharmaceutically acceptable pH regulator agent, pharmaceutically acceptable antioxidant, noble gas, the sterilization high-capacity injection is made in filtration, degerming, make that its specification is 100～800mg/ bottle, its pH value is between 3.5～7.5.

In the formulation preparation of puerarin hydrate of the present invention, employed pharmaceutically acceptable solubilizing agent or cosolvent can be water, ethanol, propylene glycol, glycerol, 1,3 butylene glycol, polysorbas20-80, Macrogol 200-1000, polyvinylpyrrolidone, sodium lauryl sulphate, pharmaceutically acceptable amide is (as nicotiamide, thiourea, urethane etc.), aminated compounds is (as ethylenediamine, diethylamine, diethanolamine, triethanolamine, meglumine, Portugal's ethamine etc.), basic amino acid (L type, the arginine D type or racemization, lysine, histidine, citrulline etc.), cyclodextrin, the preferred B-cyclodextrin of cyclodextrin, the 2-hydroxypropyl (2-HP-β-CYD), the 3-hydroxypropyl (3-HP-β-CYD), sulfobutyl ether-beta-schardinger dextrin-(one or more that SBE-β-CD) waits.

Puerarin crystalline hydrate and cyclodextrin or cyclodextrin derivative form water miscible clathrate, and wherein the quality of puerarin crystalline hydrate and cyclodextrin or cyclodextrin derivative or weight ratio are 1: 5 to 1: 60;

Pharmaceutically acceptable antibacterial can be one or more of benzalkonium chloride, benzalkonium bromide, benzyl ethamine, Xi Bailin, ethanol, phenylmercuric nitrate, thimerosal, benzyl alcohol, phenethanol, phenoxyethanol, chlorobutanol, degree rice phenol, parabens (methyl parahydroxybenzoate, ethylparaben, butyl p-hydroxybenzoate etc.), sorbic acid or its pharmaceutical salts etc.

Its pharmaceutically acceptable pH regulator agent can be pharmaceutically acceptable mineral acid or organic acid; inorganic base or organic base; also can be generalized lewis acid or alkali; can contain one or several; can be hydrochloric acid; phosphoric acid; propanoic acid; acetic acid and acetate; as sodium acetate etc.; lactic acid and lactic acid pharmaceutical salts; the citric acid pharmaceutical salts; sodium carbonate; sodium bicarbonate; potassium bicarbonate; sodium hydroxide; potassium hydroxide; phosphate; tartaric acid and pharmaceutical salts thereof; Borax; boric acid; succinic acid; caproic acid; adipic acid; fumaric acid; maleic acid; the trihydroxy aminomethane; diethanolamine; ethanolamine; isopropanolamine; diisopropanolamine (DIPA); 2-amino-2-(methylol) 1; ammediol amine; 1; the 2-hexamethylene diamine; N-methyl Fructus Vitis viniferae amine; diisopropylamine and their salt; multi-hydroxy carboxy acid and pharmaceutical salts are as glucuronic acid; gluconic acid; lactobionic acid; malic acid; threonic acid THREONIC ACID.; glucoheptonic acid; taurine; aminoacid and amino acid salts; phosphate buffer; in the borate buffer etc. one or several.

Its pharmaceutically acceptable antioxidant and stabilizing agent (comprising chelating agent) can be sulfurous acids, sulphite, bisulfites, pyrosulfite, dithionite, thiosulfate, the organosulfur compound thiourea, glutathion, dimercaptopropanol, BAL, TGA and salt, 2-mercaptopropionic acid and salt, thio-2 acid and salt, phenol compound, as gallic acid and salt, caffeic acid, the caffeiate, ferulic acid, ferulate, the di-t-butyl Pyrogentisinic Acid, 2, the 5-resorcylic acid, 2,5-resorcylic acid salt, the phenol or derivatives thereof, salicylic acid or its salt; Aminoacid with and salt; Ascorbic acid and Ascorbate, arabo-ascorbic acid and erythorbate, nicotiamide, tartaric acid, nitrate, phosphate, acetic acid pharmaceutical salts, citrate, taurine, EDTA and edta salt, as EDTA disodium, EDTA four sodium, N-two (2-ethoxy) glycine, cyclodextrin; (((SBE-β-CD) etc. is with in first-class one or several for 3-HP-β-CYD), sulfobutyl ether-beta-schardinger dextrin-for 2-HP-β-CYD), 3-hydroxypropyl for the preferred B-cyclodextrin of cyclodextrin, 2-hydroxypropyl.

Its pharmaceutically acceptable osmotic pressure regulator can be one or more in glucose, fructose, xylitol, sorbitol, mannitol, Nulomoline, maltose, dextran, sodium chloride, potassium chloride, sodium lactate, the boric acid etc.

Source and the degerming mode of reducing phlegm and internal heat can be the active carbon that adds dosing amount 0.005～3% source of reducing phlegm and internal heat, and microporous filter membrane degerming and pressure sterilizing also can adopt heat sterilization, the source of reducing phlegm and internal heat.In the hyperfiltration process, that ultrafilter can be selected for use is flat, rolling, tubular type, doughnut formula or circle boxlike etc., preferred rolling and doughnut formula ultrafilter, can adopt and hold back relative molecular mass is after 50,000 to 300,000 filter membrane is removed most of heat generation material and antibacterial, adopt the filter membrane of holding back relative molecular mass 1000～30000 to remove the residue thermal source again, preferably hold back the filter membrane of relative molecular mass 4000～20000.

Cardiovascular and cerebrovascular vessel of the present invention and opthalmological, be applicable to the application in the medicine of following treatment that caused humans and animals is infected of preparation or prevention: be used for hypertension in preparation, coronary heart disease, pulmonary heart disease, heart failure, angina pectoris, myocardial infarction, cardiogenic shock, arrhythmia, myocarditis, ischemic encephalopathy, cerebral infarction, cerebral thrombosis and sequela thereof, improve cerebral circulation, megrim caused by vertebra-fundus artery blood supply insufficiency, syndrome behind the deep vein thrombosis of lower extremity, diabetes, the diabetic complication diabetic nephropathy, diabetic peripheral neuropathy, retinopathy, retinal artery occlusion, vein obstruction, sudden deafness, reduce intraocular pressure, the control glaucoma, the application in the medicine of treatment such as blood lipid regulation or prevention.

Consumption usage: generally speaking, in the adult; get the freeze-dried powder of puerarin hydrate 0.020～0.6g of the present invention or little liquid drugs injection in 20～500 milliliters of 0.9% sodium chloride or 5～10% glucoses; do intravenous injection or instillation; every day 1～2 time; 0.9% sodium chloride of puerarin hydrate or the route of administration of 5～10% glucose great transfusion preparations are intravenous injection, and dosage is the same; Intramuscular injection: get medicine 0.020～0.6g freeze-dried powder of the present invention and be dissolved in the water for injection, intramuscular injection, every day 1～2 time; Child's amount of reducing by half is above to be used.Through gastrointestinal administration dosage, be 0.020～0.6g/ time generally speaking, every day 1～3 time.

The usage of eye drop: 1～2 of adult's usual amounts 1% puerarin eye drop one time, splash in the eyelid, closed order 3～5 minutes.Every day 1-3 time.The dosage of gel for eye use is with reference to the dosage of eye drop.

Description of drawings

Fig. 1 is the thermal analysis curue spectrum of puerarin 1 hydrate.

Fig. 2 is the thermal analysis curue spectrum of puerarin hydrate.

Fig. 3 is the thermal analysis curue spectrum of puerarin 1 hydrate.

Test condition: the Setsys of Setaram company 16, the about 5mg of sample size, programming rate: 10K/min, N ₂Flow velocity: 50ml/min is about room temperature～400 ℃.

The specific embodiment

The preparation of embodiment 1 puerarin 1 hydrate is in reaction vessel, to do powder of Radix Puerariae 80g carries 3 times with ethanol 80% aqueous solution 400ml backflow, merge extractive liquid,, coarse filtration is again with 0.22 μ m filtering with microporous membrane, filtrate concentrates, get Radix Puerariae extractum, add water, regulate PH=3-5 with dilute hydrochloric acid, it is between the 6-7 that the reuse sodium bicarbonate solution is regulated PH, filtering, is that solvent extracts with n-butyl alcohol and hexone then, and the extract evaporate to dryness gets solid, with water, methanol, formic acid, acetonitrile is a recrystallisation solvent, carry out recrystallization 3 times, filter, washing, about 80 ℃ dryings of solid 4 hours get off-white color crystalline powder 1.8g; Fusing point: 208.4～212.5 ℃ of decomposition (not proofreading and correct), ultraviolet spectra: λ ^CH3OH _Max250nm, [a] _D ²¹+ 18.14 ℃ (c=1, methanol), ESI-MS:m/z:417[M+1] ⁺Infrared spectrum: v ^KBr _MaxCm ^-13381,1627,1588,1515,1446, it is 4.19% that the Ka Shi method is measured moisture, TG-DTA: platform is weightless about 4.13%, and result's (theoretical value 4.15%) that this and sample contain 1 water of crystallization (sees accompanying drawing 1) in range of error; Elementary analysis measured value: C 57.99, H 5.26; Theoretical value: C 58.06, H 5.10.

The preparation Radix Puerariae extractum or the Radix Puerariae total flavones 30g of embodiment 2 puerarin crystalline hydrates, add water, heating is approximately to 50-70 ℃, 0.15-0.24 the ceramic membrane filter of μ m, filtrate are crossed neutral alumina chromatographic column, eluting, the D101 macroporous resin adsorption, wash with water, the TLC monitoring, reuse 30-75% ethanol water carries out eluting, finishes up to its eluting, filters, concentrating under reduced pressure, leaving standstill, is recrystallisation solvent with water, acetone, ethanol, acetonitrile, carries out recrystallization 2 times, place, filter, washing is with the solid drying of gained; About 80 ℃ dry 4-6 hour, off-white color solid 6.6 grams, fusing point: 227.8～231.8 ℃ (proofreading and correct), ultraviolet spectra: λ ^CH3OH _Max250nm; [a] _D ²¹+ 18.14 ℃ (c=1, methanol), ESI-MS:m/z:417[M+1] ⁺Infrared spectrum: v ^KBr _MaxCm ^-13380,1626,1586,1516,1446; It is 4.01% that the Ka Shi method is measured moisture, TG-DTA: platform is weightless about 3.49%, and result's (theoretical value 4.15%) that this and sample contain 1 water of crystallization (sees accompanying drawing 2) in range of error; Elementary analysis measured value: C 58.13, H 5.21; Theoretical value: C 58.06, H 5.10.

Dried powder of Radix Puerariae 90g about preparation 40 orders of embodiment 3 puerarins 1 hydrate, add the ultrasonic and reflux, extract, of water 400ml 3 times, merge extractive liquid,, through coarse filtration, again through microporous filter membrane or the ceramic membrane filter of 0.22 μ m, filtrate is then between 25-65 ℃, 0.15-2MPa down (but the pressure dynamic adjustments, or along with test is carried out and increased pressure reduction), ultrafilter membrane (the ps hollow fiber uf membrane assembly of holding back relative molecular mass 4000～50000 adopted, fiber aperture 0.011 μ m, internal diameter is 1.1mm) to filter, filtrate is filtered through holding back the NF membrane of relative molecular mass more than 200 again, concentrates, dissolve with 90% methanol aqueous solution, add acetic acid, acetonitrile is a recrystallisation solvent, places crystallization, sucking filtration, washing; As above method is solvent recrystallization 3 times with methanol, water, acetic acid, acetonitrile, with dry 4～6 hours of about 80 ℃ of the solids of gained, off-white color crystalline solid 1.6 grams, fusing point: 215.5～218.6 ℃ (proofreading and correct), ultraviolet spectra: λ ^CH3OH _Max250nm, [a] _D ²¹+ 18.14 ℃ (c=1, methanol), ESI-MS:m/z:416,399,360,350,297,267,254; Infrared spectrum: v ^KBr _MaxCm ^-13380,1625,1587,1515,1446; It is 4.21% that the Ka Shi method is measured moisture, TG-DTA: platform is weightless about 4.07%, and result's (theoretical value 4.15%) that this and sample contain 1 water of crystallization (sees accompanying drawing 3) in range of error; Elementary analysis measured value: C 58.15, H 5.02; Theoretical value: C 58.06, H 5.10.CH ₃OH

Dried powder of Radix Puerariae 90g about the preparation 20-60 order of embodiment 4 puerarin hydrates, add water 400ml supersound extraction 3 times, merge extractive liquid,, through sand mold pump coarse filtration, again through microporous filter membrane or the ceramic membrane filter of 0.22 μ m, filtrate is then between 25-65 ℃, 0.15-2MPa down (but the pressure dynamic adjustments, or along with test is carried out and increased pressure reduction), ultrafilter membrane (the ps hollow fiber uf membrane assembly of holding back relative molecular mass 3000～50000 adopted, fiber aperture 0.011 μ m, internal diameter is 1.1mm) to filter, filtrate is filtered through holding back the NF membrane of relative molecular mass more than 200 again, concentrates, dissolve with 90% methanol aqueous solution, add formic acid, acetic acid, water is recrystallisation solvent, places crystallization, sucking filtration, washing; As above method, with methanol, water, acetic acid, acetonitrile is solvent recrystallization 3 times, with dry 4～6 hours of about 50 ℃ of the solids of gained, get off-white color crystalline solid 1.6 grams, 50 ℃ of dryings got the off-white color crystalline powder about 2～4 hours again, soluble in water, fusing point: 226.5 ℃ (decompose, do not proofread and correct), ultraviolet spectra: λ ^CH3OH _Max250nm, [a] _D ²¹+ 18.14 ℃ (c=1, methanol), ESI:m/z:416[M] ⁺, 399,360,350,297,267,254; Infrared spectrum: v ^KBr _MaxCm ^-13378,1624,1585,1516,1446, it is 5.22% that the Ka Shi method is measured moisture, and the platform before heat is analyzed TG-DTA:150 ℃ is weightless about 5.32%, and this and sample contain result's (theoretical value 5.13%) of 1.25 water of crystallization in range of error.

Embodiment 5 gets puerarin 1 hydrate 20g, add arginine 10g, mannitol or xylitol 2.0～5g add fresh water for injection 400ml stirring makes dissolving, regulate pH value in 4.0～6.8 scope with citric acid or liquor sodii citratis, add activated carbon 0.01～0.5% (W/V) and stir 15～45min, filter, hold back the ultrafiltration membrance filter of relative molecular mass 3000～8000 with 0.22 micron filtering with microporous membrane or employing, by 100,200mg/ bottle or 300mg/ bottle or 400mg/ bottle or 600mg/ bottle (in puerarin) packing, jump a queue, lyophilization, tamponade, check, get finished product.

Embodiment 6 gets puerarin hydrate 20g, with mannitol 10g, meglumine 12g, Tween 80 2g, add 40～60 ℃ of water for injection 320ml stirrings and make dissolving, regulate pH value in 4.0～6.5 scope with gluconic acid or liquor sodii citratis, add activated carbon 0.01～0.5% (W/V) and stir 15～45min, filter, add water for injection to 400ml, hold back the ultrafiltration membrance filter of relative molecular mass 2000～8000 with 0.22 micron filtering with microporous membrane or employing, by 100,200mg/ bottle or 300mg/ bottle or 400mg/ bottle or 600mg/ bottle (by puerarin) packing, jump a queue, lyophilization, tamponade, be up to the standards, get finished product.

Embodiment 7 is added to puerarin hydrate 10g among the propylene glycol 90ml, adds tween 80 2ml again, stirring makes molten, add the fresh water for injection of 80ml, stir, add sodium pyrosulfite 0.1g, EDTA disodium 0.1g in the stirring, 2M lactic acid and sodium lactate are regulated pH value in 4.0～6.3 scope, add activated carbon 0.5% (W/V) and stir 15～45min, filter, measure content and pH, moisturizing to solution total amount is about 200ml, filters with 0.22 micron filtering with microporous membrane, logical nitrogen, by the embedding of 2～20ml/ bottle, sterilization, after the assay was approved finished product.

Embodiment 8 puerarins, 1 hydrates (by puerarin) 10g; glycerol 100ml; tween 80 2ml; cysteine hydrochloride 0.2g; glycine 0.4g; taurine 0.5g; 3-hydroxypropyl 2g; EDTA disodium 0.1g; be added in the fresh water for injection of 250ml; stirring makes dissolving; 2M gluconic acid and gluconic acid sodium salt are regulated pH value in 4.0～6.6 scope; add activated carbon 0.5% (W/V) and stir 15～45min; filter; moisturizing to solution total amount is 400ml; with 0.22 micron filtering with microporous membrane, logical nitrogen; by the embedding of 2～10ml/ bottle; sterilization; get finished product after the assay was approved.

After the preparation of embodiment 9 puerarin hydrate high-capacity injections is dissolved fully with the fresh water for injection of 4.5L puerarin hydrate (in dry product) 10.1g; add sodium pyrosulfite 1g; glucose 250g; taurine 2g; EDTA disodium 0.2g; sodium dihydrogen phosphate or disodium phosphate soln with 4M are regulated pH value in 4.0～6.8 scope; the active carbon that adds dosing amount 0.02～0.5% (W/V); about heated and stirred 15～30 minutes; filtering decarbonization is surveyed content; pH value, moisturizing to solution is 5L; hold back the ultrafiltration membrance filter of relative molecular mass 4000～8000 again through 0.22um microporous filter membrane fine straining or employing; through the semi-finished product chemical examination, treat its content; after pH value and clarity were qualified, logical nitrogen embedding was in the vial of 50ml or 100ml or 200ml or 250ml; pressure sterilizing 30 minutes; put cold, finished product inspection, the packing promptly.

The preparation of embodiment 10 puerarin hydrate sodium chloride transfusion: with puerarin hydrate (in puerarin) 10g, sodium chloride 45g, hydrochloric acid L-cysteine 1g, sodium pyrosulfite 1g, glycine 2g, EDTA disodium 0.2g, join in the fresh water for injection of 4.5L, the control temperature is in 80 ℃, stirring makes dissolving fully, citric acid or liquor sodii citratis with 2M are regulated pH value in 4.0～7.0 scope, the active carbon that adds dosing amount 0.1%, about heated and stirred 15～30 minutes, filtering decarbonization, survey content, pH value, moisturizing to solution is 5L, hold back the ultrafiltration membrance filter of relative molecular mass 4000～8000 again through 0.22um microporous filter membrane fine straining or employing, detect clarity, after particulate matters etc. are qualified, logical nitrogen embedding is in the vial of 50ml or 100ml or 200ml or 250ml, sterilization, finished product inspection, packing is promptly.

Embodiment 11 puerarins, 1 hydrate tablets (200mg/ sheet)

Prescription: puerarin 1 hydrate 200g

Microcrystalline Cellulose 80g

Carboxymethyl starch sodium 20g

10% 30 POVIDONE K 30 BP/USP-30 ethanol water (7: 3) solution is an amount of

Micropowder silica gel 1g

Puerarin 1 hydrate, microcrystalline Cellulose, carboxymethyl starch sodium are crossed 100 mesh sieves, 30 POVIDONE K 30 BP/USP with 10%-30 ethanol water (7: 3) solution is made soft material in right amount, crosses the 18-24 mesh sieve and granulates drying, after crossing 14-20 mesh sieve granulate, add micropowder silica gel mixing, tabletting, check, packing.

Embodiment 12 puerarins, 1 hydrate capsules (100mg/ grain)

Prescription: puerarin 1 hydrate 100g

Microcrystalline Cellulose 50g

Lactose 10g

Gelling starch 10% is an amount of

Magnesium stearate 2g

Puerarin 1 hydrate, microcrystalline Cellulose, lactose are crossed 100 mesh sieves, and the gelling starch with 10% is made soft material in right amount, and the 18-24 mesh sieve is granulated excessively, and drying behind the 14-20 mesh sieve granulate, adds magnesium stearate and mixes the fill capsule excessively.

The granule of embodiment 13 puerarin crystalline hydrates (200mg/ bag)

Prescription: puerarin crystalline hydrate 200g

Mannitol 100g

Lactose 20g

Cyclamate 2g

Solid edible essence 1g

Xanthan gum 2g

8% 30 POVIDONE K 30 BP/USP-30 ethanol water is an amount of

Puerarin crystalline hydrate, mannitol, lactose, cyclamate, edible essence are crossed 100 mesh sieves, 30 POVIDONE K 30 BP/USP with 8%-30 ethanol water is made soft material in right amount, crossing the 18-24 mesh sieve granulates, dry below 60 ℃, the xanthan gum that added 100 mesh sieves, behind the mistake 14-20 mesh sieve granulate, mixing, the xanthan gum that added 100 mesh sieves divides packing.

The eye drop of embodiment 14 puerarins 1 hydrate

Puerarin 1 hydrate 5.23g, glycerol 50ml, 30 POVIDONE K 30 BP/USP-305g are added proper amount of water for injection, after stirring makes its dissolving, again sodium sulfite 0.6g, EDTA disodium 0.3g, 5% Benza 1ml, sodium chloride 3.1g are joined in the above-mentioned solution, stirring makes dissolving, transfer to 6.0-7.0 with dibastic sodium phosphate and disodium phosphate soln, add water for injection then to 500ml, stir, detect, to clear and bright, be sub-packed in the sterilized clean eyedrops bottle sterilization with 0.45-0.2 μ m filtering with microporous membrane, put cold, promptly.

Embodiment 15 adds proper amount of water for injection with puerarin 1 hydrate 5.23g, glycerol 50ml, 30 POVIDONE K 30 BP/USP-305g, after stirring makes its dissolving, again sodium pyrosulfite 0.6g, EDTA disodium 0.3g, mannitol 4g are joined in the above-mentioned solution, stirring makes dissolving, transfer to 6.0-7.0 with borate buffer, add water for injection then to 500ml, stir, detect, to clear and bright, be sub-packed in the eyedrops bottle of sterilized clean single dose sterilization with 0.45-0.2 μ m filtering with microporous membrane, put cold, promptly.

The gel for eye use of embodiment 16 puerarins 1 compound

Prescription: puerarin 1 hydrate 5.2g, carbomer 934 or carbomer 971 2g, glycerol 10ml, mannitol 6g, EDTA disodium 0.1g, Borax 2g, boric acid 1g, ethylparaben 0.3g, dibastic sodium phosphate and disodium phosphate soln are regulated pH value in right amount, are added the injection water to 500ml.

Carbomer 934 is made its dispersion, swelling with an amount of water for injection, add glycerol, heating in water bath, stirring and evenly mixing, the puerarin hydrate, glycerol, mannitol, EDTA disodium, Borax, boric acid, the ethylparaben that add recipe quantity join in an amount of water for injection, stir, regulate about pH=6.0-6.7 with the PH regulator, make dissolving, it is joined in the gel-type vehicle of aqueous dispersion, stir, add water to full dose, be stirred to even, filtration, sterilization, packing, promptly.

Pharmacodynamics test

1, aconitine is brought out the ARR effect of rat

20 of SD rats, male and female are regardless of, and body weight 185 ± 19g is divided into 2 groups at random, and intravenous injection gives normal saline, puerarin 1 hydrate (add cosolvent etc. and be configured to injectable solution) 200mgkg respectively ^-13% pentobarbital sodium 40mgkg ^-1After the anesthesia, dorsal position is fixed on the operating-table.The sublingual vein drug administration by injection, behind the 6min, constant speed intravenous injection 0.0025% aconitine, 2 μ gmin ^-1(0.08mlmin ^-1), the record chamber of appearance early (VP), chamber speed (VT), quiver (VF) in the chamber and the time of dead (CA), calculates corresponding dosage.

The result shows that puerarin 1 hydrate group can significantly increase the consumption that aconitine causes rat VP, VT, VF and CA, and PW group compares with the normal saline group that there were significant differences, sees Table 4.

Table 4. puerarin 1 hydrate to aconitine bring out the ARR effect of rat (

N=10)

The PW group is compared (t check) with the normal saline group: p＜0.05

2, puerarin 1 hydrate is to the active inhibitory action of ACE

24 of Wistar rat 280～300g are divided into 3 groups at random, 8 every group, one group is matched group, and in addition two groups are respectively dosage 100mg/kg, the administration group of 200mg/kg (add cosolvent etc. be configured to injectable solution), intraperitoneal injection, the normal saline that matched group gives, broken behind the administration 60min with 1% pentobarbital sodium abdominal cavity fiber crops, open the breast heart extracting blood, the blood sample room temperature leaves standstill 30min, 3500r/min low-temperature centrifugation 10min gets about supernatant-65 ℃ and preserves, and is to be measured.

The active mensuration of ACE: list of references [Wang Fumin, etc. Chinese journal of medical examination, 1983,6 (3): 145～151], statistical procedures as a result: the result with Variance analysis and t check are done in expression, data.Experiment shows: rat blood serum ACE activity, and puerarin 1 hydrate is compared with matched group, and rat blood serum ACE activity is concentration dependent reduction (P＜0.01) in the test group, and puerarin 1 hydrate of variable concentrations obviously suppresses the ACE activity.The results are shown in Table 5.

Table 5. puerarin 1 hydrate is to the active inhibitory action of ACE

Since the last century the eighties, because the extensive use of angiotensin converting enzyme inhibitor (ACEI) aspect clinical cardiovascular, and the discovery and the research of local renin-angiotensin system (RAS), make people recognize RAS, especially the local RAS of cardiovascular plays crucial effects in many cardiovascular disease.ACE is positioned at the center of RAS, is the active rate-limiting enzyme of RAS, angiotensin I (Ang I) can be changed into Angiotensin II (Ang II).Confirmed that now Ang II has arteria coronaria contraction and the effect of positivity cardiac muscle power to heart; Can directly or indirectly vascular smooth muscle be shunk by promoting catecholamine to discharge; And can stimulate smooth muscle cell proliferation, impel the myocardial cell hypertrophy; Participate in ischemia reperfusion injury, cause arrhythmia.ACE can also hydrolysis delay sharp tripe, and the latter is for expanding angiogenic substance.

Under the normal physiological conditions, intravital Angiotensin II is an important modulability material of keeping circulation blood pressure, heart and vascular morphology.Under pathological states such as hypertension or insulin resistant, hypertension blood plasma or insulin level rising etc., and stimulate the movable enhancing of renin-angiotensin system (RAS), make the nervous plain H of blood plasma and local organization medium vessels generate too much.Too much Angiotensin II promotes smooth muscle cell myosin light chain phosphorylation, direct vasoconstrictive smooth muscle, and stimulate body tissue to produce too much active oxygen, the nitric oxide (NO) that the deactivation endotheliocyte discharges, make the NO lowering of concentration, increase sympathetic activity simultaneously, make the peripheral circulation resistance increase and cause elevation of the blood pressure.

On the other hand, too much Ang II promotes the formation of insulin resistant by destroying the insulin signaling transduction and to hemodynamic influence, reducing the transhipment of Skeletal Muscle Cell to glucose, forms vicious cycle.In addition, too much AngII stimulates the expression of transforming growth factor-b1 (TGF-b1) and I type Plasminogen activator, the multiple severe complication of the formation of these materials and insulin resistant such as hypertension, heart reconstruction and atherosclerotic substantial connection arranged.

1), suppress the nervous plain II invertase of circulation medium vessels system the pharmacological action that studies show that ACEI is many-sided, mainly comprises:; 2), suppress tissue and hypertensin system; 3), reduce peripheral nerve unit and discharge norepinephrine; 4), reduce endothelial system and form Endothelin; 5), the formation that increases Kallidin I and expand the prostacyclin of blood vessel; 6), reduce the secretion of aldosterone, water-sodium retention is reduced.

Angiotensin converting enzyme inhibitor can play following effect by above-mentioned effect: bring high blood pressure down; Reverse heart and blood vessel structure, alleviate left ventricular hypertrophy and weight, resistance vessel middle level/tube chamber footpath ratio is reduced, thereby reduce after hypertension, the myocardial infarction and the myocardial cell hyperplasia and the plumpness of cardiac dysfunction, improve the prognosis after the myocardial infarction, reduce unexpected incidence rate and mortality rate, reduce or prevent the infringement of hypertension target organ, treatment congestive heart failure and cardiac dysfunction, reduce the generation and the mortality rate of congestive heart failure, increase the compliance of trunk, the minimizing atherosclerosis forms and resists myocardial ischemia; Lower insulin resistant, improve sensitivity insulin; Effect with protection kidney particularly the diabetic hypertension patient, because the increased pressure of afferent glomerular arteriole, the blood flow that enters are also many, must cause the height of glomerule to filter state, and Angiotensin II generates and reduces, and reaches the effect of renal function protecting.

The present invention's experiment shows that the puerarin crystalline hydrate not only has antiarrhythmic effect; and the ACE activity all there is inhibitory action; prompting puerarin crystalline hydrate can reduce the generation of AngII and the degraded of Kallidin I; therefore; use the puerarin crystalline hydrate and help to prevent and treat arrhythmia; hypertension; congestive heart failure; the remodeling ventricle that myocardial ischemia and myocardial infarction etc. and above-mentioned reason cause; myocardial hypertrophy; hyperlipemia; the non-diabetic nephropathy; type 1 diabetes nephropathy and albuminuria; the protection renal function, and prevent that atherosclerosis from effects such as development taking place.

3, the puerarin crystalline hydrate is to the influence of rat cerebral ischemia

36 of Wistar male rats, body weight 250～300g is divided into 3 groups at random: sham operated rats, ischemia filling group again and medicine group of the present invention, 12 every group.Medicine group of the present invention: adopt the suspension oral gavage of the normal saline of puerarin 1 hydrate, dosage is 400mg/kg, and volume is 2ml, every day twice, continuous 7 days, implements cerebral ischemia hands art behind the last administration 60min; Sham operated rats and ischemia filling group are again given isometric normal saline 2ml respectively.

Rat is with 10% chloral hydrate (3.5ml/kg) intraperitoneal injection of anesthesia, and dorsal position is fixed, and reference literature is fastened improved method modeling [Shanghai Communications University's journal (medicine) 2007,27 (10): 1218-1222] with Zea Longa line; Sham operated rats bolt line only inserts 10mm, the same model group of all the other steps, and the animal back of reviving operation side Homer occurs and seeks peace and the lateral body dyskinesia is model prepares successfully.Modeling success back medicine group of the present invention administration in a day 2 times, dosage is the same, and sham operated rats and ischemia filling group are again given isometric normal saline 2ml respectively.2h extracts line out after the modeling, pours into after 24 hours again and puts to death rat, gets brain rapidly and weighs.Make 10% brain tissue homogenate's liquid with normal saline under the low temperature, low-temperature centrifugation, the centrifugal 10min of 3000r/min, abandon precipitation, get supernatant, measure SOD, MDA, NO, NOS content or activity respectively, the results are shown in Table 6 by the description of commercially available test SOD, MDA, NO, NOS corresponding reagent box.

Table 6. medicine of the present invention is to the influence of NO, NOS, MDA, SOD in the rat cerebral ischemia word perfusion cerebral tissue

The result shows, compares with sham operated rats, and SOD is active in the ischemia-reperfusion model group rat cerebral tissue obviously reduces, active obviously increase (P＜0.01) of MDA, NO content and NOS; Compare with the ischemia-reperfusion model group, SOD is active in the medicine group of the present invention rat cerebral tissue obviously raises, and NOS is active to be reduced, and MDA, NO content obviously reduce (P＜0.01)

Studies show that NO is a biologically active courier micromolecule, can pass freely through cell membrane, act on the target molecule in the born of the same parents.In vivo, very fast oxidized deactivation after NO generates is present in the inside and outside liquid of cell with the form of nitrate anion and nitrite anions.The NO of low concentration can distend the blood vessels, and suppresses platelet aggregation and adhesion, makes the ion channel downward modulation of glutamic acid regulation and control, prevent intracellular calcium overload, thereby pair cell has protective effect; But under high concentration, NO can generate super oxygen nitrite ion with the superoxide anion reaction, and super oxygen nitrite ion can be degraded to OH ^-And NO ^2-Free radical makes cell membrane generation lipid peroxidation, puts down at the film water of cell and causes intensive neurotoxicity, even cause neuronal death etc.

Studies show that NO plays an important role in the morbidity of cerebral ischemic injury, several minutes NO content obviously increases behind the ischemia, slowly descends afterwards, raises once more in flush phase NO again.Its mechanism may be neuronal damage after the cerebral ischemia, the cell membrane process of depolarization strengthens, and excitatory amino acids such as presynaptic glutamic acid generate and roll up, and born of the same parents' extracellular glutamate concentration is raise, activate N-methyl D-aspartic acid (NMDA) receptor, nmda receptor makes postsynaptic Ca after activating ²⁺Interior stream increases, and activates nitricoxide synthase NOS, promotes the generation of NO; Its two, after the cerebral ischemia, ATP consumes in a large number, the protein kinase activity that causes energy metabolism impairment and cAMP to rely on descends, because the NOS dephosphorylation makes the increased activity of NOS, thereby promotes the generation of NO.

Nitricoxide synthase is the synthetic rate-limiting enzyme NOS of nitric oxide.Early stage in cerebral ischemia, the NO that NOS mediation generates improves the blood supply of ischemic region by blood vessel dilating, has protective effect in short-term, but along with a large amount of generations of NO, because of mediation neurotoxicity effect and be dominant very soon, makes the cerebral ischemia aggravation at ischemic region NOS; To late period, cerebral ischemia brain tissue impairment, inflammatory reaction stimulate macrophage, neural microglia, neuron etc. can produce induced NOS in a large number, induced NOS can slowly and lastingly produce NO in large quantities, and excessive N O produces and discharges, and increases the weight of the neuron infringement.Therefore, the generation that reduces induced NOS can obviously reduce the release of NO, thereby reduces the cytotoxic effect of NO, plays the effect of protection brain cell.

The result of study of this experiment shows that after the cerebral ischemia, NO and NOS content significantly increase in the cerebral tissue, illustrates that NO and NOS have participated in the pathogenesis of cerebral ischemia reperfusion injury, give Drug therapy of the present invention after, NO and NOS content obviously descend.Show that thereby medicine of the present invention is by regulating the protective effect of bringing into play cerebral ischemia reperfusion injury to NOS activity and NO content.

Cerebral ischemia and reperfusion injury can cause the brain cell membrane lipid peroxidation, produce too much oxygen-derived free radicals, the damage brain cell membrane.Owing to the provisioning deficiency of tissue oxygen and energy metabolism, ATP generates minimizing during cerebral ischemia, and ionic pump lost efficacy, Na ⁺-K ⁺-atpase activity reduces, and makes a large amount of Na ⁺Interior stream, K ⁺Outflow, Cl ^-Enter in the cell with H2O is passive, cause the acute permeability swelling of neurocyte death.Na ⁺Interior stream and K ⁺Outflow causes cell membrane potential decline to produce depolarization, voltage-dependent Ca ²⁺Channel opener, Ca ²⁺Stream in a large amount of; Simultaneously because K ⁺, Protein kinase C and mediator effects such as release, receptor dependency Ca ²⁺Channel opener, Ca ²⁺Stream in a large amount of.Intracellular calcium overload can cause oxygen-derived free radicals to produce to be increased, arachidonic acid metabolic strengthens, the excitatory amino acid mediator discharges increase etc., and excitatory amino acid can cause Ca in the cell again ²⁺Overload causes neuronal apoptosis.Wherein the oxygen-derived free radicals increase is a key factor of ischemical reperfusion injury.

Various oxygen-derived free radicals are attacked the unsaturated fatty acid in the biomembrane, cause lipid peroxidation, form a series of lipid free radical and catabolite MDA thereof.MDA is as the metabolite of oxygen-derived free radicals and biomembrane unsaturated fatty acid generation lipid peroxidation, and the variation of its content can reflect oxygen-derived free radicals changes of contents in the tissue indirectly.

SOD is the main enzymatic system of defense to antioxidant radical in the cell, and it removes ultra-oxygen anion free radical by the disproportionation mode, and the protection organism is avoided the attack of free radical.The height indirect reaction of SOD vigor body remove the ability of oxygen-derived free radicals.

Therefore, medicine of the present invention can significantly reduce the lipid peroxide MDA of ischemic region cerebral tissue, and NO content improves SOD content in the rat cerebral tissue, increases the ability of removing free radical, reduces the NOS activity, can alleviate the infringement of radical reaction to cerebral tissue.This shows; medicine of the present invention suppresses the number of ways such as generation of free radical; protection cell membranous structure; help to reduce cerebrovascular permeability and improve hemorheology index. improve the brain microcirculation, oxygen-derived free radicals is brought into play its protective effect to brain to the detrimental effect of cerebral tissue behind the antagonism cerebral ischemia re-pouring.Can be used for treatment or prevention ischemic encephalopathy, cerebral infarction, cerebral thrombosis and sequela thereof, improve brain microcirculation and the cataractous effect of potential control, and be used to prepare treatment and prevent the medicine or the healthy articles for use of corresponding disease.

The beta receptor retardation puerarin crystalline hydrate of puerarin crystalline hydrate dissolves in the suitable solvent or loses water of crystallization in vivo and discharges puerarin or directly act on respective target organ or position in vivo.And puerarin can block femoral vein that isoproterenol makes cat to the diastole effect that contraction produced that methoxamedrine causes, and is dose dependent, and is similar to the propranolol effect, shows that puerarin is the beta receptor retardation to the effect of cat myocardium vessel.In addition, puerarin also has significant antagonism to guinea-pig heart room flesh β1Shou Ti, and guinea-pig tracheal strip beta 2 receptor antagonism is then needed big concentration, shows that its selectivity to β1Shou Ti is better than beta 2 receptor.Zoopery shows that from isolated organ and whole animal level puerarin is a kind of beta receptor antagonist respectively.The effector of known beta receptor is an adenyl cyclase, and receptor stimulating agent makes it to activate, and antagonist then makes it to suppress, puerarin with can suppress the activation of epinephrine fully after beta receptor combines to adenyl cyclase.

Puerarin all has certain hypotensive effect to normal and hypertension animal.Quiet notes puerarin, the blood pressure that can make the anesthesia Canis familiaris L. dose dependent occur descends fast, about effect is kept.The lumbar injection puerarin can obviously reduce the blood pressure and the decreased heart rate of clear-headed spontaneous hypertension rat.

Puerarin has the effect that reduces intraocular pressure to the rabbit ocular hypertension model that is caused by injection dexamethasone under the conjunctive bulbi, between action intensity and the drug level dependency is arranged.And puerarin eye drop intraocular pressure that rabbit ear edge vein fast injection glucose is caused raises inhibitory action arranged, and compare effect with the timolol eye drop similar, shows a kind of comparatively ideal anti-glaucoma medicine of puerarin eye drop.Have clinical experiment to find, puerarin treatment glaucoma all has the effect that reduces intraocular pressure to most primary open-angles, angle-closure and secondary glaucoma; Puerarin can also be prevented and treated cataract by microcirculation improvement and retinal function.

The beta receptor retardation of puerarin makes the puerarin crystalline hydrate show the purposes to hypertensive hypotensive effect, the glaucomatous intraocular pressure effect of reduction and antiarrhythmic effect etc.

The anesthetized mice mesentery microcirculation disturbance that the instillation puerarin can cause antiadrenergic drug; The retinal artery occlusion patient is carried out the shortening of retina fluoroscopic visualization visible microcirculation time simultaneously in the treatment of application puerarin, find that simultaneously Radix Puerariae have tangible enhancement vision, effect such as broaden one's vision, in addition, puerarin is also by improving hemorheology index as reducing whole blood contrast viscosity, erythrocyte electrophoresis, hematocrit and fibrin were prevented and treated retinal artery occlusion originally.In medication, the nail fold microcirculations are in shape always in 2 weeks with 70% patient of puerarin in treating sudden deafness, and audition has raising in various degree.

The patient of the vertebral-basilar artery insufficiency of quiet puerarin of 60 examples, doing well,improving is obvious.Puerarin can suppress atherosclerosis, impels arteriosclerosis, also can regulate cerebral blood flow by the influence to TXB2, NO, ET etc., improves cerebral circulation.Improve hemorheological index, anti-treating cervical spondylosis puerarin can reduce blood viscosity, reduces packed cell volume, shortens erythrocyte electrophoretic time, and erythrocyte sedimentation rate and reduction fibrinogen content slow down.

Someone uses puerarin injection auxiliary treatment cervical spondylosis, the result: it is 98% that clinical total effective rate is organized in treatment, with matched group comparing difference highly significant; Symptom and sign extinction time shorten after the medication of treatment group, and hemorheology detects index: whole blood viscosity, plasma viscosity, Fibrinogen and erythrocyte sedimentation rate precipitation obviously reduce after bringing under control and treating.

Puerarin improves cerebral microcirculation disturbance, control vertebro-basilar arterial ischemia vertigo, treatment cervical spondylosis, and this makes the puerarin crystalline hydrate to show to improve cerebral microcirculation disturbance, the effect of control vertebro-basilar arterial ischemia vertigo, treatment cervical spondylosis.

Discover that the puerarin lumbar injection can significantly reduce rat ethanol intake, mechanism may be relevant with its inhibition mitochondrion aldose reductase.In addition, the someone finds that puerarin is the antagonist or the partial agonist of benzodiazepine receptors, and more alcoholic acid pharmacological actions are brought into play by the brain cell benzodiazepine receptors.

Puerarin is quiet to be annotated in the coronary artery of anesthesia or clear-headed Canis familiaris L., and the coronary vasodilator blood flow is increased, and vascular resistance reduces, and its effect is strengthened along with the increase of dosage.Puerarin can make the speed of heartbeat slow down, and myocardial contraction reduces, but the blood flow of ischemic region CC is reduced, and puerarin produces by reducing the lateral branch coronary resistance the effect of ischemic myocardium.In addition, it can resist the Acute Myocardial Ischemia in Rats that pituitrin causes, may be the result of coronary artery dilator blood vessel.The generation of cardiac muscle lactic acid reduced oxygen consumption and creatine phosphokinase burst size when puerarin can obviously reduce ischemia-reperfusion, and ultrastructure myocardium behind the ischemia-reperfusion is also made moderate progress.Someone uses puerarin treatment coronary heart disease, observe hemorheology emphatically, ambulatory electrocardiogram, conventional leads electrocardiogram, the every index of result has clear improvement before the treatment, show that puerarin can effectively reduce the patients with coronary heart disease hemorheological property, blood vessel dilating improves myocardial ischemia, is a kind of medicine that resists myocardial ischemia safely and effectively.

Because the external in vivo release of puerarin crystalline hydrate with showing the characteristic of the antagonist or the partial agonist of benzodiazepine receptors, also will significantly reduce rat ethanol intake; Also will effectively reduce the patients with coronary heart disease hemorheological property, blood vessel dilating improves myocardial ischemia, becomes a kind of medicine that resists myocardial ischemia safely and effectively, and this also has corresponding evidence from test of the present invention.

Bibliographical information finds that with the quiet notes of puerarin treatment type ii diabetes patient puerarin helps to improve leaching of thrombus source trace albuminuria, delays or stops developing of diabetes glomerular microangiopathy change.48 routine type ii diabetes patients of application puerarins such as Yu Jian treatment observe, puerarin has tangible blood sugar lowering, improves the insulin resistant effect, thinks that puerarin injection is not only treatment type ii diabetes patients with insulin resistance comparatively ideal medicine; Diabetic peripheral neuropathy also there is curative effect preferably.Puerarin may be by blood vessel dilating on the one hand, microcirculation improvement, and increasing blood flow strengthens sanguimotor ability and promotes the biopotency of insulin, promptly improves the sensitivity of insulin; On the other hand, puerarin is by effectively reducing whole blood viscosity, strengthen erythrocyte deformability, improve erythrocyte membrane elasticity, its structure and physical property are improved, and then accelerate matter transportation and make glucose and insulin is striden the film ability, the insulin sensitivity face that further improves the type ii diabetes patient plays blood sugar reducing function.

The diabetes of puerarin and the therapeutical effect of complication thereof make the purposes that the puerarin crystalline hydrate shows the corresponding treatment effect and prepares corresponding prevention and medicine etc.

In addition, medicine of the present invention also will can be used for preparing corresponding treatment or prophylactic medicine to the effects such as reverse of the multidrug resistance of tumors such as gastric cancer.

The variation that is appreciated that a lot of details is possible, and therefore this do not limit the scope of the invention and spirit, and the present invention is not limited to the foregoing description.

Claims

1. cardiovascular and cerebrovascular vessel and opthalmological, it is characterized in that: puerarin crystalline hydrate, molecular formula are C ₂₁H ₂₀O ₉N H ₂O, n=0.8～1.2.

2. cardiovascular and cerebrovascular vessel according to claim 1 and opthalmological is characterized in that: puerarin crystalline hydrate, molecular formula are C ₂₁H ₂₀O ₉N H ₂O, n=0.95～1.0.

3. cardiovascular and cerebrovascular vessel according to claim 1 and opthalmological is characterized in that: the puerarin crystalline hydrate is puerarin 1 hydrate, and molecular formula is C ₂₁H ₂₀O ₉H ₂O.

4. cardiovascular and cerebrovascular vessel and opthalmological preparation method, it is characterized in that: its preparation method comprises following method,

Method A. is in reaction vessel, one or more carry out cold extraction with the low mass molecule alcohol of powder of Radix Puerariae water or C1-C6, supersound extraction, microwave extraction or backflow are carried once to merging for several times, filter, microporous filter membrane or ceramic membrane filter, filtrate concentrates, concentrate Radix Puerariae extractum, add water, regulate PH3-5 with acid, reuse alkali or sodium carbonate or sodium bicarbonate a kind of or several to regulate PH be between the 6-9, filter, use the lower ketones of low mass molecule alcohol or the C3-C8 of C3-C6 then, as hexone, or one or more of the lower member ester of C2-C8 extract for solvent, the extract evaporate to dryness gets solid, add water, the low mass molecule alcohol of reuse C3-C6 or the lower ketones of C3-C8, as hexone, or one or more of the lower member ester of C2-C8 extract for solvent, concentrate, and leave standstill, filter, with water, the lower halogenated hydrocarbon of C1-C6, the lower ketones of C3-C8 is as acetone, the lower member ester of C2-C8, the low mass molecule alcohol of C1-C6 such as methanol, ethanol, isopropyl alcohol, the lower fatty acid of C1-C6, oxolane, one or more of acetonitrile are recrystallisation solvent, carry out recrystallization once or for several times, filter, washing promptly gets the puerarin crystalline hydrate with the solid drying of gained;

Perhaps the water of method B. Radix Puerariae extractum or Radix Puerariae total flavones or Radix Puerariae is carried the pure water extraction concentrate of concentrate or Radix Puerariae, add water or C1-C6 low mass molecule alcohol one or more, microporous filter membrane (comprising ceramic membrane) filters, cross the neutral alumina chromatographic column, silica gel column chromatography and macroporous resin, or polyamide column chromatography or glucose gel resin, the low mass molecule alcohol of water or C1-C6, the lower halogenated hydrocarbon of C1-C6, one or more carry out eluting, concentrated or ultrafilter membrane concentrates, leave standstill, filter, with water, the lower halogenated hydrocarbon of C1-C6, the lower ketones of C3-C8, as acetone, the lower member ester of C2-C8, the low mass molecule alcohol of C1-C6 such as methanol, ethanol, isopropyl alcohol, the lower fatty acid of C1-C6, oxolane, one or more of acetonitrile are recrystallisation solvent, carry out recrystallization once or for several times, filter, washing promptly gets the solid drying of gained and promptly to get the puerarin crystalline hydrate;

Perhaps the water of method C. Radix Puerariae extractum or Radix Puerariae total flavones or Radix Puerariae is carried the pure water extraction concentrate of concentrate or Radix Puerariae, add water or C1-C6 low mass molecule alcohol one or more, through microporous filter membrane, the film of doughnut, ceramic membrane, ultrafilter membrane, once or for several times the filtering or concentrate of one or more of NF membrane, with water, the lower halogenated hydrocarbon of C1-C6, the lower ketones of C3-C8, as acetone, the lower member ester of C2-C8, the low mass molecule alcohol of C1-C6 such as methanol, ethanol, isopropyl alcohol, the lower fatty acid of C1-C6, oxolane, one or more of acetonitrile are recrystallisation solvent, carry out recrystallization once or for several times, filter, washing promptly gets the puerarin crystalline hydrate with the solid drying of gained;

Different step in above-mentioned each method can be used alternatingly, so that preparation puerarin hydrate.

5. the purposes of cardiovascular and cerebrovascular vessel and opthalmological, it is characterized in that: at preparation injection freeze-dried powder or little water needle injection, great transfusion preparation, through the intestinal canal administration preparation, comprise tablet, capsule, granule, through the dosing eyes preparation, comprise the application on eye drop, the gel for eye use.

6. the purposes of cardiovascular and cerebrovascular vessel and opthalmological, it is characterized in that: be used for hypertension in preparation, coronary heart disease, pulmonary heart disease, heart failure, angina pectoris, myocardial infarction, cardiogenic shock, arrhythmia, myocarditis, ischemic encephalopathy, cerebral infarction, cerebral thrombosis and sequela thereof, improve cerebral circulation, megrim caused by vertebra-fundus artery blood supply insufficiency, syndrome behind the deep vein thrombosis of lower extremity, diabetes, the diabetic complication diabetic nephropathy, diabetic peripheral neuropathy, retinopathy, retinal artery occlusion, vein obstruction, sudden deafness, reduce intraocular pressure, the control glaucoma, the application in the treatment of blood lipid regulation or the medicine of prevention.