CN101585858A - Application of glycosylated puerarin derivate and its combination for preventing and treating cardiovascular and cerebrovascular disease - Google Patents

Abstract

The invention relates to a glycosylated puerarin derivate shown in the general formula (I) and its pharmaceutically acceptable salt. In the formula, R is described as the text. A preparation method and a medicament combination containing the compound shown in the general formula (I) of the effective dose are also provided. The combination is used for treating and preventing the cardiovascular and cerebrovascular diseases, diabetic nephropathy and osteoporosis.

Description

Glycosylation puerarin derivate and composition thereof are prevented and treated cardiovascular and cerebrovascular disease in preparation and are used

Technical field

The present invention relates to field of medicaments, be specifically related to a kind of new puerarin derivate and pharmaceutical composition thereof and be used for cardiovascular and cerebrovascular disease in the preparation treatment; Prevent and treat atherosclerosis, blood fat reducing, diabetic nephropathy and to the application of insulin resistant, osteoporosis.

Background technology

Puerarin (Puerarin) is to extract the effective constituent that obtains from the root of kudzu vine (Radix Puerariae), be natural drug commonly used, record in the national drug standards, be mainly used in coronary heart disease, stenocardia, myocardial infarction, ischemic cerebrovascular disease, thrombosis of retinal artery, sudden deafness etc.Puerarin preparation has injection liquid, lyophilized injectable powder, eye drops.The actual use mainly is to be applied to clinically with drug administration by injection, and along with the prolongation of clinical duration of service, some untoward reactions reveal, and this is main because there is following shortcoming: it is fast 1) to eliminate speed in the body, and clinical using dosage is bigger, and oral administration biaavailability is little; 2) because the puerarin water solubility is low, preparation contains solubility promoter mostly to increase the stable of preparation, and insoluble impurities also increases simultaneously, and human body is produced certain untoward reaction, causes drug safety to reduce.Because solubleness is good, pharmacologically active is higher than having for product of the present invention and puerarin, the transformation period prolongs in the body, has remedied the puerarin product defects, and the security of medication is improved, curative effect increases, and is the good new drug product of a development prospect.

Summary of the invention

The derivative and the pharmacy acceptable salt thereof that the purpose of this invention is to provide the new puerarin of a class not only make its water-soluble increase, and it is fast to overcome the interior elimination of body speed, and the bad shortcoming of preparation stability.

Another object of the present invention provides a kind of derivative of the new puerarin of an above-mentioned class and method of pharmacy acceptable salt thereof of preparing, the derivative of puerarin and pharmacy acceptable salt good water solubility thereof can form pharmaceutical composition with pharmaceutically acceptable carrier.

The 3rd purpose of the present invention provides above-mentioned substance at preparation treatment coronary heart disease, stenocardia, myocardial infarction, ischemic cerebrovascular disease; Be used to prevent and treat atherosclerosis, blood fat reducing, diabetic nephropathy and the pharmaceutical composition of insulin resistant, osteoporosis is used, adopt this material to prepare oral preparations tablet and capsule, its dissolution in vitro increases, the prompting bioavailability improves, and it will be further appreciated that can make stable is the preparation of carrier with water.

For finishing above-mentioned three invention tasks, the application's technical scheme is as follows respectively:

The derivative that the purpose of this invention is to provide the new puerarin of a class, its feature is shown in structural formula (I):

Formula (I)

In the formula (I), R is monosaccharide groups or oligosaccharyl, it is characterized in that monosaccharide groups is ring-type 6 carbon sugar or 5 carbon glycosyls, 6 carbon glycosyl such as glucosyl groups, mannose group, the glucal acidic group, galactosyl, galacturonic acidic group, allose base, fructosyl, sorb glycosyl, witch hazel glycosyl, strepto-glycosyl, 2-glucosamine base, galactosamine base, or 5 carbon glycosyls are as Arabic glycosyl, the lysol glycosyl, xylosyl, ribosyl, husband's glycosyl, rhamanopyranosyl, isorhodeose base, celery glycosyl, preferred glucosyl group; Its oligosaccharyl is that 2-5 6 carbon are sugared or/and 5 carbon sugar dehydrating condensation forms oligosaccharyl, as mycose-base, and sophorosyl, new dried orange peel glycosyl, rue glycosyl, locust tree diglycosyl, malt-base, sucrose base, lactose base, rough gentian 3 glycosyls, pectin base, preferred malt-base.When the preferred glucosyl group of R, the compound of bio-transformation is puerarin-7-O-glucoside according to formula (I) compound, and promptly hemiacetal hydroxyl on the glucosyl group and puerarin A encircle 7 hydroxyls on the carbon and form glucosides; When the preferred malt-base of R, the compound of bio-transformation is puerarin-7-O-isomaltosylfructoside, promptly forms glycosidic link between 6 hydroxyls on the 7-O-glucoside on hemiacetal hydroxyl on the 2nd of group isomaltose base end the glucosyl group and the puerarin A ring.

The solution of the present invention is the glycosylated derivative that microbe transformation method prepares puerarin, promptly selecting puerarin is the parent nucleus that is transformed, and adopts somatic cells or the zyme extract of this somatic cells or the recombinant expression protein of this enzyme with puerarin glycosylated enzyme activity; Glycosylated donor is selected monosaccharide groups or oligosaccharyl; Transform the site on the C-7 position hydroxyl on the puerarin A ring, through suitable microbial conversion process, transform target compound, the preferred target compound of the present invention is puerarin-7-O-glucoside and puerarin-7-O-isomaltosylfructoside.

The present invention selects the microbial strains with puerarin glycosylated enzyme activity for use, be any other can the glycosylation puerarin microorganism or mixing microorganisms, preferred microorganism is Microbacterium oxydans CGMCC 1788, also can be that Microbacterium saperdae CGMCC 1.1906 waits all Microbacterium bacterial classifications.

The present invention to the preparation method who transforms the separation and purification of synthesizing liquid is: a) thalline in the synthetic liquid of conversion or bacterial enzyme albumen are through thermal precipitation, centrifugal removal; B) with Solid-Phase Extraction or liquid-phase extraction or column chromatography separation; C) by recrystallization difference purifying.

This programme preferably contains the synthetic liquid of conversion of puerarin-7-O-glucoside and puerarin-7-O-isomaltosylfructoside component method in the numerous converted product that transforms, separate purifying with following method:

1) transform synthetic liquid with Solid-Phase Extraction, its solid phase carrier can be to be polyamide or macroporous resin, the resin of water and/or the extraction of alcoholic solvent wash-out, and the elutriant concentrate drying is promptly;

2) separate to transform synthetic liquid with column chromatography, can adopt silica gel, or alkyl linked silica gel or macroporous resin or polyamide be stationary phase that carry out chromatographic separation, the target fraction concentrate drying promptly;

3) use the liquid-phase extraction method, it is characterized in that the solvent for use system is water-organic solvent, organic solvent is propyl carbinol or ethyl acetate or chloroform or ether or hexane, sherwood oil etc., or the mixture of above-mentioned two or more organic solvent, extraction, separating and extracting liquid, concentrate drying are promptly.

4) purification process of target compound is in the scheme: solvent can adopt water and/or alcoholic solvent, or alcohol and/or fat-soluble solvent, and its fat-soluble solvent feature is ethyl acetate or chloroform or ether or oxyethane, or hexane or sherwood oil.

The targeted thing of this programme can be further on the basis of formula (I), utilize the acidic functionality on 4 ' hydroxyl or monosaccharide groups (as glucuronic acid) or the oligosaccharyl (the glucal acid fragment that wherein contains) to prepare the salt that its pharmacy acceptable salt is metal cation salt or nitrogenous organic base, wherein, (1) metal ion is basic metal or alkaline-earth metal, preferred sodium, potassium, calcium, magnesium; (2) nitrogenous organic bases is basic aminoacids, preferred arginine, Methionin, carnitine; Or the C1-C40 nitrogenous organic base, as meglumine, glucosamine, Ligustrazine, preferred meglumine.

The preparation method of acceptable salt is on the scheme Chinese materia medica

(1) with structural formula (I) compound and nitrogenous organic base with 1: 0.5～2.0 mol ratio, in water and/or alcoholic solvent, react, or with structural formula (I) compound and metal ion with 1: 0.5～2.0 equivalence ratio, in water and/or alcoholic solvent, react.

(2) wherein nitrogenous organic base preferred bases acidic amino acid or C1-C40 organic bases, or the preferred alkaline-earth metal ions of metal ion, wherein with basic aminoacids or with the mol ratio of C1-C40 organic bases reaction be 1: 0.8-1.5, or be 1 wherein: 0.8-1.5 with the equivalence ratio of alkaline-earth metal ions reaction.

(3) wherein nitrogenous organic base preferred bases acidic amino acid or C1-C40 organic bases, or metal ion preferred as alkali ion K+, Na+ or alkaline-earth metal ions Ca++, Mg++, wherein with basic aminoacids or with the mol ratio of C1-C40 organic bases reaction be 1: 0.8-1.5, or wherein with K+, Na+, Ca++, Mg++, the equivalence ratio of reaction is 1: 0.8-1.5.

The glycosylated derivative of above-mentioned puerarin and pharmacy acceptable salt thereof add pharmaceutical excipient, are prepared into pharmaceutical preparation.

Further one of scheme is, the inventor is with containing the glycosylated derivative of puerarin and the target compound that the pharmacy acceptable salt experimental result shows formation thereof, make glycosylated derivative and the pharmacy acceptable salt thereof that puerarin has become water-soluble puerarin that contain that is slightly soluble in water originally, improved oral preparations dissolution in vitro (bioavailability improves in the body of prompting oral preparations); The water-soluble increase of the glycosylated derivative of puerarin and pharmacy acceptable salt thereof, can make with water is the injection of carrier, the pharmaceutical preparation stability of the glycosylated derivative of puerarin and pharmacy acceptable salt thereof is better than puerarin among the present invention.Therefore, can prepare the glycosylated derivative of puerarin and pharmacy acceptable salt thereof pharmaceutical application at the treatment cardiovascular and cerebrovascular diseases

The various formulations of medicinal compositions of the present invention can be according to the conventional production method preparation of pharmaceutical field.General formula (I) is mixed with one or more carriers for activeconstituents, be made into required formulation then.

Glycosylation puerarin derivate and pharmacy acceptable salt thereof are used for cardiovascular and cerebrovascular disease in the preparation treatment; Prevent and treat atherosclerosis, blood fat reducing, diabetic nephropathy and to the application of insulin resistant, osteoporosis.

Concrete application mode can be with the glycosylated derivative of described puerarin and pharmacy acceptable salt thereof the raw material as effective ingredient, then with pharmaceutically acceptable pharmaceutical excipient combination, makes and is used for the treatment of cardiovascular and cerebrovascular disease; Prevent and treat atherosclerosis, blood fat reducing, diabetic nephropathy and to the pharmaceutical composition of insulin resistant, osteoporosis, said composition contains glycosylated derivative and the pharmacy acceptable salt and the pharmaceutically acceptable carrier of above-mentioned arbitrary puerarin for the treatment of effective dose.

The glycosylated derivative of above-mentioned arbitrary puerarin and pharmacy acceptable salt thereof add pharmaceutical excipient, are prepared into pharmaceutical preparation.At preparation treatment coronary heart disease, stenocardia, myocardial infarction, ischemic cerebrovascular disease, thrombosis of retinal artery, sudden deafness; Be used to prevent and treat atherosclerosis, blood fat reducing, diabetic nephropathy and to insulin resistant, osteoporosis, anti-oxidant, anti-ageing, suppress apoptosis, suppress the medicinal application of growth of tumour cell.Described pharmaceutical composition is said any formulation on the pharmaceutics, comprises tablet, capsule, soft capsule, gelifying agent, oral preparation, suspensoid, electuary, patch, ointment, pill, powder, injection, infusion solution, freeze dried injection, vein emulsion, lipidosome injection, suppository, sustained release preparation or controlled release preparation.

Medicinal compositions of the present invention preferably contains glycosylated derivative and the pharmacy acceptable salt thereof that mol ratio is the puerarin of 0.1%～99.5% general formula (I), and wherein the glycosylated derivative of puerarin and pharmacy acceptable salt content thereof are 1.0%～95%.Medicinal compositions of the present invention removes glycosylated derivative and the pharmacy acceptable salt thereof that contains above-mentioned general formula (I) puerarin for the treatment of significant quantity, also should contain one or more pharmaceutically acceptable carriers.

Pharmaceutically acceptable carrier mentioned above is meant the pharmaceutical carrier of pharmaceutical field routine, for example: thinner, vehicle and water etc., weighting agent such as starch, sucrose, lactose, Microcrystalline Cellulose etc.; Tackiness agent such as derivatived cellulose, alginate, gelatin and polyvinylpyrrolidone; Wetting agent such as glycerine; Disintegrating agent such as sodium starch glycolate, hydroxypropylcellulose, cross-linked carboxymethyl cellulose, agar, lime carbonate and sodium bicarbonate; Absorption enhancer such as quaternary ammonium compound; Yelkin TTS; Tensio-active agent such as cetyl alcohol, sodium lauryl sulphate; Absorption carrier such as kaolin and soap clay; Lubricant such as talcum powder, calcium stearate and magnesium, micropowder silica gel and polyoxyethylene glycol etc.Can also in composition, add other assistant agent such as flavouring agent, sweeting agent etc. in addition.

Medicinal compositions of the present invention can be applied to the patient who needs this treatment by the mode of oral, rectum or administered parenterally.Be used for when oral, can be made into conventional solid preparation such as tablet, capsule, pulvis, granule etc., make liquid preparation such as water or oil-suspending agent or other liquid preparation such as syrup, oral liquid, elixir etc.; When being used for administered parenterally, can be made into solution, powder pin, water or the oiliness suspension agent etc. of injection.Preferred form is tablet, coated tablet, capsule, granule, oral liquid and injection.

Technical problem to be solved by this invention can also further realize by following technical scheme.Above-described drug combination preparation, be characterized in, its formulation is said any formulation on the pharmaceutics, comprises tablet, capsule, sprays, gelifying agent, oral preparation, suspensoid, electuary, patch, ointment, pill, powder, injection, infusion solution, freeze dried injection, sustained release preparation or controlled release preparation.

Technical problem to be solved by this invention can also further realize by following technical scheme.Above-described drug combination preparation is characterized in, it is the tablet or the capsule of the glycosylated derivative of puerarin and pharmacy acceptable salt thereof and weighting agent, disintegrating agent assembly; Or the slow releasing tablet or the capsule of the glycosylated derivative of puerarin and pharmacy acceptable salt thereof and weighting agent, hypromellose K4M assembly; Or the glycosylated derivative of puerarin is scattered in the glycosylated derivative composition soft capsule that obtains puerarin in the oil phase.

Technical problem to be solved by this invention can also further realize by following technical scheme.Above-described drug combination preparation is characterized in, it is characterized in that, described weighting agent is a sucrose, lactose, Microcrystalline Cellulose, dextrin, starch or calcium phosphate, Yelkin TTS; Described disintegrating agent is hydroxypropylcellulose, sodium starch glycolate, polyvinylpolypyrrolidone or croscarmellose sodium;

Technical problem to be solved by this invention can also further realize by following technical scheme.Above-described drug combination preparation is characterized in, also contains tackiness agent, wetting agent or lubricant in the described preparation.

Technical problem to be solved by this invention can also further realize by following technical scheme.Above-described drug combination preparation is characterized in that, it is the glycosylated derivative of puerarin and the injection of pharmacy acceptable salt and solubilizing agent or solvent formation thereof; Or the freeze-drying powder ampoule agent for injection of the glycosylated derivative of puerarin and pharmacy acceptable salt formation thereof; Or the glycosylated derivative of puerarin is scattered in the injectable emulsion that obtains in the oil phase, or suspension type injection liquid, described suspension type injection liquid be with the glycosylated derivative of puerarin and pharmacy acceptable salt micro mist thereof and Polysorbate 80 mix grind after, be dissolved into the aqueous solution of phosphoric acid potassium dihydrogen, dipotassium hydrogen phosphate, nipagin esters and Xylo-Mucine, make through grinding.

Technical problem to be solved by this invention can also further realize by following technical scheme.Above-described drug combination preparation is characterized in, described oil phase is soybean oil, poly(oxyethylene glycol) 400, Oleum Gossypii semen, peanut oil, sesame oil, Semen Maydis oil or sweet oil; In described oil phase, also can add solubilizing agent or latent solvent or oxidation inhibitor.

Technical problem to be solved by this invention can also further realize by following technical scheme.Above-described drug combination preparation is characterized in, described solubilizing agent is polyoxyethylenated castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene pyrrolidone, poloxamer, tween, polyoxyethylene glycol, pluronic F-68; Described solubility promoter is arginine, Methionin, meglumine or glucosamine, diethylamine, quadrol, urea, carnitine, Ligustrazine, niacinamide, proline(Pro), glucose, Citric Acid and sodium salt thereof.

One of preparation method of the various formulations of pharmaceutical combination preparation of the present invention is, is that activeconstituents mixes with above-described one or more carriers with the glycosylated derivative and the pharmacy acceptable salt thereof of puerarin, is made into required formulation then.

Medicinal compositions of the present invention preferably contains the glycosylated derivative that weight ratio is 0.1%～99.5% puerarin and the activeconstituents of pharmacy acceptable salt thereof, most preferably contains weight ratio and be the glycosylated derivative of 0.5%～95% puerarin and the activeconstituents of pharmacy acceptable salt thereof.

The glycosylated derivative of the present invention program's Chinese style (I) puerarin is the natural product of the novel puerarin of a class-7-O-glucosides, preferred puerarin-7-O-glucoside and puerarin-7-O-isomaltosylfructoside, similar with puerarin, have hypotensive, the coronary artery dilating and the cerebrovascular, improve the brain microcirculation, protect ischemic myocardium and myocardial ischemia-reperfusion injury, antithrombotic, decompose plasma fibrinogen, blood viscosity lowering, prevent atherosclerosis, blood fat reducing, it is normal to keep blood sugar, anti-oxidant, anti-ageing, control bone resorption and promotion bone growth, suppress apoptosis, suppress effects such as growth of tumour cell, be used for clinical treatment.But listed failing stated to the greatest extent.

Novel puerarin derivate-puerarin of the present invention-7-O-glucoside and puerarin-7-O-isomaltosylfructoside has improved physico-chemical property, pharmacokinetics and the pharmacologically active of existing puerarin.Puerarin-7-O glucoside (Puerarin-7-O-Glucoside) has than highly water-soluble, the water-soluble of puerarin-7-O-glucoside improved 18.5 times than the parent compound puerarin, the water-soluble of puerarin-7-O-isomaltosylfructoside improved 100 times than the parent compound puerarin, can directly prepare the injection water injection, significantly reduce the solubility promoter consumption, evaded the unsafe risk of medication; Puerarin-the residence time of 7-O-glucoside in blood compared with puerarin and obtains prolonging, and T1/2 is 2.85 times of puerarin, has reduced dosage; Experimental result shows that vasodilator activity significantly increases, and is the substitute of the higher puerarin medicine of security.

Embodiment

Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.

Below in conjunction with specific embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.

In following embodiment, for various processes and the method for describing in detail is common ordinary method in this area.The source of agents useful for same, trade(brand)name and be necessary to list its component person shows when occurring first that all used thereafter identical reagent if no special instructions, and is all identical with the content that shows first.

Embodiment 1.

150mL LB substratum is put into the 500mL triangular flask, altogether the 1L substratum.Behind 121 ℃ of high pressure steam sterilizations, insert Microbacterium oxydans CGMCC1788 seed liquor, 30 ℃, after the 220rpm shaking culture 12 hours, stop fermentation, somatic cells is collected in centrifugation, put into the 500mL triangular flask, adding 150mL contains the 1/15mol/L phosphoric acid buffer (pH8.0) of 0.02% puerarin-2% maltose, 30 ℃, puerarin glycosylated conversion of resting cells reaction is carried out in 200rpm vibration ventilation, behind the 48h, stop conversion reaction, centrifugal removal thalline, supernatant liquor separates with the C18 preparative high performance liquid chromatography, obtains puerarin-7-O-glucoside and puerarin-7-O-isomaltosylfructoside component respectively, the decompression thin film concentration is after proper volume, place 4 ℃ to separate out to crystallization, collect crystal, the dry back of washing is a product, puerarin-7-O-glucoside crystal and puerarin-7-O-isomaltosylfructoside crystal detects its content all greater than 99% through HPLC, asks for an interview accompanying drawing.

Above-mentioned puerarin-pure product of 7-O-glucoside are DMSO's ¹H-NMR and ¹³The feature of the chemical shift of C-NMR is as follows:

¹H-NMR(400MHz，DMSO-d6)

δ：3.28-4.05(m，glucosyl-H)，4.79(1H，d，J＝3.5Hz，glucosyl H-1″′)，4.80(1H，d，J＝9.9Hz，glucosyl H-1″)，6.80(2H，d，J＝8.6Hz，H-3′/H-5′)，6.97(1H，d，J＝8.8Hz，H-6)，7.39(2H，d，J＝8.6Hz，H-2′/H-6′)，7.92(1H，d，J＝8.8Hz，H-5)，8.29(1H，s，H-2)；

¹³C-NMR(100MHz，DMSO-d6)

δ：61.1(glc-6″，glc-6′″)，70.3(glu-4″′)，70.5(glc-4″)，71.2(glc-2″)，72.6(glc-2′″)，73.0(glc-5′″)，73.8(glc-1″)，74.0(glc-3″′)，79.2(glc-3″)，80.5(glc-5″)，99.0(glc-1″′)，113.1(C-8)，115.4(C-6/C-10，C-3′/C-5′)，123.0(C-1′)，123.6(C-3)，126.6(C-5)，153.0(C-2)，157.6(C-9，C-4′)，161.4(C-7)，175.4(C-4)。

TOF-MS mass spectrometry results: m/z:601.2[M+Na] ⁺, elementary composition is C ₂₇H ₃₁O ₁₄Na.

Puerarin-7-O-glucoside is 25 ℃ of solubleness 0.22 ± 0.003M in pure water; It is about 179.80 ℃ that DSC heat is analyzed fusing point.

Above-mentioned puerarin-pure product of 7-O-isomaltosylfructoside are DMSO's ¹H-NMR and ¹³The feature of the chemical shift of C-NMR is as follows:

¹H-NMR(400MHz，DMSO-d6)δ：3.28-4.05(m，glucosyl-H)，4.62(1H，br.S，glucosyl H-1″″)，4.76(1H，d，J＝3.2Hz，glucosyl H-1″′)，4.79(1H，d，J＝7.3Hz，glucosyl H-1″)，6.80(2H，d，J＝8.4Hz，H-3′/H-5′)，6.98(1H，d，J＝8.8Hz，H-6)，7.39(2H，d，J＝8.6Hz，H-2′/H-6′)，7.93(1H，d，J＝8.8Hz，H-5)，8.31(1H，s，H-2)；

¹³C-NMR(100MHz，DMSO-d6)δ：61.3(glc-6″″，glc-6″)，66.5(glc-6″′)，70.4(glc-4″)，70.6(glc-4′″，glc-4″″)，71.0(glc-2″″)，71.3(glc-2″)，72.4(glc-2″′)，72.5(glc-5″′)，72.9(glc-5″″)，73.6(glc-3″″)，73.9(glc-1″)，74.1(glc-3′″)，79.2(glc-3″)，80.3(glc-5″)，98.7(glc-1′″)，98.9(glc-3″″)，113.6(C-8)，115.4(C-6/C-10，C-3′/C-5′)，123.0(C-1′)，123.6(C-3)，126.7(C-5)，153.0(C-2)，157.6(C-9，C-4′)，161.4(C-7)，175.4(C-4)。

TOF-MS mass spectrometry results: m/z:763.2[M+Na] ⁺, elementary composition is C ₃₃H ₄₀O ₁₉Na.

Puerarin-7-O-isomaltosylfructoside 25 ℃ of solubleness in pure water are 1.27 ± 0.068M; It is about 188.93 ℃ that DSC heat is analyzed fusing point.

Embodiment 2.

Microbacterium oxydans CGMCC1788 bacterial classification is inserted in the 1L triangular flask of the LB substratum that contains the 300mL sterilization, 30 ℃, after the 220rpm shaking culture 24 hours, access contains in the fermentor tank of 3LLB substratum, 30 ℃, the 3L/min air flow, cultivated 18 hours, centrifugal collection thalline, with the resuspended thalline of 200mL water, adopt the broken somatic cells of Franch Press instrument 1800psi high pressure to extract glycosyltransferase, centrifugal collection zyme extract 160mL, get two parts of 40ml zyme extracts and join 2 3L respectively and contain in the 1/15mol/L PBS damping fluid (PH8.0) of 4% sucrose-0.2% puerarin, 30 ℃, stopped reaction after the 400rpm. agitation condition transforms 72 hours down.100 ℃ of heating made protein precipitation in 5 minutes, and 10000rpm removed precipitation in centrifugal 15 minutes.Supernatant liquor is leant on absorption with 35cm * 2.5cm macroporous resin chromatography, behind distilled water drip washing post, use 60% ethanol elution, concentrate, separate with chromatography, stationary phase is a silica gel, moving phase chloroform-methanol (progressively increasing methanol concentration) obtains puerarin-7-O-glucoside and puerarin-7-O-isomaltosylfructoside component respectively, the decompression thin film concentration is after proper volume, place 4 ℃ to separate out to crystallization, collect crystal, recrystallizing methanol, puerarin-7-O-glucoside crystal and and puerarin-7-O-isomaltosylfructoside crystal detect its content all greater than 95.0% through HPLC.

Embodiment 3.

150mL LB substratum is put into the 500mL triangular flask, altogether the 1L substratum.Behind 121 ℃ of high pressure steam sterilizations, insert Microbacterium oxydans CGMCC1788 seed liquor, 30 ℃, the 220rpm shaking culture is after 12 hours, stop fermentation, somatic cells is collected in centrifugation, puts into the 500mL triangular flask, adds the 1/15mol/L phosphoric acid buffer (pH8.0) that 150mL contains 0.02% puerarin-4% maltose, 30 ℃, puerarin glycosylated conversion of resting cells reaction is carried out in 200rpm vibration ventilation, behind the 72h, stops conversion reaction, centrifugal removal thalline, heating concentrates 10 times, adds 95% ethanol and transfers concentration to 20%, and thalline is removed in centrifugal or filtration, supernatant liquor passes through macroporous resin column, behind the water wash-out impurity,, collect elutriant with 60% ethanolic soln wash-out, the decompression thin film concentration is after proper volume, chromatographic column separate puerarin-7-O-isomaltosylfructoside crude product, with the smart recrystallization of 60-90% ethanol, collect crystal, the dry back of washing is a product, puerarin-7-O-isomaltosylfructoside detects its content all greater than 95% through HPLC, asks for an interview accompanying drawing.

Embodiment 4.

Microbacterium oxydans CGMCC1788 bacterial classification is inserted in the 1L triangular flask of the LB substratum that contains the 300mL sterilization, 30 ℃, after the 220rpm shaking culture 24 hours, access contains in the fermentor tank of 3L LB substratum, 30 ℃, the 3L/min air flow, cultivated 18 hours, centrifugal collection thalline, with the resuspended thalline of 200mL water, adopt the broken somatic cells of Franch Press instrument 1800psi high pressure to extract glycosyltransferase, centrifugal collection zyme extract 160mL, get two parts of 40ml zyme extracts and join 2 3L respectively and contain in the 1/15mol/L PBS damping fluid (PH8.0) of 2% sucrose-0.4% puerarin, 30 ℃, stopped reaction after the 400rpm. agitation condition transforms 32 hours down.100 ℃ of heating made protein precipitation in 5 minutes, and 10000rpm removed precipitation in centrifugal 15 minutes.Supernatant liquor adsorbs with the polyamide post, behind the water wash post, use the 20%-60% ethanol elution, the decompression thin film concentration separates with chromatographic column after proper volume, gets puerarin-7-O-glucoside crude product, use 95% ethyl alcohol recrystallization, get puerarin-7-O-glucoside, detect its content greater than 95%, ask for an interview accompanying drawing through HPLC.

Embodiment 5

The preparation of puerarin-7-O-glucoside sodium salt

Get puerarin-7-O-glucoside 2g and put in the appropriate vessel, add dissolve with ethanol, get 10% sodium carbonate solution, add in the solution of puerarin-7-O-glucoside, transfer pH to 9-11, stir and make dissolving, take out under the reduced pressure and desolvate, obtain solid 2.8g.

Embodiment 6

The preparation of puerarin-7-O-isomaltosylfructoside calcium salt

Get puerarin-7-O-isomaltosylfructoside 2g and put in the appropriate vessel, add dissolve with ethanol, get saturated limewater, add in the solution of puerarin-7-O-isomaltosylfructoside, transfer pH to 9-11, stir and make dissolving, take out under the reduced pressure and desolvate, obtain solid 3.0g.

Embodiment 7

The preparation of puerarin-7-O-glucoside arginic acid salt

Get puerarin-7-O-glucoside 2g and put in the appropriate vessel, add dissolve with ethanol, get in the solution of the water-soluble adding puerarin of L-arginase 12 .5g-7-O-glucoside, heating is stirred and is made dissolving, takes out under the reduced pressure and desolvates, and obtains solid 3.1g.

Embodiment 8

The preparation of puerarin-7-O-isomaltosylfructoside lysine salt

Get puerarin-7-O-isomaltosylfructoside 2g and put in the appropriate vessel, add dissolve with ethanol, get in the solution of the water-soluble adding puerarin of Methionin 2.3g-7-O-isomaltosylfructoside, heating is stirred and is made dissolving, takes out under the reduced pressure and desolvates, and obtains solid 2.9g.

Embodiment 9

The preparation of puerarin-7-O-isomaltosylfructoside meglumine salt

Get puerarin-7-O-isomaltosylfructoside 2g and put in the appropriate vessel, add dissolve with ethanol, get in the solution of the water-soluble adding puerarin of meglumine 3g-7-O-isomaltosylfructoside, heating is stirred and is made dissolving, takes out under the reduced pressure and desolvates, and obtains solid 3.1g.

Embodiment 8

The preparation of puerarin-7-O-glucoside meglumine salt

Get puerarin-7-O-glucoside 2g and put in the appropriate vessel, add dissolve with ethanol, get in the solution of the water-soluble adding puerarin of meglumine 3g-7-O-glucoside, heating is stirred and is made dissolving, takes out under the reduced pressure and desolvates, and obtains solid 3.1g.

Embodiment 9

The preparation of puerarin-7-O-glucoside ligustrazine salt

Get puerarin-7-O-glucoside 2g and put in the appropriate vessel, add dissolve with ethanol, get in the solution of the water-soluble adding puerarin of Ligustrazine 2.3g-7-O-glucoside, heating is stirred and is made dissolving, takes out under the reduced pressure and desolvates, and obtains solid 2.6g.

Embodiment 10

The preparation of puerarin-7-O-glucoside glucosamine salt

Get glucosamine 6.3g, be dissolved in 250ml distilled water, stir adding puerarin-7-O-glucoside 2.5g down, reflux 2h.Cooling slightly adds the 700ml dehydrated alcohol, and centrifugation discards precipitation.Supernatant liquor pressure reducing and steaming partial solvent is to the about 100ml of residual volume.Add dehydrated alcohol 70ml, solid is separated out in cooling.Filter, filter cake is with the aqueous ethanol recrystallization, and 40 degree vacuum-dryings get puerarin-7-O-glucoside glucosamine.

The medicine of the diseases such as each material conduct treatment respectively coronary heart disease, stenocardia, myocardial infarction, cerebral infarction and retina artery and vein embolism that embodiment 1-10 is prepared.

Embodiment 11

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside capsule

Puerarin-7-O-glucoside 20g

Or puerarin-7-O-isomaltosylfructoside

Microcrystalline Cellulose 60g

Lactose 100g

Sodium starch glycolate 14g

2%HPMCE5 solution is an amount of

6% tween 80 is an amount of

Magnesium Stearate 2g

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside, Microcrystalline Cellulose, lactose, sodium starch glycolate are crossed 100 mesh sieves respectively and are mixed, HPMG solution with tween 80 is tackiness agent system softwood, cross 20 mesh sieve system particles, wet granular is in 50-60.The forced air drying of C baking oven; Dried particle is crossed the whole grain of 20 mesh sieves, and with the Magnesium Stearate mixing, can is in capsule (conventional capsule or or enteric coated capsule).

Embodiment 12

The preparation of the salt capsule of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside

Puerarin-7-O-glucoside or puerarin-7-O-isomaltose 20g

The salt of glycosides (feeds intake by puerarin-7-O-glucoside calculating, down

With)

Microcrystalline Cellulose 30g

Lactose 30g

Sodium starch glycolate 5g

2%HPMCE5 solution is an amount of

6% tween 80 is an amount of

Magnesium Stearate 1g

The salt of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside, Microcrystalline Cellulose, lactose, sodium starch glycolate are crossed 100 mesh sieves respectively and are mixed, HPMC solution with tween 80 is tackiness agent system softwood, cross 20 mesh sieve system particles, wet granular is in 50-60.The forced air drying of C baking oven; Dried particle is crossed the whole grain of 20 mesh sieves, and with the Magnesium Stearate mixing, can is in capsule (conventional capsule or or enteric coated capsule).

Embodiment 13

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside tablet

Puerarin-7-O-glucoside or 20g

Puerarin-7-O-isomaltosylfructoside

Microcrystalline Cellulose 30g

Lactose 40g

Sodium starch glycolate 7g

2%HPMCE5 solution is an amount of

It is an amount of to contain 5% tween 80

Magnesium Stearate 1g

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside, Microcrystalline Cellulose, lactose, sodium starch glycolate are crossed 100 mesh sieves respectively and are mixed, HPMC solution with tween 80 is tackiness agent system softwood, cross 20 mesh sieve system particles, wet granular is in 50-60 ℃ of baking oven forced air drying; Dried particle is crossed the whole grain of 20 mesh sieves, with Magnesium Stearate mixing, compressing tablet.

Embodiment 14

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside salt tablets

The different Fructus Hordei Germinatus 20g of puerarin-7-O-glucoside or puerarin-7-O-

Glucosides salt (feed intake by puerarin-7-O-glucoside calculating,

Down together)

Microcrystalline Cellulose 30g

Lactose 40g

Sodium starch glycolate 5g

2%HPMCE5 solution is an amount of

6% tween 80 is an amount of

Magnesium Stearate 1g

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside salt, Microcrystalline Cellulose, lactose, sodium starch glycolate are crossed 100 mesh sieves respectively and are mixed, HPMC solution with tween 80 is tackiness agent system softwood, cross 20 mesh sieve system particles, wet granular is in 50-60 ℃ of baking oven forced air drying; Dried particle is crossed the whole grain of 20 mesh sieves, and with the Magnesium Stearate mixing, (1) presses ordinary tablet, and (2) press enteric coated tablet: get vinylformic acid 2g, add Viscotrol C and mix, add 95% ethanol to 600ml, with spraying turnadle pan coating dressing.

Embodiment 15

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside Yelkin TTS sheet

Puerarin-7-O-glucoside 20g

Or puerarin-7-O-isomaltosylfructoside

Microcrystalline Cellulose 30g

Soybean lecithin 40g

Sodium starch glycolate 7g

2%HPMCE5 solution is an amount of

It is an amount of to contain 5% tween 80

Magnesium Stearate 1g

Press recipe quantity, take by weighing puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside, soybean lecithin, put in the flask, add dehydrated alcohol 700ml, reflux 1-2 hour, reclaim ethanol, drying under reduced pressure grinds to form fine powder (100-200 order), Microcrystalline Cellulose, sodium starch glycolate are crossed 100 mesh sieves respectively and are mixed, HPMC solution with tween 80 is tackiness agent system softwood, crosses 20 mesh sieve system particles, and wet granular is in 50-60 ℃ of baking oven forced air drying; Dried particle is crossed the whole grain of 20 mesh sieves, and with the Magnesium Stearate mixing, (1) presses ordinary tablet, and (2) press enteric coated tablet: get vinylformic acid 2g, add Viscotrol C and mix, add 95% ethanol to 600ml, with spraying turnadle pan coating dressing.

Embodiment 16

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside salt enteric coated capsule

Get puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside salt, cellulose acetate-phthalate 209, be dissolved in acetone and ethanol (1: 1) the mixed solution 500ml solution, stir, slowly splash into normal hexane, till precipitation, sclerosis, drying is packed enteric-coated microcapsule in the common empty hard capsule again, makes enteric coated capsule.

Embodiment 17

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside slow releasing capsule

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside	50g
		Polyvidone	50g
Microcrystalline Cellulose	15g
		Hypromellose K4M	50g
3% hypromellose (E5) aqueous solution	In right amount
		Talcum powder	2g

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside and polyvidone are dissolved in the small amount of ethanol, and the decompression heating is to fling to ethanol, and the gained solid is crossed 100 mesh sieves; Above-mentioned solid is crossed 60 mesh sieves with Microcrystalline Cellulose, hypromellose K4M mix all, add 3% hypromellose (E5) aqueous solution and make softwood in right amount, cross 20 mesh sieves and granulate.40-50 ℃ of baking oven forced air drying; Dried particle is crossed the whole grain of 20 mesh sieves, adds the talcum powder of recipe quantity, mixes.Press the recipe quantity can in capsule.

Embodiment 18

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside salt sustained release capsules agent

Puerarin-7-O-glucoside or puerarin-7-O-isomaltose 80g

Glycosides salt (feeding intake by puerarin-7-O-glucoside calculating, down together)

Microcrystalline Cellulose 15g

Hypromellose K4M 100g

3% hypromellose (E5) aqueous solution is an amount of

Talcum powder 2g

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside salt, Microcrystalline Cellulose, hypromellose K4M are crossed 60 mesh sieves mix all, add 3% hypromellose (E5) aqueous solution and make softwood in right amount, cross 20 mesh sieves and granulate.40-50 ℃ of baking oven forced air drying.Dried particle is crossed the whole grain of 20 mesh sieves, adds the talcum powder of recipe quantity, mixes.Press the recipe quantity can in capsule.

Embodiment 19

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside slow releasing tablet

The different Fructus Hordei Germinatus 50g of puerarin-7-O-glucoside or puerarin-7-O-

Glucosides

Polyvidone 50g

Lactose 15g

Hypromellose K4M 100g

3% hypromellose (E5) aqueous solution is an amount of

Talcum powder 1g

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside and polyvidone are dissolved in the small amount of ethanol, and the decompression heating is to fling to ethanol, and the gained solid is crossed 100 mesh sieves; Above-mentioned solid is crossed 60 mesh sieves with lactose, hypromellose K4M mix all, add 3% hypromellose (E5) aqueous solution and make softwood in right amount, cross 20 mesh sieves and granulate.40-50 ℃ of baking oven forced air drying.Dried particle is crossed the whole grain of 20 mesh sieves, adds the talcum powder of recipe quantity, mixes, and compressing tablet promptly.

Above-mentioned example also can be selected other auxiliary material for use, and disintegrating agent is as hydroxypropylated starch, hydroxypropylcellulose, sodium starch glycolate, calcium carboxymethylcellulose, polyvinylpolypyrrolidone, croscarmellose sodium etc.; Weighting agent is as lactose, sucrose, N.F,USP MANNITOL, Microcrystalline Cellulose, dextrin, starch, calcium phosphate, secondary calcium phosphate, calcium sulfate, lime carbonate, cyclodextrin, micro mist Mierocrystalline cellulose etc.; Wetting agent and tackiness agent are as pregelatinized Starch, polyvidone, Xylo-Mucine, hypromellose; Lubricant is as talcum powder, stearic acid, Magnesium Stearate, calcium stearate, differential silica gel, hydrogenated vegetable oil, Macrogol 4000 and 6000; Wetting agent is as sodium lauryl sulphate, tween 80; Framework material is as hypromellose, ethyl cellulose etc.

Embodiment 19

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside injection

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside 10g

Water for injection adds to 5L

Get puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside, add in the water for injection, fully dissolving, add 0.1% gac, heated and boiled 15 minutes is filtered carbon removal, adjust pH to 5.0～7.0, survey intermediate content, pH value, after qualified, embedding in glass peace is cutd open, 100 ℃, 30 minutes circulation vapor sterilizations get puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside injection.

Embodiment 20

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside injection

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside	10.0g
		Arginine or Methionin	6.2g
Sodium-chlor	In right amount
		Water for injection	Add to 2.5L

Get puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside, arginine or Methionin (or getting puerarin derivate salt) are put in the appropriate vessel, add injection water 9000ml, stir, add arginine or Methionin, be heated to dissolving, add the sodium-chlor stirring and make dissolving, add water for injection to adding to 2.5L, through 0.22 μ m filtering with microporous membrane, coating-dividing sealing, in 100 ℃ of flowing steam sterilizations 30 minutes promptly.

Embodiment 21

The preparation of injection puerarin-7-O-glucoside or puerarin-7-O-isomaltose aglycone powdery injection

Puerarin-7-O-glucoside or puerarin-7-O-isomaltose

10.0g

Glycosides
		Arginine or Methionin	6.2g
N.F,USP MANNITOL	16.0g
		Water for injection	Add to 0.5L

Get puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside and arginine or Methionin and put in the appropriate vessel, add injection water 400ml, stir, ultrasonic to dissolving, add the N.F,USP MANNITOL stirring and make dissolving; Add needle-use activated carbon by 0.1%, stirred 30 minutes, and in the container of cleaning, added water for injection to 500ml through titanium core decarburization suction filtration, solution stirring was made evenly in 5 minutes, again through 0.22 μ m filtering with microporous membrane, the filtrate can in cillin bottle, every bottle of 2ml or 5ml, partly isoprene-isobutylene rubber match beyond the Great Wall then, deliver on the flaggy in the freeze drying box, insert temp probe, close chamber door.Press the freeze-drying curve lyophilize, the final drying temperature is more than 35 ℃ and kept 2 hours.Close plug, venting, outlet rolls lid.

Embodiment 22

The preparation of injection puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside salt powder injection

Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside salt	10.0g
		N.F,USP MANNITOL	16.0g
Water for injection	Add to 0.5L

Getting puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside salt (can be the salt such as arginine, Methionin, glucosamine, meglumine of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside) puts in the appropriate vessel, add injection water 400ml, stir, ultrasonic to dissolving, add the N.F,USP MANNITOL stirring and make dissolving; Add needle-use activated carbon by 0.1%, stirred 30 minutes, in the container of cleaning, add water for injection through titanium core decarburization suction filtration, solution stirring was made evenly in 5 minutes to 500ml, again through 0.22 μ m filtering with microporous membrane, the filtrate can is in cillin bottle, and partly isoprene-isobutylene rubber match is beyond the Great Wall delivered on the flaggy in the freeze drying box then, insert temp probe, close chamber door.Press the freeze-drying curve lyophilize, the final drying temperature is more than 35 ℃ and kept 2 hours.Close plug, venting, outlet rolls lid.

Embodiment 23

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltose glucoside oral liquid

Getting puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside is dissolved in the purified water, stirring makes abundant dissolving, add 85% simple syrup, regulate pH value 3.5～7.0, add 0.3% Sodium Benzoate again and make sanitas, heated and boiled 30 minutes, with the filtering with microporous membrane of 0.8 μ m, embedding in the brown oral vial of 20ml, 100 ℃, sterilization in 30 minutes gets puerarin-7-O-glucoside or puerarin-7-O-isomaltose glucoside oral liquid.

Embodiment 24

The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside injection transfusion

Get puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside, add in the water for injection, being made into concentration is 1mg/ml, fully dissolving adds 0.1% gac, heated and boiled 15 minutes, filter carbon removal, intermediate content, pH value are surveyed in adjust pH to 5.0～7.0, qualified after, embedding is in the 100ml vial, 100 ℃, 30 minutes circulation vapor sterilizations get puerarin-7-O-glucoside or puerarin-7-O-isomaltose injection transfusion.

Embodiment 25

Different puerarins-7-O-glucoside or puerarin-7-O-isomaltose glycoside injection liquid and puerarin injection are relatively

It is an amount of to get different puerarins-7-O-glucoside, puerarin-7-O-isomaltosylfructoside, puerarin, put in the 10ml volumetric flask, add water, jolting makes dissolving, constant volume, seal and promptly get the preparation that water is carrier, the results are shown in Table 4, puerarin-7-O-glucoside, puerarin-7-O-isomaltosylfructoside are stablized on preparation than puerarin.

Table 4 puerarin and derivative injection thereof are relatively

Compound	Concentration	All backs outward appearance is placed in 90 degree water-baths
			Puerarin-7-O-glucoside	4.0mg/ml	Clarification
Puerarin-7-O-isomaltosylfructoside	4.0mg/ml	Clarification
			Puerarin-7-O-glucoside: puerarin-7-O-isomaltosylfructoside (1: 1)	4.0mg/ml	Clarification
Puerarin	2.0mg/ml	Clarification has the false hair of trace
			Puerarin	4.0mg/ml	Muddy

Embodiment 26-33 explanation:

1, puerarin is called for short Pue, is provided by Qingdao Jinfeng Pharmaceutical Co., Ltd, and purity is 99% through high performance liquid chromatography (HPLC) analysis.Puerarin-7-O-glucose glycoside is called for short Pue-glu, and puerarin-7-O-isomaltosylfructoside is called for short Pue-mal, by the inventive method preparation, through high performance liquid chromatography (HPLC) purity 99%.

2, experimental animal

The Sprague-Dawley rat, body weight 200-250g, male, there is Nanjing University of Traditional Chinese Medicine's Experimental Animal Center that occupancy permit is provided: SKXK (Soviet Union) 2002-00123.

Kunming kind small white mouse, body weight 18～22g, male and female half and half are provided production licence by Nanjing Medical University's Experimental Animal Center: SCXK (Soviet Union) 2002-0031.

3, statistics

Data with

± s represents, adopts the t check, and P＜0.05 is for there being significant difference, and P＜0.01 is for there being utmost point significant difference.

4, instrument and reagent

The instrument and the reagent that relate in the method that adopts those skilled in the art to be familiar with.

Description of drawings:

Fig. 1 substrate puerarin HPLC spectrogram

The HPLC collection of illustrative plates of Fig. 2 puerarin-7-O-glucoside and puerarin-7-O-isomaltosylfructoside conversion fluid

Fig. 3 puerarin-pure product the HPLC of 7-O-glucoside collection of illustrative plates

Fig. 4 puerarin-pure product the HPLC of 7-O-isomaltosylfructoside collection of illustrative plates

The dynamic metabolism experiment of embodiment 26. puerarin derivates in blood

1 instrument and chromatographic condition

HPLC analyzes and adopts Agilent 1100 high performance liquid chromatographs (USA); Chromatographic column is Agilent Zobox OSD post (250 * 4.6 μ m, 5 μ m); Sampling volume is 20 μ L; Column temperature is room temperature; Detector is the AgilentG1314A UV-detector, and wavelength is 254nm; Mobile phase: A: water adds 0.1 ‰ (v/v) chromatographic grade acetic acid, B: methyl alcohol, A: B=70: 30; Flowing velocity 1mL/min.

2 test methods and result

2.1 sample collection

The SD rat is divided into 6 groups at random, 5 every group. Fasting 12h before the experiment. Pue, Pue-glu dissolve in respectively and form the solution that final dose is respectively 16.68mg/kg and 23.12mg/kg in the sodium chloride injection. Give rat tail vein injection Pue and Pue-glu by 4mL/kg. Contrast gives sodium chloride injection.

Take by t=1, behind 5,10,15,20,30,40,60,80,100,120,150, the 180min, the centrifuge tube that 0.5 milliliter of blood of eye socket collection places liquaemin to process, immediately with centrifugation 15min under the condition of 3000rpm, separated plasma is in order to analyzing. Control group gathers blood plasma and makes blank plasma after off-test.

2.2 sample treatment

Precision is got blood plasma 0.2mL and is put in the test tube, adds 6% perchloric acid solution 0.2mL protein precipitation again, mixes 2min on turbine mixer, then from 10min, 10000rpm puts into refrigerator and leaves standstill 2h, centrifugal 10min before measuring, supernatant is used for high performance liquid chromatography (HPLC) analysis.

2.3 data analysis

According to the described processing of 3.2.2 and use sample, the PC of Pue and Pue-glu calculates with the single-point external standard method. The data medication that obtains is analyzed for the dynamics statistical software. The result is presented at the compartment model that the in vivo metabolism of Pue and Pue-glu meets rat, and the R value is all greater than 0.99, and fitting degree is good. This explanation Pue-glu can reach balance faster between blood and extravascular tissue.

2.4 result

The main metabolic kinetic parameter is listed in the table 5. By as seen from Table 5, the holdup time of Pue-glu in blood compared with Pue and obtains prolonging, and T1/2 is 2.85 times of Pue, and being conducive to medicine has long action time in vivo. Corresponding, the AUC of evidence Pue-glu is higher than Pue, and the Cmax of Pue-glu is 1.6 times of Pue.

The dynamic metabolism in rat (Pharmacokinetic) parameter (n=5) of table 5 Pue and Pue-glu

^**: p＜0.01 difference and remarkable,^*: p＜0.05 significant difference

The half-life that t1/2=eliminates

The MRT=mean residence time

Area under the AUC=concentration time curve

The Cmax that C (max)=compound can reach

The vasodilative pharmacological effect test of embodiment 27. puerarin derivates

Test method and result

1 method

Rat is hit unconsciously with blunt, and dislocation of cervical vertebra is opened rapidly the thoracic cavity after putting to death, and gets its aorta pectoralis, puts into and fills 4 ℃, logical 100%O₂In the culture dish of saturated K-H liquid. Carefully pull out blood vessel connective tissue on every side, wash vessel inner blood, be cut into the vascular circle of long 3～4mm, insert tube chamber with finer wire, behind the destruction blood vessel endothelium that rolls, hang on and lead to 100%O₂The bath of 7mLK-H liquid in, 37 ℃ of constant temperature connect tonotransducer. Its rest tension 0.5～2g, stable equilibrium 1～2 hour, solution is once to 37 ℃ the K-H fluid exchange bath with temperature in advance for every 20min. Add NE (110^-6Mol/L), until vascular circle shrink reach stable after, change clothes to baseline with K-H liquid, stablize 20min. Repeat this operation once. Add PE 110^-5M shrinks arterial ring in advance, waits to shrink the stable acetylcholine (Ach) 10 that adds^-5M detects function of vascular endothelium, thinks that with≤20% endothelium removes, and carries out next step experiment.

Use Pue, Pue-glu, pue-mal 10^-4.5-10 ^-2The M temperature is incubated 10min, and observe the inhibitory action to various contractions: A, PE (110^-5The contraction of M) inducing, until vessel retraction reach stable after, add the Pue of cumulative dosage or the contraction that Pue-7-O-glc causes; B, KC (110^-1The contraction of M) inducing, until vessel retraction reach stable after, add the Pue of cumulative dosage or the contraction that Pue-7-O-glc causes. Record maximum collapse tension force (g) take not dosing group as control group, calculates the inhibition contraction percentage of the contraction that various medicines cause different disposal.

2 results

2.1 Pue or Pue-7-O-glc shrink the impact of aortic annulus tension force in advance on KCl

After KCl caused that the contraction of rat chest aorta ring reaches amplitude peak, cumulative bad increased the concentration (10 of Pue or Pue-7-O-glc^-4.5-10 ^-2M), find Pue, Pue-glu, Pue-mal all has the effect of diastole to the vascular circle of endothelium-denuded, and is concentration dependent, when cumulative concentration reaches 10^-2.0During M, Pue-glu, the diastole effect of pue-mal is than stronger with the Pue effect of concentration, and t checks p＜0.01. Data statistics the results are shown in Table

Table Pue and Pue-glu, Pue-mal is on the impact of the pre-shrunk aortic annulus tension force of KCl

^**Pue-glu, pue-mal compare p＜0.01 with control group,^##Pue-glu, pue-mal compares p＜0.01 with the Pue group

2.2 Pue, Pue-glc, Pue-mal shrink the impact of aortic annulus tension force in advance on PE

After PE caused that the contraction of rat chest aorta ring reaches amplitude peak, cumulative bad increased Pue, Pue-glc, the concentration (10 of Pue-mal^-4.5-10 ^-2M), find Pue, Pue-glu, Pue-mal to the vascular circle of endothelium-denuded only 10^-2M just has the effect that suppresses contraction, does not have notable difference between the two. Data statistics the results are shown in Table.

Table Pue, Pue-glu, Pue-mal is on the impact of the pre-shrunk aortic annulus tension force of PE

Pue-glu, pue-mal compares with control group^*p＜0.05， ^**p＜0.01； ^#Pue-glu, pue-mal compares p＜0.05 with the Pue group

3 conclusions

Concentration is 10^-2.5，10 ^-2The Pue-glu of M or (Pue-mal) can suppress more strongly Contraction of Aortic ring behind the removal endothelium that KCl causes than Pue does not have notable difference but the contraction that suppresses the aortic annulus behind the removal endothelium that phyenlephrinium causes compared with Pue.

Because Pue-glu, pue-mal has identical parent nucleus with Pue, and pharmacological action is similar, and prompting Pue-glu, pue-mal also have treatment coronary heart diseases and angina pectoris, myocardial infarction, ischemic cerebrovascular disease, thrombosis of retinal artery, sudden deafness; Prevent and treat atherosclerotic, reduce blood fat, diabetic nephropathy and the pharmaceutical composition of insulin resistance, anti-osteoporosis used.

Pue-glu, pue-mal compare in vasodilatory pharmacological action aspect some more excellent with Pue.

Example 28 coronary heart diseases and angina pectoris, myocardial infarction experiment

1 method

50 of Wistar rats, body weight 200～250g, male and female half and half are divided at random: sham-operation (S) group; Ischemia-reperfusion (I/R) group; Ischemic preliminary treatment (IPC) group; The Pue-glu group; The Pue-mal group. Every group 10. Reference literature [deep number etc., the preparation of big white mouse expeirmental myocardial ischemia re-perfusion model and ECG change moral characteristics, Medical University Of Chongqing's journal, 1998,23 (1): 8～11] method also improves, and copies rat heart muscle I/R model. Fixing behind 10% chloraldurate, 0.3～0.4ml/100g intraperitoneal injection of anesthesia, trachea cannula connects the toy lung ventilator. It is subcutaneous that needle electrode is inserted four limbs, by BL2420E biological function experimental system recording ecg. Open chest in left border of sternum the 4th intercostal space, cut off pericardium and expose heart, at left auricle of heart lower edge 3～4mm place inserting needle, go out pin to the pulmonary conus direction, a bit of polyethylene pipe (diameter 115mm) placed under the ligature tighten up ligature, with left front branch (LAD) blood flow that falls of blocking-up rat coronary artery. Observe electrocardiogram, take occur the tall and big broadening of QRS wave group as ligation successfully indicate (the Guo sparrow hawk, the animal model of human diseases, the People's Health Publisher, 1982:365-367). Behind the ischemic 45min, take out the i.e. again perfusion of polyethylene pipe, be reduced to gradually with QRS wave group amplitude and pour into more successfully and indicate (Wu Qixia etc., ischemia reperfusion injury and pre-adaptation, newly organized Pathological Physiology, 1999:191-210).

(1) S group: only open behind the chest at the LAD underpass and not ligation gives equivalent physiological saline tail vein injection.

(2) I/R group: ligation LAD ischemic 45min, pour into again 120min, give equivalent physiological saline tail vein injection.

(3) IPC group: ischemic 5min, pour into again 5min, 3 times repeatedly, ischemic 45min pours into 120min more subsequently, gives equivalent physiological saline tail vein injection.

(4) Pue-glu group: 5min before the ischemic, the tail vein is slowly injected.

(5) Pue-mal group: 5min before the ischemic, the tail vein is slowly injected. PUE-GLU and PUE-MAL group (100mg/kg body weight), all the other operations are organized with I/R.

2 observation index

2.1 take out rapidly heart after every group of 10 rats of myocardial infarction area are poured into and finish, original position ligation LAD through sustainer retroperfusion Azo-Blue 1～2ml, separates non-ischemic region (indigo plant is dyed) and ischemic region (dying without indigo plant). Ischemic region and non-ischemic region cardiac muscle are blotted, weigh with filter paper respectively, represent ischemic areas with ischemic region weight and the percentage of weight whole-heartedly. Again the ischemic region edge is cut perpendicular to the heart y direction, every thick 1～2mm, place 37 ℃ of 1%TTC phosphate buffers (pH 7.1) to hatch 20min, then infarcted region (canescence) is separated with non-infarcted region (brick-red), filter paper blots, weighs respectively, represents infarct size with the ratio of infarcted myocardium weight and ischemic region myocardial Mass Measured.

2.2 serum CK vigor and TnT level respectively organize rat pour into again finish after femoral artery get blood, 4 ℃, 3500r/min is centrifugal, separation of serum is then in strict accordance with kit specification time-and-motion study CK vigor. Femoral artery was got blood 1ml after perfusion finished again, anticoagulant heparin, survey TnT, each is organized the TnT concentration results and makes the frequency table data, I, II, III, IV level represent respectively TnT concentration be 0.1～0.65mg/L, 0.65～1.3mg/L, 1.3～2.0mg/L and＞2.0mg/L.

3 results

3.1 myocardial infarction area

The S group has no myocardial infarction and occurs; Except the S group, ischemic areas no significant difference (P＞0.05) between each group; With I/R group relatively, IPC group, Pue-glu group and Pue-mal group infarct size all dwindle (P＜0.05), and Pue-glu group and Pue-mal organize respectively with the IPC group difference without conspicuousness (P＞0.05). The result shows that Pue-glu and Pue-mal preliminary treatment can significantly dwindle the myocardial infarct size that ischemia-reperfusion causes, and is similar with the IPC effect. See Table 1.

3.2 serum CK vigor and TnT concentration

I/R group serum CK vigor, TnT concentration are than sham group, the obviously rising (P＜0.01 or P＜0.05) of IPC group; The IPC group is compared CK vigor and TnT concentration difference, and there are no significant with the S group; Pue-glu and Pue-mal group CK vigor, TnT concentration obviously reduce (P＜0.01 or P＜0.05) than the I/R group, and organize without significant difference (P＞0.05) with IPC. Serum CK vigor and TnT concentration when as seen, Pue-glu and Pue-mal preliminary treatment all can reduce ischemical reperfusion injury with IPC. The results are shown in Table 2.

Table 1 myocardial ischemia-area and infarct size (n=10,

±s)

# and I/R group compare P＜0.05

Table 2 serum CK vigor and TnT change in concentration (n=10,

±s)

Compare with the S group,^*P＜0.05， ^**P＜0.01; Compare #P＜0.05, ##P＜0.01 with the I/R group

The experiment of example 29 ischemic cerebrovascular diseases

1 method

The mensuration of Cerebral microcirculation blood flow

Mice by intraperitoneal injection yellow Jackets (40mg/kg) anesthesia along crown midsection skin, is gently scraped parietal bone, uses J I2200 type laser microcirculation kinetic analyzer and measures Cerebral microcirculation blood flow. Begin experiment after stablizing 5～10min. The normal brain activity microcirculatory blood flow is all measured first and recorded to the every treated animal of normal mouse, tail vein injection Pue-glu then, Pue-mal, and after the record injection at once 0,1,5,10,30, the microcirculatory blood flow during 60min. Microcirculation disorder mouse due to the high molecular dextran (HMWD), record respectively the microcirculatory blood flow that tail vein injection 10%HMWD 0.2ml causes the microcirculation disorder front and back, tail vein injection Pue-glu behind the 3min, Pue-mal, after the same record injection each the time phase Cerebral microcirculation blood flow.

2 results

2.1 Pue-glu, Pue-mal is on microcirculatory 10 every group of small white mouses, body weight 18～22g, random packet, the male and female half and half of affecting of normal mouse brain. Control group mice tail vein injection saline 0.2ml/, every animal of low dose group and high dose group is tail vein injection 1%Pue-glu, each 0.2ml of 1%Pue-mal respectively. The results are shown in Table 1. As can be seen from Table 1, intravenous injection Pue-glu, 3～5min behind the Pue-mal, Cerebral microcirculation blood flow namely significantly raises, and its effect continues 30min at least.

2.2 Pue-glu, Pue-mal affects 30 of small white mouses to microcirculation disorder mouse brain microcirculatory blood flow due to the HMWD, male and female half and half, and body weight 18～22g is divided at random: control group; The Pue-glu group; The Pue-mal group, 10 every group. Every treated animal all behind injection 10%HMWD 0.2ml 3min, tail vein injection salt solution or Pue-glu, Pue-mal, dosage is the same. The results are shown in Table 2. As seen from Table 2, behind the mouse mainline HMWD, Cerebral microcirculation blood flow obviously reduces, and keeps a reduced levels behind 3～5min, and inject Pue-glu this moment, and Pue-mal can make Cerebral microcirculation blood flow that rising is slightly arranged, and acts on more obvious.

Table 1 Pue-glu, Pue-mal on the impact of normal mouse brain microcirculatory blood flow (mV,

±s)

Annotate: compare with control group:^*P＜0.05， ^**P＜0.01

Table 2 Pue-glu, Pue-mal on the impact of microcirculation disorder mouse brain microcirculatory blood flow due to the HMWD (mV,

±s)

Annotate: compare with control group:^*P＜0.05， ^**P＜0.01

The experiment of example 30 atherosclerotics

1. method

1.1 take out rapidly the aorta pectoralis of Newborn calves under the cultivation aseptic condition of aortic endothelial cell, place the Hanks solution of high resistance, wash remaining blood stains off, be transferred in the culture dish, adopt the Trypsin Induced method to obtain endothelial cell, with the RPMI-1640 medium culture that contains 10% calf serum, put 37 ℃, 5%CO₂In the incubator, about 3d changes nutrient solution, and about a week, cell grows to the cultivation of going down to posterity when converging fully, adopts well-grown 2 generations to test.

1.2 the endothelial cell that affects of the endothelial cell damage that low-density lipoprotein (LDL) is induced is inoculated in 24 orifice plates with the density of 1 * 105cell/ml, every hole 0.5ml, be cultured to fusion after, add respectively 10^-7Mol/mlPue-glu after Pue-mal cultivates 12h, changes the culture medium that contains 50 μ g/ml LDL into and continues to cultivate 24h, draw nutrient solution, measure the content of NO in the nutrient solution, after attached cell is dissolved with 1%Triton, the activity of nitricoxide synthase in the kit measurement cell (NOS). Experiment minute control group (the RPMI-1640 culture medium that adds 0.5% NBCS (FBS) in the culture fluid of endothelial cell; Model group (adding 0.5%FBS RPMI-1640 culture medium and 50 μ g/ml LDL effect 24h in the culture fluid of endothelial cell); (12h adds Pue-glu, Pue-mal 10 to Pue-glu, Pue-mal group before the experiment in nutrient solution^-7Then mol/L adds 50 μ g/ml LDL effect 24h) and Pue-glu, Pue-mal group (12h 1mg/ml Pue-glu, Pue-mal in nutrient solution before the experiment add 50 μ g/ml LDL effect 24h).

1.3 animal and grouping adult healthy Wistar rat, body weight 220～250g, ♀Dual-purpose is provided by Nanjing Medical University's Experimental Animal Center. After feed fed for 1 week, be divided at random Control, Model, Pue-glu, Pue-mal group, 6 every group. Control: blank group, feeding standard basal feed; The Model group: model group, the high lipid food of feeding forms [Li Yikui, herbal pharmacology experimental methodology, Shanghai: Science and Technology of Shanghai publishing house, 1993:398] by 1% cholesterol, 10% leaf fat, 0.2% propylthiouracil and 88.8% basal feed. Pue-glu, Pue-mal group: experimental group, the high lipid food and add Pue-glu, Pue-mal tablet by 80mg/kg of feeding. Each organizes the feeding environment term harmonization. Lipid determination is when feeding for 6 week, the tail venous blood sampling, adopt elisa reagent (box) to use VitalabMicro semi-automatic biochemical analyzer (Holland) to measure the content of triglycerides (TG), cholesterol (TC) and HDL-C (HDL-C), calculate LDL-C (LDL-C) value according to measurement result.

2. result

2.1 the impact of NO content is compared with control group in the Human Umbilical Vein Endothelial Cells nutrient solution, 50 μ g/mlLDL group NO content obviously reduces (P＜0.05), and Pue-glu, Pue-mal make the synthetic and secretion NO rising (P＜0.01, table 1) of endothelial cell.

2.2 nitricoxide synthase in the Human Umbilical Vein Endothelial Cells (NOS) is active affect cell with the 1%Triton dissolving after, measure the activity of NOS in the cell. Compare with control group, 50 μ g/ml LDL group structural type NOS (cNOS) are active obviously to reduce (P＜0.01), and Pue-glu, Pue-mal group (10^-7Mol/L) cNOS is active in the cell obviously improves, and relatively there were significant differences (P＜0.01, table 2) with 50 μ g/ml LDL group, and Pue-glu, Pue-mal do not make significant difference to induced NOS (iNOS) activity. Pue-glu, Pue-mal group cNOS is active to organize apparent in view raising (P＜0.05, table 2) with 50 μ g/ml LDL.

2.3 experimental result shows that the model group of the high lipid food of feeding (Model) compares with Normal group (Control), brings out TG in the rat blood serum, TC, the rising of LDL-C content behind the 6W, HDL-C content obviously reduces (P＜0.01). Experimental group (Pue-glu, Pue-mal) compares with Normal group (Control), every blood lipids index also has than significant change (P＜0.05), the content of mouse serum TG, TC, LDL-C significantly reduces (P＜0.01), and HDL-C content obviously raises (P＜0.01). The results are shown in Table 3.

Table 1 Pue-glu, the impact of NO content in the Pue-mal Human Umbilical Vein Endothelial Cells nutrient solution (

±s，n＝8)

Compare with Normal group:^*Compare with model group: #P＜0.05, ##P＜0.01 P＜0.05

Table 2 Pue-glu, the impact of NOS activity in the Pue-mal Human Umbilical Vein Endothelial Cells (

±s，n＝8)

Compare with Normal group:^*Compare with model group: ##P＜0.01 P＜0.05

Table 3 Pue-glu, Pue-mal on the impact of Serum Lipids in Experimental HypercholesterolemicRats (

±s)

^*Compare P＜0.05 with Normal group, ## and model group compare: P＜0.01

The especially high TC of high fat of blood and LDL-C are for causing the one of the main reasons of atherosclerotic (AS). And high TG is the hazards [Wei Yulin etc., the preliminary observation of lipanthyl administered in essential hyperlipidemic patients treatment EHL curative effect, south of the Five Ridges cardiovascular disease magazine, 1997,2 (4): 36] of important initiation coronary heart disease. Therefore, prevent and treat hyperlipidemia prevention AS and coronary heart disease are had positive role. Originally studies show that Pue-glu, Pue-mal can effectively reduce TG in the hyperlipidemia rats serum, TC content can obviously improve HDL-C content in the rat plasma. The HDL-C/TC value is larger, then anti-AS effect stronger [punishment is vertical new for Cai Xiucheng, Wang Liguang, etc. pine-seed oil is on the impact [J] of rat fat and lipid peroxidation. Food Science, 1999,20 (6): 54,56.]

Example 31 control diabetic nephropathy experiments

1 method

1.1 behind 40 SD male rats of animal modeling precuring 1w, empty stomach 12h, lumbar injection 1% Streptozotocin (STZ) sodium citrate buffer solution (STZ 60mg/kg body weight), control group injection equivalent sodium citrate buffer solution. Blood sugar is surveyed in the blood sampling of 72h posterior vein, and blood sugar 〉=16.7mmol/L person is determined model foundation, includes this experiment in. Include 30 diabetic mice in experiment.

1.2 grouping Normal group (10); Diabetic mice is divided into each 10 of diabetic model group (10) and Pue-glu, Pue-mal treatment groups at random. Pue-glu, Pue-mal respectively organize every each 80mg/kg/d of lumbar injection Pue-glu, Pue-mal, successive administration 16w. Respectively organize for fear of ketosis and the existence of keeping diabetic mice, lumbar injection protamine zine insulin 2～4 units next day of model group and Pue-glu, Pue-mal group, and dock and get blood survey rat blood sugar, make blood glucose fluctuation about 25mmol/L, test and be total to 16w. When the 16w experiment stopped, each organized rat on the basis of fasting, collected 24h urine with metabolic cage, measured urinary albumin (UAE) content. Weigh behind the animal via etherization, femoral artery bloodletting 5ml surveys rat blood sugar, urea nitrogen, serum creatinine, calculates endogenous creatinine clearance rate, 8-iso-PGF_2α。

1.4 the observation index ordinary circumstance is observed: observe rat diet amount of drinking water, the state of mind, active situation, stool proterties and urine amount every day, the next day survey body weight and record in addition. Observe amount of drinking water, the urine amount (collecting urine with metabolic cage) of every rat, get its mean value as final data. Kidney weight in wet base: go to weigh with precision balance behind the coating.

1.5 biochemical indicator is surveyed blood sugar, urea nitrogen, serum creatinine with full automatic biochemical apparatus. Urinary albumin excretion ratio (UAER): collect the 24h urine, anticorrosion with dimethylbenzene, get 2ml behind the mixing and measure with the immunocyte calculating instrument. Glomerular filtration rate(GFR (GFR): take endogenous creatinine clearance rate as representative, GFR=(UCr * urine amount)/serum creatinine. 8-iso-PGF_2αReference reagent box specification is measured.

2 results

2.1 ordinary circumstance is observed normal group: rat body weight increases obviously, and mental status is good, and fur is glossy, moves freely, and is quick on the draw. Rat model: spirit gradually dispirited, obviously become thin, diuresis, many drinks, how moving in early days, late phase reaction is blunt, slow, the hunchbacked snake body of action, hidrosis go out, hair perpendicular matt, the urine amount is many, need change bedding and padding every day 1～2 time; 1 visual impairment is arranged; 2 cataract are arranged; There is 1 recurrent diarrhea to occur. Pue-glu, Pue-mal group: mental status is good, without cataract and tail, sufficient downright bad, moves freely, and reaction sensitivity compared with normal group is slightly poor.

2.2 Pue-glu, Pue-mal obviously descend on the rat compared with normal group body weight that affects the modeling success of rat urine amount, body weight, kidney weight/body weight, blood sugar, the urine amount increases, and blood sugar raises, and kidney weight/body weight ratio increases (P＜0.05). After the treatment, Pue-glu, Pue-mal organize than the model group hypourocrinia, and body weight increases, kidney weight/body weight ratio descends (P＜0.05), sees Table 1.

2.3 Pue-glu, Pue-mal obviously raise on the rat blood serum UAER that affects the modeling success, serum creatinine, the urea nitrogen compared with normal group of rat UAER, serum creatinine, CrCl, urea nitrogen, endogenous creatinine clearance rate descends, significant difference (P＜0.05), model group is organized than Pue-glu, Pue-mal, UAER, serum creatinine, urea nitrogen obviously raise, endogenous creatinine clearance rate descends (P＜0.05), sees Table 2.

2.4 Pue-glu, Pue-mal are to 8-iso-PGF_2αAnd the rat blood serum 8-iso-PGF that affects the modeling success of serum glycosylated hemoglobin (HbAlc)_2αObviously raise (P＜0.05) with serum HbAlc compared with normal group, model group is than Pue-glu, Pue-mal group, 8-iso-PGF_2αObviously raise with serum HbAlc (P＜0.05), see Table 3.

The variation that table 1 is respectively organized rat urine amount, body weight, kidney weight, body weight, blood sugar relatively (n=10,

±s)

^*Compare with Normal group, # and model group compare: P＜0.05, following table are together

The variation that table 2 is respectively organized rat UAER, serum creatinine, CrCl, urea nitrogen relatively (n=10,

±s)

Table 3 is respectively organized rat 8-iso-PGF_2αAnd the comparison of serum HbAlc (n=10,

±s)

The experiment of example 32 insulin resistances

1 method

1.1 foundation and the grouping of insulin resistance-hypertension (IRH) rat model

Press the rat body weight stratified random with the SPSS statistical software and extract 20 blank groups out, drink distilled water and normal diet and feed; All the other rats are drunk 5% sucrose, 22% salt solution, feed and raise the high salt high sugar feed of high fat (20% lard, 15% yolk powder, 10% sucrose, 3% salt, 52% normal diet) modeling. Every 2w detects blood pressure 1 time; In the 4th week and the 8th week, detect the rear 2h blood sugar (2hBG) of fasting blood-glucose (FBG) and oral glucose tolerance test (OGTT); 8w, the most of blood pressure compared with normal of modeling rat group rat blood pressure increases 3～5kPa (1kPa=715mmHg), and impaired glucose tolerance occurs; Select the IRH modeling rat of blood pressure and IGT, be divided at random following 6 groups with the equilibrium of SPSS statistical software: the model negative control group; Model captopril (Cap group); Each high and low 2 dosage group of Pue-glu, Pue-mal.

1.2 administrated method and index detect

Each is organized rat and all continues modeling 4w according to said method. Simultaneously, equal im 50% propane diols of blank and model negative control group, 0.18ml/kg, qd * 4w; Cap group im Cap parenteral solution 7mg/kg, qd * 4w; Pue-glu, Pue-mal be all by 60mg/kg, 30mg/kg grouping, and choose rats with bilateral Zusanli (rear three li), Pishu, shen shu and carry out acupoint injection therapy, and every day, 1 group of acupuncture point was used alternatingly qd * 4w. More than each group after administration the 2nd and each measuring blood pressure of 4w 1 time, 4w is FBG and OGTT-2hBG detects. Whole rat overnight fastings, behind the last administration 2h, all rat eye socket bloodletting, centrifugal, separation of serum, and press the operation of kit specification, measure serum cholesterol (TC), triglycerides (TG) and blood plasma blood-sugar content (FPG) with Biochemical Analyzer; Measure blood plasma FPI (Fins) content with the ELISA instrument, and calculating insulin sensitivity index (ISI): ISI=In [1/ (Fins * FPG)].

2 results

2.1 Pue-glu, Pue-mal are on the impact of IRH rat blood pressure and sugar tolerance

Table 1 result shows that IRH rat blood pressure and OGTT-2hBG are apparently higher than the blank group, and difference has highly significant (P＜0.01); Cap, Pue-glu, Pue-mal 60mg and 30mg/kg group can obviously reduce IRH rat blood pressure and OGTT-2hBG (P＜0.01); Pue-glu Pue-mal hypotensive effect is dose-effect relationship.

2.2 Pue-glu Pue-mal shows that to improvement effect table 2 result of IRH rat fat and insulin sensitivity the plasma F ins content of IRH rat, TG and TC content are apparently higher than the blank group, difference has conspicuousness (P＜0.05～0.01); ISI difference obviously descends, and highly significant (P＜0.01) is arranged; Cap group, Pue-glu, Pue-mal 60mg/kg and 30mg/kg group can reduce IRH rat TG, TC content (P＜0.05～0.01); Cap and Pue-glu Pue-mal 60mg/kg group can also reduce the blood plasma blood-sugar content (P＜0.05) of IRH rat. Cap and Pue-glu 60mg/kg group can improve low ISI, and difference has highly significant (P＜0.01), strengthens insulin sensitivity; And Pue-glu, the Pue-mal low dose group, to Fins content and ISI effect, difference is showed no conspicuousness (P＞0.05).

2.3 conclusion: experimental result as seen; Pue-glu, Pue-mal can obviously reduce IRH rat TG, TC level; prompting Pue-glu can correct the lipid-metabolism of IRH rat disorder effectively, thereby effectively protects β cell and antagonism insulin resistance, reduces the IRH rat blood pressure.

Table 1 Pue-glu, Pue-mal on the impact of IRH rat blood pressure (systolic pressure) and sugar tolerance (kPa, n=10,

±s)

Annotate: compare with the model negative control group,^*P＜0.05， ^**P＜0.01; Compare #P＜0.05, ##P＜0.01 with the blank group.

Table 2 Pue-glu to the effect of IRH rat fat and insulin sensitivity (n=10,

±s)

Annotate: compare with the model negative control group,^*P＜0.05， ^**P＜0.01; Compare #P＜0.05, ##P＜0.01 with the blank group.

Example 33 anti-sclerotin pine nuts are tested

1 method

1.1 grouping and administration: 70 rats, wherein after the anesthesia of 60 lumbar injection 40mg/kg yellow Jackets, excise according to a conventional method bilateral ovaries, 10 rats of remaininging are cooked identical operation technique, but spay not, as sham-operation group (Sham). Ovariectomized rat is divided into 6 groups, 1 week beginning gastric infusion after operation, Sham and OVX (Ovariectomization oophorectomize model control group) organize and give 0.5%CMC solution every day; Nilestriol (E3) group gives Nilestriol 0.15mg/kg, 2 times weekly (Monday and Thursday), every day, ig Pue-glu, Pue-mal were 80 and 20mg/kg to the large and small dosage group of Pue-glu, Pue-mal respectively, and the administration volume is the 110ml/100g body weight, continuous 7 months. 20 ℃～26 ℃ of receptacle temperature, humidity 50%～70% is fed with normal diet, freely drinks water. Claim weekly body weight and adjust the administration volume by new body weight.

1.2 bone densitometry: when administration 4 and 7 months, behind the rat 35mg/kg yellow Jackets intraperitoneal injection of anesthesia, upper with forearm bone density measurement software mensuration rat body bone mineral content (BMC) and Whole Body Bone Scanning mineral density (BMD) at dual intensity borne densitometers (DEXA). Instrument parameter: 134kV, 110mA, sweep length 26cm, width 1414cm.

2.3 the femur relative volume mass is measured: behind the last administration 1d, put to death rat, separate side femur and a shin bone, pick the muscle and the connective tissue that adhere to the greatest extent, after claiming weight in wet base on the electronic balance, measure its volume at homemade drainage volume measuring device, calculate its wetting phase to volume mass.

1.3 bone biomechanical property is measured: adopt the electronic universal test instrument to do the three point bending test of femur and shin bone, instrument parameter: loading velocity 6mm/min, peak load 200N, span 20mm, trace load 2 deformation curves, calculate peak load and structural strength (cause the load that the 1mm distortion needs, be equivalent to the slope on the curve).

1.4 femur ash relative volume mass: after measuring mechanical index, collect residual disconnected femur, claim ash heavy after the Muffle furnace ashing, ash ratio heavy and volume is femur ash relative volume mass.

1.5 femur calcium content and calcium salt density measurement: bone ash is dissolved in 40mL (6mol/L) hydrochloric acid, and with the EDTA compleximetry, calred is made indicator, measure calcium content, and calculate unit volume Ca content (calcium salt density).

1.6 uterus weight coefficient determination: dissect to take out the uterus, gently extrude the liquid in the uterine cavity, weigh on electronic balance, the ratio of it and body weight is the uterus weight coefficient.

3 results

3.1 Pue-glu, Pue-mal are on the impact of ovariectomized female rats BMC and BMD: rat after spay 4 and 7 months whole body BMC and BMD significantly reduce, especially with BMD for very. Pue-glu, Pue-mal be high low dosage 80 and the continuous ig 4 of 20mg/kg and 7 months respectively, significantly improve BMC and BMD. 4 and 7 months the time, the 80mg/kg of Pue-glu, Pue-mal and 20mg/kg group BMC and BMD raise respectively. And E3 group BMD is 4 be significantly increased 7 months the time, and BMC only significantly improved in the time of 4 months, but did not obviously improve (table 1) in the time of 7 months.

3.2 Pue-glu, Pue-mal are on the impact of rat femur relative volume mass and calcium salt density: compare with the Sham group, ovariectomized rats femur relative volume mass has reduced by 4.49%, and calcium element content reduces more (11.3%). PUE-GLU, each large and small dosage group of PUE-MAL increases respectively the femur relative volume mass, and the bone ash relative volume mass increases respectively. Simultaneously, femur calcium salt density is recovered fully, even also higher than the Sham group. The E3 group has similar effect, but the Amplitude Ratio PUE-GLU that increases, the heavy dose of group of PUE-MAL smaller (table 2).

3.3 Pue-glu, Pue-mal are on the impact of femur of mature ovariectomized rats and shin bone biomechanical property: spay is after 7 months, the biomechanical property of rat femur significantly descends, the peak load of femur and structural strength have descended respectively 15.0% and 17.3%, and the peak load of shin bone has also descended 13.5%. Pue-glu, Pue-mal significantly improve the bone biomechanical index, and heavy dose of group femur peak load and intensity improve respectively highly significant, and small dose group improves respectively significantly. The mechanical index of shin bone also is restored, structural strength even surpass the Sham group. The E3 group does not improve in peak load, but structural strength obviously improves (table 3).

3.4 Pue-glu, Pue-mal are on the impact of rat body weight and uterus weight: the heavy dose of group of Pue-glu, Pue-mal is the same with estrogen to cause that rat body weight obviously descends, and small dose group does not affect body weight. The obvious atrophy of rat uterus after the spay, weight significantly alleviates, and only is about 1/3 of Sham group. The E3 group then can make uterus weight return to Sham group level fully. Pue-glu, Pue-mal also can significantly increase uterus weight, and large and small dosage group all improves about 1 times the uterus weight coefficient, but than E3 group lower (table 4).

Table 1 Pue-glu, Pue-mal on the impact of ovariectomized female rats BMC and BMD (n=10,

±s)

Compare with the OVX group:^*P＜0.05  ^**P＜0.01; Compare with the Sham group: #P＜0.05 ##P＜0.01

Table 2 Pue-glu, Pue-mal are on the impact (mg/mm of femur of mature ovariectomized rats, bone ash and calcium salt relative volume mass³)(n＝10，

±s)

Compare with the OVX group:^*P＜0.05  ^**P＜0.01; Compare with the Sham group: #P＜0.05 ##P＜0.01

Table 3 Pue-glu, Pue-mal on the impact of femur of mature ovariectomized rats and shin bone biomechanical property (n=10,

±s)

Compare with the OVX group:^*P＜0.05  ^**P＜0.01; Compare with the Sham group: #P＜0.05 ##P＜0.01

Table 4 Pue-glu, Pue-mal on the impact of ovariectomized female rats body weight and uterus weight (n=10,

±s)

Compare with the OVX group:^*P＜0.05  ^**P＜0.01; Compare with the Sham group: #P＜0.05 ##P＜0.01

The biomaterial that embodiment relates to

The Classification And Nomenclature of biomaterial: oxidation microbacterium Microbacterium oxydans.

Depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center.

Address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100080.

Preservation date: on 08 25th, 2006.

Deposit number: CGMCC No.1788.

Claims (13)

1, a kind of glycosylated derivative of puerarin and pharmacy acceptable salt thereof, its feature is shown in structural formula (I):

Formula (I)

In the formula (I), parent nucleus is puerarin (R=H), and R is selected from monose (6 carbon sugar or 5 carbon sugar) base or oligosaccharides (2-5 6 carbon sugar is or/and the connection of 5 carbon sugar) base.

2,, it is characterized in that monosaccharide groups selection glucosyl group, mannose group, glucal acidic group according to the compound of claim 1, galactosyl, galacturonic acidic group, allose base, fructosyl, the sorb glycosyl, witch hazel glycosyl, strepto-glycosyl, 2-glucosamine base, galactosamine base, or Arabic glycosyl, the lysol glycosyl, xylosyl, ribosyl, husband's glycosyl, rhamanopyranosyl, isorhodeose base, celery glycosyl; Its oligosaccharyl is selected isomaltose base, mycose-base, sophorosyl, new dried orange peel glycosyl, rue glycosyl, locust tree diglycosyl, malt-base, sucrose base, lactose base, rough gentian glycosyl, pectin base.

3, according to the compound of claim 1, the compound of preferred 1 glucosyl group of R claims puerarin-7-O-glucoside, it is characterized in that hemiacetal hydroxyl and the hydroxyl on 7 carbon of puerarin on the glucosyl group forms glucosides; The compound of the preferred isomaltose base of R is puerarin-7-O-maltose glucosides, it is characterized in that forming glycosidic link between 6 hydroxyls of hemiacetal hydroxyl on the malt-base terminal glucose base and puerarin 7-O-glucoside.

4, according to the compound of claim 1, its pharmacy acceptable salt is the salt of metal cation salt or nitrogenous organic base, wherein,

(1) metal ion is basic metal or alkaline-earth metal, preferred sodium, potassium, calcium, magnesium; (2) nitrogenous organic bases is amino acid, preferred arginine, Methionin, carnitine; Or the C1-C40 nitrogenous organic base, preferred meglumine, glucosamine, Ligustrazine.

5, according to the microbial transformation synthetic method of the compound of claim 1, saccharide donor can be any saccharide donor that can be used as the glycosylase effect, and saccharide donor is monose or oligosaccharides or starch, its monose is 6 carbon sugar, as glucose, and seminose, glucuronic acid, semi-lactosi, galacturonic acid, allose, fructose, sorbose, hamamelose, streptose, 2-glucosamine, galactosamine, or 5 carbon sugar are as pectinose, lyxose, wood sugar, ribose, husband's sugar, rhamnosyl, isorhodeose, celery sugar, preferred glucose; Its oligosaccharides is that 2-5 6 carbon sugar is or/and 5 carbon sugar dehydrating condensation forms trehalose, sophorose, neohesperidose, rutinose, locust tree disaccharides, maltose, sucrose, lactose, rough gentian 3 sugar, pectin, preferred maltose.

6, according to the microbial transformation synthetic method of the compound of claim 1.

1) adopts somatic cells or the zyme extract of this somatic cells or the recombinant expression protein of this enzyme with puerarin glycosylated enzyme activity, monosaccharide groups on the saccharide donor or oligosaccharyl are transferred on the puerarin A ring on the hydroxyl of C-7 position, preferably with glucosyl group or isomaltose group-transfer to the puerarin A ring on the hydroxyl of C-7 position;

2) preferably synthetic puerarin-7-O-glucoside and/or puerarin-7-O-isomaltosylfructoside;

3) thalline in the synthetic liquid of conversion or bacterial enzyme albumen are through thermal precipitation, centrifugal removal;

4) with Solid-Phase Extraction or liquid-phase extraction or column chromatography separation:

A) transform synthetic liquid with Solid-Phase Extraction, its solid phase carrier can be to be polyamide or macroporous resin, the resin of water and/or the extraction of alcoholic solvent wash-out, and the elutriant concentrate drying is promptly;

B) use the liquid-phase extraction method, it is characterized in that the solvent for use system is water-organic agent, organic solvent is propyl carbinol or ethyl acetate or chloroform or ether or hexane, sherwood oil etc., or the mixture of above-mentioned two or more organic solvent, dividing and get extraction liquid, concentrate drying is promptly.

C) separate to transform synthetic liquid method with column chromatography, can adopt silica gel, or alkyl linked silica gel or macroporous resin or polyamide be stationary phase that carry out chromatographic separation, the target fraction concentrate drying promptly;

5) by the recrystallization purifying product, it is characterized in that: solvent can adopt water and/or alcoholic solvent, or alcohol and/or fat-soluble solvent, and its fat-soluble solvent feature is ethyl acetate or chloroform or ether or oxyethane, or hexane or sherwood oil.

7, according to claim 5,6 method, microbial strains with puerarin glycosylated enzyme activity, be any other can the glycosylation puerarin microorganism or mixing microorganisms, preferred microorganism is Microbacterium oxydans CGMCC 1788, also can be that Microbacterium saperdae CGMCC 1.1906 waits all Microbacterium bacterial classifications.

8, according to the preparation method of the described salt of claim 4: with structural formula (I) compound and nitrogenous organic base with 1: 0.5～2.0 mol ratio, in water and/or alcoholic solvent, react, or with structural formula (I) compound and metal ion with 1: 0.5～2.0 equivalence ratio, in water and/or alcoholic solvent, react.

9, according to the preparation method of the described salt of claim 4, wherein nitrogenous organic base preferred bases acidic amino acid or C1-C40 organic bases, or the preferred alkaline-earth metal ions of metal ion, wherein with basic aminoacids or with the mol ratio of C1-C40 organic bases reaction be 1: 0.8-1.5, or be 1 wherein: 0.8-1.5 with the equivalence ratio of alkaline-earth metal ions reaction.

10, according to the preparation method of the described salt of claim 4, wherein basic aminoacids is arginine or Methionin, or wherein the C1-C40 nitrogenous organic base is meglumine, glucosamine, Ligustrazine, or wherein alkalimetal ion is Na ⁺Or K ⁺Or alkaline-earth metal ions Mg ²⁺Or Ca ²⁺Ion.

11, contain the claim 1-4 compound for the treatment of significant quantity and the pharmaceutical composition of pharmaceutically acceptable carrier, be used to prevent and treat coronary heart disease, stenocardia, myocardial infarction, ischemic cerebrovascular disease, atherosclerosis, blood fat reducing, diabetic nephropathy and insulin resistant, osteoporosis.

12, drug combination preparation according to claim 12, it is characterized in that, its formulation is said any formulation on the pharmaceutics, comprises tablet, capsule, eye drops, sprays, gelifying agent, gel inhalation, oral liquid, suspensoid, electuary, ointment, pill, powder, injection, infusion solution, freeze dried injection, suppository, sustained release preparation or controlled release preparation.

13, claim 1-4 compound is prevented and treated coronary heart disease, stenocardia, myocardial infarction, ischemic cerebrovascular disease, atherosclerosis, blood fat reducing, diabetic nephropathy and the pharmaceutical composition of insulin resistant, osteoporosis is used in preparation.

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