CN101723997A - Puerarin glycosylation derivative, medicine compound, preparation method and application thereof - Google Patents
Puerarin glycosylation derivative, medicine compound, preparation method and application thereof Download PDFInfo
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- CN101723997A CN101723997A CN200810171912A CN200810171912A CN101723997A CN 101723997 A CN101723997 A CN 101723997A CN 200810171912 A CN200810171912 A CN 200810171912A CN 200810171912 A CN200810171912 A CN 200810171912A CN 101723997 A CN101723997 A CN 101723997A
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Abstract
The invention discloses a puerarin glycosylation derivative, a medicine compound thereof, a preparation method and application thereof, in particular to the puerarin glycosylation derivative of Formula (I) or a medical acceptable salt thereof, wherein R is a monose base (hexose or pentose) or an oligose base (connecting 2 to 5 hexose or pentose ). The medical acceptable salt is compounded salt of inorganic base or organic base. The puerarin glycosylation derivative or the medical acceptable salt thereof of the invention has better stability and water-solubility, promotes the bioavailability and enhances the healing effect.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to a kind of new puerarin derivate and pharmaceutical composition and preparation method, and reach purposes the medicine of insulin resistant, osteoporosis at preparation treatment prevention cardiovascular and cerebrovascular disease, atherosclerosis, blood fat reducing, diabetic nephropathy.
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, this main because: it is fast 1) to eliminate speed in its body, and clinical using dosage is bigger, and oral administration biaavailability is little; 2) the puerarin water solubility is low, and preparation contains solubility promoter mostly increasing stability of formulation, but insoluble impurities also increases, and human body is produced certain untoward reaction, causes drug safety to reduce.
Summary of the invention
At above technological deficiency, the invention provides a kind of new puerarin glycosylated derivant and its production and use, and the pharmaceutical composition that contains described puerarin glycosylated derivant or its pharmaceutically acceptable salt.
The invention provides the puerarin glycosylated derivant shown in a kind of formula (I) or its pharmacologically acceptable salts:
Formula (I)
Wherein, R is the monosaccharide groups of 6 carbon sugar or 5 carbon sugar, or is that 2-5 6 carbon sugar is or/and the oligosaccharyl that 5 carbon sugar connects; Described pharmacologically acceptable salts is the salt with mineral alkali or organic bases addition.
In puerarin glycosylated derivant of the present invention or its pharmacologically acceptable salts, further preferred described monosaccharide groups is in ring-type 6 carbon sugar or the 5 carbon glycosyls, 6 carbon glycosyls include but not limited to glucosyl group, mannose group, glucal acidic group, galactosyl, galacturonic acidic group, allose base, fructosyl, sorb glycosyl, witch hazel glycosyl, strepto-glycosyl, 2-glucosamine base or galactosamine base; Described 5 carbon glycosyls include but not limited to Arabic glycosyl, lysol glycosyl, xylosyl, ribosyl, husband's glycosyl, rhamanopyranosyl, isorhodeose base or celery glycosyl; Described oligosaccharyl is that 2-5 6 carbon are sugared or/and 5 carbon sugar dehydrating condensation forms oligosaccharyl, includes but not limited to mycose-base, sophorosyl, new dried orange peel glycosyl, rue glycosyl, locust tree diglycosyl, malt-base, sucrose base, lactose base, gentianose base or pectin base;
Described mineral alkali is the mineral alkali of basic metal or alkaline-earth metal, and organic bases is a nitrogenous organic base, and the mineral alkali of basic metal of the present invention or alkaline-earth metal includes but not limited to the mineral alkali of sodium, potassium, calcium or magnesium; Described nitrogenous organic bases can be basic aminoacids, includes but not limited to arginine, Methionin or carnitine, or the C1-C40 nitrogenous organic base except that amino acid, includes but not limited to meglumine, glucosamine or Ligustrazine.
Puerarin glycosylated thing derivative of the present invention or the acceptable salt of its medicine are preferably: puerarin-7-O-glucoside, puerarin-7-O-isomaltose glucosides, the puerarin shown in the puerarin shown in the formula (II)-7-O-fructoside or the formula (III)-7-O-isomaltulose glycosides;
Formula (II)
Formula (III)
Described its pharmacologically acceptable salts is the salt with the mineral alkali addition of potassium, calcium or magnesium, or with the salt of amino acid, meglumine, glucosamine or Ligustrazine addition, wherein, amino acid is preferably arginine, Methionin or carnitine.
Puerarin of the present invention-7-O-glucoside, the hydroxyl that is on 7 carbon of hemiacetal hydroxyl on the glucosyl group and puerarin A ring forms glucosides; Described 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; Described puerarin-7-O-fructoside, the hemiacetal hydroxyl and the hydroxyl on 7 carbon of puerarin that are on the fructosyl form glucosides; Described puerarin-7-O-isomaltulose glycosides is 6 ' on the hemiacetal hydroxyl of the terminal glucose base on the isomaltulose base on puerarin-7-O-isomaltulose glycosides and the fructosyl " form glucosides between the hydroxyl of position.
The present invention also provides the preparation method of described puerarin glycosylated derivant of formula (I) or pharmacologically acceptable salts.The present invention is transformed into monose on the saccharide donor or oligosaccharyl on the position of C-7 on the puerarin A in water, organic phase or water/organic phase two-phase by having the microbial strains of puerarin glycosylated enzyme activity, thereby obtains described derivative;
Wherein, described water is that the aqueous solution and pH are the buffered soln of 5-9, described organic phase is ethanol, butanols, acetone, ethyl acetate, dimethyl sulfoxide (DMSO) or dimethylformamide or its its mixture, and described water/organic phase two-phase is above-mentioned the do not contain water of buffered soln and the combination of above-mentioned organic phase; As required, the present invention can also add solubility promoter in mutually to above-mentioned, and described solubility promoter includes but not limited to soil temperature-80, Viscotrol C, polyoxyethylenated castor oil, polyvinylpyrrolidone, poloxamer or PluronicF-68.
Described conversion condition is: concentration of substrate is the 0.01%-saturation concentration; Invert point is 20～40 ℃; 10～400rpm shakes a bottle oscillatory reaction, or the reactor stirring reaction; Transformation time is 12～120 hours;
Wherein, described saccharide donor is monose or oligosaccharides or starch, and described monose includes but not limited to glucose, seminose, glucuronic acid, semi-lactosi, galacturonic acid, allose, fructose, sorbose, hamamelose, streptose, 2-glucosamine, galactosamine, pectinose, lyxose, wood sugar, ribose, husband's sugar, rhamnosyl, isorhodeose, celery sugar; Described oligosaccharides includes but not limited to trehalose, sophorose, neohesperidose, rutinose, locust tree disaccharides, maltose, sucrose, lactose, gentianose or pectin;
The preparation method that described its pharmacy is accepted salt is: the conversion puerarin glycosylated thing that obtains and the mineral alkali or the nitrogenous organic base that suit are reacted, thereby generate the pharmacologically acceptable salts of puerarin glycosylated derivant, wherein the acidic functionality on the glycosyl of alkali and puerarin glycosylated derivant forms salt; Affiliated mineral alkali is the mineral alkali of basic metal or alkaline-earth metal, includes but not limited to the mineral alkali of sodium, potassium, calcium or magnesium; Nitrogenous organic base can be basic aminoacids, includes but not limited to arginine, Methionin or carnitine; Or the C1-C40 nitrogenous organic base except that amino acid, include but not limited to meglumine, glucosamine or Ligustrazine, preferred meglumine.
The present invention is preferably gained puerarin glycosylated derivant and nitrogenous organic base with 1: 0.5～2.0 mol ratio, and preferred 1: the mol ratio of 0.8-1.5, in water and/or alcoholic solvent, react; Or with metal ion with 1: 0.5～2.0 equivalence ratio, preferred 1: the mineral alkali of the equivalence ratio of 0.8-1.5, in water and/or alcoholic solvent reaction promptly, affiliated metal ion includes but not limited to K
+, Na
+, Ca
++, Mg
++Ion.
The preparation method of puerarin glycosylated derivant of the present invention preferably includes following steps:
1) adopts somatic cells, microbial cells resting cell, the zyme extract of this somatic cells or the recombinant expression protein of this enzyme through osmotic treated with puerarin glycosylated enzyme activity, at water, in organic phase or water/organic phase two-phase the monosaccharide groups on the saccharide donor or oligosaccharyl transferred on the puerarin A ring on the hydroxyl of C-7 position, obtain the synthetic liquid of conversion of described glycosylated puerarin; Wherein, described water is that the aqueous solution and pH are the buffered soln of 5-9, described organic phase is ethanol, butanols, ethyl acetate, dimethyl sulfoxide (DMSO) or dimethylformamide or its its mixture, and described water/organic phase two-phase is above-mentioned the do not contain water of buffered soln and the combination of above-mentioned organic phase; Described conversion condition is: concentration of substrate is the 0.01%-saturation concentration; Invert point is 20～40 ℃; 10～400rpm shakes a bottle oscillatory reaction, or the reactor stirring reaction; Transformation time is 12～120 hours;
2) synthetic liquid be will transform through thermal precipitation, centrifugal, thalline or bacterial enzyme albumen removed;
3) utilize Solid-Phase Extraction, liquid-phase extraction or column chromatography to separate to transforming resulting solution then, wherein,
Described solid phase extraction is: solid phase carrier is polyamide or macroporous resin; The polyamide or the macroporous resin of water and/or the extraction of alcoholic solvent wash-out, then promptly with the elutriant concentrate drying;
Described liquid-phase extraction method is: solvent systems is water-organic solvent, and organic solvent is a kind of, two or more the mixture in propyl carbinol, ethyl acetate, chloroform, ether, hexane and the sherwood oil; Divide and get extraction liquid, concentrate drying promptly.
Described column chromatography is: with silica gel, alkyl linked silica gel, macroporous resin or polyamide is stationary phase, carries out chromatographic separation, collects target fraction, and concentrate drying promptly;
4) the step 3) products therefrom is carried out recrystallization purifying, wherein, recrystallization solvent is water and/or alcoholic solvent, or alcohol and/or fat-soluble solvent; Wherein, described fat-soluble solvent is ethyl acetate, chloroform, ether, oxyethane, a kind of, two or more combination in hexane and the sherwood oil.
Described step 2) in, is not particularly limited, includes but not limited to adopt angie type whizzer, 3-foot settling centrifuge, butterfly chip whizzer or tubular-bowl centrifuge to carry out centrifugal for centrifugal equipment the present invention who adopts.
Among the preparation method of the salt that puerarin glycosylated derivant of the present invention or its pharmacy are accepted, carry out the microbial cells resting cell of osmotic treated for the TritonX-100 that adopts 0.1-10% concentration through the microbial cells resting cell of osmotic treated.
Among the preparation method of puerarin glycosylated derivant of the present invention or its pharmacologically acceptable salts, microbial strains with puerarin glycosylated enzyme activity can be any microorganism or mixing microorganisms that can the glycosylation puerarin, the present invention is preferably the Microbacterium bacterial classification, more preferably Microbacterium oxydans CGMCC 1788, or Microbacterium saperdae CGMCC1.1906 or its mixture; The depositary institution of the wherein said CGMCC1788 of being numbered bacterial classification is: China Committee for Culture Collection of Microorganisms common micro-organisms center, and the depositary institution address is: No. 13, one of Zhongguangcun, Haidian District, Beijing City; Preservation date is on August 25th, 2006; Deposit number is CGMCC1788; Classification called after oxidation microbacterium, Microbacterium oxydans.
Among the preparation method of puerarin glycosylated derivant of the present invention or its pharmacologically acceptable salts, the microbial strains fermentation culture conditions is 20～40 ℃, 10～400rpm shakes bottle vibration aerated culture, or the cultivation of fermentor tank deep ventilation, and incubation time is 12～96 hours.The present invention for the employed nutritive substance of culturing micro-organisms bacterial classification without any restriction, it can be any nutrient media that is suitable for microorganism growth, include but not limited to LB nutrient solution, nutrition gravy nutrient solution or any be carbon source with starch, Semen Maydis powder, glucose, maltose, sucrose, citric acid, or be the substratum of nitrogenous source with corn steep liquor, cottonseed meal, soybean-cake flour, extractum carnis, yeast powder, peptone, ammonium nitrate, ammonium chloride or urea.
The present invention also provides a kind of puerarin glycosylated derivant of the present invention or pharmaceutical composition its pharmaceutically acceptable salt and/or pharmaceutical acceptable carrier that comprises significant quantity, and it is puerarin glycosylated derivant or its pharmacy acceptable salt shown in 0.1%～100% the general formula (I) that pharmaceutical composition of the present invention preferably contains molar content; Further preferred puerarin glycosylated derivant or its pharmacy acceptable salt content are 1.0%～95%.
Pharmaceutical composition of the present invention can be any formulation on the pharmaceutics, includes but not limited to tablet, capsule, soft capsule, gelifying agent, oral preparation, suspensoid, electuary, patch, ointment, pill, powder, injection liquid, infusion solution, freeze dried injection, vein emulsion, lipidosome injection or the suppository of conventional, quick-release, slowly-releasing or controlled release.
Pharmaceutically acceptable carrier of the present invention is meant the pharmaceutical carrier of pharmaceutical field routine, for example: thinner, vehicle and water etc., weighting agent such as starch, sucrose, lactose or 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; Sorbent material such as kaolin and soap clay; Lubricant such as talcum powder, calcium stearate and magnesium, micropowder silica gel and polyoxyethylene glycol etc.; Its assistant agent such as flavouring agent, sweeting agent etc.
Medicinal compositions of the present invention can carry out administration by the mode of oral, rectum or administered parenterally.When being used to obey administration, medicinal compositions of the present invention can be made conventional solid preparation such as tablet, capsule, pulvis, granule etc., or 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.The preferred dosage form of the present invention is tablet, coated tablet, capsule, granule, oral liquid or injection.
As required, pharmaceutical composition of the present invention can be made tablet or capsule, puerarin glycosylated derivant or its pharmacy acceptable salt and weighting agent, disintegrating agent are made conventional tablet or capsule; Perhaps puerarin glycosylated derivant or its pharmacy acceptable salt and weighting agent, hypromellose K4M are made the tablet or the capsule of slowly-releasing; Perhaps puerarin glycosylated derivant or its pharmacy acceptable salt are scattered in and make soft capsule in the oil phase.Described weighting agent includes but not limited to sucrose, lactose, Microcrystalline Cellulose, dextrin, starch or calcium phosphate, Yelkin TTS; Described disintegrating agent includes but not limited to hydroxypropylcellulose, sodium starch glycolate, polyvinylpolypyrrolidone or croscarmellose sodium; Can also contain tackiness agent, wetting agent or lubricant in the described preparation.
As required, pharmaceutical composition of the present invention can also be made injection, puerarin glycosylated derivant or its pharmacy acceptable salt and solubilizing agent or solvent are made injection; Perhaps directly puerarin glycosylated derivant or its pharmacy acceptable salt are made freeze-dried powder injection; Perhaps puerarin glycosylated derivant or its pharmacologically acceptable salts are scattered in and make injectable emulsion in the oil phase, described oil phase includes but not limited to soybean oil, poly(oxyethylene glycol) 400, Oleum Gossypii semen, peanut oil, sesame oil, Semen Maydis oil or sweet oil, also can add solubilizing agent or solubility promoter or oxidation inhibitor in described oil phase; Perhaps puerarin glycosylated derivant or its pharmacy acceptable salt micro mist and Polysorbate 80 are mixed grind after, be dissolved into the aqueous solution of phosphoric acid potassium dihydrogen, dipotassium hydrogen phosphate, nipagin esters and Xylo-Mucine, make the suspension type injection liquid through grinding.Described solubilizing agent includes but not limited to polyoxyethylenated castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene pyrrolidone, poloxamer, tween, polyoxyethylene glycol, pluronic F-68; Described solubility promoter includes but not limited to arginine, Methionin, meglumine or glucosamine, diethylamine, quadrol, urea, carnitine, Ligustrazine, niacinamide, proline(Pro), glucose or Citric Acid and sodium salt thereof.
The present invention also provides the puerarin glycosylated derivant shown in the formula (I) or its pharmacologically acceptable salts, and the pharmaceutical composition that contains described puerarin glycosylated derivant or its pharmacologically acceptable salts is in preparation treatment, prevention coronary heart disease, stenocardia, myocardial infarction, ischemic cerebrovascular disease, atherosclerosis, blood fat reducing, diabetic nephropathy and to the application in the medicine of insulin resistant, osteoporosis.That puerarin glycosylated derivant shown in the formula of the present invention (I) or its pharmacologically acceptable salts have is hypotensive, coronary artery dilating and the cerebrovascular, improves the brain microcirculation, protects ischemic myocardium and myocardial ischemia-reperfusion injury, antithrombotic, decomposition plasma fibrinogen, blood viscosity lowering, prevents atherosclerosis, blood fat reducing, keeps that blood sugar is normal, anti-oxidant, anti-ageing, control bone resorption and promote bone growth, suppress apoptosis, suppress effects such as growth of tumour cell.
Puerarin glycosylated derivant shown in (I) of the present invention or its pharmacologically acceptable salts not only have better stability and water-soluble, and have improved dissolution in vitro, have improved bioavailability.The present invention and puerarin are than solubleness is good, pharmacologically active is higher, the transformation period prolongs in the body, has remedied the puerarin product defects, and the security of medication is improved, and curative effect increases, and is the good new drug product of a development prospect.It is fast to have overcome the interior elimination of puerarin body speed, and the bad shortcoming of preparation stability
The result shows, 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.
Description of drawings
Fig. 1 is that conversion fluid HPLC analyzes collection of illustrative plates among the embodiment 1;
Fig. 2 is that substrate puerarin HPLC analyzes collection of illustrative plates among the embodiment 1;
Fig. 3 is that the product puerarin 7-O-glucoside HPLC after the separation and purification analyzes collection of illustrative plates among the embodiment 1;
Fig. 4 is that the conversion fluid HPLC among the embodiment 2 analyzes collection of illustrative plates;
Fig. 5 is that the product puerarin 7-O-isomaltose glucosides HPLC after the separation and purification among the embodiment 2 analyzes collection of illustrative plates;
Fig. 6 is that conversion fluid HPLC analyzes collection of illustrative plates among the embodiment 38;
Fig. 7 is that the product puerarin 7-O-fructoside HPLC after the separation and purification analyzes collection of illustrative plates among the embodiment 38.
Embodiment
Describe the present invention in further detail below in conjunction with specific embodiment and test example, but the present invention is not limited to this.
The component that list in the source of following examples and test used reagent in the example, trade(brand)name and being necessary 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
Bacterial classification M.oxydans CGMCC 1788 is inoculated in the 1000mL triangular flask that contains 300mL LB substratum, 30 ℃, 220rpm shaking culture 24 hours; Then cultured nutrient solution is linked in the 5L fermentor tank that contains 3L LB, rotating speed 500rpm, 30 ℃ of temperature, air flow 3L/min, tank pressure maintains 0.1MPa, fermentation 11-12h; Then, the centrifugal 10min of 8000rpm collects thalline, cleans one time with 1/15M PBS (pH8.0), recentrifuge is collected, and thalline is transferred in the 3L conversion fluid, and conversion fluid contains puerarin 4mg/mL, sucrose 6%, 1/15M PBS, pH8.0 is in the 5L reactor, 500rpm, 30 ℃, transform under the air flow 3L/min condition and carry out 48h, treat that puerarin-7-O-glucoside transformation efficiency reaches about 50% stopped reaction.100 ℃ of conversion fluids were boiled 10 minutes, the centrifugal 10min of 8000rpm removes thalline and precipitation behind the cool to room temperature, getting the supernatant conversion fluid joins on the AB-8 type macroporous resin adsorption chromatography column, with 0%-20% ethanol line style gradient elution, substep is collected effluent liquid, obtains 13.8L puerarin 7-O-glucoside elutriant altogether.The liquid underpressure distillation of collecting is concentrated into the 100ml postlyophilization, obtains puerarin-7-O-glucoside powder 5.8g at last, HPLC detects purity 99.0%, the mole rate of recovery 70%.
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，glucosylH-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.
Bacterial classification M.oxydans CGMCC 1788 is inoculated in the 250mL triangular flask that contains 00mL LB substratum, 30 ℃, 220rpm shaking culture 12 hours, then, the centrifugal 10min of 000rpm collects thalline, clean one time with 1/15M phosphate buffer soln (pH8.0), behind the recentrifuge somatic cells that obtains is suspended in 100 milliliters of 1/15M phosphate buffer solns, after using the broken instrument smudge cells of Franch Press high pressure cell, the centrifugal removal cell debris of 12000rpm is collected the supernatant cell extract, add the 100ml conversion fluid, conversion fluid contains puerarin 4mg/mL, sucrose 6%, 1/15MPBS, pH8.0,30 ℃, the 220rpm concussion transforms 48h, and the HPLC collection of illustrative plates of surveying was seen Fig. 2 after conversion fluid diluted 100 times.100 ℃ of conversion fluids were boiled 10 minutes, the centrifugal 10min of 8000rpm removes thalline and precipitation behind the cool to room temperature, getting the supernatant conversion fluid joins on the AB-8 type macroporous resin adsorption chromatography column, with 0%-30% ethanol line style gradient elution, substep is collected effluent liquid, obtains 200mL puerarin 7-O-isomaltose glucosides elutriant and 450mL puerarin 7-O-glucoside elutriant altogether.The liquid underpressure distillation of collecting is concentrated, lyophilize obtain 0.08g puerarin 7-O-isomaltose glucosides and 0.18g puerarin 7-O-glucoside at last.Puerarin 7-O-isomaltose glucosides purity 92%.
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′″，g?l?c-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 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 ℃, 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 1/15mo l/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, stops 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, and 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, and puerarin-7-O-glucoside crystal and puerarin-7-O-isomaltosylfructoside crystal detects its content all greater than 99% through HPLC.
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 ℃, 3L/mi n 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% maltose-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.
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 stopped fermentation after 12 hours, somatic cells is collected in centrifugation, put into the 500mL triangular flask, add 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, stop conversion reaction, centrifugal removal thalline, heating concentrates 10 times, add 95% ethanol and transfer concentration to 20%, thalline is removed in centrifugal or filtration, and supernatant liquor passes through macroporous resin column, behind the water wash-out impurity, with 60% ethanolic soln wash-out, collect elutriant, 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.
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 7
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.
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 9
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.
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.
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.
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 13
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 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 ℃ of baking oven forced air drying; 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 15
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 (feeding intake by puerarin-7-O-glucoside calculating, down together)
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 ℃ of baking oven forced air drying; 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 16
The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside tablet
Puerarin-7-O-glucoside or puerarin-7-O-isomaltose 20g
Glycosides
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.
The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside salt tablets
Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside 20g
Salt (feeding 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 18
The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside Yelkin TTS sheet
Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside 20g
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 19
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.
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 21
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-isomaltosylfructoside 80g
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.
The preparation of puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside slow releasing tablet
Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside 50g
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.
Embodiment 23
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 24
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 25
The preparation of injection puerarin-7-O-glucoside or puerarin-7-O-isomaltose aglycone powdery injection
| Puerarin-7-O-glucoside or puerarin-7-O-isomaltosylfructoside | ??10.0g |
| 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 26
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 27
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 28
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 29
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, puerarin-7-O-glucoside, puerarin-7-O-isomaltosylfructoside are stablized on preparation than puerarin.The result is as follows:
Puerarin and derivative injection thereof are relatively
| Compound | Concentration (mg/ml) | All backs outward appearance is placed in 90 degree water-baths |
| Puerarin-7-O-glucoside | ??4.0 | Clarification |
| Puerarin-7-O-isomaltosylfructoside | ??4.0 | Clarification |
| Puerarin-7-O-glucoside: puerarin-7-O-isomaltosylfructoside (1: 1) | ??4.0 | Clarification |
| Puerarin | ??2.0 | Clarification has the false hair of trace |
| Puerarin | ??4.0 | Muddy |
Experimental example 30-37 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 are represented with x ± s, adopt 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.
The dynamic metabolism experiment of experimental example 30 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 a room temperature; Detector is an AgilentG1314 A UV-detector, and wavelength is 254nm; Moving phase: A: water adds 0.1 ‰ (v/v) chromatographic grade acetate, B: methyl alcohol, A: B=70: 30; Velocity of flow 1mL/min.
2 test methods and result
2.1 sample collecting
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 after t=1,5,10,15,20,30,40,60,80,100,120,150, behind the 180min, the centrifuge tube that 0.5 milliliter of blood of eye socket collection places heparin sodium to handle, immediately with centrifugation 15min under the condition of 3000rpm, separated plasma is in order to analyzing.Control group is gathered blood plasma and is made blank plasma after off-test.
2.2 sample preparation
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, from 10min, 10000rpm puts into refrigerator and leaves standstill 2h then, centrifugal 10min before measuring, supernatant liquor are used for high performance liquid chromatography (HPLC) analysis.
2.3 data analysis
According to described processing of 3.2.2 and use sample, the plasma concentration of Pue and Pue-glu calculates with the single-point external standard method.The data medication that obtains is analyzed for the kinetics 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 table 5 as seen, the residence time of Pue-glu in blood compared with Pue and obtains prolonging, and T1/2 is 2.85 times of Pue, and helping medicine has long action time in vivo.Corresponding, the AUC of evidence Pue-glu is than Pue height, and the Cmax of Pue-glu is 1.6 times of Pue.
The dynamic metabolism in rat (Pharmacokinetic) parameter (n=5) of table 5Pue and Pue-glu
*: p＜0.01 difference and remarkable, *: p＜0.05 significant difference
The transformation period that t1/2=eliminates; The MRT=average retention time; Area under the AUC=concentration-time curve; The peak concentration that C (max)=compound can reach.
The vasodilative pharmacological effect test of experimental example 31 puerarin derivates
Test method and result
1 method
Rat is hit unconsciously with blunt, and dislocation of cervical vertebra is opened the thoracic cavity rapidly after putting to death, and gets its thoracic aorta, puts into and fills 4 ℃, logical 100%O
2In the culture dish of saturated K-H liquid.Carefully pull out blood vessel reticular tissue on every side, wash vessel inner blood, be cut into the vascular circle of long 3～4mm, insert tube chamber, behind the destruction blood vessel endothelium that rolls, hang on and lead to 100%O with light gage wire
2The 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), treat vascular circle shrink reach stable after, change clothes to baseline with K-H liquid, stablize 20min.Repeat this operation once.Add PE 110
-5M preshrinking arterial ring waits to shrink the stable vagusstoff (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 restraining effect to various contractions: A, PE (110
-5M) inductive shrinks, treat vasoconstriction reach stable after, add the add up Pue of dosage or the contraction that Pue-7-O-glc causes; B, KC (110
-1M) inductive shrinks, treat vasoconstriction reach stable after, add the add up Pue of dosage or the contraction that Pue-7-O-glc causes.Record maximum collapse tension force (g) is control group with not dosing group, calculates the inhibition contraction percentage of the contraction that various medicines cause different treatment.
2 results
2.1Pue or Pue-7-O-glc influences KCl preshrinking aortic annulus tensile
After KCl caused that the rat chest aorta narrowing of the ring reaches maximum amplitude, 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 to the pre-shrunk aortic annulus tensile influence 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
2.2Pue, Pue-glc, Pue-mal influences PE preshrinking aortic annulus tensile
After PE caused that the rat chest aorta narrowing of the ring reaches maximum amplitude, 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 to the pre-shrunk aortic annulus tensile influence 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 the aortic annulus that (Pue-mal) can suppress more strongly behind the removal endothelium that KCl causes than Pue shrink, and do 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 disease, stenocardia, myocardial infarction, ischemic cerebrovascular disease, thrombosis of retinal artery, sudden deafness; Prevent and treat atherosclerosis, blood fat reducing, diabetic nephropathy and the pharmaceutical composition of insulin resistant, osteoporosis is used.Pue-glu, pue-mal compare in vasodilatory pharmacological action aspect some more excellent with Pue.
Experimental example 32 coronary heart disease, stenocardia, 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 pre-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 duplicates rat heart muscle I/R model.Fixing behind 10% Chloral Hydrate, 0.3～0.4ml/100g intraperitoneal injection of anesthesia, trachea cannula connects the animalcule respirator.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,, go out pin to the pulmonary conus direction at left auricle of heart lower edge 3～4mm place inserting needle, a bit of polyethylene tube (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(ECG, with occur the tall and big broadening of QRS wave group be 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. perfusion again of polyethylene tube, 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 physiopathology, 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 120min again, give equivalent physiological saline tail vein injection.
(3) IPC group: ischemic 5min, pour into 5min again, 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
Finish back taking-up heart rapidly 2.1 every group of 10 rats of myocardial infarction area are poured into again, original position ligation LAD through aorta retroperfusion Azo-Blue 1～2ml, separates non-ischemic region (indigo plant is dyed) and ischemic region (no indigo plant is dyed).Ischemic region and non-ischemic region cardiac muscle are blotted, weigh with filter paper respectively, represent ischemic areas with the ischemic region weight and the per-cent of weight whole-heartedly.Again the ischemic region edge is cut perpendicular to the heart y direction, every thick 1～2mm, place 1%TTC phosphoric acid buffer (pH 7.1) to hatch 20min for 37 ℃, then infarcted region (canescence) is separated with non-infarcted region (brick-red), filter paper blots, weighs respectively, represents infarct size with the infarcted myocardium weight and the ratio of ischemic region cardiac muscle weight.
2.2 serum CK vigor and TnT level are respectively organized rat and poured into and finish the back femoral artery and get blood, 4 ℃, 3500r/min is centrifugal, and separation of serum is then in strict accordance with test kit specification sheets time-and-motion study CK vigor.Perfusion finishes the back femoral artery and gets blood 1ml again, and anticoagulant heparin is surveyed TnT, and 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 does not see that myocardial infarction takes place; Except that the S group, ischemic areas no significant difference (P＞0.05) between each group; Organize relatively with I/R, IPC group, Pue-glu group and Pue-ma l group infarct size all dwindle (P＜0.05), and Pue-glu organizes and the Pue-mal group does not have significance (P＞0.05) with the IPC group difference respectively.The result shows that Pue-glu and Pue-mal pre-treatment can significantly dwindle the myocardial infarct size that ischemia-reperfusion causes, and is similar with the IPC effect.The results are shown in following table.
Myocardial ischemia area and infarct size (n=10,
)
# and I/R group compare P＜0.05
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-ma l group CK vigor, TnT concentration obviously reduce (P＜0.01 or P＜0.05) than the I/R group, and organize no significant difference (P＞0.05) with IPC.Serum CK vigor and TnT concentration when as seen, Pue-glu and Pue-mal pre-treatment all can reduce ischemical reperfusion injury with IPC.Following table as a result.
Table serum CK vigor and TnT change in concentration (n=10,
)
Compare * P＜0.05, * * P＜0.01 with the S group; Compare #P＜0.05, ##P＜0.01 with the I/R group
The experiment of experimental example 33 ischemic cerebrovascular diseases
1 method
The mensuration of brain microcirculation volume of blood flow
Mice by intraperitoneal injection vetanarcol (40mg/kg) anesthesia along crown medisection skin, is gently scraped parietal bone, uses J I2200 type laser microcirculation kinetic analyzer and measures the brain microcirculation volume of blood flow.Begin experiment after stablizing 5-10min.The every treated animal of normal mouse is all measured earlier and record normal brain activity microcirculatory blood flow, 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 disturbance mouse due to the high molecular dextran (HMWD), write down the microcirculatory blood flow that tail vein injection 10%HMWD 0.2ml causes the microcirculation disturbance front and back respectively, tail vein injection Pue-glu behind the 3min, Pue-mal, the same record injection back each the time phase the brain microcirculation volume of blood flow.
2 results
2.1Pue-glu Pue-mal is to microcirculatory 10 every group of small white mouses, body weight 18-22g, random packet, the male and female half and half of influencing 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 following table.From table, can find out, intravenous injection Pue-glu, 3-5min behind the Pue-mal, the brain microcirculation volume of blood flow promptly significantly raises, and its effect continues 30min at least.
Table Pue-glu, Pue-mal to the influence of normal mouse brain microcirculatory blood flow (mV,
)
Annotate: compare with control group: * P＜0.05, * * P＜0.01
2.2Pue-glu Pue-mal is to the influence of microcirculation disturbance mouse brain microcirculatory blood flow due to the HMWD
30 of small white mouses, male and female half and half, 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 following table.By finding out that behind the mouse mainline HMWD, the brain microcirculation volume of blood flow obviously reduces, and keeps a lower level behind the 3-5min in the table, inject Pue-glu this moment, and Pue-mal can make the brain microcirculation volume of blood flow that rising is arranged slightly, acts on more obvious.
Table Pue-glu, Pue-mal to the influence of microcirculation disturbance mouse brain microcirculatory blood flow due to the HMWD (mV,
)
Annotate: compare with control group: * P＜0.05, * * P＜0.01
The experiment of experimental example 34 atherosclerosiss
1. method
1.1 the cultivation aseptic condition of aortic endothelial cell takes out the thoracic aorta of newborn cow down rapidly, place high anti-Hanks solution, wash remaining blood stains off, be transferred in the culture dish, adopt the tryptic digestion method to obtain endotheliocyte, with the RPMI-1640 culture medium culturing that contains 10% calf serum, put 37 ℃, 5%CO
2In 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 experimentize.
1.2 the endotheliocyte that influences to low-density lipoprotein (LDL) inductive endothelial cell damage is inoculated in 24 orifice plates with the density of 1 * 105cell/ml, every hole 0.5ml, be cultured to fusion after, add 10 respectively
7Mol/ml Pue-glu after Pue-mal cultivates 12h, changes the substratum 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 divides control group (the RPMI-1640 substratum that adds 0.5% new-born calf serum (FBS) in the culture fluid of endothelial cell; Model group (adding 0.5%FBS RPMI-1640 substratum and 50 μ g/mlLDL 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
-7Mol/L adds 50 μ g/ml LDL effect 24h then) 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 into Control, Model, Pue-glu, Pue-mal group at random, 6 every group.Control: blank group, feeding standard basal feed; The Model group: model group, the high lipid food of feeding is formed [Li Yikui, herbal pharmacology experimental methodology, Shanghai: Science and Technology of Shanghai press, 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 of feeding by 80mg/kg.Each organizes the feeding environment term harmonization.Lipid determination is when feeding for 6 weeks, the tail vein is got blood, adopt elisa reagent (box) to use VitalabMicro semi-automatic biochemical analyzer (Holland) to measure the content of triglyceride level (TG), cholesterol (TC) and high density lipoprotein cholesterol (HDL-C), calculate low density lipoprotein cholesterol (LDL-C) value according to measurement result.
2. result
2.1 the influence to NO content in the culture fluid of endothelial cell is compared with control group, 50 μ g/mlLDL group NO content obviously reduces (P＜0.05), and Pue-glu, Pue-mal make the synthetic and secretion NO rising (seeing the following form in P＜0.01) of endotheliocyte.
Table Pue-glu, Pue-mal to the influence of NO content in the culture fluid of endothelial cell (
, n=8)
Compare with the normal control group:
*P＜0.05, compare with model group:
#P＜0.05,
##P＜0.01
2.2 the active cell that influences of nitricoxide synthase in the endotheliocyte (NOS) with after the 1%Triton dissolving, is measured the activity of NOS in the cell.Compare with control group, 50 μ g/ml LDL group structure-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 (seeing the following form in P＜0.05) with 50 μ g/ml LDL.
The table Pue-glu, Pue-mal to the active influence of NOS in the endotheliocyte (
, n=8)
Compare with the normal control group: * P＜0.05, compare with model group: ##P＜0.01
2.3 experimental result shows that the model group of the high lipid food of feeding (Model) compares with normal control 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 control group (Control), every blood lipids index also has than considerable 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 following table.
Table Pue-glu, Pue-mal are to the influence of rat fat level (x ± s)
* compare P＜0.05 with the normal control group, ## and model group compare: P＜0.01
Especially high TC of hyperlipidemia and LDL-C are for causing the one of the main reasons of atherosclerosis (AS).And high TG is the Hazard Factor [Wei Yulin etc., micronize lipanthyl treatment Primary hyperlipemia Preliminary observation of curative, south of the Five Ridges cardiovascular diseases magazine, 1997,2 (4): 36] of important initiation coronary heart disease.Therefore, prevent and treat hyperlipidaemia prevention AS and coronary heart disease are had active effect.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 big more, then anti-AS effect strong more [punishment is upright new for Cai Xiucheng, Wang Liguang, etc. pine nut oil is to the influence [J] of rat fat and lipid peroxidation. Food science, 1999,20 (6): 54,56.]
Experimental example 35 control diabetic nephropathy experiments
1 method
1.1 behind 40 SD male rats of animal modeling precuring 1w, empty stomach 12h, abdominal 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 modelling, includes this experiment in.Include 30 diabetic mice in experiment.
1.2 grouping normal control 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 abdominal injection Pue-glu, Pue-mal, successive administration 16w.Respectively organize for fear of ketosis and the existence of keeping diabetic mice, abdominal injection protamine zine insulin 2-4 unit 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, blood urea nitrogen, serum creatinine, calculates endogenous creatinine clearance rate, 8-iso-PGF
2 α
1.4 the observation index generalized case is observed: observe rat diet amount of drinking water, the mental status, 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) of every rat, get its mean value as final data with metabolic cage.Kidney weight in wet base: go to weigh with precision balance behind the coating.
1.5 biochemical indicator is surveyed blood sugar, blood urea nitrogen, serum creatinine with full automatic biochemical apparatus.Urinary albumin excretion rate (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): with the endogenous creatinine clearance rate is representative, GFR=(uric creatinine * urine amount)/serum creatinine.8-iso-PGF
2 αReference reagent box specification sheets is measured.
2 results
2.1 generalized case 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 is dispirited gradually, obviously become thin, diuresis, many drinks, how moving in early days, and late phase reaction is blunt, action is slow, hunchbacked snake body, hidrosis go out, perpendicular matt, the urine of hair is measured manyly, and need change bedding and padding 1-2 time every day; 1 visual deterioration is arranged; 2 cataract are arranged; There is 1 recurrent diarrhea to occur.Pue-glu, Pue-mal group: mental status is good, and no cataract and tail, foot necrosis move freely, and reaction sensitivity is poor slightly than normal group.
2.2Pue-glu, Pue-mal obviously descends than the normal group body weight to the rat that influences the modeling success of rat urine amount, body weight, kidney weight/body weight, blood sugar, the urine amount increases, blood sugar increasing, kidney weight/body weight ratio increase (P＜0.05).After the treatment, Pue-glu, Pue-mal organize than the model group hypourocrinia, and weight increase, kidney weight/body weight ratio descend (P＜0.05), see the following form.
Table respectively organize rat urine amount, body weight, kidney weight, body weight, changes of blood glucose comparison (n=10,
)
* compare with the normal control group, # and model group compare: P＜0.05, following table are together
2.3Pue-glu, Pue-mal obviously raises than normal group to the rat blood serum UAER, serum creatinine, the blood urea nitrogen that influence the modeling success of rat UAER, serum creatinine, CrCl, blood urea nitrogen, endogenous creatinine clearance rate descends, significant difference (P＜0.05), model group is organized than Pue-glu, Pue-mal, UAER, serum creatinine, blood urea nitrogen obviously raise, endogenous creatinine clearance rate descends (P＜0.05), sees the following form.
The variation that table is respectively organized rat UAER, serum creatinine, CrCl, blood urea nitrogen relatively (n=10,
)
2.4Pue-glu, Pue-mal is to 8-iso-PGF
2 αAnd the rat blood serum 8-iso-PGF that influences the modeling success of serum glycolated hemoglobin (HbAlc)
2 αObviously raise (P＜0.05) than normal group with serum HbAlc, model group is than Pue-glu, Pue-mal group, 8-iso-PGF
2 αObviously raise with serum HbAlc (P＜0.05), see the following form.
Table is respectively organized rat 8-iso-PGF
2 αAnd the comparison of serum HbAlc (n=10,
)
The experiment of experimental example 36 insulin resistants
1 method
1.1 the foundation and the grouping of insulin resistant-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 fasting plasma glucose (FBG) and oral glucose tolerance test (OGTT) back 2h blood sugar (2hBG); 8w, the most of blood pressure of modeling rat increases 3-5kPa (1kPa=715mmHg) than the normal group rat blood pressure, and sugar tolerance occurs and go down; Select the IRH modeling rat of blood pressure and impaired glucose tolerance, be divided into following 6 groups at random 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 aforesaid method.Simultaneously, equal im 50% propylene glycol of blank and model negative control group,
0.18ml/kg, qd * 4w; Cap group im Cap injection liquid 7mg/kg, qd * 4w; Pue-glu, Pue-mal be all by 60mg/kg, 30mg/kg grouping, and choose rat bilateral tsu san li (back 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 test kit specification sheets, measure serum cholesterol (TC), triglyceride level (TG) and blood plasma blood-sugar content (FPG) with Biochemical Analyzer; Measure blood plasma fasting insulin (Fins) content with the enzyme immunoassay instrument, and calculating insulin sensitivity index (ISI): ISI=In[1/ (Fins * FPG)].
2 results
2.1Pue-glu, Pue-mal is to the influence of IRH rat blood pressure and sugar tolerance
Following table result shows that I RH 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); The Pue-gluPue-mal hypotensive effect is dose-effect relationship.
Table Pue-glu, Pue-mal to the influence of IRH rat blood pressure (systolic pressure) and sugar tolerance (kPa, n=10,
)
Annotate: compare * P＜0.05, * * P＜0.01 with the model negative control group; Compare #P＜0.05, ##P＜0.01 with the blank group.
2.2Pue-glu Pue-mal is to the improvement effect of IRH rat fat and insulin sensitivity
Following table result shows that the plasma F ins content of IRH rat, TG and TC content are apparently higher than the blank group, and difference has significance (P＜0.05～0.01); The ISI obvious difference descends, and highly significant (P＜0.01) is arranged; Cap group, Pue-glu, Pue-mal 60mg/ and 30mg/ 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 there is no significance (P＞0.05).
Table Pue-glu to the effect of IRH rat fat and insulin sensitivity (n=10,
)
Annotate: compare * P＜0.05, * * P＜0.01 with the model negative control group; Compare #P＜0.05, ##P＜0.01 with the blank group.
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 protects β cell and antagonism insulin resistant effectively, reduces the IRH rat blood pressure.
Experimental example 37 anti-sclerotin pine nuts are tested
1 method
1.1 grouping and administration: 70 rats, wherein after the anesthesia of 60 abdominal injection 40mg/kg vetanarcol, excise bilateral ovaries according to a conventional method, 10 rats of surplusing are cooked identical operation technique, but spay not, as sham operated rats (Sham).Ovariectomized rat is divided into 6 groups, 1 week beginning gastric infusion after operation, Sham and OVX (Ovariectomization oophorectomize model control group) group gives 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% feeds with normal diet, freely drinks water.Claim body weight weekly and adjust the administration volume by new body weight.
1.2 bone densitometry: when administration 4 and 7 months, behind the rat 35mg/kg vetanarcol intraperitoneal injection of anesthesia, go up with forearm bone density measurement software at dual intensity borne densitometers (DEXA) and to measure rat body bone mineral content (BMC) and whole body bone mineral density (BMD).Instrument parameter: 134kV, 110mA, sweep length 26cm, width 1414cm.
1.3 the femur relative volume mass is measured: behind the last administration 1d, put to death rat, separate a side femur and a shin bone, pick the muscle and the reticular tissue that adhere to the greatest extent, after claiming weight in wet base on the electronic balance, on homemade drainage stereometry device, measure its volume, calculate its wetting phase to volume mass.
1.4 bone biomechanical property is measured: adopt the electronic universal tester to do the three point bending test of femur and shin bone, instrument parameter: loading velocity 6mm/min, maximum load 200N, span 20mm, trace load 2 deformation curves, calculate maximum load and structural strength (cause the load that the 1mm distortion needs, be equivalent to the slope on the curve).
1.5 femur ash relative volume mass: after measuring mechanical index, collect residual disconnected femur, claim ash heavy after the retort furnace ashing, ash ratio heavy and volume is a femur ash relative volume mass.
1.6 femur calcium contents and calcium salt density measurement: bone ash is dissolved in 40mL (6mol/L) hydrochloric acid, and with the EDTA complexometry, calred is made indicator, measure calcium contents, and calculate unit volume Ca content (calcium salt density).
1.7 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.
2 results
2.1Pue-glu, Pue-mal is to the influence of removal ovary rat 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 (seeing the following form) in the time of 7 months.
Table Pue-glu, Pue-mal to the influence of removal ovary rat BMC and BMD (n=10,
)
Compare with the OVX group: * P＜0.05 * * P＜0.01; Compare with the Sham group: #P＜0.05##P＜0.01
2.2Pue-glu, Pue-mal is to the influence of rat femur relative volume mass and calcium salt density:
Compare with the Sham group, the rat femur relative volume mass has reduced by 4.49% behind the removal ovary, and calcium element content reduces more (11.3%).PUE-GLU, each large and small dosage group of PUE-MAL increases the femur relative volume mass respectively, and the bone ash relative volume mass increases respectively.Simultaneously, femur calcium salt density is recovered fully, even also higher than the Sham group.E3 group has similar effect, but the amplitude that increases is than PUE-GLU, the heavy dose of group of PUE-MAL smaller (seeing the following form).
Table Pue-glu, Pue-mal to the influence (mg/mm3) of removal ovary rat femur, bone ash and calcium salt relative volume mass (n=10,
)
Compare with the OVX group: * P＜0.05 * * P＜0.01; Compare with the Sham group: #P＜0.05 ##P＜0.01
2.3Pue-glu, Pue-mal is to the influence of removal ovary rat femur and shin bone biomechanical property: spay is after 7 months, the biomechanical property of rat femur significantly descends, the maximum load of femur and structural strength have descended 15.0% and 17.3% respectively, and the maximum 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 maximum load and intensity improve highly significant respectively, 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 maximum load, but structural strength obviously improves (seeing the following form).
Table Pue-glu, Pue-mal to the influence of removal ovary rat femur and shin bone biomechanical property (n=10,
)
Compare with the OVX group:
*P＜0.05
*P＜0.01; Compare with the Sham group:
#P＜0.05#
#P＜0.01
2.4Pue-glu Pue-mal is to the influence of rat body weight and uterus weight: the heavy dose of group of Pue-glu, Pue-mal is the same with oestrogenic hormon to cause that rat body weight obviously descends, and small dose group does not influence 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 (seeing the following form).
Table Pue-glu, Pue-mal to the influence of removal ovary rat body weight and uterus weight (n=10,
)
Compare with the OVX group:
*P＜0.05
*P＜0.01; Compare with the Sham group:
#P＜0.05
##P＜0.01.
Embodiment 38
Bacterial classification M.oxydans CGMCC 1788 is inoculated in the 1000mL triangular flask that contains 300mL LB substratum, 30 ℃, 220rpm shaking culture 24 hours; Then cultured nutrient solution is linked in the 5L fermentor tank that contains 3L LB, rotating speed 500rpm, 30 ℃ of temperature, air flow 3L/min, tank pressure maintains 0.1MPa, fermentation 11-12h; Then, the centrifugal 10min of 8000rpm collects thalline, clean one time with 1/15M PBS (pH8.0), recentrifuge is collected, and thalline is transferred among the 3L2%TritonX-100, stir process 2h changes the broken perviousness of cytolemma, the thalline 12000rpm that handled is centrifugal, abandon supernatant, use 3L 1/15M PBS (pH8.0) washing 10min again, the similarity condition centrifugal collecting precipitation, repeated washing 3 times.Then that osmotic treated is good somatic cells joins in the 3L conversion fluid, conversion fluid contains puerarin 4mg/mL, sucrose 6%, 1/15M PBS, pH8.0 is in the 5L reactor, 500rpm, 30 ℃, transform under the air flow 3L/min condition and carry out 48h, treat that puerarin fructoside transformation efficiency reaches 90% stopped reaction.Conversion fluid is joined on the AB-8 type macroporous resin adsorption chromatography column, and with 0%-20% ethanol line style gradient elution, substep is collected effluent liquid, obtains 13.8L puerarin 7-O-fructoside elutriant altogether.The liquid underpressure distillation of collecting is concentrated into the 100ml postlyophilization, obtains puerarin fructoside powder 7.96g at last, HPLC detects purity 100%, the mole rate of recovery 64.7%.
The molecular structure characterization of puerarin 7-O-fructoside is as follows:
Mass spectrometry results shows that the ESI-MS m/z of puerarin 7-O-fructoside is 577[M-H]
-, in conjunction with
13C NMR infers that this compound molecule formula is C
27H
30O
14, with conversion of substrate puerarin m/z 415[M-H]
-Comparing has increased by one six carbon, six oxygen glycosyls 162, infers that converted product may be the monosaccharide groups product of puerarin.
The HMBC spectrum is the result show, 7.40 (2H, d, J=8.5Hz) hydrogen signal of locating is relevant with the carbon signal at 157.6 places, 6.79 (2H, d, J=8.5Hz) hydrogen signal of locating is relevant with the carbon signal at 123.1 places, 6.97 (1H, d, J=8.8Hz) hydrogen signal of locating is relevant with the carbon signal at 115.4 places, 7.92 (hydrogen signal of J=8.8Hz) locating is relevant with the carbon signal at 161.6 places for 1H, d, show that this compound mother nucleus structure is consistent with substrate, further supported us about its inference for the monosaccharide groups product of puerarin.
According to carbon spectrum, hydrogen spectrum and two-dimensional spectrum data and in conjunction with document analysis, we infer that another glycosyl is a beta-D-fructofuranose, because it does not have terminal hydrogen, so we according to hydrogen spectrum go up on the puerarin parent nucleus 4 '-signal of OH exists, in conjunction with the position analysis that glycosyl may connect, infer that glycosyl is connected 7 of puerarin parent nucleus.Based on the above results, the structure of definite compound is puerarin-7-O-fructofuranoside at last.
Puerarin 7-O-fructoside
1H NMR (400MHz, DMSO-d6),
13(100MHz, DMSO-d6) ownership at spectrum peak is as follows for C NMR
Embodiment 39
Bacterial classification M.oxydans CGMCC 1788 is inoculated in the 1000mL triangular flask that contains 300mL LB substratum, 30 ℃, 220rpm shaking culture 24 hours; Then cultured nutrient solution is linked in the 5L fermentor tank that contains 3L LB, rotating speed 500rpm, 30 ℃ of temperature, air flow 3L/min, tank pressure maintains 0.1MPa, fermentation 11-12h; Then, the centrifugal 10min of 8000rpm collects thalline, uses washed with de-ionized water one time, and recentrifuge is collected, and thalline is transferred among the 3L 0.5%TritonX-100 stir process 4h.The thalline 12000rpm that handles is centrifugal, abandon supernatant, use 3L deionized water wash 10min again, behind the similarity condition centrifugal collecting precipitation, repeated washing 3 times, join in the 3L conversion fluid, conversion fluid contains puerarin 10mg/mL, sucrose 6%, 1/15M PBS, pH8.0, in the 5L reactor, 500rpm, 30 ℃, transform 96h under the air flow 3L/min condition, treat that puerarin fructoside transformation efficiency reaches 90% stopped reaction.Conversion fluid is joined on the AB-8 type macroporous resin adsorption chromatography column, and with 0%-30% ethanol line style gradient elution, substep is collected effluent liquid, obtains 15.8L puerarin 7-O-fructoside elutriant altogether.The liquid underpressure distillation of collecting is concentrated into the 100ml postlyophilization, obtains puerarin-7-O-fructoside powder 18.6g at last, HPLC detects purity 99.9%, the mole rate of recovery 62.7%.
In the 1000mL triangular flask that contains 300mL LB substratum, insert Microbacteriumoxydans CGMCC1788 seed liquor, 30 ℃, after the 220rpm shaking culture 12 hours, stop fermentation, somatic cells is collected in centrifugation, adds the TritonX-100 of 10% concentration, slowly rotation treatment was cleaned with 1/15mol/L phosphoric acid buffer (pH8.0) after 2 hours, centrifugal 15 minutes collecting precipitations of 12000rpm repeat twice, remove TritonX-100; Add and contain the 4mg./mL puerarin, 6% sucrose, 1/15mol/L phosphoric acid buffer (pH8.0), 30 ℃, conversion reaction is carried out in 200rpm vibration ventilation, behind the 48h, stop conversion reaction, centrifugal removal thalline contains puerarin-7-O-isomaltulose glycosides 96% in the total isoflavone in the supernatant liquor.Through the mass spectroscopy molecular weight is 740.
Experimental example 41
The hemolytic experiment of puerarin and puerarin 7-O-fructoside.
(1) test method
1. 2% red cell suspension preparation: get healthy rabbits, heart extracting blood 10ml defibrinates with the glass stick stirring to the clean dried beaker, adds 0.9% sodium chloride injection 10ml, centrifugal, abandoning supernatant adds 20ml 0.9% sodium chloride injection again and shakes up gently, and is centrifugal, abandoning supernatant, till not taking on a red color to supernatant liquor several times so repeatedly, be mixed with 2% red cell suspension by the gained red cell volume with 0.9% sodium chloride injection then, it is standby to put refrigerator.
2. test method: 1) get 7 of clean tube, be numbered, 1-5 number pipe is trial-product pipe (18mg/ml puerarin 50% propylene glycol solution), manages negative control tube No. 6, manages positive control tube No. 7.By adding 2% rabbit erythrocyte suspension, 0.9% sodium chloride injection or distilled water shown in the table 2 successively, behind the mixing, put incubation in 37 ℃ ± 0.5 ℃ water bath with thermostatic control immediately, write down the haemolysis and the aggegation situation of 15min, 30min, 45min, 1.0h, 2.0h, 3.0h, each pipe of 4.0h respectively.2) get 7 of clean tube, be numbered, 1-5 number pipe be for trial-product pipe (the 18mg/ml puerarin 7-O-fructoside aqueous solution), manages negative control tube No. 6, manages positive control tube No. 7.By adding 2% rabbit erythrocyte suspension, 0.9% sodium chloride injection or distilled water shown in the table 3 successively, behind the mixing, put incubation in 37 ℃ ± 0.5 ℃ water bath with thermostatic control immediately, write down the haemolysis and the aggegation situation of 15min, 30min, 45min, 1.0h, 2.0h, 3.0h, each pipe of 4.0h respectively.3) get 7 of clean tube, be numbered, 1-5 number pipe be for trial-product pipe (50% propylene glycol solution), manages negative control tube No. 6, manages positive control tube No. 7.By adding 2% rabbit erythrocyte suspension, 0.9% sodium chloride injection or distilled water shown in the table 4 successively, behind the mixing, put incubation in 37 ℃ ± 0.5 ℃ water bath with thermostatic control immediately, write down the haemolysis and the aggegation situation of 15min, 30min, 45min, 1.0h, 2.0h, 3.0h, each pipe of 4.0h respectively.
3. haemolysis judging criterion: a as a result: observation method of naked eye; B: spectrophotometry (by table 2,3,4 with incubation 4h respectively manage solution centrifugal, get supernatant, on spectrophotometer, the 545nm place is the OD value that blank reads each pipe with distilled water, calculates the hemolysis rate % of each developmental tube).
Table 2 adds various solution orders
| The test tube numbering | ??1 | ??2 | ??3 | ??4 | ??5 | ??6 | ??7 |
| 2% red cell suspension (ml) | ??2.5 | ??2.5 | ??2.5 | ??2.5 | ??2.5 | ??2.5 | ??2.5 |
| Physiological saline (ml) | ??2.0 | ??2.1 | ??2.2 | ??2.3 | ??2.4 | ??2.5 | |
| Distilled water (ml) | ??2.5 | ||||||
| 18mg/ml puerarin 50% propylene glycol solution | ??0.5 | ??0.4 | ??0.3 | ??0.2 | ??0.1 |
Table 3 adds various solution orders
| The test tube numbering | ??1 | ??2 | ??3 | ??4 | ??5 | ??6 | ??7 |
| 2% red cell suspension (ml) | ??2.5 | ??2.5 | ??2.5 | ??2.5 | ??2.5 | ??2.5 | ??2.5 |
| Physiological saline (ml) | ??2.0 | ??2.1 | ??2.2 | ??2.3 | ??2.4 | ??2.5 | |
| Distilled water (ml) | ??2.5 | ||||||
| The 18mg/ml puerarin 7-O-fructoside aqueous solution | ??0.5 | ??0.4 | ??0.3 | ??0.2 | ??0.1 |
Table 4 adds various solution orders
| The test tube numbering | ??1 | ??2 | ??3 | ??4 | ??5 | ??6 | ??7 |
| 2% red cell suspension (ml) | ??2.5 | ??2.5 | ??2.5 | ??2.5 | ??2.5 | ??2.5 | ??2.5 |
| Physiological saline (ml) | ??2.0 | ??2.1 | ??2.2 | ??2.3 | ??2.4 | ??2.5 | |
| Distilled water (ml) | ??2.5 | ||||||
| 50% propylene glycol solution | ??0.5 | ??0.4 | ??0.3 | ??0.2 | ??0.1 |
(2) test-results
Experimental result shows and sees Table 5-7, under the same concentrations, and the whole haemolysis of puerarin 50% propylene glycol solution group, and haemolysis does not appear in puerarin 7-O-fructoside aqueous solution group, haemolysis appears in 50% propylene glycol solution control group only 0.5ml test tube.
Table 518mg/ml puerarin 50% propylene glycol solution group
Table 6 18mg/ml puerarin 7-O-fructoside aqueous solution group
Table 7 50% propylene glycol solution control group
Illustrate: 1. 1-5 number pipe is for being subjected to reagent thing (add dose and be followed successively by 0.5-0.1ml); No. 6 pipe is physiology saline control pipe (negative control); No. 7 pipe is distilled water control tube (positive control).2. hemolysis rate＞5% promptly is judged as haemolysis.
Experimental example 42
The vasodilative pharmacological effect test of puerarin 7-O-fructoside
1, material
(1) laboratory animal: the SD rat, body weight 250-300g, male, conformity certification SCXK (Soviet Union) 2002-0004 is provided by animal reproduction field, Green Dragon mountain, Jiangning, Jiangsu Province.
(2) medicine and reagent and preparation:
1. (Norepinephrine, NA): noradrenaline bitartrate, concentration are that 2mg/ml (is 5.9 * 10 to norepinephrine
-3M), in order to activate blood vessel.
2. (Phenylephrine, Phe): distilled water is mixed with 10 to phyenlephrinium
-2M solution treats that the time spent is with distilled water diluting to 10
-5M, 2 * 10
-5M, 7 * 10
-5M, 2 * 10
-4M, 7 * 10
-4M, 2 * 10
-3M, 7 * 10
-3M is stand-by.
3. (Acetycholine, Ach): distilled water is mixed with solution to Ovisot, treats that the time spent is diluted to 10
-5M, 2 * 10
-5M, 7 * 10
-5M, 2 * 10
-4M, 7 * 10
-4M, 2 * 10
-3M, 7 * 10
-3M is stand-by.
4. puerarin: be mixed with 10-2M solution with DMSO, stand-by before distilled water diluting to 10
-5M, 2 * 10
-5M, 7 * 10
-5M, 2 * 10
-4M, 7 * 10
-4M, 2 * 10
-3M, 7 * 10
-3M.
5. puerarin-7-O-fructoside derivative: be mixed with 10 with distilled water and DMSO respectively
-2M solution, stand-by preceding distilled water diluting to 10
-5M, 2 * 10
-5M, 7 * 10
-5M, 2 * 10
-4M, 7 * 10
-4M, 2 * 10
-3M, 7 * 10
-3M.
2, test method
Rat chest aorta is got in 20% urethane anesthesia (dosage is 1g/kg, and promptly dosage is 5ml/kg), places the O of precooling
2In the saturated K-H liquid, reticular tissue around removing is prepared into the vascular strip of 2mm, and hangs on and pass to O in advance
2K-H liquid 5ml bathe in the ware, the lower end Fixation With Steel Wire, the upper end links to each other with tonotransducer, bathes to continue feeding O in the ware
2, circulator bath keeps 37 ± 0.5 ℃, and rat chest aorta gives 1.0g load, and every 15min changes damping fluid one time, and behind the balance 60min, (bathing the ware final concentration is 10 to add NA
-6M) behind the preshrinking 2 times, be eluted to baseline with K-H liquid, the beginning vasoactive is measured.
(1) puerarin-7-O-fructoside derivative is dissolved in the distilled water solution
1. the vagusstoff relaxation curves is made: arterial ring is after pre-treatment, with Phe (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) handle arterial ring and make it be contracted to platform, it is 10 that accumulative total adds concentration
-5M, 2 * 10
-5M, 7 * 10
-5M, 2 * 10
-4M, 7 * 10
-4M, 2 * 10
-3M, 7 * 10
-3The M vagusstoff, 5 μ l to 5ml bathe in the ware, make the bath final concentration be respectively 1 * 10
-8M, 1 * 10
-7.5M, 1 * 10
-7M, 1 * 10
-6.5M, 1 * 10
-6M, 1 * 10
-5.5M, 1 * 10
-5M, record diastole amplitude is made dose-effect-relaxation curves.
2. puerarin vasorelaxation action: arterial ring is after pre-treatment, with Phe (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) handle arterial ring and make it be contracted to platform, add Ach (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) the arterial ring diastole is to platform, after be eluted to baseline, its diastole is used as 100%.
With Phe (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) handle arterial ring and make it be contracted to platform, it is 10 that accumulative total adds concentration
-5M, 2 * 10
-5M, 7 * 10
-5M, 2 * 10
-4M, 7 * 10
-4M, 2 * 10
-3M, 7 * 10
-3The M puerarin, 5 μ l to 5ml bathe in the ware, make the bath final concentration be respectively 1 * 10
-8M, 1 * 10
-7.5M, 1 * 10
-7M, 1 * 10
-6.5M, 1 * 10
-6M, 1 * 10
-5.5M, 1 * 10
-5M record diastole amplitude is made the accumulative total amount effect curve.
3. puerarin-7-O-fructoside derivative vasorelaxation action: arterial ring is after pre-treatment, with Phe (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) handle arterial ring and make it be contracted to platform, add Ach (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) the arterial ring diastole is to platform, after be eluted to baseline, its diastole is used as 100%.
With Phe (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) handle arterial ring and make it be contracted to platform, it is 10 that accumulative total adds concentration
-5M, 2 * 10
-5M, 7 * 10
-5M, 2 * 10
-4M, 7 * 10
-4M, 2 * 10
-3M, 7 * 10
-3M glycosylation puerarin, 5 μ l to 5m l bathe in the ware, make the bath final concentration be respectively 1 * 10
-8M, 1 * 10
-7.5M, 1 * 10
-7M, 1 * 10
-6.5M, 1 * 10
-6M, 1 * 10
-5.5M, 1 * 10
-5M record diastole amplitude is made the accumulative total amount effect curve.
(2) puerarin-7-O-fructoside derivative is dissolved among the DMSO
1. the vagusstoff relaxation curves is made: arterial ring is after pre-treatment, with Phe (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) handle arterial ring and make it be contracted to platform, it is 10 that accumulative total adds concentration
-5M, 2 * 10
-5M, 7 * 10
-5M, 2 * 10
-4M, 7 * 10
-4M, 2 * 10
-3M, 7 * 10
-3The M vagusstoff, 5 μ l to 5ml bathe in the ware, make the bath final concentration be respectively 1 * 10
-8M, 1 * 10
-7.5M, 1 * 10
-7M, 1 * 10
-6.5M, 1 * 10
-6M, 1 * 10
-5.5M, 1 * 10
-5M record diastole amplitude is made dose-effect-relaxation curves.
2. puerarin vasorelaxation action: arterial ring is after pre-treatment, with Phe (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) handle arterial ring and make it be contracted to platform, add Ach (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) the arterial ring diastole is to platform, after be eluted to baseline, its diastole is used as 100%.
With Phe (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) handle arterial ring and make it be contracted to platform, it is 10 that accumulative total adds concentration
-5M, 2 * 10
-5M, 7 * 10
-5M, 2 * 10
-4M, 7 * 10
-4M, 2 * 10
-3M, 7 * 10
-3The M puerarin, 5 μ l to 5ml bathe in the ware, make the bath final concentration be respectively 1 * 10
-8M, 1 * 10
-7.5M, 1 * 10
-7M, 1 * 10
-6.5M, 1 * 10
-6M, 1 * 10
-5.5M, 1 * 10
-5M record diastole amplitude is made the accumulative total amount effect curve.
3. puerarin-7-O-fructoside derivative vasorelaxation action: arterial ring is after pre-treatment, with Phe (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) handle arterial ring and make it be contracted to platform, add Ach (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) the arterial ring diastole is to platform, after be eluted to baseline, its diastole is used as 100%.
With Phe (10
-2M, 5 μ l add 5ml and bathe in the ware, and making its final concentration is 10
-5M) handle arterial ring and make it be contracted to platform, it is 10 that accumulative total adds concentration
-5M, 2 * 10
-5M, 7 * 10
-5M, 2 * 10
-4M, 7 * 10
-4M, 2 * 10
-3M, 7 * 10
-3M glycosylation puerarin, 5 μ l to 5ml bathe in the ware, make the bath final concentration be respectively 1 * 10
-8M, 1 * 10
-7.5M, 1 * 10
-7M, 1 * 10
-6.5M, 1 * 10
-6M, 1 * 10
-5.5M, 1 * 10
-5M record diastole amplitude is made the accumulative total amount effect curve.
3, test-results
(1) vasodilator effect that puerarin-7-O-fructoside derivative is dissolved in the distilled water solution sees the following form, as seen puerarin (DMSO solvent) is compared with puerarin-7-O-fructoside derivative (water solvent) group, through one-way analysis of variance, diastole effect no significant difference between P=0.725＞0.5, two group.
Diastole percentage ratio under the table different concns (
, be 100% with the vagusstoff diastole)
(2) vasodilator effect that puerarin-7-O-fructoside derivative is dissolved in the DMSO solvent sees the following form, as seen in the same solvent, puerarin is compared with puerarin-7-O-fructoside derivative group, through one-way analysis of variance, diastole effect no significant difference between P=0.825＞0.5, two group.
Diastole percentage ratio under the table different concns (
, be 100% with the vagusstoff diastole)
4, conclusion
(1) in different solvents (puerarin is solvent with DMSO, and puerarin-7-O-fructoside derivative is solvent with Water), under the same concentrations, puerarin and puerarin-7-O-fructoside derivative all has diastolic blood vessel activity, and the minimum onset concentration of the two is 10
-8M, and the two vasorelaxation action does not have the significance difference opposite sex;
(2) in same solvent (all being solvent) with DMSO, under the same concentrations, puerarin and puerarin-7-O-fructoside derivative all has diastolic blood vessel activity, and the minimum onset concentration of the two is 10
-8M, and the two vasorelaxation action does not have the significance difference opposite sex.
(3) puerarin-7-O-fructoside vasodilator effect is compared with puerarin does not have notable difference, and puerarin-7-O-fructoside derivative haemolysis incidence significantly is lower than puerarin.As seen, puerarin-7-O-fructoside derivative is suitable for exploitation becomes the similar alternative medicine of puerarin of new generation.
Embodiment 43
The preparation of puerarin-7-O-fructoside sodium salt
Get puerarin-7-O-fructoside 2g and put in the appropriate vessel, add dissolve with ethanol, get 10% sodium carbonate solution, add in the solution of puerarin-7-O-fructoside, transfer pH to 9-11, stir and make dissolving, take out under the reduced pressure and desolvate, obtain solid 2.8g.
Embodiment 44
The preparation of puerarin-7-O-fructoside calcium salt
Get puerarin-7-O-fructoside 2g and put in the appropriate vessel, add dissolve with ethanol, get saturated limewater, add in the solution of puerarin-7-O-fructoside, transfer pH to 9-11, stir and make dissolving, take out under the reduced pressure and desolvate, obtain solid 3.0g.
Embodiment 45
The preparation of puerarin-7-O-fructoside arginic acid salt
Get puerarin-7-O-fructoside 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-fructoside, heating is stirred and is made dissolving, takes out under the reduced pressure and desolvates, and obtains solid 3.1g.
Embodiment 46
The preparation of puerarin-7-O-fructoside lysine salt
Get puerarin-7-O-fructoside 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-fructoside, heating is stirred and is made dissolving, takes out under the reduced pressure and desolvates, and obtains solid 2.9g.
Embodiment 47
The preparation of puerarin-7-O-fructoside meglumine salt
Get puerarin-7-O-fructoside 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-fructoside, heating is stirred and is made dissolving, takes out under the reduced pressure and desolvates, and obtains solid 3.1g.
Embodiment 48
The preparation of puerarin-7-O-fructoside ligustrazine salt
Get puerarin-7-O-fructoside 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-fructoside, heating is stirred and is made dissolving, takes out under the reduced pressure and desolvates, and obtains solid 2.6g.
Embodiment 49
The preparation of puerarin-7-O-fructoside glucosamine salt
Get glucosamine 6.3g, be dissolved in 250ml distilled water, stir adding puerarin-7-O-fructoside 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-fructoside glucosamine.
Embodiment 50
The preparation of puerarin-7-O-fructoside capsule
Puerarin-7-O-fructoside 20g
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-fructoside, Microcrystalline Cellulose, lactose, sodium starch glycolate are crossed 100 mesh sieves respectively and are mixed, and are tackiness agent system softwood with the HPMG solution of tween 80, mistake 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, can is in capsule (conventional capsule or or enteric coated capsule).
Embodiment 51
The preparation of the salt capsule of puerarin-7-O-fructoside
The salt 20g of puerarin-7-O-fructoside
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-fructoside, Microcrystalline Cellulose, lactose, sodium starch glycolate are crossed 100 mesh sieves respectively and are mixed, and are tackiness agent system softwood with the HPMC solution of tween 80, mistake 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, can is in capsule (conventional capsule or or enteric coated capsule).
Embodiment 52
The preparation of puerarin-7-O-fructoside tablet
Puerarin-7-O-fructoside 20g
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-fructoside, Microcrystalline Cellulose, lactose, sodium starch glycolate are crossed 100 mesh sieves respectively and are mixed, and are tackiness agent system softwood with the HPMC solution of tween 80, mistake 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, with Magnesium Stearate mixing, compressing tablet.
Embodiment 53
The preparation of puerarin-7-O-fructoside salt tablets
Puerarin-7-O-fructoside salt 20g
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-fructoside salt, Microcrystalline Cellulose, lactose, sodium starch glycolate are crossed 100 mesh sieves respectively and are mixed, and are tackiness agent system softwood with the HPMC solution of tween 80, mistake 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 54
The preparation of puerarin-7-O-fructoside Yelkin TTS sheet
Puerarin-7-O-fructoside 20g
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-fructoside, 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 55
The preparation of puerarin-7-O-fructoside salt enteric coated capsule
Get puerarin-7-O-fructoside salt, cellulose acetate-phthalate 209 is dissolved in acetone and ethanol (1: 1) the mixed solution 500ml solution, stirs, 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 56
The preparation of puerarin-7-O-fructoside slow releasing capsule
| Puerarin-7-O-fructoside | ??50g |
| Polyvidone | ??50g |
| Microcrystalline Cellulose | ??15g |
| Hypromellose K4M | ??50g |
| 3% hypromellose (E5) aqueous solution | In right amount |
| Talcum powder | ??2g |
Puerarin-7-O-fructoside 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 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 57
The preparation of puerarin-7-O-fructoside salt sustained release capsules agent
Puerarin-7-O-fructoside salt 80g
Microcrystalline Cellulose 15g
Hypromellose K4M 100g
3% hypromellose (E5) aqueous solution is an amount of
Talcum powder 2g
Puerarin-7-O-fructoside 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 58
The preparation of puerarin-7-O-fructoside slow releasing tablet
Puerarin-7-O-fructoside 50g
Polyvidone 50g
Lactose 15g
Hypromellose K4M 100g
3% hypromellose (E5) aqueous solution is an amount of
Talcum powder 1g
Puerarin-7-O-fructoside 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.
Embodiment 59
The preparation of puerarin-7-O-fructoside injection
Puerarin-7-O-fructoside 10g
Water for injection adds to 5L
Get puerarin-7-O-fructoside, 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-fructoside injection.
Puerarin-7-O-fructoside injection
| Puerarin-7-O-fructoside | ??10.0g |
| Arginine or Methionin | ??6.2g |
| Puerarin-7-O-fructoside | ??10.0g |
| Sodium-chlor | In right amount |
| Water for injection | Add to 2.5L |
Get puerarin-7-O-fructoside, 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 61
The preparation of injection puerarin-7-O-fructoside powder injection
| Puerarin-7-O-fructoside | ??10.0g |
| Arginine or Methionin | ??6.2g |
| N.F,USP MANNITOL | ??16.0g |
| Water for injection | Add to 0.5L |
Get puerarin-7-O-fructoside 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 62
The preparation of injection puerarin-7-O-fructoside salt powder injection
| Puerarin-7-O-fructoside salt | ??10.0g |
| N.F,USP MANNITOL | ??16.0g |
| Water for injection | Add to 0.5L |
Get puerarin-7-O-fructoside salt (can be the salt such as arginine, Methionin, glucosamine, meglumine of puerarin-7-O-fructoside) and put in the appropriate vessel, add injection water 400ml, stir, ultrasonic to dissolving, add N.F,USP MANNITOL and stir 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 63
The preparation of puerarin-7-O-fructoside oral liquid
Getting puerarin-7-O-fructoside 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-fructoside oral liquid.
Embodiment 64
The preparation of puerarin-7-O-fructoside injection transfusion
Get puerarin-7-O-fructoside, 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-fructoside or puerarin-7-O-isomaltose injection transfusion.
Embodiment 65
Puerarin-7-O-fructoside injection liquid and puerarin injection are relatively
Get puerarin-7-O-fructoside, puerarin is an amount of, puts in the 10ml volumetric flask, adds water, jolting makes dissolving, constant volume seals and promptly gets the preparation that water is carrier, the results are shown in following table, puerarin-7-O-fructoside is more stable on preparation than puerarin.
Table puerarin and puerarin-7-O-fructoside injection liquid relatively
| Compound | Concentration | All backs outward appearance is placed in 90 degree water-baths |
| Puerarin-7-O-fructoside | ??4.0mg/ml | Clarification |
| Puerarin | ??2.0mg/ml | Clarification has the false hair of trace |
| Puerarin | ??4.0mg/ml | Muddy |
Claims (10)
1. puerarin glycosylated derivant or its pharmacy acceptable salt shown in the formula (I):
Wherein, R is the monosaccharide groups of 6 carbon sugar or 5 carbon sugar, or is that 2-5 6 carbon sugar is or/and the oligosaccharyl that 5 carbon sugar connects; Described pharmacologically acceptable salts is the salt with mineral alkali or organic bases addition.
2. puerarin glycosylated derivant according to claim 1 or its pharmacologically acceptable salts, it is characterized in that described monosaccharide groups is glucosyl group, mannose group, glucal acidic group, galactosyl, galacturonic acidic group, allose base, fructosyl, sorb glycosyl, witch hazel glycosyl, strepto-glycosyl, 2-glucosamine base, galactosamine base or Arabic glycosyl, lysol glycosyl, xylosyl, ribosyl, husband's glycosyl, rhamanopyranosyl, isorhodeose base or celery glycosyl; Described oligosaccharyl is isomaltose base, mycose-base, sophorosyl, new dried orange peel glycosyl, rue glycosyl, locust tree diglycosyl, malt-base, sucrose base, lactose base, rough gentian glycosyl or pectin base; Described mineral alkali is the mineral alkali of basic metal or alkaline-earth metal, and organic bases is a nitrogenous organic base.
3. puerarin glycosylated derivant according to claim 2 or its pharmacologically acceptable salts, it is characterized in that described derivative is the puerarin-7-O-fructoside shown in puerarin-7-O-glucoside, puerarin-7-O-maltose glucosides, the formula (II) or the puerarin shown in the formula (III)-7-O-isomaltulose glycosides;
Formula (II)
Formula (III)
Described its pharmacologically acceptable salts is the salt with the mineral alkali addition of sodium, potassium, calcium or magnesium, or with the salt of amino acid, meglumine, glucosamine or Ligustrazine addition, wherein, amino acid is preferably arginine, Methionin or carnitine.
4. according to the preparation method of the arbitrary described puerarin glycosylated derivant of claim 1-3 or its pharmacologically acceptable salts, it is characterized in that, the preparation method of described derivative comprises: by having the microbial strains of puerarin glycosylated enzyme activity, at water, in organic phase or water/organic phase two-phase monose on the saccharide donor or oligosaccharyl are transformed on the position of puerarin A ring C-7, thereby obtain described derivative;
Wherein, described water is that the aqueous solution and pH are the buffered soln of 5-9; Described organic phase is ethanol, butanols, acetone, ethyl acetate, dimethyl sulfoxide (DMSO) or dimethylformamide, or their its mixture, and described water/organic phase two-phase is above-mentioned the do not contain water of buffered soln and the combination of above-mentioned organic phase; Described conversion condition is: concentration of substrate is the 0.01%-saturation concentration; Invert point is 20～40 ℃; 10～400rpm shakes a bottle oscillatory reaction, or the reactor stirring reaction; Transformation time is 12～120 hours;
Described saccharide donor is monose or oligosaccharides or starch, and described monose is glucose, seminose, glucuronic acid, semi-lactosi, galacturonic acid, allose, fructose, sorbose, hamamelose, streptose, 2-glucosamine, galactosamine, pectinose, lyxose, wood sugar, ribose, husband's sugar, rhamnosyl, isorhodeose, celery sugar; Described oligosaccharides is trehalose, sophorose, neohesperidose, rutinose, locust tree disaccharides, maltose, sucrose, lactose, gentianose or pectin;
The preparation method of described its pharmaceutically acceptable salt is: with gained puerarin glycosylated derivant and nitrogenous organic base with 1: 0.5～2.0 mol ratio, preferred 1: the mol ratio of 0.8-1.5, in water and/or alcoholic solvent, react; Or with metal ion with 1: 0.5～2.0 equivalence ratio, preferred 1: the mineral alkali of the equivalence ratio of 0.8-1.5, reaction is promptly in water and/or alcoholic solvent.
5. method according to claim 4 is characterized in that, the step that preparation method's method of described derivative comprises is as follows:
1) adopts somatic cells, microbial cells resting cell, the zyme extract of this somatic cells or the recombinant expression protein of this enzyme through osmotic treated with puerarin glycosylated enzyme activity, at water, in organic phase or water/organic phase two-phase monosaccharide groups on the saccharide donor or oligosaccharyl are transformed on the hydroxyl of puerarin A ring C-7 position, obtain the synthetic liquid of conversion of described glycosylated puerarin; Wherein, described water is that the aqueous solution and pH are the buffered soln of 5-9, described organic phase is ethanol, butanols, ethyl acetate, dimethyl sulfoxide (DMSO) or dimethylformamide or its its mixture, and described water/organic phase two-phase is above-mentioned the do not contain water of buffered soln and the combination of above-mentioned organic phase; Described conversion condition is: concentration of substrate is the 0.01%-saturation concentration; Invert point is 20～40 ℃; 10～400rpm shakes a bottle oscillatory reaction, or the reactor stirring reaction; Transformation time is 12～120 hours;
2) synthetic liquid be will transform through thermal precipitation, centrifugal, thalline or bacterial enzyme albumen removed;
3) utilize Solid-Phase Extraction, liquid-phase extraction or column chromatography to separate to transforming resulting solution then, wherein,
The method that described Solid-Phase Extraction transforms synthetic liquid is: solid phase carrier is polyamide or macroporous resin; The polyamide or the macroporous resin of water and/or the extraction of alcoholic solvent wash-out, then promptly with the elutriant concentrate drying;
The method that described liquid-phase extraction transforms synthetic liquid is: the solvent for use system is water-organic solvent, and organic solvent is a kind of, two or more the mixture in propyl carbinol, ethyl acetate, chloroform, ether, hexane and the sherwood oil; Divide and get extraction liquid, concentrate drying promptly.
Described column chromatography is separated the method that transforms synthetic liquid: with silica gel, alkyl linked silica gel, macroporous resin or polyamide is stationary phase, carries out chromatographic separation, collects target fraction, and concentrate drying promptly;
4) the step 3) products therefrom is carried out recrystallization purifying, wherein, recrystallization solvent is water and/or alcoholic solvent, or alcohol and/or fat-soluble solvent; Wherein, described fat-soluble solvent is ethyl acetate, chloroform, ether, oxyethane, a kind of, two or more combination in hexane and the sherwood oil.
6. method according to claim 5 is characterized in that, the affiliated microbial cells resting cell that carries out osmotic treated through the microbial cells resting cell of osmotic treated for the TritonX-100 that adopts 0.1-10% concentration.
7. method according to claim 6, it is characterized in that, described microbial strains with puerarin glycosylated enzyme activity is the Microbacterium bacterial classification, is preferably Microbacterium oxydansCGMCC 1788, or Microbacterium saperdae CGMCC 1.1906 or its mixture; The culture condition of described microbial strains is: 20～40 ℃, 10～400rpm shakes bottle vibration aerated culture, or the cultivation of fermentor tank deep ventilation, and incubation time is 12～96 hours.
8. the arbitrary described puerarin glycosylated derivant of claim 1-3 or its pharmacologically acceptable salts are in the application of the medicine of preparation treatment, prevention coronary heart disease, stenocardia, myocardial infarction, ischemic cerebrovascular disease, atherosclerosis, blood fat reducing, diabetic nephropathy, insulin resistant or osteoporosis.
9. a pharmaceutical composition is characterized in that, described pharmaceutical composition comprises the arbitrary described puerarin glycosylated derivant of the claim 1-3 of significant quantity or its pharmacologically acceptable salts and pharmaceutically acceptable carrier.
10. pharmaceutical composition according to claim 9, it is characterized in that the formulation of described composition is tablet, capsule, eye drops, sprays, gelifying agent, gel inhalation, oral liquid, suspensoid, electuary, ointment, pill, powder, injection, infusion solution, freeze dried injection or the suppository of routine, quick-release, slowly-releasing or controlled release.
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| CN200810171912A CN101723997A (en) | 2008-10-24 | 2008-10-24 | Puerarin glycosylation derivative, medicine compound, preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101885748A (en) * | 2010-07-22 | 2010-11-17 | 广州医学院第二附属医院 | Puerarin-7-O-glucuronide as well as preparation method and application thereof |
| WO2012048522A1 (en) * | 2010-10-13 | 2012-04-19 | 南京工业大学 | Fructosylated puerarin, and preparation method therefor and use thereof |
| KR20140027139A (en) * | 2011-07-06 | 2014-03-06 | 난징 유니버시티 오브 테크놀러지 | Fructosylated mangiferin and preparation method thereof and use thereof |
| CN110331114A (en) * | 2019-08-01 | 2019-10-15 | 山东佐田氏生物科技有限公司 | The disease-resistant Promoting bacteria longicorn microbacterium of one plant of saline-alkali tolerant and its application |
| CN114657160A (en) * | 2020-12-23 | 2022-06-24 | 浙江康恩贝制药股份有限公司 | Glycosyl transferase mutant and application thereof |
-
2008
- 2008-10-24 CN CN200810171912A patent/CN101723997A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101885748A (en) * | 2010-07-22 | 2010-11-17 | 广州医学院第二附属医院 | Puerarin-7-O-glucuronide as well as preparation method and application thereof |
| WO2012048522A1 (en) * | 2010-10-13 | 2012-04-19 | 南京工业大学 | Fructosylated puerarin, and preparation method therefor and use thereof |
| CN102443027A (en) * | 2010-10-13 | 2012-05-09 | 南京工业大学 | Fructose glycosylation puerarin, preparation method and application thereof |
| CN102443027B (en) * | 2010-10-13 | 2014-10-29 | 南京工业大学 | Fructose glycosylation puerarin, preparation method and application thereof |
| KR101622915B1 (en) | 2010-10-13 | 2016-05-20 | 난징 유니버시티 오브 테크놀러지 | Fructosylated puerarin, and preparation method therefor and use thereof |
| KR20140027139A (en) * | 2011-07-06 | 2014-03-06 | 난징 유니버시티 오브 테크놀러지 | Fructosylated mangiferin and preparation method thereof and use thereof |
| KR101653586B1 (en) | 2011-07-06 | 2016-09-02 | 난징 유니버시티 오브 테크놀러지 | Fructosylated mangiferin and preparation method thereof and use thereof |
| CN110331114A (en) * | 2019-08-01 | 2019-10-15 | 山东佐田氏生物科技有限公司 | The disease-resistant Promoting bacteria longicorn microbacterium of one plant of saline-alkali tolerant and its application |
| CN114657160A (en) * | 2020-12-23 | 2022-06-24 | 浙江康恩贝制药股份有限公司 | Glycosyl transferase mutant and application thereof |
| CN114657160B (en) * | 2020-12-23 | 2024-04-05 | 浙江康恩贝制药股份有限公司 | Glycosyltransferase mutant and application thereof |
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