CN101787061A - Application of Quzhazhigan in preparation of preparations for preventing and treating cardiac-cerebral ischemia diseases, and preparation method thereof - Google Patents
Application of Quzhazhigan in preparation of preparations for preventing and treating cardiac-cerebral ischemia diseases, and preparation method thereof Download PDFInfo
- Publication number
- CN101787061A CN101787061A CN201010116358A CN201010116358A CN101787061A CN 101787061 A CN101787061 A CN 101787061A CN 201010116358 A CN201010116358 A CN 201010116358A CN 201010116358 A CN201010116358 A CN 201010116358A CN 101787061 A CN101787061 A CN 101787061A
- Authority
- CN
- China
- Prior art keywords
- quzhazhigan
- preparation
- extract
- lhasa rhubarb
- rhizome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to application of Quzhazhigan serving as polyhydroxy stilbene glycoside compounds in preparation of preparations for preventing and treating ischemic cardio-cerebrovascular diseases, and a preparation method thereof. The Quzhazhigan is compounds (E)-1-(3,5-dihydroxy phenyl)-2(3-hydroxyl-4-O-beta-D-pyran glucose phenyl)ethane, and has a chemical structural formula. Test results show that the Quzhazhigan can significantly reduce injury caused by ischemia reperfusion after cardio-cerebral infarction and has wide application in the preparation of preparations for preventing and treating cardiac-cerebral ischemia diseases. The Quzhazhigan can be extracted from roots and/or rhizome of Rheum lhasaense and prepared in pure alcohol/water systems, thereby having the advantages of simple convenient property, environment-friendly property and strong practicability.
Description
Technical field
The present invention relates to the purposes of active components of plants Quzhazhigan in preparation control cardiac-cerebral ischemia preparation and a kind of preparation method of Quzhazhigan, Quzhazhigan extract.Described Quzhazhigan is compound (E)-1-(3, the 5-dihydroxyphenyl)-2-(3-hydroxyl-4-O--D-Glucopyranose phenyl) ethene, and its preparation method is to extract from the root of Lhasa rhubarb and/or rhizome, prepares by purification step.
Background technology
Quzhazhigan is compound (E)-1-(3, the 5-dihydroxyphenyl)-2-(3-hydroxyl-4-O-β-D-Glucopyranose phenyl) ethene, or be called 3,5,3 ', 4 '-tetrahydroxy Stilbene-3 '-the O-beta-glucoside, because of its plant origin Lhasa rhubarb rhizome has another name called bent letter (Luo Dashang chief editor in the Tibetan medicine material.Newly repair brilliant pearl book on Chinese herbal medicine, Sichuan science tech publishing house, 2004), so claim that this compound is a Quzhazhigan.Stilbene class material is that a class is by the derivative of toluylene (Fig. 1) as precursor structure, and by Gorham (Gorham J.Progress in Phytochemistry, Pergamon Press:Oxford, 1980, Vol.6 is Stilbene (Stilbenoids) in definite designation in 1980 pp.203-252).Stilbene class material exists in the spermatophyte as plant alexin more, and along with going deep into of research, its chemistry, biology diversity and continuous newfound biological activity are subject to the people's attention.The precursor structure formula of Stilbene is as follows:
After the nineties, be that the pharmacology activity research of the natural Stilbene constituents of representative is subjected to extensive concern with trans-resveratrol (Resveratrol).Mainly concentrate on anti-oxidant, antitumor to pharmacological research in the world and aspect such as phytoestrogen activity as trans-resveratrol, red sandalwood Stilbene Stilbene such as (Pterostilbene).Domestic Stilbene glycoside composition such as polygonin (Polydatin, piceid), the reducing blood-fat of rheum officinale nafenopin (Rhapontin, rhaponticin, ponticin), stilbene glucoside, the hypoglycemic and active aspect of cardiovascular and cerebrovascular carry out a series of researchs.Stilbene and Stilbene glycoside material receive increasing concern in the potential using value that many-side highlights.All disclose the anticancer compound agent that contains stilbenes compound as Chinese patent application 99806755.5 and 02806583.2, Chinese patent application 99122378.0 discloses the hypoglycemic activity of the contained Stilbene glycoside compound of ripple leaf group rheum officinale; Chinese patent application 01118193.5 discloses the hypolipidemic activity of the contained Stilbene glycosides compound of ripple leaf group rheum officinale; Chinese patent 01134283.8 discloses the anti-cardiac-cerebral ischemia effect of extracting from the Stilbene glycosides compound stilbene glycoside of Tuber Fleeceflower Root.
Quzhazhigan was in (Y.Kashiwada in 1984, et al.Isolation andCharacterization of Stilbenes.Chem.Pharm.Bull.1984,32 (9): 3501-3517) first from taro rheum officinale (trade(brand)name: obtain Imo-Diao) and finish structure and confirm, yield is about 0.4%, after this, there is report from Rheum hotaoense C. Y. Cheng et C. T. Kao, to separate and obtains a small amount of this compound (Li Junlin etc.The non-anthraquinone component research of Rheum hotaoense C. Y. Cheng et C. T. Kao, herbal medicine, 98,29:721), also contain this compound in the rheum officinale of North China, yield is about 1.8% (Wang Aiqin etc.Stilbene constituents research in the rheum officinale of North China, herbal medicine, 2001,32:878).Still the research or the report that do not have at present the relevant pharmacologically active aspect of Quzhazhigan.
The glucosides of Quzhazhigan replaces and is positioned on the phenyl ring that non-meta-hydroxyl replaces, and with glucosides the position of substitution of the Stilbene glycosides compounds such as serial stilbene glucoside that obtain in rheum officinale nafenopin, Rhapontin, deoxy-, polygonin and the Tuber Fleeceflower Root structural difference is arranged.Compare with rheum officinale nafenopin, Rhapontin, deoxy-, polygonin, Quzhazhigan has more phenolic hydroxyl group, and the present invention finds that by removing the test of DPPH free radical the radical scavenging activity of Quzhazhigan is higher than above-claimed cpd.Forming when free radical is ischemia-reperfusion, is the important root of ischemic tissue's damage, and antioxidant and free-radical scavengers can alleviate the damage of ischemic tissue, and the free radical scavenging activity that Quzhazhigan is strong makes it have more advantage in the damage of protection ischemic heart and brain tissues.Pharmacology and drug efficacy study that the inventor has carried out serial control ischemic cardio cerebrovascular diseases aspect to the Quzhazhigan extract that derives from Lhasa rhubarb and pure product thereof first.Two kinds of animal models close with human diseases are adopted in research, measure multiple pharmacodynamic index, and two kinds of samples of comprehensive evaluation are to the influence of acute cardiac-cerebral ischemia.Test-results shows that Quzhazhigan extract and pure product can obviously reduce the release that coronary artery ligation causes rats with myocardial ischemia serum CK-MB and LDH, dwindle myocardial infarct size, and anticoagulant points out it that myocardial ischemia is had obvious provide protection.Quzhazhigan extract and pure product can obviously reduce MCAO rat cerebral infarction scope, make its behavior disorder degree be improved significantly, to the significant provide protection of having of ischemic cardio cerebrovascular diseases.Therefore, Quzhazhigan extract and pure product thereof have the potential clinical value aspect the control cardiac-cerebral ischemia.
The different structure of Quzhazhigan is also brought the water miscible advantage of compound monomer.In the Stilbene class structure of matter on the phenyl ring replacement of the increase of hydroxyl and glucosyl make the water-soluble increase of compound, as trans-resveratrol solubleness in water is the 0.03g/L (normal conditions: 25 ℃, 100kPa), polygonin makes its solubleness in water be increased to about 0.5g/L because of having glycosyl, and Duoing white fir skin alcohol (Piceatannol) water solubility of a hydroxyl than trans-resveratrol is 0.5g/L.And in the structure on the phenyl ring replacement of methoxyl group compound water soluble is reduced greatly.Water-soluble as rheum officinale nafenopin and Rhapontin, deoxy-(methyl polygonin) less than 0.04g/L.Have glycosyl and more phenolic hydroxyl group in the Quzhazhigan structure, its solubleness is about 1.1g/L, is made into preparations application such as injection, oral liquid for it and offers convenience.
Stilbene class material such as trans-resveratrol can extract purifying from giant knotweed (Polygonum cuspidatum Sieb.et Zucc), also can synthesize, and extract from plant resources and Stilbene glycoside material is many.Studies show that, polygonaceae plant (Polygonaceae), particularly often contain higher Stilbene glycoside composition in Rheum (Rheum) plant, content as ponticin in Rheum hotaoense C. Y. Cheng et C. T. Kao (the Rheum hotaoense C.Y.Cheng et C.T.Kao) rhizome can reach 3% (Lu sea otter etc., the HPLC method is measured the content of ponticin in the cultivation Rheum hotaoense C. Y. Cheng et C. T. Kao, the pharmaceutical analysis magazine, 2007,27:11), the content of rheum officinale nafenopin in Rheum tanguticum (the Rheumtanguticum Maxim.ex Balf.) rhizome of some areas cultivation can reach 10% (Zhang De etc., research and development of natural products, 2005,17:217).
The inventor finds that first this plant contains abundant Stilbene glycoside composition in the composition Study of Lhasa rhubarb (Rheum lhasaense A.J.Li et P.K.Haiao) rhizome wild and plantation being carried out system, (Chinese invention patent 200510010757.X except that deoxidation ponticin, polygonin, 200710066456.8), also contain 3～5% Quzhazhigan.We also find in the process of systematic study Lhasa rhubarb chemical ingredients and preparation Quzhazhigan extract, pure product, compare with the above-mentioned rheum officinale plant variety that is rich in Stilbene glycosides composition, Lhasa rhubarb is except that the characteristics that contain high-load polyhydroxystilbene glycosides compound, contain anthraquinone and anthraquinone glycoside chemical ingredients with potential toxic side effect hardly, these characteristics have been brought practicality and application advantage for exploitation, the industrialization production of Stilbene glycoside efficient part in this plant or single component.
Summary of the invention
The objective of the invention is to overcome above-mentioned prior art deficiency, invent the application of a kind of Quzhazhigan in preparation prevention and treatment ischemic cardio cerebrovascular diseases preparation, and method for preparing Quzhazhigan and contain the Quzhazhigan extract of invention.
The present invention is achieved in that
The application of Quzhazhigan in preparation prevention and treatment ischemic cardio cerebrovascular diseases preparation, described Quzhazhigan is compound (E)-1-(3, the 5-dihydroxyphenyl)-2-(3-hydroxyl-4-O-β-D-Glucopyranose phenyl) ethene, chemical structure is:
The Quzhazhigan that uses is the Quzhazhigan extract.
Described Quzhazhigan extract, by weight percentage, the effective ingredient that contains Quzhazhigan in the Quzhazhigan extract is 50%-100%.
Described preparation method comprises the following steps:
(1) pre-treatment: pre-treatment step comprises the collection of former roots of plants of Lhasa rhubarb or rhizome, system and/or drying, chopping or pulverizing only;
(2) extract: described extraction be adopt water, moisture or not water-containing organic solvent the described Lhasa rhubarb root of step (1) or rhizome extracted obtain the Lhasa rhubarb extracting solution as extracting solvent; Described organic solvent comprises alcohol, ketone, ether and their mixture that contains 1 ~ 4 carbon atom.
(3) aftertreatment: post-processing step comprises that the organic solvent in recovery or partially recycled step (2) the gained extracting solution obtains extracting concentrated solution, gained extracts concentrated solution can add or not add water, then extracting solution is staticly settled, solid-liquid separation is removed partial impurities; Liquid phase after the removal of impurity concentrates crystallization and acquires by weight percentage through further polymeric adsorbent chromatography purification, stepwise elution, eluate, and containing Quzhazhigan is the Quzhazhigan extract of 50%-100%.
A kind of preparation method of above-mentioned Quzhazhigan extract, the described organic solvent of its step (2) is methyl alcohol, ethanol, acetone, propyl carbinol or ether; The described extraction solvent of its step (2) is 50 ~ 100% ethanol or methanol aqueous solution.
A kind of preparation method of the pure product of above-mentioned Quzhazhigan is characterized in that, described preparation method adopts Lhasa rhubarb as raw material, obtains the pure product of required Quzhazhigan by extracting purification step; The pure product purity of described Quzhazhigan is 90%-100% by weight percentage.
A kind of preparation method of described Quzhazhigan extract is characterized in that the Quzhazhigan extract is to extract from the root of Lhasa rhubarb (Rheum lhasaense A.J.Li et P.K.Haiao) of wild or cultivation or rhizome.
The purposes of Quzhazhigan in the pure product of preparation Quzhazhigan.
The purposes of Quzhazhigan extract in preparation prevention or treatment cardiovascular and cerebrovascular diseases preparation.
The Quzhazhigan preparation can be used for making medicine or protective foods as various acceptable preparation or protective foods.
The application in preparation control cardiac-cerebral ischemia diseases preparation of Quzhazhigan is provided, described Quzhazhigan is (E)-1-(3, the 5-dihydroxyphenyl)-2-(3-hydroxyl-4-O-β-D-Glucopyranose phenyl) ethene or 3,5,3 ', 4 '-tetrahydroxy Stilbene heart and brain-3 '-the O-beta-glucoside, the chemical structural formula of Quzhazhigan is as follows.
Polyhydroxystilbene glycosides compound that research at present is more such as polygonin, ponticin, Rhapontin, deoxy-etc., the C-3 position of the chemical structure of Quzhazhigan shown in its glucosyl group is substituted in, the glycosyl of Quzhazhigan then is substituted in C-3 ' position, and such Stilbene glycosides compound is not still having research aspect the anti-cardiac-cerebral ischemia activity at present.The present invention has carried out the pharmacology and the drug efficacy study of anti-cardiac-cerebral ischemia aspect to Quzhazhigan, and the extract of finding the pure product of Quzhazhigan and containing Quzhazhigan has all shown significant pharmacological activity in the model of acute cardiac-cerebral ischemia diseases.Two kinds of animal models close with human diseases are adopted in research, measure multiple pharmacodynamic index, and two kinds of samples of comprehensive evaluation are to the influence of acute cardiac-cerebral ischemia.Test-results shows; 50,100mg/kg Quzhazhigan extract and 15, the pure product abdominal injection of 30mg/kg can obviously reduce the release that coronary artery ligation causes rats with myocardial ischemia serum CK-MB and LDH; significantly dwindle myocardial infarct size, show that its damage that myocardial ischemia is brought has significant provide protection.50,100mg/kg Quzhazhigan extract and 15,30mg/kg can also obviously reduce MCAO rat cerebral infarction scope, obviously improve the neural function degree due to the focal cerebral ischemia in rats, show that its damage to cerebral ischemia has significant provide protection.Decimal thrombus in vivo due to the equal energy of the Quzhazhigan extract of a plurality of dosage and pure product anticoagulant-suprarenin forms.Above-mentioned test index shows that Quzhazhigan has the potential clinical value aspect the control cardiac-cerebral ischemia, thus, the invention provides the purposes of Quzhazhigan in preparation treatment ischemic cardio cerebrovascular diseases medicine.
Described preparation method extracts target substance from the root of Lhasa rhubarb and/or rhizome, and described Lhasa rhubarb is Rheum lhasaense A.J.Li et P.K.Haiao.
The present invention further provides Quzhazhigan and contain the Quzhazhigan preparation method of extract, described preparation method comprises the following steps:
(1) pre-treatment: pre-treatment step comprises the collection of former roots of plants of Lhasa rhubarb or rhizome, system and/or drying, chopping or pulverizing only.
(2) extract: described extraction be adopt moisture or not water-containing organic solvent the described Lhasa rhubarb root of step (1) or rhizome extracted obtain the Lhasa rhubarb extracting solution as extracting solvent; Described organic solvent comprises alcohol, ketone, ether and their mixture that contains 1 ~ 4 carbon atom.
(3) aftertreatment: post-processing step comprises that the organic solvent in recovery or partially recycled step (2) the gained extracting solution obtains extracting concentrated solution, gained extracts concentrated solution can add or not add water, then extracting solution is staticly settled, solid-liquid separation is removed partial impurities; Liquid phase after the removal of impurity is through further polymeric adsorbent chromatography purification, stepwise elution, the concentrated crystallization of eluate can obtain the described extract of claim 2 ~ 3.
In the described pre-treatment step of step (1), described Lhasa rhubarb is meant polygonaceae plant Rheum lhasaense A.J.Li et P.K.Haiao.Described Lhasa rhubarb can be wild, also can be artificial growth, and the success of artificial introducing culture rheum officinale can further enlarge the medicine source of Lhasa rhubarb or produce more excellent mutation.The present invention finds, there is high-load Quzhazhigan in the Lhasa rhubarb root and rhizome, with the difference of the time limit of gathering, season, place and other factors, the amount of the contained Quzhazhigan of Lhasa rhubarb root and rhizome can reach 3% ~ 5% (with dry weight basis), and this is very rare in plant.
In addition, the present invention is also surprised to be found, contain anthraquinone analog compound in Lhasa rhubarb root and the rhizome hardly, this is rare in rheum officinale class plant, this is a very important feature of Lhasa rhubarb: one, and use Lhasa rhubarb and be used as medicine and do not have worry the potential serious toxic side effect of anthraquinone analog compound; They are two years old, when using Lhasa rhubarb extraction toluylene active component, its extraction process can not be subjected to the particularly interference of anthraquinone glycoside of anthraquinone analog compound, therefore technology can be easier, and These characteristics is that exploitation, utilization and the industrialization production of Stilbene glycoside efficient part or single component has brought application advantage and practicality in the Lhasa rhubarb.
The diphenylethylene component content is very low in the cauline leaf of Lhasa rhubarb ground, and is relatively low as the value of extracting raw material, but still can be used as one of source of diphenylethylene compounds.The present invention preferably adopts the subterraneous root of Lhasa rhubarb and rhizome as extracting raw material, but does not get rid of with its whole plant as extracting raw material.
The Lhasa rhubarb of gathering generally need pass through pre-treatments such as clean system, cutting and/or pulverizing.Described clean system can comprise removes the foreign material such as earth that are attached in the gatherer process on Lhasa rhubarb root and the rhizome, can also comprise root and rhizome and ground cauline leaf being separated etc.The Lhasa rhubarb of being gathered can be used its bright product, can adopt also that it is air-dry, dry or the dry product of machine dry; This root of gained and rhizome or whole plant preferably carry out suitable cutting or pulverize in order to extraction.
The described organic solvent of step (2) is methyl alcohol, ethanol, acetone, propyl carbinol or ether.It extracts solvent preferred alcohol or methanol in water; The wherein special preferred alcohol aqueous solution is as extracting solvent; Further, the present invention preferably adopts 45% ~ 95% aqueous ethanolic solution as extracting solvent.
Step (2) gained Lhasa rhubarb extracting solution needs can obtain the Quzhazhigan extract through appropriate postprocessing.Because contained some main Stilbene of Lhasa rhubarb and Stilbene glycoside material such as rhapontigenin, Rhapontin, deoxy-unit, Rhapontin, deoxy-, polygonin are water-soluble limited, the present invention finds, reclaim in the extracting solution organic solvent and/or suitably concentrated extract the fat-soluble Stilbene of the contained part of extract and Stilbene glycosides compound, impurity are separated out from extracting solution, Quzhazhigan is owing to water-solublely better then be retained in the supernatant liquor.Its rate of transform can reach more than 85%.Quzhazhigan in the supernatant liquor can adopt the appropriate organic solvent extraction to carry out purifying, for example adopts propyl carbinol, ethyl acetate-methanol mixed solvent, propyl carbinol-extractions such as methylene dichloride mixed solvent; This compounds can also adopt the method abstraction purification of knowing in this areas such as continuous countercurrent extraction method.
The preferred diphenylethylene compounds that adopts in the macroporous adsorption resin chromatography method purifying supernatant liquor among the present invention.In the above-mentioned post-processing step, along with the Recovery of Organic Solvent in the extracting solution, when supernatant liquor was splined on macroporous adsorptive resins, contained Quzhazhigan was easy to by the resin column bed adsorbed in the supernatant liquor; After the last sample absorption, the resin column bed can adopt water and the washing of lower concentration aqueous solutions of organic solvent with the bigger impurity of further removal polarity, can adopt the required chemical ingredients of aqueous solutions of organic solvent stepwise elution of higher concentration subsequently, the gained elute soln can obtain described Quzhazhigan extract through concentrated, crystallization, drying.Extract can adopt pure water or simple aqueous solution recrystallization further to make with extra care the purity that improves Quzhazhigan, by recrystallization, decolouring or other means of purification can get the described pure product of claim 7 ~ 8 repeatedly.
When adopting the contained Quzhazhigan of macroporous resin adsorption supernatant liquor, column volume generally is provided with by every 100ml polymeric adsorbent (volume behind the dress post) absorption 1 ~ 3g supernatant liquor, at this moment generally can obtain the good adsorption rate of transform.On the supernatant liquor after the sample absorption, can adopt the aqueous solution, 10% ~ 15% ethanolic soln or methanol aqueous solution washing to remove most of water-soluble impurity, adopt about 25% ~ 50% ethanol or methanol aqueous solution can wash-out to be adsorbed in the Quzhazhigan of macroporous resin subsequently.Among the present invention, preferred macroporous adsorbent resin is low-pole and semipolar resin, for example marque D101, D1300, AB8 type polymeric adsorbent.Under appropriate condition, in the macroreticular resin absorbing method products therefrom, it is about 50% to 85% that the Quzhazhigan total content can reach, and the rate of transform is 80% ~ 95%.
Through behind the above-mentioned purification step, Quzhazhigan has obtained enrichment, adopts these extracts can prepare higher extract of Quzhazhigan content and pure product easily as raw material.For example, among the present invention, adopting macroreticular resin absorbing method gained wash-out partly is raw material, concentrate by suitable, it is 70% ~ 89% Quzhazhigan extract that crystallization can get content, and this extract adopts repeatedly chromatographies such as recrystallization, decolorizing with activated carbon, silica gel column chromatography, polymeric amide can obtain purity up to the pure product of the Quzhazhigan more than 90% in conjunction with the recrystallization method purifying.
Extract by the above-mentioned steps preparation is to be the mixture of main chemical compositions with the Quzhazhigan.The content that is appreciated that Quzhazhigan in this extract changes and changes to some extent with raw material batch difference, treatment process parameter.Usually, by weight percentage, contain 50% ~ 85% the Quzhazhigan of having an appointment in the described extract.
Obviously, the said extracted thing can be used for further refining purifying to obtain the pure product of Quzhazhigan.Among the present invention, so-called pure product are meant that the purity of compound is not less than 90%.
The present invention further provides Lhasa rhubarb extract thus at the pure product of preparation Quzhazhigan, and the purposes of Quzhazhigan extract in the pure product of this Quzhazhigan of preparation.
Result of study shows that the pure product of Quzhazhigan extract and Quzhazhigan are having been widely used aspect the medicine of preparation treatment cardiovascular and cerebrovascular diseases.The structure of activeconstituents is clear in the described efficient part, composition is clear and definite, helps the quality monitoring and the control of products at different levels, meets the developing direction and the requirement of natural product compound formulation.In addition, the preparation method of efficient part of the present invention is simple, and production cost is low, but operate continuously has extremely strong practicality.
According to the preparation process of routine, the present invention's extract can be prepared into any pharmaceutical preparation that is applicable to clinical application.As tablet, capsule, pill, powder, paste, dispersion agent, granule, powder injection, aqueous injection etc.
Equally, the present invention's Quzhazhigan extract also can be united the preparation that uses formation new with other activeconstituents.Obviously, these preparations that contain efficient part of the present invention are in claim scope of the present invention.
Description of drawings
Fig. 1 differentiates (iodine colour developing) for thin layer of the present invention.
The wild Lhasa rhubarb raw material of Fig. 2 trial-product liquid HPLC typical case collection of illustrative plates (Quzhazhigan, RT:7.862).
Fig. 3 plant Lhasa rhubarb raw material trial-product liquid HPLC typical case collection of illustrative plates (Quzhazhigan, RT:7.885).
Fig. 4 Quzhazhigan extract trial-product liquid HPLC typical case collection of illustrative plates (Quzhazhigan, RT:7.878).
The pure product HPLC typical case of Fig. 5 Quzhazhigan collection of illustrative plates (Quzhazhigan, RT:7.896).
Wherein in Fig. 1: 1. represent Rhapontin, deoxy-reference substance liquid; 2. represent Quzhazhigan reference substance liquid; 3. represent wild Lhasa rhubarb raw material (20080913) trial-product liquid; 4. Lhasa rhubarb raw material (20080915) trial-product liquid is planted in expression; 5. Lhasa rhubarb raw material (20080915) trial-product liquid is planted in expression; 6. Lhasa rhubarb raw material (20080915) trial-product liquid is planted in expression.
Specific embodiments
Below will further specify content of the present invention by specific embodiment.Hereinafter embodiment only is the specified otherwise in source, preparation method, chemical constitution and uses thereof with regard to efficient part of the present invention, and obviously, interest field of the present invention is not limited by these embodiment.
Embodiment 1: the qualitative identification and the detection by quantitative of Quzhazhigan in Lhasa rhubarb raw material and the Quzhazhigan extract
(1) sample source
Wild Lhasa rhubarb rhizome: purchase in the Lhasa area, remove impurity, clean, cut into slices or piece, dry.Lot number: 20080913
Plantation Lhasa rhubarb rhizome: be harvested from the Xianggelila country, Yunnan, remove impurity, clean, cut into slices or piece, dry.Lot number: 20080915
Reference substance: Quzhazhigan, self-control, purity 99%, lot number: 080602.
(2) sample and standard solution preparation
The reference substance solution preparation: precision takes by weighing with the vacuum drying Quzhazhigan reference substance of Vanadium Pentoxide in FLAKES an amount of, is made into 1mg/ml reference substance storing solution with methyl alcohol, and 27% methyl alcohol-3% acetic acid solution is diluted to 25g/ml as standard solution.
The preparation of Lhasa rhubarb raw material need testing solution: precision takes by weighing with the vacuum drying above-mentioned Lhasa rhubarb rhizome powder 10g of Vanadium Pentoxide in FLAKES, 95% alcohol reflux 3 times (120min/ time), united extraction liquid, filter paper filtering, filtrate is further with 0.45 μ m filtering with microporous membrane, abandon filtrate just, get subsequent filtrate as the Lhasa rhubarb need testing solution.
The preparation of Quzhazhigan extract need testing solution: precision takes by weighing with the vacuum drying embodiment 2 described Quzhazhigan extracts of Vanadium Pentoxide in FLAKES an amount of, be made into 10mg/ml sample storing solution with methyl alcohol, the accurate 1ml that draws, place the 25ml volumetric flask, 27% methanol constant volume, close plug, shake up, cross 0.45 μ m millipore filtration, abandon filtrate just, get subsequent filtrate as the Lhasa rhubarb extract need testing solution.
(3) qualitative identification: tlc inspection
This law adopts silica gel thin-layer chromatography.Get above-mentioned trial-product liquid and each 4l of reference substance liquid, put respectively on same silica gel thin-layer chromatography plate (silica gel G) plate, with chloroform: methyl alcohol (volume ratio, 7.5: 2.5), take out, dry for developping agent launches, place the iodine cylinder to develop the color, in the identical position of reference substance, trial-product should have corresponding spot.
(4) the contained Quzhazhigan content quantitative of trial-product detects: high performance liquid chromatography (HPLC)
Chromatographic condition: (27% methyl alcohol-3% acetic acid water is moving phase for 4.5 * 150mm, Agilent) reverse-phase chromatographic column, and O.8ml/min flow velocity detects with the DAD Ultraviolet Detector, detects wavelength 319nm to adopt Zorbax SB-C18.
Typical curve is set up: above-mentioned reference substance storing solution is diluted to 5,15,25,35,45 μ g/ml solution respectively, respectively get 10 μ l and inject liquid chromatograph respectively, measure peak area by above-mentioned chromatographic condition, with each reference substance peak area (Y, A) (X mg) carries out linear regression, regression equation: Y=6.3324X+0.1158 to sample size, r=0.9999 (Quzhazhigan, n=5).
Sample determination: above-mentioned Lhasa rhubarb raw material need testing solution, each 10 μ l of Quzhazhigan extract need testing solution inject liquid chromatograph respectively, calculate the content of Quzhazhigan in Lhasa rhubarb raw extract, the Quzhazhigan extract according to typical curve.
(4) result:
I) qualitative identification: the typical thin-layer chromatogram of Lhasa rhubarb raw extract and Quzhazhigan extract as shown in Figure 3, the result shows that in the identical position of reference substance, trial-product all has corresponding spot.
Ii) Quzhazhigan content detection: the typical HPLC spectrogram of Lhasa rhubarb raw material and Quzhazhigan extract thereof is shown in Fig. 4 ~ 6, the result shows, the content of Quzhazhigan is respectively 4.38% and 4.18% in Lhasa rhubarb natural crude drugs raw material and the plantation medicinal raw material, and the content of Quzhazhigan is 78.5% in the extract.
Embodiment 2: the preparation of Quzhazhigan extract
(1) material: Lhasa rhubarb rhizome: purchase in the Lhasa area, remove impurity, clean, cut into slices or piece, dry.Lot number: 20080913
Resin: the D101 macroporous adsorbent resin, Tianjin sea light chemical industry company limited product carries out pre-treatment according to factory's specification sheets.
(2) preparation
I) Lhasa rhubarb pre-treatment and Quzhazhigan assay thereof:
The Lhasa rhubarb plant of gathering goes mud to dry, and separates underground rhizome and ground cauline leaf, and pulverizer is ground into coarse meal with it respectively.
Get underground rhizome and ground each 100g of cauline leaf powder, with 95% alcohol immersion refluxing extraction, 120 minutes/time, the HPLC detection is carried out in sampling at interval, in extracting solution, do not contain Quzhazhigan substantially, united extraction liquid is pressed the content of the Quzhazhigan of method method described in the embodiment 1, and this detected result is as contained this total amount of compound of medicinal material.
The result: the contained Quzhazhigan total amount of compound in the Lhasa rhubarb underground rhizome of lot number 20080913 and the ground cauline leaf is respectively 5.01% and 0.001%.
Ii) extract:
Every part of 600g of Lhasa rhubarb underground rhizome powder is respectively with 50%, 75%, 95% ethanol; 50%, 90% methyl alcohol; 50% acetone; Propyl carbinol is as extracting solvent extraction.Each part medicinal material is respectively with above-mentioned different extract solvent 5.4L refluxing extraction 3 times (2.4L/1.8L/1.2L) altogether, 120min/ times.Difference united extraction liquid, Quzhazhigan mean vol (A) in the HPLC method Detection and Extraction liquid:, calculate the Quzhazhigan rate of transform (B): Quzhazhigan total amount in the extracting solution/medicinal material Quzhazhigan total amount * 100%.
The extraction test-results is as shown in table 1.
Table 1: the different extraction test-results of extracting solvent
Quzhazhigan compound amount in the extracting solution that the A:HPLC method detects; B: the Quzhazhigan rate of transform
In the said extracted test, with 50%, 75%, 95% ethanol; 50%, 90% methyl alcohol; 50% acetone; Propyl carbinol is when extracting solvent extraction, and the Quzhazhigan rate of transform is between 70% ~ 95% in the extraction step.As seen they all can be used as effective extraction solvent of this compound, and wherein, in view of alcoholic acid safety, cheapness and to extract the rate of transform higher, so the preferred alcohol aqueous solution of the present invention is as extracting solvent.
Iii) aftertreatment:
Precipitation process: respectively combining step ii) gained respectively extract solution, wherein contained organic solvent is reclaimed in decompression (0.1 ~ 0.08Mpa, 60 ℃), after adding water and being settled to 600mL, mix, place 24h for 4 ℃, centrifugal (4000rpm * 30min), get supernatant liquor and precipitation.Precipitation vacuum-drying, (C) weighs; The HPLC method detects the amount (D, E) of Quzhazhigan in supernatant liquor and the precipitation.Calculate the Quzhazhigan rate of transform (F, G) in supernatant liquor and the precipitation: supernatant liquor or precipitation Quzhazhigan total amount/extracting solution Quzhazhigan total amount * 100%.
Table 2: precipitation process test-results
C: precipitation capacity D: Quzhazhigan amount in the precipitation
E: Quzhazhigan amount E in the supernatant liquor: the Quzhazhigan rate of transform in the precipitation
F: the Quzhazhigan rate of transform in the supernatant liquor;
Table 2 data presentation, the above-mentioned extracting solution of respectively organizing concentrate to remove organic solvent and adds stand at low temperature behind the water, except that the propyl carbinol group, all the other are respectively organized sample and all produce precipitation, the rate of transform of Quzhazhigan is between 2% ~ 5% in the precipitation, and precipitation capacity is between 10 ~ 30g, and the Quzhazhigan rate of transform is between 85% ~ 95% in the supernatant liquor, as seen this step process can effectively be removed oil-soluble impurities, and the loss of Quzhazhigan is less.Therefore the present invention is the important step of this step as preparation Quzhazhigan extract.
Supernatant liquor is handled: except that n-butanol extracting liquid, merge all other supernatant liquor, filter once more, the HPLC method detects the Quzhazhigan total amount in the gained filtrate, it is divided into 4 parts, is splined on 4 D101 macroporous adsorptive resins (φ 5.0cm, 105cm respectively, column volume 2000ml) and HPLC check sample effluent liquid on the D101 resin column, do not see that diphenylethylene compounds leaks.4 D101 resin columns are respectively in turn with 1000ml water, 1000ml 15% ethanol or 10% methanol wash, and HPLC checks the scrub stream fluid, and effluent liquid does not contain or only contain a small amount of Quzhazhigan substantially.The D101 resin column is more respectively with 25,50%7 alcohol or 20% methyl alcohol, 45% methanol-eluted fractions, collect elutriant, concentrating under reduced pressure, vacuum-drying gets the Quzhazhigan extract, the First Astronautic Research Institute for Measurement and Test gets the weight (H) of end product, and the HPLC method detects Quzhazhigan total amount (I) in the end product; Calculate the content (J) of the Quzhazhigan in the end product: Quzhazhigan total amount/end product weight * 100% in the end product; Calculate the Quzhazhigan rate of transform (K): Quzhazhigan total amount * 100% in Quzhazhigan total amount in the end product/last sample filtrate.
Table 3 supernatant liquor Processing Test result
H: end product amount; I: Quzhazhigan total amount in the end product amount
J: Quzhazhigan content K in the end product amount: the Quzhazhigan rate of transform;
Quzhazhigan total amount: 33.82g in the sample filtrate (750ml) on the every resin column
Table 3 data presentation, the Quzhazhigan content of wash-out part is between 50% ~ 85% from the polymeric adsorbent, and the rate of transform of Quzhazhigan is between 85% ~ 96%.Wherein, in view of alcoholic acid safety, cheapness, therefore the preferred alcohol aqueous solution of the present invention is as eluent, between the wash-out concentration preferred 25% ~ 50%, can predict, along with the reduction of elutriant alcohol concn, the content of wash-out part Quzhazhigan can rise to some extent, and the rate of transform descends to some extent, otherwise alcohol concn raises then.
Embodiment 3: prepare pure product Quzhazhigan by Lhasa rhubarb extract and Quzhazhigan extract
(1) material: Lhasa rhubarb rhizome: purchase in the Lhasa area, remove impurity, clean, cut into slices or piece, dry.Lot number: 20080913.
(2) Lhasa rhubarb extract preparation: get Lhasa rhubarb dry rhizome powder 100g, adopt 95% ethanol to extract 3 times (2.4L/1.8L/1.2L) as extracting solvent refluxing, 120min/ time, united extraction liquid concentrates, and vacuum-drying gets Lhasa rhubarb extract.
(3) purifying: Lhasa rhubarb extract 2g, mix with 40g 200 order silica gel, adopt purification by silica gel column chromatography.Adopt chloroform/methanol (V/V, 8/1) as eluting solvent, fraction collection various flows part, thin-layer chromatography (TLC) monitoring merges the flow point that contains Quzhazhigan, decompression and solvent recovery, 15% ethyl alcohol recrystallization, vacuum-drying gets the pure product of Quzhazhigan.
The 2g of Quzhazhigan extract described in the embodiment 2 added pure water (32ml) by 16: 1, and 60 ℃ of heating for dissolving are placed 4 hours crystallizatioies, suction filtration, crystal adds suitable quantity of water, 0.5% decolorizing with activated carbon, recrystallization, get the 1.25g Quzhazhigan, vacuum-drying gets the pure product of Quzhazhigan.
(4) HPLC detects: press the purity of HPLC method detection gained Quzhazhigan among the embodiment 1, the purity of Rhapontin, deoxy-can be between 90%～99.9% in its various flows part product; Crystallization-decolouring-recrystallization gained Quzhazhigan purity is between 98%～99.6%.HPLC detects typical collection of illustrative plates and sees Fig. 7.
(5) Quzhazhigan structural confirmation
Detect the gained Quzhazhigan UV, negative FABMS, MS,
1H NMR,
13The CNMR spectrogram, it is as follows that it detects data:
Quzhazhigan: white hair shape crystal (water), mp.220-225 ℃.Be bluish voilet fluorescence under the ultraviolet lamp.UVλmax(MetOH)：217(4.38)，320(4.48)；EI-MS(m/z)：406[M]
+，244[M-glycosy]
+；negative?FAB-MS(m/z)：405[M-H]
-，243[M-H-glycosy]
-；1H?NMR(CD
3OD，500M?Hz)δppm：7.44(1H，d，J＝2.0Hz，C
2′-H)，7.05(1H，dd，J
1＝2.0Hz，J
2＝8.0Hz，C
6′-H)，6.92(1H，d，J＝16.3Hz，C-H)，6.83(1H，d，J＝16.3Hz，C-H)，6.82(1H，d，J＝8.3Hz，C
5′-H)，6.47(1H，d，J＝2.2Hz，C
2，6-H)，6.18(1H，t，J＝2.2Hz，C
4-H)，4.81(1H，d，J＝7.4Hz，anomericH)，3.95(1H，dd，J
1＝2.0，2.1Hz，J
2＝12.2Hz，C6″-H)，3.74(1H，dd，J
1＝6.0Hz，J
2＝＝12.1Hz，C6″-H)，3.41-3.56(4H，m?sugar-H)；
13C?NMR(CD
3OD，125MHz)δppm：141.1(C-1)，105.9(C-2，6)，159.4(C-3，5)，102.7(C-4)，131.3(C-1′)，116.6(C-2′)，146.9(C-3′)，148.1(C-4′)，117.2(C-5′)，123.0(C-6′)，127.8(C-α)，129.1(C-β)，104.4(C-1″)，74.9(C-2″)，78.4(C-3″)，71.4(C-4″)，77.5(C-5″)，62.5(C-6″)。
By with the comparison of document, as can be seen, in this detected result the numerical value of each proton chemical shifts numerical value and coupling constant all with document in (Y.Kashiwada, et al.I solation andCharacterization of Stilbenes.Chem.Pharm.Bull.1984,32 (9): 3501-3517; Zheng Junhua, the fruit Dean.The rheum officinale modern study. medical science press of Peking University, 2007, first version, 286) respective value coincide.Quzhazhigan is accredited as (E)-1-(3, the 5-dihydroxyphenyl)-2-(3-hydroxyl-4-O--D-Glucopyranose phenyl) ethene
[(E)-1-(3,5-dihydroxyphenyl)-2-(ethylene of 3-hydroxyl-4-O-β-D-glucopyranyphenyl)], or be called 3,5,3 ', 4 '-tetrahydroxy Stilbene-3 '-O-beta-glucoside (3,5,3 ', 4 '-tetrahydroxystilbene-3 '-O-β-D-glucopytanoside).
Embodiment 4: the pharmacotoxicological effect of Lhasa rhubarb extract
(1) test sample
Quzhazhigan extract: experiment code name QZQ-00-01.Sample is according to embodiment 2 described step preparations.Wherein Quzhazhigan content is 78.5%
Quzhazhigan: experiment code name QZQ.Sample has the preparation of Quzhazhigan extract according to embodiment 3 described steps, and purity is 99.1%.
Polygonin: available from Xi'an China collection Bioisystech Co., Ltd, lot number: POL050817, purity is 98.0%.
Above-mentioned test sample all is mixed with the test solution of different concns with-hydroxypropyl cyclodextrin.
(2) experimental animal
SPF level ICR mouse is available from unming Medical College's Experimental Animal Center.
SPF level SD rat is available from unming Medical College's Experimental Animal Center.
(3) test method
Influence to rat MCAO focal cerebral ischemia
Each organize male SD rat animal every day according to dosage gastric infusion once, continuous 5 days.30min behind the last filling stomach, with 12% Chloral Hydrate abdominal injection 350mg/kg anesthetized animal, left lateral position, cut skin along auris dextra informer mid point, separate temporalis, the disconnected cheekbone of strand is holed with dental burr in cheekbone root the place ahead, exposes MCAO, between venae cerebri inferiores and tractus olfactorius, provoke with pin, break it, after the cotton balls hemostasis by compression, layering suture muscles and skin.Sham operated rats is not except choosing disconnected MCAO, and all the other steps are identical.Animal 6h and 24h after modeling carry out the neural function scoring according to behaviors such as motion performance, with this as the disordered brain function index.Blind method is adopted in scoring, and promptly scoring person does not know the situation of administration,
Standards of grading are as follows:
The scoring of symptom behavior disorder
???????????????????????????????????????????????????????????????????????????????
Carry the about chi of mouse tail built on stilts, the wrist flexing appears in operation offside forelimb, elbow flexing, shoulder inward turning 1 ~ 4
Or wrist is arranged, the elbow flexing has inward turning again.
Animal is placed on the plane earth, push away both shoulders respectively to the inside, check resistance.1~3
Animal is placed on the metal wire side, observe the tension force of two forelimbs.1~3
Animal is placed on the plane earth, observe to have or not and turn-take.1~2
Full marks 12
???????????????????????????????????????????????????????????????????????????????
Above standard full marks are 12 minutes, and mark is high more, show that the animal behavior obstacle is serious more
Modeling 24h, after the neural function scoring, the carotid artery intubate is got blood, and a separation of serum is pressed the activity that kit method is measured AST and LDH; With anti-freezing in 1: 9, the centrifugal 5min of 1000rpm prepared platelet rich plasma (PRP) to portion with 3.8% Sodium Citrate, and the centrifugal 10min of 3500rpm prepares platelet poor plasma (PPP).By turbidimetry for Determination ADP-2Na inductive platelet aggregation rate, the final concentration of ADP is 6mol/L; Measure TT, PT, APTT and FIB respectively by kit method.Quick broken end is got brain after getting blood, puts into ice physiological saline and removes olfactory bulb, cerebellum and low brain stem, and suck dry moisture takes by weighing brain and weighs, and is cut into 5 along crown, inserts 5ml and contains 4%TTC and 1mol/LK
2HPO
40.1ml solution in, lucifuge is incubated 30min in 37 ℃ of temperature, stir once every 7-8min therebetween, after dyed, normal cerebral tissue is rose, and infarction tissue is white in color, and blocking part is cut weighed, and calculates infarction tissue's weight with the weight area method and accounts for the heavy per-cent of bilateral hemicerebrum as the cerebral infarction scope.
20～22g mouse is got in collagen protein-suprarenin induced mice thrombosis influence, and male and female half and half by sex and body weight random packet, see Table 6.Each treated animal every day according to dosage intraperitoneal injection once, continuous 5 days.30min after the last administration, the inductor that every mouse is made by collagen protein-suprarenin by the dosage intravenous injection of 0.1ml/10g body weight is observed and interior each the treated animal hemiplegia incidence of record 15min, mortality ratio and recovery rate.The significance of X2 check group difference.
Coronary ligation is caused the influence of rat heart muscle ischemic
Male SD rat, 330～385g sees Table 7 by the body weight random packet, each treated animal according to dosage every day gastric infusion once, continuous 5 days, blank group and model group waited capacity 0.5%CMC-Na 10ml/kg body weight.30min after the last administration, with 12% Chloral Hydrate abdominal injection 350mg/kg anesthetized animal, the row open chest surgery exposes heart, finds out left anterior descending coronary artery between pulmonary conus and left atrium, and apart from root 2～3mm place's ligation, heart resets and closes the thoracic cavity fast.Sham operated rats is only put the end of a thread and not ligation causes the acute myocardial ischemia model, and behind the 24h, the carotid artery intubate is got blood system is measured AST, LDH and CK-MB by kit method from serum activity; Get and core behind the blood dirtyly immediately, place ice physiological saline to pump hematocele in the chambers of the heart, remove atrium and fatty tissue, it is heavy that suck dry moisture takes by weighing ventricle, is cut into 5 along crown subsequently, and lucifuge is hatched dyeing 10min in 37 ℃ in 1%TTC solution.After dyed, the normal myocardium tissue takes on a red color, and infarction tissue is white in color, and blocking part is cut weighed, and it is myocardial infarct size that the downright bad part of calculating accounts for the heavy per-cent of ventricle.
(5) test-results
Rat medium sized artery ligation (MCAO) is caused the influence of focal cerebral ischemia
The Quzhazhigan extract is with 50,100mg/kg, the pure product of Quzhazhigan are with 15, the dosage of 30mg/kg respectively at after the MCAO operation at once with ischemia-reperfusion 6h intravenous injection 2 times, can obviously alleviate the neurological dysfunction due to the focal cerebral ischemia in rats, obviously reduce the scope of cerebral infarction, Quzhazhigan extract two dosage groups dwindle 65.87%, 40.38% respectively; The pure product two dosage groups of Quzhazhigan dwindle 75.87%, 52.21% respectively, are better than polygonin.
Pure product of table 4 Quzhazhigan and extract pour into the influence (X ± SD) of rat neurological dysfunction and cerebral infarction scope again to MCAO
Compare with sham operated rats: Δ Δ P＜0.01; Compare with model group: * P＜0.05, * * P＜0.01;
Pure product of table 5 Quzhazhigan and extract pour into the influence of rat blood clotting and platelet aggregation again to MCAO
Compare with sham operated rats:
ΔP＜0.05; Compare with model group:
*P＜0.05,
*P＜0.01;
To collagen protein-suprarenin induced mice thrombosis influence
The mouse thrombus in vivo that Quzhazhigan extract 60mg/kg, 120mg/kg dosage group, each dosage group of the pure product of Quzhazhigan all can obviously suppress due to the collagen and adrenalin forms;
The influence that pure product of table 6 Quzhazhigan and extract form the mouse thrombus in vivo
Compare with control group:
*P＜0.05,
*P＜0.01
Coronary ligation is caused the influence of rat acute myocardial infarct size
Model group rat blood serum AST, LDH and CK-MB activity have increasing in various degree, increase more obviously especially with CK-MB and LDH activity, show myocardial damage modeling success.Quzhazhigan (50mg/kg) and Quzhazhigan extract (200mg/kg) all can obviously reduce the release of CK-MB and LDH, show that to alleviate the myocardial cell injury effect remarkable; The myocardial infarct size of two administration group rats is all significantly less than model control group, compares with model group and dwindles 64.33% and 47.72% respectively.The results are shown in Table 7.
Table 7.QHQ and QZQ are to the influence of rat myocardium block scope and zymetology
The DPPH free radical scavenging activity test of embodiment 5 several Stilbene glycoside materials
(1) test sample
Quzhazhigan: self-control, sample has the preparation of Quzhazhigan extract according to embodiment 3 described steps,
Purity is 99.1%.
Polygonin: available from Xi'an China collection Bioisystech Co., Ltd, lot number: POL050817, purity is 98.0%.
Ponticin: self-control, press the pure product preparation of ponticin described in the Chinese invention patent 200510010757.X, purity: 99.5%.
Rheum officinale nafenopin: self-control, preparation from Rheum hotaoense C. Y. Cheng et C. T. Kao (Rheum hotaoense C.Y.Cheng et C.T.Kao), purity: 99.0%
Vitamin-E (Tocpherol), BHA (butylated hydroxyanisol), DPPH free radical (,-diphenyl--picrylhydrazyl) is the Sigma product.Wherein, vitamin-E is the antioxidant of natural origin commonly used, and BHA is a synthetized oxidation preventive agent commonly used in the foodstuffs industry.
Above-mentioned test sample all is mixed with the test solution of different sample concentrations with methyl alcohol.
(2) test method
Free radical scavenging activity is measured the method (M.H.Gordon with reference to people such as M.H.Gorden, et al.Antioxidant activity of hydroxytyrosol acetate compared with that of otheroliver oil polyphenols.J.Agric.Food Chem., 2001,49:2480).The test solution of each mensuration is a 0.06Mm DPPH free radical methanol solution. reference product and confession test agent all are made into the methanol solution of 2.1 (0.06 * 35) mM, are diluted to 1/10,1/4,1/2 3 different concentration respectively.During mensuration, in 3.5ml DPPH free radical methanol solution, add 0.1ml, mix, measured its absorbance value (A at 15 minutes with reference to product or sample solution
Sample), the detection wavelength is 515nm, the reference product of each different concns and sample replicate(determination) three times.Methyl alcohol is blank, and 0.06mM DPPH solution is contrast (Control).Each obtains 3-4 different concns respectively in three groups of replicate(determination) values of different time with reference to product with for test agent.Each measured value is represented the residual value of DPPH free radical in solution, with following formula it is converted into DPPH free radical scavenging activity (scavenged DPPH radical rate%): Scavenged DPPH radical rate%=(A
Control-A
Sample)/A
Control)
With this be worth make sample when the different concns DPPH free radical scavenging activity and time relation figure and under different time the relation of DPPH free radical scavenging activity and sample concentration.Sample concentration is represented with the mole number ratio (molar ratio) that adds sample and DPPH free radical, is got 0.10,0.25,0.50,1.00 in this experiment.Sample is removed the active EC of free radical
50Be to instigate the DPPH free radical to be eliminated 50% required sample mole number and the ratio that adds the DPPH mole number.
(3) test-results
Table 8copherol, BHA and each Stilbene glycosides compound DPPH free radical activity
| The sample title | ??EC 50(n＝3) |
| ??copherol | ??0.25±0.03 |
| ??0.14±0.01 | |
| Polygonin | ??0.57±0.02 |
| Rhapontin, deoxy- | ??0.78±0.01 |
| The rheum officinale nafenopin | ??0.44±0.03 |
| Quzhazhigan | ??0.28±0.01 |
Table 8 data presentation, the activity that Quzhazhigan is removed the DPPH free radical is stronger.
The Quzhazhigan extract: sample wherein extracts solvent and adopts 90% ethanol according to embodiment 2 described step preparations; Wherein the content of Quzhazhigan is 78.5%.
Method for making: take by weighing bent letter extract 100g, be dissolved in an amount of ethanol, add 900g PVP K29/32, mix, make abundant dissolving; Rotary evaporation makes ethanol volatilization, cooling, drying, solidify, pulverize cured article, get 100 grams and mix with 50 gram lactose, 30 gram pregelatinized Starchs, 10 gram sodium starch glycolatees, crossing 80 mesh sieves 2 times, mix, is tackiness agent system softwood with 1% low-substituted hydroxypropyl cellulose ethanolic soln (concentration 50%), crossing 40 mesh sieves granulates, 60 ℃ of dryings are crossed the whole grain of 30 mesh sieves, add 1.5 gram talcum powder and mix, compressing tablet can prepare 1000 of Quzhazhigan sheets.
Embodiment 7: the Quzhazhigan freeze-dried powder
The pure product of Quzhazhigan: sample wherein extracts solvent and adopts 90% ethanol according to embodiment 3 described step preparations; Wherein the content of Quzhazhigan is 99.0%.
Prescription:
The pure product 10g of Quzhazhigan
Soybean phospholipid 8g
N.F,USP MANNITOL 200g
Preparation technology: under 10,000 grades condition, qualified medicine and auxiliary material are dissolved in the water for injection together, after decolouring waits processing, add water for injection and be diluted to the 2000ml of institute.Filter, after the processing such as filter membrane coarse filtration of filtrate through filter paper and 0.4 μ m, under 100 grades condition, with the following membrane filtration of 0.2 μ m.Smart filtrate is sent into portioning machine, by every 2ml packing, covers the lid of trough of belt, sends into frozen vacuum dryer, is chilled to 35 ℃ ~ 40 ℃ fast, 2 ~ 3 hours, progressively slowly is warming up to 35 ℃ ~ 45 ℃ (needing 10 hours approximately).The jam-pack lid takes out goods, Zha Gai, packing, check, qualified pharmaceutical acceptable powder pin finished product (freeze-dried powder of the specification of 1000 10mg).
Embodiment 8: Quzhazhigan oral liquid product
Prescription: fat-soluble stilbene glycosides (content 98%) 48g
Honey 1000g
Propylene glycol 500g
Distilled water adds to 10kg
Preparation method: get recipe quantity medicine and auxiliary material,, can get 1000 oral liquid by common oral liquor preparation.
Claims (9)
1. the application of Quzhazhigan in preparation prevention and treatment ischemic cardio cerebrovascular diseases preparation, described Quzhazhigan is compound (E)-1-(3, the 5-dihydroxyphenyl)-2-(3-hydroxyl-4-O-β-D-Glucopyranose phenyl) ethene, chemical structure is:
The Quzhazhigan that uses is the Quzhazhigan extract.
2. the application of the described Quzhazhigan of claim 1 in preparation prevention and treatment ischemic cardio cerebrovascular diseases preparation is characterized in that described Quzhazhigan extract, by weight percentage, the effective ingredient that contains Quzhazhigan in the Quzhazhigan extract is 50%-100%.
3. a kind of preparation method of the described Quzhazhigan extract of claim 1 is characterized in that described preparation method comprises the following steps:
(1) pre-treatment: pre-treatment step comprises the collection of former roots of plants of Lhasa rhubarb or rhizome, system and/or drying, chopping or pulverizing only;
(2) extract: described extraction be adopt water, moisture or not water-containing organic solvent the described Lhasa rhubarb root of step (1) or rhizome extracted obtain the Lhasa rhubarb extracting solution as extracting solvent; Described organic solvent comprises alcohol, ketone, ether and their mixture that contains 1 ~ 4 carbon atom.
(3) aftertreatment: post-processing step comprises that the organic solvent in recovery or partially recycled step (2) the gained extracting solution obtains extracting concentrated solution, gained extracts concentrated solution can add or not add water, then extracting solution is staticly settled, solid-liquid separation is removed partial impurities; Liquid phase after the removal of impurity concentrates crystallization and acquires by weight percentage through further polymeric adsorbent chromatography purification, stepwise elution, eluate, and containing Quzhazhigan is the Quzhazhigan extract of 50%-100%.
4. according to a kind of preparation method of the described Quzhazhigan extract of claim 3, the described organic solvent of its step (2) is methyl alcohol, ethanol, acetone, propyl carbinol or ether; The described extraction solvent of its step (2) is 50 ~ 100% ethanol or methanol aqueous solution.
5. a kind of preparation method of the pure product of Quzhazhigan is characterized in that, described preparation method adopts Lhasa rhubarb as raw material, obtains the pure product of required Quzhazhigan by extracting purification step; The pure product purity of described Quzhazhigan is 90%-100% by weight percentage.
6. according to a kind of preparation method of the described Quzhazhigan extract of claim 3, it is characterized in that the Quzhazhigan extract is to extract from the root of the Lhasa rhubarb (Rheum lhasaense A.J.Li et P.K.Haiao) of wild or cultivation or rhizome.
7. the purposes of Quzhazhigan in preparation Quzhazhigan pure product.
8. the purposes of Quzhazhigan extract in preparation prevention or treatment cardiovascular and cerebrovascular diseases preparation.
9. the Quzhazhigan preparation can be used for making medicine or protective foods as various acceptable preparation or protective foods.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101163582A CN101787061B (en) | 2010-03-02 | 2010-03-02 | Application of Quzhazhigan in preparation of preparations for preventing and treating cardiac-cerebral ischemia diseases, and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101163582A CN101787061B (en) | 2010-03-02 | 2010-03-02 | Application of Quzhazhigan in preparation of preparations for preventing and treating cardiac-cerebral ischemia diseases, and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101787061A true CN101787061A (en) | 2010-07-28 |
| CN101787061B CN101787061B (en) | 2013-07-03 |
Family
ID=42530453
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101163582A Active CN101787061B (en) | 2010-03-02 | 2010-03-02 | Application of Quzhazhigan in preparation of preparations for preventing and treating cardiac-cerebral ischemia diseases, and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101787061B (en) |
Cited By (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102276666A (en) * | 2011-06-20 | 2011-12-14 | 昆明制药集团股份有限公司 | Quzhazhigan crystal and preparation method and application thereof |
| CN102507830A (en) * | 2011-11-21 | 2012-06-20 | 昆明制药集团股份有限公司 | High performance liquid chromatograph method for measuring content of Quzhazhigan in Rheum lhasaense |
| CN102659861A (en) * | 2012-04-18 | 2012-09-12 | 昆明制药集团股份有限公司 | Purification method of rhubarb stilbene glucoside |
| CN102847156A (en) * | 2012-10-11 | 2013-01-02 | 中国科学院西北高原生物研究所 | Fatty acid synthase inhibitor and application thereof |
| WO2013189285A1 (en) * | 2012-06-19 | 2013-12-27 | 昆明制药集团股份有限公司 | Application of piceatannol-3'-o-β-d-glucopyranoside in preparation of medicaments for improving microcirculation block |
| CN103505467A (en) * | 2012-06-15 | 2014-01-15 | 昆明制药集团股份有限公司 | Applications of stilbene compounds for preparing medicament for preventing and treating depression |
| CN104161748A (en) * | 2013-05-16 | 2014-11-26 | 昆明翔昊科技有限公司 | Application of rheum lhasaense extract A and B in preparation of biological preparation for reducing blood fat |
| CN104224813A (en) * | 2014-09-03 | 2014-12-24 | 昆明制药集团股份有限公司 | Pharmaceutical composition as well as preparation method and application thereof |
| CN104586865A (en) * | 2013-10-31 | 2015-05-06 | 昆明制药集团股份有限公司 | A pharmaceutical composition for treating cardiovascular diseases |
| CN104586864A (en) * | 2013-10-31 | 2015-05-06 | 昆明制药集团股份有限公司 | A use of a stilbene type compound in preparation of medicines treating cerebrovascular diseases |
| CN104586866A (en) * | 2013-10-31 | 2015-05-06 | 昆明制药集团股份有限公司 | A pharmaceutical composition for treating cerebrovascular diseases |
| CN104761594A (en) * | 2015-04-24 | 2015-07-08 | 昆药集团股份有限公司 | Amorphous-state 3,5,3',4'-trihydroxy-stilbene-3'-b-D-glucoside and preparation method thereof |
| CN106589007A (en) * | 2016-12-12 | 2017-04-26 | 昆药集团股份有限公司 | Preparation method and test method of CIS for Zhazhi glycoside |
| CN107602637A (en) * | 2017-09-27 | 2018-01-19 | 昆药集团股份有限公司 | Quzhazhigan anhydrous crystal forms and preparation method thereof |
| JP2018502063A (en) * | 2015-01-08 | 2018-01-25 | 昆薬集団股▲ふん▼有限公司Ｋｐｃ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ | Method for extracting stilbene compounds |
| CN107648243A (en) * | 2017-10-23 | 2018-02-02 | 昆药集团股份有限公司 | Purposes of the Quzhazhigan in the medicine of cerebral hemorrhage caused by rtPA thromboembolism treatments after preparing treatment cerebral hemorrhage or reduction cerebral ischemia |
| CN108096259A (en) * | 2018-01-12 | 2018-06-01 | 昆药集团股份有限公司 | A kind of Quzhazhigan solid lipid nano granule and preparation method thereof |
| CN108096190A (en) * | 2018-01-12 | 2018-06-01 | 昆药集团股份有限公司 | A kind of Quzhazhigan long circulating liposome and preparation method thereof |
| CN108191922A (en) * | 2018-01-09 | 2018-06-22 | 中国科学院新疆理化技术研究所 | Benzyl alcohol glycosides compound and preparation method thereof in coloquintida |
| CN112807319A (en) * | 2021-03-19 | 2021-05-18 | 大连医科大学 | Application of Quzhazhigan in treating ischemia-reperfusion injury of small intestine |
| CN114533786A (en) * | 2020-11-25 | 2022-05-27 | 昆药集团股份有限公司 | New application of Quzhazhigan |
| CN116082417A (en) * | 2021-11-05 | 2023-05-09 | 昆药集团股份有限公司 | Extraction and purification method of high-purity Quzhazhigan |
| US11744841B2 (en) | 2019-12-30 | 2023-09-05 | Kpc Pharmaceuticals, Inc. | Use of trezastilbenoside in manufacture of product for treating and/or preventing disease of respiratory system |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1611224A (en) * | 2003-11-01 | 2005-05-04 | 胡群 | Compound preparation of notoginseng total saponin and quzaqi aglycone and its preparing method |
| CN1686148A (en) * | 2005-04-20 | 2005-10-26 | 昆明翔昊科技有限公司 | Use of liposoluble stilbene glycoside kind compound in treating ischemic heart brain blood vessel disease and its preparation |
| CN101244129A (en) * | 2007-12-14 | 2008-08-20 | 昆明翔昊科技有限公司 | Lhasa rhubarb extract, preparation method, and application in preparing preparation for treating cardiovascular and cerebrovascular diseases |
-
2010
- 2010-03-02 CN CN2010101163582A patent/CN101787061B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1611224A (en) * | 2003-11-01 | 2005-05-04 | 胡群 | Compound preparation of notoginseng total saponin and quzaqi aglycone and its preparing method |
| CN1686148A (en) * | 2005-04-20 | 2005-10-26 | 昆明翔昊科技有限公司 | Use of liposoluble stilbene glycoside kind compound in treating ischemic heart brain blood vessel disease and its preparation |
| CN101244129A (en) * | 2007-12-14 | 2008-08-20 | 昆明翔昊科技有限公司 | Lhasa rhubarb extract, preparation method, and application in preparing preparation for treating cardiovascular and cerebrovascular diseases |
Cited By (38)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102276666B (en) * | 2011-06-20 | 2013-12-11 | 昆明制药集团股份有限公司 | Quzhazhigan crystal and preparation method and application thereof |
| CN102276666A (en) * | 2011-06-20 | 2011-12-14 | 昆明制药集团股份有限公司 | Quzhazhigan crystal and preparation method and application thereof |
| CN102507830B (en) * | 2011-11-21 | 2014-05-28 | 昆明制药集团股份有限公司 | High performance liquid chromatograph method for measuring content of Quzhazhigan in Rheum lhasaense |
| CN102507830A (en) * | 2011-11-21 | 2012-06-20 | 昆明制药集团股份有限公司 | High performance liquid chromatograph method for measuring content of Quzhazhigan in Rheum lhasaense |
| CN102659861B (en) * | 2012-04-18 | 2015-06-17 | 昆药集团股份有限公司 | Purification method of rhubarb stilbene glucoside |
| CN102659861A (en) * | 2012-04-18 | 2012-09-12 | 昆明制药集团股份有限公司 | Purification method of rhubarb stilbene glucoside |
| CN103505467A (en) * | 2012-06-15 | 2014-01-15 | 昆明制药集团股份有限公司 | Applications of stilbene compounds for preparing medicament for preventing and treating depression |
| CN103505467B (en) * | 2012-06-15 | 2016-02-24 | 昆明制药集团股份有限公司 | A kind of stilbenes compound is preparing the purposes in prevention and therapy antidepressant agents |
| WO2013189285A1 (en) * | 2012-06-19 | 2013-12-27 | 昆明制药集团股份有限公司 | Application of piceatannol-3'-o-β-d-glucopyranoside in preparation of medicaments for improving microcirculation block |
| CN103505468A (en) * | 2012-06-19 | 2014-01-15 | 昆明制药集团股份有限公司 | Application of 3,5,4'-trihydroxy-stilbyl-3'-O-glucoside in preparation of microcirculatory disturbance improvement medicines |
| CN103505468B (en) * | 2012-06-19 | 2016-08-31 | 昆药集团股份有限公司 | Bent letter glycosides improves the application in microcirculation disturbance medicine in preparation |
| CN102847156A (en) * | 2012-10-11 | 2013-01-02 | 中国科学院西北高原生物研究所 | Fatty acid synthase inhibitor and application thereof |
| CN104161748A (en) * | 2013-05-16 | 2014-11-26 | 昆明翔昊科技有限公司 | Application of rheum lhasaense extract A and B in preparation of biological preparation for reducing blood fat |
| CN104586866A (en) * | 2013-10-31 | 2015-05-06 | 昆明制药集团股份有限公司 | A pharmaceutical composition for treating cerebrovascular diseases |
| CN104586864A (en) * | 2013-10-31 | 2015-05-06 | 昆明制药集团股份有限公司 | A use of a stilbene type compound in preparation of medicines treating cerebrovascular diseases |
| CN104586866B (en) * | 2013-10-31 | 2017-04-26 | 昆药集团股份有限公司 | A pharmaceutical composition for treating cerebrovascular diseases |
| CN104586865A (en) * | 2013-10-31 | 2015-05-06 | 昆明制药集团股份有限公司 | A pharmaceutical composition for treating cardiovascular diseases |
| CN104586865B (en) * | 2013-10-31 | 2017-08-08 | 昆药集团股份有限公司 | A kind of pharmaceutical composition for treating angiocardiopathy |
| CN104224813A (en) * | 2014-09-03 | 2014-12-24 | 昆明制药集团股份有限公司 | Pharmaceutical composition as well as preparation method and application thereof |
| US10307453B2 (en) | 2015-01-08 | 2019-06-04 | Kpc Pharmaceuticals, Inc | Method for extracting stilbene compounds |
| JP2018502063A (en) * | 2015-01-08 | 2018-01-25 | 昆薬集団股▲ふん▼有限公司Ｋｐｃ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ | Method for extracting stilbene compounds |
| WO2016169288A1 (en) * | 2015-04-24 | 2016-10-27 | 昆药集团股份有限公司 | Amorphous piceatannol 3'-o-glucoside and preparation method thereof |
| CN104761594A (en) * | 2015-04-24 | 2015-07-08 | 昆药集团股份有限公司 | Amorphous-state 3,5,3',4'-trihydroxy-stilbene-3'-b-D-glucoside and preparation method thereof |
| CN104761594B (en) * | 2015-04-24 | 2018-01-02 | 昆药集团股份有限公司 | A kind of amorphous state Quzhazhigan and preparation method thereof |
| CN106589007A (en) * | 2016-12-12 | 2017-04-26 | 昆药集团股份有限公司 | Preparation method and test method of CIS for Zhazhi glycoside |
| CN106589007B (en) * | 2016-12-12 | 2019-04-02 | 昆药集团股份有限公司 | Cis- Quzhazhigan and preparation method thereof and detection method |
| CN107602637A (en) * | 2017-09-27 | 2018-01-19 | 昆药集团股份有限公司 | Quzhazhigan anhydrous crystal forms and preparation method thereof |
| CN107648243A (en) * | 2017-10-23 | 2018-02-02 | 昆药集团股份有限公司 | Purposes of the Quzhazhigan in the medicine of cerebral hemorrhage caused by rtPA thromboembolism treatments after preparing treatment cerebral hemorrhage or reduction cerebral ischemia |
| CN107648243B (en) * | 2017-10-23 | 2020-02-14 | 昆药集团股份有限公司 | Application of Quzhazhigan in preparation of medicine for treating cerebral hemorrhage or reducing cerebral hemorrhage caused by rtPA thrombolytic therapy after cerebral ischemia |
| CN108191922A (en) * | 2018-01-09 | 2018-06-22 | 中国科学院新疆理化技术研究所 | Benzyl alcohol glycosides compound and preparation method thereof in coloquintida |
| CN108096259A (en) * | 2018-01-12 | 2018-06-01 | 昆药集团股份有限公司 | A kind of Quzhazhigan solid lipid nano granule and preparation method thereof |
| CN108096190A (en) * | 2018-01-12 | 2018-06-01 | 昆药集团股份有限公司 | A kind of Quzhazhigan long circulating liposome and preparation method thereof |
| CN108096259B (en) * | 2018-01-12 | 2020-08-04 | 昆药集团股份有限公司 | Quzhazhigan solid lipid nanoparticle and preparation method thereof |
| CN108096190B (en) * | 2018-01-12 | 2020-08-14 | 昆药集团股份有限公司 | Quzhazhigan long-circulating liposome and preparation method thereof |
| US11744841B2 (en) | 2019-12-30 | 2023-09-05 | Kpc Pharmaceuticals, Inc. | Use of trezastilbenoside in manufacture of product for treating and/or preventing disease of respiratory system |
| CN114533786A (en) * | 2020-11-25 | 2022-05-27 | 昆药集团股份有限公司 | New application of Quzhazhigan |
| CN112807319A (en) * | 2021-03-19 | 2021-05-18 | 大连医科大学 | Application of Quzhazhigan in treating ischemia-reperfusion injury of small intestine |
| CN116082417A (en) * | 2021-11-05 | 2023-05-09 | 昆药集团股份有限公司 | Extraction and purification method of high-purity Quzhazhigan |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101787061B (en) | 2013-07-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101787061B (en) | Application of Quzhazhigan in preparation of preparations for preventing and treating cardiac-cerebral ischemia diseases, and preparation method thereof | |
| CN101244129B (en) | Lhasa rhubarb extract, preparation method, and application in preparing preparation for treating cardiovascular and cerebrovascular diseases | |
| Aissaoui et al. | Acute diuretic effect of continuous intravenous infusion of an aqueous extract of Coriandrum sativum L. in anesthetized rats | |
| KR102413705B1 (en) | Composition for improving liver function comprising Dendropanax morbifera extract | |
| EP1800685A1 (en) | Steroidal saponin pharmaceutical composition, its preparation method and use | |
| Nadumane et al. | Anticancer potential of banana flower extract: An in vitro study | |
| CN102210726A (en) | Method for combined extraction of mallow flavones and mallow saponins in seashore mallow and application | |
| WO2008121021A1 (en) | Method for extracting secoisolariciresinol and dihydroquercetin from wood | |
| CN102212093A (en) | Flavonoid glycoside compounds, method for preparing same and application | |
| KR100465113B1 (en) | Composition comprising an extract of Bambusoideae plant or tricin isolated therefrom | |
| CN1129572C (en) | Preparation and application of tanshinpolyphenolic salt | |
| JP2010528063A (en) | Method and use for obtaining an extract containing sequoyitol from a plant belonging to the genus Rhododendron, soybean, genus Ginkgo | |
| CN1943569B (en) | Chinese medicine active component composition and its preparing method and use | |
| CN104622938B (en) | Citrullus lanatus.convar megulaspemus (L.) skin extract and medical application and preparation method of composition | |
| KR101692032B1 (en) | Anti-cancer composition containing erythronium japonicum extract | |
| CN100475219C (en) | Use of liposoluble stilbene glycoside kind compound in treating ischemic heart brain blood vessel disease and preparation thereof | |
| CN107708717B (en) | Application of rhinacanthin quinone C as nerve cell apoptosis inhibitor | |
| Oudah et al. | Study the role of polyphenolic extract of CapparisSpinosa L. leaves as a hypoglycemic agent | |
| US10501472B2 (en) | Method to isolate inoscavin a from Fulviformes fastuosus and medicinal preparation thereof to treat rhabdomyosarcoma cancer conditions | |
| WO2012144711A2 (en) | Composition containing caryopteris incana extracts or a compound isolated therefrom for preventing and treating hepatotoxicity | |
| CN103110680A (en) | Preparation method of total phenolic acid of erigeron breviscapus | |
| KR100490799B1 (en) | Food comprising an extract of bambusoideae plant or tricin isolated therefrom | |
| CN103288889B (en) | Anthraquinone derivative and pharmaceutical composition thereof and its application in pharmacy | |
| CN102579527A (en) | Fructus choerospondiatis leaf total flavone and extracting method and application thereof | |
| DING | Studies on extraction and isolation of flavonoids from ginkgo leaves |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: KUNMING PHARMACEUTICAL INDUSTRY GROUP CORP., LTD. Free format text: FORMER OWNER: KUNMING XIANGHAO SCIENCE AND TECHNOLOGY CO., LTD. Effective date: 20110630 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 650104 NO. 2-1-402, DACHENGMINGYUAN, NO. 85, LINYU ROAD, KUNMING CITY, YUNNAN PROVINCE TO: 650106 NO. 166, KEYI ROAD, HIGH-TECH. DEVELOPMENT ZONE, KUNMING CITY, YUNNAN PROVINCE |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20110630 Address after: 650106 No. 166, medical Road, hi tech Development Zone, Yunnan, Kunming Applicant after: Kunming Pharmaceutical Industry Group Corp., Ltd. Address before: 650104 Yunnan Province, Kunming City Road No. 85 big famous Lin Yu Garden 2-1-402 Applicant before: Kunming Xianghao Science and Technology Co., Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee |
Owner name: KPC CO., LTD. Free format text: FORMER NAME: KUNMING PHARMACEUTICAL INDUSTRY GROUP CORP., LTD. |
|
| CP03 | Change of name, title or address |
Address after: 650106 Kunming science and Technology Industrial Development Zone, Yunnan Province Road No. 166 Patentee after: Kun Yao Group Plc Address before: 650106 No. 166, medical Road, hi tech Development Zone, Yunnan, Kunming Patentee before: Kunming Pharmaceutical Industry Group Corp., Ltd. |