CN102302615A - Effective site group of daphne giraldii nitsche leaf, preparation method, medicinal composition and application thereof - Google Patents
Effective site group of daphne giraldii nitsche leaf, preparation method, medicinal composition and application thereof Download PDFInfo
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Abstract
The invention relates to an effective site group of daphne giraldii nitsche leaf, a preparation method thereof, a medicinal composition containing the effective site group of the daphne giraldii nitsche leaf and application of the effective site group of the daphne giraldii nitsche leaf to the preparation of medicaments for treating diseases such as traumatic injury, arthritis and the like, and belongs to the technical field of pharmaceutics of traditional Chinese medicine. The effective site group of the daphne giraldii nitsche leaf is high in active ingredients and obvious in medicinal effect, medicinal sites do not damage vegetations and environment, the extraction process is easy and convenient, and the operability is high.
Description
Technical field
The present invention relates to the pharmacy of Chinese materia medica technical field, be specifically related to Daphne giraldii Nitsche leaf effective-part crowd, its preparation method, contain this Daphne giraldii Nitsche leaf effective-part crowd's pharmaceutical composition and the purposes in its medicine of treating diseases such as traumatic injury, arthritis as preparation.
Background technology
Daphne giraldii Nitsche is for having another name called Daphne giraldii Nitsche, teacher of the walking fiber crops, for thymelaeceae Daphne Giraldii Nitsche (Daphnegiraldii Nitsche), the sweet winter daphne in Shan (having another name called the Tang Gute winter daphne) (D.tanguticaMaxim), the root bark or the peel of stem of retuseleaf daphne bark (D.retusaHemsl).Acrid in the mouth, hardship, warm in nature, mild toxicity has the effect of expelling wind and removing dampness, pain relieving dissipating blood stasis, cures mainly diseases such as rheumatic arthralgia, numb limbs and tense tendons, headache, stomachache, traumatic injury.The proverb that " beat to such an extent that completely climb, be unable to do without Daphne giraldii Nitsche " arranged among the people has explained that also its analgesic activity is rooted in the hearts of the people.
All the time, explain that generally its medicinal part is root bark and peel of stem in the documents such as all kinds of pharmacy books, but be destructive to the recovery process of root bark and peel of stem that serious to local vegetation deterioration, various places have been taked the measure that restriction is excavated at present, herb resource reduces.And founder's Folium Cannabis, people think that always drug effect is undesirable, are not utilized as yet at present, its recovery process is vegetation destruction and environment not, not only meets the requirement of sustainable development, and highly beneficial for preserving the ecological environment.
At present, be that the prescribed preparation of raw material has Giraldi daphne tablet, Daphne giraldii Nitsche cream, Daphne giraldii Nitsche injection liquid, Daphne Giraldii Nitsche injection etc. with the Daphne giraldii Nitsche, the crude drug demand is bigger, can't satisfy preparation and clinical demand.
The application for a patent for invention of relevant Daphne giraldii Nitsche has (1) Daphne giraldii Nitsche leaf extract and medicinal usage (201010574800.6) thereof; This invention relates to the method for making of Daphne giraldii Nitsche leaf extract and the alcohol reflux that drug main is wanted 60-90% thereof; Macroporous resin is refining; Promptly get, this extract is used to prepare arthritic medicine.(2) a kind of active component crowd of Daphne giraldii Nitsche and its production and application (200810243322.3), this invention relates to Daphne giraldii Nitsche resisting rheumatoid disease arthritis active component crowd's preparation, its active component is total coumarins and total saponins; Process according to a certain weight ratio, its method for making is for adopting alcohol extraction, more respectively with two kinds of organic solvent extractions, recovery, concentratedly obtain two kinds of extracts; A kind of is the Diterpenes toxic component; Discard, another kind of extract is the Coumarins composition, with the medicinal liquid after the extraction of coumarin composition; The reuse macroporous resin treatment gets the total saponins composition, and total coumarins constituents and total saponins constituents are mixed according to a certain weight ratio.(3) active component of a kind of Daphne giraldii Nitsche and preparation method thereof and application (200510013864.8); This invention relates to active component of Daphne giraldii Nitsche and preparation method thereof and application; Adopt water or moisture rudimentary mellow extraction, resin purification obtains the active component of Daphne giraldii Nitsche; This medicine is made up of the Daphnetin in the handsome fiber crops of ancestral, daphnoretin, daphneticin etc., and the content of total coumarins is between 50～90%.Resulting Daphne giraldii Nitsche active component can be used as anti-inflammatory and antalgic and uses, and can be made into oral agents, injection, external preparation or the like.
Comprehensive research result shows: about the research contents of Daphne giraldii Nitsche (peel of stem and root bark) more, main pharmacodynamics definite ingredients (coumarin and saponin), preparation technology and pharmacodynamic study are more deep; And the research of founder's Folium Cannabis is almost blank; Its effective substance research is weak, not clear and definite its active component and content thereof, and pharmacodynamic experiment research is also considerably less; But the medicinal part of general Chinese medicine is different, and its chemical constituent kind, content have very big-difference.
Summary of the invention
The object of the present invention is to provide a kind of Daphne giraldii Nitsche leaf effective-part crowd, its main component is total coumarins and total flavones.
Another object of the present invention is to provide a kind of Daphne giraldii Nitsche leaf effective-part crowd's method for preparing.
Another object of the present invention is to provide a kind of pharmaceutical composition that contains Daphne giraldii Nitsche leaf effective-part crowd.
Another object of the present invention is to provide the purposes of Daphne giraldii Nitsche leaf effective-part crowd in the medicine of diseases such as preparation treatment traumatic injury, arthritis.
Particularly, the present invention provides:
(1) a kind of Daphne giraldii Nitsche leaf effective-part crowd is characterized in that containing total coumarins and total flavones, and wherein total coumarins content is 10～90%, and general flavone content is 5～60%.
(2) a kind of preparation above-mentioned (1) said Daphne giraldii Nitsche leaf effective-part crowd's method is: founder's Folium Cannabis was pulverized No. 4 sieves (65 order), with 3-15 organic solvent extraction doubly, extracting solution recovery organic solvent, add aqueous dispersion after; Centrifugal or filter, macroporous adsorbent resin on the supernatant with washing, is removed water-solubility impurity earlier; It is closely colourless to be washed till effluent, and reuse 30-90% ethanol elution is after eluent reclaims ethanol; Concentrate, drying, pulverizing promptly gets.
(3) prepare Daphne giraldii Nitsche leaf effective-part crowd's method according to above-mentioned (2), after wherein eluent reclaimed ethanol, thin up was used ethyl acetate extraction again, and the extract decompression and solvent recovery concentrates, drying, and pulverizing promptly gets.
(4) prepare Daphne giraldii Nitsche leaf effective-part crowd's method according to above-mentioned (2), after wherein eluent reclaims ethanol, can go up polyamide (60～90 order) chromatographic column again; Carry out gradient elution with Different concentrations of alcohol; Collect the 60-90% ethanol elution, the eluent decompression recycling ethanol concentrates; Drying, pulverizing promptly gets.
(5) prepare Daphne giraldii Nitsche leaf effective-part crowd's method according to above-mentioned (2), wherein said organic solvent is ethanol and/or ethyl acetate.
(6) prepare Daphne giraldii Nitsche leaf effective-part crowd's method according to above-mentioned (2), wherein said to add aqueous dispersion be that extracting solution is diluted to crude drug content is 0.1-0.8g/ml; Be preferably 0.2-0.4g/ml.
(7) above-mentioned (2) prepare Daphne giraldii Nitsche leaf effective-part crowd's method, and wherein said macroporous adsorbent resin adopts LS-300 and/or LS-303.
The inventor finds through a large amount of experimental studies; The macroporous adsorbent resin of different model is very big to Daphne giraldii Nitsche leaf effective-part crowd content influence; The inventor adopts LS-300, LS-303, LS-202, four kinds of model macroporous adsorbent resins of D101 that it has been carried out detailed research respectively, and the result sees table 1:
Four kinds of model macroporous adsorbent resins of table 1 static adsorption amount and parsing amount comparative test result
Above result of study is visible, and two kinds of models of LS-300 and LS-303 are higher to the absorption amount of total flavones and total coumarins, and desorption efficiency reaches more than 85%, so confirm to select for use the resin of these two kinds of models.
(8) above-mentioned (2) prepare Daphne giraldii Nitsche leaf effective-part crowd's method, and the concentration of alcohol when wherein using ethanol elution is 70%.
(9) prepare Daphne giraldii Nitsche leaf effective-part crowd's method according to above-mentioned (2), wherein said recovery and concentrated the finger, carry out under normal pressure or decompression.
(10) prepare Daphne giraldii Nitsche leaf effective-part crowd's method according to above-mentioned (2), wherein said drying refers to constant pressure and dry, vacuum drying etc.
(11) purposes of a kind of Daphne giraldii Nitsche leaf effective-part crowd in the medicine of diseases such as preparation treatment traumatic injury, arthritis.
(12) a kind of pharmaceutical composition of treating diseases such as traumatic injury, arthritis, it comprises Daphne giraldii Nitsche leaf effective-part crowd and drug excipient.
(13) pharmaceutical composition according to diseases such as (12) said treatment traumatic injury, arthritis is an oral solid formulation; Wherein total coumarins and total flavones mix with the conventional medicine solid excipient with 1: 0.20～15.0 weight ratios, process oral solid formulation with conventional formulation technology.Wherein additive of tablet mainly comprises starch, dextrin, cane sugar powder, magnesium stearate, Pulvis Talci etc.; The hard capsule adjuvant mainly comprises starch, dextrin, cellulose derivative etc.; The soft capsule adjuvant mainly comprises Semen Maydis oil, Cera Flava, gelatin, water, glycerol, ethyl hydroxybenzoate, Cacao pigment etc.; The granule adjuvant mainly is selected from starch, dextrin, cane sugar powder or stevioside or cyclamate or protein sugar etc.
(14) pharmaceutical composition according to diseases such as (12) said treatment traumatic injury, arthritis is an oral liquid; Wherein total coumarins and total flavones are processed oral liquid with 1: 10～50 weight ratios and conventional liq preparation medicine mixed with excipients with conventional formulation technology.Wherein the oral liquid adjuvant mainly is selected from sucrose, stevioside or cyclamate or protein sugar, tween 80, ethyl hydroxybenzoate, benzoic acid and sodium benzoate etc.;
(15) pharmaceutical composition according to diseases such as (12) said treatment traumatic injury, arthritis is the solid external application preparation; Wherein total coumarins and total flavones mix with conventional solid external application drug excipient with 1: 10～50 weight ratios, process the solid external application preparation with conventional formulation technology.Wherein external cataplasma adjuvant mainly comprises polyacrylic acid, polyvinyl alcohol, sodium carboxymethyl cellulose, carbomer, Kaolin, propylene glycol, gelatin, tartaric acid, dihydroxyaluminum aminoacetate, polyvidone, ethylenediaminetetraacetic acid (EDTA), azone, Borneolum Syntheticum etc.The externally used paste adjuvant mainly comprises gelatin, glycerol, azone, ethyl hydroxybenzoate, benzoic acid etc.; External paster adjuvant mainly comprises ethylene-vinyl acetate copolymer, silicone rubber, Polyethylene Glycol etc.
Beneficial effect of the present invention is:
(1) its analgesia of Daphne giraldii Nitsche leaf effective-part crowd provided by the invention, antiinflammatory action obviously are superior to the Daphne giraldii Nitsche extract.
(2) the present invention prepares Daphne giraldii Nitsche leaf effective-part crowd's method, and active constituent content is high, and method is easy, is easy to industrialization production.
(3) raw material used in the present invention is the leaf of Daphne giraldii Nitsche plant, and recovery process its leaf of only gathering does not influence plant strain growth, and all can gather every year, and vegetation destruction and environment do not meet the requirement of sustainable development; The position that present Daphne giraldii Nitsche uses is root bark and peel of stem, and the medicinal herb grower goes out its Lian Gen order when gathering, and what take is destructive means; Place of production vegetation is done great damage, and its resource is deficient day by day, and causes soil erosion; Environmental destruction is serious, does not meet the requirement of the Scientific Outlook on Development.
(4) all the time, the Daphne giraldii Nitsche agents area is its rhizome, and people think always that also the drug effect of leaf is undesirable, are not utilized as yet at present.The inventor finds through a large amount of experimental studies are unexpected; Active constituent content is than Daphne giraldii Nitsche medical material (root bark and peel of stem) height in founder's Folium Cannabis; Wherein: the Daphnetin of one of index components, content can reach 1.602% in the leaf, and root bark and peel of stem major part are between 0.2-1.0%; Adopt founder's Folium Cannabis as extracting raw material, it is easier to extract purification, and yield is higher.Its recovery process is vegetation destruction and environment not, not only meets the requirement of sustainable development, and highly beneficial for preserving the ecological environment.
(5) gather processing and the leaching process of founder's Folium Cannabis more easy than the Daphne giraldii Nitsche medical material, do not need special labor protection.Founder's Folium Cannabis recovery process can free-hand harvesting method carry out, and medical material is gathered and need be stripped its root bark or peel of stem, after all the company's of gathering root order plays or use the chopper felling; Strip its skin zone again; Because Daphne giraldii Nitsche skin zone contains the compositions strong to respiratory tract property such as Diterpenes, thus need certain safeguard procedures when stripping its skin, and leaf is extremely low owing to the Diterpenes component content; There is not penetrating odor, so need not take safeguard procedures; In like manner, when pulverizing, Daphne giraldii Nitsche root bark and peel of stem zest are very strong, especially boil on the nape opposite the mouth, eye, nose, thus must take the safeguard procedures of strictness, and the leaf zest is very little, does not need special protection.
The specific embodiment
Below description through the specific embodiment the present invention is described further; But this is not to be limitation of the present invention; Those skilled in the art are according to basic thought of the present invention; Can make various modifications or improvement, but only otherwise break away from basic thought of the present invention, all within scope of the present invention.
Embodiment 1: Daphne giraldii Nitsche leaf effective-part crowd's method for preparing
5 kilograms of founder's Folium Cannabis were pulverized No. 4 sieves (65 order), with 12 times 60% alcohol reflux three times, each 1 hour, merge extractive liquid; Filter, filtrate decompression (0.084Mpa) reclaims ethanol, adds aqueous dispersion to containing crude drug 0.3g/ml, centrifugal (4000 rev/mins); LS-300 type macroporous adsorptive resins on the supernatant with washing, is removed water-solubility impurity earlier; It is closely colourless to be washed till effluent, reuse 2.5BV70% ethanol elution, and the eluent decompression is (0.084Mpa) behind the recovery ethanol; Thin up is to containing crude drug 1.5g/ml, continues with equivalent ethyl acetate extraction 4 times combining extraction liquid; Reduce pressure (0.084Mpa) reclaim solvent and be concentrated into relative density 1.20 (80 ℃), vacuum drying, pulverizing promptly gets.
Embodiment 2: Daphne giraldii Nitsche leaf effective-part crowd's method for preparing
5 kilograms of founder's Folium Cannabis were pulverized No. 4 sieves (65 order), added 5 times 60% soak with ethanol 36 hours, in the percolation bucket of packing into, with 3mlmin
-1Kg
-1Flow velocity, carry out percolation, 12 times of amounts of shared solvent are collected percolate; Decompression (0.084Mpa) is reclaimed solvent, is added aqueous dispersion to containing crude drug 0.3g/ml, centrifugal (4000 rev/mins), LS-303 type macroporous adsorptive resins on the supernatant; With washing, remove water-solubility impurity earlier, it is closely colourless to be washed till effluent, reuse 2.5BV70% ethanol elution; Eluent decompression (0.084Mpa) reclaim ethanol after, thin up is to containing crude drug 1.5g/ml, continues with equivalent ethyl acetate extraction 4 times combining extraction liquid; Reduce pressure (0.084Mpa) reclaim solvent and be concentrated into relative density 1.20 (80 ℃), vacuum drying, pulverizing promptly gets.
Embodiment 3: Daphne giraldii Nitsche leaf effective-part crowd's method for preparing
5 kilograms of founder's Folium Cannabis were pulverized No. 4 sieves (65 order), extracted three times with 8 times ethyl acetate backflow, each 2 hours, merge extractive liquid; Filter, filtrate decompression (0.084Mpa) reclaims solvent, adds aqueous dispersion to containing crude drug 0.3g/ml, centrifugal (4000 rev/mins); LS-300 type macroporous adsorptive resins on the supernatant with washing, is removed water-solubility impurity earlier; It is closely colourless to be washed till effluent, and reuse 2.5BV70% ethanol elution is collected eluent; (0.084Mpa) reclaim ethanol and be concentrated into relative density 1.20 (80 ℃), vacuum drying is pulverized and is promptly got for eluent decompression.
Embodiment 4: Daphne giraldii Nitsche leaf effective-part crowd's method for preparing
5 kilograms of founder's Folium Cannabis were pulverized No. 4 sieves (65 order), added 5 times 60% soak with ethanol 36 hours, in the percolation bucket of packing into, with 3mlmin
-1Kg
-1Flow velocity, carry out percolation, 12 times of amounts of shared solvent are collected percolate; Decompression (0.084Mpa) is reclaimed solvent, is added aqueous dispersion to containing crude drug 0.3g/ml, centrifugal (4000 rev/mins), LS-300 type macroporous adsorptive resins on the supernatant; With washing, remove water-solubility impurity earlier, it is closely colourless to be washed till effluent; Reuse 2.5BV70% ethanol elution, (0.084Mpa) behind the recovery ethanol, thin up is to containing crude drug 1.5g/ml in the eluent decompression; Last polyamide (60～90 order) chromatographic column carries out gradient elution with 0-90% ethanol, collects the 60-90% ethanol elution; (0.084Mpa) reclaim solvent and be concentrated into relative density 1.20 (80 ℃), vacuum drying is pulverized and is promptly got for eluent decompression.
Embodiment 5: the method for preparing of Daphne giraldii Nitsche leaf effective-part crowd tablet
Prescription:
Daphne giraldii Nitsche leaf effective-part crowd: 100g
Starch: 100g
Dextrin: 80g
Sucrose: 15g
Magnesium stearate: an amount of
Pulvis Talci: an amount of
Process 1000
Method for making: get Daphne giraldii Nitsche leaf effective-part crowd, starch, dextrin, the sucrose mixing of recipe quantity, process granule, dry below 60 ℃, add an amount of magnesium stearate, Pulvis Talci, mixing is pressed into 1000, and sugar coating or film-coat promptly get.
Embodiment 6: the method for preparing of Daphne giraldii Nitsche leaf effective-part crowd capsule
Prescription:
Daphne giraldii Nitsche leaf effective-part crowd: 100g
Starch: 100g
Dextrin: 80g
Process 1000
Method for making: get Daphne giraldii Nitsche leaf effective-part crowd, starch, dextrin, the sucrose mixing of recipe quantity, process granule, fill and process 1000, promptly get.
Embodiment 7: the method for preparing of Daphne giraldii Nitsche leaf effective-part crowd soft capsule
Prescription:
Daphne giraldii Nitsche leaf effective-part crowd: 50g
Semen Maydis oil: 200g
Cera Flava: 4.8g
Sucrose: 15g
Process 1000
Method for making: get the Daphne giraldii Nitsche leaf effective-part crowd of recipe quantity, pulverize, cross 120 mesh sieves, mix with Semen Maydis oil, Cera Flava liquid (the heating in-depth filters) again, grind well, medicine is suspended in the Semen Maydis oil with superfine greasy particle form uniformly, process capsulation solution through colloid mill; Other gets the constituent of capsule skins such as gelatin, glycerol and puts into Sandwich pot, and the vapor interlayer heating is dissolved it, becomes rubber cement; Capsulation solution, rubber cement are put into hopper respectively, be pressed into 1000 soft capsules altogether, drying promptly gets.
Embodiment 8: the method for preparing of Daphne giraldii Nitsche leaf effective-part crowd external cream
Prescription:
Daphne giraldii Nitsche leaf effective-part crowd: 3000g
Gelatin: 75g
Glycerol: 300g
Azone: 70g
Processing 1000 pastes
Method for making: get the Daphne giraldii Nitsche leaf effective-part crowd of recipe quantity, pulverize, cross 120 mesh sieves, subsequent use; Other gets gelatin 75g, add 5 times of abundant swellings of water gaging after, drop removes unnecessary water, the glycerol that adds 300g is heated to gelatin and dissolves fully; And be concentrated into 540g, add above-mentioned fine powder, and add the azone, 0.1% ethyl hydroxybenzoate of 70g, 0.4% benzoic acid; Stir, process 4500g altogether, the stand is applied on the non-woven fabrics; Add the loam cake lining, cut into 1000 and paste, promptly get.
Embodiment 9: the method for preparing of Daphne giraldii Nitsche leaf effective-part crowd cataplasma
Prescription:
Daphne giraldii Nitsche leaf effective-part crowd: 400g
Polyacrylic acid: 200g
Dihydroxyaluminum aminoacetate: 8g
Citric acid: 8g
Polyvinylpolypyrrolidone: 80g
Propylene glycol: 1200g
Azone: 80g
Diethylamine tetraacethyl: 200g
Processing 1000 pastes
Method for making: dihydroxyaluminum aminoacetate, diethylamine tetraacethyl are added in the glycerol, fully mix, add polyacrylic acid again, continue to mix.Other gets polyvinylpolypyrrolidone and citric acid is dissolved in the water; The above-mentioned solution of getting equal portions respectively adds successively, fully mixes, and mixture is applied on the non-woven fabrics, adds the loam cake lining, cuts into 1000 and pastes, and promptly gets.
Test Example 10: Daphne giraldii Nitsche leaf effective-part crowd assay
(1) instrument and reagent
The-2001 of instrument Hitachi type ultraviolet-visible spectrophotometer; High performance liquid chromatograph (system system of U.S. TSP company
); Join the P4000 pump; The UV1000 detector, SEPU3000 data workstation (Hangzhou Pu Hui).Electronic analytical balance (Beijing Sai Duolisi balance company limited, model: BS210S d=0.1mg).Enlightening horse C
18Post (250mm * 4.6mm, 5 μ m).
Reagent methanol is chromatographically pure reagent, and water is purified water, and other reagent is analytical pure.
(supply assay usefulness, 0080-9705) Daphnetin (daphnetin) (supplies assay usefulness, lot number: 0900-200001) all available from Nat'l Pharmaceutical & Biological Products Control Institute to the reference substance rutin.
Sample: according to the homemade sample of embodiment 1,2,3,4 method for preparinies.
(2) method and result
1. total coumarins assay
Daphnetin reference substance 5mg is got in the preparation of reference substance solution, and accurate the title decides, and puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and promptly gets (containing Daphnetin 0.1mg among every 1ml).
The preparation precision of standard curve is measured reference substance solution 0.1ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml and 1.0ml, puts respectively in the 5ml measuring bottle, respectively adds methanol to scale, shakes up.With the reagent corresponding is blank, according to ultraviolet visible spectrophotometry (an appendix V of Chinese Pharmacopoeia version in 2010 A), measures trap in the 327nm wavelength, is vertical coordinate with the trap, and concentration is abscissa, the drawing standard curve.
Algoscopy is got the about 0.1g of these article fine powder, and accurate the title decides, and puts in the 100ml measuring bottle, make dissolving in right amount with methanol, and standardize solution shakes up to scale.Accurate again the absorption in the above-mentioned solution 1ml dislocation 50ml measuring bottle adds methanol to scale, shakes up.The sighting target directrix curve prepares the method under the item, measures absorbance in accordance with the law, from the weight (μ g) that standard curve is read Daphnetin the need testing solution, calculates, and promptly gets.Each embodiment sample determination result sees table 2.
2. determination of total flavonoids
Control substance of Rutin 50mg is got in the preparation of reference substance solution, accurate claims surely, puts in the 25ml measuring bottle, and it is an amount of to add methanol, puts that slight fever makes dissolving in the water-bath, puts coldly, adds methanol to scale, shakes up.Precision is measured 10ml, puts in the 100ml measuring bottle, adds water to scale, shakes up, and promptly gets (containing rutin 0.2mg among every 1ml).
The preparation precision of standard curve is measured reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml and 6ml, puts respectively in the 25ml measuring bottle, respectively adds water to 6.0ml, adds 5% sodium nitrite solution 1ml; Mixing was placed 6 minutes, added 10% aluminum nitrate solution 1ml, shook up; Placed 6 minutes, hydro-oxidation sodium test solution 10ml adds water to scale again, shakes up; Placing 15 minutes, is blank with the reagent corresponding, according to ultraviolet visible spectrophotometry (appendix V A), measures trap in the 500nm wavelength; With the trap is vertical coordinate, and concentration is abscissa, the drawing standard curve.
Algoscopy is got the about 1g of these article fine powder, accurate claims surely, puts in the apparatus,Soxhlet's, and it is an amount of to add diethyl ether, and reflux is to extracting liquid colourless, puts coldly, discards ether solution.Add methanol 90ml again, reflux is to extracting liquid colourless, and in the dislocation 100ml measuring bottle, with a small amount of washing container of methanol, washing liquid is incorporated in the same measuring bottle, adds methanol to scale, shakes up.Precision is measured 10ml, puts in the 100ml measuring bottle, adds water to scale, shakes up.Precision is measured 3ml, puts in the 25ml measuring bottle, and the sighting target directrix curve prepares method down, from " adding water to 6.0ml ", measures absorbance in accordance with the law, reads the weight (μ g) of rutin the need testing solution from standard curve, and calculating promptly gets.Each embodiment sample determination result sees table 2.
3. the assay of Daphnetin
Measure according to HPLC (an appendix VI of Chinese Pharmacopoeia version in 2010 D).
Chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler; With methanol-0.5% phosphoric acid (20: 80) is mobile phase; The detection wavelength is 327nm.Number of theoretical plate calculates by the Daphnetin peak should be not less than 3000.
The preparation precision of reference substance solution takes by weighing the Daphnetin reference substance, adds methanol and processes the solution that every 1ml contains 0.1mg, promptly gets.
The about 0.1g of these article fine powder is got in the preparation of need testing solution, and accurate the title decides, and puts in the 100ml measuring bottle, adds 85% methanol and makes dissolving, and be settled to scale, shakes up, and filters, and promptly gets.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and promptly get.Each embodiment sample determination result sees table 2.
Table 2 Daphne giraldii Nitsche leaf effective-part crowd assay result
Test Example 11: the test of pesticide effectiveness
Test objective: gained Daphne giraldii Nitsche leaf effective-part crowd of the present invention and Daphne giraldii Nitsche medicinal substances extract carry out following pharmacodynamics test:
Material:, adopt with Daphne giraldii Nitsche A same procedure to prepare Daphne giraldii Nitsche medicinal substances extract (hereinafter to be referred as Daphne giraldii Nitsche B) according to the handsome Folium Cannabis effective part group (hereinafter to be referred as Daphne giraldii Nitsche A) of embodiment 2 methods self-control ancestral.
Statistical procedures: all datas are organized a Lev-ene ' s homogeneity test of variance earlier; Adopt the independent sample t method of inspection to carry out average significance statistics then, its result representes with
.
(1) influence of Daphne giraldii Nitsche A and Daphne giraldii Nitsche B xylol induced mice ear swelling
Get 108 of female mices, be divided into 9 groups at random, 12 every group.Be administered once every day, each every mice administration 0.4mL, and successive administration 7d irritates stomach respectively and gives following medicine.The blank group: tap water 0.4mL/ only; Positive controls: aspirin, 200mg/kg; Administration group: the heavy dose of 120mg/kg of Daphne giraldii Nitsche A; Dosage 60mg/kg among the Daphne giraldii Nitsche A; The low dose of 30mg/kg of Daphne giraldii Nitsche A; The heavy dose of 120mg/kg of Daphne giraldii Nitsche B; Dosage 60mg/kg among the Daphne giraldii Nitsche B; The low dose of 30mg/kg of Daphne giraldii Nitsche B.Only evenly smear xylene 30ul/ in the wide positive and negative two sides of auris dextra behind the last administration 30min, 120min post-tensioning neck is put to death, and cuts two ears along the auricle baseline, and 8mm diameter card punch is beaten auricle, weighs respectively.Difference with two auricle weight is the swelling degree, and obtains suppression ratio, and the result sees table 1.
Suppression ratio=[(matched group swelling degree meansigma methods-administration group swelling degree meansigma methods)/matched group swelling degree meansigma methods] * 100%.
The ear swelling influence
that table 1 Daphne giraldii Nitsche A, B cause mice xylene
Annotate: carry out the t check between group, with the matched group ratio
*P＜0.05,
*P＜0.01
The result shows: Daphne giraldii Nitsche A compares with the blank group with the big or middle dose groups of Daphne giraldii Nitsche B, positive control aspirin group; All can reduce ear swelling degree due to the xylene significantly; Compare swelling degree suppression ratio with matched group and compare, all have remarkable result, and A is superior to B.
(2) Daphne giraldii Nitsche A and Daphne giraldii Nitsche B are to the influence of rat carrageenan toes swelling
Get 80 of male rats, be divided into 8 groups at random, 10 every group.Be administered once every day, presses 1.0ml/100g for each every, and 7d irritates stomach respectively and gives following medicine continuously.Blank group: tap water; Positive controls: aspirin; Administration group: the heavy dose of 100mg/kg of Daphne giraldii Nitsche A; Dosage 50mg/kg among the Daphne giraldii Nitsche A; The low dose of 25mg/kg of Daphne giraldii Nitsche A; The heavy dose of 100mg/kg of Daphne giraldii Nitsche B; Dosage 50mg/kg among the Daphne giraldii Nitsche B; The low dose of 25mg/kg of Daphne giraldii Nitsche B.After the last administration, measure its right sufficient volume with YLS-7B toes volumetric measurement appearance earlier, as administration front volume V
0, immediately at right vola subcutaneous injection 0.1% carrageenin 0.1ml.After administration 1,2,3,4,5,6h, measure its right sufficient volume V respectively
s, and calculating the swelling rate, the result sees table 2.
Swelling rate=[(V
s-V
0)/V
0] * 100%.
Table 2 Daphne giraldii Nitsche A, B on Carrageenan cause the influence
of rat toes swelling
With the matched group ratio, Daphne giraldii Nitsche A, three dose groups of B are inflammation later stage (causing scorching back 3.0～6.0 hours), and effect is obvious, all have significant difference with the matched group ratio, and A is superior to B.
(3) mensuration of mice hot plate method threshold of pain time
(5s＜lick metapedes time＜30s, is divided into 9 groups by pain threshold, 12 every group at random by 108 to choose on (55 ± 0.5) ℃ hot plate the qualified female white mice of screening.Be administered once every day, successive administration 7d.That irritates stomach administered agents and medicine respectively respectively organizes the same 1.4.1 of dosage.Measure the time that mice licks metapedes (greater than 60s in 60s, and the every jumping of mice 4 times, the time cuts 1s) behind last administration 45min, 90min, the 120min respectively.
The mensuration result of mice hot plate method threshold of pain time is as shown in table 3, the large, medium and small dose groups of Daphne giraldii Nitsche A, and the large, medium and small dose groups of Daphne giraldii Nitsche B, the threshold of pain time of positive control aspirin group is all long than the threshold of pain time of water matched group, and A is superior to B.
Table 3 Daphne giraldii Nitsche A, B are to the influence
of the mice hot plate method threshold of pain
Annotate: carry out the t check between group, and contrast the group ratio simultaneously
*P＜0.05,
*P＜0.01
(4) mensuration of acetic acid twisting method mouse writhing number of times
Choose 112 of female mices, be divided into 8 groups at random, 14 every group.Be administered once every day, successive administration 10d.That irritates stomach administered agents and medicine respectively respectively organizes the same 1.4.1 of dosage.45min lumbar injection 0.7% glacial acetic acid 0.1ml/10g after the last administration turns round the body number of times in the 20min of record injection back.The mensuration result of acetic acid twisting method mouse writhing number of times sees table 4, compares with the water matched group, and positive control morphine group has extremely strong analgesic activity.Daphne giraldii Nitsche A and Daphne giraldii Nitsche B group all can reduce turns round the body number of times, shows that Daphne giraldii Nitsche A and Daphne giraldii Nitsche B have stronger analgesic activity.
Table 4 Daphne giraldii Nitsche A, B are to the influence
of mouse writhing number of times
Annotate: carry out t check and matched group ratio between group,
*P＜0.01;
*P＜0.05
(5) the illumination tail-flick method is measured the mice TFL and is chosen 72 of male mices, is divided into 8 groups at random, 9 every group.Be administered once every day, successive administration 7d.That irritates stomach administered agents and medicine respectively respectively organizes the same 1.4.1 of dosage.Behind last administration 45min, measure the mice whipping time (2s＜whipping time＜16s is qualified data).Mensuration result to illumination tail-flick method mice TFL sees table 5, compares with matched group, and Daphne giraldii Nitsche A and Daphne giraldii Nitsche B group and positive control aspirin group can both prolong the latent time (P＜0.05) of illumination whipping.
Table 5 Daphne giraldii Nitsche A, B are to the influence
of mice TFL
Annotate: carry out t check and matched group ratio between group,
*P＜0.01;
*P＜0.05
This experimental result show Daphne giraldii Nitsche A and Daphne giraldii Nitsche B group can significance ground minimizing xylene induced mice ear swelling degree, the foot swelling rate of rat due to the carrageenin, and A is superior to B, proves that they have good anti-acute inflammation effect.In analgesic experiment, Daphne giraldii Nitsche A and Daphne giraldii Nitsche B group can increase the hot plate threshold of pain time, reduce the latent time of turning round body number of times and prolongation photoelectricity whipping that acetic acid causes by significance, and this shows that Daphne giraldii Nitsche A and Daphne giraldii Nitsche B group have the analgesic effect.Daphne giraldii Nitsche A and Daphne giraldii Nitsche B group have significant antiinflammatory and analgesic activity, and A is superior to B or equivalent with B, through this experiment, can produce evidence for enlarging the Daphne giraldii Nitsche medicine resource.
Claims (10)
1. a Daphne giraldii Nitsche leaf effective-part crowd is characterized in that containing total coumarins and total flavones, and wherein total coumarins content is 10～90%, and general flavone content is 5～60%.
2. method for preparing the said Daphne giraldii Nitsche leaf effective-part of claim 1 crowd is characterized in that preparing as follows:
Founder's Folium Cannabis was pulverized No. 4 sieves (65 order), and with 3-15 organic solvent extraction doubly, extracting solution reclaims organic solvent, add aqueous dispersion after; Centrifugal or filter, macroporous adsorbent resin on the supernatant with washing, is removed water-solubility impurity earlier; It is closely colourless to be washed till effluent, and reuse 30-90% ethanol elution is after eluent reclaims ethanol; Concentrate, drying, pulverizing promptly gets.
3. like the said method for preparing Daphne giraldii Nitsche leaf effective-part crowd of claim 2, it is characterized in that: after eluent reclaimed ethanol, thin up was used ethyl acetate extraction again, and the extract decompression and solvent recovery concentrates, drying, and pulverizing promptly gets.
4. like the said method for preparing Daphne giraldii Nitsche leaf effective-part crowd of claim 2, it is characterized in that: eluent is gone up polyamide (60～90 order) chromatographic column after reclaiming ethanol again; Carry out gradient elution with Different concentrations of alcohol; Collect the 60-90% ethanol elution, the eluent decompression recycling ethanol concentrates; Drying, pulverizing promptly gets.
5. like the said method for preparing Daphne giraldii Nitsche leaf effective-part crowd of claim 2, wherein said organic solvent is ethanol and/or ethyl acetate.
6. like the said method for preparing Daphne giraldii Nitsche leaf effective-part crowd of claim 2, wherein said to add aqueous dispersion be that extracting solution is diluted to crude drug content is 0.1-0.8g/ml, is preferably 0.2-0.4g/ml.
7. like the said method for preparing Daphne giraldii Nitsche leaf effective-part crowd of claim 2, wherein said macroporous adsorbent resin adopts LS-300 and/or LS-303.
8. like the purposes of right 1 described Daphne giraldii Nitsche leaf effective-part crowd in the medicine of diseases such as preparation treatment traumatic injury, arthritis.
9. pharmaceutical composition of treating diseases such as traumatic injury, arthritis, it comprises claim 1 said Daphne giraldii Nitsche leaf effective-part crowd and drug excipient.
10. pharmaceutical composition as claimed in claim 9, wherein said pharmaceutical composition are oral solid formulation, oral liquid, solid external application preparation, it is characterized in that:
Oral solid formulation is that total coumarins and total flavones mix with the conventional medicine solid excipient with 1: 0.20～15.0 weight ratios, processes oral solid formulation with conventional formulation technology.
Oral liquid be total coumarins and total flavones with 1: 10～50 weight ratios and conventional liq preparation medicine mixed with excipients, process oral liquid with conventional formulation technology.
The solid external application preparation is that total coumarins and total flavones mix with conventional solid external application drug excipient with 1: 10～50 weight ratios, processes the solid external application preparation with conventional formulation technology.
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| CN109045153A (en) * | 2018-11-02 | 2018-12-21 | 朗致集团万荣药业有限公司 | One plant medicinal material extracting solution and preparation method thereof |
| CN109620944A (en) * | 2019-01-25 | 2019-04-16 | 甘肃兰药药业有限公司 | It is a kind of with dispelling wind and eliminating dampness, the Chinese medicine composition of strengthening the muscles and bones and preparation method thereof |
| CN111514212A (en) * | 2020-06-03 | 2020-08-11 | 沈阳药科大学 | Preparation and application of daphne giraldii nitsche anti-liver cancer active total flavone |
| CN116650580A (en) * | 2023-06-20 | 2023-08-29 | 秦皇岛市山海关药业有限责任公司 | Daphne giraldii Nitsche product and preparation method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109045153A (en) * | 2018-11-02 | 2018-12-21 | 朗致集团万荣药业有限公司 | One plant medicinal material extracting solution and preparation method thereof |
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| CN109620944B (en) * | 2019-01-25 | 2021-07-27 | 甘肃兰药药业有限公司 | A Chinese medicinal composition with effects of dispelling pathogenic wind, removing dampness, strengthening tendons and bones, and its preparation method |
| CN111514212A (en) * | 2020-06-03 | 2020-08-11 | 沈阳药科大学 | Preparation and application of daphne giraldii nitsche anti-liver cancer active total flavone |
| CN116650580A (en) * | 2023-06-20 | 2023-08-29 | 秦皇岛市山海关药业有限责任公司 | Daphne giraldii Nitsche product and preparation method and application thereof |
| CN116650580B (en) * | 2023-06-20 | 2024-02-09 | 秦皇岛市山海关药业有限责任公司 | Daphne giraldii Nitsche product and preparation method and application thereof |
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