CN102302615B - 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 group, its preparation method, contain this Daphne giraldii Nitsche leaf effective-part group's pharmaceutical composition and the purposes in its medicine for the treatment of the 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), root bark or the peel of stem of retuseleaf daphne bark (D.retusaHemsl).Acrid in the mouth, hardship, warm in nature, mild toxicity, have the effect of expelling wind and removing dampness, pain relieving dissipating blood stasis, cures mainly the 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, also illustrated that its analgesic activity is rooted in the hearts of the people.
All the time, generally illustrate that 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, serious to local vegetation deterioration, various places have been taked the measure that restriction is excavated at present, and herb resource reduces.And founder's Folium Cannabis, people think that drug effect is undesirable always, not yet are utilized 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, the prescribed preparation that the Daphne giraldii Nitsche of take is raw material has Giraldi daphne tablet, Daphne giraldii Nitsche cream, Daphne giraldii Nitsche injection liquid, Daphne Giraldii Nitsche injection etc., and the crude drug demand is larger, can't meet 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, obtain, this extract is for the preparation of arthritic medicine.(2) active component group of a kind of Daphne giraldii Nitsche and its preparation method and application (200810243322.3), this invention relates to the preparation of Daphne giraldii Nitsche resisting rheumatoid disease arthritis active component group, its active component is total coumarins and total saponins, make according to a certain weight ratio, its method for making is for adopting alcohol extraction, use respectively again two kinds of organic solvent extractions, reclaim, concentrate and obtain two kinds of extracts, a kind of is the Diterpenes toxic component, discard, another kind of extract is the Coumarins composition, by the medicinal liquid after the coumarinoids extraction, obtain the total saponins composition by macroporous resin treatment again, total content of coumarin in Angelica 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, obtain the active component of Daphne giraldii Nitsche, the Daphnetin of this medicine in the handsome fiber crops of ancestral, daphnoretin, daphneticin etc. form, and the content of total coumarins is between 50～90%.Resulting Daphne giraldii Nitsche active component can be used as the anti-inflammatory and antalgic application, can be made into oral agents, injection, external preparation etc.
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, a little less than its effective substance Study of Thin, not clear and definite its active component and content thereof, pharmacodynamic experiment research is also considerably less, but the medicinal part difference of general Chinese medicine, its chemical composition 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 group, 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 group's preparation method.
Another object of the present invention is to provide a kind of pharmaceutical composition that contains Daphne giraldii Nitsche leaf effective-part group.
Another object of the present invention is to provide the purposes of Daphne giraldii Nitsche leaf effective-part group in the medicine of the diseases such as preparation treatment traumatic injury, arthritis.
Particularly, the invention provides:
(1) a kind of Daphne giraldii Nitsche leaf effective-part group, is characterized in that containing total coumarins and total flavones, and wherein total coumarin content is 10～90%, and general flavone content is 5～60%.
(2) a kind of method for preparing above-mentioned (1) described Daphne giraldii Nitsche leaf effective-part group is: founder's Folium Cannabis was pulverized to No. 4 sieves (65 order), and with the organic solvent extraction of 3-15 times, extracting solution reclaims organic solvent, after adding aqueous dispersion, centrifugal or filtration, macroporous adsorbent resin on supernatant, first wash with water, remove water-solubility impurity, be washed till effluent closely colourless, then use the 30-90% ethanol elution, after eluent reclaims ethanol, concentrated, dry, pulverize and get final product.
(3) prepare Daphne giraldii Nitsche leaf effective-part group's method according to above-mentioned (2), after wherein eluent reclaims ethanol, thin up, be extracted with ethyl acetate again, and the extract decompression and solvent recovery is concentrated, dry, pulverize and get final product.
(4) prepare Daphne giraldii Nitsche leaf effective-part group's method according to above-mentioned (2), after wherein eluent reclaims ethanol, can go up again polyamide (60～90 order) chromatographic column, ethanol with variable concentrations carries out gradient elution, collect the 60-90% ethanol elution, the eluent decompression recycling ethanol, concentrated, dry, pulverize and get final product.
(5) prepare Daphne giraldii Nitsche leaf effective-part group'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 group'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 group's method, and wherein said macroporous adsorbent resin adopts LS-300 and/or LS-303.
The inventor is through lot of experiments research discovery, the macroporous adsorbent resin of different model is very large to Daphne giraldii Nitsche leaf effective-part group content influence, the inventor adopts respectively LS-300, LS-303, LS-202, tetra-kinds of model macroporous adsorbent resins of D101 to be studied in detail it, the results are shown in Table 1:
Four kinds of model macroporous adsorbent resin static adsorbances of table 1 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 uptake of total flavones and total coumarins, and desorption efficiency reaches more than 85%, therefore determine the resin of selecting these two kinds of models.
(8) above-mentioned (2) prepare Daphne giraldii Nitsche leaf effective-part group's method, and the concentration of alcohol during wherein with ethanol elution is 70%.
(9) prepare Daphne giraldii Nitsche leaf effective-part group'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 group'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 group in the medicine of the diseases such as preparation treatment traumatic injury, arthritis.
(12) a kind of pharmaceutical composition for the treatment of the diseases such as traumatic injury, arthritis, it comprises Daphne giraldii Nitsche leaf effective-part group and drug excipient.
(13) according to the pharmaceutical composition of the diseases such as (12) described treatment traumatic injury, arthritis, be oral solid formulation, wherein total coumarins and total flavones mix with the conventional medicine solid excipient with 1: 0.20～15.0 weight ratios, with conventional formulation technique, make oral solid formulation.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) according to the pharmaceutical composition of the diseases such as (12) described treatment traumatic injury, arthritis, be oral liquid, wherein total coumarins and total flavones, with 1: 10～50 weight ratios and conventional liq preparation medicine mixed with excipients, are made oral liquid with conventional formulation technique.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) according to the pharmaceutical composition of the diseases such as (12) described treatment traumatic injury, arthritis, be 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, with conventional formulation technique, make the solid external application preparation.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 group provided by the invention, antiinflammatory action obviously are better than the Daphne giraldii Nitsche extract.
(2) the present invention prepares Daphne giraldii Nitsche leaf effective-part group's method, and active constituent content is high, and method is easy, is easy to industrialization production.
(3) leaf that raw material used in the present invention is the Daphne giraldii Nitsche plant, recovery process its leaf of only gathering, do not affect plant strain growth, often can gather every year, and vegetation destruction and environment, do not meet the requirement of sustainable development; The position that Daphne giraldii Nitsche is used at present is root bark and peel of stem, and while gathering, the medicinal herb grower goes out its Lian Gen order, and what take is destructive means, place of production vegetation is done great damage, and its resource is day by day deficient, 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 also think that the drug effect of leaf is undesirable always, not yet are utilized at present.The inventor studies unexpected the discovery by lot of experiments, in founder's Folium Cannabis, active constituent content is than Daphne giraldii Nitsche medical material (root bark and peel of stem) height, wherein: the Daphnetin of one of index components, in leaf, content can reach 1.602%, and root bark and peel of stem major part are between 0.2-1.0%; Adopt founder's Folium Cannabis as extracting raw material, extract purification easier, 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) founder's Folium Cannabis gather processing and leaching process 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 Gen of the Lian of gathering order plays or uses the chopper felling, strip again its skin zone, because the composition that Diterpenes etc. is strong to respiratory tract irritation is contained in Daphne giraldii Nitsche skin zone, thus need certain safeguard procedures while stripping its skin, and leaf is extremely low due to Diterpenoids from bulbus content, there is no penetrating odor, therefore do not need to 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 strict safeguard procedures, and the leaf zest is very little, does not need special protection.
The specific embodiment
Below the invention will be further described for the description by the specific embodiment, but this is not 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 the scope of the present invention.
Embodiment 1: Daphne giraldii Nitsche leaf effective-part group's preparation method
5 kilograms of founder's Folium Cannabis were pulverized to No. 4 sieves (65 order), with 60% alcohol reflux of 12 times three times, each 1 hour, merge extractive liquid, filter, filtrate decompression (0.084Mpa) reclaims ethanol, add aqueous dispersion to containing crude drug 0.3g/ml, centrifugal (4000 rev/mins), LS-300 type macroporous adsorptive resins on supernatant, first wash with water, remove water-solubility impurity, be washed till effluent closely colourless, use again the 2.5BV70% ethanol elution, after ethanol is reclaimed in eluent decompression (0.084Mpa), be diluted with water to containing crude drug 1.5g/ml, continue with equivalent ethyl acetate extraction 4 times, combining extraction liquid, decompression (0.084Mpa) is reclaimed solvent and is concentrated into relative density 1.20 (80 ℃), vacuum drying, pulverize and get final product.
Embodiment 2: Daphne giraldii Nitsche leaf effective-part group's preparation method
5 kilograms of founder's Folium Cannabis were pulverized to No. 4 sieves (65 order), added 60% soak with ethanol 36 hours of 5 times, in the percolation bucket of packing into, with 3mlmin
-1kg
-1flow velocity, carry out percolation, share 12 times of amounts of solvent, collect percolate, solvent is reclaimed in decompression (0.084Mpa), add aqueous dispersion to containing crude drug 0.3g/ml, centrifugal (4000 rev/mins), LS-303 type macroporous adsorptive resins on supernatant, first wash with water, remove water-solubility impurity, be washed till effluent closely colourless, use again the 2.5BV70% ethanol elution, after ethanol is reclaimed in eluent decompression (0.084Mpa), be diluted with water to containing crude drug 1.5g/ml, continue with equivalent ethyl acetate extraction 4 times, combining extraction liquid, decompression (0.084Mpa) is reclaimed solvent and is concentrated into relative density 1.20 (80 ℃), vacuum drying, pulverize and get final product.
Embodiment 3: Daphne giraldii Nitsche leaf effective-part group's preparation method
5 kilograms of founder's Folium Cannabis were pulverized to No. 4 sieves (65 order), by the ethyl acetate backflow of 8 times, extract three times, each 2 hours, merge extractive liquid, filter, filtrate decompression (0.084Mpa) reclaims solvent, add aqueous dispersion to containing crude drug 0.3g/ml, centrifugal (4000 rev/mins), LS-300 type macroporous adsorptive resins on supernatant, first wash with water, remove water-solubility impurity, be washed till effluent closely colourless, use again the 2.5BV70% ethanol elution, collect eluent, eluent decompression (0.084Mpa) is reclaimed ethanol and is concentrated into relative density 1.20 (80 ℃), vacuum drying, pulverize and get final product.
Embodiment 4: Daphne giraldii Nitsche leaf effective-part group's preparation method
5 kilograms of founder's Folium Cannabis were pulverized to No. 4 sieves (65 order), added 60% soak with ethanol 36 hours of 5 times, in the percolation bucket of packing into, with 3mlmin
-1kg
-1flow velocity, carry out percolation, share 12 times of amounts of solvent, collect percolate, solvent is reclaimed in decompression (0.084Mpa), add aqueous dispersion to containing crude drug 0.3g/ml, centrifugal (4000 rev/mins), LS-300 type macroporous adsorptive resins on supernatant, first wash with water, remove water-solubility impurity, be washed till effluent closely colourless, use again the 2.5BV70% ethanol elution, after ethanol is reclaimed in eluent decompression (0.084Mpa), be diluted with water to containing crude drug 1.5g/ml, upper polyamide (60～90 order) chromatographic column, carry out gradient elution with 0-90% ethanol, collect the 60-90% ethanol elution, eluent decompression (0.084Mpa) is reclaimed solvent and is concentrated into relative density 1.20 (80 ℃), vacuum drying, pulverize and get final product.
Embodiment 5: the preparation method of Daphne giraldii Nitsche leaf effective-part group tablet
Prescription:
Daphne giraldii Nitsche leaf effective-part group: 100g
Starch: 100g
Dextrin: 80g
Sucrose: 15g
Magnesium stearate: appropriate
Pulvis Talci: appropriate
Make 1000
Method for making: Daphne giraldii Nitsche leaf effective-part group, starch, dextrin, the sucrose of getting recipe quantity mix, and granulation, dry below 60 ℃, adds appropriate magnesium stearate, Pulvis Talci, mixes, and is pressed into 1000, and sugar coating or film-coat, obtain.
Embodiment 6: the preparation method of Daphne giraldii Nitsche leaf effective-part group capsule
Prescription:
Daphne giraldii Nitsche leaf effective-part group: 100g
Starch: 100g
Dextrin: 80g
Make 1000
Method for making: Daphne giraldii Nitsche leaf effective-part group, starch, dextrin, the sucrose of getting recipe quantity mix, and granulation is filled and made 1000, obtains.
Embodiment 7: the preparation method of Daphne giraldii Nitsche leaf effective-part group soft capsule
Prescription:
Daphne giraldii Nitsche leaf effective-part group: 50g
Semen Maydis oil: 200g
Cera Flava: 4.8g
Sucrose: 15g
Make 1000
Method for making: get the Daphne giraldii Nitsche leaf effective-part group of recipe quantity, pulverize, cross 120 mesh sieves, mix with Semen Maydis oil, Cera Flava liquid (the heating in-depth, filter) again, grind well through colloid mill, medicine is suspended in Semen Maydis oil uniformly with superfine greasy particle form, makes capsulation solution; Separately get the constituent of the capsule skins such as gelatin, glycerol and put into Sandwich pot, the vapor interlayer heating, dissolve it, becomes rubber cement; Capsulation solution, rubber cement are put into respectively to hopper, be pressed into altogether 1000 soft capsules, drying, obtain.
Embodiment 8: the preparation method of Daphne giraldii Nitsche leaf effective-part group external plaster
Prescription:
Daphne giraldii Nitsche leaf effective-part group: 3000g
Gelatin: 75g
Glycerol: 300g
Azone: 70g
Make 1000 subsides
Method for making: get the Daphne giraldii Nitsche leaf effective-part group of recipe quantity, pulverize, cross 120 mesh sieves, standby; Separately get gelatin 75g, after adding 5 times of abundant swellings of water gaging, drain unnecessary water, adding the glycerol of 300g to be heated to gelatin 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, make altogether 4500g, spread onto on non-woven fabrics, add the upper cover lining, cut into 1000 subsides, obtain.
Embodiment 9: the preparation method of Daphne giraldii Nitsche leaf effective-part group cataplasma
Prescription:
Daphne giraldii Nitsche leaf effective-part group: 400g
Polyacrylic acid: 200g
Dihydroxyaluminum aminoacetate: 8g
Citric acid: 8g
Polyvinylpolypyrrolidone: 80g
Propylene glycol: 1200g
Azone: 80g
Diethylamine tetraacethyl: 200g
Make 1000 subsides
Method for making: dihydroxyaluminum aminoacetate, diethylamine tetraacethyl are added in glycerol, fully mix, then add polyacrylic acid, continue to mix.Separately getting polyvinylpolypyrrolidone and citric acid is dissolved in water; The above-mentioned solution of getting respectively equal portions adds successively, fully mixes, and mixture is applied on non-woven fabrics, adds the upper cover lining, cuts into 1000 subsides, obtains.
Test example 10: Daphne giraldii Nitsche leaf effective-part group assay
(1) instrument and reagent
The-2001 of instrument Hitachi type ultraviolet-visible spectrophotometer; High performance liquid chromatograph (U.S. TSP company
The system system), join the P4000 pump, 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.
The reference substance rutin (for assay, 0080-9705) Daphnetin (daphnetin) (for assay, lot number: 0900-200001) all purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
Sample: according to the homemade sample of embodiment 1,2,3,4 preparation method.
(2) method and result
1. total coumarin content is measured
Daphnetin reference substance 5mg is got in the preparation of reference substance solution, accurately weighed, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and obtains (containing Daphnetin 0.1mg in every 1ml).
The preparation precision of standard curve measures 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.Take corresponding reagent as blank, according to ultraviolet visible spectrophotometry (appendix V A of Chinese Pharmacopoeia version in 2010), measure trap at 327nm wavelength place, take trap as vertical coordinate, concentration is abscissa, the drawing standard curve.
Algoscopy is got the about 0.1g of this product fine powder, accurately weighed, puts in the 100ml measuring bottle, with methanol, make in right amount to dissolve, and standardize solution shakes up to scale.The accurate absorption in above-mentioned solution 1ml dislocation 50ml measuring bottle, add methanol to scale again, shakes up.Method under sighting target directrix curve preparation is measured absorbance in accordance with the law, and the weight (μ g) of from standard curve, reading Daphnetin in need testing solution is calculated, and obtains.Each embodiment sample determination the results are shown in Table 2.
2. determination of total flavonoids
Control substance of Rutin 50mg is got in the preparation of reference substance solution, accurately weighed, puts in the 25ml measuring bottle, adds methanol appropriate, puts slight fever in water-bath and makes to dissolve, and lets cool, and adds methanol to scale, shakes up.Precision measures 10ml, puts in the 100ml measuring bottle, adds water to scale, shakes up, and obtains (containing rutin 0.2mg in every 1ml).
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml and 6ml, put respectively in the 25ml measuring bottle, respectively add water to 6.0ml, add 5% sodium nitrite solution 1ml, mix, place 6 minutes, add 10% aluminum nitrate solution 1ml, shake up, place 6 minutes, hydro-oxidation sodium test solution 10ml, add water to again scale, shake up, place 15 minutes, take corresponding reagent as blank, according to ultraviolet visible spectrophotometry (appendix V A), measure trap at 500nm wavelength place, take trap as vertical coordinate, concentration is abscissa, the drawing standard curve.
Algoscopy is got the about 1g of this product fine powder, accurately weighed, puts in apparatus,Soxhlet's, adds diethyl ether appropriate, and reflux, to extracting liquid colourless, lets cool, and discards ether solution.Add methanol 90ml, reflux is to extracting liquid colourless again, and in dislocation 100ml measuring bottle, with a small amount of washing container of methanol, washing liquid is incorporated in same measuring bottle, adds methanol to scale, shakes up.Precision measures 10ml, puts in the 100ml measuring bottle, adds water to scale, shakes up.Precision measures 3ml, puts in the 25ml measuring bottle, and the method under sighting target directrix curve preparation from " adding water to 6.0ml ", is measured absorbance in accordance with the law, from standard curve, reads the weight (μ g) of rutin in need testing solution, calculates, and obtains.Each embodiment sample determination the results are shown in Table 2.
3. the assay of Daphnetin
According to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2010), measure.
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler; Methanol-0.5% phosphoric acid (20: 80) of take is mobile phase; The detection wavelength is 327nm.Number of theoretical plate calculates and should be not less than 3000 by the Daphnetin peak.
The preparation precision of reference substance solution takes the Daphnetin reference substance, adds methanol and makes the solution of every 1ml containing 0.1mg, obtains.
The about 0.1g of this product fine powder is got in the preparation of need testing solution, accurately weighed, puts in the 100ml measuring bottle, adds 85% methanol and makes to dissolve, and be settled to scale, shakes up, and filters, and obtains.
Algoscopy is accurate reference substance solution and each 10 μ l of need testing solution of drawing respectively, and the injection liquid chromatography, measure, and obtains.Each embodiment sample determination the results are shown in Table 2.
Table 2 Daphne giraldii Nitsche leaf effective-part group assay result
Test example 11: the test of pesticide effectiveness
Test objective: gained Daphne giraldii Nitsche leaf effective-part group of the present invention and Daphne giraldii Nitsche medicinal substances extract carry out following pharmacodynamics test:
Material: according to the handsome Folium Cannabis effective part group (hereinafter to be referred as Daphne giraldii Nitsche A) of embodiment 2 method self-control ancestral, adopt with Daphne giraldii Nitsche A same procedure and prepare Daphne giraldii Nitsche medicinal substances extract (hereinafter to be referred as Daphne giraldii Nitsche B).
Statistical procedures: all datas first organized between Lev-ene ' s homogeneity test of variance, then adopt the independent sample t method of inspection to carry out average significance statistics, its result with
Mean.
(1) impact of Daphne giraldii Nitsche A and Daphne giraldii Nitsche B xylol induced mice ear swelling
Get 108 of female mices, be divided at random 9 groups, 12 every group.Be administered once every day, each every mice administration 0.4mL, and successive administration 7d, gavage gives following medicine respectively.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 in 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 in Daphne giraldii Nitsche B; The low dose of 30mg/kg of Daphne giraldii Nitsche B.Only evenly smear dimethylbenzene 30ul/ in the wide positive and negative two sides of auris dextra after last administration 30min, 120min post-tensioning neck is put to death, and along the auricle baseline, cuts two ears, and 8mm diameter card punch is beaten auricle, weighs respectively.The difference of two auricle weight of take is swelling, and obtains suppression ratio, the results are shown in Table 1.
Suppression ratio=[(matched group swelling meansigma methods-administration group swelling meansigma methods)/matched group swelling meansigma methods] * 100%.
The ear swelling impact that table 1 Daphne giraldii Nitsche A, B cause mice dimethylbenzene
Annotate: carry out the t check between group, with the matched group ratio
*P<0.05,
*P<0.01
Result shows: Daphne giraldii Nitsche A compares with the blank group with the big or middle dosage group of Daphne giraldii Nitsche B, positive control aspirin group, all can reduce significantly ear swelling degree due to dimethylbenzene, compare the swelling suppression ratio with matched group and compare, all there is remarkable result, and A is better than B.
(2) Daphne giraldii Nitsche A and the Daphne giraldii Nitsche B impact on the swelling of rat carrageenan toes
Get 80 of male rats, be divided at random 8 groups, 10 every group.Be administered once every day, presses 1.0ml/100g for each every, continuous 7d, and gavage gives following medicine respectively.Blank group: tap water; Positive controls: aspirin; Administration group: the heavy dose of 100mg/kg of Daphne giraldii Nitsche A; Dosage 50mg/kg in 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 in Daphne giraldii Nitsche B; The low dose of 25mg/kg of Daphne giraldii Nitsche B.After the last administration, first with YLS-7B toes capacity measurer, measure its right sufficient volume, 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 respectively its right sufficient volume V
s, and calculate swelling rate, the results are shown in Table 2.
Swelling rate=[(V
s-V
0)/V
0] * 100%.
Table 2 Daphne giraldii Nitsche A, B on Carrageenan cause the impact of rat toes swelling
With the matched group ratio, Daphne giraldii Nitsche A, tri-dosage groups of B are inflammation later stage (causing scorching latter 3.0～6.0 hours), and successful, all have significant difference with the matched group ratio, and A is better than B.
(3) mensuration of hot plate method in mice 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.The medicine that gavage gives respectively and medicine respectively organize the same 1.4.1 of dosage.Measure respectively the time that mice licks metapedes (be greater than 60s in 60s, and the every jumping of mice 4 times, the time cuts 1s) after last administration 45min, 90min, 120min.
The measurement result of hot plate method in mice threshold of pain time is as shown in table 3, the large, medium and small dosage group of Daphne giraldii Nitsche A, and the large, medium and small dosage group 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 better than B.
Table 3 Daphne giraldii Nitsche A, the B impact on the hot plate method in mice 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 at random 8 groups, 14 every group.Be administered once every day, successive administration 10d.The medicine that gavage gives respectively and medicine respectively organize the same 1.4.1 of dosage.45min lumbar injection 0.7% glacial acetic acid 0.1ml/10g after the last administration, the writhing number of times after the record injection in 20min.The measurement result of acetic acid twisting method mouse writhing number of times, in Table 4, is compared 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 the writhing number of times, show that Daphne giraldii Nitsche A and Daphne giraldii Nitsche B have stronger analgesic activity.
Table 4 Daphne giraldii Nitsche A, the B impact on the 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 at random 8 groups, 9 every group.Be administered once every day, successive administration 7d.The medicine that gavage gives respectively and medicine respectively organize the same 1.4.1 of dosage.Measure the mice whipping time (2s<whipping time<16s is qualified data) after last administration 45min.The measurement result of illumination tail-flick method mice TFL, in Table 5, is compared with matched group, the latent time (P<0.05) that Daphne giraldii Nitsche A and Daphne giraldii Nitsche B group and positive control aspirin group can the prolonging light whippings.
Table 5 Daphne giraldii Nitsche A, the B impact on the 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 dimethylbenzene induced mice ear swelling degree, the foot swelling rate of rat due to carrageenin, and A is better than B, proves that they have good anti-inflammation effect.In analgesic experiment, the writhing number of times that Daphne giraldii Nitsche A and Daphne giraldii Nitsche B group energy significance increase hot plate threshold of pain time, minimizing acetic acid cause and the latent time that extends the photoelectricity whipping, 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, A be better than B or with the B equivalence, by this experiment, can produce evidence for enlarging the Daphne giraldii Nitsche medicine resource.
Claims (8)
1. a Daphne giraldii Nitsche leaf effective-part group, contain total coumarins and total flavones, and wherein total coumarin content is 10～90%, and general flavone content is 5～60%, it is characterized in that described Daphne giraldii Nitsche leaf effective-part group is prepared from as follows:
1) founder's Folium Cannabis was pulverized No. 4 to sieve, by the ethanol of 3-15 times and/or ethyl acetate, extract, extracting solution reclaims ethanol and/or ethyl acetate, after adding aqueous dispersion, and centrifugal or filtration, macroporous adsorbent resin on supernatant, first wash with water, remove water-solubility impurity, be washed till effluent closely colourless, use the 30-90% ethanol elution, eluent reclaims ethanol again;
2), after eluent reclaims ethanol, thin up, be extracted with ethyl acetate, the extract decompression and solvent recovery is concentrated, dry, pulverize and get final product; Or
3), after eluent reclaims ethanol, upper polyamide chromatography post, carry out gradient elution with the ethanol of variable concentrations, collects the 60-90% ethanol elution, the eluent decompression recycling ethanol is concentrated, dry, pulverize and get final product.
2. prepare as claimed in claim 1 Daphne giraldii Nitsche leaf effective-part group, wherein said preparation process 3) in polyamide chromatography post be the 60-90 order.
3. prepare as claimed in claim 1 Daphne giraldii Nitsche leaf effective-part group, wherein said preparation process 1) to add aqueous dispersion be that extracting solution is diluted to crude drug content is 0.1-0.8g/ml.
4. prepare as claimed in claim 1 Daphne giraldii Nitsche leaf effective-part group, wherein said preparation process 1) to add aqueous dispersion be that extracting solution is diluted to crude drug content is 0.2-0.4g/ml.
5. prepare as claimed in claim 1 Daphne giraldii Nitsche leaf effective-part group, wherein said preparation process 1) in macroporous adsorbent resin adopt LS-300 and/or LS-303.
6. the purposes of Daphne giraldii Nitsche leaf effective-part group as claimed in claim 1 in the medicine of the diseases such as preparation treatment traumatic injury, arthritis.
7. a pharmaceutical composition for the treatment of the diseases such as traumatic injury, arthritis, it comprises claim 1 described Daphne giraldii Nitsche leaf effective-part group and drug excipient.
8. pharmaceutical composition as claimed in claim 7, wherein said pharmaceutical composition is oral solid formulation, oral liquid, solid external application preparation, it is characterized in that:
Oral solid formulation is that Daphne giraldii Nitsche leaf effective-part group mixes with the conventional medicine solid excipient, with conventional formulation technique, makes oral solid formulation.
Oral liquid is Daphne giraldii Nitsche leaf effective-part group and conventional liq preparation medicine mixed with excipients, with conventional formulation technique, makes oral liquid.
The solid external application preparation is that Daphne giraldii Nitsche leaf effective-part group mixes with conventional solid external application drug excipient, with conventional formulation technique, makes the solid external application preparation.
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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 |
| 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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| CN1094627A (en) * | 1993-05-03 | 1994-11-09 | 周惠仁 | Giraldi daphne tablet and preparation method thereof |
| CN1883618A (en) * | 2005-06-21 | 2006-12-27 | 天津药物研究院 | Effective parts of cirald daphne bark, preparation method and application thereof |
| CN101856435A (en) * | 2009-04-07 | 2010-10-13 | 天津药物研究院 | Preparation method and content measurement method of Daphne giraldii Nitsche total coumarin extract |
| CN102008599A (en) * | 2010-12-06 | 2011-04-13 | 吉林圣亚医药科技有限公司 | Girald daphne bark leaf extract and medicinal application thereof |
| CN102078443A (en) * | 2009-12-01 | 2011-06-01 | 天津药物研究院 | Medicine composition, application and preparation thereof |
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| CN1094627A (en) * | 1993-05-03 | 1994-11-09 | 周惠仁 | Giraldi daphne tablet and preparation method thereof |
| CN1883618A (en) * | 2005-06-21 | 2006-12-27 | 天津药物研究院 | Effective parts of cirald daphne bark, preparation method and application thereof |
| CN101856435A (en) * | 2009-04-07 | 2010-10-13 | 天津药物研究院 | Preparation method and content measurement method of Daphne giraldii Nitsche total coumarin extract |
| CN102078443A (en) * | 2009-12-01 | 2011-06-01 | 天津药物研究院 | Medicine composition, application and preparation thereof |
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