CN1305512C - Traditional Chinese medicine compound recipe for treating hyperplasia of mammary glands and preparation method thereof - Google Patents
Traditional Chinese medicine compound recipe for treating hyperplasia of mammary glands and preparation method thereof Download PDFInfo
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- CN1305512C CN1305512C CNB2004100374063A CN200410037406A CN1305512C CN 1305512 C CN1305512 C CN 1305512C CN B2004100374063 A CNB2004100374063 A CN B2004100374063A CN 200410037406 A CN200410037406 A CN 200410037406A CN 1305512 C CN1305512 C CN 1305512C
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Abstract
The present invention relates to a compound Chinese medicine preparation for treating the hyperplasia of mammary glands, a preparation method thereof and a quality control method thereof. The compound Chinese medicine preparation is mainly composed of green tangerine peel, bupleurum root, cowherb seed (parched) common prism tuber, zedoray rhizome, corydalis tuber (prepared by vinegar) cattail pollen, thunberg fritillary bulb, spica prunellae, dahurian angelica and borneol through alcohol extraction, concentration, adding auxiliary materials, heating to be dissolved and obtain extract, painting and stripping to obtain a pasting membrane. At the same time, the present invention also provides the quality control method of the compound Chinese medicine preparation through ingredient identification and content measurement. The present invention has the advantage of convenient use and has the functions of inhibiting hyperplasia, removing stasis, removing blood stasis, relieving pain, reducing the content of estradiol in blood serum, plasma viscosity and platelet aggregation. The present invention is an effective medicine for treating the hyperplasia of mammary glands.
Description
Technical field
The present invention relates to a kind of compound Chinese medicinal preparation and preparation method thereof, method of quality control, particularly relate to a kind of compound Chinese medicinal preparation for the treatment of cyclomastopathy and preparation method thereof, method of quality control.
Background technology
Cystic hyperplasia of breast is the chronic outgrowth general name of mammary gland master, a matter, claims mammary dysplasia again, belongs to motherland's medical science " nodules of the breast ", " nodule in the breast " category.Clinical symptoms is a principal character with lump in breast and pain, good sending out in 20～40 years old women, and male and child have the patient by chance.Primary disease sickness rate height occupy the first in mastopathy.The main method of modern medicine treatment primary disease is to implement excision for bigger lump, but postoperative has possibility of recurrence, and a lot of patients are for to the feared state of mind of operation and to the worry of postoperative recurrence, and often refusal is performed the operation.And, early stage at primary disease, when lump hour, also be unsuitable for operation.The at present employing of the method for Chinese medicine primary disease taken orally more, be extensive use of the decoction except that principle clinically according to determination of treatment based on pathogenesis obtained through differentiation of symptoms and signs, Chinese patent medicine such as RUKUAIXIAO PIAN, BUPIXIAO PIAN, RUZENGNING PIAN, Traditional Chinese medicinal liquid for eliminating module in breast, BAIXIAO DAN etc. are treated the nodules of the breast of different pattern of syndrome respectively and are demonstrate,proved, and certain effect is all arranged.Because the part takes place primary disease, its sick position is limitation comparatively, relatively is fit to external treatment, but the Chinese patent medicine that suitable at present primary disease external treatment uses still belongs to blank.
Summary of the invention
The object of the invention is to provide a kind of compound Chinese medicinal preparation; A kind of compound Chinese medicinal preparation and externally used pad pasting agent preparation method thereof for the treatment of cyclomastopathy, another purpose of the present invention is to provide the method for quality control of this compound Chinese medicinal preparation.
The present invention seeks to be achieved through the following technical solutions:
The present invention forms (by weight) by following raw material:
Pericarpium Citri Reticulatae Viride 80-120 Radix Bupleuri 80-120 Semen Vaccariae 80-120
Rhizoma sparganic 80-120 Rhizoma Curcumae 80-120 Rhizoma Corydalis 80-120
Pollen Typhae 80-120 Bulbus Fritillariae Thunbergii 80-120 Spica Prunellae 80-120
Radix Angelicae Dahuricae 80-120 Borneolum Syntheticum 8-12
The above-mentioned raw materials optimum ratio is (by weight):
Pericarpium Citri Reticulatae Viride 100 Radix Bupleuri 100 Semen Vaccariae
(stir-fry)100
Rhizoma sparganic 100 Rhizoma Curcumae 100 Rhizoma Corydalis
(vinegar system)100
Pollen Typhae 100 Bulbus Fritillariae Thunbergiis 100 Spica Prunellaes 100
The Radix Angelicae Dahuricae 100 Borneolum Syntheticums 10
Pharmaceutical composition of the present invention can be made into dosage form clinically any or that pharmaceutically accept.
Described dosage form is tablet, soft capsule, capsule, granule, drop pill, oral liquid or membrane.
Membrane preparation technology of the present invention is as follows:
More than ten simply, except that Borneolum Syntheticum, ten flavors such as all the other Pericarpium Citri Reticulatae Virides add 50-70% alcohol reflux twice, each 1-3 hour, filter, merging filtrate, decompression recycling ethanol also are concentrated into relative density 1.10～1.11 (60 ℃), Chinese medicine extraction extractum is standby; Other gets polyvinyl alcohol 65-95 weight portion, with twice of washing with alcohol, add water 320-480 weight portion, heating makes dissolving, adds polyvinylpyrrolidone 8-12 weight portion, glycerol 40-60 weight portion, azone 7-11 weight portion and Borneolum Syntheticum (using 80% dissolve with ethanol), mixing merges with above-mentioned concentrated solution, stir evenly, get the pad pasting drug extract; Even all coatings are dried, demoulding, and film-making, packing promptly gets pad pasting; Or with the drug extract drying, pulverize, adding starch and/or other suitable adjuvants, mixing is granulated, encapsulated or direct compression or make suitable preparation such as granule.The method of quality control that this compositions is made medicament comprises discriminating and/or assay.
Discrimination method comprises a kind of and/or several in the following method:
A, get 4 of pad pastings, shred, add methanol 20-30ml, put in the water-bath reflux 1-1.5 hour, put coldly, filter, filtrate evaporate to dryness, residue add water 20-30ml makes dissolving, extract 2 times with the ether jolting, each 15-25ml discards ether solution, the reuse water-saturated n-butanol extracts 3 times, and each 20-30ml merges n-butyl alcohol liquid, add isopyknic ammonia solution, jolting is placed and is made layering, get n-butyl alcohol liquid evaporate to dryness, residue adds methanol 1-3ml makes dissolving, as need testing solution.Other gets Radix Bupleuri control medicinal material 1g, the 20-30ml that adds diethyl ether, and reflux 30-60 minute, discard ether solution, medicinal residues volatilize, and add methanol 30-40ml, and reflux 30-60 minute, filter, filtrate evaporate to dryness, residue add methanol 1-3ml makes dissolving, in contrast medical material solution.Test according to thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 10-15: 40-50: 20-25: lower floor's solution that 8-12 chloroform-ethyl acetate-methanol-water is placed below 10 ℃ is developing solvent, launches, and takes out, dry, spray is with 1% paradime thylaminobenzaldehyde ethanol solution of sulfuric acid (1 → 10), and it is clear to be heated to the speckle colour developing at 70 ℃, puts respectively under 365nm daylight and the ultra-violet lamp and inspects.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color or the principal spot of yellow fluorescence.
B, get 2 of pad pastings, add water 20-30ml, heating makes dissolving, the 15-25ml that adds diethyl ether again, and supersound process 10-15 minute, divide and get ether layer, volatilize, residue adds ethyl acetate 1-3ml makes dissolving, as need testing solution.Other gets Rhizoma Curcumae control medicinal material 0.5 gram, adds 30～60 ℃ of petroleum ether 15-25ml, and supersound process 20-30 minute, filter, filtrate is concentrated into about 1ml, in contrast medical material solution.Test according to thin layer chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 8-10: 1-2 normal hexane-ethyl acetate is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color.
C, get 5 of pad pastings, shred, add ethanol 40-60ml, put in the water-bath reflux 1-1.5 hour, and put coldly, filter, filtrate evaporate to dryness, residue add 0.1mol/L hydrochloric acid 15-25ml makes dissolving, with 60～90 ℃ of Petroleum ether extraction 2 times, each 10-15ml, the water intaking layer is regulated pH value to 9～10 with strong aqua ammonia, reuse ether extraction 3 times, each 15-25ml merges ether solution, volatilize, residue adds chloroform 0.5ml makes dissolving, as need testing solution.Other gets the tetrahydropalmatine reference substance, adds ethanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin layer chromatography, draw each 3 μ 1 of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose that contains 2% sodium hydroxide, with 7-9: 3-5: 1-1.5 normal hexane-chloroform-methanol is developing solvent, launches, take out, dry, put in the iodine vapor and smoked 5-10 minute, take out, placed 30-40 minute, and put under the 365nm ultra-violet lamp and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
D, get and differentiate need testing solution under the c item, as need testing solution.Other gets peimine, peiminine reference substance, adds chloroform and makes the solution that every 1ml contains 0.5mg respectively, in contrast product solution.Test according to thin layer chromatography, draw need testing solution 10 μ 1, each 5 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 7-9: 3-5: 1-1.5 benzene-ethyl acetate-diethylamine is developing solvent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution and sodium nitrite ethanol test solution.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
E, get and differentiate need testing solution under the b item, as need testing solution.Other gets the imperatorin reference substance, adds ethyl acetate and makes the solution that every 1ml contains 1mg, in contrast product solution.Test according to thin layer chromatography, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 30～60 ℃ of 3-4: 2-3 petroleum ether-ether is developing solvent, launching below 25 ℃, take out, dry, put under the 365nm ultra-violet lamp and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
F, get and differentiate need testing solution under the b item, as need testing solution.Other gets the Borneolum Syntheticum reference substance, and chlorination is copied into the solution that every 1ml contains 2mg, in contrast product solution.Test according to thin layer chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 15-18: 2-4 cyclohexane extraction-ethyl acetate is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to the speckle colour developing at 105 ℃.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
Content assaying method comprises following method:
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; Acetonitrile-water-phosphoric acid (15-20: 80-85: 0.01) be mobile phase; Detect wavelength 284nm.Number of theoretical plate calculates by Hesperidin standard substance peak should be not less than 2000.
The preparation of reference substance solution: it is an amount of that precision takes by weighing 105 ℃ of Hesperidin reference substances that are dried to constant weight, adds methanol and make the solution that every 1ml contains 0.1mg, shakes up, promptly.
The preparation of need testing solution: get this product under the weight differential item, shred, get 0.2-0.5g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, claim to decide weight, supersound process 1-1.5 hour, put cold, claim to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly.
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly.
Every of this product contains Pericarpium Citri Reticulatae Viride with Hesperidin (C
28H
34O
15) meter, must not be less than 6.0mg.
Product clinic trial by preparation technology's gained of the present invention is effective, can increase local blood drug level, use as membrane, easy to carry, clean, sanitary, has the inhibition hypertrophy, eliminating stagnation blood stasis removing, analgesia, reducing effects such as serum estradiol content, plasma viscosity and platelet aggregation number, is the active drug of treatment cyclomastopathy.Following experimental example further specifies the preparation technology's of the present invention advanced and application in the medicine of treatment cyclomastopathy.
Experimental example 1: the design of extraction process route
This prescription was that clinical decocting uses originally, and the treatment cyclomastopathy is effective, but large usage quantity, considered the anatomical structure characteristics of this disease, and we change it into external preparation.Hesperidin, saikoside, peimisine, tetrahydropalmatine, angelica dahurica coumarin, prunellin etc. are pure soluble components, again according to the characteristics of skin to the easier absorption of pure soluble components, extract solvent and select Diluted Alcohol so determine technology.
Though Pericarpium Citri Reticulatae Viride, Radix Bupleuri, rhizoma sparganic, Rhizoma Curcumae, the Radix Angelicae Dahuricae contain volatile oil in the prescription, but experiment shows, except that Rhizoma Curcumae, all the other flavour of a drug lists are carried and all are difficult to obtain free volatile oil, this 5 flavor medicine is merged the also not enough one thousandth of oil-collecting ratio that vapor distillation extracts volatile oil, extract meaninglessly, and the water-solubility impurity extraction rate is increased, cause certain difficulty for the preparation aspect; Simultaneously, Rhizoma Curcumae has circulation of qi promoting removing blood stasis, removing food stagnancy analgesic effect, be the further above-mentioned design of checking, with the analgesic activity is index, replenished that independent alcohol extraction process is made preparation (technology I) and alcohol extraction process adds the pharmacodynamics contrast experiment that volatile matter distillation oil such as Rhizoma Curcumae are made preparation (technology II), method and result are as follows:
Get 80 of mices, male and female half and half, 18-22 gram.In testing preceding 24 hours in the mouse back depilation, depilation area 2 * 2cm
2After 24 hours mice is divided into 8 groups at random, 10 every group.Matched group is vehicle group, positive drug indomethacin, the high, medium and low dosage group of technology I, the high, medium and low dosage group of technology II.After the administration 1.5 hours, the equal lumbar injection 0.6% acetic acid 0.2ml/ of each Mus only.Observe the writhing response number that each Mus occurs in 15 minutes.
The result shows that technology I and technology II have certain analgesic activity, and high dose group and matched group relatively have significant difference (P＜0.05).But there was no significant difference between technology I and technology II group.See Table 1.
Table 1: the analgesic activity of Dichlorodiphenyl Acetate writhing response
| Group | Dosage (containing crude drug g/kg) | Number of animals (only) | Writhing response number (only) | The writhing response number (x ± s) |
| Matched group | - | 10 | 10 | 29.60±10.43 |
| The positive drug group | 8.0mg/kg | 10 | 4*** | 0.6±0.97*** |
| High dose group (technology II) | 8.0 | 10 | 10 | 15.8±7.86* |
| Middle dosage group (technology II) | 4.0 | 10 | 9 | 18.8±8.76 |
| Low dose group (technology II) | 2.0 | 10 | 10 | 20.0±11.51 |
| High dose group (technology I) | 8.0 | 10 | 10 | 14.1±7.43* |
| Middle dosage group (technology I) | 4.0 | 10 | 10 | 17.0±10.51 |
| Low dose group (technology I) | 2.0 | 10 | 10 | 17.0±12.05 |
Annotate: compare * * * P＜0.001 with the blank group, * P＜0.05
This shows,, reduce production costs that volatile oil can not extract separately separately for simplifying technical process.
Experimental example 2: extraction process technology Study on Conditions
Positive quadraturing design test
Table 2: orthogonal test factor level
| Factor | Level | ||
| Concentration of alcohol | 60％ | 70％ | 80％ |
| The ethanol consumption | 5 times | 8 times | 10 times |
| Extraction time | 1.0 hour | 1.5 hour | 2.0 hour |
Index components measuring principle, method and experimental result
This test is that the result judges one of index with the Radix Bupleuri total saponin, has ten flavor medicines compositions because this prescription extracts flavour of a drug, and is impure more, so use 1%KH
2PO
4And the ammonia solution washing, to remove impurity, exempted negative interference.
The saikoside assay method is as follows: precision takes by weighing saikoside a standard substance 1.00mg, with dissolve with ethanol and standardize solution in the 10ml volumetric flask, as standard solution.Get this standard solution, 70 ℃ of water bath methods add 1% paradime thylaminobenzaldehyde alcoholic solution 0.1ml, 70 ℃ of warm 10min of water-bath, take out, the cooling back adds phosphatase 24 ml, reacts 30min in 70 ℃ of water-baths, takes out, place cooling, behind phosphoric acid standardize solution 5ml, carry out full wavelength scanner.And to record its maximum absorption wavelength be 545nm.
Orthogonal experiments and variance analysis
The Radix Bupleuri total saponin variance analysis sees Table 3;
80% ethanol soluble extraction variance analysis sees Table 4.
Table 3: Radix Bupleuri total saponin variance analysis
| Soruces of variation | Sum of sguares of deviation from mean | Degree of freedom | Variance | The F value | Marginal value | Significance |
| A | 0.0109 | 2 | 0.0055 | 5.0230 | F 0.05(2，20)＝3.49 | *0.01＜P＜0.05 |
| B | 0.0085 | 2 | 0.0043 | 3.9171 | F 0.01(2，20)＝5.85 | *0.01＜P＜0.05 |
| C | 0.0028 | 2 | 0.0014 | 1.2903 | ||
| e＝e1+e2 | 0.0217 | 20 | 0.0011 |
The variance analysis of table 4:80% ethanol soluble extraction
| Soruces of variation | Sum of sguares of deviation from mean | Degree of freedom | Variance | The F value | Marginal value | Significance |
| A | 14.58 | 2 | 7.29 | 6.87 | F 0.05(2，20)＝3.49 | **(P＜0.01) |
| B | 13.84 | 2 | 6.92 | 6.52 | F 0.01(2，20)＝5.85 | **(P＜0.01) |
| C | 3.58 | 2 | 1.79 | 1.69 | ||
| e＝e1+e2 | 21.22 | 20 | 1.06 |
By above-mentioned experimental result and variance analysis as can be known, A (concentration of alcohol), B (ethanol consumption) two factors have appreciable impact to experimental result, can tentatively think and select A for use
1, B
3, promptly the ethanol of 10 times of amounts 60% is preferable extraction process; C (extraction time) influence is little, there was no significant difference between three levels, but C as a result directly perceived
3＞C
2And C
1, consider that proper extension extraction time helps the stripping of ingredient, so extraction time is selected C
3, optimum extraction process is defined as A
1B
3C
3, the flavour of a drug of promptly writing out a prescription are measured 60% alcohol reflux 2 times, each 2 hours with 8～10 times.
Experimental example 3: preparations shaping technical study
1, the comparative test of polyvinyl alcohol (PVA05-88) different amounts
Table 5
| The PVA consumption | Filming performance | Drug loading |
| 30％ | Good | Greatly |
| 40％ | Good | Little |
| 50％ | Good | Little |
PVA is an optimum amount with 30%.
2, the comparative test of glycerol different amounts
Table 6
| The glycerol consumption | Toughness | The demoulding situation | Medicine film content of dispersion |
| 10％ | Difference | Difference | Greatly |
| 15％ | Good | Good | Bigger |
| 20％ | Good | Good | Little |
Glycerol is optimum amount with 15%.
3, the use of polyvinylpyrrolidone (PVP): PVP has multifrequency nature and effects such as bonding, thickening, suspending, dispersion, hydrotropy, complexation, film forming, in this product, add a small amount of PVP, help dispersion and the hydrotropy of Chinese medicine extraction extractum in macromolecule filming material solution, but its consumption what concern little with the membrane molding, and PVP belongs to the import pharmaceutic adjuvant, price is higher, for minimizing cost, so select for use 1% consumption to add.
4, azone is as Percutaneous absorption enhancer, and general consumption is 0.5%～2%, and what concern not quite with the membrane molding because of its consumption, so select for use 1% consumption to add.
Experimental example 4: the qualitative identification test of Radix Bupleuri
This discriminating is with the positive contrast of Radix Bupleuri medical material, differentiates the qualitative identification that contains Radix Bupleuri in the preparation.Get 4 of this product, shred, add methanol 30ml, put in the water-bath reflux 1 hour, put coldly, filter, filtrate evaporate to dryness, residue add water 20ml makes dissolving, extract 2 times with the ether jolting, each 15ml discards ether solution, the reuse water-saturated n-butanol extracts 3 times, and each 20ml merges n-butyl alcohol liquid, add isopyknic ammonia solution, jolting is placed and is made layering, get n-butyl alcohol liquid evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution.Other gets Radix Bupleuri control medicinal material 1g, the 30ml that adds diethyl ether, and reflux 30 minutes discards ether solution, and medicinal residues volatilize, and add methanol 30ml, and reflux 30 minutes filters, and filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, in contrast medical material solution.Draw each 5 μ 1 of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethyl cellulose, (15: 40: 22: 10) lower floor's solution of placing below 10 ℃ was developing solvent with chloroform-ethyl acetate-methanol-water, launch, take out, dry, spray is with 1% paradime thylaminobenzaldehyde ethanol solution of sulfuric acid (1 → 10), it is clear to be heated to speckle colour developing at 70 ℃, puts respectively under daylight and the ultra-violet lamp (365nm) and inspects.In the test sample chromatograph, with the control medicinal material relevant position on, show the principal spot of same color or the principal spot of yellow fluorescence.The negative control test liquid is noiseless.
In the experiment, adopt saikoside a, d chemical reference substance in contrast, but identification result is not as adopting the Radix Bupleuri control medicinal material, so the Radix Bupleuri control medicinal material has been used in a discriminating.
Experimental example 5: the qualitative identification test of Rhizoma Curcumae
This discriminating is with the positive contrast of Rhizoma Curcumae control medicinal material, differentiates the qualitative identification that has Rhizoma Curcumae in the preparation.Get 2 of this product, add water 15ml, heating makes dissolving, the 20ml that adds diethyl ether again, and supersound process 10 minutes is divided and is got ether layer, volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution.Get Rhizoma Curcumae control medicinal material 0.5 gram, add petroleum ether (30-60 ℃) 20ml, supersound process 20 minutes is divided and is got petroleum ether layer, is concentrated into about 1ml, in contrast medical material solution.Draw each 10 μ 1 of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, be developing solvent with normal hexane-ethyl acetate (8.5: 1.5), launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and hot blast blows to clear spot.Under daylight, inspect.In the test sample chromatograph, with the control medicinal material relevant position on, show the speckle of same color.
In the experiment,, be developing solvent with petroleum ether (60～90 ℃)-ethyl acetate (9: 1) with the silica gel G plate of 0.5%CMC-Na, the colour developing of 2% vanillin sulphuric acid test solution, test sample shows identical aubergine speckle with positive control in same position, and negative control is noiseless; Once used petroleum ether (60～90 ℃)-chloroform-water (6: 1: 0.5); Petroleum ether developing solvents such as (60～90 ℃), but identification result is developing solvent not as adopting normal hexane-ethyl acetate (8.5: 1.5).
Experimental example 6: the qualitative identification test of Rhizoma Corydalis
This discriminating is with the positive contrast of tetrahydropalmatine, differentiates the qualitative identification that contains Rhizoma Corydalis in the preparation.Get 5 of this product, shred, add ethanol 50ml, put in the water-bath reflux 1 hour, and put coldly, filter, filtrate evaporate to dryness, residue add 0.1mol/L hydrochloric acid 20ml makes dissolving, extracts 2 times with petroleum ether (60～90 ℃), each 10ml, the water intaking layer is regulated pH value to 9～10 with strong aqua ammonia, reuse ether extraction 3 times, each 20ml merges ether solution, volatilize, residue adds chloroform 0.5ml makes dissolving, as need testing solution.Get tetrahydropalmatine, add ethanol and make the solution that every 1ml contains 1mg, in contrast product solution.Draw each 3 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 2% sodium hydroxide, with normal hexane-chloroform-methanol (7.5: 4: 1) is developing solvent, launch, take out, dry, put in the iodine vapor and smoked 5 minutes, take out, placed 30 minutes, put under the uviol lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
Silica gel G plate with 0.5%CMC-Na, with ether-cyclohexane extraction-methanol (5: 3: 0.5) is developing solvent, develop the color with iodine vapor, after volatilizing, under uviol lamp 365nm, inspect, test sample shows identical yellow fluorescence speckle with positive control in same position, and negative control is noiseless, but identification result is developing solvent not as adopting normal hexane-chloroform-methanol (7.5: 4: 1).
Experimental example 7: the qualitative identification test of Bulbus Fritillariae Thunbergii
This discriminating is with peimine, the positive contrast of second, differentiates the qualitative identification that has Bulbus Fritillariae Thunbergii in the preparation.Get peimine, peiminine reference substance, chlorination is copied into the solution that every 1ml contains 0.5mg, in contrast product solution.Draw need testing solution 10 μ l and above-mentioned reference substance solution 5 μ l under the Rhizoma Corydalis discriminating item, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with benzene-ethyl acetate-diethylamine (8: 4: 1) is developing solvent, launch, take out, dry, spray is inspected under the daylight with rare bismuth potassium iodide test solution and sodium nitrite ethanol test solution.Test sample has identical color speckle with positive control at the same position place.Peimine, second and tetrahydropalmatine all are the alkaloids compositions, and the sample extraction method is identical, so the need testing solution in this test has adopted the need testing solution under the Rhizoma Corydalis discriminating item.
Also select the silica gel G plate of the 0.5%CMC-Na of 2%NaOH in the experiment for use, with normal hexane-chloroform-methanol (7.5: 4: 1) is developing solvent, twice expansion, respectively with rare bismuth potassium iodide test solution and the colour developing of sodium nitrite ethanol test solution, test sample shows identical yellow spotting with positive control in same position, negative control is noiseless, but the speckle separating degree as above method is not good.Also use peracetic acid ethyl ester-methanol-strong ammonia solution (17: 2: 1); (3: 4: 1.5: 1) lower floor's solution was developing solvent to chloroform-ethyl acetate-methanol-water, but identification result is developing solvent not as adopting benzene-ethyl acetate-diethylamine (8: 4: 1).
Experimental example 8: the qualitative identification test of the Radix Angelicae Dahuricae
This discriminating is with the positive contrast of imperatorin, differentiates the qualitative identification that contains the Radix Angelicae Dahuricae in the preparation.Get the imperatorin reference substance, add ethyl acetate and make the solution that every 1ml contains 1mg, in contrast product solution.Draw Rhizoma Curcumae and differentiate item need testing solution 10ul and above-mentioned reference substance solution 5ul down, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with petroleum ether (30～60 ℃)-ether (3: 2) is developing solvent, launching below 25 ℃, take out, dry, put under the uviol lamp (365nm) and inspect.Test sample has identical color fluorescence speckle with positive control at the same position place.
In the experiment, also used the silica gel H plate, silica gel G F
254Plate used toluene-ethyl acetate-formic acid (6.5: 1.5: 0.5) to be developing solvent, but identification result is developing solvent not as adopting petroleum ether (30～60 ℃)-ether (3: 2).
Experimental example 9: the qualitative identification test of Borneolum Syntheticum
This discriminating is with the positive contrast of Borneolum Syntheticum reference substance, differentiates the qualitative identification that contains Borneolum Syntheticum in the preparation.Get the Borneolum Syntheticum reference substance, chlorination is copied into the solution that 1ml contains 2mg, in contrast product solution.Draw Rhizoma Curcumae and differentiate item need testing solution and each 2 μ l of above-mentioned reference substance solution down, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with cyclohexane extraction-ethyl acetate (17: 3) is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, 105 ℃ of heating several minutes, to clear spot.On test sample and the reference substance chromatograph relevant position, show identical color speckle.
Experimental example 10: the experimentation of assay
1, assay object and measure into the component selections foundation
This preparation by Pericarpium Citri Reticulatae Viride, Radix Bupleuri, Semen Vaccariae, rhizoma sparganic, Rhizoma Curcumae, Rhizoma Corydalis, Pollen Typhae, Bulbus Fritillariae Thunbergii, Spica Prunellae, the Radix Angelicae Dahuricae and Borneolum Syntheticum ten simply Chinese medicine form, wherein Pericarpium Citri Reticulatae Viride is a monarch drug, and soothing the liver dispelling the stagnated QI, the stagnant merit of removing food stagnancyization are arranged.The chemical effective ingredient of Pericarpium Citri Reticulatae Viride is its contained Hesperidin.This preparation is chosen the monarch drug Pericarpium Citri Reticulatae Viride, and its contained Hesperidin is worked out assay.
2, the standard curve and the range of linearity
Precision takes by weighing Hesperidin reference substance 2.08mg, puts in the 10ml volumetric flask, adds dissolve with methanol and is diluted to scale, shakes up, and as standard solution, concentration is 0.208mg/ml.Above-mentioned Hesperidin solution 2,5,8,10, the 15 μ l of accurate absorption, inject chromatograph of liquid respectively, measure peak area by above-mentioned condition, with the peak area is vertical coordinate, the Hesperidin sample size is an abscissa, drawing standard curve (see figure 1), and the calculating regression equation is: A=1610.1x+50.055R=0.9997
Table 7: the Hesperidin range of linearity is investigated the result
| Sample size (μ l) | 2 | 5 | 8 | 10 | 15 |
| Sample size (μ g) | 0.416 | 1.040 | 1.664 | 2.080 | 3.120 |
| The peak area average | 694.26 | 1719.35 | 2799.63 | 3377.45 | 5505.54 |
As shown in Figure 1, Hesperidin is in linear relation between 0.416～3.120 μ g.
3, carry out precision test, stability test, repeatability test, recovery test, blank assay, reference substance and medical material controlled trial, 3 batch samples are measured by the described content assaying method of quality standard draft, its result is as follows:
Table 8: sample size measurement result
| Lot number | Content (mg/ sheet) | Average content (mg/ sheet) |
| 991116 | 9.64 9.71 | 9.67 |
| 991214 | 9.92 9.57 | 9.75 |
| 000117 | 9.86 9.95 | 9.91 |
According to last table result, this product content limit can be made, but considers the practical situation of medical material and production, and tentative this product content limit is every and contains Pericarpium Citri Reticulatae Viride with Hesperidin (C
28H
34O
15) meter, must not be less than 6.0mg.
Experimental example 11:FRANZ diffusion cell method is carried out the transdermal test in vitro permeability test
The preparation of animal isolated skin: get rat (body weight 185～205g), mice (the about 30g of body weight), slough the hair of rat abdomen and mouse back respectively with depilatory, raised one day, before the test they are put to death, peel off the skin of rat abdomen and mouse back, carefully reject subcutaneous fat, clean skin with distilled water and normal saline, be soaked in the normal saline, standby.
Experimental provision and method: adopt circulating circulation disperser, with being cut into and identical small pieces (about 0.1355g, the area 4.4cm of receiving chamber oral thermometer area size of known content by preparation technology's gained pad pasting of the present invention
2), the accurate title, decided weight, will be affixed on the rat skin in vitro for the reagent film, makes it to contact closely with skin, is placed between the diffuser casing and receiving chamber that improves the Franz diffusion cell, and levels clamps, and as shown in Figure 1, the receiving chamber effective area is 4.4cm
2, dischargeable capacity is 14.2ml.Add the normal saline 14.2ml that contains 0.5% Tween 80 in receiving chamber, drive away bubble, open the magnetic stirring apparatus continuous stirring and regulate 37 ℃ ± 1 ℃ constant temperature of bath temperature, the magnetic stirring apparatus rotating speed is about 120r/min.The accurate respectively acceptable solution 1ml that draws added isopyknic acceptable solution simultaneously at 1,2,4,6,8,10,12,24 hour.Sample is through filtering with microporous membrane, and the accurate 50 μ l sample liquid of drawing are injected high performance liquid chromatograph, determine Determination of Hesperidin Content in the acceptable solution, calculate accumulation infiltration capacity (Q), and calculate the transmitance of transdermal experiment 12 hours and 24 hours Hesperidins, the results are shown in Table 9, table 10 and Fig. 2.
Table 9: the different time rat skin is received Hesperidin accumulation infiltration capacity (n=3) in the liquid pool
| Time | 1h | 2h | 4h | 6h | 8h | 10h | 12h | 24h |
| Peak area | 10061 | 44499 | 177017 | 305389 | 418601 | 535121 | 599481 | 933839 |
| Accumulation infiltration capacity (μ g/cm 2) | 0.363 | 1.63 | 6.52 | 11.5 | 16.0 | 20.5 | 23.1 | 35.4 |
| RSD(％) | 1.11 | 0.94 | 0.71 | 0.64 | 0.66 | 0.68 | 0.79 | 0.94 |
| 12 hours Hesperidins accumulation infiltration capacity always accounts for the percentage rate of the amount of sticking=5.40% 24 a hour Hesperidin accumulation infiltration capacity and accounts for the percentage rate of the amount of sticking=8.28% always | ||||||||
Table 10: the different time mouse skin is received Hesperidin accumulation infiltration capacity (n=3) in the liquid pool
| Time | 1h | 2h | 4h | 6h | 8h | 10h | 12h | 24h |
| Peak area | 87220 | 237521 | 354171 | 556154 | 725797 | 809573 | 884833 | 1167814 |
| Accumulation infiltration capacity (μ g/cm 2) | 3.15 | 8.81 | 13.4 | 21.0 | 27.7 | 31.1 | 34.1 | 44.5 |
| RSD(％) | 1.52 | 0.97 | 0.79 | 0.83 | 0.67 | 0.98 | 0.76 | 0.88 |
| 24 hours Hesperidins accumulation infiltration capacity always accounts for the percentage rate of the amount of sticking=7.98% 24 a hour Hesperidin accumulation infiltration capacity and accounts for the percentage rate of the amount of sticking=10.4% always | ||||||||
By table 9,10, Fig. 2 as can be seen, be affixed on rat and mouse skin outward by preparation technology's gained pad pasting of the present invention after, the index components Hesperidin can see through skin in 1 hour in the film, and transit dose in time prolongation and increase, and can keep more than 24 hours.Show by preparation technology's gained pad pasting Chinese medicine effective ingredient of the present invention and can and play a role, be suitable for transdermal administration through skin.
Always accounting for by the Hesperidin in preparation technology's gained pad pasting of the present invention through 12 hours, 24 hours of rat abdomen skin accumulation infiltration capacities, the amount of sticking is respectively 5.4%, 8.28%; Always accounting for through 12 hours, 24 hours of mouse back skin accumulation infiltration capacities, the amount of sticking is respectively 7.98%, 10.4%.Show by preparation technology's gained pad pasting of the present invention and have transdermal performance preferably.
Experimental example 12: thin layer chromatography is analyzed ingredient
1, standard control liquid:
1. getting the tetrahydropalmatine reference substance adds ethanol and makes the solution that every 1ml contains 0.5mg.
2. get Borneolum Syntheticum reference substance chlorination and copy into the solution that every 1ml contains 2mg.
2, the preparation of sample liquid:
1. collection is got by 24 hours Transdermal absorption liquid 50ml of preparation technology's gained pad pasting of the present invention, adds 0.1mol/L hydrochloric acid and regulates pH value to 2～3, extracts 3 times with petroleum ether (60～90 ℃), each 20ml divides and gets petroleum ether layer, volatilizes in the water-bath, residue adds chloroform 0.5ml, as sample liquid A.
2. divide and get above-mentioned water layer, regulate pH value to 9～10 with strong aqua ammonia, reuse ether extraction 3 times, each 30ml merges ether solution, volatilizes, and residue adds chloroform 0.5ml makes dissolving, as sample liquid B.
3, thin layer chromatography: condition sees the following form
Table 11
| Lamellae | Point sample | Developing solvent | Developer/uviol lamp | The chromatograph speckle | |
| I | Silica gel G | Borneolum Syntheticum contrast liquid 3ul, sample liquid A 5ul | Cyclohexane extraction-ethyl acetate (17: 3) | 5% vanillin sulfuric acid solution spraying baking colour developing | Two purple dots (Borneolum Syntheticum, isoborneol) |
4, result
As seen the thin-layer chromatogram of test I, II, clear spots such as Borneolum Syntheticum, tetrahydropalmatine illustrates by alkaloids compositions such as liposoluble constituent such as the Borneolum Syntheticum in preparation technology's gained pad pasting of the present invention and tetrahydropalmatines and can be absorbed through skin.
Experimental example 13: rabbit hyperplasia of mammary gland model test
30 of Female rabbits, after normal two weeks of raising, get 5 as normal control group intramuscular injection normal saline 0.5ml (kgd), all the other 25 intramuscular injection estradiol benzoate 0.3ml (kgd), continuous 25 days, stop injection, be divided into height at random, in, low dose group, 5 groups of model group and positive control drug group, every group 5, low dose group, in dosage group and high dose group give respectively with by preparation technology's gained pad pasting drug extract 1.8g crude drug/kg of the present invention, 3.6g crude drug/kg and 5.4g crude drug/kg (evenly spreading upon on 6 mammary gland of rabbit), fix 4～6 hours with the plastic foil covering and with gauze, administration every day 1 time, each mammary gland of every rabbit of positive drug control group is given and 2 * 5cm
2RUPIXIAO TIEGAO, model group and normal control group are given and normal saline, continuous 20 days.
1, perusal classification results sees Table 12.
Table 12: by preparation technology's gained pad pasting drug extract of the present invention to the cyclomastopathy result that detects by an unaided eye
| Group | Dosage G/kg | Number of animals (only) | Classification | The U value | ||
| - | + | ++ | ||||
| Model control group normal control group drug extract group drug extract group drug extract group RUPIXIAO group | 1.8 3.6 5.4 2×2.5cm 2/ mammary gland | 5 5 5 5 5 5 | 0 5 0 0 4 3 | 1 0 2 2 0 1 | 4 0 3 3 1 1 | 2.74 ** 0.55 0.55 2.08 * 1.97 * |
Annotate: compare with model control group,
*P＜0.05,
*P＜0.01
From perusal rabbit nipple general form, (5.4g crude drug/kg) can obviously suppress the enlargement (P＜0.01) of mammary gland, and ((3.6g crude drug/kg) and model group be no significant difference (P＞0.05) relatively for 1.8g crude drug/kg) and middle dosage group by preparation technology's gained pad pasting drug extract low dose group of the present invention by preparation technology's gained pad pasting drug extract high dose group of the present invention.
2, light microscopic result:
In model group 5 examples 4 examples are arranged, cyclomastopathy, quantity increases, and the acinus epithelial proliferation is bilayer or multilamellar, and the ductal epithelium hypertrophy has epithelium bunch formation, ductal ectasia, number increases, and interstitial fibers hamartoplasia is fine and close; 1 routine rarely seen cyclomastopathy in addition.
Similar to model group by preparation technology's gained pad pasting drug extract low dose group of the present invention with middle dosage group.
By in preparation technology's gained pad pasting drug extract high dose group 5 examples of the present invention 4 examples being arranged, mammary gland ductule epithelial proliferation is lighter, and ductal ectasia is not obvious, and the acinus epithelium is monolayer or bilayer, does not have obvious hypertrophy.The expansion of visible cyclomastopathy of 1 example and conduit in addition.
It is monolayer or bilayer that 3 routine mammary gland ductule epitheliums are arranged in RUPIXIAO group 5 examples, bunch formation of no epithelium, and ductal ectasia is not obvious, and acinus does not also have obvious hypertrophy; In addition 2 routine breast duct epithelial proliferations are multiple layer or multilamellar, epithelium bunch occurs, and ductal ectasia is more obvious, and acinus does not have obvious hypertrophy.
Normal control group 5 routine mammary gland ductules are the simple columnar epithelium cell, and the acinus epithelium is a simple cuboidal epithelium, outward by basement membrane, and visible therebetween myoepithelial cell, a matter is a loose connective tissue, a lobules of mammary gland body of gland and a matter ratio are normal.
The light microscopic result shows that tentatively high dose can suppress cyclomastopathy more significantly by preparation technology's gained pad pasting drug extract of the present invention, and low dosage also has certain inhibition cyclomastopathy effect by preparation technology's gained pad pasting drug extract of the present invention is similar to positive control drug.
3, Electronic Speculum result:
Normal control group mammary gland acinus epithelial cell cube or column, the chamber face has less or middle amount microvillus, and iuntercellular has junctional complex, visible myoepithelial cell in cell based bottom and sarolemma, kytoplasm inner cell tolerance is medium, and mitochondrion, Golgi body, endoplasmic reticulum, secretory vacuole are normal.
Model group mammary gland body of gland and ductal epithelial cell level increase, polarity does not have disorder, as seen microvillus is irregular, junctional complex is normal, rough endoplasmic reticulum, mitochondrion, Golgi body number increase in the kytoplasm, and the sub-secretory granule number of cipher telegram increases, and fibrocyte and collagenocyte quantity increase in the endoplasmic reticulum, myoepithelial cell increases, and base stage thickens.
High dose group mammary gland body of gland and ductal epithelial cell level are normal or increase; polarity is normal; microvillus is arranged rule; junctional complex is normal; kytoplasm inner cell organ number is roughly normal, the visible sub-distributed granule of cipher telegram, and fibrocyte and collagen fiber quantity normally or slightly increase in the matter; myoepithelial cell is normal, and base stage slightly thickens.
Electronic Speculum result shows that high dose can make outgrowth mammary gland recover normal substantially by preparation technology's gained pad pasting drug extract of the present invention.
Experimental example 14: serum estradiol assay experiment.
Table 13 serum estradiol content (X ± s)
| Group | Animal | Dosage g/kg | Estradiol content (pg/ml) |
| Normal control group model matched group drug extract group drug extract group drug extract group positive controls | 5 5 5 5 5 5 | - - 1.8 3.6 5.4 4×2.5cm 2/ mammary gland | 33.62±5.42 ** 48.59±6.74 34.93±1.71 ** 35.16±1.92 ** 33.22±3.88 ** 29.97±7.06 ** |
Annotate: compare with model control group
*P＜0.01
By table 13 as seen, high, medium and low dosage group, positive controls, normal control group serum estradiol content and model control group all have significant difference, show that high, medium and low dosage group and positive controls all can significantly reduce serum estradiol content.
Experimental example 15: blood viscosity test experience
By table 14 as seen, the plasma viscosity of model group, plasma fibrinogen and platelet aggregation number and normal control group more all change, all there were significant differences (P＜0.05) and the plasma viscosity of the platelet aggregation number of low dose group and middle dosage group and high dose, platelet aggregation number are compared with model control group, other every indexs and normal control group or model control group there was no significant difference (P＞0.05), prompting is to have eliminating stagnation blood stasis dispelling effect by reducing plasma viscosity and platelet aggregation number by preparation technology's gained pad pasting of the present invention, thereby suppresses cyclomastopathy.
Table 14: the blood viscosity testing result (X ± s)
| Group | Blood viscosity (mpas) | Plasma viscosity (mpas) | Packed cell volume (%) | Plasma fibrinogen (G/L) | Platelet aggregation | ||||
| 1(5s) | 2(10s) | 3(35s) | 4(120s) | Expansion type (%) | Aggregation number (individual) | ||||
| Model control drug medicinal extract group (low) drug extract group (in) drug extract group (height) positive control normal control | 17.0±6.5 14.1±6.6 14.8±7.1 13.0±3.7 11.1±1.3 12.6±1.6 | 10.5±4.0 8.2±1.8 8.6±1.7 9.5±2.8 8.4±0.8 9.0±0.7 | 6.8±2.0 5.2±0.8 5.1±0.9 5.9±1.6 5.6±0.5 7.0±2.0 | 5.3±1.9 4.1±0.7 * 4.2±1.1 4.8±1.7 4.3±0.5 5.3±0.9 | 1.79±0.06 * 1.73±0.07 1.72±0.09 1.69±0.03 * 1.69±0.02 ** 1.70±0.04 * | 42.7±3.2 41.6±2.5 41.9±1.9 39.7±3.0 42.2±3.0 43.4±2.9 | 4.18±0.18 ** 3.62±0.55 3.69±0.61 3.57±0.52 3.44±0.74 3.20±0.38 ** | 21.8±1.9 * 19.6±1.1 19.7±1.3 19.8±1.9 20.3±1.7 19.4±1.5 * | 47.6±2.4 * 42.7±3.6 * 42.9±2.5 * 43.9±2.3 * 44.7±3.3 42.8±2.8 * |
Annotate: 1. compare with the normal control group
*P＜0.01;
*P＜0.05
2. compare with model control group
##P＜0.01;
#P＜0.05
Can suppress the cyclomastopathy that rabbit intramuscular injection estradiol causes by preparation technology's gained pad pasting drug extract of the present invention, reduce serum estradiol content, reduce blood plasma viscosity and platelet aggregation number.
Experimental example 16: the swollen test of rat granuloma
Get 60 of 120～150g male rats, be divided into 6 groups at random, every group 10, etherization, dorsal position is fixed, the chest iodine disinfection, and 75% cotton ball soaked in alcohol takes off iodine, cut 1cm left and right sides osculum, with the autoclaving cotton balls (contain mycillin mixed liquor 0.2ml, soak 70 ℃ oven dry) of ophthalmology tweezer with 20mg, it is subcutaneous to implant left and right sides axillary fossa from incision, skin suture immediately from operation beginning administration on the same day, is about to drug extract and is coated in and is affixed on the plastic foil on the scalpel edge surrounding skin, with immobilization with adhesive tape 4～6 hours, not administration of negative control, high dose group are coated with by preparation technology's gained pad pasting drug extract 8.0g crude drug/kg of the present invention, and middle dosage group is coated with by preparation technology's gained pad pasting drug extract 4.0g crude drug/kg of the present invention, low dose group is coated with by preparation technology's gained pad pasting drug extract 2.0g crude drug/kg of the present invention, and the RUPIXIAO group is applied 3.3 * 4cm
2/ only, the diclofenac group gives 0.3g/kg diclofenac, and with the gauze immobilization with adhesive tape, each organizes administration every day 1 time, successive administration six days.Opened former otch on the 7th day, cotton balls taken out together with connective tissue, reject fatty tissue, put 70 ℃ of oven dry in the baking oven, weigh, with claim weight deduct the former weight of cotton balls and promptly get granuloma weight, t checks and respectively organizes granuloma weight.
Table 15: the pad pasting drug extract is to the bullate influence of rat granuloma (X ± s)
| Group | Animal (only) | Dosage (g/kg) | Granuloma weight (mg) |
| Negative control group drug extract group drug extract group drug extract group diclofenac group RUPIXIAO group | 10 10 10 10 10 10 | 8 4 2 0.3 3.3×4cm 2/ only | 80.8±19.3 62.2±19.2 * 72.6±18.5 * 83.5±67.0 39.7±29.6 ** 64.5±27.1 |
Annotate: compare with negative control
*P＜0.01,
*P＜0.05
High, middle dosage group and negative control group relatively have significant difference (P＜0.05), show that it has the effect of certain inhibition granulation hyperplasia.
Experimental example 17: mouse writhing method analgesic experiment;
Mice is divided into 6 groups at random by body weight, sex, 10 every group: (1) matched group is coated with distilled water 0.1ml/20g; (2) the low dose group mice is coated with 1.0g/kg; (3) dosage group mice is coated with 2.0g/kg in; (4) the high dose group mice is coated with 4.0g/kg; (5) RUPIXIAO group mice is pasted RUPIXIAO TIEGAO 2.5 * 4cm
2/ only; (5) diclofenac group mice is coated with Vitalin Emulgel 2.0g/kg.Respectively organize mouse web portion before the experiment and lose hair or feathers, will be tried thing during experiment and be applied in mouse web portion depilation district skin according to the medication needs, and with gauze and immobilization with adhesive tape; The 40min pneumoretroperitoneum is only injected 0.5% acetic acid 0.2ml/, observes mouse writhing number of times in the 15min, and the result carries out the t check.
Table 16: pad pasting drug extract extractum is to the influence of mouse writhing number of times
| Group | Dosage (g/kg) | Number of animals (only) | Turn round the body number of times |
| Matched group drug extract group drug extract group drug extract group diclofenac group RUPIXIAO group | 1.0 2.0 4.0 2.0 2.5×4cm 2/ only | 10 10 10 10 10 10 | 23.6±13.9 5.9±7.56 ** 5.5±7.91 * 5.2±5.71 * 5.0±5.96 ** 11.9±12.6 |
Compare with matched group,
*P＜0.05, * * p＜0.01
RUPIXIAO has the trend that the mouse writhing reaction is reduced, but no statistical significance, diclofenac can obviously reduce the mouse writhing number of times, by three dosage groups of preparation technology's gained pad pasting drug extract of the present invention and diclofenac same purpose is arranged.
Experimental example 18: mice hot plate experiment
Hot plate preheating 10 minutes makes temperature at 55 ℃ ± 5 ℃.Screen qualified mice: get 18-23g female mice number, each 1 is placed on the hot plate, and mice is from being placed on the hot plate to the pain threshold of metapedes required time (second) as this Mus occurring licking.Allly lick the metapedes time less than 5 seconds or give it up greater than 30 seconds or leaper.(totally 60) are divided into 6 groups at random with qualified mice, repeat to survey its normal pain threshold, get two subnormal threshold of pain meansigma methodss, as pain threshold before this Mus administration.Before the experiment mouse web portion is lost hair or feathers according to the medication needs, before the experiment medicine is applied to depilation place, cover plastic foil, immobilization with adhesive tape.Give 4g crude drug/kg by preparation technology's gained pad pasting drug extract high dose group of the present invention, middle dosage group 2g crude drug/kg, low dose group 1g crude drug/kg, the equal abdominal part of diclofenac group 2g/kg are smeared, and the RUPIXIAO TIEGAO abdominal part is pasted, and survey the mice pain threshold after the administration in 15,30,60 minutes respectively.As 60 seconds still reactionless, mice is taken out, in order to avoid scald, its pain threshold was in 60 seconds.The result carries out the t check.
Table 17: the influence of the pain that pad pasting drug extract extractum causes the mice hot plate method
| Group | Dosage (g/kg) | Number of animals (only) | The preceding threshold of pain of administration (s) | After the administration different time threshold of pain (s) | |||
| 15min | 30min | 60min | 120min | ||||
| The matched group drug extract group drug extract group drug extract group diclofenac breast addiction that disappears | - 1.0 2.0 4.0 2.0 2.5×4cm 2/ only | 10 10 10 10 10 10 | 14.1±4.9 11.9±2.8 11.1±2.6 10.4±2.4 14.5±4.2 15.1±2.7 | 15.7±5.9 13.6±7.2 12.8±8.1 11.3±3.8 29.9±17.4 * 16.3±7.6 | 16.4±8.7 15.1±6.6 15.1±7.1 15.1±7.0 23.8±14.7 13.6±7.6 | 17.3±9.2 17.1±7.0 * 17.5±7.6 * 17.8±8.7 * 16.3±9.1 15.7±6.6 | 17.7±8.8 16.2±9.7 16.3±8.9 15.9±5.0 16.4±5.7 16.1±7.2 |
Annotate: with compare *＜0.05 before the administration, * * P＜0.01
When 60min, the 15min with before the administration, compare that all there were significant differences (P＜0.05) respectively by the basic, normal, high dosage group of preparation technology's gained pad pasting drug extract of the present invention and Vitalin Emulgel group, show that each group has significant analgesia role by preparation technology's gained pad pasting drug extract of the present invention, compares before RUPIXIAO and the administration and does not demonstrate analgesic activity (P＞0.05).
Description of drawings:
Fig. 1 Hesperidin reference substance canonical plotting
Hesperidin accumulation infiltration capacity changing trend diagram in Fig. 2 different time absorption liquid
Embodiment 1:
Pericarpium Citri Reticulatae Viride 250g Radix Bupleuri 250g Semen Vaccariae (stir-fry) 250g
Rhizoma sparganic 250g Rhizoma Curcumae 250g Rhizoma Corydalis (vinegar) 250g
Pollen Typhae 250g Bulbus Fritillariae Thunbergii 250g Spica Prunellae 250g
Radix Angelicae Dahuricae 250g Borneolum Syntheticum 25g
More than ten simply, except that Borneolum Syntheticum, ten flavors such as all the other Pericarpium Citri Reticulatae Virides add 60% alcohol reflux twice, each 10 times of amounts 2 hours filter, merging filtrate, decompression recycling ethanol also are concentrated into relative density 1.10～1.11 (60 ℃), Chinese medicine extraction extractum is standby; Other gets polyvinyl alcohol 200g, with washing with alcohol twice, adds water 1000ml, heating makes dissolving, adds polyvinylpyrrolidone 25g, glycerol 100ml, azone 25ml and Borneolum Syntheticum (using 80% dissolve with ethanol), mixing merges with above-mentioned concentrated solution, stir evenly, demoulding is dried in coating, (4cm * 6cm), every medicine film contains crude drug 2.525g to make 1000.
Embodiment 2:
Pericarpium Citri Reticulatae Viride 80g Radix Bupleuri 120g Semen Vaccariae
(stir-fry)80g
Rhizoma sparganic 120g Rhizoma Curcumae 80g Rhizoma Corydalis
(vinegar system)120g
Pollen Typhae 80g Bulbus Fritillariae Thunbergii 120g Spica Prunellae 80g
Radix Angelicae Dahuricae 120g Borneolum Syntheticum 8g
More than ten simply, except that Borneolum Syntheticum, ten flavors such as all the other Pericarpium Citri Reticulatae Virides add 60% alcohol reflux twice, each 2 hours, filter, merging filtrate, decompression recycling ethanol also are concentrated into relative density 1.10～1.11 (60 ℃), Chinese medicine extraction extractum is standby; Other gets polyvinyl alcohol 65g, with washing with alcohol twice, adds water 320g, heating makes dissolving, adds polyvinylpyrrolidone 8g, glycerol 40g, azone 7g and Borneolum Syntheticum (using 80% dissolve with ethanol), mixing merges with above-mentioned concentrated solution, stir evenly, get the pad pasting drug extract, even all coatings, dry demoulding, film-making, packing promptly gets pad pasting.
Embodiment 3:
Pericarpium Citri Reticulatae Viride 120g Radix Bupleuri 80g Semen Vaccariae
(stir-fry)120g
Rhizoma sparganic 80g Rhizoma Curcumae 120g Rhizoma Corydalis
(vinegar system)80g
Pollen Typhae 120g Bulbus Fritillariae Thunbergii 80g Spica Prunellae 120g
Radix Angelicae Dahuricae 80g Borneolum Syntheticum 12g
More than ten simply, except that Borneolum Syntheticum, ten flavors such as all the other Pericarpium Citri Reticulatae Virides add 60% alcohol reflux twice, each 2 hours, filter, merging filtrate, decompression recycling ethanol also are concentrated into relative density 1.10～1.11 (60 ℃), Chinese medicine extraction extractum is standby; Other gets polyvinyl alcohol 95g, with washing with alcohol twice, adds water 480g, heating makes dissolving, adds polyvinylpyrrolidone 12g, glycerol 60g, azone 11g and Borneolum Syntheticum (using 80% dissolve with ethanol), mixing merges with above-mentioned concentrated solution, stir evenly, get the pad pasting drug extract, even all coatings, dry demoulding, film-making, packing promptly gets pad pasting.
Embodiment 4:
A, get 4 of pad pastings, shred, add methanol 30ml, put in the water-bath reflux 1 hour, put coldly, filter, filtrate evaporate to dryness, residue add water 20ml makes dissolving, extract 2 times with the ether jolting, each 15ml discards ether solution, the reuse water-saturated n-butanol extracts 3 times, and each 20ml merges n-butyl alcohol liquid, add isopyknic ammonia solution, jolting is placed and is made layering, get n-butyl alcohol liquid evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution.Other gets Radix Bupleuri control medicinal material 1g, the 30ml that adds diethyl ether, and reflux 30 minutes discards ether solution, and medicinal residues volatilize, and add methanol 30ml, and reflux 30 minutes filters, and filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, in contrast medical material solution.Test according to thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 15: 40: 22: lower floor's solution that 10 chloroforms-ethyl acetate-methanol-water is placed below 10 ℃ was developing solvent, launched, and took out, dry, spray is with 1% paradime thylaminobenzaldehyde ethanol solution of sulfuric acid 1 → 10, and it is clear to be heated to the speckle colour developing at 70 ℃, puts respectively under 365nm daylight and the ultra-violet lamp and inspects.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color or the principal spot of yellow fluorescence.
B, get 2 of pad pastings, add water 15ml, heating makes dissolving, the 20ml that adds diethyl ether again, and supersound process 10 minutes is divided and is got ether layer, volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution.Other gets Rhizoma Curcumae control medicinal material 0.5 gram, adds 30～60 ℃ of petroleum ether 20ml, and supersound process 20 minutes filters, and filtrate is concentrated into about 1ml, in contrast medical material solution.Test according to thin layer chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 8.5: 1.5 normal hexane-ethyl acetates was developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear that hot blast blows to the speckle colour developing.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color.
C, get 5 of pad pastings, shred, add ethanol 50ml, put in the water-bath reflux 1 hour, and put coldly, filter, filtrate evaporate to dryness, residue add 0.1mol/L hydrochloric acid 20ml makes dissolving, with 60～90 ℃ of Petroleum ether extraction 2 times, each 10ml, the water intaking layer is regulated pH value to 9～10 with strong aqua ammonia, reuse ether extraction 3 times, each 20ml merges ether solution, volatilize, residue adds chloroform 0.5ml makes dissolving, as need testing solution.Other gets the tetrahydropalmatine reference substance, adds ethanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin layer chromatography, draw each 3 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose that contains 2% sodium hydroxide, be developing solvent with 7.5: 4: 1 normal hexane-chloroform-methanols, launch, take out, dry, put in the iodine vapor and smoked 5 minutes, take out, placed 30 minutes, and put under the 365nm ultra-violet lamp and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
D, get and differentiate need testing solution under the c item, as need testing solution.Other gets peimine, peiminine reference substance, adds chloroform and makes the solution that every 1ml contains 0.5mg respectively, in contrast product solution.Test according to thin layer chromatography, draw need testing solution 10 μ l, each 5 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 8: 4: 1 benzene-ethyl acetate-diethylamine was developing solvent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution and sodium nitrite ethanol test solution.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
E, get and differentiate need testing solution under the b item, as need testing solution.Other gets the imperatorin reference substance, adds ethyl acetate and makes the solution that every 1ml contains 1mg, in contrast product solution.Test according to thin layer chromatography, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 30～60 ℃ of following 3: 2 petroleum ether-ether is developing solvent, launching below 25 ℃, take out, dry, put under the 365nm ultra-violet lamp and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
F, get and differentiate need testing solution under the b item, as need testing solution.Other gets the Borneolum Syntheticum reference substance, and chlorination is copied into the solution that every 1ml contains 2mg, in contrast product solution.Test according to thin layer chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 17: 3 cyclohexane extraction-ethyl acetates was developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to the speckle colour developing at 105 ℃.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
Embodiment 5:
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; Acetonitrile-water-phosphoric acid (17: 83: 0.01) is mobile phase; Detect wavelength 284nm.Number of theoretical plate calculates by Hesperidin standard substance peak should be not less than 2000.
The preparation of reference substance solution: it is an amount of that precision takes by weighing 105 ℃ of Hesperidin reference substances that are dried to constant weight, adds methanol and make the solution that every 1ml contains 0.1mg, shakes up, promptly.
The preparation of need testing solution: get this product under the weight differential item, shred, get about 0.3g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds claims decide weight, and supersound process 1 hour is put coldly, and weight decided in title again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly.
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly.
Every of this product contains Pericarpium Citri Reticulatae Viride with Hesperidin (C
28H
34O
15) meter, must not be less than 6.0mg.
Embodiment 6:
Pericarpium Citri Reticulatae Viride 100g Radix Bupleuri 100g Semen Vaccariae
(stir-fry)100g
Rhizoma sparganic 100g Rhizoma Curcumae 100g Rhizoma Corydalis
(vinegar system)100g
Pollen Typhae 100g Bulbus Fritillariae Thunbergii 100g Spica Prunellae 100g
Radix Angelicae Dahuricae 100g Borneolum Syntheticum 10g
PVA
05-8880g; PVP
K-3010g; Glycerol 4ml (50g); Azone 1ml (9g); Water 400g
(4cm * 6cm), every medicine film contains crude drug 2.525g to make 400 of pad pastings by preparation technology of the present invention.Cyclomastopathy patient uses, every side hypertrophy place 1 subsides every day.
Claims (10)
1, a kind of pharmaceutical composition for the treatment of cyclomastopathy is characterized in that this pharmaceutical composition is to be made by following bulk drugs:
Pericarpium Citri Reticulatae Viride 80-120 Radix Bupleuri 80-120 does not stay capable 80-120
Rhizoma sparganic 80-120 Rhizoma Curcumae 80-120 Rhizoma Corydalis 80-120
Pollen Typhae 80-120 Bulbus Fritillariae Thunbergii 80-120 Spica Prunellae 80-120
Radix Angelicae Dahuricae 80-120 Borneolum Syntheticum 8-12.
2, pharmaceutical composition as claimed in claim 1 is characterized in that this pharmaceutical composition is to be made by following bulk drugs:
Pericarpium Citri Reticulatae Viride 100 Radix Bupleuri 100 Semen Vaccariae (parched)s 100
Rhizoma sparganic 100 Rhizoma Curcumae 100 Rhizoma Corydalis (processed with vinegar) 100
Pollen Typhae 100 Bulbus Fritillariae Thunbergiis 100 Spica Prunellaes 100
The Radix Angelicae Dahuricae 100 Borneolum Syntheticums 10.
3, pharmaceutical composition according to claim 1 and 2 is characterized in that said composition makes the tablet of clinical acceptance, soft capsule, capsule, granule, drop pill, oral liquid or membrane.
4, the preparation method of pharmaceutical composition membrane as claimed in claim 1 or 2 is characterized in that this method may further comprise the steps:
Get Pericarpium Citri Reticulatae Viride, Radix Bupleuri, Semen Vaccariae (parched), rhizoma sparganic, Rhizoma Curcumae, Rhizoma Corydalis (processed with vinegar), Pollen Typhae, Bulbus Fritillariae Thunbergii, Spica Prunellae and the Radix Angelicae Dahuricae and add twice of 50-70% alcohol reflux, each 1-3 hour, filter, merging filtrate, decompression recycling ethanol and when being concentrated into 60 ℃ relative density be 1.10～1.11, Chinese medicine extraction extractum is standby; Other gets polyvinyl alcohol 65-95 weight portion, with washing with alcohol twice, adds water 320-480 weight portion, heating makes dissolving, adds polyvinylpyrrolidone 8-12 weight portion, glycerol 40-60 weight portion, azone 7-11 weight portion and with the Borneolum Syntheticum of 80% dissolve with ethanol, mixing merges with above-mentioned concentrated solution, stir evenly, get the pad pasting drug extract, even all coatings, dry demoulding, film-making, packing promptly gets pad pasting.
5, preparation of drug combination method as claimed in claim 4 is characterized in that this method may further comprise the steps:
Get Pericarpium Citri Reticulatae Viride, Radix Bupleuri, Semen Vaccariae (parched), rhizoma sparganic, Rhizoma Curcumae, Rhizoma Corydalis (processed with vinegar), Pollen Typhae, Bulbus Fritillariae Thunbergii, Spica Prunellae and the Radix Angelicae Dahuricae and add twice of 60% alcohol reflux, each 2 hours, filter, merging filtrate, decompression recycling ethanol and when being concentrated into 60 ℃ relative density be 1.10～1.11, Chinese medicine extraction extractum is standby; Other gets polyvinyl alcohol 80 weight portions, with washing with alcohol twice, adds water 400 weight portions, heating makes dissolving, adds polyvinylpyrrolidone 10 weight portions, glycerol 50 weight portions, azone 9 weight portions and with the Borneolum Syntheticum of 80% dissolve with ethanol, mixing merges with above-mentioned concentrated solution, stir evenly, get the pad pasting drug extract, even all coatings, dry demoulding, film-making, packing, promptly.
6, the method for quality control of pharmaceutical composition as claimed in claim 1 or 2 is characterized in that discrimination method in this method comprises one or more in the following discriminating:
A, get 4 of pad pastings, shred, add methanol 20-30ml, put in the water-bath reflux 1-1.5 hour, put coldly, filter, filtrate evaporate to dryness, residue add water 20-30ml makes dissolving, extract 2 times with the ether jolting, each 15-25ml discards ether solution, the reuse water-saturated n-butanol extracts 3 times, and each 20-30ml merges n-butyl alcohol liquid, the ammonia solution that adds equal volume, jolting is placed and is made layering, get n-butyl alcohol liquid evaporate to dryness, residue adds methanol 1-3ml makes dissolving, as need testing solution; Other gets Radix Bupleuri control medicinal material 1g, the 20-30ml that adds diethyl ether, and reflux 30-60 minute, discard ether solution, medicinal residues volatilize, and add methanol 30-40ml, and reflux 30-60 minute, filter, filtrate evaporate to dryness, residue add methanol 1-3ml makes dissolving, in contrast medical material solution; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 10-15: 40-50: 20-25: lower floor's solution that 8-12 chloroform-ethyl acetate-methanol-water is placed below 10 ℃ is developing solvent, launches, and takes out, dry, spray is with 1% paradime thylaminobenzaldehyde ethanol solution of sulfuric acid 1 → 10, and it is clear to be heated to the speckle colour developing at 70 ℃, puts respectively under 365nm daylight and the ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color or the principal spot of yellow fluorescence;
B, get 2 of pad pastings, add water 20-30ml, heating makes dissolving, the 15-25ml that adds diethyl ether again, and supersound process 10-15 minute, divide and get ether layer, volatilize, residue adds ethyl acetate 1-3ml makes dissolving, as need testing solution; Other gets Rhizoma Curcumae control medicinal material 0.5 gram, adds 30～60 ℃ of petroleum ether 15-25ml, and supersound process 20-30 minute, filter, filtrate is concentrated into 1ml, in contrast medical material solution; Test according to thin layer chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 8-10: 1-2 normal hexane-ethyl acetate is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear that hot blast blows to the speckle colour developing; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color;
C, get 5 of pad pastings, shred, add ethanol 40-60ml, put in the water-bath reflux 1-1.5 hour, and put coldly, filter, filtrate evaporate to dryness, residue add 0.1mol L hydrochloric acid 15-25ml makes dissolving, with 60～90 ℃ of Petroleum ether extraction 2 times, each 10-15ml, the water intaking layer is regulated pH value to 9～10 with strong aqua ammonia, reuse ether extraction 3 times, each 15-25ml merges ether solution, volatilize, residue adds chloroform 0.5ml makes dissolving, as need testing solution; Other gets the tetrahydropalmatine reference substance, adds ethanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 3 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose that contains 2% sodium hydroxide, with 7-9: 3-5: 1-1.5 normal hexane-chloroform-methanol is developing solvent, launches, take out, dry, put in the iodine vapor and smoked 5-10 minute, take out, placed 30-40 minute, and put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
D, get and differentiate need testing solution under the c item, as need testing solution; Other gets peimine, peiminine reference substance, adds chloroform and makes the solution that every 1ml contains 0.5mg respectively, in contrast product solution; Test according to thin layer chromatography, draw need testing solution 10 μ l, each 5 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 7-9: 3-5: 1-1.5 benzene-ethyl acetate-diethylamine is developing solvent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution and sodium nitrite ethanol test solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
E, get and differentiate need testing solution under the b item, as need testing solution; Other gets the imperatorin reference substance, adds ethyl acetate and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 30～60 ℃ of 3-4: 2-3 petroleum ether-ether is developing solvent, launching below 25 ℃, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
F, get and differentiate need testing solution under the b item, as need testing solution; Other gets the Borneolum Syntheticum reference substance, and chlorination is copied into the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin layer chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 15-18: 2-4 cyclohexane extraction-ethyl acetate is developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
7, the method for quality control of pharmaceutical composition as claimed in claim 6 is characterized in that discrimination method in this method comprises one or more in the following discriminating:
A, get 4 of pad pastings, shred, add methanol 30ml, put in the water-bath reflux 1 hour, put coldly, filter, filtrate evaporate to dryness, residue add water 20ml makes dissolving, extract 2 times with the ether jolting, each 15ml discards ether solution, the reuse water-saturated n-butanol extracts 3 times, and each 20ml merges n-butyl alcohol liquid, the ammonia solution that adds equal volume, jolting is placed and is made layering, get n-butyl alcohol liquid evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution; Other gets Radix Bupleuri control medicinal material 1g, the 30ml that adds diethyl ether, and reflux 30 minutes discards ether solution, and medicinal residues volatilize, and add methanol 30ml, and reflux 30 minutes filters, and filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, in contrast medical material solution; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 15: 40: 22: lower floor's solution that 10 chloroforms-ethyl acetate-methanol-water is placed below 10 ℃ was developing solvent, launched, and took out, dry, spray is with 1% paradime thylaminobenzaldehyde ethanol solution of sulfuric acid 1 → 10, and it is clear to be heated to the speckle colour developing at 70 ℃, puts respectively under 365nm daylight and the ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color or the principal spot of yellow fluorescence;
B, get 2 of pad pastings, add water 15ml, heating makes dissolving, the 20ml that adds diethyl ether again, and supersound process 10 minutes is divided and is got ether layer, volatilizes, and residue adds ethyl acetate 1ml makes dissolving, as need testing solution; Other gets Rhizoma Curcumae control medicinal material 0.5 gram, adds 30～60 ℃ of petroleum ether 20ml, and supersound process 20 minutes filters, and filtrate is concentrated into 1ml, in contrast medical material solution; Test according to thin layer chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 8.5: 1.5 normal hexane-ethyl acetates was developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear that hot blast blows to the speckle colour developing; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the principal spot of same color;
C, get 5 of pad pastings, shred, add ethanol 50ml, put in the water-bath reflux 1 hour, and put coldly, filter, filtrate evaporate to dryness, residue add 0.1mol L hydrochloric acid 20ml makes dissolving, with 60～90 ℃ of Petroleum ether extraction 2 times, each 10ml, the water intaking layer is regulated pH value to 9～10 with strong aqua ammonia, reuse ether extraction 3 times, each 20ml merges ether solution, volatilize, residue adds chloroform 0.5ml makes dissolving, as need testing solution; Other gets the tetrahydropalmatine reference substance, adds ethanol and makes the solution that every 1ml contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 3 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose that contains 2% sodium hydroxide, be developing solvent with 7.5: 4: 1 normal hexane-chloroform-methanols, launch, take out, dry, put in the iodine vapor and smoked 5 minutes, take out, placed 30 minutes, and put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
D, get and differentiate need testing solution under the c item, as need testing solution; Other gets peimine, peiminine reference substance, adds chloroform and makes the solution that every 1ml contains 0.5mg respectively, in contrast product solution; Test according to thin layer chromatography, draw need testing solution 10 μ l, each 5 μ l of reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 8: 4: 1 benzene-ethyl acetate-diethylamine was developing solvent, launch, take out, dry, spray is with rare bismuth potassium iodide test solution and sodium nitrite ethanol test solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
E, get and differentiate need testing solution under the b item, as need testing solution; Other gets the imperatorin reference substance, adds ethyl acetate and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw need testing solution 10 μ l, reference substance solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 30～60 ℃ of following 3: 2 petroleum ether-ether is developing solvent, launching below 25 ℃, take out, dry, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color;
F, get and differentiate need testing solution under the b item, as need testing solution; Other gets the Borneolum Syntheticum reference substance, and chlorination is copied into the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin layer chromatography, draw each 2 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with 17: 3 cyclohexane extraction-ethyl acetates was developing solvent, launch, take out, dry, spray is with 5% vanillin sulfuric acid solution, and it is clear to be heated to the speckle colour developing at 105 ℃; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
8, the method for quality control of pharmaceutical composition as claimed in claim 1 or 2 is characterized in that the assay in this method is:
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; 15-20: 80-85: acetonitrile-water-phosphoric acid of 0.01 is mobile phase; Detect wavelength 284nm; Number of theoretical plate calculates by Hesperidin standard substance peak should be not less than 2000;
The preparation of reference substance solution: it is an amount of that precision takes by weighing 105 ℃ of Hesperidin reference substances that are dried to constant weight, adds methanol and make the solution that every 1ml contains 0.1mg, shakes up, promptly;
The preparation of need testing solution: get this product under the weight differential item, shred, get 0.2-0.5g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds, claim to decide weight, supersound process 1-1.5 hour, put cold, claim to decide weight again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly;
Every of this product contains Pericarpium Citri Reticulatae Viride with Hesperidin C
28H
34O
15Meter must not be less than 6.0mg.
9, the method for quality control of pharmaceutical composition as claimed in claim 8 is characterized in that the assay in this method is:
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; Acetonitrile-water-phosphoric acid of 17: 83: 0.01 is mobile phase; Detect wavelength 284nm; Number of theoretical plate calculates by Hesperidin standard substance peak should be not less than 2000;
The preparation of reference substance solution: it is an amount of that precision takes by weighing 105 ℃ of Hesperidin reference substances that are dried to constant weight, adds methanol and make the solution that every 1ml contains 0.1mg, shakes up, promptly;
The preparation of need testing solution: get this product under the weight differential item, shred, get 0.3g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds claims decide weight, and supersound process 1 hour is put coldly, and weight decided in title again, supply with methanol and to subtract weight loss, shake up, filter, get subsequent filtrate, promptly;
Algoscopy: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly;
Every of this product contains Pericarpium Citri Reticulatae Viride with Hesperidin C
28H
34O
15Meter must not be less than 6.0mg.
10, the application of pharmaceutical composition as claimed in claim 1 or 2 in the medicine of preparation treatment cyclomastopathy.
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| CNB2004100374063A CN1305512C (en) | 2004-04-30 | 2004-04-30 | Traditional Chinese medicine compound recipe for treating hyperplasia of mammary glands and preparation method thereof |
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| CNB2004100374063A CN1305512C (en) | 2004-04-30 | 2004-04-30 | Traditional Chinese medicine compound recipe for treating hyperplasia of mammary glands and preparation method thereof |
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| Publication Number | Publication Date |
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| CN1689626A CN1689626A (en) | 2005-11-02 |
| CN1305512C true CN1305512C (en) | 2007-03-21 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100475904C (en) * | 2005-07-11 | 2009-04-08 | 佛山市顺德区汉达精密电子科技有限公司 | Polycarbonate/ABS composition |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101564521B (en) * | 2009-05-20 | 2011-06-22 | 石振刚 | External plaster for treating hyperplasia of mammary glands |
| CN102698204B (en) * | 2012-06-19 | 2014-01-15 | 广西嘉进药业有限公司 | Zhuang nationality transdermal patch for treating hyperplasia of mammary glands and preparation method of Zhuang nationality transdermal patch |
| CN105954388A (en) * | 2016-04-22 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Method for determining peimine and peininine in Zhejiang fritillaria bulb by using ultrasonic extraction process |
| CN106539945A (en) * | 2016-12-08 | 2017-03-29 | 遵义医学院 | A kind of Chinese medicine and preparation method for treating cyclomastopathy |
| CN108434339A (en) * | 2018-06-11 | 2018-08-24 | 孙丽琴 | Eye cold compress patch and preparation method thereof |
| CN109078154A (en) * | 2018-10-31 | 2018-12-25 | 中山市六安堂健康管理有限公司 | A kind of pharmaceutical composition and its preparation method and application for treating the proliferation of mammary gland |
| CN109045250A (en) * | 2018-11-08 | 2018-12-21 | 汤凤池 | A kind of cyclomastopathy Chinese medicine and preparation method thereof |
| CN113588815B (en) * | 2021-07-13 | 2023-02-28 | 西安千禾药业股份有限公司 | Method for simultaneously detecting content of multiple active ingredients in Rupishu capsule |
| CN113866298B (en) * | 2021-09-22 | 2023-05-23 | 山东中医药大学 | Quality evaluation method for distinguishing green tangerine peel from vinegar green tangerine peel |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1246354A (en) * | 1999-08-20 | 2000-03-08 | 张四岩 | Medicinal composition for treating hyperplasia of mammary gland disease |
| CN1468609A (en) * | 2002-07-18 | 2004-01-21 | 王泽淇 | Bolus for curing mastosis quickly |
-
2004
- 2004-04-30 CN CNB2004100374063A patent/CN1305512C/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1246354A (en) * | 1999-08-20 | 2000-03-08 | 张四岩 | Medicinal composition for treating hyperplasia of mammary gland disease |
| CN1468609A (en) * | 2002-07-18 | 2004-01-21 | 王泽淇 | Bolus for curing mastosis quickly |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100475904C (en) * | 2005-07-11 | 2009-04-08 | 佛山市顺德区汉达精密电子科技有限公司 | Polycarbonate/ABS composition |
Also Published As
| Publication number | Publication date |
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| CN1689626A (en) | 2005-11-02 |
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