CN102288701B - Method for detecting Chinese medicinal composition for freeing lung and relieving asthma - Google Patents
Method for detecting Chinese medicinal composition for freeing lung and relieving asthma Download PDFInfo
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- CN102288701B CN102288701B CN201110212969.1A CN201110212969A CN102288701B CN 102288701 B CN102288701 B CN 102288701B CN 201110212969 A CN201110212969 A CN 201110212969A CN 102288701 B CN102288701 B CN 102288701B
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Abstract
The invention discloses a method for detecting a Chinese medicinal composition for freeing lung and relieving asthma. The Chinese medicinal composition consists of the following raw material medicines: ephedra, ginger, cassia twig, bitter apricot seed, Chinese magnoliavine fruit (prepared) and roasted liquorice. The detection method comprises the steps of detecting the ephedrine content of the ephedra and detecting the Schizandrol content of the Chinese magnoliavine fruit.
Description
The present invention is for dividing an application, and original bill application number is 200710122488.5, and the original bill applying date is on 09 26th, 2007, and original bill denomination of invention is: Chinese medicine composition of freeinging lung and relieving asthma and preparation method thereof and method of quality control.
Technical field
The present invention relates to a kind ofly for declaring lung, the detection method of the Chinese medicine composition of relievining asthma, relates in particular to a kind ofly for declaring lung, and the detection method of the capsule of relievining asthma, belongs to technical field of traditional Chinese medicines.
Background technology
Chronic obstructive pulmonary disease is a kind of commonly encountered diseases of internal department, frequently-occurring disease, is a kind of important chronic respiratory disease, and ill patient's number is many, dead rate is high, due to its protracted inflammation, have a strong impact on patient's labour capacity and quality of life, and be subject to the generally attention of countries in the world.
At present, medical circle there is no the development that way is controlled the chronic obstructive pulmonary disease state of an illness effectively, seeks to prevent that the effective ways of chronic obstructive pulmonary disease from being domestic and international medical circle problem anxious to be resolved.Pathogenesis of chronic obstructive pulmonary disease mechanism is not yet completely clear, generally believe that at present chronic obstructive pulmonary disease is that to take air flue, pulmonary parenchyma and pulmonary vascular chronic inflammation be feature, at different parts alveolation macrophage, T lymphocyte and the neutrophil leucocyte of lung, increase.The inflammatory cell activating discharges medium, destroys the structure of lung and (or) promotes neutrophil leucocyte inflammatory reaction.Suck deleterious particle or gas and can cause lung inflammation; Smoking can be induced inflammation and directly be damaged lungs.Except inflammation, the proteinase of lung and antiprotease system imbalance, and oxidation antioxidation unbalance also play a part important.Modern medicine is by drug therapy for chronic obstructive pulmonary disease at present, and prevention and complication, improve symptom, is mainly expiratory dyspnea, improves the quality of living, and removes the inducement of acute exacerbations in patients with chronic obstructive pulmonary disease, enjoins patient's smoking cessation.Because this patient's immunity of organisms reduces and to breathe defense mechanism impaired, easily there is respiratory tract infection, repeated infection causes bronchial mucosa congestion and edema, and glandular hyperplasia is loose, and secreting function is hyperfunction, and tube wall thickens, narrow, increases the weight of airflow obstruction.The pathogen that infects is common is bacterium, virus, mycoplasma etc.Western modern medicine mainly adopts antibacterials, hormone for bacterium, and there is no active drug for infecting due to virus, in addition antibacterials, hormone have again certain toxic and side effect, in addition, even if acute stage is eased through treatment, still can not effectively control its recurrence.Therefore, for better relieve chronic obstructive disease of lung development, control its recurrence, improve life quality, Treatment of COPD is standardized more, the prescription medicine of finding safe and effective treatment Patients with Chronic Obstructive Pulmonary Disease is very necessary.
Summary of the invention
The object of the invention is to provide a kind of a surname's of having lung, the Chinese medicine composition of antiasthmatic effect;
The object of the invention is also to provide a kind of a surname's of having lung, the Chinese medicine composition preparation method of antiasthmatic effect;
The object of the invention is also to provide a kind of a surname's of having lung, the method for quality control of the Chinese medicine composition of antiasthmatic effect.
The present invention seeks to be achieved through the following technical solutions:
Of the present invention have a surname's lung, and the Chinese medicine composition of antiasthmatic effect is to be made by the bulk drug of following weight ratio:
Chinese ephedra 3000-4000 weight portion, ginger 2000-3000 weight portion, cassia twig 2000-3000 weight portion, semen armeniacae amarae 1000-1500 weight portion, the fruit of Chinese magnoliavine (system) 250-500 weight portion, honey-fried licorice root 80-160 weight portion
Above-mentioned raw materials optimum ratio is:
Chinese ephedra 3000-3200 weight portion, ginger 2800-3000 weight portion, cassia twig 2800-3000 weight portion, semen armeniacae amarae 1400-1500 weight portion, the fruit of Chinese magnoliavine (system) 300-400 weight portion, honey-fried licorice root 100-120 weight portion
Above-mentioned raw materials optimum ratio is:
Chinese ephedra 3000g, ginger 3000g, cassia twig 3000g, semen armeniacae amarae 1500g, the fruit of Chinese magnoliavine (system) 375g, honey-fried licorice root 112.5g
The preparation method of Chinese medicine composition of the present invention comprises the following steps:
Above medicinal material is except semen armeniacae amarae, and other gomi herbs, adopt circulated in countercurrent extraction process boiling 1-3 time, each 2-4 hour, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), let cool to room temperature, add equivalent ethanol, stir evenly, standing 12-36 hour, get supernatant, reclaim ethanol and be concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃); Semen armeniacae amarae rinsing dedust soil, drains the water and splits dish above, puts into pressure sterilizing pot, and control vapor pressure is 0.01～0.03kpa/cm
2(103 ℃), hot pressing boiling 20-40 minute, taking-up is put into a small amount of cold water and is soaked, and removes kind of a skin, dries, and obtains almond processed product.By above-mentioned thick paste and the vacuum drying of almond processed product, pulverize, according to this area routine techniques, make the preparation of clinical acceptance, as: capsule, tablet, granule, soft capsule, pill.
The preparation method of Chinese medicinal composition capsules agent of the present invention is: get bulk drug Chinese ephedra 3000g, ginger 3000g, cassia twig 3000g, semen armeniacae amarae 1500g, the fruit of Chinese magnoliavine (system) 375g, honey-fried licorice root 112.5g
It is characterized in that the method comprises the following steps:
Above medicinal material is except semen armeniacae amarae, and other gomi herbs, adopt circulated in countercurrent extraction process boiling 2 times, each 2 hours, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), let cool to room temperature, add equivalent ethanol, stir evenly, standing 24 hours, get supernatant, reclaim ethanol and be concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃); Semen armeniacae amarae rinsing dedust soil, drains the water and splits dish above, puts into pressure sterilizing pot, and control vapor pressure is 0.01～0.03kpa/cm
2(103 ℃), hot pressing boiling 30 minutes, taking-up is put into a small amount of cold water and is soaked, and removes kind of a skin, dries, and obtains almond processed product.By above-mentioned thick paste and the vacuum drying of almond processed product, pulverize, add dextrin appropriate, mix, incapsulate, make 1000, obtain.
Pharmaceutical composition method of quality control of the present invention comprises one or more in following discrimination method and/or assay:
(1) get medicine of the present invention and be equivalent to crude drug 9-11g, add strong ammonia solution 1-3ml, then add methenyl choloride 30-50ml, add hot reflux 0.5-2 hour, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get ephedrine hydrochloride, add methyl alcohol and make every 1ml containing the solution of 1mg, in contrast product solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each 3～5 μ l of above-mentioned two kinds of solution, put respectively in same and take on the silica gel g thin-layer plate that sodium carboxymethyl cellulose is binder, take methenyl choloride-methyl alcohol-strong ammonia solution (15-25: 3-10: 0.5-2) be developping agent, launch, take out, dry, spray, with 5% ethanol solution of ninhydrin, is heated to spot colour developing at 105 ℃ clear.In test sample chromatogram, with the corresponding position of reference substance chromatogram on, aobvious identical aubergine spot.
(2) get medicine of the present invention and be equivalent to crude drug 200-220g, the 20-40ml that adds diethyl ether, ultrasonic 20-40 minute (power 300W, frequency 50kHz), filters, and filtrate volatilizes, and residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get rhizoma zingiberis control medicinal material 1g, add water 80-100ml and decoct 1 hour, filter, filtrate is concentrated into 20-30ml, adds equal-volume sherwood oil (60～90 ℃) extraction, gets upper strata liquid, evaporate to dryness, and residue adds 1ml methyl alcohol to be made to dissolve, in contrast medicinal material solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw control medicinal material solution 5 μ l, need testing solution 15 μ l, put respectively on same silica gel g thin-layer plate, take sherwood oil (60～90 ℃)-ethyl acetate (3-7: 0.5-2) be developping agent, second outspread, takes out, and dries, spray, with 5% vanillic aldehyde sulfuric acid solution, is heated to spot colour developing at 105 ℃ clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
(3) get medicine of the present invention and be equivalent to crude drug 200-220g, add sherwood oil (60～90 ℃) 40-60ml, add hot reflux 0.5-2 hour, filter, residue adds methyl alcohol 40-60ml, adds hot reflux 0.5-2 hour, filter, filtrate evaporate to dryness, residue adds water 30-50ml to be made to dissolve, and with water saturated normal butyl alcohol jolting, extracts 2-4 time, each 10-30ml, merge normal butyl alcohol liquid, with the saturated water washing of normal butyl alcohol 2-4 time, at every turn 20-40ml, get normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Another extracting Radix Glycyrrhizae control medicinal material 0.5g, is made in the same way of control medicinal material solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each 3～5 μ l of above-mentioned two kinds of solution, put respectively in same and take on the silica gel g thin-layer plate that sodium carboxymethyl cellulose is binder, take methenyl choloride-methanol-water (10-15: 5-10: lower floor's solution 1-3) is developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot colour developing at 105 ℃ clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
[assay]
(1) according to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2005), measure.
Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; Take 0.1% phosphoric acid (containing 0.1% triethylamine)-acetonitrile (90-100: 0-10) be mobile phase; Detection wavelength is 210nm.Number of theoretical plate calculates and should be not less than 2500 by ephedrine hydrochloride peak.
It is appropriate that ephedrine hydrochloride reference substance is got in the preparation of reference substance solution, accurately weighed, with methyl alcohol, dissolves and make every 1ml containing the solution of 50 μ g, and product solution, obtains in contrast.
The preparation of need testing solution is got medicine of the present invention and is equivalent to crude drug 3-5g, accurately weighed, and to 50ml volumetric flask, precision adds the hydrochloric acid solution 1ml of 0.5mol/L, add again methyl alcohol to scale, jam-pack, ultrasonic processing 20-40 minute, let cool, miillpore filter (0.45 μ m) filters, and obtains.
Determination method is accurate reference substance solution and each 5 μ l～10 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains.
The daily metering of medicine of the present invention contains Chinese ephedra with ephedrine hydrochloride (C
10h
15nOHCl) amount meter, must not be less than 8.0mg.
(2) according to high performance liquid chromatography (appendix VI D), measure.
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Acetonitrile-methyl alcohol-0.1% phosphoric acid (35-40: 15-20: 40-50) be mobile phase; Detection wavelength is 250nm.Number of theoretical plate calculates and should be not less than 3000 by schizandrin peak.
Schizandrin reference substance 1.5mg is got in the preparation of reference substance solution, accurately weighed, puts in 50ml measuring bottle, with methyl alcohol, dissolves and is diluted to scale, shakes up, and obtains (every 1ml is containing schizandrin 0.03mg).
The preparation of need testing solution is got medicine of the present invention and is equivalent to crude drug 100-110g, accurately weighed, puts in tool plug conical flask, and precision adds methyl alcohol 50ml, weighed weight, ultrasonic processing (power 250W, frequency 20kHz) 10-30min, take out, let cool, more weighed weight, add methyl alcohol and supply the weight of minimizing, filter, the accurate subsequent filtrate 20ml that draws, evaporate to dryness, residue adds methyl alcohol to be made to dissolve, and is dissolved to 5ml, with miillpore filter (0.45 μ m), filter, get subsequent filtrate, obtain.
Determination method is accurate reference substance solution and each 10 μ l of test solution of drawing respectively, and injection liquid chromatography, measures, and obtains.
Medicine of the present invention is containing schizandrin (C
24h
32o
7) must not be less than 0.40%.
Pharmaceutical composition method of quality control of the present invention is preferably as follows one or more in discrimination method and/or assay:
(1) get medicine of the present invention and be equivalent to crude drug 10.8g, add strong ammonia solution 2ml, then add methenyl choloride 40ml, add hot reflux 1 hour, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get ephedrine hydrochloride, add methyl alcohol and make every 1ml containing the solution of 1mg, in contrast product solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each 3～5 μ l of above-mentioned two kinds of solution, put respectively in same and take on the silica gel g thin-layer plate that sodium carboxymethyl cellulose is binder, methenyl choloride-methyl alcohol-the strong ammonia solution (20: 5: 0.5) of take is developping agent, launch, take out, dry, spray, with 5% ethanol solution of ninhydrin, is heated to spot colour developing at 105 ℃ clear.In test sample chromatogram, with the corresponding position of reference substance chromatogram on, aobvious identical aubergine spot.
(2) get medicine of the present invention and be equivalent to crude drug 216.25g, the 30ml that adds diethyl ether, ultrasonic 30 minutes (power 300W, frequency 50kHz), filters, and filtrate volatilizes, and residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get rhizoma zingiberis control medicinal material 1g, add water 80ml and decoct 1 hour, filter, filtrate is concentrated into 20ml, adds equal-volume sherwood oil (60～90 ℃) extraction, gets upper strata liquid, evaporate to dryness, and residue adds 1ml methyl alcohol to be made to dissolve, in contrast medicinal material solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw control medicinal material solution 5 μ l, need testing solution 15 μ l, put respectively on same silica gel g thin-layer plate, sherwood oil (60～90 ℃)-ethyl acetate (5: 1) of take are developping agent, second outspread, takes out, and dries, spray, with 5% vanillic aldehyde sulfuric acid solution, is heated to spot colour developing at 105 ℃ clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
(3) get medicine of the present invention and be equivalent to crude drug 216.25g, add sherwood oil (60～90 ℃) 50ml, add hot reflux 1 hour, filter, residue adds methyl alcohol 50ml, adds hot reflux 1 hour, filter, filtrate evaporate to dryness, residue adds water 40ml to be made to dissolve, with water saturated normal butyl alcohol jolting, extract 3 times, each 20ml, merges normal butyl alcohol liquid, with the saturated water washing of normal butyl alcohol 3 times, each 30ml, gets normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Another extracting Radix Glycyrrhizae control medicinal material 0.5g, is made in the same way of control medicinal material solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw each 3～5 μ l of above-mentioned two kinds of solution, put respectively in same and take on the silica gel g thin-layer plate that sodium carboxymethyl cellulose is binder, the lower floor's solution of methenyl choloride-methanol-water (13: 7: 2) of take is developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot colour developing at 105 ℃ clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.
[assay]
(1) according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2005), measure.
Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; 0.1% phosphoric acid (containing 0.1% the triethylamine)-acetonitrile (97: 3) of take is mobile phase; Detection wavelength is 210nm.Number of theoretical plate calculates and should be not less than 2500 by ephedrine hydrochloride peak.
It is appropriate that ephedrine hydrochloride reference substance is got in the preparation of reference substance solution, accurately weighed, with methyl alcohol, dissolves and make every 1ml containing the solution of 50 μ g, and product solution, obtains in contrast.
The preparation of need testing solution is got medicine of the present invention and is equivalent to crude drug 4.3g, accurately weighed, and to 50ml volumetric flask, precision adds the hydrochloric acid solution 1ml of 0.5mol/L, add again methyl alcohol to scale, jam-pack, ultrasonic processing 30 minutes, let cool, miillpore filter (0.45 μ m) filters, and obtains.
Determination method is accurate reference substance solution and each 5 μ l～10 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains.
The daily metering of medicine of the present invention contains Chinese ephedra with ephedrine hydrochloride (C
10h
15nOHCl) amount meter, must not be less than 8.0mg.
(2) according to high performance liquid chromatography (appendix VI D), measure.
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filling agent; Acetonitrile-methyl alcohol-0.1% phosphoric acid (39: 16.7: 44.3) is mobile phase; Detection wavelength is 250nm.Number of theoretical plate calculates and should be not less than 3000 by schizandrin peak.
Schizandrin reference substance 1.5mg is got in the preparation of reference substance solution, accurately weighed, puts in 50ml measuring bottle, with methyl alcohol, dissolves and is diluted to scale, shakes up, and obtains (every 1ml is containing schizandrin 0.03mg).
The preparation of need testing solution is got medicine of the present invention and is equivalent to crude drug 108.1g, accurately weighed, puts in tool plug conical flask, and precision adds methyl alcohol 50ml, weighed weight, ultrasonic processing (power 250W, frequency 20kHz) 20min, take out, let cool, more weighed weight, add methyl alcohol and supply the weight of minimizing, filter, the accurate subsequent filtrate 20ml that draws, evaporate to dryness, residue adds methyl alcohol to be made to dissolve, and is dissolved to 5ml, with miillpore filter (0.45 μ m), filter, get subsequent filtrate, obtain.
Determination method is accurate reference substance solution and each 10 μ l of test solution of drawing respectively, and injection liquid chromatography, measures, and obtains.
Medicine of the present invention is containing schizandrin (C
24h
32o
7)must not be less than 0.40%.
Embodiment
Following experimental example and embodiment further illustrate but are not limited to down the present invention
Experimental example 1. Chinese drug-treated group of the present invention and composite capsule preparation technology's optimization experiment
1. the screening of water extraction amount of water
Configure every part of 648.8g of 5 parts of medicinal materials, dividing three groups tests, 6 times of amounts of first group of amount of water, 4 times of amounts, 8 times of amounts of second group of amount of water, 6 times of amounts, 10 times of amounts of the 3rd group of amount of water, 8 times of amounts, the 4th group of circulated in countercurrent extraction process adds 2 times of amounts of water, the 5th group of circulated in countercurrent extraction process adds 4 times of amounts of water, and the 6th group of circulated in countercurrent extraction process adds 6 times of amounts of water, take that to go out cream amount clearly after water extraction be index, determine amount of water, the results are shown in Table 1:
Table 1: water extraction amount of water
Take paste volume as index, can find out that the water consumption of circulated in countercurrent extraction process obviously reduces, paste-forming rate is improved, and can reduce concentration time, selects circulated in countercurrent extraction process to add 4 times of amounts of water in production.
2, the screening of alcohol precipitation alcohol adding amount
Configure every part of 648.8g of 3 parts of medicinal materials, minutes three groups test, first group adds 0.5 times of amount of amount of alcohol, and second group adds 1 times of amount of amount of alcohol, and the 3rd group adds 2 times of amounts of amount of alcohol, take after alcohol precipitation and goes out thick paste amount as index, determines alcohol adding amount, the results are shown in Table 2:
Table 2: alcohol precipitation alcohol adding amount
Take paste volume as index, can find out that to add 1 times of ethanol amount paste volume better, in production, select the 1 times of amount of amount of alcohol that adds.
Table 3: important technological parameters
3, pilot scale
Table 4: batch pilot scale production data
The optimization experiment of amygdalate processing procedure in experimental example 2. Chinese drug-treated group of the present invention and thing
Chinese medicine semen armeniacae amarae contains antitussive component amarogentin, under proper condition, as make moist, decoction and water logging etc., itself contained amygdalase easily makes amarogentin decompose, thereby reduce, even loses drug effect.Therefore, semen armeniacae amarae need reach the object that the enzyme that goes out is protected glycosides through concocting.Due to current traditional concocting method: decocting cooking method and the method for the frying enzyme that goes out is protected glycosides poor effect, and quality is restive, thus steam heat platen press introduced herein and classic method is carried out contrast experiment, to explore better concocting method.
1 instrument and reagent
1.1 instruments: YXQ.GY22.600 type horizontal circular pressurized steam sterilizer (Hengyang medical apparatus and instruments factory).
1.2 reagents: silver nitrate is AR level; Potassium iodide, liquor ammoniae fortis is CP level; The raw product of semen armeniacae amarae and goods.
2 methods and result
The assay of amarogentin in 2.1 semen armeniacae amaraes.
Get semen armeniacae amarae (raw product) the about 15g of meal, precise weighing, puts in kjeldahl flask, add water 150ml, close plug, puts in 37 ℃ of water-baths and is incubated 2h immediately, connect condenser pipe, water flowing steam distillation, distillate imports in the absorption liquid of water 10ml and ammonia solution 2ml, and receiving flask is put in ice bath cooling, when reaching 60ml, distillate stops distillation, in distillate, add potassium iodide test solution (16.5%) 2ml, with slowly titration of silver nitrate solution (0.1mol/L), the yellow muddiness of showing to solution does not disappear.1ml silver nitrate solution (0.1mol/L) is equivalent to 91.48mg amarogentin (C
20h
27nO
11).The results are shown in Table 5.
Amarogentin assay in table 5 semen armeniacae amarae
* silver nitrate titration liquid is 0.1008mol/L
As shown in Table 1, in raw semen armeniacae amarae, the average content of amarogentin is 4.343%.
In 2.2 traditional concocted method goods, amarogentin and assay, respectively by decocting cooking method system with fry legal system semen armeniacae amarae and be ground into meal, are respectively got the about 15g of meal.According to said method, carry out assay.The results are shown in Table 6.
Table 6 semen armeniacae amarae tradition concocted method amarogentin measurement result
As shown in Table 2, the average content of frying amarogentin in legal system semen armeniacae amarae is 3.061%; In decocting cooking method semen armeniacae amarae processed, the average content of amarogentin is 2.989%.
The assay of amarogentin in 2.3 steam heat platen press goods
Semen armeniacae amarae 200g cleans post rinse dedust soil, drains the water and splits dish above, puts into pressure sterilizing pot, and control vapor pressure is 0.03kpa/cm
2(103 ℃), hot pressing boiling 30 minutes, taking-up is put into a small amount of cold water and is soaked, and removes kind of a skin, dries.And measure as stated above the content of amarogentin, the results are shown in Table 7.
Amarogentin measurement result after table 7 steam heat platen press process of preparing Chinese medicine semen armeniacae amarae
As shown in Table 3, in steam heat platen press process of preparing Chinese medicine semen armeniacae amarae, the average content of amarogentin is 4.059%.
3 results
3.1 semen armeniacae amaraes at present conventional concocting method are decocting cooking method and fry method, and these two kinds method is simple, and applicability is strong, but the enzyme weak effect that goes out, residual enzyme can also continue to decompose glycosides.Decocting cooking method is concocted semen armeniacae amarae and is dropped in boiling water, causes water temperature to decline, and severe patient can be down to 70 ℃, and this provides chance for enzymolysis, and water consumption is large, and part glycosides is water-soluble and lose.The method of frying will be fried to full Huang, just can reach the enzyme effect of going out, and duration and degree of heating temperature is wayward, and products appearance yellowing, affects drug quality.Because when operation human factor (temperature, time are by artificially judging) impact is larger, quality is difficult to homogeneous.
The 3.2 steam hot pressing enzyme process that goes out is concocted semen armeniacae amarae, and temperature is high compared with decocting cooking method, and is not soaked in water, and the enzyme that goes out is effective, and glycosides runs off few.According to experimental result, hot pressing (103 ℃) the enzyme 30min that goes out, the enzyme rate of going out can reach 97.5%.
Experimental example 3. pharmacodynamic experiments
In order to prove curative effect of the present invention, we have carried out following pharmacodynamic experiment.
Following test of pesticide effectiveness medicine group of the present invention used is the capsule according to embodiment 4 preparations;
Positive control drug: the granule that following methods makes:
Chinese ephedra 1200g, ginger 1200g, the fruit of Chinese magnoliavine (system) 150g, honey-fried licorice root 45g
Sucrose 627g, dextrin 260g make 1000g
Above four traditional Chinese medicine material, boiling secondary, 3 hours for the first time, 2 hours for the second time, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), let cool to room temperature, add equivalent ethanol, stir evenly, standing 24 hours, get supernatant, decompression recycling ethanol is also concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃).With sucrose, dextrin, mix, granulation, dry, obtain.
1 material
1.1 reagent ammoniacal liquor, inflexible rafter acid, phenol red, histamine phosphate, acecoline, histamine phosphate.
1.2 instrument 721 spectrophotometers, Shanghai San analytical instrument factory produces.
1.3 animal Kunming mouses, male and female half and half, weight 18-22g; Rat, male and female half and half, weight 200-250g.Cavy 150～200g, ♀ ♂ dual-purpose.
The impact that 2 antitussive effects are cough caused on mouse ammoniacal liquor.48 of mouse, male and female half and half, be divided at random 4 groups, according to the form below dosage gastric infusion, the 1st the sky, afternoon each 1 time, be placed in mouse in 5000ml glass bell jar during 30min after administration in the 2nd day, and constant voltage is by ammoniacal liquor (28%) spray people bell jar, spraying 5s, observes the cough latent period of mouse and the number of times (3min) of coughing.The results are shown in Table 8.
Table 8 medicine group of the present invention on mouse because of the cough caused impact of ammoniacal liquor (x ± s)
Note: compare with physiological saline group
*p < 0.05,
*p < 0.01.
Result: medicine group of the present invention can make the cough caused prolongation of latency of mouse ammoniacal liquor, 3min cough number of times reduces.The impact of 3 phlegm-dispelling functions on the phenol red expectoration amount of Respiratory Tract of Mice, 48 of mouse, male and female half and half, are divided into 4 groups at random, by the various dose gavage medicine of table 1, the 1st the sky, afternoon each 1 time, within the 2nd day, give medicine after 0.5h lumbar injection phenol red solution 0.5ml/ only.After 30min, de-vertebra is put to death, and from thyroid cartilage, inserts about 0.3cm in people's tracheae, with 1ml syringe, draws NaHCO
3solution 0.5ml injects in tracheae, repeatedly push away continuously and take out 3 times, finally with syringe, irrigating solution is extracted out in note people test tube, operate as stated above 3 times, rinse 9 times, draw altogether sodium carbonate liquor 1.5ml, merge the about 1.2-1.5ml of eluate, after centrifugal, get supernatant, the phenol red secretory volume of 721 spectrophotometer eudiometer, the results are shown in Table 9.
Table 9 medicine group of the present invention is on the impact of the phenol red expectoration amount of Respiratory Tract of Mice (x ± s)
Note: compare with physiological saline group:
*p < 0.05,
*p < 0.01, compares ※ P < 0.05 with positive controls.
Result: medicine group of the present invention can make the phenol red expectoration amount of Respiratory Tract of Mice increase.
4 antiasthmatic effects
4.2 medicine groups of the present invention are drawn and are breathed heavily preclinical impact cavy
Guinea pig asthmatic model preparation causes by spraying the method for breathing heavily, select Baby guinea pig, body weight < 200g, insert in the organic glass case of 20cm * 20cm * 150cm, ultrasonic atomizatio sprays into 2% acetylcholine and 0.2% histamine phosphate mixed liquor 15s, screen, draw to breathe heavily and surpass 120s person latent period and will not select.Next day, cavy random packet, medication group per os gives medicine group of the present invention and positive controls (calculating by crude drug amount, lower same), and negative control group gives isometric physiological saline, aminophylline group lumbar injection aminophylline (25mgkg
-1).After medication, 1h ultrasonic atomizatio sprays into 2% acetylcholine and 0.2% histamine phosphate mixed liquor 15s and measures to draw and breathe heavily latent period (from spraying, start to asthma attack, expiratory dyspnea, until the time of twitching and falling down to the ground), the results are shown in Table 10.
Table 10 medicine group of the present invention is on the preclinical impact of Experimental Asthma In Guinea-pigs (x ± s, n=8)
With the comparison of physiological saline group
*p < 0.01
Result: medicine group of the present invention can make Experimental Asthma In Guinea-pigs obviously extend latent period, due to histamine acetylcholine mix is stimulated, asthma has antagonism.
4.3 impacts that guinea-pig isolated tracheal smooth muscle bar is shunk
Isolated helical strips of guinea is prepared in the shrinkage test of guinea-pig isolated tracheal smooth muscle bar, and be placed in and fill in oxygenation Ke-Heng Shi liquid isolated organ mensuration bath (37 ℃), load 2.0g balance, (final concentration is respectively 2.5 * 10 to inject respectively choline or histamine
-7with 5 * 10
-7mmolL
-1), record maximum collapse height, add respectively on this basis medicine group of the present invention and positive controls, aminophylline group, after balance, record shrinks height, the results are shown in Table 11.
On this basis, set up according to a certain percentage 4 dosage groups separately, record it and shrink height.With the log10 dose of the oral liquid contraction inhibiting rate corresponding with it, return calculating, show that its IC50 is respectively 0.095gL
-1(choline, n=4, r=0.989) and 0.345gL
-1(histamine, n=4, r=0.965).
The impact (x ± s, n=6) that table 11 medicine group of the present invention is shunk guinea-pig isolated tracheal smooth muscle
Acetylcholine final concentration: 2.5 * 10
-7mmolL
-1; Histamine final concentration: 5 * 10
-7mmolL
-1; With the comparison of physiological saline group
*p < 0.05,
*p < 0.01
Result: acetylcholine or histamine can cause Airway smooth muscle and strongly shrink, and medicine group of the present invention has obvious relexation to the tracheal smooth muscle of spasm.
4.4 impacts that isolated ileum segments in guinea pigs smooth muscle is shunk
Isolated ileum segments in guinea pigs 2cm is prepared in the shrinkage test of isolated ileum segments in guinea pigs smooth muscle, be placed in and fill in oxygenation tyrode's solution organ mensuration bath (37 ℃), trace its shrinkage curve, start experiment after the spontaneous easypro contracting stable equilibrium of intestinal tube, (final concentration is 5 * 10 to inject respectively choline or histamine
-7mmolL
-1), record maximum collapse height, add respectively on this basis medicine group of the present invention and positive controls, atropine group, after balance, record shrinks height, the results are shown in Table 12.
On this basis, set up according to a certain percentage 4 dosage groups separately, record it and shrink height, with log10 dose and its corresponding contraction inhibiting rate of medicine group of the present invention, return calculating, show that its IC50 is respectively 0.183gL
-1(choline, n=4, r=0.982) and 0.249gL
-1(histamine, n=4, r=0.948).
Table 12 medicine group of the present invention is on the impact of isolated ileum segments in guinea pigs smooth muscle contraction (x ± s, n=6)
Acetylcholine final concentration: 5 * 10
-7mmolL
-1; Histamine final concentration 5 * 10
-7mmolL
-1; With control group comparison
*p < 0.01
Result: the contraction that medicine group of the present invention can be to Ileum From A White smooth muscle due to anti-acetylcholine or histamine, has obvious diastole effect.
4.5 cause the impact of guinea-pig isolated trachea volume to acetylcholine
The imitative in vitro complete tracheae capillary tube technique of guinea-pig isolated trachea volume test, stunning cavy, prepares in vitro complete tracheae sample, is placed in the bath of oxygenation Ke-Heng Shi liquid of constant temperature (37 ℃), observes and connects liquid level in 0.1cm kapillary.Inject after choline, tracheae shrinks, and tracheal volume reduces, and liquid level is raised, and reinject medicine group of the present invention or physiological saline record after administration the liquid level of 5min poor, the results are shown in Table 13.
Table 13 medicine group of the present invention causes the impact (x ± s, n=6) of guinea-pig isolated trachea volume on acetylcholine
Acetylcholine final concentration: 2.5 * 10
-7mmolL
-1; With control group comparison
*p < 0.05,
*p < 0.01
Result: acetylcholine shrinks tracheal smooth muscle, and volume reduces, medicine group of the present invention can make the liquid level of raising decline, and has the remarkable effect that guinea-pig isolated trachea volume dwindles due to pair anti-acetylcholine.
4.6 impacts in vitro cavy lung airway perfusion
In vitro Guinea pig lung bronchus perfusion experimen cavy hits dizzy, arteria carotis bloodletting, and separated tracheae also takes out in the lump together with cardiopulmonary, with 37 ℃, gives containing oxygen locke solution and carries out perfusion, by perfusion bottle height degree, regulates perfusion flow (10mlmin
-1left and right).After perfusion flow is stable, inject a certain amount of acetylcholine solution (3 * 10
-7mmolL
-1), perfusion flow obviously reduces, then injects respectively a certain amount of medicine group (0.16gL of the present invention
-1) and aminophylline (0.025gL
-1), observe the variation of perfusion flow, the results are shown in Table 14.
Table 14 medicine group of the present invention is on the impact of guinea pig in vitro lung airway perfusion (x ± s, n=6)
Choline concentration: 3 * 10
-7mmolL
-1; Medicine group concentration of the present invention: 0.16gml
-1; Aminophylline concentration: 0.025gml
-1; With the comparison of control group+choline group
*p < 0.01.
Result: add after choline, because trachea and bronchus smooth muscle contraction Isolated-lung bronchus perfusion flow obviously reduces, medicine group of the present invention has the effect of obviously anti-acetylcholine being shunk and increasing lung airway perfusion amount.
Experimental example 4 discrimination test screenings
One, rhizoma zingiberis discrimination test screening
The preparation of need testing solution:
Need testing solution one: get medicine of the present invention and be equivalent to crude drug 216.25g, the 30ml that adds diethyl ether, ultrasonic 30 minutes (power 300W, frequency 50kHz), filters, and filtrate volatilizes, and residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution;
Need testing solution two: get medicine of the present invention and be equivalent to crude drug 216.25g, add sherwood oil (60～90 ℃) 50ml, add hot reflux 1 hour, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution;
Need testing solution three: get medicine of the present invention and be equivalent to crude drug 216.25g, the 50ml that adds diethyl ether, adds hot reflux 1 hour, filters, filtrate evaporate to dryness, and residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution;
Need testing solution four: get medicine of the present invention and be equivalent to crude drug 216.25g, add sherwood oil (60～90 ℃) 30ml, ultrasonic 30 minutes (power 300W, frequency 50kHz), filter filtrate evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.
Separately get rhizoma zingiberis control medicinal material 1g, add water 80ml and decoct 1 hour, filter, filtrate is concentrated into 20ml, adds equal-volume sherwood oil (60～90 ℃) extraction, gets upper strata liquid, evaporate to dryness, and residue adds 1ml methyl alcohol to be made to dissolve, in contrast medicinal material solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw control medicinal material solution 5 μ l, need testing solution 15 μ l, put respectively on same silica gel g thin-layer plate, sherwood oil (60～90 ℃)-ethyl acetate (5: 1) of take are developping agent, second outspread, takes out, and dries, spray, with 5% vanillic aldehyde sulfuric acid solution, is heated to spot colour developing at 105 ℃ clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, the results are shown in Table 15.
The selection result of table 15 need testing solution
The preparation of control medicinal material solution:
Control medicinal material solution one: rhizoma zingiberis control medicinal material 1g, add water 80ml and decoct 1 hour, filter, filtrate is concentrated into 20ml, adds equal-volume sherwood oil (60～90 ℃) extraction, gets upper strata liquid, evaporate to dryness, residue adds 1ml methyl alcohol to be made to dissolve, in contrast medicinal material solution;
Control medicinal material solution two: rhizoma zingiberis control medicinal material 1g, add sherwood oil (60～90 ℃) 50ml, add hot reflux 1 hour, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, in contrast medicinal material solution.
According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, draw control medicinal material solution one, two each 5 μ l, put respectively on same silica gel g thin-layer plate, sherwood oil (60～90 ℃)-ethyl acetate (5: 1) of take are developping agent, second outspread, takes out, and dries, spray, with 5% vanillic aldehyde sulfuric acid solution, is heated to spot colour developing at 105 ℃ clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, the results are shown in Table 16.
The selection result of table 16 control medicinal material solution
| control |
1 | 2 |
| color developing effect | color developing effect is good | develop the color unintelligible, have interference. |
The selection of developping agent
Developping agent one: sherwood oil (60～90 ℃)-ethyl acetate (5: 1)
Developping agent two: sherwood oil (60～90 ℃)-ethyl acetate (5: 1)
Developping agent three: cyclohexane-ether (1: 1)
Developping agent four: cyclohexane-ether (1: 1)
Get medicine of the present invention and be equivalent to crude drug 216.25g, the 30ml that adds diethyl ether, ultrasonic 30 minutes (power 300W, frequency 50kHz), filters, and filtrate volatilizes, and residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Separately get rhizoma zingiberis control medicinal material 1g, add water 80ml and decoct 1 hour, filter, filtrate is concentrated into 20ml, adds equal-volume sherwood oil (60～90 ℃) extraction, gets upper strata liquid, evaporate to dryness, and residue adds 1ml methyl alcohol to be made to dissolve, in contrast medicinal material solution.According to thin-layered chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), test, on four silica gel g thin-layer plates, point control medicinal material solution 5 μ l, need testing solution 15 μ l, respectively with developping agent one, two, three, four, second outspread, takes out, and dries, spray, with 5% vanillic aldehyde sulfuric acid solution, is heated to spot colour developing at 105 ℃ clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, the results are shown in Table 17.
The selection result of table 17 developping agent
| |
1 | 2 | 3 | 4 |
| Launch effect | Poor | Poor | Good | Poor |
Two, Radix Glycyrrhizae discrimination test screening,
The selection of developping agent
Developping agent one: lower floor's solution of methenyl choloride-methanol-water (13: 7: 2);
Developping agent two: normal butyl alcohol-3mol/L ammonia solution-ethanol (5: 2: 1);
Developping agent three: the upper solution of methenyl choloride-methanol-water (13: 7: 2).
Get medicine of the present invention and be equivalent to crude drug 216.25g, add sherwood oil (60～90 ℃) 50ml, add hot reflux 1 hour, filter, residue adds methyl alcohol 50ml, adds hot reflux 1 hour, filter, filtrate evaporate to dryness, residue adds water 40ml to be made to dissolve, with water saturated normal butyl alcohol jolting, extract 3 times, each 20ml, merges normal butyl alcohol liquid, with the saturated water washing of normal butyl alcohol 3 times, each 30ml, gets normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution.Another extracting Radix Glycyrrhizae control medicinal material 0.5g, is made in the same way of control medicinal material solution.According to thin-layered chromatography (appendix VI B of Chinese Pharmacopoeia version in 2005), test, get and take two of the silica gel g thin-layer plates that sodium carboxymethyl cellulose is binder, draw each 3～5 μ l of above-mentioned two kinds of solution, put respectively on thin layer plate, respectively with developping agent one, two, three, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot colour developing at 105 ℃ clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, the results are shown in Table 18.
The selection result of table 18 developping agent
| |
1 | 2 | 3 |
| Launch effect | Good | Poor | Poor |
Experimental example 4 Chinese ephedra content assaying methods are learned and are investigated
Detecting instrument: the SPD-10ATvp of Shimadzu company type high performance liquid chromatograph
Chromatographic column: Di Ma company (ZorbaxC184.6 * 250mm, 5 μ m)
Mobile phase: 0.1% phosphoric acid (containing 0.1% triethylamine)-acetonitrile (97: 3)
Detect wavelength: 210nm column temperature: room temperature flow rate: 1.000ml/min
Reference substance source: ephedrine hydrochloride reference substance, is purchased from Nat'l Pharmaceutical & Biological Products Control Institute
Assay method: get preparation sample liquid; And preparation lacks the blank preparation of Chinese ephedra, preparation negative controls.With miillpore filter (0.45 μ m), filter, precision is drawn each 5～10 μ l of negative controls, reference substance solution and need testing solution respectively, and injection liquid chromatography is measured, and obtains.
1. content assaying method is investigated:
(1) preparation of blank test blank solution is to get the group's medicine that lacks Chinese ephedra, by technique, make blank preparation, press again need testing solution preparation method preparation, above-mentioned chromatographic condition is measured, result blank solution at retention time identical with ephedrine hydrochloride reference substance solution place without chromatographic peak, therefore think noiseless.
(2) need testing solution is got in stability test, respectively at latter 0,2,4,6,12,24 hour of preparation, measures in accordance with the law, and injection same volume, the results are shown in Table 19.
Table 19 stability test result
Result shows, ephedrine hydrochloride is basicly stable in 24 hours.
(3) linear relationship is investigated and to be got reference substance solution (51.28 μ g/ml) and shake up, accurate 2,4,6,8, the 10 μ l that draw inject high performance liquid chromatograph respectively, measure peak area, the results are shown in Table 20, and drawing standard curve, see accompanying drawing 1, result shows that ephedrine hydrochloride is good in 0.103 μ g～0.513 μ g scope internal linear relation, and its regression equation is: Y=1942305X+23805 (r=0.9995).
Table 20 linear relationship is investigated result
(4) the accurate ephedrine hydrochloride reference substance solution of drawing of precision test, repeats sample introduction 5 times, and each 5 μ l, try to achieve relative standard deviation < 2%, the results are shown in Table 21:
Table 21 Precision test result
(5) text method is pressed in reappearance test, gets 5 parts of the same batch samples of lab scale, and every part is measured, and tries to achieve relative standard deviation < 2%, the results are shown in Table 22:
Table 22 reproducible test results
(6) recovery test takes the lab scale sample 0.2g of known content, accurately weighed, and precision adds ephedrine hydrochloride reference substance solution (0.4024mg/ml) 5ml, press preparation method's operation of text need testing solution, measure its content, and calculate its recovery, measurement result is in Table 23:
Table 23 recovery test result
Result shows: recovery test is qualified, and this test method can be used for detecting the content of ephedrine hydrochloride in this product.
2, sample size is measured
According to text content assaying method, measure 3 prepared batch samples of embodiment 5.The results are shown in Table 24:
Table 24 Determination of ephedrine hydrochloride measurement result table
Conclusion: by the assay of ephedrine hydrochloride in 3 crowdes of embodiment 5, in every preparation, Chinese ephedra is counted 2.70mg～2.85mg with ephedrine hydrochloride amount, contains Chinese ephedra in ephedrine hydrochloride amount in the daily metering of medicine group of the present invention, must not be less than 8.0mg.
Experimental example 5 fruit of Chinese magnoliavine content assaying methods are learned and are investigated
1. instrument and reagent
Japan Shimadzu LC-10AT high performance liquid chromatograph; SPD-10A UV-detector; KQ-250 type processor for ultrasonic wave; Schizandrin reference substance; Methyl alcohol is chromatographically pure, and it is pure that other reagent are analysis.
2. method and result
2.1 chromatographic condition
Chromatographic column: Shim-pack-C18 post (5 μ m, 4.6mmX150mm), mobile phase: acetonitrile-methyl alcohol-0.1% phosphoric acid (39: 16.7: 44.3); Flow velocity 1ml/min; Detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 3000 by schizandrin peak; Column temperature is 30 ℃.
The preparation of 2.2 reference substance solution
It is appropriate that precision takes schizandrin reference substance, adds methyl alcohol and be mixed with every 1ml containing 0.03mg solution, obtains.
The preparation of 2.3 need testing solutions
Get medicine of the present invention and be equivalent to crude drug 108.1g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, weighed weight, ultrasonic processing 20min, take out, let cool, more weighed weight, add methyl alcohol and supply the weight of minimizing, filter, the accurate subsequent filtrate 20ml that draws, evaporate to dryness, residue adds methyl alcohol to be made to dissolve, and is dissolved to 5ml, with miillpore filter (0.45 μ m), filter, get subsequent filtrate, obtain.
The preparation of 2.4 negative control solutions
In the preparation of prescription ratio, not containing the negative sample of schisandra chinensis medicinal material, by the preparation method of need testing solution, prepare negative control solution.Accurate each 10 μ l of negative control solution, need testing solution and reference substance solution that draw respectively, in injection liquid chromatography, by liquid phase chromatogram condition mensuration, result shows, in negative control solution chromatogram, retention time identical with reference substance place is noiseless.
2.5 linear relationships are investigated
Accurate schizandrin reference substance solution (0.03mg/ml) 4,8,12,16, the 20 μ l that draw, inject respectively high performance liquid chromatograph analysis, the reference substance sample size (μ g) of take is horizontal ordinate, the long-pending integrated value in sharp side of reference substance of take is ordinate mapping, drawing standard curve, its regression equation is: Y=1327476.55X-9683.109, r=0.9998, result shows, schizandrin is good linear relationship between 0.11792-0.5896 μ g.
Table 25 linear relationship is investigated result
2.6 precision test
The accurate need testing solution of drawing repeats sample introduction continuous 6 times, and each 10 μ l, try to achieve relative standard deviation < 2%, the results are shown in Table 26:
Table 26 Precision test result
2.7 stability test
Get need testing solution, in placement 0,2,4 and 6h, sample introduction 10 μ l, record chromatogram respectively, and the RSD=0.49% of result schizandrin peak area, the results are shown in Table 27.
Table 27 stability test result
Result shows, schizandrin is basicly stable in 6 hours.
2.8 reappearance tests
Get same batch sample, by this paper method is independent, measure five times, sample introduction is measured successively, schizandrin content in sample, and, try to achieve relative standard deviation < 2%, in Table 28.
Table 28 reproducible test results
Result shows that its reappearance is good.
2.9 recovery test
Get schizandrin reference substance appropriate, join in the medicine group sample of the present invention of surveying schizandrin content, by preparation method's preparation of need testing solution, and record schizandrin content, calculate recovery rate, measurement result is in Table 29.
Table 29 average recovery measurement result
Average recovery rate=98.22%, RSD=0.60%
2.10 sample determination
By herein preparing reference substance solution and need testing solution under 2.2 and 2.3, by above-mentioned chromatographic condition, measure in accordance with the law, measure altogether three batch samples, the results are shown in Table 30.
Table 30 sample determination result
Conclusion
(1) fruit of Chinese magnoliavine is the main flavour of a drug in medicine group of the present invention, and its contained schizandrin is one of main effective constituent, therefore usings this product schizandrin content as medicine group quality control index of the present invention.Adopt in this article the content of schizandrin in Preparations by HPLC, this method is simple to operate, favorable reproducibility, and the recovery is high.
(2) in the relevant report of schizandrin HPLC content assaying method, mobile phase adopts the systems such as methyl alcohol, water more.We grope by experiment, determine that take methyl alcohol, acetonitrile, 0.1% tricresyl phosphate kind system is mobile phase, and in sample chromatogram, schizandrin separating effect is comparatively satisfied, and degree of separation is high, and peak shape symmetry is better.
Accompanying drawing explanation
Fig. 1: ephedrine hydrochloride typical curve
Following embodiment all can realize above invention effect
The Chinese ephedra 3000g ginger 3000g fruit of Chinese magnoliavine (system) 375g honey-fried licorice root 112.5g
Above four traditional Chinese medicine material, boiling secondary, 3 hours for the first time, 2 hours for the second time, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), let cool to room temperature, add equivalent ethanol, stir evenly, standing 24 hours, get supernatant, reclaim ethanol and be concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃).Vacuum drying, pulverizes, and makes the preparation of clinical acceptance according to this area routine techniques, as: capsule, tablet, granule, soft capsule, pill.
Embodiment 2
Chinese ephedra 3000g, ginger 3000g, cassia twig 2800 weight portions, semen armeniacae amarae 1000 weight portions, the fruit of Chinese magnoliavine (system) 375g, honey-fried licorice root 112.5g
Above medicinal material except semen armeniacae amarae, other gomi herbs, boiling 3 times, each 2 hours, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), let cool to room temperature, add equivalent ethanol, stir evenly, standing 24 hours, get supernatant, reclaim ethanol and be concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃); Semen armeniacae amarae rinsing dedust soil, drains the water and splits dish above, puts into pressure sterilizing pot, and control vapor pressure is 0.03kpa/cm2 (103 ℃), hot pressing boiling 30 minutes, and taking-up is put into a small amount of cold water and is soaked, and removes kind of a skin, dries, and obtains almond processed product.By above-mentioned thick paste and the vacuum drying of almond processed product, pulverize, according to this area routine techniques, make the preparation of clinical acceptance, as: capsule, tablet, granule, soft capsule, pill.
Embodiment 3
Chinese ephedra 3000g, ginger 3000g, the fruit of Chinese magnoliavine (system) 375g, honey-fried licorice root 112.5g
Above four traditional Chinese medicine material, boiling secondary, 3 hours for the first time, 2 hours for the second time, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), let cool to room temperature, add equivalent ethanol, stir evenly, standing 24 hours, get supernatant, reclaim ethanol and be concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃).Vacuum drying, pulverizes, and adds dextrin 300g, cane sugar powder 600g, mixes, and obtains medicinal mixture, and granulation is dry, makes 1000 bags, obtains.
Embodiment 4 tablets
Chinese ephedra 3000g, ginger 3000g, the fruit of Chinese magnoliavine (system) 375g, honey-fried licorice root 112.5g
Above four traditional Chinese medicine material, adopts circulated in countercurrent extractive technique boiling secondary, 3 hours for the first time, 2 hours for the second time, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), let cool to room temperature, add equivalent ethanol, stir evenly, standing 24 hours, get supernatant, reclaim ethanol and be concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃).Vacuum drying, pulverizes, and adds CMC 20g, mixes, and obtains medicinal mixture, and granulation is dry, whole grain, and compressing tablet, dressing, makes 1000, obtains.
Embodiment 5 capsules
Chinese ephedra 3000g, ginger 3000g, the fruit of Chinese magnoliavine (system) 375g, honey-fried licorice root 112.5g
Above four traditional Chinese medicine material, boiling secondary, 3 hours for the first time, 2 hours for the second time, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), let cool to room temperature, add equivalent ethanol, stir evenly, standing 24 hours, get supernatant, reclaim ethanol and be concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃).Vacuum drying, pulverizes, and adds dextrin 150g, mixes, and obtains medicinal mixture, and granulation, encapsulated, makes 1000, obtains.
Embodiment 6 soft capsules
Chinese ephedra 3000g, ginger 3000g, the fruit of Chinese magnoliavine (system) 375g, honey-fried licorice root 112.5g,
Above four traditional Chinese medicine material, boiling secondary, 3 hours for the first time, 2 hours for the second time, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), lets cool to room temperature, add equivalent ethanol, stir evenly, standing 24 hours, get supernatant, reclaim ethanol and be concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃), drying under reduced pressure, pulverize, mix with fine powder, add vegetable oil 200g, stir evenly, make 660 of soft capsules, obtain.
Embodiment 7 granules
Chinese ephedra 3000g, ginger 3000g, the fruit of Chinese magnoliavine (system) 375g, honey-fried licorice root 112.5g
Above four traditional Chinese medicine material, boiling secondary, 3 hours for the first time, 2 hours for the second time, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), let cool to room temperature, add equivalent ethanol, stir evenly, standing 24 hours, get supernatant, reclaim ethanol and be concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃).Vacuum drying, pulverizes, and adds cane sugar powder 600g, dextrin 300g, mixes, and obtains medicinal mixture, and granulation is dry, makes 1000g, obtains.
Embodiment 8 pills
Chinese ephedra 3000g, ginger 3000g, the fruit of Chinese magnoliavine (system) 375g, the above four traditional Chinese medicine material of honey-fried licorice root 112.5g, boiling secondary, 3 hours for the first time, 2 hours for the second time, collecting decoction, filters, and filtrate decompression is concentrated into the clear cream that relative density is 1.08 (60 ℃), let cool to room temperature, add equivalent ethanol, stir evenly, standing 24 hours, get supernatant, reclaim ethanol and be concentrated into the thick paste that relative density is 1.34～1.40 (60～70 ℃).60 ℃ of drying under reduced pressure, pulverize, and mix and take the ethanolic solution of 3%PVP and make 1000 balls as bonding agent with above-mentioned fine powder, obtain.
Claims (1)
1. have a surname's lung, the detection method of the pharmaceutical composition of antiasthmatic effect, is characterized in that the method comprises the steps:
Differentiate: (1) compositions of getting it filled is equivalent to crude drug 10.8g, adds strong ammonia solution 2ml, then adds methenyl choloride 40ml, adds hot reflux 1 hour, filter, filtrate evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution A; Separately get ephedrine hydrochloride, add methyl alcohol and make every 1ml containing the solution of 1mg, in contrast product solution A; According to appendix VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2005, draw each 3～5 μ l of above-mentioned two kinds of solution, put respectively in same and take on the silica gel g thin-layer plate that sodium carboxymethyl cellulose is binder, methenyl choloride-methyl alcohol-the strong ammonia solution of take 20: 5: 0.5 is developping agent, launch, take out, dry, spray, with 5% ethanol solution of ninhydrin, is heated to spot colour developing at 105 ℃ clear; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, aobvious identical aubergine spot;
(2) compositions of getting it filled is equivalent to crude drug 216.25g, the 30ml that adds diethyl ether, ultrasonic 30 minutes, power 300W, frequency 50kHz; Filter, filtrate volatilizes, and residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution B; Separately get rhizoma zingiberis control medicinal material 1g, add water 80ml and decoct 1 hour, filter, filtrate is concentrated into 20ml, adds equal-volume boiling range and be the petroleum ether extraction of 60～90 ℃, gets upper strata liquid, evaporate to dryness, and residue adds 1ml methyl alcohol to be made to dissolve, in contrast medicinal material solution B; According to appendix VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2005, draw control medicinal material solution B 5 μ l, need testing solution B15 μ l, put respectively on same silica gel g thin-layer plate, take 5: 1 boiling range as 60～90 ℃ of petroleum ether-ethyl acetates be developping agent, second outspread, takes out, and dries, spray, with 5% vanillic aldehyde sulfuric acid solution, is heated to spot colour developing at 105 ℃ clear; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
(3) compositions of getting it filled is equivalent to crude drug 216.25g, and adding boiling range is 60～90 ℃ of sherwood oil 50ml, adds hot reflux 1 hour, filter, residue adds methyl alcohol 50ml, adds hot reflux 1 hour, filter, filtrate evaporate to dryness, residue adds water 40ml to be made to dissolve, with water saturated normal butyl alcohol jolting, extract 3 times, each 20ml, merges normal butyl alcohol liquid, with the saturated water washing of normal butyl alcohol 3 times, each 30ml, gets normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 1ml to be made to dissolve, as need testing solution C; Another extracting Radix Glycyrrhizae control medicinal material 0.5g, is made in the same way of control medicinal material solution C; According to appendix VI B thin-layered chromatography test of Chinese Pharmacopoeia version in 2005, draw each 3～5 μ l of above-mentioned two kinds of solution, put respectively in same and take on the silica gel g thin-layer plate that sodium carboxymethyl cellulose is binder, the lower floor's solution of 13: 7: 2 methenyl choloride-methanol-waters of take is developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot colour developing at 105 ℃ clear; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;
Assay
(1) according to appendix VID high effective liquid chromatography for measuring of Chinese Pharmacopoeia version in 2005;
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; 0.1% phosphoric acid-the acetonitrile of 97:3 of take is mobile phase, and described 0.1% phosphoric acid is containing 0.1% triethylamine; Detection wavelength is 210nm; Number of theoretical plate calculates and should be not less than 2500 by ephedrine hydrochloride peak;
The preparation of reference substance solution: get ephedrine hydrochloride reference substance appropriate, accurately weighed, with methyl alcohol, dissolve and make every 1ml containing the solution of 50 μ g, product solution D, obtains in contrast;
The preparation of need testing solution: the compositions of getting it filled is equivalent to crude drug 4.3g, accurately weighed, to 50ml volumetric flask, precision adds the hydrochloric acid solution 1ml of 0.5mol/L, add again methyl alcohol to scale, jam-pack, ultrasonic processing 30 minutes, let cool, 0.45 μ m filtering with microporous membrane, obtains need testing solution D;
Determination method: precision is drawn reference substance solution D and each 5 μ l～10 μ l of need testing solution D respectively, and injection liquid chromatography, measures, and obtains;
The daily metering of pharmaceutical composition of the present invention in ephedrine hydrochloride amount, must not be less than 8.0mg containing Chinese ephedra;
(2) according to appendix VID high effective liquid chromatography for measuring of 2005 editions pharmacopeia;
Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica, be filling agent; Acetonitrile-methyl alcohol-0.1% phosphoric acid of 39:16.7:44.3 is mobile phase; Detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 3000 by schizandrin peak;
The preparation of reference substance solution: get schizandrin reference substance 1.5mg, accurately weighed, put in 50ml measuring bottle, with methyl alcohol, dissolve and be diluted to scale, shake up, obtain every 1ml containing the reference substance solution E of schizandrin 0.03mg;
The preparation of need testing solution: the compositions of getting it filled is equivalent to crude drug 108.1g, accurately weighed, puts in tool plug conical flask, and precision adds methyl alcohol 50ml, weighed weight, ultrasonic processing 20min, power 250W, frequency 20kHz; Take out, let cool, more weighed weight, add the weight that methyl alcohol is supplied minimizing, filter, the accurate subsequent filtrate 20ml that draws, evaporate to dryness, residue adds methyl alcohol to be made to dissolve, and is dissolved to 5ml, with 0.45 μ m miillpore filter, filters, and gets subsequent filtrate, obtains need testing solution E;
Determination method: precision is drawn reference substance solution E and each 10 μ l of need testing solution E respectively, and injection liquid chromatography, measures, and obtains;
Pharmaceutical composition of the present invention must not be less than 0.40% containing schizandrin;
Described pharmaceutical composition bulk drug consists of:
Chinese ephedra 3000-4000 weight portion, ginger 2000-3000 weight portion, cassia twig 2000-3000 weight portion, semen armeniacae amarae 1000-1500 weight portion, Fructus Schisandrae (processed) 250-500 weight portion, honey-fried licorice root 80-160 weight portion.
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| CN102879514A (en) * | 2012-08-07 | 2013-01-16 | 何述金 | Mass control method for medicinal composition for treating hepatitis |
| CN104436129A (en) * | 2014-11-26 | 2015-03-25 | 黑龙江省智诚医药科技有限公司 | Ephedra and ginger dispersible tablet and preparation method thereof |
| CN107764924B (en) * | 2016-08-19 | 2020-01-31 | 江苏康缘药业股份有限公司 | Detection method of effective components in asthma granules |
| CN109991359B (en) * | 2018-01-02 | 2021-07-13 | 苏州玉森新药开发有限公司 | Quality control method of Chinese medicinal composition with cough relieving and phlegm eliminating effects |
| CN108693298A (en) * | 2018-06-15 | 2018-10-23 | 四川逢春制药有限公司 | A kind of detection method for treating the Chinese medicine preparation of flu |
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