CN1565613A - Chinese medicinal composition for treating acute pharyngitis and gingivitis and preparing method thereof - Google Patents
Chinese medicinal composition for treating acute pharyngitis and gingivitis and preparing method thereof Download PDFInfo
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Abstract
The invention discloses a Chinese medicinal composition for treating acute pharyngitis and gingivitis and preparing method thereof, wherein the composition is prepared from rheum officinale, bear gall, baikal skullcap root, root of balloonflower, dahurian angelica root, cordate houttuynia, and mint.
Description
Invention field
The present invention relates to a kind of Chinese medicine composition, particularly be used for the treatment of the Chinese medicine composition of acute pharyngitis, gingivitis, relate to the preparation method and the method for quality control of said composition simultaneously.
Background technology
Acute pharyngitis, gingivitis all belong to commonly encountered diseases, the frequently-occurring disease in clinical, and be wherein common with the card of lung sthenia gastropyrexia and wind heat again.But normal double the holding under the arm mutually of two cards risen at the beginning of its disease clinically, and be how evil because of the affection due to external wind and heat poison, follows through superinverse and violate lung; Or improper diet, have a liking for the good wine of cigarette, the smoked lung stomach that burns; Or have a liking for and eat pungent rich product, the heat-transformation fire-transformation of processing; Or the lung stomach have long-pending heat, and is multiple because of being invaded by exogenous pathogen, fire and wind stirring up each other and causing.The fire belongs with yang heresy, scorching on its property, so pathogenic heat the easiest follow through on disturb.Throat is the door of lung, accumulation of heat in the lung swollen the resembling of larynx pain or show of then vomitting, and the stomach network is burned with anger and is followed through last smoked, the corruption of intenseness of heat meat, stagnation of QI-blood, then red swelling of gingiva, pain in gum; Lung gastric heat poison, pathogenic heat stop up known widely in, burning hot rising, then pain can radiation and the basal part of the ear, jaw under the cheek; Lung gastric heat poison is flourishing, and the absurd row of forcing blood then can go out to see gingival hemorrhage; Wind heat is violated table, and the heat stagnation flesh natural fibre line of meat is then seen fever of the body, or slight chill, and also to be that the lung sthenia gastropyrexia is double hold levying of wind heat under the arm for big dry stool, yellowish or reddish urine, red tongue, white or yellow thin fur, floating-rolling pulse number etc.
Summary of the invention
One object of the present invention is to disclose a kind of new treatment acute pharyngitis, the Chinese medicine composition of gingivitis; Another object of the present invention is the method for the Chinese medicine composition of a kind of new treatment acute pharyngitis of open preparation, gingivitis; The object of the invention also is to disclose a kind of method of quality control of new Chinese medicine composition.
The crude drug of pharmaceutical composition of the present invention is formed and proportioning following (by weight):
Radix Et Rhizoma Rhei 160-240 weight portion Fel Ursi powder 5-18 weight portion Radix Scutellariae 160-240 weight portion
Radix Platycodonis 160-240 weight portion Radix Angelicae Dahuricae 160-240 weight portion Herba Houttuyniae 350-450 weight portion
Herba Menthae 60-140 weight portion.
The preferred Radix et Rhizoma Rhei (processed with wine) of described Radix Et Rhizoma Rhei.
The above-mentioned prescription medicine of the present invention can add excipient, is prepared into pharmaceutical dosage form commonly used according to the preparation process of routine, for example, and tablet, granule, capsule, pill, oral liquid, masticatory, aerosol, soft capsule, suppository, drop pill etc.
The preparation method of this medicament composition capsule agent:
More than seven the flavor, Radix et Rhizoma Rhei (processed with wine) soaks in right amount with 60-85% ethanol, 60-80 ℃ of drying is ground into fine powder, Fel Ursi powder is ground into fine powder, sieving for standby; The Radix Angelicae Dahuricae, Herba Houttuyniae, Herba Menthae add 9-11 times of water extraction volatile oil 4-6 hour, collect volatile oil, and the aqueous solution after distillation device is in addition collected, and 2-4 times of water gaging washing of medicinal residues reuse 1-3 time filters, and merges aqueous solution, and be standby; Other gets beta-schardinger dextrin-10.5 weight portions, adds 9-11 times of water dissolution, slowly drips the alcoholic solution of above-mentioned volatile oil, volatile oil: ethanol=1-2: 1-2, under the ultrasound wave of 20-40KHz ultrasonic 1-2 hour, cold preservation was spent the night, sucking filtration, the room temperature decompression is dry down, crosses the 70-90 mesh sieve, and is standby; All the other two flavors are cut into segment with Radix Scutellariae, add 6-8 times of decocting with Radix Platycodonis and boil 1-3 time, and each 1-2 hour, collecting decoction filtered, and the aqueous solution of filtrate and above-mentioned collection merges, and being concentrated into 70-85 ℃ of relative density is 1.10-1.15, and spray drying gets dry extract; Get dry extract and Radix et Rhizoma Rhei (processed with wine) powder, Fel Ursi powder, and volatile oil beta-cyclodextrin inclusion and 0-20 weight portion starch, mixing incapsulates, and makes 1000, promptly.
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 this composite preparation 1g, add methanol 20ml, supersound process 10-20 minute, filter, filtrate is concentrated into 1ml, as need testing solution; Get Radix Et Rhizoma Rhei control medicinal material powder 0.5g, make control medicinal material solution with the method operation; Other gets the emodin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the 0.4-0.6% sodium carboxymethyl cellulose, with 6-8: 2-4: 0.1-0.2 benzene-Ethyl formate-formic acid is developing solvent, launch, take out, dry; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show the speckle of same color;
B. get this composite preparation 2.5g and add methanol ultrasonic 1-3 time, each 30ml, 10-20 minute, filter, filtrate evaporate to dryness, residue add that 8-12% sodium hydroxide solution 20ml is ultrasonic to make dissolving, 95-105 ℃ heating hydrolysis 1-3 hour, put cold, with the hydrochloric acid adjust pH to 2-3, drink extraction 2-4 time with acetic acid second, each 15ml merges ethyl acetate liquid, wash each 40ml, ethyl acetate liquid water bath method with water 2-4 time, residue makes dissolving with methanol 1ml, and is centrifugal, gets supernatant as need testing solution; Other gets chenodeoxycholic acid and ursodesoxycholic acid reference substance, adds methanol respectively and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the 0.4-0.6% sodium carboxymethyl cellulose, with 9-11: 4-6: 4-6: 2-4: the upper solution of 1-2 isobutyltrimethylmethane .-ether-glacial acetic acid-n-butanol-water is developing solvent, launch, take out, dry, spray is with the 15-25% sulfuric acid solution, in 100-110 ℃ of baking 4-7 minute; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
C. get this composite preparation 4g, add methanol 40ml, ultrasonic 15-25 minute, filter the filtrate evaporate to dryness, residue adds water 20ml makes dissolving, transfers pH to 3-4 with hydrochloric acid, adds ethyl acetate extraction 1-3 time, each 20ml, merge ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the baicalin reference substance, adds methanol and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5 μ l of above-mentioned two kinds of solution, putting in same 0.4-0.6% sodium carboxymethyl cellulose with the preparation of 1% sodium acetate respectively is on the silica gel g thin-layer plate of adhesive, with 4-6: 2-4: 1-2: 1-2 ethyl acetate-acetone-formic acid-water is developing solvent, launch, take out, dry, spray is with 2% ferric chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
D. get this composite preparation 4g, put in the round-bottomed flask, add water 250ml, connect volatile oil determination apparatus according to volatile oil extraction method (an appendix X of Chinese Pharmacopoeia version in 2000 D); Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 3-5 hour, stop heating, place a moment, divide and get the ethyl acetate layer, as need testing solution; Other gets the methylnonanone reference substance, adds ethyl acetate and makes the solution that every 1ml contains 10 μ g, in contrast product solution; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), 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 0.4-0.6% sodium carboxymethyl cellulose, with 18-20: 1-2 benzene-ethyl acetate is developing solvent, launch, take out, dry, spray is with the dinitrophenylhydrazine test solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
E. get this composite preparation 4g, put in the round-bottomed flask, add water 250ml, connect volatile oil determination apparatus according to volatile oil extraction method (an appendix X of Chinese Pharmacopoeia version in 2000 D); Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 3-5 hour, stop heating, place a moment, divide and get the ethyl acetate layer, as need testing solution; Get the Mentholum reference substance, add ethyl acetate and make the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), get 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 0.4-0.6% sodium carboxymethyl cellulose, with 7-10: 1-2 normal hexane-ethyl acetate is developing solvent, launch, take out, dry, spray is with 1-3: 7-9 vanillin sulphuric acid test solution-alcoholic acid mixed solution, and it is clear to be heated to the speckle colour developing at 95-110 ℃; In the test sample chromatograph, on the corresponding position of reference substance chromatograph, show the speckle of same color;
Content assaying method comprises a kind of and/or several in the following method:
A. emodin, chrysophanol: measure according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000); Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 80-90: 10-20 methanol-0.1% phosphoric acid solution is a mobile phase; The detection wavelength is 254nm; Number of theoretical plate calculates by the emodin peak should be not less than 1500; The preparation of reference substance solution, precision take by weighing emodin, each 10mg of chrysophanol reference substance, put respectively in the 100ml measuring bottle, with dissolve with methanol and be diluted to scale, shake up as emodin chrysophanol reference substance stock solution; Precision is measured emodin reference substance stock solution 1ml, chrysophanol reference substance stock solution 2ml respectively, puts respectively in the 25ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets to contain among the every 1ml of emodin among 4 μ g, the every 1ml of chrysophanol to contain 8 μ g; This composite preparation 0.2g is got in the preparation of need testing solution, and accurate the title decides, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, under the ultrasound wave of 20-40KHz supersound process 1-2 hour, put cold, claim to decide weight again, replenish the weight that subtracts mistake, shake up with methanol, centrifugal, get supernatant as need testing solution; Algoscopy, accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly; This composite preparation per unit amount contains Radix Et Rhizoma Rhei with emodin (C
15H
10O
5) and chrysophanol (C
15H
10O
4) the total amount meter, must not be less than 6.5-8mg;
B. baicalin: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D); Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 43-53: 50-58: the 0.1-0.3 methanol-water-phosphoric acid is a mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the baicalin peak should be not less than 2500; The preparation of reference substance solution, precision take by weighing at 55-65 ℃ of drying under reduced pressure 3-5 hour baicalin reference substance an amount of, add methanol and make every 1ml and contain 20 μ g solution, promptly; This composite preparation 28.5mg is got in the preparation of need testing solution, accurate claims surely, puts in the 25ml measuring bottle, and it is an amount of to add 45-55% ethanol, and supersound process 10-25 minute, put coldly, add the 45-55% ethanol dilution to scale, shake up, centrifugal, get supernatant as need testing solution; Survey the shallow lake method, accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly; This composite preparation per unit amount contains Radix Scutellariae by baicalin (C
12H
18O
11) meter, must not be less than 55-65mg.Described per unit amount is meant the finished medicines dosage of suitable crude drug 13.1g.
The weight that above-mentioned composite preparation differentiated, taken by weighing during assay is that capsule is removed capsule shells, and tablet is then removed coating, and liquid preparation is dried to the weight behind the powder.
Present composition preparation has eliminating fire and detoxication, the wind-dispelling heat-dissipating effect, to acute pharyngitis, pharyngalgia due to the gingivitis (the double wind heat of holding under the arm of lung sthenia gastropyrexia), dry pharynx is scorching hot or diseases such as gingival swelling and pain, thirsty, fever of the body or slight chill, big dry stool, yellow urine have very good effect, and takes safety.
Following experimental example is used to further specify the present invention.The used present composition preparation of following experimental example is than visiing gram medicated powder.
Experimental example 1The antibacterium effect
The test of 1 in-vitro antibacterial
1.1 experiment material
1.1.1 medicine
(1) than visiing gram medicated powder: provide by SiChuan JinHui Pharmacy Co., Ltd, brown ceramic powder, lot number: 990222, every Ke Bibaike medicated powder is equivalent to crude drug in whole 2.789 grams.Take by weighing 20 Ke Bibaike medicated powder during test, add to 50 ℃ of m-H agar culture medium 66.8ml mixings of thawing after, 1000mg/ml concentration, take out again this medicinal liquid of 40ml be used for the test.Promptly sucking-off 20ml pastille culture medium is toppled over plate from 40ml, add 20ml in the remaining 20ml pastille culture medium again and do not have the dilution of medicine culture medium, behind the mixing again sucking-off 20ml topple over plate, and the like, contain in the preparation of this doubling dilution promptly that to be that 1000mg/ml, 500,250,125,62.5,31.2,15.6,8,4,2,1,0.5,0.25,0.125,0.06,0.03mg/ml a series of close the medicine plate stand-by than visiing gram medicated powder final concentration (with the crude drug amount).
(2) cefoperazone for inj sodium: specification: 1.0g/ bottle, content 88%, lot number: 65835061.Pfizer inc, DaLian, China.With sterilized water dissolving preparation, final concentration is that 128,64,32,16,8,4,2,1,0.5,0.25,0.125,0.06,0.03,0.015,0.008 μ g/ml series pastille plate is stand-by during test.
(3) tinidazole injection: specification 0.5g/250ml is produced lot number: 970417 by Qili Pharaceutical Co., Ltd., Sichuan.The serial pastille agar plate that is mixed with final concentration and is 128,64,32,16.8,4,2,1,0.5,0.25,0.125,0.06,0.03,0.015,0.008,0.004 μ g/ml is stand-by.
1.1.2 antibacterial
(1) clinical isolates strain: the test bacterial strain uses therefor is the clinical bacteria that 1998.1-1999.4 collects from the attached First Academy of Chengdu Huaxi Medical Univ, Annex II institute, and identifies again through the conventional method of inspection.
Staphylococcus aureus 26 strains, coagulase negative staphylococcus 2 strains (imitation staphylococcus 1 strain, 1 strain of corn staphylococcus), staphylococcus epidermidis 22 strains, bacillus pyocyaneus 10 strains, 10 strains of Ke Shi pneumobacillus, Bacillus proteus 10 strains (proteus mirabilis 4 strains, proteus vulgaris 6 strains), escherichia coli 6 strains, acinetobacter calcoaceticus 8 strains (Acinetobacter junii 3 strains, luxuriant and rich with fragrance acinetobacter calcoaceticus 5 strains fall), anaerobe 5 strains (bacteroides fragilis 3 strains, propionibacterium 2 strains).
(2) standard Quality Control bacterial strain: staphylococcus aureus ATCC25923, escherichia coli ATCC25922, bacillus pyocyaneus NCTC10662 are that Sichuan Industrial Institute of Antibiotics preserves bacterial strain.
1.1.3 culture medium
(1) M-H culture medium: caseinhydrolysate 17.5g, beef powder 5g, soluble starch 1.5g, adding distil water 1000ml, pH7.2.Add the 15g agar powder in the solid medium.Be used for Gram-positive, negative aerobe.
(2) anaerobic culture base: commercially available anaerobe special culture media.Shanghai Vaccine and Serum Institute produces, lot number: 980501, be used for anaerobe.
1.2 experimental technique
Adopt the agar doubling dilution to measure than the minimum inhibitory concentration of visiing gram medicated powder.With multiple spot inoculation instrument with microbionation on the agar plate surface that contains different pharmaceutical concentration, every some bacteria containing amount is about 10
5CFU/ml, hatching 18-20 hour (anaerobe insert 37 ℃ of anaerobism incubators (Yiwu Refrigerator General Factory, Zhejiang Prov.'s manufacturing) cultivate 48 hours) observed result for 37 ℃, is the minimum inhibitory concentration (MIC value) of medicine to this bacterium with the least concentration of institute's composite medicine in the no bacterial growth plate culture medium.
1.3 experimental result
Experimental result sees Table 1, table 2.
1.4 conclusion
By in the table 1 as seen, than visit the gram medicated powder examination Gram-positive, negative bacteria are all had certain antibacterial vigor.Is 2-125mg/ml than visiing gram medicated powder to the MIC scope of staphylococcus aureus, and the MIC value scope of his-and-hers watches Portugal coccus is at 4-500mg/ml; Than visit gram medicated powder to bacillus pyocyaneus to press down the Seedling vigor strong to staphylococcus aureus, table Portugal coccus, MIC value scope is at 0.06-62.5mg/ml, to the MIC position scope of Ke Shi pneumobacillus at 0.06-62.5mg/ml, antibacterial vigor to Bacillus proteus and acinetobacter calcoaceticus is also strong, and the MIC value is 0.06mg/ml-0.25mg/ml altogether.Than visiing gram medicated powder the examination anaerobe is also had certain Seedling vigor that presses down, MIC is 15.6-62.5mg/ml, sees Table 2.
Table 1 is than visiing gram medicated powder antibacterial activity in vitro
Antibacterial (strain number) is than visiing gram (mg/ml) cefoperazone (μ g/ml)
Staphylococcus aureus (26) MICRange 2-125 0.06-〉128
MIC
50 15.6 32
MIC
90 62.5 64
Form staph (22) MICRange 4-500 0.5-64
MIC
50 15.6 4
MIC
90 125 32
Coagulase negative staphylococcus (2) MICRange 15.6 32
(1 strain of corn staphylococcus)
(imitation Portugal's coccus 1 strain)
Bacillus pyocyaneus (10) MICRange 0.06-62.5 0.5-4
MIC
50 0.06 4
MIC
90 15.6 4
Ke Shi pneumobacillus (10) MICRange 0.06-62.5 0.06-〉128
MIC
50 62.5 0.5
MIC
90 62.5 128
Proteus (10) MICRange 0.25 0.125-4
MIC
50 0.06 0.5
MIC
90 0.06 1
Acinetobacter calcoaceticus (10) MICRange 0.06 0.06-16
MIC
50 0.06 0.06
MIC
90 0.06 8
Escherichia coli (5) MICRange 15.6-62.5 0.5-4
MIC
50 31.2 1
MIC
90 62.5 4
MTC
The Quality Control bacterial strain
Than visiing gram (mg/ml) cefoperazone (μ g/ml)
Escherichia coli ATCC25922 15.6 0.25
Bacillus pyocyaneus NCTC10662 0.06 0.5
Staphylococcus aureus ATCC25923 15.6 32
Table 2 is than visiing the antibacterial action of gram medicated powder to anaerobe
MTC
Antibacterial
Than visiing gram (crude drug mg/ml) tinidazole (μ g/ml)
Bacteroides fragilis BfATCC27285 31.2 0.25
Bacteroides fragilis Bf961 15.6 0.125
Bacteroides fragilis Bf965 15.6 0.06
Propionibacterium 62.5 0.5
Propionibacterium granulosum 62.5 2
The test of 2 endogenous protectives
2.1 experiment material
1, laboratory animal: select healthy Kunming mouse for use, body weight 18-22 gram, male and female half and half are provided by Sichuan Industrial Institute of Antibiotics animal reproduction chamber.No. 85, the real moving Guan Zhidi in animal quality certification river.
2, experimental drug: than visiing gram medicated powder, provide by SiChuan JinHui Pharmacy Co., Ltd, brown ceramic powder, lot number: 990222, every Ke Bibai fills medicated powder and is equivalent to crude drug in whole 2.789 grams.
FUPAISUAN JIAONANG: fairy house pharmaceutical factory of Zhejiang medicine limited company produces lot number 981104 specification 0.1g/ grains.
3, experimental strain
The antibacterial that is used for infection animal is staphylococcus aureus 991915, escherichia coli 9912, is the clinical separation pathogenic bacterium of collecting from the area, Chengdu in 1999.
2.2 experimental technique
1, the preparation of test organisms liquid
Test strain is inoculated in the MH meat soup, cultivated 18 hours for 37 ℃, with the suitable diluted for use of 5% sterilization dry yeast liquid.
2, the mensuration of the minimum bacterium amount (MLD) that causes death
Get healthy Kunming mouse, body weight 18-22 gram, random packet, every group of 10 mices, male and female half and half, draw the dilution bacterium liquid of above-mentioned difference respectively lumbar injection go in the mice body, every Mus 0.5ml, infect the continuous observation in back 7 days, and record dead mouse number, measure (MLD) with the minimum bacterium amount that causes mice 100% death as minimum deadly bacterium.With the infection dosage of this bacterium amount as the endogenous protective test.
3, the preparation of medicinal liquid
The test medication is all with the preparation of 0.5%CMC and normal saline, is 2.80mg/ml, 2.24mg/ml, 1.8mg/ml, 1.44mg/ml, 1.15mg/ml, 0.92mg/ml, 0.74mg/ml than visiing the concentration that gram medicated powder is mixed with the medicinal liquid that concentration is 235.8mg/ml, 15.2mg/ml, 78.6mg/ml, 39.3mg/ml, 19.65mg/ml (in the crude drug amount), norfloxacin.
4, infection and treatment experiment
Laboratory animal is evenly divided into groups at random by sex, body weight, male and female half and half, every group of 10 mices, each organizes respectively, and dosage is: 5895mg/kg, 3930mg/kg, 1965mg/kg, 983mg/kg, 491mg/kg (in the crude drug amount, are respectively 30,20,10,5,2.5 times of clinical Coming-of-Age Day consumption.Clinical consumption per day: 196.5mg (crude drug)/kg), the dosage of positive control drug FUPAISUAN JIAONANG is: 70mg/kg, 56mg/kg, 45mg/kg, 36mg/kg, 28.8mg/kg, 23.0mg/kg, 18.5mg/kg.
The mouse stomach ratio is visitd gram medicated powder, once a day, successive administration four days, 0.5ml/ 20g of every mice respectively organizes the mouse peritoneal infection and tried bacterium liquid after administration in the 5th day, and every Mus infection dosage is 0.5ml (1MLD).FUPAISUAN JIAONANG each gastric infusion after reaching 4 hours at once behind the infectious bacteria liquid is once established the infection matched group simultaneously, and record infects dead mouse number in back seven days, calculates its median effective dose (ED according to infecting back seven days animals survived numbers
50) and 95% fiducial limit.
5, Data Processing in Experiment
Than visiing gram medicated powder and contrast medicine FUPAISUAN JIAONANG; program software to its 95% fiducial limit of median effective dose ED50 of staphylococcus aureus 991915, escherichia coli 9912 infecting mouse endogenous protective effects is all worked out by the Bliss method utilization Chinese Academy of Medical Sciences uses a computer and calculates and statistical procedures.
2.3 experimental result
Than visit gram medicated powder, FUPAISUAN JIAONANG gastric infusion to the protective effect of infecting in the mice body the results are shown in Table 3, table 4, than visit gram medicated powder, FUPAISUAN JIAONANG respectively gastric infusion to the ED of staphylococcus aureus 991915 infecting mouses
50Value is respectively 2027mg/kg and 28.67mg/kg, to the ED of escherichia coli 9912 infecting mouses
50Be respectively 4162mg/kg and 45.87mg/kg.
2.4 conclusion
Can find out from above experimental result, also present certain vivo bacteria corrosion action than visiing gram medicated powder, the ED of staphylococcus aureus and coli-infection mice
50Be respectively the 2027mg{ crude drug }/kg and 4162mg (crude drug)/kg.
Table 3 is than visiing gram medicated powder and the protective effect (staphylococcus aureus) of FUPAISUAN JIAONANG to infecting in the mice body
Infection strain (bacterial strain mortality rate (only)
Number) dosage mortality rate ED
50And 95% fiducial limit
Medicine
(intoxicating bacterium amount) be number of animals (only) % (mg/kg) (mg/kg)
(CFU/ml)
The staphylococcus aureus ratio is visitd gram medicine 5,859 1,/10 10
The No.991915 powder is (by giving birth to 3,930 4,/10 40
(5.2 * 10
7) dose meter 1,965 5,/10 50
Calculate) 983 7,/10 70 2027
491 9/10 90 (1208-3466)
45.00 1/10 10
36.00 3/10 30
Norfloxacin
28.80 5/10 50
Capsule
23.0 7/10 70 28.67
18.5 9/10 90 (24.22-33.97)
Matched group----10,/10 100
Table 4 is than visiing gram medicated powder and the protective effect (escherichia coli) of FUPAISUAN JIAONANG to infecting in the mice body
Infection strain (bacterial strain mortality rate (only)
Number) dosage mortality rate ED
50And 95% fiducial limit
Medicine
(intoxicating bacterium amount) be number of animals (only) % (mg/kg) (mg/kg)
(CFU/ml)
The escherichia coli ratio is visitd gram 5,859 3,/10 30
No.9912 medicated powder is (by 3,930 6,/10 60
(2.2 * 10
4) crude drug amount 1,965 8,/10 80
Calculate) 983 9,/10 90 4162
491 10/10 100 (2782-9010)
70.00 1/10 10
56.00 3/10 30
Norfloxacin
45.00 6/10 60
Capsule
36.00 7/10 70 45.87
28.80 9/10 90 (38.94-54.61)
Matched group----10,/10 100
Experimental example 2Antivirus action
Experiment material:
1, than visiing gram medicated powder brown ceramic powder, provide lot number by SiChuan JinHui Pharmacy Co., Ltd: 990222, the suitable crude drug in whole 2.789g of 1g powder.
2, positive control drug virazole (ribavirin) injection, specification 100mg/ml, lot number: 980605, the Yichang three gorges pharmaceutical factory produces.
3, Strain adenovirus (AdV) 3 types, 7 types, respiratory syncytial virus (Rsv) are all introduced from Shoudu Inst. of Pediatrics, and Rsv, Adv3,7 type TCID50 are measured in People's Hospital, Sichuan Prov.'s cultivation of going down to posterity the Viral Laboratory respectively before the experiment.
4, cell strain HeLa and Hep-2 cell are provided by Chengdu Inst. of Biological Products and Sichuan Province health and epidemic prevention station respectively, and the recovery of People's Hospital, Sichuan Prov. Viral Laboratory is gone down to posterity, and growth-promoting media is 10% calf serum Eagles liquid.
2 experimental techniques
1, cell toxicity test
To be subjected to reagent and positive control drug (virazole) from 1 with Hanks liquid: 10-1: 160 make doubling dilution (is subjected to the reagent ratio to visit gram medicated powder concentration and is respectively 10,5,2,5,1.25,0.625mg/ml, contrast medicine virazole concentration is respectively 1,0.5,0.25,0.125,0.0625mg/ml).Get than visiing gram medicated powder and virazole 1: 10-1: 160 medicinal liquids, inoculate each 2 of sub-HeLa cell and Hep-2 cell pipes respectively, treat that cell absorption is after 1 hour, add 2% calf serum Eagles respectively at each cell pipe and keep liquid, putting 37 ℃ of calorstats cultivates, day by day the observation of cell toxic reaction is established the cell contrast simultaneously.
2, antivirus test
Get than visit gram medicated powder and virazole each 1: 10-1: 80 medicinal liquids, respectively with 100TCID50Rsv and Adv3,7 type mixed in equal amounts, put the direct effect of room temperature after 1 hour, get each 2 of each mixed liquor inoculation HeLa cell and Hep-2 cell pipes respectively, treat cell absorption after 1 hour, add 2% calf serum Eagles and keep liquid (establishing each virus control, cell contrast, blank synchronously) and put 37 ℃ of calorstats and cultivate.37 ℃ of anti-Rsv cell pipes were cultivated after 24 hours, moved to put 35 ℃ of rotating and culturing casees and cultivate (10-12 change/hour), observed anti-Rsv, anti-Adv3,7 type cytopathys day by day.When 3+ (+++) appears in virus control, the cell contrast is during for (-), but termination test.When 2+ (++) pathological changes appears in test cell pipe, can be judged as medicine to this virus unrestraint effect.
Experimental result:
1, cell toxicity test result
Be subjected to the reagent ratio to visit gram medicated powder 1: 10-1: 160 medicinal liquids do not have any toxic reaction to the Hela cell.Except that 1: 10 medicinal liquid can cause section H ep-2 cell circle contracts, 1: 20-1: 160 medicinal liquids do not have any toxic reaction to the Hep-2 cell.Positive control drug (virazole) 1: 10-1: 160 medicinal liquids and blank (Hanks liquid replaces medicine) are to HeLa and the equal avirulence of Hep-2 cell.See Table 5.
2, antiviral result
Can suppress pathological changes in the Rsv cell than visiing 1: 10 medicinal liquid of gram medicated powder, to occur that circle contracts be for due to the drug toxicity to few part cell in this concentration liquid, and 1: 40 medicinal liquid can suppress the Adv3 type, 1: 10 medicinal liquid can suppress pathological changes in the Adv7 type cell.1: 20-1: 80 medicinal liquids are to Rsv, 1: the 80 pair of Adv3 type, 1: 20-1: 80 medicinal liquids all can not suppress cytopathy to the Adv7 type.
1: 40 medicinal liquid of pathological changes azoles can suppress pathological changes in the Rsv cell, and 1: 10 medicinal liquid can suppress pathological changes in the Adv3 type cell, and 1: 10-1: 80 medicinal liquids can not suppress pathological changes in the Adv7 type cell.Blank all can not suppress cytopathy.See Table 6.
Table 5 cell toxicity test result
Drug level
The contrast of drug cell cell
1∶10 1∶20 1∶40 1∶80 1∶160
Than visit the gram HeLa------
Hep-2 + - - - - -
Virazole HeLa------
Hep-2 - - - - - -
Blank HeLa------
Hep-2 - - - - - -
Annotate: "-" expression 2 solencyte avirulences, "+" expression part cell circle contracts.
Than visiing gram medicated powder in actual weight.
Table 6 extracorporeal antivirus effect result of the test
The contrast of drug disease cytotoxic drug concentration virus control cell
1∶10 1∶20 1∶40 1∶80
Adv3 - ± ± +++ +++ -
Than visit the gram Adv7-++ ++ ++++++-
Rsv ± ++ ++ +++ +++ -
Adv3 - ++ ++ +++ +++ -
Virazole Adv7 ++ ++ ++ ++++++-
Rsv - - - +++ +++ -
Adv3 +++ +++ +++ +++ +++ -
Blank Adv7 +++++++++++++++-
Rsv +++ +++ +++ +++ +++ -
Annotate: "-" expression cell does not have pathological changes, and " ± " expression part cell circle contracts, and " ++ "-" +++" expression cytopathy degree.
Than visiing gram medicated powder in actual weight.
Conclusion:
Than visit the gram medicated powder 1: 10-1: 40 concentration (10-2.5mg/ml) to the Adv3 type, 1: 10 concentration (10mg/ml) also Adv7 type and Rsv are had inhibitory action.Positive control drug virazole 1: 10-1: 40 concentration (1.0-0.25mg/ml) to Rsv, 1: 10 concentration (1.0mg/ml) also the Adv3 type is had inhibitory action, but 1: 10-1: 40 concentration (1-0.125mg/ml) all can not suppress pathological changes in the cell to the Adv7 type.Blank all can not suppress pathological changes in the cell.Be subjected to reagent and slightly be better than virazole than visiing the inhibitory action of gram medicated powder to Adv3 type and Adv7 type, virazole is better than than visiing gram medicated powder the inhibitory action of Rsv.
Experimental example 3 antiinflammatory actions
The test of 1 mouse dimethylbenzene ear expanding
The test of rat carrageenan pedal swelling
1, experiment material
Medicine: than visiing gram medicated powder, brown ceramic powder, every gram is equivalent to the 2.789g crude drug, and lot number 990222 is provided by SiChuan JinHui Pharmacy Co., Ltd.With distilled water be mixed with 35.2,70.4,140.9mg/ml (suitable respectively 98.2,196.3,393.0mg (crude drug)/ml) for low, in, high dose group is used.
Aspirin Enteric-coated Tablets, white tablet, 50mg/ sheet.Hainan Bikai Pharmaceutical Co., Ltd produces, lot number 970915.It is standby to be mixed with 50mg/ml with distilled water.
Carrageenin is provided by institute of materia medica, Liaoning Province, and it is standby to be mixed with 1% concentration.
Animal: the SD rat, male and female half and half, 170-300g is provided by Chengdu University of Traditional Chinese Medicine's Experimental Animal Center, the quality certification number: No. 8, the real moving Guan Zhidi in river.
2, experimental technique
58 of rats, be divided into 5 groups (seeing Table 7) at random according to the sex body weight, gram medicated powder basic, normal, high dosage group irritates respectively that stomach gives 0.98,1.96 than visiing, 3.93g (crude drug)/kg (be equivalent to respectively clinical consumption per day 5 times, 10 times, 20 times), the administration volume is 1ml/100g, negative group replaces to wait capacity distilled water, once a day, continuous 7 days, positive group is irritated stomach and is given Aspirin Enteric-coated Tablets 1g/kg (be equivalent to clinical rheumatism consumption per day 20 times), the administration volume is 2ml/100g, once a day, continuous three days.Cause inflammation with the left back sufficient sole of the foot of 0.1ml/ rat of 1% chondrus ocellatus Holmes skin middle part subcutaneous injection half an hour after the last administration, cause scorching preceding and back 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours sufficient sole of the foot volumes with volumetric determination respectively, so that the difference of sufficient sole of the foot volume is relatively respectively organized the difference with the distilled water group as the swelling degree before and after scorching.
3, experimental result
The results are shown in Table 7.
The result shows that aspirin and Bi Bai gram medicated powder high dose all has tangible antiinflammatory action in the table, and is more not obvious than visiing in the gram medicated powder dosage and low dosage antiinflammatory action.
4, conclusion
Pitch certain glue and cause the rat paw inflammation and (have tangible antiinflammatory action during 3.93g (crude drug)/kg) than visiing gram medicated powder diagonal angle at high dose.
Table 7 is than the influence of visiing gram medicated powder on Carrageenan rat paw proinflammatory effect
Animal
Dosage
The packet count swelling degree of the paw (ml, X ± SD)
(g/kg)
(only) 5 30 60 120 240 360 (branch)
Distilled water--12 0.14 ± 0.11 0.30 ± 0.16 0.42 ± 0.19 0.69 ± 0.29 0.74 ± 0.28 0.76 ± 0.31
Ah Si
1 10 0.07±0.07 0.12±0.12** 0.10±0.10*** 0.19±0.19*** 0.28±0.31** 0.35±0.33**
Woods
Than visiing gram 3.93 12 0.08 ± 0.09 0.09 ± 0.09***, 0.10 ± 0.10*** 0.44 ± 0.21* 0.47 ± 0.21* 0.49 ± 0.24*
Than visiing gram 1.96 12 0.12 ± 0.12 0.27 ± 0.21 0.39 ± 0.21 0.46 ± 0.28 0.63 ± 0.31 0.73 ± 0.25
Than visiing gram 0.98 12 0.15 ± 0.15 0.26 ± 0.20 0.32 ± 0.16 0.52 ± 0.29 0.70 ± 0.22 0.72 ± 0.31
Annotate: compare * p＜0.05, * * p＜0.01, * * * p＜0.001 with distilled water
Than visiing the gram dosage dose of making a living.
Experimental example 4 analgesic activities
1 writhing method
1.1 experiment material
Medicine: than visiing gram medicated powder, brown ceramic powder, every gram is equivalent to the 2.789g crude drug, and lot number 990222 is provided by SiChuan JinHui Pharmacy Co., Ltd.With distilled water be mixed with 17.5,35.0, (suitable respectively 48.8,97.6,196.7mg (crude drug)/ml) uses for basic, normal, high dosage group 70.5mg/ml.
Meperidine hydrochloride inj, colourless transparent liquid, 1ml/ props up, every hydrochloric Pethidine 0.05g, lot number 950606, Qinghai Pharmaceutic Plant produces.It is standby to be mixed with 1.67mg/ml with normal saline.
Animal: Kunming mouse, male and female half and half, 18-22g is provided by Huaxi Medical Univ's Experimental Animal Center, the quality certification number: No. 67, the real moving Guan Zhidi in river.
1.2 experimental technique
50 of mices, be divided into 5 groups (seeing Table 8) at random according to the sex body weight, gram medicated powder is low than visiing, in, high dose group is irritated stomach respectively and is given 0.98,1.95,3.93g (crude drug)/kg (is equivalent to 5 times of clinical consumption per day respectively, 10 times, 20 times), the administration volume is 0.4ml/20g, negative group replaces with the distilled water that waits capacity, be administered once every day, successive administration 3 days, positive group only at experiment lumbar injection meperidine hydrochloride inj 0.033g/kg on the same day (be equivalent to clinical consumption per day 20 times) once, the administration volume is 0.4ml/20g, half an hour is to acetic acid 0.2ml/ of mouse peritoneal injection 0.6% after the last administration, mouse writhing number of times in 15 minutes behind the record injection acetic acid, the difference of each knob of comparison and distilled water group.
1.3 experimental result
The results are shown in Table 8.
The result shows that pethidine hydrochloride and Bi Bai gram medicated powder low dosage can reduce obviously that acetic acid causes turns round the body number of times and has significant analgesic activity, than visiing gram medicated powder height, middle dosage the trend of analgesic activity is arranged, but with the distilled water group than no significant difference.
Table 8 restrains the influence that the medicated powder Dichlorodiphenyl Acetate causes mice pain than visiing
Group dosage (g/kg) number of animals (only)
Turn round body number of times (X ± SD)
Distilled water-10 47.2 ± 10.8
Pethidine hydrochloride 0.033 10 4.7 ± 6.7***
Than visiing gram 3.93 10 34.7 ± 16.5
Than visiing gram 1.95 10 40.4 ± 10.0
Than visiing gram 0.98 10 36.6 ± 10.1*
Annotate: compare * p＜0.05, * * * p＜0.001 with distilled water
Than visiing the gram dosage dose of making a living
1.4 conclusion
(pain that 0.98g (crude drug)/kg) causes mice acetic acid has significant analgesic activity than visiing gram medicated powder low dosage.
5 refrigeration functions
1 rat 2, the test of 4-dinitrophenol pyrogenicity
1.1 experiment material
Medicine: than visiing gram medicated powder, brown ceramic powder, every gram is equivalent to the 3.62g crude drug, and lot number 990521 is provided by SiChuan JinHui Pharmacy Co., Ltd.With distilled water be mixed with 27.1,54.3, (suitable respectively 98.2,196.5,393.0mg (crude drug)/ml) uses for basic, normal, high dosage group 108.6mg/ml.
Aspirin Enteric-coated Tablets, white tablet, 0.3g/ sheet.Northwest Synthetic Pharmaceutics Factory No.2 produces, lot number 981201.It is standby to be mixed with 60mg/ml with distilled water.
2, the 4-dinitrophenol, yellow crystals, reagent three factories in Shanghai are mixed with 1.25% solution for standby with dehydrated alcohol.
Animal: the SD rat, male and female half and half, 200-290g is provided by Chengdu University of Traditional Chinese Medicine's Experimental Animal Center, the quality certification number: No. 8, the real moving Guan Zhidi in river.
Instrument: WMZ-03 temperature indicator, Shanghai Medical Instrument and Meter Factory.
1.2 experimental technique
Twice of temperature of experiment before measurement rat anus (temperature indicator probe inserts anus 2cm place), select 60 of normal body temperature rats to be divided into 5 groups (seeing Table 11) at random according to the sex body weight, gram medicated powder is low than visiing, in, high dose group is irritated stomach respectively and is given 0.98,1.96,3.93g (crude drug)/kg (is equivalent to 5 times of clinical consumption per day respectively, 10 times, 20 times), positive group is irritated stomach and is given aspirin 0.6g/kg (be equivalent to clinical consumption per day 20 times), the administration volume is 1ml/100g, negative group replaces with the distilled water that waits capacity, half an hour is in rat back subcutaneous injection 2 after the administration, 4-dinitrophenol 25mg/kg (volume 0.2ml/100g) pyrogenicity, measure after the pyrogenicity 0.5 respectively, 1,2,2.5,3 hours anus temperature, as observation index, relatively respectively organize difference with anus temperature approach (anus temperature after the pyrogenicity-pyrogenicity fore-anus temperature) before and after the pyrogenicity with the distilled water group.
1.3 experimental result
The results are shown in Table 9.
Table 9 is than visiing gram medicated powder to rat 2, the influence of 4-dinitrophenol pyrogenic action
Number of animals-anus temperature approach
Dosage
Group (℃, X ± SD)
(g/kg) (only)
0.5 1.0 2.0 2.5 3.0 hours
Distillation
-- 12 1.0±0.4 1.6±0.5 1.6±0.5 1.9±0.4 1.8±0.4
Water
Ah Si
0.6 12 0.3±0.3*** 0.8±0.7** 2.0±0.9 2.1±1.1 2.1±1.2※
Woods
Than visiing
3.93 12 0.5±0.3** 0.7±0.4*** 1.0±0.8* 1.2±0.4*** 1.1±0.4***
Gram
Than visiing
1.96 12 0.6±0.3* 0.8±0.4*** 0.7±0.5*** 1.0±0.6*** 0.9±0.5***
Gram
Than visiing
0.98 12 0.4±0.3*** 0.4±0.4*** 0.9±0.9* 1.3±0.8* 1.4±0.7
Gram
Annotate: compare * P＜0.05, * * p＜0.01, * * * p＜0.001 with distilled water
Than visiing the gram dosage dose of making a living
※ has two male rat death in the time of 180 minutes, the number of animals of this moment is that 10 results show that aspirin is in injection 2,0.5 and 1 hour meter reveals refrigeration function behind the 4-dinitrophenol, than visiing gram medicated powder height, middle dosage in injection 2, all showed as refrigeration function behind the 4-dinitrophenol in 0.5,1,2,2.5,3 hour, than visiing gram medicated powder low dosage in injection 2, behind the 4-dinitrophenol 0.5,1,2,2.5 hour also performance refrigeration function is arranged.
1.4 conclusion
Than visiing the high, medium and low dosage of gram medicated powder to 2,4-dinitrophenol pyrogenicity rat has significant refrigeration function, and holds time longer than the refrigeration function of aspirin.
The test of 2 rabbit peptone pyrogenicity
2.1 experiment material
Medicine: than visiing gram medicated powder, brown ceramic powder, every gram is equivalent to the 3.62g crude drug, and lot number 390521 is provided by SiChuan JinHui Pharmacy Co., Ltd.With distilled water be mixed with 54.1,108.3, (suitable respectively 196,392,784mg (crude drug)/ml) uses for basic, normal, high dosage group 216.6mg/ml.
Aspirin Enteric-coated Tablets, white tablet, the 0.3g/ sheet, Northwest Synthetic Pharmaceutics Factory No.2 produces, lot number 9812022.Be mixed with distilled water: 120mg/ml is standby.
Peptone F403, Shanghai chemical reagents corporation of Chinese Medicine group, lot number F990812.Be mixed with 10% standby with distilled water.
Animal: Japanese white big ear rabbit, 1.6-2.95kg is provided by Chengdu University of Traditional Chinese Medicine's Experimental Animal Center.
Instrument: Omron MC-3B type computer numeral formula clinical thermometer, Dalian, Omron company limited product.
2.2 experimental technique
Twice of temperature of experiment before measurement rabbit anus (thermometer inserts anus 2cm place), select the normal body temperature rabbit to be divided into 6 groups (seeing Table 10) at random, inject 10% peptone F403 10ml/kg pyrogenicity in leg muscle, after 1 hour, irritate stomach respectively and give 0.98.1.96,3.92g (crude drug)/kg (be equivalent to respectively clinical consumption per day 5 times, 10 times, 20 times) than visiing gram medicated powder basic, normal, high dosage group, positive group is irritated stomach and is given aspirin.
Table 10 than visit gram medicated powder to the influence of rabbit peptone pyrogenic action (℃, X ± SD)
Liver temperature approach after the number of animals administration
Dosage
Group (only) (♀, normal body temperature
(g/kg) 1 2 3 4 5 6h
♂)
Distilled water--7 (3,4) 38.8 ± 0.2 0.7 ± 0.3 1.0 ± 0.4 1.3 ± 0.3 1.3 ± 0.3 1.1 ± 0.6 1.1 ± 0.5
Ah Si 0.2 ± 0.4**, 0.3 ± 0.5*, 0.4 ± 0.3*
0.6 9(5，4) 38.6±0.3 0.2±0.4* 0.6±0.7 1.0±0.6
Woods * * * * *
Than visiing gram 3.92 8 (4,4) 38.9 ± 0.3 0.0 ± 0.7*, 0.4 ± 0.8* 0.7 ± 0.9 1.0 ± 0.5 1.0 ± 0.5 1.0 ± 0.5
Than visiing gram 1.96 8 (5,3) 38.6 ± 0.2 0.1 ± 0.5*, 0.7 ± 0.4* 0.9 ± 0.5 1.0 ± 0.5 1.2 ± 0.6 1.3 ± 0.5
Than visiing gram 0.98 8 (5,3) 38.7 ± 0.2 0.4 ± 0.5 0.6 ± 0.5 0.8 ± 0.6 1.0 ± 0.6 1.1 ± 0.6 1.1 ± 0.6
Annotate: compare * P＜0.05, * * * P＜0.001 with distilled water
Than visiing the gram dosage dose of making a living
(0.6g/kg be equivalent to clinical consumption per day 20 times), the administration volume is 5ml/kg, and negative group replaces with the distilled water that waits capacity, measures 1,2,3,4,5,6 hour anus temperature after the administration respectively, the difference of calculating and normal body temperature, the relatively difference of each group and distilled water group.
2.3 experimental result
The results are shown in Table 10.
The result shows that aspirin is after administration) the rabbit anus temperature of peptone pyrogenicity is significantly reduced, has tangible refrigeration function, than visit gram medicated powder height, middle dosage group 1 hour meter after administration reveals the trivial solution heat effect, all the other time points and low dose group only had analgesic trend in 4 hours in the past after administration, compare there was no significant difference with the distilled water group.
2.4 conclusion
Than visit gram medicated powder height, middle dosage has certain refrigeration function to peptone pyrogenicity rabbit.
Experimental example 6 defecating feces excretions
The test of 1 intestinal propulsion
1.1 experiment material
Medicine: than visiing gram medicated powder, brown ceramic powder, every gram is equivalent to the 2.789g crude drug, and lot number 990222 is provided by SiChuan JinHui Pharmacy Co., Ltd.With 10% charcoal end suspension be mixed with 17.5,35.0,70.5mg/ml (suitable respectively 48.8,97.6,196.7mg (crude drug)/ml) for low, in, high dose group is used.
The dahuang tansuanqingna sheet, pale brown color chips agent, 0.15g/ sheet.Sichuan wide lean pharmaceutical Co. Ltd produces, lot number 970335.It is standby to be mixed with 0.45g/20ml with 10% charcoal end suspension.
Animal: Kunming mouse, 18-22g is provided by Huaxi Medical Univ's Experimental Animal Center, the quality certification number: No. 67, the real moving Guan Zhidi in river.
1.2 experimental technique
52 of Kunming mouses, be divided into 5 groups (seeing Table 11) at random according to the sex body weight, positive group is irritated stomach and is given dahuang tansuanqingna sheet 0.45g/kg (be equivalent to clinical consumption per day 20 times), gram medicated powder basic, normal, high dosage group irritates respectively that stomach gives 0.98,1.95 than visiing, 3.93g (crude drug)/kg (be equivalent to respectively clinical consumption per day 5 times, 10 times, 20 times), the administration volume is 0.4ml/20g, and cloudy domestic animal group is to wait 10% of capacity.
Table 11 is than visiing gram medicated powder to the propulsive influence of mouse small intestine
Group dosage (g/kg) number of animals (only, ♀, ♂) charcoal end propelling rate %
Negative group----11 (6,5) 49.8 ± 10.1
Dahuang tansuanqingna 0.45 10 (5,5) 79.8 ± 19.3***
Than visiing gram 3.93 10 (5,5), 74.3 ± 22.1**
Than visiing gram 1.95 11 (6,5), 67.1 ± 10.2***
Than visiing gram 0.98 10 (5,5) 57.6 ± 15.6
Annotate: compare * * p＜0.01, * * * p＜0.001 with the feminine gender group
Than visiing the gram dosage dose of making a living
Charcoal end suspension replaces, after the administration 15 minutes, disconnected neck is put to death, dissect, isolate the small intestinal of pylorus to ileocecus, measure pylorus to the small intestinal total length of ileocecus and pylorus to charcoal end length foremost, calculate intestinal propulsion rate=charcoal end front end and pylorus apart from (cm)/small intestinal total length (cm) * 100%.
1.3 experimental result
The results are shown in Table 11.
1.4 conclusion
Than visit gram medicated powder height, middle dosage can significantly promote the mouse small intestine propulsion functions, and low dosage promotes that the intestinal propulsion function is not obvious.
2 mice excrement peritonitis model tests
2.1 experiment material
Medicine: than visiing gram medicated powder, brown ceramic powder, every gram is equivalent to the 2.789g crude drug, and lot number 990222 is provided by SiChuan JinHui Pharmacy Co., Ltd.Be mixed with 17.5,35.0 with 10% charcoal end suspension, (suitable respectively 48.8,97.6,196.7mg (crude drug)/ml) uses for basic, normal, high dosage group 70.5mg/ml.
The dahuang tansuanqingna sheet, pale brown color chips agent, 0.15g/ sheet.Sichuan wide lean pharmaceutical Co. Ltd produces, lot number 970335.It is standby to be mixed with 0.45g/20ml with 10% charcoal end suspension.
Animal: Kunming mouse, male and female half and half, 18-22g is provided by Huaxi Medical Univ's Experimental Animal Center, the quality certification number: No. 67, the real moving Guan Zhidi in river.
2.2 experimental technique
Get oneself excrement of mice a little, grind, add the normal saline suspendible, with four layers of gauze elimination dregs, get every mouse peritoneal injection of this kind filtrate 0.5ml, the perpendicular hair of visible mice after 18 hours, it is few moving to curl, and does not eat food, dissect 2 and see that intraperitoneal has a little liquid, the hyperemia of part intestinal tube, most inflations, part spasm.Get 50 and thus much plant mice, be divided into 5 groups at random, 10 mices do not make normal control (seeing Table 12) to liquid manure in addition, positive group is irritated stomach and is given dahuang tansuanqingna sheet 0.45g/kg (be equivalent to clinical consumption per day 20 times), the gram medicine is low than visiing, in, high dose group is irritated stomach respectively and is given 0.98,1.96,3.93g (crude drug)/kg (is equivalent to 5 times of clinical consumption per day respectively, 10 times, 20 times), the administration volume is 0.4ml/20g, model group and normal control group with etc. the 10% charcoal end suspension of capacity replace, a defecation grain number in time (surpass 7 hours in 7 hours) and 7 hours of melena arranged first in record, and the difference of each group and model group relatively.
Table 12 is than visiing the influence of gram medicated powder to excrement peritonitis mice charcoal end excrement efflux time and faecal pellet number (X ± SD)
Group dosage (g/kg) number of animals (only) melena time of occurrence (divides defecation grain in 7 hours
Clock) counts (grain)
Normal control group----10 148.0 ± 36.6*** 6.8 ± 2.6***
Model group----10 305.1 ± 91.2 2.4 ± 2.3
Radix Et Rhizoma Rhei bicarbonate 0.45 10 199.5 ± 96.6***, 7.3 ± 4.8***
Sodium
Than visiing gram 3.93 10 210.7 ± 91.6***, 6.9 ± 4.3***
Than visiing gram 1.95 10 261.4 ± 87.0**, 3.6 ± 3.2**
Than visiing gram 0.98 10 313.9 ± 105.2 2.10 ± 2.8
Annotate: compare * * P＜0.01, * * * P＜0.001 with model group
Than visiing the gram dosage dose of making a living
2.3 experimental result
Knot is that fruit sees Table 12.
Model group and normal control group compare, melena time of occurrence significant prolongation, and faecal pellet digital display work reduces in 7 hours, shows the modeling success.Dahuang tansuanqingna sheet and than visiing melena time of occurrence that gram medicated powder height, middle dosage can make excrement peritonitis mice in advance makes that the faecal pellet number increases in 7 hours, has significant defecating feces excretion.
2.4 conclusion
Than visit gram medicated powder height, middle dosage has significant defecating feces excretion to excrement peritonitis mice.
Following embodiment all can realize the effect of above-mentioned experimental example.
Embodiment 1 capsule
Radix Et Rhizoma Rhei (processed with wine) 200g Fel Ursi powder 10g Radix Scutellariae 200g Radix Platycodonis 200g
Radix Angelicae Dahuricae 200g Herba Houttuyniae 400g Herba Menthae 100g
More than seven the flavor, Radix et Rhizoma Rhei (processed with wine) soaks in right amount with 75% ethanol, 70 ± 5 ℃ of dryings are ground into fine powder, Fel Ursi powder is ground into fine powder, sieving for standby; The Radix Angelicae Dahuricae, Herba Houttuyniae, Herba Menthae add 10 times of water extraction volatile oil 5 hours, collect volatile oil, and the aqueous solution after distillation device is in addition collected, and 3 times of water gaging washings of medicinal residues reuse 2 times filter, and merge 3 times aqueous solution, and are standby; Other gets beta-schardinger dextrin-10.5g, adds 10 times of water dissolutioies, slowly drips the alcoholic solution of above-mentioned volatile oil, volatile oil: ethanol=1: 1, and under the ultrasound wave of 30 ± 2KHz ultrasonic 1 hour, cold preservation was spent the night, sucking filtration, the room temperature decompression is dry down, crosses 80 mesh sieves, and is standby; All the other two flavors are cut into segment with Radix Scutellariae, decoct with water twice with Radix Platycodonis, add 8 times of amount boiling water for the first time, add 6 times of water gagings for the second time, and each 1.5 hours, collecting decoction filtered.The aqueous solution of filtrate and above-mentioned collection merges, and being concentrated into 80 ℃ of relative densities is 1.10-1.15, and spray drying gets dry extract.Get dry extract and Radix et Rhizoma Rhei (processed with wine) powder, Fel Ursi powder, and volatile oil beta-cyclodextrin inclusion and appropriate amount of starch, mixing incapsulates, and makes 1000, promptly.
Embodiment 2 granules
Radix Et Rhizoma Rhei (processed with wine) 230g Fel Ursi powder 15g Radix Scutellariae 230g Radix Platycodonis 230g
Radix Angelicae Dahuricae 230g Herba Houttuyniae 440g Herba Menthae 130g
More than seven the flavor, Radix et Rhizoma Rhei (processed with wine) soaks in right amount with 70% ethanol, 80 ℃ of dryings are ground into fine powder, Fel Ursi powder is ground into fine powder, sieving for standby; The Radix Angelicae Dahuricae, Herba Houttuyniae, Herba Menthae add 11 times of water extraction volatile oil 5 hours, collect volatile oil, and the aqueous solution after distillation device is in addition collected, and 3 times of water gaging washings of medicinal residues reuse 2 times filter, and merge 3 times aqueous solution, and are standby; Other gets beta-schardinger dextrin-10.5g, adds 10 times of water dissolutioies, slowly drips the alcoholic solution of above-mentioned volatile oil, volatile oil: ethanol=1: 1, and under the ultrasound wave of 35KHz ultrasonic 1 hour, cold preservation was spent the night, sucking filtration, the room temperature decompression is dry down, crosses 85 mesh sieves, and is standby; All the other two flavors are cut into segment with Radix Scutellariae, decoct with water twice with Radix Platycodonis, add 8 times of amount boiling water for the first time, add 6 times of water gagings for the second time, and each 1.5 hours, collecting decoction filtered.The aqueous solution of filtrate and above-mentioned collection merges, and being concentrated into 80 ℃ of relative densities is 1.10-1.15, and spray drying gets dry extract.Get dry extract and Radix et Rhizoma Rhei (processed with wine) powder, Fel Ursi powder, and volatile oil beta-cyclodextrin inclusion and appropriate amount of starch are made granule 380g.
Embodiment 3Tablet
Radix Et Rhizoma Rhei (processed with wine) 180g Fel Ursi powder 8g Radix Scutellariae 180g Radix Platycodonis 180g
Radix Angelicae Dahuricae 180g Herba Houttuyniae 380g Herba Menthae 80g
More than seven the flavor, Radix et Rhizoma Rhei (processed with wine) soaks in right amount with 78% ethanol, 65 ℃ of dryings are ground into fine powder, Fel Ursi powder is ground into fine powder, sieving for standby; The Radix Angelicae Dahuricae, Herba Houttuyniae, Herba Menthae add 9 times of water extraction volatile oil 5 hours, collect volatile oil, and the aqueous solution after distillation device is in addition collected, and 3 times of water gaging washings of medicinal residues reuse 2 times filter, and merge 3 times aqueous solution, and are standby; Other gets beta-schardinger dextrin-10.5g, adds 10 times of water dissolutioies, slowly drips the alcoholic solution of above-mentioned volatile oil, volatile oil: ethanol=1: 1, and under the ultrasound wave of 25KHz ultrasonic 1.5 hours, cold preservation was spent the night, sucking filtration, the room temperature decompression is dry down, crosses 75 mesh sieves, and is standby; All the other two flavors are cut into segment with Radix Scutellariae, decoct with water twice with Radix Platycodonis, add 8 times of amount boiling water for the first time, add 6 times of water gagings for the second time, and each 1.5 hours, collecting decoction filtered.The aqueous solution of filtrate and above-mentioned collection merges, and being concentrated into 80 ℃ of relative densities is 1.10-1.15, and spray drying gets dry extract.Get dry extract and Radix et Rhizoma Rhei (processed with wine) powder, Fel Ursi powder, and volatile oil beta-cyclodextrin inclusion and appropriate amount of starch, often regulation becomes 1000 in tablet.
Embodiment 4The method of quality control of capsule
Differentiate: a. gets this product content 1g, adds methanol 20ml, and supersound process 15 minutes filters, and filtrate is concentrated into 1ml, as need testing solution; Get Radix Et Rhizoma Rhei control medicinal material powder 0.5g, make control medicinal material solution with the method operation; Other gets the emodin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with 0.5% sodium carboxymethyl cellulose, with 7: 3: 0.1 benzene-Ethyl formate-formic acid was developing solvent, launch, take out, dry; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show the speckle of same color;
B. get this product content 2.5g and add methanol ultrasonic 2 times, each 30ml, 15 minutes, filter, filtrate evaporate to dryness, residue add that 10% sodium hydroxide solution 20ml is ultrasonic to make dissolving, 100 ℃ of heating hydrolysis 2 hours, put cold, with the hydrochloric acid adjust pH to 2-3, drink extraction 3 times with acetic acid second, each 15ml merges ethyl acetate liquid, wash each 40ml, ethyl acetate liquid water bath method with water 3 times, residue makes dissolving with methanol 1ml, and is centrifugal, gets supernatant as need testing solution; Other gets chenodeoxycholic acid and ursodesoxycholic acid reference substance, adds methanol respectively and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with 0.5% sodium carboxymethyl cellulose, with 10: 5: 5: the upper solution of 3: 1 isobutyltrimethylmethane .-ether-glacial acetic acid-n-butanol-waters was developing solvent, launch, take out, dry, spray is with 20% sulfuric acid solution, in 105 ℃ of bakings 5 minutes; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
C. get this product content 4g, add methanol 40ml, ultrasonic 20 minutes, filter the filtrate evaporate to dryness, residue adds water 20ml makes dissolving, transfers pH to 3-4 with hydrochloric acid, adds ethyl acetate extraction 2 times, each 20ml, merge ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the baicalin reference substance, adds methanol and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5 μ l of above-mentioned two kinds of solution, putting in same 0.5% sodium carboxymethyl cellulose with the preparation of 1% sodium acetate respectively is on the silica gel g thin-layer plate of adhesive, with 5: 3: 1: 1 ethyl acetate-acetone-formic acid-water was developing solvent, launch, take out, dry, spray is with 2% ferric chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
D. get this product content 4g, put in the round-bottomed flask, add water 250ml, extract Division A League Matches of French Football methods (an appendix X of Chinese Pharmacopoeia version in 2000 D) according to volatile oil and connect volatile oil determination apparatus; Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 4 hours stops heating, places a moment, divides and gets the ethyl acetate layer, as need testing solution; Other gets the methylnonanone reference substance, adds ethyl acetate and makes the solution that every 1ml contains 10 μ g, in contrast product solution; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), 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 0.5% sodium carboxymethyl cellulose, with 19: 1 benzene-ethyl acetates was developing solvent, launch, take out, dry, spray is with the dinitrophenylhydrazine test solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
E. get this product content 4g, put in the round-bottomed flask, add water 250ml, extract Division A League Matches of French Football methods (an appendix X of Chinese Pharmacopoeia version in 2000 D) according to volatile oil and connect volatile oil determination apparatus; Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 4 hours stops heating, places a moment, divides and gets the ethyl acetate layer, as need testing solution; Get the Mentholum reference substance, add ethyl acetate and make the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), get 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 0.5% sodium carboxymethyl cellulose, with 9: 1 normal hexane-ethyl acetates was developing solvent, launch, take out, dry, spray is with 2: 8 vanillin sulphuric acid test solution-alcoholic acid mixed solutions, and it is clear to be heated to the speckle colour developing at 100 ℃; In the test sample chromatograph, on the corresponding position of reference substance chromatograph, show the speckle of same color;
Assay: a. emodin, chrysophanol: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D); Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol-0.1% phosphoric acid solution was a mobile phase in 85: 15; The detection wavelength is 254nm; Number of theoretical plate calculates by the emodin peak should be not less than 1500; The preparation of reference substance solution, precision take by weighing emodin, each 10mg of chrysophanol reference substance, put respectively in the 100ml measuring bottle, with dissolve with methanol and be diluted to scale, shake up; As emodin, chrysophanol reference substance stock solution; Precision is measured emodin reference substance stock solution 1ml, chrysophanol reference substance stock solution 2ml respectively, puts respectively in the 25ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets to contain among the every 1ml of emodin among 4 μ g, the every 1ml of chrysophanol to contain 8 μ g; The preparation of need testing solution is got the content under this product content uniformity item, mixing, get 0.2g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, and supersound process is 1 hour under the ultrasound wave of 30 ± 2KHz, put cold, claim to decide weight again, replenish the weight that subtracts mistake, shake up with methanol, centrifugal, get supernatant as need testing solution; 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 Radix Et Rhizoma Rhei with emodin (C
15H
10O
5) and chrysophanol (C
15H
10O
4) the total amount meter, must not be less than 0.7mg;
B. baicalin: measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D); Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 47: 53: 0.2 methanol-water-phosphoric acid are mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the baicalin peak should be not less than 2500; The preparation of reference substance solution, precision take by weighing at 4 hours baicalin reference substance of 60 ℃ of drying under reduced pressure an amount of, add methanol and make every 1ml and contain 20 μ g solution, promptly; The content under the content uniformity item is got in the preparation of need testing solution, and mixing is got 28.5mg, and accurate the title decides, put in the 25ml measuring bottle, it is an amount of to add 50% ethanol, and supersound process 15 minutes is put cold, add 50% ethanol dilution to scale, shake up, centrifugal, get supernatant as need testing solution; Survey the shallow lake method, 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 Radix Scutellariae by baicalin (C
12H
18O
11) meter, must not be less than 6.0mg.
Claims (13)
1. Chinese medicine composition for the treatment of acute pharyngitis, gingivitis is characterized in that this Chinese medicine composition made by following raw material medicaments:
Radix Et Rhizoma Rhei 160-240 weight portion Fel Ursi powder 5-18 weight portion Radix Scutellariae 160-240 weight portion
Radix Platycodonis 160-240 weight portion Radix Angelicae Dahuricae 160-240 weight portion Herba Houttuyniae 350-450 weight portion
Herba Menthae 60-140 weight portion.
2. Chinese medicine composition as claimed in claim 1 is characterized in that this Chinese medicine composition made by following raw material medicaments:
Radix Et Rhizoma Rhei 200 weight portion Fel Ursi powders 10 weight portion Radix Scutellariaes 200 weight portion Radix Platycodoniss 200 weight portions
The Radix Angelicae Dahuricae 200 weight portion Herba Houttuyniae 400 weight portion Herba Menthaes 100 weight portions.
3. Chinese medicine composition as claimed in claim 1 is characterized in that this Chinese medicine composition made by following raw material medicaments:
Radix Et Rhizoma Rhei 230 weight portion Fel Ursi powders 15 weight portion Radix Scutellariaes 230 weight portion Radix Platycodoniss 230 weight portions
The Radix Angelicae Dahuricae 230 weight portion Herba Houttuyniae 440 weight portion Herba Menthaes 130 weight portions.
4. as the described Chinese medicine composition of claim 1-3, it is characterized in that the preferred Radix et Rhizoma Rhei (processed with wine) of Radix Et Rhizoma Rhei in this Chinese medicine composition.
5. Chinese medicine composition as claimed in claim 4 is characterized in that said composition adds excipient and is prepared into a kind of in tablet, granule, capsule, pill, oral liquid, masticatory, aerosol, the soft capsule.
6. the preparation method of Chinese medicinal composition capsules agent as claimed in claim 5, it is characterized in that this method is: Radix et Rhizoma Rhei (processed with wine) soaks in right amount with 60-85% ethanol, and 60-80 ℃ of drying is ground into fine powder, and Fel Ursi powder is ground into fine powder, sieving for standby; The Radix Angelicae Dahuricae, Herba Houttuyniae, Herba Menthae add 9-11 times of water extraction volatile oil 4-6 hour, collect volatile oil, and the aqueous solution after distillation device is in addition collected, and 2-4 times of water gaging washing of medicinal residues reuse 1-3 time filters, and merges aqueous solution, and be standby; Other gets beta-schardinger dextrin-10.5 weight portions, adds 9-11 times of water dissolution, slowly drips the alcoholic solution of above-mentioned volatile oil, volatile oil: ethanol=1-2: 1-2, under the ultrasound wave of 20-40KHz ultrasonic 1-2 hour, cold preservation was spent the night, sucking filtration, the room temperature decompression is dry down, crosses the 70-90 mesh sieve, and is standby; All the other two flavors are cut into segment with Radix Scutellariae, add 6-8 times of decocting with Radix Platycodonis and boil 1-3 time, and each 1-2 hour, collecting decoction filtered, and the aqueous solution of filtrate and above-mentioned collection merges, and being concentrated into 70-85 ℃ of relative density is 1.10-1.15, and spray drying gets dry extract; Get dry extract and Radix et Rhizoma Rhei (processed with wine) powder, Fel Ursi powder, and volatile oil beta-cyclodextrin inclusion and an amount of 0-20 weight portion starch, mixing incapsulates, and makes 1000, promptly.
7. the preparation method of Chinese medicinal composition capsules agent as claimed in claim 6, it is characterized in that this method is: Radix et Rhizoma Rhei (processed with wine) soaks in right amount with 75% ethanol, and 65-75 ℃ of drying is ground into fine powder, and Fel Ursi powder is ground into fine powder, sieving for standby; The Radix Angelicae Dahuricae, Herba Houttuyniae, Herba Menthae add 10 times of water extraction volatile oil 5 hours, collect volatile oil, and the aqueous solution after distillation device is in addition collected, and 3 times of water gaging washings of medicinal residues reuse 2 times filter, and merge 3 times aqueous solution, and are standby; Other gets beta-schardinger dextrin-10.5 weight portions, adds 10 times of water dissolutioies, slowly drips the alcoholic solution of above-mentioned volatile oil, volatile oil: ethanol=1: 1, and under the ultrasound wave of 28-32KHz ultrasonic 1 hour, cold preservation was spent the night, sucking filtration, the room temperature decompression is dry down, crosses 80 mesh sieves, and is standby; All the other two flavors are cut into segment with Radix Scutellariae, decoct with water twice with Radix Platycodonis, add 8 times of amount boiling water for the first time, add 6 times of water gagings for the second time, and each 1.5 hours, collecting decoction filtered; The aqueous solution of filtrate and above-mentioned collection merges, and being concentrated into 80 ℃ of relative densities is 1.10-1.15, and spray drying gets dry extract; Get dry extract and Radix et Rhizoma Rhei (processed with wine) powder, Fel Ursi powder, and volatile oil beta-cyclodextrin inclusion and appropriate amount of starch, mixing incapsulates, and makes 1000, promptly.
8. as the sharp method of quality control that requires 5 described Chinese medicinal composition preparation, it is characterized in that discrimination method in this method comprises one or more in the following discriminating:
A. get this composite preparation 1g, add methanol 20ml, supersound process 10-20 minute, filter, filtrate is concentrated into 1ml, as need testing solution; Get Radix Et Rhizoma Rhei control medicinal material powder 0.5g, make control medicinal material solution with the method operation; Other gets the emodin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to thin layer chromatography test, draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the 0.4-0.6% sodium carboxymethyl cellulose, with 6-8: 2-4: 0.1-0.2 benzene-Ethyl formate-formic acid is developing solvent, launch, take out, dry; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show the speckle of same color;
B. get this composite preparation 2.5g and add methanol ultrasonic 1-3 time, each 30ml, 10-20 minute, filter, filtrate evaporate to dryness, residue add that 8-12% sodium hydroxide solution 20ml is ultrasonic to make dissolving, 95-105 ℃ heating hydrolysis 1-3 hour, put cold, with the hydrochloric acid adjust pH to 2-3, drink extraction 2-4 time with acetic acid second, each 15ml merges ethyl acetate liquid, wash each 40ml, ethyl acetate liquid water bath method with water 2-4 time, residue makes dissolving with methanol 1ml, and is centrifugal, gets supernatant as need testing solution; Other gets chenodeoxycholic acid and ursodesoxycholic acid reference substance, adds methanol respectively and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography, draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the 0.4-0.6% sodium carboxymethyl cellulose, with 9-11: 4-6: 4-6: 2-4: the upper solution of 1-2 isobutyltrimethylmethane .-ether-glacial acetic acid-n-butanol-water is developing solvent, launch, take out, dry, spray is with the 15-25% sulfuric acid solution, in 100-110 ℃ of baking 4-7 minute; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
C. get this composite preparation 4g, add methanol 40ml, ultrasonic 15-25 minute, filter the filtrate evaporate to dryness, residue adds water 20ml makes dissolving, transfers pH to 3-4 with hydrochloric acid, adds ethyl acetate extraction 1-3 time, each 20ml, merge ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the baicalin reference substance, adds methanol and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, putting in same 0.4-0.6% sodium carboxymethyl cellulose with the preparation of 1% sodium acetate respectively is on the silica gel g thin-layer plate of adhesive, with 4-6: 2-4: 1-2: 1-2 ethyl acetate-acetone-formic acid-water is developing solvent, launch, take out, dry, spray is with 2% ferric chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
D. get this composite preparation 4g, put in the round-bottomed flask, add water 250ml, an appendix XD connects volatile oil determination apparatus according to volatile oil extraction method Chinese Pharmacopoeia version in 2000; Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 3-5 hour, stop heating, place a moment, divide and get the ethyl acetate layer, as need testing solution; Other gets the methylnonanone reference substance, adds ethyl acetate and makes the solution that every 1ml contains 10 μ g, in contrast product solution; According to thin layer chromatography test, 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 0.4-0.6% sodium carboxymethyl cellulose, with 18-20: 1-2 benzene-ethyl acetate is developing solvent, launches, and takes out, dry, spray is with the dinitrophenylhydrazine test solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
E. get this composite preparation 4g, put in the round-bottomed flask, add water 250ml, an appendix XD connects volatile oil determination apparatus according to volatile oil extraction method Chinese Pharmacopoeia version in 2000; Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 3-5 hour, stop heating, place a moment, divide and get the ethyl acetate layer, as need testing solution; Get the Mentholum reference substance, add ethyl acetate and make the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin layer chromatography, get 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 0.4-0.6% sodium carboxymethyl cellulose, with 7-10: 1-2 normal hexane-ethyl acetate is developing solvent, launch, take out, dry, spray is with 1-3: 7-9 vanillin sulphuric acid test solution-alcoholic acid mixed solution, and it is clear to be heated to the speckle colour developing at 95-110 ℃; In the test sample chromatograph, on the corresponding position of reference substance chromatograph, show the speckle of same color.
9. as the sharp method of quality control that requires 8 described Chinese medicinal composition preparation, the discrimination method that it is characterized in that capsule comprises one or more in the following discriminating:
A. get this product content 1g, add methanol 20ml, supersound process 15 minutes filters, and filtrate is concentrated into 1ml, as need testing solution; Get Radix Et Rhizoma Rhei control medicinal material powder 0.5g, make control medicinal material solution with the method operation; Other gets the emodin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to thin layer chromatography test, draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with 0.5% sodium carboxymethyl cellulose, be developing solvent with 7: 3: 0.1 benzene-Ethyl formate-formic acid, launch, take out, dry; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show the speckle of same color;
B. get this product content 2.5g and add methanol ultrasonic 2 times, each 30ml, 15 minutes, filter, filtrate evaporate to dryness, residue add that 10% sodium hydroxide solution 20ml is ultrasonic to make dissolving, 100 ℃ of heating hydrolysis 2 hours, put cold, with the hydrochloric acid adjust pH to 2-3, drink extraction 3 times with acetic acid second, each 15ml merges ethyl acetate liquid, wash each 40ml, ethyl acetate liquid water bath method with water 3 times, residue makes dissolving with methanol 1ml, and is centrifugal, gets supernatant as need testing solution; Other gets chenodeoxycholic acid and ursodesoxycholic acid reference substance, adds methanol respectively and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography, draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with 0.5% sodium carboxymethyl cellulose, with 10: 5: 5: the upper solution of 3: 1 isobutyltrimethylmethane .-ether-glacial acetic acid-n-butanol-waters was developing solvent, launch, take out, dry, spray is with 20% sulfuric acid solution, in 105 ℃ of bakings 5 minutes; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
C. get this product content 4g, add methanol 40ml, ultrasonic 20 minutes, filter the filtrate evaporate to dryness, residue adds water 20ml makes dissolving, transfers pH to 3-4 with hydrochloric acid, adds ethyl acetate extraction 2 times, each 20ml, merge ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the baicalin reference substance, adds methanol and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, putting in same 0.5% sodium carboxymethyl cellulose with the preparation of 1% sodium acetate respectively is on the silica gel g thin-layer plate of adhesive, with 5: 3: 1: 1 ethyl acetate-acetone-formic acid-water was developing solvent, launch, take out, dry, spray is with 2% ferric chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
D. get this product content 4g, put in the round-bottomed flask, add water 250ml, extract the Division A League Matches of French Football method according to volatile oil and connect volatile oil determination apparatus; Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 4 hours stops heating, places a moment, divides and gets the ethyl acetate layer, as need testing solution; Other gets the methylnonanone reference substance, adds ethyl acetate and makes the solution that every 1ml contains 10 μ g, in contrast product solution; According to thin layer chromatography test, 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 0.5% sodium carboxymethyl cellulose, with 19: 1 benzene-ethyl acetates was developing solvent, launched, and took out, dry, spray is with the dinitrophenylhydrazine test solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
E. get this product content 4g, put in the round-bottomed flask, add water 250ml, extract the Division A League Matches of French Football method according to volatile oil and connect volatile oil determination apparatus; Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 4 hours stops heating, places a moment, divides and gets the ethyl acetate layer, as need testing solution; Get the Mentholum reference substance, add ethyl acetate and make the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin layer chromatography, get each 10 μ l of above-mentioned two kinds of solution and above-mentioned reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with 0.5% sodium carboxymethyl cellulose, with 9: 1 normal hexane-ethyl acetates was developing solvent, launch, take out, dry, spray is with 2: 8 vanillin sulphuric acid test solution-alcoholic acid mixed solutions, and it is clear to be heated to the speckle colour developing at 100 ℃; In the test sample chromatograph, on the corresponding position of reference substance chromatograph, show the speckle of same color.
10. as the sharp method of quality control that requires 5 described Chinese medicinal composition preparation, it is characterized in that content assaying method in this method comprises one or both in the following assay:
A. emodin, chrysophanol: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 80-90: 10-20 methanol-0.1% phosphoric acid solution is a mobile phase; The detection wavelength is 254nm; Number of theoretical plate calculates by the emodin peak should be not less than 1500; The preparation of reference substance solution, precision take by weighing emodin, each 10mg of chrysophanol reference substance, put respectively in the 100ml measuring bottle, with dissolve with methanol and be diluted to scale, shake up as emodin chrysophanol reference substance stock solution; Precision is measured emodin reference substance stock solution 1ml, chrysophanol reference substance stock solution 2ml respectively, puts respectively in the 25ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets to contain among the every 1ml of emodin among 4 μ g, the every 1ml of chrysophanol to contain 8 μ g; This composite preparation 0.2g is got in the preparation of need testing solution, and accurate the title decides, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, under the ultrasound wave of 20-40KHz supersound process 1-2 hour, put cold, claim to decide weight again, replenish the weight that subtracts mistake, shake up with methanol, centrifugal, get supernatant as need testing solution; Algoscopy, accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly; This composite preparation per unit amount contains the total amount of Radix Et Rhizoma Rhei in emodin and chrysophanol, must not be less than 6.5-8mg;
B. baicalin: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 43-53: 50-58: the 0.1-0.3 methanol-water-phosphoric acid is a mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the baicalin peak should be not less than 2500; The preparation of reference substance solution, precision take by weighing at 55-65 ℃ of drying under reduced pressure 3-5 hour baicalin reference substance an amount of, add methanol and make every 1ml and contain 20 μ g solution, promptly; This composite preparation 28.5mg is got in the preparation of need testing solution, accurate claims surely, puts in the 25ml measuring bottle, and it is an amount of to add 45-55% ethanol, and supersound process 10-25 minute, put coldly, add the 45-55% ethanol dilution to scale, shake up, centrifugal, get supernatant as need testing solution; Survey the shallow lake method, accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly; This composite preparation per unit amount contains Radix Scutellariae by baicalin, must not be less than 55-65mg.
11. as the sharp method of quality control that requires 10 described Chinese medicinal composition preparation, the content assaying method that it is characterized in that capsule comprises one or both in the following assay:
A. emodin, chrysophanol: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol-0.1% phosphoric acid solution was a mobile phase in 85: 15; The detection wavelength is 254nm; Number of theoretical plate calculates by the emodin peak should be not less than 1500; The preparation of reference substance solution, precision take by weighing emodin, each 10mg of chrysophanol reference substance, put respectively in the 100ml measuring bottle, with dissolve with methanol and be diluted to scale, shake up; As emodin, chrysophanol reference substance stock solution; Precision is measured emodin reference substance stock solution 1ml, chrysophanol reference substance stock solution 2ml respectively, puts respectively in the 25ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets to contain among the every 1ml of emodin among 4 μ g, the every 1ml of chrysophanol to contain 8 μ g; The preparation of need testing solution is got the content under this product content uniformity item, mixing, get 0.2g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, and supersound process is 1 hour under the ultrasound wave of 30 ± 2KHz, put cold, claim to decide weight again, replenish the weight that subtracts mistake, shake up with methanol, centrifugal, get supernatant as need testing solution; 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 the total amount of Radix Et Rhizoma Rhei in emodin and chrysophanol, must not be less than 0.7mg;
B. baicalin: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 47: 53: 0.2 methanol-water-phosphoric acid are mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the baicalin peak should be not less than 2500; The preparation of reference substance solution, precision take by weighing at 4 hours baicalin reference substance of 60 ℃ of drying under reduced pressure an amount of, add methanol and make every 1ml and contain 20 μ g solution, promptly; The content under the content uniformity item is got in the preparation of need testing solution, and mixing is got 28.5mg, and accurate the title decides, put in the 25ml measuring bottle, it is an amount of to add 50% ethanol, and supersound process 15 minutes is put cold, add 50% ethanol dilution to scale, shake up, centrifugal, get supernatant as need testing solution; Survey the shallow lake method, 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 Radix Scutellariae by baicalin, must not be less than 6.0mg.
12. the method for quality control of Chinese medicinal composition preparation as claimed in claim 5 is characterized in that this method may further comprise the steps:
Differentiate: a. gets this composite preparation 1g, adds methanol 20ml, and supersound process 10-20 minute, filter, filtrate is concentrated into 1ml, as need testing solution; Get Radix Et Rhizoma Rhei control medicinal material powder 0.5g, make control medicinal material solution with the method operation; Other gets the emodin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to thin layer chromatography test, draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the 0.4-0.6% sodium carboxymethyl cellulose, with 6-8: 2-4: 0.1-0.2 benzene-Ethyl formate-formic acid is developing solvent, launch, take out, dry; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show the speckle of same color;
B. get this composite preparation 2.5g and add methanol ultrasonic 1-3 time, each 30ml, 10-20 minute, filter, filtrate evaporate to dryness, residue add that 8-12% sodium hydroxide solution 20ml is ultrasonic to make dissolving, 95-105 ℃ heating hydrolysis 1-3 hour, put cold, with the hydrochloric acid adjust pH to 2-3, drink extraction 2-4 time with acetic acid second, each 15ml merges ethyl acetate liquid, wash each 40ml, ethyl acetate liquid water bath method with water 2-4 time, residue makes dissolving with methanol 1ml, and is centrifugal, gets supernatant as need testing solution; Other gets chenodeoxycholic acid and ursodesoxycholic acid reference substance, adds methanol respectively and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography, draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the 0.4-0.6% sodium carboxymethyl cellulose, with 9-11: 4-6: 4-6: 2-4: the upper solution of 1-2 isobutyltrimethylmethane .-ether-glacial acetic acid-n-butanol-water is developing solvent, launch, take out, dry, spray is with the 15-25% sulfuric acid solution, in 100-110 ℃ of baking 4-7 minute; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
C. get this composite preparation 4g, add methanol 40ml, ultrasonic 15-25 minute, filter the filtrate evaporate to dryness, residue adds water 20ml makes dissolving, transfers pH to 3-4 with hydrochloric acid, adds ethyl acetate extraction 1-3 time, each 20ml, merge ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the baicalin reference substance, adds methanol and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, putting in same 0.4-0.6% sodium carboxymethyl cellulose with the preparation of 1% sodium acetate respectively is on the silica gel g thin-layer plate of adhesive, with 4-6: 2-4: 1-2: 1-2 ethyl acetate-acetone-formic acid-water is developing solvent, launch, take out, dry, spray is with 2% ferric chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
D. get this composite preparation 4g, put in the round-bottomed flask, add water 250ml, an appendix XD connects volatile oil determination apparatus according to volatile oil extraction method Chinese Pharmacopoeia version in 2000; Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 3-5 hour, stop heating, place a moment, divide and get the ethyl acetate layer, as need testing solution; Other gets the methylnonanone reference substance, adds ethyl acetate and makes the solution that every 1ml contains 10 μ g, in contrast product solution; According to thin layer chromatography test, 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 0.4-0.6% sodium carboxymethyl cellulose, with 18-20: 1-2 benzene-ethyl acetate is developing solvent, launches, and takes out, dry, spray is with the dinitrophenylhydrazine test solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
E. get this composite preparation 4g, put in the round-bottomed flask, add water 250ml, an appendix XD connects volatile oil determination apparatus according to volatile oil extraction method Chinese Pharmacopoeia version in 2000; Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 3-5 hour, stop heating, place a moment, divide and get the ethyl acetate layer, as need testing solution; Get the Mentholum reference substance, add ethyl acetate and make the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin layer chromatography, get 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 0.4-0.6% sodium carboxymethyl cellulose, with 7-10: 1-2 normal hexane-ethyl acetate is developing solvent, launch, take out, dry, spray is with 1-3: 7-9 vanillin sulphuric acid test solution-alcoholic acid mixed solution, and it is clear to be heated to the speckle colour developing at 95-110 ℃; In the test sample chromatograph, on the corresponding position of reference substance chromatograph, show the speckle of same color;
Assay: f. emodin, chrysophanol: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 80-90: 10-20 methanol-0.1% phosphoric acid solution is a mobile phase; The detection wavelength is 254nm; Number of theoretical plate calculates by the emodin peak should be not less than 1500; The preparation of reference substance solution, precision take by weighing emodin, each 10mg of chrysophanol reference substance, put respectively in the 100ml measuring bottle, with dissolve with methanol and be diluted to scale, shake up as emodin chrysophanol reference substance stock solution; Precision is measured emodin reference substance stock solution 1ml, chrysophanol reference substance stock solution 2ml respectively, puts respectively in the 25ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets to contain among the every 1ml of emodin among 4 μ g, the every 1ml of chrysophanol to contain 8 μ g; This composite preparation 0.2g is got in the preparation of need testing solution, and accurate the title decides, put in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, under the ultrasound wave of 20-40KHz supersound process 1-2 hour, put cold, claim to decide weight again, replenish the weight that subtracts mistake, shake up with methanol, centrifugal, get supernatant as need testing solution; Algoscopy, accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly; This composite preparation per unit amount contains the total amount of Radix Et Rhizoma Rhei in emodin and chrysophanol, must not be less than 6.5-8mg;
G. baicalin: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 43-53: 50-58: the 0.1-0.3 methanol-water-phosphoric acid is a mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the baicalin peak should be not less than 2500; The preparation of reference substance solution, precision take by weighing at 55-65 ℃ of drying under reduced pressure 3-5 hour baicalin reference substance an amount of, add methanol and make every 1ml and contain 20 μ g solution, promptly; This composite preparation 28.5mg is got in the preparation of need testing solution, accurate claims surely, puts in the 25ml measuring bottle, and it is an amount of to add 45-55% ethanol, and supersound process 10-25 minute, put coldly, add the 45-55% ethanol dilution to scale, shake up, centrifugal, get supernatant as need testing solution; Survey the shallow lake method, accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly; This composite preparation per unit amount contains Radix Scutellariae by baicalin, must not be less than 55-65mg.
13. the method for quality control of Chinese medicinal composition preparation as claimed in claim 12 is characterized in that the capsule method may further comprise the steps:
Differentiate: a. gets this product content 1g, adds methanol 20ml, and supersound process 15 minutes filters, and filtrate is concentrated into 1ml, as need testing solution; Get Radix Et Rhizoma Rhei control medicinal material powder 0.5g, make control medicinal material solution with the method operation; Other gets the emodin reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to thin layer chromatography test, draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with 0.5% sodium carboxymethyl cellulose, be developing solvent with 7: 3: 0.1 benzene-Ethyl formate-formic acid, launch, take out, dry; In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, show the speckle of same color;
B. get this product content 2.5g and add methanol ultrasonic 2 times, each 30ml, 15 minutes, filter, filtrate evaporate to dryness, residue add that 10% sodium hydroxide solution 20ml is ultrasonic to make dissolving, 100 ℃ of heating hydrolysis 2 hours, put cold, with the hydrochloric acid adjust pH to 2-3, drink extraction 3 times with acetic acid second, each 15ml merges ethyl acetate liquid, wash each 40ml, ethyl acetate liquid water bath method with water 3 times, residue makes dissolving with methanol 1ml, and is centrifugal, gets supernatant as need testing solution; Other gets chenodeoxycholic acid and ursodesoxycholic acid reference substance, adds methanol respectively and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography, draw each 5-8 μ l of above-mentioned three kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with 0.5% sodium carboxymethyl cellulose, with 10: 5: 5: the upper solution of 3: 1 isobutyltrimethylmethane .-ether-glacial acetic acid-n-butanol-waters was developing solvent, launch, take out, dry, spray is with 20% sulfuric acid solution, in 105 ℃ of bakings 5 minutes; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
C. get this product content 4g, add methanol 40ml, ultrasonic 20 minutes, filter the filtrate evaporate to dryness, residue adds water 20ml makes dissolving, transfers pH to 3-4 with hydrochloric acid, adds ethyl acetate extraction 2 times, each 20ml, merge ethyl acetate liquid, evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets the baicalin reference substance, adds methanol and makes the reference substance solution that every 1ml contains 1mg; Test according to thin layer chromatography, draw each 5 μ l of above-mentioned two kinds of solution, putting in same 0.5% sodium carboxymethyl cellulose with the preparation of 1% sodium acetate respectively is on the silica gel g thin-layer plate of adhesive, with 5: 3: 1: 1 ethyl acetate-acetone-formic acid-water was developing solvent, launch, take out, dry, spray is with 2% ferric chloride alcoholic solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
D. get this product content 4g, put in the round-bottomed flask, add water 250ml, extract the Division A League Matches of French Football method according to volatile oil and connect volatile oil determination apparatus; Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 4 hours stops heating, places a moment, divides and gets the ethyl acetate layer, as need testing solution; Other gets the methylnonanone reference substance, adds ethyl acetate and makes the solution that every 1ml contains 10 μ g, in contrast product solution; According to thin layer chromatography test, 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 0.5% sodium carboxymethyl cellulose, with 19: 1 benzene-ethyl acetates was developing solvent, launched, and took out, dry, spray is with the dinitrophenylhydrazine test solution; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;
E. get this product content 4g, put in the round-bottomed flask, add water 250ml, extract the Division A League Matches of French Football method according to volatile oil and connect volatile oil determination apparatus; Add water from determinator upper end and make and be full of the scale part, add ethyl acetate 0.5ml again, connect reflux condensing tube, reflux 4 hours stops heating, places a moment, divides and gets the ethyl acetate layer, as need testing solution; Get the Mentholum reference substance, add ethyl acetate and make the solution that every 1ml contains 2mg, in contrast product solution; Test according to thin layer chromatography, get 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 0.5% sodium carboxymethyl cellulose, with 9: 1 normal hexane-ethyl acetates was developing solvent, launch, take out, dry, spray is with 2: 8 vanillin sulphuric acid test solution-alcoholic acid mixed solutions, and it is clear to be heated to the speckle colour developing at 100 ℃; In the test sample chromatograph, on the corresponding position of reference substance chromatograph, show the speckle of same color;
Assay: f. emodin, chrysophanol: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol-0.1% phosphoric acid solution was a mobile phase in 85: 15; The detection wavelength is 254nm; Number of theoretical plate calculates by the emodin peak should be not less than 1500; The preparation of reference substance solution, precision take by weighing emodin, each 10mg of chrysophanol reference substance, put respectively in the 100ml measuring bottle, with dissolve with methanol and be diluted to scale, shake up; As emodin, chrysophanol reference substance stock solution; Precision is measured emodin reference substance stock solution 1ml, chrysophanol reference substance stock solution 2ml respectively, puts respectively in the 25ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets to contain among the every 1ml of emodin among 4 μ g, the every 1ml of chrysophanol to contain 8 μ g; The preparation of need testing solution is got the content under this product content uniformity item, mixing, get 0.2g, the accurate title, decide, and puts in the tool plug conical flask, the accurate methanol 50ml that adds claims to decide weight, and supersound process is 1 hour under the ultrasound wave of 30 ± 2KHz, put cold, claim to decide weight again, replenish the weight that subtracts mistake, shake up with methanol, centrifugal, get supernatant as need testing solution; 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 the total amount of Radix Et Rhizoma Rhei in emodin and chrysophanol, must not be less than 0.7mg;
G. baicalin: according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 47: 53: 0.2 methanol-water-phosphoric acid are mobile phase; The detection wavelength is 280nm; Number of theoretical plate calculates by the baicalin peak should be not less than 2500; The preparation of reference substance solution, precision take by weighing at 4 hours baicalin reference substance of 60 ℃ of drying under reduced pressure an amount of, add methanol and make every 1ml and contain 20 μ g solution, promptly; The content under the content uniformity item is got in the preparation of need testing solution, and mixing is got 28.5mg, and accurate the title decides, put in the 25ml measuring bottle, it is an amount of to add 50% ethanol, and supersound process 15 minutes is put cold, add 50% ethanol dilution to scale, shake up, centrifugal, get supernatant as need testing solution; Survey the shallow lake method, 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 Radix Scutellariae by baicalin, must not be less than 6.0mg.
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| CN101603953B (en) * | 2009-07-20 | 2011-09-28 | 韩桂茹 | Method for quantitatively detecting polytypes of chrysophanol and emodin in rhubarb and compound preparation of rhubarb |
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