CN101554417B - Quality control method of antitumor Chinese medicine composition - Google Patents
Quality control method of antitumor Chinese medicine composition Download PDFInfo
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- CN101554417B CN101554417B CN2008100233256A CN200810023325A CN101554417B CN 101554417 B CN101554417 B CN 101554417B CN 2008100233256 A CN2008100233256 A CN 2008100233256A CN 200810023325 A CN200810023325 A CN 200810023325A CN 101554417 B CN101554417 B CN 101554417B
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Abstract
The invention discloses a quality control method of antitumor Chinese medicine composition which is prepared according to the weight portion by 20-60 of gamboges, 700-1000 agrimony and 500-800 of umbellate pore furgus. In the quality control method, the content of gambogicacid in the composition is measured by adopting a high performance liquid chromatography, and an agrimony thin layer chromatography discrimination method and a gamboge thin layer chromatography discrimination are built. The quality control method has high separation efficiency, good stability and high analysis speed, and controls the quality reliability of oral solid preparations of the antitumor Chinese medicine composition.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, relate to a kind of method of quality control of antitumor Chinese.
Background technology
Malignant tumor serious threat human health and life, its mortality rate occupies first of the various diseases.At present, early discovery, the early diagnosis of malignant tumor are not properly settled as yet, also lacked effective Therapeutic Method.Nearly more than two decades comes, and Chinese medicine more and more causes people's attention in the effect aspect the anti-curing oncoma.Along with to the going deep into of Chinese medicine tumor research, the raising of curative effect is used Chinese medicine and is cooperated doctor trained in Western medicine method treatment malignant tumor to obtain extensively to carry out.Treat the therapeutic purposes of malignant tumor " no tumor existence " in the past, forward " existence of band tumor ", the direction transformation that improves life quality.Give full play to the advantage of Chinese medicine, can transfer the regulation and control of host's potential participation, promote the rehabilitation of tumor patient tumor.
By Resina garciniae 20～60 weight portions, Herba Agrimoniae 700～1000 weight portions, the prepared Chinese medicine composition of Polyporus 500～800 weight portions has the effect of strengthening vital QI to eliminate pathogenic factors, be applicable to before and after treatment operation, the chemicotherapy and should not perform the operation and the malignant tumor patient of chemicotherapy, and safety, no obvious toxic-side effects.At present also there is not complete quality standard to control the quality of said composition preparation, the invention provides the method for quality control of said composition oral solid formulation, can comprehensively, effectively control its quality, further ensure the effectiveness and the safety of this pharmaceutical composition.
Summary of the invention
The method of quality control that the purpose of this invention is to provide a kind of antitumor Chinese oral solid formulation.
The crude drug of pharmaceutical composition involved in the present invention is formed and proportioning is: Resina garciniae 20～60 weight portions, Herba Agrimoniae 700～1000 weight portions, Polyporus 500～800 weight portions, optimum ratio are Resina garciniae 40 weight portions, Herba Agrimoniae 833 weight portions, Polyporus 667 weight portions.
Press the pharmacy preparation method, above-mentioned raw materials medicine dosage form can be said dosage form on any medicament, includes but are not limited to granule, tablet, oral solid formulation, powder, pill, oral liquid etc.
This preparation of drug combination method can for:
A. take by weighing Resina garciniae 20～60 weight portions and add 95% alcohol reflux three times, merge backflow, filter, filtrate concentrate extractum A;
B. take by weighing Herba Agrimoniae 700～1000 weight portions, Polyporus 500～800 weight portions, decoct with water three times, collecting decoction filters, filtrate concentrate extractum B;
C. extractum A and extractum B are dried to dried cream respectively, are ground into fine powder, merge, add adjuvant, make various dosage forms according to common process.
The method of quality control of this composition oral solid preparation comprises to be differentiated and assay.
A kind of is the method for quality control that crude drug is made the oral solid formulation of antineoplastic pharmaceutical compositions by Resina garciniae 20～60 weight portions, Herba Agrimoniae 700～1000 weight portions and Polyporus 500～800 weight portions three flavor Chinese medicines, it is characterized in that content assaying method is in this method:
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With volume ratio is 70～90: 10～20: methanol-water-glacial acetic acid of 0.01～1 is a mobile phase; The detection wavelength is 350～380nm; Number of theoretical plate calculates by the gamlogic acid peak should be not less than 3000;
The preparation of reference substance solution: it is an amount of to get the gamlogic acid reference substance, and accurate the title decides, and adds dehydrated alcohol and makes the solution that every 1ml contains 0.3～0.5mg, promptly;
The preparation of need testing solution: it is an amount of to get the aforementioned pharmaceutical compositions oral solid formulation, and accurate the title decided porphyrize, get 0.14～0.15g, the accurate title, decide, and puts in the 10ml measuring bottle, it is an amount of to add dehydrated alcohol, ultrasonic 3～15 minutes, adds dehydrated alcohol to scale, shake up, filter, the accurate filtrate 2.0ml that draws is in the 10ml measuring bottle, and mobile phase is diluted to scale, shake up, promptly;
Algoscopy: accurate respectively reference substance solution and each 10～20ul of need testing solution of drawing, inject chromatograph of liquid, measure, promptly.
A kind of is the method for quality control that crude drug is made the oral solid formulation of antineoplastic pharmaceutical compositions by Resina garciniae 20～60 weight portions, Herba Agrimoniae 700～1000 weight portions and Polyporus 500～800 weight portions three flavor Chinese medicines, it is characterized in that content assaying method is specially in this method:
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With volume ratio is that methanol-water-glacial acetic acid of 83: 17: 0.1 is a mobile phase; The detection wavelength is 362nm; Number of theoretical plate calculates by the gamlogic acid peak should be not less than 3000;
The preparation of reference substance solution: it is an amount of to get the gamlogic acid reference substance, and accurate the title decides, and adds dehydrated alcohol and makes the solution that every 1ml contains 0.4mg, promptly;
The preparation of need testing solution: it is an amount of to get the aforementioned pharmaceutical compositions oral solid formulation, and accurate the title decided porphyrize, get about 0.15g, the accurate title, decide, and puts in the 10ml measuring bottle, it is an amount of to add dehydrated alcohol, ultrasonic 6 minutes, adds dehydrated alcohol to scale, shake up, filter, the accurate filtrate 2.0ml that draws is in the 10ml measuring bottle, and mobile phase is diluted to scale, shake up, promptly;
Algoscopy: accurate respectively reference substance solution and each 20ul of need testing solution of drawing, inject chromatograph of liquid, measure, promptly.
In the above-mentioned content assaying method, the composition oral solid preparation is the taking dose meter per diem, contains Resina garciniae with gamlogic acid C
38H
44O
8Meter must not be less than 23.2mg.
This method of above-mentioned method of quality control also comprises one or both in the following discrimination method:
A, get aforementioned pharmaceutical compositions oral solid formulation content 0.3～0.5 gram, porphyrize adds methanol 2～10ml, and supersound process 20～40 minutes is put coldly, filters, and gets supernatant as need testing solution.Other gets Resina garciniae control medicinal material 5～15mg, shines medical material solution in pairs with legal system.According to the thin layer chromatography test, draw each 2～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume ratio is 10～20: 0.5～3: chloroform-methanol of 0.5～3-triethylamine is developing solvent, launches, and takes out, dry, put under the ultra-violet lamp and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color, negative sample is at the relevant position immaculate.
B, get aforementioned pharmaceutical compositions oral solid formulation 1～5 gram, porphyrize adds chloroform 20～60ml, supersound process 5～20 minutes is put coldly, filters, discard filtrate, residue adds methanol 20～60ml, and water-bath refluxed 10～30 minutes, put cold, filter, filtrate evaporate to dryness, residue add methanol 0.5～1.5ml makes dissolving, as need testing solution.Other gets Herba Agrimoniae control medicinal material 1～3g, shines medical material solution in pairs with legal system.According to the thin layer chromatography test, draw each 3～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume ratio is 3～8: 2～7: toluene-ethyl acetate-formic acid of 0.5～2 is developing solvent, launches, and takes out, dry, put under the ultra-violet lamp and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical speckle, negative sample is at the relevant position immaculate.
Above-mentioned method of quality control specifically can comprise one or both in the following discrimination method:
A, get aforementioned pharmaceutical compositions oral solid formulation 0.4 gram, porphyrize adds methanol 5ml, and supersound process 30 minutes is put coldly, filters, and gets supernatant as need testing solution.Other gets Resina garciniae control medicinal material 10mg, shines medical material solution in pairs with legal system.According to thin layer chromatography (" 2005 editions appendix IVB of Chinese pharmacopoeia) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume ratio is that chloroform-methanol-triethylamine of 15: 1: 1 is developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color, negative sample is at the relevant position immaculate.
B, get aforementioned pharmaceutical compositions oral solid formulation 3 gram, porphyrize adds chloroform 40ml, and supersound process 10 minutes is put cold, filter, discard filtrate, residue adds methanol 40ml, and water-bath refluxed 20 minutes, put cold, filter, filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.Other gets Herba Agrimoniae control medicinal material 2g, shines medical material solution in pairs with legal system.According to thin layer chromatography (" 2005 editions appendix IVB of Chinese pharmacopoeia) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume ratio is that toluene-ethyl acetate-formic acid of 5: 4: 0.8 is developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical speckle, negative sample is at the relevant position immaculate.
Method of quality control of the present invention can also for:
Assay: with the octadecylsilane chemically bonded silica is filler, is that methanol-water-glacial acetic acid of 83: 17: 0.1 is a mobile phase with volume ratio, and the detection wavelength is 362nm, and number of theoretical plate calculates by the gamlogic acid peak should be not less than 3000; It is an amount of to get the gamlogic acid reference substance, and accurate the title decides, and adds dehydrated alcohol and makes the solution that every 1ml contains 0.4mg, promptly gets reference substance solution; It is an amount of to get the composition oral solid preparation, and porphyrize is got about 0.15g, the accurate title, decide, and puts in the 10ml measuring bottle, and it is an amount of to add dehydrated alcohol, ultrasonic 6 minutes, add dehydrated alcohol to scale, shake up, filter, the accurate filtrate 2.0ml that draws is in the 10ml measuring bottle, mobile phase is diluted to scale, shakes up, and promptly gets need testing solution; Accurate respectively reference substance solution and each 20ul of need testing solution of drawing injects chromatograph of liquid, measures, promptly.The composition oral solid preparation is the taking dose meter per diem, contains Resina garciniae with gamlogic acid C
38H
44O
8Meter must not be less than 23.2mg.
Differentiate: a, get aforementioned pharmaceutical compositions oral solid formulation 0.4 gram, porphyrize adds methanol 5ml, and supersound process 30 minutes is put coldly, filters, and gets supernatant as need testing solution.Other gets Resina garciniae control medicinal material 10mg, shines medical material solution in pairs with legal system.According to thin layer chromatography (" 2005 editions appendix IVB of Chinese pharmacopoeia) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume ratio is that chloroform-methanol-triethylamine of 15: 1: 1 is developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color, negative sample is at the relevant position immaculate.B, get aforementioned pharmaceutical compositions oral solid formulation 3 gram, porphyrize adds chloroform 40ml, and supersound process 10 minutes is put cold, filter, discard filtrate, residue adds methanol 40ml, and water-bath refluxed 20 minutes, put cold, filter, filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.Other gets Herba Agrimoniae control medicinal material 2g, shines medical material solution in pairs with legal system.According to thin layer chromatography (" 2005 editions appendix IVB of Chinese pharmacopoeia) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with volume ratio is that toluene-ethyl acetate-formic acid of 5: 4: 0.8 is developing solvent, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical speckle, negative sample is at the relevant position immaculate.
The main strengthening vital QI to eliminate pathogenic factors of this pharmaceutical composition is applied to prepare antitumor drug.Be applicable to before and after treatment operation, the chemicotherapy and should not perform the operation and the malignant tumor patient of chemicotherapy, evident in efficacy, and safety, no obvious toxic-side effects.
Method of quality control of the present invention is applicable to the clinical acceptable oral solid formulation of this antineoplastic pharmaceutical compositions, includes but not limited to capsule, tablet, granule, pill etc., preferred capsule, and every day, dose was equivalent to primary dose 4g～8g.Method of quality control separation efficiency height of the present invention, good stability, analysis speed are fast, are this antitumor Chinese quality reliability of oral solid preparations methods of control.The present invention is specifically addressed (to call the compound agrimony capsule in the following text) by testing example.
Test example 1. assays are selected experiment
Resina garciniae is a monarch drug in the prescription, and wherein gamlogic acid is its main active, and therefore selecting gamlogic acid for use is the index components of assay.
Instrument and reagent
High performance liquid chromatograph: SPD-10Avp; UV-detector: PDA2996; Reference substance: gamlogic acid (Hanbon Sci. ﹠ Tech. Co., Ltd. provides, and uses for assay); Test sample: compound agrimony capsule (adopting the preparation of embodiment 1 method), lot number: 20060201,20060202,20060203.
(1) selection of chromatographic condition test
1. detect wavelength determination
Detect according to UV-detector, the ultraviolet maximum absorption wavelength of gamlogic acid is 362nm, so select for use 362nm to detect wavelength.
2. the investigation of chromatographic column, column temperature and mobile phase:
System suitability is carried out following test investigated, the results are shown in Table 1.
Table 1. system suitability is investigated result of the test
3. extract the investigation of solvent and time
It is an amount of to get this product content, and porphyrize is got about 0.15g, and accurate the title decides, and puts in the 10ml measuring bottle, makes extractant with mobile phase and dehydrated alcohol respectively, and the ultrasonic back of different time measurement result sees Table 2.
The investigation of table 2. extractant and time
Determine the preparation of need testing solution:
It is an amount of to get this product content, and porphyrize is got about 0.15g, and accurate the title decides, and puts in the 10ml measuring bottle, it is an amount of to add dehydrated alcohol, and ultrasonic 6 minutes, add dehydrated alcohol to scale, shake up, filter, get filtrate 2.0ml in the 10ml measuring bottle, mobile phase is diluted to scale, shakes up, and gets need testing solution.
Chromatographic condition:
Chromatographic column: C
18Post; Column temperature: 20 ℃; Mobile phase: methanol: water: glacial acetic acid is 83: 17: 0.1; Flow velocity: 1.0ml/min; Detect wavelength: 362nm.
(2) specificity is investigated
Get the about 0.15g of negative sample (not containing Resina garciniae), the accurate title, decide, and presses the preparation of need testing solution preparation method, and the accurate 20 μ l of absorption inject chromatograph of liquid, presses above-mentioned chromatographic condition and measure.The result does not have absworption peak and occurs in the gamlogic acid corresponding position.
(3) investigation of linear relationship
Accurate absorption reference substance stock solution (1.2mg/ml) is an amount of respectively, and being diluted to concentration with mobile phase is 13.13 μ g/ml, 26.25 μ g/ml, 52.5 μ g/ml, 105 μ g/ml, 210 μ g/ml, the reference substance solution of 420 μ g/ml.Each accurate 20 μ l of absorption injects chromatograph of liquid, continuous sample introduction 2 times, measure peak area by above-mentioned chromatographic condition, with the peak area integrated value is vertical coordinate (Y), is abscissa (X) with reference substance concentration, the drawing standard curve, calculate regression Calculation, get regression equation: Y=26198X+53122, (correlation coefficient r=1, n=6).The range of linearity is 13.13-420ug/ml, the results are shown in Table 3.
Table 3. standard curve is drawn the result of the test table
(4) precision test
According to above-mentioned chromatographic condition, accurate reference substance solution (concentration is 1.2mg/ml) the 20 μ l that draw inject chromatograph of liquid, and continuous sample introduction 6 times is measured.Calculating average peak area is 1410951, and its RSD value is 1.41%, the Pass Test requirement.The results are shown in Table 4.
Table 4. Precision test result table
(5) replica test
Getting this product content, an amount of (lot number: 20060201), porphyrize takes by weighing 6 parts, and every part of about 0.15g accurately claims surely, prepares according to the need testing solution preparation method.According to above-mentioned chromatographic condition, accurate each 20 μ l that draw inject chromatograph of liquid, and continuous sample introduction is measured for 2 times.The RSD value is 1.18%, meets requirement of experiment.The results are shown in Table 5.
Table 5. replica test is table as a result
(6) stability test
(lot number: 20060201), porphyrize is got about 0.15g in right amount to get this product content, the accurate title, decide, according to the preparation of need testing solution preparation method, according to above-mentioned chromatographic condition, the accurate 20 μ l of absorption inject chromatograph of liquid, every 2.5h sample introduction 1 time, measure the stability of need testing solution 12h.Its RSD value is .0.2%, meets requirement of experiment.The results are shown in Table 6.
Table 6. stability test is table as a result
(7) average recovery test
Get the sample (lot number 20060201) of known content, porphyrize is got about 0.075g, and accurate the title decides, and each adds 80,100,120% reference substance stock solution of known content.Press the preparation of need testing solution preparation method again, totally 9 parts.Accurate each 20 μ l that draw inject high performance liquid chromatograph, and continuous sample introduction 3 times is measured.Calculating average average recovery is 99.94%, and its RSD value is 1.57%, meets each requirement of experiment.The results are shown in Table 7.
Table 7. average recovery result of the test table
(8) medical material assay
Get the about 0.025g of each medicinal material coarse powder, the accurate title, decide, and presses the preparation of need testing solution preparation method, and each sample introduction 20 μ l measure.The results are shown in Table 8.
Table 8. medical material assay result
(9) sample size determination test
1. the preparation of reference substance solution
Precision takes by weighing puts the gamlogic acid reference substance 10.5mg of exsiccator drying under reduced pressure more than 48 hours, puts in the 25ml volumetric flask, adds anhydrous alcohol solution and is diluted to scale, shakes up, and concentration is 0.420mg/ml.
2. the preparation of need testing solution
It is an amount of to get this product content, and porphyrize is got about 0.15g, and accurate the title decides, and puts in the 10ml measuring bottle, it is an amount of to add dehydrated alcohol, and ultrasonic 6 minutes, add dehydrated alcohol to scale, shake up, filter, get filtrate 2.0ml in the 10ml measuring bottle, mobile phase is diluted to scale, shakes up, and gets need testing solution.
Get lot number 20060201,20060202 respectively, 3 in 20060203 sample, porphyrize is got about 0.15g, and accurate the title, decide, and presses the preparation of need testing solution preparation method, promptly.Accurate each the 20 μ l of need testing solution that draw inject high performance liquid chromatograph, and continuous sample introduction 2 times is measured, promptly.The results are shown in Table 9.
Table 9. three batch sample assay results
Experimental result is as seen: this method accuracy is higher, can be used for the assay of gamlogic acid in the compound agrimony capsule.Three batch samples (20060201,20060202,20060203) its content is respectively 7.09mg/ grain, 7.41mg/ grain 7.14mg/ grain, average content is the 7.21mg/ grain, medical material gamlogic acid content is 229.33mg/g, the rate of transform of gamlogic acid is respectively 77.3%, 80.7%, 77.8% in its preparation, and the mean transferred rate is 78.6%.According to average content 7.21mg/ grain, consider that the extraction of medical material and technology may cause floating of content, therefore sample average content is floated downward 20%, the content of every gamlogic acid is 5.77mg, round numbers be the 5.8mg/ grain as limit, the finished product content limit is defined as: promptly every of this composition capsule contains Resina garciniae with gamlogic acid (C
38H
44O
8) meter, must not be less than 5.8mg.
Test example 2, the capsular pharmacodynamic experiment research of compound agrimony:
1, the compound agrimony capsule is irritated stomach to mice-transplanted tumor S
180Inhibitory action
Test material
Title: compound agrimony capsule, lot number: 20060201; Cyclophosphamide (CTX), specification: the 200mg/ bottle, lot number: 06060121, Hengrui Medicine Co., Ltd., Jiangsu Prov..
Experimental technique:
Get 50 of ICR kind white mice, 18-22g, male and female half and half are pressed transplanted tumor organon inoculation S
180Solid type is inoculated back 24 hours and is claimed Mus heavy, and is divided into 5 groups at random, and 10 every group, male and female half and half, blank group and CTX group are respectively the positive and negative matched group.Inoculate administration after 24 hours, the ig administration, administration volume: 0.4ml/20g, once a day, administration is 7 times altogether, and the 2nd day mice weighed after drug withdrawal, puts to death tumor-bearing mice and separates the tumor piece, claims tumor heavy, and the gained data are carried out statistical procedures (t check).
The result shows, compares with the blank group, and (300,150mg/kg) group all can suppress animal inhibition sarcoma S to the compound agrimony capsule significantly
180Growth (P＜0.01), less to the influence of the body weight of experiment mice simultaneously.Positive drug CTX group is to S
180The inhibitory action of tumor and to the influence of the body weight of laboratory animal significantly (P＜0.01).The experiment triplicate, the result is close.See Table 10.
Table 10 compound agrimony capsule ig is to mice-transplanted tumor S
180Inhibitory action
(n=10)
*P＜0.05
*Compare with the blank group P＜0.01
2, the compound agrimony capsule is irritated the inhibitory action of stomach to mice-transplanted tumor EC
Experimental technique:
Get 50 of above-mentioned specification mices and inoculate back 24 hours and claim Mus heavy, and be divided into 5 groups at random by transplanted tumor organon inoculation EC solid type, 10 every group, male and female half and half, blank group and CTX group are respectively the positive and negative matched group.Inoculate administration after 24 hours, ig administration, administration volume: 0.4ml/20g
Once a day, administration is 7 times altogether, and the 2nd day mice weighed after drug withdrawal, puts to death tumor-bearing mice and separates the tumor piece, claims tumor heavy, and the gained data are carried out statistical procedures (t check).
The result shows, compares with the blank group, and (300,150mg/kg) group all can suppress the growth (P＜0.01) that animal suppresses tumor EC to the compound agrimony capsule significantly, and is less to the body weight influence of experiment mice simultaneously.Positive drug CTX group is to the inhibitory action of EC tumor with to the body weight influence of laboratory animal significantly (P＜0.01).The experiment triplicate, the result is close.See Table 11.
Table 11 compound agrimony capsule ig is to the inhibitory action of mice-transplanted tumor EC
(n=10)
*P＜0.05
*Compare with the blank group P＜0.01
3, the compound agrimony capsule is irritated the inhibitory action of stomach to mice-transplanted tumor Heps
Experimental technique:
Get 50 of above-mentioned specification mices and inoculate back 24 hours and claim Mus heavy, and be divided into 5 groups at random by transplanted tumor organon inoculation Heps solid type, 10 every group, male and female half and half, blank group and CTX group are respectively the positive and negative matched group.Inoculate administration after 24 hours, ig administration, administration volume: 0.4ml/20g
Once a day, administration is 7 times altogether, and the 2nd day mice weighed after drug withdrawal, puts to death tumor-bearing mice and separates the tumor piece, claims tumor heavy, and the gained data are carried out statistical procedures (t check).
The result shows, compares with the blank group, and (300,150mg/kg) group all can suppress the growth (P＜0.01) that animal suppresses hepatocarcinoma tumor Heps to the compound agrimony capsule significantly, and is less to the body weight influence of experiment mice simultaneously.Positive drug CTX group is to the inhibitory action of Heps tumor with to the body weight influence of laboratory animal significantly (P＜0.01).The experiment triplicate, the result is close.See Table 12.
Table 12 compound agrimony capsule ig is to the inhibitory action of mice-transplanted tumor Heps
(n=10)
*P＜0.05
*Compare with the blank group P＜0.01
Conclusion:
The compound agrimony capsule (300,150,75mg/kg) irritate stomach (ig) administration to mice transplanted tumor S
180, EC, the growth of Heps all has the obvious suppression effect, and wherein compound agrimony capsule (300mg/kg) ig is to mice transplanted tumor S
180, EC, the growth inhibition ratio best result Bie Keda 53.4%, 55.98% and 56.62% of Heps, less to the body weight influence of experiment mice simultaneously.But CTX is to transplanted tumor S under the similarity condition
180, EC, the inhibitory action of Heps and remarkable to the body weight influence of laboratory animal.
Test example 3, medicine acute toxicity testing of the present invention
Experiment material:
Compound agrimony capsule, every gram extract powder are equivalent to 9.49 gram raw medicinal herbs, lot number: 20060201; 0.5% sodium carboxymethyl cellulose (CMC-Na) solution.
Experimental animal:
40 of healthy ICR mices, cleaning level, male and female half and half, body weight 18-22g; 40 of healthy SD rats, cleaning level, male and female half and half, body weight 130～150g.By the supply of Zhejiang Province's Experimental Animal Center, the animal quality quality certification: SCXK (Zhejiang) 2003-0001.
Dosage group and definite foundation:
According to mice, the pre-test result of rat acute toxicity and toxic reaction situation, the complete dead dosage of the anxious malicious prerun oral administered compound XIANHECAO JIAONANG of mice is 10.0g/kg, and the dosage of living entirely is 6.0g/kg; The complete dead dosage of the anxious malicious prerun oral administered compound XIANHECAO JIAONANG of rat is 10.0g/kg, and the dosage of living entirely is 4.5g/kg.Mice respectively is provided with following dosage group with group apart from 0.8 with group distance 0.84, rat.
Mice:
The 1st group of compound agrimony capsule 6.0g/kg
The 2nd group of compound agrimony capsule 7.1g/kg
The 3rd group of compound agrimony capsule 8.4g/kg
The 4th group of compound agrimony capsule 10.0g/kg
Rat:
The 1st group of compound agrimony capsule 5.1g/kg
The 2nd group of compound agrimony capsule 6.4g/kg
The 3rd group of compound agrimony capsule 8.0g/kg
The 4th group of compound agrimony capsule 10.0g/kg
Above dosage group is faced the time spent and is mixed with the compound agrimony capsule suspension of variable concentrations, equal capacity with 0.5% sodium carboxymethyl cellulose (CMC-Na) solution, uses for mice, rat oral gavage.Administration capacity: mice: 0.8ml/20g body weight; Rat: 2.0ml/100g body weight.
Test method:
Get 40 of ICR mices, 40 of SD rats are divided into above each dosage group at random, and 10 every group, male and female half and half.Fasting be can't help water more than 12 hours (beginning fasting ten eight: 30 evening before that day, 9 preceding administrations in morning next day) before each dosage group administration.Irritating stomach gives above each dosage group compound agrimony capsule on an empty stomach respectively, at once observe the response situation of animal after the administration, comprise that animal appearance, behavioral activity, the mental status, appetite, defecation and color thereof, fur, nose, eye, mouth have or not abnormal secretion thing and death condition, the timely postmortem of dead animal is if perusal has obvious pathological changes internal organs then to carry out histopathologic examination.And administration the 1st day observed 1 time every 30 minutes to 1 hour, observed 6h continuously, observed later every day 1 time, observed reaction of record animal toxicity and dead distribution situation continuously 14 days.Handle with the NDST statistical software according to mortality rate, calculate mice, the oral acute half lethal dose (LD of rat by the Bliss method
50) and 95% credible limit value, tabulation expression.
Result of the test:
1. mice difference single oral (po) (consistent with the clinical administration approach) gives compound agrimony capsule 6.0,7.1,8.4,10.0g/kg dosage; abnormal response does not all appear in administration each group at once; medicine sample loose stool, urinary incontinence (dripping urine), movable digestion, urinary system and psychologic nervous system reaction such as reduce, close one's eyes appear in administration after 2～3 hours; 24 hours and 48 hours are observed respectively after the administration, and 6.0,7.1, each survival mice of 8.4g/kg dosage group progressively recovers normal activity.Administration begins to occur dead after 23 hours, each was organized mortality rate and was respectively 0%, 30%, 70%, 100% after the administration between 23 hours to 72 hours the death time.Its reaction occurrence rate, extent of reaction, duration of the reaction, dead occurrence rate and dead time of occurrence all are corresponding relation with dosage, and the high reaction of dosage weighs, time of occurrence morning, longer duration, mortality rate are also high, and dosage hangs down then opposite.Handle with the NDST statistical software, calculate the mice single oral by the Bliss method and give compound agrimony capsular acute half lethal dose (LD
50) and 95% credible limit value be 7.7257 (7.2403～8.2437) g/kg.Dead animal pathological anatomy macroscopy shows no obvious abnormalities except that the gastric drug residue, and off-test is cutd open all survival mice of inspection and also do not seen obvious macroscopic internal organs pathological change.
2. rat difference single oral (po) gives compound agrimony capsule 5.1,6.4,8.0,10.0g/kg dosage, at once respectively organize after the administration and abnormal response all do not occur, digestion, urinary system and psychiatric system reactions such as sialorrhea, medicine sample loose stool, urinary incontinence (dripping urine), movable minimizing, lethargy appear in administration after 30 minutes～1 hour, perpendicular hair, reaction such as hunchbacked, prostrate appear in administration after 2 hours, the oral cavity nasal cavity appearred in 6 hours later in administration color secretions phenomenon, and administration is each dosage group rat phenomenon (especially 10.0g/kg dosage is obvious) that all occurs becoming thin after the 2nd day.5.1g/kg after the administration of dosage group 48 hours, 6.4,72 hours above-mentioned toxic reactions of each survival rats fade away after the administration of 8.0g/kg dosage group.Administration begins to occur dead after 8 hours, each was organized mortality rate and was respectively 10%, 30%, 80%, 90% after the administration between 8 hours to 72 hours the death time.Its reaction occurrence rate, extent of reaction, duration of the reaction, mortality rate and dead time of occurrence all are corresponding relation with dosage, and the high reaction of dosage weighs, time of occurrence morning, longer duration, mortality rate are also high, and dosage hangs down then opposite.Handle with the NDST statistical software, calculate the acute half lethal dose (LD of rat oral administered compound XIANHECAO JIAONANG by the Bliss method
50) and 95% credible limit value be 6.9931 (6.2545～7.819) g/kg.Dead animal pathological anatomy macroscopy shows no obvious abnormalities except that the gastric drug residue, and off-test is cutd open all survival rats of inspection and do not seen obvious macroscopic internal organs pathological change.
Test example 4, medicine long term toxicity test of the present invention
Test material:
Compound agrimony capsule, every gram extract powder are equivalent to 9.49 gram raw medicinal herbs, and lot number 20060201 is provided by China Medicine University's development; 0.5% sodium carboxymethyl cellulose (CMC-Na) solution.
Experimental animal:
The SD rat, cleaning level, age 5-6 age in week, body weight: ♂ 86～104g, ± s:94.9 ± 4.6; ♀ 85～103g, ± s:94.6 ± 4.8; Totally 120, each 60 of ♀ ♂.By Zhejiang Province's Experimental Animal Center supply, the animal quality quality certification number: SCXK (Zhejiang) 2003-0001.
The dosage grouping:
High dose group compound agrimony capsule 1.0g/ (kgd) * 180d
Middle dosage group compound agrimony capsule 0.5g/ (kgd) * 180d
Low dose group compound agrimony capsule 0.25g/ (kgd) * 180d
Matched group 0.5%CMC-Na solution 10ml/ (kgd) * 180d
More than each group face with preceding and be mixed with variable concentrations, the equal suspension of capacity with 0.5%CMC-Na solution, confession rat oral gavage usefulness, the administration capacity is the 1.0ml/100g body weight, it is identical that each organizes the administration capacity.
Dosage is determined foundation:
1. according to the clinical plan of people dosage 1.953g/ people d (press the 60kg body weight and calculate, be i.e. 0.03g/kgd);
2. be 6.0g/kg according to acute toxicity trial test mice single oral (po) administration dosage alive entirely, rat single oral (po) administration dosage alive entirely is 4.5g/kg.
Test method:
Get 120 of cleaning level SD rats, numbering, male be odd number, female is even number, is divided into four test group by randomized blocks, i.e. high, medium and low three the dosage groups of compound agrimony capsule and a matched group, 30 every group, respectively 15 of male and female.Observe a week before the test, the behavioral activity of record rat, ingest, drink water, spirit, hair, body weight etc.Duration of test, per os of same time of every morning is irritated stomach (po) and is administered once (consistent with the clinical administration approach), and administration is 6 days weekly, continuous 180 days (6 months).Weigh weekly once, adjust dosage with the body weight increase and decrease.Every morning the observed and recorded rat general signs (comprising behavior, motor function, breathing, hair color, mouth, eye, nose, ear, excrement, urine etc.), food ration, amount of drinking water and death condition, discovery has the animal of toxic reaction to take out single cage raising, primary part observation, discovery has dead or the timely postmortem of dying animal, does to see substantially and histopathologic examination.
Hematology, blood biochemical, organ weights, coefficient and pathomorphology and histological examination are carried out in administration mid-term (administration three months), drug withdrawal next day (administration six months) and drug withdrawal one month (convalescent period end) respectively.Fasting be can't help water more than 12 hours (preceding a whole night of 20 beginnings fasting, blood sampling about 8: 30 morning of next day) before checking, behind etherization, and ventral aorta blood sampling, EDTA-K
2Anticoagulated whole blood is measured erythrocyte (RBC) with Sha jasmine Automatic Blood Cell Analyzer, leukocyte (WBC), haemachrome (HGB), platelet (PLT), packed cell volume (HCT), mean corpuscular volume (MCV), average hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), erythrocyte volume distribution density (RDW), mean platelet volume (MPV), MPW (PDW), thrombocytocrit (PCT) and lymphocyte (Lym#), intermediate value cell (Mid#) and granulocyte (Grn#).Measure prothrombin time (PT) with the CA-100 coagulo meter.Measure reticulocyte (Ret) with brilliant cresyl blue test tube staining.Serum is measured aspartic acid transferring enzyme (AST), alanine aminotransferase (ALT), alkali phosphatase (ALP), blood urea nitrogen (BUN), creatinine (Crea), total protein (T.P), albumin (ALB), blood glucose (GLU), total bilirubin (T.BIL), T-CHOL (T.CHO) with HITACHI7020 automatic clinical chemistry analyzer and pairing reagent, creatine phosphokinase (CK), triglyceride (TG) are measured Na with NOVA 10 electrolyte instrument and pairing reagent
+, K
+, Cl
-, TCa concentration.Administration mid-term, drug withdrawal next day and convalescent period finish to put to death 1/3 rat respectively and do system's postmortem and histopathologic examination, measure the organ weights and the coefficient (%) of the heart, liver, spleen, lung, kidney, brain, thymus, adrenal gland, thyroid, testis, epididymis, prostate, uterus, ovary; Core simultaneously, liver, spleen, lung, kidney, adrenal gland, thyroid, pancreas, stomach, duodenum, ileum, colon, hypophysis, brain, spinal cord, breastbone, thymus, lymph node, bladder, uterus, ovary, the attached testis of testis, prostate make paraffin section, HE dyeing, do pathology histological examination, observation and analysis with Nikon fluorescence photography microscope and histopathology image analysis system.More than every observation index (initial data) all carry out statistical analysis and processing with NDST software kit and the corresponding statistical procedure of EXCEL, with the basic data that statistical significance, general response situation, the result of histopathologic examination set up in conjunction with this laboratory, overall merit result of the test.
Result of the test:
The result shows, under this test dose condition, continuous six months of SD rat respectively per os to give lot number be 20060201 compound agrimony capsule 1.0,0.5 and 0.25g/ (kgd) dosage, toxicities such as its main toxicity is the minimizing of high dose group part activities in rats, medicine sample loose stool, dripping urine, perpendicular hair, depilation, body weight gain is slow or alleviate are mainly liver, intestinal, stomach, thymus, spleen, lymph node and bone marrow to the poisoning target organ of dead rat during the administration; Poisoning target organ to the biopsy rat is liver, small intestinal, and belongs to reversibility influence (drug withdrawal was checked and do not seen analogue in month).The dosage of being poisoned to death is 1.0g/ (kgd), and safe dose is 0.5g/ (kgd), and non-toxic is 0.25g/ (kgd).
The specific embodiment
The present invention is further elaborated by the following examples.
The method of quality control of embodiment 1 composition capsule
Resina garciniae 40g, Herba Agrimoniae 833g, Polyporus 667g, more than three flavor a. take by weighing Resina garciniae and add 95% alcohol reflux three times, for the first time 6 times of amounts, the second time 3 times of amounts, 3 times of amounts for the third time, each 0.5 hour, merge backflow, filter, filtrate recycling ethanol, relative density is the extractum A of 1.25-1.30 when being evaporated to 50 ℃.B. take by weighing Herba Agrimoniae, Polyporus, decoct with water three times, 12 times of amounts for the first time, 10 times of amounts for the second time, 10 times of amounts for the third time, each 2 hours, collecting decoction filtered, and relative density was the extractum B of 1.25-1.30 when filtrate was concentrated into 50 ℃.C. extractum A and extractum B are dried to dried cream respectively, are ground into fine powder, merge, add adjuvant, make 1000 seed lac wafers, to call the compound agrimony capsule in the following text.
Assay:
With the octadecylsilane chemically bonded silica is filler, is that methanol-water-glacial acetic acid of 83: 17: 0.1 is a mobile phase with volume ratio, and the detection wavelength is 362nm, and number of theoretical plate calculates by the gamlogic acid peak should be not less than 3000; It is an amount of to get the gamlogic acid reference substance, and accurate the title decides, and adds dehydrated alcohol and makes the solution that every 1ml contains 0.42mg, promptly gets reference substance solution; It is tolerant to get compound agrimony capsule 20 intragranulars, and accurate the title decided porphyrize, get about 0.15g, the accurate title, decide, and puts in the 10ml measuring bottle, it is an amount of to add dehydrated alcohol, ultrasonic 6 minutes, adds dehydrated alcohol to scale, shake up, filter, the accurate filtrate 2.0ml that draws is in the 10ml measuring bottle, and mobile phase is diluted to scale, shake up, promptly get need testing solution; Accurate respectively reference substance solution and each 20ul of need testing solution of drawing injects chromatograph of liquid, measures, promptly.Every contains Resina garciniae with gamlogic acid C in this composite preparation
38H
44O
8Meter must not be less than 5.8mg.
Differentiate:
A. get compound agrimony capsule 's content 0.4 gram, porphyrize adds methanol 5ml, and supersound process 30 minutes is put coldly, filters, and gets supernatant as need testing solution.Other gets Resina garciniae control medicinal material 10mg, shines medical material solution in pairs with legal system.According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be that chloroform-methanol-triethylamine of 15: 1: 1 is developing solvent with volume ratio, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color, negative sample is at the relevant position immaculate.
B. get compound agrimony capsule 's content 3 grams, porphyrize adds chloroform 40ml, and supersound process 10 minutes is put cold, filter, discard filtrate, residue adds methanol 40ml, and water-bath refluxed 20 minutes, put cold, filter, filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.Other gets Herba Agrimoniae control medicinal material 2g, shines medical material solution in pairs with legal system.According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be that toluene-ethyl acetate-formic acid of 5: 4: 0.8 is developing solvent with volume ratio, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical speckle, negative sample is at the relevant position immaculate.
The method of quality control of embodiment 2 composition tablets
Resina garciniae 40g, Herba Agrimoniae 833g, Polyporus 667g, more than three flavor a. take by weighing Resina garciniae and add 95% alcohol reflux three times, for the first time 6 times of amounts, the second time 3 times of amounts, 3 times of amounts for the third time, each 0.5 hour, merge backflow, filter, filtrate recycling ethanol, relative density is the extractum A of 1.25-1.30 when being evaporated to 50 ℃.B. take by weighing Herba Agrimoniae, Polyporus, decoct with water three times, 12 times of amounts for the first time, 10 times of amounts for the second time, 10 times of amounts for the third time, each 2 hours, collecting decoction filtered, and relative density was the extractum B of 1.25-1.30 when filtrate was concentrated into 50 ℃.C. extractum A and extractum B are dried to dried cream respectively, are ground into fine powder, merge, add adjuvant, be pressed into 1000, to call the compound agrimony sheet in the following text.
Assay:
With the octadecylsilane chemically bonded silica is filler, is that methanol-water-glacial acetic acid of 83: 17: 0.1 is a mobile phase with volume ratio, and the detection wavelength is 362nm, and number of theoretical plate calculates by the gamlogic acid peak should be not less than 3000; It is an amount of to get the gamlogic acid reference substance, and accurate the title decides, and adds dehydrated alcohol and makes the solution that every 1ml contains 0.42mg, promptly gets reference substance solution; It is an amount of to get the compound agrimony sheet, and porphyrize is got about 0.15g, and accurate the title decides, and puts in the 10ml measuring bottle, it is an amount of to add dehydrated alcohol, and ultrasonic 6 minutes, add dehydrated alcohol to scale, shake up, filter, the accurate filtrate 2.0ml that draws is in the 10ml measuring bottle, and mobile phase is diluted to scale, shakes up, and promptly gets need testing solution; Accurate respectively reference substance solution and each 20ul of need testing solution of drawing injects chromatograph of liquid, measures, promptly.This composition tablet is the taking dose meter per diem, contains Resina garciniae with gamlogic acid C
38H
44O
8Meter must not be less than 23.2mg.
Differentiate:
A. get compound agrimony sheet 0.4 gram, porphyrize adds methanol 5ml, and supersound process 30 minutes is put coldly, filters, and gets supernatant as need testing solution.Other gets Resina garciniae control medicinal material 10mg, shines medical material solution in pairs with legal system.According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be that chloroform-methanol-triethylamine of 15: 1: 1 is developing solvent with volume ratio, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color, negative sample is at the relevant position immaculate.
The method of quality control of embodiment 3 composition granules
Resina garciniae 40g, Herba Agrimoniae 833g, Polyporus 667g, more than three flavor a. take by weighing Resina garciniae and add 95% alcohol reflux three times, for the first time 6 times of amounts, the second time 3 times of amounts, 3 times of amounts for the third time, each 0.5 hour, merge backflow, filter, filtrate recycling ethanol, relative density is the extractum A of 1.25-1.30 when being evaporated to 50 ℃.B. take by weighing Herba Agrimoniae, Polyporus, decoct with water three times, 12 times of amounts for the first time, 10 times of amounts for the second time, 10 times of amounts for the third time, each 2 hours, collecting decoction filtered, and relative density was the extractum B of 1.25-1.30 when filtrate was concentrated into 50 ℃.C. extractum A and extractum B are dried to dried cream respectively, are ground into fine powder, merge, add adjuvant, make 1000 bags of granules, to call the compound agrimony granule in the following text.
Assay:
With the octadecylsilane chemically bonded silica is filler, is that methanol-water-glacial acetic acid of 83: 17: 0.1 is a mobile phase with volume ratio, and the detection wavelength is 362nm, and number of theoretical plate calculates by the gamlogic acid peak should be not less than 3000; It is an amount of to get the gamlogic acid reference substance, and accurate the title decides, and adds dehydrated alcohol and makes the solution that every 1ml contains 0.42mg, promptly gets reference substance solution; It is an amount of to get the compound agrimony granule, and porphyrize is got about 0.15g, and accurate the title decides, and puts in the 10ml measuring bottle, it is an amount of to add dehydrated alcohol, and ultrasonic 6 minutes, add dehydrated alcohol to scale, shake up, filter, the accurate filtrate 2.0ml that draws is in the 10ml measuring bottle, and mobile phase is diluted to scale, shakes up, and promptly gets need testing solution; Accurate respectively reference substance solution and each 20ul of need testing solution of drawing injects chromatograph of liquid, measures, promptly.This composite preparation is the taking dose meter per diem, contains Resina garciniae with gamlogic acid C
38H
44O
8Meter must not be less than 23.2mg.
The method of quality control of embodiment 4 compositions pills
Resina garciniae 40g, Herba Agrimoniae 833g, Polyporus 667g, more than three flavor a. take by weighing Resina garciniae and add 95% alcohol reflux three times, for the first time 6 times of amounts, the second time 3 times of amounts, 3 times of amounts for the third time, each 0.5 hour, merge backflow, filter, filtrate recycling ethanol, relative density is the extractum A of 1.25-1.30 when being evaporated to 50 ℃.B. take by weighing Herba Agrimoniae, Polyporus, decoct with water three times, 12 times of amounts for the first time, 10 times of amounts for the second time, 10 times of amounts for the third time, each 2 hours, collecting decoction filtered, and relative density was the extractum B of 1.25-1.30 when filtrate was concentrated into 50 ℃.C. extractum A and extractum B are dried to dried cream respectively, merge, be ground into fine powder, add adjuvant, make 1000 balls, to call the compound agrimony ball in the following text.
Differentiate:
A. get compound agrimony ball 0.4 gram, porphyrize adds methanol 5ml, and supersound process 30 minutes is put coldly, filters, and gets supernatant as need testing solution.Other gets Resina garciniae control medicinal material 10mg, shines medical material solution in pairs with legal system.According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be that chloroform-methanol-triethylamine of 15: 1: 1 is developing solvent with volume ratio, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color, negative sample is at the relevant position immaculate.
B. get compound agrimony ball 3 gram, porphyrize adds chloroform 40ml, and supersound process 10 minutes is put coldly, filters, and discards filtrate, and residue adds methanol 40ml, and water-bath refluxed 20 minutes, puts coldly, filters, and filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.Other gets Herba Agrimoniae control medicinal material 2g, shines medical material solution in pairs with legal system.According to thin layer chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be that toluene-ethyl acetate-formic acid of 5: 4: 0.8 is developing solvent with volume ratio, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical speckle, negative sample is at the relevant position immaculate.
Claims (6)
1. one kind is the quality determining method that crude drug is made the oral solid formulation of antineoplastic pharmaceutical compositions by Resina garciniae 20～60 weight portions, Herba Agrimoniae 700～1000 weight portions and Polyporus 500～800 weight portions three flavor Chinese medicines, it is characterized in that content assaying method is in this method:
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With volume ratio is that methanol-water-glacial acetic acid of 83: 17: 0.1 is a mobile phase; The detection wavelength is 362nm; Number of theoretical plate calculates by the gamlogic acid peak should be not less than 3000;
The preparation of reference substance solution: it is an amount of to get the gamlogic acid reference substance, and accurate the title decides, and adds dehydrated alcohol and makes the solution that every 1ml contains 0.3～0.5mg, promptly;
The preparation of need testing solution: it is an amount of to get the aforementioned pharmaceutical compositions oral solid formulation, and accurate the title decided porphyrize, get 0.14～0.15g, the accurate title, decide, and puts in the 10ml measuring bottle, it is an amount of to add dehydrated alcohol, ultrasonic 3～15 minutes, adds dehydrated alcohol to scale, shake up, filter, the accurate filtrate 2.0ml that draws is in the 10ml measuring bottle, and mobile phase is diluted to scale, shake up, promptly;
Algoscopy: accurate respectively reference substance solution and each 10～20ul of need testing solution of drawing, inject chromatograph of liquid, measure, promptly.
2. according to claim 1 a kind of be the quality determining method that crude drug is made the oral solid formulation of antineoplastic pharmaceutical compositions by Resina garciniae 20～60 weight portions, Herba Agrimoniae 700～1000 weight portions and Polyporus 500～800 weight portions three flavor Chinese medicines, it is characterized in that content assaying method is in this method:
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With volume ratio is that methanol-water-glacial acetic acid of 83: 17: 0.1 is a mobile phase; The detection wavelength is 362nm; Number of theoretical plate calculates by the gamlogic acid peak should be not less than 3000;
The preparation of reference substance solution: it is an amount of to get the gamlogic acid reference substance, and accurate the title decides, and adds dehydrated alcohol and makes the solution that every 1ml contains 0.4mg, promptly;
The preparation of need testing solution: it is an amount of to get the aforementioned pharmaceutical compositions oral solid formulation, and accurate the title decided porphyrize, get 0.15g, the accurate title, decide, and puts in the 10ml measuring bottle, it is an amount of to add dehydrated alcohol, ultrasonic 6 minutes, adds dehydrated alcohol to scale, shake up, filter, the accurate filtrate 2.0ml that draws is in the 10ml measuring bottle, and mobile phase is diluted to scale, shake up, promptly;
Algoscopy: accurate respectively reference substance solution and each 20ul of need testing solution of drawing, inject chromatograph of liquid, measure, promptly.
3. quality determining method according to claim 1 and 2 is characterized in that in the content assaying method that the composition oral solid preparation is the taking dose meter per diem, contains Resina garciniae with gamlogic acid C
38H
44O
8Meter must not be less than 23.2mg.
4. quality determining method according to claim 1 and 2 is characterized in that this method comprises one or both in the following discrimination method:
A, get aforementioned pharmaceutical compositions oral solid formulation 0.3～0.5 gram, porphyrize adds methanol 2～10ml, and supersound process 20～40 minutes is put coldly, filters, and gets supernatant as need testing solution; Other gets Resina garciniae control medicinal material 5～15mg, shines medical material solution in pairs with legal system, according to the thin layer chromatography test, draw each 2～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be 10～20: 0.5～3 with volume ratio: chloroform-methanol of 0.5～3-triethylamine is developing solvent, launch, take out, dry, put under the ultra-violet lamp and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color, negative sample is at the relevant position immaculate;
B, get aforementioned pharmaceutical compositions oral solid formulation 1～5 gram, porphyrize adds chloroform 20～60ml, supersound process 5～20 minutes is put coldly, filters, discard filtrate, residue adds methanol 20～60ml, and water-bath refluxed 10～30 minutes, put cold, filter, filtrate evaporate to dryness, residue add methanol 0.5～1.5ml makes dissolving, as need testing solution; Other gets Herba Agrimoniae control medicinal material 1～3g, shines medical material solution in pairs with legal system, according to the thin layer chromatography test, draw each 3～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be 3～8: 2～7 with volume ratio: toluene-ethyl acetate-formic acid of 0.5～2 is developing solvent, launch, take out, dry, put under the ultra-violet lamp and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical speckle, negative sample is at the relevant position immaculate.
5. quality determining method according to claim 4 is characterized in that this method comprises one or both in the following discrimination method:
A, get aforementioned pharmaceutical compositions oral solid formulation 0.4 gram, porphyrize adds methanol 5ml, and supersound process 30 minutes is put coldly, filters, and gets supernatant as need testing solution; Other gets Resina garciniae control medicinal material 10mg, shines medical material solution in pairs with legal system; Test according to thin layer chromatography, drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is that chloroform-methanol-triethylamine of 15: 1: 1 is developing solvent with volume ratio, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color, negative sample is at the relevant position immaculate;
B, get aforementioned pharmaceutical compositions oral solid formulation 3 gram, porphyrize adds chloroform 40ml, and supersound process 10 minutes is put cold, filter, discard filtrate, residue adds methanol 40ml, and water-bath refluxed 20 minutes, put cold, filter, filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets Herba Agrimoniae control medicinal material 2g, shines medical material solution in pairs with legal system; Test according to thin layer chromatography, drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is that toluene-ethyl acetate-formic acid of 5: 4: 0.8 is developing solvent with volume ratio, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical speckle, negative sample is at the relevant position immaculate.
6. quality determining method according to claim 1 and 2 is characterized in that this method is:
Assay: with the octadecylsilane chemically bonded silica is filler, is that methanol-water-glacial acetic acid of 83: 17: 0.1 is a mobile phase with volume ratio, and the detection wavelength is 362nm, and number of theoretical plate calculates by the gamlogic acid peak should be not less than 3000; It is an amount of to get the gamlogic acid reference substance, and accurate the title decides, and adds dehydrated alcohol and makes the solution that every 1ml contains 0.4mg, promptly gets reference substance solution; Get the content of 20 of this product, the accurate title, decided porphyrize, get 0.15g, the accurate title, decide, and puts in the 10ml measuring bottle, it is an amount of to add dehydrated alcohol, ultrasonic 6 minutes, adds dehydrated alcohol to scale, shake up, filter, the accurate filtrate 2.0ml that draws is in the 10ml measuring bottle, and mobile phase is diluted to scale, shake up, promptly get need testing solution; Accurate respectively reference substance solution and each 20ul of need testing solution of drawing injects chromatograph of liquid, measures, that is, this composition oral solid preparation is the taking dose meter per diem, contains Resina garciniae with gamlogic acid C
38H
44O
8Meter must not be less than 23.2mg;
Differentiate: a, get aforementioned pharmaceutical compositions oral solid formulation 0.4 gram, porphyrize adds methanol 5ml, and supersound process 30 minutes is put coldly, filters, and gets supernatant as need testing solution; Other gets Resina garciniae control medicinal material 10mg, shines medical material solution in pairs with legal system; Test according to thin layer chromatography, drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is that chloroform-methanol-triethylamine of 15: 1: 1 is developing solvent with volume ratio, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color, negative sample is at the relevant position immaculate; B, get aforementioned pharmaceutical compositions oral solid formulation 3 gram, porphyrize adds chloroform 40ml, and supersound process 10 minutes is put cold, filter, discard filtrate, residue adds methanol 40ml, and water-bath refluxed 20 minutes, put cold, filter, filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution; Other gets Herba Agrimoniae control medicinal material 2g, shines medical material solution in pairs with legal system; Test according to thin layer chromatography, drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is that toluene-ethyl acetate-formic acid of 5: 4: 0.8 is developing solvent with volume ratio, launch, take out, dry, put under the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical speckle, negative sample is at the relevant position immaculate.
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| CN1709316A (en) * | 2005-06-13 | 2005-12-21 | 郭延宝 | Medicine for treating cancer and its preparing method |
| CN101011559A (en) * | 2007-02-01 | 2007-08-08 | 南中兴 | Traditional Chinese medicine preparation for treating malignant tumor and its preparation method |
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