CN101320027B - Fingerprint pattern quality determination method of american cockroaches medicinal materials and their extract - Google Patents
Fingerprint pattern quality determination method of american cockroaches medicinal materials and their extract Download PDFInfo
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Abstract
The invention relates to a fingerprint quality determining method of American cockroach drug material and extract thereof. With the fingerprint technology applied, the method controls the quality of the American cockroach drug material and extract thereof. The method uses the fingerprint technology to effectively control raw materials from the American cockroach drug material, guarantees the stable quality of the American cockroach drug material under the certain preparing process, thereby further effectively controlling the quality of preparations made from the American cockroach extract, including but not confined to rehabilitation new liquid, rehabilitation new capsule, rehabilitation new pill, rehabilitation new spray, etc. The fingerprint technology provided by the invention can be used as a key means for evaluating the Chinese herbal medicine quality, which is beneficial to the modernization of Chinese herbal medicine.
Description
Technical field
The present invention relates to a kind of measuring method of Chinese crude drug, particularly relate to the fingerprint pattern quality determination method of American cockroach medicinal material and extract thereof.
Background technology
The American cockroach medicinal material records in " Yunnan Province's drug standards " version in 1996, and principal ingredient is amino acid (alanine, leucine, isoleucine etc.) and alkaloid (uracil etc.).Only carried out the assay and the amino acid whose discriminating of total amino acid in this quality of medicinal material standard, above method all can not be controlled the American cockroach medicinal material preferably, the particularly main alkaloidal quality control of effective constituent.And alkaloidal HPLC fingerprint spectrum method mensuration in American cockroach medicinal material and the extract is not still had report so far.Therefore, by research, set up the assay method of alkaloidal HPLC finger-print in high American cockroach medicinal material of repeatability and the extract, can be used as and measure alkaloidal chemical composition method in American cockroach medicinal material and the extract, also can be used as and identify American cockroach medicinal material and extraction of substance amount control method.
Summary of the invention
The measuring method that the objective of the invention is to the finger-print of open American cockroach medicinal material and extract thereof.
The present invention seeks to be achieved through the following technical solutions.
The fingerprint pattern quality determination method of American cockroach medicinal material of the present invention, this method comprises the steps:
The preparation of A, need testing solution: take by weighing American cockroach 1～10 weight portion, add 50～95% ethanol, 20～200 parts by volume, refluxed 1～8 hour, put cold, filter, filtrate recycling ethanol is put in the 25 parts by volume measuring bottles, thin up shakes up to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 40～60 mesh sieves and remove coarse grain, remove particulate after 140～160 mesh sieves, be soaked in water 18～30 hours, soaked 1～3 hour with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 3～5 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 3～5 times of amounts soaked 1～3 hour, stirred 3～5 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 2～4 times of amounts soaked 1～3 hour, stirred 3～5 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10 parts by volume, be added on the cation exchange resin column of having handled well, collect effluent, with 5-10 parts by volume water washing cation exchange resin column, collect cleansing solution, merge with effluent, be transferred in the 25 parts by volume measuring bottles, constant volume shakes up, and is standby;
B. the preparation of reference substance solution: take by weighing uracil, add water and make the solution that every parts by volume contains uracil 0.0000002～0.0001 weight portion, in contrast product solution;
C. measure: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.01～0.2mol/L is a moving phase; The detection wavelength is 240～280nm; Draw each 0.005～0.05 parts by volume of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American cockroach medicinal material according to high effective liquid chromatography for measuring; The test sample finger-print should should be greater than 0.90 with the standard finger-print similarity; The American cockroach medicinal material has 11 total fingerprint peakses, is the uracil chromatographic peak with reference to peak S, and the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.480 ± 0.048, No. 2 peaks 0.508 ± 0.051,0.629 ± 0.063, No. 5 peaks 0.780 ± 0.078,0.556 ± 0.056, No. 4 peaks, No. 3 peaks, S peak 1.000 ± 0.000,1.901 ± 0.190, No. 8 peaks 2.108 ± 0.211,1.295 ± 0.130, No. 7 peaks, No. 6 peaks, 2.655 ± 0.266, No. 10 peaks 3.515 ± 0.352, No. 9 peaks; In 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peak=(0.223～0.414): (0.188～0.349): (0.996～1.494): 1.000: (0.376～0.626).
The fingerprint pattern quality determination method of American cockroach medicinal material of the present invention is preferably as follows detection method:
The preparation of A, need testing solution: take by weighing American cockroach 5 weight portions, add 85% ethanol, 50 parts by volume, refluxed 3 hours, put coldly, filter, filtrate recycling ethanol is put in the 25 parts by volume measuring bottles, and thin up shakes up to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10 parts by volume, be added on the cation exchange resin column of having handled well, collect effluent, with 10 parts by volume water washing cation exchange resin columns, collect cleansing solution, merge with effluent, be transferred in the 25 parts by volume measuring bottles, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every parts by volume contains uracil 0.00001 weight portion, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.05mol/L is a moving phase; The detection wavelength is 259nm; Draw each 0.02 parts by volume of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American cockroach medicinal material according to high effective liquid chromatography for measuring; The test sample finger-print should should be greater than 0.90 with the standard finger-print similarity; The American cockroach medicinal material has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.480 ± 0.048, No. 2 peaks 0.508 ± 0.051, No. 3 peaks 0.556 ± 0.056, No. 4 peaks 0.629 ± 0.063, No. 5 peaks 0.780 ± 0.078, S peak 1.000 ± 0.000, No. 6 peaks 1.295 ± 0.130, No. 7 peaks 1.901 ± 0.190,2.655 ± 0.266, No. 10 peaks 3.515 ± 0.352,2.108 ± 0.211, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peak=(0.223～0.414): (0.188～0.349): (0.996～1.494): 1.000: (0.376～0.626).
The fingerprint pattern quality determination method of American-cockroach-extract of the present invention, this method comprises the steps:
The preparation of A, need testing solution: take by weighing American-cockroach-extract 0.5～5 weight portion, put in the 25 parts by volume measuring bottles, add 20 parts by volume water, sonicated 5～30 minutes, thin up is to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 40～60 mesh sieves and remove coarse grain, remove particulate after 140～160 mesh sieves, be soaked in water 24 hours, soaked 1～3 hour with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 1～3 time at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 3～5 times of amounts soaked 1～3 hour, stirred 3～5 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 1～3 hour, stirred 3～5 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10 parts by volume, be added on the cation exchange resin column of having handled well, collect effluent, with 5-10 parts by volume water washing cation exchange resin column, collect cleansing solution, merge with effluent, be transferred in the 25 parts by volume measuring bottles, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every parts by volume contains uracil 0.0000002～0.0001 weight portion, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.01～0.2mol/L is a moving phase; The detection wavelength is 240～280nm; Draw each 0.005～0.05 parts by volume of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American-cockroach-extract according to high effective liquid chromatography for measuring; The test sample finger-print should should be greater than 0.90 with the standard finger-print similarity; American-cockroach-extract has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.480 ± 0.048, No. 2 peaks 0.508 ± 0.051, No. 3 peaks 0.556 ± 0.056, No. 4 peaks 0.629 ± 0.063, No. 5 peaks 0.780 ± 0.078, S peak 1.000 ± 0.000, No. 6 peaks 1.295 ± 0.130, No. 7 peaks 1.901 ± 0.190,2.655 ± 0.266, No. 10 peaks 3.515 ± 0.352,2.108 ± 0.211, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peak=(0.234～0.434): (0.191～0.354): (1.000～1.500): 1.000: (0.377～0.628);
Wherein above-mentioned American-cockroach-extract can be prepared by conventional method, can also prepare by following method: get American cockroach, pulverize, alcohol reflux with 50%～95% three times, the alcohol reflux 1～3 hour that adds for the first time 5～10 times of weight portions, the alcohol reflux 1～3 hour that adds for the second time 3～8 times of weight portions, the alcohol reflux 0.5～2 hour that adds 2～8 times of weight portions for the third time merges extract, filters, reclaiming ethanol and being condensed into relative density is 1.10～1.40 thick paste, add the pure water of 10～30 times of thick paste weight, heated and stirred leaves standstill, cooling, grease is fully floated, take off clear liquid, filter, it is 1.10～1.40 thick paste that filtrate is condensed into relative density, promptly gets American-cockroach-extract.
The fingerprint pattern quality determination method of American-cockroach-extract of the present invention is preferably as follows detection method:
The preparation of A, need testing solution: take by weighing American-cockroach-extract 1 weight portion, put in the 25 parts by volume measuring bottles, add 20 parts by volume water, sonicated 20 minutes, thin up is to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10 parts by volume, be added on the cation exchange resin column of having handled well, collect effluent, with 10 parts by volume water washing cation exchange resin columns, collect cleansing solution, merge with effluent, be transferred in the 25 parts by volume measuring bottles, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every parts by volume contains uracil 0.00001 weight portion, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.05mol/L is a moving phase; The detection wavelength is 259nm; Draw each 0.02 parts by volume of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American-cockroach-extract according to high effective liquid chromatography for measuring; The test sample finger-print should should be greater than 0.90 with the standard finger-print similarity; American-cockroach-extract has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.480 ± 0.048, No. 2 peaks 0.508 ± 0.051, No. 3 peaks 0.556 ± 0.056, No. 4 peaks 0.629 ± 0.063, No. 5 peaks 0.780 ± 0.078, S peak 1.000 ± 0.000, No. 6 peaks 1.295 ± 0.130, No. 7 peaks 1.901 ± 0.190,2.655 ± 0.266, No. 10 peaks 3.515 ± 0.352,2.108 ± 0.211, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peak=(0.234～0.434): (0.191～0.354): (1.000～1.500): 1.000: (0.377～0.628);
Wherein above-mentioned American-cockroach-extract can be prepared by conventional method, can also prepare by following method: get American cockroach, pulverize, the alcohol reflux with 80%～90% three times adds the alcohol reflux 2 hours of 8 times of weight portions the first time, the alcohol reflux 1.5 hours that adds for the second time 5 times of weight portions, the alcohol reflux 1 hour that adds 3 times of weight portions for the third time merges extract, filters, reclaiming ethanol and being condensed into relative density is 1.30 thick paste, add the pure water of 20 times of thick paste weight, heated and stirred leaves standstill, cooling, grease is fully floated, take off clear liquid, filter, it is 1.30 thick paste that filtrate is condensed into relative density, promptly gets American-cockroach-extract.
The pass of foregoing invention weight portion and parts by volume is the relation of g/ml.
The present invention uses fingerprint pattern technology effectively to control the quality of American cockroach medicinal material and extract thereof; The present invention uses fingerprint pattern technology to begin raw material is effectively controlled from the American cockroach medicinal material, guaranteed under certain preparation technology's situation, gained American-cockroach-extract steady quality, thus the quality of making by American-cockroach-extract can further effectively be controlled including but not limited to preparations such as the new liquid of rehabilitation, rehabilitation new capsule, rehabilitation new film and the new sprays of rehabilitation; Fingerprint pattern technology provided by the invention can be used as the key means of estimating traditional Chinese medicine quality.
Following experimental example and embodiment are used to further specify but are not limited to the present invention.
Determining of the total peak of experimental example 1 American cockroach medicinal materials fingerprint
1, finger-print
According to 10 batches of given correlation parameters of American cockroach medicinal material HPLC collection of illustrative plates, the American cockroach medicinal material all occurred in 30 minutes by the main chromatographic peak that aforementioned preparation method measures gained; 1 hour need testing solution chromatogram shows 30 minutes and does not occur chromatographic peak later on.Relatively the chromatogram of each batch sample finds that it is that each batch is total that 11 peaks are arranged, and sets up standard finger-print thus.
2. the demarcation of total fingerprint peaks
According to the result of calculation of 10 batches of American cockroach medicinal material test sample finger-print relevant datas, the average relative retention time in total peak (peak number) is followed successively by 0.480, No. 2 peak 0.510, No. 1 peak, 0.631, No. 5 peak 0.784,0.557, No. 4 peak, No. 3 peaks, S peak 1.000,1.926, No. 8 peaks 2.104,1.295, No. 7 peaks, No. 6 peaks, No. 9 peaks 2.665, No. 10 peaks 3.517, as the foundation that total fingerprint peaks is demarcated, allowing relative deviation is ± 10% with this.
3. total fingerprint peaks relative retention time
The relative retention time table 1 of total fingerprint peaks in the 10 batches of test sample finger-prints.
Table 1 American cockroach medicinal material has fingerprint peaks relative retention time (n=10)
The RSD of the relative retention time at 10 batches of total peaks of American cockroach medicinal material shows that all less than 2.0% the consistance of each batch sample is better.
4. total fingerprint peaks relative retention time
The peak area of total fingerprint peaks sees Table 2 in the 10 batches of test sample finger-prints.
Table 2 American cockroach medicinal material has fingerprint peaks peak area (n=10)
With reference to the related request of " technical requirement (provisional) of traditional Chinese medicine finger-print research ", the area ratio of each total fingerprint peaks must relative fixed.To the odds ratio of each total peak area in the ratio of each total peak area in the test sample collection of illustrative plates and the finger-print, retention time is less than or equal to 30 minutes total peak: unimodal area accounts for total peak area more than or equal to 20% total peak, and its difference must not be greater than ± 20%; Unimodal area accounts for total peak area more than or equal to 10%, and less than 20% total peak, its difference must not be greater than ± 25%; Unimodal area accounts for total peak area more than or equal to 5%, and less than 10% total peak, its difference must not be greater than ± 30%; Unimodal area accounts for total peak area less than 5% total peak, and peak area ratio is not made the regulation that requires.Two total peak-to-peak areas such as total peak 2, total peak 3 account for 5%～10% of total peak area in the total peak of American cockroach medicinal material, and average relative peak area is respectively 0.319 and 0.268, and RSD is 1.84% and 2.26%; Total peak 4 peak areas account for total peak area greater than 20%, and average relative peak area is 1.245, and RSD is 1.60%; Total peak 9 peak areas account for 10%～20% of total peak area, and average relative peak area is 0.501, and RSD is 1.71%.Thereby determined the relative peak area at above five total peaks: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peak=(0.223～0.414): (0.188～0.349): (0.996～1.494): 1.000: (0.376～0.626).
5. non-total peak area
Remove the peak that keeps without post, the non-total peak total area that has calculated 10 batch samples accounts for the ratio of total peak area, the results are shown in Table 3.
The non-total peak total area of table 3 American cockroach medicinal material accounts for the number percent (n=10) of total peak area
| Sample number into spectrum | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | On average |
| Proportion % | 4.77 | 3.19 | 3.59 | 3.84 | 4.04 | 4.38 | 5.64 | 3.54 | 3.54 | 3.13 | 3.97 |
The ratio that the non-total peak of each batch American cockroach medicinal material total area accounts for the total area meets the regulation of " technical requirement (provisional) of traditional Chinese medicine finger-print research " all less than 10%.
6, American cockroach medicinal materials fingerprint similarity evaluation
Get ten batches of American cockroach medicinal materials, detect as stated above, draw the finger-print of ten batch samples, the area of computer aided similarity evaluation software (Central South University's modernization of cmm center establishment) that adopts the Chinese Pharmacopoeia council to recommend calculates, and similarity sees Table 4.
Table 4 American cockroach medicinal materials fingerprint similarity
Determining of the total peak of experimental example 2 American-cockroach-extract finger-prints
1, finger-print
According to 10 batches of given correlation parameters of American-cockroach-extract HPLC collection of illustrative plates, American-cockroach-extract all occurred in 30 minutes by the main chromatographic peak that aforementioned preparation method measures gained; 1 hour need testing solution chromatogram shows 30 minutes and does not occur chromatographic peak later on.Relatively the chromatogram of each batch sample finds that it is that each batch is total that 11 peaks are arranged, and sets up standard finger-print thus.
2. the demarcation of total fingerprint peaks
According to the result of calculation of 10 batches of American-cockroach-extract test sample finger-print relevant datas, the average relative retention time in total peak (peak number) is followed successively by 0.480, No. 2 peak 0.508, No. 1 peak, 0.629, No. 5 peak 0.780,0.556, No. 4 peak, No. 3 peaks, S peak 1.000,1.901, No. 8 peaks 2.108,1.295, No. 7 peaks, No. 6 peaks, No. 9 peaks 2.655, No. 10 peaks 3.515, as the foundation that total fingerprint peaks is demarcated, allowing relative deviation is ± 10% with this.
3. total fingerprint peaks relative retention time
The relative retention time table 5 of total fingerprint peaks in the 10 batches of test sample finger-prints.
Table 5 American-cockroach-extract has fingerprint peaks relative retention time (n=10)
The RSD of the relative retention time at 10 batches of total peaks of American-cockroach-extract shows that all less than 2.0% the consistance of each batch sample is better.
4. total fingerprint peaks relative retention time
The peak area of total fingerprint peaks sees Table 6 in the 10 batches of test sample finger-prints.
Table 6 American-cockroach-extract has fingerprint peaks peak area (n=10)
With reference to the related request of " technical requirement (provisional) of traditional Chinese medicine finger-print research ", the area ratio of each total fingerprint peaks must relative fixed.To the odds ratio of each total peak area in the ratio of each total peak area in the test sample collection of illustrative plates and the finger-print, retention time is less than or equal to 30 minutes total peak: unimodal area accounts for total peak area more than or equal to 20% total peak, and its difference must not be greater than ± 20%; Unimodal area accounts for total peak area more than or equal to 10%, and less than 20% total peak, its difference must not be greater than ± 25%; Unimodal area accounts for total peak area more than or equal to 5%, and less than 10% total peak, its difference must not be greater than ± 30%; Unimodal area accounts for total peak area less than 5% total peak, and peak area ratio is not made the regulation that requires.Two total peak-to-peak areas such as total peak 2, total peak 3 account for 5%～10% of total peak area in the total peak of American-cockroach-extract, and average relative peak area is respectively 0.334 and 0.272, and RSD is 1.96% and 1.50%; Total peak 4 peak areas account for total peak area greater than 20%, and average relative peak area is 1.250, and RSD is 1.03%; Total peak 9 peak areas account for 10%～20% of total peak area, and average relative peak area is 0.502, and RSD is 1.60%.Thereby determined the relative peak area at above five total peaks: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peak=(0.234～0.434): (0.191～0.354): (1.000～1.500): 1.000: (0.377～0.628).
5. non-total peak area
Remove the peak that keeps without post, the non-total peak total area that has calculated 10 batch samples accounts for the ratio of total peak area, the results are shown in Table 7.
The non-total peak total area of table 7 American-cockroach-extract accounts for the number percent (n=10) of total peak area
| Sample number into spectrum | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | On average |
| Proportion % | 3.43 | 3.58 | 3.50 | 3.63 | 3.55 | 3.54 | 3.62 | 3.56 | 3.55 | 3.38 | 3.53 |
The ratio that the non-total peak of each batch American-cockroach-extract total area accounts for the total area meets the regulation of " technical requirement (provisional) of traditional Chinese medicine finger-print research " all less than 5%.
6, American-cockroach-extract finger-print similarity evaluation
Get ten batches of American-cockroach-extracts, detect as stated above, draw the finger-print of ten batch samples, the area of computer aided similarity evaluation software (Central South University's modernization of cmm center establishment) that adopts the Chinese Pharmacopoeia council to recommend calculates, and similarity sees Table 8.
Table 8 American-cockroach-extract finger-print similarity
Experimental example 3 American cockroach medicinal material need testing solution stability tests
Getting American cockroach medicinal material need testing solution of the present invention is determination object, the same day, second day, the 4th day measured (sample is put in the refrigerator and preserved) in preparation respectively according to aforementioned chromatographic condition, write down each total chromatographic peak retention time and peak area, retention time with the object of reference uracil is reference, converse the ratio of the relative retention time and the relative peak area at each total peak, as a result table 9～10.The result shows the relative standard deviation of each total peak relative retention time less than 2%, and is very stable; The relative standard deviation of the relative peak area of main peaks is less than 2%.Above result shows that need testing solution is stable in 4 days.
Table 9 American cockroach medicinal material stability test relative retention time
Table 10 American cockroach medicinal material stability test relative peak area
The test of experimental example 4 American cockroach medicinal material need testing solution reappearances
Prepare 5 parts of American cockroach medicinal material need testing solutions of the present invention, write down each total chromatographic peak retention time and peak area respectively, retention time with the object of reference uracil is reference, converses the ratio of the relative retention time and the relative peak area at each total peak, as a result table 11～12.The result shows the relative standard deviation of each total peak relative retention time less than 2%, and is very stable; The relative standard deviation of the relative peak area of main peaks is less than 2%.It is good that above result shows that need testing solution prepares reappearance.
Table 11 American cockroach medicinal material reappearance test relative retention time
Table 12 American cockroach medicinal material reappearance test relative peak area
Experimental example 5 American-cockroach-extract need testing solution stability tests
Getting American-cockroach-extract need testing solution of the present invention is determination object, the same day, second day, the 4th day measured (sample is put in the refrigerator and preserved) in preparation respectively according to aforementioned chromatographic condition, write down each total chromatographic peak retention time and peak area, retention time with the object of reference uracil is reference, converse the ratio of the relative retention time and the relative peak area at each total peak, as a result table 13～14.The result shows the relative standard deviation of each total peak relative retention time less than 2%, and is very stable; The relative standard deviation of the relative peak area of main peaks is less than 2%.Above result shows that need testing solution is stable in 4 days.
Table 13 American-cockroach-extract stability test relative retention time
Table 14 American-cockroach-extract stability test relative peak area
The test of experimental example 6 American-cockroach-extract need testing solution reappearances
Prepare 5 parts of American-cockroach-extract need testing solutions of the present invention, write down each total chromatographic peak retention time and peak area respectively, retention time with the object of reference uracil is reference, converses the ratio of the relative retention time and the relative peak area at each total peak, as a result table 15～16.The result shows the relative standard deviation of each total peak relative retention time less than 2%, and is very stable; The relative standard deviation of the relative peak area of main peaks is less than 2%.It is good that above result shows that need testing solution prepares reappearance.
Table 15 American-cockroach-extract reappearance test relative retention time
Table 16 American-cockroach-extract reappearance test relative peak area
Following embodiment is used to further specify but is not limited to the present invention.
Embodiment
Embodiment 1: the fingerprint pattern quality determination method of American cockroach medicinal material
The preparation of A, need testing solution: take by weighing American cockroach 5g, add 80% ethanol 100ml, refluxed 3 hours, put coldly, filter, filtrate recycling ethanol is put in the 25ml measuring bottle, and thin up shakes up to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10ml, being added on the internal diameter of having handled well is 1.5cm, and the height of bed is on the cation exchange resin column of 2cm, collects effluent, with 10ml water washing cation exchange resin column, collect cleansing solution, merge with effluent, be transferred in the 25ml measuring bottle, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every 1ml contains uracil 1 μ g, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.01mol/L is a moving phase; The detection wavelength is 250nm; Survey and draw need testing solution and each 20 μ l injection liquid chromatograph of reference substance solution,, obtain the finger-print of American cockroach medicinal material according to high effective liquid chromatography for measuring; Test sample finger-print and standard finger-print similarity are 0.989; The American cockroach medicinal material has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.482, No. 2 peaks 0.511, No. 3 peaks 0.556, No. 4 peaks 0.633, No. 5 peaks 0.788,1.000, No. 6 peaks 1.290, S peak, No. 7 peaks 1.923,2.667, No. 10 peaks 3.512,2.100, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively the peak No. 2: No. 3 peaks: No. 4 peaks: S peak: No. 8 peaks=0.341: 0.289: 1.294: 1.000: 0.526.
Embodiment 2: the fingerprint pattern quality determination method of American cockroach medicinal material
The preparation of A, need testing solution: take by weighing American cockroach 10g, add 90% ethanol 200ml, refluxed 1 hour, put coldly, filter, filtrate recycling ethanol is put in the 25ml measuring bottle, and thin up shakes up to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10ml, being added on the internal diameter of having handled well is 1.5cm, and the height of bed is on the 4cm cation exchange resin column, collects effluent, with 5ml water washing cation exchange resin column, collect cleansing solution, merge with effluent, be transferred in the 25ml measuring bottle, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every 1ml contains uracil 10 μ g, as reference product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.05mol/L is a moving phase; The detection wavelength is 260nm; Draw each 5 μ l of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American cockroach medicinal material according to high effective liquid chromatography for measuring; Test sample finger-print and standard finger-print similarity are 0.992; The American cockroach medicinal material has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.487, No. 2 peaks 0.513, No. 3 peaks 0.552, No. 4 peaks 0.639, No. 5 peaks 0.780,1.000, No. 6 peaks 1.292, S peak, No. 7 peaks 1.925,2.662, No. 10 peaks 3.510,2.103, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively the peak No. 2: No. 3 peaks: No. 4 peaks: S peak: No. 8 peaks=0.324: 0.299: 1.214: 1.000: 0.461.
Embodiment 3: the fingerprint pattern quality determination method of American cockroach medicinal material
The preparation of A, need testing solution: take by weighing American cockroach, take by weighing 1g, add 60% ethanol 50ml, refluxed 8 hours, put coldly, filter, filtrate recycling ethanol is put in the 25ml measuring bottle, and thin up shakes up to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10ml, being added on the internal diameter of having handled well is 1.5cm, and the height of bed is on the 1cm cation exchange resin column, collects effluent, with 10ml water washing cation exchange resin column, collect cleansing solution, merge with effluent, be transferred in the 25ml measuring bottle, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every 1ml contains uracil 5 μ g, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.2mol/L is a moving phase; The detection wavelength is 280nm; Draw each 50 μ l of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American cockroach medicinal material according to high effective liquid chromatography for measuring; Test sample finger-print and standard finger-print similarity are 0.983; The American cockroach medicinal material has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.476, No. 2 peaks 0.505, No. 3 peaks 0.566, No. 4 peaks 0.623, No. 5 peaks 0.778,1.000, No. 6 peaks 1.280, S peak, No. 7 peaks 1.937,2.654, No. 10 peaks 3.524,2.117, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively the peak No. 2: No. 3 peaks: No. 4 peaks: S peak: No. 8 peaks=0.352: 0.267: 1.283: 1.000: 0.517.
Embodiment 4: the fingerprint pattern quality determination method of American cockroach medicinal material
The preparation of A, need testing solution: take by weighing American cockroach, take by weighing 5g, add 85% ethanol 50ml, refluxed 3 hours, put coldly, filter, filtrate recycling ethanol is put in the 25ml measuring bottle, and thin up shakes up to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10ml, be added on the cation exchange resin column of having handled well, collect effluent, with 10ml water washing cation exchange resin column, collect cleansing solution, merge with effluent, be transferred in the 25ml measuring bottle, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every 1ml contains uracil 10 μ g, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.05mol/L is a moving phase; The detection wavelength is 259nm; Draw each 20 μ l of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American cockroach medicinal material according to high effective liquid chromatography for measuring; Test sample finger-print and standard finger-print similarity are 0.992; The American cockroach medicinal material has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.480, No. 2 peaks 0.513, No. 3 peaks 0.559, No. 4 peaks 0.635, No. 5 peaks 0.786,1.000, No. 6 peaks 1.298, S peak, No. 7 peaks 1.924,2.669, No. 10 peaks 3.518,2.107, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peaks=0.354: 0.269: 1.287: 1.000: 0.527.
Embodiment 5: the fingerprint pattern quality determination method of American-cockroach-extract
The preparation of A, need testing solution: take by weighing American-cockroach-extract 0.5g, put in the 25ml measuring bottle, add 20ml water, sonicated 5 minutes, thin up is to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10ml, being added on the internal diameter of having handled well is 1.5cm, and the height of bed is on the 4cm cation exchange resin column, collects effluent, with 5ml water washing cation exchange resin column, collect cleansing solution, merge with effluent, be transferred in the 25ml measuring bottle, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every 1ml contains uracil 1 μ g, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.01mol/L is a moving phase; The detection wavelength is 250nm; Draw need testing solution and inject liquid chromatograph,, obtain the finger-print of American-cockroach-extract according to high effective liquid chromatography for measuring with reference to each 50 μ l of product solution; Test sample finger-print and standard finger-print similarity are 0.974; American-cockroach-extract has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.485, No. 2 peaks 0.507, No. 3 peaks 0.558, No. 4 peaks 0.622, No. 5 peaks 0.789,1.000, No. 6 peaks 1.296, S peak, No. 7 peaks 1.903,2.653, No. 10 peaks 3.517,2.107, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peaks=0.334: 0.254: 1.246: 1.000: 0.497;
Wherein above-mentioned American-cockroach-extract can be prepared by conventional method, can also prepare by following method: get American cockroach, pulverize, the alcohol reflux with 80%～90% three times adds the alcohol reflux 2 hours of 8 times of weight portions the first time, the alcohol reflux 1.5 hours that adds for the second time 5 times of weight portions, the alcohol reflux 1 hour that adds 3 times of weight portions for the third time merges extract, filters, reclaiming ethanol and being condensed into relative density is 1.30 thick paste, add the pure water of 20 times of thick paste weight, heated and stirred leaves standstill, cooling, grease is fully floated, take off clear liquid, filter, it is 1.30 thick paste that filtrate is condensed into relative density, promptly gets American-cockroach-extract.
Embodiment 6: the fingerprint pattern quality determination method of American-cockroach-extract
The preparation of A, need testing solution: take by weighing American-cockroach-extract 1g, put in the 25ml measuring bottle, add 20ml water, sonicated 20 minutes, thin up is to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10ml, being added on the internal diameter of having handled well is 1.5cm, and the height of bed is on the 1cm cation exchange resin column, collects effluent, with 10ml water washing cation exchange resin column, collect cleansing solution, merge with effluent, be transferred in the 25ml measuring bottle, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every 1ml contains uracil 10 μ g, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.05mol/L is a moving phase; The detection wavelength is 259nm; Draw each 20 μ l of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American-cockroach-extract according to high effective liquid chromatography for measuring; Test sample finger-print and standard finger-print similarity are 0.996; American-cockroach-extract has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.483, No. 2 peaks 0.502, No. 3 peaks 0.551, No. 4 peaks 0.637, No. 5 peaks 0.776,1.000, No. 6 peaks 1.292, S peak, No. 7 peaks 1.909,2.656, No. 10 peaks 3.517,2.104, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peaks=0.364: 0.274: 1.248: 1.000: 0.528;
Wherein above-mentioned American-cockroach-extract can be prepared by conventional method, can also prepare by following method: get American cockroach, pulverize, the alcohol reflux with 80%～90% three times adds the alcohol reflux 2 hours of 8 times of weight portions the first time, the alcohol reflux 1.5 hours that adds for the second time 5 times of weight portions, the alcohol reflux 1 hour that adds 3 times of weight portions for the third time merges extract, filters, reclaiming ethanol and being condensed into relative density is 1.30 thick paste, add the pure water of 20 times of thick paste weight, heated and stirred leaves standstill, cooling, grease is fully floated, take off clear liquid, filter, it is 1.30 thick paste that filtrate is condensed into relative density, promptly gets American-cockroach-extract.
Embodiment 7: the fingerprint pattern quality determination method of American-cockroach-extract
The preparation of A, need testing solution: take by weighing American-cockroach-extract 5g, put in the 25ml measuring bottle, add 20ml water, sonicated 30 minutes, thin up is to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10ml, being added on the internal diameter of having handled well is 1.5cm, and the height of bed is on the 2cm cation exchange resin column, collects effluent, with 10ml water washing cation exchange resin column, collect cleansing solution, merge with effluent, be transferred in the 25ml measuring bottle, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every 1ml contains uracil 20 μ g, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.2mol/L is a moving phase; The detection wavelength is 270nm; Draw each 5 μ l of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American-cockroach-extract according to high effective liquid chromatography for measuring; Test sample finger-print and standard finger-print similarity are 0.983; American-cockroach-extract has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.482, No. 2 peaks 0.511, No. 3 peaks 0.566, No. 4 peaks 0.633, No. 5 peaks 0.785,1.000, No. 6 peaks 1.300, S peak, No. 7 peaks 1.912,2.666, No. 10 peaks 3.521,2.114, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peaks=0.363: 0.284: 1.257: 1.000: 0.526;
Wherein above-mentioned American-cockroach-extract can be prepared by conventional method, can also prepare by following method: get American cockroach, pulverize, the alcohol reflux with 80%～90% three times adds the alcohol reflux 2 hours of 8 times of weight portions the first time, the alcohol reflux 1.5 hours that adds for the second time 5 times of weight portions, the alcohol reflux 1 hour that adds 3 times of weight portions for the third time merges extract, filters, reclaiming ethanol and being condensed into relative density is 1.30 thick paste, add the pure water of 20 times of thick paste weight, heated and stirred leaves standstill, cooling, grease is fully floated, take off clear liquid, filter, it is 1.30 thick paste that filtrate is condensed into relative density, promptly gets American-cockroach-extract.
Claims (6)
1. the fingerprint pattern quality determination method of an American cockroach medicinal material is characterized in that this method comprises the steps:
The preparation of A, need testing solution: take by weighing American cockroach 1～10 weight portion, add 50～95% ethanol, 20～200 parts by volume, refluxed 1～8 hour, put cold, filter, filtrate recycling ethanol is put in the 25 parts by volume measuring bottles, thin up shakes up to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 40～60 mesh sieves and remove coarse grain, remove particulate after 140～160 mesh sieves, be soaked in water 24 hours, soaked 1～3 hour with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 3～5 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 3～5 times of amounts soaked 1～3 hour, stirred 3～5 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 2～4 times of amounts soaked 1～3 hour, stirred 3～5 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10 parts by volume, be added on the cation exchange resin column of having handled well, collect effluent, with 5-10 parts by volume water washing cation exchange resin column, collect cleansing solution, merge with effluent, be transferred in the 25 parts by volume measuring bottles, constant volume shakes up, and is standby;
B. the preparation of reference substance solution: take by weighing uracil, add water and make the solution that every parts by volume contains uracil 0.0000002～0.0001 weight portion, in contrast product solution;
C. measure: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.01～0.2mol/L is a moving phase; The detection wavelength is 240～280nm; Draw each 0.005～0.05 parts by volume of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American cockroach medicinal material according to high effective liquid chromatography for measuring; Test sample finger-print and standard finger-print similarity should be greater than 0.90; The American cockroach medicinal material has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.480 ± 0.048, No. 2 peaks 0.508 ± 0.051, No. 3 peaks 0.556 ± 0.056, No. 4 peaks 0.629 ± 0.063, No. 5 peaks 0.780 ± 0.078, S peak 1.000 ± 0.000, No. 6 peaks 1.295 ± 0.130, No. 7 peaks 1.901 ± 0.190,2.655 ± 0.266, No. 10 peaks 3.515 ± 0.352,2.108 ± 0.211, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peak=(0.223～0.414): (0.188～0.349): (0.996～1.494): 1.000: (0.376～0.626).
2. fingerprint pattern quality determination method as claimed in claim 1 is characterized in that this method comprises the steps:
The preparation of A, need testing solution: take by weighing American cockroach 5 weight portions, add 85% ethanol, 50 parts by volume, refluxed 3 hours, put coldly, filter, filtrate recycling ethanol is put in the 25 parts by volume measuring bottles, and thin up shakes up to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10 parts by volume, being added on the internal diameter of having handled well is 1.5cm, and the height of bed is on the cation exchange resin column of 2cm, collects effluent, with 10 parts by volume water washing cation exchange resin columns, collect cleansing solution, merge with effluent, be transferred in the 25 parts by volume measuring bottles, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every parts by volume contains uracil 0.00001 weight portion, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.05mol/L is a moving phase; The detection wavelength is 259nm; Draw each 0.02 parts by volume of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American cockroach medicinal material according to high effective liquid chromatography for measuring; Test sample finger-print and standard finger-print similarity should be greater than 0.90; The American cockroach medicinal material has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.480 ± 0.048, No. 2 peaks 0.508 ± 0.051, No. 3 peaks 0.556 ± 0.056, No. 4 peaks 0.629 ± 0.063, No. 5 peaks 0.780 ± 0.078, S peak 1.000 ± 0.000, No. 6 peaks 1.295 ± 0.130, No. 7 peaks 1.901 ± 0.190,2.655 ± 0.266, No. 10 peaks 3.515 ± 0.352,2.108 ± 0.211, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peak=(0.223～0.414): (0.188～0.349): (0.996～1.494): 1.000: (0.376～0.626).
3. the fingerprint pattern quality determination method of an American-cockroach-extract is characterized in that this method comprises the steps:
The preparation of A, need testing solution: take by weighing American-cockroach-extract 1 weight portion, put in the 25 parts by volume measuring bottles, add 20 parts by volume water, sonicated 20 minutes, thin up is to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10 parts by volume, be added on the cation exchange resin column of having handled well, collect effluent, with 10 parts by volume water washing cation exchange resin columns, collect cleansing solution, merge with effluent, be transferred in the 25 parts by volume measuring bottles, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every parts by volume contains uracil 0.00001 weight portion, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.05mol/L is a moving phase; The detection wavelength is 259nm; Draw each 0.02 parts by volume of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American-cockroach-extract according to high effective liquid chromatography for measuring; Test sample finger-print and standard finger-print similarity should be greater than 0.90; American-cockroach-extract has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.480 ± 0.048, No. 2 peaks 0.508 ± 0.051, No. 3 peaks 0.556 ± 0.056, No. 4 peaks 0.629 ± 0.063, No. 5 peaks 0.780 ± 0.078, S peak 1.000 ± 0.000, No. 6 peaks 1.295 ± 0.130, No. 7 peaks 1.901 ± 0.190,2.655 ± 0.266, No. 10 peaks 3.515 ± 0.352,2.108 ± 0.211, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peak=(0.234～0.434): (0.191～0.354): (1.000～1.500): 1.000: (0.377～0.628);
Wherein above-mentioned American-cockroach-extract is prepared by conventional method.
4. fingerprint pattern quality determination method as claimed in claim 3 is characterized in that this method comprises the steps:
The preparation of A, need testing solution: take by weighing American-cockroach-extract 1 weight portion, put in the 25 parts by volume measuring bottles, add 20 parts by volume water, sonicated 20 minutes, thin up is to scale, filter subsequent filtrate; Get Zeo-karb, grind, cross 50 mesh sieves and remove coarse grain, remove particulate after 150 mesh sieves, be soaked in water 24 hours, soaked 2 hours with the 2mol/L hydrochloric acid solution of 2～3 times of amounts the incline back of anhydrating, and stir 4 times at the interval, removes acid solution, wash with water to nearly neutrality, the 1mol/L sodium hydroxide solution that adds 4 times of amounts soaked 2 hours, stirred 4 times at interval, removed alkali lye, wash with water to nearly neutrality, the 2mol/L hydrochloric acid solution that adds 3 times of amounts soaked 2 hours, stirred 4 times at interval, remove acid solution, wash with water to nearly neutrality, soak in the water and place, standby; Get subsequent filtrate 10 parts by volume, being added on the internal diameter of having handled well is 1.5cm, and the height of bed is on the cation exchange resin column of 2cm, collects effluent, with 10 parts by volume water washing cation exchange resin columns, collect cleansing solution, merge with effluent, be transferred in the 25 parts by volume measuring bottles, constant volume shakes up, and is standby;
The preparation of B, reference substance solution: take by weighing uracil, add water and make the solution that every parts by volume contains uracil 0.00001 weight portion, in contrast product solution;
C, mensuration: chromatographic column is that octadecylsilane chemically bonded silica is a filling agent; Ammonium dibasic phosphate solution with 0.05mol/L is a moving phase; The detection wavelength is 259nm; Draw each 0.02 parts by volume of need testing solution and reference substance solution and inject liquid chromatograph,, obtain the finger-print of American-cockroach-extract according to high effective liquid chromatography for measuring; Test sample finger-print and standard finger-print similarity should be greater than 0.90; American-cockroach-extract has 11 total fingerprint peakses, with reference to peak S is the uracil chromatographic peak, the relative retention time of 11 total fingerprint peakses is respectively: No. 1 peak 0.480 ± 0.048, No. 2 peaks 0.508 ± 0.051, No. 3 peaks 0.556 ± 0.056, No. 4 peaks 0.629 ± 0.063, No. 5 peaks 0.780 ± 0.078, S peak 1.000 ± 0.000, No. 6 peaks 1.295 ± 0.130, No. 7 peaks 1.901 ± 0.190,2.655 ± 0.266, No. 10 peaks 3.515 ± 0.352,2.108 ± 0.211, No. 9 peaks, No. 8 peaks, in 11 total fingerprint peakses, the ratio of 5 peak areas is respectively: the ratio of peak area is respectively: No. 2 peaks: No. 3 peaks: No. 4 peaks: S peak: No. 8 peak=(0.234～0.434): (0.191～0.354): (1.000～1.500): 1.000: (0.377～0.628);
Wherein above-mentioned American cockroach is prepared by conventional method.
5. as claim 3,4 described fingerprint pattern quality determination methods, it is characterized in that American-cockroach-extract is prepared by following method:
Get American cockroach, pulverize, alcohol reflux with 50%～95% three times, the alcohol reflux 1～3 hour that adds for the first time 5～10 times of weight portions adds the alcohol reflux 1～3 hour of 3～8 times of weight portions for the second time, adds the alcohol reflux 0.5～2 hour of 2～8 times of weight portions for the third time, merge extract, filter, reclaiming ethanol and being condensed into relative density is 1.10～1.40 thick paste, adds the pure water of 10～30 times of thick paste weight, heated and stirred, leave standstill, cooling is fully floated grease, take off clear liquid, filter, it is 1.10～1.40 thick paste that filtrate is condensed into relative density, promptly gets American-cockroach-extract.
6. fingerprint pattern quality determination method as claimed in claim 5 is characterized in that American-cockroach-extract is prepared by following method:
Get American cockroach, pulverize, alcohol reflux with 80%～90% three times, the alcohol reflux 2 hours that adds for the first time 8 times of weight portions adds the alcohol reflux 1.5 hours of 5 times of weight portions for the second time, adds the alcohol reflux 1 hour of 3 times of weight portions for the third time, merge extract, filter, reclaiming ethanol and being condensed into relative density is 1.30 thick paste, adds the pure water of 20 times of thick paste weight, heated and stirred, leave standstill, cooling is fully floated grease, take off clear liquid, filter, it is 1.30 thick paste that filtrate is condensed into relative density, promptly gets American-cockroach-extract.
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| CN106324134A (en) * | 2016-08-18 | 2017-01-11 | 四川好医生攀西药业有限责任公司 | HPLC-DAD fingerprint quality determination method of Periplaneta americana drug material |
| CN106338556A (en) * | 2016-08-18 | 2017-01-18 | 四川好医生攀西药业有限责任公司 | GC-MS coupling technology-based method for analysis of components in cockroaches in different growing stages |
| CN106370738B (en) * | 2016-08-18 | 2020-04-17 | 四川好医生攀西药业有限责任公司 | Fingerprint quality determination method for periplaneta americana medicinal material |
| CN109100460A (en) * | 2018-10-18 | 2018-12-28 | 大理大学 | A kind of Toxicity Analysis detection method of the active site of American cockroach pulmonary fibrosis resistant |
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Assignee: Hunan Central South Kelun Medicine Co., Ltd. Assignor: Sichuan Kelun Pharmaceutical Co., Ltd. Contract record no.: 2011510000143 Denomination of invention: Fingerprint pattern quality determination method of american cockroaches medicinal materials and their extract Granted publication date: 20110629 License type: Exclusive License Open date: 20081210 Record date: 20110812 |