CN105181852A - Method for identifying raw corydalis yanhusuo material and decocted corydalis yanhusuo material - Google Patents
Method for identifying raw corydalis yanhusuo material and decocted corydalis yanhusuo material Download PDFInfo
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Abstract
A method for identifying a raw corydalis yanhusuo material and a decocted corydalis yanhusuo material comprises the steps that firstly, test article solutions are prepared from the corydalis yanhusuo materials; secondly, the test article solutions are subjected to chromatographic analysis, and obtained chromatograms serves as fingerprint spectrums of samples; thirdly, the obtained fingerprint spectrums are subjected to retention time drift correction through a dynamic time warping method; fourthly, the chromatography fingerprint spectrums obtained after all the corydalis yanhusuo samples are corrected through the dynamic time warping method serves as row vectors to form a data matrix X, and principle component analysis operation is conducted after the data matrix X is standardized; finally, an artificial neural network classification model is established to identify the vinegar-processed corydalis yanhusuo material and the non-vinegar-processed corydalis yanhusuo material. The method has the advantages of being free of influences of retention time drift in the fingerprint spectrums, objective, and accurate.
Description
Technical field
The present invention relates to Chinese crude drug Technique of Qualitative Analysis field, particularly a kind of method differentiating the raw ripe medicinal material of corydalis tuber.
Background technology
Corydalis tuber (corydalis tuber) is the dry tuber of papaveraceae plant corydalis (CorydalisyanhusuoW.T.Wang), there is analgesia, invigorate blood circulation, the loose stasis of blood, the effect such as to regulate the flow of vital energy, calm, its clinical efficacy is definite, is widely used in Chinese patent drug and produces and clinical prescription.Corydalis tuber is the typical medicaments of synergy after vinegar system, and vinegar system can make its alkaloids composition stripping quantity increase, and significantly improves drug effect.Therefore, be necessary to propose a kind ofly to carry out mirror method for distinguishing to the ripe corydalis tuber medicinal material through vinegar and the raw corydalis tuber medicinal material without vinegar.
The discrimination method of the vinegar system proposed in current all kinds of document and non-Rhizoma Corydalis (processed with vinegar) medicinal material is the method (Dou Zhiying that proposes such as Dou Zhi English mainly, Jin Zuxiang, Wei Siwen, Wu Zijun, Luo Chenyan, the method for the raw ripe medicine materical crude slice of a kind of quick discriminating corydalis tuber, Chinese patent, CN103543235B, 2015.07.15).This patent proposition thin layer chromatography measures the content of Tetrahydropalmatine in Rhizoma Corydalis, take Content determination of dl-tetrahydropalmatine as index, observes the difference difference vinegar system of bright spot in chromatographic sheet and non-vinegar pharmacy material.But corydalis tuber is as a kind of Chinese crude drug, its quality and drug effect are determined by the mass action of each component, only considers that the content of one or more active components has the possibility of inaccurate evaluation quality of medicinal material.In addition, only measure the content of a kind of active component tetrahydropalmatine (or several active component) in corydalis tuber, by intentional or unintentionally mix respective substance, directly can change the evaluation result to medicinal material.As can be seen here, the discrimination method of existing vinegar system and non-Rhizoma Corydalis (processed with vinegar) medicinal material is subject to larger restriction in actual applications, needs to set up a kind ofly to consist of the discriminating vinegar system on basis and the method for non-Rhizoma Corydalis (processed with vinegar) medicinal material with corydalis tuber medicinal material overall chemical.
Chromatographic fingerprinting directly can reflect the overall chemical composition of Chinese crude drug.Using the chemometrics method such as Chemical Pattern Recognition to analyze chromatographic fingerprinting can the quality of accurate evaluation Chinese crude drug.But shift of retention time phenomenon can have a strong impact on the Chemical Pattern Recognition result of chromatographic fingerprinting, need to correct.Shift of retention time bearing calibration conventional is at present comparatively large by the subjective impact of analyst, and objectivity and standard deficiency, be subject to larger restriction in actual applications.Therefore, a kind of applied range, chromatographic fingerprints of Chinese materia medica shift of retention time bearing calibration that objectivity is strong is needed to set up.This has very important meaning to the market surpervision of Chinese crude drug and standardized production.
Summary of the invention
In order to overcome the shortcoming of above-mentioned prior art, the object of the present invention is to provide a kind of method differentiating the raw ripe medicinal material of corydalis tuber, integrated use dynamic time warping (Dynamictimewarping, and Chemical Pattern Recognition method DTW), by correcting and pattern recognition analysis the shift of retention time of chromatographic fingerprinting, differentiate the raw ripe medicinal material of corydalis tuber, the method has not by the advantage that shift of retention time in finger-print affects, and has objective and accurate feature.
In order to achieve the above object, the technical scheme that the present invention takes is:
Differentiate a method for the raw ripe medicinal material of corydalis tuber, step is:
S1, prepare need testing solution: get corydalis tuber medicinal material, grind into powder, add organic solvent and solvend in medicinal powder is all dissolved; Gained potpourri is placed after 16-24 hour and is added ammoniacal liquor, ultrasonic extraction in 40-45 DEG C of water-bath; Then carry out suction filtration, heating gained filtrate, after filtrate evaporate to dryness, remaining solid matter adds proper amount of methanol dissolving, filters, obtain need testing solution through 0.45 μm of filter membrane;
S2, stratographic analysis is carried out to need testing solution, using the chromatogram obtained as the finger-print of sample;
S3, by dynamic time warping method, shift of retention time correction is carried out to gained finger-print;
S4, with each corydalis tuber sample through DTW correct after gained chromatographic fingerprinting form a data matrix X as row vector, here the often row of matrix X corresponds to the chromatographic fingerprinting of a sample, often row correspond to a sampled point in chromatography experiment, namely in the chromatographic signal value recorded per second; Principal component analysis (PCA) (Principalcomponentanalysis, PCA) computing is carried out to after matrix X standardization; Reservation can make accumulative variance contribution ratio be greater than the major component of 85%, with these major components resolute, namely the respective column in score matrix (scorematrix), as the input variable of artificial neural network (Artificialneuralnetwork, ANN) model;
S5, set up multilayer perceptron (MultilayerPerceptron) artificial nerve network model; Input variable corresponds to the major component that principal component analysis (PCA) computing retains; Output variable corresponds to two classification of corydalis tuber sample, i.e. vinegar system and non-Rhizoma Corydalis (processed with vinegar) medicinal material; Classification thresholds setting is not less than 0.90; Artificial nerve network model is set up with Feedback error (backpropagation, BP) Algorithm for Training; Namely this model can be used for the taxonomic history of corydalis tuber medicinal material sample.
In described step S1, the organic solvent dissolution of every 1.0g corydalis tuber medicinal material powder total amount 50-60mL, wherein, the ratio of organic solvent to be chloroform with methyl alcohol with volume ratio be 5:1 carries out the mixed solvent mixed;
In the described ammoniacal liquor added and described organic solvent, the volume ratio of methyl alcohol is 0.15:1;
Consumption for the methyl alcohol of remaining solid matter after dissolving filtrate evaporate to dryness is that every 1.0g corydalis tuber medicinal material powder correspondence uses methyl alcohol 10mL.
In described step S4, described artificial neural network is the multilayer perceptron artificial neural network with Feedback error Algorithm for Training.
Beneficial effect of the present invention is:
The present invention proposes and a kind ofly easy to use mirror method for distinguishing is carried out to vinegar system and non-Rhizoma Corydalis (processed with vinegar) medicinal material.By using DTW to carry out shift of retention time correction, well solve shift of retention time problem common in chromatographic fingerprinting analysis.By principal component analysis (PCA) and Artificial Neural Network being combined, can accurately distinguish vinegar system and non-Rhizoma Corydalis (processed with vinegar) sample according to the chromatographic fingerprinting after shift of retention time correction.The drug effect difference of the corydalis tuber medicinal material of vinegar system and non-vinegar is comparatively remarkable, and propose the chemical method can differentiated it, the market surpervision and the standardized production that can be corydalis tuber medicinal material provide required method, have very important practical significance.This method can also be applied to the quality assessment of other products in traditional Chinese medicine quality control.
Accompanying drawing explanation
Fig. 1 is the chromatographic fingerprinting of each corydalis tuber sample in embodiments of the invention one.
Fig. 2 is the chromatographic fingerprinting of each corydalis tuber sample in embodiments of the invention two.
Fig. 3 is the chromatographic fingerprinting of each corydalis tuber sample in embodiments of the invention three.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
See accompanying drawing, the present invention is a kind of method differentiating the raw ripe medicinal material of corydalis tuber, and step is:
S1, prepare need testing solution: get corydalis tuber medicinal material, grind into powder, add organic solvent and solvend in medicinal powder is all dissolved; Gained potpourri is placed after 16-24 hour and is added ammoniacal liquor, ultrasonic extraction in 40-45 DEG C of water-bath; Then carry out suction filtration, heating gained filtrate, after filtrate evaporate to dryness, remaining solid matter adds proper amount of methanol dissolving, filters, obtain need testing solution through 0.45 μm of filter membrane;
S2, stratographic analysis is carried out to need testing solution, using the chromatogram obtained as the finger-print of sample;
S3, by dynamic time warping method, shift of retention time correction is carried out to gained finger-print; Especially, shift of retention time correction must be carried out by dynamic time warping method to all finger-prints;
S4, with each corydalis tuber sample through DTW correct after gained chromatographic fingerprinting form a data matrix X as row vector, here the often row of matrix X corresponds to the chromatographic fingerprinting of a sample, often row correspond to a sampled point in chromatography experiment, namely in the chromatographic signal value recorded per second; Principal component analysis (PCA) computing is carried out to after matrix X standardization; Reservation can make accumulative variance contribution ratio be greater than the major component of 85%, with these major components resolute, the respective column namely in score matrix, as the input variable of artificial nerve network model;
S5, set up multilayer perceptron artificial nerve network model; Input variable corresponds to principal component analysis (PCA) major component that computing retains; Output variable corresponds to two classification of corydalis tuber sample, i.e. vinegar system and non-Rhizoma Corydalis (processed with vinegar) medicinal material; Classification thresholds setting is not less than 0.90; Artificial nerve network model is set up with Feedback error Algorithm for Training; Namely this model can be used for the taxonomic history of corydalis tuber medicinal material sample.
Concrete, in described step S1, the organic solvent dissolution of every 1.0g corydalis tuber medicinal material powder total amount 50-60mL, wherein, the ratio of organic solvent to be chloroform with methyl alcohol with volume ratio be 5:1 carries out the mixed solvent mixed.
Concrete, in the described ammoniacal liquor added and described organic solvent, the volume ratio of methyl alcohol is 0.15:1.
Concrete, the consumption for the methyl alcohol of remaining solid matter after dissolving filtrate evaporate to dryness is that every 1.0g corydalis tuber medicinal material powder correspondence uses methyl alcohol 10mL.
Especially, in described step S4, described artificial neural network is the multilayer perceptron artificial neural network with Feedback error Algorithm for Training.
Embodiment one
Collect 13 corydalis tuber medicinal material samples, all samples information is in table 1.
Table 1 corydalis tuber medicinal material sample message
The first step, prepares need testing solution by corydalis tuber medicinal material: get corydalis tuber medicinal material powder, accurately weighed, is placed in triangular flask; Add organic solvent, this organic solvent is chloroform and the methyl alcohol preparation of 5:1 by volume ratio, every 1.0g corydalis tuber medicinal material powder organic solvent total amount 50-60mL; Place and add ammoniacal liquor after 16 hours, wherein the volume ratio of ammoniacal liquor and methyl alcohol is 0.15:1, ultrasonic extraction 1 hour in 40 DEG C of water-baths, suction filtration after taking out, heating gained filtrate, after filtrate evaporate to dryness, remaining solid matter adds methyl alcohol dissolving, and every 1.0g corydalis tuber medicinal material powder correspondence uses methyl alcohol 10mL, filter through 0.45 μm of filter membrane, obtain need testing solution.
Second step, sets up the finger-print of corydalis tuber sample.Chromatographic condition used is: PhenomenexLunaC18 chromatographic column (250mm × 4.6mm, ID:5 μm); Column temperature 30 DEG C; Gradient elution, flowing used is met each other table 2; Flow velocity: 1.0mLmin
-1; Spectral scan scope: 240-400nm, it is 289nm that chromatogram gathers wavelength; Working time: 75min, per second sampling should be carried out; Sample size: 5 μ L.
Table 2 measures the mobile phase composition of corydalis tuber finger-print
3rd step, with dynamic time warping method to set up 13 samples chromatographic fingerprinting carry out shift of retention time correction.Dynamic time warping is a kind of common method solving voice signal coupling and identification problem.The basic thought of algorithm is by regular in time series for processed signal (Warping), namely stretches or shortens, making its characteristic quantity corresponding with reference signal, simultaneously consistent with the length of reference signal.This process is called Time alignment.In the process, the corresponding time relationship of signal and the reference signal of being processed with the Time alignment function representation meeting certain condition, the similarity between two time serieses is weighed with consolidation path distance, cumulative distance minimum corresponding warping function when solving two Signal Matching with dynamic programming (dynamicprogramming), namely this function corresponds to best regular path.The chromatographic fingerprinting of Chinese medicine is similar to voice signal, and the not homogeneous that same sample carries out under same experimental conditions is tested the time series signal obtained and generally had difference.That is, these signals have closely similar shape on the whole, but these shapes are not alignment in time series.When the finger-print of comparison each sample, must shift of retention time correction be carried out, mutually be alignd by the regular characteristic peak of each sample that makes of time shaft.
In DTW method, setting parameter is: PadLength:[00]; Span:20; MaxStep:2; Band:0.05.Figure 1 shows that the chromatographic fingerprinting of each corydalis tuber sample in the present embodiment.Be not calibrated finger-print more than " ← " in figure, correspond respectively to 1-13 sample from top to bottom." ← " corrects gained finger-print through DTW, corresponds respectively to 1-13 sample from top to bottom.As seen from Figure 1, if not calibrated, in the finger-print of these 13 samples, the retention time of multiple characteristic peak has obvious difference, and after DTW corrects, shift of retention time obtains good correction.
4th step, with 1-12 sample correction gained finger-print for row vector forms a matrix.Principal component analysis (PCA) computing is carried out to this matrix, using first three major component of gained (PC1, PC2 and PC3) resolute as 3 input variables of artificial neural network.
5th step, sets up multilayer perceptron artificial nerve network model.Artificial neural network is made up of multiple processing unit (also referred to as neuron or node), and the information that each neuron is loaded by input channel forms output information through mathematics conversion, then is delivered to other neurons.The basic thought of artificial neural network is by constantly adjusting connection weight between each neuron and biased, thus makes error function reach minimum value.Multilayer perceptron artificial neural network is a kind of neural network with forward-propagating mechanism containing one or more layers implicit node between input layer and output layer.Artificial neural network is a kind of conventional Chemical Pattern Recognition method having supervision, these class methods utilize oneself a collection of composition of sample calibration set of knowing of classification, by setting up the pattern recognition model of classifying to sample to the study of calibration set, then with gained model, unknown sample is classified.The output variable of network is set as two classification, i.e. vinegar system and non-Rhizoma Corydalis (processed with vinegar) medicinal material.Classification thresholds is set as 0.95.Here artificial neural network is used for Chemical Pattern Recognition, and the threshold value of its output node has appreciable impact to classification results.If threshold value arranges too high, model is too harsh, lacks " ambiguity ", does not have actual application value; Threshold value arranges too low, then model is too loose, and often make the mistake classification.Common, when using a standardized output node to classify, threshold value is set as that the model obtained between 0.90-0.95 is better.Network training is carried out by Feedback error (backpropagation, BP) method.Learning rate and study momentum are set as 0.6 and 0.3 respectively.Carry out training the artificial nerve network model obtained for classifying.With gained model, No. 13 samples are classified.This model judges that No. 13 samples are as non-vinegar sample preparation product, coincide with actual conditions.
Embodiment two
Collect 15 corydalis tuber medicinal material samples, all samples information is in table 3.
Table 3 corydalis tuber medicinal material sample message
The first step, prepares need testing solution by corydalis tuber medicinal material: get corydalis tuber medicinal material powder, accurately weighed, is placed in triangular flask; Add organic solvent, this organic solvent is chloroform and the methyl alcohol preparation of 5:1 by volume ratio, every 1.0g corydalis tuber medicinal material powder organic solvent total amount 50-60mL; Place and add ammoniacal liquor after 24 hours, wherein the volume ratio of ammoniacal liquor and methyl alcohol is 0.15:1, ultrasonic extraction 1 hour in 45 DEG C of water-baths, suction filtration after taking out, heating gained filtrate, after filtrate evaporate to dryness, remaining solid matter adds methyl alcohol dissolving (every 1.0g corydalis tuber medicinal material powder correspondence uses methyl alcohol 10mL), filters, obtain need testing solution through 0.45 μm of filter membrane.
Second step, sets up the finger-print of corydalis tuber sample: chromatographic condition used is: PhenomenexLunaC18 chromatographic column (250mm × 4.6mm, ID:5 μm); Column temperature 30 DEG C; Gradient elution, flowing used is met each other table 2; Flow velocity: 1.0mLmin
-1; Spectral scan scope: 240-320nm, it is 289nm that chromatogram gathers wavelength; Working time: 75min, per second sampling should be carried out; Sample size: 5 μ L.
3rd step, with DTW method to set up 15 samples chromatographic fingerprinting carry out shift of retention time correction: in DTW method, setting parameter is: PadLength:[00]; Span:20; MaxStep:2; Band:0.05.Figure 2 shows that the chromatographic fingerprinting of each corydalis tuber sample in the present embodiment.Be not calibrated finger-print more than " ← " in figure, 1-15 sample from top to bottom." ← " corrects gained finger-print through DTW, corresponds respectively to 1-15 sample from top to bottom.As seen from Figure 2, if not calibrated, in the finger-print of these 15 samples, the retention time of multiple characteristic peak has obvious difference, and after DTW corrects, shift of retention time obtains good correction.
4th step, corrects gained finger-print for row vector with 1-14 sample through DTW and forms a matrix.Principal component analysis (PCA) computing is carried out to this matrix: using first three major component of gained (PC1, PC2 and PC3) resolute as 3 input variables of artificial neural network.
5th step, sets up multilayer perceptron artificial nerve network model.The output variable of network is set as two classification, i.e. vinegar system and non-Rhizoma Corydalis (processed with vinegar) medicinal material: classification thresholds is set as 0.90.Network training is carried out by Feedback error method.Learning rate and study momentum are set as 0.6 and 0.3 respectively.Carry out training the artificial nerve network model obtained for classifying.With gained model, No. 15 samples are classified.This model judges that No. 15 samples are as vinegar sample preparation product, and this and actual conditions are coincide.
Embodiment three
Collect 22 corydalis tuber medicinal material samples, all samples information is in table 4.
Table 4 corydalis tuber medicinal material sample message
The first step, prepares need testing solution by corydalis tuber medicinal material: get corydalis tuber medicinal material powder, accurately weighed, is placed in triangular flask; Add organic solvent, this organic solvent is chloroform and the methyl alcohol preparation of 5:1 by volume ratio, every 1.0g corydalis tuber medicinal material powder organic solvent total amount 50-60mL; Place and add ammoniacal liquor after 20 hours, wherein the volume ratio of ammoniacal liquor and methyl alcohol is 0.15:1, ultrasonic extraction 1 hour in 42 DEG C of water-baths, suction filtration after taking out, heating gained filtrate, after filtrate evaporate to dryness, remaining solid matter adds methyl alcohol dissolving (every 1.0g corydalis tuber medicinal material powder correspondence uses methyl alcohol 10mL), filters, obtain need testing solution through 0.45 μm of filter membrane.
Second step, sets up the finger-print of corydalis tuber sample: chromatographic condition used is: PhenomenexLunaC18 chromatographic column (250mm × 4.6mm, ID=5 μm); Column temperature 30 DEG C; Gradient elution, flowing used is met each other table 2; Flow velocity: 1.0mLmin
-1; Spectral scan scope: 240-350nm, it is 289nm that chromatogram gathers wavelength; Working time: 75min, per second sampling should be carried out; Sample size: 5 μ L.
3rd step, with DTW method to set up 20 samples chromatographic fingerprinting carry out shift of retention time correction: in DTW method, setting parameter is: PadLength:[00]; Span:20; MaxStep:2; Band:0.05.Figure 3 shows that the chromatographic fingerprinting of each corydalis tuber sample in the present embodiment.Be not calibrated finger-print more than " ← " in figure, correspond respectively to 1-22 sample from top to bottom." ← " corrects gained finger-print through DTW, corresponds respectively to 1-22 sample from top to bottom.As seen from Figure 3, if not calibrated, in the finger-print of these 22 samples, the retention time of multiple characteristic peak has obvious difference, and after DTW corrects, shift of retention time obtains good correction.
4th step, corrects gained finger-print for row vector with 1-18 sample through DTW and forms a data matrix.Principal component analysis (PCA) computing is carried out to this matrix: using first three major component of principal component analysis (PCA) gained (PC1, PC2 and PC3) resolute as the input variable of artificial neural network.
5th step, sets up multilayer perceptron artificial nerve network model: the output variable of network is set as two classification, i.e. vinegar system and non-Rhizoma Corydalis (processed with vinegar) medicinal material.Classification thresholds is set as 0.95.Network training is carried out by Feedback error method.Learning rate and study momentum are set as 0.9 and 0.3 respectively.Train the artificial nerve network model obtained for classifying.With gained model, 19-22 sample is classified.Result is all correct to the classification of these 4 samples.
The present invention does not limit to above-mentioned cited embodiment, those skilled in the art can according to principle of work of the present invention and the embodiment provided above, various equivalent amendment, equivalent replacement, content increase and decrease can be made and reconfigure, thus forming how new embodiment.
Claims (2)
1. differentiate a method for the raw ripe medicinal material of corydalis tuber, it is characterized in that, step is:
S1, prepare need testing solution: get corydalis tuber medicinal material, grind into powder, add organic solvent and solvend in medicinal powder is all dissolved; Gained potpourri is placed after 16-24 hour and is added ammoniacal liquor, ultrasonic extraction in 40-45 DEG C of water-bath; Then carry out suction filtration, heating gained filtrate, after filtrate evaporate to dryness, remaining solid matter adds proper amount of methanol dissolving, filters, obtain need testing solution through 0.45 μm of filter membrane;
S2, stratographic analysis is carried out to need testing solution, using the chromatogram obtained as the finger-print of sample;
S3, by dynamic time warping method, shift of retention time correction is carried out to gained finger-print;
S4, with each corydalis tuber sample through DTW correct after gained chromatographic fingerprinting form a data matrix X as row vector; Principal component analysis (PCA) computing is carried out to after matrix X standardization; Reservation can make accumulative variance contribution ratio be greater than the major component of 85%, with the input variable obtaining resolute behaviour artificial neural networks model of these major components;
S5, set up multilayer perceptron artificial nerve network model; Input variable corresponds to the major component that principal component analysis (PCA) computing retains; Output variable corresponds to two classification of corydalis tuber sample, i.e. vinegar system and non-Rhizoma Corydalis (processed with vinegar) medicinal material; Classification thresholds setting is not less than 0.90; Artificial nerve network model is set up with Feedback error Algorithm for Training; Namely this model can be used for the taxonomic history of corydalis tuber medicinal material sample.
2. a kind of method differentiating the raw ripe medicinal material of corydalis tuber according to claim 1, is characterized in that:
In described step S1, the organic solvent dissolution of every 1.0g corydalis tuber medicinal material powder total amount 50-60mL, wherein, the ratio of organic solvent to be chloroform with methyl alcohol with volume ratio be 5:1 carries out the mixed solvent mixed;
In the described ammoniacal liquor added and described organic solvent, the volume ratio of methyl alcohol is 0.15:1;
Consumption for the methyl alcohol of remaining solid matter after dissolving filtrate evaporate to dryness is that every 1.0g corydalis tuber medicinal material powder correspondence uses methyl alcohol 10mL.
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| CN113138248A (en) * | 2021-04-25 | 2021-07-20 | 贵州中烟工业有限责任公司 | Characteristic spectrum extraction method and detection method for feed liquid preparation quality stability |
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| CN106841418A (en) * | 2016-12-26 | 2017-06-13 | 哈尔滨珍宝制药有限公司 | The detection method and method of quality control of the characteristic spectrum of corydalis tuber granule |
| CN113138248A (en) * | 2021-04-25 | 2021-07-20 | 贵州中烟工业有限责任公司 | Characteristic spectrum extraction method and detection method for feed liquid preparation quality stability |
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Application publication date: 20151223 |