CN105267355A - Extraction method of pharmaceutic preparation and building method of its prediction model - Google Patents
Extraction method of pharmaceutic preparation and building method of its prediction model Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicine extraction and particularly relates to an extraction method of a pharmaceutic preparation function in nourishing blood to relieve restlessness and calming heart to soothe the nerves, and a building method of its prediction model. The extraction method uses extraction rate, geniposide content and spinosin content as inspection indicators, geniposide and spinosin are extracted as maximally as possible by optimizing the extraction time, the types of extraction solvents and usage of extraction solvents, their contents are further measured, and thus giving overall control for the quality of the pharmaceutic preparation and assisting in improving the application safety and stability of the pharmaceutic preparation.
Description
Technical field
The invention belongs to field of traditional Chinese medicine extraction, be specifically related to a kind of to there is relieving restlessness of nourishing blood, the extracting method of pharmaceutical preparation of mind tranquilizing and the heart calming effect and the method for building up of forecast model thereof.
Background technology
Xinshen Ning tablets by the Eastern Han Dynasty cure holy Zhang Zhongjing show suanzaoren decoctionization in " Medical Treasures of the Golden Chamber " and cut out.Be made up of Six-element Chinese medicines such as Semen Ziziphi Spinosae (parched), Radix Polygalae, Poria, Fructus Gardeniae, Massa Medicata Fermentata, Radix Glycyrrhizaes, record in the national drug standards (WS-10410 (ZD-0410) 2002-2012Z), there is relieving restlessness of nourishing blood, effect of mind tranquilizing and the heart calming, for heart-liver blood deficiency, insomnia and dreamful sleep, irritated and shy, the diseases such as tired lack of appetite.Xinshen Ning tablets is evident in efficacy, and clinical practice is comparatively extensive.Wherein monarch drug in the Semen Ziziphi Spinosae (parched) side of being, plays vital effect, and the quality of the quality of medicinal material that feeds intake, the height of component content, directly affect the drug effect of Xinshen Ning tablets.
" screening of the extraction of Xinshen Ning capsule and moulding process and optimization " (" Shandong medicine ", 2008,48 (28), 103) a kind of extracting method of Xinshen Ning capsule is disclosed, be investigate target with jasminoidin, comprise following extraction step: get described pharmaceutical preparation in right amount, extract 3 times, add 10 times of water gagings 1st time, extract 2h; 2nd, respectively add 8 times of water gagings for 3 times, extract 1h at every turn.But, because Xinshen Ning capsule is made up of Six-element Chinese medicines such as Semen Ziziphi Spinosae (parched), Radix Polygalae, Poria, Fructus Gardeniae, Massa Medicata Fermentata, Radix Glycyrrhizaes, extract jasminoidin only by this extracting method, measure its content further, the object comprehensively controlling its quality can not be reached.
Therefore, set up a kind of extracting method of Xinshen Ning tablets, it is significant that the quality for Xinshen Ning tablets carries out control comprehensively.
Summary of the invention
Therefore, the present invention proposes a kind of extracting method of pharmaceutical preparation with relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, and then provides the method for building up of its forecast model.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions:
The invention provides a kind of extracting method of pharmaceutical preparation with relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, the crude drug of described pharmaceutical preparation consists of: Semen Ziziphi Spinosae (parched), Radix Polygalae, Poria, Fructus Gardeniae, Massa Medicata Fermentata, Radix Glycyrrhizae;
This extracting method comprises the extraction step as follows to jasminoidin and spinosin:
Get described pharmaceutical preparation appropriate, Extraction solvent is 60%-80% ethanol, and extraction time is 1-5 time, extracts 0.5h-3h at every turn, and the quantity of solvent at every turn added is 6-9 times of the weight times amount of described pharmaceutical preparation.
Preferably, the present invention is above-mentioned has the extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, and this extracting method comprises the extraction step as follows to jasminoidin and spinosin:
Get described pharmaceutical preparation appropriate, Extraction solvent is 65%-75% ethanol, and extraction time is 2-4 time, extracts 1h-2h at every turn, and the quantity of solvent at every turn added is 7-8 times of the weight times amount of described pharmaceutical preparation.
Further preferably, the present invention is above-mentioned has the extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, and this extracting method comprises the extraction step as follows to jasminoidin and spinosin:
Get described pharmaceutical preparation appropriate, Extraction solvent is 70% ethanol, and extraction time is 3 times, extracts 1.5h at every turn, and the quantity of solvent at every turn added is 7.44 times of the weight times amount of described pharmaceutical preparation.
Further preferably, the present invention is above-mentioned has the extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect,
The crude drug of described pharmaceutical preparation consists of: Semen Ziziphi Spinosae (parched) 200-300 weight portion, Radix Polygalae 120-210 weight portion, Poria 120-210 weight portion, Fructus Gardeniae 30-140 weight portion, Massa Medicata Fermentata 30-140 weight portion, Radix Glycyrrhizae 30-140 weight portion.
Further preferably, the present invention is above-mentioned has the extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect,
The crude drug of described pharmaceutical preparation consists of: Semen Ziziphi Spinosae (parched) 220-280 weight portion, Radix Polygalae 140-190 weight portion, Poria 140-190 weight portion, Fructus Gardeniae 50-110 weight portion, Massa Medicata Fermentata 50-110 weight portion, Radix Glycyrrhizae 50-110 weight portion.
Further preferably, the present invention is above-mentioned has the extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect,
The crude drug of described pharmaceutical preparation consists of: Semen Ziziphi Spinosae (parched) 250 weight portion, Radix Polygalae 167 weight portion, Poria 167 weight portion, Fructus Gardeniae 83 weight portion, Massa Medicata Fermentata 83 weight portion, Radix Glycyrrhizae 83 weight portion.
The present invention also provides the above-mentioned method for building up of forecast model of extracting method of pharmaceutical preparation with relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, comprises the following steps:
(1) Plackett-Burman design screening major influence factors
Carry out Plackett-Burman design, selected soak time, in the extraction time of total extraction time, single-trial extraction time, boiling degree, Extraction solvent for influence factor, with dry cream rate, jasminoidin and spinosin content for evaluation index, significance analysis is carried out to described influence factor;
Test according to Plackett-BurmanDesign experimental design, often parallel 3 times of group experiment, results averaged, use Designexpert software to carry out significance analysis to described influence factor;
(2) Box-Behnken effect surface method Optimized Extraction Process
Setting empirical factor X
1for in the extraction time of total extraction time, empirical factor X
2for Extraction solvent-ol water ratio, empirical factor X
3for solubilization dosage, setting evaluation index Y
1for dry cream rate, evaluation index Y
2for Determination of Gardenoside, evaluation index Y
3for spinosin content, Box-BehnkenDesign is used to test preferred described empirical factor;
The index that the index the smaller the better to value and value are the bigger the better, adopts Hassan method respectively, asks " normalizing value " OD value, d
min=(y
max-y
i)/(y
max-y
min), d
max=(y
i-y
min)/(y
max-y
min), OD=(d
1d
2d
3... .d
n) 1/n, n be index number; By the data obtained Design-Expert8.0.0 software experiment Analysis, take OD as response value, respectively multiple linear regression and binomial fitting are carried out to each empirical factor, through the inspection of F value, general comment OD value the results of analysis of variance;
(3) process certification experiment
With the Extraction technique after optimization, with described have relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect pharmaceutical preparation for experimental subject, carry out 3 parallel laboratory tests, carry out dry cream rate mensuration, the assay of jasminoidin and the assay of spinosin, and calculate general comment OD value, with the accuracy of confirmatory experiment model.
Preferably, the present invention is above-mentioned has the method for building up of forecast model of extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, and the assay of described spinosin comprises the following steps:
(1) preparation of need testing solution: get pharmaceutical preparation to be measured, accurately weighed, porphyrize, get 0.5 weight portion, accurately weighed, put in tool plug conical flask, precision adds 70% methanol 25 parts by volume, weighed weight, ultrasonic 10 minutes, let cool, weighed weight, supply the weight of less loss with 70% methanol, shake up, filter, get subsequent filtrate, to obtain final product;
(2) preparation of reference substance solution: it is appropriate that precision takes spinosin reference substance, adds methanol appropriate, makes the reference substance solution containing spinosin 0.010mg/mL;
(3) chromatographic condition: with 4.6mm × 250mm, the PerkinElmerC18 of 5 μm is chromatographic column, take acetonitrile as mobile phase A, with water-0.1% glacial acetic acid for Mobile phase B, carries out gradient elution according to following program: 0min, A:B are 10%:90%; 0min-15min, A:B are 10%:90% → 17%:83%; 15min, A:B are 17%:83%; 15min-25min, A:B are 17%:83% → 23%:77%; 25min, A:B are 23%:77%; 25min-30min, A:B are 23%:77% → 35%:65%; 30min, A:B are 35%:65%; 30min-33min, A:B are 35%:65% → 10%:90%; 33min-37min, A:B are 10%:90%; Determined wavelength 335nm; Column temperature 25 DEG C; Flow velocity 1.0mL/min;
(4) precision draws need testing solution and reference substance solution 10 μ L respectively, injects high performance liquid chromatograph, measures;
The pass of described weight portion and parts by volume is g/mL.
Preferably, the present invention is above-mentioned has the method for building up of forecast model of extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, and the assay of described jasminoidin comprises the following steps:
(1) preparation of need testing solution: get pharmaceutical preparation to be measured, accurately weighed, porphyrize, get 0.5 weight portion, accurately weighed, put in tool plug conical flask, precision adds methanol 50 parts by volume, weighed weight, ultrasonic 30 minutes, let cool, weighed weight, supply the weight of less loss with methanol, shake up, filter, get subsequent filtrate, to obtain final product;
(2) preparation of reference substance solution: it is appropriate that precision takes jasminoidin reference substance, adds methanol appropriate, makes containing jasminoidin 0.08mg/mL reference substance solution;
(3) chromatographic condition: be filler with octadecylsilane chemically bonded silica take volume ratio as the acetonitrile-water of 12:88 is mobile phase, determined wavelength 238nm, flow velocity 1.0mL/min;
(4) precision draws need testing solution and reference substance solution 10 μ L respectively, injects high performance liquid chromatograph, measures;
The pass of described weight portion and parts by volume is g/mL.
Technical scheme tool of the present invention has the following advantages:
The present invention has the extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, with dry cream rate, the content of jasminoidin and the content of spinosin for inspection target, by optimizing extraction time, the kind of Extraction solvent, the consumption of Extraction solvent, jasminoidin and spinosin are as far as possible farthest extracted, a pacing of going forward side by side its content fixed, thus the quality of this pharmaceutical preparation can be controlled comprehensively, contribute to the safety and stability improving this drug use.
Accompanying drawing explanation
In order to be illustrated more clearly in the specific embodiment of the invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in detailed description of the invention or description of the prior art below.Apparently, the accompanying drawing in the following describes is some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, can also obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 is extraction time X in experimental example 2 of the present invention
1(alcohol water compares X for (in total extraction time) and Extraction solvent
2) contour map that OD value (Y) is affected;
Fig. 2 is extraction time X in experimental example 2 of the present invention
1(alcohol water compares X for (in total extraction time) and Extraction solvent
2) diagram of block that OD value (Y) is affected;
Fig. 3 is extraction time X in experimental example 2 of the present invention
1(in total extraction time) and solubilization dosage (X
3) contour map that OD value (Y) is affected;
Fig. 4 is extraction time X in experimental example 2 of the present invention
1(in total extraction time) and solubilization dosage (X
3) diagram of block that OD value (Y) is affected;
Fig. 5 be in experimental example 2 of the present invention Extraction solvent (alcohol water compares X
2) and solubilization dosage X
3on the contour map that OD value (Y) affects;
Fig. 6 be in experimental example 2 of the present invention Extraction solvent (alcohol water compares X
2) and solubilization dosage X
3on the diagram of block that OD value (Y) affects.
Detailed description of the invention
Be clearly and completely described technical scheme of the present invention below in conjunction with accompanying drawing, obviously, described embodiment is the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
embodiment 1the preparation of Xinshen Ning tablets
[prescription] Semen Ziziphi Spinosae (parched) 250g, Radix Polygalae 167g, Poria 167g, Fructus Gardeniae 83g, Massa Medicata Fermentata 83g, Radix Glycyrrhizae 83g.
[preparation method] described tablet is prepared by following methods:
(1) get Radix Glycyrrhizae and the Massa Medicata Fermentata of selected weight portion, pulverize, sieve, mixing, obtains fine powder;
(2) get the Semen Ziziphi Spinosae (parched) of selected weight portion, Fructus Gardeniae, Radix Polygalae and Poria, decoct with water twice, first time decocts 3 hours, and second time decocts 2 hours, collecting decoction, and filter, it is the clear paste of 1.05-1.20 that filtrate is concentrated into 50 DEG C of relative densities;
(3) be added in described clear paste by described fine powder, mixing, adds right amount of auxiliary materials, granule processed, adds Magnesium Stearate proper quantity, mixing, and dry, tabletted, coating, obtains tablet.
embodiment 2
The present embodiment has the extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, comprises the extraction step as follows to jasminoidin and spinosin:
Get described pharmaceutical preparation appropriate, Extraction solvent is 70% ethanol, and extraction time is 3 times, extracts 1.5h at every turn, and the quantity of solvent at every turn added is 7.44 times of the weight times amount of described pharmaceutical preparation.
embodiment 3
The present embodiment has the extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, comprises the extraction step as follows to jasminoidin and spinosin:
Get described pharmaceutical preparation appropriate, Extraction solvent is 65% ethanol, and extraction time is 4 times, extracts 1h at every turn, and the quantity of solvent at every turn added is 8 times of the weight times amount of described pharmaceutical preparation.
embodiment 4
The present embodiment has the extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, comprises the extraction step as follows to jasminoidin and spinosin:
Get described pharmaceutical preparation appropriate, Extraction solvent is 75% ethanol, and extraction time is 2 times, extracts 2h at every turn, and the quantity of solvent at every turn added is 7 times of the weight times amount of described pharmaceutical preparation.
embodiment 5
The present embodiment has the method for building up of forecast model of extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, comprises the following steps:
(1) Plackett-Burman design screening major influence factors
Designexpert software is used to carry out Plackett-Burman design, selected soak time, in the extraction time of total extraction time, single-trial extraction time, boiling degree, Extraction solvent for influence factor, with dry cream rate, jasminoidin and spinosin content for evaluation index, significance analysis is carried out to described influence factor;
Test according to Plackett-BurmanDesign experimental design, often parallel 3 times of group experiment, results averaged, use Designexpert software to carry out significance analysis to described influence factor;
(2) Box-Behnken effect surface method Optimized Extraction Process
Setting empirical factor X
1for in the extraction time of total extraction time, empirical factor X
2for Extraction solvent-ol water ratio, empirical factor X
3for solubilization dosage, setting evaluation index Y
1for dry cream rate, evaluation index Y
2for Determination of Gardenoside, evaluation index Y
3for spinosin content, Box-BehnkenDesign is used to test preferred described empirical factor;
The index that the index the smaller the better to value and value are the bigger the better, adopts Hassan method respectively, asks " normalizing value " OD value, d
min=(y
max-y
i)/(y
max-y
min), d
max=(y
i-y
min)/(y
max-y
min), OD=(d
1d
2d
3... .d
n) 1/n, n be index number; By the data obtained Design-Expert8.0.0 software experiment Analysis, take OD as response value, respectively multiple linear regression and binomial fitting are carried out to each empirical factor, through the inspection of F value, general comment OD value the results of analysis of variance;
(3) process certification experiment
With the Extraction technique after optimization, with described have relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect pharmaceutical preparation for experimental subject, carry out 3 parallel laboratory tests, carry out dry cream rate mensuration, the assay of jasminoidin and the assay of spinosin, and calculate general comment OD value, with the accuracy of confirmatory experiment model.
embodiment 6
The present embodiment has the method for building up of forecast model of extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, and the assay of described spinosin comprises the following steps:
(1) preparation of need testing solution: get pharmaceutical preparation to be measured, accurately weighed, porphyrize, get 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methanol 25mL, weighed weight, ultrasonic 10 minutes, let cool, weighed weight, supply the weight of less loss with 70% methanol, shake up, filter, get subsequent filtrate, to obtain final product;
(2) preparation of reference substance solution: it is appropriate that precision takes spinosin reference substance, adds methanol appropriate, makes the reference substance solution containing spinosin 0.010mg/mL;
(3) chromatographic condition: with 4.6mm × 250mm, the PerkinElmerC18 of 5 μm is chromatographic column, take acetonitrile as mobile phase A, with water-0.1% glacial acetic acid for Mobile phase B, carries out gradient elution according to following program: 0min, A:B are 10%:90%; 0min-15min, A:B are 10%:90% → 17%:83%; 15min, A:B are 17%:83%; 15min-25min, A:B are 17%:83% → 23%:77%; 25min, A:B are 23%:77%; 25min-30min, A:B are 23%:77% → 35%:65%; 30min, A:B are 35%:65%; 30min-33min, A:B are 35%:65% → 10%:90%; 33min-37min, A:B are 10%:90%; Determined wavelength 335nm; Column temperature 25 DEG C; Flow velocity 1.0mL/min;
(4) precision draws need testing solution and reference substance solution 10 μ L respectively, injects high performance liquid chromatograph, measures.
embodiment 7
The present embodiment has the method for building up of forecast model of extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, and the assay of described jasminoidin comprises the following steps:
(1) preparation of need testing solution: get pharmaceutical preparation to be measured, accurately weighed, porphyrize, get 0.5g, accurately weighed, put in tool plug conical flask, precision adds methanol 50mL, weighed weight, ultrasonic 30 minutes, let cool, weighed weight, supply the weight of less loss with methanol, shake up, filter, get subsequent filtrate, to obtain final product;
(2) preparation of reference substance solution: it is appropriate that precision takes jasminoidin reference substance, adds methanol appropriate, makes containing jasminoidin 0.08mg/mL reference substance solution;
(3) chromatographic condition: be filler with octadecylsilane chemically bonded silica take volume ratio as the acetonitrile-water of 12:88 is mobile phase, determined wavelength 238nm, flow velocity 1.0mL/min;
(4) precision draws need testing solution and reference substance solution 10 μ L respectively, injects high performance liquid chromatograph, measures.
comparative example 1
This comparative example has the extracting method of pharmaceutical preparation of relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, comprises following extraction step:
Get described pharmaceutical preparation appropriate, extract 3 times, add 10 times of water gagings the 1st time, extract 2h; 2nd, respectively add 8 times of water gagings for 3 times, extract 1h at every turn.
experimental example 1
1, instrument and material
Agilent1260 high performance liquid chromatograph, evaporative light detector detector; VWD detector; KQ-500DE medical digital controlled ultrasonic cleaner; AE240 100,000/electronic analytical balance, Switzerland's prunus mume (sieb.) sieb.et zucc. Teller.
Spinosin reference substance (lot number 111869-201203) is bought by National Institute for Food and Drugs Control; Acetonitrile (U.S. Fisher), methanol (U.S. Fisher) are chromatographically pure; Methanol (good Xinghua, Tianjin work glass apparatus Trade Co., Ltd.), glacial acetic acid (Tianjin recovery development in science and technology company limited) is analytical pure, water (three grades of reverse osmosis legal systems obtain purified water).
Xinshen Ning tablets (lot number 492043), Xinshen Ning tablets negative controls, the raw material preparing Xinshen Ning tablets negative controls used provide by Jingfukang Pharmaceutical Group Co., Ltd., are accredited as genuine piece by Jingfukang Pharmaceutical Group Co., Ltd. engineer TCD identification teacher.Xinshen Ning tablets is prepared according to the method for embodiment 1.
1.1 chromatographic condition
Adopt PerkinElmerC18 chromatographic column (4.6mm × 250mm, 5 μm); A (acetonitrile)-B (0.1% glacial acetic acid) is mobile phase, online gradient elution, and 0-15min-25min-30min-33min-37min, A phase is 10%-17%-23%-35%-10%-10%; Determined wavelength: 335nm; Column temperature: 25 DEG C; Flow velocity: 1.0mL/min; Sampling volume: 10 μ L.
The preparation of 1.2 reference substance solution
It is appropriate that precision takes spinosin reference substance, adds methanol appropriate, make the reference substance solution containing spinosin 0.01mg/mL.
The preparation of 1.3 need testing solutions
Get Xinshen Ning tablets several pieces, removing coating, accurately weighed, porphyrize, gets about 0.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methanol 25mL, weighed weight, ultrasonic 10 minutes, lets cool, weighed weight, supplies the weight of less loss, shakes up with 70% methanol, filter, get subsequent filtrate and get final product.
The preparation of 1.4 negative controls solution
By the flavour of a drug proportions in prescription not containing the negative sample of Semen Ziziphi Spinosae medical material, according to legal system below " 1.3 " item for negative controls.
1.5 specificity experiments
By above-mentioned experiment condition, respectively negative controls solution, need testing solution and reference substance solution are injected hplc determination.
1.6 linear relationships are investigated
It is appropriate that precision takes reference substance, adds methanol and make reference substance liquid containing spinosin 0.053424mg/mL, adopt coubling dilution to make series mass strength solution, the concentration of spinosin is made to be respectively 0.026712mg/mL, 0.013356mg/mL, 0.006678mg/mL, 0.003339mg/mL.HPLC analysis is carried out according to chromatographic condition under " 1.1 " item.Regression equation Y=20043X+4.4708 is obtained, r=0.9999 (n=5) with the concentration X drawing standard curve of the peak area Y of spinosin to spinosin.
Result shows, the spinosin in Xinshen Ning tablets is good in 3.339-53.424 μ g/mL scope internal linear relation.
1.7 Precision Experiment
Get the reference substance solution that content is 0.013356mg/mL, according to the chromatographic condition under " 1.1 " item, repeat sample introduction 6 times, record each chromatographic peak area, calculate RSD value.
Result shows, the spinosin RSD value in Xinshen Ning tablets is 0.75%.That is, this detection method precision is good.
1.8 repeated experiment
Precision takes same lot number Xinshen Ning tablets (lot number 492043), by legal system available test sample solution 6 parts below " 1.3 " item, measures by chromatographic condition under " 1.1 " item, record chromatographic peak area.
Result shows, the average content of the spinosin in Xinshen Ning tablets is 0.1623mg/ sheet, and RSD is 1.46% (n=6).That is, this detection method favorable reproducibility.
1.9 stability experiment
Get according to the standby need testing solution of legal system below " 1.3 " item, difference 0,2,4,6,8,10,12h sample introduction 10 μ L after the production, record chromatographic peak area.
Result shows, the spinosin chromatographic peak area RSD value in Xinshen Ning tablets is 1.46%.That is, need testing solution is good at 12h internal stability.
1.10 average recovery is tested
Precision takes the Xinshen Ning tablets (lot number 492043) 9 parts measuring spinosin (0.1530mg/ sheet) content, accurately weighed 0.25g, add a certain amount of reference substance, according to legal system available test sample solution below " 1.3 " item, measure by chromatographic condition under " 1.1 " item, calculate average recovery with absolute mass mark in need testing solution.Known, the spinosin average recovery rate in Xinshen Ning tablets is 100.91%, RSD value is 1.36%.
experimental example 2
1, Plackett-Burman design screening major influence factors
1.1 factors and level
Use Designexpert8.0.6 software to carry out Plackett-Burman design (PBD), investigate the significance that each factor affects dry cream rate, jasminoidin and spinosin content.In reality produces greatly, Semen Ziziphi Spinosae, Fructus Gardeniae etc., for pulverizing feeds intake, temporarily do not consider the impact of medical material granularity on extraction process at this.
According to the large knowhow of reality, experimental design 6 empirical factors (A: soak time, B: extraction time (in total extraction time), C: single-trial extraction time, D: boiling degree, E: Extraction solvent (alcohol water ratio), F: solubilization dosage) and 6 blank factor (G, H, I, J, K, L), totally 12 groups of experiments.Each factor high (code: 1), low (code :-1) 2 levels are determined according to preliminary result.Designed experiment is as shown in table 1.
Table 1Plackett-Burman designs influence factor and level
1.2Plackett-Burman designs (PBD) experimental result
Test according to PBD experimental design, often parallel 3 times of group experiment, results averaged.PBD experimental design table and the results are shown in Table 2.
Table 2PBD experimental design table and result
Use Designexpert8.0.6 software to carry out significance analysis to each factor, specific experiment the results are shown in Table 3.Wherein, that response value P < 0.05 is significance influence factor.
Table 3PBD interpretation
note: P < 0.05 is pole significant factors
From analysis result, the impact order of each influence factor on dry cream rate is: extraction time (in total extraction time) > Extraction solvent (alcohol water ratio) > boiling degree > solubilization dosage > single-trial extraction time > soak time; Each influence factor on the impact of jasminoidin order is: extraction time (in total extraction time) > Extraction solvent (alcohol water than) > solubilization dosage > soak time > single-trial extraction time > seethes with excitement degree; The impact order of each influence factor on spinosin is: extraction time (in total extraction time) > Extraction solvent > solubilization dosage > seethes with excitement the degree > soak time > single-trial extraction time.
Get each influence factor to occur simultaneously comprehensive single index analysis, extraction time (in total extraction time), Extraction solvent (alcohol water ratio) and solubilization dosage are for significant factors, soak time, single-trial extraction time and boiling degree are non-limiting factor, carry out Box-Behnken design (BBD) on this basis.
2, Box-Behnken effect surface method Optimized Extraction Process
2.1BBD empirical factor, level and result
Namely because extraction time is discontinuous variable, so this variable factors of extraction time is in total extraction time, be converted into continuous variable, total extraction time is 1.5h to extract 1 time, extracts 2 times and total extraction time is 3h, extracting be for 3 times total extraction time is 4.5h.Be fixed as 0h in soak time, the single-trial extraction time is fixed as 1.5h, under boiling degree gets violent fluidized state, and setting X
1(extraction time (in total extraction time)), X
2(Extraction solvent (alcohol water ratio)), X
3(solubilization dosage) is variable factors, Y
1(dry cream rate), Y
2(Determination of Gardenoside), Y
3(spinosin content) is evaluation index, uses BBD to test preferred optimum extraction process parameter.
BBD empirical factor and level are in table 4, and gained specific experiment the results are shown in Table 5.
Table 4BBD empirical factor and level
Table 5BBD experiment and result
2.2 models fittings and date processing
The date processing of multi objective generally adopts overall desirability method.The index that the index the smaller the better to value and value are the bigger the better, adopts Hassan method respectively, asks " normalizing value " OD value.D
min=(y
max-y
i)/(y
max-y
min), d
max=(yi-ymin)/(y
max-y
min), OD=(d
1d
2d
3... .d
n) 1/n, n be index number.By the data obtained Design-Expert8.0.0 software experiment Analysis, be that response value carries out multiple linear regression and binomial fitting to each factor respectively with OD, check through F value.General comment OD value the results of analysis of variance is in table 6.
Model significantly (P < 0.01), loses and intends item not significantly (P > 0.05), R
2=0.94, result shows that this Regression Model Simulator is all right, and the representativeness of regression equation is better, can Accurate Prediction practical situation.Regression equation is as follows: Y=0.70+0.27X
1+ 0.071X
2+ 0.19X
3+ 0.029X
1x
2-0.059X
1x
3-0.18X
2x
3-0.13X
1 2-0.057X
2 2-0.100X
3 2
The variance analysis of table 6BBD experimental result
2.3 effect surface analyses and the prediction of BBD experimental model
According to Regression Analysis Result, application Design-Expert8.0.6 software, fixes certain variable in 3 variablees, and draw three-dimension curved surface and contour map that two other variable affects general comment OD value, experimental result is shown in Fig. 1,2,3,4,5 and 6.
As can be seen from Fig. 1 and Fig. 2, total extraction time and Extraction solvent comparatively not remarkable to the reciprocal action of response value; As can be seen from Fig. 3 and Fig. 4, total extraction time and solubilization dosage have certain reciprocal action to response value; As can be seen from Fig. 5 and Fig. 6, Extraction solvent and solubilization dosage are to the significant interaction of response value.
Through the screening of DesignExpert8.0.6 software analysis, obtaining Xinshen Ning tablets Extraction technique optimal value is: extraction time is 3 times (total extraction time is 4.5h), and Extraction solvent is 70% ethanol, and solubilization dosage is 7.44 times, predictive value Y
1(dry cream rate %)=16.78, Y
2(jasminoidin mg/ sheet)=3.6234, Y
3(OD value)=0.8848, (spinosin mg/ sheet)=0.2392, Y.
2.4 process certification experiments
By the accuracy of confirmatory experiment model, consider practical situation, be 3 times (total extraction time is for 4.5h) with extraction time, Extraction solvent is 70% ethanol, solubilization dosage is 7.5 times, with the Extraction technique after optimizing, carries out 3 parallel laboratory tests in the ratio of Xinshen Ning tablets prescription 500, measure the content of dry cream rate, jasminoidin, spinosin, and calculate general comment OD value.Experimental results is in table 7 and table 8.
Table 7 process certification experimental result
Table 8 process certification experimental result
From table 7 and table 8, measured value and the predictive value deviation of dry cream rate, Determination of Gardenoside and spinosin content are all less than 2%, and actual measurement OD value is less than 3% with the average deviation of prediction OD value.This shows, the result with predict the outcome comparatively close.
2.5 comparative study
By this selection process pilot product and market circulation handicraft product (Jingfukang Pharmaceutical Group Co., Ltd.: lot number 392170,492009,492016,492043; Certain Pharmaceutical Co., Ltd: lot number 140519,140802,140804) contrast.Specific experiment the results are shown in Table 9 and table 10.
Rear technological experiment result optimized by table 9
Remarks: 1,2,3 is the pilot product optimizing rear technique, and 4,5,6 (530011 batches, 530012 batches and 530013 batches) are mass production products, and the Chinese crude drug used with pilot product is same lot number.
The experimental result of table 10 market circulation handicraft product
From table 9 and table 10, the content of geniposide in Xinshen Ning tablets and spinosin all increases (in Chinese medicine, target component content generally only has lower limit, does not have the upper limit, except some toxic components have regulation).
In sum, the forecast model that this experimental example is set up has good predictability, and the optimum extraction process parameter credibility that BBD-effect surface method analyzes gained is higher.
experimental example 3
Extract jasminoidin and the spinosin of Xinshen Ning tablets prepared by embodiment 1 respectively according to the method for embodiment 2 and comparative example 1, and carry out further carrying out assay to it, specific experiment result is as shown in table 11.
The content results of table 11 jasminoidin and spinosin
As shown in Table 11, compared with adopting the method for comparative example 1, adopt the method for embodiment 2 to extract jasminoidin and the spinosin of Xinshen Ning tablets, its content is higher.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (9)
1. have the extracting method of pharmaceutical preparation for relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect, it is characterized in that, the crude drug of described pharmaceutical preparation consists of: Semen Ziziphi Spinosae (parched), Radix Polygalae, Poria, Fructus Gardeniae, Massa Medicata Fermentata, Radix Glycyrrhizae;
This extracting method comprises the extraction step as follows to jasminoidin and spinosin:
Get described pharmaceutical preparation appropriate, Extraction solvent is 60%-80% ethanol, and extraction time is 1-5 time, extracts 0.5h-3h at every turn, and the quantity of solvent at every turn added is 6-9 times of the weight times amount of described pharmaceutical preparation.
2. the extracting method of pharmaceutical preparation with relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect according to claim 1, is characterized in that, this extracting method comprises the extraction step as follows to jasminoidin and spinosin:
Get described pharmaceutical preparation appropriate, Extraction solvent is 65%-75% ethanol, and extraction time is 2-4 time, extracts 1h-2h at every turn, and the quantity of solvent at every turn added is 7-8 times of the weight times amount of described pharmaceutical preparation.
3. the extracting method of pharmaceutical preparation with relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect according to claim 2, is characterized in that, this extracting method comprises the extraction step as follows to jasminoidin and spinosin:
Get described pharmaceutical preparation appropriate, Extraction solvent is 70% ethanol, and extraction time is 3 times, extracts 1.5h at every turn, and the quantity of solvent at every turn added is 7.44 times of the weight times amount of described pharmaceutical preparation.
4. having according to any one of claim 1-3 is nourished blood the extracting method of pharmaceutical preparation of relieving restlessness, mind tranquilizing and the heart calming effect, it is characterized in that,
The crude drug of described pharmaceutical preparation consists of: Semen Ziziphi Spinosae (parched) 200-300 weight portion, Radix Polygalae 120-210 weight portion, Poria 120-210 weight portion, Fructus Gardeniae 30-140 weight portion, Massa Medicata Fermentata 30-140 weight portion, Radix Glycyrrhizae 30-140 weight portion.
5. the extracting method of pharmaceutical preparation with relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect according to claim 4, is characterized in that,
The crude drug of described pharmaceutical preparation consists of: Semen Ziziphi Spinosae (parched) 220-280 weight portion, Radix Polygalae 140-190 weight portion, Poria 140-190 weight portion, Fructus Gardeniae 50-110 weight portion, Massa Medicata Fermentata 50-110 weight portion, Radix Glycyrrhizae 50-110 weight portion.
6. the extracting method of pharmaceutical preparation with relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect according to claim 5, is characterized in that,
The crude drug of described pharmaceutical preparation consists of: Semen Ziziphi Spinosae (parched) 250 weight portion, Radix Polygalae 167 weight portion, Poria 167 weight portion, Fructus Gardeniae 83 weight portion, Massa Medicata Fermentata 83 weight portion, Radix Glycyrrhizae 83 weight portion.
7. having described in any one of claim 1-6 is nourished blood the method for building up of forecast model of extracting method of pharmaceutical preparation of relieving restlessness, mind tranquilizing and the heart calming effect, it is characterized in that, comprises the following steps:
(1) Plackett-Burman design screening major influence factors
Carry out Plackett-Burman design, selected soak time, in the extraction time of total extraction time, single-trial extraction time, boiling degree, Extraction solvent for influence factor, with dry cream rate, the content of jasminoidin and the content of spinosin for evaluation index, significance analysis is carried out to described influence factor;
Test according to Plackett-BurmanDesign experimental design, often parallel 3 times of group experiment, results averaged, use Designexpert software to carry out significance analysis to described influence factor;
(2) Box-Behnken effect surface method Optimized Extraction Process parameter
Setting empirical factor X
1for in the extraction time of total extraction time, empirical factor X
2for Extraction solvent-ol water ratio, empirical factor X
3for solubilization dosage, setting evaluation index Y
1for dry cream rate, evaluation index Y
2for Determination of Gardenoside, evaluation index Y
3for spinosin content, Box-BehnkenDesign is used to test preferred described empirical factor;
The index that the index the smaller the better to value and value are the bigger the better, adopts Hassan method respectively, asks " normalizing value " OD value, d
min=(y
max-y
i)/(y
max-y
min), d
max=(y
i-y
min)/(y
max-y
min), OD=(d
1d
2d
3... .d
n) 1/n, n be index number; By the data obtained Design-Expert8.0.0 software experiment Analysis, take OD as response value, respectively multiple linear regression and binomial fitting are carried out to each empirical factor, through the inspection of F value, general comment OD value the results of analysis of variance;
(3) process certification experiment
With the Extraction technique after optimization, with described have relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect pharmaceutical preparation for experimental subject, carry out 3 parallel laboratory tests, carry out dry cream rate mensuration, the assay of jasminoidin and the assay of spinosin, and calculate general comment OD value, with the accuracy of confirmatory experiment model.
8. the method for building up of forecast model of extracting method of pharmaceutical preparation with relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect according to claim 7, is characterized in that,
The assay of described spinosin comprises the following steps:
(1) preparation of need testing solution: get pharmaceutical preparation to be measured, accurately weighed, porphyrize, get 0.5 weight portion, accurately weighed, put in tool plug conical flask, precision adds 70% methanol 25 parts by volume, weighed weight, ultrasonic 10 minutes, let cool, weighed weight, supply the weight of less loss with 70% methanol, shake up, filter, get subsequent filtrate, to obtain final product;
(2) preparation of reference substance solution: it is appropriate that precision takes spinosin reference substance, adds methanol appropriate, makes the reference substance solution containing spinosin 0.010mg/mL;
(3) chromatographic condition: with 4.6mm × 250mm, the PerkinElmerC18 of 5 μm is chromatographic column, take acetonitrile as mobile phase A, with water-0.1% glacial acetic acid for Mobile phase B, carries out gradient elution according to following program: 0min, A:B are 10%:90%; 0min-15min, A:B are 10%:90% → 17%:83%; 15min, A:B are 17%:83%; 15min-25min, A:B are 17%:83% → 23%:77%; 25min, A:B are 23%:77%; 25min-30min, A:B are 23%:77% → 35%:65%; 30min, A:B are 35%:65%; 30min-33min, A:B are 35%:65% → 10%:90%; 33min-37min, A:B are 10%:90%; Determined wavelength 335nm; Column temperature 25 DEG C; Flow velocity 1.0mL/min;
(4) precision draws need testing solution and reference substance solution 10 μ L respectively, injects high performance liquid chromatograph, measures;
The pass of described weight portion and parts by volume is g/mL.
9. the method for building up of forecast model of extracting method of pharmaceutical preparation with relieving restlessness of nourishing blood, mind tranquilizing and the heart calming effect according to claim 7, is characterized in that,
The assay of described jasminoidin comprises the following steps:
(1) preparation of need testing solution: get pharmaceutical preparation to be measured, accurately weighed, porphyrize, get 0.5 weight portion, accurately weighed, put in tool plug conical flask, precision adds methanol 50 parts by volume, weighed weight, ultrasonic 30 minutes, let cool, weighed weight, supply the weight of less loss with methanol, shake up, filter, get subsequent filtrate, to obtain final product;
(2) preparation of reference substance solution: it is appropriate that precision takes jasminoidin reference substance, adds methanol appropriate, makes containing jasminoidin 0.08mg/mL reference substance solution;
(3) chromatographic condition: be filler with octadecylsilane chemically bonded silica take volume ratio as the acetonitrile-water of 12:88 is mobile phase, determined wavelength 238nm, flow velocity 1.0mL/min;
(4) precision draws need testing solution and reference substance solution 10 μ L respectively, injects high performance liquid chromatograph, measures;
The pass of described weight portion and parts by volume is g/mL.
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| CN110580550A (en) * | 2019-09-11 | 2019-12-17 | 北京华润高科天然药物有限公司 | Method for establishing prediction model of production process of scutellaria baicalensis extract and application of prediction model |
| CN111429978A (en) * | 2020-03-20 | 2020-07-17 | 广东药科大学 | Method for rapidly discovering effective components of traditional Chinese medicine compound and application |
| CN112782330A (en) * | 2020-12-28 | 2021-05-11 | 浙江绿晶生物科技股份有限公司 | Method for extracting hovenia dulcis thunb flavor substance |
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