CN105267355B - A kind of extracting method of pharmaceutical preparation and its method for building up of prediction model - Google Patents
A kind of extracting method of pharmaceutical preparation and its method for building up of prediction model Download PDFInfo
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Abstract
The invention belongs to field of traditional Chinese medicine extraction, and in particular to a kind of extracting method of the pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect and its method for building up of prediction model.The extracting method, using the content of dry cream rate, the content of Gardenoside and spinosin as inspection target, by optimizing extraction time, the type of Extraction solvent, the dosage of Extraction solvent, so that Gardenoside and spinosin are farthest extracted as far as possible, its content is determined in a pacing of going forward side by side, so as to control the quality of the pharmaceutical preparation comprehensively, the safety and stability that the drug uses is helped to improve.
Description
Technical field
The invention belongs to field of traditional Chinese medicine extraction, and in particular to a kind of drug system with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect
The extracting method of agent and its method for building up of prediction model.
Background technique
From suanzaoren decoctionization in Xinshen Ning tablets " Synopsis Golden Chamber " written by Eastern Han Dynasty's holy doctor Zhang Zhongjing is cut out.By stir-fry wild jujube
The Six-elements Chinese medicinal composition such as benevolence, Radix Polygalae, Poria cocos, cape jasmine, Divine Comedy, Radix Glycyrrhizae is recorded in the national drug standards (WS-10410 (ZD-
0410) 2002-2012Z), there is blood-nourishing relieving restlessness, the effect of antitoxic heart-soothing and sedative, be used for heart-liver blood deficiency, insomnia and dreamful sleep is irritated and shy,
The diseases such as tired deficiency of food.Xinshen Ning tablets are significant in efficacy, and clinical application is relatively broad.Wherein Semen Ziziphi Spinosae (parched) be side in monarch drug in a prescription, play to
Important role is closed, the height of the superiority and inferiority of the quality of medicinal material that feeds intake, component content directly affects the drug effect of Xinshen Ning tablets.
" mind Yiganning capsule extracts and the screening and optimization of moulding process " (" Shandong medicine ", 2008,48 (28), 103) are public
A kind of extracting method of mind Yiganning capsule has been opened, has been to investigate target, including following extraction step with Gardenoside: taking the drug system
3 times, the 1st time plus 10 times of amount water are extracted in appropriate agent, extract 2h；2nd, 3 time respectively plus 8 times of amount water, extracts 1h every time.However, due to
Mind Yiganning capsule is by the Six-elements Chinese medicinal composition such as Semen Ziziphi Spinosae (parched), Radix Polygalae, Poria cocos, cape jasmine, Divine Comedy, Radix Glycyrrhizae, only by the extraction side
Method extracts Gardenoside, further measures its content, can not achieve the purpose that control its quality comprehensively.
Therefore, the extracting method for establishing a kind of Xinshen Ning tablets, carrying out control comprehensively for the quality of Xinshen Ning tablets has weight
Want meaning.
Summary of the invention
Therefore, the present invention proposes a kind of extracting method of pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, in turn
The method for building up of its prediction model is provided.
In order to solve the above technical problems, the present invention is achieved through the following technical solutions:
The present invention provides a kind of extracting method of pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, the drug
The bulk pharmaceutical chemicals of preparation form are as follows: Semen Ziziphi Spinosae (parched), Radix Polygalae, Poria cocos, cape jasmine, Medicated Leaven, Radix Glycyrrhizae；
The extracting method includes the extraction step to Gardenoside and spinosin as follows:
Take the pharmaceutical preparation appropriate, Extraction solvent is 60%-80% ethyl alcohol, and extraction time is 1-5 times, is extracted every time
0.5h-3h, the quantity of solvent being added every time are 6-9 times of the times of weight amount of the pharmaceutical preparation.
Preferably, the extracting method of the above-mentioned pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect of the present invention, the extraction
Method includes the extraction step to Gardenoside and spinosin as follows:
Take the pharmaceutical preparation appropriate, Extraction solvent is 65%-75% ethyl alcohol, and extraction time is 2-4 times, is extracted every time
1h-2h, the quantity of solvent being added every time are 7-8 times of the times of weight amount of the pharmaceutical preparation.
It is further preferred that the extracting method of the above-mentioned pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect of the present invention,
The extracting method includes the extraction step to Gardenoside and spinosin as follows:
Take the pharmaceutical preparation appropriate, Extraction solvent is 70% ethyl alcohol, and extraction time is 3 times, extracts 1.5h every time, every time
The quantity of solvent of addition is 7.44 times of the times of weight amount of the pharmaceutical preparation.
It is further preferred that the extracting method of the above-mentioned pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect of the present invention,
The bulk pharmaceutical chemicals of the pharmaceutical preparation form are as follows: Semen Ziziphi Spinosae (parched) 200-300 parts by weight, Radix Polygalae 120-210 parts by weight, Fu
Siberian cocklebur 120-210 parts by weight, cape jasmine 30-140 parts by weight, Medicated Leaven 30-140 parts by weight, Radix Glycyrrhizae 30-140 parts by weight.
It is further preferred that the extracting method of the above-mentioned pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect of the present invention,
The bulk pharmaceutical chemicals of the pharmaceutical preparation form are as follows: Semen Ziziphi Spinosae (parched) 220-280 parts by weight, Radix Polygalae 140-190 parts by weight, Fu
Siberian cocklebur 140-190 parts by weight, cape jasmine 50-110 parts by weight, Medicated Leaven 50-110 parts by weight, Radix Glycyrrhizae 50-110 parts by weight.
It is further preferred that the extracting method of the above-mentioned pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect of the present invention,
The bulk pharmaceutical chemicals of the pharmaceutical preparation form are as follows: 250 parts by weight of Semen Ziziphi Spinosae (parched), 167 parts by weight of Radix Polygalae, 167 weight of Poria cocos
Measure part, 83 parts by weight of cape jasmine, 83 parts by weight of Medicated Leaven, 83 parts by weight of Radix Glycyrrhizae.
The present invention also provides the prediction moulds of the extracting method of the above-mentioned pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect
The method for building up of type, comprising the following steps:
(1) Plackett-Burman design screening major influence factors
Plackett-Burman design is carried out, selected soaking time, the extraction time in terms of total extraction time, single mention
Taking time, boiling degree, Extraction solvent is influence factor, right using dry cream rate, Gardenoside and spinosin content as evaluation index
The influence factor carries out significance analysis；
It is tested according to Plackett-Burman Design experimental design, every group of experiment is 3 times parallel, is as a result averaged
Value carries out significance analysis to the influence factor with Design expert software；
(2) Box-Behnken effect surface method Optimized Extraction Process
Set empirical factor X1For extraction time, the empirical factor X in terms of total extraction time2For Extraction solvent -ol water ratio,
Empirical factor X3For solubilization dosage, evaluation index Y is set1For dry cream rate, evaluation index Y2For Determination of Gardenoside, evaluation index Y3
For spinosin content, the preferably empirical factor is tested with Box-Behnken Design；
The index that the index the smaller the better to value and value are the bigger the better is respectively adopted Hassan method, seeks " normalizing
Value " OD value, dmin=(ymax-yi)/(ymax-ymin), dmax=(yi-ymin)/(ymax-ymin), OD=(d1d2d3....dn) 1/n, n
For index number；The data obtained is subjected to experimental analysis with Design-Expert8.0.0 software, using OD as response, respectively to each
Empirical factor carries out multiple linear regression and binomial fitting, examines through F value, general comment OD value the results of analysis of variance；
(3) process certification is tested
It is real with the pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect with the Extraction technique after optimization
Object is tested, 3 parallel laboratory tests are carried out, carries out the measurement of dry cream rate, the assay of Gardenoside and the assay of spinosin, and
General comment OD value is calculated, with the accuracy of confirmatory experiment model.
Preferably, the prediction of the extracting method of the above-mentioned pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect of the present invention
The method for building up of model, the assay of the spinosin the following steps are included:
(1) preparation of test solution: taking pharmaceutical preparation to be measured, accurately weighed, finely ground, takes 0.5 parts by weight, and precision claims
It is fixed, it sets in stuffed conical flask, 70% methanol, 25 parts by volume, weighed weight is added in precision, and ultrasound 10 minutes is let cool, weighed weight,
The weight that less loss is supplied with 70% methanol, shakes up, filtering, take subsequent filtrate to get；
(2) preparation of reference substance solution: it is appropriate that precision weighs spinosin reference substance, adds methanol appropriate, is made containing this skin
The reference substance solution of promise element 0.010mg/mL；
(3) chromatographic condition: with 4.6mm × 250mm, 5 μm of Perkin Elmer C18 is chromatographic column, is flowing with acetonitrile
Phase A carries out gradient elution: 0min, A:B 10%:90% using -0.1% glacial acetic acid of water as Mobile phase B according to the procedure below；
0min-15min, A:B are 10%:90% → 17%:83%；15min, A:B 17%:83%；15min-25min, A:B are
17%:83% → 23%:77%；25min, A:B 23%:77%；25min-30min, A:B are 23%:77% → 35%:
65%；30min, A:B 35%:65%；30min-33min, A:B are 35%:65% → 10%:90%；33min-37min,
A:B is 10%:90%；Detection wavelength 335nm；25 DEG C of column temperature；Flow velocity 1.0mL/min；
(4) accurate respectively to draw 10 μ L of test solution and control solution, inject high performance liquid chromatograph, measurement；
The relationship of the parts by weight and parts by volume is g/mL.
Preferably, the prediction of the extracting method of the above-mentioned pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect of the present invention
The method for building up of model, the assay of the Gardenoside the following steps are included:
(1) preparation of test solution: taking pharmaceutical preparation to be measured, accurately weighed, finely ground, takes 0.5 parts by weight, and precision claims
It is fixed, it sets in stuffed conical flask, 50 parts by volume of methanol, weighed weight is added in precision, and ultrasound 30 minutes is let cool, weighed weight uses first
Alcohol supplies the weight of less loss, shakes up, filtering, take subsequent filtrate to get；
(2) preparation of reference substance solution: it is appropriate that precision weighs Gardenoside reference substance, adds methanol appropriate, is made containing Gardenoside
0.08mg/mL reference substance solution；
(3) chromatographic condition: using octadecylsilane chemically bonded silica as filler, it is for the acetonitrile-water of 12:88 with volume ratio
Mobile phase, Detection wavelength 238nm, flow velocity 1.0mL/min；
(4) accurate respectively to draw 10 μ L of test solution and control solution, inject high performance liquid chromatograph, measurement；
The relationship of the parts by weight and parts by volume is g/mL.
Technical solution of the present invention has the advantages that
The present invention has the extracting method of the pharmaceutical preparation of blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, with dry cream rate, Gardenoside
Content and the content of spinosin are inspection target, by optimizing extraction time, the type of Extraction solvent, the use of Extraction solvent
Amount, so that Gardenoside and spinosin are farthest extracted as far as possible, its content is determined in a pacing of going forward side by side, so as to comprehensive
The quality for controlling the pharmaceutical preparation helps to improve the safety and stability that the drug uses.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described.It should be evident that in being described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is extraction time X in experimental example 2 of the present invention1(in terms of total extraction time) and Extraction solvent (alcohol water ratio X2) to OD
It is worth the contour map that (Y) influences；
Fig. 2 is extraction time X in experimental example 2 of the present invention1(in terms of total extraction time) and Extraction solvent (alcohol water ratio X2) to OD
It is worth the diagram of block that (Y) influences；
Fig. 3 is extraction time X in experimental example 2 of the present invention1(in terms of total extraction time) and solubilization dosage (X3) to OD value (Y)
The contour map of influence；
Fig. 4 is extraction time X in experimental example 2 of the present invention1(in terms of total extraction time) and solubilization dosage (X3) to OD value (Y)
The diagram of block of influence；
Fig. 5 is Extraction solvent (alcohol water ratio X in experimental example 2 of the present invention2) and solubilization dosage X3OD value (Y) is influenced contour
Line chart；
Fig. 6 is Extraction solvent (alcohol water ratio X in experimental example 2 of the present invention2) and solubilization dosage X3The three-dimensional that OD value (Y) is influenced
Surface chart.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation
Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
Embodiment 1The preparation of Xinshen Ning tablets
[prescription] Semen Ziziphi Spinosae (parched) 250g, Radix Polygalae 167g, Poria cocos 167g, cape jasmine 83g, Medicated Leaven 83g, Radix Glycyrrhizae 83g.
[preparation method] described tablet is prepared by following methods:
(1) Radix Glycyrrhizae and the Medicated Leaven of selected parts by weight are taken, is crushed, is sieved, mixes, obtains fine powder；
(2) Semen Ziziphi Spinosae (parched), cape jasmine, Radix Polygalae and the Poria cocos for taking selected parts by weight, add water to cook twice, and it is small to decoct 3 for the first time
When, second of decoction 2 hours, collecting decoction filters, and filtrate is concentrated into the clear cream that 50 DEG C of relative densities are 1.05-1.20；
(3) fine powder is added into the clear cream, is mixed, right amount of auxiliary materials, pelleting is added, it is suitable that magnesium stearate is added
Amount mixes, dry, tabletted, is coated to get tablet.
Embodiment 2
The present embodiment has the extracting method of the pharmaceutical preparation of blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, including as follows to cape jasmine
The extraction step of glycosides and spinosin:
Take the pharmaceutical preparation appropriate, Extraction solvent is 70% ethyl alcohol, and extraction time is 3 times, extracts 1.5h every time, every time
The quantity of solvent of addition is 7.44 times of the times of weight amount of the pharmaceutical preparation.
Embodiment 3
The present embodiment has the extracting method of the pharmaceutical preparation of blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, including as follows to cape jasmine
The extraction step of glycosides and spinosin:
Take the pharmaceutical preparation appropriate, Extraction solvent is 65% ethyl alcohol, and extraction time is 4 times, extracts 1h every time, adds every time
The quantity of solvent entered is 8 times of the times of weight amount of the pharmaceutical preparation.
Embodiment 4
The present embodiment has the extracting method of the pharmaceutical preparation of blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, including as follows to cape jasmine
The extraction step of glycosides and spinosin:
Take the pharmaceutical preparation appropriate, Extraction solvent is 75% ethyl alcohol, and extraction time is 2 times, extracts 2h every time, adds every time
The quantity of solvent entered is 7 times of the times of weight amount of the pharmaceutical preparation.
Embodiment 5
The present embodiment has the foundation of the prediction model of the extracting method of the pharmaceutical preparation of blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect
Method, comprising the following steps:
(1) Plackett-Burman design screening major influence factors
With Design expert software carry out Plackett-Burman design, select soaking time, always to extract when
Between count extraction time, the single-trial extraction time, boiling degree, Extraction solvent be influence factor, with dry cream rate, Gardenoside and Si Pi
Promise cellulose content is evaluation index, carries out significance analysis to the influence factor；
It is tested according to Plackett-Burman Design experimental design, every group of experiment is 3 times parallel, is as a result averaged
Value carries out significance analysis to the influence factor with Design expert software；
(2) Box-Behnken effect surface method Optimized Extraction Process
Set empirical factor X1For extraction time, the empirical factor X in terms of total extraction time2For Extraction solvent -ol water ratio,
Empirical factor X3For solubilization dosage, evaluation index Y is set1For dry cream rate, evaluation index Y2For Determination of Gardenoside, evaluation index Y3
For spinosin content, the preferably empirical factor is tested with Box-Behnken Design；
The index that the index the smaller the better to value and value are the bigger the better is respectively adopted Hassan method, seeks " normalizing
Value " OD value, dmin=(ymax-yi)/(ymax-ymin), dmax=(yi-ymin)/(ymax-ymin), OD=(d1d2d3....dn) 1/n, n
For index number；The data obtained is subjected to experimental analysis with Design-Expert8.0.0 software, using OD as response, respectively to each
Empirical factor carries out multiple linear regression and binomial fitting, examines through F value, general comment OD value the results of analysis of variance；
(3) process certification is tested
It is real with the pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect with the Extraction technique after optimization
Object is tested, 3 parallel laboratory tests are carried out, carries out the measurement of dry cream rate, the assay of Gardenoside and the assay of spinosin, and
General comment OD value is calculated, with the accuracy of confirmatory experiment model.
Embodiment 6
The present embodiment has the foundation of the prediction model of the extracting method of the pharmaceutical preparation of blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect
Method, the assay of the spinosin the following steps are included:
(1) preparation of test solution: taking pharmaceutical preparation to be measured, accurately weighed, finely ground, takes 0.5g, accurately weighed, sets tool
It fills in conical flask, 70% methanol 25mL, weighed weight is added in precision, and ultrasound 10 minutes is let cool, weighed weight, with 70% methanol
The weight for supplying less loss, shakes up, filtering, take subsequent filtrate to get；
(2) preparation of reference substance solution: it is appropriate that precision weighs spinosin reference substance, adds methanol appropriate, is made containing this skin
The reference substance solution of promise element 0.010mg/mL；
(3) chromatographic condition: with 4.6mm × 250mm, 5 μm of Perkin Elmer C18 is chromatographic column, is flowing with acetonitrile
Phase A carries out gradient elution: 0min, A:B 10%:90% using -0.1% glacial acetic acid of water as Mobile phase B according to the procedure below；
0min-15min, A:B are 10%:90% → 17%:83%；15min, A:B 17%:83%；15min-25min, A:B are
17%:83% → 23%:77%；25min, A:B 23%:77%；25min-30min, A:B are 23%:77% → 35%:
65%；30min, A:B 35%:65%；30min-33min, A:B are 35%:65% → 10%:90%；33min-37min,
A:B is 10%:90%；Detection wavelength 335nm；25 DEG C of column temperature；Flow velocity 1.0mL/min；
(4) accurate respectively to draw 10 μ L of test solution and control solution, inject high performance liquid chromatograph, measurement.
Embodiment 7
The present embodiment has the foundation of the prediction model of the extracting method of the pharmaceutical preparation of blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect
Method, the assay of the Gardenoside the following steps are included:
(1) preparation of test solution: taking pharmaceutical preparation to be measured, accurately weighed, finely ground, takes 0.5g, accurately weighed, sets tool
It fills in conical flask, methanol 50mL, weighed weight is added in precision, and ultrasound 30 minutes is let cool, weighed weight supplies less loss with methanol
Weight, shake up, filter, take subsequent filtrate to get；
(2) preparation of reference substance solution: it is appropriate that precision weighs Gardenoside reference substance, adds methanol appropriate, is made containing Gardenoside
0.08mg/mL reference substance solution；
(3) chromatographic condition: using octadecylsilane chemically bonded silica as filler, it is for the acetonitrile-water of 12:88 with volume ratio
Mobile phase, Detection wavelength 238nm, flow velocity 1.0mL/min；
(4) accurate respectively to draw 10 μ L of test solution and control solution, inject high performance liquid chromatograph, measurement.
Comparative example 1
This comparative example has the extracting method of the pharmaceutical preparation of blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, including extracts step as follows
It is rapid:
It takes the pharmaceutical preparation appropriate, extracts 3 times, the 1st time plus 10 times of amount water, extract 2h；2nd, 3 time is respectively plus 8 times of amount water,
1h is extracted every time.
Experimental example 1
1, instrument and material
Agilent1260 high performance liquid chromatograph evaporates photodetector detector；VWD detector；The medical number of KQ-500DE
Control ultrasonic cleaner；2,400 a ten thousandth electronic analytical balance of AE, Switzerland's plum Teller.
Spinosin reference substance (lot number 111869-201203) is bought by National Institute for Food and Drugs Control；Acetonitrile (beauty
State Fisher), methanol (U.S. Fisher) be chromatographically pure；Methanol (the good Xinghua work glass apparatus Trade Co., Ltd. in Tianjin), ice
Acetic acid (development in science and technology Co., Ltd is recovered in Tianjin) is to analyze pure, water (three-level reverse osmosis legal system obtains purified water).
Xinshen Ning tablets (lot number 492043), are prepared used in Xinshen Ning tablets negative controls Xinshen Ning tablets negative controls
Raw material is provided by Jingfukang Pharmaceutical Group Co., Ltd., by Jingfukang Pharmaceutical Group Co., Ltd. engineer TCD identificafion Shi Jian
It is set to genuine piece.Xinshen Ning tablets are prepared according to the method for embodiment 1.
1.1 chromatographic condition
Using Perkin Elmer C18 chromatographic column (4.6mm × 250mm, 5 μm)；A (acetonitrile)-B (0.1% glacial acetic acid) is
Mobile phase, online gradient elution, 0-15min-25min-30min-33min-37min, A phase are 10%-17%-23%-35%-
10%-10%；Detection wavelength: 335nm；Column temperature: 25 DEG C；Flow velocity: 1.0mL/min；Sampling volume: 10 μ L.
The preparation of 1.2 reference substance solutions
It is appropriate that precision weighs spinosin reference substance, adds methanol appropriate, the reference substance of the 0.01mg/mL containing spinosin is made
Solution.
The preparation of 1.3 test solutions
Take Xinshen Ning piece several pieces, remove coating, it is accurately weighed, it is finely ground, take about 0.5g, it is accurately weighed, set stuffed conical flask
In, 70% methanol 25mL, weighed weight is added in precision, and ultrasound 10 minutes lets cool, weighed weight, supplies less loss with 70% methanol
Weight, shake up, filter, take subsequent filtrate to obtain the final product.
The preparation of 1.4 negative controls solution
The negative sample for being free of semen ziziphi spinosae medicinal material is prepared in the flavour of a drug ratio in prescription, prepares yin according to method below " 1.3 " item
Property comparison liquid.
The experiment of 1.5 specificities
By above-mentioned experiment condition, negative controls solution, test solution and reference substance solution are injected into liquid phase color respectively
Spectrometer measurement.
1.6 linear relationships are investigated
Precision weighs that reference substance is appropriate, adds methanol that the reference substance liquid of the 0.053424mg/mL containing spinosin is made, using again
Series mass strength solution is made than dilution method, making the concentration of spinosin is 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.With spinosin
Peak area Y draws standard curve to the concentration X of spinosin and obtains regression equation Y=20043X+4.4708, r=0.9999 (n=
5)。
The result shows that the linear relationship within the scope of 3.339-53.424 μ g/mL of the spinosin in Xinshen Ning tablets is good.
1.7 Precision Experiment
Taking content is the reference substance solution of 0.013356mg/mL, according to the chromatographic condition under " 1.1 " item, repeats sample introduction 6
It is secondary, each chromatographic peak area is recorded, RSD value is calculated.
The result shows that the spinosin RSD value in Xinshen Ning tablets is 0.75%.That is, the detection method precision is good.
1.8 repeated experiment
Precision weighs same lot number Xinshen Ning tablets (lot number 492043), by legal system available test sample solution 6 below " 1.3 " item
Part, it is measured by chromatographic condition under " 1.1 " item, records chromatographic peak area.
The result shows that the average content of the spinosin in Xinshen Ning tablets is 0.1623mg/ piece, RSD is 1.46% (n=
6).That is, the detection method favorable reproducibility.
1.9 stability experiment
The test solution according to method preparation below " 1.3 " item is taken, respectively 0,2,4,6,8,10, the 12h sample introduction after preparation
10 μ L record chromatographic peak area.
The result shows that the spinosin chromatographic peak area RSD value in Xinshen Ning tablets is 1.46%.That is, test solution exists
12h internal stability is good.
1.10 sample recovery rate is tested
Precision weighs 9 parts of Xinshen Ning tablets (lot number 492043) for having measured spinosin (0.1530mg/ piece) content, accurate
A certain amount of reference substance is added in weighed 0.25g, according to legal system available test sample solution below " 1.3 " item, by chromatostrip under " 1.1 " item
Part is measured, and calculates sample recovery rate with absolute mass score in test solution.It is found that the spinosin in Xinshen Ning tablets
Average recovery rate is that 100.91%, RSD value is 1.36%.
Experimental example 2
1, Plackett-Burman design screening major influence factors
1.1 factors and level
Plackett-Burman design (PBD) is carried out with Design expert8.0.6 software, investigates each factor pair
The conspicuousness that dry cream rate, Gardenoside and spinosin content influence.Since in practical mass production, semen ziziphi spinosae, cape jasmine etc. has been powder
It is broken to feed intake, do not consider influence of the medicinal material granularity to extraction process temporarily herein.
According to practical mass production experience, (A: soaking time, B: extraction time is (always to extract for 6 empirical factors of experimental design
Time meter), C: single-trial extraction time, D: boiling degree, E: Extraction solvent (alcohol water ratio), F: solubilization dosage) and 6 blank factors
(G, H, I, J, K, L), totally 12 groups of experiments.According to preliminary result determine each factor it is high (code: 1), low (code: -1) 2
It is horizontal.Designed experiment is as shown in table 1.
1 Plackett-Burman of table designs influence factor and level
1.2 Plackett-Burman design (PBD) experimental result
It is tested according to PBD experimental design, every group of experiment is 3 times parallel, and results are averaged.PBD experimental design table and
It the results are shown in Table 2.
2 PBD experimental design table of table and result
Significance analysis is carried out to each factor with Design expert 8.0.6 software, specific experiment the results are shown in Table
3.Wherein, response P < 0.05 is conspicuousness influence factor.
3 PBD analysis of experimental results of table
Note: P < 0.05 is extremely significant sexual factor
From the point of view of analyzing result, influence sequence of each influence factor to dry cream rate are as follows: extraction time is (with total extraction time
Meter) > Extraction solvent (alcohol water ratio) > boiling degree > solubilization dosage > single-trial extraction time > soaking time；Each influence factor
Influence sequence to Gardenoside are as follows: extraction time (in terms of total extraction time) > Extraction solvent (alcohol water ratio) > solubilization dosage > leaching
Steep > time, > single-trial extraction time boiling degree；Influence sequence of each influence factor to spinosin are as follows: extraction time is (with total
Extraction time meter) the > Extraction solvent > solubilization dosage > boiling degree > soaking time > single-trial extraction time.
Take the comprehensive single index analysis of each influence factor intersection, extraction time (in terms of total extraction time), Extraction solvent (alcohol water
Than) and solubilization dosage be significant factors, soaking time, the single-trial extraction time and boiling degree be non-limiting factor, with this
For according to progress Box-Behnken design (BBD).
2, Box-Behnken effect surface method Optimized Extraction Process
2.1BBD empirical factor, level and result
Since extraction time is discontinuous variable, so this variable factors of extraction time in terms of total extraction time, convert
For continuous variable, extracting i.e. 1 total extraction time is 1.5h, it extracts 2 times and total extraction time is 3h, extracting 3 times is total extract
Time is 4.5h.It is fixed as 0h in soaking time, the single-trial extraction time is fixed as 1.5h, and boiling degree takes violent fluidized state
Under, set X1(extraction time (in terms of total extraction time)), X2(Extraction solvent (alcohol water ratio)), X3(solubilization dosage) is factor change
Amount, Y1(dry cream rate), Y2(Determination of Gardenoside), Y3(spinosin content) is evaluation index, is preferably most preferably mentioned with BBD experiment
Taking technique parameter.
BBD empirical factor and level are shown in Table 4, and gained specific experiment the results are shown in Table 5.
4 BBD empirical factor of table and level
5 BBD of table experiment and result
2.2 models fittings and data processing
The data processing of multi objective generally uses overall desirability method.The index the smaller the better to value and value are bigger
Hassan method is respectively adopted in better index, seeks " normalizing value " OD value.dmin=(ymax-yi)/(ymax-ymin), dmax=
(yi-ymin)/(ymax-ymin), OD=(d1d2d3....dn) 1/n, n is index number.By the data obtained Design-Expert
8.0.0 software carries out experimental analysis, carries out multiple linear regression and binomial fitting, warp to each factor respectively using OD as response
F value is examined.General comment OD value the results of analysis of variance is shown in Table 6.
Model is significant (P < 0.01), loses quasi- item not significantly (P > 0.05), R2=0.94, the results showed that the regression model is quasi-
Close all right, representative preferable, the energy Accurate Prediction actual conditions of regression equation.Regression equation is as follows: Y=0.70+
0.27X1+0.071X2+0.19X3+0.029X1X2-0.059X1X3-0.18X2X3-0.13X1 2-0.057X2 2-0.100X3 2
6 BBD experimental result variance analysis of table
2.3 effect surface analysis and the prediction of BBD experimental model
Some variable in 3 variables is fixed using Design-Expert8.0.6 software according to Regression Analysis Result,
Three-dimension curved surface and contour map that other two variable influences general comment OD value are drawn, experimental result is shown in Fig. 1,2,3,4,5 and 6.
It is more not significant to the reciprocation of response to can be seen that total extraction time and Extraction solvent from Fig. 1 and Fig. 2；
From figs. 3 and 4 it can be seen that total extraction time and solubilization dosage have certain reciprocation to response；From Fig. 5 and Fig. 6
Extraction solvent and solubilization dosage be can be seen that the significant interaction of response.
Through Design Expert8.0.6 software Analysis and Screening, Xinshen Ning tablets Extraction technique optimal value is obtained are as follows: mention
Taking number is 3 times (total extraction time is 4.5h), and Extraction solvent is 70% ethyl alcohol, and solubilization dosage is 7.44 times, predicted value Y1It is (dry
Cream rate %)=16.78, Y2(Gardenoside mg/ piece)=3.6234, Y3(spinosin mg/ piece)=0.2392, Y (OD value)=
0.8848。
2.4 process certifications experiment
By the accuracy of confirmatory experiment model, actual conditions are comprehensively considered, with extraction time for 3 (total extraction times
For 4.5h), Extraction solvent is 70% ethyl alcohol, and solubilization dosage is 7.5 times, with the Extraction technique after optimization, by Xinshen Ning tablets
The ratio that prescription is 500 carries out 3 parallel laboratory tests, measures dry cream rate, the content of Gardenoside, spinosin, and calculate general comment OD
Value.Experimental results are shown in Table 7 and table 8.
7 process certification experimental result of table
8 process certification experimental result of table
By table 7 and table 8 it is found that dry cream rate, the measured value of Determination of Gardenoside and spinosin content and predicted value deviation are equal
Less than 2%, the average deviation of actual measurement OD value and prediction OD value is less than 3%.This shows that verification result more connects with prediction result
Closely.
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 medicine company Co., Ltd: lot number 140519,140802,140804) it carries out
Comparison.Specific experiment the results are shown in Table 9 and table 10.
Technological experiment result after table 9 optimizes
Remarks: 1,2,3 be technique after optimization pilot product, 4,5,6 (530011 batches, 530012 batches and 530013 batches) are
Mass production product is same lot number with the Chinese medicine that pilot product uses.
The experimental result of 10 market circulation handicraft product of table
By table 9 and table 10 it is found that the content of geniposide in Xinshen Ning tablets and spinosin all increase (in Chinese medicine,
Target component content generally only has lower limit, without the upper limit, other than some toxic components have regulation).
In conclusion the prediction model that this experimental example is established has preferable predictive, BBD- effect surface method analysis institute
The optimum extraction process parameter confidence level obtained is higher.
Experimental example 3
The Gardenoside and Si Pi of Xinshen Ning tablets prepared by embodiment 1 are extracted according to the method for embodiment 2 and comparative example 1 respectively
Promise element, and further progress carries out assay to it, specific experiment result is as shown in table 11.
The content results of table 11 Gardenoside and spinosin
As shown in Table 11, compared with using the method for comparative example 1, the Cape jasmine of Xinshen Ning tablets is extracted using the method for embodiment 2
Sub- glycosides and spinosin, content are higher.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (8)
1. a kind of extracting method of the pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, which is characterized in that the drug system
The bulk pharmaceutical chemicals of agent form are as follows: Semen Ziziphi Spinosae (parched) 200-300 parts by weight, Radix Polygalae 120-210 parts by weight, Poria cocos 120-210 parts by weight, Cape jasmine
Sub- 30-140 parts by weight, Medicated Leaven 30-140 parts by weight, Radix Glycyrrhizae 30-140 parts by weight；
The extracting method includes the extraction step to Gardenoside and spinosin as follows:
Take the pharmaceutical preparation appropriate, Extraction solvent is 60%-80% ethyl alcohol, and extraction time is 1-5 times, extracts 0.5h- every time
3h, the quantity of solvent being added every time are 6-9 times of the times of weight amount of the pharmaceutical preparation.
2. the extracting method of the pharmaceutical preparation according to claim 1 with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, feature
It is, which includes the extraction step to Gardenoside and spinosin as follows:
Take the pharmaceutical preparation appropriate, Extraction solvent is 65%-75% ethyl alcohol, and extraction time is 2-4 times, 1h-2h is extracted every time,
The quantity of solvent being added every time is 7-8 times of the times of weight amount of the pharmaceutical preparation.
3. the extracting method of the pharmaceutical preparation according to claim 2 with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, feature
It is, which includes the extraction step to Gardenoside and spinosin as follows:
Take the pharmaceutical preparation appropriate, Extraction solvent is 70% ethyl alcohol, and extraction time is 3 times, extracts 1.5h every time, is added every time
Quantity of solvent be 7.44 times of times of weight amount of the pharmaceutical preparation.
4. the extracting method of the pharmaceutical preparation according to claim 1 with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, feature
It is, the bulk pharmaceutical chemicals composition of the pharmaceutical preparation are as follows: Semen Ziziphi Spinosae (parched) 220-280 parts by weight, Radix Polygalae 140-190 parts by weight, Poria cocos
140-190 parts by weight, cape jasmine 50-110 parts by weight, Medicated Leaven 50-110 parts by weight, Radix Glycyrrhizae 50-110 parts by weight.
5. the extracting method of the pharmaceutical preparation according to claim 4 with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect, feature
It is, the bulk pharmaceutical chemicals composition of the pharmaceutical preparation are as follows: 250 parts by weight of Semen Ziziphi Spinosae (parched), 167 parts by weight of Radix Polygalae, 167 weight of Poria cocos
Part, 83 parts by weight of cape jasmine, 83 parts by weight of Medicated Leaven, 83 parts by weight of Radix Glycyrrhizae.
6. a kind of extraction side of the described in any item pharmaceutical preparations with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect of claim 1-5
The method for building up of the prediction model of method, which comprises the following steps:
(1) Plackett-Burman design screening major influence factors
Plackett-Burman design is carried out, when selecting soaking time, the extraction time in terms of total extraction time, single-trial extraction
Between, boiling degree, Extraction solvent be influence factor, with the content of dry cream rate, the content of Gardenoside and spinosin be evaluation refer to
Mark carries out significance analysis to the influence factor；
It is tested according to Plackett-Burman Design experimental design, every group of experiment is 3 times parallel, and results are averaged,
Significance analysis is carried out to the influence factor with Design expert software；
(2) Box-Behnken effect surface method Optimized Extraction Process parameter
Set empirical factor X1For extraction time, the empirical factor X in terms of total extraction time2For Extraction solvent -ol water ratio, experiment
Factor X3For solubilization dosage, evaluation index Y is set1For dry cream rate, evaluation index Y2For Determination of Gardenoside, evaluation index Y3For this
Skin promise cellulose content tests the preferably empirical factor with Box-Behnken Design；
Hassan method is respectively adopted in the index that the index the smaller the better to value and value are the bigger the better, and seeks " normalizing value " OD
Value, dmin=(ymax-yi)/(ymax-ymin), dmax=(yi-ymin)/(ymax-ymin), OD=(d1d2d3....dn) 1/n, n is to refer to
Mark number；The data obtained is subjected to experimental analysis with Design-Expert8.0.0 software, using OD as response, respectively to each experiment
Factor carries out multiple linear regression and binomial fitting, examines through F value, general comment OD value the results of analysis of variance；
(3) process certification is tested
It is experiment pair with the pharmaceutical preparation with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect with the Extraction technique after optimization
As carrying out 3 parallel laboratory tests, carrying out the measurement of dry cream rate, the assay of Gardenoside and the assay of spinosin, and calculate
General comment OD value, with the accuracy of confirmatory experiment model.
7. the prediction of the extracting method of the pharmaceutical preparation according to claim 6 with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect
The method for building up of model, which is characterized in that
The assay of the spinosin the following steps are included:
(1) preparation of test solution: taking pharmaceutical preparation to be measured, accurately weighed, finely ground, takes 0.5 parts by weight, accurately weighed, sets
In stuffed conical flask, 70% methanol, 25 parts by volume, weighed weight is added in precision, and ultrasound 10 minutes is let cool, weighed weight, is used
70% methanol supplies the weight of less loss, shakes up, filtering, take subsequent filtrate to get；
(2) preparation of reference substance solution: it is appropriate that precision weighs spinosin reference substance, adds methanol appropriate, is made containing spinosin
The reference substance solution of 0.010mg/mL；
(3) chromatographic condition: with 4.6mm × 250mm, 5 μm of Perkin Elmer C18 is chromatographic column, using acetonitrile as mobile phase A,
Using -0.1% glacial acetic acid of water as Mobile phase B, gradient elution: 0min, A:B 10%:90% is carried out according to the procedure below；0min-
15min, A:B are 10%:90% → 17%:83%；15min, A:B 17%:83%；15min-25min, A:B 17%:
83% → 23%:77%；25min, A:B 23%:77%；25min-30min, A:B are 23%:77% → 35%:65%；
30min, A:B 35%:65%；30min-33min, A:B are 35%:65% → 10%:90%；33min-37min, A:B are
10%:90%；Detection wavelength 335nm；25 DEG C of column temperature；Flow velocity 1.0mL/min；
(4) accurate respectively to draw 10 μ L of test solution and control solution, inject high performance liquid chromatograph, measurement；
The relationship of the parts by weight and parts by volume is g/mL.
8. the prediction of the extracting method of the pharmaceutical preparation according to claim 6 with blood-nourishing relieving restlessness, antitoxic heart-soothing and sedative effect
The method for building up of model, which is characterized in that
The assay of the Gardenoside the following steps are included:
(1) preparation of test solution: taking pharmaceutical preparation to be measured, accurately weighed, finely ground, takes 0.5 parts by weight, accurately weighed, sets
In stuffed conical flask, 50 parts by volume of methanol, weighed weight is added in precision, and ultrasound 30 minutes is let cool, weighed weight is mended with methanol
The weight of sufficient less loss, shakes up, filtering, take subsequent filtrate to get；
(2) preparation of reference substance solution: it is appropriate that precision weighs Gardenoside reference substance, adds methanol appropriate, is made containing Gardenoside
0.08mg/mL reference substance solution；
(3) chromatographic condition: using octadecylsilane chemically bonded silica as filler, using volume ratio for 12:88 acetonitrile-water as flowing
Phase, Detection wavelength 238nm, flow velocity 1.0mL/min；
(4) accurate respectively to draw 10 μ L of test solution and control solution, inject high performance liquid chromatograph, measurement；
The relationship of the parts by weight and parts by volume is g/mL.
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