CN107607665A - One seedling medicine subprostrate sophora and its Chinese medicine preparation method of quality control - Google Patents
One seedling medicine subprostrate sophora and its Chinese medicine preparation method of quality control Download PDFInfo
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- CN107607665A CN107607665A CN201710912033.7A CN201710912033A CN107607665A CN 107607665 A CN107607665 A CN 107607665A CN 201710912033 A CN201710912033 A CN 201710912033A CN 107607665 A CN107607665 A CN 107607665A
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- matrine
- oxymatrine
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Abstract
The invention belongs to field of traditional Chinese medicine technology, discloses a seedling medicine subprostrate sophora and its Chinese medicine preparation method of quality control, extracts the active component in medicinal material and preparation；Isolated and purified using a variety of chromatogram means, assay, Structural Identification are carried out to isolated compound；Extraction and separation detection technique are optimized according to experimental result；Establish Guizhou seedling medicine bicolor and open the method for quality control of larynx sword；Isolated compound structure is accredited as：Matrine, oxymatrine；Matrine content is 0.6100%；Oxymatrine concentration is 0.4576%.The present invention is to evaluating the quality and stability of each place of production medicinal material and Chinese medicine preparation；And plantation can be developed for GAP from now on certain scientific basis is provided；The present invention is swift to operate, easy, reproducible, and the quality restriction available for subprostrate sophora medicinal material and its preparation is analyzed.
Description
Technical field
The invention belongs to field of traditional Chinese medicine technology, more particularly to a seedling medicine subprostrate sophora and its Chinese medicine preparation quality control
Method.
Background technology
Subprostrate sophora be legume sophora tonkinensis Gapnep (Sophora tonkinensis Gagnep) drying root and rhizome, rhizome
It is in irregular nodular pattern to expand, and there is stem foot residual upper end, and lower end, which has, takes root several, and root is in cylinder, and slightly curved song, main product is in wide
West.Alias：Potato root, root of subprostrate sophora, the small coptis, meadowrueleaf corydalis root, be the conventional traditional valuable ingredient of traditional Chinese medicine of China, be used in northeast, North China,
The ground such as Central China.First recorded in Song《Kaibao Bencao》, " subprostrate sophora, raw sword south and Yizhou, Guo Zhou, modern Guangxi also has Su Songwei, with loyalty
State, ten thousand state persons are preferred.Seedling is climing, and as vertical, Ye Qing is uncomfortable through the winter, adopts root August, for example small Chinese scholartree of Guangxi person .. " more than high chi ..., mainly
It is distributed in Jingxi, Napo County, Debao, Longzhou, the county such as reach the clouds.Wild, bitter is cold in nature, poisonous, return lung, stomach, have it is clearing heat and detoxicating,
The effect of relieving sore-throat of subsiding a swelling.《Book on Chinese herbal medicine is looked for the truth》Record：" solution abscess of throat first key medicine ".Its medicine pents up available for fiery poison, acute tonsillitis larynx
Numbness, abscess of throat, gingivitis, aphthae, insect-bite.Alkaloid is based on matrine, oxymatrine, and first containing N-
Base sparteine and Anagyrine etc., Recent study find that matrine alkaloid is in central nervous system, cardiovascular system
System, antiviral, anti-inflammatory, immune and antitumor etc. have important pharmacological activity and using future.Open the master of larynx sword spray
It is matrine to want active ingredient, and the color of liquid medicine is light brown to brown；Sweet, micro- fiber crops, slight bitter, there is the salubrious ice of peppermint
Cool feeling, swelling and pain relieving is cured mainly, it is clearing heat and detoxicating.
Larynx sword spray is opened, Main Ingredients and Appearance has subprostrate sophora, Crisped Ardisia Ardisia gigantifolia, menthol etc..It is mainly used in clearing heat and detoxicating, detumescence
Analgesic, the abscess of throat caused by lung stomach intrinsic heat, dry mouth with bitter taste, swelling and aching of gum and canker sore etc..But measure is originally opened in larynx sword
The content measuring method operability of oxymatrine is poor, cumbersome.
Subprostrate sophora founds Chinese medicine plantation Co., Ltd and carried out forever via Guiyang Medical Plant Garden, Guizhou Province Anlong County at present
Introducing and planting.But the raw material of larynx sword spray is opened as important ethnic drug, at present substantially using Guangxi as raw material site.One
Aspect is that its wild resource is very rare due to excessively excavating.The material base of another aspect mesh first two medicinal material is not deep enough
Enter and the scope of finished product exploitation is narrower, constrain the further industrialization of the resource.So going deep into its chemical active ingredient, sieve
Publish existing New function and new application；The quality control standard of bicolor and its preparation is formulated simultaneously, contributes to dominant plant resource
Deep development and utilization.
The main matter of subprostrate sophora pharmacological action is various alkaloids, including matrine, oxymatrine, Anagyrine.
Research finds matrine alkaloid in central nervous system, and cardiovascular system is antiviral, anti-inflammatory, is immunized and antitumor waits just
There is important pharmacological activity in face and using future[3], wherein alkaloid using the matrine of quinolizidine kind and oxymatrine as
It is main.R＆D of modern TCM shows, containing alkaloid, Quercetin, polysaccharide and other multiple active components in subprostrate sophora, wherein matrine with
Medicinal active ingredient is wanted based on oxymatrine.
Medical industry by the use of subprostrate sophora as raw material, a large amount of injections for developing treatment hepatitis, abscess of throat tablet with
And antitumor Chinese patent drug, thus matrine in subprostrate sophora is studied, extracting method and the optimum extraction condition tool of oxymatrine
There are very high pharmaceutical value and economic benefit.Alkaloid method mainly has thin-layered chromatography, hair in report measure subprostrate sophora at present
Cons electrophoresis method, high performance liquid chromatography (HPLC) etc..Thin-layered chromatography is limited to separative efficiency and detection sensitivity, general multi-purpose
In detection total alkaloid or qualitative detection.When capillary electrophoresis detects medicinal material, often impurity serious interference.Simultaneous quantitative detects
Matrine and the most widely used method of oxymatrine are HPLC.
The theoretical foundation of ultrasonic wave extraction is to utilize ultrasonic wave calorifics mechanism, ultra sonic machinery mechanism and cavitation.It is super
Compared with traditional leaching method, the speed of its extraction process greatly improves sound wave extraction method, and the impurity in extract solution is more traditional
Method is low.
In summary, the problem of prior art is present be：Subprostrate sophora wild resource is due to excessively having excavated very at present
It is rare；Material base is not deep enough and the scope of finished product exploitation is narrower, constrains industrialization；
Existing subprostrate sophora and its Chinese medicine preparation method of quality control, not to subprostrate sophora and open sword larynx spray and use efficient liquid
Phase chromatography, determine the content of matrine；Make prior art operation cumbersome, poor repeatability；
The existing quantitatively speed of detection matrine and oxymatrine extraction process is low, and the impurity in extract solution is high；Not yet
Have and use L9 (34) orthogonal test carried out to subprostrate sophora matrine, the analysis of the extraction conditions of oxymatrine.
The content of the invention
The problem of existing for prior art, the invention provides a seedling medicine subprostrate sophora and its Chinese medicine preparation quality control
Method.
The present invention is achieved in that a seedling medicine subprostrate sophora and its Chinese medicine preparation method of quality control, including：Extract medicine
Active component in material and preparation；Isolated and purified using high performance liquid chromatography, while isolated compound is carried out
Assay, Structural Identification；
Extraction conditions and separation detection technological parameter are optimized according to experimental result；
Establish seedling medicine bicolor and open the method for quality control of larynx sword.
Further, the separation method of active component and the optimization of separation condition in the subprostrate sophora, including：
Matrine in root of subprostrate sophora and its preparation is extracted using ultrasonic extraction, matrine content, color are determined from HPLC
Spectrum post is symmetry C18；Mobile phase is methanol：Water=80 of 0.025% phosphoric acid:20；Flow velocity：0.7mL.min-1, detect ripple
It is long：210nm, column temperature：20℃.
Further, it is described that isolated compound structure is accredited as：Matrine, oxymatrine；Matrine content
For 0.6100%；Oxymatrine concentration is 0.4576%.
Further, the method that extraction is optimized according to experimental result includes：
Utilize ultrasonic extraction subprostrate sophora ingredient matrine, oxymatrine；Matrine extraction conditions soak not sieve
Temperature raising degree is 30 DEG C, and ultrasonic time is 30 minutes；
For oxymatrine extraction conditions not sieve, extraction temperature is 20 DEG C, and ultrasonic time is 30 minutes.
Further, the method for quality control established seedling medicine bicolor and open larynx sword, by establish measure subprostrate sophora and
The HPLC methods for opening oxymatrine concentration in larynx sword carry out quality control, and the HPLC methods include：
Chloroform-methanol-conc. aq. ammonia=40 are selected by volume：10：1 carries out ultrasonic extraction, using high-efficient liquid phase color
Law popularization determines oxymatrine concentration, and from symmetry C18 chromatographic columns, mobile phase is methanol：Water containing 0.025% phosphoric acid
=95:5th, methanol：Water=0 containing 0.025% phosphoric acid:100 gradient；Elution flow rate 0.70mlmin-1, Detection wavelength is
210nm, 24.8 DEG C of column temperature.
Advantages of the present invention and good effect are：The profound active principle basis excavated in ethnic drug, and simultaneously with
The genunie medicinal materials in other places of production are contrasted, and by being contrasted to the active component result in two places of production, establish Gui Zhoushan
The quantitative detecting method and quality control standard of beans root.Optimization Guizhou subprostrate sophora optimum extraction process and key parameter values are (molten
Agent pH value, extraction temperature time, extracting times etc.), the GAP kinds for local medicinal material are planted and second development and utilization lays the foundation, such as
It can be extended to provincial standard, the performance rating standard of unified current two big planting base of Guizhou subprostrate sophora (Guiyang Medical Plant Garden,
Chinese medicine plantation Co., Ltd is found forever in Guizhou Province Anlong County), it can be provided for the popularization of Guizhou Province's ethnic drug and plantation development bigger
Opportunity.
The present invention is to subprostrate sophora and opens sword larynx spray and uses high performance liquid chromatography, determines the content of matrine.Matrine
Sample size is in 0.6250mg.mL-1-1.000mg.mL-1In the range of with peak area value be in good linear relationship (r=0.9991),
Average recovery rate is 98.36%, RSD=1.11% (n=5).
This method is swift to operate, easy, reproducible, and the quality restriction available for subprostrate sophora medicinal material and its preparation is analyzed.
The present invention chose grit number, ultrasonic wave extraction time, ultrasonic wave extraction temperature, using L9 (34) orthogonal experiment plan
Meter, Three factors-levels experiment is carried out, to subprostrate sophora matrine, the extraction conditions of oxymatrine are analyzed, and are subprostrate sophora
Intensive processing with using and subprostrate sophora analysis of biological activity with application offer foundation.
The present invention determines subprostrate sophora by foundation and opens the HPLC methods of oxymatrine concentration in larynx sword, as a result shows,
Oxymatrine carries out sample introduction in 0.0185mgml-1-0.2590mg·ml-1In the range of peak area value present, good linear close
It is y=21993x+244505 (r=0.9971), average recovery rate is that 97.6% standard deviation (RSD) is 1.47%.This method
Accurately, quickly, high sensitivity, favorable reproducibility, subprostrate sophora can be determined and open oxymatrine concentration in larynx sword, subprostrate sophora can be used as
And the detection method of content of quality of the pharmaceutical preparations control.
Brief description of the drawings
Fig. 1 is seedling medicine subprostrate sophora and its Chinese medicine preparation method of quality control flow chart provided in an embodiment of the present invention.
Fig. 2 is resulting standard working curve figure provided in an embodiment of the present invention.
Fig. 3 is the chromatogram of standard matrine provided in an embodiment of the present invention.
Fig. 4 is that Guangxi subprostrate sophora provided in an embodiment of the present invention and adult, children open matrine chromatogram in larynx sword spray
Figure.
In figure：A, subprostrate sophora matrine chromatogram；B, adult opens larynx sword spray matrine chromatogram；C, children open larynx sword
Spray matrine chromatogram.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The present invention opens active component in larynx sword as object using Guizhou seedling medicine subprostrate sophora and its Chinese medicine preparation.In main Chinese medicinal materials
Alkaloids, volatile oil chemical composition.With reference to tradition and modernity extractive technique extract activity in medicinal material and preparation into
Point；Isolated and purified using a variety of chromatogram means, assay, Structural Identification are carried out to isolated compound.According to
Experimental result optimization extraction and separation detection technique；Establish Guizhou seedling medicine bicolor and open the method for quality control of larynx sword
(HPLC), to evaluating the quality and stability of each place of production medicinal material and Chinese medicine preparation；And plantation can be developed for GAP from now on to provide
Certain scientific basis.
The application principle of the present invention is explained in detail below in conjunction with the accompanying drawings.
As shown in figure 1, seedling medicine subprostrate sophora provided in an embodiment of the present invention and its Chinese medicine preparation method of quality control include with
Lower step：
S101：Extract the active component in medicinal material and preparation；Isolated and purified using a variety of chromatogram means, to separating
The compound arrived carries out assay, Structural Identification；
S102：Extraction and separation detection technique are optimized according to experimental result；
S103：Establish seedling medicine bicolor and open the method for quality control of larynx sword.
The optimization of the separation method of active component and separation condition in the subprostrate sophora, including：
Matrine in root of subprostrate sophora and its preparation is extracted using ultrasonic extraction, matrine content, color are determined from HPLC
Spectrum post is symmetry C18；Mobile phase is methanol：Water=80 of 0.025% phosphoric acid:20；Flow velocity：0.7mL.min-1, detect ripple
It is long：210nm, column temperature：20℃.
It is described that isolated compound structure is accredited as：Matrine, oxymatrine；Matrine content is
0.6100%；Oxymatrine concentration is 0.4576%.
The method that extraction is optimized according to experimental result includes：
Utilize ultrasonic extraction subprostrate sophora ingredient matrine, oxymatrine；Matrine extraction conditions soak not sieve
Temperature raising degree is 30 DEG C, and ultrasonic time is 30 minutes；
For oxymatrine extraction conditions not sieve, extraction temperature is 20 DEG C, and ultrasonic time is 30 minutes.
The method of quality control established seedling medicine bicolor and open larynx sword, by establishing measure subprostrate sophora and opening in larynx sword
The HPLC methods of oxymatrine concentration carry out quality control, and the HPLC methods include：
Chloroform-methanol-conc. aq. ammonia=40 are selected by volume：10：1 carries out ultrasonic extraction, using high-efficient liquid phase color
Law popularization determines oxymatrine concentration, and from symmetry C18 chromatographic columns, mobile phase is methanol：Water containing 0.025% phosphoric acid
=95:5th, methanol：Water=0 containing 0.025% phosphoric acid:100 gradient；Elution flow rate 0.70mlmin-1, Detection wavelength is
210nm, 24.8 DEG C of column temperature.
(1) specific embodiment is further described to the application principle of the present invention below.
1st, discrimination method
With reference to the method in Chinese Pharmacopoeia version one in 2000, by the investigation to extracting method, the thin of subprostrate sophora is established
Layer chromatography discrimination method.
2nd, subprostrate sophora and the extracting and developing and Structural Identification of main active in larynx sword are opened
Using the traditional and new extraction and separation technology of effective component of chinese medicine, mainly extract and isolate subprostrate sophora (Guizhou
Production, Guangxi production), open alkaloids, volatile oil material in larynx sword (adult type, child form)；Using GC/MS, HPLC to dividing
Qualitative and quantitative analysis is carried out from compound after purification.
According to the place of production composition of Comparison of experiment results two or the similarities and differences of content, and the analysis of experimental results medicinal material for combining preparation exists
Contribution in prescription；Further optimization optimum extraction process and key parameter values.
3rd, Guizhou seedling medicine subprostrate sophora and the quality control of larynx sword is opened
According to result, most important active component is chosen as standard, Guizhou seedling medicine subprostrate sophora is built and Chinese medicine preparation is opened
Larynx sword HPLC quantitative detecting methods, establish quality control standard.Laid the foundation for subprostrate sophora second development and utilization, and be Guizhou Province
The construction of ethnic drug chemical libraries provides valid data.
4th, the isolation and purification method of main active：
Isolated and purified using a variety of chromatogram means, mainly using chromatography separating method (including HPLC, GC etc.), for
Two effective constituents (alkaloids, volatile oil) are isolated and purified repeatedly.
5th, the identification of compound structure：
Mainly using a variety of wave spectrums or GC-MS such as MS, ultraviolet absorption spectroscopies, with reference to document and standard items spectrogram pair
According to the structure of authenticating compound.
6th, the factor selection of medicinal material extract technique and key parameter values.
Conditional parameter (solvent PH value, extraction temperature time, the extraction time of extraction process are mainly investigated by orthogonal experiment method
Number etc.), it is that drug quality and deep development (Chinese medicine preparation) provide scientific basis.
7th, establish Guizhou subprostrate sophora and open the quality control standard of larynx sword：
Active component and its pharmacological effect main in subprostrate sophora are illustrated with reference to document, be its as quality control reference substance
Establish theory support.Build Guizhou seedling medicine subprostrate sophora and open larynx sword (adult type, child form) HPLC quantitative detecting methods, establish matter
Measure control standard.Laid the foundation for the second development and utilization of subprostrate sophora, and the construction for Guizhou ethnic drug chemical libraries provides effectively
Data.
(2) specific embodiment is further described to the application principle of the present invention below.
1 instrument and reagent
1.1 instrument
The accurately weighed electronic balance of FA2004N types (Shanghai Jing Hai Instrument Ltd.)；KQ3200DE type ultrasonic cleaning machines
(Kunshan Ultrasonic Instruments Co., Ltd.)；Electric-heated thermostatic water bath (Beijing Xicheng District medical machinery factory)；Standard screen：(10 mesh, hole
Footpath is 2mm；20 mesh, aperture 0.84mm), Dao Xuzhongtuo instrument plants of Shangyu city；Polyvinylidene fluoride microporous filtering film：Organic system aperture
It is 0.45 μm for 0.45 μm of (the grand board in Tianjin), miillpore filter aqueous phase aperture, originates in Haixing County Asia scavenging material factory；It is disposable
Asepsis injector (band syringe needle) (Jiangxi Hongda Medical Equipment Group Corp., Ltd.)；Waters high performance liquid chromatographs,
Symmetry C18 (4.6*250mm, 0.5 μm) chromatographic column, Model Code515 pumps, ultraviolet 2487 detectors of Waters
1.2 reagent
Methanol is the pure and mild chromatographically pure of analysis；Chloroform, phosphoric acid, concentrated ammonia liquor are that analysis is pure；Matrine reference substance (batch
Number：110805-200508, ZD:ON49-X8EQ)；Subprostrate sophora originates in Guilin, is provided by the power medicine company of Guizhou three；Open the spray of larynx sword
Mist agent is produced in Sanli Pharmaceutical Co., Ltd., Guizhou.
2 methods and result
2.1 testing conditions
Chromatographic column is symmetry C18, and mobile phase is Methanol-water (0.025% phosphoric acid) (80:20), use is isocratic
Elution disengaging time is 15min.The wavelength of detection is 210nm, flow velocity 0.70mL.min-1, column temperature be 25 DEG C, number of theoretical plate with
It is 10 μ L that matrine, which calculates the sample size that subprostrate sophora and adult open larynx sword spray in about 2300. experiments, and children open larynx sword
Spray sample size for 15 μ L.
The preparation of 2.2 reference substances
The appropriate matrine reference substance by phosphorus pentoxide drying to constant weight of precision weighing, is configured to methanol
Per solution of the 1mL containing 2.5mg.With 0.45 μm of syringe-type organic phase filtering with microporous membrane after shaking up, reference substance solution is produced, is placed in
It is labelled in sample bottle, it is sealed.
The preparation of 2.3 need testing solutions
Precision weighs three parts of 0.55-0.60g Vietnamese Sophora Roots, is placed in the conical flask with plug, and precision measures addition three
Chloromethanes-methanol-strong ammonia solution (40:10:1) mixed solution 50ml, is stoppered, weighed weight, after placing 30 minutes, using super
Sound device processing (power 250W, frequency 40kHz) 40 minutes, weighed weight again after cooling, weight chloroform-first of reduction
Alcohol-strong ammonia solution (40:10:1) mixed solution is supplied, and is filtered after shaking up with fast grade filter paper, accurate measures filtrate
10ml, for recycling design to medicinal extract shape, into residue plus about 5ml methanol makes its dissolving, is transferred to 10ml appearances under 40 DEG C of heating water baths
In measuring bottle, with methanol constant volume to scale, with 0.45 μm of filtering with microporous membrane after shaking up, its subsequent filtrate is taken, it is molten to produce test sample
Liquid, be placed in sample bottle it is labelled, be sealed.Adult's medicine, children's medicine are operated according to above-mentioned method.
2.4 methodological study
2.4.1 linear relationship is investigated
Precision weighs appropriate matrine reference substance, with methanol dilution constant volume into storing solutions of every 1mL containing 2.5mg, afterwards
Stand-by storage solution allocation determines 5 concentration needed for working curve, 62.5 μ g.mL-1、125μg.mL-1、250μg.mL-1、500μ
g.mL-1、1000μg.mL-1.It is that mobile phase carries out 80 with methanol-water (0.025% phosphoric acid):20 isocratic elutions, the wavelength of detection
For 210nm, flow velocity 0.70mL.min-1, column temperature is 25 DEG C, and number of theoretical plate 2300, sample size is 10 μ L.
Resulting standard working curve such as Fig. 2, equation of linear regression y=7335.5x+136487R^2=0.9991.
The chromatogram of standard matrine is as shown in Figure 3.
2.4.2 precision test
Accurate standard matrine of drawing the μ L of sample size 10, passes through the integration peak face of calculating with No.1 sample continuous sample introduction 5 times
Product result, the RSD for obtaining them is respectively 0.89%, and this result shows that this method has good precision.
2.4.3 reappearance test
Subprostrate sophora is taken respectively and opens five parts of the Duplicate Samples of larynx sword spray, is determined its content according to method, as a result can be obtained mountain
The average content of matrine is 0.83%, RSD 1.7% in beans root；Open the average content of matrine in larynx sword spray
0.18%, RSD 1.2%.As a result show that this method has good reappearance.
2.4.4 stability experiment
The accurate same No.1 sample drawn subprostrate sophora and open larynx sword spray, peak area is determined respectively at 0,2,4,8,16h
And record, the RSD that their integrated peak areas is calculated is respectively 1.8% and 1.3%.As a result show, two in 16 hours
The stability of kind sample is all fine.
2.4.5 average recovery is tested
Using sample-adding recovery method, five parts of the sample of a certain amount of known content is taken, adds appropriate standard items, according to method
Prepare and determine their content, calculate their rate of recovery respectively.The rate of recovery measures respectively 97.66%, 99.83%,
97.18%th, 98.65%, 98.48%, average recovery rate 98.36%, RSD=1.11%.Measurement result shows returning for this method
Yield is good.It the results are shown in Table 1.
The matrine average recovery measurement result of table 1
2.5 sample sizes are tested
The content for opening subprostrate sophora and adult, children matrine in larynx sword spray according to testing conditions " 2.1 " detects.
Chromatogram is shown in Fig. 4, and the result of measure and data processing are shown in Table 2.
The determination of matrine result of table 2
(remarks：A is subprostrate sophora sample, and B is that adult opens larynx sword spray sample, and C is that children open larynx sword spray sample.)
3 using Methanol-water (0.025% phosphoric acid) (95:5) mobile phase is used as, its separating effect is found not in preliminary experiment
Ideal, the rear mobile phase that adjusts is Methanol-water (0.025% phosphoric acid) (80:20), good separation.It can be seen that by experimental result
Adult opens matrine content in larynx sword spray and is more than subprostrate sophora matrine content, and children open matrine in larynx sword spray and contained
Amount is about 1/20 in the subprostrate sophora of Guangxi, and about adult opens 1/30 in larynx sword spray.Mean percent content is respectively
0.8468% (adult preparation), 0.6141% (Guangxi subprostrate sophora), 0.0366% (children preparation).Experiment is used and carried using ultrasound
The content with matrine in high effective liquid chromatography for measuring sample is followed the example of, understands that this method is accurate through methodology proof list, quickly,
High sensitivity, favorable reproducibility, the assay side of larynx sword (adult type, child form) quality control can be opened as subprostrate sophora and preparation
Method.
(3) specific embodiment is further described to the application principle of the present invention below.
1 instrument and reagent
1.1 instrument
The accurately weighed electronic balance of FA2004N types (Shanghai Jing Hai Instrument Ltd.)；KQ3200DE type ultrasonic cleaning machines
(Kunshan Ultrasonic Instruments Co., Ltd.)；Electric-heated thermostatic water bath (Beijing Xicheng District medical machinery factory)；Miillpore filter：It is (organic
System and water system) grand Tianjin board, aperture 0.45um；(Jiangxi flood has disposable sterilized syringe (band pin) up to medicine equipment group
Limit company)；Waters high performance liquid chromatographs；Standard screen：10 mesh, 20 mesh.
1.2 reagent
Methanol (chromatographically pure, analysis are pure), chloroform, phosphoric acid, ammoniacal liquor are pure to analyze, and water is pure water.
2 experimental methods
2.1HPLC determines matrine content
Accurately weighed 0.55-0.60g matrines, are placed in conical flask with cover, and precision adds chloroform-methanol-dense ammonia
Solution (40:10：1) the close plugs of 50ml, weighed weight, place 30 minutes, be ultrasonically treated (power 250W, frequency 40kHz, temperature 25
DEG C), conical flask is placed in ultrasonic processor 40 minutes, then weighed weight, with chloroform-methanol-liquor ammoniae fortis (40:10:
1) mixed solution supplies less loss weight.Reagent is shaken up, filtered using fast grade filter paper, 10ml filtrates are pipetted with pipette, 40
Solution is reclaimed at a temperature of DEG C to medicinal extract shape, residue, which adds methanol about 5ml, makes its dissolving, is transferred in 10ml volumetric flasks, adds methanol
To graduation mark, shake up, with 0.45 filtering with microporous membrane, take subsequent filtrate, produce, put sample bottle label, be sealed.Chromatostrip
Part：Chromatographic column is C18.The methanol as mobile phase of matrine has maximum purple to absorb at 210nm, Methanol-water (0.025% phosphorus
Acid) (80: 20) be mobile phase isocratic elution, disengaging time 15 minutes.Detection wavelength is 210nm, flow velocity 0.70ml/min,
Column temperature is 25 DEG C.Theoretical cam curve is calculated as 2300 with matrine.
It is appropriate that precision weighs standard substance matrine reference substance, and solution of every 1ml containing 2.5mg is diluted to methanol constant volume,
5 concentration of stand-by storage liquid configuration work curve, 62.5ug/ml, 125ug/ml, 250ug/ml, 500ug/ml, 1000ug/ml.
By standard reserving solution by above-mentioned chromatographic condition difference sample introduction, sample size 10ul.Concentration is 1000ug/ml standard appearance.Institute
The equation of linear regression y=7335.5x+136487R obtained2=0.9991.
2.2HPLC determines oxymatrine concentration
Accurately weighed 0.55-0.60g oxymatrines, are placed in conical flask with cover, and precision addition chloroform-methanol-
Liquor ammoniae fortis (40:10：1) the close plugs of 50ml, weighed weight, place 30 minutes, be ultrasonically treated (power 250W, frequency 40kHz, temperature
25 DEG C of degree), conical flask is placed in ultrasonic processor 40 minutes, then weighed weight, with chloroform-methanol-liquor ammoniae fortis
(40:10:1) mixed solution supplies less loss weight.Reagent is shaken up, filtered using fast grade filter paper, 10ml filters are pipetted with pipette
Liquid, solution being reclaimed at a temperature of 40 DEG C to medicinal extract shape, residue, which adds methanol about 5ml, makes its dissolving, is transferred in 10ml volumetric flasks,
Methanol is added to shake up to graduation mark, with 0.45 filtering with microporous membrane, take subsequent filtrate, produce, put sample bottle label, be sealed.
Chromatographic condition：The methanol solution of oxymatrine has maximal ultraviolet absorption at 210nm, and Methanol-water (0.025% phosphoric acid) is
Mobile phase, gradient elution is carried out from Methanol-water (0.025% phosphoric acid) 95: 5 to 0: 100, detection time is 15 minutes.Wavelength is
210nm, flow velocity 0.70ml/min, column temperature are 25 DEG C, theoretical cam curve 2300.
It is appropriate that precision weighs oxymatrine reference substance, is diluted to every 1ml with methanol constant volume and contains 3.7mg, stand-by storage solution
5 concentration of configuration work curve, respectively 18.5 μ g/ml, 37 μ g/ml, 92.5 μ g/ml, 222 μ g/ml, 259 μ g/ml.With
Methanol-water (0.025% phosphoric acid) (80:20) it is mobile phase, Detection wavelength 210nm, flow velocity 0.70ml/min, column temperature is
24.8 DEG C of number of theoretical plates are 2300, and sample size is 10 μ l.It is by above-mentioned chromatographic condition difference sample introduction, sample size by standard reserving solution
10ul.Concentration is 222ug/ml standard appearance.The equation of linear regression of gained is y=21993x+244505R2=0.9971.
2.3 Orthogonal Experiment and Design
2.3.1 the Orthogonal Experiment and Design of matrine
Three factors (crossing grit number, extraction temperature, ultrasonic time) are selected in this experiment, three levels make orthogonal test.It is horizontal because
Plain table is shown in Table 1.
2.3.2 the Orthogonal Experiment and Design of bitter oxidation ginseng alkali
Three factors (crossing grit number, extraction temperature, ultrasonic time) are selected in this experiment, three levels make orthogonal test.It is horizontal because
Plain table is shown in Table 3.
The experimental factor water-glass of table 3
3 results are with discussing
The range analysis result of 3.1 orthogonal tests
Orthogonal experiment results are shown in Table 4,6.K1, K2, K3 are the content summation of respective horizontal in table, and k1, k2, k3 are corresponding water
The average value of flat K1, K2, K3 content summation, R are extreme difference, i.e. R=Kmax—Kmin.(A) K1 as can be seen from Table 4>K2>
K3、(B)K3>K1>K2、(C)K2>K1>K3, therefore, the optimum condition of extraction subprostrate sophora matrine is A1B3C2, i.e., product is only
Sieve, extraction temperature are 30 DEG C, and ultrasonic time is 30 minutes；(A) K1 as can be seen from Table 6>K3>K2、(B)K1>K3>K2、(C)K2
>K1>K3, therefore, the optimum condition of extraction subprostrate sophora oxymatrine is A1B1C2, i.e. product do not sieve, extraction temperature 20
DEG C, ultrasonic time is 30 minutes.
The results of analysis of variance of 3.2 orthogonal tests
Table 5,7 is the analysis of variance table of orthogonal experiments.S is variance quadratic sum in table, and f is the free degree, and V is equal
Side, F are S and D ratio.In table 5, SB>SA>Sc, so influence of three factors to matrine experimental result is followed successively by extraction temperature
Degree, cross grit number, ultrasonic time.Analysis result shows that extraction temperature has highly significant influence, mistake on content in scope of experiment
Grit number has highly significant influence on content, and ultrasonic time is not notable on content results influence；In table 7, SB>SA>Sc, institute
Extraction temperature is followed successively by with influence of three factors to oxymatrine experimental result, crosses grit number, ultrasonic time.Analysis result table
Bright, extraction temperature has highly significant influence on content in scope of experiment, and crossing grit number has highly significant influence on content, and surpasses
The sound time influences not notable on content results.
3.3 the checking test of result of the test
Under optimum test condition, repeating verification experimental verification, the optimum condition according to extraction matrine is A1B3C2,
I.e. product does not sieve, and extraction temperature is 30 DEG C, and ultrasonic time is 30 minutes, and by this condition through 3 batches of checking tests, with orthogonal examination
It is basically identical to test result, average content 0.6100%；Optimum condition according to extraction oxymatrine is A1B1C2, i.e., product is not
Sieving, extraction temperature are 20 DEG C, and ultrasonic time is 30 minutes, and by this condition through 3 batches of checking tests, with orthogonal experiments base
This is consistent, average content 0.4576%.
The L9 (3 of table 44) orthogonal test matrine result
The variance analysis of table 5
F [0.05] (2,4)=6.94, F [0.1] (2,4)=4.325
The L9 (3 of table 64) orthogonal test oxymatrine result
The variance analysis of table 7
F [0.05] (2,4)=6.94, F [0.1] (2,4)=4.325
The extreme difference and variance analysis that 4 pairs of orthogonal experiments are carried out draw ultrasonic assistant extraction subprostrate sophora matrine most
Good extraction conditions A1B3C2, i.e. product do not sieve, and extraction temperature is 30 DEG C, and ultrasonic time is 30 minutes；Factor B height is notable, because
Plain A is notable, and factor C is not notable, and factor primary and secondary is ordered as B-A-C, extraction rate reached to 0.61%.Extract the optimal of oxymatrine
Condition is A1B1C2, i.e. product do not sieve, and extraction temperature is 20 DEG C, and ultrasonic time is 30 minutes；Factor B height is notable, factor A
Significantly, factor C is not notable, and factor primary and secondary is ordered as B-A-C, extraction rate reached to 0.4576%.
(4) below specific embodiment to the present invention the method for quality control established seedling medicine bicolor and open larynx sword make into
The description of one step.
1 instrument and reagent
1.1 instrument
KQ3200DE type instrument ultrasonic cleaning machines, Kunshan Ultrasonic Instruments Co., Ltd.；FA2004N type electronic balances, on
Hai Jinghai Instrument Ltd.；Standard screen：(10 mesh, aperture 2mm, 20 mesh, aperture 0.84mm), Shangyu city opens up instrument in road ruins
Device factory；Polyvinylidene fluoride microporous filtering film：Organic system aperture is 0.45 μm, Haixing County Asia scavenging material factory on (the grand board in Tianjin)；Micropore is filtered
Film：Aqueous phase aperture is 0.45 μm, upper Haixing County Asia scavenging material factory；Syringe-type miillpore filter：Aperture is 0.45 μm (the grand board in Tianjin)；One
Secondary property uses asepsis injector (band syringe needle) Jiangxi Hongda Medical Equipment Group Corp., Ltd.；Electric-heated thermostatic water bath, West Beijing
City medical machinery factory；Waters2487 type detectors；Model Code515 pumps；Waters high performance liquid chromatographs；Chromatogram
Post：symmetry C18(4.6*250mm,0.5um).
1.2 reagent
Place of production Guilin subprostrate sophora；There is provided by the power medicine company of Guizhou three, opening larynx sword spray and originating in the power pharmacy of Guizhou three has
Limit responsible company；Oxymatrine reference substance (lot number 1170780-201508ID：PH4F-X8FQ Chinese food drug assays are ground
Study carefully institute)；Methanol chromatographically pure and analysis are pure；Chloroform, phosphoric acid, concentrated ammonia liquor are that analysis is pure.
2 methods and result
2.1 testing conditions
Chromatographic column used in experiment is symmetry C18 (4.6*250mm, 0.5um).Liquid phase chromatogram condition oxymatrine
Methanol solution have maximal ultraviolet absorption at 210nm, methanol-water (0.025% phosphoric acid) is mobile phase, methanol-water
(0.025% phosphoric acid) is from 95:5 to 0:100 carry out gradient elution, and detection time is 20 minutes.Wavelength is 210nm, and flow velocity is
0.70ml·min-1, the standard items amount of 24.8 DEG C of column temperature, wherein working curve is 10 μ l, and Guangxi subprostrate sophora sample size is 10 μ l,
It is 50 μ l to open larynx sword spray (adult's medicine) sample size, and it is 50 μ l to open larynx sword spray (children's medicine) sample size, and number of theoretical plate is
2300。
The preparation of 2.2 reference substances
It is appropriate that precision weighs the matrine reference substance through phosphorus pentoxide drying to constant weight, adds methanol to be configured to every 1ml and contains
2.5mg solution.Shake up, be 0.45 μm of syringe-type organic phase filtering with microporous membrane with aperture, produce, put sample bottle label,
It is sealed.
The preparation of 2.3 test liquid samples
Guangxi subprostrate sophora sample powder is taken to sieve (10 mesh), tri- parts of precision weighing Guangxi subprostrate sophora sample 0.55-0.60g,
It is positioned in conical flask with cover.Precision adds chloroform-methanol-liquor ammoniae fortis (40:10:1) mixed solution 50ml, close plug,
Precisely weigh, place 30 minutes.(power 250W, frequency 40kHz, temperature are 25 DEG C) is ultrasonically treated by three tapers
Bottle is placed in ultrasonic processor 40 minutes, again accurately weighed weight, with chloroform-methanol-liquor ammoniae fortis (40:10:1) mix
Close the weight that solution supplies less loss.Shake up, filtered with fast grade filter paper, precision measures 10ml filtrates, 40 DEG C of recovery solution to medicinal extract
Shape, adding methanol about 5ml makes dissolving, is transferred in 10ml volumetric flasks, adds methanol to be shaken up, with 0.45 μm of syringe-type to graduation mark
Filtering with microporous membrane (aqueous phase) (nylon66 fiber), takes subsequent filtrate, produces, put sample bottle label, be sealed.Open larynx sword spray
(adult's medicine), open larynx sword spray (children's medicine) and also operated as stated above.
2.4 methodological study
2.4.1 the investigation of linear relationship
It is appropriate that precision weighs oxymatrine reference substance, is diluted to every 1ml with methanol constant volume and contains 3.7mg, stand-by storage solution
5 concentration of configuration work curve, respectively 18.5 μ gml-1, 37 μ gml-1, 92.5 μ gml-1, 222 μ gml-1, 259
μg·ml-1.With methanol-water (0.25% phosphoric acid) mobile phase, methanol-water (0.25% phosphoric acid) (95:5) -- methanol-water
(0.025% phosphoric acid) (0:100) gradient elution, Detection wavelength 210nm, flow velocity 0.70ml/min, column temperature are 24.8 DEG C of reasons
It is 2300 by plate number, sample size is 10 μ l.It is y=21993x+244505 r=0.9971 to obtain equation of linear regression.
2.4.2 stability test
Same subprostrate sophora test liquid sample is taken in 0,2,4,6,8,10h determination sample oxymatrine concentration, to investigate
Its stability, determine peak area and record, its integrated peak areas RSD values are calculated and show need testing solution extremely for 1.6%
It is few stable in 10h.
2.4.3 precision test
It is 92.5 μ gml that precision, which draws concentration,-1The μ l of oxymatrine reference substance solution 10, liquid chromatograph is injected, repeatedly
Sample introduction 5 times, pass through the integrated peak areas result of calculating, RSD 0.94%.This result shows that the precision of this method is good.
2.4.4 reappearance test
Take same lot number opens 5 parts of larynx sword spray sample, prepares need testing solution according to " 2.3 " laxative remedy, sample size is
10 μ l, determine peak area and record, calculate oxymatrine concentration, its RSD is 1.2%, shows that this method reappearance is good.
2.4.5 average recovery is tested
Using sample-adding absorption method, five parts of the sample of a certain amount of known content is taken, appropriate standard items is added, prepares survey in accordance with the law
They fixed content, calculates its rate of recovery.The rate of recovery is respectively 98.5%, 99.1%, 95.9%, 97.8%, 96.7%.It is average
The rate of recovery is 97.6%, and standard deviation (RSD) is 1.47%.As a result show that the rate of recovery of this method is good.It the results are shown in Table 8.
The oxymatrine average recovery measurement result of table 8
2.5 sample sizes determine
According to testing conditions " 2.1 " item Guangxi subprostrate sophora and adult children are opened with the content of oxymatrine in larynx sword spray
Detected.Measurement result is shown in Table 9.Guangxi subprostrate sophora, adult and children open being averaged for oxymatrine in larynx sword spray and contained
Measure (μ gg-1) it is respectively 42.50 μ gg-1、35.30μg·g-1、1.37μg·g-1；Standard deviation (RSD%) is respectively
0.96%th, 1.70%, 2.34%.
The oxymatrine concentration measurement result of table 9
(remarks：A is Guangxi subprostrate sophora sample, and B is that adult opens larynx sword spray sample, and C is that children open larynx sword spray sample
Product.)
3 conclusions
Mobile phase is used in experiment as methanol-water (0.025% phosphoric acid) 20:80 isocratic elution, separate under the conditions of this
It is ineffective, and detect when opening larynx sword spray (children's medicine) oxymatrine and negative peak occur.Then use mobile phase for methanol-
Water (0.025% phosphoric acid), methanol-water (0.025% phosphoric acid) (5:95) -- methanol-water (0.025% phosphoric acid) (0:100) gradient is washed
De-, separating effect is notable, and negative peak does not occur.
By experimental result it can be seen that the content ratio of oxymatrine opens larynx sword spray (adult's medicine) in the subprostrate sophora of Guangxi
The content of middle oxymatrine is slightly higher, wherein the content for opening larynx sword spray (children's medicine) oxymatrine is minimum, opens the spray of larynx sword
The content of oxymatrine is approximately 1/35 of oxymatrine concentration in the subprostrate sophora of Guangxi in mist agent (children's medicine), is approximately that adult opens
The 1/30 of oxymatrine concentration in larynx sword spray.
This experiment uses infusion process combination ultrasonic extraction, and for extracting method, high performance liquid chromatography is detection method, determines sample
Oxymatrine concentration in product.Subprostrate sophora is measured, larynx sword spray (adult's medicine) is opened, opens the middle oxidation of larynx sword spray (children's medicine)
Matrine content is respectively 42.50 μ gg-1、35.30μg·g-1、1.13μg·g-1.Guangxi subprostrate sophora, adult open the spraying of larynx sword
It is respectively 0.96%, 1.70%, 2.34% that oxymatrine standard deviation (RSD) in larynx sword spray is opened in agent, children.Experiment knot
Fruit shows that this method is accurate, quickly, high sensitivity, favorable reproducibility, can determine subprostrate sophora and open oxymatrine concentration in larynx sword,
The detection method of content that can be controlled as subprostrate sophora and the quality of the pharmaceutical preparations.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (5)
1. a seedling medicine subprostrate sophora and its Chinese medicine preparation method of quality control, it is characterised in that the seedling medicine subprostrate sophora and wherein
Medicine quality of the pharmaceutical preparations control method extracts the active component in medicinal material and preparation；Isolated and purified using a variety of chromatogram means, it is right
Isolated compound carries out assay, Structural Identification；
Extraction and separation detection technique are optimized according to experimental result；
Establish seedling medicine bicolor and open the method for quality control of larynx sword.
2. seedling medicine subprostrate sophora as claimed in claim 1 and its Chinese medicine preparation method of quality control, it is characterised in that the mountain beans
The optimization of the separation method of active component and separation condition in root, including：
Matrine in root of subprostrate sophora and its preparation is extracted using ultrasonic extraction, matrine content, chromatographic column are determined from HPLC
For symmetry C18；Mobile phase is methanol：Water=80 of 0.025% phosphoric acid:20；Flow velocity：0.7mL.min-1, Detection wavelength：
210nm, column temperature：20℃.
3. seedling medicine subprostrate sophora as claimed in claim 1 and its Chinese medicine preparation method of quality control, it is characterised in that described pair point
It is accredited as from obtained compound structure：Matrine, oxymatrine；Matrine content is 0.6100%；Oxymatrine contains
Measure as 0.4576%.
4. seedling medicine subprostrate sophora as claimed in claim 1 and its Chinese medicine preparation method of quality control, it is characterised in that the basis
The method that experimental result optimizes extraction includes：
Utilize ultrasonic extraction subprostrate sophora ingredient matrine, oxymatrine；Matrine extraction conditions is do not sieve, extraction temperature
Spend for 30 DEG C, ultrasonic time is 30 minutes；
For oxymatrine extraction conditions not sieve, extraction temperature is 20 DEG C, and ultrasonic time is 30 minutes.
5. seedling medicine subprostrate sophora as claimed in claim 1 and its Chinese medicine preparation method of quality control, it is characterised in that the foundation
Seedling medicine bicolor and the method for quality control for opening larynx sword, by establishing measure subprostrate sophora and opening oxymatrine concentration in larynx sword
HPLC methods carry out quality control, and the HPLC methods include：
Chloroform-methanol-conc. aq. ammonia=40 are selected by volume：10：1 carries out ultrasonic extraction, using high-efficient liquid phase color law popularization
Oxymatrine concentration is determined, from symmetry C18 chromatographic columns, mobile phase is methanol：Water=95 containing 0.025% phosphoric acid:
5th, methanol：Water=0 containing 0.025% phosphoric acid:100 gradient；Elution flow rate 0.70mlmin-1, Detection wavelength 210nm, post
24.8 DEG C of temperature.
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