CN102419350B - Method for carrying out simultaneous quantitative analysis on four lignan components in Chinese magnoliavine raw material and Chinese magnoliavine extract - Google Patents
Method for carrying out simultaneous quantitative analysis on four lignan components in Chinese magnoliavine raw material and Chinese magnoliavine extract Download PDFInfo
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Abstract
The invention discloses a method for carrying out simultaneous determination on contents of schisandrin, schisantherin, deoxyschizandrin and schisandrin B in a Chinese magnoliavine raw material or a Chinese magnoliavine extract. The method employs means of crushing, sieving, methanol ultrasonic extraction, calibrating volume and filtering to treat schisandra chinensis or a Chinese magnoliavine extract to obtain a sample solution for testing; a high performance liquid chromatography is utilized for simultaneous determination on contents of four lignan components. The method utilizes a reverse direction distribution chromatographic theory to carry out analysis on the sample by gradient elution, under conditions of a column temperature of 25-45 DEG C, mobile phases of methanol and 0.05-0.2% trifluoroacetic acid aqueous solution (methanol and trifluoroacetic acid aqueous solution in a volume ratio of 50:50-100:0), an elution time of 30-60 min, a flow velocity of 0.7-1.5 ml / min and a detection wavelength of 210-280 nm. The quality detection method is simple, rapid, sensitive and accurate.
Description
Technical field
The invention belongs to technical field of traditional Chinese medicines, relate to a kind of quantitative analysis method of traditional Chinese medicine ingredients, be specifically related to a kind of method with schizandrin, Schisantherin C, schizandrin A, deoxyschizandrin content in efficient liquid-phase chromatography method quantitative test schisandra raw material and/or Fructus Schisandrae Chinensis extractum.
Background technology
The fruit of Chinese magnoliavine is commonly called as mountain flower green pepper, sliding weight of steelyard, the medicine fruit of Chinese magnoliavine, face rattan, five plums etc., is the dry mature fruit of magnoliaceae schisandra (Schisandra chinesnsis Baill)." the sweet acid of five tastes skin and flesh, works hard in core, has into taste " carried in Tang etc. " Tang materia medica ", therefore there is the name of the fruit of Chinese magnoliavine.Ancient medical book Cheng Ta Chi Zhu, profound and, meeting and, list in the earliest Sheng Nong's herbal classic top grade, herbal function is the power of strengthening by means of tonics, medical value is high, clinically be mainly used in that chronic cough void is breathed heavily, Tianjin wound is thirsty, the deficiency of Yin is quenched one's thirst, spontaneous perspiration, night sweat, seminal emission, endless diarrhea, palpitaition, insomnia, dreaminess etc., is one of most widely used tonic Chinese medicine of China's traditional Chinese medical science.
Li Shizhen (1518-1593 A.D.) was once called: " there be dividing of north and south five tastes the present, and southern product person look red, and northern product person look black, and people's tonic must be good with northern product person ".It is two kinds that Chinese Pharmacopoeia version in 2000 is recorded fructus schisandrae and kadsura longepedunculata respectively, and the fruit of Chinese magnoliavine is only refered in particular to the dry mature fruit of fructus schisandrae, and kadsura longepedunculata refers in particular to the dry mature fruit of schisandra chinensis.
Fructus schisandrae is famous medicinal plant, the mountain areas such as NATURAL DISTRIBUTION Xiaoxing ' anling Mountains of Heilongjiang Province, Wanda Mountain, Zhang Guangcai ridge, master ridge.Main product is in the schisandra fruit dry product on the ground such as northeast and Hebei, and commodity custom is called " fructus schisandrae ".Fresh fructus schisandrae fruit be the berry of hunting pink, and succulence, taste are sour and micro-puckery, have Chinese prickly ash smell.Fruit drink is the raw material of processing natural health drink; The black aubergine of dry fruit, has crape wrinkle, is traditional Chinese crude drug, and central nervous system, respiratory system to human body have excitation, and heart, liver, blood pressure are had to regulating action, and eyesight, hearing to people are strengthened function; Promote choleresis, improve antibacterial ability.
Fructus schisandrae principal ingredient is lignanoid, volatile oil, triterpene, organic acid, vitamin and polysaccharide etc.Wherein Lignanoids compounds is mainly cyclohexyl biphenyl octene compounds, and modern medicine shows, lignanoids is topmost drug activity composition in the fruit of Chinese magnoliavine, and central nervous system, cardiovascular system, digestive system etc. are had to multiple pharmacological effect.At present, for the Study on Physiological Activity of lignanoid more be to the effective composition of liver.To 7 kinds of effective constituent pharmacological researches of the fruit of Chinese magnoliavine and clinical proof, all there is the effect that reduces hepatitis serum GPT levels, wherein wuweizi ester B effect is the strongest, is secondly alcohol second, the third element, second element, first element, ester first and alcohol first.Its chemical structural formula is as follows respectively:
(I) wuweizi ester B
(II) wuweizi alcohol B
(III) schisandrin C
(IV) deoxyschizandrin:
(V) schizandrin A
(VI) Schisantherin C
(VII) schizandrin
It is mainly to hepatocellular protective effect that enzyme mechanism falls in the fruit of Chinese magnoliavine; can promote the hepatocellular reparation of damage and suppress liver cell lesion, make cell membrane that certain functional occur and change, permeability reduces; reduce thereby make to drain to transaminase in blood plasma, to the synthetic inhibiting effect that there is no of transaminase.Find that in addition fruit of Chinese magnoliavine lignans also has antioxidation, the strongest with the antioxidation activity of deoxyschizandrin.
The method for separating and detecting of fruit of Chinese magnoliavine lignanoid chemical composition mainly contains spectrophotometric method, Capillary Electrochromatography and high performance liquid chromatography, and wherein applying maximum analytical approachs is high performance liquid chromatography.
CN 101373182 A provide a kind of quality determining method of Chinese medicine fructus schisandrae, after its utilization is pulverized, is sieved, the ultrasonic extraction of methyl alcohol, constant volume, filtration treatment means process fructus schisandrae, obtain the finger-print of lignan component and organic acid composition by electrospray ionization mass spectrum, determine lignanoid contained in fructus schisandrae and organic acid kind according to its finger-print, utilize the content of high effective liquid chromatography for measuring Lignanoids compounds, comprehensive its finger-print and assay data, can evaluate the quality of fructus schisandrae.
CN 1866026 A disclose the quality detection method of Kadsura japonica alcohol-extractum.Described Kadsura japonica alcohol-extractum adopts following at least one method implementation quality prosecution: with dry cream weighing scale, be no less than 5.6% with its total lignan content of determined by ultraviolet spectrophotometry (with schizandrin A weighing scale); Or be no less than 0.7% with its Schisantherin C content of high effective liquid chromatography for measuring; Or be no less than 1.0% with its schizandrin A content of high effective liquid chromatography for measuring.The present invention, by the content detection control to Kadsura japonica alcohol-extractum effective constituent in production process, can control the inherent quality of the medicine of being made up of Kadsura japonica alcohol-extractum effectively.
At present mainly that even measure same component for different analytical instrument, analysis condition also can not be completely applicable, need to adjust or again grope analytical approach taking single or certain several composition as main about the quality control of the fruit of Chinese magnoliavine.And report all imperfections for the method research of determining best efficient liquid phase chromatographic analysis condition in different analytical instrument at present, not yet had about being that (150 × 4.6mm, 5 μ are m) taking the relevant report of methyl alcohol-0.1% trifluoroacetic acid aqueous solution schizandrin, Schisantherin C, schizandrin A and deoxyschizandrin in mobile phase Simultaneous Determination schisandra raw material and/or Fructus Schisandrae Chinensis extractum for ZORBAX Extend-C18 with anti-phase distribution post.
Summary of the invention
At present, not yet have about being that (150 × 4.6mm, 5 μ are m) taking the relevant report of methyl alcohol-0.1% trifluoroacetic acid aqueous solution schizandrin, Schisantherin C, schizandrin A and deoxyschizandrin in mobile phase Simultaneous Determination schisandra raw material and/or Fructus Schisandrae Chinensis extractum for ZORBAX Extend-C18 with anti-phase distribution post.Therefore object of the present invention is just to provide schizandrin, Schisantherin C, schizandrin A and deoxyschizandrin method in a kind of easy, quick, sensitive, accuracy is high, degree of accuracy is good Simultaneous Determination schisandra raw material and/or Fructus Schisandrae Chinensis extractum.
In Simultaneous Determination schisandra raw material of the present invention and/or Fructus Schisandrae Chinensis extractum, the method for schizandrin, Schisantherin C, schizandrin A and deoxyschizandrin, comprises the steps:
(1) test sample solution preparation
Test sample solution I: the low-carbon alcohols of selection 50%～100%, for extracting solvent, is pulverized fructus schisandrae, adds described extraction solvent after being dried, and carries out ultrasonic extraction, through micro-pore-film filtration acquisition sample solution I.Or:
Test sample solution II: through alcohol extracting, the medicinal extract after drying under reduced pressure dissolves by low-carbon alcohols, obtains sample solution II through micro-pore-film filtration.
The low-carbon alcohols of the preferred 1-5 of a described low-carbon alcohols carbon atom, as methyl alcohol, ethanol, propyl alcohol or its potpourri, more preferably adopts methyl alcohol.
Being dried described in test sample solution I, its baking temperature is preferably 40-70 DEG C, further preferred 50-60 DEG C, most preferably 55 DEG C; Its drying time preferred 6-48h, further preferred 12～24h.
Dried Fructus Schisandrae Chinensis powder described in test sample solution I minces, and preferably and then crosses 30～80 mesh sieves.
The mixing ratio of described Chinese Magnolivine Fruit and low-carbon alcohols is: (0.25～0.5g) Chinese Magnolivine Fruit/(25～50mL) low-carbon alcohols.
Described ultrasonic extraction time is 10～90min, preferably 30～60min, preferred room temperature～60 DEG C of ultrasonic extraction temperature, further preferably 20～40 DEG C.
Described medicinal extract preferably, in the ratio constant volume of (150～250mg) medicinal extract/10ml, filters after constant volume after dissolving by low-carbon alcohols.
Described filtration preferably adopts aperture to be less than the microporous barrier filter membrane of 0.60 μ m, more preferably adopts aperture to be less than the microporous barrier filter membrane of 0.45 μ m.
(2) mensuration of fruit of Chinese magnoliavine lignanoid content
High performance liquid chromatography experiment condition: ZORBAX Extend-C18 liquid-phase chromatographic column (150 × 4.6mm, 5 μ are m); 25 DEG C～45 DEG C of column temperatures; Mobile phase: low-carbon alcohols and 0.05%～0.2% trifluoroacetic acid aqueous solution; Elution time 30～60min; Flow velocity 0.7～1.5ml/min; Detect wavelength 210～280nm; Sample feeding amount 10～50 μ l.
The volume ratio of described low-carbon alcohols and trifluoroacetic acid aqueous solution is 50: 50～100: 0, preferably 50: 50～80: 20.Within 100: 0 of the present invention, represent that mobile phase is only low-carbon alcohols, do not comprise trifluoroacetic acid aqueous solution, but the present invention preferably includes trifluoroacetic acid aqueous solution, the content that is trifluoroacetic acid aqueous solution is greater than 0, low-carbon alcohols content is less than 100, and then the volume ratio of low-carbon alcohols and trifluoroacetic acid aqueous solution is less than 100: 0.
The mensuration of lignanoid's content: (I) accurately take the schizandrin 5mg after drying under reduced pressure, Schisantherin C 1.4mg, schizandrin A 2mg, deoxyschizandrin 3.6mg, dissolves by chromatographic grade low-carbon alcohols, is settled to 10ml, through micro-pore-film filtration, preparation standard reference substance solution; (II) then the standard reference material solution of step (I) is carried out to stratographic analysis, sample introduction 2 μ l respectively, 5 μ l, 15 μ l, 25 μ l, 35 μ l, 45 μ l, calculate gained concentration as horizontal ordinate taking sample introduction 25 μ l, and going out peak area is ordinate drawing standard curve; (III) the described test sample solution of being prepared by step (1) carries out stratographic analysis; (IV) according to the content of four kinds of fruit of Chinese magnoliavine lignanoids in typical curve calculating test sample.
The present invention chooses specific mobile phase: low-carbon alcohols and 0.05%～0.2% trifluoroacetic acid aqueous solution, specific ZORBAX Extend-C18 liquid-phase chromatographic column, and specific analysis condition, when having realized schizandrin in schisandra raw material and Fructus Schisandrae Chinensis extractum, Schisantherin C, schizandrin A and/or deoxyschizandrin, measure, solved the technical barrier that cannot simultaneously measure schizandrin, Schisantherin C, schizandrin A and deoxyschizandrin in schisandra raw material and Fructus Schisandrae Chinensis extractum in prior art.
In the determination step of the fruit of Chinese magnoliavine of the present invention lignanoid content, the configuration standard reference substance adopting also obtains typical curve, subsequently sample is carried out stratographic analysis and calculates concrete component concentration according to typical curve, it is the prior art of affiliated technical field, person of ordinary skill in the field has the ability to obtain this technology and applies it in the determination step of fruit of Chinese magnoliavine lignanoid content, this is person of ordinary skill in the field's required skill, therefore the present invention is no longer with regard to the determination step outside described particular analysis condition, sample introduction step and calculation procedure are described in detail.
According to assay method of the present invention, learn as calculated, schisandra raw material Zhong Ge lignanoid content range is respectively schizandrin 4～8mg/g, Schisantherin C 0.2～0.5mg/g, schizandrin A 0.5～1mg/g, deoxyschizandrin 2.5～3mg/g, and Fructus Schisandrae Chinensis extractum Zhong Ge lignanoid content is respectively schizandrin 9～12mg/g, Schisantherin C 0.6～1mg/g, schizandrin A 1～2mg/g, deoxyschizandrin 4～6mg/g.Therefore, the scope drawing according to described result of calculation, after repeatedly measuring, can give up the not concrete content within the scope of this obtaining, thereby remainder averages and obtains mean value, and then complete the quantitative test of four kinds of lignanoid's content in schisandra raw material and Fructus Schisandrae Chinensis extractum.
Sample pretreatment process of the present invention is simple, and extraction efficiency is high.Can be easy, quick, sensitive, accurate, stable time, measure the content of four kinds of lignanoids in schisandra raw material and/or Fructus Schisandrae Chinensis extractum.
Brief description of the drawings
The ultraviolet-visible absorption spectroscopy figure of Fig. 1, four kinds of fruit of Chinese magnoliavine lignanoids
Fig. 2, elution program 0～60min:2%～100% methanol-water solution
Fig. 3, elution program 0～30min:50%～80% methanol-water solution 30～40min:80%～100% methanol-water solution
Fig. 4, elution program are: 0～5min:55% methanol-water solution, 5～20min:55%～60% methanol-water solution, 20～25min:60%～62% methanol-water solution, 25～60min:75% methanol-water solution
Fig. 5, elution program are: 0～15min, 58～63%; 15～20min, 63～65%; 20～55min, 65～78%
Fig. 6, the impact of column temperature on retention time
The impact of the flow velocity of Fig. 7, mobile phase on retention time
Fig. 8, mobile phase are the chromatogram of methyl alcohol-0.1% trifluoroacetic acid solution
Fig. 9, quantitative test canonical plotting
Embodiment
(1) selection of chromatographic column and mobile phase
Reversed-phase liquid chromatography pattern, first-selected C18 post are selected in the analysis of general medicinal material or compound total extract.For general reversed-phase column, with methanol-water or acetonitrile water, as mobile phase, fruit of Chinese magnoliavine lignanoid dissolves in methyl alcohol, ethanol, acetonitrile, is insoluble in water, can be dissolved in sherwood oil, but solubleness is smaller.General methanol/water or acetonitrile/water are that mobile phase is done stratographic analysis, compare from several respects such as separating effect and peak types, and employing methanol/water is mobile phase.
(2) selection of detection wavelength
Get schizandrin, Schisantherin C, schizandrin A and the analysis of deoxyschizandrin standard solution sample introduction, within the scope of 190～700nm, carried out uv scan.All there is good absorption peak at 218nm and tetra-kinds of components of 254nm as seen from uv absorption spectra (Fig. 1), because the following mobile phase of 230nm has absorption, can cause unstability of base line, thereby affect quantitative test.And solvent disturbs minimum under this wavelength, be that the first-selection of simultaneously measuring four kinds of lignanoid's content detects wavelength so select 254nm.
(3) selection of gradient elution program
Change gradually gradient elution program according to going out peak effect, compare from several respects such as retention time, separating effect, chromatographic peak types, determine suitable gradient.
(4) selection of column temperature
The chromatogram of gained sample under more different column temperature conditions, select the suitable column temperature composition that makes to coexist in component to be measured and sample to obtain optimal separation, and analysis efficiency is high.
(5) selection of flow velocity
Get different flow rate of mobile phase and done the stratographic analysis of test sample, compare from several respects such as retention time, separating effect, chromatographic peak types, determine suitable flow velocity.
(6) selection of modifier
Add the different modifying agent of debita spissitudo, compare from peak type improvement situation, determine modifying agent.
To be the test sample prepared taking Fructus Schisandrae Chinensis extractum establish as object above-described analysis condition, there is the interference of many impurity components, in the practical application of method, run into other samples and be mostly this complex component, utilize this method of testing generally can make component to be measured effectively be separated with interfering component, adjustment analysis condition combination that to some extent can be suitable in difference at testing tool, realizes quantitative test quickly and accurately as proportion of mobile phase, column temperature, flow velocity and gradient elution program etc.
Determining of embodiment 1:HPLC analysis condition
The present embodiment adopts Agilent highly effective liquid phase chromatographic system: quaternary pump G1311A, online degasser G1379A, and automatic sampler G1329A, variable wavelength detects 1314A; Reagent: it is special that methyl alcohol and acetonitrile are HPLC, water is deionized water.
1. the selection of chromatographic column and mobile phase
Due to the complicated variety of traditional Chinese medicine ingredients, determining of analytical approach first will be for its feature, find chromatogram mode and best column system, reversed-phase liquid chromatography pattern is selected in the analysis of general medicinal material or compound total extract, set up the analytical approach that is inverted to the anti-phase full concentration range of the non-water of Chinese medicine high performance liquid chromatography from pure water, ionic and strong polar compound does not retain, non-polar compound can flow out under the anti-phase concentration of non-water from pillar, and on reverse-phase chromatographic column, obtains separation between the compound at these the two poles of the earth.For reverse-phase chromatography, first-selected C18 post.For general reversed-phase column, with methanol-water or acetonitrile water, as mobile phase, fruit of Chinese magnoliavine lignanoid dissolves in methyl alcohol, ethanol, acetonitrile, is insoluble in water, can be dissolved in sherwood oil, but solubleness is smaller.General methanol/water or acetonitrile/water are that mobile phase is done stratographic analysis, compare from several respects such as separating effect and peak types, and employing methanol/water is mobile phase.
2. detect the selection of wavelength
Get schizandrin, Schisantherin C, schizandrin A and the analysis of deoxyschizandrin standard solution sample introduction, within the scope of 190～700nm, carried out uv scan.All there is good absorption peak at 218nm and tetra-kinds of components of 254nm as seen from uv absorption spectra (Fig. 1), because the following mobile phase of 230nm has absorption, can cause unstability of base line, thereby affect quantitative test.And solvent disturbs minimum under this wavelength, be that the first-selection of simultaneously measuring four kinds of lignanoid's content detects wavelength so select 254nm.
3. the selection of gradient elution program program
Change gradient elution program, compare from several respects such as retention time, separating effect, chromatographic peak types, determine suitable gradient.
First use 2% methanol aqueous solution as initial concentration, rise to 100% at 60min methanol concentration, fruit of Chinese magnoliavine test sample is carried out to gradient elution, after composing as seen peak and mainly concentrate on 40min from chromatogram (Fig. 2).
Because three target peak separating effects are poor below, therefore consider to reduce concentration gradient, extend appearance time, gradient elution program is adjusted to 0～30min:50%～80% methanol-water solution, 30～40min:80%～100% methanol-water solution.From chromatogram (Fig. 3) can find out first aim peak and the peak-to-peak degree of separation of first impurity very large, baseline wander is too large, rear three target peak analytical effects are bad.
Consider to improve methyl alcohol initial concentration, reduce concentration gradient, and by going out Feng Chu and slow down the change of concentration.Adjusting gradient elution program is: 0～5min:55% methanol-water solution, 5～20min:55%～60% methanol-water solution, 20～25min:60%～62% methanol-water solution, 25～60min:75% methanol-water solution.Better (Fig. 4) of separating effect under this gradient elution, first aim peak can continue reach, and the 4th peak separating effect is slightly poor.
Consideration reduces wash-out concentration herein again, readjusts gradient elution program and is: 0～15min, 58～63%; 15～20min, 63～65%; 20～55min, 65～78%; 56～62min, 95%.Good separating effect (Fig. 5), but there is conditions of streaking at peak, and baseline wander is larger.
4. the selection of column temperature
Respectively 25 DEG C of column temperatures, 30 DEG C, 35 DEG C, 40 DEG C, under 45 DEG C of conditions, sample solution is done to stratographic analysis, within the scope of 20 DEG C～40 DEG C, chromatographic peak all can be separated preferably, variation diagram (Fig. 6) from the retention time of target components along with column temperature, column temperature raises, and retention time significantly moves forward.Simultaneously the degree of separation of impurity peaks changes also and obviously changes with column temperature, and in the time of 35 DEG C, impurity peaks has reached good separation, 40 DEG C and 45 DEG C of degree of separation slightly poor.Comprehensive analysis efficiency and separating effect are considered, select 35 DEG C as stratographic analysis column temperature.
5. the selection of flow rate of mobile phase
Respectively at flow rate of mobile phase 0.5ml/min, 0.75ml/min, 1.0ml/min, 1.25ml/min, has done the stratographic analysis of sample when 1.5ml/min.In the time that flow velocity is too low, although each component can reach good separation, chromatographic peak is wide, and peak type is poor, and analysis efficiency is low; In the time that flow velocity is too high, although it is fast to go out peak, analysis efficiency is high, and degree of separation is low, and some component can not be separated preferably.Variation diagram (Fig. 7) from the retention time of target components with flow rate of mobile phase, the retention time of target components is along with the increase of flow velocity obviously reduces, in the time that flow velocity is 1.0ml/min, component to be measured in sample is separated preferably, consider based on several respects such as retention time, separating effect, chromatographic peak types, select the detection flow velocity that 1.0ml/min is mobile phase.
6. the selection of modifier
The reason of peak hangover is generally because Si-OH hydroxyl is not reacted with solute by complete bonding on silica matrix, the conventional methylic material of little molecule carries out bonding sealing silicon hydroxyl to silica gel again, or in mobile phase, add the material to hydroxyl sensitivity, residual hydroxy groups is sheltered.Investigate and in water, added the impact on peak type of 0.1% phosphoric acid, acetic acid and trifluoroacetic acid, add phosphoric acid and acetic acid little to the improvement of peak type, and add trifluoroacetic acid to improve the conditions of streaking (Fig. 8) of chromatographic peak, trifluoroacetic acid volatility is good simultaneously, do not hinder pillar, so selective flow is methyl alcohol and 0.1% trifluoroacetic acid aqueous solution mutually.
Embodiment 2: quantitative correlation test
The standard solution of preparation is changed to sample size by above-mentioned analysis condition and carry out stratographic analysis, the chromatographic peak area obtaining and solution concentration are done to linear regression analysis, the regression curve obtaining has good linear relationship (Fig. 9).
Embodiment 3: instrument precision experiment
Under optimum chromatogram condition, the parallel sample introduction of accurate absorption reference substance solution 25 μ l 10 times, can be found out by measurement result, the relative deviation of four kinds of materials is all less than 3.0%, and the reliability of measurement result can meet analysis requirement.
Embodiment 4: the recovery and repeated experiment
Getting respectively standard items 0.5mg adds in 0.100g medicinal extract, preparation test sample solution is analyzed, three calculate recovery rates of parallel laboratory test, and the recovery of fruit of Chinese magnoliavine lignanoid is respectively schizandrin 102.5%, Schisantherin C 106.6%, schizandrin A 99.4%, deoxyschizandrin 99.6%, relative standard deviation is respectively schizandrin 2.9%, Schisantherin C 4.6%, schizandrin A 2.6%, deoxyschizandrin 1.9%, measurement result meets analyzes requirement.
Embodiment 5:
Test sample solution I: the fruit of Chinese magnoliavine is pulverized, at 55 DEG C of dry 12h, crosses 30 mesh sieve, accurately takes equivalent Chinese Magnolivine Fruit 0.25g, adds 25mL methyl alcohol, weighs, and at 25 DEG C of ultrasonic 30min, takes out, and weighs and supplies weight with methyl alcohol, shakes up.
Test sample solution II: precision takes medicinal extract 183mg after alcohol extracting, drying under reduced pressure, with methyl alcohol dissolving, and is settled to 10ml, and 0.45 μ m filter membrane filters and get final product.
High performance liquid chromatography experiment condition: ZORBAX Extend-C18 liquid-phase chromatographic column (150 × 4.6mm, 5 μ are m); 35 DEG C of column temperatures; Mobile phase: methyl alcohol and 0.1% trifluoroacetic acid aqueous solution; Gradient elution program is: be 0～15min, and 58%A～63% linear gradient; 15～20min, 63%A～65%A linear gradient; 20～55min, 65%A～78%A linear gradient; Flow velocity 1ml/min; Detect wavelength 254nm; Sample feeding amount 25 μ l.
Stratographic analysis obtains schisandra raw material Zhong Ge lignanoid content and is respectively schizandrin 5.8mg/g, Schisantherin C 0.38mg/g, schizandrin A 0.75mg/g, deoxyschizandrin 2.6mg/g by marking curve calculation, and Fructus Schisandrae Chinensis extractum Zhong Ge lignanoid content is respectively schizandrin 10.7mg/g, Schisantherin C 0.72mg/g, schizandrin A 1.38mg/g, deoxyschizandrin 0.45mg/g.
Embodiment 6:
Test sample solution I: the fruit of Chinese magnoliavine is pulverized, at 55 DEG C of dry 12h, crosses 30 mesh sieve, accurately takes equivalent Chinese Magnolivine Fruit 0.5g, adds 30mL methyl alcohol, weighs, and at 35 DEG C of ultrasonic 45min, takes out, and weighs and supplies weight with methyl alcohol, shakes up.
Test sample solution II: precision takes medicinal extract 205mg after alcohol extracting, drying under reduced pressure, with methyl alcohol dissolving, and is settled to 10ml, and 0.45 μ m filter membrane filters and get final product.
High performance liquid chromatography experiment condition: ZORBAX Extend-C18 liquid-phase chromatographic column (150 × 4.6mm, 5 μ are m); 35 DEG C of column temperatures; Mobile phase: methyl alcohol and 0.1% trifluoroacetic acid aqueous solution; Gradient elution program is: be 0～15min, and 58%A～63% linear gradient; 15～20min, 63%A～65%A linear gradient; 20～55min, 65%A～78%A linear gradient; Flow velocity 1ml/min; Detect wavelength 254nm; Sample feeding amount 25 μ l.
Stratographic analysis obtains schisandra raw material Zhong Ge lignanoid content and is respectively schizandrin 5.6mg/g, Schisantherin C 0.39mg/g, schizandrin A 0.68mg/g, deoxyschizandrin 2.4mg/g by marking curve calculation, and Fructus Schisandrae Chinensis extractum Zhong Ge lignanoid content is respectively schizandrin 11.3mg/g, Schisantherin C 0.92mg/g, schizandrin A 1.31mg/g, deoxyschizandrin 0.49mg/g.
Applicant's statement, the present invention illustrates detailed process equipment and process flow process of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned detailed process equipment and process flow process, do not mean that the present invention must rely on above-mentioned detailed process equipment and process flow process and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, and the selections of the equivalence replacement to the each raw material of product of the present invention and the interpolation of auxiliary element, concrete mode etc., within all dropping on protection scope of the present invention and open scope.
Claims (13)
1. a method for schizandrin, Schisantherin C, schizandrin A and deoxyschizandrin in Simultaneous Determination schisandra raw material or Fructus Schisandrae Chinensis extractum, comprises the steps:
(1) test sample solution preparation
Test sample solution I: the low-carbon alcohols of selection 50%～100% is for extracting solvent, fructus schisandrae is pulverized, after dry, add described extraction solvent, the mixing ratio of described Chinese Magnolivine Fruit and low-carbon alcohols is (0.25～0.5g) Chinese Magnolivine Fruit/(25～50mL) low-carbon alcohols, carry out ultrasonic extraction, obtain sample solution I through micro-pore-film filtration; Or
Test sample solution II: through alcohol extracting, the medicinal extract after drying under reduced pressure dissolves by low-carbon alcohols, described medicinal extract in the ratio constant volume of (150～250mg) medicinal extract/10mL, filters after constant volume after dissolving by low-carbon alcohols, obtains sample solution II through micro-pore-film filtration;
(2) mensuration of fruit of Chinese magnoliavine lignanoid content
High performance liquid chromatography experiment condition: 150 × 4.6mm, 5 μ m ZORBAX Extend-C18 liquid-phase chromatographic columns; 25 DEG C～45 DEG C of column temperatures; Mobile phase: low-carbon alcohols and 0.05%～0.2% trifluoroacetic acid aqueous solution; Elution time 30～60min; Flow velocity 0.7～1.5mL/min; Detect wavelength 210～280nm; Sample feeding amount 10～50 μ L;
The mensuration of lignanoid's content: (I) accurately take the schizandrin 5mg after drying under reduced pressure, Schisantherin C 1.4mg, schizandrin A 2mg, deoxyschizandrin 3.6mg, dissolves by chromatographic grade low-carbon alcohols, is settled to 10mL, through micro-pore-film filtration, preparation standard reference substance solution; (II) then the standard reference material solution of step (I) is carried out to stratographic analysis, sample introduction 2 μ L respectively, 5 μ L, 15 μ L, 25 μ L, 35 μ L, 45 μ L, calculate gained concentration as horizontal ordinate taking sample introduction 25 μ L, and going out peak area is ordinate drawing standard curve; (III) the described test sample solution of being prepared by step (1) carries out stratographic analysis; (IV) according to the content of four kinds of fruit of Chinese magnoliavine lignanoids in typical curve calculating test sample.
2. the method for claim 1, is characterized in that, dry described in test sample solution I, and its baking temperature is 40-70 DEG C; Be 6-48h its drying time;
Dried Fructus Schisandrae Chinensis powder described in test sample solution I minces, and and then crosses 30～80 mesh sieves;
Described ultrasonic extraction time is 10～90min, and ultrasonic extraction temperature is room temperature～60 DEG C.
3. method as claimed in claim 2, is characterized in that, dry described in test sample solution I, and its baking temperature is 50-60 DEG C; Be 12～24h drying time;
Described ultrasonic extraction time is 30～60min, and ultrasonic extraction temperature is 20～40 DEG C.
4. method as claimed in claim 3, is characterized in that, dry described in test sample solution I, and its baking temperature is 55 DEG C.
5. the method as described in one of claim 1-3, is characterized in that, described low-carbon alcohols is methyl alcohol, ethanol, propyl alcohol or its potpourri.
6. method as claimed in claim 5, is characterized in that, described low-carbon alcohols is methyl alcohol.
7. the method as described in one of claim 1-4, is characterized in that, described filtration adopts aperture to be less than the microporous barrier filter membrane of 0.60 μ m.
8. method as claimed in claim 7, is characterized in that, described filtration adopts aperture to be less than the microporous barrier filter membrane of 0.45 μ m.
9. the method as described in one of claim 1-5, is characterized in that, states in step (1), and extracting solvent is 80% methyl alcohol, and ultrasonic time is 40min.
10. the method as described in one of claim 1-6, is characterized in that, in described step (2), column temperature is 35 DEG C, and mobile phase is methyl alcohol and 0.1% trifluoroacetic acid aqueous solution.
11. methods as described in one of claim 1-7, is characterized in that, in described step (2), the volume ratio of described low-carbon alcohols and trifluoroacetic acid aqueous solution is 50: 50～100: 0.
12. methods as claimed in claim 11, is characterized in that, in described step (2), the volume ratio of described low-carbon alcohols and trifluoroacetic acid aqueous solution is 50: 50～80: 20.
13. methods as described in one of claim 1-8, it is characterized in that, by the rejection of data of content outside following ranges in measurement result repeatedly: schisandra raw material Zhong Ge lignanoid content range is respectively schizandrin 4～8mg/g, Schisantherin C 0.2～0.5mg/g, schizandrin A 0.5～1mg/g, deoxyschizandrin 2.5～3mg/g, or Fructus Schisandrae Chinensis extractum Zhong Ge lignanoid content is respectively schizandrin 9～12mg/g, Schisantherin C 0.6～1mg/g, schizandrin A 1～2mg/g, deoxyschizandrin 4～6mg/g, and by average all the other measurement results, obtain schizandrin, Schisantherin C, the mean value of schizandrin A and deoxyschizandrin.
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