CN104749306B - A kind of method of the finger printing building Herba Epimedii extract - Google Patents
A kind of method of the finger printing building Herba Epimedii extract Download PDFInfo
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Abstract
The invention belongs to field of traditional Chinese, be specifically related to the construction method of the finger printing of the extract of a kind of epimedium herb。The method of the finger printing of structure Herba Epimedii extract of the present invention develops the construction method suitable in Herba Epimedii extract targetedly, it is determined that its total fingerprint characteristic, obtains standard finger-print。This method is easy, stable, precision is high, reproducible, can be effectively used to the quality control of Herba Epimedii extract。
Description
Technical field
The invention belongs to field of traditional Chinese, the method being specifically related to the finger printing of a kind of extract building epimedium herb。
Background technology
Herba Epimedii is the plant of Berberidaceae Epimedium, it is the parts of generic medicinal plants of the traditional Chinese medical science, is generally the dried leaves of Herba Epimedii (EpimediumbrevicornuMaxim.), Epimedium sagittatum (Epimediumsagittatum (Sieb.etZucc) Maxim.), E. Pubescens (EpimediumpubescensMaxim.) or Herba Epimedii (EpimediumkorenumNakai)。
Pharmacological research shows, Herba Epimedii has the effect of kidney-replenishing, bone and muscle strengthening, wind-damp dispelling。And growing along with Chinese medicine extraction technology, the extract of Herba Epimedii, as the processing product of epimedium herb, is increasingly becoming the important source material of numerous Chinese medicine preparation。
At present, through resin purification gained after Herba Epimedii extract many employings solvent extraction, and there is good medical value。But it is comparatively single to be limited to the detection means about Herba Epimedii extract, substantially based on the content of testing index composition, the method also making the quality control about Herba Epimedii extract is comparatively single。But, owing to Chinese medicine extract is as the processing product of Chinese crude drug, the complex system being made up of numerous compounds, it is only the quality condition being difficult to comprehensively reflect Chinese medicine extract by the detection of single or several compositions, this also becomes a difficult problem for Chinese medicine quality detection。
Chinese medicine fingerprint technology is a kind of modernization key technology controlling Chinese medicine quality, in the world it have been recognised that, what it was maximum is characterized in that the overall chemical information that can comprehensively reflect Chinese medicine, thus reach control Chinese medicine quality purpose。Therefore, setting up corresponding finger printing for Chinese crude drug, Chinese medicine extract, Chinese medicine preparation, be effective means present stage controlling Chinese medicine quality, also the quality control for solving Herba Epimedii extract provides effective way。
Summary of the invention
For this, the technical problem to be solved is in that a kind of method providing finger printing building Herba Epimedii extract, and utilizes fingerprint pattern technology that the quality of described Herba Epimedii extract is control effectively。
A kind of method that present invention also offers finger printing building Herba Epimedii extract, it includes following operating procedure:
The preparation of need testing solution: accurately weighed Herba Epimedii extract 0.05-0.15 weight portion, put in conical flask, accurate addition volumetric concentration is the methanol solution 15-50 parts by volume of 35%-100%, weighed, ultrasonic 10-50min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 35%-100%, cross microporous filter membrane, take subsequent filtrate, to obtain final product；
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.05-0.50mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol；
High-efficient liquid phase chromatogram condition: chromatographic column is with octadecylsilane chemically bonded silica for filler；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 0.05-2%；Column temperature: 20 DEG C-40 DEG C；Detection wavelength: 260nm-280nm；Flow velocity: 0.5-1.5ml min-1, carrying out gradient elution, concrete gradient elution program is: from 0-10min, mobile phase A: Mobile phase B is 15%:85%；From 10-20min, mobile phase A: Mobile phase B is by 15%:85% → 20%:80%；From 20-45min, mobile phase A: Mobile phase B is by 20%:80% → 24%:76%；From 45-65min, mobile phase A: Mobile phase B is 24%:76%；From 65-70min, mobile phase A: Mobile phase B is by 24%:76% → 30%:70%；From 70-90min, mobile phase A: Mobile phase B is by 30%:70% → 40%:60%；From 90-105min, mobile phase A: Mobile phase B is by 40%:60% → 90%:10%；
Sample determination method: draw need testing solution and reference substance solution 2-25 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing；
The relation that relation is g/mL of described weight portion and parts by volume。
The described finger printing being built the Herba Epimedii extract obtained by said method there are 16 common characteristic peaks, contrast with reference substance finger printing reference substance appearance time, determine that No. 6 peak is Epimedin A chromatographic peak by appearance time order, No. 7 peak is Epimedin B chromatographic peak, No. 8 peak is epimedin chromatographic peak, No. 9 peak is icariin chromatographic peak, No. 16 peak is baohuoside Ⅰ chromatographic peak, with No. 9 peak for reference to peak, 16 chromatographic peak retention time ratios are followed successively by: 0.110-0.140, 0.490-0.520, 0.525-0.555, 0.560-0.600, 0.810-0.840, 0.850-0.880, 0.890-0.920, 0.940-0.970, 1.000, 1.290-1.320, 1.350-1.380, 1.450-1.480, 1.540-1.570, 1.595-1.625, 1.605-1.635, 1.670-1.700。
Further, the method for the described finger printing building Herba Epimedii extract, including following operating procedure:
The preparation of need testing solution: accurately weighed Herba Epimedii extract 0.1 weight portion, put in conical flask, accurate addition volumetric concentration is methanol solution 25 parts by volume of 50%, weighed, ultrasonic 15min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 50%, cross microporous filter membrane, take subsequent filtrate, to obtain final product；
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.1mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol；
High-efficient liquid phase chromatogram condition: chromatographic column is with octadecylsilane chemically bonded silica for filler；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 0.1%；Column temperature: 30 DEG C；Detection wavelength: 270nm；Flow velocity: 1.0ml min-1, carrying out gradient elution, concrete gradient elution program is: from 0-10min, mobile phase A: Mobile phase B is 15%:85%；From 10-20min, mobile phase A: Mobile phase B is by 15%:85% → 20%:80%；From 20-45min, mobile phase A: Mobile phase B is by 20%:80% → 24%:76%；From 45-65min, mobile phase A: Mobile phase B is 24%:76%；From 65-70min, mobile phase A: Mobile phase B is by 24%:76% → 30%:70%；From 70-90min, mobile phase A: Mobile phase B is by 30%:70% → 40%:60%；From 90-105min, mobile phase A: Mobile phase B is by 40%:60% → 90%:10%；
Sample determination method: draw need testing solution and reference substance solution 10 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing。
Detection gained Herba Epimedii extract finger printing there are 16 common characteristic peaks, contrast with reference substance finger printing reference substance appearance time, determine that No. 6 peak is Epimedin A chromatographic peak by appearance time order, No. 7 peak is Epimedin B chromatographic peak, No. 8 peak is epimedin chromatographic peak, No. 9 peak is icariin chromatographic peak, No. 16 peak is baohuoside Ⅰ chromatographic peak, with No. 9 peak for reference to peak, 16 chromatographic peak retention time ratios are followed successively by: 0.126, 0.510, 0.532, 0.570, 0.821, 0.865, 0.907, 0.955, 1.000, 1.308, 1.363, 1.467, 1.559, 1.609, 1.618, 1.685。
Further, the method for the described finger printing building Herba Epimedii extract, including following operating procedure:
The preparation of need testing solution: accurately weighed Herba Epimedii extract 0.05 weight portion, put in conical flask, accurate addition volumetric concentration is methanol solution 50 parts by volume of 35%, weighed, ultrasonic 10min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 35%, cross microporous filter membrane, take subsequent filtrate, to obtain final product；
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.05mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol；
High-efficient liquid phase chromatogram condition: chromatographic column is with octadecylsilane chemically bonded silica for filler；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 0.05%；Column temperature: 20 DEG C；Detection wavelength: 260nm；Flow velocity: 0.5ml min-1, carrying out gradient elution, concrete gradient elution program is: from 0-10min, mobile phase A: Mobile phase B is 15%:85%；From 10-20min, mobile phase A: Mobile phase B is by 15%:85% → 20%:80%；From 20-45min, mobile phase A: Mobile phase B is by 20%:80% → 24%:76%；From 45-65min, mobile phase A: Mobile phase B is 24%:76%；From 65-70min, mobile phase A: Mobile phase B is by 24%:76% → 30%:70%；From 70-90min, mobile phase A: Mobile phase B is by 30%:70% → 40%:60%；From 90-105min, mobile phase A: Mobile phase B is by 40%:60% → 90%:10%；
Sample determination method: draw need testing solution and reference substance solution 2 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing。
Detection gained Herba Epimedii extract finger printing there are 16 common characteristic peaks, contrast with reference substance finger printing reference substance appearance time, determine that No. 6 peak is Epimedin A chromatographic peak by appearance time order, No. 7 peak is Epimedin B chromatographic peak, No. 8 peak is epimedin chromatographic peak, No. 9 peak is icariin chromatographic peak, No. 16 peak is baohuoside Ⅰ chromatographic peak, with No. 9 peak for reference to peak, 16 chromatographic peak retention time ratios are followed successively by: 0.115, 0.495, 0.525, 0.565, 0.810, 0.855, 0.890, 0.945, 1.000, 1.295, 1.355, 1.455, 1.545, 1.595, 1.610, 1.675。
Further, the method for the described finger printing building Herba Epimedii extract, including following operating procedure:
The preparation of need testing solution: accurately weighed Herba Epimedii extract 0.15 weight portion, put in conical flask, accurate addition volumetric concentration is methanol solution 15 parts by volume of 100%, weighed, ultrasonic 50min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 100%, cross microporous filter membrane, take subsequent filtrate, to obtain final product；
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.50mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol；
High-efficient liquid phase chromatogram condition: chromatographic column is with octadecylsilane chemically bonded silica for filler；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 2%；Column temperature: 40 DEG C；Detection wavelength: 280nm；Flow velocity: 1.5ml min-1, carrying out gradient elution, concrete gradient elution program is: from 0-10min, mobile phase A: Mobile phase B is 15%:85%；From 10-20min, mobile phase A: Mobile phase B is by 15%:85% → 20%:80%；From 20-45min, mobile phase A: Mobile phase B is by 20%:80% → 24%:76%；From 45-65min, mobile phase A: Mobile phase B is 24%:76%；From 65-70min, mobile phase A: Mobile phase B is by 24%:76% → 30%:70%；From 70-90min, mobile phase A: Mobile phase B is by 30%:70% → 40%:60%；From 90-105min, mobile phase A: Mobile phase B is by 40%:60% → 90%:10%；
Sample determination method: draw need testing solution and reference substance solution 25 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing。
Detection gained Herba Epimedii extract finger printing there are 16 common characteristic peaks, contrast with reference substance finger printing reference substance appearance time, determine that No. 6 peak is Epimedin A chromatographic peak by appearance time order, No. 7 peak is Epimedin B chromatographic peak, No. 8 peak is epimedin chromatographic peak, No. 9 peak is icariin chromatographic peak, No. 16 peak is baohuoside Ⅰ chromatographic peak, with No. 9 peak for reference to peak, 16 chromatographic peak retention time ratios are followed successively by: 0.138, 0.518, 0.550, 0.585, 0.835, 0.880, 0.915, 0.965, 1.000, 1.315, 1.380, 1.475, 1.565, 1.625, 1.635, 1.695。
Herba Epimedii extract of the present invention extracts by the following method and obtains: takes epimedium herb and pulverizes, weigh 1 weight portion, add the water of 20-40 parts by volume and/or the ethanol of 50-95% is solvent extraction 1-3 hour, filter after extracting solution is reclaimed ethanol, pass through JD-1(WLD) type or D101 type macroporous adsorptive resins, then 60-80% ethanol elution is used, start to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete, and eluent reclaims ethanol, concentration, dry obtains Herba Epimedii extract；The relation that relation is g/mL of described weight portion and parts by volume。
Further, described Herba Epimedii extract extracts by the following method and obtains: takes epimedium herb and pulverizes, weigh 1 weight portion, the ethanol adding water and/or 50-95% is solvent extraction twice, first time adds 10-20 parts by volume (g/mL) solvent extraction 1 hour, second time adds 10-20 parts by volume (g/mL) solvent extraction 1 hour, extracting solution is merged, filter after reclaiming ethanol, pass through JD-1(WLD) type or D101 type macroporous adsorptive resins, then 70% ethanol elution is used, start to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete, eluent reclaims ethanol, concentration, dry and obtain Herba Epimedii extract。
Preferably, described Herba Epimedii extract extracts by the following method and obtains: takes epimedium herb and pulverizes, weigh 1 weight portion, extracting in water twice, first time adds 15 parts by volume (g/mL) and extracts 1 hour, second time adds 10 parts by volume (g/mL) and extracts 1 hour, twice extracting solution is merged, filter, pass through JD-1(WLD) separation of type macroporous adsorptive resins, then 70% ethanol elution is used, start to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete, eluent reclaims ethanol, concentration, dry and obtain Herba Epimedii extract。
Preferably, described Herba Epimedii extract extracts by the following method and obtains: takes epimedium herb and pulverizes, weigh 1 weight portion, add 50% ethanol extraction twice, first time adds 20 parts by volume (g/mL) and extracts 1 hour, second time adds 10 parts by volume (g/mL) and extracts 1 hour, twice extracting solution is merged, filter after reclaiming ethanol, separated by D101 type macroporous adsorptive resins, then 70% ethanol elution is used, start to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete, eluent reclaims ethanol, concentration, dry and obtain Herba Epimedii extract。
Preferably, described Herba Epimedii extract extracts by the following method and obtains: takes epimedium herb and pulverizes, weigh 1 weight portion, add 95% ethanol extraction twice, first time adds 10 parts by volume (g/mL) and extracts 1 hour, second time adds 20 parts by volume (g/mL) and extracts 1 hour, twice extracting solution is closed, filter after reclaiming ethanol, pass through JD-1(WLD) separation of type macroporous adsorptive resins, then using 70% ethanol elution, start to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete。Eluent reclaims ethanol, concentration, dry obtains Herba Epimedii extract。
Present invention also offers and extract, according to said method, the Herba Epimedii extract obtained。
The invention also discloses the application at detection Herba Epimedii content and field of quality control of the method for above-mentioned structure Herba Epimedii extract finger printing。
The method of the finger printing of structure Herba Epimedii extract of the present invention, with Herba Epimedii extract for detection object, develop the construction method of the finger printing suitable in this extract targetedly, obtain comparatively comprehensively profile information, and can in conjunction with the information of chromatographic peaks multiple in finger printing, reflect the quality of Herba Epimedii extract more comprehensively, the problem of the one-sidedness being different from prior art from independent one or several chemical composition to judge Herba Epimedii extract entirety and arrange, it is possible to more efficient, reasonably product quality is monitored。Meanwhile, the method for structure finger printing of the present invention has stability height, precision height, reproducible advantage。
Herba Epimedii extract of the present invention is preferably by water and/or ethanol extraction that volumetric concentration is 50-95% and via JD-1(WLD) type or D101 type macroporous adsorptive resins be isolated, and the information of the finger printing obtained is more fully effective。
The foundation experiment of experimental example Herba Epimedii extract finger printing
1, instrument and reagent
1.1 instruments
BS110S type electronic balance (MadebySartorius, d=0.1mg)；
CP225D type precision electronic balance (MadebySartorius, d=0.01mg)；
Agilent1200 type high performance liquid chromatograph (band DAD detector)；
AgilentZORBAXXDB-C18(250 × 4.6mm, 5 μm) chromatographic column；
SB-5200DTD processor for ultrasonic wave (NingBo XinZhi Biology Science Co., Ltd)。
1.2 reagents
Epimedin A (lot number 100723, purchased from Chengdu Purification Technology Development Co., Ltd., purity > 98%)；
Epimedin B (lot number 100806, purchased from Chengdu Purification Technology Development Co., Ltd., purity > 98%)；
Epimedin (lot number 100412, purchased from Chengdu Purification Technology Development Co., Ltd., purity > 98%)；
Icariin (lot number 110737-200312, Nat'l Pharmaceutical & Biological Products Control Institute provides)；
Baohuoside Ⅰ (lot number 113558-15-9, purchased from SHANGHAITAUTOBIOTECHCO., LTD, purity > 98%)；
Acetonitrile, chromatographically pure (Dikma company of the U.S.)；
Water is Watson pure water；
Other reagent are analytical pure。
1.3 samples
Described Herba Epimedii extract sample obtains by the following method: take epimedium herb 10g, pulverize, extracting in water twice, first time adds 150ml and extracts 1 hour, second time adds 100ml and again extracts 1 hour, twice extracting solution is merged, filter, and pass through JD-1(WLD) type macroporous adsorptive resins, then 70% ethanol elution is used, starting to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete, and eluent reclaims ethanol, concentration, dry obtains Herba Epimedii extract。
It is that S1-S12(lot number is followed successively by 11016,11025,11036,11040,11045,12004,12012,12022,12036,12040,12051,12060 by extract number consecutively obtained above) detect。
2, method and result
The preparation of 2.1 reference substance solution
Accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, put in 25mL measuring bottle, it is made into every 1ml Epimedin A containing 0.1mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance solution with methanol, to obtain final product。
The preparation of 2.2 need testing solutions
Weighing above-mentioned dried Herba Epimedii extract 0.1g, accurately weighed, accurate addition volumetric concentration is the methanol solution 25mL of 50%, weighed, ultrasonic 10min, weighs after cooling, and supplies the weight of less loss with the methanol solution that volumetric concentration is 50%, shakes up, cross 0.45 μm of filter membrane, take subsequent filtrate, to obtain final product。2.3 chromatographic conditions
Chromatographic column: AgilentZORBAXXDB-C18(250 × 4.6mm, 5 μm) chromatographic column；Mobile phase: with acetonitrile for mobile phase A, with volumetric concentration be 0.1% phosphoric acid water for Mobile phase B, carry out gradient elution, elution program is as follows；Flow velocity is 1ml/min；Column temperature: 30 DEG C；Detection wavelength: 270nm；Gradient elution program:
0-10 minute, mobile phase A was 15%, and Mobile phase B is 85%；
10-20 minute, mobile phase A became 20% from 15%, and Mobile phase B becomes 80% from 85%；
20-45 minute, mobile phase A became 24% from 20%, and Mobile phase B becomes 76% from 80%；
45-65 minute, mobile phase A was 24%, and Mobile phase B is 76%；
65-70 minute, mobile phase A became 30% from 24%, and Mobile phase B becomes 70% from 76%；
70-90 minute, mobile phase A became 40% from 30%, and Mobile phase B becomes 60% from 70%；
90-105 minute, mobile phase A became 90% from 40%, and Mobile phase B becomes 10% from 60%。2.4 sample determination methods
Drawing need testing solution and reference substance solution 10 μ l respectively, inject high performance liquid chromatograph, measure, by test sample record collection of illustrative plates, Fig. 1 is shown in by the collection of illustrative plates of wherein said reference substance solution, and Fig. 2 is shown in by the collection of illustrative plates of detection sample。Each characteristic peak visible is clearly separated。
2.5 methodological studies
2.5.1 precision test
Taking same need testing solution, continuous sample introduction 6 times, gained finger printing stacking chart sees accompanying drawing 3。Adopting " chromatographic fingerprints of Chinese materia medica the system of analysis and appraisal " (A version in 2004) that its finger printing is analyzed, compared with the reference fingerprint generated, the similarity of 6 parts of samples is all higher than 0.950。With icariin peak, No. 9 peaks for reference to peak, the relative retention time at its total peak and the RSD of relative peak area are respectively less than 5%, it was shown that instrument stabilizer, and precision is good, meets the requirement of fingerprint pattern technology。
2.5.2 replica test
Taking same lot number test sample, by the parallel preparation of test sample preparation method 6 parts, sample introduction, gained finger printing stacking chart sees Fig. 4。Adopting " chromatographic fingerprints of Chinese materia medica the system of analysis and appraisal " (A version in 2004) that its finger printing is analyzed, compared with the reference fingerprint generated, the similarity of 6 parts of samples is all higher than 0.950。With No. 9 peaks for reference to peak, the relative retention time at its total peak and the RSD of relative peak area are respectively less than 5%, it was shown that repeatability is good, meets the requirement of fingerprint pattern technology。
2.5.3 stability test
Taking same need testing solution, respectively at 0,2,8,12,16,24,48 little sample introductions constantly, gained finger printing stacking chart sees Fig. 5。Adopting " chromatographic fingerprints of Chinese materia medica the system of analysis and appraisal " (A version in 2004) that its finger printing is analyzed, compared with the reference fingerprint generated, the similarity of 6 parts of samples is all higher than 0.950。With icariin peak, No. 9 peaks for reference to peak, the relative retention time at its total peak and the RSD of relative peak area are respectively less than 5%, it was shown that sample stability is good, meets the requirement of fingerprint pattern technology。
The foundation of 2.6 finger printing
2.6.1 the foundation of common pattern
The finger printing of 12 batch samples is imported " chromatographic fingerprints of Chinese materia medica the system of analysis and appraisal " (A version in 2004), form the finger printing stacking chart (Fig. 6) of described each extract sample, and set up the common pattern (Fig. 7) of Herba Epimedii extract finger printing, from the chromatographic peak of common pattern, demarcate 16 total peaks。Compare through reference substance finger printing, chromatographic peak is belonged to, identify peak 6 for Epimedin A, peak 7 is Epimedin B, peak 8 is epimedin, and peak 9 is icariin, and peak 16 is baohuoside Ⅰ, with No. 9 peak for reference to peak, each chromatographic peak retention time is: 0.126,0.510,0.532,0.570,0.821,0.865,0.907,0.955,1.000,1.308,1.363,1.467,1.559,1.609,1.618,1.685。
2.6.2 similarity analysis
12 batch sample finger printing are imported " chromatographic fingerprints of Chinese materia medica the system of analysis and appraisal " (A version in 2004), calculate similarity, see table 1 below,
Table 112 batch sample fingerprint similarity
Visible, the method for structure Herba Epimedii extract finger printing of the present invention, can the comprehensive information of accurate response Herba Epimedii extract, and method is accurate, feasible。
Accompanying drawing explanation
In order to make present disclosure be more likely to be clearly understood, below according to specific embodiments of the invention and in conjunction with accompanying drawing, the present invention is further detailed explanation, wherein
Fig. 1 is the detection collection of illustrative plates of reference substance solution；
Fig. 2 is the finger printing of detection sample；
Fig. 3 is the Precision Experiment stacking chart of the finger printing of described extract；
Fig. 4 is the repeated experiment stacking chart of the finger printing of described extract；
Fig. 5 is the stability experiment stacking chart of the finger printing of described extract；
Fig. 6 is the finger printing stacking chart of described each extract sample；
Fig. 7 is the finger printing common pattern figure of described each extract sample。
Detailed description of the invention
Embodiment 1
Described in the present embodiment, Herba Epimedii extract obtains by the following method: takes epimedium herb 10g and pulverizes, add 50% ethanol extraction twice, first time adds 200mL and extracts 1 hour, second time adds 100mL and extracts 1 hour, twice extracting solution is merged, filter after reclaiming ethanol, separated by D101 type macroporous adsorptive resins, then 70% ethanol elution is used, start to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete, and eluent reclaims ethanol, concentration, dry obtains Herba Epimedii extract。
The method building Herba Epimedii finger printing described in the present embodiment, comprises the steps:
The preparation of need testing solution: accurately weighed above-mentioned Herba Epimedii extract 0.5g, put in conical flask, accurate addition volumetric concentration is the methanol solution 500ml of 35%, weighed, ultrasonic 10min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 35%, cross microporous filter membrane, take subsequent filtrate, to obtain final product。
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.05mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol。
High-efficient liquid phase chromatogram condition: with AgilentZORBAXXDB-C18(250 × 4.6mm, 5 μm) chromatographic column；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 0.05%；Column temperature: 20 DEG C；Detection wavelength: 260nm；Flow velocity: 0.5ml min-1, carrying out gradient elution, concrete gradient elution program is:
0-10 minute, mobile phase A was 15%, and Mobile phase B is 85%；
10-20 minute, mobile phase A became 20% from 15%, and Mobile phase B becomes 80% from 85%；
20-45 minute, mobile phase A became 24% from 20%, and Mobile phase B becomes 76% from 80%；
45-65 minute, mobile phase A was 24%, and Mobile phase B is 76%；
65-70 minute, mobile phase A became 30% from 24%, and Mobile phase B becomes 70% from 76%；
70-90 minute, mobile phase A became 40% from 30%, and Mobile phase B becomes 60% from 70%；
90-105 minute, mobile phase A became 90% from 40%, and Mobile phase B becomes 10% from 60%。
Sample determination method: draw need testing solution and reference substance solution 2 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing。
Detection gained Herba Epimedii extract finger printing there are 16 common characteristic peaks, contrast with reference substance finger printing reference substance appearance time, determine that No. 6 peak is Epimedin A chromatographic peak by appearance time order, No. 7 peak is Epimedin B chromatographic peak, No. 8 peak is epimedin chromatographic peak, No. 9 peak is icariin chromatographic peak, No. 16 peak is baohuoside Ⅰ chromatographic peak, with No. 9 peak for reference to peak, 16 chromatographic peak retention time ratios are followed successively by: 0.115, 0.495, 0.525, 0.565, 0.810, 0.855, 0.890, 0.945, 1.000, 1.295, 1.355, 1.455, 1.545, 1.595, 1.610, 1.675。
Embodiment 2
Described in the present embodiment, Herba Epimedii extract obtains by the following method: takes epimedium herb 10g and pulverizes, add 95% ethanol extraction twice, first time adds 100mL and extracts 1 hour, second time adds 200mL and extracts 1 hour, is merged by twice extracting solution, filters after reclaiming ethanol, pass through JD-1(WLD) separation of type macroporous adsorptive resins, then using 70% ethanol elution, start to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete。Eluent reclaims ethanol, concentration, dry obtains Herba Epimedii extract。
The method building Herba Epimedii finger printing described in the present embodiment, comprises the steps:
The preparation of need testing solution: accurately weighed above-mentioned Herba Epimedii extract 0.15g, put in conical flask, accurate addition volumetric concentration is the methanol solution 15ml of 100%, weighed, ultrasonic 50min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 100%, cross microporous filter membrane, take subsequent filtrate, to obtain final product。
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.50mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol。
High-efficient liquid phase chromatogram condition: with AgilentZORBAXXDB-C18(250 × 4.6mm, 5 μm) chromatographic column；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 2%；Column temperature: 40 DEG C；Detection wavelength: 280nm；Flow velocity: 1.5ml min-1, carrying out gradient elution, concrete gradient elution program is:
0-10 minute, mobile phase A was 15%, and Mobile phase B is 85%；
10-20 minute, mobile phase A became 20% from 15%, and Mobile phase B becomes 80% from 85%；
20-45 minute, mobile phase A became 24% from 20%, and Mobile phase B becomes 76% from 80%；
45-65 minute, mobile phase A was 24%, and Mobile phase B is 76%；
65-70 minute, mobile phase A became 30% from 24%, and Mobile phase B becomes 70% from 76%；
70-90 minute, mobile phase A became 40% from 30%, and Mobile phase B becomes 60% from 70%；
90-105 minute, mobile phase A became 90% from 40%, and Mobile phase B becomes 10% from 60%。
Sample determination method: draw need testing solution and reference substance solution 25 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing。
Detection gained Herba Epimedii extract finger printing there are 16 common characteristic peaks, contrast with reference substance finger printing reference substance appearance time, determine that No. 6 peak is Epimedin A chromatographic peak by appearance time order, No. 7 peak is Epimedin B chromatographic peak, No. 8 peak is epimedin chromatographic peak, No. 9 peak is icariin chromatographic peak, No. 16 peak is baohuoside Ⅰ chromatographic peak, with No. 9 peak for reference to peak, 16 chromatographic peak retention time ratios are followed successively by: 0.138, 0.518, 0.550, 0.585, 0.835, 0.880, 0.915, 0.965, 1.000, 1.315, 1.380, 1.475, 1.565, 1.625, 1.635, 1.695。
Embodiment 3
Described in the present embodiment, Herba Epimedii extract obtains by the following method: takes epimedium herb 10g and pulverizes, extracting in water twice, first time adds 100mL and extracts 1 hour, second time adds 200mL and extracts 1 hour, twice extracting solution is merged and filters, pass through JD-1(WLD) separation of type macroporous adsorptive resins, then use 70% ethanol elution, starting to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete。Eluent reclaims ethanol, concentration, dry obtains Herba Epimedii extract。
The method building Herba Epimedii finger printing described in the present embodiment, comprises the steps:
The preparation of need testing solution: accurately weighed above-mentioned Herba Epimedii extract 0.1g, put in conical flask, accurate addition volumetric concentration is the methanol solution 25ml of 50%, weighed, ultrasonic 15min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 50%, cross microporous filter membrane, take subsequent filtrate, to obtain final product。
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.1mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol。
High-efficient liquid phase chromatogram condition: with AgilentZORBAXXDB-C18(250 × 4.6mm, 5 μm) chromatographic column；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 0.1%；Column temperature: 30 DEG C；Detection wavelength: 270nm；Flow velocity: 1.0ml min-1, carrying out gradient elution, concrete gradient elution program is:
0-10 minute, mobile phase A was 15%, and Mobile phase B is 85%；
10-20 minute, mobile phase A became 20% from 15%, and Mobile phase B becomes 80% from 85%；
20-45 minute, mobile phase A became 24% from 20%, and Mobile phase B becomes 76% from 80%；
45-65 minute, mobile phase A was 24%, and Mobile phase B is 76%；
65-70 minute, mobile phase A became 30% from 24%, and Mobile phase B becomes 70% from 76%；
70-90 minute, mobile phase A became 40% from 30%, and Mobile phase B becomes 60% from 70%；
90-105 minute, mobile phase A became 90% from 40%, and Mobile phase B becomes 10% from 60%。
Sample determination method: draw need testing solution and reference substance solution 10 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing。
Detection gained Herba Epimedii extract finger printing there are 16 common characteristic peaks, contrast with reference substance finger printing reference substance appearance time, determine that No. 6 peak is Epimedin A chromatographic peak by appearance time order, No. 7 peak is Epimedin B chromatographic peak, No. 8 peak is epimedin chromatographic peak, No. 9 peak is icariin chromatographic peak, No. 16 peak is baohuoside Ⅰ chromatographic peak, with No. 9 peak for reference to peak, 16 chromatographic peak retention time ratios are followed successively by: 0.126, 0.510, 0.532, 0.570, 0.821, 0.865, 0.907, 0.955, 1.000, 1.308, 1.363, 1.467, 1.559, 1.609, 1.618, 1.685。
Obviously, above-described embodiment is only for clearly demonstrating example, and is not the restriction to embodiment。For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description。Here without also cannot all of embodiment be given exhaustive。And the apparent change thus extended out or variation are still among the protection domain of the invention。
Claims (9)
1. the method for the finger printing building Herba Epimedii extract, it is characterised in that include following operating procedure:
The preparation of need testing solution: accurately weighed Herba Epimedii extract 0.05-0.15 weight portion, put in conical flask, accurate addition volumetric concentration is the methanol solution 15-50 parts by volume of 35%-100%, weighed, ultrasonic 10-50min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 35%-100%, cross microporous filter membrane, take subsequent filtrate, to obtain final product；
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.05-0.50mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol；
High-efficient liquid phase chromatogram condition: chromatographic column is with octadecylsilane chemically bonded silica for filler；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 0.05-2%；Column temperature: 20 DEG C-40 DEG C；Detection wavelength: 260nm-280nm；Flow velocity: 0.5-1.5ml min-1, carrying out gradient elution, concrete gradient elution program is: from 0-10min, mobile phase A: Mobile phase B is 15%:85%；From 10-20min, mobile phase A: Mobile phase B is by 15%:85% → 20%:80%；From 20-45min, mobile phase A: Mobile phase B is by 20%:80% → 24%:76%；From 45-65min, mobile phase A: Mobile phase B is 24%:76%；From 65-70min, mobile phase A: Mobile phase B is by 24%:76% → 30%:70%；From 70-90min, mobile phase A: Mobile phase B is by 30%:70% → 40%:60%；From 90-105min, mobile phase A: Mobile phase B is by 40%:60% → 90%:10%；
Sample determination method: draw need testing solution and reference substance solution 2-25 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing；
The relation that relation is g/mL of described weight portion and parts by volume。
2. the method for the finger printing of structure Herba Epimedii extract according to claim 1, it is characterised in that include following operating procedure:
The preparation of need testing solution: accurately weighed Herba Epimedii extract 0.1 weight portion, put in conical flask, accurate addition volumetric concentration is methanol solution 25 parts by volume of 50%, weighed, ultrasonic 15min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 50%, cross microporous filter membrane, take subsequent filtrate, to obtain final product；
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.1mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol；
High-efficient liquid phase chromatogram condition: chromatographic column is with octadecylsilane chemically bonded silica for filler；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 0.1%；Column temperature: 30 DEG C；Detection wavelength: 270nm；Flow velocity: 1.0ml min-1, carrying out gradient elution, concrete gradient elution program is: from 0-10min, mobile phase A: Mobile phase B is 15%:85%；From 10-20min, mobile phase A: Mobile phase B is by 15%:85% → 20%:80%；From 20-45min, mobile phase A: Mobile phase B is by 20%:80% → 24%:76%；From 45-65min, mobile phase A: Mobile phase B is 24%:76%；From 65-70min, mobile phase A: Mobile phase B is by 24%:76% → 30%:70%；From 70-90min, mobile phase A: Mobile phase B is by 30%:70% → 40%:60%；From 90-105min, mobile phase A: Mobile phase B is by 40%:60% → 90%:10%；
Sample determination method: draw need testing solution and reference substance solution 10 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing。
3. the method for the finger printing of structure Herba Epimedii extract according to claim 1, it is characterised in that include following operating procedure:
The preparation of need testing solution: accurately weighed described Herba Epimedii extract 0.05 weight portion, put in conical flask, accurate addition volumetric concentration is methanol solution 50 parts by volume of 35%, weighed, ultrasonic 10min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 35%, cross microporous filter membrane, take subsequent filtrate, to obtain final product；
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.05mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol；
High-efficient liquid phase chromatogram condition: chromatographic column is with octadecylsilane chemically bonded silica for filler；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 0.05%；Column temperature: 20 DEG C；Detection wavelength: 260nm；Flow velocity: 0.5ml min-1, carrying out gradient elution, concrete gradient elution program is: from 0-10min, mobile phase A: Mobile phase B is 15%:85%；From 10-20min, mobile phase A: Mobile phase B is by 15%:85% → 20%:80%；From 20-45min, mobile phase A: Mobile phase B is by 20%:80% → 24%:76%；From 45-65min, mobile phase A: Mobile phase B is 24%:76%；From 65-70min, mobile phase A: Mobile phase B is by 24%:76% → 30%:70%；From 70-90min, mobile phase A: Mobile phase B is by 30%:70% → 40%:60%；From 90-105min, mobile phase A: Mobile phase B is by 40%:60% → 90%:10%；
Sample determination method: draw need testing solution and reference substance solution 2 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing。
4. the method for the finger printing of structure Herba Epimedii extract according to claim 1, it is characterised in that include following operating procedure:
The preparation of need testing solution: accurately weighed described Herba Epimedii extract 0.15 weight portion, put in conical flask, accurate addition volumetric concentration is methanol solution 15 parts by volume of 100%, weighed, ultrasonic 50min, weighs after cooling, the weight of less loss is supplied with the methanol that volumetric concentration is 100%, cross microporous filter membrane, take subsequent filtrate, to obtain final product；
The preparation of reference substance solution: accurately weighed Epimedin A, Epimedin B, epimedin, icariin, baohuoside Ⅰ reference substance are appropriate, the solution of every 1ml part Epimedin A containing 0.50mg, Epimedin B, epimedin, icariin, baohuoside Ⅰ it is made into, as reference substance solution with methanol；
High-efficient liquid phase chromatogram condition: chromatographic column is with octadecylsilane chemically bonded silica for filler；Mobile phase A is acetonitrile, and Mobile phase B is the phosphate aqueous solution of volumetric concentration 2%；Column temperature: 40 DEG C；Detection wavelength: 280nm；Flow velocity: 1.5ml min-1, carrying out gradient elution, concrete gradient elution program is: from 0-10min, mobile phase A: Mobile phase B is 15%:85%；From 10-20min, mobile phase A: Mobile phase B is by 15%:85% → 20%:80%；From 20-45min, mobile phase A: Mobile phase B is by 20%:80% → 24%:76%；From 45-65min, mobile phase A: Mobile phase B is 24%:76%；From 65-70min, mobile phase A: Mobile phase B is by 24%:76% → 30%:70%；From 70-90min, mobile phase A: Mobile phase B is by 30%:70% → 40%:60%；From 90-105min, mobile phase A: Mobile phase B is by 40%:60% → 90%:10%；
Sample determination method: draw need testing solution and reference substance solution 25 μ l respectively, inject high performance liquid chromatograph, measure, obtain finger printing。
5. the method for the finger printing building Herba Epimedii extract according to any one of claim 1-4, it is characterised in that described Herba Epimedii extract extracts by the following method and obtains:
Take epimedium herb to pulverize, weigh 1 weight portion, add the water of 20-40 parts by volume and/or the ethanol of 50-95% is solvent extraction 1-3 hour, filter after extracting solution is reclaimed ethanol, by JD-1 (WLD) type or D101 type macroporous adsorptive resins, then use 60-80% ethanol elution, start to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete, and eluent reclaims ethanol, concentration, dry obtains Herba Epimedii extract；
The relation that relation is g/mL of described weight portion and parts by volume。
6. the method for the finger printing of structure Herba Epimedii extract according to claim 5, it is characterised in that described Herba Epimedii extract extracts by the following method and obtains:
Take epimedium herb to pulverize, weigh 1 weight portion, the ethanol adding water and/or 50-95% is solvent extraction twice, first time adds 10-20 parts by volume solvent extraction 1 hour, second time adds 10-20 parts by volume solvent extraction 1 hour, extracting solution is merged, filter after reclaiming ethanol, by JD-1 (WLD) type or D101 type macroporous adsorptive resins, then 70% ethanol elution is used, starting to collect eluent when effluent color substantially deepens, when wash water color becomes extremely shallow, eluting is complete, and eluent reclaims ethanol, concentration, dry obtains Herba Epimedii extract。
7. according to claim 1, 2, 3, the method of the finger printing building Herba Epimedii extract described in 4 or 6, it is characterized in that, described Herba Epimedii extract finger printing has 16 common characteristic peaks, contrast with reference substance finger printing reference substance appearance time, determine that No. 6 peak is Epimedin A chromatographic peak by appearance time order, No. 7 peak is Epimedin B chromatographic peak, No. 8 peak is epimedin chromatographic peak, No. 9 peak is icariin chromatographic peak, No. 16 peak is baohuoside Ⅰ chromatographic peak, with No. 9 peak for reference to peak, 16 chromatographic peak retention time ratios are followed successively by: 0.110-0.140, 0.490-0.520, 0.525-0.555, 0.560-0.600, 0.810-0.840, 0.850-0.880, 0.890-0.920, 0.940-0.970, 1.000, 1.290-1.320, 1.350-1.380, 1.450-1.480, 1.540-1.570, 1.595-1.625, 1.605-1.635, 1.670-1.700。
8. the method for the finger printing of structure Herba Epimedii extract according to claim 5, it is characterized in that, described Herba Epimedii extract finger printing has 16 common characteristic peaks, contrast with reference substance finger printing reference substance appearance time, determine that No. 6 peak is Epimedin A chromatographic peak by appearance time order, No. 7 peak is Epimedin B chromatographic peak, No. 8 peak is epimedin chromatographic peak, No. 9 peak is icariin chromatographic peak, No. 16 peak is baohuoside Ⅰ chromatographic peak, with No. 9 peak for reference to peak, 16 chromatographic peak retention time ratios are followed successively by: 0.110-0.140, 0.490-0.520, 0.525-0.555, 0.560-0.600, 0.810-0.840, 0.850-0.880, 0.890-0.920, 0.940-0.970, 1.000, 1.290-1.320, 1.350-1.380, 1.450-1.480, 1.540-1.570, 1.595-1.625, 1.605-1.635, 1.670-1.700。
9. the method building Herba Epimedii extract finger printing described in any one of claim 1-6 is in the application of detection Herba Epimedii content and field of quality control。
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| CN107655994B (en) * | 2017-09-30 | 2020-07-03 | 天津中医药大学 | Method for measuring content of 16 chemical components in epimedium extract |
| CN109459515B (en) * | 2018-07-05 | 2021-09-17 | 广州科曼生物科技有限公司 | Herba epimedii control extract (arrow leaf) and application thereof |
| CN109187810B (en) * | 2018-10-29 | 2021-06-08 | 广州白云山陈李济药厂有限公司 | Detection method of traditional Chinese medicine composition for treating rheumatoid arthritis |
| CN110672747A (en) * | 2019-10-23 | 2020-01-10 | 劲牌生物医药有限公司 | Method for detecting epimedium component, method for identifying variety of epimedium and application |
| CN111474276B (en) * | 2020-05-08 | 2022-02-25 | 亚宝药业集团股份有限公司 | Quality control method of yang invigorating tablet preparation |
| CN113834879B (en) * | 2020-06-23 | 2023-10-20 | 国药集团同济堂（贵州）制药有限公司 | Establishment and application of epimedium flavone component fingerprint based on SPE technology |
| CN113281439B (en) * | 2021-07-25 | 2021-11-26 | 江西汇仁药业股份有限公司 | Quality control detection method of Shenbao tablets |
| CN115480001A (en) * | 2022-07-29 | 2022-12-16 | 丹东药业集团有限公司 | Herba epimedii particle two-dimensional multi-information reference internal standard quality control method |
| CN116115663A (en) * | 2022-11-17 | 2023-05-16 | 劲牌有限公司 | Preparation method and application of epimedium herb active extract |
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