CN101703557A - Method for detecting quality of persimmon leaf extract - Google Patents
Method for detecting quality of persimmon leaf extract Download PDFInfo
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Abstract
The invention provides a method for detecting the quality of a persimmon leaf extract, which comprises the following steps of: mixing a to-be-detected sample of the persimmon leaf extract with N,N-dimethylformamide to obtain mixed solution serving as test sample solution; mixing acetic ether with the N,N-dimethylformamide to obtain mixed solution serving as reference substance solution; and according to a gas chromatography, using cyanopropyl phenyl-dimethyl polysiloxane as a chromatographic column of a stationary liquid, and injecting the test sample solution and the reference substance solution into a gas chromatograph respectively to measure the content of the acetic ether in the test sample solution. The method selects the N,N-dimethylformamide as a solvent which is mixed with the to-be-detected sample and a reference substance respectively to prepare sample solution, then selects the cyanopropyl phenyl-dimethyl polysiloxane as the chromatographic column of the stationary liquid, and can accurately measure the residual quantity of the acetic ether in the persimmon leaf extract according to the gas chromatography so as to accurately evaluate the quality of the persimmon leaf extract.
Description
Technical field
The present invention relates to the drug detection field, be specifically related to a kind of detection method of quality of persimmon leaf extract.
Background technology
Folium Kaki extract is used as in " the southern regions of the Yunnan Province book on Chinese herbal medicine " of medicine before more than 600 years just on the books, and its good cough-relieving is breathed heavily, the effect of lung account, internal hemorrhage and blood pressure lowering makes it have important position in Chinese medicine extended family.
In the prior art, Folium Kaki extract is prepared as follows: get dried Folium Kaki, decoct with water secondary, 2 hours for the first time, 1 hour for the second time, collecting decoction filters, and it is 1.12～1.15 (60 ℃) that filtrate is concentrated into relative density, add ethanol and reach 85wt% to containing the alcohol amount, standing over night, the leaching supernatant, standby; The precipitate that filtration obtains merges cleaning mixture, standing over night with 65wt% washing with alcohol twice, the leaching supernatant merges supernatant twice, reclaims ethanol, add an amount of water, mixing filters, and filtrate is used ethyl acetate extraction four times, combined ethyl acetate liquid, reclaim ethyl acetate and be condensed into thick paste, cold drying promptly obtains Folium Kaki extract.Adding the Chinese medicine preparation that adjuvant is made by Folium Kaki extract, is Guangzhou Baiyunshan Heji Huangpu Chinese Medicine Co., Ltd.'s exclusive Chinese medicine protection kind, records in the 20 the 294th page in " Drug Standard of Ministry of Public Health of the Peoples Republic of China " Chinese traditional patent formulation preparation.
When preparing Folium Kaki extract according to the method described above, owing to repeatedly use ethyl acetate, therefore can residual ethyl acetate in the finished product Folium Kaki extract.Under prior art, the ethyl acetate residual quantity in the Folium Kaki extract is not had clear and definite detection method, therefore can't confirm the amount of the residual ethyl acetate in the Folium Kaki extract, cause quality of persimmon leaf extract can accurately not estimate.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is, a kind of detection method of Folium Kaki extract is provided, to determine the residual quantity of ethyl acetate in the Folium Kaki extract product to be tested.
In order to solve above technical problem, the invention provides a kind of detection method of quality of persimmon leaf extract, comprising:
Get Folium Kaki extract product to be tested and N, dinethylformamide mixes, and the mixed liquor that obtains is as need testing solution;
Get ethyl acetate reference substance and N, dinethylformamide mixes, and the mixed liquor that obtains is product solution in contrast;
According to gas chromatography, use with the chromatographic column of cyanogen propyl group phenyl-dimethyl polysiloxane as fixative, with described need testing solution and reference substance solution difference inject gas chromatograph, measure the residual quantity of ethyl acetate in the described need testing solution.
Preferably, in the described step according to ethyl acetate residual quantity in the described need testing solution of gas chromatography determination: the initial temperature that described chromatographic column heats up is 49 ℃～51 ℃, and temperature retention time is 4min～6min; And then chromatographic column risen to 218 ℃～222 ℃ with the speed of 34 ℃～36 ℃ of per minutes, insulation 1min～3min.
Preferably, in the described step according to ethyl acetate residual quantity in the described need testing solution of gas chromatography determination: the detected temperatures of the flame ionization detector in the gas chromatograph is that 245 ℃～255 ℃, injector temperature are that 215 ℃～225 ℃, head space temperature are that 75 ℃～85 ℃, temperature retention time are 28～32min.
Preferably, described cyanogen propyl group phenyl-dimethyl polysiloxane consists of 6wt% cyanogen propyl group phenyl-94wt% dimethyl polysiloxane.
Preferably, described detection method further comprises the step of using kaempferol and quercetin content in the described Folium Kaki extract product to be tested of high effective liquid chromatography for measuring:
Get kaempferol and Quercetin and methanol mixed, the mixed liquor that obtains is as the high performance liquid chromatography reference substance solution;
The high performance liquid chromatography that contains Folium Kaki extract product to be tested need testing solution is provided;
Use kaempferol and quercetin content in the described Folium Kaki extract product to be tested of high effective liquid chromatography for measuring.
Preferably, the described high performance liquid chromatography that contains the Folium Kaki extract product to be tested that provides is specially with need testing solution:
Get described Folium Kaki extract product to be tested powder methanol constant volume, obtain first mixed liquor, filter;
Get and filter that filtrate that described first mixed liquor obtains adds methanol and hydrochloric acid solution obtains second mixed liquor, shake up and reflux, put coldly, then described second mixed liquor is transferred in the measuring bottle, mistake leaching filtrate obtains need testing solution.
Preferably, describedly get described Folium Kaki extract product to be tested powder and be specially with methanol constant volume:
Get described Folium Kaki extract product to be tested powder and methanol mixed and place tool plug container, sealing bottle cork, mixed liquor in the described tool plug container is claimed to decide weight and process supersound process, put cold, claim to decide weight once more, supply the weight that mixed liquor lacks in the described tool plug container with methanol, shake up after-filtration and obtain described first mixed liquor.
Preferably, describedly use that kaempferol and the used filler of quercetin content are methanol-0.2wt% phosphoric acid solution as octadecylsilane chemically bonded silica, mobile phase in the described Folium Kaki extract product to be tested of high effective liquid chromatography for measuring.
Preferably, describedly use that the detection wavelength of kaempferol and quercetin content is 360nm in the described Folium Kaki extract product to be tested of high effective liquid chromatography for measuring.
Preferably, the number of theoretical plate of kaempferol and quercetin content calculates by the Quercetin peak and is not less than 2500 in the described Folium Kaki extract product to be tested of described use high effective liquid chromatography for measuring.
The invention provides a kind of detection method of quality of persimmon leaf extract, in this detection method, with Folium Kaki extract product to be tested and N, dinethylformamide mixes, obtain need testing solution, with ethyl acetate reference substance and N, the dinethylformamide mixing obtains reference substance solution, reuse cyanogen propyl group phenyl-dimethyl polysiloxane is the chromatographic column of fixative, by gas chromatography determination ethyl acetate residual quantity wherein.The present invention selects N, dinethylformamide is mixed with sample solution with product to be tested and reference substance respectively as solvent, selecting cyanogen propyl group phenyl-dimethyl polysiloxane then is the chromatographic column of fixative, can detect the residual quantity of ethyl acetate in the Folium Kaki extract accurately according to gas chromatography, thereby quality of persimmon leaf extract is estimated accurately.
A kind of preferred embodiment in, detection method provided by the invention further comprises uses high efficiency chromatography to measure kaempferol and quercetin content step in the Folium Kaki extract product to be tested, the present invention is dissolved in methanol product solution in contrast with kaempferol and Quercetin, can measure kaempferol in the Folium Kaki extract product to be tested and Quercetin, can further determine the medicine effective content in the described Folium Kaki extract product to be tested like this.
Description of drawings
Fig. 1; The disclosed reference substance liquid of embodiment of the invention gas chromatogram;
Fig. 2; The disclosed need testing solution gas chromatogram of the embodiment of the invention;
Fig. 3; The high-efficient liquid phase chromatogram of the disclosed reference substance solution of the embodiment of the invention;
Fig. 4; The high-efficient liquid phase chromatogram of the disclosed need testing solution of the embodiment of the invention.
The specific embodiment
In order further to understand the present invention, below in conjunction with embodiment the preferred embodiment of the invention is described, but should be appreciated that these describe just to further specifying the features and advantages of the present invention, rather than to the restriction of claim of the present invention.
At first specific term is once explained
Appendix: the appendix that refers to 2005 editions Chinese Pharmacopoeias.
High performance liquid chromatography (HPLC, High Performance Liquid Chromatography) adopts high performance liquid chromatograph that the chemical constituent of sample is carried out a kind of technical method qualitative, quantitative analysis.
Gas chromatography (GC, Gas Chromatography) adopts the gas liqud chromatography instrument that the chemical constituent of sample is carried out a kind of technical method qualitative, quantitative analysis.
Owing in the leaching process of Folium Kaki extract, repeatedly used ethyl acetate, so can the residual quality that has ethyl acetate to influence Folium Kaki extract in Folium Kaki extract, and the present invention detects ethyl acetate residual quantity in the Folium Kaki extract product to be tested by gas chromatography.
Concrete steps are: precision takes by weighing Folium Kaki extract product to be tested 0.2g, grinds, and places first empty bottle then, and precision takes by weighing N again, and dinethylformamide 2ml places first empty bottle, shakes up, and after the sealing, obtains need testing solution.
Precision is measured ethyl acetate reference substance 2ml, uses N, and the solution that contains 0.5ml among every 1ml is made in the dinethylformamide dilution, and precision is measured above-mentioned diluent 2ml, places second empty bottle, and sealing is product solution in contrast.
Measuring according to gas chromatography (appendix V E), is the capillary column of fixative with 6wt% cyanogen propyl group phenyl-94wt% dimethyl polysiloxane, and the capillary column specification is 0.25mm * 30m; Column temperature: initial temperature is 50 ℃, keeps 5min, and the capillary column after will being incubated then rises to 220 ℃ with the speed of 35 ℃ of per minutes, keeps 2min.The detected temperatures of flame ionization detector is 250 ℃, and injector temperature is 220 ℃.The head space temperature is 80 ℃, and temperature retention time is 30min, sample injection time 1min.The reference substance gas chromatogram as shown in Figure 1, the need testing solution gas chromatogram, contains ethyl acetate and surpasses 0.5wt% with calculated by peak area by external standard method as shown in Figure 2.
Flavone compound is the main effective ingredient in the Folium Kaki extract, and the Folium Kaki extract flavone is based on the aglycon and the glycoside thereof of Quercetin and kaempferol.The aglycon total amount of Quercetin after the Folium Kaki extract hydrolysis and kaempferol can reflect the general flavone content in the Folium Kaki extract substantially, the same general flavone content that also can reflect in the Folium Kaki extract is so also comprised the assay method that uses Folium Kaki extract kaempferol and quercetin content in the high effective liquid chromatography for measuring Folium Kaki extract product to be tested in the present invention.Concrete steps are as follows:
With the octadecylsilane chemically bonded silica is filler, and the mixed that with methanol and 0.2wt% phosphoric acid according to weight ratio is 50: 50 is as being mobile phase.
Get kaempferol and Quercetin respectively, the accurate title, decide, and with methanol mixed, is configured to the mixed solution that every 1ml contains kaempferol and each 50 μ g of Quercetin, and the mixed liquor that obtains is as the high performance liquid chromatography reference substance solution.
Get Folium Kaki extract product to be tested powder O.1g, the accurate title, be placed in the tool plug conical flask surely, the accurate methanol 20ml that adds in described tool plug conical flask, the sealing back claims to decide weight, through the supersound process of power 250W, frequency 40kHz 30 minutes, room temperature is placed cooling then, claims to decide weight once more, uses methanol to supply the weight of disappearance if any deficiency.
The mixed liquor of standardize solution is shaken up, and after the filtration, precision is measured subsequent filtrate 10ml to conical flask, add methanol 10ml, percentage by volume and be 25% hydrochloric acid solution 5ml after, shook up reflux 30 minutes, the room temperature cooling is placed, and is transferred in the volumetric flask of 50ml.With methanol with the washing of all dispensing containers after, washing liquid is transferred to described volumetric flask, add methanol to scale, sealing shakes up filtration, gets filtrate, promptly obtains the high performance liquid chromatography need testing solution of Folium Kaki extract product to be tested.
High-performance liquid chromatogram determination method is: accurate respectively described need testing solution and each 10 μ l of reference substance solution of drawing, inject chromatograph of liquid, and to measure, the detection wavelength is 360nm, number of theoretical plate calculates and should all be not less than 2500 by the Quercetin peak.The high-efficient liquid phase chromatogram of reference substance solution as shown in Figure 3, the high-efficient liquid phase chromatogram of need testing solution is pressed the Folium Kaki extract dry product and is calculated, with Quercetin (C as shown in Figure 4
15H
10O
7) count greater than 3.8wt%, with kaempferol (C
15H
10O
6) count greater than 4.8wt%.
The present invention kaempferol and the quercetin content, also comprises the step of using thin layer chromatography to identify Folium Kaki extract in the gas chromatography determination ethyl acetate residual quantity of Folium Kaki extract and high effective liquid chromatography for measuring Folium Kaki extract product to be tested.
Concrete steps are: get Folium Kaki extract product to be tested 0.2g, porphyrize is with the 25ml methanol mixed, supersound process 30 minutes is filtered above-mentioned methyl alcohol mixed liquor the filtrate evaporate to dryness, remaining residue adds 15ml water, fully stirs and makes dissolving, filters, filtrate adds hydrochloric acid 0.5ml, extract with absolute ether 30ml jolting, divide and get ether layer, volatilize, residue adds methanol 1ml makes dissolving, as need testing solution.
Other gets Folium Kaki extract control medicinal material 8g, according to making control medicinal material solution with the identical compound method of need testing solution.
Get the protocatechuic acid reference substance again, make the solution that every 1ml contains 1mg with methanol mixed, in contrast product solution.According to thin layer chromatography (appendix VI B) test, draw each 10 μ l of above-mentioned three kinds of solution, put in same silica gel G F respectively
254On the lamellae, be that methylene chloride-methanol-formic acid mixed liquor of 10: 0.3: 0.3 is developing solvent, launch, take out, dry, put under the ultra-violet lamp that wavelength is 254nm and inspect with weight ratio.In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, observe the speckle whether the Folium Kaki extract product to be tested shows same color, if can confirm that then the Folium Kaki extract product to be tested is a Folium Kaki extract, and contain protocatechuic acid.
Use thin layer chromatography to identify to Folium Kaki extract and can also use other reference substance.
Concrete steps are: precision takes by weighing Folium Kaki extract product to be tested 0.2g, porphyrize, and with the 25ml methanol mixed, supersound process 30 minutes is filtered above-mentioned methyl alcohol mixed liquor, and filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution.
Other gets Folium Kaki extract control medicinal material 2g, according to making control medicinal material solution with the identical compound method of need testing solution.
Even up pier fruit acid reference substance again, make the solution that every 1ml contains 1mg with methanol mixed, in contrast product solution.Test according to thin layer chromatography (appendix VI B), draw each 10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with weight ratio is that toluene-ethyl acetate-formic acid mixed liquor of 7: 2: 0.3 is developing solvent, launches, and exhibition is apart from 12cm, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to speckle colour developing at 105 ℃.In the test sample chromatograph, with control medicinal material and the corresponding position of reference substance chromatograph on, observe the speckle whether the Folium Kaki extract product to be tested shows same color, if can confirm that then the Folium Kaki extract product to be tested is a Folium Kaki extract, and contain oleanolic acid.
Embodiment 5
This present invention is in the gas chromatography determination ethyl acetate residual quantity of Folium Kaki extract and high effective liquid chromatography for measuring Folium Kaki extract product to be tested kaempferol and the quercetin content, also comprise the mensuration of the ethyl acetate extractum of Folium Kaki extract, concrete steps are:
Get this product, be ground into coarse powder, get about 0.5g, the accurate title, decide, and is solvent with ethyl acetate, measures according to the hot dipping (appendix X A) in the ethanol-soluble extractives algoscopy, surpasses 75wt%.
The present invention also provides the determination step of the moisture of Folium Kaki extract.
Measure according to aquametry (appendix IX H first method), water content must not surpass 5.0wt%
Embodiment 7
The present invention also provides the determination step of content of beary metal in the Folium Kaki extract.
Step is: get product to be tested product 1g, check (appendix IX E second method) in accordance with the law, must not surpass by weight less than 20/1000000ths.
More than the detection method of a kind of quality of persimmon leaf extract provided by the invention is described in detail.Used specific case herein principle of the present invention and embodiment are set forth, the explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (10)
1. the detection method of a quality of persimmon leaf extract is characterized in that, comprising:
Get Folium Kaki extract product to be tested and N, dinethylformamide mixes, and the mixed liquor that obtains is as need testing solution;
Get ethyl acetate reference substance and N, dinethylformamide mixes, and the mixed liquor that obtains is product solution in contrast;
According to gas chromatography, use with the chromatographic column of cyanogen propyl group phenyl-dimethyl polysiloxane as fixative, with described need testing solution and reference substance solution difference inject gas chromatograph, measure the residual quantity of ethyl acetate in the described need testing solution.
2. detection method according to claim 1, it is characterized in that, in the described step according to ethyl acetate residual quantity in the described need testing solution of gas chromatography determination: the initial temperature that described chromatographic column heats up is 49 ℃～51 ℃, and temperature retention time is 4min～6min; And then chromatographic column risen to 218 ℃～222 ℃ with the speed of 34 ℃～36 ℃ of per minutes, insulation 1min～3min.
3. detection method according to claim 2, it is characterized in that in the described step according to ethyl acetate residual quantity in the described need testing solution of gas chromatography determination: the detected temperatures of the flame ionization detector in the gas chromatograph is that 245 ℃～255 ℃, injector temperature are that 215 ℃～225 ℃, head space temperature are that 75 ℃～85 ℃, temperature retention time are 28～32min.
4. detection method according to claim 1 is characterized in that, described cyanogen propyl group phenyl-dimethyl polysiloxane consist of 6wt% cyanogen propyl group phenyl-94wt% dimethyl polysiloxane.
5. according to any described detection method of claim 1 to 4, it is characterized in that, further comprise the step of using kaempferol and quercetin content in the described Folium Kaki extract product to be tested of high effective liquid chromatography for measuring:
Get kaempferol and Quercetin and methanol mixed, the mixed liquor that obtains is as the high performance liquid chromatography reference substance solution;
The high performance liquid chromatography that contains Folium Kaki extract product to be tested need testing solution is provided;
Use kaempferol and quercetin content in the described Folium Kaki extract product to be tested of high effective liquid chromatography for measuring.
6. detection method according to claim 5 is characterized in that, the described high performance liquid chromatography that contains the Folium Kaki extract product to be tested that provides is specially with need testing solution:
Get described Folium Kaki extract product to be tested powder methanol constant volume, obtain first mixed liquor, filter;
Get and filter that filtrate that described first mixed liquor obtains adds methanol and hydrochloric acid solution obtains second mixed liquor, shake up and reflux, put coldly, then described second mixed liquor is transferred in the measuring bottle, mistake leaching filtrate obtains need testing solution.
7. detection method according to claim 5 is characterized in that, describedly gets described Folium Kaki extract product to be tested powder and is specially with methanol constant volume:
Get described Folium Kaki extract product to be tested powder and methanol mixed and place tool plug container, sealing bottle cork, mixed liquor in the described tool plug container is claimed to decide weight and process supersound process, put cold, claim to decide weight once more, supply the weight that mixed liquor lacks in the described tool plug container with methanol, shake up after-filtration and obtain described first mixed liquor.
8. detection method according to claim 5, it is characterized in that, describedly use that kaempferol and the used filler of quercetin content are methanol-0.2wt% phosphoric acid solution as octadecylsilane chemically bonded silica, mobile phase in the described Folium Kaki extract product to be tested of high effective liquid chromatography for measuring.
9. detection method according to claim 5 is characterized in that, describedly uses that the detection wavelength of kaempferol and quercetin content is 360nm in the described Folium Kaki extract product to be tested of high effective liquid chromatography for measuring.
10. detection method according to claim 5 is characterized in that, the number of theoretical plate of kaempferol and quercetin content calculates by the Quercetin peak and is not less than 2500 in the described Folium Kaki extract product to be tested of described use high effective liquid chromatography for measuring.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507826A (en) * | 2011-11-11 | 2012-06-20 | 广州白云山和记黄埔中药有限公司 | HPLC (High Performance Liquid Chromatography) analysis method of persimmon leaf extract and preparation of persimmon leaf extract |
| CN106706788A (en) * | 2016-12-26 | 2017-05-24 | 合肥智汇医药科技有限公司 | Qualitative and quantitative analysis method for flavonoid effective components in persimmon leaf extract |
| CN109662986A (en) * | 2017-12-12 | 2019-04-23 | 广州白云山和记黄埔中药有限公司 | A kind of novel medical use of extractive from leaves of persimmon and its preparation |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100477985C (en) * | 2005-12-06 | 2009-04-15 | 福寿堂制药有限公司 | Dispersible tablet for clearing away heat of brain and heart and preparation process thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507826A (en) * | 2011-11-11 | 2012-06-20 | 广州白云山和记黄埔中药有限公司 | HPLC (High Performance Liquid Chromatography) analysis method of persimmon leaf extract and preparation of persimmon leaf extract |
| CN106706788A (en) * | 2016-12-26 | 2017-05-24 | 合肥智汇医药科技有限公司 | Qualitative and quantitative analysis method for flavonoid effective components in persimmon leaf extract |
| CN106706788B (en) * | 2016-12-26 | 2019-05-17 | 合肥智汇医药科技有限公司 | A kind of quantification and qualification method of flavonoids effective constituent in extractive from leaves of persimmon |
| CN109662986A (en) * | 2017-12-12 | 2019-04-23 | 广州白云山和记黄埔中药有限公司 | A kind of novel medical use of extractive from leaves of persimmon and its preparation |
| CN109662986B (en) * | 2017-12-12 | 2022-05-31 | 广州白云山和记黄埔中药有限公司 | Persimmon leaf extract and new medical application of preparation thereof |
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