CN105651879A - Method for measuring content of quercetin 3-O-beta-D-glucoside in parthenocissus dalzielii medicine - Google Patents
Method for measuring content of quercetin 3-O-beta-D-glucoside in parthenocissus dalzielii medicine Download PDFInfo
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Abstract
The invention discloses a method for measuring the content of quercetin 3-O-beta-D-glucoside in parthenocissus dalzielii medicine. The method is characterized in that high performance liquid chromatography is adopted; the quercetin 3-O-beta-D-glucoside is used for contrast; methanol:0.05-percent phosphoric acid=(40 to 60):(50 to 50) is used as a mobile phase. Experiments prove that the content measuring method has the advantages that the precision is high; the reproducibility is good; the stability is good; the recovery rate is high; the measuring result is accurate; the quality detection of the parthenocissus dalzielii medicine is completed; the foundation is laid for the quality standard building of the parthenocissus dalzielii medicine.
Description
Technical field
The invention belongs to Chinese medicinal material Quality Control Technology field, particularly relate to the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in a kind of different leaf parthenocissus medicinal material.
Background technology
Different leaf parthenocissus is the dry stem leaf of Vitaceae Parthenocissus different leaf parthenocissus (Parthenocissusheterophylla (BI.) Merr.), is distributed in Guangxi, Anhui, zhejiang and other places district. Have and dispel rheumatism, Tong Longlu, Huo Lu, hemostasis, the effects such as removing toxic substances. The Guangxi treatment being used for the illnesss such as rheumatic arthralgia, migraine, rheumatism sore poison, fracture among the people. At present, about the research of different leaf parthenocissus, the research of haematochrome in rarely seen different leaf parthenocissus pericarp, about the research of its pharmacologically active and chemical composition, content assaying method, there is not been reported.
Applicant's pre-stage test shows in different leaf parthenocissus medicinal material main containing flavones ingredient, and flavonoid compound generally has good anti-oxidant activity, adopt colorimetric method for determining different leaf parthenocissus difference extraction position to the scavenging(action) of the bitter hydrazide group free radical [DPPH] of phenylbenzene, illustrate that it has anti-oxidant activity. The chemical composition of different leaf parthenocissus anti-oxidant activity position-Ethyl acetate fraction is furtherd investigate, adopt multiple chromatographic column separation method, separation obtains 6 compounds, is respectively beans Gona-4-en-3-one, daucosterol, apigenin, luteolin, Quercetin, Quercetin 3-O-��-D-Glucose glycosides. Wherein, Quercetin 3-O-��-D-Glucose glycosides has the physiologically active for the treatment of myocardial ischemia, anoxic.
Summary of the invention
The technical problem to be solved in the present invention is to provide the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in a kind of accurate, reliable, stable different leaf parthenocissus medicinal material, to guarantee the quality of different leaf parthenocissus medicinal material, ensure the safe, effective, stable of its clinical application.
For solving the problems of the technologies described above, the present invention is by the following technical solutions: the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in different leaf parthenocissus medicinal material, adopt high performance liquid chromatography, taking Quercetin-3-O-��-D-Glucose glycosides as comparison, taking methyl alcohol: 0.05% phosphoric acid=40��60: 50��50 as moving phase.
Chromatographic condition and system suitability are tested and are: taking C18 bonded silica gel as weighting agent; Taking methyl alcohol: 0.05% phosphoric acid=40��60: 50��50 as moving phase;Flow velocity is 1��2ml min-1; Post temperature is 25��40 DEG C; Determined wavelength is 258��370nm; Theoretical stage number should be not less than 3000.
Chromatographic condition and system suitability are tested and are: taking C18 bonded silica gel as weighting agent; Taking methyl alcohol: 0.05% phosphoric acid=46: 54 as moving phase; Flow velocity is 1ml min-1; Post temperature is 25 DEG C; Determined wavelength is 258nm; Theoretical stage number should be not less than 3000.
The preparation of reference substance solution is undertaken by following operation: precision takes Quercetin-3-O-��-D-Glucose glycosides reference substance, makes 0.02��0.20mg ml with methyl alcohol-1Solution.
Reference substance solution concentration is 0.20mg ml-1��
The preparation of need testing solution is undertaken by following operation: get different leaf parthenocissus medicinal powder 2.0g, accurately weighed, puts in tool plug Erlenmeyer flask, adds methyl alcohol 15ml, ultrasonic 40min, lets cool, and supplies weightlessness with methyl alcohol, filters, get continuous filtrate, cross 0.45 ��m of millipore filtration, to obtain final product.
Measure and undertaken by following operation: accurate absorption reference substance solution and each 10��20 �� L of need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product.
Measure and undertaken by following operation: accurate absorption reference substance solution and each 10 �� L of need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product.
Activeconstituents in Quercetin 3-O-��-different leaf parthenocissus medicinal material of D-Glucose glycosides system, for guaranteeing the quality of different leaf parthenocissus medicinal material, ensure the safe, effective, stable of its clinical application, the selected Quercetin 3-O-��-D-Glucose glycosides of contriver is as the index of different leaf parthenocissus medicinal material assay, by the content of Quercetin-3-O-��-D-Glucose glycosides in different leaf parthenocissus medicinal material is carried out quantitative examination, establish the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in a kind of different leaf parthenocissus medicinal material. This method adopts high performance liquid chromatography, taking Quercetin-3-O-��-D-Glucose glycosides as comparison, taking methyl alcohol: 0.05% phosphoric acid=40��60: 50��50 as moving phase. Experiment confirms, the content assaying method precision height of the present invention, favorable reproducibility, good stability, rate of recovery height, and measurement result is accurate, and the perfect quality examination of different leaf parthenocissus medicinal material, for the foundation of different leaf parthenocissus quality of medicinal material standard is laid a good foundation.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of Quercetin 3-O-��-D-Glucose glycosides reference substance.
Fig. 2 is the high-efficient liquid phase chromatogram of different leaf parthenocissus medicinal material.
Embodiment
One, in different leaf parthenocissus medicinal material, the content assaying method of Quercetin-3-O-��-D-Glucose glycosides is studied
1. instrument and reagent
1200 series of high efficiency liquid chromatographs (U.S. Agilent), inersilC18 chromatographic column (4.6mm �� 250mm, 5 ��m), BS110S type 1,/10 ten thousand electronic analytical balance (Mei Tele company). DFY-500 pulverizer (woods great machinery company limited of Wenling, Zhejiang province city), 9200S ultrasonic cleaner (Shanghai).
Different leaf parthenocissus medicinal material (picks up from seven Different sources); Quercetin-3-O-��-D-Glucose glycosides reference substance (self-control), purity 98.0%,13CNMR��1HNMR, MS spectral data is consistent with document; Methyl alcohol, acetonitrile are chromatographically pure, and other reagent are analytical pure.
2. the selection of chromatographic condition
When carrying out the selection of Quercetin-3-O-��-D-Glucose glycosides chromatographic condition, especially determined wavelength and flow phase system are selected. The chromatographic condition finally determined is: inersilC18 (4.6*250mm, 5 ��m) chromatographic column; Methyl alcohol: 0.05% phosphoric acid (46: 54) is moving phase;Flow velocity is 1ml min-1; Post temperature is 25 DEG C; Determined wavelength is 258nm.
(1) selection of determined wavelength
Quercetin-3-O-��-D-Glucose glycosides is carried out full wavelength scanner, and result shows it has maximum absorption at 258nm, therefore selects 258nm to be determined wavelength.
(2) selection of moving phase
Investigated different ratios acetonitrile-water, methanol-water, methyl alcohol-phosphoric acid water be that moving phase is on the impact of separating effect. Result shows, methyl alcohol-0.05% phosphoric acid (46: 54) is more suitable, target peak in sample can be made to reach with impurity peaks and be separated, and peak type is better, therefore elects moving phase as.
3. the selection of trial-product extraction conditions
(1) selection of Extraction solvent
Having investigated methyl alcohol, 60% ethanol and 95% ethanol as Extraction solvent, result shows that the extraction efficiency of methyl alcohol is higher.
(2) selection of extracting method
To supersound extraction 40min, circumfluence distillation 1h, surname extraction 4h, three kinds of extracting method extraction yields compare, and extraction yield is basically identical, it is contemplated that ultrasonic extracting method is easy and simple to handle, when economizing, therefore select supersound extraction.
(3) selection of extraction time
Supersound extraction 30min, 40min, 60min, compare the extraction yield of three, it has been found that 30min extraction yield is slightly low, and 40min and 60min extraction yield is basically identical.
Finally select trial-product extracting method for adding methyl alcohol, supersound extraction 40min.
4. linear relationship is investigated
Accurate absorption Quercetin-3-O-��-D-Glucose glycosides reference substance solution 0.5ml respectively, 1.0ml, 2.0ml, 4.0ml, 5.0ml, in 5ml measuring bottle, add methanol solution to scale. Enter sample 10 �� l respectively, measure peak area by above-mentioned chromatographic condition. Repeat into sample 3 times respectively; Taking the reference substance concentration of correspondence as X-coordinate, its peak area mean value is ordinate zou, drawing standard curve. Obtain regression equation Y=14.774X-13.525 (r=0.9999), linearity range 0.02��0.20mg mL-1��
5. precision test
Accurate above-mentioned Quercetin-3-O-��-D-Glucose glycosides of drawing is according to each 10 �� l of product solution, and continuous sample introduction 6 times, the RSD of peak area is 1.42%.
6. repeated experiment test
Precision takes with 6 parts, different leaf parthenocissus sample totally, prepares by the preparation method of aforementioned trial-product, and the RSD measuring Quercetin 3-O-��-D-Glucose glycosides content is respectively 0.56%.
7. stability test
The same need testing solution of accurate absorption enters sample respectively at 0,2,4,6,8,24h, and the RSD measuring Quercetin-3-O-��-D-Glucose glycosides content is respectively 0.67%, shows that test liquid is stable in 24h.
8. average recovery test
Employing adds sample absorption method, gets the sample of known content, accurately weighed, precision adds a certain amount of Quercetin-3-O-��-D-Glucose glycosides respectively, prepares test liquid according to the preparation method of aforementioned trial-product, measures according to said determination method, calculate the rate of recovery, the results are shown in Table 1.
Table 1 average recovery test (n=6)
9. sample determination
Prepare trial-product and reference substance solution by aforementioned, enter sample respectively, record color atlas (Fig. 1 and Fig. 2), calculate the content of Quercetin-3-O-��-D-Glucose glycosides, the results are shown in Table 2.
Quercetin 3-O-��-D-Glucose glycosides assay result (%) in table 27 batch medicinal material
| Sample number into spectrum | The place of production, source | Gather the date | Peak area | Quercetin 3-O-��-D-Glucose glycosides |
| 1 | Guangxi Ning Chuanxian | 2024-5-17 | 356.5 | 0.04 |
| 2 | Guangxi Yulin 1 | 2014-5-18 | 247.9 | 0.02 |
| 3 | Tiane County, Guangxi | 2014-5-21 | 412.3 | 0.04 |
| 4 | Tiane County, Guangxi eight La Xiang | 2014-5-21 | 284.7 | 0.02 |
| 5 | Guangxi Yulin 2 | 2014-5-1 | 326.2 | 0.02 |
| 6 | Guigang, Guangxi | 2014-6-18 | 726.5 | 0.06 |
| 7 | Guangxi guest | 2014-2-19 | 1382 | 0.07 |
Two, the application of content assaying method of the present invention
Application examples 1
Different leaf parthenocissus medicinal material is from Guigang, Guangxi, with reference to the inventive method, according to " Chinese Pharmacopoeia " 2015 editions one annex VID high effective liquid chromatography for measuring, specific as follows:
Chromatographic condition and system suitability experiment: taking C18 bonded silica gel as weighting agent;Taking methyl alcohol: 0.05% phosphoric acid=46: 54 as moving phase; Flow velocity is 1ml min-1; Post temperature is 25 DEG C; Determined wavelength is 258nm; Theoretical stage number should be not less than 3000.
The preparation of reference substance solution: it is appropriate that precision takes Quercetin-3-O-��-D-Glucose glycosides reference substance, makes 0.20mg ml with methyl alcohol-1Solution.
The preparation of need testing solution: get different leaf parthenocissus medicinal powder (crossing 40 orders) 2.0g, accurately weighed, put in tool plug Erlenmeyer flask, add methyl alcohol 15ml, ultrasonic 40min, lets cool, and supplies weightlessness with methyl alcohol, filters, get continuous filtrate, cross 0.45 ��m of millipore filtration, to obtain final product.
Assay method: accurate absorption reference substance solution and each 10 �� L of need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product.
Result: in this routine different leaf parthenocissus medicinal material, the content of Quercetin 3-O-��-D-Glucose glycosides is 0.06%.
Application examples 2
Different leaf parthenocissus medicinal material is from Laibin City, Guangxi, with reference to the inventive method, according to " Chinese Pharmacopoeia " 2015 editions one annex VID high effective liquid chromatography for measuring, specific as follows:
Chromatographic condition and system suitability experiment: taking C18 bonded silica gel as weighting agent; Taking methyl alcohol: 0.05% phosphoric acid=48: 52 as moving phase; Flow velocity is 1ml min-1; Post temperature is 30 DEG C; Determined wavelength is 270nm; Theoretical stage number should be not less than 3000.
The preparation of reference substance solution: it is appropriate that precision takes Quercetin-3-O-��-D-Glucose glycosides reference substance, makes 0.20mg ml with methyl alcohol-1Solution.
The preparation of need testing solution: get different leaf parthenocissus medicinal powder (crossing 40 orders) 2.0g, accurately weighed, put in tool plug Erlenmeyer flask, add methyl alcohol 15ml, ultrasonic 40min, lets cool, and supplies weightlessness with methyl alcohol, filters, get continuous filtrate, cross 0.45 ��m of millipore filtration, to obtain final product.
Assay method: accurate absorption reference substance solution and each 20 �� L of need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product.
Result: in this routine different leaf parthenocissus medicinal material, the content of Quercetin 3-O-��-D-Glucose glycosides is 0.07%.
Application examples 3
Different leaf parthenocissus medicinal material is from Tiane County, Guangxi, with reference to the inventive method, according to " Chinese Pharmacopoeia " 2015 editions one annex VID high effective liquid chromatography for measuring, specific as follows:
Chromatographic condition and system suitability experiment: taking C18 bonded silica gel as weighting agent; Taking methyl alcohol: 0.05% phosphoric acid=40: 60 as moving phase; Flow velocity is 1ml min-1; Post temperature is 25 DEG C; Determined wavelength is 332nm; Theoretical stage number should be not less than 3000.
The preparation of reference substance solution: it is appropriate that precision takes Quercetin-3-O-��-D-Glucose glycosides reference substance, makes 0.20mg ml with methyl alcohol-1Solution.
The preparation of need testing solution: get different leaf parthenocissus medicinal powder (crossing 40 orders) 2.0g, accurately weighed, put in tool plug Erlenmeyer flask, add methyl alcohol 15ml, ultrasonic 40min, lets cool, and supplies weightlessness with methyl alcohol, filters, get continuous filtrate, cross 0.45 ��m of millipore filtration, to obtain final product.
Assay method: accurate absorption reference substance solution and each 10 �� L of need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product.
Result: in this routine different leaf parthenocissus medicinal material, the content of Quercetin 3-O-��-D-Glucose glycosides is 0.04%.
Application examples 4
Different leaf parthenocissus medicinal material is from Yulin City of Guangxi Zhuang Autonomous, with reference to the inventive method, according to " Chinese Pharmacopoeia " 2015 editions one annex VID high effective liquid chromatography for measuring, specific as follows:
Chromatographic condition and system suitability experiment: taking C18 bonded silica gel as weighting agent;Taking methyl alcohol: 0.05% phosphoric acid=50: 50 as moving phase; Flow velocity is 1ml min-1; Post temperature is 30 DEG C; Determined wavelength is 258nm; Theoretical stage number should be not less than 3000.
The preparation of reference substance solution: it is appropriate that precision takes Quercetin-3-O-��-D-Glucose glycosides reference substance, makes 0.20mg ml with methyl alcohol-1Solution.
The preparation of need testing solution: get different leaf parthenocissus medicinal powder (crossing 40 orders) 2.0g, accurately weighed, put in tool plug Erlenmeyer flask, add methyl alcohol 15ml, ultrasonic 40min, lets cool, and supplies weightlessness with methyl alcohol, filters, get continuous filtrate, cross 0.45 ��m of millipore filtration, to obtain final product.
Assay method: accurate absorption reference substance solution and each 10 �� L of need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product.
Result: in this routine different leaf parthenocissus medicinal material, the content of Quercetin 3-O-��-D-Glucose glycosides is 0.02%.
Application examples 5
Different leaf parthenocissus medicinal material is from Guangxi Ning Chuanxian, with reference to the inventive method, according to " Chinese Pharmacopoeia " 2015 editions one annex VID high effective liquid chromatography for measuring, specific as follows:
Chromatographic condition and system suitability experiment: taking C18 bonded silica gel as weighting agent; Taking methyl alcohol: 0.05% phosphoric acid=47: 53 as moving phase; Flow velocity is 1ml min-1; Post temperature is 30 DEG C; Determined wavelength is 270nm; Theoretical stage number should be not less than 3000.
The preparation of reference substance solution: it is appropriate that precision takes Quercetin-3-O-��-D-Glucose glycosides reference substance, makes 0.20mg ml with methyl alcohol-1Solution.
The preparation of need testing solution: get different leaf parthenocissus medicinal powder (crossing 40 orders) 2.0g, accurately weighed, put in tool plug Erlenmeyer flask, add methyl alcohol 15ml, ultrasonic 40min, lets cool, and supplies weightlessness with methyl alcohol, filters, get continuous filtrate, cross 0.45 ��m of millipore filtration, to obtain final product.
Assay method: accurate absorption reference substance solution and each 10 �� L of need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product.
Result: in this routine different leaf parthenocissus medicinal material, the content of Quercetin 3-O-��-D-Glucose glycosides is 0.04%.
Claims (8)
1. the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in a different leaf parthenocissus medicinal material, it is characterized in that adopting high performance liquid chromatography, taking Quercetin-3-O-��-D-Glucose glycosides as comparison, taking methyl alcohol: 0.05% phosphoric acid=40��60: 50��50 as moving phase.
2. the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in different leaf parthenocissus medicinal material according to claim 1, it is characterised in that chromatographic condition and system suitability experiment be: taking C18 bonded silica gel as weighting agent; Taking methyl alcohol: 0.05% phosphoric acid=40��60: 50��50 as moving phase; Flow velocity is 1��2ml min-1; Post temperature is 25��40 DEG C; Determined wavelength is 258��370nm; Theoretical stage number should be not less than 3000.
3. the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in different leaf parthenocissus medicinal material according to claim 1, it is characterised in that chromatographic condition and system suitability experiment be: taking C18 bonded silica gel as weighting agent; Taking methyl alcohol: 0.05% phosphoric acid=46: 54 as moving phase; Flow velocity is 1ml min-1; Post temperature is 25 DEG C; Determined wavelength is 258nm; Theoretical stage number should be not less than 3000.
4. the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in different leaf parthenocissus medicinal material according to claim 1, it is characterized in that the preparation of reference substance solution is undertaken by following operation: precision takes Quercetin-3-O-��-D-Glucose glycosides reference substance, makes 0.02��0.20mg ml with methyl alcohol-1Solution.
5. the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in different leaf parthenocissus medicinal material according to claim 1, it is characterised in that: described reference substance solution concentration is 0.20mg ml-1��
6. the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in different leaf parthenocissus medicinal material according to claim 1, it is characterized in that the preparation of need testing solution is undertaken by following operation: get different leaf parthenocissus medicinal powder 2.0g, accurately weighed, put in tool plug Erlenmeyer flask, add methyl alcohol 15ml, ultrasonic 40min, lets cool, and supplies weightlessness with methyl alcohol, filter, get continuous filtrate, cross 0.45 ��m of millipore filtration, to obtain final product.
7. the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in different leaf parthenocissus medicinal material according to claim 1, it is characterized in that measuring and undertaken by following operation: accurate absorption reference substance solution and each 10��20 �� L of need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product.
8. the content assaying method of Quercetin 3-O-��-D-Glucose glycosides in different leaf parthenocissus medicinal material according to claim 1, it is characterized in that measuring and undertaken by following operation: accurate absorption reference substance solution and each 10 �� L of need testing solution respectively, inject high performance liquid chromatograph, measure, to obtain final product.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106404955A (en) * | 2016-09-10 | 2017-02-15 | 长治市食品药品检验所 | Method for measuring content of quercetin-7-glucoside in sorghum red pigment |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106404955A (en) * | 2016-09-10 | 2017-02-15 | 长治市食品药品检验所 | Method for measuring content of quercetin-7-glucoside in sorghum red pigment |
| CN106404955B (en) * | 2016-09-10 | 2019-12-17 | 长治医学院 | Method for determining content of quercetin-7-glucoside in sorghum red pigment |
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