CN103149299A - Method for quickly measuring content of flavonoid constituents in paniculata - Google Patents
Method for quickly measuring content of flavonoid constituents in paniculata Download PDFInfo
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- CN103149299A CN103149299A CN2013100687245A CN201310068724A CN103149299A CN 103149299 A CN103149299 A CN 103149299A CN 2013100687245 A CN2013100687245 A CN 2013100687245A CN 201310068724 A CN201310068724 A CN 201310068724A CN 103149299 A CN103149299 A CN 103149299A
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- buzhaye
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- saponaretin
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Abstract
The invention discloses a method for quickly measuring the content of flavonoid constituents in paniculata. The method is used for measuring the contents of epicatechin, vitexin, isovitexin and narcissoside in paniculata, wherein the contents can be quickly measured by using an HPLC (High Performance Liquid Chromatography) method. The method can be used for measuring the contents of the four flavonoid constituents, namely the epicatechin, the vitexin, the isovitexin and the narcissosid, in the paniculata simultaneously through one step. The measurement method provided by the invention has the advantages of high speed, simplicity, accuracy, high sensitivity and good stability, and is very beneficial to the application in quick quality control of medicinal materials of the paniculata in a drug preparation process.
Description
Technical field
The present invention relates to the method for quality control of Chinese medicine, be specifically related to the rapid assay methods of flavones ingredient content in buzhaye, can measure simultaneously the Fast Measurement of the content of epicatechin in buzhaye, Vitexina, Saponaretin and 4 kinds of flavones ingredients of narcissin, belong to traditional Chinese medicine quality detection technique field.
Background technology
Buzhaye is the dry leaf of Tiliaceae plant Fallopia nervosa Microcos paniculata L., originates in the ground such as Guangdong, Guangxi, Hainan, Yunnan, and also there are distribution in India, Indonesia; In China, especially abundant with the Guangdong and Guangxi Provinces regions resources, wherein the Yangxi in Guangdong, Zhanjiang are main product ground, all take wild as main.The little acid of this taste, cool in nature, nontoxic, have clearing heat and promoting diuresis, the stomach invigorating stagnant effect that disappears, be used for the treatment of cold, fever, jaundice, poor appetite, indigestion, abdominal distention, have loose bowels, sore, centipde-bite etc.
Buzhaye mainly contains the compositions such as flavones, alkaloid, organic acid, volatile oil, tannin, phenols.Flavones ingredient wherein has many-sided biologically active, as anti peroxidation of lipid, anti-ageing, remove free radical, reduce blood fat and cholesterolemia, anti-inflammation, antiviral, strengthen immunologic function etc.
Available technology adopting HPLC method is only open carries out respectively assay to epicatechin, Vitexina, Saponaretin and 4 kinds of flavones ingredients of narcissin in buzhaye, the content quick determination method of known HPLC is all that one or both flavones are wherein measured, the content quick determination method of the HPLC that unexposed record is measured four kinds of flavonoids compositions simultaneously, so its process is long, the used time is more, efficient is not high.
Therefore, need badly at present and set up a kind of quick rapid assay methods that can measure simultaneously 4 kinds of flavones ingredients of buzhaye, the method satisfies the requirement of the quick quality control of buzhaye medicinal material in pharmacy procedure.
Summary of the invention
The object of the present invention is to provide a kind of quick rapid assay methods that can measure simultaneously 4 kinds of flavones ingredients of buzhaye.
The technical scheme that the present invention provides for achieving the above object is:
In a kind of buzhaye, the rapid assay methods of flavones ingredient content, is characterized in that, it adopts the HPLC method, and the method is the disposable content of measuring simultaneously epicatechin in buzhaye, Vitexina, Saponaretin and 4 kinds of flavones ingredients of narcissin.
It specifically comprises the following steps:
(1) chromatographic condition
Chromatographic column: Shiseido Capcell pak MG C
18(4.6 * 250mm, 5 μ m) post; Mobile phase: take methyl alcohol as mobile phase A, take water as Mobile phase B, carry out gradient elution; Detect wavelength: 280nm; Flow velocity: 1.0mL/min; Column temperature: 25 ℃;
Condition of gradient elution is:
0～35 minute, mobile phase A 21～35%, Mobile phase B 79～65%;
35～60 minutes, mobile phase A 35～48%, Mobile phase B 65～52%;
60～62 minutes, mobile phase A 48～80%, Mobile phase B 52～20%;
(2) preparation of reference substance solution
Get epicatechin, Vitexina, Saponaretin and narcissin reference substance appropriate, accurately weighed, add 70% methyl alcohol and make the mixed solution that every mL contains epicatechin 60 μ g, Vitexina 25 μ g, Saponaretin 40 μ g and narcissin 60 μ g, and get final product;
(3) preparation of need testing solution
Get approximately 1g of buzhaye medicinal powder (crossing sieve No. three), accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50mL, close plug, weighed weight, ultrasonic processing (power 200W, frequency 40kHz) 1 hour lets cool, weighed weight again, supply the weight of less loss with 70% methyl alcohol, shake up, filter, get subsequent filtrate, and get final product;
(4) determination method
Precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, and the injection liquid chromatography is measured, and gets the content of epicatechin, Vitexina, Saponaretin and narcissin.
Rapid assay methods of the present invention is simple, quick, accurate, highly sensitive, precision, good stability, can disposablely measure simultaneously epicatechin, Vitexina, Saponaretin and narcissin content in the buzhaye sample of unknown content, can be used for the quick quality control of buzhaye medicinal material in pharmacy procedure.
Description of drawings
Fig. 1, buzhaye HPLC spectrogram in the embodiment of the present invention, A. test sample; B. mix reference substance; 1 epicatechin; 2 Vitexinas; 3 Saponaretins; 4 narcissins.
Embodiment
Employing HPLC method provided by the invention, the step of measuring simultaneously the rapid assay methods of 4 kinds of flavones ingredients of buzhaye is:
1 instrument and reagent
The Agilent1200 high performance liquid chromatograph, Mettler XS205DU type electronic analytical balance (Switzerland).
The buzhaye sample is gathered respectively, and each cities and counties reach in each large pharmaceuticals purchase from Guangdong Province, collect altogether 10 parts, all samples is accredited as through professor Liu Fajin of Academy of Traditional Chinese Medicine, Guangdong Province: the dry leaf of Tiliaceae plant Fallopia nervosa Microcos paniculata L. is the " kind that Chinese pharmacopoeia version in 2010 is recorded.Epicatechin (lot number: 878-200102), Vitexina (lot number: 111687-200602) be purchased from Nat'l Pharmaceutical ﹠ Biological Products Control Institute; Saponaretin (lot number: Y-127-110705), narcissin (lot number: S-063-110926) be purchased from Chengdu Rui Fensi bio tech ltd; Methyl alcohol is chromatographically pure, and the liquid phase water is Watson distilled water, and it is pure that all the other reagent are analysis.
2 methods and result
2.1 chromatographic condition
Chromatographic column: Shiseido Capcell pak MG C
18(4.6 * 250mm, 5 μ m) post; Mobile phase: take methyl alcohol as mobile phase A, take water as Mobile phase B, according to the form below carries out gradient elution; Detect wavelength: 280nm; Flow velocity: 1.0mL/min; Column temperature: 25 ℃.
Condition of gradient elution is:
0～35 minute, mobile phase A 21～35%, Mobile phase B 79～65%;
35～60 minutes, mobile phase A 35～48%, Mobile phase B 65～52%;
60～62 minutes, mobile phase A 48～80%, Mobile phase B 52～20%;
2.2 the preparation of reference substance solution
Get epicatechin, Vitexina, Saponaretin and narcissin reference substance appropriate, accurately weighed, add 70% methyl alcohol and make the mixed solution that every mL contains epicatechin 145.80 μ g, Vitexina 58.10 μ g, Saponaretin 95.80 μ g and narcissin 144.40 μ g, as storing solution.
2.3 the preparation of need testing solution
Get approximately 1g of buzhaye medicinal powder (crossing sieve No. three), accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50mL, close plug, weighed weight, ultrasonic processing (power 200W, frequency 40kHz) 1 hour lets cool, weighed weight again, supply the weight of less loss with 70% methyl alcohol, shake up, filter, get subsequent filtrate, and get final product.
2.4 determination method
Precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, and the injection liquid chromatography is measured.
2.5 linear relationship is investigated
precision measures mixes reference substance solution 0.5, 1, 2, 4, 6, 8, 10ml, put respectively in the 10ml measuring bottle, add 70% methyl alcohol to dissolve and be diluted to scale, getting the epicatechin mass concentration is 7.290, 14.580, 29.160, 58.320, 87.480, 116.640, 145.800 μ g/ml, the Vitexina mass concentration is 2.905, 5.810, 11.620, 23.240, 34.860, 46.480, 58.100 μ g/ml, the Saponaretin mass concentration is 4.790, 9.580, 19.160, 38.320, 57.480, 76.640, 95.800 μ g/ml, the narcissin mass concentration is 7.220, 14.440, 28.880, 57.760, 86.640, 115.520, 144.400 the serial solution of μ g/ml.Accurate each the 10 μ l injection liquid chromatographies of above-mentioned serial solution of drawing are measured by " 2.1 " lower chromatographic condition.Take peak area integrated value (Y) as ordinate, sample size (X, μ g/ml) is horizontal ordinate, carries out linear regression, and getting regression equation is Y
The table youngster Theine=6.1847X-9.3822(r=0.9999, n=7); Y
Vitexina=14.671X-12.085(r=0.9999, n=7); Y
Saponaretin=19.632X-26.722(r=0.9999, n=7); Y
Narcissin=8.1311X-13.749(r=0.9999, n=7).Result shows, the sample size of Vitexina and Saponaretin is the good linear relation with peak area integrated value separately respectively in 7.290-145.8,2.905-58.10,4.790-95.80,7.220-144.4 μ g/ml scope.
2.6 precision test
Accurate same mixing reference substance solution (epicatechin 58.32 μ g, Vitexina 23.24 μ g, Saponaretin 38.32 μ g and narcissin 57.76 μ g) the 10 μ l that draw, the injection liquid chromatography, repeat sample introduction 6 times by " 2.1 " lower chromatographic condition, record chromatogram.As a result, the RSD of epicatechin, Vitexina, Saponaretin and narcissin peak area be respectively 0.96%, 1.22%, 1.04% and 0.80%(n be 6), show that instrument precision is good.
2.7 stability test
Accurate same need testing solution (S10) the 10 μ l that draw are respectively at 0,1,2,4,8,12, the 24h sample introduction measures, and records peak area.As a result, the RSD of epicatechin, Vitexina, Saponaretin and narcissin peak area be respectively 1.08%, 0.87%, 0.56% and 0.67%(n be 7), show that need testing solution is good at the 24h internal stability.
2.8 replica test
Precision takes with a collection of buzhaye sample (S9) appropriate, totally 6 parts, respectively by " a 2.3 " below legal system available test sample solution, then measure by " 2.1 " lower chromatographic condition sample introduction, record peak area, the massfraction of epicatechin, Vitexina, Saponaretin and narcissin in calculation sample.As a result, the average quality mark of epicatechin is 0.0929%, RSD=1.18%(n=6); The average quality mark of Vitexina is 0.1224%, RSD=0.89%(n=6); The average quality mark of Saponaretin is 0.1604%, RSD=0.92%(n=6); The average quality mark of narcissin is 0.2189%, RSD=0.98%(n=6), shows that this method repeatability is good.
2.9 average recovery test
Get with 9 parts, the buzhaye sample (S9) of a collection of known quality mark, every part of about 0.5g, accurately weighed, precision adds 70% methanol solution 50ml of epicatechin, Vitexina, Saponaretin and the narcissin mixing reference substance of certain mass concentration respectively, by " a 2.3 " below legal system available test sample solution, measure by " 2.1 " lower chromatographic condition sample introduction, calculate average recovery, result sees Table respectively 1～4.
Table 1 epicatechin average recovery test findings (n=9)
Table 2 Vitexina average recovery test findings (n=9)
Table 3 Saponaretin average recovery test findings (n=9)
Table 4 narcissin average recovery test findings (n=9)
2.10 sample size is measured
The buzhaye sample of getting separate sources is appropriate, respectively by " a 2.3 " below legal system available test sample solution, then carries out assay by " 2.1 " lower chromatographic condition, the results are shown in Table 5; Chromatogram is seen Fig. 1.
Table 5 sample size measurement result
Can be drawn by above experimental result, rapid assay methods of the present invention is simple, quick, accurate, highly sensitive, precision, good stability, can measure simultaneously epicatechin, Vitexina, Saponaretin and narcissin content in the buzhaye sample of unknown content, can be used for the quick quality control of buzhaye medicinal material in pharmacy procedure.
According to above preferred embodiment, the present invention has been made description.Should be understood that the description of front and embodiment are just to illustrating the present invention.Under prerequisite without departing from the spirit and scope of the present invention, those skilled in the art can design multiple alternative of the present invention and improvement project, within it all should be understood to be in protection scope of the present invention.
Claims (2)
1. the rapid assay methods of flavones ingredient content in a buzhaye, is characterized in that, it adopts the HPLC method, and the method is the disposable content of measuring simultaneously epicatechin in buzhaye, Vitexina, Saponaretin and 4 kinds of flavones ingredients of narcissin.
2. the rapid assay methods of flavones ingredient content in buzhaye claimed in claim 1 is characterized in that it comprises the following steps:
(1) chromatographic condition
Chromatographic column: Shiseido Capcell pak MG C
18, 4.6 * 250mm, 5 μ m posts; Mobile phase: take methyl alcohol as mobile phase A, take water as Mobile phase B, carry out gradient elution; Detect wavelength: 280nm; Flow velocity: 1.0mL/min; Column temperature: 25 ℃;
Condition of gradient elution:
0～35 minute, mobile phase A 21～35%, Mobile phase B 79～65%;
35～60 minutes, mobile phase A 35～48%, Mobile phase B 65～52%;
60～62 minutes, mobile phase A 48～80%, Mobile phase B 52～20%;
(2) preparation of reference substance solution
Get epicatechin, Vitexina, Saponaretin and narcissin reference substance appropriate, accurately weighed, add 70% methyl alcohol and make the mixed solution that every mL contains epicatechin 60 μ g, Vitexina 25 μ g, Saponaretin 40 μ g and narcissin 60 μ g, and get final product;
(3) preparation of need testing solution
Get the buzhaye medicinal powder and cross No. three and sieve approximately 1g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50mL, close plug, weighed weight, ultrasonic power 200W frequency 40kHz processed 1 hour, let cool, weighed weight again, supply the weight of less loss with 70% methyl alcohol, shake up, filter, get subsequent filtrate, and get final product;
(4) determination method
Precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, and the injection liquid chromatography is measured, and gets the content of epicatechin, Vitexina, Saponaretin and narcissin.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108375646A (en) * | 2018-02-01 | 2018-08-07 | 贵州维康子帆药业股份有限公司 | A kind of oxalic detection method |
| CN111929400A (en) * | 2020-09-28 | 2020-11-13 | 广东省第二中医院（广东省中医药工程技术研究院） | Method for determining content of main components in Bushao lipid-regulating capsule |
Citations (1)
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| CN102836188A (en) * | 2012-09-25 | 2012-12-26 | 孙冬梅 | Folium microcotis total flavone extract and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102836188A (en) * | 2012-09-25 | 2012-12-26 | 孙冬梅 | Folium microcotis total flavone extract and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
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| 卢鹏 等: "布渣叶HPLC指纹图谱研究", 《中药新药与临床药理》, vol. 23, no. 5, 30 September 2012 (2012-09-30), pages 567 - 569 * |
| 卢鹏 等: "布渣叶药材质量标准的初步研究", 《广东药学院学报》, vol. 28, no. 4, 31 August 2012 (2012-08-31), pages 419 - 422 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108375646A (en) * | 2018-02-01 | 2018-08-07 | 贵州维康子帆药业股份有限公司 | A kind of oxalic detection method |
| CN111929400A (en) * | 2020-09-28 | 2020-11-13 | 广东省第二中医院（广东省中医药工程技术研究院） | Method for determining content of main components in Bushao lipid-regulating capsule |
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Address after: 510000 Hanford Road, Guangzhou, Guangdong, No. 60, No. Patentee after: GUANGDONG SECOND TRADITIONAL CHINESE MEDICINE HOSPITAL (GUANGZHOU PROVINCE ENGINEERING TECHNOLOGY RESEARCH INSTITUTE OF T.C.M.) Address before: 510000 Hanford Road, Guangzhou, Guangdong, No. 60, No. Patentee before: Guangdong Institute of Traditional Chinese Medicine |
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