CN103149299B - 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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- CN103149299B CN103149299B CN201310068724.5A CN201310068724A CN103149299B CN 103149299 B CN103149299 B CN 103149299B CN 201310068724 A CN201310068724 A CN 201310068724A CN 103149299 B CN103149299 B CN 103149299B
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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, the Fast Measurement that can simultaneously measure the content of epicatechin in buzhaye, Vitexina, Saponaretin and 4 kinds of flavones ingredients of narcissin, belongs 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 Guangdong and Guangxi Provinces regions resources, wherein the Yangxi in Guangdong, Zhanjiang are main product ground, all take wild as main.This samples micro-acid, cool in nature, nontoxic, has clearing heat and promoting diuresis, the stomach invigorating stagnant effect that disappears, and is used for the treatment of cold, fever, jaundice, poor appetite, indigestion, abdominal distention, has 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, efficiency is not high.
Therefore, need badly at present and set up a kind of quick rapid assay methods that can simultaneously measure 4 kinds of flavones ingredients of buzhaye, the method meets 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 simultaneously measure 4 kinds of flavones ingredients of buzhaye.
The technical scheme that the present invention provided is for achieving the above object:
In buzhaye, a rapid assay methods for flavones ingredient content, is characterized in that, it adopts HPLC method, and the method is the disposable content of simultaneously measuring 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 every mL containing the mixed solution of epicatechin 60 μ g, Vitexina 25 μ g, Saponaretin 40 μ g and narcissin 60 μ g, obtain;
(3) preparation of need testing solution
Get the about 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, the weight of supplying less loss with 70% methyl alcohol, shakes up, and filters, get subsequent filtrate, obtain;
(4) determination method
Precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, and injection liquid chromatography is measured, and obtains 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 epicatechin, Vitexina, Saponaretin and narcissin content in the buzhaye sample of unknown content simultaneously, can be used for the quick quality control of buzhaye medicinal material in pharmacy procedure.
Accompanying drawing explanation
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 simultaneously measuring the rapid assay methods of 4 kinds of flavones ingredients of buzhaye is:
1 instrument and reagent
Agilent1200 high performance liquid chromatograph, Mettler XS205DU type electronic analytical balance (Switzerland).
Buzhaye sample each cities and counties Ji Ge great pharmaceuticals that gathers respectively from Guangdong Province buys, 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., the kind of recording for < < Chinese Pharmacopoeia > > version in 2010.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 Rui Fensi bio tech ltd, Chengdu; Methyl alcohol is chromatographically pure, and 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%;
The preparation of 2.2 reference substance solution
Get epicatechin, Vitexina, Saponaretin and narcissin reference substance appropriate, accurately weighed, add 70% methyl alcohol and make every mL containing the mixed solution of epicatechin 145.80 μ g, Vitexina 58.10 μ g, Saponaretin 95.80 μ g and narcissin 144.40 μ g, as storing solution.
The preparation of 2.3 need testing solutions
Get the about 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, the weight of supplying less loss with 70% methyl alcohol, shakes up, and filters, get subsequent filtrate, obtain.
2.4 determination method
Precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, and injection liquid chromatography is measured.
2.5 linear relationships are investigated
Precision measures mixes reference substance solution 0.5, 1, 2, 4, 6, 8, 10ml, put respectively in 10ml measuring bottle, add 70% methyl alcohol to dissolve and be diluted to scale, obtaining epicatechin mass concentration is 7.290, 14.580, 29.160, 58.320, 87.480, 116.640, 145.800 μ g/ml, Vitexina mass concentration is 2.905, 5.810, 11.620, 23.240, 34.860, 46.480, 58.100 μ g/ml, Saponaretin mass concentration is 4.790, 9.580, 19.160, 38.320, 57.480, 76.640, 95.800 μ g/ml, 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, measure by " 2.1 " lower chromatographic condition.The peak area integrated value (Y) of take is ordinate, and sample size (X, μ g/ml) is horizontal ordinate, carries out linear regression, and obtaining regression equation is Y
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 good linear relation with peak area integrated value separately respectively within the scope of 7.290-145.8,2.905-58.10,4.790-95.80,7.220-144.4 μ g/ml.
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, injection liquid chromatography, by " 2.1 " lower chromatographic condition, repeat sample introduction 6 times, 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, respectively at 0,1,2,4,8,12,24h 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 24h internal stability.
2.8 replica test
Precision takes with a collection of buzhaye sample (S9) appropriate, totally 6 parts, respectively by " 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 tests
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 " 2.3 " below legal system available test sample solution, by " 2.1 " lower chromatographic condition sample introduction, measure, calculate average recovery, result is respectively in Table 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 " 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 shown in Fig. 1.
Table 5 sample size measurement result
By above experimental result, can be drawn, rapid assay methods of the present invention is simple, quick, accurate, highly sensitive, precision, good stability, can measure epicatechin, Vitexina, Saponaretin and narcissin content in the buzhaye sample of unknown content simultaneously, 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 to description.Should be understood that description above 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 (1)
1. a rapid assay methods for flavones ingredient content in buzhaye, 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 every mL containing the mixed solution of epicatechin 60 μ g, Vitexina 25 μ g, Saponaretin 40 μ g and narcissin 60 μ g, obtain;
(3) preparation of need testing solution
Get buzhaye medicinal powder and cross No. three and sieve about 1g, accurately weighed, put in tool plug conical flask, precision adds 70% methyl alcohol 50mL, close plug, weighed weight, ultrasonic power 200W frequency 40kHz, processes 1 hour, lets cool, weighed weight again, the weight of supplying less loss with 70% methyl alcohol, shakes up, and filters, get subsequent filtrate, obtain;
(4) determination method
Precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, and injection liquid chromatography is measured, and obtains the content of epicatechin, Vitexina, Saponaretin and narcissin.
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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 |
Non-Patent Citations (6)
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| HPLC测定布渣叶中牡荆苷的含量;罗文汇 等;《中国实验方剂学杂志》;20120331;第18卷(第5期);第110-111页 * |
| 卢鹏 等.布渣叶HPLC指纹图谱研究.《中药新药与临床药理》.2012,第23卷(第5期),第567-569页. |
| 卢鹏 等.布渣叶药材质量标准的初步研究.《广东药学院学报》.2012,第28卷(第4期),第419-422页. |
| 布渣叶HPLC指纹图谱研究;卢鹏 等;《中药新药与临床药理》;20120930;第23卷(第5期);第567-569页 * |
| 布渣叶药材质量标准的初步研究;卢鹏 等;《广东药学院学报》;20120831;第28卷(第4期);第420-421页的2.2 * |
| 罗文汇 等.HPLC测定布渣叶中牡荆苷的含量.《中国实验方剂学杂志》.2012,第18卷(第5期),第110-111页. |
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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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