CN103175804A - Method for determining contents of flavonoid constituents in microcos paniculata based on near infrared spectrum technology - Google Patents
Method for determining contents of flavonoid constituents in microcos paniculata based on near infrared spectrum technology Download PDFInfo
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- CN103175804A CN103175804A CN2013100671694A CN201310067169A CN103175804A CN 103175804 A CN103175804 A CN 103175804A CN 2013100671694 A CN2013100671694 A CN 2013100671694A CN 201310067169 A CN201310067169 A CN 201310067169A CN 103175804 A CN103175804 A CN 103175804A
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Abstract
The invention discloses a method for determining contents of flavonoid constituents in microcos paniculata based on a near infrared spectrum technology. The method is used for determining the contents of four flavonoid constituents, namely epicatechin, vitexin, isovitexin and narcissoside in the microcos paniculata based on the near infrared spectrum technology. The method is a rapid determination method and is simple, correct, high in sensitivity, good in precision and stability, and very suitable for application in the rapid quality control on the microcos paniculata medicinal material during medicine preparation.
Description
Technical field
The present invention relates to the method for quality control of Chinese medicine; Be specifically related to the assay method of flavones ingredient content in a kind of buzhaye, based on the rapid assay methods of epicatechin, Vitexina, Saponaretin and 4 kinds of flavones ingredients of narcissin in the buzhaye of near-infrared spectrum technique, 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.Near-infrared spectral analysis technology (near infrared spectroscopy, NIRS) have analysis speed fast, to the sample nondestructive evil, without remarkable advantages such as chemical contaminations, be widely used in recent years the every field such as agricultural, tobacco, petrochemical complex, medicine.The present invention utilizes near-infrared spectrum technique in conjunction with partial least square method (Partical Least Square, PLS), epicatechin, Vitexina, Saponaretin and 4 kinds of flavones ingredients of narcissin in buzhaye are carried out assay, be intended to set up the rapid assay methods of 4 kinds of flavones ingredients of a kind 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 the rapid assay methods of epicatechin in buzhaye, Vitexina, Saponaretin and 4 kinds of flavones ingredients of narcissin, described rapid assay methods is based on near-infrared spectrum technique.
The technical scheme that the present invention provides for achieving the above object is:
The assay method of flavones ingredient content in a kind of buzhaye is characterized in that the method is to adopt near-infrared spectrum technique to measure.It specifically comprises the following steps:
Step 1: the mensuration of epicatechin, Vitexina, Saponaretin and narcissin content
Measure with the HPLC method content that epicatechin, Vitexina, Saponaretin and narcissin in the buzhaye sample are used in modeling;
Step 2: the collection of near infrared spectrum
Gather the modeling near infrared spectrum of buzhaye sample;
Step 3: the foundation of model
According to the near infrared spectrum of modeling with the buzhaye sample, the epicatechin that itself and HPLC record, Vitexina, Saponaretin and narcissin content, choose representational sample composition calibration set, use partial least square method and set up calibration model, buzhaye NIR spectrum is level and smooth and carry out the ordinate normalized with 9, and the modeling wave band of epicatechin, Saponaretin and narcissin is 7800～9999cm
-1, the modeling wave band of Vitexina is 7100～7800cm
-1, get the buzhaye model;
Step 4: the Fast Measurement of sample
Gather the near infrared spectrum data of buzhaye sample to be measured, it is imported in model, carry out Fast Measurement, namely get the content of epicatechin, Vitexina, Saponaretin and narcissin in the buzhaye sample.
The mensuration of epicatechin, Vitexina, Saponaretin and narcissin content in abovementioned steps one can adopt the content assaying method of the HPLC of the known epicatechin of prior art, Vitexina, Saponaretin and narcissin.Preferred employing " measuring simultaneously the HPLC content determination of epicatechin in buzhaye, Vitexina, Saponaretin and 4 kinds of flavones ingredients of narcissin ", concrete assay method is as follows:
(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 58.32 μ g, Vitexina 23.24 μ g, Saponaretin 38.32 μ g and narcissin 57.76 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.
The method that the invention has the advantages that is simple, accurate, highly sensitive, precision, good stability, the near infrared forecast model is in case set up, just can be rapidly and accurately the buzhaye sample of unknown content be carried out Fast Measurement, can be used for the quick quality control of buzhaye medicinal material in pharmacy procedure.
Description of drawings
Fig. 1,49 parts of buzhaye sample epicatechin content distribution figure in the embodiment of the present invention.
Fig. 2,49 parts of buzhaye sample Vitexina content distribution figure in the embodiment of the present invention.
Fig. 3,49 parts of buzhaye sample Saponaretin content distribution figure in the embodiment of the present invention.
Fig. 4,49 parts of buzhaye sample narcissus chaste tree glycosides content distribution figure in the embodiment of the present invention.
Fig. 5, the NIR spectrum stacking diagram of 49 parts of buzhaye samples in the embodiment of the present invention.
Fig. 6, the epicatechin content prediction value of 49 parts of buzhaye samples and the correlogram between measured value in the embodiment of the present invention.
Fig. 7, the Vitexina content prediction value of 49 parts of buzhaye samples and the correlogram between measured value in the embodiment of the present invention.
Fig. 8, the Saponaretin content prediction value of 49 parts of buzhaye samples and the correlogram between measured value in the embodiment of the present invention.
Fig. 9, the narcissin content prediction value of 49 parts of buzhaye samples and the correlogram between measured value in the embodiment of the present invention.
Embodiment
One, the foundation of near infrared forecast model
1 instrument and reagent
Nicolet6700 Fourier transformation infrared spectrometer (U.S. Thermo Nicolet company), is furnished with integrating sphere diffuse reflection sampling system and Omnic signals collecting software, the Agilent1200 high performance liquid chromatograph, Mettler XS205DU type electronic analytical balance (Switzerland), data processing software are SAS JMP Statistical Discovery V9.0.2 statistical study great master.
The buzhaye sample is gathered respectively, and each cities and counties reach in each large pharmaceuticals purchase from Guangdong Province, collect altogether 49 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
2.1HPLC the content of epicatechin, Vitexina, Saponaretin and narcissin in the mensuration buzhaye
2.1.1 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%;
2.1.2 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 58.32 μ g, Vitexina 23.24 μ g, Saponaretin 38.32 μ g and narcissin 57.76 μ g, and get final product.
2.1.3 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.
2.1.4 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.The content distribution figure of the epicatechin of 49 parts of buzhaye samples, Vitexina, Saponaretin and narcissin in embodiment sees Fig. 1～4.
2.2 the collection of near infrared spectrum
Get approximately 5g of buzhaye sample powder (crossing 60 mesh sieves), put into sample cup, evenly pave, make powder closely knit under vibration number.Take air as reference, scan the test sample mode by following experiment condition: integrating sphere diffuse reflection, resolution are 4cm
-1, automatic gain, the spectra collection scope is 10000～4000cm
-1, scanning times is 32 times, and temperature is 20～25 ℃, and relative humidity is 10%～20%.Each sample is got 3 parts and is done parallel laboratory test, asks for its averaged spectrum.In embodiment, Fig. 5 is seen in the stack of the near infrared spectrum of 49 parts of buzhaye samples.
2.3 the foundation of model
2.3.1 the selection of calibration set and checking collection sample
From 49 samples collecting, according to its epicatechin, Vitexina, Saponaretin and narcissin content, choose 39 representational sample composition calibration sets, remain 10 samples and be checking collection sample, table 1 has been listed calibration set and checking collection sample epicatechin, Vitexina, Saponaretin and narcissin content range.
Table 1 calibration set and checking collection sample epicatechin, Vitexina, Saponaretin and narcissin Content scope
2.3.2 spectroscopic data pre-service
This test is by the related coefficient (R of comparison model
2) and root-mean-square error (RMSE), select proper preprocessing procedures.Data can be found out from table 2, the best preprocess method of buzhaye epicatechin, Vitexina, Saponaretin and narcissin calibration model be 9 smoothly in conjunction with ordinate normalization.
The different preprocessing procedures of table 2 is to R
2Impact with RMSE
2.3.3 the selection of modeling wave band
This effects different spectral ranges to model related coefficient (R
2) and the impact of root-mean-square error (RMSE).Result is as shown in table 3, and the best modeled wave band of epicatechin, Saponaretin and narcissin is 7800～9999cm
-1, the best modeled wave band of Vitexina is 7100～7800cm
-1, can make R like this
2When reaching maximum, RMSE is minimum.
When adopting the PLS modeling, the selection of main cause subnumber is determined by reciprocation and the matrix effect of measured matter in system.Therefore, calibration result preferably be should obtain when choosing, match deficiency and overfitting avoided again.This test selects the predicted root mean square error value (RMSE) that cross validation obtains to determine the main cause subnumber.
Table 3 band selection is to R
2Impact with RMSE
2.3.4 the foundation of calibration model
In application SAS JMP Statistical Discovery V9.0.2 statistical study great master, partial least square method is set up calibration model.Buzhaye NIR spectrum is level and smooth and carry out the ordinate normalized with 9, and the modeling wave band of epicatechin, Saponaretin and narcissin is 7800～9999cm
-1, the modeling wave band of Vitexina is 7100～7800cm
-1, determine that through cross validation the main cause subnumber of epicatechin, Vitexina, Saponaretin and narcissin is respectively 10,7,9,8, obtain the R of model
2 Epicatechin=0.9756, RMSE
Epicatechin=0.0075; R
2 Vitexina=0.9756, RMSE
Vitexina=0.0075; R
2 Saponaretin=0.9756, RMSE
Saponaretin=0.0075; R
2 Narcissin=0.9756, RMSE
Narcissin=0.0075.Correlogram between the NIR predicted value of epicatechin, Vitexina, Saponaretin and narcissin and pharmacopeia method measured value is seen Fig. 6～9.
Two, sample Fast Measurement
To remain 10 samples as checking collection sample, the content of checking collection sample is all in the estimation range of model.According to the above-mentioned calibration model of building, in the near infrared spectrum data importing model with testing sample, can carry out fast prediction to its content.The coefficient R of epicatechin, Vitexina, Saponaretin and narcissin fitting a straight line
2Be respectively 0.9276,0.9084,0.9252,0.9305.Result shows, the deviation between NIR predicted value and HPLC measured value is less, and the predictive ability of institute's established model is better, sees Table 1.
Table 1 buzhaye sample Fast Measurement result
Can be drawn by above experimental result, simple, accurate, highly sensitive, precision, good stability, the near infrared forecast model is in case set up, and just can be rapidly and accurately the buzhaye sample of unknown content be carried out Fast Measurement, 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 assay method of flavones ingredient content in a buzhaye, described method is measured based on near-infrared spectrum technique, it is characterized in that it comprises the following steps:
(1) mensuration of epicatechin, Vitexina, Saponaretin and narcissin content
Measure with the HPLC method content that epicatechin, Vitexina, Saponaretin and narcissin in the buzhaye sample are used in modeling;
(2) collection of near infrared spectrum
Gather the modeling near infrared spectrum of buzhaye sample;
(3) foundation of model
According to the near infrared spectrum of modeling with the buzhaye sample, and epicatechin, Vitexina, Saponaretin and narcissin content that in step (1), the HPLC method records, choose representational sample composition calibration set, use partial least square method and set up calibration model, buzhaye NIR spectrum is level and smooth and carry out the ordinate normalized with 9, and the modeling wave band of epicatechin, Saponaretin and narcissin is 7800～9999cm
-1, the modeling wave band of Vitexina is 7100～7800cm
-1, get the buzhaye model;
(4) Fast Measurement of sample
Gather the near infrared spectrum data of buzhaye sample to be measured, it is imported in model, carry out Fast Measurement, namely get the content of epicatechin, Vitexina, Saponaretin and narcissin in the buzhaye sample.
2. the assay method of flavones ingredient content in buzhaye claimed in claim 1 is characterized in that the concrete grammar of the mensuration of epicatechin, Vitexina, Saponaretin and narcissin content in step (1) is:
(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 58.32 μ g, Vitexina 23.24 μ g, Saponaretin 38.32 μ g and narcissin 57.76 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, to put in tool plug conical flask, precision adds 70% methyl alcohol 50mL, close plug, weighed weight, ultrasonic processing 1 hour lets cool, more weighed weight, supplies 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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| 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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Application publication date: 20130626 |