CN109856264B - Method for detecting content of effective components in blumea balsamifera medicinal material - Google Patents
Method for detecting content of effective components in blumea balsamifera medicinal material Download PDFInfo
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Abstract
The scheme discloses a detection method for the content of active ingredients in a blumea balsamifera medicinal material, which belongs to the technical field of detection of the medicinal ingredients of traditional Chinese medicinal materials, and the method comprises the following steps: 1) respectively dissolving 18 kinds of effective components such as Rutin (Rutin) and Hyperoside (Hyperoside) in methanol water solution to obtain reference solution; 2) extracting a blumea balsamifera test sample with a methanol water solution, and filtering to prepare a test sample solution; 3) respectively injecting a reference substance solution and a test solution with the same volume into a high performance liquid chromatograph, measuring the peak areas of the reference substance solution and the test solution, and calculating to obtain the contents of 18 effective components such as rutin and hyperin in the blumea balsamifera test sample. The invention provides a rapid and accurate method for comprehensively controlling the quality of the blumea balsamifera medicinal material and the preparation thereof.
Description
Technical Field
The invention relates to the technical field of detection of medicinal components of traditional Chinese medicinal materials, in particular to a method for detecting the content of effective components in a blumea balsamifera medicinal material.
Background
Blumea balsamifera is dry aerial parts of Blumea balsamifera (L.) DC. The Chinese medicine is also called as Dafengai. Mainly produced in Guangxi and Guizhou provinces. According to records of Guangxi Chinese medicinal material standards (1990 edition), blumea balsamifera (blumea balsamifera) is pungent, bitter and warm in nature and has the effects of warming middle-jiao and activating blood, regulating menstruation, dispelling wind and removing dampness. Modern pharmacodynamic studies show that flavonoids compounds in the blumea balsamifera have multiple effects of oxidation resistance, cancer resistance, radiation resistance, acute liver injury protection and the like [ section earthquake, periwiny, chemical components and pharmacological research progress [ J ]. China journal of modern clinical medicine, 2006,4(21):1941-1945 ].
At present, there are many reports on the determination of the content of active ingredients of blumea balsamifera as follows: [ Huang Yonglin, Zhao Shi, Wenyongxin, determination of total flavone content in blumea balsamifera at different positions [ J ]. Guangxi plant, 2006,26(4):453 one 455.], [ ginger Jian Lian, Duxiu, Qiu Qin, blue-kernel cyan, Li Shengmao, screening Han deep, determination of total flavone content in blumea balsamifera at different production places [ J ]. Guangxi institute of traditional Chinese medicine, 2009,12(4):44-46.], [ Tankang, Seiko, Kanghui, etc. ], a method for preparing xanthoxylin using natural blumea balsamifera powder as raw material [ ZL201210184682.7], [ Raleiting, Lincui Guajian, Chenhai Haiyi, etc. ], reverse phase high performance liquid chromatography method for determining chlorogenic acid content in blumea balsamifera Ayunnanensis [ J ]. Shiniguchi medicine, 2008,19(9): 3. 2074.] and [ Huangyuan, Zhonghao Liaozhao, Chiao, Konghao, J. D. Yunnan Ying Xian acid method for determination of blumea acid content, 2012,40(19):99-100 ], and the like. There are also many reports on fingerprint of effective components of blumea balsamifera, such as: [ Yangbubo, Yangthenhubei, Wangxiangbe, etc. ] the UPLC fingerprint spectrum identification of blumea balsamifera medicinal material [ J ]. the Chinese medicine of time treasure, 2016,27(9): 2174. 2176.], [ Chinese millet, Zhazhi, Anjun, etc. ] the GC fingerprint spectrum research of blumea balsamifera medicinal material of different production places [ J ]. Chinese medicine, 2011,25(12): 1191. 1194.], [ von Hua, Yang Ye, Wangxiangbe, etc. ] the GC-MS fingerprint spectrum and the correlation research of blumea balsamifera and its pseudodongfeng grass [ J ]. Chinese pharmacy, 2017,28(9): 1257. 1261.], [ Sunwu, Pausihong, pandan, etc. ], the GC-MS fingerprint spectrum and the correlation research of blumea balsamifera and its extract [ J ]. the Chinese herbal medicine, 2017,48(4): 693. 699 ].
The existing method for measuring the content of effective components in the blumea balsamifera medicinal materials is mainly divided into three types: 1) measuring total flavonoids by an ultraviolet spectrophotometry, wherein the ultraviolet measurement of the total flavonoids has larger errors due to the influence of absorption wavelengths and the interference of other components; 2) the single component quantitative analysis cannot comprehensively reflect the quality of the medicinal materials; 3) the fingerprint spectrum is used for qualitative analysis, and the fingerprint spectrum can only qualitatively evaluate the quality of the medicinal materials. Therefore, the existing quality analysis methods of blumea balsamifera medicinal materials have certain limitations. In order to better develop and utilize the blumea balsamifera medicinal material and control the quality of the blumea balsamifera medicinal material, the quality control of the blumea balsamifera medicinal material is deeply researched.
Disclosure of Invention
The invention aims to provide a method for detecting the content of active ingredients in a blumea balsamifera medicinal material, and aims to solve the technical problem that the existing detection method is incomplete in detection and analysis of the content of active ingredients.
The method for detecting the content of the effective components in the blumea balsamifera medicinal material comprises the following steps:
step 1), preparation of 18 reference solutions: respectively dissolving rutin, hyperoside, isoquercitrin, 3',5, 7-tetrahydroxy-4' -methoxyflavanone, 3',5,5', 7-tetrahydroxyflavanone, quercetin, 3',4', 5-tetrahydroxy-7-methoxyflavanone, felwort phenol C, diosmetin, tamarigenin, 3,5, 7-trihydroxy-3 ',4' -dimethoxyflavone, 3', 5-trihydroxy-4', 7-dimethoxyflavanone, blumea balsamifera, rhamnetin, 3',4', 5-trihydroxy-3, 7-dimethoxyflavone, xanthoxylin, phytolaccaicin, 3',4', 5-dihydroxy-3 ',4', 7-trimethoxyflavone reference substances in methanol water solution, preparing corresponding 18 reference substance solutions;
step 2), extracting a blumea balsamifera medicinal material sample with a methanol water solution, and filtering to prepare a sample solution;
step 3), respectively injecting the corresponding 18 kinds of reference substance solutions and test substance solutions with the same volume into a high performance liquid chromatograph, then measuring the peak areas of each reference substance solution and each test substance solution and calculating to obtain the contents of the above 18 kinds of effective components in the blumea balsamifera test substance medicinal material;
wherein the stationary phase of the high performance liquid chromatograph is carbon octadecyl bonded silica gel, and the particle size of the carbon octadecyl bonded silica gel is less than or equal to 5 mu m; the mobile phase is acetonitrile-acid water solution, and the acetonitrile-acid water solution is as follows: 20 to 65 percent of acetonitrile by volume percentage.
The method comprises the steps of firstly obtaining chromatographic conditions for basically and completely separating the active ingredients of the blumea balsamifera through groping and optimizing the chromatographic conditions of the high-performance liquid analysis of the active ingredients of the blumea balsamifera, and carrying out reference substance control attribution on the active ingredients in the high-performance liquid chromatography 18 to clearly obtain the specific active ingredients of the blumea balsamifera; further, by researching the extraction conditions, the extraction conditions which can extract the 18 effective components basically and completely at the same time are optimized; the method for measuring the content is verified by a methodology through sample adding recovery rate, a linear range, precision, repeatability, stability and the like, and the result shows that the method established by the invention, namely the method for measuring the content of 18 effective components in the blumea balsamifera medicinal material simultaneously, can comprehensively analyze the effective components in the blumea balsamifera medicinal material.
Further, the particle size of the carbon octadecyl bonding silica gel in the step 3) is 5 μm or 4 μm or 3.5 μm. The small-particle size of the carbon octadecyl bonded silica gel is beneficial to the separation of components, the smaller the particle size is, the better the separation degree is, but the particle size of 5 mu m can meet the separation requirement of each chromatographic peak.
Further, the acid water in the mobile phase in the step 3) is one of phosphoric acid, acetic acid, formic acid and trifluoroacetic acid, and the volume concentration of the acid water is 0.01-0.5%. Under the condition, the separation of the chromatographic peaks of the effective components to be detected can be promoted, a better peak shape is presented, and tailing is avoided.
Further, the acid water in the mobile phase in the step 3) is acetic acid with the volume concentration of 0.2%. Under the condition, the chromatographic peaks of the effective components to be detected can be completely separated, and the chromatographic peaks have good shapes and no tailing.
Further, the mobile phase in the step 3) is as follows: in volume percent, the following table shows:
under the gradient condition, 18 effective components to be measured can be completely separated, and the analysis result is more reliable.
Further, the methanol aqueous solution in the step 2) is 60-80% by volume concentration.
Further, the method for detecting the content of the effective components in the blumea balsamifera medicinal material comprises the following steps:
step 1), preparation of 18 reference solutions: respectively dissolving rutin, hyperoside, isoquercitrin, 3',5, 7-tetrahydroxy-4' -methoxyflavanone, 3',5,5', 7-tetrahydroxyflavanone, quercetin, 3',4', 5-tetrahydroxy-7-methoxyflavanone, felwort phenol C, diosmetin, tamarigenin, 3,5, 7-trihydroxy-3 ',4' -dimethoxyflavone, 3', 5-trihydroxy-4', 7-dimethoxyflavanone, blumea balsamifera, rhamnetin, 3',4', 5-trihydroxy-3, 7-dimethoxyflavone, xanthoxylin, phytolaccaicin, 3, 5-dihydroxy-3 ',4', 7-trimethoxyflavone reference substances in 80% methanol aqueous solution, preparing a reference substance solution of 0.01-0.50 mg/ml;
step 2), preparation of a test solution: accurately weighing 0.6g of blumea balsamifera medicinal material powder, placing in a 50ml round bottom flask, accurately adding 25ml of 80% methanol aqueous solution, weighing, heating and refluxing for 30 minutes, cooling, complementing the weight, shaking up, and filtering to obtain a test solution;
step 3), respectively and precisely absorbing 10 mu l of reference substance solution and test substance solution, injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph, measuring peak areas of the reference substance solution and the test substance solution, and calculating the contents of the above 18 effective components in the sample according to an external standard method;
wherein, the stationary phase is 250 multiplied by 4.6mm, the carbon octadecyl bonding silica gel column with the diameter of 5 μm, the detection wavelength is respectively as follows: 254nm and 289nm, the theoretical plate number is not less than 3000 according to the peak calculation of 3,3',5, 7-tetrahydroxy-4' -methoxyflavanone, the mobile phase is acetonitrile-acid water solution, and the acetonitrile-acid water solution is: 20 to 65 percent of acetonitrile by volume percentage.
Drawings
FIG. 1 is HPLC chromatogram of herba Blumeae Balsamiferae and 18 kinds of reference substances.
In the figure, A is a chromatogram of a reference substance at the wavelength of 289 nm; b, chromatogram of a reference substance at the wavelength of 254 nm; c, carrying out chromatogram of blumea balsamifera medicinal material at wavelength of 289 nm; d, carrying out chromatogram of blumea balsamifera medicinal material under the wavelength of 254 nm; 1, rutin and 2: hyperin, 3: isoquercitrin, 4: 3,3',5, 7-tetrahydroxy-4' -methoxyflavanone, 5: 3',5,5', 7-tetrahydroxyflavanone, 6: quercetin, 7: 3,3',4', 5-tetrahydroxy-7-methoxyflavanone, 8: felwort propyl, 9: diosmetin, 10: tamarix chinensis flavin, 11: 3,5, 7-trihydroxy-3 ',4' -dimethoxyflavone, 12: 3,3', 5-trihydroxy-4', 7-dimethoxyflavanone, 13: blumea balsamifera, 14: rhamnonin, 15: 3',4', 5-trihydroxy-3, 7-dimethoxyflavone, 16: xanthoxylin, 17: merchant lute, 18: 3, 5-dihydroxy-3 ',4', 7-trimethoxy flavone.
Detailed Description
The following is further detailed by the specific embodiments:
the method for detecting the content of the effective components in the blumea balsamifera medicinal material comprises the following steps:
step 1), preparation of 18 reference solutions: respectively dissolving rutin, hyperoside, isoquercitrin, 3',5, 7-tetrahydroxy-4' -methoxyflavanone, 3',5,5', 7-tetrahydroxyflavanone, quercetin, 3',4', 5-tetrahydroxy-7-methoxyflavanone, felwort phenol C, diosmetin, tamarigenin, 3,5, 7-trihydroxy-3 ',4' -dimethoxyflavone, 3', 5-trihydroxy-4', 7-dimethoxyflavanone, blumea balsamifera, rhamnetin, 3',4', 5-trihydroxy-3, 7-dimethoxyflavone, xanthoxylin, phytolaccaicin, 3, 5-dihydroxy-3 ',4', 7-trimethoxyflavone reference substances in 80% methanol aqueous solution, preparing a reference solution;
the concentration of each control solution is 0.968mg/g of rutin, 0.908mg/g of hyperoside, 0.868mg/g of isoquercitrin, 6.540mg/g of 3,3',5, 7-tetrahydroxy-4' -methoxyflavanone, 1.510mg/g of 3',5,5', 7-tetrahydroxyflavanone, 0.315mg/g of quercetin, 2.071mg/g of 3,3',4', 5-tetrahydroxy-7-methoxyflavanone, 0.274mg/g of felwort C, 0.074mg/g of diosmetin, 0.133mg/g of tamarigenin, 0.289mg/g of 3,5, 7-trihydroxy-3 ',4' -dimethoxyflavone, 2.336mg/g of 3,3', 5-trihydroxy-4', 7-dimethoxyflavanone, 1.871mg/g of blumea balsamifera, 1.871mg/g of isoquercitin, 0.655mg/g of rhamnosine, 0.060mg/g of 3',4', 5-trihydroxy-3, 7-dimethoxy flavone, 0.534mg/g of xanthoxyline, 0.025mg/g of phytoxanthin and 0.090mg/g of 3, 5-dihydroxy-3 ',4', 7-trimethoxy flavone;
step 2), preparation of a test solution: accurately weighing 0.6g of blumea balsamifera medicinal material powder, placing in a 50ml round bottom flask, accurately adding 25ml of 80% methanol aqueous solution, weighing, heating and refluxing for 30 minutes, cooling, complementing the weight, shaking up, and filtering to obtain a test solution;
step 3), respectively and precisely absorbing 10 mu l of reference substance solution and test substance solution, injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph, measuring peak areas of the reference substance solution and the test substance solution, and calculating the contents of the above 18 effective components in the sample according to an external standard method;
wherein, the chromatographic conditions of the method are as follows:
octadecylsilane chemically bonded silica is used as a stationary phase (250 multiplied by 4.6mm,5 mu m); the mobile phase was acetonitrile-acetic acid aqueous solution, and the elution was carried out in a gradient manner as shown in the following table.
The detection wavelengths are respectively: 254nm and 289nm, and the theoretical plate number is not less than 3000 calculated according to the peak of 3,3',5, 7-tetrahydroxy-4' -methoxyflavanone.
Methodology validation
1) The specificity is as follows: as shown in figure 1, under the preferable chromatographic condition, the spectral peaks of the above 18 effective components in the blumea balsamifera medicinal material are well separated and have no interference, which shows that the method has better specificity.
2) Linearity: precisely preparing the above 18 effective component reference solutions with different concentrations. Accurately sucking 10 μ L of the above control solution, measuring according to the above determined chromatographic conditions, drawing a standard curve with the sample amount (X) of each control as abscissa and the peak area integral value (Y) as ordinate, and obtaining the linear regression equation and linear range of the above 18 effective components, as shown in Table 1 below.
Table 1: linear regression equation and linear range of 18 effective components
3) Precision degree
The same sample solution 10 μ l is accurately absorbed with precision (n is 5) in day, sample introduction is carried out for 5 times, peak areas of the above 18 effective components are measured, and the calculated relative standard deviation is less than 3.0%.
The precision (n is 15) in the daytime is accurately absorbed by 10 mul of the same test solution, the continuous measurement is carried out for three days, the measurement is carried out for 5 times every day, the peak areas of the above 18 effective components are measured, and the calculated relative standard deviation is less than 3.0 percent.
4) Repeatability of
6 portions of the same sample (batch number: GZ20161001) are taken, the operation is carried out under the item of 'preparation of test solution', the measurement is carried out under the chromatographic conditions, the peak areas of the 18 effective components are measured, and the calculated relative standard deviations are all less than 3.0%.
5) Sample recovery rate
Taking 0.6g of blumea balsamifera (batch number: GZ20161001) with known contents of the 18 active ingredients, precisely weighing 6 parts, placing in a 50ml volumetric flask, adding the same amount of the 18 active ingredient reference substances into each part, precisely adding 50ml of 80% methanol aqueous solution, heating and refluxing for 30 min, cooling, supplementing the weight, shaking, filtering, operating according to the method of content determination, determining the peak areas of each spectrum, calculating the sample addition recovery rate to be 95-105%, and the relative standard deviation to be less than 3.0%.
6) Stability of
Accurately sucking 10 mu l of the same test solution, measuring at 0h, 1h, 2h, 4h, 8h, 12h, 16h, 24h and 48h, observing by the peak areas of the 18 effective components, and calculating the relative standard deviation to be less than 3.0 percent, which indicates that the sample has good stability within 48 h.
Claims (3)
1. The method for detecting the content of the active ingredients in the blumea balsamifera medicinal material is characterized by comprising the following steps: the method comprises the following steps:
step 1), preparation of 18 reference solutions: respectively dissolving rutin, hyperoside, isoquercitrin, 3',5, 7-tetrahydroxy-4' -methoxyflavanone, 3',5,5', 7-tetrahydroxyflavanone, quercetin, 3',4', 5-tetrahydroxy-7-methoxyflavanone, felwort phenol C, diosmetin, tamarigenin, 3,5, 7-trihydroxy-3 ',4' -dimethoxyflavone, 3', 5-trihydroxy-4', 7-dimethoxyflavanone, blumea balsamifera, rhamnetin, 3',4', 5-trihydroxy-3, 7-dimethoxyflavone, xanthoxylin, phytolaccaicin, 3',4', 5-dihydroxy-3 ',4', 7-trimethoxyflavone reference substances in methanol water solution, preparing corresponding 18 reference substance solutions;
step 2), preparation of a test solution: accurately weighing 0.6g of blumea balsamifera medicinal material powder, placing the powder into a 50ml round-bottom flask, accurately adding 25ml of 60-80% methanol aqueous solution, weighing, heating and refluxing for 30 minutes, cooling, complementing the weight, shaking up, and filtering to obtain a test solution;
step 3), respectively injecting the corresponding 18 kinds of reference substance solutions and test substance solutions with the same volume into a high performance liquid chromatograph, then measuring the peak areas of each reference substance solution and each test substance solution and calculating to obtain the contents of the above 18 kinds of effective components in the blumea balsamifera test substance medicinal material;
wherein the stationary phase of the high performance liquid chromatograph is a carbon octadecyl bonding silica gel column with the particle size of 5 mu m or 4 mu m or 3.5 mu m, and the particle size of the carbon octadecyl bonding silica gel column is 250 multiplied by 4.6 mm; the detection wavelength is 254nm, the theoretical plate number is not less than 3000 according to the peak of 3,3',5, 7-tetrahydroxy-4' -methoxyflavanone, the mobile phase is acetonitrile-acid water solution, wherein the mobile phase is acetonitrile-acid water solution, the acid water in the mobile phase is one of phosphoric acid, acetic acid, formic acid and trifluoroacetic acid, and the volume concentration of the acid water is 0.01-0.5%; the gradient of the mobile phase, in volume percent, is shown in the following table:
2. the method for detecting the content of the active ingredients in the blumea balsamifera medicinal material according to claim 1, which is characterized by comprising the following steps: the acid water is acetic acid with the volume concentration of 0.2 percent.
3. The method for detecting the content of the active ingredients in the blumea balsamifera medicinal material according to claim 2, which is characterized by comprising the following steps: the methanol aqueous solution in the step 2) is 80% methanol aqueous solution by volume concentration.
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| CN113552242B (en) * | 2020-09-03 | 2023-04-07 | 广东一方制药有限公司 | Method for constructing Chinese rose fingerprint, detection method and content determination method |
| CN115453024B (en) * | 2022-09-26 | 2023-10-31 | 贵州汇腾萃取技术应用研究院有限责任公司 | Method for measuring content of 1, 2-dimethyl-1-ethyl formate-2-cyclopentene in blumea balsamifera extract |
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