CN102788854A - Method for measuring contents of three flavonoid compounds in rhododendron seniavinii drug simultaneously - Google Patents
Method for measuring contents of three flavonoid compounds in rhododendron seniavinii drug simultaneously Download PDFInfo
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- CN102788854A CN102788854A CN2012102764186A CN201210276418A CN102788854A CN 102788854 A CN102788854 A CN 102788854A CN 2012102764186 A CN2012102764186 A CN 2012102764186A CN 201210276418 A CN201210276418 A CN 201210276418A CN 102788854 A CN102788854 A CN 102788854A
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Abstract
The invention provides a method for measuring the contents of three flavonoid compounds in a rhododendron seniavinii drug simultaneously. The method comprises the following steps of: firstly preparing standard solution and liquid to be measured, then carrying out high-performance liquid chromatography detection on the standard solution and the liquid to be measured, wherein the chromatographic condition is as follows: the chromatographic column is a C18 chromatographic column; the mobile phase is acetonitrile and water; the column temperature is 25 DEG C; the detection wavelength is 360nm; the sample size is 20muL; the change process of the mobile phase is 0-25min; the volume ratio of the acetonitrile and the water is 70:30; in 25-26min, the volume ratio of the acetonitrile and the water is changed from 70:30 isocratic gradient to 60:40; and in 26-55min, the volume ratio of the acetonitrile and the water is 60:40. The method has the advantages that the contents of the three flavonoid compounds in the rhododendron seniavinii drug can be measured simultaneously, so that the measurement steps are reduced and the time consumed by measurement is reduced.
Description
[technical field]
The present invention relates to the method for simultaneous determination of multiponents in the kind of plant, relate in particular to three kinds of flavonoid content methods for measuring simultaneously in a kind of Rhododendron seniavinii medicinal material.
[background technology]
Rhododendron seniavinii is dry spray and the leaf of Cuculidae plant root or twig of Seniavin Rhododendron (Rhododendron senniavinii Maxim.), has the expelling phlegm and arresting coughing effect, is the local medicinal material in Fujian Province; Rhododendron seniavinii cough syrup series of products are with the clinical practice Chinese patent drug of Rhododendron seniavinii medicinal material as primary raw material.Flavone compounds such as the Hyperoside in the Rhododendron seniavinii medicinal material, quercitin and Quercetin are the material bases of its pharmacologically active; Hyperoside, quercitin and Quercetin three's contents level directly has influence on the quality of medicinal raw material, and finally influences the quality of patent medicine.The quantitative determination method of flavone compound is the necessary means of Rhododendron seniavinii quality of medicinal material science monitoring in exploitation and the application Rhododendron seniavinii medicinal material.
At present in the Rhododendron seniavinii medicinal material there be the bibliographical information of the related detecting method of Hyperoside, quercitin and Quercetin: " HPLC measures the content of quercitin in the root or twig of Seniavin Rhododendron ", " HPLC measures the content of Hyperoside in the root or twig of Seniavin Rhododendron ", and " RP-HPLC measures several kinds of Fujian and produces the quercetin contents in the cuckoo ".Institute's reported method is to use efficient liquid-phase chromatography method respectively Hyperoside, quercitin and Quercetin to be carried out independent quantitative measurement, and the required consumed time of multiple content of flavonoids is longer in the feasible mensuration Rhododendron seniavinii medicinal material.Therefore, the practitioner requires study and a kind ofly can be simultaneously the content of the multiple brass compounds in the Rhododendron seniavinii medicinal material be carried out method for measuring.
[summary of the invention]
Technical matters to be solved by this invention is to provide three kinds of flavonoid content methods for measuring simultaneously in a kind of Rhododendron seniavinii medicinal material, has reduced the step of measuring, and has saved the time.
The present invention is three kinds of flavonoid content methods for measuring simultaneously in a kind of Rhododendron seniavinii medicinal material that solves the problems of the technologies described above through following technical scheme, this method operation as follows:
(1) preparation of standard solution: to take by weighing an amount of purity respectively be 98% Hyperoside, quercitin, Quercetin reference substance and mix with 2: 1: 1 mass ratioes; Adopt methyl alcohol that mixed reference substance is dissolved afterwards and constant volume to obtain the mother liquor of standard solution; The total concentration of this mother liquor is 4.000mg/mL, then mother liquor is diluted to the standard solution of variable concentrations gradient;
(2) preparation of extract: take by weighing an amount of Rhododendron seniavinii medicinal powder and add methyl alcohol; And the feed liquid mass ratio is 1: 5-1: 20; Ultrasonic Extraction 0.5-3 hour afterwards, after ultrasonic Extraction finishes solution is filtered evaporate to dryness, and the sample that evaporate to dryness is obtained with methanol constant volume to 100-500mL; Adopt 45nm aperture membrane filtration to get the extract of Rhododendron seniavinii medicinal material flavone compound then, this extract is liquid to be measured;
(3) high performance liquid chromatography detects: on chromatographic column, be moving phase with the acetonitrile-water, and gradient elution separating flavone compounds, chromatographic condition is following:
Detecting instrument: high performance liquid chromatograph;
Chromatographic column: C18 chromatographic column;
Moving phase: acetonitrile-water;
Column temperature: 25 ℃;
Detect wavelength: 360nm;
Sample size: 20 μ L;
The moving phase change procedure: in 0min～25min, acetonitrile: the volume ratio of water is 70:30; In 25min～26min, the volume ratio of acetonitrile and water is converted to 60:40 by degree gradients such as 70:30; In 26min～55min, the volume ratio of acetonitrile and water is 60:40;
(4) measure: get the standard solution and the liquid to be measured that prepare respectively; And adopt above-mentioned chromatographic condition to carry out sample detection; The record chromatogram carries the software integration through chromatographic apparatus and obtains peak area, calculates the concentration of Hyperoside, quercitin, Quercetin in the liquid to be measured;
Wherein, three kinds of flavone compounds are respectively Hyperoside, quercitin, Quercetin.
Further, ultrasonic Extraction adopts filter paper to filter solution after finishing in the preparation manipulation of said extract.
The beneficial effect of three kinds of flavonoid contents methods for measuring simultaneously is in the Rhododendron seniavinii medicinal material of the present invention: can measure simultaneously the content that three kinds of flavone compounds in the Rhododendron seniavinii medicinal material are Hyperoside, quercitin and Quercetin, thereby reduce the step of measuring, reduced and measured required consumed time.
[description of drawings]
Combine embodiment that the present invention is done further description with reference to the accompanying drawings.
Fig. 1 is the high-efficient liquid phase chromatogram of standard solution of the present invention.
Fig. 2 is embodiment one a liquid high-efficient liquid phase chromatogram to be measured among the present invention.
Fig. 3 is embodiment two liquid high-efficient liquid phase chromatograms to be measured among the present invention.
Fig. 4 is embodiment three liquid high-efficient liquid phase chromatograms to be measured among the present invention.
[embodiment]
Method for measuring simultaneously further specifies to three kinds of flavone compounds (being Hyperoside, quercitin and Quercetin) content in the Rhododendron seniavinii medicinal material of the present invention in conjunction with following embodiment.
Embodiment one
1. the preparation of standard solution: take by weighing 20mg Hyperoside, 10mg quercitin, 10mg Quercetin reference substance and mixing respectively; Constant volume is the mother liquor of 4.000mg/mL to obtain total concentration to adopt methyl alcohol to dissolve also to mixed reference substance afterwards; Then mother liquor is diluted to 6 concentration gradients respectively, concentration gradient is respectively 0.01,0.1,0.2,0.5,0.8 and 1.0 times of mother liquid concentration; Wherein Hyperoside, quercitin, and the purity of Quercetin reference substance be 98%.
2. the preparation of liquid to be measured: take by weighing 10g Rhododendron seniavinii medicinal powder; The methyl alcohol (being that the feed liquid mass ratio is 1: 5) that adds 5 times of its quality; Ultrasonic Extraction is 3 hours afterwards, after ultrasonic Extraction finishes solution is carried out filter paper filtering and evaporate to dryness, the sample that evaporate to dryness is obtained with methanol constant volume to 100mL; Adopt 45nm aperture membrane filtration to get the extract of Rhododendron seniavinii medicinal material flavone compound then, this extract is liquid to be measured.
3. high performance liquid chromatography detects: on chromatographic column, be moving phase with the acetonitrile-water, and gradient elution separating flavone compounds, chromatographic condition is following:
Detecting instrument: Agilent 1200 type high performance liquid chromatographs;
Chromatographic column: C18 chromatographic column;
Moving phase: acetonitrile-water;
Column temperature: 25 ℃;
Detect wavelength: 360nm;
Sample size: 20 μ L;
The moving phase change procedure: in 0min～25min, acetonitrile: the volume ratio of water is 70:30; In 25min～26min, the volume ratio of acetonitrile and water is converted to 60:40 by degree gradients such as 70:30; In 26min～55min, the volume ratio of acetonitrile and water is 60:40.
4. measure: get the high performance liquid chromatograph that the standard solution for preparing and liquid to be measured injects above-mentioned chromatographic condition respectively and carry out assay determination; Obtain the equation of linear regression of three kinds of reference substances and the high-efficient liquid phase chromatogram of sample; Particularly, the equation of linear regression of three kinds of reference substances is as shown in table 1 below, and the high-efficient liquid phase chromatogram of standard solution is as shown in Figure 1; The high-efficient liquid phase chromatogram of liquid to be measured is as shown in Figure 2; And carry the software integration through chromatographic apparatus and obtain peak area, the concentration that calculates Hyperoside, quercitin, Quercetin in the liquid to be measured is respectively 0.8092mg/mL, 0.7908mg/mL and 0.0853mg/mL.
Three kinds of reference substance equations of linear regression of table 1, related coefficient, the range of linearity
In order not repeat burden, following examples are only set forth explanation to difference.
Embodiment two
The preparation of liquid to be measured: take by weighing 10g Rhododendron seniavinii medicinal powder; The methyl alcohol (being that the feed liquid mass ratio is 1: 20) that adds 20 times of its quality; Ultrasonic Extraction is 0.5 hour afterwards, after ultrasonic Extraction finishes solution is carried out filter paper filtering and evaporate to dryness, the sample that evaporate to dryness is obtained with methanol constant volume to 250mL; Adopt 45nm aperture membrane filtration to get the extract of Rhododendron seniavinii medicinal material flavone compound then, this extract is liquid to be measured.
Measure: get liquid to be measured that present embodiment prepares and inject the high performance liquid chromatograph consistent and carry out assay determination with embodiment one chromatographic condition; Obtain the high-efficient liquid phase chromatogram of liquid to be measured; Particularly, the high-efficient liquid phase chromatogram of sample liquid is as shown in Figure 3, and carries the software integration and obtain peak area through chromatographic apparatus; The concentration that calculates Hyperoside, quercitin, Quercetin in the liquid to be measured is respectively 0.3772mg/mL, 0.0938mg/mL and 0.0105mg/mL.
Embodiment three
The preparation of liquid to be measured: take by weighing 10g Rhododendron seniavinii medicinal powder; The methyl alcohol (being that the feed liquid mass ratio is 1: 15) that adds 15 times of its quality; Ultrasonic Extraction is 2 hours afterwards, after ultrasonic Extraction finishes solution is carried out filter paper filtering and evaporate to dryness, the sample that evaporate to dryness is obtained with methanol constant volume to 500mL; Adopt 45nm aperture membrane filtration to get the extract of Rhododendron seniavinii medicinal material flavone compound then, this extract is liquid to be measured.
Measure: get sample liquid that present embodiment prepares and inject the high performance liquid chromatograph consistent and carry out assay determination with embodiment one chromatographic condition; Obtain the high-efficient liquid phase chromatogram of sample; Particularly, the high-efficient liquid phase chromatogram of sample liquid is as shown in Figure 4, and carries the software integration and obtain peak area through chromatographic apparatus; Calculate the concentration 0.0539mg/mL of Hyperoside, quercitin, Quercetin in the liquid to be measured, 0.0333mg/mL and 0.0707mg/mL.
To sum up, can measure simultaneously, thereby reduce the step of measuring, reduced and measured required consumed time the content that three kinds of flavone compounds in the Rhododendron seniavinii medicinal material are Hyperoside, quercitin and Quercetin.
Claims (2)
1. three kinds of flavonoid content methods for measuring simultaneously in the Rhododendron seniavinii medicinal material, it is characterized in that: said three kinds of flavone compounds are respectively Hyperoside, quercitin, Quercetin; This method is operated as follows:
(1) preparation of standard solution: to take by weighing an amount of purity respectively be 98% Hyperoside, quercitin, Quercetin reference substance and mix with 2: 1: 1 mass ratioes; Adopt methyl alcohol that mixed reference substance is dissolved afterwards and constant volume to obtain the mother liquor of standard solution; The total concentration of this mother liquor is 4.000mg/mL, then mother liquor is diluted to the standard solution of variable concentrations gradient;
(2) preparation of extract: take by weighing an amount of Rhododendron seniavinii medicinal powder and add methyl alcohol; And the feed liquid mass ratio is 1: 5-1: 20; Ultrasonic Extraction 0.5-3 hour afterwards, after ultrasonic Extraction finishes solution is filtered evaporate to dryness, and the sample that evaporate to dryness is obtained with methanol constant volume to 100-500mL; Adopt 45nm aperture membrane filtration to get the extract of Rhododendron seniavinii medicinal material flavone compound then, this extract is liquid to be measured;
(3) high performance liquid chromatography detects: on chromatographic column, be moving phase with the acetonitrile-water, and gradient elution separating flavone compounds, chromatographic condition is following:
Detecting instrument: high performance liquid chromatograph;
Chromatographic column: C18 chromatographic column;
Moving phase: acetonitrile-water;
Column temperature: 25 ℃;
Detect wavelength: 360nm;
Sample size: 20 μ L;
The moving phase change procedure: in 0min～25min, acetonitrile: the volume ratio of water is 70:30; In 25min～26min, the volume ratio of acetonitrile and water is converted to 60:40 by degree gradients such as 70:30; In 26min～55min, the volume ratio of acetonitrile and water is 60:40;
(4) measure: get the standard solution and the liquid to be measured that prepare respectively; And adopt above-mentioned chromatographic condition to carry out sample detection; The record chromatogram carries the software integration through chromatographic apparatus and obtains peak area, calculates the concentration of Hyperoside, quercitin, Quercetin in the liquid to be measured.
2. according to three kinds of flavonoid content methods for measuring simultaneously in the said Rhododendron seniavinii medicinal material of claim 1, it is characterized in that: ultrasonic Extraction adopts filter paper to filter solution after finishing in the preparation manipulation of said extract.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1700463A1 (en) * | 1988-08-09 | 1991-12-23 | Кемеровский государственный медицинский институт | Method for separating flavonoids |
| CN102012405A (en) * | 2010-09-16 | 2011-04-13 | 陕西省中医药研究院汉唐制药有限公司 | Detection method for flavonoids compounds in cotton rose general flavone |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1700463A1 (en) * | 1988-08-09 | 1991-12-23 | Кемеровский государственный медицинский институт | Method for separating flavonoids |
| CN102012405A (en) * | 2010-09-16 | 2011-04-13 | 陕西省中医药研究院汉唐制药有限公司 | Detection method for flavonoids compounds in cotton rose general flavone |
Non-Patent Citations (4)
| Title |
|---|
| LORENZO B ET AL: "Quantitative Characterization of Flavonoid Compounds in", 《JOURNAL OF AGRICULTURE AND FOOD CHEMISTRY》 * |
| 张圆圆等: "反相高效液相色谱法测定普通鹿蹄草中的黄酮苷类成分", 《色谱》 * |
| 彭全材等: "高效液相色谱法同时测定鱼腥草中7黄酮的含量", 《江西师范大学学报(自然科学版)》 * |
| 黄兹英等: "HPLC 法同时测定夏枯草中芦丁、金丝桃苷、槲皮素和山柰酚", 《中成药》 * |
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Application publication date: 20121121 |