CN103175910A - Method for controlling quality of liquorice and liquorice preparation - Google Patents

Abstract

The invention relates to a method for controlling quality of a liquorice medicinal material and a liquorice preparation. The method concretely relates to the step of simultaneously determining the content of the liquorice medicinal material and the content of six main active ingredients, namely liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizic acid and glycyrrhetinic acid in the liquorice preparation by utilizing a high performance liquid chromatography. A new method for controlling the quality of the liquorice and the liquorice preparation is established and the six main active ingredients, namely liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizic acid and glycyrrhetinic acid are quantitatively determined simultaneously by utilizing the high performance liquid chromatography. Confirmed by methodological investigation and test, the method is simple and convenient, correct in result and good in reproducibility, the defect of the existing quality control is overcome, and the quality control on the liquorice and the liquorice preparation is perfect and scientific.

Description

The method of quality control of a kind of Radix Glycyrrhizae and preparation thereof

Technical field:

The present invention relates to the method for quality control of licorice medicinal materials and preparation thereof, be specifically related to: the content of the liquiritin in this medicinal material of hplc simultaneous determination and preparation thereof, isoliquiritin, glycyrrhizin, isoliquiritigenin, glycyrrhizic acid, six kinds of main active of enoxolone.

Background technology:

Radix Glycyrrhizae is the dry root and rhizome of pulse family glycyrrhiza genus Radix Glycyrrhizae Glycyrrhiza uralensis Fisch, glycyrrhiza inflate bat Glycyrrhiza inflata Bat or glycyrrhiza glabra Glycyrrhiza glabra L, is one of the most frequently used Important Chinese Medicinal Herbs material.Before two thousand years, Shennong's Herbal is just classified Radix Glycyrrhizae on medicine as and is taken advantage of.China's traditional Chinese medical science thinks, Radix Glycyrrhizae is invigorated the spleen and benefited qi, and grows to cough moistening lung, and the emergency detoxifcation is in harmonious proportion hundred medicines, therefore saying of " ten side's nine grass " is arranged, and enjoys the good reputation of " king of Chinese herbal medicine ".

The chemical composition of Radix Glycyrrhizae is very complicated, scholar both domestic and external is studied it in different fields, so far from Radix Glycyrrhizae isolated compound more than the 100 kind of compounds such as Coumarins, triterpenes and flavonoids such as glycyrrhizin, enoxolone, liquiritin, isoliquiritin, Neoliquiritin, Neoisoliquiritin, glycyrrhizin, isoliquiritigenin and glycyrol, isoglycyrol, 7-methylcoumarin essence, umbelliferone are arranged, all have physiologically active widely.

At present Radix Glycyrrhizae and preparation thereof are carried out the research of quality control a lot, also set up a lot of quantitative analysis methods simultaneously, as gas phase-mass spectrometry, liquid phase-mass spectrometry, high-speed countercurrent chromatography etc.But these methods are just carried out quality control to a or two composition in Radix Glycyrrhizae, can't embody licorice medicinal materials and contain the full-quality of Radix Glycyrrhizae Chinese patent medicine preparation, and very high to the instrument of instrument, find in the practice examining operation simultaneously, said method exists that specificity is poor, complex operation, poor controllability, the assay deficiency such as poor stability as a result, but affects the generalization of standard.

Summary of the invention

The objective of the invention is to formulate new Radix Glycyrrhizae and the method for quality control of preparation thereof, utilize hplc simultaneous determination liquiritin, isoliquiritin, glycyrrhizin, isoliquiritigenin, glycyrrhizic acid, six kinds of main active of enoxolone to carry out quantitative measurement, investigate test through methodology, confirm that the method is easy, result is accurate, favorable reproducibility, made up the deficiency of existing quality control, the quality control of Radix Glycyrrhizae and preparation thereof is improved and science more.

The assay method of Radix Glycyrrhizae of the present invention and the quality control of preparation high performance liquid chromatography thereof comprises the steps:

The method of quality control of a kind of Radix Glycyrrhizae and preparation thereof is:

(1) efficient liquid phase chromatographic analysis: chromatographic condition chromatographic column: Agilent HC-C ₁₈Post (4.6mm * 250mm, 5 μ m); Mobile phase: acetonitrile: 0.04% formic acid solution; Adopt the gradient elution mode: 0min → 4min → 20min → 25min → 38min → 50min → 60min, acetonitrile 20% → 20% → 38% → 55% → 90% → 90% → 20%; Flow velocity: 1mlmin ^-1Column temperature: 30 ℃; Detect wavelength: 250nm, 365nm, number of theoretical plate should be not less than 2000 by glycyrrhizic acid;

(2) preparation of need testing solution: the test sample of Radix Glycyrrhizae or glycyrrhiza preparation is ground into the 20-40 order, precision takes Radix Glycyrrhizae powder 0.15g or glycyrrhiza preparation powder 0.25g, precision adds 8ml50% alcohol immersion 30min, then ultrasonic extraction 30min, filter after being placed to room temperature, filtrate is settled to 10ml with 50% ethanol, filters with miillpore filter (0.45 μ m), get subsequent filtrate, as need testing solution;

(3) preparation of reference substance solution: precision takes liquiritin, glycyrrhizin, glycyrrhizic acid, isoliquiritin, isoliquiritigenin, six kinds of reference substances of enoxolone respectively, adds methyl alcohol and dissolves and be made into liquiritin 1.728 mgml ^-1, glycyrrhizin 2.736 mgml ^-1, glycyrrhizic acid 4.680 mgml ^-1, isoliquiritin 2.016 mgml ^-1, isoliquiritigenin 0.612 mgml ^-1, enoxolone 0.540 mgml ^-1Storing solution;

(4) content assaying method: precision is drawn reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, the content of six kinds of compositions of mensuration respectively.

It is testing index that the present invention has selected six kinds of active components in Radix Glycyrrhizae and preparation thereof, and carries out following methods and learn the investigation test:

1. accurate reference substance solution 0.2ml, 0.4ml, 0.6ml, 0.8ml, the 1.0ml of drawing of the range of linearity, add methanol constant volume to 1ml.By above-mentioned chromatographic condition 10 μ l sample introductions, take the peak area x that records as horizontal ordinate, sample quality y is ordinate, calculates regression equation.Result shows, reference substance peak area and concentration are good linear relationship.The results are shown in Table 1.

Regression equation and the range of linearity of six kinds of main active of table 1

2. precision is got reference substance solution, measures by the method under the drafting item of typical curve, and RSD is respectively 0.917%, 1.128%, 1.052%, 3.884%, 1.945%, 3.487% as a result, shows that method precision is good.

3. stability is got need testing solution, presses the method under the drafting item of typical curve, is measuring at 0,4,8,12,24h respectively.RSD is respectively 2.069%, 2.129%, 1.484%, 3.197%, 2.565%, 3.457% as a result, shows that sample is good at 24 hours internal stabilities.

4. reappearance is got with a collection of test sample appropriate (n=5), accurately weighed respectively, after processing according to above-mentioned test sample preparation method, carry out assay, RSD is respectively 1.389%, 3.015%, 2.551%, 1.494%, 2.540%, 1.882% as a result, and result shows that the method reappearance is good.

5. the accurate sample that takes known content of average recovery test is 3 parts, and precision adds reference substance appropriate respectively, after processing according to above-mentioned test sample preparation method, carries out assay, and experimental result sees Table 2.

Table 2 average recovery result

By above-mentioned 5 tests as can be known, the assay method of Radix Glycyrrhizae of the present invention and the quality control of preparation high performance liquid chromatography thereof has advantages of that linear relationship is good, precision is high, good stability, favorable reproducibility, average recovery be high.

Description of drawings

Fig. 1 represents in a reason six kinds of main active assay liquid chromatograms in ball;

Fig. 2 represents six kinds of main active assay liquid chromatograms in b compound glycyrrhizin sheet;

Fig. 3 represents six kinds of main active assay liquid chromatograms in the c Liu Yi San;

Fig. 4 represents six kinds of main active assay liquid chromatograms in the d Brown Mixtura.

Embodiment

The present invention will be described in detail below in conjunction with embodiment, and embodiment does not consist of any restriction of the present invention.

Embodiment:

Content with liquiritin, isoliquiritin, glycyrrhizin, isoliquiritigenin, glycyrrhizic acid, six kinds of main active of enoxolone in ball, b compound glycyrrhizin sheet, c Liu Yi San, four kinds of glycyrrhiza preparations of d Brown Mixtura in high effective liquid chromatography for measuring a reason.

The instrument that uses has: high performance liquid chromatograph (Agilent 1100 series: Agilent G1311A pump, Agilent G1316A UV-detector, Agilent G1313A automatic sampler); SL4001N electronic balance (the accurate scientific and technological Instr Ltd. of Shanghai Min Qiao); Ultrasonic oscillator (power 300W, frequency 40kHz).

(1) efficient liquid phase chromatographic analysis chromatographic condition chromatographic column: Agilent HC-C ₁₈Post (4.6mm * 250mm, 5 μ m); Mobile phase: acetonitrile: 0.04% formic acid solution; Adopt the gradient elution mode: 0min → 4min → 20min → 25min → 38min → 50min → 60min, acetonitrile 20% → 20% → 38% → 55% → 90% → 90% → 20%; Flow velocity: 1mlmin ^-1Column temperature: 30 ℃; Detect wavelength: 250nm, 365nm, number of theoretical plate should be not less than 2000 by glycyrrhizic acid.

(2) preparation of reference substance solution: accurately respectively take six kinds of reference substances in right amount in the volumetric flask of 10ml, add methyl alcohol and dissolve and be made into liquiritin 1.728 mgml ^-1, glycyrrhizin 2.736 mgml ^-1, glycyrrhizic acid 4.680 mgml ^-1, isoliquiritin 2.016 mgml ^-1, isoliquiritigenin 0.612 mgml ^-1, enoxolone 0.540 mgml ^-1Storing solution.

(3) preparation of need testing solution: four kinds of glycyrrhiza preparations are ground to form fine powder, precision takes above-mentioned each 0.25g of preparation powder, precision adds 8ml50% alcohol immersion 30min, then ultrasonic extraction 30min, filter after being placed to room temperature, filtrate is settled to 10ml with 50% ethanol, filters with miillpore filter (0.45 μ m), get subsequent filtrate, as need testing solution.

(4) content assaying method: precision is drawn reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, the content of six kinds of compositions of mensuration respectively.

Measurement result is referring to Fig. 1 ~ 4, and in each figure, the other arabic numeral 1 ~ 6 of each peak value of curve represent respectively liquiritin, isoliquiritin, glycyrrhizin, isoliquiritigenin, glycyrrhizic acid, six kinds of active components of enoxolone.

Claims (1)

1. the method for quality control of a Radix Glycyrrhizae and preparation thereof is characterized in that:

(1) efficient liquid phase chromatographic analysis: chromatographic condition chromatographic column: Agilent HC-C ₁₈Post (4.6mm * 250mm, 5 μ m); Mobile phase: acetonitrile: 0.04% formic acid solution; Adopt the gradient elution mode: 0min → 4min → 20min → 25min → 38min → 50min → 60min, acetonitrile 20% → 20% → 38% → 55% → 90% → 90% → 20%; Flow velocity: 1mlmin ^-1Column temperature: 30 ℃; Detect wavelength: 250nm, 365nm, number of theoretical plate should be not less than 2000 by glycyrrhizic acid;

(2) preparation of need testing solution: licorice powder is broken into the 20-40 order, perhaps the test sample with glycyrrhiza preparation grinds to form fine powder, precision takes Radix Glycyrrhizae powder 0.15g or glycyrrhiza preparation powder 0.25g, precision adds 8ml50% alcohol immersion 30min, and ultrasonic extraction 30min then filters after being placed to room temperature, filtrate is settled to 10ml with 50% ethanol, filter with miillpore filter (0.45 μ m), get subsequent filtrate, as need testing solution;

(3) preparation of reference substance solution: precision takes liquiritin, glycyrrhizin, glycyrrhizic acid, isoliquiritin, isoliquiritigenin, six kinds of reference substances of enoxolone respectively, adds methyl alcohol and dissolves and be made into liquiritin 1.728 mgml ^-1, glycyrrhizin 2.736 mgml ^-1, glycyrrhizic acid 4.680 mgml ^-1, isoliquiritin 2.016 mgml ^-1, isoliquiritigenin 0.612 mgml ^-1, enoxolone 0.540 mgml ^-1Storing solution;

(4) content assaying method: precision is drawn reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, the content of six kinds of compositions of mensuration respectively.

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