CN113049724B - Fingerprint construction method and detection method of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition - Google Patents
Fingerprint construction method and detection method of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition Download PDFInfo
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Abstract
The invention discloses a fingerprint construction method and a detection method of a cassia twig, peony and rhizoma anemarrhenae decoction composition, wherein the fingerprint construction method comprises the following steps: (1) Precisely weighing the cassia twig, peony root and rhizoma anemarrhenae decoction composition, adding water, and performing ultrasonic treatment to obtain a test solution; (2) Preparing reference solution from 22 reference substances such as gallic acid, catechin, albiflorin, ephedrine hydrochloride and pseudoephedrine hydrochloride; (3) Precisely absorbing the test solution and each reference solution, injecting into an ultra high performance liquid chromatograph for chromatographic analysis, detecting the wavelength at 210nm to obtain a test sample fingerprint and a reference chromatogram, and formulating a standard fingerprint of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition. The quality of the product to be detected can be comprehensively reflected by the standard fingerprint spectrum, and the product quality of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction preparation can be effectively controlled.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine quality detection, in particular to a fingerprint construction method and a detection method of a cassia twig, peony and rhizoma anemarrhenae decoction composition.
Background
The cassia twig, peony and rhizoma anemarrhenae decoction belongs to one of 100 classical Chinese parties in the directory of ancient classical Chinese predetermined orders (the first group) formulated by the national drug administration in 2018. From the book of Zhang Zhongjing of east Han famous family, jin Kui Yao L ü e, the prescription is composed of nine medicines of cassia twig, prepared aconite root (processed), peony, ephedra, ginger, white atractylodes rhizome, anemarrhena rhizome, ledebouriella root and licorice. Wherein the cassia twig and the monkshood are monarch drugs, and the cassia twig is pungent, sweet and warm, warms and dredges the channels and collaterals, and is good at easing the joints to eliminate wind evil; fu Zi is pungent, sweet and hot, and is the key herb for dredging twelve meridians and purifying yang; the two medicines are used together to expel wind and remove dampness to dredge vessels, warm meridians and dispel cold to strengthen yang. The ephedra herb, the divaricate saposhnikovia root and the largehead atractylodes rhizome are ministerial drugs, the ephedra herb Xin Weigu is warm, the divaricate saposhnikovia root is pungent, sweet and slightly warm, and the largehead atractylodes rhizome is bitter, sweet and warm, and the combination of the ephedra herb, the divaricate saposhnikovia root and the largehead atractylodes rhizome can dispel wind and cold, dispel dampness and relieve pain. The rhizoma anemarrhenae, the peony and the ginger are adjuvant drugs, the rhizoma anemarrhenae clears heat and nourishes yin, the white peony root nourishes blood and nourishes nutrient, and the ginger harmonizes stomach and prevents vomiting. The liquorice is used as a guiding drug to coordinate the effects of the other drugs in the recipe. The formula has the effects of dispelling wind and removing dampness, warming meridians and relieving arthralgia, and nourishing yin and clearing heat, and is named after the particularity of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae in the formula, so that the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction is called.
The classical famous prescription is a prescription recorded in ancient Chinese medical record which is widely applied, has definite curative effect and obvious characteristics and advantages. The traditional Chinese medicine decoction is the mainstream of clinical medication of traditional Chinese medicine, the classical famous prescription is the crystal of thousands of years of clinical practice of traditional Chinese medicine, has the characteristics of reasonable formula, definite curative effect, high safety and the like, is one of essences of the theory of traditional Chinese medicine, is inconvenient to decoct and carry, has difficult unified standard, and seriously influences the clinical application of the traditional Chinese medicine, so the traditional Chinese medicine decoction is simple and easy to use, and has great research and development significance on the standard granules of the classical famous prescription, which meet the rhythm of modern life. The clinical curative effect of the classical famous prescription is exact, and the quality of the compound preparation is controlled by using a modern analysis technology, so that the stable quality of the compound preparation is ensured, and the clinical application of the classical famous prescription can be better promoted.
In the prior art, researches on a cassia twig, peony and rhizoma anemarrhenae Shang Zhiwen map detection method are also related, for example, a detection method of ZL 201910285906.5 cassia twig, peony and rhizoma anemarrhenae decoction uses gallic acid, mangiferin, paeoniflorin, linarin, liquiritin, 5-O-methylvisammol glycoside, cinnamic acid and ammonium glycyrrhizinate as reference substances, extracts components of the cassia twig, peony and rhizoma anemarrhenae decoction by using 50% methanol, and the detection wavelength of chromatographic conditions is 254nm. However, the Guizhi Shaoyao Zhimu Tang is made from nine herbs, the chemical components of the decoction are dozens or hundreds, and the quality of the preparation is only detected by eight chemical reference substances, which cannot effectively control the quality. Moreover, the extraction of the cassia twig, peony and rhizoma anemarrhenae decoction with 50% methanol and the detection with 254nm wavelength in the patent also affect the effectiveness of quality control.
Disclosure of Invention
The invention aims to provide a fingerprint construction method and a detection method of a cassia twig, peony and rhizoma anemarrhenae decoction composition, which can effectively control the product quality of a cassia twig, peony and rhizoma anemarrhenae decoction preparation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fingerprint construction method of a cassia twig, peony and rhizoma anemarrhenae decoction composition comprises the following steps:
(1) Precisely weighing the cassia twig, peony and rhizoma anemarrhenae decoction composition, adding water, and performing ultrasonic treatment to obtain a test solution;
(2) Gallic acid, catechin, albiflorin, paeoniflorin, 1,2,3,4,6-O-pentagalloyl glucose, cinnamyl alcohol, cinnamic acid, cimicidin, 5-O-methyl visammol glycoside, sec-O-sec, sec-O-pentagalloyl glucose, 5-hydroxymethyl furfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and atractylenolide
Atractylenolide
6-gingerol, apioside liquiritin, liquiritigenin, isoliquiritin, ammonium glycyrrhizinate, neomangiferin, mangiferin, ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances;
(3) Precisely absorbing the test solution and each reference solution, injecting into an ultra high performance liquid chromatograph for chromatographic analysis, detecting the wavelength at 210nm to obtain a test sample fingerprint and a reference chromatogram, and formulating a standard fingerprint of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition.
Further, in the step (3), the chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica chromatographic column, flow rate 0.25ml/min, column temperature: 30 ℃;
gradient elution is carried out by taking acetonitrile-0.1% phosphoric acid solution as a mobile phase as a system, and the elution sequence is as follows:
0-6 min, 3% of acetonitrile and 97% of 0.1% phosphoric acid solution;
6-8 min, acetonitrile 3% → 5%,0.1% phosphoric acid solution 97% → 95%;
8-16 min, acetonitrile 5% → 8%,0.1% phosphoric acid solution 95% → 92%;
16-23 min, acetonitrile 8% → 14%,0.1% phosphoric acid solution 92% → 86%;
23-29 min, acetonitrile 14% → 16%,0.1% phosphoric acid solution 86% → 84%;
29-33 min, 16% of acetonitrile and 84% of 0.1% phosphoric acid solution;
33-45 min, acetonitrile 16% → 35%,0.1% phosphoric acid solution 84% → 65%;
45-49 min, acetonitrile 35% → 60%,0.1% phosphoric acid solution 65% → 40%;
49-53 min, acetonitrile 60% → 95%,0.1% phosphoric acid solution 40% → 5%.
Further, the standard fingerprint comprises 33 characteristic peaks, a 15 peak mangiferin chromatographic peak is used as an S peak, and the relative retention time of each characteristic peak is as follows: peak 1 relative retention time RRT of 0.142,2 Peak relative retention time RRT of 0.272,3 Peak relative retention time RRT of 0.309,4 Peak relative retention time RRT of 0.336,5 Peak relative retention time RRT of 0.380,6 Peak relative retention time RRT of 0.429,7 Peak relative retention time RRT of 0.488,8 Peak relative retention time RRT of 0.523,9 Peak relative retention time RRT of 0.573, peak 10 relative retention time RRT of 0.626, peak 11 relative retention time RRT of 0.672, peak 12 relative retention time RRT of 0.9, peak 13 relative retention time RRT of 0.792, peak 14 relative retention time RRT of 0.945, peak 15 (S) Peak relative retention time RRT of 1.000, peak 16 relative retention time RRT of 1.043, peak relative retention time RRT of 1.013, the peak relative retention time RRT No. 18 is 1.053, the peak relative retention time RRT No. 19 is 1.067, the peak relative retention time RRT No. 20 is 1.081, the peak relative retention time RRT No. 21 is 1.129, the peak relative retention time RRT No. 22 is 1.178, the peak relative retention time RRT No. 23 is 1.197, the peak relative retention time RRT No. 24 is 1.253, the peak relative retention time RRT No. 25 is 3524 zxft No. 26 is 1.327, the peak relative retention time RRT No. 27 is 1.396, the peak relative retention time RRT No. 28 is 1.608, the peak relative retention time RRT No. 29 is 1.895, the peak relative retention time RRT No. 30 is 3754 zxft No. 31 is 49xft RRT No. 84, the peak relative retention time RRT No. 32 is 52xft No. 72, the peak relative retention time RRT No. 7945 is 7972 zxft No. 7945.
Furthermore, chromatographic peaks 1, 7, 8, 12, 13, 15, 18, 21, 23, 25, 26, 28, 29, 31, 32 and 33 in the standard fingerprint correspond to retention time of chromatographic peaks of gallic acid, ephedrine hydrochloride, pseudoephedrine hydrochloride, catechin, neomangiferin, mangiferin, albiflorin, paeoniflorin, cimicin, apigenin, liquiritin, 5-O-methylvisammioside, cinnamic acid, sec-O-cogongrasin, ammonium glycyrrhizinate and a 6-gingerol reference substance in sequence.
Further, in the step (1), 0.18g of the cassia twig, peony and rhizoma anemarrhenae decoction composition is precisely weighed, placed in a 10ml measuring flask, added with water for ultrasonic treatment for 10-30 minutes, cooled, diluted to 10ml scale by adding water, and filtered to obtain the test solution.
Further, the preparation method of the cassia twig, peony and rhizoma anemarrhenae decoction composition comprises the following steps:
putting 12g of cassia twig, 9g of white paeony root, 12g of rhizoma anemarrhenae, 6g of ephedra herb, 6g of radix aconiti lateralis preparata, 12g of divaricate saposhnikovia root, 15g of ginger, 5g of largehead atractylodes rhizome and 6g of liquorice root into a 5L casserole, adding 1400ml of water, soaking for 30 minutes, and decocting to obtain 400ml of decoction;
filtering the hot decoction liquid with 350-mesh filter cloth, concentrating the filtrate, freeze-drying and crushing to obtain the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition.
Further, in the step (2), the method for preparing the reference solution comprises:
precisely weighing gallic acid, catechin, albiflorin, paeoniflorin and 1,2,3,4,6-O-pentagalloylglucose as reference substances, and adding methanol to obtain radix Paeoniae alba mixed reference substance solution;
accurately weighing cinnamyl alcohol and cinnamic acid reference substances, and adding methanol to obtain a cassia twig mixed reference substance solution;
accurately weighing cimicidin, cimicifugal, 5-O-methylvisammioside and hamaudol glycoside, and adding methanol to obtain radix Saposhnikoviae mixed reference solution;
precisely weighing 5-hydroxymethyl furfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and atractylenolide
And atractylenolide
Adding methanol into the reference substance to obtain mixed reference solution of Atractylodis rhizoma;
precisely weighing 6-gingerol reference substance, and adding methanol to obtain rhizoma Zingiberis recens reference substance solution;
precisely weighing apigenin liquiritin, liquiritigenin, isoliquiritin and ammonium glycyrrhizinate reference, and adding methanol to obtain Glycyrrhrizae radix mixed reference solution;
precisely weighing new mangiferin and mangiferin reference substances, and adding methanol to obtain rhizoma anemarrhenae mixed reference substance solution;
precisely weighing ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances, and adding methanol to obtain herba Ephedrae mixed reference substance solution.
Further, the gallic acid, catechin, albiflorin, paeoniflorin, 1,2,3,4,6-O-pentagalloylglucose, cinnamyl alcohol, cinnamic acid, cimicidin, 5-O-methylvisammol, hamaudolin, 5-hydroxymethyl furfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and atractylenolide
Atractylenolide
6-gingerol, apigenin, liquiritin, liquiritigenin, isoliquiritin, ammonium glycyrrhizinate, neomangiferin, mangiferin, ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances, wherein the content of each reference substance solution is 10-100 mu g per 1 ml.
A fingerprint detection method of a cassia twig, peony and rhizoma anemarrhenae decoction composition comprises the following steps:
preparing a to-be-detected sample solution from the to-be-detected cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition by adopting a water extraction method;
injecting the sample solution to be detected into an ultra-high performance liquid chromatograph for chromatographic analysis to generate a sample map to be detected;
and (3) taking the standard fingerprint established by the fingerprint construction method of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition as a reference map, and calculating the similarity between the to-be-detected sample map and the standard fingerprint, wherein the quality is qualified if the similarity is more than 0.9.
Further, the chromatographic conditions for injecting the sample solution to be detected into the ultra-high performance liquid chromatograph for chromatographic analysis are as follows:
a chromatographic column: octadecylsilane chemically bonded silica chromatographic column, flow rate 0.25ml/min, column temperature: 30 ℃;
gradient elution is carried out by taking acetonitrile-0.1% phosphoric acid solution as a mobile phase as a system, and the elution sequence is as follows:
0-6 min, 3% of acetonitrile and 97% of 0.1% phosphoric acid solution;
6-8 min, acetonitrile 3% → 5%,0.1% phosphoric acid solution 97% → 95%;
8-16 min, acetonitrile 5% → 8%,0.1% phosphoric acid solution 95% → 92%;
16-23 min, acetonitrile 8% → 14%,0.1% phosphoric acid solution 92% → 86%;
23-29 min, acetonitrile 14% → 16%,0.1% phosphoric acid solution 86% → 84%;
29-33 min, 16% of acetonitrile and 84% of 0.1% phosphoric acid solution;
33-45 min, acetonitrile 16% → 35%,0.1% phosphoric acid solution 84% → 65%;
45-49 min, acetonitrile 35% → 60%,0.1% phosphoric acid solution 65% → 40%;
49-53 min, acetonitrile 60% → 95%,0.1% phosphoric acid solution 40% → 5%.
The invention has the beneficial effects that:
the invention prepares the test solution by a water extraction method, has better reaction effect on components with good water solubility, can obtain more chromatographic peaks by reaction, more comprehensively characterizes the sample, and is beneficial to improving the accuracy of product quality detection.
The detection wavelength adopted by the invention is 210nm, and the peak information content of the cassia twig, peony and rhizoma anemarrhenae decoction composition at the wavelength of 210nm is maximum. The chromatogram obtained at the wavelength can represent ephedrine hydrochloride and pseudoephedrine hydrochloride components in the ephedra, and ephedrine substances and single preparations thereof are taken as easily-toxic chemicals to be strictly controlled in China, so that the detection wavelength of 210nm, which is the fingerprint of the cassia twig, peony and rhizoma anemarrhenae decoction composition, can more comprehensively reflect the product components.
The types of the reference substances in the invention reach 27 types, and correspond to seven traditional Chinese medicinal materials except the bighead atractylodes rhizome and the monkshood in the cassia twig, peony and rhizoma anemarrhenae decoction composition. The fingerprint spectrum of the cassia twig, peony and rhizoma anemarrhenae decoction composition provided by the invention can comprehensively reflect the quality information of the cassia twig, peony and rhizoma anemarrhenae decoction, thereby comprehensively and effectively controlling the quality of a cassia twig, peony and rhizoma anemarrhenae decoction preparation product.
Drawings
FIG. 1 is a three-dimensional map of a test solution;
FIG. 2 is a fingerprint of a sample solution at different wavelengths;
FIG. 3 is a finger print of a test solution detected by different mobile phases;
FIG. 4 shows finger prints obtained by testing test solutions prepared with different extraction agents;
FIG. 5 is a chromatogram of a mixed reference solution of white peony root;
FIG. 6 is a chromatogram of a mixed reference solution of Cassia twig;
FIG. 7 is a chromatogram of a solution of a mixed reference of Saposhnikovia divaricata;
FIG. 8 is a chromatogram of a mixed reference solution of Atractylodis rhizoma;
FIG. 9 is a chromatogram of a ginger reference solution;
FIG. 10 is a chromatogram of a mixed reference solution of Glycyrrhiza glabra;
FIG. 11 is a chromatogram of a mixed reference solution of rhizoma anemarrhenae;
FIG. 12 is a chromatogram of a mixed reference solution of Ephedra sinica Stapf;
FIG. 13 is a graph of 5 batches of Guizhi Shaoyao Zhimu Tang composition;
FIG. 14 shows a fingerprint of a composition of Guizhi Shaoyao Zhimu Tang.
Detailed Description
The technical solution of the present invention will be further described with reference to the following embodiments.
The invention provides a fingerprint construction method of a cassia twig, peony and rhizoma anemarrhenae decoction composition, which comprises the following steps:
(1) Precisely weighing the cassia twig, peony and rhizoma anemarrhenae decoction composition, adding water, and performing ultrasonic treatment to obtain a test solution;
(2) Gallic acid, catechin, albiflorin, paeoniflorin, 1,2,3,4,6-O-pentagalloyl glucose, cinnamyl alcohol, cinnamic acid, cimicidin, 5-O-methyl visammol glycoside, sec-O-sec, sec-O-pentagalloyl glucose, 5-hydroxymethyl furfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and atractylenolide
Atractylenolide
6-gingerol, apioside liquiritin, liquiritigenin, isoliquiritin, ammonium glycyrrhizinate, neomangiferin, mangiferin, ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances;
(3) Precisely absorbing the test solution and each reference solution, injecting into an ultra high performance liquid chromatograph for chromatographic analysis, and detecting the wavelength at 210nm to obtain a test sample fingerprint and a reference chromatogram, and formulating a standard fingerprint of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition.
The invention prepares the test solution by a water extraction method, has better reaction effect on components with good water solubility, can obtain more chromatographic peaks by reaction, more comprehensively characterizes the sample, and is beneficial to improving the accuracy of product quality detection.
The detection wavelength adopted by the invention is 210nm, and the peak information content of the cassia twig, peony and rhizoma anemarrhenae decoction composition at the wavelength of 210nm is maximum. The chromatogram obtained at the wavelength can represent ephedrine hydrochloride and pseudoephedrine hydrochloride components in the ephedra, and ephedrine substances and single preparations thereof are taken as easily-toxic chemicals to be strictly controlled in China, so that the detection wavelength of 210nm, which is the fingerprint of the cassia twig, peony and rhizoma anemarrhenae decoction composition, can more comprehensively reflect the product components.
The types of the reference substances in the invention reach 27 types, and correspond to seven traditional Chinese medicinal materials except the bighead atractylodes rhizome and the monkshood in the cassia twig, peony and rhizoma anemarrhenae decoction composition. The fingerprint spectrum of the cassia twig, peony and rhizoma anemarrhenae decoction composition provided by the invention can comprehensively reflect the quality information of the cassia twig, peony and rhizoma anemarrhenae decoction, thereby comprehensively and effectively controlling the quality of a cassia twig, peony and rhizoma anemarrhenae decoction preparation product.
Further, in the step (3), the chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica chromatographic column, flow rate 0.25ml/min, column temperature: 30 ℃;
gradient elution is carried out by taking acetonitrile-0.1% phosphoric acid solution as a mobile phase as a system, and the elution sequence is as follows:
0-6 min, 3% of acetonitrile and 97% of 0.1% phosphoric acid solution;
6-8 min, acetonitrile 3% → 5%,0.1% phosphoric acid solution 97% → 95%;
8-16 min, acetonitrile 5% → 8%,0.1% phosphoric acid solution 95% → 92%;
16-23 min, acetonitrile 8% → 14%,0.1% phosphoric acid solution 92% → 86%;
23-29 min, acetonitrile 14% → 16%,0.1% phosphoric acid solution 86% → 84%;
29-33 min, 16% of acetonitrile and 84% of 0.1% phosphoric acid solution;
33-45 min, acetonitrile 16% → 35%,0.1% phosphoric acid solution 84% → 65%;
45-49 min, acetonitrile 35% → 60%,0.1% phosphoric acid solution 65% → 40%;
49-53 min, acetonitrile 60% → 95%,0.1% phosphoric acid solution 40% → 5%.
The above-described elution procedure has a short run time and low mobile phase consumption. The invention adopts the chromatographic conditions and UPLC for detection, so that the fingerprint construction method has shorter operation time and less mobile phase consumption, and can save time and material cost.
Further, the standard fingerprint comprises 33 characteristic peaks, a 15 peak mangiferin chromatographic peak is used as an S peak, and the relative retention time of each characteristic peak is as follows: peak 1 relative retention time RRT of 0.142,2 Peak relative retention time RRT of 0.272,3 Peak relative retention time RRT of 0.309,4 Peak relative retention time RRT of 0.336,5 Peak relative retention time RRT of 0.380,6 Peak relative retention time RRT of 0.429,7 Peak relative retention time RRT of 0.488,8 Peak relative retention time RRT of 0.523,9 Peak relative retention time RRT of 0.573, peak 10 relative retention time RRT of 0.626, peak 11 relative retention time RRT of 0.672, peak 12 relative retention time RRT of 0.9, peak 13 relative retention time RRT of 0.792, peak 14 relative retention time RRT of 0.945, peak 15 (S) Peak relative retention time RRT of 1.000, peak 16 relative retention time RRT of 1.043, peak relative retention time RRT of 1.013, the peak relative retention time RRT No. 18 is 1.053, the peak relative retention time RRT No. 19 is 1.067, the peak relative retention time RRT No. 20 is 1.081, the peak relative retention time RRT No. 21 is 1.129, the peak relative retention time RRT No. 22 is 1.178, the peak relative retention time RRT No. 23 is 1.197, the peak relative retention time RRT No. 24 is 1.253, the peak relative retention time RRT No. 25 is 3524 zxft No. 26 is 1.327, the peak relative retention time RRT No. 27 is 1.396, the peak relative retention time RRT No. 28 is 1.608, the peak relative retention time RRT No. 29 is 1.895, the peak relative retention time RRT No. 30 is 3754 zxft No. 31 is 49xft RRT No. 84, the peak relative retention time RRT No. 32 is 52xft No. 72, the peak relative retention time RRT No. 7945 is 7972 zxft No. 7945.
According to the invention, 5 batches of chromatogram of the cassia twig, peony and rhizoma anemarrhenae decoction composition are led into a similarity evaluation system, a chromatographic peak with stable relative retention time and good separation degree is selected as a characteristic peak and determined as a common peak, and 33 characteristic peaks are finally determined through multiple experimental researches, so that the purpose of more comprehensively and effectively controlling the quality of the preparation is achieved.
Furthermore, chromatographic peaks 1, 7, 8, 12, 13, 15, 18, 21, 23, 25, 26, 28, 29, 31, 32 and 33 in the standard fingerprint correspond to retention time of chromatographic peaks of gallic acid, ephedrine hydrochloride, pseudoephedrine hydrochloride, catechin, neomangiferin, mangiferin, albiflorin, paeoniflorin, cimicin, apigenin, liquiritin, 5-O-methylvisammioside, cinnamic acid, sec-O-cogongrasin, ammonium glycyrrhizinate and a 6-gingerol reference substance in sequence. According to the peak sequence of each component, the ultraviolet absorption spectrum of each component can be compared with the chromatographic peak in the cassia twig, peony and anemarrhena decoction by full-wavelength scanning of a diode array detector, so that 16 known peaks can be identified.
Further, in the step (1), 0.18g of the cassia twig, peony and rhizoma anemarrhenae decoction composition is precisely weighed, placed in a 10ml measuring flask, added with water for ultrasonic treatment for 10-30 minutes, cooled, diluted to 10ml scale by adding water, and filtered to obtain the test solution. The test solution obtained by the method can contain more chemical components, and is beneficial to comprehensively detecting the cassia twig, peony root and rhizoma anemarrhenae decoction composition.
Further, the preparation method of the cassia twig, peony and rhizoma anemarrhenae decoction composition comprises the following steps:
putting 12g of cassia twig, 9g of white paeony root, 12g of rhizoma anemarrhenae, 6g of ephedra herb, 6g of radix aconiti lateralis preparata, 12g of divaricate saposhnikovia root, 15g of ginger, 5g of largehead atractylodes rhizome and 6g of liquorice root into a 5L casserole, adding 1400ml of water, soaking for 30 minutes, and decocting to obtain 400ml of decoction; filtering the hot decoction liquid with 350-mesh filter cloth, concentrating the filtrate, freeze-drying and crushing to obtain the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition.
Further, in the step (2), the method for preparing the reference solution comprises the following steps:
precisely weighing gallic acid, catechin, albiflorin, paeoniflorin and 1,2,3,4,6-O-pentagalloylglucose as reference substances, and adding methanol to obtain radix Paeoniae alba mixed reference substance solution;
accurately weighing cinnamyl alcohol and cinnamic acid reference substances, and adding methanol to obtain a cassia twig mixed reference substance solution;
accurately weighing cimicidin, cimicifugal, 5-O-methylvisammioside and hamaudol glycoside, and adding methanol to obtain radix Saposhnikoviae mixed reference solution;
precisely weighing 5-hydroxymethyl furfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and atractylenolide
And atractylenolide
Adding methanol into the reference substance to obtain mixed reference solution of Atractylodis rhizoma;
precisely weighing 6-gingerol reference substance, and adding methanol to obtain rhizoma Zingiberis recens reference substance solution;
precisely weighing apigenin liquiritin, liquiritigenin, isoliquiritin and ammonium glycyrrhizinate reference, and adding methanol to obtain Glycyrrhrizae radix mixed reference solution;
precisely weighing new mangiferin and mangiferin reference substances, and adding methanol to obtain rhizoma anemarrhenae mixed reference substance solution;
precisely weighing ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances, and adding methanol to obtain herba Ephedrae mixed reference substance solution.
In the fingerprint spectrum construction method, a plurality of reference substances are prepared into a mixed reference substance solution, so that the experimental time is saved compared with that of a single reference solution, and the characteristic peak in the standard spectrum corresponding to the reference substances can be obtained through the peak emergence sequence of each component, the ginger belonging peak of the fingerprint spectrum is only one, and the 6-gingerol is a common and easily detected component of the ginger, so that only the 6-gingerol is selected as the reference solution of the ginger.
Further, the gallic acid, catechin, albiflorin, paeoniflorin, 1,2,3,4,6-O-pentagalloylglucose, cinnamyl alcohol, meatCinnamic acid, cimicifugal glycoside, 5-O-methylvisammioside, helometasol glycoside, 5-hydroxymethyl furfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and atractylenolide
Atractylenolide
6-gingerol, apigenin, liquiritin, liquiritigenin, isoliquiritin, ammonium glycyrrhizinate, neomangiferin, mangiferin, ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances, wherein the content of each reference substance solution is 10-100 mu g per 1 ml. The content of the reference substance in the range can have better detection effect.
Correspondingly, the invention also provides a fingerprint detection method of the cassia twig, peony and rhizoma anemarrhenae decoction composition, which comprises the following steps:
preparing a to-be-detected sample solution from the to-be-detected cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition by adopting a water extraction method;
injecting the sample solution to be detected into an ultra-high performance liquid chromatograph for chromatographic analysis to generate a sample map to be detected;
and (3) taking the standard fingerprint established by the fingerprint construction method of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition as a reference map, and calculating the similarity between the to-be-detected sample map and the standard fingerprint, wherein the quality is qualified if the similarity is more than 0.9.
The quality of the cassia twig, peony and rhizoma anemarrhenae decoction composition is detected by taking the standard fingerprint as a reference map, and the quality information of the cassia twig, peony and rhizoma anemarrhenae decoction can be comprehensively reflected, so that the quality of a cassia twig, peony and rhizoma anemarrhenae decoction preparation product is comprehensively and effectively controlled
Further, the chromatographic conditions for injecting the sample solution to be detected into the ultra-high performance liquid chromatograph for chromatographic analysis are as follows:
a chromatographic column: octadecylsilane chemically bonded silica chromatographic column, flow rate 0.25ml/min, column temperature: 30 ℃;
gradient elution is carried out by taking acetonitrile-0.1% phosphoric acid solution as a mobile phase as a system, and the elution sequence is as follows:
0-6 min, 3% of acetonitrile and 97% of 0.1% phosphoric acid solution;
6-8 min, acetonitrile 3% → 5%,0.1% phosphoric acid solution 97% → 95%;
8-16 min, acetonitrile 5% → 8%,0.1% phosphoric acid solution 95% → 92%;
16-23 min, acetonitrile 8% → 14%,0.1% phosphoric acid solution 92% → 86%;
23-29 min, acetonitrile 14% → 16%,0.1% phosphoric acid solution 86% → 84%;
29-33 min, 16% of acetonitrile and 84% of 0.1% phosphoric acid solution;
33-45 min, acetonitrile 16% → 35%,0.1% phosphoric acid solution 84% → 65%;
45-49 min, acetonitrile 35% → 60%,0.1% phosphoric acid solution 65% → 40%;
49-53 min, acetonitrile 60% → 95%,0.1% phosphoric acid solution 40% → 5%.
The chromatographic conditions in the fingerprint detection method are consistent with those established by the standard fingerprint, so that the detection error can be reduced.
The invention is further illustrated by the following examples.
The reagents and apparatus of the following examples are as follows.
Reagent testing:
gallic acid (batch No. 110831-201605, china institute for food and drug testing);
a catechin control substance (batch number 877-200001, national institute for testing food and drug);
paeontolactone reference (batch No. 5583, shanghai Shidande Standard technology services Co., ltd.);
paeoniflorin reference (batch number: 110736-201943, china institute for food and drug testing);
1,2,3,4,6-O-pentagalloylglucose (lot No. 3367, shanghai podan standard technical service ltd);
cinnamyl alcohol (batch number: 18070404, chengdu Philide biotechnology limited)
Cinnamic acid reference (batch No. 110786-201604, china institute for food and drug testing);
cimicifugal hormone reference substance (batch No. 111710-200602, china pharmaceutical biological products institute);
shengma glucoside (batch number: 111522-201712, china institute for food and drug testing);
5-O-methylvisammioside control (batch No. 111523-201610, china institute for foodstuff and drug testing);
heliomorphin reference (batch No. 111714-200501, china pharmaceutical and biological products institute);
5-hydroxymethylfurfural (batch No. 111626-201610, national institute for food and drug testing);
neochlorogenic acid (batch: DST180130-015, doudster Biotechnology Co., ltd.);
chlorogenic acid (batch number: 110753-201817, institute for testing food and drug, china);
cryptochlorogenic acid (batch number: DST170210-035, doudster Biotechnology Co., ltd.);
atractylodes macrocephala lactone III (batch number: 11978-201501, china institute for food and drug testing);
atractylodes macrocephala lactone II (batch number: 111976-201501, china institute for food and drug testing);
6-gingerol control (batch No. 111833-201505, national institute for food and drug testing);
apigenin liquiritin reference (batch No. 5569, shanghai Shidande Standard technical services, ltd.);
liquiritin reference (batch No. 111610-201607, china institute for food and drug testing);
glycyrrhizin (batch No. 4280, shanghai Shidande Standard technology services, inc.);
isoliquiritin (batch number: 6387, shanghai Shidande Standard technical services, inc.);
ammonium glycyrrhizinate reference (batch number: 110731-201720, china institute for food and drug testing);
a new mangiferin control (batch No. 3671, shanghai shidan standard technical service ltd);
mangiferin control (batch No. 111607-201704, china institute for testing food and drug);
ephedrine hydrochloride reference (batch number: 0714-990, china institute for testing biological products of drugs);
pseudoephedrine hydrochloride reference (batch No. 171237-200807, china institute for drug and biological products);
cassia twig, peony and anemarrhena decoction (self-made), methanol (BCR company, USA, pure chromatogram),
Acetonitrile (BCR corporation, usa, chromatographically pure), water is ultrapure water.
The instrument comprises the following steps:
waters H-class ultra high performance liquid chromatograph; waters PDA detector; an Empower workstation;
a Waters Acquity UPLC BEH C18 (2.1X 150mm,1.7 μm) chromatography column;
one-ten-thousandth analytical balance (AL 104, mettler-toledo);
one-tenth-ten-thousandth analytical balance (MS 105DU, mettler-toledo);
ultrasonic cleaning machines (KQ-500 DE ultrasonic instruments Co., ltd., kunshan city);
ultrapure water systems (Merck company, MILLIPORE Synergy UV).
Model TRL-05 Freeze dryer (Dalianshuangri technologies, inc.).
Example 1
White peony root mixed reference solution: taking a proper amount of gallic acid, catechin, albiflorin, paeoniflorin, 1,2,3,4,6-O-pentagalloylglucose reference substances, precisely weighing, adding methanol to prepare a solution containing 100 μ g of gallic acid, 100 μ g of catechin, 10 μ g of albiflorin, 20 μ g of paeoniflorin, 1,2,3,4,6-O-pentagalloylglucose and 80 μ g of gallic acid, catechin, albiflorin and 1,2,3,4,6-O-pentagalloylglucose in each 1ml, and taking the solution as a mixed reference substance solution;
cassia twig mixed reference solution: taking a proper amount of cinnamyl alcohol and cinnamic acid reference substances, precisely weighing, and adding methanol to prepare a solution containing 30 micrograms of cinnamyl alcohol and 20 micrograms of cinnamic acid per 1ml, wherein the solution is used as a mixed reference substance solution;
wind-break mixed reference solution: taking a proper amount of cimicifugaside, cimicifugatin, 5-O-methylvisammioside and hamaudol glycoside reference substances, precisely weighing, adding methanol to prepare a solution containing 60 mu g of cimicifugaside, 10 mu g of cimicifugatin, 60 mu g of 5-O-methylvisammioside and 10 mu g of hamaudol glycoside per 1ml, and taking the solution as a mixed reference substance solution;
mixed reference solution of white atractylodes rhizome: taking a proper amount of reference substances of 5-hydroxymethylfurfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, atractylenolide III and atractylenolide II, precisely weighing, and respectively adding methanol to prepare solutions containing 100 micrograms of 5-hydroxymethylfurfural, 50 micrograms of neochlorogenic acid, 50 micrograms of chlorogenic acid, 50 micrograms of cryptochlorogenic acid, 100 micrograms of atractylenolide III and 100 micrograms of atractylenolide II in each 1ml as mixed reference solutions;
ginger reference solution: taking a proper amount of 6-gingerol reference substance, precisely weighing, adding methanol to prepare a solution containing 50 microgram of 6-gingerol per 1ml as a reference substance solution;
licorice mixed reference solution: taking appropriate amount of apiose liquiritin, liquiritigenin, isoliquiritin and ammonium glycyrrhizinate as reference substances, accurately weighing, adding methanol to obtain solution containing apiose liquiritin 20 μ g, liquiritigenin 20 μ g, isoliquiritin 20 μ g and ammonium glycyrrhizinate 20g per 1ml, and using as mixed reference substance solution;
rhizoma anemarrhenae mixed reference solution: taking appropriate amount of neomangiferin and mangiferin reference substances, precisely weighing, adding methanol to obtain solution containing neomangiferin 50 μ g and mangiferin 50 μ g per 1ml, and using as mixed reference substance solution;
herba ephedrae mixed reference solution: taking appropriate amount of ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances, precisely weighing, and adding methanol to obtain solutions containing ephedrine hydrochloride 50 μ g and pseudoephedrine hydrochloride 100 μ g per 1ml as mixed reference substance solutions.
By comparing with the above mixed reference solution, as shown in fig. 5-12, 16 known peaks in fingerprint spectrum of the decoction composition of ramulus Cinnamomi, radix Paeoniae and rhizoma anemarrhenae are obtained, including gallic acid, ephedrine hydrochloride, pseudoephedrine hydrochloride, catechin, neomangiferin, mangiferin, albiflorin, paeoniflorin, linarin, apioside, liquiritin, 5-O-methylvisammol glycoside, cinnamic acid, heliomin, ammonium glycyrrhizinate, and 6-gingerol.
Example 2 preparation of Cassia twig, peony and Anemarrhena decoction composition and preparation of test solution
Weighing the following decoction pieces in parts by weight: 12g of cassia twig, 9g of white paeony root, 12g of rhizoma anemarrhenae, 6g of ephedra herb, 6g of sliced aconite, 12g of divaricate saposhnikovia root, 15g of ginger, 5g of largehead atractylodes rhizome and 6g of liquorice; placing nine-ingredient decoction pieces in a 5L casserole, adding 1400ml of water, soaking for 30 min, covering, boiling, keeping slightly boiling to 400ml of decoction, filtering with 350 mesh filter cloth while hot, concentrating the filtrate under reduced pressure to appropriate amount, lyophilizing, pulverizing, and mixing to obtain the composition.
Taking about 0.18g of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition, precisely weighing, putting into a 10ml measuring flask, adding a proper amount of water, carrying out ultrasonic treatment for 10-30 minutes (power is 250W and frequency is 40 kHz), cooling, adding water to dilute to a scale, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution.
Example 3 examination of chromatographic conditions of finger print
(1) Selection of detection wavelength
Octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; flow rate 0.25ml/ml, column temperature: at 30 ℃.
Table 1: fingerprint mobile phase gradient program
| Time (minutes) | Acetonitrile (%) | 0.1% phosphoric acid (%) |
| 0~6 | 3 | 97 |
| 6~8 | 3→5 | 97→95 |
| 8~16 | 5→8 | 95→92 |
| 16~23 | 8→14 | 92→86 |
| 23~29 | 14→16 | 86→84 |
| 29~33 | 16 | 84 |
| 33~45 | 16→35 | 84→65 |
| 45~49 | 35→60 | 65→40 |
| 49~53 | 60→95 | 40→5 |
The sample solution of example 2 was subjected to full-wavelength detection using a diode array detector, based on the collected three-dimensional spectra (see FIG. 1), and simultaneously collected spectra at wavelengths of 210nm, 230nm, 254nm, 270nm, and 280nm (see FIG. 2). Through comprehensive investigation, compared with other wavelengths, the peak information amount of the cassia twig, peony and rhizoma anemarrhenae decoction composition at the wavelength of 210nm is the largest, and ephedrine hydrochloride and pseudoephedrine hydrochloride components in ephedra can be characterized, so that 210nm is determined as the detection wavelength of the fingerprint of the cassia twig, peony and rhizoma anemarrhenae decoction composition (see fig. 1 and fig. 2).
(2) Investigation of chromatographic systems
Based on the chromatographic conditions in the step (1), the detection wavelength of the fingerprint spectrum of the cassia twig, peony and rhizoma anemarrhenae decoction composition is selected to be 210nm, if acetonitrile-formic acid aqueous solution or acetonitrile-acetic acid aqueous solution is used as a mobile phase, interference exists at the wavelength of 210nm, so that an acetonitrile-phosphoric acid aqueous solution and an acetonitrile-water system are respectively considered: the sample solution of example 2 was precisely extracted and injected into the chromatograph for chromatographic analysis, and the fingerprint detection is shown in fig. 3.
The results show that: the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition has stable baseline in an acetonitrile-phosphoric acid water solution system, and the chromatographic peak type and the separation effect are obviously superior to other mobile phase systems, so that the mobile phase system is determined to be the acetonitrile-phosphoric acid water solution.
EXAMPLE 4 examination of the method for preparing a test article
Based on the optimal chromatographic conditions examined in example 3, the extraction effects of different solvents, namely 60% methanol, 60% ethanol and water, on the fingerprint of the cassia twig, peony and rhizoma anemarrhenae decoction composition are respectively examined.
Preparation of test solutions in different solvents:
taking 0.18g of the cassia twig, peony and rhizoma anemarrhenae decoction composition of the embodiment 2, precisely weighing, putting into a 10ml measuring flask, respectively adding a proper amount of 60% methanol, 60% ethanol and water, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooling, respectively diluting to a scale with corresponding solvents, shaking up, filtering, and taking a subsequent filtrate to obtain the cassia twig, peony and rhizoma anemarrhenae decoction composition; each 1. Mu.l of the resulting extract was precisely aspirated, and the resulting extract was injected into a liquid chromatograph to measure the optimum chromatographic conditions as examined in example 3.
The result is shown in fig. 4, the number of extraction peaks of the cassia twig, peony and rhizoma anemarrhenae decoction composition by water is the largest, the operation is convenient and the solvent is safe in comprehensive consideration, so that the water is selected as the extraction solvent of the fingerprint spectrum of the cassia twig, peony and rhizoma anemarrhenae decoction composition.
Example 5 methodological examination
5.1 specificity test
And precisely sucking 1 mu l of each of the reference solution in the embodiment 1, the test solution in the embodiment 2 and the blank solvent, injecting the solution into a liquid chromatograph, and performing fingerprint spectrum method determination according to the optimal chromatographic conditions in the embodiment 3, wherein corresponding chromatographic peaks appear at corresponding positions of the chromatogram of the test solution and the chromatogram of the reference solution, and no corresponding peak exists on the blank sample, which indicates that the sample is negative and has no interference.
5.2 precision test
The method is operated according to the fingerprint precision, samples of the S1 batch are taken to prepare a sample solution by the method of the embodiment 2, the sample introduction is carried out for 6 times continuously according to the optimal chromatographic conditions of the embodiment 3, the chromatogram is led into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) to calculate the similarity, the result similarity is more than 0.95, and the instrument precision is good.
5.3 stability test
According to the operation of the method for measuring the stability of the finger-print, samples of S1 batches are taken to prepare a test sample solution by the method of the embodiment 2, samples are respectively injected and measured according to the optimal chromatographic conditions of the embodiment 3 in 0 th, 4 th, 8 th, 12 th, 20 th and 24 th hours after the preparation is finished, the chromatograms are led into software of a traditional Chinese medicine chromatogram finger-print similarity evaluation system (2012 edition) to calculate the similarity, the result similarity is more than 0.95, and the test sample solution is proved to have good stability in 24 hours after the preparation is finished.
5.4 repeatability test
According to the operation of the fingerprint repeatability determination method, samples of S1 batches are taken to prepare 6 test solution portions by the method of the embodiment 2, sample introduction and determination are respectively carried out according to the optimal chromatographic conditions of the embodiment 3, the chromatogram is led into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) to calculate the similarity, and the result similarity is over 0.95, so that the method is good in repeatability.
5.5 intermediate precision test
The method comprises the steps of operating by different experimenters on different dates according to a fingerprint repeatability determination method, taking S1 batches of samples to prepare 6 parts of test solution by the method of the embodiment 2, respectively carrying out sample injection determination according to the optimal chromatographic conditions of the embodiment 3, introducing chromatograms into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) to calculate similarity, and leading the result similarity to be more than 0.95, thereby indicating that the method has good intermediate precision.
The test results show that the analysis method has good stability and repeatability.
Example 6 determination of common peaks and establishment of control fingerprints
Preparing 5 batches of cassia twig, peony and rhizoma anemarrhenae decoction compositions by adopting different batches of decoction pieces respectively, preparing a sample solution by the method of example 2, detecting the sample solution by the optimal chromatographic condition of example 3 to obtain fingerprints, introducing the fingerprints of the 5 batches of sample solution into a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition), performing full spectrum matching by adopting an average method, determining that the common characteristic peaks are 33, taking a No. 15 mangiferin chromatographic peak as an S peak, and calculating the relative retention time of the common peaks of the 5 batches of samples. The relative retention time deviation RSD of the 33 common characteristic peaks is less than 2 percent, namely:
the relative retention time RRT of the No. 1 peak is 0.142, and the RSD% is 0.78%;
the relative retention time RRT of the No. 2 peak is 0.272, and the RSD% is 1.11%;
the relative retention time RRT of the No. 3 peak is 0.309, and the RSD% is 1.20%;
peak 4 relative retention time RRT 0.336, rsd% 0.75%;
the relative retention time RRT of the No. 5 peak is 0.380, and the RSD% is 1.16%;
the relative retention time RRT of the No. 6 peak is 0.429, and the RSD% is 0.90%;
the relative retention time RRT of the No. 7 peak is 0.488, and the RSD% is 0.87%;
the relative retention time RRT of the No. 8 peak is 0.523, and the RSD% is 0.76%;
peak 9 relative retention time RRT 0.573, RSD% 0.87%;
the 10 peak relative retention time RRT is 0.626 and the RSD% is 0.76%;
the relative retention time RRT of the No. 12 peak is 0.749, and the RSD% is 0.39%;
peak 17 relative retention time RRT is 1.043, rsd% is 0.02%;
the relative retention time RRT of the 18 th peak is 1.053, and the RSD% is 0.08%;
the 19 th peak relative retention time RRT is 1.067, and the RSD% is 0.19%;
the relative retention time RRT of the No. 20 peak is 1.081, and the RSD% is 0.12%;
peak 22 relative retention time RRT is 1.178 and rsd% is 0.27%;
relative retention time RRT of No. 24 peak is 1.253, and RSD% is 0.19%;
peak 26 relative retention time RRT of 1.327 and rsd% of 0.14%;
peak 27 relative retention time RRT 1.396 and rsd% 0.32%;
the relative retention time RRT of the 28 th peak is 1.608, and the RSD% is 0.40%;
the relative retention time RRT of the 31 # peak is 1.926 and the RSD% is 0.37%;
peak 32 relative retention time RRT is 2.190 and rsd% is 0.49%;
the relative retention time RRT of the No. 33 peak is 2.243, and the RSD% is 0.48%; among them, the S peak No. 15 is a chromatographic peak of the reference.
5 batches of cassia twig, peony and rhizoma anemarrhenae decoction composition samples are fitted by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) (see figure 13) to generate a cassia twig, peony and rhizoma anemarrhenae decoction control fingerprint (see figure 14). The sample similarity is calculated by adopting an included angle cosine method, and the result shows that the similarity of 5 batches of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition is between 0.900 and 0.991, the similarity is higher, and the chemical components are stable and the quality is good.
TABLE 2 evaluation results of similarity of 5 Guizhi Shaoyao Zhimu Tang compositions
| Numbering | S1 | S2 | S3 | S4 | S5 |
| S1 | 1.000 | 0.939 | 0.970 | 0.991 | 0.867 |
| S2 | 0.939 | 1.000 | 0.971 | 0.934 | 0.766 |
| S3 | 0.970 | 0.971 | 1.000 | 0.975 | 0.834 |
| S4 | 0.991 | 0.934 | 0.975 | 1.000 | 0.874 |
| S5 | 0.867 | 0.766 | 0.834 | 0.874 | 1.000 |
| Comparison fingerprint | 0.990 | 0.955 | 0.985 | 0.991 | 0.900 |
Other configurations and operations of the fingerprint construction method and the detection method of the Guizhi Shaoyao Zhimu Tang composition according to the embodiment of the present invention are known to those skilled in the art and will not be described in detail herein.
In the description herein, references to the description of "an embodiment," "an example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (8)
1. A fingerprint construction method of a cassia twig, peony and rhizoma anemarrhenae decoction composition is characterized by comprising the following steps:
(1) Precisely weighing the cassia twig, peony and rhizoma anemarrhenae decoction composition, adding water, and performing ultrasonic treatment to obtain a test solution, wherein the prescription of the cassia twig, peony and rhizoma anemarrhenae decoction composition comprises 12g of cassia twig, 9g of white paeony root, 12g of rhizoma anemarrhenae, 6g of ephedra, 6g of prepared aconite, 12g of divaricate saposhnikovia root, 15g of ginger, 5g of bighead atractylodes rhizome and 6g of liquorice;
(2) Preparing reference solution from gallic acid, catechin, albiflorin, paeoniflorin, 1,2,3,4,6-O-pentagalloyl glucose, cinnamyl alcohol, cinnamic acid, cimicidin, 5-O-methyl visammol glycoside, sec-hamaudol glycoside, 5-hydroxymethyl furfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, atractylenolide III, atractylenolide II, 6-gingerol, apioside, liquiritin, liquiritigenin, isoliquiritin, ammonium glycyrrhizic acid, neomangiferin, mangiferin, ephedrine hydrochloride and pseudoephedrine hydrochloride;
(3) Precisely absorbing the test solution and each reference solution, injecting into an ultra-high performance liquid chromatograph for chromatographic analysis, wherein the detection wavelength is 210nm, obtaining a test sample fingerprint and a reference chromatogram, and establishing a standard fingerprint of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition;
in the step (3), the chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica chromatographic column, flow rate 0.25ml/min, column temperature: 30 ℃;
gradient elution is carried out by taking acetonitrile-0.1% phosphoric acid solution as a mobile phase as a system, and the elution sequence is as follows:
0-6 min, 3% of acetonitrile and 97% of 0.1% phosphoric acid solution;
6-8 min, acetonitrile 3% → 5%,0.1% phosphoric acid solution 97% → 95%;
8-16 min, acetonitrile 5% → 8%,0.1% phosphoric acid solution 95% → 92%;
16-23 min, acetonitrile 8% → 14%,0.1% phosphoric acid solution 92% → 86%;
23-29 min, acetonitrile 14% → 16%,0.1% phosphoric acid solution 86% → 84%;
29-33 min, 16% of acetonitrile and 84% of 0.1% phosphoric acid solution;
33-45 min, acetonitrile 16% → 35%,0.1% phosphoric acid solution 84% → 65%;
45-49 min, acetonitrile 35% → 60%,0.1% phosphoric acid solution 65% → 40%;
49-53 min, acetonitrile 60% → 95%,0.1% phosphoric acid solution 40% → 5%.
2. The fingerprint construction method of Guizhi Shaoyao Zhimu Tang composition as claimed in claim 1, wherein the standard fingerprint comprises 33 characteristic peaks, the 15 peak mangiferin chromatographic peak is the S peak, the relative retention time of each characteristic peak is: peak 1 relative retention time RRT of 0.142,2 Peak relative retention time RRT of 0.272,3 Peak relative retention time RRT of 0.309,4 Peak relative retention time RRT of 0.336,5 Peak relative retention time RRT of 0.380,6 Peak relative retention time RRT of 0.429,7 Peak relative retention time RRT of 0.488,8 Peak relative retention time RRT of 0.523,9 Peak relative retention time RRT of 0.573, peak 10 relative retention time RRT of 0.626, peak 11 relative retention time RRT of 0.672, peak 12 relative retention time RRT of 0.9, peak 13 relative retention time RRT of 0.792, peak 14 relative retention time RRT of 0.945, peak 15 (S) Peak relative retention time RRT of 1.000, peak 16 relative retention time RRT of 1.043, peak relative retention time RRT of 1.013, the peak relative retention time RRT No. 18 is 1.053, the peak relative retention time RRT No. 19 is 1.067, the peak relative retention time RRT No. 20 is 1.081, the peak relative retention time RRT No. 21 is 1.129, the peak relative retention time RRT No. 22 is 1.178, the peak relative retention time RRT No. 23 is 1.197, the peak relative retention time RRT No. 24 is 1.253, the peak relative retention time RRT No. 25 is 3524 zxft No. 26 is 1.327, the peak relative retention time RRT No. 27 is 1.396, the peak relative retention time RRT No. 28 is 1.608, the peak relative retention time RRT No. 29 is 1.895, the peak relative retention time RRT No. 30 is 3754 zxft No. 31 is 49xft RRT No. 84, the peak relative retention time RRT No. 32 is 52xft No. 72, the peak relative retention time RRT No. 7945 is 7972 zxft No. 7945.
3. The method of claim 2, wherein peaks at positions 1, 7, 8, 12, 13, 15, 18, 21, 23, 25, 26, 28, 29, 31, 32 and 33 in the standard fingerprint correspond to peaks at retention times of peaks at gallic acid, ephedrine hydrochloride, pseudoephedrine hydrochloride, catechin, neomangiferin, mangiferin, albiflorin, paeoniflorin, glucoroniside, apigenin, liquiritin, 5-O-methylvisammioside, cinnamic acid, sec-O, ammonium glycyrrhizinate, and 6-gingerol control.
4. The fingerprint construction method of Guizhi Shaoyao Zhimu Tang composition as claimed in claim 1, wherein in step (1), 0.18g of Guizhi Shaoyao Zhimu Tang composition is precisely weighed, put into a 10ml measuring flask, added with water for ultrasonic treatment for 10-30 minutes, cooled, diluted to 10ml scale with water, and filtered to obtain the test solution.
5. The fingerprint construction method of the Guizhi Shaoyao Zhimu Tang composition of claim 4, wherein the preparation method of the Guizhi Shaoyao Zhimu Tang composition comprises:
putting 12g of cassia twig, 9g of white paeony root, 12g of rhizoma anemarrhenae, 6g of ephedra herb, 6g of sliced aconite, 12g of divaricate saposhnikovia root, 15g of ginger, 5g of largehead atractylodes rhizome and 6g of liquoric root into a 5L marmite, adding 1400ml of water, soaking for 30 minutes, and decocting to obtain 400ml of decoction;
filtering the hot decoction liquid with 350-mesh filter cloth, concentrating the filtrate, freeze-drying and crushing to obtain the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction composition.
6. The fingerprint spectrum construction method of the cinnamon twig peony and rhizoma anemarrhenae decoction composition according to claim 1, wherein the method for preparing the reference solution in the step (2) comprises:
precisely weighing gallic acid, catechin, albiflorin, paeoniflorin and 1,2,3,4,6-O-pentagalloylglucose as reference substances, and adding methanol to obtain radix Paeoniae alba mixed reference substance solution;
accurately weighing cinnamyl alcohol and cinnamic acid as reference substances, and adding methanol to obtain a mixed reference substance solution of ramulus Cinnamomi;
accurately weighing cimicidin, cimicifugal, 5-O-methylvisammioside and hamaudol glycoside, and adding methanol to obtain radix Saposhnikoviae mixed reference solution;
accurately weighing 5-hydroxymethylfurfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, atractylenolide III and atractylenolide II as reference substances, and adding methanol to obtain mixed reference substance solution of Atractylodis rhizoma;
precisely weighing 6-gingerol reference substance, and adding methanol to obtain rhizoma Zingiberis recens reference substance solution;
precisely weighing apigenin liquiritin, liquiritigenin, isoliquiritin and ammonium glycyrrhizinate reference, and adding methanol to obtain Glycyrrhrizae radix mixed reference solution;
precisely weighing new mangiferin and mangiferin reference substances, and adding methanol to obtain rhizoma anemarrhenae mixed reference substance solution;
precisely weighing ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances, and adding methanol to obtain herba Ephedrae mixed reference substance solution.
7. The method of claim 6, wherein the content of gallic acid, catechin, albiflorin, paeoniflorin, 1,2,3,4,6-O-pentagalloylglucose, cinnamyl alcohol, cinnamic acid, cimicidin, 5-O-methylvisammioside, sec-O-Helioside, 5-hydroxymethylfurfural, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, atractylenolide III, atractylenolide II, 6-gingerol, apigenin, liquiritin, liquiritigenin, isoliquiritin, ammonium glycyrrhizic acid, neomangiferin, mangiferin, ephedrine hydrochloride, and pseudoephedrine hydrochloride in each reference solution is 10-100 μ g/1 ml.
8. A fingerprint detection method of a cassia twig, peony and rhizoma anemarrhenae decoction composition is characterized by comprising the following steps:
preparing a to-be-detected sample solution from the to-be-detected cassia twig, peony and rhizoma anemarrhenae decoction composition by adopting a water extraction method, wherein the prescription of the to-be-detected cassia twig, peony and rhizoma anemarrhenae decoction composition comprises 12g of cassia twig, 9g of radix paeoniae alba, 12g of rhizoma anemarrhenae, 6g of ephedra, 6g of processed radix aconiti lateralis preparata, 12g of radix sileris, 15g of ginger, 5g of rhizoma atractylodis macrocephalae and 6g of liquorice;
injecting the sample solution to be detected into an ultra-high performance liquid chromatograph for chromatographic analysis;
taking a standard fingerprint prepared by the fingerprint construction method of the cassia twig, peony and rhizoma anemarrhenae decoction composition as claimed in any one of claims 1 to 7 as a reference, and calculating the similarity between the sample spectrum to be tested and the standard fingerprint, wherein the quality is qualified if the similarity is more than 0.9;
wherein, the chromatographic conditions for chromatographic analysis by injecting the sample solution to be tested into the ultra-high performance liquid chromatograph are as follows:
and (3) chromatographic column: octadecylsilane chemically bonded silica chromatographic column, flow rate 0.25ml/min, column temperature: 30 ℃;
gradient elution is carried out by taking acetonitrile-0.1% phosphoric acid solution as a mobile phase as a system, and the elution sequence is as follows:
0-6 min, 3% of acetonitrile and 97% of 0.1% phosphoric acid solution;
6-8 min, acetonitrile 3% → 5%,0.1% phosphoric acid solution 97% → 95%;
8-16 min, acetonitrile 5% → 8%,0.1% phosphoric acid solution 95% → 92%;
16-23 min, acetonitrile 8% → 14%,0.1% phosphoric acid solution 92% → 86%;
23-29 min, acetonitrile 14% → 16%,0.1% phosphoric acid solution 86% → 84%;
29-33 min, 16% of acetonitrile and 84% of 0.1% phosphoric acid solution;
33-45 min, acetonitrile 16% → 35%,0.1% phosphoric acid solution 84% → 65%;
45-49 min, acetonitrile 35% → 60%,0.1% phosphoric acid solution 65% → 40%;
49-53 min, acetonitrile 60% → 95%,0.1% phosphoric acid solution 40% → 5%.
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| CN114113432B (en) * | 2021-07-27 | 2023-08-25 | 南京海昌中药集团有限公司 | Detection method of ginseng and poria cocos wine fingerprint |
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| CN114814069B (en) * | 2022-06-08 | 2023-05-09 | 国药集团广东环球制药有限公司 | Thin-layer identification method for 8 medicinal herbs in 9 medicinal herbs in whole formulation of cassia twig, chinese herbaceous peony and anemarrhena decoction |
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