CN113341052A - Method for measuring content of effective components of ephedra decoction substance standard - Google Patents
Method for measuring content of effective components of ephedra decoction substance standard Download PDFInfo
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- CN113341052A CN113341052A CN202110761770.8A CN202110761770A CN113341052A CN 113341052 A CN113341052 A CN 113341052A CN 202110761770 A CN202110761770 A CN 202110761770A CN 113341052 A CN113341052 A CN 113341052A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a method for measuring the content of effective components of ephedra decoction based on substance, belonging to the technical field of traditional Chinese medicine detection. The method for measuring the content of the effective components of the ephedra decoction substance standard comprises the following steps: step one, preparing a material standard: processing the medicinal materials by the method recorded in ancient books, decocting, concentrating, and drying to obtain a substance standard; step two, preparing a test solution; step three, preparing a reference substance solution; and step four, qualitative and quantitative testing of the chromatogram. The determination method provided by the invention can be simultaneously suitable for decoction pieces, intermediates and material bases, the material base preparation process is scientific and reasonable, the material base can not be obviously changed, the determination method is utilized to establish the characteristic map of the material base effective components of the ephedra decoction, the content of the material base effective components of the ephedra decoction, the paste yield and the transfer rate of the material base in the preparation process of the ephedra decoction are given, and the foundation is laid for the development of the ephedra decoction.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for measuring the content of effective components of ephedra decoction based on a substance standard.
Background
In the ancient classical famous prescription catalog (first batch) published by the State administration of traditional Chinese medicine, the origin of the ephedra decoction is given and comes from the Shang Han Lun of Zhang Zhongjing in the Han Dynasty. The ephedra decoction comes from Zhang Zhongjing of the Han Dynasty, the original text of which is: ' Ephedra herb three two (with nodes removed), cassia twig two (with peel removed), licorice root one two (with roast), almond seventy (with peel and tip removed). And nine liters of water is used for decocting the other four medicines, the weight of the medicines is reduced by two liters, the upper foam is removed, the medicines are taken internally, two liters of the medicines are decocted and half is taken, the dregs are removed, the medicines are taken warmly and eighty percent, the medicines are taken again to slightly sweat without sipping the porridge, and the rest is treated by the cassia twig method. The method is mainly used for treating exterior excess syndrome of wind-cold type, such as aversion to cold, fever, headache, body pain, arthralgia, asthma without sweat, thin and white tongue coating, floating and tense pulse, and the pathogenesis of the exterior excess syndrome of wind-cold type is wind-cold external restraint, wei-defensive qi stagnation, hair orifice blockage, lung qi loss, and the like.
The traditional Chinese medicine registration management supplementary provisions (hereinafter referred to as supplementary provisions) released in 2008 from the national food and drug administration and administration (CFDA) brings opportunities for the inheritance and development of classical famous prescriptions. The prescription screening and demonstration has various requirements, and the research needs to be carried out from the aspects of history record, years of completed books, prescription evolution, examination of measuring units, usage and dosage, modern research, clinical application and the like, and in addition, the research needs to carry out document arrangement, analysis, innovation and the like on the aspects of basic sources, species, effective components, pharmacology, modern application, processing, quality control and the like of the used medicinal materials. The greatest characteristic of classical famous prescription research is that the traditional Chinese medicine preparation is in line with the ancient way, and the key of the ancient way is that the processing method, the original prescription dosage, the medicinal materials are based on the source, and the decocting process is consistent with the ancient medical collection literature. The rationality of the modern preparation process is considered while following the ancient process, so that the modern quality standard and clinical curative effect of the ancient classical prescription are realized, and the treasure left by the ancient Chinese medicine is better inherited.
The traditional Chinese medicine quality control method also uses a chemical medicine quality control mode for reference, and the method uses a blank solution to make a standard calibration curve so as to measure the content of one or more index components in the Chinese medicine. The method plays an irreplaceable role in the development process of traditional Chinese medicine research, and is still used as a standard method for measuring the chemical component content of different traditional Chinese medicine varieties until the pharmacopoeia of the people's republic of China. For example, CN105116086A discloses a method for measuring the content of paeoniflorin and albiflorin in red peony root by HPLC-ELSD detection with an ELSD detector, and plotting curves to obtain a standard curve equation; also, for example, CN104345110A discloses a method for measuring the contents of seven ingredients in a Chinese medicinal composition preparation, which adopts UPLC detection, formulation of a standard curve and result calculation.
The above examples all illustrate that the methods for detecting components and measuring contents of traditional Chinese medicine are qualitative and quantitative analysis methods which are mature in the field of pharmaceutical analysis, but because traditional Chinese medicine is a complex system which is quite different from chemical medicine and comprises complex chemical components with different structures, unpredictable interfering compounds and analytes coexist in a complex matrix, a standard calibration curve obtained by using a series of concentration standard solutions diluted by pure solvents does not consider the existence of matrix effect, so that the environments for detecting the analytes in the pure solvents and traditional Chinese medicine extracting solutions are greatly different, thereby affecting the accuracy, reproducibility and selectivity of the content measuring method. Therefore, the key point of the quality control of the traditional Chinese medicine is to find a content determination method suitable for the characteristics of the traditional Chinese medicine.
In addition, no determination method for the content of the effective components of the ephedra decoction substance standard exists in the prior art. The method prepares the ephedra decoction substance standard to be researched into a test article, prepares a single effective component into a series of reference solution, measures the content of the effective components of the two solutions by chromatography to determine a characteristic map of the substance standard, and calculates the transfer rate and the paste yield of the effective components of the substance standard in the preparation process, thereby carrying out the process and quality control analysis of the preparation process and evaluating the scientificity and rationality of the method.
Disclosure of Invention
The invention aims to provide a method for measuring the content of active ingredients of a ephedra decoction substance base, which aims to solve the problem that the conventional method for measuring the content of the active ingredients of the ephedra decoction substance base is lack.
The purpose of the invention can be realized by the following technical scheme:
a method for measuring the content of active ingredients of a ephedra decoction substance standard comprises the following steps:
step one, preparing a material standard: processing, decocting, concentrating and drying the medicinal materials by the method recorded in ancient books to prepare a substance standard, specifically taking 41.25g of ephedra herb, putting the ephedra herb in a 3L casserole, adding 1800mL of water, putting the ephedra herb on an electric ceramic stove (opening a cover), adjusting to strong fire (1800W), decocting for about 70min (reducing the decoction by 400mL), and removing floating foams; adding 27.5g of cassia twig, burning 23.64g of semen armeniacae amarae (pounding and sieving by a second sieve) and 13.75g of fried liquorice, adjusting to slow fire (600-00W), decocting for about 80min (500 mL of decoction is remained), filtering by a 150-mesh filter cloth, concentrating the filtered liquid medicine under reduced pressure (40-50 ℃ and-0.08-0.1 MPa) to about 50mL, cooling, freezing in a refrigerator at (-20 ℃) for 12 hours, drying in a freeze dryer at (-45 ℃) for 24 hours, taking out, crushing, sieving by a 80-mesh sieve, and mixing uniformly to obtain the traditional Chinese medicine composition;
step two, preparation of a test solution: precisely weighing the substance obtained in the first step, grinding, dissolving the substance by using a methanol or ethanol solution with the mass concentration of 50%, weighing, ultrasonically treating for 30min, cooling, weighing again, complementing the weight loss by using the methanol or ethanol solution with the mass concentration of 50%, shaking up, filtering, and taking the subsequent filtrate to obtain a sample solution, wherein the ultrasonic treatment condition is that the power is 250W, the frequency is 50kHz, and the mass ratio of the substance to the methanol or ethanol solution with the mass concentration of 50% is 1: 100, respectively;
step three, preparing a reference substance solution: preparing a reference solution by using the effective components as a reference and a solvent;
step four, qualitative and quantitative testing of the chromatogram: the chromatographic experimental conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent (hydrophilic column); taking a solvent A-a solvent B as a mobile phase, and performing gradient elution; the detection wavelength is 210 nm; precisely sucking 10 μ L of reference solution and sample solution respectively, and injecting into liquid chromatograph; the calculation method comprises the following steps: and (3) putting the reference sample solution into a chromatograph, taking the chromatographic peak area as the ordinate and the sample amount as the abscissa to obtain a standard curve of each reference sample solution, putting the sample into the chromatograph, and bringing the peak area of each component into the standard curve to obtain the content of the effective component of the ephedra decoction substance standard.
Further, the effective components in the three steps comprise ephedrine, pseudoephedrine, amygdalin, liquiritin, glycyrrhizic acid and cinnamic acid.
Further, when the effective component is ephedrine or pseudoephedrine, ephedrine hydrochloride or pseudoephedrine hydrochloride and methanol are prepared into control solution with concentration of 40 μ g/mL.
Further, when the active ingredient is amygdalin, the amygdalin and 70% ethanol solution are prepared into a control solution with the concentration of 0.35 mg/mL.
Further, when the effective component is liquiritin or glycyrrhizic acid, the effective component and 70% ethanol solution are prepared into a control solution with the concentration of 40 mg/mL.
Further, when the effective component is cinnamic acid, the cinnamic acid and an ethanol solution with the mass fraction of 70% are prepared into a control solution, and the concentration is 10 mug/mL.
Further, the mobile phase in the fourth step is selected according to the effective components in the control solution, when the effective components are ephedrine or pseudoephedrine, the solvent A is acetonitrile, and the solvent B is 0.1% phosphoric acid solution (both mass fractions) containing 0.04% triethylamine; when the active ingredient is amygdalin, the solvent A is methanol and the solvent B is water; when the active ingredient is liquiritin or glycyrrhizic acid, the solvent A is acetonitrile, and the solvent B is a phosphoric acid solution with the mass fraction of 0.05%; when the active ingredient is amygdalin, the solvent A is acetonitrile, and the solvent B is phosphoric acid solution with the mass fraction of 0.1%.
Further, the content of the effective components of the ephedra decoction substance standard prepared by randomly combining 15 batches of genuine medicinal materials is determined according to the method for determining the content of the effective components of the ephedra decoction substance standard, and the content of the effective components of the ephedra decoction substance standard of 15 batches is statistically analyzed to obtain the following results:
the ephedra decoction comprises the following effective components in terms of substance standard: the total content range of ephedrine and pseudoephedrine is 13.14mg/g-24.40mg/g, the content range of liquiritin is 3.90mg/g-7.23mg/g, the content range of glycyrrhizic acid is 5.30mg/-9.84mg/g, the content range of amygdalin is 19.05mg/g-17.071mg/g, and the content range of cinnamic acid is 0.79mg/g-1.47 mg/g;
the specified values of 9 retention times of the total peaks of the standard effective component characteristic spectrum of the ephedra decoction are as follows: 0.52 (peak 1), 0.55 (peak 2), 0.86 (peak 3), 0.89 (peak 4), 0.98 (peak 5), 1.00[ peak 6(S) ], 1.35 (peak 7), 1.67 (peak 8), 1.75 (peak 9), 9 peaks which are identified cover all the flavors in the ephedrine soup material standard, and the quality characteristics of the ephedrine soup material standard are better shown.
Further, the paste yield and transfer rate of the standard effective components of the ephedra decoction in the preparation process are calculated according to the following formulas:
the paste yield is 100 percent of the material standard dry powder amount/the material feeding amount of the decoction pieces;
the transfer rate is the content of the effective components in the sample/the content of the effective components in the decoction pieces is 100 percent;
and the range of the calculated plaster yield of the standard effective components of 15 batches of ephedra decoction materials is as follows: 8.43% -15.64%;
the transfer rate of the effective components of 15 batches of ephedra decoction material standard is as follows: ephedrine and pseudoephedrine 43.25%, liquiritin 63.13%, glycyrrhizic acid 55.39%, amygdalin 39.0%, and cinnamic acid 34.63%.
The invention has the beneficial effects that:
the method for measuring the content of the effective components of the ephedra decoction substance standard is reasonable and feasible, can be simultaneously suitable for decoction pieces, intermediates and substance standards, has scientific and reasonable substance standard preparation process, and does not cause obvious change of the substance standard.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a reference characteristic spectrum of 15 batches of the ephedra decoction;
FIG. 2 is a control profile of the active ingredient;
FIG. 3 is a comparison graph of a reference substance and a full-scale feature map;
FIG. 4 is a comparison chart of the characteristic spectrum of the herbs and the whole formula.
In the drawings, each reference numeral represents the following:
in FIG. 1, S1-S15 represent 15 batches of lyophilized powder;
in FIG. 2, ephedrine hydrochloride 1; 2 pseudoephedrine hydrochloride; 6(s) amygdalin; 7, liquiritin; 8 cinnamic acid; 9 glycyrrhizic acid.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
materials: high performance liquid chromatograph, LC-2030, Shimadzu corporation, Japan. Ephedrine reference (110752-. Purchased from the institute of food and drug testing, China, and the water is purified water.
Decoction pieces of Ephedra decoction were purchased from the stalk of the grass of Ephedra sinica Stapf belonging to Ephedraceae, 3 production areas. Ramulus Cinnamomi is twig of Cinnamomum cassia Presl of Lauraceae, and Almond is dried mature seed of Prunus Armeniaca L.var.ansu Maxim. of Rosaceae, and dried root and rhizome of Glycyrrhiza Glabra Fisch. of Glycyrrhiza of Leguminosae.
The decoction pieces of the ephedra decoction are checked according to 2020 edition Chinese pharmacopoeia and all meet the requirements. The information of the origin is shown in table 1.
TABLE 1 Ephedra decoction 15 batches of substance-based information on producing area of each decoction piece
Example 2:
preparation of a material standard: putting 41.25g of ephedra herb in example 1 into a 3L casserole, adding 1800mL of water, putting the casserole on an electric ceramic stove (uncovering), adjusting to strong fire (1800W), decocting for about 70min (reducing the decoction by 400mL), and removing froth; adding 27.5g of cassia twig in example 1, 23.64g of bitter apricot seed in example 1 (smashed and sieved by a No. two sieve) and 13.75g of fried licorice root in example 1, adjusting to slow fire (600 and 800W), decocting for about 80min (500 mL of decoction remains), filtering through a 150-mesh filter cloth, concentrating the filtered liquid medicine under reduced pressure (40-50 ℃ and-0.1 MPa) to about 50mL, cooling, freezing for 12 hours in a refrigerator (20 ℃), drying for 24 hours in a freeze dryer (45 ℃), taking out, crushing, sieving with an 80-mesh sieve, mixing uniformly to obtain a ephedra decoction substance standard, randomly combining the component medicines in example 1 to obtain 15 batches of ephedra decoction substance standard, and marking MHT-1, MHT-2, MHT-3, MHT-4, MHT-5, MHT-6, MHT-8 and MHT-6, MHT-9, MHT-10, MHT-11, MHT-12, MHT-13, MHT-14, and MHT-15.
Example 3:
determination of ephedrine and pseudoephedrine contents:
(1) preparation of a test solution: respectively taking obtained substance references in MHT-1-MHT-15, grinding, taking 0.25g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 50% methanol in mass fraction, weighing, ultrasonically treating (power 250W, frequency 50kHz) for 30min, cooling, weighing again, complementing the weight loss by 50% methanol in mass fraction, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution;
(2) preparation of control solutions: taking appropriate amount of ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances, precisely weighing, and adding methanol to obtain solutions containing 40 μ g of each 1 mL;
(3) qualitative and quantitative testing of the chromatogram: octadecylsilane chemically bonded silica is used as a filling agent (hydrophilic column); using acetonitrile (A) -phosphoric acid (containing 0.04% triethylamine) (B) with the mass fraction of 0.1% as a mobile phase, and performing gradient elution; the detection wavelength was 210 nm. The number of theoretical plates is not less than 3000 calculated according to ephedrine hydrochloride peak, gradient elution is shown in table 2, and the test method comprises: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring the contents of ephedrine hydrochloride and pseudoephedrine hydrochloride to obtain 15 batches of ephedrine hydrochloride and pseudoephedrine total contents shown in Table 6.
TABLE 2
Example 4:
determination of amygdalin content
(1) Preparation of a test solution: respectively taking obtained substance references in MHT-1-MHT-15, grinding, taking 0.25g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 50% methanol in mass fraction, weighing, ultrasonically treating (power 250W, frequency 50kHz) for 30min, cooling, weighing again, complementing the weight loss by 50% methanol in mass fraction, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution;
(2) preparation of control solutions: taking appropriate amount of amygdalin reference substance, precisely weighing, and adding methanol to obtain 0.35mg solution per 1 mL;
(3) qualitative and quantitative testing of the chromatogram: octadecylsilane chemically bonded silica is used as a filling agent; taking methanol (A) -water (B) as a mobile phase, and performing gradient elution; the detection wavelength is 210nm, the number of theoretical plates is not less than 7000 calculated according to amygdalin peak, gradient elution is shown in Table 3, respectively and precisely absorbing 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring the content of the reference amygdalin in the obtained 15 batches of the ephedrine soup material, wherein the content of the reference amygdalin is shown in Table 6.
TABLE 3
Example 5:
content determination of liquiritin/glycyrrhizic acid
(1) Preparation of a test solution: respectively taking obtained substance references in MHT-1-MHT-15, grinding, taking 0.25g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 50% methanol in mass fraction, weighing, ultrasonically treating (power 250W, frequency 50kHz) for 30min, cooling, weighing again, complementing the weight loss by 50% methanol in mass fraction, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution;
(2) preparation of control solutions: taking appropriate amount of liquiritin and ammonium glycyrrhizinate as reference substances, precisely weighing, and adding 70% ethanol by mass fraction to obtain solution containing 0.04mg per 1 mL;
(3) qualitative and quantitative testing of the chromatogram: octadecylsilane chemically bonded silica is used as a filling agent; taking acetonitrile (A) -phosphoric acid (B) with the mass fraction of 0.05 percent as a mobile phase, and performing gradient elution; the detection wavelength is 237nm, the number of theoretical plates is not less than 5000 calculated according to the liquiritin peak, the gradient elution is shown in the table 4, and the test method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring the content of glycyrrhizin/glycyrrhizic acid based on the herba Ephedrae decoction to obtain 15 batches of herba Ephedrae decoction with content of glycyrrhizin/glycyrrhizic acid based on the material shown in Table 6.
TABLE 4
Example 6:
determination of cinnamic acid content:
(1) preparation of a test solution: respectively taking obtained substance references in MHT-1-MHT-15, grinding, taking 0.25g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 50% ethanol in mass fraction, weighing, ultrasonically treating (with power of 250W and frequency of 50kHz) for 30min, cooling, weighing again, complementing the lost weight with 50% ethanol in mass fraction, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution;
(2) preparation of control solutions: taking a proper amount of a cinnamic acid reference substance, precisely weighing, and respectively adding 70% ethanol by mass fraction to prepare solutions containing 10 micrograms per 1 mL;
(3) qualitative and quantitative testing of the chromatogram: octadecylsilane chemically bonded silica is used as a filling agent; taking acetonitrile (A) -phosphoric acid (B) with the mass fraction of 0.1% as a mobile phase, and performing gradient elution; the detection wavelength is 237nm, the number of theoretical plates is not less than 5000 calculated according to the liquiritin peak, the gradient elution is shown in the table 5, and the test method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring the content of glycyrrhizin/glycyrrhizic acid based on the herba Ephedrae decoction to obtain 15 batches of herba Ephedrae decoction with content of glycyrrhizin/glycyrrhizic acid based on the material shown in Table 6.
TABLE 5
Example 6:
methodology investigation:
the peak area (Y) of the active ingredient is used for carrying out linear regression on the mass concentration (X), wherein the linear regression equations of ephedrine, pseudoephedrine, liquiritin, glycyrrhizic acid and amygdalin and cinnamic acid are respectively that Y is 2120097.3866X-106916.8296(r is 1.0000), 9996Y is 2064346.1536X-46226.3659(r is 0.9995), Y is 1963296X-9753(r is 0.9999), Y is 513051X-27128(r is 0.9992), Y is 953691X-5833.3(r is 1.0000) and Y is 8263858.6031X-44294.4592(r is 1.0000).
By the examination of sample recovery rate, the sample recovery rates of ephedrine, pseudoephedrine hydrochloride, liquiritin, glycyrrhizic acid, amygdalin and cinnamic acid are respectively 93.18%, 94.41%, 95.53%, 103.6%, 95.10% and 98.80%. And the RSD is less than 5 percent, which shows that the establishment of the method for measuring the content of each index component of the ephedra decoction is reasonable, and the accuracy, the repeatability and the stability are good.
Example 7:
the results of qualitative and quantitative tests on the contents of the effective components of the reference substances in MHT-1, MHT-2, MHT-3, MHT-4, MHT-5, MHT-6, MHT-8, MHT-9, MHT-10, MHT-11, MHT-12, MHT-13, MHT-14 and MHT-15 by using a chromatograph are shown in Table 6;
the paste yield of the standard active ingredients of the ephedra decoction material is as follows: the cream yield was calculated according to the following formula:
the extract yield is 100% of the material-based dry powder amount/the amount of the fed tablet, and the results are shown in table 6.
TABLE 6 determination of content of effective components and extraction rate of herba Ephedrae decoction
Example 8:
the transmission of each index component in the material standard is as follows:
the transfer rate was performed according to the following formula:
the transfer rate is the content of the effective components in the sample/the content of the effective components in the decoction pieces is 100%.
The transfer rate of the decoction pieces to the corresponding material objects can reflect whether the condition of damaging the index components exists in the concentration and drying mode, and the transfer rate of the decoction pieces to the corresponding material objects can reflect the whole transfer condition of the index components, so that the transfer rates of the decoction pieces to the corresponding material objects and the decoction pieces to the corresponding material objects are respectively calculated. The results are shown in tables 7 and 8.
TABLE 7 transfer rate of each index component decoction to material standard
TABLE 8 transfer rate of each index ingredient decoction pieces to material basis
The results showed that the average transfer rates of ephedrine hydrochloride, pseudoephedrine hydrochloride, liquiritin, glycyrrhizic acid, amygdalin and cinnamic acid in the decoction pieces were 43.25%, 63.13%, 55.39%, 39.0% and 34.63%, respectively. The average transfer rates of ephedrine hydrochloride, pseudoephedrine hydrochloride, liquiritin, glycyrrhizic acid, amygdalin and cinnamic acid in the decoction of the ephedra decoction to the substance standard are respectively 90.05%, 96.07%, 93.55%, 93.87% and 86.46%.
Example 9:
high performance liquid chromatography feature profile analysis
(1) Preparation of a test solution: respectively taking obtained substance references in MHT-1-MHT-15, grinding, taking 0.25g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 50% methanol in mass fraction, weighing, ultrasonically treating (power 250W, frequency 50kHz) for 30min, cooling, weighing again, complementing the weight loss by 50% methanol in mass fraction, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution;
(2) preparation of reference solutions: taking appropriate amount of semen Armeniacae amarum as reference, precisely weighing, and adding 70% ethanol respectively to obtain solutions containing 0.04mg per 1 mL;
(3) chromatographic qualitative and quantitative analysis: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile (A) -0.1% phosphoric acid (B) was used as a mobile phase, and gradient elution was performed under the conditions shown in Table 9, and the measurement method was: precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, and measuring.
TABLE 9
(4) Researching the characteristic spectrum methodology of the ephedra decoction:
through methodology investigation on precision, repeatability, stability and the like, RSD of relative retention time of each peak to a reference peak is calculated respectively, results are all less than 2%, and RSD of relative peak area of each peak to the reference peak is calculated, and results are all less than 5%. The instrument has good precision, the method has good repeatability, and the sample has good stability within 24 hours.
(5) Establishing a characteristic spectrum of the ephedra decoction:
taking 15 batches of freeze-dried powder, carrying out sample injection determination analysis according to chromatographic experimental conditions, recording chromatograms, counting retention time and relative retention time of characteristic peaks (see table 10 and table 11), and stacking the chromatograms by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012) (see fig. 1). S1-S15 in figure 1 represent 15 batches of freeze-dried powder, and the contrast characteristic spectrum of the generated effective components is shown in figure 2.
9 characteristic peaks should be presented in the characteristic map of the test article, the peak corresponding to the reference peak is the S peak, the relative retention time of each characteristic peak and the S peak is calculated, and should be within +/-10% of the specified value, the specified value of the retention time is: 0.52 (peak 1), 0.55 (peak 2), 0.86 (peak 3), 0.89 (peak 4), 0.98 (peak 5), 1.00[ peak 6(S) ], 1.35 (peak 7), 1.67 (peak 8), 1.75[ peak 9(S) ].
TABLE 10 Retention time of characteristic map of ephedra decoction
TABLE 11 relative retention time of characteristic spectrum of ephedra decoction
| Peak 1 | |
Peak 3 | |
|
|
Peak 7 | |
|
|
| S1 | 0.5224 | 0.5498 | 0.8635 | 0.8819 | 0.9790 | 1.0000 | 1.3723 | 1.6979 | 1.7831 |
| S2 | 0.5243 | 0.5517 | 0.8657 | 0.8823 | 0.9790 | 1.0000 | 1.3630 | 1.6862 | 1.7704 |
| S3 | 0.5241 | 0.5516 | 0.8659 | 0.8823 | 0.9790 | 1.0000 | 1.3651 | 1.6891 | 1.7734 |
| S4 | 0.5234 | 0.5508 | 0.8657 | 0.8816 | 0.9791 | 1.0000 | 1.3690 | 1.6944 | 1.7794 |
| S5 | 0.5238 | 0.5512 | 0.8503 | 0.8824 | 0.9790 | 1.0000 | 1.3635 | 1.6873 | 1.7719 |
| S6 | 0.5256 | 0.5520 | 0.8659 | 0.8816 | 0.9792 | 1.0000 | 1.3705 | 1.6964 | 1.7816 |
| S7 | 0.5252 | 0.5520 | 0.8658 | 0.8818 | 0.9792 | 1.0000 | 1.3683 | 1.6939 | 1.7791 |
| S8 | 0.5255 | 0.5519 | 0.8658 | 0.8818 | 0.9791 | 1.0000 | 1.3681 | 1.6936 | 1.7786 |
| S9 | 0.5245 | 0.5513 | 0.8654 | 0.8818 | 0.9787 | 1.0000 | 1.3659 | 1.6904 | 1.7748 |
| S10 | 0.5246 | 0.5517 | 0.8661 | 0.8837 | 0.9791 | 1.0000 | 1.3551 | 1.6753 | 1.7585 |
| S11 | 0.5329 | 0.5625 | 0.8700 | 0.8927 | 0.9827 | 1.0000 | 1.3148 | 1.6211 | 1.7000 |
| S12 | 0.5321 | 0.5618 | 0.8699 | 0.8924 | 0.9825 | 1.0000 | 1.3166 | 1.6236 | 1.7027 |
| S13 | 0.5324 | 0.5615 | 0.8697 | 0.8924 | 0.9826 | 1.0000 | 1.3144 | 1.6205 | 1.6995 |
| S14 | 0.5297 | 0.5590 | 0.8690 | 0.8903 | 0.9818 | 1.0000 | 1.3239 | 1.6333 | 1.7130 |
| S15 | 0.5302 | 0.5595 | 0.8694 | 0.8916 | 0.9821 | 1.0000 | 1.3185 | 1.6261 | 1.7052 |
Example 10:
the comparison of the control with the full-scale characteristic map is shown in figure 3.
Example 11:
the comparison of the characteristic maps of the herbs and the whole formula is shown in FIG. 4.
In the description herein, references to the description of "one embodiment," "an example," "a specific 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.
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.
Claims (5)
1. A method for measuring the content of active ingredients of a ephedra decoction substance standard is characterized by comprising the following steps:
step one, preparing a material standard: mixing herba Ephedrae and water, decocting with strong fire for 70min, and removing froth; adding ramulus Cinnamomi, semen Armeniacae amarum and parched Glycyrrhrizae radix, decocting with slow fire for 80min, filtering with 150 mesh filter cloth, concentrating the filtrate at 40-50 deg.C under-0.08-0.1 MPa, cooling, freezing at-20 deg.C for 12 hr, freeze drying at-45 deg.C for 24 hr, taking out, pulverizing, sieving with 80 mesh sieve, and mixing to obtain basic material;
step two, preparation of a test solution: weighing the substance obtained in the step one, grinding, dissolving with 50% methanol or 50% ethanol solution, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss with 50% methanol or 50% ethanol solution, shaking, filtering, and collecting the filtrate to obtain the sample solution;
step three, preparing a reference substance solution: preparing a reference solution by using the effective components as a reference and a solvent;
step four, qualitative and quantitative testing of the chromatogram: the chromatographic experimental conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; taking a solvent A-a solvent B as a mobile phase, and performing gradient elution; the detection wavelength is 210 nm; respectively sucking 10 μ L of reference solution and sample solution, and injecting into liquid chromatograph; the calculation method comprises the following steps: and (3) putting the reference sample solution into a chromatograph, taking the chromatographic peak area as the ordinate and the sample amount as the abscissa to obtain a standard curve of the reference sample solution, putting the sample into the chromatograph, and bringing the peak area of the component into the standard curve to obtain the content of the effective component of the ephedra decoction substance standard.
2. The method for measuring the content of the standard effective components of the ephedra decoction substance according to claim 1, wherein in the step one, the dosage ratio of the ephedra, the water, the cassia twig, the blanched bitter almond and the fried liquorice is 41.25 g: 1800 mL: 27.5 g: 23.64 g: 13.75 g; the effective components of the three steps comprise ephedrine, pseudoephedrine, amygdalin, liquiritin, glycyrrhizic acid and cinnamic acid.
3. The method for measuring the content of active ingredients in the ephedra decoction substance base according to claim 2, wherein when the active ingredients are ephedrine or pseudoephedrine, ephedrine hydrochloride or pseudoephedrine hydrochloride and methanol are prepared into a control solution with the concentration of 40 μ g/mL; when the effective component is amygdalin, the amygdalin and 70% ethanol solution are prepared into a control solution with the concentration of 0.35 mg/mL; when the effective component is liquiritin or glycyrrhizic acid, the liquiritin or glycyrrhizic acid is mixed with 70% ethanol solution to prepare a control solution with the concentration of 40 mg/mL; when the effective component is cinnamic acid, the cinnamic acid and 70% ethanol solution are prepared into a control solution with the concentration of 10 mug/mL.
4. The method for measuring the content of the effective ingredients of the ephedra decoction substance base according to claim 1, wherein the solvent A-solvent B mobile phase in the step four is as follows: when the effective component is ephedrine or pseudoephedrine, the solvent A is acetonitrile, the solvent B is phosphoric acid solution with mass fraction of 0.1%, and the phosphoric acid solution contains triethylamine with mass fraction of 0.04%; when the active ingredient is amygdalin, the solvent A is methanol and the solvent B is water; when the active ingredient is liquiritin or glycyrrhizic acid, the solvent A is acetonitrile, and the solvent B is a phosphoric acid solution with the mass fraction of 0.05%; when the active ingredient is amygdalin, the solvent A is acetonitrile, and the solvent B is phosphoric acid solution with the mass fraction of 0.1%.
5. The content of the standard effective components of the ephedrine decoction substance determined by the method as claimed in claim 1, is characterized in that the total content of ephedrine and pseudoephedrine is within the range of 13.14mg/g-24.40mg/g, the content of liquiritin is within the range of 3.90mg/g-7.23mg/g, the content of glycyrrhizic acid is within the range of 5.30mg/-9.84mg/g, the content of amygdalin is within the range of 19.05mg/g-17.071mg/g, and the content of cinnamic acid is within the range of 0.79mg/g-1.47 mg/g.
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