CN115266961A - Method for constructing characteristic spectrum of perilla stem medicinal preparation - Google Patents
Method for constructing characteristic spectrum of perilla stem medicinal preparation Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly provides a construction method of a characteristic spectrum of a perilla stem medicinal preparation, which comprises the following steps of (1) preparing a test solution; (2) Taking a test sample solution, detecting by adopting a high performance liquid chromatography, adopting a Waters CORTEC UPLC T3 chromatographic column, wherein a mobile phase comprises an acid-containing aqueous solution and acetonitrile, and a gradient elution program comprises the following steps: 0 → 3min → 10min → 16min → 26min → 35min → 40min → 45-48min, the volume percentage of acetonitrile in the mobile phase is: 2% → 5% → 12% → 14% → 16% → 16% → 18% → 18%. The invention can realize effective separation of 11 common characteristic peaks, and the obtained characteristic spectrum has stable baseline, good peak shape of the characteristic peaks and short detection time, thereby providing scientific basis for comprehensively establishing the quality control standard of the perilla stem formula granules. But also can accurately position the peak positions of caffeic acid, rosmarinic acid, viscapine-II and apigenin-7-O-diglucuronide, and fully reflect the integrity and the characteristics of perilla stem medicinal preparations (such as preparations like formula granules).
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a characteristic spectrum of a perilla stem medicinal preparation.
Background
The caulis Perillae is dry stem of Perilla frutescens (L.) Brit of Perilla of Labiatae, has warm stem, pungent taste and aromatic smell, and has effects of regulating qi-flowing, relieving epigastric distention, relieving pain, preventing miscarriage, etc. The overground part of the perilla mainly contains various chemical components such as volatile oil, flavone and glycosides thereof, terpenes, lipoid and the like. Wherein, the flavone is a component generally contained in plants, and the perilla flavone has pharmacological actions of resisting inflammation, resisting oxidation, reducing blood fat, resisting thrombus, resisting bacteria and the like. Research shows that the flavonoid compounds in the stem of the perilla are similar to the components in the leaves, and all contain various flavonoid compounds such as flavonoid, flavanone, flavonol, flavanonol and the like. Perilla stem is a variety collected in Chinese pharmacopoeia (2020 edition one), but only the character items and content determination are specified in the quality standard.
Common preparation types of the perilla stem preparation include powder, formula granules and the like, for example, the formula granules are prepared by extracting, concentrating, drying, preparing and the like perilla stem decoction pieces, and the material basis of the formula granules is greatly different from that of medicinal materials. At present, no report is found on the study of the characteristic map of the perilla stem formula granules. Literature research reports research on high performance liquid chromatography fingerprint of perilla stem, and authors' Moscong, korea and so on. The document mainly focuses on the research on the medicinal materials of perilla stem. The characteristic peak-off time of the established atlas is relatively dense after the atlas is compared from multiple batches intuitively. And most chromatographic peaks have poor resolution and are not separated effectively. In addition, because the traditional Chinese medicine formula particles do not have the characteristics of medicinal material property identification, the method is not suitable for quality detection of preparations prepared from perilla stem medicines such as perilla stem formula particles, and the like, and has the defects of few characteristic peaks, poor separation effect and long detection and analysis time.
Disclosure of Invention
Therefore, the invention aims to provide a construction method of a perilla stem medicinal preparation characteristic map, which is used for establishing the characteristic map of the variety according to the characteristics of the perilla stem medicinal preparation, realizing the effective separation of each characteristic peak, improving the number of the characteristic peaks, shortening the detection and analysis time, improving the separation effect and providing scientific basis for comprehensively establishing the quality control standard of perilla stem formula granules.
Therefore, the invention provides a method for constructing a characteristic map of a perilla stem medicinal preparation, which comprises the following steps of,
(1) Preparing a test solution;
(2) Detecting the sample solution by high performance liquid chromatography, adopting a Waters CORTEC UPLC T3 chromatographic column, wherein the mobile phase comprises an acid-containing aqueous solution and acetonitrile, and the gradient elution procedure comprises the following steps: 0 → 3min → 10min → 16min → 26min → 35min → 40min → 45-48min, the volume percentage of acetonitrile in the mobile phase is: 2% → 5% → 12% → 14% → 16% → 16% → 18% → 18%.
Further, the step (2) also satisfies at least one of the following 1) to 5):
1) The detection wavelength is 325-335nm, the flow rate is 0.24-0.26mL/min, the column temperature is 20-30 ℃, and the sample injection volume is 1-10 mul;
2) The mobile phase is a mixed solution of acetonitrile and an acid-containing aqueous solution;
3) The gradient elution procedure further comprises: 45-48min → 50min, the volume percentage of acetonitrile in the mobile phase is: 18% → 20%;
4) The acid-containing aqueous solution is an aqueous solution containing 0.1-0.3% of phosphoric acid or 0.1-0.3% of formic acid by volume percentage;
5) The Waters CORTEC UPLC T3 chromatographic column is a Waters CORTEC UPLC T3 (2.1 mm. Times.150mm, 1.6 μm) chromatographic column.
Further, the step (1) comprises weighing the sample, adding a solvent for extraction to obtain an extracting solution, carrying out solid-liquid separation, and taking liquid, namely the sample solution.
Further, the step (1) also satisfies any one or more of the following A-E:
A. the ratio of the mass of the sample to the volume of the solvent is 0.25-3.0:5-20 parts of; the relation between the mass and the volume is g/mL;
B. the extraction mode is reflux extraction or ultrasonic extraction;
C. the extraction time is more than or equal to 10min, preferably 15-40min;
D. the solid-liquid separation is selected from centrifugation or filter membrane filtration;
E. the solvent is selected from 40-80% methanol aqueous solution or 40-80% acetone aqueous solution.
Further, the step (1) comprises weighing 0.25-1.0g of perilla stem formula particles, placing the perilla stem formula particles in a conical flask with a plug, precisely adding 40-80% by volume of methanol aqueous solution or 5-20ml of 40-80% by volume of acetone aqueous solution, sealing the plug, weighing the mixture, carrying out ultrasonic treatment for 15-40min, weighing the mixture again, supplementing the reduced weight by 40-80% by volume of methanol aqueous solution or 40-80% by volume of acetone aqueous solution, shaking up, and filtering to obtain a sample solution.
Further, the construction method also comprises a step of preparing a reference substance solution by adopting at least one of caffeic acid, rosmarinic acid, viscapine-II and apigenin-7-O-diglucuronide as a solvent, and a step of detecting the reference substance solution by high performance liquid chromatography in any one of the construction methods to obtain a reference substance map.
Further, each 1mL of the reference solution contains at least one of 1-100 μ g caffeic acid, 1-100 μ g rosmarinic acid, 1-100 μ g visanin-II and 1-100 μ g apigenin-7-O-diglucuronide; and/or, the solvent used in the preparation of the control solution is selected from methanol or methanol water solution.
Further, the construction method also comprises the steps of preparing a reference medicinal material solution of the reference medicinal material by using an extract obtained by adding water into the perilla stem reference medicinal material for extraction according to the preparation method of the test solution in any one of the construction methods, and detecting the reference medicinal material solution of the reference medicinal material by using the high performance liquid chromatography in any one of the construction methods to obtain a reference map of the reference medicinal material.
Preferably, the perilla stem reference drug is extracted by water, filtered, dried and processed by liquid drying, and then the reference drug solution is prepared according to the preparation method of the test solution in the construction method of any one of claims 1 to 4.
Further, taking 1.0-3.0g of perilla stem as a reference medicinal material, placing the reference medicinal material into a conical flask with a plug, adding 20-100ml of water, heating and refluxing for 10-60 minutes, filtering, evaporating filtrate to dryness, adding 40-80% by volume of methanol aqueous solution or 5-20ml of 40-80% by volume of acetone aqueous solution into residue, carrying out ultrasonic treatment for 15-40min, cooling, shaking up, filtering, and taking the subsequent filtrate as a reference medicinal material solution.
Further, the characteristic map of the perilla stem medicinal preparation has 11 common characteristic peaks, the peak corresponding to the reference substance peak of the caffeic acid reference substance is an S peak, and the relative retention time of each characteristic peak and the S peak is within the range of +/-10% of a specified value; the specified values are: 0.32 (peak 1), 0.86 (peak 2), 1.00 (peak 3), 1.29 (peak 4), 1.40 (peak 5), 1.48 (peak 6), 1.76 (peak 7), 1.81 (peak 8), 2.06 (peak 9), 2.85 (peak 10), 3.22 (peak 11).
In the invention, the medicinal preparation of the perilla stem can be common preparations prepared by extracting the perilla stem with water, such as perilla stem formula granules or perilla stem standard decoction freeze-dried powder and the like.
In some embodiments, the method further comprises the construction of a comparison characteristic spectrum of the perilla stem medicinal preparation, and the comparison characteristic spectrum of the perilla stem medicinal preparation is generated by utilizing a traditional Chinese medicine chromatogram characteristic spectrum similarity evaluation system for characteristic spectrums obtained by detecting a plurality of batches of perilla stem medicinal preparation samples. At least 3 batches of perilla stem granules are used, for example 3 batches, 7 batches, 15 batches, 17 batches of perilla stem granules.
In some embodiments, the step of marking the common characteristic peak is further included after the traditional Chinese medicine chromatogram characteristic spectrum similarity evaluation software is used for generating the comparison characteristic spectrum of the perilla stem medicinal preparation.
The invention also provides application of the construction method of the characteristic spectrum of the perilla stem medicinal preparation in quality detection of the perilla stem medicinal preparation.
The invention also provides a quality detection method of the perilla stem medicinal preparation, which comprises the step of comparing the characteristic spectrum of a perilla stem product to be detected with the contrast characteristic spectrum of the perilla stem medicinal preparation; the characteristic spectrum of the perilla stem product to be detected is obtained by constructing the perilla stem product to be detected according to any one of the construction methods, and the perilla stem medicinal preparation contrast characteristic spectrum is selected from any one of the following (1) to (3):
(1) It has 11 common characteristic peaks, the peak corresponding to the caffeic acid reference substance peak is S peak, and the relative retention time of each characteristic peak and S peak is within the range of + -10% of the specified value; the specified values are: 0.32 (peak 1), 0.86 (peak 2), 1.00 (peak 3), 1.29 (peak 4), 1.40 (peak 5), 1.48 (peak 6), 1.76 (peak 7), 1.81 (peak 8), 2.06 (peak 9), 2.85 (peak 10), 3.22 (peak 11);
(2) Using a characteristic map of the perilla stem medicinal preparation obtained by a single batch or multiple batches of perilla stem medicinal preparations according to any one of the construction methods;
(3) And (3) using the characteristic maps obtained by the multi-batch perilla stem medicinal preparation according to any one of the construction methods, and comparing the characteristic maps by using a mean value method or a median method.
In the present invention, the "peak corresponding to the caffeic acid reference peak" is a characteristic peak having a degree of coincidence with the caffeic acid reference peak of not less than 50%.
% acetone represents the volume percent of acetone in the aqueous acetone solution and% methanol represents the volume percent of methanol in the aqueous methanol solution.
The technical scheme of the invention has the following advantages:
1. the method for constructing the characteristic spectrum of the perilla stem medicinal preparation adopts a Waters CORTEC UPLC T3 chromatographic column, a mobile phase comprises an acid-containing aqueous solution and acetonitrile, and a gradient elution program comprising the following programs is adopted through continuously optimizing the gradient elution program: 0 → 3min → 10min → 16min → 26min → 35min → 40min → 45-48min, the volume percentage of acetonitrile in the mobile phase is: 2% → 5% → 12% → 14% → 16% → 16% → 18% → 18%; finally, 11 common characteristic peaks are obtained, effective separation of the 11 common characteristic peaks is realized, the obtained characteristic spectrum baseline is stable, the characteristic peak shape is good, the detection time is short, and a scientific basis is provided for comprehensively establishing a quality control standard of perilla stem formula particles. But also can accurately position the peak positions of caffeic acid, rosmarinic acid, viscapine-II and apigenin-7-O-diglucuronide, and fully reflect the integrity and the characteristics of perilla stem medicinal preparations (such as preparations like formula granules).
2. According to the construction method of the characteristic spectrum of the perilla stem medicinal preparation, the optimal extraction process and chromatographic conditions are determined by observing the optimized chromatographic conditions, the extraction solvent, the dosage of the extraction solvent and other extraction conditions, so that the peak area is higher, the separation effect is better, and the quality monitoring of perilla stem formula particles can be more comprehensively carried out.
3. According to the quality detection method of the perilla stem medicinal preparation, the quality of perilla stem formula particles can be comprehensively, clearly and effectively detected by comparing the characteristic spectrum of a perilla stem formula particle product to be detected with the reference characteristic spectrum of the perilla stem formula particles.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a chromatogram of method one of the mobile phase gradient optimization experiments of Experimental example 1;
FIG. 2 is a chromatogram of method two in a mobile phase gradient optimization experiment of Experimental example 1;
FIG. 3 is a chromatogram of method three in a mobile phase gradient optimization experiment of Experimental example 1;
FIG. 4 is a chromatogram of method four in a mobile phase gradient optimization experiment of Experimental example 1;
FIG. 5 is a chromatogram of acetonitrile-water in a mobile phase investigation experiment in Experimental example 1;
FIG. 6 is a chromatogram of acetonitrile-0.2% phosphoric acid water in a mobile phase investigation experiment of Experimental example 1;
FIG. 7 is a chromatogram of acetonitrile-0.2% formic acid water in a mobile phase investigation experiment of Experimental example 1;
FIG. 8 is a chromatogram of acetonitrile-0.1% formic acid water in an investigation experiment of mobile phase acid concentration in Experimental example 1;
FIG. 9 is a chromatogram of acetonitrile-0.2% formic acid water in an investigation experiment of mobile phase acid concentration in Experimental example 1;
FIG. 10 is a chromatogram of acetonitrile-0.3% formic acid water in an experiment for investigating the concentration of mobile phase acid in Experimental example 1;
FIG. 11 is a chromatogram at 20 ℃ in a column temperature investigation experiment;
FIG. 12 is a chromatogram at 25 ℃ in a column temperature investigation experiment;
FIG. 13 is a chromatogram at 30 ℃ in a column temperature investigation experiment;
FIG. 14 is a chromatogram at a flow rate of 0.24ml/min in a flow rate investigation experiment;
FIG. 15 is a chromatogram at a flow rate of 0.25ml/min in a flow rate investigation experiment;
FIG. 16 is a chromatogram at a flow rate of 0.26ml/min in a flow rate investigation experiment;
FIG. 17 is a chromatogram of a Waters CORTEC UPLC T3 column in a study experiment of the column;
FIG. 18 is a chromatogram from an Agilent InfinityLab Poroshell 120SB-Aq column in a column investigation experiment;
FIG. 19 is Thermo Hypersil GOLD in a chromatographic column investigation experimentTMChromatogram of AQ chromatographic column;
FIG. 20 is a comparison of the spectra of the control and the characteristics of the perilla stem granules;
FIG. 21 is a chromatogram of perilla stem as a control drug;
FIG. 22 is a characteristic and control profile of 15 batches of perilla stem granules;
FIG. 23 is a control profile of perilla stem granules;
FIG. 24 is a characteristic spectrum of perilla stem granules in example 1;
FIG. 25 is a reference map of a control in example 1;
FIG. 26 is a characteristic spectrum of lyophilized powder of standard decoction of perilla stem in example 2;
Detailed Description
The following examples are provided to better understand the present invention, not to limit the best mode, and not to limit the content and protection scope of the present invention, and any product that is the same or similar to the present invention and is obtained by combining the present invention with other features of the prior art and the present invention falls within the protection scope of the present invention. The examples do not indicate specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are conventional reagent products which are commercially available, and manufacturers are not indicated.
Experimental example 1 examination of construction method
1. Instrument, reagent and reagent
High performance liquid chromatograph 1: waters Arc UPLC, PDA detector. High performance liquid chromatograph 2: thermo Ultimate3000, comprising Pump: LPG-3400SD; colum component: TCC-3000RS; autosumpler: WPS-3000SL; photometer: DAD-3000. Other instruments: KQ-700DE digital control ultrasonic cleaner (available from ultrasonic instruments, kunshan city); one-ten-thousandth balance (BS 224s, sartorius); one-ten-thousandth balance (ME 204, METTLER TOLEDO); one in ten thousand balance (XR 205SM-DR, precisa); a rotary evaporator (Shanghai Iran instruments Co., ltd., OSB-2200); and (3) chromatographic column: waters CORTEC UPLC T3 (2.1 mm. Times.150mm, 1.6 μm) chromatography column;
rosmarinic acid reference (China institute for testing food and drug, batch No. 111871-202007); caulis Perillae as reference material (China institute for food and drug assay, lot # 121172-201403); caffeic acid reference (China institute for food and drug assay, batch No. 110885-201703). Acetonitrile is chromatographic pure, and water is ultrapure water; other reagents were analytically pure.
Reagent testing: the perilla stem granules can be prepared by the conventional method in the field, for example, the perilla stem granules are prepared according to the following steps: taking 10000g of perilla stem decoction pieces, adding water for decoction, filtering, concentrating the filtrate into clear paste, drying, uniformly mixing and granulating to obtain the traditional Chinese medicine composition. Perilla stem formula granules with the batch numbers of 1904001W, 1907002S, 1912001S, 2004001W, 2005001S, 2007001S, 2008001W, 2009002S, 2011001W, 2102001S, 2104001W, 2106001S, 2108002W, 2109001W and 2111001S are prepared by adopting different batches of perilla stem decoction pieces.
The freeze-dried powder of the standard decoction of perilla stem can be prepared by adopting a conventional method in the field, for example, the invention is prepared according to the following steps: taking a perilla stem medicinal material, processing according to relevant regulations under the item of perilla stem medicinal materials of 2020 edition of Chinese pharmacopoeia (processing technology, taking a proper amount of perilla stem medicinal materials, removing impurities, slightly soaking, moistening thoroughly, cutting into thick slices (2-4 mm), drying to prepare perilla stem decoction pieces, taking 100g of perilla stem decoction pieces, placing in a casserole, extracting twice, adding 1000ml of water, soaking for 30 minutes, boiling with strong fire (5 grades), decocting with slow fire (3 grades), filtering while hot with slow fire (3 grades), adding 800ml of water into dregs, boiling with strong fire (5 grades), decocting with slow fire (3 grades) for 25 minutes, filtering while hot with 200 meshes of gauze (nylon), combining filtrates, rapidly cooling the filtrates to room temperature, concentrating the filtrates under reduced pressure (the temperature of 50 ℃, the vacuum degree of 20-50 mbar) to a concentrated extract with the relative density of 1.08 g/ml-1.12 g/ml, placing the concentrated extract in a freeze dryer, freeze-drying at the temperature of (-82 ℃, drying under the pressure of 0MPa, weighing, grinding to powder, and filling in a bottle to obtain freeze-dried perilla stem powder.
2. Preparation of test solution
Precisely weighing perilla stem formula particles, precisely weighing about 0.5g, placing the particles in a conical flask with a plug, precisely adding 25ml of 60% acetone, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, weighing again, supplementing the weight loss by using 60% acetone, shaking up, and filtering to obtain a test solution.
3. Optimization of chromatographic conditions
(1) Mobile phase gradient optimization experiment
The test sample solution prepared as described in item 2 of this experimental example was subjected to detection by high performance liquid chromatography using a Waters CORTEC UPLC T3 (2.1 mm. Times.150mm, 1.6 μm) column, and eluted under the following gradient conditions, respectively, at a flow rate of 0.25 ml/min; the column temperature was 25 ℃, the detection wavelength was 330nm, and the sample volume was 1. Mu.l.
TABLE 1 gradient conditions under methods one to three
TABLE 2 gradient conditions under method four
TABLE 3 suitability parameters for each gradient chromatographic peak system
As shown in fig. 1-4 and table 3, the characteristic peak information of method four is large, the separation effect is good, and the peak shape is good, although method one also has 11 peaks, 1 of which has a low degree of separation, and the peak shape is poor.
(2) Examination of Mobile phase
A test solution of perilla stem granules was prepared from the perilla stem granules according to the method described in item 2 of this experimental example. The chromatographic conditions of the fourth method under item 3 (1) of the experimental example are respectively adopted to detect the sample solution of the perilla stem formula particle, the difference is only that the mobile phase B is different, the gradient procedures are the same, the influence of different mobile phase systems on the characteristic spectrum separation effect of the perilla stem formula particle is compared, and the mobile phase systems are arranged as follows: (1) a mobile phase A: acetonitrile, mobile phase B: water, (2) mobile phase a: acetonitrile, mobile phase B:0.2% (volume percent) formic acid water, (3) mobile phase a: acetonitrile, mobile phase B:0.2% (volume percent) phosphoric acid water.
The results are shown in the following table and figures 5-7, and the results show that the separation effect of each chromatographic peak of the mobile phase system of acetonitrile-0.2% phosphoric acid water and acetonitrile-0.2% formic acid water is better, and the mobile phase of acetonitrile-0.2% formic acid water is the best choice in consideration of the protection of the chromatographic column.
TABLE 4 System Adaptation parameters
(3) Investigation experiment of mobile phase acid concentration
A test solution of perilla stem granules was prepared from the perilla stem granules according to the method described in item 2 of this experimental example. The chromatographic conditions of the fourth method under the item 3 (1) of the experimental example are respectively adopted to detect the test solution of the perilla stem formula particles, the difference is only that the concentration of a mobile phase B is different, the gradient program is the same, the influence of the concentration of different mobile phases B on the characteristic spectrum separation effect of the perilla stem formula particles is compared, and a mobile phase system is set as follows: (1) a mobile phase A: acetonitrile, mobile phase B:0.1% formic acid water, (2) mobile phase a: acetonitrile, mobile phase B:0.2% formic acid water, (3) mobile phase a: acetonitrile, mobile phase B:0.3% formic acid water.
The results are shown in the following table and fig. 8-10, and the results show that different acetonitrile-formic acid water (volume percentage is 0.1-0.3%) mobile phase systems have similar separation effects on various spectral peaks, and the adaptability parameters of various spectral peak systems are also similar.
TABLE 5 investigation of different mobile phase acid concentrations
(4) Investigation experiment of column temperature
A test solution of perilla stem granules was prepared from the perilla stem granules according to the method described in item 2 of this experimental example. The chromatographic conditions of the fourth method under the item 3 (1) of the experimental example are respectively adopted to detect the test solution of the perilla stem formula particle, the difference is only that the column temperature is different, the influence of different column temperatures on the durability of the characteristic spectrum of the perilla stem formula particle is compared, and the column temperature is set as follows: 20 deg.C, 25 deg.C, 30 deg.C. The results show that the separation degree effect is better at 20-30 ℃, wherein the separation degree effect is optimal at 20-25 ℃.
TABLE 6 investigation results of different column temperatures
(5) Investigation experiment of flow velocity
A test solution of perilla stem granules was prepared from the perilla stem granules according to the method described in item 2 of this experimental example. The chromatographic conditions of the fourth method under the item 3 (1) of the experimental example are respectively adopted to detect the test solution of the perilla stem formula particle, the difference is only that the flow rates are different, the influence of different flow rates on the durability of the characteristic spectrum of the perilla stem formula particle is compared, and the flow rates are respectively set as follows: 0.24ml/min, 0.25ml/min, 0.26ml/min. The results show that the separation degree effect is better under 0.24-0.26ml/min, wherein 0.24-0.25ml/min is the best.
TABLE 7 investigation results of different flow rates
(5) Investigation experiment of chromatographic column
The perilla stem granules were taken to prepare a test solution of the perilla stem granules according to the method in item 2 of this experimental example. The chromatographic conditions of the fourth method in the item 3 (1) of the experimental example are respectively adopted to detect the test solution of the perilla stem formula particles, the difference is only that chromatographic columns are different, and the durability of the characteristic spectra of the perilla stem formula particles of different chromatographic columns is comparedInfluence, different brands of octadecylsilane bonded silica columns (column 1TMAQ, 2.1X 150mm,1.9 μm. The results show that: the characteristic spectrum measured by comparing chromatographic columns of different brands can find that the chromatographic columns 2 and 3 have poor separation effect on each chromatographic peak, the chromatographic peak information content of the chromatographic column 1 is obviously increased, and the separation effect is obviously improved, so that the chromatographic column adopting the Waters CORTEC UPLC T3 (2.1 mm multiplied by 150mm,1.6 mu m) chromatographic column as the construction method of the characteristic spectrum of the perilla stem formula particles is adopted.
TABLE 8 investigation results of different chromatography columns
4. Preparation of test solution
(1) Investigation of extraction solvent
Taking perilla stem formula particles, weighing about 0.5g precisely, placing the particles in a conical flask with a plug, respectively and precisely adding 25ml of extraction solvents (60% acetone, 40% methanol, 60% methanol and 80% methanol, all in volume percentage), sealing the plug, weighing the particles, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, weighing the particles again, complementing the lost weight with corresponding extraction solvents, shaking up, filtering, precisely sucking 1 mu l of subsequent filtrate, and determining according to the chromatographic conditions of the method IV under the item 3 (1) of the experimental example. The result shows that the sample system extracted by 60% acetone, 40% methanol, 60% methanol and 80% methanol has better adaptability parameters.
TABLE 9 examination of different extraction solvents
(2) Examination of the amount of extraction solvent
Taking perilla stem formula particles, about 0.5g, precisely weighing, placing the particles in a conical flask with a plug, precisely adding 5ml, 10ml and 20ml of extraction solvent 60% methanol respectively, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, weighing again, complementing the loss weight with 60% methanol, shaking up, filtering, precisely sucking 1 mu l of subsequent filtrate, and determining according to the chromatographic conditions of the method IV under the item 3 (1) of the experimental example. As can be seen from the following table, 5-20ml of extraction solvent can satisfy the requirements of chromatographic separation.
TABLE 10 examination of the amount of different extraction solvents
(3) Investigation of sample size
Taking perilla stem formula particles, investigating the influence of different sampling amounts (0.25 g, 0.50g and 1.0 g) on the extraction effect, respectively placing the particles into conical flasks with stoppers, respectively and precisely adding 10ml of 60% methanol as an extraction solvent, sealing the stoppers, weighing the weight, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, weighing the weight again, supplementing the lost weight with 60% methanol, shaking up, filtering, precisely absorbing 1 mu l of subsequent filtrate, and measuring according to the chromatographic condition of the method IV under the item 3 (1) of the experimental example. As can be seen from the following table, the sample volumes of 0.25g, 0.50g and 1.0g satisfy the requirements of chromatographic separation.
TABLE 11 examination of different sample volumes
(4) Investigation of sample volume
Taking perilla stem formula particles, precisely weighing about 0.5g, placing the particles into conical flasks with stoppers, precisely adding 10ml of extraction solvent 60% methanol, sealing the conical flasks, weighing, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, weighing again, supplementing the lost weight with 60% methanol, shaking up, filtering, precisely sucking 1 mu l, 2 mu l and 3 mu l of subsequent filtrate respectively, and measuring according to the chromatographic conditions of the method IV under the item 3 (1) of the experimental example. As can be seen from the following table, the sampling volumes of 1. Mu.l, 2. Mu.l and 3. Mu.l can meet the requirements of chromatographic separation, and the results of the sampling amounts are slightly different.
TABLE 12 investigation results of different sample injection volumes
(5) Determination of preparation method of test solution
According to the research result, the preparation method of the determined test solution comprises the following steps: taking a proper amount of a test sample, grinding, taking 0.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of 60% methanol, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 20 minutes, cooling, shaking up, filtering, and taking a subsequent filtrate to obtain the product.
5. Identification of characteristic peaks
Precisely weighing appropriate amount of caffeic acid reference substance, rosmarinic acid reference substance, visanin-II reference substance, and apigenin-7-O-diglucuronide reference substance, and adding 60% methanol to obtain reference substance solution containing 40 μ g of each reference substance per 1 ml.
A perillartine formula granule sample solution is prepared according to the preparation method of example 1, and a caffeic acid reference substance solution, a rosmarinic acid reference substance solution, a viscapine-II reference substance solution, an apigenin-7-O-diglucuronide reference substance solution and a perillartine formula granule sample solution are detected and compared by high performance liquid chromatography according to the method of example 1, and the result is shown in fig. 20, wherein S1 is perillartine formula granules, S2 is viscapine-II, S3 is apigenin-7-O-diglucuronide, S4 is caffeic acid, and S5 is rosmarinic acid.
And (3) knotting: the retention time of chromatogram peaks of No. 3 peak, no. 4 peak, no. 10 peak and No. 11 peak in chromatogram of caulis Perillae formula granule sample is respectively consistent with those of chromatogram peaks of caffeic acid, viscapine-II, apigenin-7-O-diglucuronide and rosmarinic acid reference substance, and it can be confirmed that No. 3 peak is caffeic acid, no. 4 peak is viscapine-II, no. 10 peak is apigenin-7-O-diglucuronide, and No. 11 peak is rosmarinic acid.
6. Determination of characteristic peak and establishment of contrast map
(1) Construction method
Taking 3.0g of perilla stem as a reference medicinal material, placing the reference medicinal material in a conical flask with a plug, adding 50ml of water, heating and refluxing for 45 minutes, filtering, evaporating the filtrate to dryness, adding 10ml of 60% methanol into the residue, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 20 minutes, cooling, shaking up, filtering, and taking the subsequent filtrate as a reference medicinal material solution. Taking 15 batches of perilla stem formula particles to prepare a test solution according to the following method respectively, precisely weighing the test solution, grinding, taking 0.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of 60% methanol, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 20 minutes, cooling, shaking up, filtering, and taking a subsequent filtrate to obtain the perilla stem formula particle.
Respectively detecting 15 batches of perilla stem formula particle sample solutions and reference medicinal material reference substance solutions by high performance liquid chromatography: waters CORTEC UPLC T3 (2.1 mm. Times.150mm, 1.6 μm) column; the acetonitrile mobile phase A and the 0.2 percent formic acid aqueous solution are taken as the mobile phase B, and gradient elution is carried out according to the specification in the following table; flow rate 0.25ml per minute; the column temperature was 25 ℃; the detection wavelength was 330nm. The number of theoretical plates should be not less than 10000 calculated according to caffeic acid peak.
TABLE 13 gradient elution procedure
The results are shown in FIGS. 21-23, in which S1 (11) to S15 (11) in FIG. 22 are the feature maps of 15 batches of perilla stem granules 1904001W to 2111001S, and R (11) is the comparison feature map.
TABLE 14 relative retention time of Perilla Stem batch formulations
TABLE 15 relative peak areas of Perilla Stem batches
(2) A traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) is adopted to synthesize 15 batches of perilla stem formula particle samples, a comparison spectrum (namely a comparison characteristic spectrum) of a perilla stem formula particle characteristic spectrum is established, as shown in figure 23, 11 peaks are selected as characteristic peaks in total. The peak No. 3 is a caffeic acid reference substance peak (S peak), and the relative retention times of each characteristic peak and S peak are 0.32 (peak 1), 0.86 (peak 2), 1.00 (peak 3), 1.29 (peak 4), 1.40 (peak 5), 1.48 (peak 6), 1.76 (peak 7), 1.81 (peak 8), 2.06 (peak 9), 2.85 (peak 10), and 3.22 (peak 11). The results of the characteristic maps of 15 batches of perilla stem formula granules show that 11 common peaks have good transmissibility, and the relative retention time of each characteristic peak of 15 batches of perilla stem formula granules is within +/-10% of a specified value.
Therefore, it is specified that: the test sample characteristic spectrum should present 11 characteristic peaks, the peak corresponding to the caffeic acid reference substance peak is S peak, and the relative retention time of each characteristic peak and S peak should be within + -10% of the specified value. The specified values are: 0.32 (peak 1), 0.86 (peak 2), 1.00 (peak 3), 1.29 (peak 4), 1.40 (peak 5), 1.48 (peak 6), 1.76 (peak 7), 1.81 (peak 8), 2.06 (peak 9), 2.85 (peak 10), 3.22 (peak 11).
Experimental example 2 methodological verification
1. Precision of the instrument
Taking perilla stem formula particles (lot number: 2008001W) to prepare a sample solution according to the method of example 1, taking 6 parts of the sample solution, testing according to the high performance liquid chromatography of example 1, repeatedly injecting samples for 6 times, calculating the RSD of the relative retention time of 11 characteristic peaks to be 0-0.08%, and indicating that the precision of the instrument is good.
TABLE 16 relative retention time results for instrument precision tests
2. Repeatability
A perilla stem formula particle (batch number: 2008001W) sample is taken, 6 parts of test solution are prepared in parallel according to the method of the example 1 and are measured, the RSD of the relative retention time of each characteristic peak is calculated to be less than 2.0%, and the result shows that the method has good repeatability.
TABLE 17 repeatability test relative retention time results
3. Intermediate precision (personnel)
Three experimenters respectively prepare a test solution of the perilla stem formula particle (batch number: 2008001W) according to the method of the example 1 and measure, and the RSD of the relative retention time of the calculated characteristic peak is less than 2.0 percent, and the intermediate precision is good.
TABLE 18 relative retention time with intermediate precision (person)
4. Durability
(1) Stability survey
Taking perilla stem formula particles (batch number: 2008001W) to prepare a test solution according to the method of the embodiment 1, and respectively measuring the solution at 0h, 2h, 4h, 8h, 12h and 24h according to the high performance liquid chromatography of the embodiment 1, wherein the RSD of the calculated relative retention time of the characteristic peak is less than 2.0%. The result shows that the test solution is stable within 24h and meets the determination requirement.
TABLE 19 stability test relative Retention time results Table
(2) Different instruments
Comparing the durability influence of different high performance liquid chromatographs on the characteristic spectrum of the perilla stem formula granules (batch number: 2008001W), and detecting by using different instruments. An appropriate amount of perilla stem granules was taken and tested on a test sample of perilla stem granules using different instruments (Waters Arc UPLC and Thermo Ultimate 3000) according to the method of example 1. The results show that the RSD of the relative retention time of each characteristic peak should be less than 5.0%. Indicating that the method is relatively durable.
TABLE 20 relative retention time results for different instruments
Example 1
The embodiment provides a method for constructing a characteristic spectrum of perilla stem formula granules, which comprises the following steps:
preparation of a test solution: precisely weighing perilla stem formula particles, grinding, precisely weighing 0.5g, placing in a conical flask with a plug, precisely adding 10ml of 60% methanol, carrying out ultrasonic treatment (power 250W and frequency 40 kHz) for 20 minutes, cooling, shaking up, filtering, and taking subsequent filtrate to obtain the perilla stem formula particles.
Preparation of control solutions: precisely weighing appropriate amount of caffeic acid and rosmarinic acid reference substances, and adding 60% methanol to obtain mixed solution containing 0.2mg of caffeic acid and rosmarinic acid per 1ml as reference substance solution;
and (3) testing by high performance liquid chromatography: precisely sucking 1 μ l of reference substance solution of reference substance and 1 μ l of test solution of caulis Perillae, injecting into liquid chromatograph, and measuring with the following chromatographic conditions: octadecylsilane chemically bonded silica was used as a filler (Waters CORTEC UPLC T3 (2.1 mm. Times.150mm, 1.6 μm) column chromatography); acetonitrile is taken as a mobile phase A, 0.2% formic acid aqueous 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 per minute; the column temperature is 25 ℃; the detection wavelength was 330nm. The number of theoretical plates should be not less than 10000 calculated according to caffeic acid peak.
TABLE 21 gradient program
TABLE 22 Peak results
As can be seen from the above table and FIGS. 24-25, 11 characteristic peaks in FIG. 24 have good separation effect, high peak area, short detection and analysis time, and peak 1 in FIG. 25 is caffeic acid; peak 2 is rosmarinic acid.
Example 2
The embodiment provides a method for constructing a feature map of perilla stem standard decoction freeze-dried powder, and the difference from the embodiment 1 is only that perilla stem standard decoction freeze-dried powder is adopted to replace perilla stem formula granules, and peak results are shown in the following table.
TABLE 23 Peak results
| Serial number | Retention time | Peak area | Peak height | Degree of separation | Symmetry factor | Number of |
| 1 | 4.083 | 68188 | 20439 | - | 0.96 | 34710 |
| 2 | 11.011 | 75686 | 21764 | 76.66 | 1.11 | 244631 |
| 3 | 12.762 | 553736 | 128904 | 18.38 | 1.54 | 244664 |
| 4 | 16.469 | 122976 | 21756 | 30.69 | 1.48 | 207442 |
| 5 | 17.871 | 25783 | 5035 | 10.2 | 1.05 | 272067 |
| 6 | 18.884 | 131930 | 24627 | 7.65 | 1.49 | 331501 |
| 7 | 22.562 | 30679 | 4734 | 24.71 | 1.27 | 285084 |
| 8 | 23.203 | 54312 | 8686 | 3.81 | 1.06 | 303775 |
| 9 | 26.298 | 71701 | 9285 | 16.29 | 0.95 | 253475 |
| 10 | 36.550 | 47722 | 3109 | 33.24 | 1.18 | 127668 |
| 11 | 41.184 | 1274007 | 91689 | 12.09 | 1.23 | 203837 |
As can be seen from the above table and FIG. 26, the separation effect of 11 characteristic peaks is good, and the detection and analysis time is short.
Claims (10)
1. A method for constructing a characteristic map of a perilla stem medicinal preparation is characterized by comprising the following steps of,
(1) Preparing a test solution;
(2) Taking a test sample solution, detecting by adopting a high performance liquid chromatography, adopting a Waters CORTEC UPLC T3 chromatographic column, wherein a mobile phase comprises an acid-containing aqueous solution and acetonitrile, and a gradient elution program comprises the following steps: 0 → 3min → 10min → 16min → 26min → 35min → 40min → 45-48min, the volume percentage of acetonitrile in the mobile phase is: 2% → 5% → 12% → 14% → 16% → 16% → 18% → 18%.
2. The building method according to claim 1, wherein the step (2) further satisfies at least one of the following 1) to 5):
1) The detection wavelength is 325-335nm, the flow rate is 0.24-0.26mL/min, the column temperature is 20-30 ℃, and the sample injection volume is 1-10 mul;
2) The mobile phase is a mixed solution of acetonitrile and an acid-containing aqueous solution;
3) The gradient elution procedure further comprises: 45-48min → 50min, the volume percentage of acetonitrile in the mobile phase is: 18% → 20%;
4) The acid-containing aqueous solution is an aqueous solution containing 0.1-0.3% of phosphoric acid or 0.1-0.3% of formic acid by volume percentage;
5) The Waters CORTEC UPLC T3 chromatographic column is a Waters CORTEC UPLC T3 (2.1 mm. Times.150mm, 1.6 μm) chromatographic column.
3. The construction method according to claim 1 or 2, wherein the step (1) comprises weighing the sample, adding a solvent for extraction to obtain an extract, performing solid-liquid separation, and taking the liquid to obtain a sample solution.
4. The construction method according to claim 3, wherein the step (1) further satisfies any one or more of the following A-E:
A. the ratio of the mass of the sample to the volume of the solvent is 0.25-3.0:5-20; the relation between the mass and the volume is g/mL;
B. the extraction mode is reflux extraction or ultrasonic extraction;
C. the extraction time is more than or equal to 10min, preferably 15-40min;
D. the solid-liquid separation is selected from centrifugation or filter membrane filtration;
E. the solvent is selected from 40-80% methanol aqueous solution or 40-80% acetone aqueous solution.
5. The method according to any one of claims 1 to 4, further comprising a step of preparing a reference solution by using a solvent for at least one of caffeic acid, rosmarinic acid, viscapin-II and apigenin-7-O-diglucuronide, and a step of detecting the reference solution by high performance liquid chromatography according to any one of claims 1 to 4 to obtain a reference map.
6. The constructing method according to claim 5, wherein each 1mL of the control solution contains at least one of 1-100 μ g caffeic acid, 1-100 μ g rosmarinic acid, 1-100 μ g viscapine-II, and 1-100 μ g apigenin-7-O-diglucuronide; and/or, the solvent used in the preparation of the control solution is selected from methanol or methanol water solution.
7. The method according to any one of claims 1 to 6, further comprising the steps of preparing a reference solution of the perilla stem by using an extract obtained by extracting a reference perilla stem with water according to the method for preparing a test solution in the method according to any one of claims 1 to 4, and detecting the reference solution of the reference perilla stem by using the high performance liquid chromatography in the method according to any one of claims 1 to 4 to obtain a reference map of the reference perilla stem, preferably, extracting the reference perilla stem with water, filtering, drying the filtrate, and preparing the reference solution of the reference perilla stem according to the method for preparing the test solution in the method according to any one of claims 1 to 4.
8. The method according to any one of claims 1 to 7, wherein the perilla stem drug preparation has a characteristic spectrum with 11 common characteristic peaks, the peak corresponding to the caffeic acid reference substance peak is an S peak, and the relative retention time of each characteristic peak to the S peak is within the range of ± 10% of the specified value; the specified values are: 0.32 (peak 1), 0.86 (peak 2), 1.00 (peak 3), 1.29 (peak 4), 1.40 (peak 5), 1.48 (peak 6), 1.76 (peak 7), 1.81 (peak 8), 2.06 (peak 9), 2.85 (peak 10), 3.22 (peak 11).
9. The use of the method for constructing a characteristic map of a perilla stem pharmaceutical preparation according to any one of claims 1 to 8 for the quality control of a perilla stem pharmaceutical preparation.
10. A quality detection method of a perilla stem medicinal preparation is characterized by comprising the step of comparing a characteristic spectrum of a perilla stem product to be detected with a reference characteristic spectrum of the perilla stem medicinal preparation; the characteristic map of the perilla stem product to be detected is obtained by constructing the perilla stem product to be detected according to the construction method of any one of claims 1 to 8, and the reference characteristic map of the perilla stem medicinal preparation is selected from any one of the following (1) to (3):
(1) It has 11 common characteristic peaks, the peak corresponding to the caffeic acid reference substance peak is S peak, and the relative retention time of each characteristic peak and S peak is in the range of + -10% of the specified value; the specified values are: 0.32 (peak 1), 0.86 (peak 2), 1.00 (peak 3), 1.29 (peak 4), 1.40 (peak 5), 1.48 (peak 6), 1.76 (peak 7), 1.81 (peak 8), 2.06 (peak 9), 2.85 (peak 10), 3.22 (peak 11);
(2) A characteristic map of a medicinal preparation of perilla stem obtained by the method for constructing according to any one of claims 1 to 8 using a single lot or a plurality of lots of medicinal preparations of perilla stem;
(3) A control feature map by a mean or median method using a plurality of batches of the perilla stem drug preparation obtained by the construction method according to any one of claims 1 to 8.
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| 亢倩丽;李壮壮;范珊珊;容蓉;蒋海强;姜晓琳;张继秋;巩丽丽;: "基于UPLC-Q-Exactive-Orbitrap-MS的紫苏叶与紫苏梗化学成分分析", 中国实验方剂学杂志, vol. 26, no. 13, pages 156 - 162 * |
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