CN112730688B - Method for identifying characteristic spectrum of dogbane leaf formula particle - Google Patents
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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
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Abstract
The invention discloses a method for identifying a characteristic spectrum of a dogbane leaf formula particle, which comprises the following steps of: taking 0.25g of herba Apocyni Veneti leaf formula granule, precisely weighing, placing in a conical flask with a stopper, precisely adding extraction solvent, extracting for 10-30min, cooling, shaking, filtering, and collecting the filtrate; taking appropriate amount of hyperoside, caffeic acid, chlorogenic acid, astragalin, neochlorogenic acid, cryptochlorogenic acid, and isoquercitrin as reference substances, and adding 70% methanol to make into solution; the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the silica gel is 5 μm; a diode array detector with the detection wavelength of 220-360 nm; column temperature: 20-30 ℃; flow rate: 0.8-1.2 mL/min; mobile phase: acetonitrile is taken as a mobile phase A, 0.2 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out; precisely sucking 10 μ L of reference solution, reference solution and sample solution respectively, injecting into liquid chromatograph, and measuring.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for identifying a characteristic spectrum of dogbane leaf formula particles.
Background
The apocynum venetum is erect half shrub, the height is 1.5-3 meters, the height is about 2 meters generally, and the maximum height can reach 4 meters; the branches are in butt or intertidal, cylindrical, smooth and hairless, purple or light red. Leaf pairing, only near pairing at the branch, the ellipse of the leaf is in a needle shape to oval long circle shape with the length of 1-5 cm and the width of 0.5-1.5 cm, the tip is sharp to blunt, the leaf is short-pointed, the base is sharp to blunt, the margin of the leaf is provided with fine teeth, and the two sides are hairless; the leaf veins are fine, slightly convex or flat on the leaf back and unobvious on the leaf surface, 10-15 side veins are arranged on each side and are knotted in front of the leaf margin; the leaf stalk is 3-6 mm long; glands are located between petioles, and the leaves fall off in old age. Dogbane leaf, name of Chinese medicinal material. The product is leaf of Apocynum venetum L of Apocynaceae. Collected in summer, removed of impurities and dried. The main functional indications are as follows: clear liver fire, calm liver and stop wind. Can be used for treating headache, vertigo, infantile convulsion, and tetany due to excessive liver fire. It has the effect of lowering blood pressure. At present, qualitative research literature for the dogbane leaf formula particles is relatively lacked, and quality monitoring of the dogbane leaf formula particles is difficult.
Disclosure of Invention
In order to solve the problems, the invention provides a method for identifying the characteristic spectrum of the dogbane leaf formula particle.
The purpose of the invention is realized by the following technical scheme:
a method for identifying a characteristic spectrum of a particle formula of dogbane leaf is characterized by comprising the following steps:
(1) and preparing a test solution: taking 0.25g of dogbane leaf formula particles, precisely weighing, placing in a conical flask with a plug, precisely adding an extraction solvent, weighing, extracting for 10-30min, cooling, weighing again, supplementing the lost weight with the extraction solvent, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the product;
(2) and preparing a reference substance solution: taking 0.5g of folium Apocyni Veneti as a reference medicinal material, precisely weighing, placing in a conical flask with a stopper, precisely adding 70% methanol, sealing the stopper, weighing, ultrasonically treating, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and taking the subsequent filtrate; accurately weighing appropriate amount of hyperoside, caffeic acid, chlorogenic acid, astragalin, neochlorogenic acid, cryptochlorogenic acid, and isoquercitrin as reference substances, and adding 70% methanol to obtain solution;
(3) and chromatographic conditions: a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the silica is 5 microns; a detector: a diode array detector with the detection wavelength of 220-360 nm; column temperature: 20-30 ℃; flow rate: 0.8-1.2 mL/min; mobile phase: acetonitrile is taken as a mobile phase A, 0.2 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out;
(4) and testing: precisely sucking 10 μ L of reference solution, reference medicinal material solution and sample solution respectively, injecting into liquid chromatograph, and measuring.
Preferably, in step (1), the sample extraction solvent is ethanol or 50% methanol or 70% methanol or water.
Preferably, in the step (1), the extraction mode is reflux extraction or ultrasonic extraction.
Preferably, in the step (1), the extraction solvent is 50 mL.
Preferably, in the step (1), the extraction time is 10min or 20min or 30 min.
Preferably, in the step (2), a proper amount of hyperin, caffeic acid, chlorogenic acid, astragalin, neochlorogenic acid, cryptochlorogenic acid and isoquercitrin reference substances are precisely weighed and added with 70% methanol to prepare solutions each containing 30 mug per 1mL, so as to obtain the product.
Preferably, in step (3), the chromatographic conditions are as follows: a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the silica is 5 microns; a detector: diode array detector with detection wavelength of 320nm and theoretical plate number not less than 8000 calculated according to hyperin; column temperature: 25 ℃; flow rate: 1.0 mL/min; mobile phase: acetonitrile is taken as a mobile phase A, 0.2 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out; the gradient elution procedure was:
the mobile phase A rises from 11% (v/v) to 15% (v/v) within 0-10 min;
keeping the mobile phase A for 15% (v/v) for 10-15 min;
the mobile phase A rises from 15% (v/v) to 20% (v/v) within 15-25 min;
the mobile phase A rises from 20% (v/v) to 25% (v/v) within 25-40 min;
the mobile phase A rises from 25% (v/v) to 40% (v/v) for 40-55 min.
Preferably, the characteristic map should present 7 characteristic peaks, wherein the peak corresponding to the hyperin reference is the S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ± 8% of the specified value, and the specified value of each peak is: peak 10.272, peak 20.408, peak 30.443, peak 40.541, peak 51.000 (S), peak 61.025, peak 71.194.
The beneficial effects of this technical scheme are as follows:
(1) the HPLC characteristic spectrum detection method can comprehensively reflect the types and the amounts of chemical components contained in the dogbane leaf formula particles, and can more effectively embody the integrity and the comprehensive action of the components in the dogbane leaf formula particles.
(2) According to the HPLC characteristic spectrum detection method of the apocynum venetum leaf formula particles, corresponding apocynum venetum leaf reference medicinal materials and chemical reference products are used as references, the chemical component corresponding relation between the apocynum venetum leaf formula particles and the apocynum venetum leaf reference medicinal materials is shown on the one hand, and the method is simple, good in stability and high in precision; on the other hand, the apocynum venetum leaf formula particles are possibly similar to other traditional Chinese medicine formula particles in properties, and whether the apocynum venetum leaves are the apocynum venetum leaves cannot be identified from the properties.
Drawings
FIG. 1 is a graph of the ultra-violet absorption spectrum of hyperin;
FIG. 2 is a diagram of the UV absorption spectrum of caffeic acid;
FIG. 3 is a graph of the ultraviolet absorption spectrum of chlorogenic acid;
FIG. 4 is an ultraviolet absorption spectrum of astragalin;
FIG. 5 is a graph of the UV absorption spectrum of neochlorogenic acid;
FIG. 6 is a graph of the UV absorption spectrum of cryptochlorogenic acid;
FIG. 7 is a diagram of the UV absorption spectrum of isoquercitrin;
FIG. 8 is a chromatogram of different wavelengths of folium Apocyni Veneti granule;
FIG. 9 is a chromatogram of dogbane leaf formula granules at different column temperatures;
FIG. 10 is a chromatogram of dogbane leaf formula granules at different flow rates;
FIG. 11 is a chromatogram of dogbane leaf formulation granules in different solvents;
FIG. 12 is a chromatogram of dogbane leaf formula granules obtained by different extraction methods;
FIG. 13 is a chromatogram of dogbane leaf formula granules taken at different extraction times;
FIG. 14 shows the chromatogram peak identification of folium Apocyni Veneti granule;
FIG. 15 is a chromatogram of dogbane leaf formula granules obtained using different instruments;
FIG. 16 is a chromatogram of dogbane leaf formula granules using different chromatographic columns;
FIG. 17 is a graph of a characteristic spectrum of 3 batches of dogbane leaf formula granules;
FIG. 18 is a comparison feature spectrum of dogbane leaf formula granule; peak 1: chlorogenic acid; peak 2: chlorogenic acid; peak 3: cryptochlorogenic acid; peak 4: caffeic acid; peak 5: hyperin; peak 6: isoquercitrin; peak 7: astragalin.
Detailed Description
1. Laboratory instruments and materials
High performance liquid chromatograph: shimadzu 20-AT HPLC, Agilent 1260 HPLC, Waters2998-e2695 HPLC;
an electronic balance: ME204E/02, MS205DU, XP26 (Mettler-Tollido instruments, Inc.);
an ultra-pure water machine: cell type 1810A (Shanghai Mohler scientific instruments, Inc.);
an ultrasonic cleaner: model KQ5200DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);
a chromatographic column: AgiLent 5TC-C18(2) 250X 4.6mm (Agilent), Kromasil C185 μm 4.6X 250mm, Shimadzu 5 μm C18250X 4.60 mm.
Hyperin (China institute for testing food and drug; lot number: 111521-,
caffeic acid (China institute for testing and testing food and drug; batch No. 110885-,
chlorogenic acid (China institute for food and drug assay, batch No. 110753-201415, content in 96.2%),
astragalin (Vickqi Biotech Co., Ltd., Sichuan, lot number wkq17111511),
chlorogenic acid (Dopperfeld Biotechnology Co., Ltd., lot number: 17062003, content 98%),
cryptochlorogenic acid (Dopperfeld Biotechnology Co., Ltd., lot number: 17061401, content 98%),
isoquercitrin (China institute for testing and testing food and drug, lot number: 111809-,
apocynum venetum leaf reference drug (China food and drug testing research institute, lot number: 120979-201505).
Acetonitrile (SIGMA, chromatographically pure); the water is ultrapure water, and other reagents are analytically pure.
The dogbane leaf formula granules are GR-LBMY-01, GR-LBMY-02 and GR-LBMY-03.
2. Method for preparing high-efficiency liquid effect detection
Preparing reference solution by accurately weighing appropriate amount of hyperoside, caffeic acid, chlorogenic acid, astragalin, neochlorogenic acid, cryptochlorogenic acid, and isoquercitrin, and adding 70% methanol to obtain solutions each containing 30 μ g per 1 mL.
Preparation of a reference medicinal material solution, taking 0.5g of dogbane leaf reference medicinal material, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of 70% methanol, sealing, weighing, ultrasonically treating (power 600W, frequency 40kHz) for 20 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the apocynum venetum extract.
The preparation method of the test solution comprises mixing the above materials, grinding, weighing herba Apocyni Veneti leaf granule 0.25g, precisely weighing, placing in conical flask with plug, precisely adding 50mL of 70% methanol, weighing, ultrasonic treating for 20min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the filtrate.
Chromatographic conditions are as follows: a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the silica is 5 microns; a detector: diode array detector with detection wavelength of 320nm and theoretical plate number not less than 8000 calculated according to hyperin peak; column temperature: 25 ℃; flow rate: 1.0 mL/min; mobile phase: acetonitrile is taken as a mobile phase A, 0.2 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out; the gradient elution procedure was:
the mobile phase A rises from 11% (v/v) to 15% (v/v) within 0-10 min;
keeping the mobile phase A for 15% (v/v) for 10-15 min;
the mobile phase A rises from 15% (v/v) to 20% (v/v) within 15-25 min;
the mobile phase A rises from 20% (v/v) to 25% (v/v) within 25-40 min;
the mobile phase A rises from 25% (v/v) to 40% (v/v) for 40-55 min.
The determination method comprises precisely sucking reference solution, reference medicinal material solution and sample solution 10 μ L respectively, injecting into liquid chromatograph, and determining.
3. Inspection of chromatographic conditions
3.1 wavelength investigation
Based on the experimental conditions, a diode array detector is utilized to respectively carry out full-wave-band scanning on hyperoside, caffeic acid, chlorogenic acid, astragalin, neochlorogenic acid, cryptochlorogenic acid, isoquercitrin and the test solution, and chromatograms of the test solution at wavelengths of 220nm, 250nm, 270nm, 286nm, 300nm, 320nm, 340nm and 360nm are respectively extracted. As shown in fig. 1-8.
The result shows that the chromatographic peak information content is larger when the detection wavelength is 320nm, the chromatogram base line is more stable, and therefore the detection wavelength is determined to be 320 nm.
3.2 column temperature investigation
Based on the experimental conditions set forth above, the column temperatures were examined at 20 ℃, 25 ℃ and 30 ℃. As shown in fig. 9 and table 1.
TABLE 1 column temperature investigation-ratio of characteristic peaks to retention time
The result shows that the influence of the column temperature on the relative retention time of each chromatogram peak is small, the chromatogram peak shapes are symmetrical and the separation degrees are good under each column temperature condition, so that the temperature of 25 ℃ is finally selected as the column temperature of the apocynum venetum leaf formula particle characteristic spectrum method.
3.3 investigation of flow Rate
Based on the experimental conditions set forth above, the flow rates were examined at 0.8mL/min, 1mL/min, and 1.2mL/min, respectively. As shown in fig. 10 and table 2.
Table 2 flow rate investigation-characteristic peak relative retention time
The result shows that when the flow rates are respectively 0.8mL/min, 1.0mL/min and 1.2mL/min, the relative retention time RSD of each characteristic peak is 0.22-9.96%, and when the flow rate is 1.0mL/min, the chromatogram peak shape is good and the separation degree is moderate. Therefore, the flow rate was determined to be 1.0 mL/min.
4. Investigation of preparation of test solution
4.1 examination of extraction solvent
The results of examining ethanol, 50% methanol, 70% methanol, methanol and water as extraction solvents show that the chromatographic peak information obtained by using 70% methanol as the extraction solvent is large and the separation degree is good, so 70% methanol is selected as the extraction solvent. As shown in fig. 11.
4.2 examination of extraction methods
Compared with the reflux extraction and the ultrasonic extraction, the chromatographic peak information is approximately the same, and finally, the ultrasonic extraction with simple operation is selected for extraction. As shown in fig. 12.
4.3 investigation of extraction time
Ultrasonic extraction was compared for 10, 20, 30 minutes, and finally selected for 20 minutes. As shown in fig. 13.
4.4 method for preparing final definite sample
Taking 0.25g of the product granules (batch number: GR-LBMY-01), grinding, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of 70% methanol, weighing, ultrasonically treating for 20 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
5. Methodology investigation
5.1 chromatographic peak assignment
Preparation of a test solution: preparing the test solution of the dogbane leaf formula particle according to the experimental conditions.
Preparation of reference solutions: taking appropriate amount of hyperoside, quercetin, caffeic acid, chlorogenic acid, astragalin, neochlorogenic acid, cryptochlorogenic acid, and isoquercitrin as reference substances, precisely weighing, and adding 70% methanol to make into solutions each containing 30 μ g per 1 mL.
Preparation of reference drug solution: taking about 0.5g of dogbane leaf as a reference medicinal material, precisely weighing, placing into a conical flask with a plug, precisely adding 50mL of 70% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 600W and frequency 40kHz) for 20 minutes, cooling, weighing again, supplementing the weight loss with 70% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the apocynum venetum extract.
Preparation of negative control solution: and preparing a negative control solution of the apocynum venetum leaf formula granules according to the experimental conditions drawn up above.
And positioning the peak of the characteristic diagram spectrum of the dogbane leaf formula particle. As shown in fig. 14.
5.2 precision test
Taking a test sample solution of the dogbane leaf formula particle (batch number: GR-LBMY-01), continuously injecting samples for 6 times according to a formulated experimental method, wherein 10 mu L of samples are injected each time, and calculating the relative retention time and the relative peak area of each characteristic peak. As shown in tables 3 and 4.
TABLE 3 precision investigation-Retention time
TABLE 4 precision investigation-Peak area
The results show that the instrument is accurate.
5.3 repeatability test
6 parts of dogbane leaf formula particles (batch number: GR-LBMY-01) are precisely weighed, and the preparation and the determination are carried out according to a formulated experimental method. As shown in tables 5 and 6.
TABLE 5 repeatability test-relative retention time ratio
TABLE 6 repeatability test-relative peak area ratio
The result shows that the method has good repeatability.
5.4 intermediate precision investigation
5.41 investigation of different instruments
Based on the experimental conditions, accurately weighing two parts of folium Apocyni Veneti formula granules (batch number: GR-LBMY-01), preparing test solution, and measuring on Shimadzu 20-AT, Waters2998-e2695 and Agilen 1260 type high performance liquid chromatograph. As shown in fig. 15, table 7 and table 8.
TABLE 7 Instrument durability test-relative Retention time ratio
TABLE 8 Instrument durability test-relative Peak area ratio
The results show that the RSD range of each characteristic peak relative retention time is 0.13-5.32% when the test sample is detected by the 3 instruments.
5.42 different personnel and time review
Based on the experimental conditions, two parts of dogbane leaf formula particles (batch number: GR-LBMY-01) are precisely weighed by different persons (A, B) at different time (T1 and T2) respectively, and a test sample is prepared and measured. As shown in tables 9 and 10.
TABLE 9 ratio of person and time review-relative retention time
TABLE 10 personnel and time survey-relative Peak area ratios
The result shows that different people can determine the same sample at different time, and the method has better stability.
5.5 durability test
5.51 durability test of column
On the basis of the experimental conditions set forth above, the chromatographic columns were 250X 4.6mm (AgiLent 5TC-C18(2), 4.6X 250mm (Agilent), Kromasil C185. mu.m, 4.6X 250mm, and Shimadzu 5. mu. m C18250X 4.60mm, respectively. And (6) carrying out a survey. As shown in fig. 16, table 11 and table 12.
TABLE 11 column durability test-relative retention time ratio
TABLE 12 column durability test-relative peak area ratio
The results show that the RSD of the characteristic peak relative retention time is 0.15-4.46%, and the RSD of the characteristic peak relative peak area is 0.82-13.46% when the sample is detected by the 3 chromatographic columns.
5.52 stability test
Based on the experimental conditions, the same test solution is taken and respectively measured at 0h, 3h, 6h, 9h, 12h and 24 h. As shown in tables 13 and 14.
Table 13 stability investigation-retention time
TABLE 14 stability investigation-Peak area
The result shows that the RSD of the retention time of the corresponding characteristic peak is 0.16-0.26%, and the sample solution is stable within 24 hours.
In summary, the RSD of each characteristic peak relative retention time meets the requirements in the above studies, and the method is good. The 7 characteristic peaks are included in the subsequent examination.
The technical solutions for achieving the objects of the present invention are further illustrated by the following specific examples, and it should be noted that the technical solutions claimed in the present invention include, but are not limited to, the following examples.
Example 1
Verification result of 3 batches of apocynum venetum leaf formula granules
And (3) measuring the characteristic spectrum of the 3 batches of samples of the product by a drawn-up method, and calculating the relative retention time and the relative peak area. As shown in fig. 17, table 15 and table 16.
TABLE 153 batch Apocynum venetum leaf formulation relative Retention time
Relative peak area of particles of formula of Table 163 batches of Apocynum venetum L
According to the principle that the relative retention time is stable, samples of each batch can be detected, and the peaks are relatively high, 7 peaks with good repeatability are selected as characteristic peaks. The result shows that when the peak 5 is taken as an S peak, the relative retention time RSD of the characteristic peak of 3 batches of apocynum venetum leaf formula particles is 0.01-0.06%, and the relative retention time RSD of 7 characteristic peaks of 3 batches of apocynum venetum leaf formula particles is less than 4%.
Example 2
Making of specified value limit of relative retention time
Methodology the various survey items and the validation results are summarized in tables 17 and 18:
TABLE 17 methodology results RSD% summary criteria for each item-relative retention time
TABLE 18 methodology results for each item of the methodology RSD% summary Standard-relative Peak area
From the above table, it can be seen that the flow rate has the greatest effect on each characteristic peak, so the flow rate is set to 1mL/min, and in order to increase the reproducibility and applicability of the method, the specified value of the relative retention time of each peak is temporarily set to 8% with reference to the instrument and column durability results; the durability and the verification result have a large influence on the relative peak area result, so that the relative peak area is not listed as a standard.
Finally, the following steps are provided: 7 characteristic peaks are presented in the characteristic map of the test article, wherein the peak corresponding to the hyperin reference substance is an S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within +/-8% of a specified value. The specified values are: 0.272 (peak 1), 0.408 (peak 2), 0.443 (peak 3), 0.541 (peak 4), 1.000 (peak 5, S), 1.025 (peak 6), 1.194 (peak 7).
Synthesizing 3 batches of dogbane leaf formula particles by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and establishing a control spectrum of the characteristic spectrum of the dogbane leaf formula particles. As shown in fig. 18.
The above-mentioned embodiments are further described in detail for the purpose of illustrating the invention, the technical solutions and the advantages, it should be understood that the above-mentioned embodiments are only exemplary of the invention, and are not intended to limit the invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the invention should be included in the protection scope of the invention.
Claims (7)
1. A method for identifying a characteristic spectrum of a particle formula of dogbane leaf is characterized by comprising the following steps:
(1) and preparing a test solution: taking 0.25g of dogbane leaf formula particles, precisely weighing, placing in a conical flask with a plug, precisely adding an extraction solvent, weighing, extracting for 10-30min, cooling, weighing again, supplementing the lost weight with the extraction solvent, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the product;
(2) and preparing a reference substance solution: taking 0.5g of folium Apocyni Veneti as a reference medicinal material, precisely weighing, placing in a conical flask with a stopper, precisely adding 70% methanol, sealing the stopper, weighing, ultrasonically treating, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and taking the subsequent filtrate; accurately weighing appropriate amount of hyperoside, caffeic acid, chlorogenic acid, astragalin, neochlorogenic acid, cryptochlorogenic acid, and isoquercitrin as reference substances, and adding 70% methanol to obtain solution;
(3) the chromatographic conditions were as follows: a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size of the silica is 5 microns; a detector: diode array detector with detection wavelength of 320nm and theoretical plate number not less than 8000 calculated according to hyperin; column temperature: 25 ℃; flow rate: 1.0 mL/min; mobile phase: acetonitrile is taken as a mobile phase A, 0.2 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out; the gradient elution procedure was:
the mobile phase A is increased from 11 percent by volume to 15 percent by volume within 0 to 10 min;
keeping the volume ratio of the mobile phase A at 15% for 10-15 min;
15-25min, the volume ratio of the mobile phase A is increased from 15% to 20%;
the mobile phase A is increased from 20 percent by volume to 25 percent by volume within 25-40 min;
the mobile phase A is increased from 25 percent by volume to 40 percent by volume within 40-55 min;
(4) and testing: precisely sucking 10 μ L of reference solution, reference medicinal material solution and sample solution respectively, injecting into liquid chromatograph, and measuring.
2. The method for identifying the characteristic spectrum of the dogbane leaf formula particle as claimed in claim 1, is characterized in that: in the step (1), the sample extraction solvent is ethanol or 50% methanol or 70% methanol or water.
3. The method for identifying the characteristic spectrum of the dogbane leaf formula particle as claimed in claim 1, is characterized in that: in the step (1), the extraction mode is reflux extraction or ultrasonic extraction.
4. The method for identifying the characteristic spectrum of the dogbane leaf formula particle as claimed in claim 1, is characterized in that: in the step (1), the extraction solvent is 50 mL.
5. The method for identifying the characteristic spectrum of the dogbane leaf formula particle as claimed in claim 1, is characterized in that: in the step (1), the extraction time is 10min or 20min or 30 min.
6. The method for identifying the characteristic spectrum of the dogbane leaf formula particle as claimed in claim 1, is characterized in that: in the step (2), a proper amount of hyperin, caffeic acid, chlorogenic acid, astragalin, neochlorogenic acid, cryptochlorogenic acid and isoquercitrin reference substances are precisely weighed, and 70% methanol is added to prepare solutions containing 30 mu g of each 1 mL.
7. The method for identifying the characteristic spectrum of the dogbane leaf formula particle as claimed in claim 1, is characterized in that: the characteristic map should present 7 characteristic peaks, wherein the peak corresponding to the hyperin reference is the S peak, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-8% of a specified value, and the specified value of each peak is as follows: peak 10.272, peak 20.408, peak 30.443, peak 40.541, peak 51.000 (S), peak 61.025, peak 71.194.
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| CN101357147B (en) * | 2008-08-29 | 2012-08-22 | 周亚球 | Extract of dogbane leaf with finger print and preparation and analytical method thereof |
| CN104237416B (en) * | 2014-09-27 | 2015-08-26 | 中国人民解放军兰州军区乌鲁木齐总医院 | The HPLC method of 6 kinds of flavones ingredient content in Simultaneously test poacynum hendersonii woodson leaf |
| CN110927311B (en) * | 2019-12-03 | 2022-04-01 | 广东一方制药有限公司 | Construction method of UPLC (ultra performance liquid chromatography) characteristic spectrum of dogbane leaf medicinal material and method for measuring content of flavonoid component of dogbane leaf medicinal material |
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