CN112213404A - Fingerprint construction and content determination method of pseudo-ginseng medicinal liquor - Google Patents
Fingerprint construction and content determination method of pseudo-ginseng medicinal liquor Download PDFInfo
- Publication number
- CN112213404A CN112213404A CN201910629060.2A CN201910629060A CN112213404A CN 112213404 A CN112213404 A CN 112213404A CN 201910629060 A CN201910629060 A CN 201910629060A CN 112213404 A CN112213404 A CN 112213404A
- Authority
- CN
- China
- Prior art keywords
- peak
- fingerprint
- retention time
- content
- psoralen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
-
- 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
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
-
- 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
-
- 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
- G01N2030/042—Standards
- G01N2030/047—Standards external
Landscapes
- Physics & Mathematics (AREA)
- 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)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for constructing a fingerprint spectrum of pseudo-ginseng medicinal liquor and measuring the content of the pseudo-ginseng medicinal liquor, which comprises the following steps of: filtering Notoginseng radix medicated liquor, and collecting filtrate as test solution; taking psoralen reference substance, precisely weighing, and adding ethanol to obtain reference substance solution; performing high performance liquid chromatography analysis on the test solution to obtain HPLC fingerprint, determining common peaks according to relative retention time of the test solution and the reference, adopting chemical components of LC-MS fingerprint characteristic fingerprint peaks, and establishing HPLC fingerprint of the Notoginseng radix medicated liquor by using standard control; and calculating the content of the component to be detected according to the standard product. The invention provides a fingerprint method capable of simultaneously detecting 14 pharmacodynamic ingredients in the pseudo-ginseng medicinal liquor, can quantitatively detect the content of each main pharmacodynamic ingredient, provides a method for objectively evaluating the quality level of a medicine, and ensures the clinical curative effect of the medicine; the method has good precision, stability and reproducibility, and can meet the requirement of fingerprint spectrum measurement of Notoginseng radix medicated liquor.
Description
Technical Field
The invention relates to a quality detection method of a compound traditional Chinese medicine preparation, in particular to a fingerprint construction method and a content determination method of pseudo-ginseng medicinal liquor.
Background
The pseudo-ginseng medicinal liquor is a traditional Chinese medicine compound preparation consisting of 21 traditional Chinese medicinal materials such as pseudo-ginseng, epimedium herb, Chinese angelica, sappan wood, malaytea scurfpea fruit and the like, the monarch drug pseudo-ginseng is warm and slightly bitter in property, can activate blood circulation and remove meridian obstruction to relieve pain, and can tonify qi and blood to supplement deficiency, and the four-piece shingles, phyllanthus urinaria, ligusticum wallichii, frankincense and sappan wood also have strong effects of dissipating blood stasis and removing meridian obstruction to relieve swelling and relieving; herba Epimedii and fructus Psoraleae are representative for tonifying liver and kidney and strengthening tendons and bones, and radix Angelicae sinensis is representative for nourishing blood and regulating blood; the medicines have the effects of relaxing tendons and activating collaterals, dissipating blood stasis and relieving pain, dispelling wind and removing dampness, strengthening muscles and bones and the like, and are mainly used for treating traumatic injury, rheumatic osteodynia, numbness of limbs and other symptoms.
The inventor adopts High Performance Liquid Chromatography (HPLC) in pharmacopoeia to detect 21 traditional Chinese medicinal materials and base liquor in the pseudo-ginseng medicinal liquor, and combines the existing literature reports to determine that the main major components in the pseudo-ginseng medicinal liquor comprise polysaccharide, amino acid, saponin, flavone, coumarin, organic acid, phenols and the like, and the content of most components is lower; relevant pharmacodynamic tests show that the pseudo-ginseng saponin, psoralen, isopsoralen, icariin, original brazilin and other components have good pharmacological activity, and are effective components of the pseudo-ginseng medicinal liquor for treating rheumatic arthralgia. However, the current standard of the pseudo-ginseng medicinal liquor is collected in volume 11 (WS3-B-2079-96) of the standard Chinese medicinal prescription preparation of the medicine of the Ministry of health, except for character inspection, only the characteristics of pseudo-ginseng, epimedium herb, malaytea scurfpea fruit and the like are qualitatively identified, no quantitative detection method is available, and the integral quality of the product cannot be reflected. In addition, Teqingde et al (HPLC method for directly determining the content [ M ] of psoralen and isopsoralen in the notoginseng medicated wine, Chinese medicine standard, 2017,188(3):202-206) determine the content of psoralen and isopsoralen in the notoginseng medicated wine by HPLC method, a chromatographic column is Sino Chrom ODS.BP (200mm × 4.6mm, 5 μ M), a mobile phase is methanol-acetonitrile-water, and a detection wavelength is 246nm, but because a detection object only aims at two active ingredients of psoralen and isopsoralen, the separation degree of other drug effect ingredients such as notoginsenoside is very poor, the peak emergence time of other drug effect ingredients such as notoginsenoside is mainly concentrated in the first 10 minutes, and the interference of each ingredient is serious, so that quantitative detection cannot be carried out.
The pseudo-ginseng medicinal liquor has a complex prescription, the quality of the medicine is evaluated by simply detecting one or two index components, and the overall control of the integrity and the internal quality of the traditional Chinese medicine compound preparation is difficult to reflect comprehensively. Although the literature reports that HPLC (high performance liquid chromatography) is adopted to detect psoralen and isopsoralen in the pseudo-ginseng medicinal liquor, the spectral absorption characteristics of each component in the pseudo-ginseng medicinal liquor formula are different, pseudo-ginseng saponin as a characteristic component is absorbed at the tail end under the ultraviolet condition, and compared with flavone, coumarin, organic acid and other components in the pseudo-ginseng medicinal liquor, the absorption wavelength is lower, and the components are difficult to detect at the same time under a single wavelength; in addition, component analysis reports of similar compositions are few, peaks detected under conventional chromatographic conditions are few, and the problems of separation degree of each characteristic component, chromatographic peak front delay, tailing and the like cannot be solved at present.
Therefore, the establishment of the fingerprint analysis method capable of reflecting the quality characteristics of the pseudo-ginseng medicinal liquor can realize quantitative detection of the effective components, achieve quality control and effectively ensure the clinical curative effect of the medicine.
The invention content is as follows:
aiming at the defects of the prior art, the invention provides a method for constructing a fingerprint spectrum of pseudo-ginseng medicinal liquor and a method for measuring the content of the fingerprint spectrum of the pseudo-ginseng medicinal liquor. The method has the advantages of good stability and reliability, good reproducibility and high precision, makes up for the defects of the existing quality control method, and has guiding significance for quality detection and evaluation of Notoginseng radix medicated liquor.
The technical scheme adopted by the invention is as follows:
a fingerprint spectrum construction and content measurement method for pseudo-ginseng medicinal liquor comprises the following steps:
(1) preparation of a test solution: filtering Notoginseng radix medicated liquor, and collecting filtrate as test solution;
(2) preparation of control solutions: taking a proper amount of psoralen reference substance, precisely weighing, and adding ethanol to prepare a reference substance solution;
(3) performing high performance liquid chromatography analysis on the test solution to obtain HPLC fingerprint, determining common peaks according to relative retention time of the test solution and the reference, adopting chemical components of LC-MS fingerprint characteristic fingerprint peaks, and establishing HPLC fingerprint of the Notoginseng radix medicated liquor by using standard control;
the conditions of the high performance liquid chromatography analysis comprise:
welch Ultimate XB-C18 (4.6X 250mm, 5-Micron) as a test chromatographic column;
mass spectrum conditions: an electrospray positive ion scanning mode and an electrospray negative ion scanning mode are adopted, the scanning range m/z is 100-3000, the temperature of drying gas is 350 ℃, and the flow rate of a dryer is 12L-min-1Atomizer pressure 35psig, capillary voltage 4000V, lysis voltage 175V, cone voltage 65V, collision energy 50.00V.
Wherein, the notoginseng medicinal liquor is prepared by 21 traditional Chinese medicines such as notoginseng, sappan wood, psoralea fruit, epimedium herb and the like, and has more chemical component types and larger polarity difference, so that gradient elution is considered. Since there are few reports of component analysis of similar formulations, acetonitrile (a) -water (B), methanol (a) -water (B), acetonitrile (a) -0.05% acetic acid (B), acetonitrile (a) -0.05% formic acid (B) were investigated during mobile phase gradient exploration; as a result, it was found that the separation degree can be improved by gradually increasing the organic phase ratio with time; when acetonitrile-water is used as a mobile phase, except notoginsenoside, most of chromatographic peaks have better peak shapes, better separation degree and uniform distribution; aiming at the phenomenon that the forward delay, the tailing and other peak shapes of partial chromatographic peaks are poor, the influence of adding a proper amount of weak acid into a mobile phase on the overall chromatographic behavior is further examined, and the results show that when 0.05 percent of acetic acid is added into water and 0.05 percent of formic acid is added into water, the peak shapes of the chromatographic peaks are sharp, the degrees of separation are good, the retention time of the peaks are basically consistent, but when 0.05 percent of acetic acid is added into acetonitrile, the base line is more stable under the low-wavelength detection condition, the acetonitrile-0.05 percent of acetic acid is selected as the mobile phase, and the final chromatographic condition is obtained after optimization.
Therefore, in order to further improve the separation degree of notoginsenoside and other components, gradient elution is adopted, and the steps comprise: 0 to (20-30) min, 14% A → 20% A by volume fraction; (20-30) - (35-45) min, 20% A → 45% A; (35-45) - (45-55) min, 45% A → 55% A; (45-55) - (65-75) min, 55% A → 65% A; (70-75 min, 65% A → 90% A; 75-90 min, 90% A;
according to the preferable technical scheme, the high performance liquid chromatography mobile phase comprises the following components: acetonitrile (a) -0.05% acetic acid (B) by volume fraction. The step of gradient elution comprises: according to the volume fraction, the time is 0-24 min, 14% A → 20% A; 24-40 min, 20% A → 45% A; 40-50 min, 45% A → 55% A; 50-70 min, 55% A → 65% A; 70-75 min, 65% A → 90% A; 75-90 min, 90% A.
Flow rate 1ml min-1(ii) a The column temperature is 21-29 ℃;
secondly, the notoginseng medicinal liquor has larger polarity difference, such as polysaccharide, amino acid, saponin, flavone, coumarin, organic acid, phenols and the like; the psoralen and isopsoralen are measured by using an HPLC method through isocratic elution in the prior art, but the measurement of main pharmacodynamic components such as notoginsenoside is not realized, because the spectral absorption characteristics of various pharmacodynamic components are different, particularly, the difference of the maximum absorption wavelength of the notoginsenoside and other components is large, the notoginsenoside is easily affected by the gradient elution, the baseline drift is serious, the reproducibility is poor, the quantification is inaccurate and the like, so that the literature report that the notoginsenoside and other components are simultaneously qualitatively or quantitatively detected is basically not found. So that its detection wavelength is 0-30 min-240 nm, and its detection wavelength is 20-30 min-203 nm, and its detection wavelength is 30-40-85-95 min-240 nm; the invention further adjusts the detection wavelength as follows: 240n m for 0-26min, 203nm for 26-36min, and 240nm for 36-90 min.
The HPLC fingerprint of the pseudo-ginseng medicinal liquor is determined by taking ginsenoside rg1, ferulic acid, icariin, psoralen, isopsoralen, protobrazilin B, neopsoralen isoflavone, psoralen flavanone, bakuchiol, ligustilide, psoralen B and 11-carbonyl-beta-acetyl boswellic acid as references.
The method determines 19 peaks as characteristic fingerprint peaks through HPLC analysis of 21 batches of sample solutions according to retention time of each chromatographic peak; and identifying compounds corresponding to 14 characteristic peaks, wherein the peak 1 is original brazilin B, and the retention time is 10.95 min; peak 4 is ferulic acid, retention time is 21.79 min; peak 6 is ginsenoside rg1, retention time is 34.85 min; peak 7 is icariin, retention time is 37.60 min; peak 8 is psoralen, retention time is 41.52 min; peak 9 is isopsoralen, retention time is 42.46 min; peak 10 is neobavachinin, retention time is 50.95 min; peak 11 is psoralen, retention time is 52.62 min; peak 12 is psoralen, retention time is 57.32 min; peak 13 is ligustilide, retention time is 59.17 min; peak 14 is psoralen B, retention time is 62.72 min; peak 16 is bavachinin methyl ether, retention time is 65.08 min; peak 18 is bakuchiol, retention time is 78.82 min; peak 19 is 11-carbonyl- β -acetyl boswellic acid with a retention time of 87.64 min.
(4) And calculating the content of the component to be detected according to the standard product.
Therefore, based on the physical and chemical properties of the main medicinal components of the pseudo-ginseng medicinal liquor, the problem of separation degree of each medicinal component is effectively solved by combining the comprehensive consideration of multiple factors such as chromatographic columns, elution conditions, detection wavelengths and the like, and the purpose of synchronously and quantitatively detecting 14 medicinal components with different physical and chemical properties such as pseudo-ginseng saponin and the like is realized; provides a method for objectively evaluating the quality level of a medicine, and ensures the clinical curative effect of the medicine. The method has good precision, stability and reproducibility, and can meet the requirement of fingerprint spectrum measurement of Notoginseng radix medicated liquor.
Description of the drawings:
FIG. 1 shows the fingerprint of Notoginseng radix medicated liquor.
Wherein peak 1 is protobrazilein B, peak 4 is ferulic acid, peak 6 is ginsenoside rg1, peak 7 is icariin, peak 8 is psoralen, peak 9 is isopsoralen, peak 10 is neobavachin, peak 11 is psoralen A, peak 12 is psoralen, peak 13 is ligustilide, peak 14 is psoralen B, peak 16 is psoralen flavanonol methyl ether, peak 18 is bakuchiol, and peak 19 is 11-carbonyl-beta-acetyl boswellic acid.
FIG. 2 is a chromatogram of a methodological inspection system applicability test in the detection method published by "HPLC method for directly determining the content of psoralen and isopsoralen in pseudo-ginseng medicinal liquor" of Teqingde et al, wherein A is a chromatogram of a mixed reference, 1 is a psoralen peak, and 2 is an isopsoralen peak; FIG. B is a chromatogram of the sample, wherein 1 is a peak of Psoralea corylifolia and 2 is a peak of Psoralea corylifolia; panel C is a negative control chromatogram.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
33 batches of notoginseng medicinal liquor (information is shown in table 1) are collected, S1-S21 are commercially available samples provided by Yunnan Xitao green pharmaceutical industry GmbH, S22-S27 are self-made notoginseng medicinal liquor samples, and S28-S33 are based on the current medicine standard of the notoginseng medicinal liquor (WS)3-B-2079-96) to prepare a single medicated wine.
TABLE 1 Notoginseng radix medicated liquor sample information
Example 1: fingerprint detection method for pseudo-ginseng medicinal liquor
1. Preparation of test and control solutions
Preparation of a test solution: filtering Notoginseng radix medicated liquor S16 sample, and collecting filtrate as test solution.
Preparation of control solutions: taking a proper amount of psoralen reference substance, precisely weighing, and adding ethanol to obtain psoralen reference substance solution.
2. Chromatographic conditions are as follows: the chromatographic column is Welch Ultimate XB-C18, 4.6 multiplied by 250mm, 5-Micron; the mobile phase is as follows: acetonitrile (a) -0.05% acetic acid (B) by volume ratio. The step of gradient elution comprises: 0-24 min, 14% A → 20% A; 24-40 min, 20% A → 45% A; 40-50 min, 45% A → 55% A; 50-70 min, 55% A → 65% A; 70-75 min, 65% A → 90% A; 75-90 min, 90% A.
The detection wavelength is 240nm in 0-26min, 203nm in 26-36min and 240nm in 36-90 min; flow rate 1ml min-1(ii) a The column temperature is 25 ℃; the number of theoretical plates is not less than 8000 according to psoralen peak.
Mass spectrum conditions: an electrospray positive ion scanning mode and an electrospray negative ion scanning mode are adopted, the scanning range m/z is 100-3000, the temperature of drying gas is 350 ℃, and the flow rate of a dryer is 12L-min-1Atomizer pressure 35psig, capillary voltage 4000V, lysis voltage 175V, cone voltage 65V, collision energy 50.00V.
3. And (3) determination: precisely absorbing the test solution and the reference solution respectively, injecting into a high performance liquid chromatograph for determination, recording the chromatogram, obtaining the HPLC fingerprint of the notoginseng medicinal liquor, determining common peaks according to the relative retention time of each chromatogram peak in the chromatogram and the reference, and selecting 19 common peaks as characteristic fingerprint peaks to establish the reference fingerprint of the notoginseng medicinal liquor (figure 1).
The most main chromatographic peaks are identified by LC-MS and standard substance control, and compounds corresponding to 14 common peaks are determined, wherein peak 1 is original brazilein B, peak 4 is ferulic acid, peak 6 is ginsenoside rg1, peak 7 is icariin, peak 8 is psoralen, peak 9 is isopsoralen, peak 10 is neopsoralen isoflavone, peak 11 is psoralen A, peak 12 is psoralen, peak 13 is ligustilide, peak 14 is psoralen B, peak 16 is psoralen methyl ether, peak 18 is bakuchiol, and peak 19 is 11-carbonyl-beta-acetyl boswellic acid.
And calculating the content of the above components, wherein the content of peak 1 original brazilein B is 45.0 μ g/ml, the content of peak 4 ferulic acid is 10.5 μ g/ml, the content of peak 6 ginsenoside rg1 is 1200.0 μ g/ml, the content of peak 7 icariin is 22.4 μ g/ml, the content of peak 8 psoralen is 45.9 μ g/ml, the content of peak 9 isopsoralen is 40.0 μ g/ml, the content of peak 10 neopsoralen is 16.2 μ g/ml, the content of peak 11 psoralen is 10.2 μ g/ml, the content of peak 12 psoralen is 6.7 μ g/ml, the content of peak 13 ligustilide is 118.4 μ g/ml, the content of peak 14 psoralen is 12.2 μ g/ml, the content of peak 16 dihydroflavone methyl ether is 18.2 μ g/ml, and the content of peak 18.6 μ g/ml, peak 1911-carbonyl-beta-acetyl boswellic acid content 63.5. mu.g/ml.
Example 2: fingerprint detection method for pseudo-ginseng medicinal liquor
1. Preparation of test and control solutions
Preparation of a test solution: filtering Notoginseng radix medicated liquor S19 sample, and collecting filtrate as test solution.
Preparation of control solutions: taking a proper amount of psoralen reference substance, precisely weighing, and adding ethanol to obtain psoralen reference substance solution.
2. Chromatographic conditions are as follows: the chromatographic column is Welch Ultimate XB-C18, 4.6 multiplied by 250mm, 5-Micron; the mobile phase is as follows: acetonitrile (a) -water (B) in volume ratio. The step of gradient elution comprises: 0-24 min, 14% A → 20% A; 24-40 min, 20% A → 45% A; 40-50 min, 45% A → 55% A; 50-70 min, 55% A → 65% A; 70-75 min, 65% A → 90% A; 75-90 min, 90% A.
The detection wavelength is 240nm in 0-26min, 203nm in 26-36min and 240nm in 36-90 min; flow rate 1ml min-1(ii) a The column temperature is 25 ℃; the number of theoretical plates is not less than 8000 according to psoralen peak.
Mass spectrum conditions: an electrospray positive ion scanning mode and an electrospray negative ion scanning mode are adopted, the scanning range m/z is 100-3000, the temperature of drying gas is 350 ℃, and the flow rate of a dryer is 12L-min-1Atomizer pressure 35psig, capillary voltage 4000V, lysis voltage 175V, cone voltage 65V, collision energy 50.00V.
3. And (3) determination: precisely absorbing the test solution and the reference solution respectively, injecting into a high performance liquid chromatograph for determination, recording the chromatogram, obtaining the HPLC fingerprint of the notoginseng medicinal liquor, determining common peaks according to the relative retention time of each chromatogram peak and the reference in the chromatogram, selecting 19 common peaks as characteristic fingerprint peaks, and establishing the reference fingerprint of the notoginseng medicinal liquor (figure 1).
The most main chromatographic peaks are identified by LC-MS and standard substance control, and compounds corresponding to 14 common peaks are determined, wherein peak 1 is original brazilein B, peak 4 is ferulic acid, peak 6 is ginsenoside rg1, peak 7 is icariin, peak 8 is psoralen, peak 9 is isopsoralen, peak 10 is neopsoralen isoflavone, peak 11 is psoralen A, peak 12 is psoralen, peak 13 is ligustilide, peak 14 is psoralen B, peak 16 is psoralen methyl ether, peak 18 is bakuchiol, and peak 19 is 11-carbonyl-beta-acetyl boswellic acid.
And calculating the content of the above components, wherein the content of peak 1 original brazilein B is 35.2 μ g/ml, the content of peak 4 ferulic acid is 9.4 μ g/ml, the content of peak 6 ginsenoside rg1 is 1140.0 μ g/ml, the content of peak 7 icariin is 19.6 μ g/ml, the content of peak 8 psoralen is 37.3 μ g/ml, the content of peak 9 isopsoralen is 31.9 μ g/ml, the content of peak 10 neopsoralen is 15.2 μ g/ml, the content of peak 11 psoralen is 7.7 μ g/ml, the content of peak 12 psoralen is 5.8 μ g/ml, the content of peak 13 ligustilide is 92.4 μ g/ml, the content of peak 14 psoralen is 11.9 μ g/ml, the content of peak 16 dihydroflavone is 15.3 μ g/ml, and the content of peak 18 bakuchiol is 174.6 μ g/ml, peak 1911-carbonyl-beta-acetyl boswellic acid content 47.5. mu.g/ml.
Example 3: fingerprint detection method for pseudo-ginseng medicinal liquor
1. Preparation of test and control solutions
Preparation of a test solution: filtering Notoginseng radix medicated liquor S20 sample, and collecting filtrate as test solution.
Preparation of control solutions: taking a proper amount of psoralen reference substance, precisely weighing, and adding ethanol to obtain psoralen reference substance solution.
2. Chromatographic conditions are as follows: the chromatographic column is Welch Ultimate XB-C18, 4.6 multiplied by 250mm, 5-Micron; the mobile phase is as follows: methanol (a) -water (B) in volume ratio. The step of gradient elution comprises: 0-24 min, 14% A → 20% A; 24-40 min, 20% A → 45% A; 40-50 min, 45% A → 55% A; 50-70 min, 55% A → 65% A; 70-75 min, 65% A → 90% A; 75-90 min, 90% A.
The detection wavelength is 240nm in 0-26min, 203nm in 26-36min and 240nm in 36-90 min; flow rate 1ml min-1(ii) a The column temperature is 25 ℃; the number of theoretical plates is not less than 8000 according to psoralen peak.
Mass spectrum conditions: an electrospray positive ion scanning mode and an electrospray negative ion scanning mode are adopted, the scanning range m/z is 100-3000, the temperature of drying gas is 350 ℃, and the flow rate of a dryer is 12L-min-1Atomizer pressure 35psig, capillary voltage 4000V, lysis voltage 175V, cone voltage 65V, collision energy 50.00V.
3. And (3) determination: precisely absorbing the test solution and the reference solution respectively, injecting into a high performance liquid chromatograph for determination, recording the chromatogram, obtaining the HPLC fingerprint of the notoginseng medicinal liquor, determining common peaks according to the relative retention time of each chromatogram peak and the reference in the chromatogram, selecting 19 common peaks as characteristic fingerprint peaks, and establishing the reference fingerprint of the notoginseng medicinal liquor (figure 1).
The most main chromatographic peaks are identified by LC-MS and standard substance control, and compounds corresponding to 14 common peaks are determined, wherein peak 1 is original brazilein B, peak 4 is ferulic acid, peak 6 is ginsenoside rg1, peak 7 is icariin, peak 8 is psoralen, peak 9 is isopsoralen, peak 10 is neopsoralen isoflavone, peak 11 is psoralen A, peak 12 is psoralen, peak 13 is ligustilide, peak 14 is psoralen B, peak 16 is psoralen methyl ether, peak 18 is bakuchiol, and peak 19 is 11-carbonyl-beta-acetyl boswellic acid.
And calculating the content of the above components, wherein the content of peak 1 original brazilein B is 37.3 μ g/ml, the content of peak 4 ferulic acid is 8.2 μ g/ml, the content of peak 6 ginsenoside rg1 is 972.0 μ g/ml, the content of peak 7 icariin is 18.0 μ g/ml, the content of peak 8 psoralen is 32.6 μ g/ml, the content of peak 9 isopsoralen is 37.5 μ g/ml, the content of peak 10 neopsoralen is 13.9 μ g/ml, the content of peak 11 psoralen is 7.2 μ g/ml, the content of peak 12 psoralen is 5.0 μ g/ml, the content of peak 13 ligustilide is 85.6 μ g/ml, the content of peak 14 psoralen is 10.2 μ g/ml, the content of peak 16 dihydroflavone is 14.1 μ g/ml, and the content of peak 18 bakuchiol is 153.3 μ g/ml, peak 1911-carbonyl-beta-acetyl boswellic acid content 42.3. mu.g/ml.
Example 4: method for detecting fingerprint characteristic components of pseudo-ginseng medicinal liquor
1. Preparation of test and control solutions
Preparation of a test solution: filtering Notoginseng radix medicated liquor S21 sample, and collecting filtrate as test solution.
Preparation of control solutions: taking a proper amount of psoralen reference substance, precisely weighing, and adding ethanol to obtain psoralen reference substance solution.
2. Chromatographic conditions are as follows: the chromatographic column is Welch Ultimate XB-C18, 4.6 multiplied by 250mm, 5-Micron; the mobile phase is as follows: acetonitrile (a) -0.05% formic acid (B) by volume. The step of gradient elution comprises: 0-24 min, 14% A → 20% A; 24-40 min, 20% A → 45% A; 40-50 min, 45% A → 55% A; 50-70 min, 55% A → 65% A; 70-75 min, 65% A → 90% A; 75-90 min, 90% A.
The detection wavelength is 240nm in 0-26min, 203nm in 26-36min and 240nm in 36-90 min; flow rate 1ml min-1(ii) a The column temperature is 25 ℃; the number of theoretical plates is not less than 8000 according to psoralen peak.
Mass spectrum conditions: an electrospray positive ion scanning mode and an electrospray negative ion scanning mode are adopted, the scanning range m/z is 100-3000, the temperature of drying gas is 350 ℃, and the flow rate of a dryer is 12L-min-1Atomizer pressure 35psig, capillary voltage 4000V, lysis voltage 175V, cone voltage 65V, collision energy 50.00V.
3. And (3) determination: precisely absorbing the test solution and the reference solution respectively, injecting into a high performance liquid chromatograph for determination, recording the chromatogram, obtaining the HPLC fingerprint of the notoginseng medicinal liquor, determining common peaks according to the relative retention time of each chromatogram peak and the reference in the chromatogram, selecting 19 common peaks as characteristic fingerprint peaks, and establishing the reference fingerprint of the notoginseng medicinal liquor (figure 1).
The most main chromatographic peaks are identified by LC-MS and standard substance control, and compounds corresponding to 14 common peaks are determined, wherein peak 1 is original brazilein B, peak 4 is ferulic acid, peak 6 is ginsenoside rg1, peak 7 is icariin, peak 8 is psoralen, peak 9 is isopsoralen, peak 10 is neopsoralen isoflavone, peak 11 is psoralen A, peak 12 is psoralen, peak 13 is ligustilide, peak 14 is psoralen B, peak 16 is psoralen methyl ether, peak 18 is bakuchiol, and peak 19 is 11-carbonyl-beta-acetyl boswellic acid.
And calculating the content of the above components, wherein the content of peak 1 original brazilein B is 41.2 μ g/ml, the content of peak 4 ferulic acid is 9.6 μ g/ml, the content of peak 6 ginsenoside rg1 is 1090.0 μ g/ml, the content of peak 7 icariin is 20.7 μ g/ml, the content of peak 8 psoralen is 39.7 μ g/ml, the content of peak 9 isopsoralen is 42.0 μ g/ml, the content of peak 10 neopsoralen is 16.2 μ g/ml, the content of peak 11 psoralen is 9.4 μ g/ml, the content of peak 12 psoralen is 6.3 μ g/ml, the content of peak 13 ligustilide is 106.2 μ g/ml, the content of peak 14 psoralen is 12.0 μ g/ml, the content of peak 16 dihydroflavone is 16.4 μ g/ml, and the content of peak 18 bakuchiol is 200.0 μ g/ml, peak 1911-carbonyl-beta-acetyl boswellic acid content 53.1. mu.g/ml.
Example 5: investigation of influence factors of fingerprint of pseudo-ginseng medicinal liquor
1. Preparation of test and control solutions
Preparation of a test solution: filtering Notoginseng radix medicated liquor S1 sample, and collecting filtrate as test solution.
Preparation of control solutions: taking a proper amount of psoralen reference substance, precisely weighing, and adding ethanol to obtain psoralen reference substance solution.
2. Chromatographic conditions are as follows: the chromatographic column is Welch Ultimate XB-C18, 4.6 multiplied by 250mm, 5-Micron; the mobile phase is as follows: acetonitrile (a) -0.05% acetic acid (B) by volume ratio. The step of gradient elution comprises: 0-24 min, 14% A → 20% A; 24-40 min, 20% A → 45% A; 40-50 min, 45% A → 55% A; 50-70 min, 55% A → 65% A; 70-75 min, 65% A → 90% A; 75-90 min, 90% A.
The detection wavelength is 240nm at 0-26min and 26-3 nm203nm for 6min and 240nm for 36-90 min; flow rate 1ml min-1(ii) a The column temperature is 21-29 ℃; the number of theoretical plates is not less than 8000 according to psoralen peak.
Mass spectrum conditions: an electrospray positive ion scanning mode and an electrospray negative ion scanning mode are adopted, the scanning range m/z is 100-3000, the temperature of drying gas is 350 ℃, the flow rate of a dryer is 12 L.min < -1 >, the pressure of an atomizer is 35psig, the voltage of a capillary tube is 4000V, the cracking voltage is 175V, the voltage of a conical hole is 65V, and the collision energy is 50.00V.
3. And (3) determination: respectively and precisely absorbing the test solution and the reference solution, injecting the test solution and the reference solution into a high performance liquid chromatograph for determination, detecting at different column temperatures, recording a chromatogram to obtain an HPLC fingerprint of the notoginseng medicinal liquor, introducing the HPLC fingerprint into traditional Chinese medicine fingerprint similarity evaluation system (2012 edition) software with the fingerprint obtained in the embodiment 1, and performing similarity analysis and comparison, wherein the similarity is not less than 0.90 (see table 2).
TABLE 2 degree of similarity of finger-print of pseudo-ginseng medicinal liquor with different column temperatures
Example 6: methodology investigation of fingerprint spectrum of pseudo-ginseng medicinal liquor
1. And (3) precision test: taking a sample solution of the sample No. S14, carrying out continuous sample injection for 6 times, recording a chromatogram, introducing the chromatogram into software of a traditional Chinese medicine fingerprint similarity evaluation system (2012 edition), and carrying out similarity evaluation, wherein the result shows that the similarity of common peaks of the samples measured at each time is greater than 0.99, which indicates that the precision of the method is good.
2. And (3) repeatability test: taking 6 parts of sample No. S14, preparing a test solution according to the method of example 1, injecting samples respectively, recording chromatograms, introducing the chromatograms into software of a traditional Chinese medicine fingerprint similarity evaluation system (2012 edition), and carrying out similarity evaluation, wherein the result shows that the similarity of the common peak of each measured sample is greater than 0.99, which indicates that the method has good repeatability.
3. Intermediate precision: taking a sample of No. S14, preparing a test sample solution by 3 groups of personnel at time points of 3 different dates by the method of example 1, detecting on different liquid chromatographs (an Agilent 1260 binary pump of the liquid chromatograph and a quaternary pump of the Agilent 1260 of the liquid chromatograph) by using the same chromatographic column, recording chromatograms, introducing the chromatograms into software of a traditional Chinese medicine fingerprint similarity evaluation system (2012 edition), and carrying out similarity evaluation, wherein the result shows that the similarity of common peaks of the samples measured at each time is greater than 0.99, and the intermediate precision of the method is good.
4. And (3) solution stability test: taking the sample solution of the sample No. S14, respectively measuring in 0, 2, 4, 8, 12, 16, 24 and 48h according to the chromatographic conditions of the example 1, recording a chromatogram, introducing the chromatogram into the software of a traditional Chinese medicine fingerprint similarity evaluation system (2012 edition) for similarity evaluation, and displaying that the similarity of the common peak of each measured sample is more than 0.99, which indicates that the sample solution is stable in 48 h.
The methodology investigation test shows that the method has good precision, stability and reproducibility, and can meet the fingerprint spectrum determination requirement of the pseudo-ginseng medicinal liquor.
Example 7: fingerprint similarity evaluation of pseudo-ginseng medicinal liquor of different batches
The quality monitoring of the pseudo-ginseng medicinal liquor is carried out by utilizing the fingerprint spectrum, and the specific method comprises the following steps: (1) preparing a sample solution from 21 batches of the pseudo-ginseng medicinal liquor according to the method in the embodiment 1, and constructing a fingerprint of the pseudo-ginseng medicinal liquor to be detected according to the method in the embodiment 1;
(2) the spectra were imported into the software of the fingerprint similarity evaluation system (2012 edition) of chinese traditional medicine, and the similarity of samples of each batch was calculated using the common pattern of characteristic spectra as a control (see table 3). The similarity between the 21 batches of the pseudo-ginseng medicinal liquor and the reference fingerprint is more than 0.99, which indicates that the quality of each batch of the pseudo-ginseng medicinal liquor is stable.
Similarity value of fingerprint of notoginseng medicinal liquor in Table 321 batch
Similarity value of fingerprint of pseudo-ginseng medicinal liquor in Table 321 batch
Claims (5)
1. A fingerprint spectrum construction and content determination method of pseudo-ginseng medicinal liquor is characterized by comprising the following steps:
(1) preparation of a test solution: filtering Notoginseng radix medicated liquor, and collecting filtrate as test solution;
(2) preparation of control solutions: taking a proper amount of psoralen reference substance, precisely weighing, and adding ethanol to prepare a reference substance solution;
(3) performing high performance liquid chromatography analysis on the test solution to obtain HPLC fingerprint, determining common peaks according to relative retention time of the test solution and the reference, adopting chemical components of LC-MS fingerprint characteristic fingerprint peaks, and establishing HPLC fingerprint of the Notoginseng radix medicated liquor by using standard control; the conditions of the high performance liquid chromatography analysis are as follows:
the chromatographic column is Welch Ultimate XB-C18, 4.6 multiplied by 250mm, 5-Micron;
mass spectrum conditions: an electrospray positive ion scanning mode and an electrospray negative ion scanning mode are adopted, the scanning range m/z is 100-3000, the temperature of drying gas is 350 ℃, and the flow rate of a dryer is 12L-min-1Atomizer pressure 35psig, capillary voltage 4000V, lysis voltage 175V, cone voltage 65V, collision energy 50.00V;
the mobile phase is any one of acetonitrile (A) -water (B), methanol (A) -water (B), acetonitrile (A) -0.05% acetic acid (B) and acetonitrile (A) -0.05% formic acid (B);
gradient elution: 0 to (20-30) min, 14% A → 20% A by volume fraction; (20-30) - (35-45) min, 20% A → 45% A; (35-45) - (45-55) min, 45% A → 55% A; (45-55) - (65-75) min, 55% A → 65% A; 70-75 min, 65% A → 90% A; 75-90 min, 90% A;
the flow rate of the mobile phase is 1 ml/min-1；
The column temperature is 21-29 ℃;
the detection wavelength is as follows:
is a light-emitting diode (LED) with the wavelength of 240nm,
is the light wave with the wavelength of 203nm,
is 240 nm;
(4) and calculating the content of the component to be detected according to the standard product.
2. The method for constructing the fingerprint spectrum and measuring the content of the pseudo-ginseng medicinal liquor according to claim 1, which is characterized in that: the gradient elution step described in step (3) comprises: according to the volume fraction, the time is 0-24 min, 14% A → 20% A; 24-40 min, 20% A → 45% A; 40-50 min, 45% A → 55% A; 50-70 min, 55% A → 65% A; 70-75 min, 65% A → 90% A; 75-90 min, 90% A.
3. The method for constructing the fingerprint of the notoginseng medicinal liquor and measuring the content of the notoginseng medicinal liquor according to claim 1, which is characterized in that: the detection wavelength in the step (3) is 240nm in 0-26min, 203nm in 26-36min and 240nm in 36-90 min.
4. The method for constructing the fingerprint of the notoginseng medicinal liquor and measuring the content of the notoginseng medicinal liquor according to any one of claims 1 to 3, which is characterized in that: the mobile phase in step (3) is acetonitrile (a) -0.05% acetic acid (B).
5. The method for constructing the fingerprint spectrum and measuring the content of the pseudo-ginseng medicinal liquor according to claim 1, which is characterized in that: the number of the characteristic peaks in the step (3) is 14, wherein the peak 1 is the original brazilein B, and the retention time is 10.95 min; peak 4 is ferulic acid, retention time is 21.79 min; peak 6 is ginsenoside rg1, retention time is 34.85 min; peak 7 is icariin, retention time is 37.60 min; peak 8 is psoralen, retention time is 41.52 min; peak 9 is isopsoralen, retention time is 42.46 min; peak 10 is neobavachinin, retention time is 50.95 min; peak 11 is psoralen, retention time is 52.62 min; peak 12 is psoralen, retention time is 57.32 min; peak 13 is ligustilide, retention time is 59.17 min; peak 14 is psoralen B, retention time is 62.72 min; peak 16 is bavachinin methyl ether, retention time is 65.08 min; peak 18 is bakuchiol, retention time is 78.82 min; peak 19 is 11-carbonyl- β -acetyl boswellic acid with a retention time of 87.64 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910629060.2A CN112213404B (en) | 2019-07-12 | 2019-07-12 | Fingerprint construction and content determination method of pseudo-ginseng medicinal liquor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910629060.2A CN112213404B (en) | 2019-07-12 | 2019-07-12 | Fingerprint construction and content determination method of pseudo-ginseng medicinal liquor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112213404A true CN112213404A (en) | 2021-01-12 |
| CN112213404B CN112213404B (en) | 2022-11-15 |
Family
ID=74047841
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910629060.2A Active CN112213404B (en) | 2019-07-12 | 2019-07-12 | Fingerprint construction and content determination method of pseudo-ginseng medicinal liquor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112213404B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113406233A (en) * | 2021-06-20 | 2021-09-17 | 无锡济煜山禾药业股份有限公司 | Method for determining content of ferulic acid and catalpol in spleen-tonifying and waist-strengthening medicinal liquor |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103969346A (en) * | 2013-01-25 | 2014-08-06 | 天士力制药集团股份有限公司 | Panax notoginseng saponin component analysis method |
| CN108362800A (en) * | 2018-05-08 | 2018-08-03 | 亳州中药材商品交易中心有限公司 | The construction method and HPLC finger-prints of HPLC finger-prints |
| CN108872417A (en) * | 2018-05-08 | 2018-11-23 | 亳州中药材商品交易中心有限公司 | A kind of construction method and HPLC finger-print of finger-print |
| CN109486621A (en) * | 2017-09-08 | 2019-03-19 | 刘志彪 | A kind of Bulbilus boussingaultiae pseudobaselloidis liquid medicine and preparation method thereof |
| CN109692302A (en) * | 2017-10-20 | 2019-04-30 | 云南希陶绿色药业股份有限公司 | A kind of preparation process of radix notoginseng medicated wine for oral administration that treating treating rheumatic ostealgia |
-
2019
- 2019-07-12 CN CN201910629060.2A patent/CN112213404B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103969346A (en) * | 2013-01-25 | 2014-08-06 | 天士力制药集团股份有限公司 | Panax notoginseng saponin component analysis method |
| CN109486621A (en) * | 2017-09-08 | 2019-03-19 | 刘志彪 | A kind of Bulbilus boussingaultiae pseudobaselloidis liquid medicine and preparation method thereof |
| CN109692302A (en) * | 2017-10-20 | 2019-04-30 | 云南希陶绿色药业股份有限公司 | A kind of preparation process of radix notoginseng medicated wine for oral administration that treating treating rheumatic ostealgia |
| CN108362800A (en) * | 2018-05-08 | 2018-08-03 | 亳州中药材商品交易中心有限公司 | The construction method and HPLC finger-prints of HPLC finger-prints |
| CN108872417A (en) * | 2018-05-08 | 2018-11-23 | 亳州中药材商品交易中心有限公司 | A kind of construction method and HPLC finger-print of finger-print |
Non-Patent Citations (1)
| Title |
|---|
| 崔庆德等: "HPLC法直接测定三七药酒中补骨脂素与异补骨脂素的含量", 《中国药品标准》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113406233A (en) * | 2021-06-20 | 2021-09-17 | 无锡济煜山禾药业股份有限公司 | Method for determining content of ferulic acid and catalpol in spleen-tonifying and waist-strengthening medicinal liquor |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112213404B (en) | 2022-11-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yan et al. | Advancement in the chemical analysis of Paeoniae Radix (Shaoyao) | |
| Yu et al. | Determination of seventeen main flavonoids and saponins in the medicinal plant Huang‐qi (Radix Astragali) by HPLC‐DAD‐ELSD | |
| Qi et al. | Quality evaluation of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors | |
| Qiao et al. | Chemical fingerprint and quantitative analysis of Fructus Psoraleae by high‐performance liquid chromatography | |
| Yan et al. | Simultaneous determination of puerarin, daidzin, daidzein, paeoniflorin, albiflorin, liquiritin and liquiritigenin in rat plasma and its application to a pharmacokinetic study of Ge-Gen Decoction by a liquid chromatography–electrospray ionization-tandem mass spectrometry | |
| CN105353053B (en) | The content assaying method of scutellarin and scutellarin in a kind of Sculellaria barbata medicinal material or its granule | |
| Wang et al. | Quantitative analysis of flavonoids, alkaloids and saponins of Banxia Xiexin decoction using ultra-high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry | |
| Zhang et al. | Development of a matrix solid-phase dispersion extraction combined with high-performance liquid chromatography for determination of five lignans from the Schisandra chinensis | |
| Li et al. | Simultaneous determination of main phytoecdysones and triterpenoids in radix achyranthis bidentatae by high-performance liquid chromatography with diode array-evaporative light scattering detectors and mass spectrometry | |
| CN110412155B (en) | Detection method of HPLC (high performance liquid chromatography) characteristic spectrum of Erdi decoction | |
| CN114152686B (en) | Fingerprint construction method of traditional Chinese medicine compound containing cinnamon and application of fingerprint construction method | |
| Lee et al. | Quantification of isoflavonoids and triterpene saponins in Astragali Radix, the root of Astragalus membranaceus, via reverse-phase high-performance liquid chromatography coupled with integrated pulsed amperometric detection | |
| Bardakci et al. | Simultaneous quantification of six flavonoids in four Scutellaria taxa by HPLC-DAD method | |
| CN110108827B (en) | Method for simultaneously determining eight active ingredients in antipyretic and antitoxic tablet | |
| Ong et al. | Evaluation of pressurized liquid extraction and pressurized hot water extraction for tanshinone I and IIA in Salvia miltiorrhiza using LC and LC-ESI-MS | |
| CN102028773A (en) | Method for identifying radix rosae gigantea extract | |
| Chen et al. | Quality control of a herbal medicinal preparation using high-performance liquid chromatographic and capillary electrophoretic methods | |
| CN112213404B (en) | Fingerprint construction and content determination method of pseudo-ginseng medicinal liquor | |
| Du et al. | Chemical profiling and marker characterization of Huangqin decoction prepared with three types of peony root by liquid chromatography with electrospray ionization mass spectrometry | |
| CN110441413B (en) | Construction method and detection method of HPLC fingerprint of Qianbai rhinitis tablets | |
| Shakya et al. | Development and validation of a high-performance thin-layer chromatography method for the quantification of rutin in the fruit pulp of Benincasa hispida (Thunb.) Cogniaux | |
| Huang et al. | Full component analysis of Tianma-Gouteng-Yin | |
| CN113341007B (en) | HPLC (high Performance liquid chromatography) characteristic spectrum-based method for measuring content of all ingredients of jujube kernel nerve-soothing capsules | |
| CN110687219B (en) | Detection method of suhuang cough-relieving capsule fingerprint and application thereof | |
| CN114636779A (en) | Method for constructing sanhua decoction reference sample freeze-dried powder fingerprint spectrum and fingerprint spectrum thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 655000 Ruihe West Road, Qujing Economic and Technological Development Zone, Yunnan Province Applicant after: Yunnan kangenbeixitao Pharmaceutical Co.,Ltd. Address before: 655000 Ruihe West Road, Qujing Economic and Technological Development Zone, Yunnan Province Applicant before: YUNNAN XITAO GREEN PHARMACEUTICAL Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |