CN115236242A - Fingerprint spectrum of four-ingredient powder of Angongniuhuang pill and detection method of components of four-ingredient powder - Google Patents
Fingerprint spectrum of four-ingredient powder of Angongniuhuang pill and detection method of components of four-ingredient powder Download PDFInfo
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- CN115236242A CN115236242A CN202211024547.6A CN202211024547A CN115236242A CN 115236242 A CN115236242 A CN 115236242A CN 202211024547 A CN202211024547 A CN 202211024547A CN 115236242 A CN115236242 A CN 115236242A
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Abstract
The invention provides a method for detecting fingerprint spectrums of four-ingredient powder of Angongniuhuang pills. The invention also provides application of the fingerprint detection method of the four-ingredient powder of the AngongNiuhuang pill in quality detection of components in the four-ingredient powder of the AngongNiuhuang pill and a quality detection method thereof. The invention further provides a method for measuring the content of 9 components in the four-component powder of the AngongNiuhuang pill. The invention further provides a screening method of the fingerprint spectrums of the multiple medicinal materials in the four-flavor powder of the Angong Niuhuang Wan. According to the fingerprint spectrum and the detection method of the ingredients of the four-ingredient powder of the AngongNiuhuang pill, the high performance liquid phase fingerprint spectrum of the four-ingredient powder of the AngongNiuhuang pill is established, 9 chemical ingredients in the four-ingredient powder of the AngongNiuhuang pill can be quantitatively analyzed, the attribution of each single medicinal material chromatographic peak in the four-ingredient powder of the AngongNiuhuang pill is confirmed, the source of the raw materials can be effectively monitored, and the quality of the raw medicinal materials is strictly controlled.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine component detection, relates to fingerprint spectrums of four kinds of powder of Angongniuhuang pills and a detection method of components of the four kinds of powder, and particularly relates to fingerprint spectrums of four kinds of powder of Angongniuhuang pills and 9 main effective components of the four kinds of powder of Angongniuhuang pills: detection method of berberine hydrochloride, palmatine hydrochloride, coptisine, epiberberine, baicalin, wogonoside, baicalein, wogonin, and geniposide.
Background
The Angong Niuhuang Wan, first recorded in Qing. Wu Jutong "Wen Bing tiao Bian (diagnosis of Wen disease), is a famous and precious Chinese patent medicine commonly used in treating high fever apoplexy in traditional Chinese medicine, is the first of the" Wen Bing san Bao "prescription for clearing heat and inducing resuscitation in traditional Chinese medicine, is well known as" rescue emergency in time, and is really dangerous to be marked in hectare "and recorded in the Chinese pharmacopoeia of the calendar edition. The medicine is prepared from calculus bovis, pulvis Cornus Bubali Concentratus, moschus or artificial Moschus, margarita, cinnabaris, realgar, coptidis rhizoma, scutellariae radix, fructus Gardeniae, radix Curcumae and Borneolum Syntheticum by refining Margarita with water or pulverizing into fine powder; respectively refining Cinnabaris and Realgar with water to obtain superfine powder; pulverizing Coptidis rhizoma, scutellariae radix, fructus Gardeniae, and radix Curcumae into fine powder; grinding calculus bovis, pulvis Cornus Bubali Concentratus, moschus or artificial Moschus, and Borneolum Syntheticum, mixing with the above powder, grinding, sieving, mixing, adding appropriate amount of refined honey, and making into big honeyed pill or gold coating.
The four-ingredient powder of the Angongniuhuang pill is one of main intermediates in the production process of the Angongniuhuang pill preparation, and the intermediate is prepared by mixing and crushing four Chinese medicinal materials of coptis root, scutellaria root, gardenia and curcuma root. The traditional Chinese medicine composition is mainly used for treating diseases such as heat-clearing and detoxifying, pathogenic factors entering pericardium, febrile convulsion, coma and delirium, stroke coma and the like, is mainly used for treating viral encephalitis, cerebral ischemia, stroke and hyperpyrexia and syncope caused by the stroke, intractable epilepsy and infantile convulsion clinically, and has better clinical effect.
Quality control identification items of Angongniuhuang pill from the 2020 edition of Chinese pharmacopoeia include microscopic characteristic identification of buffalo horn concentrated powder, margarita, cinnabaris, realgar, coptidis rhizoma, scutellariae radix, fructus Gardeniae, and radix Curcumae, and thin layer identification of calculus bovis, scutellariae radix, coptidis rhizoma, borneolum Syntheticum, and Moschus; the content measurement items include bilirubin in calculus bovis, baicalin and berberine hydrochloride in Scutellariae radix and Coptidis rhizoma. However, the finger print and the components of the four-ingredient powder of the Angong Niuhuang Wan have not been studied comprehensively.
The traditional Chinese medicine fingerprint has the characteristics of integrity, macroscopicity, fuzzy analysis and the like, can comprehensively reflect the types and the quantity of chemical components contained in the traditional Chinese medicine by describing the integral characteristics of the traditional Chinese medicine and adopting a proper fuzzy processing mode, and achieves the aim of integral quality control, thereby becoming an effective means for the quality control of the traditional Chinese medicine and being widely applied to the field of the quality monitoring and evaluation of the traditional Chinese medicine. The chromatographic fingerprint analysis can enable the overall characteristics of various chemical components contained in the traditional Chinese medicine to be visualized, thereby revealing the quality problem which is difficult to find by conventional inspection. The fingerprint spectrum is a modern Chinese medicine quality control method from the perspective of 'full components' according to the characteristics of the overall comprehensive action of multiple components and multiple target points of the Chinese medicine, and the quality control of the non-single component medicine is more comprehensive. At present, systematic qualitative and quantitative research on four kinds of powder of Angongniuhuang pills is lacked. Therefore, it is necessary to establish a fingerprint spectrum and a multi-component content determination method of the four powders of the Angongniuhuang Wan, which can complete qualitative and quantitative analysis of the components in the four powders of the Angongniuhuang Wan so as to effectively control the quality of the four powders of the Angongniuhuang Wan.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a fingerprint spectrum of the four-component powder of the Angong Niuhuang Wan and a detection method of components of the four-component powder of the Angong Niuhuang Wan, wherein the pretreatment and high performance liquid chromatography methods of optimized conditions are adopted to establish the high performance liquid fingerprint spectrum of the four-component powder of the Angong Niuhuang Wan, and the systematic contribution of different chemical components to the fingerprint spectrum of the four-component powder of the Angong Niuhuang Wan is highlighted from different sides, so that the detection of the full-range chemical components of the four-component powder of the Angong Niuhuang Wan is realized, the current situation of each component in the four-component powder of the Angong Niuhuang Wan is comprehensively reflected, and a reference basis is provided for the overall control and evaluation of the quality of the four-component powder of the Angong Niuhuang Wan.
In order to achieve the above and other related objects, a first aspect of the present invention provides a method for detecting fingerprint of four powders of Angongniuhuang Wan, comprising the steps of:
1) Preparation of a test solution: dissolving four kinds of powder samples of Angongniuhuang pill in solvent, ultrasonic extracting, cooling, shaking, and filtering to obtain test solution;
2) Preparation of control solutions: dissolving berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, baicalein, wogonin, geniposide, and wogonoside in solvent to desired volume to obtain reference solution;
3) And (3) determination: respectively measuring the test solution and the reference solution by adopting a High Performance Liquid Chromatography (HPLC) method with the same chromatographic conditions to obtain a fingerprint of the test solution and a fingerprint of the reference solution, comparing the fingerprint of the test solution with the fingerprint of the reference solution, and performing attribution positioning on index components in the fingerprint of the test solution, thereby obtaining the fingerprint of the four-flavor powder of the AngongNiuhuang pill.
Preferably, in the step 1), the four-ingredient powder of the bezoar chest functioning pill is in a powder shape.
Preferably, in the step 1), the four-ingredient powder sample of the bezoar chest functioning pill is prepared before the test solution is prepared 60 Co-gamma ray irradiation.
More preferably, the 60 The dosage of Co-gamma ray irradiation is 0-10KGy, preferably one of 0KGy, 6KGy and 10 KGy. When said 60 When the dosage of Co-gamma ray irradiation is 0KGy, the irradiation is not carried out.
More preferably, the 60 The maximum absorbed dose of Co-gamma ray irradiation is less than or equal to 6KGy.
The described 60 Co-gamma ray irradiation sterilization technology refers to the utilization of 60 The gamma ray generated by Co can kill the microbes on the surface and inside of the material. Because the traditional Chinese medicine is applied to the root and the rhizome, the traditional Chinese medicine grows in the strainIn a soil environment with abundant and complicated microorganisms, the medicinal materials still carry a certain amount of microorganisms after being cleaned and cut. Especially for Chinese patent medicine varieties which are directly powdered for use, the requirement on the initial bacteria content of the medicinal materials is strict, or the bacteria content of finished products exceeds the standard. The irradiation with a certain radiation amount can effectively reduce the content of microorganisms in the traditional Chinese medicine, and simultaneously reduce the microbial load, thereby achieving the effect of sterilizing the traditional Chinese medicine. Therefore, it is necessary to perform irradiation sterilization on crude drug powder of raw medicinal materials of a Chinese medicinal preparation, thereby further reducing the microbial load.
Preferably, in the step 1), the ratio of the added weight (g) of the four-ingredient powder sample of the bezoar chest functioning pill to the added volume (mL) of the solvent is 0.05:15-25.
More preferably, the ratio of the added weight (g) of the four-flavor powder sample of the bezoar chest functioning pill to the added volume (mL) of the solvent is 0.05:20.
preferably, in step 1) or 2), the solvent is 50-100% methanol. More preferably, the solvent is 75% methanol.
The 50-100% methanol is 50-100% methanol water solution. The 75% methanol is 75% methanol water solution by volume percentage.
Preferably, in the step 1), the four powder samples of the bezoar chest functioning pill need to be precisely weighed after the solvent is added.
Preferably, in the step 1), the ultrasonic extraction time is 30-60min. More preferably, the ultrasound extraction time is 45min.
Preferably, in step 1), the power of the ultrasonic extraction is 200-300W, and the frequency of the ultrasonic extraction is 30-50kHz. More preferably, the power of the ultrasonic extraction is 250W, and the frequency of the ultrasonic extraction is 40kHz.
Preferably, in step 1), the cooling is followed by reweighing and top-up loss with solvent.
Preferably, in the step 1), standing is needed for a moment after shaking up.
More preferably, the standing is for a moment to allow the solution after ultrasonic extraction to stratify.
Preferably, in step 1), the filtration is a supernatant filtration membrane, and after discarding the primary filtrate, a subsequent filtrate is taken.
More preferably, the filter is a 0.45 μm filter.
Preferably, in the step 2), the reference solution is prepared by stepwise dilution.
More preferably, in the reference stock solution adopted by the stepwise dilution, the content range of berberine hydrochloride is 43.60-87.20 μ g/mL, the content range of palmatine hydrochloride is 8.98-17.96 μ g/mL, the content range of epiberberine is 8.04-16.08 μ g/mL, the content range of coptisine is 15.10-30.20 μ g/mL, the content range of baicalin is 101.95-203.90 μ g/mL, the content range of wogonin is 18.01-36.02 μ g/mL, the content range of baicalein is 3.01-6.02 μ g/mL, the content range of wogonin is 2.25-4.50 μ g/mL, and the content range of geniposide is 40.51-81.02 μ g/mL.
Preferably, in the step 2), the CAS number of the berberine hydrochloride is 633-65-8, the CAS number of the palmatine hydrochloride is 10605-02-4, the CAS number of the epiberberine is 6873-09-2, the CAS number of the coptisine is 6020-18-4, the CAS number of the baicalin is 21967-41-9, the CAS number of the wogonin is 51059-44-0, the CAS number of the baicalin is 491-67-8, the CAS number of the wogonin is 632-85-9, and the CAS number of the geniposide is 24514-63-8.
Preferably, in the step 2), the content range of berberine hydrochloride in the reference solution is 0.087-1.744 μ g/mL, the content range of palmatine hydrochloride is 0.018-0.359 μ g/mL, the content range of epiberberine is 0.016-0.322 μ g/mL, the content range of coptisine is 0.030-0.604 μ g/mL, the content range of baicalin is 0.203-4.078 μ g/mL, the content range of wogonin is 0.036-0.720 μ g/mL, the content range of baicalein is 0.006-0.120 μ g/mL, the content range of wogonin is 0.005-0.100 μ g/mL, and the content range of geniposide is 0.081-1.620 μ g/mL.
Preferably, in the step 3), the fingerprint of the test solution is compared with the fingerprint of the reference solution, and the corresponding characteristic peak in the fingerprint of the test solution is identified through the relative retention time according to the known characteristic peak in the fingerprint of the reference solution, so as to perform attribution positioning on the index component in the fingerprint of the test solution.
Preferably, in the step 3), the column in the high performance liquid chromatography is a C18 column. More preferably, the column in the high performance liquid chromatography is an Agilent TC-C18 column (4.6 mm. Times.250mm, 5 μm).
Preferably, in step 3), the detector in the high performance liquid chromatography is a photodiode array detector (DAD).
Preferably, in the step 3), the column temperature in the high performance liquid chromatography is 20 to 30 ℃. More preferably, the column temperature in the high performance liquid chromatography is 26 ℃.
Preferably, in the step 3), the sample amount in the high performance liquid chromatography is 1-20 μ L. More preferably, the sample size in the high performance liquid chromatography is 10. Mu.L.
Preferably, in the step 3), the flow rate in the high performance liquid chromatography is 0.5-1.0mL/min. More preferably, in the high performance liquid chromatography, the flow rate is 0.8mL/min.
Preferably, in the step 3), the detection wavelength in the high performance liquid chromatography is 220-240nm. More preferably, in the high performance liquid chromatography, the detection wavelength is 230nm.
Preferably, in the step 3), the mobile phase in the high performance liquid chromatography is acetonitrile-0.1-0.3% phosphoric acid water solution, wherein the phase A is acetonitrile, and the phase B is 0.1-0.3% phosphoric acid water solution; the analysis time is 90min; and (4) gradient elution.
More preferably, in the high performance liquid chromatography, the mobile phase is acetonitrile-0.2% phosphoric acid aqueous solution, wherein the phase A is acetonitrile, and the phase B is 0.2% phosphoric acid aqueous solution; the analysis time is 90min; gradient elution.
The 0.1-0.3% phosphoric acid aqueous solution is 0.1-0.3% phosphoric acid aqueous solution by volume percentage. The 0.2% phosphoric acid aqueous solution is 0.2% phosphoric acid aqueous solution in volume percentage.
More preferably, the specific procedure of the gradient elution is:
0-5min, phase A: the volume ratio of the phase B is 5:95-11:89;
5-25min, phase A: the volume ratio of the phase B is 11:89-23:77;
25-35min, phase A: the volume ratio of the phase B is 23:77-25:75;
35-45min, phase A: phase B volume ratio is 25:75-26:74;
45-55min, phase A: the volume ratio of the phase B is 26:74-31:69;
55-60min, phase A: the volume ratio of the phase B is 31:69-39:61;
60-70min, phase A: the volume ratio of the phase B is 39:61-69:31;
70-80min, phase A: the volume ratio of the phase B is 69:31-89:11;
80-90min, phase A: the volume ratio of the phase B is 89:11-95:5.
the second aspect of the invention provides application of a fingerprint spectrum detection method of the four-ingredient powder of the Angong Niuhuang Wan in quality detection of components in the four-ingredient powder of the Angong Niuhuang Wan.
The third aspect of the invention provides a quality detection method of the four-component powder of the AngongNiuhuang pill, which comprises the steps of obtaining the fingerprint of the four-component powder of the AngongNiuhuang pill by adopting the detection method of the fingerprint of the four-component powder of the AngongNiuhuang pill, and comparing the similarity of the obtained fingerprint of the four-component powder of the AngongNiuhuang pill with the comparison fingerprint of the four-component powder of the AngongNiuhuang pill obtained under the same fingerprint detection condition.
Preferably, when the measured fingerprint of the four-flavor powder of the bezoar chest functioning pill and the comparison fingerprint of the four-flavor powder of the bezoar chest functioning pill are compared in similarity, software of 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system' 2012 edition issued by the national pharmacopoeia committee is adopted for comparison. More preferably, the similarity between the fingerprint of the four powders of Angong Niuhuang Wan measured by the method and the comparison fingerprint of the four powders of Angong Niuhuang Wan measured by the method is more than or equal to 0.99.
More preferably, when fingerprint common peaks of the four-flavor powder of the bezoar chest functioning pill and the comparison fingerprint of the four-flavor powder of the bezoar chest functioning pill are matched, automatic full spectrum matching is performed when the time window width is 0.2min after multipoint correction, and the fingerprint and the comparison fingerprint are generated by an average method.
Preferably, the fingerprint of the four powders of the AngongNiuhuang pill is obtained by the same conditions as the detection method of the fingerprint of the four powders of the AngongNiuhuang pill, the fingerprint of the four powders of the AngongNiuhuang pill comprises 23 common fingerprint peaks, and the No. 16 peak is taken as a reference peak (S peak, relative retention time is 1.0000), the relative retention time of other 22 common fingerprint peaks is sequentially No. 1 peak (0.2327 +/-0.0010), no. 2 peak (0.2783 +/-0.0010), no. 3 peak (0.3705 +/-0.0010), no. 4 peak (0.4204 +/-0.0010), no. 5 peak (0.4473 +/-0.0010), no. 6 peak (0.4957 +/-0.0019), no. 7 peak (0.5485 +/-0.0022), no. 8 peak (0.5909 +/-0.0012), no. 9 peak (0.6286 +/-0.0040), no. 10 peak (0.7213 +/-0.0029), no. 11 peak (0.8085 +/-0.0010), no. 12 peak (0.8560 +/-0.0043), no. 13 peak (0.8716 +/-0.0045), no. 14 peak (0.0048874 +/-0.0045), no. 15 peak (0.0050.00529 +/-0.0031 peak (621.0041) and No. 1.0071 peak (1 +/-19), no. 1.0023) peak (No. 1.00419), no. 1.00419, no. 3 +/-0.00719), no. 1.9), no. 3.9, no. 3 peak (No. 1.00419).
The specific data of the reference fingerprint of the four powders of ANGONGNIUHUANG pill is shown in FIG. 2A.
More preferably, the comparison fingerprint of the four-ingredient powder of the AngongNiuhuang pill is compared with the fingerprint of a comparison product solution, and the peak 5 is located and determined to be the fingerprint of geniposide, the peak 13 is the fingerprint of epiberberine, the peak 15 is the fingerprint of coptisine, the peak 16 is the fingerprint of baicalin, the peak 17 is the fingerprint of palmatine hydrochloride, the peak 18 is the fingerprint of berberine hydrochloride, the peak 20 is the fingerprint of wogonin, the peak 21 is the fingerprint of baicalin, and the peak 22 is the fingerprint of wogonin.
The fourth aspect of the invention provides a method for measuring the content of 9 components in the four-component powder of AngongNiuhuang Wan, which comprises the following steps:
a) Preparation of a test solution: the detection method is the same as the step 1) of the fingerprint detection method of the four-ingredient powder of the Angongniuhuang pill;
b) Preparation of control solutions: the detection method is the same as the step 2) of the fingerprint detection method of the four-ingredient powder of the Angongniuhuang pill;
c) And (3) determination: respectively measuring the test solution in the step a) and the reference solution in the step b) by adopting a high performance liquid chromatography with the same chromatographic conditions as those in the fingerprint detection method of the four-ingredient powder of the bezoar chest functioning pill, and calculating the content of 9 components in the test solution by adopting an external standard method.
Preferably, the external standard method is as follows: respectively transferring a series of reference substance solutions with different volumes in the step b) to prepare a series of solutions with different concentrations, carrying out sample injection analysis by adopting a high performance liquid chromatograph to obtain a linear relation between the content of 9 components in the reference substance solution and the peak area, drawing corresponding standard working curves according to the peak area of each component chromatographic spectrum corresponding to the corresponding content, and calculating to obtain a regression equation of each standard working curve. And detecting the sample solution by using a high performance liquid chromatograph, and substituting the chromatographic peak areas of the 9 components in the obtained sample solution into the regression equation of each standard working curve respectively to obtain the content of the corresponding component.
More preferably, in the standard working curve, the peak area is taken as the ordinate, and the content of each control solution is taken as the abscissa.
The fifth aspect of the invention provides a method for screening the fingerprint spectrums of a plurality of medicinal materials in four-flavor powder of Angong Niuhuang Wan, which comprises the following steps:
a) Preparing a sample solution of a single medicinal material: preparing any one or more of 4 medicinal material samples of coptis chinensis, scutellaria baicalensis, gardenia and radix curcumae in the four-ingredient powder of the bezoar chest functioning pill according to the step 1) of the detection method of fingerprint spectrums of the four-ingredient powder of the bezoar chest functioning pill to respectively obtain at least one single medicinal material sample solution;
b) And (3) determination: determining the sample solution of the single medicinal material by adopting a High Performance Liquid Chromatography (HPLC) method with the same chromatographic conditions as the step 3) of the detection method of fingerprint of the four-ingredient powder of the bezoar chest functioning pill to obtain the fingerprint of the sample solution of the single medicinal material;
c) Obtaining of a contrast fingerprint spectrum: obtaining a comparison fingerprint of the four powders of the AngongNiuhuang pill by adopting the same steps as the detection method of the fingerprint of the four powders of the AngongNiuhuang pill;
d) And (3) quality detection: and comparing the fingerprint of the single medicinal material sample solution with the comparison fingerprint of the four-flavor powder of the AngongNiuhuang pill, and identifying the corresponding characteristic peaks of the single medicinal material sample solution in the comparison fingerprint of the four-flavor powder of the AngongNiuhuang pill through relative retention time, thereby performing attribution positioning on the characteristic peaks in the fingerprint of the single medicinal material sample solution.
Preferably, in step a), the Coptis chinensis is dried rhizome of Coptis chinensis francis franch, coptis deltoidea c.y.cheng et Hsiao or Coptis teta wall. The Scutellariae radix is dried root of Scutellaria baicalensis Georgi of Labiatae. The fructus Gardeniae is dried mature fruit of Gardenia jasminoides Ellis of Rubiaceae. The radix Curcumae is dried root tuber of Curcuma wenyujin Y.H.Chen et C.Ling, curcuma longa L.and Curcuma kwangsiensis S.G.Lee et C.F.Liang or Curcuma zedoaria phaeocauli Val.
Preferably, in the step D), the attribution of the characteristic peaks of the measured fingerprint spectrum of the single-medicinal-material sample solution and the comparison fingerprint spectrum of the four-ingredient powder of the Angong Niuhuang Wan is positioned, the analysis and the processing are performed by adopting software of 2012 edition of traditional Chinese medicine chromatography fingerprint spectrum similarity evaluation System issued by the State pharmacopoeia Commission, and the attribution of the characteristic peaks of the single medicinal materials of the four-ingredient powder of the Angong Niuhuang Wan is confirmed by the relative retention time of the characteristic peaks on the comparison fingerprint spectrum of the four-ingredient powder of the Angong Niuhuang Wan.
Preferably, in the step D), the fingerprint of the coptis sample solution includes 11 common fingerprint peaks, and the 11 common fingerprint peaks include peak 1, peak 4, peak 6, peak 7, peak 10, peak 12, peak 13, peak 14, peak 15, peak 17 and peak 18.
Preferably, in the step D), the fingerprint of the scutellaria baicalensis sample solution in the single-herb sample solution comprises 8 common fingerprint peaks, and the 8 common fingerprint peaks are 8 peaks, 9 peaks, 16 peaks, 19 peaks, 20 peaks, 21 peaks, 22 peaks and 23 peaks.
Preferably, in the step D), the fingerprint of the gardenia sample solution in the single-ingredient drug sample solution includes 4 common fingerprint peaks, and the 4 common fingerprint peaks are a peak 2, a peak 3, a peak 5 and a peak 11.
Preferably, in the step D), the fingerprint of the curcuma aromatica sample solution does not include common fingerprint peaks in the single medicinal material sample solution.
Among the common fingerprint peaks, the 5 peak is determined to be the fingerprint peak of jasminoidin, the 13 peak is determined to be the fingerprint peak of epiberberine, the 15 peak is determined to be the fingerprint peak of coptisine, the 16 peak is determined to be the fingerprint peak of baicalin, the 17 peak is determined to be the fingerprint peak of palmatine hydrochloride, the 18 peak is determined to be the fingerprint peak of berberine hydrochloride, the 20 peak is determined to be the fingerprint peak of wogonoside, the 21 peak is determined to be the fingerprint peak of baicalein, and the 22 peak is determined to be the fingerprint peak of wogonin.
The water used in the invention is pure water.
As described above, the fingerprint spectrum and the component detection method of the four-ingredient powder of the Angong Niuhuang Wan provided by the invention adopt the pretreatment and high performance liquid chromatography methods of optimizing reaction conditions to establish the high performance liquid chromatography fingerprint spectrum of the four-ingredient powder of the Angong Niuhuang Wan, and the contributions of different types of chemical components to the fingerprint spectrum system of the four-ingredient powder of the Angong Niuhuang Wan are highlighted from different side surfaces, so that the detection of the chemical components of the whole formula of the four-ingredient powder of the Angong Niuhuang Wan is realized, and a reference basis is provided for the quality standard of the four-ingredient powder of the Angong Niuhuang Wan. The standard curves of the 9 components measured by the method have good linear relation in respective ranges, the average sample recovery rate is 97.09-103.23%, the RSD is less than 3.58%, and the method has good repeatability, high accuracy and high precision.
The method provided by the invention can also carry out quantitative analysis on 9 chemical components (berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, baicalein, wogonin, geniposide and wogonoside) in the four-ingredient powder of the bezoar chest functioning pill, thereby realizing one-time sample analysis and simultaneously completing qualitative and quantitative analysis.
The method provided by the invention can also carry out attribution confirmation on the chromatographic peaks of a plurality of single medicinal materials in the four-ingredient powder of the bezoar chest functioning pill, and the fingerprint embodies the characteristic peaks of the coptis chinensis, the scutellaria baicalensis, the gardenia and the radix curcumae. The method can realize effective monitoring from the source of raw materials, strictly control the quality of the raw materials, and can be used for quality control in the production process, thereby ensuring the safety and effectiveness of clinical medication.
Drawings
FIG. 1 shows the fingerprint spectra of four powders of Angong Niuhuang Wan in four batches before and after irradiation.
Fig. 2 shows a comparison fingerprint of the four powders of the bezoar chest functioning pill and fingerprints 2A and 2B of a comparison product, wherein fig. 2A is the comparison fingerprint of the four powders of the bezoar chest functioning pill, fig. 2B is the fingerprint of the comparison product, and 5: jasminoidin, 13: epiberberine, 15: coptisine, 16: baicalin, 17: palmatine hydrochloride, 18: berberine hydrochloride, 20: wogonoside, 21: baicalein, 22: wogonin.
Fig. 3 shows a comparative superposition chromatogram of the comparison fingerprint of the four-ingredient powder of the bezoar chest functioning pill and the fingerprints of each single medicinal material, wherein, 5: jasminoidin, 13: epiberberine, 15: coptisine, 16: baicalin, 17: palmatine hydrochloride, 18: berberine hydrochloride, 20: wogonoside, 21: baicalein, 22: wogonin.
Fig. 4 shows the fingerprint of the reference substance of the four-ingredient powder of the Angong Niuhuang Wan and the fingerprints 4A and 4B of the test sample, wherein fig. 4A is the fingerprint of the reference substance of the four-ingredient powder of the Angong Niuhuang Wan, fig. 4B is the fingerprint of the test sample, 1: geniposide, 2: epiberberine, 3: coptisine, 4: baicalin, 5: palmatine hydrochloride, 6: berberine hydrochloride, 7: wogonoside, 8: baicalein, 9: wogonin.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The reagents and equipment used in the following examples are as follows:
1. reagent
Sample preparation: four-flavor powder of Angong Niuhuang Wan (batch Nos. 190201, 190301, 190401, 190501, 190601, 190701, 190801, 190901, respectively) is provided by Shanghai and Huangyao limited. Single medicinal materials: the coptis root, the scutellaria baicalensis, the gardenia and the radix curcumae are provided by Shanghai and Huangyao limited companies, are all conventional Chinese medicinal materials and can be purchased from the market.
Berberine hydrochloride (batch No. 110713-201814, mass fraction 86.7%), palmatine hydrochloride (batch No. 110732-201812, mass fraction 97.6%), baicalin (batch No. 110715-201821, mass fraction 95.4%), baicalein (batch No. 111595-201808, mass fraction 97.9%), wogonin (batch No. 112002-201702, mass fraction 98.5%), wogonin (batch No. 111514-201706, mass fraction not less than 98.0%), and geniposide (batch No. 110749-201718, mass fraction 97.6%) all purchased from China food and drug testing institute; epiberberine (batch No. 19031923, mass fraction of not less than 98.0%) and coptisine (batch No. 19031922, mass fraction of not less than 98.0%) were purchased from Shanghai Tongtian Biotechnology, inc.
Methanol (analytical grade AR, national institute of chemical Co., ltd.), ethanol (analytical grade AR, shanghai Borol chemical Co., ltd.), acetonitrile (chromatographic grade, TEDIA, USA), phosphoric acid (chromatographic grade, TEDIA, USA), and ultrapure water (produced by Milli-Q ultrapure water treatment system).
2. Instrument for measuring the position of a moving object
Agilent1260 high performance liquid chromatograph (OpenLAB CDS 2.1 chromatography workstation, G1312B binary pump, G1322A auto degasser, G7116A column oven, G7115ADAD detector, G7129A autosampler, agilent, usa); SB-5200DTD ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., ltd.); an electric hot blast drying oven (shanghai-heng scientific instruments ltd); analytical electronic balances of the AL204 and XS205 types (mettler-toledo instruments); milli-Q Advantage A10 ultra-pure water system (Merck Millipore, germany).
Example 1
1. Sample pretreatment
Preparation of a test solution: taking 0.05g of AngongNiuhuang pill four-flavor powder sample, precisely weighing, placing in a 50mL conical flask with a plug, precisely adding 20mL of 75% methanol, sealing, weighing, ultrasonically extracting (power 250W, frequency 40 kHz) for 45 minutes, cooling, weighing again, complementing weight loss with 75% methanol, shaking up, standing for a moment, taking supernatant, filtering with a 0.45 mu m filter membrane, and taking subsequent filtrate to obtain sample solution No. 1.
Preparation of control solutions: precisely weighing berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, baicalein, wogonin, geniposide and wogonoside as reference substances, adding 75% methanol, dissolving in a 100mL volumetric flask, diluting to constant volume, shaking, and making into reference substance stock solution. In the reference substance stock solution, the content range of berberine hydrochloride is 487.20 mu g/mL, the content range of palmatine hydrochloride is 17.96 mu g/mL, the content range of epiberberine is 16.08 mu g/mL, the content range of coptisine is 30.20 mu g/mL, the content range of baicalin is 203.90 mu g/mL, the content range of wogonin is 36.02 mu g/mL, the content range of baicalein is 6.02 mu g/mL, the content range of wogonin is 4.50 mu g/mL, and the content range of geniposide is 81.02 mu g/mL.
And precisely measuring the reference substance stock solution, diluting with 75% methanol step by step, and fixing the volume to prepare a series of reference substance solutions with different concentrations. In a series of reference substance solutions with different concentrations, the content range of berberine hydrochloride is 0.087-1.744 μ g/mL, the content range of palmatine hydrochloride is 0.018-0.359 μ g/mL, the content range of epiberberine is 0.016-0.322 μ g/mL, the content range of coptisine is 0.030-0.604 μ g/mL, the content range of baicalin is 0.203-4.078 μ g/mL, the content range of wogonin is 0.036-0.720 μ g/mL, the content range of baicalein is 0.006-0.120 μ g/mL, the content range of wogonin is 0.005-0.100 μ g/mL, and the content range of geniposide is 0.081-1.620 μ g/mL.
2. Chromatographic conditions
The chromatographic conditions of the high performance liquid chromatography are as follows: agilent TC-C18 column (4.6 mm. Times.250mm, 5 μm); the detector is a photodiode array detector (DAD); column temperature: 26 ℃; sample introduction amount: 10 mu L of the solution; the flow rate is 0.8mL/min; the detection wavelength is 230nm; the mobile phase is acetonitrile-0.2% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.2% phosphoric acid water solution; the analysis time is 90min; and (4) gradient elution.
The specific procedure of the gradient elution is as follows:
0-5min, phase A: the volume ratio of the phase B is 5:95-11:89;
5-25min, phase A: the volume ratio of the phase B is 11:89-23:77;
25-35min, phase A: the volume ratio of the phase B is 23:77-25:75;
35-45min, phase A: phase B volume ratio is 25:75-26:74;
45-55min, phase A: the volume ratio of the phase B is 26:74-31:69;
55-60min, phase A: the volume ratio of the phase B is 31:69-39:61;
60-70min, phase A: the volume ratio of the phase B is 39:61-69:31;
70-80min, phase A: the volume ratio of the phase B is 69:31-89:11;
80-90min, phase A: the volume ratio of the phase B is 89:11-95:5.
3. measurement of
And (3) respectively measuring the sample solution and the reference solution in the step (1) by adopting the high performance liquid chromatography of the chromatographic conditions in the step (2) to obtain the fingerprint of the sample solution and the fingerprint of the reference solution. The fingerprint of the test solution is compared with the fingerprint of the reference solution, and the corresponding characteristic peak in the fingerprint of the test solution is identified through the relative retention time according to the known characteristic peak in the fingerprint of the reference solution, so that the attribution positioning is carried out on the index components in the fingerprint of the test solution, and the fingerprint of the four-flavor powder of the Angong Niuhuang Wan is obtained.
Example 2
1. Sample pretreatment
Preparation of a test solution: taking 0.05g of AngongNiuhuang pill four-flavor powder sample, precisely weighing, placing in a 50mL conical flask with a plug, precisely adding 15mL of 75% methanol, sealing, weighing, ultrasonically extracting (power 240W, frequency 30 kHz) for 40 minutes, cooling, weighing again, complementing weight loss with 75% methanol, shaking up, standing for a moment, taking supernatant, filtering with a 0.45 mu m filter membrane, and taking subsequent filtrate to obtain sample solution No. 2.
Preparation of control solutions: precisely weighing berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, baicalein, wogonin, geniposide and wogonoside as reference substances, adding 75% methanol, dissolving in a 100mL volumetric flask, diluting to constant volume, shaking, and making into reference substance stock solution. And precisely measuring the reference substance stock solution, diluting with 75% methanol step by step, and fixing the volume to prepare a series of reference substance solutions with different concentrations.
The concentration ranges of berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, baicalein, wogonin, geniposide and wogonoside in the control stock solution and a series of control solutions with different concentrations are the same as the step 1 in the example 1.
2. Chromatographic conditions
The chromatographic conditions of the high performance liquid chromatography are as follows: agilent TC-C18 column (4.6 mm. Times.250mm, 5 μm); the detector is a photodiode array detector (DAD); column temperature: 20 ℃; sample injection amount: 5 mu L of the solution; the flow rate is 1.0mL/min; the detection wavelength is 225nm; the mobile phase is acetonitrile-0.1% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.1% phosphoric acid water solution; the analysis time is 90min; and (4) gradient elution.
The procedure for gradient elution was the same as in step 2 of example 1.
3. Measurement of
The procedure was as in step 3 of example 1.
Example 3
1. Sample pretreatment
Preparing a test solution: taking 0.05g of AngongNiuhuang pill four-flavor powder sample, precisely weighing, placing in a 50mL conical flask with a plug, precisely adding 25mL of 75% methanol, sealing, weighing, ultrasonically extracting (power 260W, frequency 50 kHz) for 50 minutes, cooling, weighing again, complementing weight loss with 75% methanol, shaking up, standing for a moment, taking supernatant, filtering with a 0.45 μm filter membrane, and taking subsequent filtrate to obtain sample solution No. 3.
Preparation of control solutions: precisely weighing berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, baicalein, wogonin, geniposide and wogonoside, dissolving in 75% methanol in a 100mL volumetric flask, adding water, and shaking to obtain reference stock solution. And precisely measuring the reference substance stock solution, diluting with 75% methanol step by step, and fixing the volume to prepare a series of reference substance solutions with different concentrations.
The concentration ranges of berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, baicalein, wogonin, geniposide and wogonoside in the reference stock solution and a series of reference solutions with different concentrations are the same as the concentration range in step 1 of example 1.
2. Chromatographic conditions
The chromatographic conditions of the high performance liquid chromatography are as follows: agilent TC-C18 column (4.6 mm. Times.250mm, 5 μm); the detector is a photodiode array detector (DAD); column temperature: 30 ℃; sample injection amount: 15 mu L of the solution; the flow rate is 0.6mL/min; the detection wavelength is 235nm; the mobile phase is acetonitrile-0.3% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.3% phosphoric acid water solution; the analysis time is 90min; and (4) gradient elution.
The procedure for gradient elution was the same as in step 2 of example 1.
3. Measurement of
The procedure was as in step 3 of example 1.
Example 4
The fingerprint detection method of the four powders of the Angong Niuhuang Wan established in the embodiment 1 is adopted to detect 12 batches of the four powders of the Angong Niuhuang Wan, a chromatogram is recorded, and fingerprint data of the four batches of the four powders of the Angong Niuhuang Wan are respectively imported into similarity of traditional Chinese medicine chromatogram fingerprints issued by the Committee of national pharmacopoeia at 0kGy (non-radiation before irradiation), 6kGy and 10kGy of samplesIn software of 2012 edition of the degree evaluation system, 190201-0kGy as a reference spectrum is adopted, automatic full spectrum matching is carried out after multipoint correction (time window is 0.2 min), a fingerprint spectrum and a comparison fingerprint spectrum are generated by using an average number method, the similarity comparison is carried out on the fingerprint spectrum of a test sample and the comparison fingerprint spectrum of the four-flavor powder of the Angong Niuhuang Wan obtained under the same fingerprint spectrum detection condition, the similarity of the fingerprint spectrum of the four-flavor powder of each batch of Angong Niuhuang Wan is calculated, and the result shows that the similarity of the fingerprint spectrum of the four-flavor powder of the twelve batches of Angong Niuhuang Wan is more than 0.99, which indicates that the similarity of the fingerprint spectrums of the four-flavor powder of the twelve batches of Angong Niuhuang Wan is more than 0.99 60 The overall quality of each batch of samples before and after Co-gamma ray irradiation is stable, the information of the irradiated samples is shown in table 1, and the specific similarity condition is shown in tables 1 and 2.
TABLE 1 irradiation sample information Table
TABLE 2 similarity change chart of fingerprint before and after irradiation of four powders of ANGONGNIUHUANG pill
The reference fingerprint spectrum of the specific four-ingredient powder of the Angong Niuhuang Wan is shown in figure 2A, as can be seen from figure 2A, common peaks are determined according to the relative retention time of each chromatographic peak in a chromatogram, wherein the proportion of 23 common peaks of an HPLC characteristic spectrum to the total peak area is up to more than 95%, the 23 common peaks are selected as characteristic fingerprint peaks, and the specific data are shown in table 3. Wherein the peak emergence time of the No. 16 peak is moderate, the resolution is good, and the peak area is large, so the No. 16 peak is taken as a reference peak (S peak, the relative retention time is 1.0000), the relative retention time of other 22 common fingerprint peaks is sequentially No. 1 peak (0.2327 +/-0.0010), no. 2 peak (0.2783 +/-0.0010), no. 3 peak (0.3705 +/-0.0010), no. 4 peak (0.4204 +/-0.0010), no. 5 peak (0.4473 +/-0.0010), no. 6 peak (0.4957 +/-0.0019), no. 7 peak (0.5485 +/-0.0022), no. 8 peak (0.5909 +/-0.0012), no. 9 peak (0.6286 +/-0.0040), no. 10 peak (0.7213 +/-0.0029) peak 11 (0.8085 + -0.0010), peak 12 (0.8560 + -0.0043), peak 13 (0.8716 + -0.0045), peak 14 (0.8874 + -0.0045), peak 15 (0.9029 + -0.0051), peak 17 (1.0934 + -0.0072), peak 18 (1.1473 + -0.0097), peak 19 (1.2587 + -0.0010), peak 20 (1.3433 + -0.0015), peak 21 (1.5119 + -0.0036), peak 22 (1.6255 + -0.0046) and peak 23 (1.6347 + -0.0047).
Table 3 fingerprint consensus peak relative retention time n =6,x ± SD
Note: no. 16 peak is a positioning peak (S peak)
Comparing the comparison fingerprint spectrum of the four-ingredient powder of the Angong Niuhuang Wan with the fingerprint spectrum of the comparison product solution, identifying corresponding characteristic peaks in the comparison fingerprint spectrum of the four-ingredient powder of the Angong Niuhuang Wan according to the known characteristic peaks in the fingerprint spectrum of the comparison product solution in figure 2B through relative retention time, locating and determining that the No. 5 peak is the fingerprint peak of geniposide, the No. 13 peak is the fingerprint peak of epiberberine, the No. 15 peak is the fingerprint peak of coptisine, the No. 16 peak is the fingerprint peak of baicalin, the No. 17 peak is the fingerprint peak of palmatine hydrochloride, the No. 18 peak is the fingerprint peak of berberine hydrochloride, the No. 20 peak is the fingerprint peak of wogonin, the No. 21 peak is the fingerprint peak of baicalin, and the No. 22 peak is the fingerprint peak of wogonin.
Example 5
The detection method of fingerprint spectra of the four-ingredient powder of the bezoar chest functioning pill is verified by methodology, and the performance index results are as follows.
1. Precision degree
Taking 1 part of four-flavor powder (batch number 190301-0 kGy) of Angong Niuhuang Wan, preparing a test sample solution according to the step 1 in the example 1, detecting according to the chromatographic condition of the step 2 in the example 1, continuously injecting samples for 6 times, recording a chromatogram, and observing the relative retention time of specified common peaks in a fingerprint by taking a No. 16 peak (baicalin) as a reference peak, wherein the results show that the RSD of the relative retention time of 23 common peaks is less than 1.0%, the RSD of the relative retention area is less than 3.0%, and the results show that the precision of the instrument is good.
2. Repeatability of
Taking 6 parts of Angong Niuhuang Wan Siwei powder (batch No. 190301-0 kGy) sample, preparing a sample solution according to the step 1 in the example 1, detecting according to the chromatographic condition of the step 2 in the example 1, taking a No. 16 peak (baicalin) as a reference peak, calculating the relative retention time of each common peak and the RSD of the relative peak area, and as a result, the RSD of 23 common peaks is less than 1.0%, and the RSD of the relative peak area is less than 3.0%, which indicates that the method has good repeatability.
3. Stability of
Taking the same batch of four-ingredient powder (batch No. 190301-0 kGy) of the AngongNiuhuang pill, preparing a test solution according to the step 1 in the above example 1, then respectively placing for 0h, 2h, 4h, 8h, 12h and 24h, and then detecting according to the chromatographic condition of the step 2 in the above example 1, taking the No. 16 peak (baicalin) as a reference peak, and as a result, the relative retention time RSD of 23 common peaks is less than 1.0%, and the RSD of the relative peak area is less than 3.0%, which indicates that the test solution has good stability in 24 h.
Example 6
1. Sample pretreatment
Preparing a test solution: the procedure was the same as that for preparing the test solution in step 1 of example 1.
Preparation of control solutions: the same procedure was followed as in step 1 of example 1 to prepare a control solution.
2. Chromatographic conditions
The chromatographic conditions of the high performance liquid chromatography were the same as those of the high performance liquid chromatography in step 2 of example 1.
3. Measurement of
And (3) respectively transferring a series of reference substance solutions with different volumes by adopting an external standard method to prepare a series of solutions with different concentrations, and carrying out sample injection analysis by adopting a high performance liquid chromatograph to draw a standard working curve. And then, carrying out sample injection analysis on the obtained test solution by adopting a high performance liquid chromatograph, and substituting the analysis result into a standard working curve to obtain the content of the 9 components in the test solution.
Specifically, a series of reference substance solutions with different volumes are respectively transferred to prepare a series of solutions with different concentrations, a high performance liquid chromatograph is adopted for sample injection analysis to obtain the linear relation between the content of 9 components in the reference substance solution and the peak area, each component chromatographic peak area corresponds to the corresponding content, a corresponding standard working curve is drawn, and the regression equation of each standard working curve is calculated. And detecting the sample solution by using a high performance liquid chromatograph, and substituting the chromatographic peak areas of the 9 components in the obtained sample solution into the regression equation of each standard working curve respectively to obtain the content of the corresponding component.
Example 7
Precisely sucking 1, 2, 5, 10, 15 and 20 mu L of reference substance stock solutions, respectively preparing the 9 reference substance series solutions with different concentrations according to the embodiment 4, adopting the HPLC detection conditions of the embodiment 4, carrying out high performance liquid chromatography analysis by injecting 10 mu L of the reference substance, drawing a standard curve by taking the content (mu g) as an X axis of a horizontal coordinate and a peak area as a Y axis of a vertical coordinate, and obtaining a regression equation, a correlation coefficient and a linear range, wherein the specific result is shown in the table 4.
As can be seen from Table 4, the regression equation has a good linear relationship when samples are injected in the corresponding concentration range, and the correlation coefficient r is more than or equal to 0.9999. And taking the concentration of the reference substance as a limit of quantitation (LOQ) when the signal-to-noise ratio of the peak area is 10 times (S/N = 10), and converting the concentration into the content of the sample to obtain the target substance with higher sensitivity.
TABLE 4 Linear relationship and quantitative limits
Example 8
1. System applicability
The sample solution and the control solution were prepared according to step 1 of example 4, and sample injection was carried out according to the chromatographic conditions of step 2 of example 4, and the chromatograms are shown in FIG. 4A and FIG. 4B. As can be seen from FIGS. 4A and 4B, the retention time of the sample and the reference is consistent, the peak patterns of the chromatographic peaks of the components are symmetrical, the separation degrees are all greater than 1.5, the theoretical plate numbers are all greater than 10000, and the system applicability is good.
2. Precision degree
Taking 1 part of the control solution prepared in the step 1 of the example 4, injecting the sample according to the chromatographic condition of the step 2 of the example 4, repeating the sample injection for 6 times, recording a chromatogram, wherein the RSDs (n = 6) of berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, wogonoside, baicalein, wogonin and geniposide are respectively 0.21%, 0.37%, 0.68%, 0.15%, 0.47%, 0.41%, 0.87%, 0.90% and 0.25% by peak area, which indicates that the precision of the instrument is good.
3. Repeatability of
The four-ingredient powder samples of the AngongNiuhuang pill of batch No. 190301-0kGy are precisely weighed to 6 parts, the samples are prepared according to the preparation method of the test solution in the step 1 in the example 4, the sample is injected according to the chromatographic conditions in the step 2 in the example 4, and the chromatogram is recorded. The RSDs of berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, wogonoside, baicalein, wogonin and geniposide are respectively 1.44%, 0.66%, 2.50%, 2.12%, 0.73%, 0.67%, 2.66%, 2.67% and 0.89% by content, which indicates that the method has good repeatability.
4. Stability of
The sample of the four-ingredient powder of the bezoar bovis anglicarpae pills of lot 190301-0kGy is taken, the sample solution is prepared according to the step 1 in the above example 4, and then is placed for 0h, 2h, 4h, 8h, 12h and 24h, and then is detected according to the chromatographic condition of the step 2 in the above example 4, and the RSDs of the peak areas of the No. 16 peak (baicalin) as the reference peak and the peak areas of berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, wogonoside, baicalein, wogonin and jasminoidin are 2.19%, 0.31%, 2.01%, 2.23%, 0.45%, 0.40%, 2.39%, 2.07% and 0.29%, respectively, which indicates that the sample solution is stable within 24 h.
5. Recovery rate of sample
9 parts of an Angong Niuhuang Wan Siwei powder (batch No. 190301-0 kGy) are precisely weighed to be 0.025g, and control solutions of the components are precisely added according to three different concentration levels (1: 0.5, 1:1, 1: 1.5), a test solution is prepared according to the step 1 in the example 4, sample injection analysis is respectively carried out according to the chromatographic conditions of the step 2 in the example 4, and the sample adding recovery rate and the RSD of the components are calculated according to the measured amount and the added amount, and the results are shown in a table 5. As can be seen from Table 5, the average sample recovery rates and RSDs of berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, wogonoside, baicalein, wogonin, and geniposide were 99.63% (1.83%), 101.48% (1.08%), 97.09% (2.40%), 98.76% (1.84%), 100.83% (1.69%), 101.80% (1.73%), 103.23% (3.58%), 97.99% (2.91%), and 101.05% (2.40%), respectively. Indicating that the accuracy of the method is good.
TABLE 5 sample recovery test results (n = 3)
| Index component | Average loading recovery /) | RSD/％ |
| Berberine hydrochloride | 99.63 | 1.83 |
| Palmatine hydrochloride | 101.48 | 1.08 |
| Epeberrine | 97.09 | 2.40 |
| Coptisine | 98.76 | 1.84 |
| Baicalin | 100.83 | 1.69 |
| Wogonoside | 101.80 | 1.73 |
| Baicalein | 103.23 | 3.58 |
| Wogonin | 97.99 | 2.91 |
| Gardenia glycosides | 101.05 | 2.40 |
Example 9
The test sample solution is prepared according to the step 1 in the example 1 after the irradiation treatment of the multi-batch-number four-ingredient powder of the bezoar chest functioning pill with the doses of 0, 6 and 10kGy respectively, the sample injection analysis is carried out according to the chromatographic conditions of the step 3 in the example 1, and the contents of berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, wogonin, baicalein, wogonin and geniposide in the four-ingredient powder of the bezoar chest functioning pill before and after the irradiation are calculated respectively, and the results are shown in the table 6.
TABLE 6 measurement results of the contents of the respective components (mg. G) -1 ,n＝8)
As can be seen from table 6, it is, 60 the content of 9 components in the four-ingredient powder of the bezoar chest functioning pill before and after Co irradiation is subjected to group t test analysis to investigate whether the content difference of the components (berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, wogonin, baicalein, wogonin and geniposide) between different irradiation dose groups has statistical significance. As a result, the P values of the 9 components are all greater than 0.05, which indicates that the difference in mean between groups is not statistically significant, i.e., the 9 components are subjected to different dosages (0 kGy, 6kGy, 10 kGy) 60 There was no significant difference in the change in Co content after irradiation, i.e. the dose used in this study 60 Co irradiation sterilization does not affect the content of 9 components in the four-ingredient powder of the Angong Niuhuang Wan.
Example 10
The test solution was prepared by the above step 1 of example 1.
Taking coptis chinensis, scutellaria baicalensis, gardenia and radix curcumae respectively, and preparing 4 single medicinal material sample solutions by adopting the step 1 in the embodiment 1.
By adopting the step 2 in the embodiment 1, the sample solution to be tested and the sample solutions of 4 single medicinal materials are respectively measured, and the fingerprint spectrums of the sample solution to be tested and the sample solutions of 4 single medicinal materials are respectively obtained. The obtained test solution and the fingerprints of the 4 single-medicinal-material sample solutions are introduced into software of 2012 edition "traditional Chinese medicine chromatography fingerprint similarity evaluation system" issued by the committee of national pharmacopoeia for analysis and treatment, and meanwhile, the reference fingerprints of the four-flavor powder of the Angong Niuhuang pill are obtained according to the step 3 in the embodiment 1. The fingerprint spectrums of the sample solution to be tested and the 4 single medicinal material sample solutions are respectively compared with the comparison fingerprint spectrums of the four powders of the Angong Niuhuang Wan, and the corresponding characteristic peaks of the 4 single medicinal material sample solutions in the comparison fingerprint spectrums of the four powders of the Angong Niuhuang Wan are identified through relative retention time, so that the attribution positioning is carried out on the characteristic peaks in the fingerprint spectrums of the 4 single medicinal material sample solutions, and the specific result is shown in a figure 3 and a table 7.
As can be seen from fig. 3 and table 7, in the sample solution of the single drug material, the fingerprint of the sample solution of the coptis includes 11 common fingerprint peaks, and the 11 common fingerprint peaks are peak No. 1, peak No. 4, peak No. 6, peak No. 7, peak No. 10, peak No. 12, peak No. 13, peak No. 14, peak No. 15, peak No. 17, and peak No. 18; the fingerprint spectrum of the scutellaria baicalensis sample solution comprises 8 common fingerprint peaks, wherein the 8 common fingerprint peaks are No. 8 peak, no. 9 peak, no. 16 peak, no. 19 peak, no. 20 peak, no. 21 peak, no. 22 peak and No. 23 peak; the fingerprint spectrum of the gardenia sample solution comprises 4 common fingerprint peaks, wherein the 4 common fingerprint peaks are a No. 2 peak, a No. 3 peak, a No. 5 peak and a No. 11 peak; the fingerprint of the radix curcumae sample solution does not comprise common fingerprint peaks.
TABLE 7 chromatographic peak attribution of each single medicinal material of four-ingredient powder of Angong Niuhuang Wan
Among the above common fingerprint peaks, as shown in fig. 3, after comparison, the 5 th peak is identified as the fingerprint peak of geniposide, the 13 th peak is identified as the fingerprint peak of epiberberine, the 15 th peak is identified as the fingerprint peak of coptisine, the 16 th peak is identified as the fingerprint peak of baicalin, the 17 th peak is identified as the fingerprint peak of palmatine hydrochloride, the 18 th peak is identified as the fingerprint peak of berberine hydrochloride, the 20 th peak is identified as the fingerprint peak of wogonoside, the 21 st peak is the fingerprint peak of baicalein, and the 22 th peak is identified as the fingerprint peak of wogonin. As can be seen from FIG. 3 and Table 7, the compound chromatographic peaks of other medicinal materials except for Curcuma aromatica are well characterized and embodied in the fingerprint, and the attribution is confirmed.
In conclusion, the fingerprint spectrum and the detection method of the ingredients of the four-ingredient powder of the Angongniuhuang pill provided by the invention establish the high performance liquid fingerprint spectrum of the four-ingredient powder of the Angongniuhuang pill, can perform quantitative analysis on 9 chemical ingredients in the four-ingredient powder of the Angongniuhuang pill, and can perform attribution confirmation on chromatographic peaks of each single medicinal material in the four-ingredient powder of the Angongniuhuang pill, thereby effectively monitoring from the source of the raw materials and strictly controlling the quality of the raw medicinal materials. Therefore, the present invention overcomes various disadvantages of the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims (10)
1. A detection method of fingerprint spectra of four-ingredient powder of Angongniuhuang pill comprises the following steps:
1) Preparing a test solution: dissolving the four-flavor powder sample of the bezoar chest functioning pill in a solvent, performing ultrasonic extraction, cooling, shaking up, and filtering to obtain a test solution;
2) Preparation of control solutions: dissolving berberine hydrochloride, palmatine hydrochloride, epiberberine, coptisine, baicalin, baicalein, wogonin, geniposide, and wogonoside in solvent, and diluting to desired volume to obtain control solution;
3) And (3) determination: respectively measuring the test solution and the reference solution by adopting a high performance liquid chromatography with the same chromatographic condition to obtain a fingerprint of the test solution and a fingerprint of the reference solution, comparing the fingerprint of the test solution with the fingerprint of the reference solution, and performing attribution positioning on index components in the fingerprint of the test solution, thereby obtaining the fingerprint of the AngongNiuhuang Wan four-ingredient powder.
2. The method for detecting fingerprint spectrums of the four-ingredient powder of AngongNiuhuang Wan according to claim 1, wherein the step 1) or 2) comprises any one or more of the following conditions:
a1 In step 1), the four-ingredient powder sample of the bezoar chest functioning pill is prepared before the test solution is prepared 60 Irradiating with Co-gamma rays;
a2 In step 1), the ratio of the weight of the four kinds of powder of the bezoar chest functioning pill to the volume of the solvent is 0.05:15-25,g/mL;
a3 In step 1) or 2), the solvent is 50-100% methanol;
a4 In the step 1), the ultrasonic extraction time is 30-60min;
a5 In the step 1), the power of ultrasonic extraction is 200-300W, and the frequency of ultrasonic extraction is 30-50kHz.
3. The method for detecting fingerprint of the four-flavor powder of AngongNiuhuang Wan according to claim 1, wherein in the step 3), the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 chromatographic column; the detector is a photodiode array detector; the column temperature is 20-30 ℃; the sample injection amount is 1-20 mu L; the flow rate is 0.5-1.0mL/min; the detection wavelength is 220-240nm; the mobile phase is acetonitrile-0.1-0.3% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.1-0.3% phosphoric acid water solution; the analysis time is 90min; gradient elution.
4. The method for detecting fingerprint of Angongniuhuang Wan four-ingredient powder according to claim 3, wherein the specific procedure of gradient elution is as follows:
0-5min, phase A: the volume ratio of the phase B is 5:95-11:89;
5-25min, phase A: the volume ratio of the phase B is 11:89-23:77;
25-35min, phase A: the volume ratio of the phase B is 23:77-25:75;
35-45min, phase A: the volume ratio of the phase B is 25:75-26:74;
45-55min, phase A: the volume ratio of the phase B is 26:74-31:69;
55-60min, phase A: the volume ratio of the phase B is 31:69-39:61;
60-70min, phase A: the volume ratio of the phase B is 39:61-69:31;
70-80min, phase A: the volume ratio of the phase B is 69:31-89:11;
80-90min, phase A: the volume ratio of the phase B is 89:11-95:5.
5. the use of the detection method of fingerprint of the four powders of Angongniuhuang Wan according to any one of claims 1 to 4 in quality detection of the components in the four powders of Angongniuhuang Wan.
6. A quality detection method for Angongniuhuang Wan four-flavor powder comprises the steps of obtaining Angongniuhuang Wan four-flavor powder fingerprint spectrum by using the detection method for Angongniuhuang Wan four-flavor powder fingerprint spectrum according to any one of claims 1 to 4, and comparing the similarity of the obtained Angongniuhuang Wan four-flavor powder fingerprint spectrum with a comparison fingerprint spectrum of Angongniuhuang Niuhuang Wan four-flavor powder obtained under the same fingerprint spectrum detection condition.
7. The quality detection method of the Angongniuhuang Wan Siwei powder according to claim 6, wherein the comparison fingerprint of the Angongniuhuang Wan Siwei powder is obtained under the same conditions as the detection method of the Angongniuhuang Wan Siwei powder fingerprint of any one of claims 1 to 4, and comprises 23 common fingerprint peaks, wherein the 16 th peak is taken as a reference peak S peak, the relative retention time is 1.0000, the relative retention time of other 22 common fingerprint peaks is 0.2327 +/-0.0010 of peak 1, 0.2783 +/-0.0010 of peak 2, 0.3705 +/-0.0010 of peak 3, 0.4204 +/-0.0010 of peak 4, 0.4473 +/-0.0010 of peak 5, 0.4957 +/-0.0019 of peak 6, 0.5485 +/-0.0022 of peak 7, 0.5909 +/-0.0012 of peak 8, 0.6286 +/-0.0040 of peak 9, 0.7213 +/-0.0029 of peak 10, 0.8085 +/-0.0010 of peak 11, 0.8560 +/-0.0043 of peak 12, 0.626 +/-0.0048715 of peak 14, 0.8874 +/-0.0045 of peak 15, 0.0059029 +/-0.0051 of peak 17, 0.091.00734 of peak 1, 620.0041 +/-0.00419 of peak 5119 of peak 1.00119, and 0.3433 +/-0.0023 of peak 1.0023 of peak 1.9 of peak 1.7 +/-0.0049 of peak 1.0049 of peak 15, and No. 1.0049 of peak 1.0043, and No. 7 +/-0.0043.0043.00119 of peak 1.7 +/-0.7.7.
8. A method for measuring the content of 9 components in four-ingredient powder of Angongniuhuang pill comprises the following steps:
a) Preparation of a test solution: the method is the same as the step 1) of the fingerprint detection method of the four-ingredient powder of the AngongNiuhuang pill of any one of the claims 1 to 4;
b) Preparation of control solutions: the method is the same as the step 2) of the fingerprint detection method of the four-ingredient powder of the AngongNiuhuang pill of any one of the claims 1 to 4;
c) And (3) determination: respectively measuring the test solution in the step a) and the reference solution in the step b) by adopting a high performance liquid chromatography with the same chromatographic conditions as in the fingerprint spectrum detection method of the four-ingredient powder of the bezoar chest functioning pill according to any one of claims 1 to 4, and calculating the content of 9 ingredients in the test solution by adopting an external standard method.
9. A method for screening fingerprints of multiple medicinal materials in four-flavor powder of Angongniuhuang pill comprises the following steps:
a) Preparing a sample solution of a single medicinal material: preparing any one or more of 4 medicinal material samples of coptis chinensis, scutellaria baicalensis, gardenia and radix curcumae in the four-ingredient powder of the bezoar chest functioning pill according to the step 1) of the detection method of the fingerprint of the four-ingredient powder of the bezoar chest functioning pill in any one of claims 1 to 4, and respectively obtaining at least one single medicinal material sample solution;
b) And (3) determination: determining the sample solution of the single medicinal material by adopting the high performance liquid chromatography with the same chromatographic conditions in the step 3) of the fingerprint detection method of the four-ingredient powder of the AngongNiuhuang Wan according to any one of claims 1 to 4, and obtaining the fingerprint of the sample solution of the single medicinal material;
c) Obtaining a comparison fingerprint: obtaining a comparison fingerprint spectrum of the four powders of the bezoar chest functioning pill according to the same steps as the detection method of the fingerprint spectrum of the four powders of the bezoar chest functioning pill according to the claim 1 to 4;
d) And (3) quality detection: and comparing the fingerprint of the single medicinal material sample solution with the comparison fingerprint of the four powders of the Angongniuhuang pill, and identifying the corresponding characteristic peaks of the single medicinal material sample solution in the comparison fingerprint of the four powders of the Angongniuhuang pill by relative retention time, thereby performing attribution positioning on the characteristic peaks in the fingerprint of the single medicinal material sample solution.
10. The screening method of fingerprint spectra of multiple medicinal materials in the four-flavor powder of AngongNiuhuang Wan according to claim 9, wherein the step D) comprises any one or more of the following conditions:
b1 The fingerprint of the coptis sample solution comprises 11 common fingerprint peaks, wherein the 11 common fingerprint peaks are No. 1 peak, no. 4 peak, no. 6 peak, no. 7 peak, no. 10 peak, no. 12 peak, no. 13 peak, no. 14 peak, no. 15 peak, no. 17 peak and No. 18 peak;
b2 ) the fingerprint spectrum of the scutellaria baicalensis sample solution comprises 8 common fingerprint peaks, wherein the 8 common fingerprint peaks are No. 8 peak, no. 9 peak, no. 16 peak, no. 19 peak, no. 20 peak, no. 21 peak, no. 22 peak and No. 23 peak;
b3 The fingerprint of the gardenia sample solution comprises 4 common fingerprint peaks, wherein the 4 common fingerprint peaks are a No. 2 peak, a No. 3 peak, a No. 5 peak and a No. 11 peak;
b4 ) the fingerprint of the turmeric sample solution does not include common fingerprint peaks.
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