Eight-treasure cream fingerprint spectrum construction method, fingerprint spectrum and application

Abstract

The invention discloses a construction method of an eight-treasure paste fingerprint spectrum, the fingerprint spectrum and application. The construction method comprises the following steps: weighing eight-treasure paste, and adding the weighed eight-treasure paste into a methanol solution to obtain a test sample; 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizinate and liquiritin are weighed and added into a methanol solution, and a reference substance is obtained; respectively carrying out high performance liquid chromatography on the test sample and the reference substances to respectively obtain chromatograms of the test sample and the reference substances; determining a common peak according to the chromatogram of the test sample, comparing the common peak with the chromatogram of the reference substance, identifying the chemical components of the common peak, then selecting a reference peak, calculating the relative retention time of the common peak, and constructing to obtain the eight-treasure cream fingerprint spectrum. The construction method of the Bazhen cream fingerprint spectrum is beneficial to obtaining of more chromatographic peaks so as to realize overall detection of multiple components of the Bazhen cream, and the number of the chromatographic peaks of a liquid chromatogram obtained under the condition of gradient elution is large and the degree of separation is good.

Description

Technical Field

[0001] The present application relates to the technical field of quality control of Bazhen Paste, and in particular to a method for constructing a fingerprint spectrum of Bazhen Paste, a fingerprint spectrum and an application thereof. Background Art

[0002] Bazhen Gao is composed of eight herbs: Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Licorice root, Angelica sinensis, White Peony Root, Chuanxiong Rhizome, and Rehmannia glutinosa. It has the effect of replenishing qi and blood, and is suitable for people with qi and blood deficiency, sallow complexion, and weakness in the limbs. The quality control of traditional Chinese patent medicine compound preparations currently uses thin layer chromatography and high performance liquid chromatography (HPLC) to conduct qualitative and quantitative analysis of the active ingredients therein. The above technologies are mainly aimed at detecting a single effective ingredient, but there are many types of Chinese medicine compounds. Different medicinal material ratios, medicinal material origins, and medicinal material pretreatment methods in the prescription will affect the composition and efficacy of Chinese medicinal materials. Therefore, a single, small number of chemical components cannot fully characterize the pharmacological and pharmacodynamic effects of Chinese medicinal materials. In addition, the component analysis of Chinese medicinal materials has problems such as complex components and difficulty in separation.

[0003] Currently, Bazhen Paste only has internal enterprise standards. Its ingredients are relatively complex. Qualitative identification and quantitative analysis of simple indicator components are difficult to fully control the intrinsic quality of Bazhen Paste. Its quality standards should strengthen specific identification and multi-component and overall quality control. Summary of the invention

[0004] The invention provides a method for constructing a fingerprint of Bazhen Paste, a fingerprint and an application thereof, so as to solve the technical problem that the analysis method in the prior art is difficult to comprehensively control the intrinsic quality of Bazhen Paste.

[0005] To achieve the above object, the technical solution provided by the present invention is as follows:

[0006] The first aspect of the present invention provides a method for constructing a fingerprint of Bazhen Gao, comprising the following steps:

[0007] S1. Weigh Bazhen Gao and add methanol solution to obtain a test sample; weigh 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizinate, and liquiritin respectively, and add them to the methanol solution to obtain reference substances containing the above six components respectively;

[0008] S2, performing high performance liquid chromatography analysis on the test sample and the reference substance obtained in step S1, respectively, to obtain chromatograms of the test sample and each of the reference substances;

[0009] The conditions of the high performance liquid chromatography analysis are as follows: the stationary phase of the chromatographic column is octadecylsilane bonded silica gel as filler, a 0.1% to 0.2% by volume inorganic acid solution is used as mobile phase A, and acetonitrile is used as mobile phase B for gradient elution; the step of the gradient elution is carried out according to the following procedure, calculated by volume fraction:

[0010] From 0 to 10 min, mobile phase A was reduced from 93% to 85%, and mobile phase B was increased from 7% to 15%;

[0011] From 10 to 25 min, mobile phase A decreased from 85% to 76%, and mobile phase B increased from 15% to 24%;

[0012] From 25 to 30 min, mobile phase A was reduced from 76% to 70%, and mobile phase B was increased from 24% to 30%;

[0013] 30-35 min, mobile phase A was reduced from 70% to 55%, and mobile phase B was increased from 30% to 45%;

[0014] 35-40 min, mobile phase A was reduced from 55% to 10%, and mobile phase B was increased from 45% to 90%;

[0015] 40-50 min, mobile phase A is 10%, mobile phase B is 90%;

[0016] 50-51 min, mobile phase A increased from 10% to 93%, and mobile phase B decreased from 90% to 7%;

[0017] 51-60 min, mobile phase A: 93%, mobile phase B: 7%;

[0018] S3. Determine the common peaks from the chromatogram of the test sample, compare the common peaks with the chromatogram of the reference sample, identify the chemical components of the common peaks, then select reference peaks, calculate the relative retention time of the common peaks, and construct the fingerprint of the Eight Treasures Paste.

[0019] Furthermore, a similarity evaluation was performed on the fingerprint of the Eight Treasures Paste, and the similarity evaluation method was: importing the chromatograms of multiple batches of the test samples and the fingerprint of the Eight Treasures Paste into a similarity evaluation software for similarity evaluation analysis, and the similarities were all greater than 0.9; the number of common peaks was 19.

[0020] Furthermore, in the step of identifying the chemical components of the common peaks, 5 peaks were identified, namely, the peaks corresponding to 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, ammonium glycyrrhizinate, and liquiritin.

[0021] Furthermore, during the high performance liquid chromatography analysis in step S2, the injection flow rate is 1.0 mL / min to 2.0 mL / min, the column temperature is 25° C. to 35° C., the injection volume is 10 μL to 20 μL, the detection wavelength is 230 nm to 400 nm, and the analysis time is 50 min to 90 min.

[0022] Furthermore, in the high performance liquid chromatography analysis in step S2, the injection flow rate is 1.0 mL / min, the column temperature is 30° C., the injection volume is 10 μL, the detection wavelength is 230 nm, and the analysis time is 60 min.

[0023] Furthermore, before the high performance liquid chromatography analysis in step S2, the test sample and the reference sample are filtered through a 0.22 μm to 0.45 μm microporous filtration membrane respectively.

[0024] Furthermore, the volume percentage of the methanol solution in step S1 is 70% to 100%.

[0025] Furthermore, in step S1, the eight-treasure paste is added to the methanol solution in a material-liquid ratio of 1:5-10; and the six components are respectively added to the methanol solution in a material-liquid ratio of 1:5-10.

[0026] The second aspect of the present invention provides the fingerprint of Bazhen Paste obtained by the above construction method.

[0027] The third aspect of the present invention provides the application of the fingerprint of Bazhen Paste obtained by the above construction method in the quality control process of Bazhen Paste.

[0028] The method for constructing the fingerprint of Bazhen Paste provided by the present invention is conducive to obtaining a larger number of chromatographic peaks so as to realize the overall detection of multiple components of Bazhen Paste. The number of chromatographic peaks of the liquid chromatography obtained by the present invention using the conditions of gradient elution is large and the separation is good. The fingerprint of Bazhen Paste obtained by the above construction method has a high similarity with the chromatograms of multiple batches of test samples, which can more comprehensively reflect the component information contained in Bazhen Paste and better characterize the quality of Bazhen Paste.

[0029] Bazhen Gao is mainly made of eight herbs, namely, Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Licorice root, Angelica sinensis, White peony root, Ligusticum chuanxiong and Rehmannia glutinosa, through a complex process. Its chemical composition is relatively complex, and the content determination of a single ingredient is one-sided and cannot reflect the overall composition information of the product. The above construction method has realized the quality control of the whole Bazhen Gao for the first time. It does not identify a single compound or medicinal material, and can more effectively guide the feeding, strictly regulate the production operation, and ensure the safety and effectiveness of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The chromatograms of 10 batches of Bazhen Gao test samples in the embodiments of the present invention;

[0032] Figure 2 The eight treasures paste spectrum and the reference fingerprint spectrum after common peak matching and correction in the embodiment of the present invention (S1-S10 are the eight treasures paste samples after peak matching and correction, and R is the reference fingerprint spectrum);

[0033] Figure 3 The chromatogram is a graph showing the common peaks of the reference substances and the test substances in the embodiments of the present invention;

[0034] Figure 4 This is a chromatogram of the sample solution of 8 kinds of medicinal materials decoction in the embodiment of the present invention;

[0035] Figure 5 This is a graph showing the results of investigating the effects of different solvents on the construction of fingerprints in the embodiments of the present invention;

[0036] Figure 6 This is a graph showing the results of investigating the effects of different column temperatures on the construction of fingerprints in an embodiment of the present invention;

[0037] Figure 7 This is a graph showing the results of investigating the effects of different chromatographic columns on the construction of fingerprint spectra in the embodiments of the present invention. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0039] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the conditions under which this application can be implemented. Therefore, they have no substantive technical significance. Any structural modification, change in proportional relationship or adjustment of size should still fall within the scope of the technical content disclosed in this application without affecting the effects and purposes that can be achieved by this application.

[0040] In a first aspect of the embodiment of the present application, a method for constructing a fingerprint of Bazhen Paste is provided, comprising the following steps:

[0041] S1. Weigh Bazhen Gao and add methanol solution to obtain a test sample; weigh 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizinate, and liquiritin respectively, and add them to the methanol solution to obtain reference substances containing the above six components respectively;

[0042] S2, performing high performance liquid chromatography analysis on the test sample and reference substance obtained in step S1, respectively, to obtain chromatograms of the test sample and each reference substance;

[0043] The conditions for high performance liquid chromatography analysis are as follows: the stationary phase of the chromatographic column is octadecylsilane bonded silica gel as filler, a 0.1% to 0.2% by volume inorganic acid solution is used as mobile phase A, and acetonitrile is used as mobile phase B for gradient elution; the steps of gradient elution are carried out according to the following procedure, calculated by volume fraction:

[0044] From 0 to 10 min, mobile phase A was reduced from 93% to 85%, and mobile phase B was increased from 7% to 15%;

[0045] From 10 to 25 min, mobile phase A decreased from 85% to 76%, and mobile phase B increased from 15% to 24%;

[0046] From 25 to 30 min, mobile phase A was reduced from 76% to 70%, and mobile phase B was increased from 24% to 30%;

[0047] 30-35 min, mobile phase A was reduced from 70% to 55%, and mobile phase B was increased from 30% to 45%;

[0048] 35-40 min, mobile phase A was reduced from 55% to 10%, and mobile phase B was increased from 45% to 90%;

[0049] 40-50 min, mobile phase A is 10%, mobile phase B is 90%;

[0050] 50-51 min, mobile phase A increased from 10% to 93%, and mobile phase B decreased from 90% to 7%;

[0051] 51-60 min, mobile phase A: 93%, mobile phase B: 7%;

[0052] S3. Determine the common peaks from the chromatogram of the test sample, compare the common peaks with the chromatogram of the reference sample, identify the chemical components of the common peaks, then select the reference peaks, calculate the relative retention time of the common peaks, and construct the fingerprint of Bazhen Paste.

[0053] The embodiment of the present application uses 0.2% by volume phosphoric acid solution-acetonitrile as the elution system, so that most of the effective ingredients in the Eight Treasures Paste are well separated, and then a complete high-performance liquid chromatography spectrum is obtained, which lays the foundation for the construction of the fingerprint spectrum.

[0054] The embodiments of the present application used multiple batches of Bazhen Paste as samples for high performance liquid chromatography analysis. During the research process, the effects of different mobile phases, different concentrations and types of mixed solvents on the extraction of chemical components in Bazhen Paste were explored, and a chromatographic method suitable for Bazhen Paste test samples was explored.

[0055] In the examples of the present application, similarity evaluation software (2012.130723) is used, and a reference spectrum is selected from the chromatograms of multiple batches of test samples, and a control fingerprint spectrum is generated by the average method, with a time width of 0.1. The chromatograms of the above multiple batches of test samples and the above control fingerprint spectrum are imported into the similarity evaluation software for similarity evaluation analysis, and it is determined that the similarities are all greater than 0.9, indicating that the control fingerprint spectrum is representative.

[0056] The chromatograms of the above batches of test products were processed, and a total of 19 groups of common peaks were marked with reference to factors such as peak shape and retention time. The 19 groups of peaks were numbered from small to large from 1 to 19 according to the retention time in the reference fingerprint. The chromatograms of the above 19 groups of common peaks were compared with those of the reference product, and the chemical components of the common peaks could be identified. The reference peaks were selected, the relative retention time of the common peaks was calculated, and the fingerprint of the Eight Treasures Paste was constructed.

[0057] The method for constructing the fingerprint of Bazhen Paste provided in the embodiment of the present application is conducive to obtaining a larger number of chromatographic peaks so as to realize the overall detection of multiple components of Bazhen Paste. The number of chromatographic peaks of the liquid chromatography obtained by the present invention using the conditions of gradient elution is large and the separation is good. The fingerprint of Bazhen Paste obtained by the above construction method has a high similarity with the chromatograms of multiple batches of test samples, which can more comprehensively reflect the component information contained in Bazhen Paste and better characterize the quality of Bazhen Paste.

[0058] Bazhen Gao is mainly made of eight herbs, namely, Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Licorice root, Angelica sinensis, White peony root, Ligusticum chuanxiong and Rehmannia glutinosa, through a complex process. Its chemical composition is relatively complex, and the content determination of a single ingredient is one-sided and cannot reflect the overall composition information of the product. The above construction method has realized the quality control of the whole Bazhen Gao for the first time. It does not identify a single compound or medicinal material, and can more effectively guide the feeding, strictly regulate the production operation, and ensure the safety and effectiveness of the drug.

[0059] In the embodiment of the present application, in the step of identifying the chemical components of the common peaks, a total of 5 peaks were identified, namely, the peaks corresponding to 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, ammonium glycyrrhizate, and liquiritin. The identified peaks are 5-hydroxymethylfurfural (peak 1), paeoniflorin (peak 11), chlorogenic acid (peak 8), ammonium glycyrrhizate (peak 16), and liquiritin (peak 12).

[0060] The second aspect of the embodiments of the present application provides a fingerprint of Bazhen Paste obtained by the above-mentioned method for constructing the fingerprint of Bazhen Paste.

[0061] The third aspect of the embodiments of the present application provides the application of the fingerprint of Bazhen Paste obtained by the above-mentioned method for constructing the fingerprint of Bazhen Paste in the quality control process of Bazhen Paste.

[0062] In order to better explain the present application and facilitate understanding, the present application is described in detail below in conjunction with the accompanying drawings through specific embodiments. The chromatographic columns, reagents, and batch numbers of the eight-treasure paste used in the following embodiments are as follows:

[0063] Bazhen Paste (National Medicine Approval No. Z20093561) is produced by Hunan Kelun Pharmaceutical Co., Ltd.

[0064] Bazhen Paste (bottle, 150g): There are 10 batches in total, with batch numbers X211115, X230201, X230202, X230401, X230402, X230403, X230601, X230601, X240101, X240102, and X240702.

[0065] Medicinal materials (bagged, sealed): Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Licorice root, Angelica sinensis, White Peony Root, Chuanxiong Rhizome, Rehmannia glutinosa, which are commercially available.

[0066] Reagents: acetonitrile, phosphoric acid, methanol are all commercially available. 5-Hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizinate, and liquiritin are all commercially available.

[0067] Chromatographic columns: CAPCELL PAK C18MGⅡ (4.6mm*250mm, 5μm); ZORBAX SB-C18.

[0068] Example 1

[0069] A method for constructing a fingerprint spectrum of Bazhen Paste comprises the following steps:

[0070] 1. Take 2 mL of each of 10 batches of Bazhen Gao, dissolve them in 5 times the volume of 70% methanol solution, filter them through a 0.22 μm microporous filter membrane for later use, and obtain 10 batches of Bazhen Gao test samples.

[0071] 5-Hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizinate, and liquiritin were weighed respectively, and methanol solution was added to prepare solutions containing 50 μg, 50 μg, 20 μg, 20 μg, 40 μg, and 50 μg per milliliter to obtain reference substances. The peak areas of chromatographic peaks of 5-Hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizinate, and liquiritin accounted for a large proportion and were relatively stable, so these 6 compounds were selected as references to prepare reference substances.

[0072] 2. The above test products and reference products were analyzed by high performance liquid chromatography, and the chromatographic conditions were as follows: the chromatographic column was CAPCELLPAK C18MGⅡ (4.6mm*250mm, 5μm). Mobile phase: 0.2% sulfuric acid aqueous solution (A)-acetonitrile (B); flow rate 1.0mL / min, column temperature 30℃, injection volume 10μL; detection wavelength: 230nm; total analysis time 60min. The gradient elution procedure is shown in Table 1.

[0073] Table 1 Gradient elution procedures for test and reference substances

[0074]

[0075] 3. Analyze the chromatograms of the 10 batches of test products obtained, referring to Figure 1 , the chromatographic peaks with good stability and appropriate response values ​​in the fingerprints of 10 batches of Bazhen Gao samples were selected as common peaks. The Chinese medicine fingerprint similarity evaluation system (2012.130723) software was used to select Figure 1 The chromatogram of S1 in the figure was used as a reference spectrum, and the control fingerprint spectrum was generated by the average method. Figure 2 In R, the time width is 0.1, and a total of 19 common peaks are calibrated. The 19 common peaks are numbered from 1 to 19 from small to large according to the retention time in the reference fingerprint.

[0076] The similarity between the chromatograms of 10 batches of Bazhen Gao and the reference fingerprint R is shown in Table 2. The similarity between the two is greater than 0.9. The chromatograms of Bazhen Gao samples from different batches have good similarity, indicating that the reference fingerprint is representative. Figure 2 R is used as the evaluation standard for the samples of Bazhen Gao. See Table 3, taking the peak No. 13 with good separation, the largest peak area and peak height as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak, and the calculation results are shown in Tables 4 and 5 respectively.

[0077] Table 2 Similarity between the chromatogram of 10 batches of Bazhen Gao and the reference fingerprint

[0078]

[0079]

[0080] Table 3 Peaks, peak areas and retention times in the fingerprints

[0081]

[0082] Table 4 Relative retention time of common peaks of 10 batches of Bazhen Gao

[0083]

[0084]

[0085] Table 5. Relative peak areas of the common peaks of 10 batches of Bazhen Gao

[0086]

[0087] Compare the chromatogram of the test sample with that of the reference sample, refer to Figure 3 According to the retention time of each reference substance chromatographic peak, 5 of the 15 common peaks on the chromatogram of the Bazhen Gao sample were identified, namely 5-hydroxymethylfurfural (peak 1), paeoniflorin (peak 11), chlorogenic acid (peak 8), ammonium glycyrrhizinate (peak 16), and liquiritin (peak 12). In addition, there is 1 common peak that cannot be identified. It may be a component produced by the process or reaction between chemical components during the production of Chinese patent medicines, and is not contained in the 8 medicinal materials of Bazhen Gao alone. Further exploration is needed.

[0088] The test sample and 8 single herb solutions (Dangshen, Atractylodes macrocephala (fried), Poria, Licorice, Angelica sinensis, White Peony Root, Chuanxiong, Rehmannia glutinosa) were prepared according to the test sample preparation method of this embodiment. Specifically, the above 8 herbs were crushed and decocted twice with 10 times and 8 times water respectively, each time for 1 hour, filtered, the above two filtrates were combined, and concentrated to 150mL to 200mL for standby use; then the single decoction sample solution was prepared according to the test sample preparation method of this embodiment.

[0089] The above single decoction sample solutions were analyzed by HPLC using the chromatographic conditions of this embodiment to obtain fingerprints of 8 single herbs. Figure 4 , from bottom to top in the spectrum are: Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Licorice root, Angelica sinensis, White Peony Root, Chuanxiong, Rehmannia glutinosa. The 19 common peaks of the fingerprint of Bazhen Gao calibrated in this example are compared with the above Figure 4 The fingerprint spectra of 8 single herbs were compared and analyzed, and the source herbs of 19 common peaks in the fingerprint spectra of Bazhen Paste were attributed. The attribution results are shown in Table 6.

[0090] Table 6 The medicinal properties of the 19 peaks in the chromatogram of the test sample

[0091]

[0092] As can be seen from Table 6, the 19 common peaks of the Bazhen Paste samples are attributed to the eight herbs in the Bazhen Paste, namely, Codonopsis pilosula, Atractylodes macrocephala (fried), Poria cocos, Licorice root, Angelica sinensis, White Peony Root, Ligusticum chuanxiong, and Rehmannia glutinosa. This shows that the fingerprint method can be used to comprehensively control the quality of the Bazhen Paste.

[0093] Example 2

[0094] This example conducts a methodological investigation on the construction method of the determined Bazhen Gao fingerprint spectrum, including the investigation of precision, repeatability, and stability. The specific investigation method and conclusion are as follows:

[0095] 1. Precision inspection

[0096] The method of this embodiment was used to prepare the sample of Bazhen Gao, and the same chromatographic conditions were used for continuous injection for 6 times. 19 common peaks in the spectrum were manually selected for multi-point correction. After matching the Mark peak, the average method was used to generate a control fingerprint spectrum, with a time width of 0.1, and the similarity between the spectra in the comparison result data was compared. The results showed that the similarity between the results of the 6 injections was between 99.9% and 100%, see Table 7, indicating that the method of this application has good precision.

[0097] Table 7 Precision spectrum similarity evaluation

[0098]

[0099] 2. Repeatability test

[0100] The method of this embodiment was used to prepare 6 groups of test samples from the same batch of Bazhen Gao, and the same chromatographic conditions were used for sample injection and detection. 19 common peaks in the spectrum were manually selected for multi-point correction. After matching the Mark peak, the average method was used to generate a control fingerprint spectrum with a time width of 0.1, and the similarity between the spectra in the comparison result data was compared. The results showed that the similarity between the results of the 6 injections was between 99.9% and 100%, see Table 8, indicating that the method of this application has good repeatability.

[0101] Table 8 Repeatability verification results

[0102]

[0103] 3. Stability inspection

[0104] The method of this embodiment was used to prepare the Bazhen Gao test sample, and the same chromatographic conditions were used. The sample was injected 8 times at 8 time points (0h, 2h, 4h, 6h, 8h, 10h, 12h, 24h), and 19 common peaks in the spectrum were manually selected for multi-point correction. After matching the Mark peak, the average method was used to generate a control fingerprint spectrum, and the time width was 0.1. The similarity between the spectra in the comparison result data was compared. The results showed that the similarity between the results of the 8 injections was between 98.7% and 99.9%, as shown in Table 9, indicating that the sample prepared by the method of this application maintained stable quality within 24 hours after preparation.

[0105] Table 9 Stability verification results

[0106]

[0107] Example 3 Parameter optimization in the construction of the fingerprint of Bazhen Gao

[0108] The study examined the effects of different brands of chromatographic columns, different column temperatures, different flow rates, and different solvents on the construction of fingerprints.

[0109] (1) Effect of different solvents

[0110] The effects of different solvents (100% methanol, 70% methanol, 100% ethanol, 70% ethanol) on the construction of fingerprints were investigated. The results are shown in Figure 5 , the spectra from bottom to top are: 100% methanol group, 70% methanol group, 100% ethanol group, 70% ethanol group, among which 70% methanol has the best extraction effect, with a large number of peaks and high separation.

[0111] (2) Effect of different column temperatures

[0112] The effects of different column temperatures (35°C, 30°C, 25°C, 20°C) on the construction of fingerprints were investigated. The results are shown in Figure 6 , the spectra from bottom to top are: 35℃, 30℃, 25℃, 20℃, among which 30℃ has the best separation.

[0113] (3) Influence of different chromatographic columns

[0114] The effects of different chromatographic columns ZORBAXSB-C18 (S1) and CAPCELLPAKC18MGⅡ (S2) on the construction of fingerprints were investigated. The results are shown in Figure 7 , the spectra from bottom to top are: ZORBAX SB-C18 chromatogram, CAPCELLPAKC18MGⅡ chromatogram, among which CAPCELLPAKC18MGⅡ has better separation.

[0115] (4) Effect of different gradient elution conditions

[0116] The proportion of the organic phase was set from low to high. According to the results of the spectrum, the peaks of the chemical components in the extract of Bazhen Gao were mainly concentrated in the range of 93% 0.2% phosphoric acid aqueous solution-7% acetonitrile to 70% 0.2% phosphoric acid aqueous solution-30% acetonitrile. According to the density of peaks in different retention time regions, the change rate of the mobile phase ratio in different unit time was set, the total method time was increased, and the peak information in the fingerprint spectrum was presented as completely and clearly as possible. The specific experiments of some gradient elution settings are shown in Tables 10 to 13.

[0117] Table 10 First gradient elution settings

[0118]

[0119] Table 11 Second gradient elution settings

[0120]

[0121] Table 12 The third gradient elution setting

[0122]

[0123] Table 13 Fourth gradient elution settings

[0124]

[0125] Finally, the optimized gradient elution program was obtained as follows:

[0126] From 0 to 10 min, mobile phase A was reduced from 93% to 85%, and mobile phase B was increased from 7% to 15%;

[0127] From 10 to 25 min, mobile phase A decreased from 85% to 76%, and mobile phase B increased from 15% to 24%;

[0128] From 25 to 30 min, mobile phase A was reduced from 76% to 70%, and mobile phase B was increased from 24% to 30%;

[0129] 30-35 min, mobile phase A was reduced from 70% to 55%, and mobile phase B was increased from 30% to 45%;

[0130] 35-40 min, mobile phase A was reduced from 55% to 10%, and mobile phase B was increased from 45% to 90%;

[0131] 40-50 min, mobile phase A is 10%, mobile phase B is 90%;

[0132] 50-51 min, mobile phase A increased from 10% to 93%, and mobile phase B decreased from 90% to 7%;

[0133] 51-60min, mobile phase A is 93%, mobile phase B is 7%.

[0134] The fingerprint method established in this application can simultaneously detect many fingerprint components in Bazhen Paste, and can more accurately reflect the quality of the preparation. At the same time, the fingerprint method constructed in this application has good precision, repeatability and stability, rich chromatographic peak information and good separation. The established fingerprint can effectively characterize the quality of Bazhen Paste, which is helpful to improve the quality evaluation system of Bazhen Paste and provide a theoretical and practical basis for the comprehensive and effective control of the quality of Bazhen Paste.

[0135] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a fingerprint of Bazhen Paste, characterized in that: The following steps are involved: S1. Weigh Bazhen Gao and add methanol solution to obtain a test sample; weigh 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, baicalin, ammonium glycyrrhizinate, and liquiritin respectively, and add them to the methanol solution to obtain reference substances containing the above six components respectively; S2, performing high performance liquid chromatography analysis on the test sample and the reference substance obtained in step S1, respectively, to obtain chromatograms of the test sample and each of the reference substances; The conditions of the high performance liquid chromatography analysis are as follows: the stationary phase of the chromatographic column is octadecylsilane bonded silica gel as filler, a 0.1% to 0.2% by volume inorganic acid solution is used as mobile phase A, and acetonitrile is used as mobile phase B for gradient elution; the step of the gradient elution is carried out according to the following procedure, calculated by volume fraction: From 0 to 10 min, mobile phase A decreased from 93% to 85%, and mobile phase B increased from 7% to 15%; From 10 to 25 min, mobile phase A decreased from 85% to 76%, and mobile phase B increased from 15% to 24%; From 25 to 30 min, mobile phase A was reduced from 76% to 70%, and mobile phase B was increased from 24% to 30%; 30-35 min, mobile phase A was reduced from 70% to 55%, and mobile phase B was increased from 30% to 45%; 35-40 min, mobile phase A was reduced from 55% to 10%, and mobile phase B was increased from 45% to 90%; 40-50 min, mobile phase A is 10%, mobile phase B is 90%; 50-51 min, mobile phase A increased from 10% to 93%, and mobile phase B decreased from 90% to 7%; 51-60 min, mobile phase A: 93%, mobile phase B: 7%; S3. Determine the common peaks from the chromatogram of the test sample, compare the common peaks with the chromatogram of the reference sample, identify the chemical components of the common peaks, then select reference peaks, calculate the relative retention time of the common peaks, and construct the fingerprint of the Eight Treasures Paste.

2. The method for constructing the fingerprint of Bazhen Gao according to claim 1, characterized in that: A similarity evaluation was performed on the fingerprint of the Eight Treasures Paste. The similarity evaluation method was as follows: the chromatograms of multiple batches of the test samples and the fingerprint of the Eight Treasures Paste were imported into a similarity evaluation software for similarity evaluation analysis. The similarities were all greater than 0.

9. The number of common peaks was 19.

3. The method for constructing the fingerprint of Bazhen Gao according to claim 1, characterized in that: In the step of identifying the chemical components of the common peaks, a total of 5 peaks were identified, which were the peaks corresponding to 5-hydroxymethylfurfural, paeoniflorin, chlorogenic acid, ammonium glycyrrhizinate, and liquiritin.

4. The method for constructing the fingerprint of Bazhen Gao according to claim 1, characterized in that: During the high performance liquid chromatography analysis in step S2, the injection flow rate is 1.0 mL / min to 2.0 mL / min, the column temperature is 25° C. to 35° C., the injection volume is 10 μL to 20 μL, the detection wavelength is 230 nm to 400 nm, and the analysis time is 50 min to 90 min.

5. The method for constructing the fingerprint of Bazhen Gao according to claim 4, characterized in that: During the high performance liquid chromatography analysis in step S2, the injection flow rate is 1.0 mL / min, the column temperature is 30° C., the injection volume is 10 μL, the detection wavelength is 230 nm, and the analysis time is 60 min.

6. The method for constructing the fingerprint of Bazhen Gao according to claim 1, characterized in that: Before the high performance liquid chromatography analysis in step S2, the test sample and the reference sample are filtered through a 0.22 μm to 0.45 μm microporous filtration membrane respectively.

7. The method for constructing the fingerprint of Bazhen Gao according to any one of claims 1 to 6, characterized in that: The volume percentage of the methanol solution in step S1 is 70% to 100%.

8. The method for constructing the fingerprint of Bazhen Gao according to any one of claims 1 to 6, characterized in that: In step S1, the eight-treasure paste is added to the methanol solution in a material-liquid ratio of 1:5-10; and the six components are respectively added to the methanol solution in a material-liquid ratio of 1:5-10.

9. The fingerprint of Bazhen Paste obtained by the method for constructing the fingerprint of Bazhen Paste according to any one of claims 1 to 8.

10. Application of the fingerprint of Bazhen Paste obtained by the method for constructing the fingerprint of Bazhen Paste according to any one of claims 1 to 8 in the quality control process of Bazhen Paste.

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