Establishment method of HPLC (High Performance Liquid Chromatography) fingerprint spectrum of detoxification

Abstract

The invention belongs to the field of preparation analysis, and discloses a method for establishing an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of detoxification and hematogenesis granules. According to the invention, a method of combining the HPLC fingerprint spectrum and the chemical mode of the detoxification and hematogenesis granules is established, the contents of the deacetylasperuloside, the rosmarinic acid, the scutellarin, the luteolin, the apigenin and the kaempferol in the detoxification and hematogenesis granules are detected at the same time, and a comprehensive quality evaluation model of the detoxification and hematogenesis granules with different batch numbers is established, so that the analysis result is comprehensive and objective; the method is simple, convenient, reliable, good in repeatability, accurate, stable and high in sensitivity, the multi-component content determination method provides scientific support for quality supervision of the Zhuang medicine preparation detoxification and hematopoiesis granules, and the method can be used for comprehensive quality evaluation and control of the detoxification and hematopoiesis granules. Therefore, the production stability of product quality and the safety and effectiveness of clinical medication are ensured.

Description

Technical Field

[0001] The invention belongs to the field of preparation analysis, and more specifically relates to a method for establishing an HPLC fingerprint spectrum of detoxification and blood-generating granules. Background Art

[0002] The detoxification and blood-generating granules are composed of 13 Zhuang herbs, including Hedyotis diffusa, Scutellaria barbata, Solanum nigrum, Portulaca oleracea, Prunella vulgaris, Herba Artemisiae chinensis, Fritillaria thunbergii, Panax notoginseng, Curcuma zedoaria, Astragalus, Red ginseng, Angelica sinensis, and Licorice. Approval number: Guiyaozhizi M20100002. It has been used in the form of a decoction in Guangxi International Zhuang Medicine Hospital since 1998. It is mainly used in the clinical treatment of acute lymphocytic leukemia, acute myeloid leukemia, and chronic myeloid leukemia, in combination with chemotherapy drugs or alone. After long-term clinical verification, the total effective rates for acute lymphocytic leukemia, acute myeloid leukemia, and chronic myeloid leukemia reached 78.13%, 87.80%, and 63.64%, respectively. It is safe and reliable for clinical use, has a definite effect, has no toxic side effects, and is well tolerated by patients.

[0003] Traditional Chinese medicine believes that the Herba Hedyotis diffusae in this preparation clears away heat and detoxifies, the Scutellaria barbata clears heat and detoxifies, disperses blood stasis and relieves pain, the Solanum nigrum clears heat and detoxifies, and reduces swelling, and the Herba Portulacae oleraceae clears heat and detoxifies, and cools blood and reduces swelling. The four herbs are used together to achieve the effect of clearing heat and detoxifying, and are collectively the monarch herbs; the Herba Prunellae clears heat, disperses stagnation, and reduces swelling, the Herba Agrimoniae Smallis clears heat, promotes dampness, detoxifies, and stops bleeding, the Bulbus Fritillariae Thunbergii clears heat and resolves phlegm, relieves depression and disperses stagnation, the Herba Notoginseng disperses blood stasis and stops bleeding, reduces swelling and relieves pain, and the Rhizoma Curcumae (prepared with vinegar) promotes qi, resolves blood stasis, eliminates accumulation and relieves pain. The five herbs are collectively the minister herbs, which assist the main herbs in clearing heat and detoxifying, and have the effects of cooling blood and stopping bleeding, dispersing blood stasis and dispersing stagnation, and regulating the qi of the dragon path and the fire path; the Herba Red Ginseng replenishes qi and strengthens the spleen, the Herba Astragali replenishes qi and raises yang, benefits the Defence and consolidates the exterior, and the Herba Angelicae Sinensis replenishes blood and regulates menstruation. The three herbs are also adjuvant herbs that invigorate qi and nourish blood; and the Radix Licorice harmonizes the various herbs and resolves all toxins, and is the guiding herb. The combination of these medicines can clear away heat and toxins, dissipate blood stasis, replenish qi and blood, and open up the dragon path, fire path, and regulate the valley path and the air path. Chinese medicine believes that its functions and indications are clearing away heat and toxins, dissipating blood stasis and dissipating stagnation, and replenishing qi and blood.

[0004] However, this Zhuang medicine preparation has many ingredients and complex components, and has the characteristics of multi-target, multi-pathway, and multi-component synergistic effects. It lacks modern quality control and evaluation standard methods, which restricts its further application and development.

[0005] Therefore, how to provide a method for establishing the HPLC fingerprint of Jiedu Shengxue Granules so as to comprehensively, macroscopically and comprehensively reflect and control the quality of the preparation is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies in the prior art, the present invention provides a method for establishing an HPLC fingerprint of detoxification and blood-generating granules.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for establishing an HPLC fingerprint of detoxification and blood-generating granules comprises the following steps:

[0009] (1) Preparation of reference solution: weigh appropriate amounts of deacetylated oxalic acid methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol and apigenin reference substances respectively, dissolve them in methanol to prepare a mixed reference solution;

[0010] (2) Preparation of test solution: Take an appropriate amount of Jiedu Shengxue granules, grind them into powder, weigh them accurately, place them in a stoppered conical flask, add methanol accurately, stopper them tightly, weigh them, treat with ultrasound, cool them, weigh them again, make up the loss with methanol, shake them well, filter them, evaporate the filtrate to dryness, dissolve the residue in distilled water, extract them with ethyl acetate, evaporate the ethyl acetate layer to dryness, dissolve the residue in methanol, place them in a volumetric flask, dilute to the mark, and filter them with a microporous membrane to obtain the test solution;

[0011] (3) Determination of HPLC chromatographic conditions: chromatographic column TYPEMG II, methanol as mobile phase A, 0.4% phosphoric acid solution as mobile phase B, gradient elution;

[0012] (4) Preparation of HPLC standard fingerprint: Analyze and compare the reference solution and the test solution according to the chromatographic conditions of step (3) to obtain the HPLC standard fingerprint of Jiedu Shengxue Granules consisting of the common characteristic peaks of the samples;

[0013] (5) The peak areas of the common characteristic peaks of the HPLC standard fingerprints of multiple batches of samples were imported into SPSS23.0 software for cluster analysis and principal component analysis, and then imported into SIMCA 14.1 software for OPLS-DA analysis.

[0014] Preferably, in step (2), an appropriate amount of detoxification and blood-generating granules is taken, ground, accurately weighed to 2.5 g, placed in a stoppered conical flask, accurately added with 25 mL of 80% methanol, sealed, weighed, ultrasonically treated for 40 min, cooled, weighed again, supplemented with 80% methanol to make up the lost mass, shaken well, filtered, the filtrate is evaporated to dryness, the residue is dissolved in 20 mL of distilled water, extracted three times with 20 mL of ethyl acetate, the ethyl acetate layer is evaporated to dryness, the residue is dissolved in methanol, placed in a 2 mL volumetric flask, fixed to the scale line, and filtered with a 0.45 um microporous filter membrane to obtain the product.

[0015] Preferably, the chromatographic column in step (3) has a specification of 4.6×250 mm and a particle size of 5 μm.

[0016] Preferably, the gradient elution conditions in step (3) are: 0-10 min, 6%-12% A; 10-20 min, 12%-28% A; 20-30 min, 28%-52% A; 30-50 min, 52%-60% A; 50-60 min, 60%-73% A; 60-62 min, 73%-6% A; 62-77 min, 6% A.

[0017] Preferably, in step (3), the flow rate is 1.0 mL / min, the column temperature is 25° C., the detection wavelength is 254 nm, and the injection volume is 10 μL.

[0018] Preferably, the number of samples is 10 batches.

[0019] Preferably, the fingerprint comprises 16 common peaks.

[0020] Preferably, the fingerprint spectrum includes 16 common peaks, among which peak 3 is deacetylcaryophylle methyl ester, peak 8 is scutellarin, peak 9 is rosmarinic acid, peak 14 is luteolin, peak 15 is kaempferol, and peak 16 is apigenin.

[0021] It can be seen from the above technical solution that, compared with the prior art, the present invention provides a method for establishing the HPLC fingerprint of Jiedu Shengxue granules, which has the following beneficial effects:

[0022] 1. The present invention establishes a method for combining the HPLC fingerprint of Jiedu Shengxue Granules with a chemical model, and simultaneously detects the contents of deacetylcarbamate methyl ester, rosmarinic acid, scutellariae glycoside, luteolin, apigenin, and kaempferol therein, and establishes a comprehensive quality evaluation model for Jiedu Shengxue Granules of different batches, the analysis results are comprehensive and objective, the method is simple and reliable, has good repeatability, is accurate and stable, and has high sensitivity, and the multi-component content determination method provides scientific support for the quality supervision of the Zhuang medicine preparation Jiedu Shengxue Granules, and can be used for comprehensive quality comprehensive evaluation and control of the Jiedu Shengxue Granule preparation, thereby ensuring the production stability of product quality and the safety and effectiveness of clinical medication.

[0023] 2. When screening the mobile phase composition, since the proportion of water phase in the mobile phase of the chromatographic elution method is relatively large, the Shiseido TYPEMG II 5μm (4.6mmL.D.×250mm) chromatographic column with better water resistance was selected for fingerprint methodological investigation; and in the selection of mobile phase water phase and organic phase, when the water phase is 0.4% phosphoric acid solution and the organic phase is methanol, the number of chromatographic peaks is large and the separation is better. Therefore, methanol-0.4% phosphoric acid solution was selected as the mobile phase. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention 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 embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0025] Figure 1 This is an overlay of HPLC fingerprints of 10 batches of Jiedu Shengxue granules.

[0026] Figure 2 This is the standard fingerprint of Jiedu Shengxue Granules HPLC, in which: 1-16 are 16 common peaks.

[0027] Figure 3 It is the HPLC spectrum of the mixed reference solution, wherein: Peak 3-deacetylcaryophylle methyl ester, Peak 8-scutellarin, Peak 9-rosmarinic acid, Peak 14-luteolin, Peak 15-kaempferol, Peak 16-apigenin.

[0028] Figure 4 This is the dendrogram of cluster analysis of 10 batches of Jiedu Shengxue granules.

[0029] Figure 5 This is the OPLS-DA model score graph for 10 batches of Detoxification and Blood-Generating Granules.

[0030] Figure 6 VIP values ​​of 16 chromatographic peaks of 10 batches of Detoxification and Blood-producing Granules. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The instruments used in the present invention include: LC-2030c plus high performance liquid chromatograph (Shimadzu Corporation, Japan), SB-5200D ultrasonic cleaning machine (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.), EVERTECH ultrapure water instrument (Guangxi Aiwei Technology Co., Ltd.), and AS220.R2 analytical balance (made in Poland by Suzhou Peco Laboratory Instrument Technology Co., Ltd.).

[0033] The materials used in the present invention are as follows:

[0034] Reference substances: deacetylated oxalic acid methyl ester (batch number: 170100-202202, purity greater than 98%), baicalin (batch number: 250025-202403, purity greater than 98%), rosmarinic acid (batch number: 130013-202301, purity greater than 98%), luteolin (batch number: 130057-202403, purity greater than 98%), kaempferol (batch number: 190035-202403, purity greater than 98%), and apigenin (batch number: 170072-202403, purity greater than 98%) were all purchased from Shanghai Hongyong Biotechnology Co., Ltd.

[0035] Reagents: 85% phosphoric acid (Tianjin Damao Chemical Reagent Factory, product batch number: 20220801); chromatographic methanol (Thermo Fisher Scientific (China) Co., Ltd., product batch number: F2403D203); ethyl acetate (Sinopharm Chemical Reagent Co., Ltd. 20220927); analytical methanol (Chengdu Cologne Chemicals Co., Ltd., product batch number: 2023101302); water was prepared by the ultrapure water manufacturing system of Chengdu Yuechun Technology Co., Ltd.

[0036] Test sample: Zhuang medicine preparation Jiedu Shengxue granules, purchased from Guangxi International Zhuang Medicine Hospital. A total of 10 batches of Zhuang medicine preparation Jiedu Shengxue granules were collected, and the product batch numbers are shown in Table 1.

[0037] Table 110 Batch No. of Zhuang Medicine Detoxification and Blood-Rising Granules

[0038]

[0039] Example 1

[0040] A method for establishing an HPLC fingerprint of detoxification and blood-generating granules comprises the following steps:

[0041] (1) Preparation of reference solution: weigh appropriate amounts of deacetylated oxalic acid methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol and apigenin reference substances respectively, accurately weigh them, and dissolve them in methanol to prepare a mixed reference solution containing 0.2, 0.104, 0.051, 0.007, 0.00515 and 0.02 mg / ml of the above six components respectively;

[0042] (2) Preparation of test solution: Take an appropriate amount of Jiedu Shengxue granules, grind them into powder, accurately weigh 2.5 g, place in a stoppered conical flask, accurately add 25 mL of 80% methanol, stopper, weigh, ultrasonically treat for 40 min, cool, weigh again, make up the loss with 80% methanol, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 20 mL of distilled water, extract three times with 20 mL of ethyl acetate, evaporate the ethyl acetate layer to dryness, dissolve the residue in methanol, place in a 2 mL volumetric flask, dilute to the mark, and filter with a 0.45 μm microporous filter membrane to obtain the test solution;

[0043] (3) Determination of HPLC chromatographic conditions: the chromatographic column was Shiseido TYPEMG II, with a particle size of 5 μm (4.6 mm L.D.×250 mm); methanol was used as mobile phase A, 0.4% phosphoric acid solution was used as mobile phase B, and gradient elution was performed (0-10 min, 6%-12% A; 10-20 min, 12%-28% A; 20-30 min, 28%-52% A; 30-50 min, 52%-60% A; 50-60 min, 60%-73% A; 60-62 min, 73%-6% A; 62-77 min, 6% A); the flow rate was 1.0 mL / min; the column temperature was 25°C; the detection wavelength was 254 nm; and the injection volume was 10 μL.

[0044] (4) Preparation of HPLC standard fingerprint: Take 2.5g of each of 10 batches of Jiedu Shengxue Granules with different product batch numbers (numbers: S1~S10), prepare the test solution according to step (2), and obtain a total of 10 batches of test solution. According to the chromatographic conditions of step (3), inject 10μL respectively, perform the measurement, obtain HPLC chromatograms of different batches, and collect the chromatograms. Use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) for analysis to obtain the Jiedu Shengxue Granule HPLC standard fingerprint. Figure 1 and Figure 2 shown.

[0045] There are 16 common peaks in 10 batches of Jiedu Shengxue Granules with different batch numbers. By comparing the spectrum with the mixed reference solution ( Figure 3 ), a total of 6 chromatographic peaks were identified, namely, peak 3 for deacetylcaryophylle methyl ester, peak 8 for scutellarin, peak 9 for rosmarinic acid, peak 14 for luteolin, peak 15 for kaempferol, and peak 16 for apigenin. Deacetylcaryophylle methyl ester was used as the reference peak because of its larger peak area and better separation. The relative retention times and relative peak areas of other components were calculated, as shown in Tables 2 and 3 for details.

[0046] Take 10 batches of test sample solutions prepared under the conditions of step (2) and measure them respectively under the chromatographic conditions of step (3). Import the data into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition), and use the HPLC control fingerprint of the sample as a reference to perform an overall similarity evaluation. Results The similarity of 10 batches of different batches of Jiedu Shengxue Granules (numbers: S1 to S10) is between 0.991 and 0.998, indicating that there are slight quality differences between different batches of Jiedu Shengxue Granules, as shown in Table 4.

[0047] Table 2 Relative retention time of common peaks in HPLC fingerprints of 10 different batches of Jiedu Shengxue Granules

[0048]

[0049]

[0050] Table 3 Relative peak areas of common peaks in HPLC fingerprints of 10 batches of different batches of Jiedu Shengxue Granules

[0051]

[0052] Table 4 Similarity results of 10 batches of different batches of Jiedu Shengxue granules

[0053]

[0054] 1. Methodological Investigation

[0055] 1.1 Precision test

[0056] Take the test solution S5, inject according to the chromatographic conditions of step (3), inject 6 times continuously, and collect the chromatogram. Take the retention time and peak area of ​​deacetylmosin methyl ester as the reference peak, and record the relative retention time and relative peak area of ​​each common peak. The results show that the RSD of the relative retention time of the 16 common peaks is less than 0.35% (n=6), and the RSD of the relative peak area is less than 2.50% (n=6). The similarity calculated by the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) is greater than 0.99, indicating that the instrument has good precision.

[0057] 1.2 Repeatability test

[0058] Take the test solution S5, divide it into 6 parts in parallel, inject and measure according to the chromatographic conditions of step (3), and collect chromatograms. Take the retention time and peak area of ​​deacetylmosin methyl ester as the reference peak, and calculate the relative retention time and relative peak area of ​​each common peak. The results show that the relative retention time RSD of the 16 common peaks is less than 0.1%, and the relative peak area RSD is less than 2.5% (n=6). The similarity calculated by the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) is greater than 0.99, indicating that the method has good repeatability.

[0059] 1.3 Stability test

[0060] Take the test solution S5, and place it at room temperature for 0, 4, 8, 12, 18, and 24 hours, and then inject and measure according to the chromatographic conditions of step (3), and record the chromatogram. The retention time and peak area of ​​deacetyl oxalic acid methyl ester peak are used as reference peaks to calculate the relative retention time and relative peak area of ​​each common peak. The results show that the relative retention time RSD of the 16 common peaks is less than 0.61%, and the relative peak area RSD is less than 2.40% (n=6). The similarity calculated by the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) is greater than 0.99, indicating that the test solution is stable within 24 hours at room temperature.

[0061] 2. Chemometric Analysis

[0062] 2.1 Cluster analysis

[0063] The peak areas of the 16 common peaks of the HPLC fingerprints of 10 batches of Jiedu Shengxue granules were characterized by cluster analysis using SPSS23.0 analysis software. The intergroup linkage method was used to measure the Euclidean square distance to perform cluster analysis on the samples. The results are as follows: Figure 4 When the classification distance is 7, 10 batches of detoxification and blood-generating granules can be clustered into four categories, among which S4, S5, S1, are clustered into Class I, S2, S3, are clustered into Class II, S8, S9, S10, are clustered into Class III, and S6, S7 are clustered into Class IV. When the classification distance is 20, 10 batches of detoxification and blood-generating granules can be clustered into two categories, among which S4, S5, S1, S2, S3, are clustered into Class I, and S8, S9, S10, S6, S7 are clustered into Class II.

[0064] 2.2 Principal Component Analysis

[0065] The peak areas of 16 common peaks of 10 batches of Jiedushengxue granules were used as variables, and data were standardized by SPSS23.0 software, and then imported into SPSS23.0 software for principal component analysis. The eigenvalues ​​and variance contribution rates of the principal components are shown in Table 5. As can be seen from the table, the eigenvalues ​​of components 1 and 2 in the principal component analysis are both >1, and their cumulative variance contribution rate is 88.610%, indicating that extracting two principal components 1 and 2 can represent 88.610% of the information of the common peaks of Jiedushengxue granules samples, which is representative and can be used to evaluate the quality of Jiedushengxue granules. Further analysis of the principal component load matrix (see Table 6) shows that the principal component load matrix reflects the contribution size and direction of each variable to the principal component. The greater the absolute value of the load, the greater the contribution to the principal component. The information of principal component 1 comes from chromatographic peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, of which peak 3 is deacetylcaryophyllene methyl ester, peak 8 is scutellarin, and peak 9 is rosmarinic acid. The information of principal component 2 comes from chromatographic peaks 12, 14, 15, and 16, of which peak 14 is luteolin, peak 15 is kaempferol, and peak 16 is apigenin. In order to distinguish the quality differences among the 10 batches of Jiedu Shengxue granules, the scores of each principal component were calculated according to the principal component matrix data, and then the eigenvectors were standardized. According to the formula y1=0.2744*x7+0.2721*x6+0.2699*x11+0.2685*x16+0.2662*x13+0.2656*x9+0.2651*x10+0.2639*x5+0.2568*x14+0.2549*x15+0.2523*x12+0.2503*x8+0.2399*x1+0.1994*x2+0.1824*x3+0.1909*x4;

[0066] y2=-0.1538*x7+0.0291*x6-0.1989*x11-0.166*x16-0.0865*x13+0.1217*x9+

[0067] 0.1653*x10+0.264*x5+0.2104*x14+0.0138*x15-0.1354*x12-0.3321*x8-0.1936*x1-0.3267*x2+0.4354*x3+0.5318*x4;

[0068] Y=0.77936×y1+0.10374×y2.

[0069] The comprehensive scores of the principal components were calculated. It can be seen from Table 7 that the comprehensive scores of the four batches of Zhuang medicine preparation Jiedu Shengxue Granules, S7, S6, S10, and S9, ranked in the top four.

[0070] Table 5 Principal component eigenvalues ​​and variance contribution rates

[0071]

[0072]

[0073] Table 6 Principal component loading matrix

[0074]

[0075] Table 7 Principal component factor scores and rankings

[0076]

[0077]

[0078] 2.3 Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA)

[0079] Orthogonal partial least squares discriminant analysis is a powerful chemometric tool that plays an important role in the quality control of traditional Chinese medicine. OPLS-DA can effectively distinguish different batches or sources of traditional Chinese medicine by analyzing the chemical composition data of traditional Chinese medicine, thereby evaluating the consistency and reliability of its quality. This method can help identify key quality markers in traditional Chinese medicine, which are key indicators of the effectiveness, safety and overall quality of traditional Chinese medicine. This tool is widely used in the quality control of traditional Chinese medicine and its preparations. The peak areas of 16 common peaks of 10 batches of Jiedu Shengxue granules were used as variables and imported into SIMCA 14.1 software for OPLS-DA analysis to obtain the corresponding model. The score graph is shown in Figure 5 As can be seen from the figure, the samples of S1, S2, S3, S4, and S5 are clustered into one category, and the samples of S6, S7, S8, S9, and S10 are clustered into one category. The separation of each category of samples is obvious, indicating that the clustering is good. The explanation rate parameter R2X (cum) of the model is 0.897 (> 0.5), and the model discrimination parameter R2Y (cum) is 0.945 (> 0.5), indicating that the established model has a good fit and high stability. The cumulative prediction ability parameter Q2 (cum) is 0.852 (> 0.5), indicating that the prediction model has good accuracy and predictive power.

[0080] The VIP diagram can directly reflect the weight of each component on the difference between groups. VIP>1 means that the contribution of the variable to the established model is higher than the average level, which is used as the difference standard. A total of 7 components with large contributions were screened out by the VIP diagram, and they were ranked in order of size as peak 16 (apigenin), peak 12, peak 11 and peak 8 (scutellaria baicalin), peak 15 (kaempferol), peak 13 and peak 7. The results are shown in Figure 6 .

[0081] 3. Determination of multi-component content

[0082] 3.1 Linear Relationship

[0083] Accurately measure the single reference substance stock solution under step (2) and place it in a 25mL volumetric flask, mix it, dilute it to the mark with methanol, and shake it. Then accurately measure 1.75, 2.25, 2.5, 3.25, and 4mL of the mixed reference solution to a 5mL volumetric flask, dilute it to the mark with methanol, and shake it to obtain a reference substance mixed solution of each series concentration, filter it with a 0.45μm microporous filter membrane, take the filtrate, and inject it. According to the chromatographic conditions of step (3), the sample is injected and measured, and the peak areas of deacetylated oxalic acid methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol, and apigenin are recorded. The peak area (Y) is used as the ordinate and the concentration (X) of deacetylated oxalic acid methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol, and apigenin are used as the abscissa for linear regression. The linear regression equation and linear correlation coefficient are shown in Table 8. The results showed that deacetylcarbamate methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol and apigenin had good linearity within the detection concentration range.

[0084] Table 2-7 Standard curve regression equation and linearity

[0085]

[0086] 3.2 Precision test

[0087] The mixed reference solution of step (1) was accurately aspirated and injected six times continuously under the chromatographic conditions of step (3). The peak areas of deacetylcarbamate methyl ester, baicalin, rosmarinic acid, luteolin, kaempferol and apigenin were recorded. The RSDs of the peak areas of deacetylcarbamate methyl ester, baicalin, rosmarinic acid, luteolin, kaempferol and apigenin were calculated to be 0.84%, 0.56%, 0.77%, 1.14%, 1.04% and 0.49% (n=6), respectively, indicating that the precision of the instrument was good.

[0088] 3.3 Repeatability

[0089] Take the same batch of samples (batch number 210103), accurately weigh 6 portions, prepare the test solution according to the method of step (2), and parallel 6 portions, sample injection and determination according to the chromatographic conditions of step (3), record the peak areas of deacetylcarbamate methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol and apigenin, and calculate the content. Results The RSDs of the contents of deacetylcarbamate methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol and apigenin in the samples were 0.88%, 1.05%, 0.56%, 1.07%, 1.11% and 0.99% (n=6), respectively, indicating that the method has good repeatability.

[0090] 3.4 Stability test

[0091] Take a sample (batch number: 210103) and prepare a test solution according to the method of step (2). After preparation, place it at room temperature for 0, 4, 8, 12, 16, 20, and 24 hours, and then inject and measure it according to the chromatographic conditions of step (3). Record the peak areas of deacetylcarbamate methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol and apigenin. The RSDs of the peak areas of deacetylcarbamate methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol and apigenin were calculated to be 0.87%, 1.11%, 0.87%, 0.85%, 0.85%, and 1.06% (n=7), respectively, indicating that the test solution has good stability within 24 hours.

[0092] 3.5 Sample recovery test

[0093] Take 6 portions of the measured Jiedu Shengxue granule sample powder (batch number: 210103), each 1.25 g, accurately weigh, and accurately add reference solution containing deacetylcarbamate methyl ester (g), baicalin (g), rosmarinic acid (g), luteolin (g), kaempferol (g) and apigenin (g) respectively, prepare the test solution according to the method of step (2), and then inject and measure according to the chromatographic conditions of step (3), record the peak areas of deacetylcarbamate methyl ester, baicalin, rosmarinic acid, luteolin, kaempferol and apigenin to calculate the sample recovery rate and RSD value. The results showed that the average recoveries of deacetylcarbamate methyl ester, baicalin, rosmarinic acid, luteolin, kaempferol and apigenin were 100.58%, 98.65%, 95.16%, 91.93%, 98.35% and 97.06% (n=6), respectively, and the RSDs were 0.85%, 1.34%, 1.05%, 1.32%, 1.31% and 1.46%, respectively.

[0094] 3.6 Content determination

[0095] Take 10 batches of Jiedu Shengxue Granules, prepare the test solution according to the method of step (2), inject and measure according to the chromatographic conditions of step (3), and record the peak areas of deacetylcarbamate methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol and apigenin. The unit is mg / g. The results are shown in Table 9. The contents of deacetylcarbamate methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol and apigenin in the 10 batches of Jiedu Shengxue Granules are 0.0155-0.0208, 0.0269-0.0535, 0.0704-0.1195, 0.0039-0.0090, 0.0033-0.0083 and 0.0090-0.0327 mg / g, respectively.

[0096] Table 9 Contents of 6 active ingredients in 10 batches of Jiedu Shengxue Granules

[0097]

[0098] Therefore, the present invention studies the fingerprints of 10 batches of detoxification and blood-producing granules, establishes the HPLC control fingerprints of detoxification and blood-producing granules, obtains 16 common peaks, identifies 6 of them, and carries out similarity evaluation, respectively combines cluster analysis, principal component analysis, orthogonal partial least squares discriminant analysis, and comprehensively and objectively evaluates the quality of detoxification and blood-producing granules of different batches. The overall similarity is high in the similarity evaluation of the fingerprints of 10 batches of detoxification and blood-producing granules, and the similarity of 10 batches of samples is greater than 0.90, indicating that the chemical composition of the detoxification and blood-producing granules is relatively similar. There are 16 common peaks in the 10 batches of detoxification and blood-producing granules, but the RSD of the relative retention time is 0-0.34%, and the RSD of the relative peak area is 0-38.25%, indicating that although the chemical composition of these 10 batches of samples is relatively similar, there are still differences in the content. In cluster analysis, 3 out of 10 batches of Jiedushengxue granules were clustered into Class I, 2 into Class II, 3 into Class III, and 2 into Class IV. In the analysis of principal component eigenvalue and variance contribution rate, 2 principal components were extracted, with a cumulative contribution rate of 88.610%. The principal component factor comprehensive score ranked the 10 batches of samples, with S7, S6, S10, and S9 batches of Jiedushengxue granules at the front, indicating that there was an obvious classification trend between different batches of Jiedushengxue granules, which indirectly indicated that the preparation may be affected by factors such as the source of medicinal materials, the storage process, or the processing method during production, and the internal chemical composition of each batch may change. Therefore, in the production process of traditional Chinese medicine preparations, it is necessary not only to pay attention to standardized production during the production process, but also to strictly control the source of medicinal materials and quality control. These practices can provide important guarantees for the uniformity and stability of different batches of Jiedushengxue granules. This experiment also established an OPLS-DA model for analysis and concluded that the VIP values ​​of chromatographic peaks 16, 12, 11, 8, 15, 13, and 7 were greater than 1, which made a great contribution to the fingerprint of the detoxification and blood-generating granules, indicating that these components should be highly valued in quality analysis. However, chromatographic peaks 12, 11, 13, and 7 have yet to be identified and require further study.

[0099] Based on the 6 selected index components, content determination was carried out to further analyze and evaluate the Zhuang medicine preparation. The results showed that the components with the highest content in the same batch of preparations were rosmarinic acid, baicalin, and deacetylcarbamate methyl ester, indicating that these 3 components should be given high attention in quality control. However, due to different batches, the content of the same component in different batches was different, but the content of luteolin, kaempferol, and apigenin was still above 0.003 mg / g, indicating that luteolin, kaempferol, and apigenin can also be regarded as important factors in the comprehensive evaluation of the overall quality of the Zhuang medicine preparation Jiedu Shengxue Granules.

[0100] In summary, the HPLC fingerprint established in the present invention combined with the differential chemical components screened by orthogonal partial least squares discriminant analysis and the content determination and statistical analysis of components such as acetylcaryophylle methyl ester, baicalin, rosmarinic acid, luteolin, kaempferol and apigenin can illustrate the overall quality characteristics and differences of Jiedu Shengxue Granules, and provide a reference for the quality control of Jiedu Shengxue Granules.

[0101] 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 establishing an HPLC fingerprint of Jiedu Shengxue granules, characterized in that: The following steps are involved: (1) Preparation of reference solution: Weigh appropriate amounts of deacetylated oxalic acid methyl ester, scutellarin, rosmarinic acid, luteolin, kaempferol and apigenin reference substances respectively, dissolve them in methanol to prepare a mixed reference solution; (2) Preparation of test solution: Take an appropriate amount of Jiedu Shengxue granules, grind them into powder, weigh them accurately, place them in a stoppered conical flask, add methanol accurately, stopper them tightly, weigh them, treat with ultrasound, cool them, weigh them again, make up the loss with methanol, shake them well, filter them, evaporate the filtrate to dryness, dissolve the residue in distilled water, extract them with ethyl acetate, evaporate the ethyl acetate layer to dryness, dissolve the residue in methanol, place them in a volumetric flask, dilute to the mark, and filter them with a microporous membrane to obtain the test solution; (3) Determination of HPLC chromatographic conditions: chromatographic column TYPEMG II, methanol as mobile phase A, 0.4% phosphoric acid solution as mobile phase B, gradient elution; (4) Preparation of HPLC standard fingerprint: Analyze and compare the reference solution and the test solution according to the chromatographic conditions of step (3) to obtain the HPLC standard fingerprint of Jiedu Shengxue Granules consisting of the common characteristic peaks of the samples; (5) The peak areas of the common characteristic peaks of the HPLC standard fingerprints of multiple batches of samples were imported into SPSS23.0 software for cluster analysis and principal component analysis, and then imported into SIMCA 14.1 software for OPLS-DA analysis.

2. The method for establishing the HPLC fingerprint of the detoxification and blood-generating granules according to claim 1, characterized in that: Take an appropriate amount of Jiedu Shengxue Granules in step (2), grind them into powder, accurately weigh 2.5 g, place them in a stoppered conical flask, accurately add 25 mL of 80% methanol, stopper, weigh, ultrasonically treat for 40 min, cool, weigh again, make up the lost mass with 80% methanol, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 20 mL of distilled water, extract three times with 20 mL of ethyl acetate, evaporate the ethyl acetate layer to dryness, dissolve the residue with methanol, place in a 2 mL volumetric flask, dilute to the mark, and filter with a 0.45 um microporous filter membrane to obtain the product.

3. The method for establishing the HPLC fingerprint of the detoxification and blood-generating granules according to claim 1, characterized in that: The specifications of the chromatographic column in step (3) are 4.6×250 mm and the particle size is 5 μm.

4. The method for establishing the HPLC fingerprint of Jiedu Shengxue Granule according to claim 1, characterized in that: The gradient elution conditions in step (3) are: 0-10 min, 6%-12% A; 10-20 min, 12%-28% A; 20-30 min, 28%-52% A; 30-50 min, 52%-60% A; 50-60 min, 60%-73% A; 60-62 min, 73%-6% A; 62~77min, 6%A.

5. The method for establishing the HPLC fingerprint of Jiedu Shengxue Granule according to claim 1, characterized in that: In step (3), the flow rate was 1.0 mL / min, the column temperature was 25° C., the detection wavelength was 254 nm, and the injection volume was 10 μL.

6. The method for establishing the HPLC fingerprint of Jiedu Shengxue Granule according to claim 1, characterized in that: The number of samples was 10 batches.

7. The method for establishing the HPLC fingerprint of Jiedu Shengxue Granule according to claim 1, characterized in that: The fingerprint spectrum includes 16 common peaks.

8. The method for establishing the HPLC fingerprint of Jiedu Shengxue Granule according to claim 7, characterized in that: The fingerprint spectrum includes 16 common peaks, among which peak 3 is deacetylcaryophylle methyl ester, peak 8 is scutellarin, peak 9 is rosmarinic acid, peak 14 is luteolin, peak 15 is kaempferol, and peak 16 is apigenin.

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