Construction method and application of coughing capsule characteristic chromatogram

Abstract

The invention relates to a construction method and application of a coughing capsule characteristic chromatogram, which can effectively solve the problem of effective overall control and evaluation of the quality of a coughing capsule product. The method comprises the following steps: (1) preparing solutions: respectively preparing a test solution, a reference solution, a single medicinal material sample solution and a single medicinal material-lacking negative sample solution; (2) determining main components for constructing the characteristic spectrum; (3) constructing a characteristic spectrum: determining a characteristic peak and a reference peak, and establishing the characteristic spectrum and a control spectrum R; the method is simple and easy to operate, the product quality of the coughing capsule can be clearly judged by comparing the characteristic peak graph of the coughing capsule to be detected with the characteristic spectrum of the coughing capsule constructed by the method disclosed by the invention, and the problems of effective overall control and evaluation of the product quality of the coughing capsule can be effectively solved; the method is a great innovation in quality control and evaluation of the cough-relieving capsules, and has remarkable economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a method for constructing a characteristic spectrum of a Kening capsule and applications thereof. Background Art

[0002] Kening Capsules are a traditional Chinese medicine developed based on a proven folk remedy for whooping cough and colds in children. Made from pig bile extract, Fritillaria thunbergii, and Platycodon grandiflorum, they are primarily used to clear heat and promote lung function, resolve phlegm, and relieve cough. They are used to treat wind-heat, phlegm-heat cough, expectoration, and yellowish-white or thick sputum. Their quality directly impacts their efficacy, and to ensure quality, analysis and research of the capsule's active ingredients is essential. However, research on the individual ingredients in Kening Capsules is currently lacking. Current research demonstrates that Kening Capsules contain nucleosides, bile acids, and saponins, primarily uracil, cytidine, guanosine, adenine, uridine, thymidine, adenosine, and deoxyadenosine. Establishing a characteristic profile of the active ingredients, as a quality control technique, can ensure product quality and efficacy.

[0003] Characteristic spectrum is an efficient quality control model based on the overall understanding of traditional Chinese medicine. With the help of certain modern technologies such as spectroscopy and chromatography, information spectra or chromatograms of the chemical components of traditional Chinese medicine are obtained. The main characteristic peaks in the spectrum can fully reflect the relationship between the components contained. It has strong characteristic properties, can effectively identify the authenticity of samples, has a large amount of information, consistency, stability, integrity and other characteristics. The nucleoside components in the medicinal materials of Kening Capsules were analyzed by HPLC, and the characteristic spectrum of Kening Capsules was established using the traditional Chinese medicine fingerprint spectrum similarity evaluation system. It reflects the transfer relationship of the nucleoside components in Kening Capsules during the preparation process, reduces batch differences, can identify the traditional Chinese medicine components from a holistic perspective, and comprehensively reflect the relative relationship of the chemical components of traditional Chinese medicine. It can accurately and effectively control and evaluate the quality of traditional Chinese medicine and its preparations as a whole, and provide a scientific basis for better control of the quality of Kening Capsules products. However, there has been no public report so far. Summary of the Invention

[0004] In view of the above situation, in order to overcome the shortcomings of the existing technology, the purpose of the present invention is to provide a method for constructing a characteristic map of Kening Capsules and its application, which can effectively solve the problem of effective overall control and evaluation of the quality of Kening Capsules products.

[0005] The technical solution provided by the present invention is a method for constructing a characteristic spectrum of Kening Capsules, which is achieved by the following steps:

[0006] (1). Make solution

[0007] 1.1 Preparation of test solution

[0008] Place the Kening capsule powder in a stoppered conical flask, add 20% methanol solution by volume, mix well, heat in a water bath, cool, centrifuge, and filter to obtain the first filtrate. Repeat the above operation on the residue to obtain the second filtrate. Combine the filtrates and filter to obtain the test solution.

[0009] 1.2 Preparation of reference solution

[0010] Add uracil, cytidine, guanosine, adenine, uridine, thymidine, adenosine, and deoxyadenosine to purified water to prepare reference solutions;

[0011] 1.3 Preparation of single herbal sample solution

[0012] Accurately weigh the single medicinal materials in the test sample prescription, and then prepare the sample solutions of each single medicinal material according to "1.1";

[0013] 1.4. Preparation of negative sample solution for missing single medicinal material

[0014] Accurately weigh the negative sample of the missing single herbal ingredient in the prescription, and then prepare the negative sample solution of the missing single herbal ingredient according to "1.1";

[0015] (2) Determine the main components of constructing the feature map

[0016] The main medicinal ingredients of Kening Capsules are pig bile extract, Fritillaria thunbergii, and Platycodon grandiflorum. Using a high-performance liquid chromatograph, a 1 / 100,000 electronic balance, and a centrifuge, liquid chromatography (HPLC) was used to extract the characteristic spectrum of the active main ingredients in Kening Capsules under specific chromatographic conditions (known technology). The 12 main active ingredients of Kening Capsules were identified as: deoxyadenosine, thymidine, cytidine, adenine, adenosine, guanosine, uridine, uracil, and four common peaks with unidentified components.

[0017] (3) Constructing feature maps

[0018] 3.1. First determine the characteristic peak and reference peak

[0019] The characteristic peak of thymidine was used as the reference peak to calculate the relative retention time and relative peak area of the common peak;

[0020] 3.2. Establishment of feature maps

[0021] According to the method under "1.1", 15 batches of samples were prepared, numbered S1 to S15 as test solution, and injected according to the chromatographic conditions under "2". The chromatograms were recorded and saved in AIA format. The data were then imported into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition) software to construct the characteristic spectrum and the reference spectrum R.

[0022] Application of the characteristic map of Kening Capsules constructed by the method of the present invention in the overall control and evaluation of the quality of Kening Capsules products.

[0023] The method of the present invention is simple and easy to operate. The characteristic peak graph of the Kening capsule to be tested can be compared with the characteristic spectrum of the Kening capsule constructed by the method of the present invention, so that the product quality of the Kening capsule can be judged at a glance. The method can effectively solve the problem of effective overall control and evaluation of the product quality of the Kening capsule, is a major innovation in the quality control and evaluation of the Kening capsule, and has significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The chromatograms are obtained under different solvent conditions;

[0025] Figure 2 The chromatogram of the test solution is (Note: 1. uracil; 2. cytidine; 3. uridine; 6. adenine; 7. guanosine; 9. thymidine; 11. adenosine; 12. deoxyadenosine);

[0026] Figure 3 is the chromatogram of the mixed reference solution;

[0027] Figure 4 This is the chromatogram of single medicinal material of pig bile extract, Fritillaria thunbergii, and Platycodon grandiflorum;

[0028] Figure 5 The chromatograms of negative medicinal materials are missing pig bile extract, missing Fritillaria thunbergii, and missing Platycodon grandiflorum;

[0029] Figure 6 is the blank solution chromatogram;

[0030] Figure 7 This is the precision test chart of Kening Capsules finished product;

[0031] Figure 8 This is the repeatability test diagram of the finished product of Kening Capsules;

[0032] Figure 9 This is the stability test chart of the finished product of Kening Capsules;

[0033] Figure 10 This is the common pattern spectrum of characteristic spectra of 15 batches of Kening Capsules finished products;

[0034] Figure 11 This is the reference characteristic spectrum of the finished product of Kening Capsules;

[0035] Figure 12 is the chromatogram of mixed reference substance;

[0036] Figure 13 This is the clustering tree analysis diagram of 15 batches of Kening Capsules products;

[0037] Figure 14This is the lithotripsy picture of 15 batches of Kening Capsules;

[0038] Figure 15 This is the principal component analysis score diagram of 15 batches of Kening Capsule samples;

[0039] Figure 16 This is the OPLS-DA score graph of 15 batches of Kening Capsule samples;

[0040] Figure 17 This is the variable projection importance (VIP) value diagram of the components of 15 batches of Kening Capsules samples. DETAILED DESCRIPTION

[0041] The specific implementation methods of the present invention are described in detail below with reference to examples and specific situations.

[0042] The present invention can be specifically implemented by the following specific embodiments:

[0043] The technical solution provided by the present invention is a method for constructing a characteristic spectrum of Kening Capsules, which is achieved by the following steps:

[0044] (1). Make solution

[0045] 1.1 Preparation of test solution

[0046] Accurately weigh 2 g of Kening capsule powder and place it in a stoppered conical flask. Accurately measure 40 mL of 20% methanol solution and add it to the stoppered conical flask. Mix it evenly with the Kening capsule powder. Heat in a 60°C water bath for 0.5 h, let it cool, and add 20% methanol solution to make up the lost mass. Pour it into a centrifuge tube and centrifuge at 4000 r / min. -1 Centrifuge for 10 min, filter to obtain the first filtrate, repeat the above operation once to obtain the second filtrate, combine the filtrates, and filter with a 0.22 μm microporous membrane to obtain the test solution;

[0047] 1.2 Preparation of reference solution

[0048] Accurately weigh appropriate amounts of uracil, cytidine, guanosine, adenine, uridine, thymidine, adenosine, and deoxyadenosine reference substances and add purified water to prepare reference substance solutions with mass concentrations of 5.0136, 6.5175, 7.4925, 5.0555, 5.0645, 4.9705, 5.0455, and 5.0152 μg / mL, respectively;

[0049] 1.3 Preparation of single herbal sample solution

[0050] Accurately weigh the single medicinal materials with the same prescription as the test sample, and then prepare the sample solutions of each single medicinal material according to "1.1";

[0051] 1.4. Preparation of negative sample solution for missing single medicinal material

[0052] Accurately weigh the negative samples of the missing single herbal medicine in the prescription, and then prepare the sample solutions of each single herbal medicine according to "1.1";

[0053] (2) Determine the main components of constructing the feature map

[0054] The main drug components of Kening Capsules are pig bile extract, Fritillaria thunbergii and Platycodon grandiflorum. The characteristic spectrum active main components in Kening Capsules are extracted by liquid chromatography (HPLC) using a high performance liquid chromatograph, a 1 / 100,000 electronic balance and a centrifuge (known technology). The main active ingredients of Kening Capsules are determined to be: hyodeoxycholic acid, taurohyodeoxycholic acid, platycodon saponin deoxyadenosine, thymidine, cytidine, adenine, adenosine, guanosine, uridine and uracil, among which the main components of pig bile extract are hyodeoxycholic acid, taurohyodeoxycholic acid, platycodon saponin deoxyadenosine, thymidine, cytidine, adenine, adenosine, guanosine, uridine and uracil. The main components of Fritillaria thunbergii are deoxyadenosine and thymidine, and the main components of Platycodon grandiflorum are platycodon saponins. The common components of Fritillaria thunbergii and Platycodon grandiflorum are cytidine, adenine, adenosine, guanosine, uridine and uracil. When the characteristic spectrum was established, the chromatographic conditions were not compatible with the components of hyodeoxycholic acid, taurohyodeoxycholic acid and platycodon saponins. Only the 12 components under the chromatographic conditions were assigned to the characteristic spectrum. Among them, deoxyadenosine and thymidine were assigned to Fritillaria thunbergii, cytidine, adenine, adenosine, guanosine, uridine and uracil were assigned to Fritillaria thunbergii and Platycodon grandiflorum, and the remaining 4 common peaks were not assigned to a specific peak.

[0055] The chromatographic conditions are as follows: Chromatographic column: Agilent TC-C 18 Column: 4.6 mm × 250 mm, 5 μm; mobile phase: water (A): methanol (B) by volume, gradient elution as specified in the following table:

[0056] Time (min) Mobile phase A (%) Mobile phase B (%) 0～15 95 5 15～20 95→90 5→10 20～25 90→80 10→20 25～35 80→72 20→28 35～45 72→95 28→5

[0057] Detection wavelength: 260 nm; flow rate: 0.8 mL min -1 ; Injection volume: 20 μL; Column temperature: 25°C;

[0058] (3) Constructing feature maps

[0059] 3.1. First determine the characteristic peak and reference peak

[0060] In the characteristic spectrum, the thymidine chromatographic peak is stable and has moderate retention time, resolution, and peak area. Therefore, the characteristic peak of thymidine is selected as the reference peak to calculate the relative retention time and relative peak area of the common peak.

[0061] 3.2. Establishment of feature maps

[0062] According to the method under "1.1", 15 batches of samples were prepared, numbered S1 to S15 as test solution, and the samples were injected according to the chromatographic conditions under "2". The chromatograms were recorded and saved in AIA format. The data were then imported into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) software to obtain the characteristic spectra and reference spectra R of the 15 batches of samples. The method was: using S1 as the reference spectrum, the chromatographic peak time window width was 0.1, the median method, multi-point correction and automatic matching of each chromatographic peak to form a common pattern Figure 1. According to the characteristic spectrum analysis, 12 common peaks were identified in 15 batches of Kening Capsules finished products. The relative retention time and UV spectrum were compared with the reference peaks, which were peak 1-uracil, peak 2-cytidine, peak 3-uridine, peak 6-adenine, peak 7-guanosine, peak 9-thymidine, peak 11-adenosine and peak 12-deoxyadenosine. Among them, the four common peaks of peaks 4, 5, 8 and 10 were not identified. The 12 common peaks were used as characteristic peaks to construct the control characteristic spectrum R of Kening Capsules finished products and the chromatogram of the mixed reference (see Figure 11 、 12 ).

[0063] Application of the characteristic map of Kening Capsules constructed by the method of the present invention in the overall control and evaluation of the quality of Kening Capsules products.

[0064] The method of the present invention is simple, easy to operate, stable and reliable. By comparing the characteristic peak graph of the Kening capsule to be tested with the characteristic spectrum of the Kening capsule constructed by the method of the present invention, the overall quality of the Kening capsule product can be quickly and conveniently effectively controlled and evaluated. After testing, the effect is very good. The relevant information is as follows:

[0065] 1. Instruments and Materials

[0066] 1.1 Instrument

[0067] Table 1 Experimental instruments

[0068]

[0069] 1.2 Materials

[0070] Table 2 Experimental materials

[0071]

[0072] Fifteen batches of finished Kening capsules (batch numbers are 221231, 230101, 2301031, 2301032, 2212212, 2301052, 230106, 230109, 231207, 2401151, 2401152, 2403061, 2404301, and 2404302, numbered S1 to S15) were provided by Henan Lingrui Pharmaceutical Co., Ltd.

[0073] 2. Experimental Methods

[0074] 2.1 Chromatographic conditions

[0075] Chromatographic column: Agilent TC-C 18 Column (4.6 mm × 250 mm, 5 μm); Mobile phase: A (water)-B (methanol) gradient elution: 0-15 min (B 5%-5%); 15-20 min (B 5%-10%); 20-25 min (B 10%-20%); 25-35 min (B 20%-28%); 35-45 min (B 28%-5%). Detection wavelength: 260 nm; Flow rate: 0.8 mL min-1. 1 ; Injection volume: 20μL; Column temperature: 25℃.

[0076] 2.2 Solution preparation

[0077] 2.2.1 Preparation of test solution

[0078] Accurately weigh 2 g of Kening capsule powder and place it in a stoppered conical flask. Accurately measure 40 mL of 20% methanol solution, weigh the mass, mix well, heat in a 60°C water bath for 0.5 h, let cool, and add 20% methanol solution to make up the lost mass. Pour the mixture into a centrifuge tube and centrifuge at 4000 r / min. -1 Centrifuge for 10 minutes, filter, repeat the above operation once for the residue, and filter the combined filtrate through a 0.22 μm microporous membrane to obtain the product.

[0079] 2.2.2 Preparation of reference solution

[0080] Accurately weigh appropriate amounts of uracil, cytidine, guanosine, adenine, uridine, thymidine, adenosine, and deoxyadenosine reference substances, and add purified water to prepare mixed solutions with mass concentrations of 5.0136, 6.5175, 7.4925, 5.0555, 5.0645, 4.9705, 5.0455, and 5.0152 μg / mL, respectively.

[0081] 2.2.3 Preparation of single herbal medicine sample solution

[0082] Accurately weigh the single medicinal materials in the same prescription as the test sample, and prepare the sample solutions of each single medicinal material according to the subsequent processing method under "2.2.1".

[0083] 2.2.4 Preparation of negative sample solution for missing single medicinal material

[0084] Accurately weigh the negative samples of the missing single medicinal materials in the same prescription as the test sample, and prepare the sample solutions of each single medicinal material according to the subsequent processing method under "2.2.1".

[0085] 3. Method Development and Validation

[0086] 3.1 Investigation of test solution preparation method

[0087] 3.1.1 Investigation of test solution treatment methods

[0088] According to the tentative test solution preparation method, different test solution treatment methods were selected to investigate their effects on the stability of the test solution. Accurately weigh 2g of Kening Capsule powder and place it in a stoppered conical flask. The test was conducted using water and ultrasound, 10% methanol and ultrasound, 20% methanol and ultrasound, 10% methanol and water bath heating, and 20% methanol and water bath heating. The ultrasound power was 300W and the operating frequency was 40kHz. The water bath was heated at 60℃ for 0.5h. The solution was then poured into a centrifuge tube and heated at 4000r·min- 1 Centrifuge for 10 minutes, filter, and repeat the above steps once for the residue. Filter the combined filtrates through a 0.22 μm microporous membrane to obtain the product. Experimental results indicate that the 20% methanol and water bath heating method can ensure good stability of the test solution within 48 hours. Other methods can result in instability of the test solution and poor peak symmetry. Therefore, the 20% methanol and water bath heating method is suitable for preparing the test solution.

[0089] 3.1.2 Investigation of test sample solvent

[0090] According to the tentative method for preparing the test solution, different test solvents were selected to further investigate the effect on the peak shape of each reference substance in the chromatogram. 2 g of Kening Capsule powder was accurately weighed and placed in a stoppered conical flask. 40 mL of water, 20% methanol, 40% methanol, 50% methanol, 80% methanol, and methanol solution were accurately measured and weighed respectively. The mixture was evenly mixed and heated in a 60°C water bath for 0.5 h. The mixture was cooled and the amount of the lost mass was supplemented with 20% methanol. The mixture was poured into a centrifuge tube and centrifuged at 4000 r·min- 1 Centrifuge for 10 minutes, filter, and repeat the above operation once for the residue. The combined filtrate is filtered through a 0.22μm microporous filter membrane to obtain the product. The experimental results show that when 20% methanol is used as the solvent for the test sample, its peak shape is well symmetrical and the accuracy is relatively appropriate. It can be used as a solvent for the preparation of the test sample solution. Figure 1 )

[0091] 3.1.3 Investigation of test sample dosage

[0092] According to the tentative test solution preparation method, different test sample dosages were selected to further investigate the effect of the chromatogram on the peak shape of each reference substance. 1 g, 1.5 g, 2 g, 2.5 g, and 3 g of Kening Capsule powder were accurately weighed and placed in a stoppered conical flask. 40 mL of 20% methanol solution was accurately measured and weighed, mixed evenly, heated in a 60°C water bath for 0.5 h, cooled, and supplemented with 20% methanol to make up the lost mass. The mixture was poured into a centrifuge tube and centrifuged at 4000 r / min. -1Centrifuge for 10 minutes, filter, and repeat the above operation once for the residue. Filter the combined filtrate through a 0.22 μm microporous filter membrane to obtain the product. The experimental results show that when the sample weight is 2 g, the peak shape is obvious and the symmetry is good, which can be used as the starting sample weight for the test sample.

[0093] IV. Methodological Investigation

[0094] 4.1 Specificity Investigation

[0095] Take the test solution, reference solution, single herbal medicine solution, negative herbal medicine solution and blank solution respectively, and inject them according to the chromatographic conditions under "2.1" to detect the characteristic spectrum, and examine the chromatographic conditions and the specificity of the conditions. Figure 2-4 As shown, the negative medicinal material solution and blank solution have no interference with the common peaks of the characteristic spectrum.

[0096] 4.2 Precision test

[0097] Take Kening Capsules (S10, batch number 2401151), prepare the test solution according to the method of "2.2.1" and inject the sample continuously for 6 times according to the chromatographic conditions under "2.1". Take Peak 9 as the reference peak, calculate the relative retention time and relative peak area respectively, and the RSD values are all within 3% (n=6). The instrument precision is good. The results are shown in Tables 3, 4 and Figure 7 .

[0098] Table 3 Relative retention time precision test results of Kening capsule finished product

[0099]

[0100] Table 4 Relative peak area precision test results of Kening capsule finished product

[0101]

[0102] 4.3 Repeatability test

[0103] Six samples of Kening Capsule (S10, batch number 2401151) test solution were prepared in parallel. The sample solution preparation method under "2.2.1" was followed and the chromatographic conditions under "2.1" were used for injection. Peak 9 was used as the reference peak. The relative retention time and relative peak area were calculated. The RSD values were all within 3% (n=6), indicating that the method had good reproducibility and met the technical requirements of the characteristic spectrum. The results are shown in Tables 5, 6 and Figure 8 .

[0104] Table 5 Relative retention time repeatability test results of Kening capsule finished product

[0105]

[0106] Table 6 Relative peak area repeatability test results of Kening capsule finished product

[0107]

[0108] 4.4 Stability test

[0109] Take Kening Capsules (S10, batch number 2401151), according to the test solution preparation method under "2.2.1" and the chromatographic conditions under "2.1", the samples were injected at 0h, 2h, 4h, 8h, 12h, 16h, 24h, 36h, and 48h, respectively. Peak 9 was used as the reference peak, and the relative retention time and relative peak area were calculated. The RSD values were all within 3% (n=9). The test solution had good stability within 48h. The results are shown in Tables 7, 8 and Figure 9 .

[0110] Table 7 Kening Capsule finished product relative retention time test sample solution stability test results

[0111]

[0112]

[0113] Table 8 Kening Capsule finished product relative peak area test sample solution stability test results

[0114]

[0115] 5. Establishment of the Characteristic Spectrum of Kening Capsules

[0116] 5.1 Determination of characteristic peaks and reference peaks

[0117] In the characteristic spectrum, the thymidine chromatographic peak is stable and the retention time, separation degree, and peak area are moderate. Therefore, thymidine (peak 9) is selected as a reference to calculate the relative retention time and relative peak area of the common peak.

[0118] 5.2 Establishment of feature maps

[0119] According to the method under "2.2.1", 15 batches of sample solutions (batch numbers are 221231, 230101, 230106, 230109, 231207, 2212212, 2301031, 2301032, 2301052, 2401151, 2401152, 2403061, 2403062, 2404301, 2404302, numbered S1 to S15) were prepared respectively. The samples were injected according to the chromatographic conditions under "2.1", and the chromatograms were recorded and saved in AIA format. The data were then imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) software to obtain the characteristic spectra of the 15 batches of samples (see Figure 8 ) and reference pattern R (see Figure 9). The specific method is to set S1 as the reference spectrum, the chromatographic peak time window width is 0.1, the median method, multi-point correction and automatic matching of each chromatographic peak to form a common pattern diagram. According to the characteristic spectrum analysis, 12 common peaks were identified in 15 batches of Kening Capsules finished products. The relative retention time and ultraviolet spectrum were compared with the reference peaks, and a total of 8 peaks were identified, namely uracil (peak 1), cytidine (peak 2), uridine (peak 3), adenine (peak 6), guanosine (peak 7), thymidine (peak 9), adenosine (peak 11), and deoxyadenosine (peak 12). These 12 common peaks are used as characteristic peaks to generate a reference spectrum R.

[0120] 5.3 Common Peak Sources of Characteristic Spectra of Kening Capsules Finished Product

[0121] According to the method under "2.2.1", the finished product S1 of Kening Capsule was prepared respectively. The negative sample solutions of each single herbal medicine and the missing single herbal medicine were injected according to the chromatographic conditions under "2.1". The chromatogram was obtained. Figure 4 、 5 . After superposition and comparison, 12 peaks were obtained, and the attribution is as follows: Peaks 4, 5, 8, 9, and 12 are from Fritillaria thunbergii, and Peaks 1, 2, 3, 6, 7, 10, and 11 are common peaks of Fritillaria thunbergii and Platycodon grandiflorum. Fritillaria thunbergii and Platycodon grandiflorum contribute the most to the characteristic spectrum. Due to the limitations of the chromatographic conditions and the influence of the pig bile preparation process, the pig bile extract did not produce any peaks under the chromatographic conditions, so it did not contribute to this characteristic spectrum. The relative retention time and UV spectrum were compared with the reference peaks, and a total of 8 peaks were identified, namely uracil (peak 1), cytidine (peak 2), uridine (peak 3), adenine (peak 6), guanosine (peak 7), thymidine (peak 9), adenosine (peak 11), and deoxyadenosine (peak 12).

[0122] 5.4 Comparison of characteristic spectra of different batches of Kening Capsules

[0123] According to the test solution preparation method under "2.2.1", 15 batches of Kening Capsule test solution were prepared respectively. Samples were injected according to the chromatographic conditions under "2.1". Taking thymidine (peak 9) as the reference, the relative retention time and relative peak area of each common peak were calculated. The results are shown in Tables 9-1, 9-2 and Tables 10-1, 10-2.

[0124] Table 9-1 Relative retention time of common peaks in Kening Capsules finished product

[0125]

[0126] Table 3-9-2 Relative retention time of common peaks in Kening Capsules finished product

[0127]

[0128] Table 10-1 Relative peak areas of common peaks in the finished product of Kening Capsules

[0129]

[0130]

[0131] Table 10-2 Relative peak areas of common peaks in the finished product of Kening Capsules

[0132]

[0133] From the results in Table 3-9, it can be seen that the RSD values of the relative retention times of the common peaks of each batch of Kening Capsules are between 0% and 2.0189%, the peak time is relatively stable, the RSD difference of Peak 2 is relatively large, and the other 11 common characteristic peaks have good reproducibility; from the results in Table 3-10, it can be seen that the RSD values of the relative peak areas of each characteristic peak are between 0% and 35.9424%, indicating that the types of nucleoside components of the 15 batches of Kening Capsules are consistent, but the content of the components varies greatly between batches, among which the RSD differences of Peak 4 and Peak 10 are the largest.

[0134] 5.5 Similarity Evaluation

[0135] The test solutions of 15 batches of Kening Capsule samples (S1-S15) were injected according to the chromatographic conditions under item "2.1", and the chromatograms were recorded and saved in AIA format. They were then imported into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition) software for matching and correction, and the similarity of each batch of products was calculated. The results are shown in Table 11.

[0136] Table 11 Similarity evaluation results of 15 batches of Kening Capsules (n=15)

[0137]

[0138]

[0139] As shown in Table 11, the similarity between the 15 batches of Kening Capsules finished product samples and the established standard reference characteristic profiles ranged from 0.939 to 0.99, all greater than 0.90. This indicates that the chemical composition of the prepared Kening Capsules is relatively stable, and the overall profiles of each batch are essentially consistent. In summary, the Kening Capsules medicinal material characteristic profile established in this experiment is highly reliable and can be used as a reference profile for the Kening Capsules finished product.

[0140] 5.6 Determination of the specified value of relative retention time

[0141] Taking thymidine (peak 9) as the reference peak, the relative retention time RSD of other common peaks in 15 batches of Kening Capsules was calculated to be between 0% and 2.0189%. The results are shown in Table 9. The relative deviation range of the relative retention time of each characteristic peak of the test sample and the characteristic peak in the control characteristic spectrum is shown in Table 13. According to Table 13, in order to eliminate the difference caused by the peak time offset of different components in the common peak on the chromatographic column and better overcome the systematic error in the experimental process, it is temporarily stipulated that the relative deviation of the relative retention time of each characteristic peak of the test sample of Kening Capsule product and the standard spectrum is within ± 5%, which indicates that the quality of the Kening Capsule product is qualified, otherwise it is unqualified.

[0142] Table 12 Relative retention time of the characteristic spectrum of the finished product of Kening Capsules

[0143]

[0144] Table 13 Relative retention time deviation range of common peaks of 15 batches of Kening Capsules and corresponding peaks in the control characteristic spectrum

[0145]

[0146]

[0147] According to the above results, the relative retention time values of other peaks in the control characteristic spectrum are tentatively set to 0.2343 (peak 1), 0.2805 (peak 2), 0.3775 (peak 3), 0.5377 (peak 4), 0.6897 (peak 5), 0.7307 (peak 6), 0.8192 (peak 7), 0.9457 (peak 8), 1.0000 (peak 9), 1.0655 (peak 10), 1.1263 (peak 11), and 1.1669 (peak 12). There should be 12 characteristic peaks in the characteristic spectrum of Kening Capsules, and the peak corresponding to thymidine is the S peak. The relative retention time of each characteristic peak and the S peak should be within ±5% of the specified value.

[0148] 5.7 Chemometric Analysis Characteristic Spectra

[0149] 5.7.1 Cluster Analysis (HCA)

[0150] The common peak area data of 15 batches of Kening Capsule samples were imported into IBM SPSS Statistics 26, and the intergroup linkage method was adopted to determine the distance. The squared Euclidean distance was selected. The results are as follows: Figure 11When the cluster distance was 9, 15 batches of Kening Capsules were clustered into 4 categories: S2, S3, S6, S7, and S8 were clustered into one category, S4, S9, S10, S11, S12, S13, and S14 were clustered into one category, and S1 and S5 were clustered into separate categories. The cluster analysis results showed that there were certain differences between the batches of samples, and the fluctuation in the quality of the medicinal materials used to produce Kening Capsules was the main reason for the above results. Therefore, stabilizing the quality of medicinal materials and selecting authentic medicinal materials from appropriate production areas can effectively reduce the differences between batches of medicinal materials.

[0151] 5.7.2 Principal Component Analysis (PCA)

[0152] The 12 common peak area data from 15 batches of Kening Capsules samples were imported into IBM SPSS Statistics 26 software and standardized before principal component analysis. The eigenvalues and cumulative variance contributions of the principal component analysis were recorded (see Table 14). The results show that three principal components with eigenvalues greater than 1 were extracted, with a cumulative contribution rate of 81.130%. This indicates that the analysis results of these three principal components can generally reflect the similarities and differences between different batches of Kening Capsules, and that the variability in product quality is influenced by multiple components.

[0153] Table 3-14 Eigenvalues and cumulative variance contribution rates

[0154]

[0155] Table 15 shows that in the first principal component, peaks 4 and 12 have higher contribution rates, in the second principal component, peaks 9 and 10 have higher contribution rates, and in the third principal component, peak 3 has higher contribution rates. This shows that the above components may be the reasons for the differences between different batches of products.

[0156] Table 15 PCA factor loading matrix of Kening Capsules

[0157]

[0158] 5.7.3 Orthogonal Least Partial Squares-Discriminant Analysis (OPLS-DA)

[0159] To further investigate the differences in chemical composition between different batches of Kening Capsules, the 12 common peak areas in 15 batches of Kening Capsules samples were normalized and then imported into SIMCA 14.1 software for orthogonal least squares-discriminant analysis (OPLS-DA) analysis. The OPLS-DA score matrix and variable importance (VIP) projection value diagram were obtained. Figure 16 、 17 Its key parameters R2X=0.654、R2Y=0.711、Q2=0.502, all greater than 0.5, the model has good fitting and prediction capabilities. Figure 16 It can be seen that 15 batches of Kening capsule samples are divided into 3 categories, which is consistent with the PCA classification results, and the separation is significant, indicating that there are certain differences in the components of different batches of samples; the VIP diagram can intuitively reflect the influence of each chromatographic peak. VIP values greater than 1 are more important variables. Figure 17 It can be seen that the VIP values of the six components, namely Peak 11, Peak 5, Peak 6, Peak 12, Peak 4, and Peak 9, are greater than 1 when sorted by VIP value. Among them, a total of four peaks were identified, Peak 6 (adenine), Peak 9 (thymidine), Peak 11 (adenosine), and Peak 12 (deoxyadenosine). Therefore, it is speculated that these four components are the main differential components in Kening Capsules.

[0160] VI. Conclusion

[0161] This section established characteristic spectra for 15 batches of Kening Capsules samples, with similarities >0.9. The methodological findings are favorable and could provide a new evaluation metric for the quality standards of Kening Capsules. However, the characteristics of porcine bile extract were not reflected, primarily due to limitations in chromatographic conditions and the influence of the porcine bile extract preparation process. No corresponding compounds were found in the porcine bile extract, and further research is needed.

[0162] Using a characteristic spectrum combined with chemical pattern recognition, it was determined that the quality of different batches of Kening Capsules samples varied. PCA and OPLS-DA analyses classified 15 batches of Kening Capsules into three categories. Based on the VIP value plot, four major differential components were identified: Peak 6 (adenine), Peak 9 (thymidine), Peak 11 (adenosine), and Peak 12 (deoxyadenosine). Therefore, monitoring and controlling relevant components is crucial during the procurement of medicinal materials, production operations, and product testing. This allows companies to more scientifically and comprehensively control drug quality and improve product stability. In this experiment, the results of HCA, PCA, and OPLS-DA analyses were largely consistent with the similarity assessment results, confirming each other. This study established a characteristic spectrum for Kening Capsules products. The method is stable and reliable, providing a scientific basis for improving the quality evaluation standards for Kening Capsules.

Claims

1. A method for constructing a characteristic spectrum of Kening Capsules, characterized by being achieved by the following steps: (1). Make solution 1.1 Preparation of test solution Accurately weigh 2 g of Kening capsule powder and place it in a stoppered conical flask. Accurately measure 40 mL of 20% methanol solution by volume and add it to the stoppered conical flask. Mix evenly with the Kening capsule powder. Heat in a 60°C water bath for 0.5 h, let cool, and make up the lost mass with 20% methanol solution. Pour the mixture into a centrifuge tube and centrifuge at 4000 r·min- 1 Centrifuge for 10 minutes and filter to obtain the first filtrate. Repeat the above operation once to obtain the second filtrate. Combine the filtrates and filter with a 0.22 μm microporous membrane to obtain the test solution. 1.2 Preparation of reference solution Accurately weigh appropriate amounts of uracil, cytidine, guanosine, adenine, uridine, thymidine, adenosine, and deoxyadenosine reference substances, and add purified water to prepare reference substance solutions with mass concentrations of 5.0136, 6.5175, 7.4925, 5.0555, 5.0645, 4.9705, 5.0455, and 5.0152 μg / mL, respectively; 1.3 Preparation of single herbal sample solution Accurately weigh the single medicinal materials with the same prescription as the test sample, and then prepare the sample solutions of each single medicinal material according to "1.1"; 1.

4. Preparation of negative sample solution for missing single medicinal material Accurately weigh the negative samples of the missing single herbal ingredients in the prescription, and then prepare the sample solutions of each single herbal ingredient according to "1.1"; (2) Determine the main components of constructing the feature map The main drug components of Kening Capsules are pig bile extract, Fritillaria thunbergii and Platycodon grandiflorum. The characteristic spectrum active main components in Kening Capsules are extracted by liquid chromatography (HPLC) using a high performance liquid chromatograph, a 100,000th electronic balance and a centrifuge (known technology). The main active ingredients of Kening Capsules are determined to be: hyodeoxycholic acid, taurohyodeoxycholic acid, platycodon saponin, deoxyadenosine, thymidine, cytidine, adenine, adenosine, guanosine, uridine and uracil, among which the main components of pig bile extract are hyodeoxycholic acid, taurohyodeoxycholic acid, platycodon saponin, deoxyadenosine, thymidine, cytidine, adenine, adenosine, guanosine, uridine and uracil. Acid, the main components of Fritillaria thunbergii are deoxyadenosine and thymidine, the main components of Platycodon grandiflorum are platycodon saponins, and the common components of Fritillaria thunbergii and Platycodon grandiflorum are cytidine, adenine, adenosine, guanosine, uridine and uracil. When the characteristic spectrum was established, the chromatographic conditions were not compatible with the components of hyodeoxycholic acid, taurohyodeoxycholic acid and platycodon saponins. Only the 12 components under the chromatographic conditions were attributed to the characteristic spectrum. Among them, deoxyadenosine and thymidine were attributed to Fritillaria thunbergii, cytidine, adenine, adenosine, guanosine, uridine and uracil were attributed to Fritillaria thunbergii and Platycodon grandiflorum, and the remaining 4 common peaks were not attributed; The chromatographic conditions are as follows: Chromatographic column: Agilent TC-C 18 Column: 4.6 mm × 250 mm, 5 μm; mobile phase: water (A): methanol (B) by volume, gradient elution as specified in the following table: Detection wavelength: 260 nm; flow rate: 0.8 mL min -1 ; Injection volume: 20 μL; Column temperature: 25°C; (3) Constructing feature maps 3.

1. First determine the characteristic peak and reference peak In the characteristic spectrum, the thymidine chromatographic peak is stable and has moderate retention time, resolution, and peak area. Therefore, the characteristic peak of thymidine is selected as the reference peak to calculate the relative retention time and relative peak area of the common peak. 3.

2. Establishment of feature maps According to the method under "1.1", 15 batches of samples were prepared, numbered S1 to S15 as test solution, and the samples were injected according to the chromatographic conditions under "2". The chromatograms were recorded and saved in AIA format. The data were then imported into the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System" (2012 version) software to obtain the characteristic spectra and reference spectra R of the 15 batches of samples. The method was: using S1 as the reference spectrum, the chromatographic peak time window width was 0.1, the median method, multi-point correction and automatic matching of each chromatographic peak to form a common pattern Figure, according to the characteristic spectrum analysis, 12 common peaks were identified in 15 batches of Kening Capsules finished products. The relative retention time and UV spectrum were compared with the reference peaks, namely peak 1-uracil, peak 2-cytidine, peak 3-uridine, peak 6-adenine, peak 7-guanosine, peak 9-thymidine, peak 11-adenosine and peak 12-deoxyadenosine. Among them, the four common peaks of peaks 4, 5, 8 and 10 were not identified. The 12 common peaks were used as characteristic peaks to construct the control characteristic spectrum R of Kening Capsules finished products and the chromatogram of the mixed reference product.

2. Application of the characteristic spectrum of Kening Capsules constructed by the method of claim 1 in the overall control and evaluation of the quality of Kening Capsules products.

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