A method for constructing a characteristic map of a cough-relieving capsule and its application

Abstract

A method for constructing a characteristic spectrum of Ke Ning capsules and its application can effectively solve the problem of overall quality control and evaluation of Ke Ning capsule products. It is achieved through the following steps: (1) Solution preparation: Prepare test solution, reference solution, single-herb sample solution, and negative sample solution lacking a single herb; (2) Determine the main components for constructing the characteristic spectrum; (3) Construct the characteristic spectrum: Determine the characteristic peaks and reference peaks, and establish the characteristic spectrum and reference spectrum R. This method is simple and easy to operate. By comparing the characteristic peak diagram of the Ke Ning capsule to be tested with the characteristic spectrum of Ke Ning capsules constructed by this method, the product quality of Ke Ning capsules can be clearly judged. This effectively solves the problem of overall quality control and evaluation of Ke Ning capsule products, representing a major innovation in the quality control and evaluation of Ke Ning capsules, with significant economic and social benefits.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to a method for constructing a characteristic spectrum of a cough-relieving capsule and its application. Background Technology

[0002] Ke Ning Capsules are a traditional Chinese medicine developed based on folk remedies for treating whooping cough and colds in children. They are made from pig bile extract, Fritillaria thunbergii, and Platycodon grandiflorus, primarily used to clear heat, promote lung function, resolve phlegm, and relieve cough. They are indicated for coughs due to wind-heat or phlegm-heat, characterized by yellow-white or thick, difficult-to-explode phlegm. The quality of the capsules directly affects their efficacy. To ensure quality, it is essential to analyze and study the effective active ingredients. However, current research lacks specific studies on the individual herbal components in Ke Ning Capsules. Current research demonstrates that Ke Ning Capsules contain nucleoside, bile acid, and saponin components, mainly uracil, cytidine, guanosine, adenine, uridine, thymidine, adenosine, and deoxyadenosine. Establishing a characteristic spectrum of the effective components can serve as a quality control technique to guarantee product quality and efficacy.

[0003] Characteristic chromatograms are an efficient quality control model based on a holistic understanding of traditional Chinese medicine (TCM). Utilizing modern technologies such as spectroscopy and chromatography, informational spectra or chromatograms of the chemical components of TCM are obtained. The main characteristic peaks in the chromatogram comprehensively reflect the relationships between the contained components. This method is characterized by strong specificity, effective identification of sample authenticity, large information content, consistency, stability, and integrity. By analyzing nucleoside components in the raw materials of Ke Ning capsules using HPLC, and establishing a characteristic chromatogram for Ke Ning capsules using a TCM fingerprint chromatogram similarity evaluation system, the method reflects the transfer relationships of nucleoside components during the preparation process, reducing batch-to-batch variations. It allows for the holistic identification of TCM components, comprehensively reflecting the relative relationships of TCM chemical components, and enabling accurate and effective overall control and evaluation of the quality of TCM and its preparations. This provides a scientific basis for better quality control of Ke Ning capsule products, but no publicly reported examples have been found to date. Summary of the Invention

[0004] In view of the above situation and to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for constructing a characteristic spectrum of Ke Ning capsules and its application, which can effectively solve the problem of effective overall control and evaluation of the quality of Ke Ning capsules.

[0005] The technical solution provided by this invention is a method for constructing a feature map of a cough-relieving capsule, which is achieved through the following steps:

[0006] (1). Make solution

[0007] 1.1 Preparation of the test solution

[0008] Place the cough-relieving capsule powder in a stoppered conical flask, add a 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 filter residue to obtain the second filtrate. Combine the filtrates, filter, and obtain the test solution.

[0009] 1.2 Preparation of reference solution

[0010] Uracil, cytidine, guanosine, adenine, uridine, thymidine, adenosine, and deoxyadenosine were added to purified water to prepare reference solutions.

[0011] 1.3 Preparation of single-herb sample solutions

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

[0013] 1.4 Preparation of negative sample solutions for samples lacking a single medicinal herb

[0014] Accurately weigh a negative sample of a single herb missing from the prescription, and then prepare a negative sample solution of the single herb missing as described in section "1.1";

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

[0016] The main drug components of Ke Ning capsules are pig bile extract, Fritillaria thunbergii and Platycodon grandiflorus. Using high performance liquid chromatography (HPLC), an electronic balance with an index of 1 / 100,000 and a centrifuge, the main active components with characteristic chromatograms in Ke Ning capsules were extracted under specific chromatographic conditions (known technology). The 12 main active components of Ke Ning capsules were identified as: deoxyadenosine, thymidine, cytidine, adenine, adenosine, guanosine, uridine, uracil and 4 components with common peaks whose attribution was not determined.

[0017] (3) Constructing feature maps

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

[0019] Using the characteristic peak of thymidine as a reference peak, the relative retention time and relative peak area of ​​the common peaks were calculated.

[0020] 3.2 Establishment of Feature Maps

[0021] Prepare 15 batches of test solutions, numbered S1 to S15, according to the method in section "1.1". Inject the samples under the chromatographic conditions in section "2", record the chromatograms, save the data in AIA format, and import them into the software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) to construct characteristic chromatograms and reference chromatograms R.

[0022] The application of the characteristic map of Ke Ning capsule constructed by the method of this invention in the overall control and evaluation of the quality of Ke Ning capsule product.

[0023] The method of this invention is simple and easy to operate. By comparing the characteristic peak diagram of the cough capsule to be tested with the characteristic spectrum of the cough capsule constructed by the method of this invention, the product quality of the cough capsule can be clearly judged at a glance. It can effectively solve the problem of effective overall control and evaluation of the product quality of cough capsule. It is a major innovation in the quality control and evaluation of cough capsule, and has significant economic and social benefits. Attached Figure Description

[0024] Figure 1 Chromatograms under different solvent conditions;

[0025] Figure 2 Chromatogram of the test solution (Note: 1. Uracil; 2. Cytidine; 3. Urate; 6. Adenine; 7. Guanosine; 9. Thymidine; 11. Adenosine; 12. Deoxyadenosine);

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

[0027] Figure 4 Chromatograms of pig bile extract, Fritillaria thunbergii, and Platycodon grandiflorus as single medicinal materials;

[0028] Figure 5 Chromatograms of negative-negative medicinal materials lacking pig bile extract, Fritillaria thunbergii, and Platycodon grandiflorus;

[0029] Figure 6 This is the chromatogram of a blank solution;

[0030] Figure 7 Precision test spectrum of Ke Ning capsules;

[0031] Figure 8 The repeatability test spectrum of the finished product of Ke Ning capsules;

[0032] Figure 9 Stability test chromatogram of the finished Ke Ning capsules;

[0033] Figure 10 The characteristic chromatograms of 15 batches of Ke Ning capsules are shared pattern chromatograms;

[0034] Figure 11 A characteristic chromatogram of the finished product of Ke Ning Capsules;

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

[0036] Figure 13 Clustering tree diagram of 15 batches of Ke Ning capsules;

[0037] Figure 14The image shows lithotripsy from 15 batches of Ke Ning capsules.

[0038] Figure 15 Principal component analysis score chart for 15 batches of Ke Ning capsules;

[0039] Figure 16 OPLS-DA score charts for 15 batches of Ke Ning capsules;

[0040] Figure 17 Projected importance (VIP) values ​​of component variables for 15 batches of Ke Ning capsules. Detailed Implementation

[0041] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0042] In specific implementation, the present invention can be given by the following specific embodiments:

[0043] The technical solution provided by this invention is a method for constructing a feature map of a cough-relieving capsule, which is achieved through the following steps:

[0044] (1). Make solution

[0045] 1.1 Preparation of the test solution

[0046] Accurately weigh 2g of cough suppressant capsule powder and place it in a stoppered conical flask. Accurately measure 40mL of 20% methanol solution and add it to the stoppered conical flask. Mix the cough suppressant capsule powder thoroughly and heat in a 60℃ water bath for 0.5h. After cooling, replenish the lost mass with 20% methanol solution and pour into a centrifuge tube. Centrifuge at 4000r·min. -1 Centrifuge for 10 min, filter to obtain the first filtrate, repeat the above operation once for the filter residue to obtain the second filtrate, combine the filtrates, 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 standards, and add them to purified water to prepare reference standard 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-herb sample solutions

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

[0051] 1.4 Preparation of negative sample solutions for samples lacking a single medicinal herb

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

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

[0054] The main components of Ke Ning capsules are porcine bile extract, Fritillaria thunbergii, and Platycodon grandiflorus. Using high-performance liquid chromatography (HPLC), an electronic balance with a density of 1 / 100,000, and a centrifuge, the main active components with characteristic chromatograms in Ke Ning capsules were extracted (a known technique). Based on the main components of the three drugs, the main active components of Ke Ning capsules were determined to be: porcine deoxycholic acid, taurine deoxycholic acid, Platycodon grandiflorus saponin deoxyadenosine, thymidine, cytidine, adenine, adenosine, guanosine, uridine, and uracil. Among these, the main components of the porcine bile extract are porcine deoxycholic acid and taurine deoxycholic acid. The main components of Fritillaria thunbergii are deoxyadenosine and thymidine, while the main components of Platycodon grandiflorus are platycodins. The common components of Fritillaria thunbergii and Platycodon grandiflorus are cytidine, adenine, adenosine, guanosine, uridine, and uracil. When establishing the characteristic spectrum, the chromatographic conditions were not compatible with the components of porcine deoxycholic acid, taurine deoxycholic acid, and platycodins. Only the 12 components under the chromatographic conditions were assigned characteristic spectra. Among them, deoxyadenosine and thymidine were assigned to Fritillaria thunbergii, while cytidine, adenine, adenosine, guanosine, uridine, and uracil were shared by Fritillaria thunbergii and Platycodon grandiflorus. The assignment of the remaining 4 common peaks was not determined.

[0055] The chromatographic conditions were as follows: Column: Agilent TC-C 18 Column: 4.6 mm × 250 mm, 5 μm; mobile phase: water (A): methanol (B) by volume ratio, gradient elution performed according to the table below:

[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℃;

[0058] (3) Constructing feature maps

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

[0060] In the characteristic chromatogram, 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, and the relative retention time and relative peak area of ​​the common peak are calculated.

[0061] 3.2 Establishment of Feature Maps

[0062] Fifteen batches of samples, numbered S1 to S15, were prepared according to the method described in section "1.1". The samples were injected under the chromatographic conditions described in section "2", and the chromatograms were recorded. The data was saved in AIA format and imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) software to obtain the characteristic chromatograms and reference chromatograms R of the 15 batches of samples. The method used was as follows: using S1 as the reference chromatogram, with a peak time window width of 0.1, the median method, multi-point correction, and automatic matching of each chromatographic peak to form a common pattern. Figure 1 shows that, based on characteristic chromatogram analysis, 12 common peaks were identified in 15 batches of Ke Ning capsules. The relative retention times and UV spectra were compared with those of the reference standard peaks, revealing them to be 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. The assignments of four common peaks (4, 5, 8, and 10) were not determined. Using these 12 common peaks as characteristic peaks, a characteristic chromatogram R of the Ke Ning capsules reference standard and a chromatogram of the mixed reference standard were constructed (see Figure 1). Figure 11 , 12 ).

[0063] The application of the characteristic map of Ke Ning capsule constructed by the method of this invention in the overall control and evaluation of the quality of Ke Ning capsule product.

[0064] The method of this invention is simple, easy to operate, stable and reliable. It can compare the characteristic peak diagram of the cough-relieving capsule to be tested with the characteristic spectrum of the cough-relieving capsule constructed by the method of this invention, so as to quickly and conveniently achieve effective overall control and evaluation of the quality of the cough-relieving capsule product. Experiments have shown that the effect is very good. Relevant information is as follows:

[0065] I. Instruments and Materials

[0066] 1.1 Instruments

[0067] Table 1 Experimental Instruments

[0068]

[0069] 1.2 Materials

[0070] Table 2 Experimental Materials

[0071]

[0072] Fifteen batches of Ke Ning capsules (batch numbers 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] II. Experimental Methods

[0074] 2.1 Chromatographic conditions

[0075] 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: 20 μL; Column temperature: 25℃.

[0076] 2.2 Solution Preparation

[0077] 2.2.1 Preparation of the test solution

[0078] Accurately weigh 2g of cough suppressant capsule powder and place it in a stoppered conical flask. Accurately measure 40mL of 20% methanol solution, weigh it, mix thoroughly, heat in a 60℃ water bath for 0.5h, cool, and replenish the lost mass with 20% methanol solution. Pour the mixture into a centrifuge tube and centrifuge at 4000r·min. -1 Centrifuge for 10 min, filter, repeat the above operation once with the filter residue, combine the filtrates and filter with a 0.22 μm microporous membrane to obtain the final 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 standards, and add them to 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-herb sample solutions

[0082] Accurately weigh the single medicinal materials in the prescription, including the test sample, and prepare the sample solution of each single medicinal material according to the method described in section "2.2.1".

[0083] 2.2.4 Preparation of negative sample solutions for samples lacking a single medicinal herb

[0084] Accurately weigh the negative samples of the missing single medicinal materials in the prescription, and prepare the sample solutions of each single medicinal material according to the method described in section "2.2.1".

[0085] III. Method Development and Validation

[0086] 3.1 Investigation of the preparation method of the test solution

[0087] 3.1.1 Investigation of the treatment method for the test sample solution

[0088] Following the provisional method for preparing the test solution, different treatment methods were selected to investigate their effects on the stability of the test solution. 2g of Ke Ning capsule powder was accurately weighed and placed in a stoppered conical flask. The solutions were tested 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. Ultrasound was performed at 300W power and 40kHz frequency. Water bath heating was conducted at 60℃ for 0.5h. The solutions were then transferred to centrifuge tubes and centrifuged at 4000 rpm. 1 Centrifuge for 10 min, filter, repeat the above operation once with the filter residue, combine the filtrates and filter through a 0.22 μm microporous membrane to obtain the final product. Experimental results show that the treatment method using 20% ​​methanol and water bath heating ensures good stability of the test solution within 48 h, while other methods result in instability and poor peak symmetry. Therefore, the treatment method using 20% ​​methanol and water bath heating can be selected as the preparation method for the test solution.

[0089] 3.1.2 Investigation of the solvent for the test sample

[0090] Following the provisional method for preparing the test solution, different solvents were selected to further investigate their effect on the peak shape of each reference standard in the chromatogram. 2g of cough suppressant capsule powder was accurately weighed and placed in a stoppered conical flask. 40mL of water, 20% methanol, 40% methanol, 50% methanol, 80% methanol, and methanol solution were accurately measured and weighed. The mixtures were thoroughly mixed and heated in a 60℃ water bath for 0.5h. After cooling, 20% methanol was added to replenish the lost mass. The mixture was then poured into centrifuge tubes and centrifuged at 4000 rpm. 1 Centrifuge for 10 min, filter, repeat the above operation once with the filter residue, combine the filtrates and filter through a 0.22 μm microporous membrane to obtain the final product. Experimental results show that 20% methanol, as the solvent for the test sample, exhibits good peak symmetry and suitable accuracy, and can be used as the solvent for preparing the test sample solution. Figure 1 )

[0091] 3.1.3 Investigation on the dosage of test sample

[0092] Following the provisional method for preparing the test solution, different amounts of the test sample were selected to further investigate the effect of the chromatogram on the peak shape of each reference standard. 1g, 1.5g, 2g, 2.5g, and 3g of cough suppressant capsule powder were accurately weighed and placed in stoppered conical flasks. 40mL of 20% methanol solution was accurately measured and weighed, mixed thoroughly, and heated in a 60℃ water bath for 0.5h. After cooling, the lost mass was replenished with 20% methanol, and the mixture was poured into centrifuge tubes and centrifuged at 4000 rpm. -1Centrifuge for 10 min, filter, repeat the above operation once with the filter residue, combine the filtrates and filter through a 0.22 μm microporous membrane to obtain the final product. 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 initial sample weight.

[0093] IV. Methodological Investigation

[0094] 4.1 Specificity Examination

[0095] Take the test solution, reference solution, single herb solution, negative control herb solution, and blank solution respectively, and inject them according to the chromatographic conditions under section "2.1" to detect the characteristic chromatographic patterns, and examine the chromatographic conditions and their specificity. For example... Figure 2-4 As shown, the solution lacking negative medicinal materials and the blank solution do not interfere with the common peaks of the characteristic spectrum.

[0096] 4.2 Precision Test

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

[0098] Table 3. Results of the precision test on the relative retention time of Ke Ning capsules.

[0099]

[0100] Table 4. Results of the precision test on the relative peak area of ​​Ke Ning capsules.

[0101]

[0102] 4.3 Repeatability Test

[0103] Six parallel solutions of Ke Ning capsules (S10, batch number 2401151) were prepared according to the preparation method of the test solution in section "2.2.1" and the chromatographic conditions in section "2.1". Peak 9 was used as the reference peak, and the relative retention time and relative peak area were calculated for each solution. The RSD values ​​were all within 3% (n=6), indicating that the method has good repeatability and meets the technical requirements of the characteristic chromatogram. The results are shown in Tables 5 and 6. Figure 8 .

[0104] Table 5. Results of the repeatability test on the relative retention time of the finished Ke Ning capsules.

[0105]

[0106] Table 6. Results of the repeatability test on the relative peak area of ​​the finished Ke Ning capsules.

[0107]

[0108] 4.4 Stability Test

[0109] Ke Ning capsules (S10, batch number 2401151) were prepared according to the test solution preparation method in section "2.2.1" and the chromatographic conditions in section "2.1" were followed. Injections were performed at 0h, 2h, 4h, 8h, 12h, 16h, 24h, 36h, and 48h, respectively. 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=9). The test solution showed good stability within 48h. The results are shown in Tables 7 and 8. Figure 9 .

[0110] Table 7. Relative retention time of Ke Ning capsules and results of stability test of test solution.

[0111]

[0112]

[0113] Table 8. Relative peak area of ​​Ke Ning capsules and results of stability test of test solution.

[0114]

[0115] V. Establishment of the Characteristic Map of Ke Ning Capsules

[0116] 5.1 Determination of characteristic peaks and reference peaks

[0117] In the characteristic chromatogram, the thymidine chromatographic peak is stable and has moderate retention time, resolution and peak area. Therefore, thymidine (peak 9) is selected as the reference to calculate the relative retention time and relative peak area of ​​the common peak.

[0118] 5.2 Establishment of Feature Maps

[0119] Following the method described in section "2.2.1", 15 batches of samples (batch numbers 221231, 230101, 230106, 230109, 231207, 2212212, 2301031, 2301032, 2301052, 2401151, 2401152, 2403061, 2403062, 2404301, and 2404302, numbered S1 to S15) were prepared as test solutions. These solutions were injected under the chromatographic conditions described in section "2.1", and the chromatograms were recorded. The data was saved in AIA format and imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) software to obtain the characteristic chromatograms of the 15 batches of samples (see...). Figure 8 ) and reference spectrum R (see Figure 9The specific method is as follows: S1 is the reference chromatogram, the chromatographic peak time window width is 0.1, the median method is used, multi-point correction is applied, and each chromatographic peak is automatically matched to form a common pattern chromatogram. Based on the characteristic chromatogram analysis, 12 common peaks were identified from 15 batches of Ke Ning capsules. By comparing the relative retention times and UV spectra with the reference peaks, 8 peaks were identified: 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). A reference chromatogram R was generated using these 12 common peaks as characteristic peaks.

[0120] 5.3 Source of common peaks in the characteristic chromatogram of Ke Ning capsules finished product

[0121] Following the method described in section "2.2.1", the finished product S1 of the cough-relieving capsules, as well as negative sample solutions of each single herb and those lacking a single herb, were prepared. These were injected under the chromatographic conditions described in section "2.1" to obtain chromatograms, as shown in the figure. Figure 4 , 5 After superposition and comparison, 12 peaks were obtained, and their assignments are as follows: peaks 4, 5, 8, 9, and 12 are from Fritillaria thunbergii; peaks 1, 2, 3, 6, 7, 10, and 11 are common peaks of Fritillaria thunbergii and Platycodon grandiflorus. Fritillaria thunbergii and Platycodon grandiflorus contribute significantly to the characteristic chromatogram. Due to the limitations of the chromatographic conditions and the influence of the pig bile preparation process, no peaks were eluted under these chromatographic conditions, and therefore, no peaks were contributed to this characteristic chromatogram. By comparing the relative retention time and UV spectrum with the reference peaks, a total of 8 peaks were identified: 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 chromatograms of different batches of Ke Ning capsules

[0123] Fifteen batches of Ke Ning capsule test solutions were prepared according to the test solution preparation method in section “2.2.1”. The samples were injected according to the chromatographic conditions in section “2.1”. The relative retention time and relative peak area of ​​each common peak were calculated with thymidine (peak 9) as a reference. The results are shown in Tables 9-1, 9-2 and Tables 10-1, 10-2.

[0124] Table 9-1 Relative retention times of peaks in Ke Ning capsules (finished product)

[0125]

[0126] Table 3-9-2 Relative retention times of peaks in Ke Ning capsules (finished product)

[0127]

[0128] Table 10-1 Relative peak area of ​​Ke Ning Capsules (finished product)

[0129]

[0130]

[0131] Table 10-2 Relative peak areas of the finished Ke Ning capsules

[0132]

[0133] As shown in Table 3-9, the relative retention time (RSD) values ​​of the common peaks in all batches of Ke Ning capsules ranged from 0% to 2.0189%, indicating relatively stable elution times. The RSD of peak 2 showed relatively large differences, while the other 11 common characteristic peaks exhibited good reproducibility. As shown in Table 3-10, the relative peak area (RSD) values ​​of each characteristic peak ranged from 0% to 35.9424%, indicating good consistency in the types of nucleoside components among the 15 batches of Ke Ning capsules. However, the content of components varied significantly between batches, with peaks 4 and 10 showing the greatest RSD differences.

[0134] 5.5 Similarity Evaluation

[0135] The test solutions of 15 batches of Ke Ning capsules (S1-S15) were injected according to the chromatographic conditions in section “2.1”, the chromatograms were recorded and saved in AIA format. Then, they were imported into the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” (2012 version) 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 Ke Ning capsules (n=15)

[0137]

[0138]

[0139] As shown in Table 11, the similarity between the 15 batches of Ke Ning capsules and the established standard control characteristic chromatograms in this experiment ranged from 0.939 to 0.99, all > 0.90. This indicates that the prepared Ke Ning capsules are relatively stable in chemical composition, and the overall appearance of each batch is basically consistent. In summary, the characteristic chromatogram of Ke Ning capsules established in this experiment is highly reliable and can be used as a reference chromatogram for the finished Ke Ning capsules.

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

[0141] Using thymidine (peak 9) as a reference peak, the relative retention times (RSD) of other common peaks in 15 batches of Ke Ning capsules were calculated to be between 0% and 2.0189%, as shown in Table 9. The relative deviation range of the relative retention times of each characteristic peak in the test sample and the characteristic peak in the reference chromatogram is shown in Table 13. According to Table 13, in order to eliminate the differences caused by peak time shifts of different components in the common peaks on the chromatographic column and to better overcome systematic errors in the experimental process, it is temporarily stipulated that the relative deviation of the relative retention times of each characteristic peak in the test sample of Ke Ning capsules and the standard chromatogram should be within ±5%, which indicates that the quality of Ke Ning capsules is qualified; otherwise, it is unqualified.

[0142] Table 12. Comparative Characteristic Chromatography of Ke Ning Capsules and Their Relative Retention Time

[0143]

[0144] Table 13. Range of relative retention time deviation between the common peaks and corresponding peaks in the characteristic chromatograms of 15 batches of Ke Ning capsules and the control.

[0145]

[0146]

[0147] Based on the above results, the tentatively set relative retention times for other peaks in the control characteristic chromatogram are 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). The characteristic chromatogram of Ke Ning capsules should have 12 characteristic peaks, with the peak corresponding to thymidine being the S peak. The relative retention times of each characteristic peak and the S peak should be within ±5% of the specified values.

[0148] 5.7 Chemometric Analysis Characteristic Spectra

[0149] 5.7.1 Cluster Analysis (HCA)

[0150] The peak area data of 15 batches of Ke Ning capsules were imported into IBM SPSS Statistics 26. The inter-group linkage method was used, and the squared Euclidean distance was selected for measurement. The results are as follows: Figure 11When the cluster distance was 9, the 15 batches of Ke Ning capsules were clustered into 4 groups: S2, S3, S6, S7, and S8 clustered into one group; S4, S9, S10, S11, S12, S13, and S14 clustered into another group; and S1 and S5 each clustered into a separate group. The cluster analysis results indicate that there are certain differences between the batches of samples, and the fluctuation in the quality of the medicinal materials used to produce Ke Ning capsules is the main reason for these results. Therefore, stabilizing the quality of the medicinal materials and selecting authentic medicinal materials from suitable producing areas can effectively reduce batch-to-batch differences.

[0151] 5.7.2 Principal Component Analysis (PCA)

[0152] The common peak area data of 12 samples from 15 batches of Ke Ning capsules were imported into IBM SPSS Statistics 26 software. After standardization, principal component analysis was performed, and the eigenvalues ​​and cumulative variance contribution rates of the principal component analysis were recorded (see Table 14). The results show that three principal components with eigenvalues ​​> 1 were extracted, with a cumulative contribution rate of 81.130%. This indicates that the analysis results of these three principal components can basically show the similarities and differences between different batches of Ke Ning capsules, and that the differences in product quality are influenced by multiple components.

[0153] Table 3-14 Eigenvalues ​​and Cumulative Variance Contribution Rate

[0154]

[0155] As shown in Table 15, peaks 4 and 12 have relatively high contribution rates in the first principal component, peaks 9 and 10 have relatively high contribution rates in the second principal component, and peak 3 has a relatively high contribution rate in the third principal component. Therefore, these components may be the cause of the differences between different batches of products.

[0156] Table 15 PCA factor loading matrix for Ke Ning capsules

[0157]

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

[0159] To further investigate the differences in chemical composition among different batches of Ke Ning capsules, the areas of 12 common peaks from 15 batches of Ke Ning capsule samples were standardized and imported into SIMCA 14.1 software for orthogonal partial least squares-discriminant analysis (OPLS-DA). The OPLS-DA score matrix and variable importance (VIP) projection value plot were obtained, as shown below. Figure 16 , 17 Its key parameters R²X = 0.654, R²Y = 0.711, and Q² = 0.502 are all greater than 0.5, indicating that the model has good fitting and predictive ability. Figure 16 It can be seen that the 15 batches of Ke Ning capsules samples were divided into 3 categories, consistent with the PCA classification results, and the separation was significant, indicating that there are certain differences in the components among different batches of samples; the VIP chromatogram can intuitively reflect the influence of each chromatographic peak, and a VIP value greater than 1 indicates a more important variable. Figure 17 It can be seen that, according to the size of the VIP value, the six components with VIP values ​​greater than 1 are peak 11, peak 5, peak 6, peak 12, peak 4, and peak 9. Among them, 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 Ke Ning capsules.

[0160] VI. Conclusion

[0161] This section established characteristic chromatograms for 15 batches of Ke Ning capsule samples, with a similarity > 0.9, indicating good methodological results. This provides a novel evaluation index for the quality standards of Ke Ning capsules. However, the characteristics of the porcine bile extract were not observed, mainly 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 combination of characteristic spectral mapping and chemical pattern recognition, the study revealed certain quality differences among different batches of Ke Ning capsules. PCA and OPLS-DA analyses categorized 15 batches of Ke Ning capsules into three groups, and based on the VIP value map, four main differentially expressed components were identified: peak 6 (adenine), peak 9 (thymidine), peak 11 (adenosine), and peak 12 (deoxyadenosine). Therefore, monitoring and controlling relevant components is crucial in the procurement of medicinal materials, production operations, and product testing. This allows companies to control drug quality more scientifically and comprehensively, thereby improving product stability. In this experiment, the results of HCA, PCA, and OPLS-DA analyses were largely consistent with the similarity assessment results, mutually reinforcing each other. This study established a characteristic spectral mapping method for Ke Ning capsules, which is stable and reliable, providing a scientific basis for improving the quality evaluation standards of Ke Ning capsules.

Claims

1. A method for constructing a feature map of a cough-relieving capsule, characterized by the following steps: (1), making solution 1.1 Preparation of the test solution Accurately weigh 2g of cough suppressant capsule powder and place it in a stoppered conical flask. Accurately measure 40 mL of 20% methanol solution and add it to the flask. Mix the cough suppressant capsule powder thoroughly and heat in a 60℃ water bath for 0.5h. After cooling, replenish the lost mass with 20% methanol solution and pour the mixture into a centrifuge tube. Centrifuge at 4000 rpm. -1 Centrifuge for 10 min, filter to obtain the first filtrate, repeat the above operation once for the filter residue to obtain the second filtrate, combine the filtrates, 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 standards, and add them to purified water to prepare reference standard 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-herb sample solutions Accurately weigh the single medicinal materials in the prescription, including the test sample, and then prepare sample solutions of each single medicinal material according to section "1.1"; 1.4 Preparation of negative sample solutions for samples lacking a single medicinal herb Accurately weigh negative samples of each missing herb in the prescription, and then prepare sample solutions of each herb according to section "1.1"; (2) Determine the main components for constructing the feature map The main components of Ke Ning capsules are porcine bile extract, Fritillaria thunbergii, and Platycodon grandiflorus. High-performance liquid chromatography (HPLC) was used with a 1 / 100,000 electronic balance and centrifuge to separate the main active components from Ke Ning capsules using characteristic chromatograms. Based on the main components of the three drugs, the main active components of Ke Ning capsules were determined to be: porcine deoxycholic acid, taurine deoxycholic acid, Platycodon grandiflorus saponin, deoxyadenosine, thymidine, cytidine, adenine, adenosine, guanosine, uridine, and uracil. Among these, the main components of porcine bile extract are porcine deoxycholic acid, taurine deoxycholic acid, and Fritillaria thunbergii. The main components of Fritillaria thunbergii are deoxyadenosine and thymidine, while the main components of Platycodon grandiflorus are platycodins. Fritillaria thunbergii and Platycodon grandiflorus share components such as cytidine, adenine, adenosine, guanosine, uridine, and uracil. When establishing the characteristic chromatogram, the chromatographic conditions were not compatible with the components of porcine deoxycholic acid, taurine deoxycholic acid, and platycodins. Only 12 components under these chromatographic conditions were assigned to the characteristic chromatogram. Among them, deoxyadenosine and thymidine were assigned to Fritillaria thunbergii, while cytidine, adenine, adenosine, guanosine, uridine, and uracil were shared by Fritillaria thunbergii and Platycodon grandiflorus. The assignments of the remaining 4 shared peaks were not determined. The chromatographic conditions were as follows: Column: Agilent TC-C 18 Column: 4.6 mm × 250 mm, 5 μm; mobile phase: water A: methanol B (volume ratio), gradient elution performed according to the table below: Detection wavelength: 260 nm; Flow rate: 0.8 mL / min -1 Injection volume: 20 μL; Column temperature: 25℃; (3) Constructing feature maps 3.1 First, determine the characteristic peak and the reference peak. In the characteristic chromatogram, 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, and the relative retention time and relative peak area of ​​the common peak are calculated. 3.2 Establishment of Feature Maps Prepare 15 batches of test solutions, numbered S1 to S15, according to the method in section "1.1". Inject the samples under the chromatographic conditions in section "2", record the chromatograms, save the data in AIA format, and import them into the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software to obtain the characteristic chromatograms and reference chromatograms R of the 15 batches of samples. The method is as follows: using S1 as the reference chromatogram, the chromatographic peak time window width is 0.1, the median method is used, multi-point correction is performed, and each chromatographic peak is automatically matched to form a common pattern chromatogram. Based on the characteristic chromatogram analysis, a total of 12 common peaks were identified in 15 batches of Ke Ning capsules. The relative retention times and UV spectra 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. The four common peaks 4, 5, 8, and 10 were not assigned. Using these 12 common peaks as characteristic peaks, a characteristic chromatogram R of the Ke Ning capsules and a chromatogram of the mixed reference standard were constructed.

2. The application of the characteristic map of Ke Ning capsule constructed by the method of claim 1 in the overall control and evaluation of the quality of Ke Ning capsule product.

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