A method for detecting the content of kaixin san ingredients

Abstract

This invention discloses a method for detecting the content of components in Kaixin San (a traditional Chinese medicine formula). Based on high-performance liquid chromatography (HPLC), a method for detecting the content of components in Kaixin San is constructed. By adopting a suitable gradient elution program, especially by strictly controlling the initial mobile phase ratio of the gradient elution program, and by combining the selection of chromatographic column and mobile phase, the method can rapidly and comprehensively determine the content of six chemical components in Kaixin San under dual-wavelength conditions: polygalactosidone III, 3,6-disinyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone, and α-asarone. The method has high sensitivity, good specificity, accurate results, and good reproducibility.

Description

Technical Field

[0001] This invention relates to the field of component analysis technology, and in particular to a method for detecting the content of the components in Kaixin San (a traditional Chinese medicine formula). Background Technology

[0002] Kaixin San is one of the first 100 prescriptions included in the "List of Famous Ancient Prescriptions (First Batch)" formulated by the State Administration of Traditional Chinese Medicine and the State Drug Administration in 2018. It was first recorded in "Essential Prescriptions Worth a Thousand Pieces of Gold" written by Sun Simiao, a medical scientist of the Tang Dynasty. Kaixin San is composed of four Chinese herbs: ginseng, polygala, calamus, and poria. The prescription is simple yet powerful. According to the basic theory of traditional Chinese medicine, the prescription is as follows: ginseng greatly replenishes the vital energy, calms the mind and improves intelligence; poria strengthens the spleen, calms the mind, and promotes diuresis and dampness removal; calamus resolves phlegm, opens the orifices and awakens the mind; polygala calms the mind, resolves phlegm and opens the orifices. It can be combined with calamus to enhance the effect of resolving phlegm and opening the orifices, and can be combined with poria to enhance the function of connecting the heart and kidneys (Jian Wenxuan, Zhang Zhao. Research progress on the tonifying effect of Kaixin San [J]. Journal of Neuropharmacology, 2018, 8(06):38.). Modern research shows that Kaixin San has a variety of pharmacological activities, including anti-dementia, improving learning and memory, anti-oxidation, anti-depression, anti-aging, anti-fatigue, and promoting nerve cell differentiation and regeneration. It has significant therapeutic effects on Alzheimer's disease, depression, anxiety and other diseases (Wang Haixia, Jiang Ning, Lü Jingwei, Liu Sisi, Liu Xinmin. Research progress on the anti-depression and learning and memory improvement effects and mechanisms of Kaixin San [J]. Chinese Traditional and Herbal Drugs, 2020, 51(14):3802-3813.).

[0003] Traditional Chinese patent medicines have complex chemical compositions, making it difficult to conduct comprehensive quality evaluations. The same problem also exists with KaiXinSan. At present, the analysis of the index components of KaiXinSan is relatively single, unable to comprehensively cover multiple index components in KaiXinSan and lacking an overall quality control method for multi-index components associated with its efficacy. It cannot reflect the content of the active ingredients in KaiXinSan as a whole. Through literature and patent searches, patents related to the determination of the components of KaiXinSan are found. Among them, the patent related to the determination of active ingredients is "A Detection Method for the Contents of Multi-Index Components in KaiXinSan" (CN115267005A). This patent established a method for determining the contents of 5 components, namely polygalaxanthone III, 3,6'-di-O-sinapoyl-sucrose, ginsenoside Rg1, pachymic acid, and dehydro-pachymic acid, using high-performance liquid chromatography (HPLC). However, it does not include the determination of the contents of the main pharmacodynamic components β-asarone and α-asarone. Moreover, this detection method uses four-wavelength detection and requires analyzing the contents of five components at four wavelengths, namely 203nm, 210nm, 242nm, and 320nm. The analysis method is not simple enough. In addition, the highest content of polygalaxanthone III detected in the sample by this method is 0.1529mg / g, and the highest content of 3,6'-di-O-sinapoyl-sucrose is 1.4372mg / g, which is lower than the results detected in the existing literature (Shang Bingxian, Zhao Zhenxia, Zeng Qi, Su Jian, Xu Bing, Liu Yongli, Lei Haimin, etc. Research on the Key Quality Attributes of the Benchmark Sample of the Classical Famous Prescription KaiXinSan [J]. China Journal of Chinese Materia Medica, 2023, 48(02): 382-389.). This method does not fully extract and detect the components in the sample and cannot most truly reflect the content in traditional Chinese medicine. Therefore, this condition has certain limitations. In addition, the patent "A Detection Method for the HPLC Characteristic Chromatogram of a Pharmaceutical Preparation" (CN113866306B) also involves the detection method of KaiXinSan. However, the detection time of this method is 102 minutes, which is relatively long. And it only uses a single wavelength. The wavelength range involved in this patent is 200-205nm, which is not the maximum absorption wavelength of polygalaxanthone III and 3,6'-di-O-sinapoyl-sucrose. Therefore, this method cannot most truly reflect the content in traditional Chinese medicine.

[0004] The components selected for the detection of KaiXinSan by the existing methods lack representativeness, and the detection methods are not simple enough, unable to most truly reflect the content of the components in traditional Chinese medicine. In order to promote the development of KaiXinSan and ensure that enterprises can control the quality of finished patent medicines, it is urgent to establish an analytical method with high sensitivity, good specificity, accurate results, and good reproducibility for the multiple index components contained in KaiXinSan, provide a basis for the selection of raw materials of traditional Chinese patent medicines, guarantee the product quality, provide a basis for the quality control and evaluation of KaiXinSan, and provide a reference for establishing a content determination method for traditional Chinese patent medicine preparations containing Polygala tenuifolia, Panax ginseng, and Acorus tatarinowii. Summary of the Invention

[0005] Based on the technical problems existing in the background technology, this invention proposes a method for detecting the content of Kaixin San components. The method uses high performance liquid chromatography under dual wavelength conditions to simultaneously determine the content of six chemical components in Kaixin San, namely, polygalactosidone III, 3,6-disinoyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone, and α-asarone, within 70 min. The method has high sensitivity, good specificity, accurate results, and good reproducibility.

[0006] The present invention proposes a method for detecting the content of Kaixin San (a traditional Chinese medicine formula), comprising the following steps:

[0007] S1. After adding Kaixin San to methanol solution, ultrasonic treatment and filtration are performed. The filtrate is then used to prepare the test solution.

[0008] S2. Take the reference standards of polygala tenuifolia succinate III, 3,6-disinyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone and α-asarone, dissolve them in methanol solution, and make up to volume to prepare the reference standard solution.

[0009] S3. The test solution and the reference solution are detected by high performance liquid chromatography at 203 nm and 245 nm. The detection method is as follows: set the running parameters program, inject the test solution or the reference solution into the injection valve, and the solution in the injection valve flows into the chromatographic column together with the mobile phase for separation. After complete separation, it enters the detector for detection.

[0010] Preferably, in S1 and S2, the volume percentage of the methanol solution is 75%.

[0011] Preferably, in S1, the mass ratio of Kaixin powder to the volume of methanol solution is 1:25 g / mL.

[0012] Preferably, in step S1, the ultrasonic treatment time is 30 minutes.

[0013] Preferably, in S1, the pore size of the filter membrane is 0.22 μm.

[0014] Preferably, in S2, the CAS number of the polygalactosanone III is 162857-78-5, the CAS number of 3,6-disinyl sucrose is 139891-98-8, the CAS number of ginsenoside Re is 52286-59-6, the CAS number of ginsenoside Rb1 is 41753-43-9, the CAS number of β-asarone is 5273-86-9, and the CAS number of α-asarone is 2883-98-9.

[0015] Since the main active components of Polygala tenuifolia include a variety of effective chemical components such as saponins, ketones, oligosaccharide esters, and alkaloids, Polygala tenuifolia sinomenol III and 3,6-disinyl sucrose represent ketones and oligosaccharide esters, respectively, and have the effects of calming the mind and improving intelligence, and antidepressant effects; the main active component of ginseng is ginsenoside, which has the effects of improving intelligence, strengthening the heart, and anti-aging, and ginsenoside Re and ginsenoside Rb1 are used as the standard for measuring ginsenosides; the main active components of Acorus tatarinowii are β-asarone and α-asarone, which have antispasmodic, anticonvulsant, and synergistic effects with sodium pentobarbital. Therefore, this invention selects Polygala tenuifolia sinomenol III, 3,6-disinyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone, and α-asarone as the indicator components of Kaixin San (a traditional Chinese medicine formula).

[0016] Preferably, in step S3, the mobile phase is acetonitrile-0.05% phosphoric acid solution, wherein phase A is acetonitrile and phase B is 0.05% phosphoric acid solution.

[0017] Preferably, in step S3, the flow rate of the solution in the injection valve flowing into the chromatographic column together with the mobile phase is 1.0 mL / min.

[0018] Preferably, in step S3, the detector is a DAD detector.

[0019] Preferably, in step S3, the chromatographic column is a C18 chromatographic column, and the packing agent in the chromatographic column is octadecylsilane-bonded silica gel.

[0020] More preferably, the chromatographic column is a Thermo-Acclaim™ 120-C18 (250*4.6mm, 5μm).

[0021] Preferably, in step S3, the column temperature of the chromatographic column is 30°C.

[0022] Preferably, in step S3, the injection volume of the test solution or reference solution into the injection valve is 10 μL.

[0023] Preferably, in step S3, the high-performance liquid chromatography (HPLC) employs gradient elution, and the specific procedure is as follows:

[0024] From 0 to 3 minutes, the volume percentage of phase B was 85-84%, with the remainder being phase A;

[0025] After 3-5 minutes, the volume percentage of phase B is 84-80%, with the remainder being phase A;

[0026] 5-16 min, the volume percentage of phase B is 80%, and the remainder is phase A;

[0027] 16-21 min, the volume percentage of phase B is 80-75%, and the remainder is phase A;

[0028] 21-31 min, the volume percentage of phase B is 75-73%, and the remainder is phase A;

[0029] 31-38 min, the volume percentage of phase B is 73-67%, and the remainder is phase A;

[0030] 38-47 min, the volume percentage of phase B is 67-60%, and the remainder is phase A;

[0031] From 47 to 59 minutes, the volume percentage of phase B was 60%, with the remainder being phase A.

[0032] 59-62 min, the volume percentage of phase B is 60-52%, and the remainder is phase A;

[0033] After 62-70 minutes, the volume percentage of phase B is 52-48%, with the remainder being phase A.

[0034] Beneficial Effects: This invention, based on high-performance liquid chromatography (HPLC), constructs a method for detecting the content of components in Kaixin San (a traditional Chinese medicine formula). By employing a suitable gradient elution program, especially by strictly controlling the initial mobile phase ratio, and by selecting the chromatographic column and mobile phase, a rapid and comprehensive determination of the content of six active components of Kaixin San—polygalactosidone III, 3,6-disinyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone, and α-asarone (including lipid-soluble and water-soluble components)—is achieved. The detection method of this invention features short analysis time; detection limits for all six components are below 0.008 μg / mL, indicating high sensitivity; no interfering peaks are observed in the blank medium, demonstrating good specificity; and the linear relationship r of the six indicator components is well established. 2 All values ​​were greater than 0.999, indicating accurate results. In all methodological investigations, the peak area RSDs of the six indicator components were all less than 2%, demonstrating good reproducibility. This invention constructs a multi-indicator component detection method related to the efficacy of Kaixin San, which can accurately determine the content of indicator components, comprehensively reflect the efficacy of the compound, provide a basis for the quality control and evaluation of Kaixin San, and provide a reference for establishing content determination methods in traditional Chinese medicine preparations containing Polygala tenuifolia, ginseng, and Acorus tatarinowii, thus providing effective support for the high-quality development of products. Attached Figure Description

[0035] Figure 1 The HPLC chromatogram of Kaixin San at a wavelength of 203 nm is shown by the detection method proposed in this invention.

[0036] Peak 1: Polygala tenuifolia ketone III; Peak 2: 3,6-disinyl sucrose; Peak 3: Ginsenoside Re; Peak 4: Ginsenoside Rb1; Peak 5: β-asarone; Peak 6: α-asarone.

[0037] Figure 2 The HPLC chromatogram of Kaixin San at a wavelength of 245 nm is shown by the detection method proposed in this invention.

[0038] Peak 1: Polygala tenuifolia ketone III; Peak 2: 3,6-disinyl sucrose; Peak 5: β-asarone; Peak 6: α-asarone.

[0039] Figure 3 This is an HPLC chromatogram of a blank medium at a wavelength of 203 nm, representing the detection method proposed in this invention.

[0040] Figure 4 This is an HPLC chromatogram of a blank medium at a wavelength of 245 nm, representing the detection method proposed in this invention.

[0041] Figure 5 The HPLC chromatogram of Kaixin San at a wavelength of 203 nm is shown for the detection method proposed in Comparative Example 1.

[0042] Peak 1: Polygala tenuifolia ketone III; Peak 2: 3,6-disinyl sucrose; Peak 3: Ginsenoside Re; Peak 4: Ginsenoside Rb1; Peak 5: β-asarone; Peak 6: α-asarone.

[0043] Figure 6 The HPLC chromatogram of Kaixin San at a wavelength of 245 nm is shown for the detection method proposed in Comparative Example 1.

[0044] Peak 1: Polygala tenuifolia ketone III; Peak 2: 3,6-disinyl sucrose; Peak 5: β-asarone; Peak 6: α-asarone. Detailed Implementation

[0045] The technical solution of the present invention will be described in detail through specific embodiments.

[0046] Example 1

[0047] 1. Preparation of the test solution

[0048] Accurately weigh 1g of Kaixin San reference sample, place it in a stoppered conical flask, accurately add 25mL of 75% methanol solution, seal, ultrasonically extract for 30min, shake well, take the supernatant and filter it through a 0.22μm filter membrane, take the filtrate, and obtain the test solution.

[0049] 2. Preparation of reference solution

[0050] Accurately weigh the reference standards of polygala tenuifolia succinate III, 3,6-disinyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone and α-asarone, dissolve them in 75% methanol solution and dilute to volume to prepare a series of reference standard solutions of different concentrations.

[0051] 3. Measurement

[0052] High-performance liquid chromatography (HPLC) test conditions: mobile phase was acetonitrile-0.05% phosphoric acid aqueous solution, where phase A was acetonitrile and phase B was 0.05% phosphoric acid aqueous solution; flow rate was 1.0 mL / min; detector was a DAD detector; detection wavelengths were 203 nm and 245 nm; column was a C18 column; the column packing material was octadecylsilane-bonded silica gel; column temperature was 30℃; injection volume was 10 μL; analysis time was 70 min; gradient elution was used.

[0053] Gradient elution, the specific procedure is as follows:

[0054] From 0 to 3 minutes, the volume percentage of phase B was 85-84%, with the remainder being phase A;

[0055] After 3-5 minutes, the volume percentage of phase B is 84-80%, with the remainder being phase A;

[0056] 5-16 min, the volume percentage of phase B is 80%, and the remainder is phase A;

[0057] 16-21 min, the volume percentage of phase B is 80-75%, and the remainder is phase A;

[0058] 21-31 min, the volume percentage of phase B is 75-73%, and the remainder is phase A;

[0059] 31-38 min, the volume percentage of phase B is 73-67%, and the remainder is phase A;

[0060] 38-47 min, the volume percentage of phase B is 67-60%, and the remainder is phase A;

[0061] From 47 to 59 minutes, the volume percentage of phase B was 60%, with the remainder being phase A.

[0062] 59-62 min, the volume percentage of phase B is 60-52%, and the remainder is phase A;

[0063] After 62-70 minutes, the volume percentage of phase B is 52-48%, with the remainder being phase A.

[0064] The reference solution was injected for determination using the external standard single-point method; the fifteen batches of Kaixin San reference samples were measured using a double-sample double-needle method.

[0065] Table 1. Results of determination of six components in fifteen batches of Kaixin San reference samples.

[0066]

[0067]

[0068] Verification of the method in this application

[0069] The methodological verification was carried out on the detection methods of polygalaxanthone III, 3,6-disinapoyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone and α-asarone in the Kāixīn Sǎn sample of the present invention, and the performance index results are as follows.

[0070] 1. Linear relationship of the detection method

[0071] Weigh accurately appropriate amounts of the reference substances of polygalaxanthone III, 3,6-disinapoyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone and α-asarone, and prepare a series of reference substance solutions with different concentrations by adding 75% methanol solution according to Step 2 in Example 1. According to the chromatographic conditions in Step 3 of Example 1, accurately pipette 10 μL of the reference substance solution and inject it into the high-performance liquid chromatograph. Taking the reference substance concentration as the ordinate and the peak area of each index component as the abscissa, draw the standard curve. The specific data of the standard curve are shown in Table 1. At the same time, calculate the standard regression equations, correlation coefficients and linear ranges of polygalaxanthone III, 3,6-disinapoyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone and α-asarone. The specific results are shown in Table 2. It can be seen from Table 2 that the linear relationships of the 6 index components are good within their respective injection mass concentration ranges, indicating that the method of the present invention has a wide linear range and high accuracy.

[0072] Table 2 Standard curve data of six index components

[0073]

[0074] 2. Stability

[0075] Take the Kāixīn Sǎn sample with batch number KXS202301, prepare the test solution according to Step 1 in Example 1, and carry out injection analysis at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h respectively according to the chromatographic conditions in Step 3 of Example 1. The specific results are shown in Table 3. The results show that the RSDs of the chromatographic peak areas of polygalaxanthone III, 3,6-disinapoyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone and α-asarone within 24 h are all less than 2%, indicating that the detection of the test solution within 24 h has no effect on the results, and the stability of the detection method is good.

[0076] Table 3 Stability

[0077]

[0078] 3. Precision

[0079] 3.1 Instrument precision

[0080] Reference standards of polygalactosidone III, 3,6-disinyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone, and α-asarone were accurately weighed and prepared into reference solutions of a certain concentration by adding 75% methanol solution according to step 2 in Example 1. The solutions were then analyzed six times consecutively under the chromatographic conditions described in step 3 of Example 1. The specific results are shown in Table 4. The results indicate that the peak area RSD of the six index components in the six consecutive injections was less than 2%, indicating good instrument precision.

[0081] Table 4 Instrument Precision

[0082]

[0083] 3.2 Repeatability

[0084] Six test solutions were prepared in parallel according to step 1 of Example 1 using a sample of Kaixin San (batch number KXS202301). Each test solution was measured twice under the chromatographic conditions described in step 3 of Example 1. The specific results of peak area repeatability are shown in Table 6. The results show that the RSD of the peak areas of the five index components were all less than 2%, meeting the requirements of the 2020 edition of the Chinese Pharmacopoeia, indicating good method repeatability.

[0085] Table 5 Repeatability

[0086]

[0087] 3.3 Intermediate Precision

[0088] Samples of Kaixin San (a traditional Chinese medicine) from the same batch, batch number KXS202301, were collected by different experimenters. Three test solutions were prepared for each sample using step 1 of Example 1. The solutions were analyzed using the same HPLC system under the chromatographic conditions described in step 3 of Example 1. The peak area results are shown in Table 7. The results indicate that the RSD of the peak areas of the six indicator components in Kaixin San measured by different experimenters using the same instrument was less than 2%, indicating good intermediate precision.

[0089] Table 6 Intermediate Precision

[0090]

[0091] 4. Recovery rate

[0092] Accurately weigh 1.0 g of 1-kJ of Kaixin San (batch number KXS202301) of known concentration. Add a mixed reference solution of 6 indicator components at three levels: 50%, 100%, and 150% of the content, with 3 replicates for each level. Prepare the test solution according to step 1 in Example 1, and analyze it under the chromatographic conditions in step 3 of Example 1. The results are shown in Table 7. As shown in Table 7, the recoveries of the 6 indicator components all meet the recovery limits specified in the 2020 edition of the Chinese Pharmacopoeia (Part IV), indicating that the determination method has good accuracy.

[0093] Table 7 Recovery Rate

[0094]

[0095]

[0096] 5. Durability

[0097] Samples of Kaixin San (a traditional Chinese medicine) from the same batch, batch number KXS202301, were tested using the sample solutions prepared in step 1 of Example 1. Chromatographic conditions were applied according to step 3 of Example 1, with different column types, column temperatures, flow rates, mobile phase ratios, and sample extraction times. Peak area results are shown in Table 8-12. The results indicate that after changing the conditions, the RSD of the peak areas of the six indicator components was less than 2%, indicating that the content determination method has good robustness.

[0098] Table 8. Robustness (Column Type)

[0099]

[0100]

[0101] Table 9 Durability (Column Temperature)

[0102]

[0103] Table 10 Durability (Flow Rate)

[0104]

[0105] Table 11 Durability (Acid-Water Mobile Phase Ratio)

[0106]

[0107] Table 12 Durability (Sample Extraction Time)

[0108]

[0109] Comparative Example 1

[0110] The difference between this comparative example and Example 1 is that the ratio of phase AB data in the elution procedure is:

[0111] From 0 to 10 minutes, the volume percentage of phase B is 95-85%, with the remainder being phase A;

[0112] After 10-20 minutes, the volume percentage of phase B is 85-84%, with the remainder being phase A.

[0113] 20-31 min, the volume percentage of phase B is 84-82%, and the remainder is phase A;

[0114] From 31 to 33 minutes, the volume percentage of phase B was 82-77%, with the remainder being phase A;

[0115] 33-52 min, the volume percentage of phase B is 77-60%, and the remainder is phase A;

[0116] 52-70 min, the volume percentage of phase B is 60-48%, and the remainder is phase A;

[0117] 70-76 min, the volume percentage of phase B is 48-27%, and the remainder is phase A;

[0118] 76-93 min, the volume percentage of phase B is 27-20%, and the rest is phase A;

[0119] 93-100 min, the volume percentage of phase B is 20-5%, and the rest is phase A;

[0120] 100-102 min, the volume percentage of phase B is 5%, and the rest is phase A.

[0121] Everything else is the same as in Example 1.

[0122] The accuracy of the detection method in Comparative Example 1 was verified using the recovery rate of the spiking sample, as follows:

[0123] Table 13 Recovery Rate

[0124]

[0125]

[0126] The results showed that the detection method of the present invention has a short analysis time; the detection limits for all six components are below 0.008 μg / mL, indicating high sensitivity; the blank medium showed no interfering peaks, indicating good specificity; and the linear relationship r of the six index components was [not specified]. 2All values ​​were greater than 0.999, indicating accurate results. In all methodological investigations, the peak area RSDs of the six indicator components were all less than 2%, demonstrating good reproducibility. Comparative Example 1 data showed that after changing the AB phase data ratio in the elution procedure, the average recovery rate of the assay method was lower than that in Example 1, and the RSD value of Comparative Example 1 was greater than 2%, meaning that the accuracy of Comparative Example 1 was lower than that of Example 1.

[0127] from Figure 1 It can be clearly seen that the detection method proposed in this invention detects six characteristic peaks in the HPLC chromatogram of Kaixin San at a wavelength of 203 nm. Among them, peak 1 is the characteristic peak of Polygala tenuifolia ketone III, peak 2 is the characteristic peak of 3,6-disinoyl sucrose, peak 3 is the characteristic peak of ginsenoside Re, peak 4 is the characteristic peak of ginsenoside Rb1, peak 5 is the characteristic peak of β-asarone, and peak 6 is the characteristic peak of α-asarone. Figure 2 It can be seen that the detection method proposed in this invention detects the HPLC chromatogram of Kaixin San at a wavelength of 245 nm, showing the characteristic peaks of polygala tenuifolia ketone III, 3,6'-disinyl sucrose, β-asarone, and α-asarone. Moreover, 245 nm is the maximum absorption wavelength of polygala tenuifolia ketone III and 3,6'-disinyl sucrose, which can most accurately reflect the content in traditional Chinese medicine. Figure 3 , Figure 4 The images show the HPLC chromatograms of the blank medium at wavelengths of 203 nm and 245 nm, respectively, for the detection method proposed in this invention. It can be seen that the blank medium has no interfering peaks and good specificity. Figure 5 The HPLC chromatogram of Kaixin San at a wavelength of 203 nm is shown in Comparative Example 1. The characteristic peaks 1, 3, and 4 are not obvious and are accompanied by impurity peaks, which interfere with the accuracy of the detection data. Figure 6 The HPLC chromatogram of Kaixin San at a wavelength of 245 nm was obtained using the detection method proposed in Comparative Example 1. Characteristic peaks of polygala tenuifolia ketone III, 3,6-disinyl sucrose, β-asarone, and α-asarone were detected. However, the characteristic peak of polygala tenuifolia ketone III was weak and indistinct, and was accompanied by the appearance of impurity peaks, which interfered with the accuracy of the detection data.

[0128] In summary, the method for detecting the content of components in Kaixin San provided by this invention exhibits good precision, repeatability, and stability. The results of sample recovery and robustness are accurate and reliable, truly reflecting the quality differences of various indicator components in Kaixin San and improving the quality control system of Kaixin San. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and has high industrial application value.

[0129] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting the content of Kaixin San (a traditional Chinese medicine formula), comprising the following steps: S1. After adding Kaixin San to methanol solution, ultrasonic treatment and filtration are performed. The filtrate is then used to prepare the test solution. S2. Take the reference standards of polygala tenuifolia succinate III, 3,6-disinyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone and α-asarone, dissolve them in methanol solution, and make up to volume to prepare the reference standard solution. S3. The test solution and the reference solution are detected by high performance liquid chromatography at 203 nm and 245 nm. The steps are as follows: Set the operating parameters program, inject the test solution or the reference solution into the injection valve, and the solution in the injection valve flows into the chromatographic column together with the mobile phase for separation. After complete separation, the solution enters the detector for detection. The packing material in the chromatographic column is octadecylsilane-bonded silica gel. In step S3, the high-performance liquid chromatography method employs gradient elution, and the specific procedure is as follows: From 0 to 3 minutes, the volume percentage of phase B is 85-84%, with the remainder being phase A; After 3-5 minutes, the volume percentage of phase B is 84-80%, with the remainder being phase A; 5-16 min, the volume percentage of phase B is 80%, and the remainder is phase A; 16-21 min, the volume percentage of phase B is 80-75%, and the remainder is phase A; 21-31 min, the volume percentage of phase B is 75-73%, and the remainder is phase A; 31-38 min, the volume percentage of phase B is 73-67%, and the remainder is phase A; 38-47 min, the volume percentage of phase B is 67-60%, and the remainder is phase A; 47-59 min, the volume percentage of phase B is 60%, and the remainder is phase A; 59-62 min, the volume percentage of phase B is 60-52%, and the remainder is phase A; After 62-70 minutes, the volume percentage of phase B is 52-48%, with the remainder being phase A; Phase A is acetonitrile, and phase B is 0.05% phosphoric acid solution; The above detection method was used to determine the contents of six active ingredients of Kaixin San: polygalactosanone III, 3,6-disinyl sucrose, ginsenoside Re, ginsenoside Rb1, β-asarone and α-asarone.

2. The method for detecting the content of the Kaixin San component according to claim 1, characterized in that, In S1, the mass ratio of Kaixin powder to the volume of methanol solution is 1:25 g / mL; the ultrasonic treatment time is 30 min; and the pore size of the filter membrane is 0.22 μm.

3. The method for detecting the content of the Kaixin San component according to claim 1, characterized in that, In S1 and S2, the volume percentage of the methanol solution is 75%.

4. The method for detecting the content of the Kaixin San component according to claim 1, characterized in that, In step S3, the injection volume of the test solution or reference solution into the injection valve is 10 μL.

5. The method for detecting the content of the Kaixin San component according to claim 1, characterized in that, In S3, the mobile phase is acetonitrile-0.05% phosphoric acid solution.

6. The method for detecting the content of the Kaixin San component according to claim 1, characterized in that, In step S3, the flow rate of the solution in the injection valve flowing into the chromatographic column together with the mobile phase is 1.0 mL / min.

7. The method for detecting the content of the Kaixin San component according to claim 1, characterized in that, In S3, the detector is a DAD detector.

8. The method for detecting the content of the Kaixin San component according to claim 1, characterized in that, In S3, the chromatographic column is a C18 column; the column temperature is 30℃.

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