Method for determining contents of ester saikosides and d in radix bupleuri

Abstract

The invention belongs to the technical field of quality control of traditional Chinese medicine preparations, and relates to a method for determining the content of ester saikosides a and d in radix bupleuri, which comprises the following steps of: performing ultrasonic treatment on a radix bupleuri sample to be detected by taking methanol as a solvent to obtain a test solution I; performing heating reflux extraction on a radix bupleuri sample to be detected by taking methanol containing ammonia water as a solvent to obtain a test solution II; respectively detecting the contents of saikoside a and saikoside d in the test solution I and the test solution II by adopting a high performance liquid chromatography-ultraviolet detector; and calculating the difference between the two values to obtain the contents of ester-type saikosaponin a and d. According to the method, a high performance liquid chromatography-ultraviolet detector is adopted for determining the content of saikoside a and saikoside d after free and ester saikoside in radix bupleuri encounters alkali and ester bonds are broken and hydrolyzed, the content of the ester saikoside is obtained indirectly through a differential method, and the problem that quantitative determination can still be carried out under the condition that no reference substance exists is solved; a reference is provided for deeply researching chemical components of the radix bupleuri medicinal material.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology for Chinese medicinal materials and processed medicinal slices, and relates to a method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense. Background Technology

[0002] Bupleurum is a plant of the Apiaceae family. Bupleurum chinense DC. or Narrow-leaved Bupleurum Bupleurum scorzonerifolium The dried root of Willd. is commonly known as "Northern Bupleurum" and "Southern Bupleurum" depending on its characteristics. It has the effects of relieving exterior syndromes, soothing the liver, raising yang and reducing fever, and is one of the commonly used medicinal ingredients in traditional Chinese medicine prescriptions.

[0003] The main components of Bupleurum chinense include saponins, volatile oils, and fatty acids. Saponins are both its effective and toxic components. Among them, saponin a and saponin d are included in the 2025 edition of the Chinese Pharmacopoeia as quality control indicators for content determination. However, Bupleurum chinense also contains a class of ester-type saponins a and d, formed by the combination of small-molecule organic acids such as malonic acid and acetic acid with the hydroxyl groups on the terminal sugars of saponins a and d. These esters are also present in relatively high amounts in Bupleurum chinense, comparable to or even higher than the content of saponin a / d. Therefore, ester-type saponins a and ester-type saponins d are also an important class of saponins that cannot be ignored.

[0004] In the current edition of the Chinese Pharmacopoeia, among the quantitative analysis methods using high performance liquid chromatography, whether it is the internal standard method, the external standard method, the principal component self-comparison method with correction factors, or the principal component self-comparison method without correction factors and the area normalization method, all of the above determination methods require corresponding reference standards for the exploration of methods for the determination of constant quantities.

[0005] Ester-type saikosaponin a and ester-type saikosaponin d possess both unstable ester and ether bonds. The ester bond breaks under acidic or alkaline conditions to form saikosaponin a and d, while the ether bond breaks under acidic conditions, resulting in the formation of secondary metabolites. Aqueous methanol can also alter both bonding modes. The structures of ester-type saikosaponin a and d make it difficult to extract and separate individual compounds, hindering direct content determination.

[0006] Even in the determination of the content of saikosaponins a and d, the pharmacopoeia method and most literature methods (based on the pharmacopoeia method) cannot accurately reflect the content of the original saikosaponins a and d, nor can they accurately reflect the content of saikosaponins a and d after complete hydrolysis.

[0007] Therefore, a simple, easy-to-operate, and highly accurate method is needed to determine the content of ester-type saikosaponins in Bupleurum chinense. Summary of the Invention

[0008] The main objective of this invention is to overcome the deficiencies in the prior art and provide a method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense.

[0009] To achieve the above objectives, the specific technical solution is as follows: This invention provides a method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense. The method involves treating the Bupleurum chinense sample with methanol as a solvent via ultrasonication to obtain test solution one; extracting the Bupleurum chinense sample with a methanol solution containing ammonia via reflux to obtain test solution two; detecting the content of saikosaponins a and d in test solution one and test solution two respectively using high-performance liquid chromatography-ultraviolet detection; and calculating the difference between the two solutions to obtain the content of ester-type saikosaponins a and d.

[0010] This invention uses methanol to treat Bupleurum chinense samples, and employs high-performance liquid chromatography-ultraviolet (HPLC-UV) to determine the contents of free saikosaponin a and saikosaponin d. Based on the characteristic of ester-type saikosaponins breaking ester bonds upon contact with alkali, the invention uses methanol containing ammonia for extraction under reflux. The total amounts of hydrolyzed saikosaponin a and saikosaponin d are then determined using HPLC-UV, and the content of ester-bonded saikosaponins is calculated by the difference between the two values. This provides a reference for further research on the chemical composition of Bupleurum chinense.

[0011] Furthermore, the chromatographic conditions for the detection are as follows: Chromatographic column: octadecylsilane-bonded silica gel column; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is water.

[0012] The chromatographic column and mobile phase used in this invention help to achieve better separation and detection of target components at lower absorption wavelengths.

[0013] Furthermore, the elution mode of the chromatographic column is gradient elution, and the gradient elution program for detecting the test solution is: 0-4 min, 36%A; 20-21 min, 40%→43%A; 38 min, 43%A.

[0014] The present invention employs the above-mentioned gradient elution procedure, which can achieve better separation of the target component from the test solution in a shorter time.

[0015] Furthermore, the elution mode of the chromatographic column is gradient elution, and the gradient elution program for detecting the second test solution is: 0-30 min, 25%→64%A; 31-44 min, 100%A.

[0016] After hydrolysis, the concentration of the target component in the test solution 2 increases before the retention time. Different gradient elutions are used to ensure better separation of the target component.

[0017] Furthermore, the chromatographic conditions for determining the test solution one are as follows: flow rate 1.0 mL / min. -1 The column temperature was 29 ℃, and the injection volume was 5–15 µL.

[0018] Furthermore, the chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 µm; preferably, it is an Agilent ZORBAX SB-C18 (4.6 × 250 mm, 5 µm).

[0019] Furthermore, the ultraviolet wavelength is 209–211 nm, preferably 210 nm.

[0020] The chromatographic conditions of this invention, under liquid chromatography with a UV detector, achieve target peak resolution of 1.52 and 2.74, which is greater than 1.5. The chromatographic conditions of this invention can effectively solve the resolution problem.

[0021] Further, the methanol solution containing ammonia is a solution of 55% to 65% methanol containing 15% to 20% concentrated ammonia test solution; preferably, it is a mixed solution with a volume ratio of concentrated ammonia test solution to 60% methanol solution of 5:1.

[0022] Furthermore, the preparation method of the test solution one is as follows: The Bupleurum sample to be tested is sieved, accurately weighed, and sealed with methanol. The weight is then measured. The sample is ultrasonically treated at 15–30°C for 20–30 minutes. The weight loss is replenished with methanol, the sample is shaken well, filtered, and the filtrate is collected. Preferably, the sample is ultrasonically treated at 20°C for 30 minutes.

[0023] This invention employs ultrasonic extraction with methanol to determine the content of free saikosaponins a and d in Bupleurum chinense. The extraction is performed with pure methanol, without water or evaporation, which minimizes the breakage of ester bonds and slows down the hydrolysis rate. Furthermore, the methanol environment is non-acidic, preventing the ether bonds in saikosaponins a and d from breaking and forming diene structures. This ensures the accuracy and stability of the measurement results, and more accurately reflects the content of protosaikosaponins a / d in Bupleurum chinense.

[0024] Furthermore, the mass-to-volume ratio of the Bupleurum chinense sample to the methanol is 0.5g:15ml, or 0.5g:20ml, or 0.5g:25ml, preferably 0.5g:20ml.

[0025] Preferably, the Bupleurum chinense sample to be tested is passed through a No. 3 sieve, accurately weighed to 0.5g, placed in a stoppered conical flask, accurately pipetted 20ml of methanol, sealed tightly, weighed, ultrasonically treated at 20℃ for 30 minutes, the weight loss is replenished with methanol, shaken well, filtered, and the filtrate is collected to obtain the final product.

[0026] Furthermore, the preparation method of the second test solution is as follows: The Bupleurum sample to be tested is sieved, accurately weighed, and then sealed with 55%–65% methanol containing 15%–20% concentrated ammonia solution as solvent. The sample is weighed again. The sample is heated under reflux for 60–70 minutes, cooled, and weighed again. The weight loss is made up with methanol, the sample is shaken well, filtered, and the filtrate is collected.

[0027] This invention employs reflux extraction of total saikosaponins a and d using an ammonia-containing methanol solution. The ammonia ratio and water bath method provide a rapid ester bond breaking environment for the ester-type saikosaponins. The methanol solution simultaneously ensures the solubility of saikosaponins a and d, while the alkaline environment protects the stability of the ether bonds in saikosaponins a and d, maximizing the hydrolysis of the ester-type saikosaponins.

[0028] Furthermore, the mass-to-volume ratio of the Bupleurum chinense sample to be tested and the solvent of 55%–65% methanol containing 15%–20% concentrated ammonia solution is 0.5g:20ml or 0.5g:25ml, preferably 0.5g:25ml.

[0029] Preferably, the Bupleurum chinense sample to be tested is passed through a No. 3 sieve, accurately weighed to 0.5 g, placed in a stoppered conical flask, and accurately added to 25 ml of a mixed solution of 60% methanol solution and concentrated ammonia test solution with a volume ratio of 5:1. The flask is then sealed tightly, weighed, heated under reflux for 1 hour, cooled, and weighed again. The weight loss is made up with methanol, shaken well, filtered, and the filtrate is collected to obtain the final product.

[0030] Furthermore, the reference standards in the mixed reference solution of the test method are saikosaponin a and saikosaponin d; the diluent is methanol.

[0031] In a specific embodiment of the present invention, the method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense includes the following steps: (1) Preparation of mixed reference solution: accurately weigh saikosaponin a and saikosaponin d respectively, add methanol to dissolve and dilute to obtain the solution; (2) Preparation of test solution one: The sample of Bupleurum to be tested is sieved, accurately weighed, added to methanol and sealed, and weighed; at a temperature of 15-30℃, it is ultrasonically treated for 20-30 minutes, the weight loss is made up with methanol, shaken well, filtered, and the filtrate is taken to obtain the test solution. (3) Preparation of test solution II: The sample of Bupleurum to be tested is sieved, weighed accurately, and 55% to 65% methanol containing 15% to 20% ammonia water is added as solvent and sealed and weighed; heated under reflux for 60 to 70 minutes, cooled, weighed again, and the weight lost is made up with methanol, shaken well, filtered, and the filtrate is taken to obtain the test solution. (4) Detection of free saikosaponins: The contents of saikosaponin a and saikosaponin d in Bupleurum were determined by high performance liquid chromatography-ultraviolet detector using test solution I and mixed reference solution respectively; (5) Detection of total saikosaponins: The contents of saikosaponin a and saikosaponin d in Bupleurum were determined by high performance liquid chromatography-ultraviolet detector using test solution II and mixed reference solution respectively; (6) Difference calculation: Calculate the difference between the contents of saikosaponin a and saikosaponin d determined in step (5) and step (4) to obtain the contents of ester-type saikosaponin a and d.

[0032] Compared with the prior art, the present invention has the following significant advantages: This invention employs high-performance liquid chromatography-ultraviolet detection to determine the contents of free saikosaponin a and saikosaponin d in Bupleurum chinense, and to determine the total amount of saikosaponin a and saikosaponin d after the hydrolysis of ester-type saikosaponins upon alkali bond cleavage. The content of ester-type saikosaponins is obtained indirectly by calculating the difference between the two, solving the problem of quantitative determination even in the absence of reference standards, and providing a reference for in-depth research on the chemical composition of Bupleurum chinense. Furthermore, the testing method of this invention is not limited to application in Bupleurum chinense, but is also applicable to other medicinal materials containing triterpenoids, such as ginseng and astragalus membranaceus, providing research ideas for these types of medicinal materials. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is the HPLC chromatogram of the mixed reference solution corresponding to the test sample of this invention; Figure 2 This is the HPLC chromatogram of the test solution of the present invention; Figure 3 This is the HPLC chromatogram of the mixed reference solution corresponding to test sample two of this invention; Figure 4 This is the HPLC chromatogram of the test solution II of this invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] Unless otherwise specified in the embodiments of the present invention, the techniques or conditions described in the literature in this field or the product instructions shall be followed; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through legitimate channels.

[0037] Instruments and materials used in the embodiments and comparative examples of this invention Instruments: Agilent 1260 liquid chromatograph, DTC-27J ultrasonic cleaner.

[0038] Reference standards: Saikosaponin a, purity 94.8%, batch number 110777-201912; Saikosaponin d, purity 96.3%, batch number 110778-201912, both provided by the China National Institutes for Food and Drug Control.

[0039] Reagents and reagents: Acetonitrile was of chromatographic grade, methanol and ammonia were of analytical grade, and water was ultrapure water.

[0040] For details of Bupleurum samples and their origins, please refer to Table 1: Table 1. Information on Bupleurum Samples

[0041] Note: " / " indicates that the place of origin is unknown.

[0042] Example 1. Detection of free saikosaponins 1.1 Chromatographic conditions The chromatographic column was an Agilent ZORBAX SB-C18 (4.6 × 250 mm, 5 μm). Acetonitrile was used as mobile phase A, and water as mobile phase B. Gradient elution was performed: 0–4 min, 36% A; 20–21 min, 40% → 43% A; 38 min, 43% A. The detection wavelength was 210 nm, and the flow rate was 1.0 mL / min. -1 The column temperature was 29 ℃, and the injection volume was 15 μL for the reference standard and 15 μL for the test sample.

[0043] 1.2 Preparation of mixed reference solution Accurately weigh 15.46 mg of saikosaponin a and 12.83 mg of saikosaponin d, place them in the same 50 ml volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and this is the mixed reference solution ①. Accurately pipette 5 ml of mixed reference solution ① into a 50 ml volumetric flask, add methanol to the mark, shake well, and this is the mixed reference solution ②.

[0044] 1.3 Preparation of Test Solution 1 Take 0.5g of the powder (self-numbered O) through a No. 3 sieve and place it in a stoppered conical flask. Accurately pipette 20ml of methanol, seal tightly, weigh, and sonicate at 20℃ for 30 minutes. Make up the weight loss with methanol, shake well, filter, and collect the filtrate to obtain the product.

[0045] 1.4 Methodological Examination 1.4.1 Examination of Linear Relationships Accurately pipette 2, 3, 5, and 10 μl of mixed reference solution ① and 2, 5, 10, and 15 μl of mixed reference solution ② into the liquid chromatograph and measure the peak area. Plot a standard curve with the injection volume as the x-axis and the peak area as the y-axis. Calculate the regression equations for saikosaponins a and d as follows: y = 340.48x + 1.8393 and y = 359.88x + 1.5233, respectively, with correlation coefficients of 0.9999 for both.

[0046] 1.4.2 Precision Experiment Accurately pipette 15 μl of the mixed reference solution ② and inject it into 6 injections consecutively. Measure the peak areas of saikosaponins a and d. The average peak areas were 150.03 and 133.92, respectively, with RSD values ​​of 0.73% and 0.84%, respectively.

[0047] 1.4.3 Stability Test Accurately pipette the same test solution (self-labeled O) and measure the peak area at 1h, 6h, 12h, 18h, 24h, 32h, and 48h after preparation. Within 32h, the average peak areas of saikosaponins a and d were 142.21 and 123.71, respectively, with RSD values ​​of 1.68% and 1.38%. At 48h, the average peak areas of saikosaponins a and d were 143.18 and 124.63, respectively, with RSD values ​​of 2.35% and 2.33%. This indicates that saikosaponins a and d are relatively stable within 32h, but tend to be unstable after 32h.

[0048] 1.4.4 Repeatability Experiment Take the same sample (self-numbered O), and prepare 6 parallel samples according to the preparation method of test solution under section "1.3". The average contents of saikosaponin a and saikosaponin d were 1.0753 mg / g and 0.8834 mg / g, respectively, with RSDs of 0.85% and 0.71%, respectively.

[0049] 1.4.5 Accuracy Experiment The spiking recovery method was used. Three portions of each mixture were accurately pipetted: 5 ml of mixed reference solution ②, 1 ml of mixed reference solution ①, and 2 ml of mixed reference solution ①. These were placed in Erlenmeyer flasks and allowed to evaporate to dryness. Three parallel samples (numbered O) were then added to each sample, weighed accurately, and prepared according to the preparation method for the test solution under section "1.3". The average recoveries were 98.99% and 99.57%, with RSDs of 2.03% and 1.96%, respectively.

[0050] The HPLC chromatogram of the mixed reference solution corresponding to test sample 1 is as follows: Figure 1 As shown, the HPLC chromatogram of test solution one is as follows: Figure 2 As shown.

[0051] 2. Detection of total saikosaponins a and d 2.1 Chromatographic conditions The chromatographic column was an Agilent ZORBAX SB-C18 (4.6 × 250 mm, 5 μm). Acetonitrile was used as mobile phase A, and water as mobile phase B. Gradient elution was performed: 0–30 min, 25% → 64% A; 31–44 min, 100% A. The detection wavelength was 210 nm, the column temperature was 30 °C, and the flow rate was 1.0 mL / min. -1 Injection volume: 10-15 μL for reference standard and 5-15 μL for test sample.

[0052] 2.2 Preparation of mixed reference solution Accurately weigh 10.54 mg of saikosaponin a and 14.53 mg of saikosaponin d, place them in the same 50 ml volumetric flask, dissolve them in methanol and dilute to the mark, shake well, and this is the mixed reference solution ①. Accurately pipette 5 ml of mixed reference solution ① into a 10 ml volumetric flask, dilute to the mark with methanol, shake well, and this is the mixed reference solution ②. Accurately pipette 2 ml of mixed reference solution ② into a 5 ml volumetric flask, dilute to the mark with methanol, shake well, and this is the mixed reference solution ③.

[0053] 2.3 Preparation of Test Solution II Take 0.5g of the powder (self-numbered cq1) through a No. 3 sieve and place it in a stoppered conical flask. Accurately add 25ml of a mixed solution of 60% methanol solution and concentrated ammonia test solution (5:1), seal tightly, weigh, heat under reflux for 1 hour, cool, weigh again, make up the lost weight with methanol, shake well, filter, and collect the filtrate.

[0054] 2.4 Methodological Examination 2.4.1 Examination of Linear Relationships Accurately pipette 15 and 20 μl of mixed reference solution ①, 10 and 20 μl of mixed reference solution ②, and 2, 5, 10, 15, and 20 μl of reference solution ③, and inject them into the liquid chromatograph to determine the peak area. Plot a standard curve with the injection volume as the x-axis and the peak area as the y-axis. Calculate the regression equations for saikosaponins a and d as follows: y = 330.17x - 0.7947 and y = 358.81x - 1.0532, respectively, with correlation coefficients of 0.9999 for both.

[0055] 2.4.2 Precision Experiment Accurately pipette 10 μl of the mixed reference solution ③ and inject it into 6 injections consecutively. Measure the peak areas of saikosaponins a and d. The average peak areas were 132.36 and 201.58, respectively, with RSD values ​​of 0.73% for both.

[0056] 2.4.3 Stability Test Accurately pipette the same test solution II (self-numbered cq1) and measure the peak area at 1h, 12h, 24h, 48h, and 72h after preparation. The average peak areas of saikosaponins a and d are 165.87 and 254.87, respectively, with RSD values ​​of 0.32% and 0.83%, respectively. Saikosaponins a and d are stable within 72 hours, indirectly indicating that the hydrolysis is relatively complete.

[0057] 2.4.4 Repeatability Experiment Take the same sample (self-numbered cq1), and prepare 6 parallel samples according to the preparation method of test solution 2 under "2.3". The average contents of saikosaponin a and saikosaponin d were 2.4686 mg / g and 3.5122 mg / g, respectively, with RSDs of 1.14% and 1.15%, respectively.

[0058] 2.4.5 Accuracy Experiment The spiking recovery method was used. 3 ml of the mixed reference solution ① was accurately pipetted into a conical flask, and 6 parallel portions were prepared. After evaporation, 0.25 g of the sample was added to each portion, accurately weighed, and prepared according to the preparation method of test solution 2 under section "2.3". The average recovery rates were 101.46% and 100.58%, with RSDs of 1.94% and 2.00%, respectively.

[0059] 2.4.6 Conversion Rate Experiment To confirm the hydrolysis rate of the test sample solution preparation method under section "2.3" of this invention, high-resolution mass spectrometry LCQ Orbittrap Velos Pro was used, with the molecular ion peaks of the ester-type saikosaponins (821.4693, 863.4798, 865.4591) as the detection targets, with an allowable error of ±5 ppm. The peak area of ​​the ester-type saikosaponins in the ultrasonic and hydrolyzed samples was determined using the area normalization method. The hydrolysis rate was found to be 96.33%, which is greater than 95%, indicating relatively complete hydrolysis.

[0060] The HPLC chromatogram of the mixed reference solution corresponding to test sample 2 is as follows: Figure 3 As shown, the HPLC chromatogram of test solution two is as follows. Figure 4 As shown.

[0061] 3. Sample Measurement Results The collected samples were prepared according to the test solution preparation method one or two under "1.3" and "2.3". The results of determination and final calculation of ester saikosaponin a and ester saikosaponin d are detailed in Table 2.

[0062] Table 2. Determination results of ester-type saikosaponins a and d (mg / g)

[0063] Comparative Example The 2010 edition of the Chinese Pharmacopoeia added a content determination item for saikosaponins a and d in Bupleurum chinense, and this content determination item has been retained to the current edition of the Pharmacopoeia. Currently, most literature on the determination of saikosaponin content adds or subtracts the extraction solvent concentration based on the Pharmacopoeia method or uses the Pharmacopoeia method.

[0064] Five batches of samples were tested according to the content determination method under the Bupleurum entry in the 2025 edition of the Chinese Pharmacopoeia for comparison. The test solution was prepared as follows: 0.5 g of the powder (numbered JC1, JC2, JC3, JC4, and JC5) was accurately weighed and passed through a No. 4 sieve. The powder was placed in a stoppered conical flask, and 25 ml of methanol solution containing 5% concentrated ammonia was added. The flask was sealed tightly and ultrasonically treated at 30°C (200 W, 40 kHz) for 30 minutes. The solution was filtered, and the container and residue were washed twice with 20 ml of methanol. The washings and filtrate were combined, and the solvent was recovered to dryness. The residue was dissolved in methanol and transferred to a 5 ml volumetric flask. Methanol was added to the mark, and the solution was shaken well and filtered. The filtrate was collected as the test solution.

[0065] The measurement results are detailed in Table 3.

[0066] Table 3 Comparison of results from different methods (mg / g)

[0067] The pharmacopoeia method uses ultrasonic treatment with an ammonia-containing methanol solution, which is equivalent to hydrolyzing a portion of the ester-type saikosaponins. However, based on the ultrasonic conditions, complete hydrolysis is not achieved. Therefore, the measurement results fall between those of the ultrasonic method and the ammonia hydrolysis method of this invention. The results show that the content of protosaikosaponin a is slightly higher than that of saikosaponin d, while the content of saikosaponin a is lower than that of saikosaponin d after hydrolysis. This indicates that the content and conversion rate of ester-type saikosaponin d are both higher than those of ester-type saikosaponin a. The pharmacopoeia method represents a certain stage in the hydrolysis process, and the measurement results are consistent with the conversion pattern.

[0068] Results Discussion This invention employs ultrasonic extraction of free saikosaponins a and d from Bupleurum chinense using methanol at temperatures below 30°C. The extraction is performed with pure methanol, without anhydrous or evaporation steps, which minimize ester bond breakage and thus slow down the hydrolysis rate. Furthermore, the methanol environment provides a non-acidic pH, reducing the likelihood of ether bonds in saikosaponins a and d breaking to form dienes. These conditions ensure the accuracy and stability of the results, providing a more accurate reflection of the original saikosaponin a / d content in Bupleurum chinense.

[0069] In this embodiment of the invention, total saikosaponins a and d are extracted by reflux extraction using a 60% methanol solution containing 1 / 6 ammonia. The ammonia ratio and water bath method provide a rapid ester bond breaking environment for the ester-type saikosaponins. The 60% methanol solution simultaneously ensures the solubility of saikosaponins a and d, while the alkaline environment protects the stability of the ether bonds in saikosaponins a and d. These determination conditions maximize the hydrolysis of the ester-type saponins.

[0070] The chromatographic conditions of this invention, under liquid chromatography with a UV detector, achieve target peak resolution of 1.52 and 2.74, which is greater than 1.5. The chromatographic conditions of this invention can effectively solve the resolution problem.

[0071] The results showed that ester-type saikosaponins were present in Bupleurum chinense at levels comparable to saikosaponins a and d, representing a significant portion of the chemical composition of the plant. This invention, through an indirect method, determined the composition of ester-type saikosaponins, solving the challenge of quantitative determination even without reference standards, and paving the way for further research into the fundamental components of Bupleurum chinense.

[0072] The testing method of this invention is not limited to its application in Bupleurum chinense, but is also applicable to other medicinal materials containing triterpenoids, such as ginseng and astragalus, providing research ideas for this type of medicinal material.

[0073] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense, characterized in that, The sample of Bupleurum chinense to be tested was subjected to ultrasonic treatment with methanol as solvent to obtain test solution one; the sample of Bupleurum chinense to be tested was subjected to extraction by heating and reflux with methanol solution containing ammonia as solvent to obtain test solution two; the contents of saikosaponin a and saikosaponin d in test solution one and test solution two were detected by high performance liquid chromatography-ultraviolet detection, respectively; the difference between the two was calculated to obtain the contents of ester-type saikosaponin a and d.

2. The method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense according to claim 1, characterized in that, The chromatographic conditions for the detection are as follows: Chromatographic column: octadecylsilane-bonded silica gel column; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is water.

3. The method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense according to claim 1 or 2, characterized in that, The elution mode of the chromatographic column is gradient elution. The gradient elution program for detecting the test solution is: 0-4 min, 36% A; 20-21 min, 40% → 43% A; 38 min, 43% A.

4. The method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense according to claim 1 or 2, characterized in that, The elution mode of the chromatographic column is gradient elution. The gradient elution program for detecting the test solution II is: 0-30 min, 25%→64%A; 31-44 min, 100%A.

5. The method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense according to claim 1 or 2, characterized in that, The chromatographic conditions for determining the test solution were as follows: flow rate 1.0 mL / min. -1 The column temperature was 29 ℃, and the injection volume was 5–15 µL.

6. The method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense according to claim 5, characterized in that, The chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 µm. And / or, the ultraviolet wavelength is 209–211 nm, preferably 210 nm.

7. The method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense according to claim 1 or 2, characterized in that, The methanol solution containing ammonia is a solution of 55% to 65% methanol containing 15% to 20% concentrated ammonia test solution.

8. The method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense according to claim 1, 2, 6, or 7, characterized in that, The preparation method of the test solution is as follows: sieve the Bupleurum chinense sample to be tested, weigh it accurately, add methanol and seal it, weigh it again; sonicate it at 15-30℃ for 20-30 minutes, replenish the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution; preferably, sonicate it for 30 minutes. And / or, the mass-to-volume ratio of the Bupleurum chinense sample to the methanol is 0.5g:15ml, or 0.5g:20ml, or 0.5g:25ml, preferably 0.5g:20ml.

9. The method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense according to claim 7, characterized in that, The preparation method of the second test solution is as follows: The Bupleurum sample to be tested is sieved, accurately weighed, and then sealed with 55%–65% methanol containing 15%–20% concentrated ammonia solution as solvent. The sample is then weighed again. The sample is heated under reflux for 60–70 minutes, cooled, and weighed again. The weight loss is replenished with methanol, the sample is shaken well, filtered, and the filtrate is collected. And / or, the mass-to-volume ratio of the Bupleurum chinense sample to be tested to the solvent of 55%-65% methanol containing 15%-20% ammonia is 0.5g:20ml or 0.5g:25ml, preferably 0.5g:25ml.

10. The method for determining the content of ester-type saikosaponins a and d in Bupleurum chinense according to claim 1, 2, 6, or 7, characterized in that, The reference standards for the mixed reference solution of the assay method are saikosaponin a and saikosaponin d; the diluent is methanol.

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