Method for determining contents of multiple components of radix bupleuri in radix bupleuri and rhizoma cyperi medicine pair

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for determining the content of multiple components of radix bupleuri in a radix bupleuri and rhizoma cyperi medicine pair. According to the method, a high performance liquid chromatography method is adopted, and chromatographic conditions for detection are as follows: a chromatographic column adopts SUPERIOREX ODS (4.6 * 150 mm, 5 [mu] m); mobile phase purified water (A)-acetonitrile (B) is used for gradient elution (0-30 min, 30%-55% of B); the flow rate is 1.0 mL / min, and the ultraviolet detection wavelength is 210 nm; the column temperature is 30 DEG C; the sample size is 20 mu L; obtaining content information of the medicine pair according to a detection result; the medicine pair combination is radix bupleuri and rhizoma cyperi, and reference substances are saikoside a, saikoside c and saikoside d. According to the method disclosed by the invention, saikoside a, saikoside c and saikoside d in the radix bupleuri and rhizoma cyperi medicine pair can be rapidly and simultaneously separated, the separation effect is good, the method can be used for measuring the content of the components, and a theoretical basis is provided for improving the quality control level of a traditional Chinese medicine preparation containing the radix bupleuri and rhizoma cyperi medicine pair, new medicine development and clinical reasonable medication.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for determining the content of multiple components of bupleurum in a bupleurum and cyperus rotundus drug pair. Background Art

[0002] Drug pair, also known as drug pair, is a relatively fixed combination of two drugs in TCM clinical practice. In formulating and selecting drugs, drug pair compatibility follows the theory of TCM properties (mainly four qi, five flavors, meridians, ascending and descending, floating and sinking, and toxic and non-toxic) and the theory of seven emotions and harmony (mutual support, mutual fear and mutual killing), etc., to achieve the purpose of syndrome differentiation and treatment, improve efficacy, increase drug efficacy, and alleviate toxicity through compatibility. As the smallest unit of TCM compatibility, drug pair is closely linked to TCM and prescriptions, reflecting the most basic compatibility theory and the core connotation of prescriptions; as the key drug combination for the clinical treatment of diseases, syndromes and symptoms, it also reflects the pertinence and flexibility of TCM diagnosis and treatment, and is the main form of TCM clinical medication and the unique advantage of diagnosis and treatment of diseases. Studying the compatibility of drug pairs is helpful to analyze the compatibility mechanism of prescriptions and their mechanism of action in treating diseases. Studying the compatibility rules of drug pairs can be used as one of the important entry points for studying the compatibility rules of compound prescriptions, thereby revealing their compatibility rules and scientific connotations, deeply exploring and improving the compatibility theory of Chinese medicine compound prescriptions, and providing clues and basis for the study of the compatibility mechanism of compound prescriptions.

[0003] The drug pair of Bupleurum and Cyperus comes from the monarch-minister combination of Bupleurum Soothing Liver Powder in Volume 56 of Jingyue Complete Works of the Ming Dynasty. Bupleurum is the monarch, regulating liver qi and dispersing depression; Cyperus is the minister, specifically entering the liver meridian, soothing the liver and relieving depression, and can also regulate qi and relieve pain. It is a commonly used drug pair in the clinical treatment of liver depression and qi stagnation in traditional Chinese medicine. In clinical treatment of traditional Chinese medicine, Bupleurum and Cyperus are often used in combination as liver-soothing and qi-regulating drugs. Studies have shown that in the 649 prescriptions counted, the frequency of the combination of Bupleurum and Cyperus appeared as many as 196 times. After studying the etiology, pathogenesis and medication rules of traditional Chinese medicine in the treatment of post-stroke depression, it was found that among the 181 Chinese medicines in 154 prescriptions, Bupleurum and Cyperus were frequently used qi-regulating drugs, with a frequency of ≥30 times. In clinical prescriptions, there are many ratios of Bupleurum and Cyperus, such as Zishen Tong'er Decoction (Bupleurum: Cyperus = 1:1), Kaiyu Shunqi Pills (Bupleurum: Cyperus = 2:1), Tiaojing Paste (Bupleurum: Cyperus = 1:2), etc. The combination of Bupleurum and Cyperus has the effect of soothing the liver and regulating qi, and can be used clinically to treat infertility, depression, epilepsy, Alzheimer's disease, liver depression, gastroesophageal reflux disease and other diseases.

[0004] The combination of mutually beneficial and mutually dependent drugs has a synergistic effect, which can increase the dissolution of the effective ingredients of the two drugs and reduce the dissolution of the toxic ingredients, thereby exerting a stronger efficacy and improving the therapeutic efficacy to achieve the purpose of "synergy". This is the principle of application of the preferred drug pair for combination. The determination of the content of Chinese medicine ingredients by high-performance liquid chromatography can control the quality of Chinese medicinal materials and Chinese medicine products, explain the interaction after the combination of Chinese medicines, identify the authenticity and quality of medicinal materials, and can also be used for quality control of the production process of Chinese medicine. It is currently one of the effective means of quality control of Chinese medicine and its preparations.

[0005] The applicant conducted a chemical composition analysis on the drug pair of Bupleurum and Cyperus rotundus, and established a high-performance liquid chromatography method to simultaneously determine the contents of saikosaponin a, saikosaponin c, and saikosaponin d in the extract of the drug pair, providing a theoretical basis for the quality control of compound preparations containing this drug pair and laying a foundation for the clinical application research of the compatibility of this drug pair. Summary of the invention

[0006] Based on this, the present invention provides a method for determining the content of multiple components of Bupleurum in a Bupleurum and Cyperus drug pair. In this method, the extract of the drug pair is used as the test solution, and the standard solution of the target active ingredient to be quantitatively studied is diluted into a series of standard working solutions of concentration, and then detected by a detector to determine the standard calibration curve of the target active ingredient, thereby determining the content of the target active ingredient; this method can reproduce the detection environment of different active ingredients in the drug pair when using different detectors to the greatest extent, thereby deducting the interference caused by co-eluted endogenous substances, effectively eliminating the influence of matrix effect, and making the content determination result more accurate. The method is a method for determining the content of active ingredients in the drug pair that conforms to the characteristics of traditional Chinese medicine.

[0007] A method for determining the content of multiple components of bupleurum in a bupleurum and cyperus rotundus drug pair, the method comprising the following steps: using high performance liquid chromatography; using Shiseido SUPERIOREX ODS (4.6×150 mm, 5 μm) as a chromatographic column; gradient elution of purified water (A)-acetonitrile (B) as a mobile phase (0-30 min, 30%-55% B); a flow rate of 1.0 mL / min, an ultraviolet detection wavelength of 210 nm; a column temperature of 30°C; an injection volume of 20 μL; drawing a standard curve based on the detection results, and calculating and obtaining the content information of the traditional Chinese medicine pair; wherein the traditional Chinese medicine pair is a combination of bupleurum and cyperus rotundus, and the reference substances are saikosaponin a, saikosaponin c, and saikosaponin d.

[0008] Furthermore, accurately measure an appropriate amount of equal drug pair powder, add 70% ethanol at a solid-liquid ratio of 1:10, let stand at room temperature for 15 min after weighing, ultrasonicate at 50 ℃ for 45 min, cool, weigh again and make up the weight, filter through a shaken membrane, take 2 ml of the filtrate, and dilute to volume with 70% ethanol in a 10 mL volumetric flask to obtain the alcohol extract of the drug pair test sample.

[0009] Furthermore, 70% ethanol was used to dissolve and prepare the stock solutions of saikosaponin a (0.5 mg / mL), saikosaponin c (0.698 mg / mL), and saikosaponin d (0.529 mg / mL) as reference standard solutions.

[0010] Furthermore, the mother solution of the standard solution of the active ingredient to be tested was diluted with 70% ethanol to obtain a series of concentration standard working solutions.

[0011] Inject into high performance liquid chromatography instrument and detect with ultraviolet detector. Each standard curve contains at least 6 concentration points, and each concentration is measured 3 times to make a standard calibration curve.

[0012] Furthermore, the determination of the contents of the multiple components is to simultaneously determine the contents of saikosaponin a, saikosaponin c and saikosaponin d in the drug pair of Bupleurum and Cyperus rotundus.

[0013] Furthermore, when the flow rate was 0.8 ml / min and the column temperature was 35°C, the peak at the retention time of 17 minutes was saikosaponin a, the peak at the retention time of 12 minutes was saikosaponin c, and the peak at the retention time of 25 minutes was saikosaponin .

[0014] According to another aspect of the present invention, a quality control method for a substance containing saikosaponin a, saikosaponin c and saikosaponin d is provided, and the quality control method comprises the following steps: (1) establishing a high performance liquid phase content determination method; (2) taking a sample test solution and testing it according to the chromatographic conditions in the above-mentioned construction method to obtain a fingerprint of the sample to be tested; (3) calculating the content according to a regression equation, and a product that meets the requirements is a qualified product, and a product that does not meet the requirements is an unqualified product.

[0015] According to another aspect of the present invention, there is provided a use of the above-mentioned determination method, the above-mentioned construction method or the above-mentioned quality control method in quality detection and / or quality evaluation and / or quality control of the Chinese herbal medicine pair of Bupleurum and Cyperus rotundus.

[0016] According to another aspect of the present invention, there is provided a use of the above-mentioned determination method, the above-mentioned construction method or the above-mentioned quality control method in the study of the compatibility ratio of the Chinese medicine pair of Bupleurum and Cyperus rotundus and / or clinical treatment.

[0017] The invention provides a method for determining the contents of multiple components of bupleurum in a bupleurum and cyperus herb pair. The method has simple conditions and short analysis time, solves the problems of difficulty in separating fingerprint characteristic peaks and interference of impurity peaks, ensures the chemical composition stability and use safety of a reference sample, and can quickly and simultaneously separate saikosaponin a, saikosaponin c and saikosaponin d in the bupleurum and cyperus herb pair, with good separation effect.

[0018] The method of the invention has good stability, high precision, good reproducibility, convenience and is easy to master, and can be used for determining the contents of saikosaponin a, saikosaponin c and saikosaponin d in the Chinese medicine pair of bupleurum and cyperus, providing a theoretical and experimental basis for the research on the Chinese medicine pair of bupleurum and cyperus, so that the Chinese medicine pair can better serve human life and health. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following are illustrations of the drawings required for use in the description of the embodiments. The drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0020] Figure 1 HPLC chromatogram of mixed reference substances (1, saikosaponin c; 2, saikosaponin a; 3, saikosaponin d).

[0021] Figure 2 HPLC chromatogram of the extracts of Bupleurum and Cyperus rotundus (1, saikosaponin c; 2, saikosaponin a; 3, saikosaponin d). DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below in conjunction with specific implementation methods so that the present invention can be understood in more detail, but the scope of protection required is not limited to the description. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0023] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0024] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0025] Example

[0026] 1. Instruments and Materials

[0027] 1.1 Instrument

[0028] Shimadzu LC-20A high performance liquid chromatograph (Shimadzu Corporation, Japan); TYHD-360 ultrasonic cleaning machine (Tianyou Hengda Technology Co., Ltd.); PRACTUM224-1CN electronic analytical balance (Sartorius, Germany); WP-UP-III-20 laboratory-specific ultrapure water machine (Sichuan Wottel Technology Development Co., Ltd.).

[0029] 1.2 Reagents and test drugs

[0030] The reference substances were saikosaponin a (batch number: Yz050723, purity ≥98%), saikosaponin c (batch number: 03008-202205, purity ≥98%), and saikosaponin d (batch number: Yz082923, purity ≥98%), all of which were collected from Nanjing Yuanzhi Biotechnology Co., Ltd.; the medicinal materials were bupleurum (batch number: 80802681, originating in Hubei) and cyperus rotundus (batch number: 40301301, originating in Guangdong), all of which were purchased from Kangmei Pharmaceutical Pharmacy, and after testing, they all met the requirements of the 2020 edition of the Chinese Pharmacopoeia for medicinal bupleurum and cyperus rotundus; the reagents were acetonitrile chromatographic grade (Merck, Germany), ethanol analytical grade, and purified water.

[0031] 2 Methods and Results

[0032] 2.1 Sample processing

[0033] 2.1.1 Preparation of reference solution

[0034] Prepare the stock solutions of saikosaponin a (0.5 mg / mL), saikosaponin c (0.698 mg / mL), and saikosaponin d (0.529 mg / mL) reference standard solutions by dissolving in 70% ethanol.

[0035] 2.1.2 Preparation of sample solution

[0036] Crush the Radix Bupleuri and Cyperus rotundus and sieve to obtain fine powder. Accurately weigh 0.1 g of the powder of the Radix Bupleuri and Cyperus rotundus, add 70% ethanol solution at a solid-liquid ratio of 1:10, let stand at room temperature for 15 min after weighing, ultrasonicate at 50 °C for 45 min, cool, weigh again and make up the weight, shake well and filter through a 0.45 μm microporous filter membrane, take 2 mL of the filtrate, and dilute to volume with 70% ethanol in a 10 mL volumetric flask to obtain the alcohol extract of the Radix Bupleuri and Cyperus rotundus test sample.

[0037] 2.2 Chromatographic conditions

[0038] The chromatographic column used was Shiseido SUPERIOREX ODS (4.6×150 mm, 5 μm); the mobile phase was purified water (A)-acetonitrile (B) gradient elution (0-30 min, 30%-55% B); the flow rate was 1.0 mL / min, the UV detection wavelength was 210 nm; the column temperature was 30°C; and the injection volume was 20 μL.

[0039] 2.3 Preparation of standard curve

[0040] The prepared reference solution saikosaponin a was diluted with 70% ethanol to prepare standard solutions with concentrations of 0.05, 0.025, 0.01, 0.005, and 0.002 mg / mL in sequence; the saikosaponin c reference solution was diluted to prepare standard solutions with concentrations of 0.01745, 0.008725, 0.00349, 0.001745, and 0.000698 mg / mL; the saikosaponin d reference solution was diluted to prepare standard solutions with concentrations of 0.0529, 0.02645, 0.01058, 0.00529, and 0.002116 mg / mL.

[0041] The above-mentioned reference substance solutions were accurately pipetted and injected into HPLC to obtain the peak areas and retention times of saikosaponins a, c, and d. The vertical axis was the peak area (Y), the horizontal axis was the reference substance mass concentration (X), and the standard curves and correlation coefficients (r) of each reference substance were obtained by linear regression. The standard curves of saikosaponins a were f(x)=0.00012754x-0.196818 (r=0.9993), saikosaponins c were f(x)=0.000174721x+0.0986522 (r=0.9999), and saikosaponins d were f(x)=0.000122688x-0.135875 (r=0.9999).

[0042] 2.4 Determination of content of each component

[0043] Accurately weigh the powder of Bupleurum and Cyperus to prepare the sample solution, inject it into the high performance liquid chromatography for determination, and calculate the contents of saikosaponins a, c, and d in the alcohol extract of Bupleurum and Cyperus.

[0044] 2.5 Methodological Review

[0045] 2.5.1 System suitability test

[0046] Under the above chromatographic conditions, the alcohol extract of the Bupleurum and Cyperus herb pair prepared in a ratio of 1:1 was injected into the chromatograph and the chromatogram was recorded ( Figure 1 , Figure 2 ), the system adaptability was calculated according to the chromatographic parameters. As a result, saikosaponin a, saikosaponin c, and saikosaponin d could all achieve baseline separation, with a resolution greater than 1.5 and a theoretical plate number greater than 5000.

[0047] 2.5.2 Precision test

[0048] The same reference solution prepared in the previous step was accurately drawn and injected into HPLC. The sample was injected 6 times continuously under the determined chromatographic conditions, and the peak areas of the three components were recorded and the RSD values ​​were calculated. The RSD values ​​of the peak areas of saikosaponin a, c, and d were 0.11%, 0.35%, and 0.15%, respectively (n=6), indicating that the instrument had good precision.

[0049] 2.5.3 Repeatability test

[0050] Take the powder of Bupleurum and Cyperus rotundus, prepare the sample solution (6 parallels in total) according to the determined extraction method, and then inject and measure the sample under the above determined chromatographic conditions, record the data of each component, and calculate the average content and RSD value of each component. As a result, the average content of saikosaponin a, c, and d in the sample solution was 1.72, 0.72, and 2.23 mg / g, respectively, and the calculated RSD values ​​were 1.06%, 1.74%, and 1.78% (n=6), all less than 2%, indicating that the detection method has good repeatability.

[0051] 2.5.4 Stability test

[0052] The prepared test solution of the same Bupleurum and Cyperus rotundus drug pair (1:1) was accurately aspirated and placed for 0, 2, 4, 8, 16, and 24 hours for sampling, measurement, recording, and calculation. The results showed that the RSD of the peak area of ​​each component was 1.57%, 1.17%, and 0.83% (n=6), respectively, all less than 2.0%, indicating that the sample alcohol extract at room temperature was stable within 24 hours.

[0053] 2.5.5 Sample recovery test

[0054] Six portions of ethanol extracts of Bupleurum and Cyperus rotundus (1:1) with measured component contents were accurately measured, and the reference solution prepared in advance was accurately added respectively. The extraction was prepared according to the determined extraction method, and the extract was sampled and measured to calculate the content and recovery rate of each component. The results showed that the average recovery rates of saikosaponin a, c, and d were 94.84%, 97.20%, and 103.16%, respectively, and the RSDs were 1.29%, 1.39%, and 1.83%, respectively (n=6).

[0055] 3 Discussion and Conclusion

[0056] Bupleurum is a commonly used drug in clinical practice. It has complex chemical composition and rich medicinal value. Its extract has a high content of saikosaponin and also has a variety of pharmacological properties. As early as in the "Shennong Bencao Jing", it was pointed out that Bupleurum can treat the heart and abdomen, dispel cold evil, remove qi stagnation, and resolve accumulation. Studies have shown that saikosaponin can inhibit the growth of liver, pancreatic, and kidney tumor cells, inhibit the frequency and duration of epileptic seizures, protect neurons, reduce immune liver fibrosis, protect the liver, and have anti-inflammatory and antioxidant effects. In clinical practice, Bupleurum and Cyperus can be used to treat infertility in combination; "Yue Ke Xin De Ji" says "there is a nucleus in the breast, ... liver qi is not dredged, and swelling and hardness are formed", Bupleurum and Cyperus can regulate endocrine, inhibit and eliminate lumps to treat liver depression; it can also improve spleen and stomach qi stagnation, relieve stomach qi and digestion, and be used for chronic gastritis.

[0057] By analyzing the prescriptions containing the Bupleurum and Cyperus rotundus drug pair in the "Chinese Medicine Prescription Database" and the 2020 edition of the "Chinese Pharmacopoeia", it is found that the Bupleurum and Cyperus rotundus drug pair is the most commonly used in a 1:1 equal ratio, and its efficacy and main indications are related to its compatibility dose and compatibility ratio. When the dose of Cyperus rotundus in the Bupleurum and Cyperus rotundus drug pair is larger than that of Bupleurum, it is mostly used for gynecological diseases, such as irregular menstruation, abdominal pain during menstruation, metrorrhagia and leukorrhea. With the increase in the dosage of Bupleurum, the heat-clearing effect of the Bupleurum and Cyperus rotundus drug pair gradually becomes prominent. Bupleurum and Cyperus rotundus can be used together to achieve synergistic effects, thereby playing the role of soothing the liver and relieving depression and clearing away heat from the surface. In traditional Chinese medicine prescriptions, the size of the drug dosage and the compatibility ratio play a decisive role in the efficacy and indications of the prescription, and are important links that must be considered in clinical prescription and medication. Therefore, studying the compatibility relationship of drug pairs is very important for improving the clinical efficacy and rational use of traditional Chinese medicine. Therefore, this experiment established a method for determining the contents of three components, namely, saikosaponin a, saikosaponin c, and saikosaponin d, in the alcohol extract of the Bupleurum and Cyperus rotundus drug pair, conducted corresponding quality control research, and provided a theoretical basis for the clinical treatment direction of different compatibility ratios of the drug pair.

[0058] In the early stage of the experiment, the solvent, solvent concentration, solvent volume, extraction time and other factors of the test sample were investigated based on the content of the component to be determined, and the corresponding extraction method was determined; the chromatographic method was used to compare the separation of the mobile phases of acetonitrile-water and methanol-water, flow rates of 0.8, 1.0, 1.2 mL / min, column temperatures of 25, 30 and 35°C, and injection volumes of 10 and 20 μL. The results showed that the best separation effect was achieved under the conditions of mobile phase acetonitrile-water, gradient elution, flow rate of 1.0 mL / min, column temperature of 30°C, and injection volume of 20 μL. In this experiment, a standard curve was established to determine the content of the components, and various methodological investigations were carried out, and the results met the methodological requirements.

[0059] The invention can be used to determine the content of substances containing saikosaponin a, saikosaponin c, and saikosaponin d. The method can reproduce the detection environment of the target active ingredient in the detector to the maximum extent, and the results of the determination of the active ingredient content are more accurate and the separation effect is good. It is an effective ingredient content determination method that conforms to the characteristics of traditional Chinese medicine, provides an experimental basis for improving the quality control level of the drug pair of Bupleurum and Cyperus, and also lays the foundation for the development of new drugs containing this drug pair and the rational use of drugs in clinical practice.

[0060] The first innovation of this invention is the addition of a method for determining the contents of saikosaponin a, saikosaponin c and saikosaponin d, which provides a scientific, reasonable and feasible quality control method for the drug pair of bupleurum and cyperus rotundus, and provides sufficient basis for the harmonizing and balancing effect, safety and effectiveness of the Chinese medicine; the second innovation is that this method is based on the principle that the ultraviolet absorption conditions of saikosaponin a, saikosaponin c and saikosaponin d are different, and adopts different detection wavelengths while keeping other chromatographic conditions the same, which effectively shortens the detection time, saves time, raw materials, reagents and energy consumption costs, and plays a significant beneficial role in reducing costs, increasing efficiency and improving quality.

[0061] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The above embodiments are only representative examples of the present invention. At the same time, the technical scheme of the present invention is not limited to the above embodiments. Changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention should be considered as the protection scope of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for determining the content of multiple components of Radix Bupleuri in the Radix Cyperi herb pair, characterized in that: The high performance liquid chromatography method was used for determination; method steps: the test solution and the reference solution of the Chinese herbal medicine pair were taken for detection; the chromatographic conditions for the detection were: the chromatographic column was Shiseido SUPERIOREX ODS (4.6×150 mm, 5 μm); the mobile phase was purified water (A)-acetonitrile (B), gradient elution (0-30 min, 30%-55% B); the flow rate was 1.0 mL / min, the ultraviolet detection wavelength was 210 nm; the column temperature was 30 ℃; the injection volume was 20 μL; according to the test results, a standard curve was drawn to obtain the content information of the Chinese herbal medicine pair; the Chinese herbal medicine pair was composed of Bupleurum and Cyperus, and the reference substances were saikosaponin a, saikosaponin c, and saikosaponin d.

2. The method for determining the content of multiple components of Radix Bupleuri in the Radix Cyperi drug pair according to claim 1, characterized in that: The high performance liquid chromatography method uses a high performance liquid chromatography system equipped with an ultraviolet detector and a chromatographic column using Shiseido SUPERIOREX ODS (4.6×150 mm, 5 μm).

3. The method for determining the content of multiple components of Radix Bupleuri in the Radix Cyperi drug pair according to claim 1, characterized in that: The reference solution is prepared by the following steps: using 70% ethanol to prepare saikosaponin a solution with a concentration of 0.5 mg / mL to obtain a saikosaponin a reference solution; using 70% ethanol to prepare saikosaponin c with a concentration of 0.698 mg / mL to obtain a saikosaponin c reference solution; using 70% ethanol to prepare saikosaponin d with a concentration of 0.529 mg / mL to obtain a saikosaponin d reference solution.

4. The method for determining the content of multiple components of Radix Bupleuri in the Radix Cyperi drug pair according to claim 1, characterized in that: The test solution is prepared by the following steps: crushing and sieving the medicinal materials of Bupleurum and Cyperus, accurately measuring an appropriate amount of powder of the same proportion of Bupleurum and Cyperus, adding 70% ethanol at a solid-liquid ratio of 1:10, standing at room temperature for 15 min after weighing, ultrasonicating at 50°C for 45 min, cooling, weighing again and making up the weight, shaking and filtering through a membrane, taking 2 mL of the filtrate, and diluting to 70% ethanol in a 10 mL volumetric flask to obtain the test sample alcohol extract.

5. The method for determining the content of multiple components of Radix Bupleuri in the Radix Bupleuri and Rhizoma Cyperi drug pair according to claim 1, characterized in that: The reference substance used for drawing the standard curve was prepared by the following steps: the prepared reference substance solution saikosaponin a was diluted with 70% ethanol to concentrations of 0.05, 0.025, 0.01, 0.005, and 0.002 mg / mL standard solutions in sequence; the saikosaponin c reference substance solution was diluted to concentrations of 0.01745, 0.008725, 0.00349, 0.001745, and 0.000698 mg / mL standard solutions; the saikosaponin d reference substance solution was diluted to concentrations of 0.0529, 0.02645, 0.01058, 0.00529, and 0.002116 mg / mL standard solutions.

6. The method for determining the content of multiple components of Radix Bupleuri in the Radix Cyperi drug pair according to claim 1, characterized in that: The series of standard working solutions may have 1 to 6 concentrations, or more than 6 concentrations, and should be able to ensure that the standard calibration curve of the target analyte is accurate and reliable.

7. The method for determining the content of multiple components of Radix Bupleuri in the Radix Cyperi drug pair according to claim 1, characterized in that: The component content determination is to simultaneously determine the contents of saikosaponin a, saikosaponin c, and saikosaponin d.

8. The method for determining the content of multiple components of Radix Bupleuri in the Radix Cyperi drug pair according to claim 1, characterized in that: Method Determination steps: Prepare test solution and reference solution, inject them into high performance liquid chromatography, detect under chromatographic conditions, record retention time and peak area respectively, and calculate the content of the component to be measured by standard curve method.

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