Quantitative analysis method for effective components of common goldenrop medicinal material

Abstract

The invention belongs to the technical field of quality detection of traditional Chinese medicinal materials, and particularly relates to a quantitative analysis method for effective components of common goldenrop medicinal materials. The method comprises the following steps: a, preparation of a reference substance solution: precisely weighing neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3, 5-O-dicaffeoylquinic acid, quercitrin and 4, 5-O-dicaffeoylquinic acid reference substances, and adding methanol to prepare a mixed reference substance solution; b, preparation of a test solution: crushing a common goldenrop herb, adding a methanol solution for extraction, and filtering to obtain the test solution; and c, determination: respectively and precisely sucking the reference substance solution and the test solution, injecting into an ultra-high performance liquid chromatograph, determining, and calculating the content by adopting an external standard method. The method can simultaneously detect and analyze the contents of seven chemical components in the common goldenrop herb, has good linearity, repeatability, stability and recovery rate, and can effectively control the quality of the common goldenrop herb.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicinal materials, and particularly relates to a quantitative analysis method for effective components of Radix Bupleuri medicinal materials. Background Art

[0002] Bupleurum is a traditional Chinese medicinal material with a history of more than 2,000 years of application. It was first recorded in the Shennong Bencao Jing and listed as a top-grade medicine. It has the effects of dispersing fever, relieving liver depression, and raising yang energy. The plants of the genus Bupleurum in the Apiaceae family are mainly distributed in the temperate and subtropical regions of the Northern Hemisphere. There are about 200 genera and 2,500 species. There are 36 species in China, of which 25 are used as medicine. The 2020 edition of the Chinese Pharmacopoeia only includes two origins: Bupleurum chinense DC. and Bupleurumscorzonerifolium Willd. Among the existing drug standards in my country, 590 formula preparations use Bupleurum as raw materials. The market demand has increased year by year, while the wild resources have decreased significantly, resulting in a shortage of Bupleurum, and its resources are far from meeting market demand.

[0003] At present, most other plants of the genus Bupleurum, such as Bupleurum chinense, Bupleurum equisetum, and Bupleurum malkangense, are also used as Bupleurum in their production areas. These plants have a long history of medicinal use and rich resource reserves in their production areas, which greatly alleviates the supply demand of Bupleurum chinense and Bupleurum serrata. Existing research results show that other medicinal plants of the genus Bupleurum differ from Bupleurum chinense in terms of medicinal parts, types and contents of active ingredients. Therefore, studying the material basis of local traditional medicinal materials, exploring and promoting new high-quality resources, and establishing effective quality control methods can better meet the market demand for medicines and ensure the rational development and application of medicinal materials.

[0004] The medicinal material "Bupleurum hamiltonii" is included in the Yunnan Province Traditional Chinese Medicine Standard under "Dian Bupleurum" (Yun YNZYC-0343-2010). It is the dried whole herb of Bupleurum hamiltonii N.P.Balakr., a plant of the Umbelliferae family. It is distributed in Yunnan, Guizhou, Sichuan, Hubei, Guangxi and other provinces. Its above-ground parts are abundant and easy to collect, and have good utilization prospects.

[0005] The existing standards for the quality control of Radix Bupleuri only include simple thin layer identification, moisture and ash inspection, and extracts, but no content determination method, making it difficult to effectively control its quality. At present, there is very little research data on the chemical composition and quantitative control methods of Radix Bupleuri. Experimental studies have found that Radix Bupleuri mainly contains a large number of organic acids and flavonoids, which have multiple pharmacological activities such as antipyretic and analgesic, anti-inflammatory, antiviral, and immune-enhancing. Therefore, it is very necessary to develop a method that can quantitatively detect the content of multiple active ingredients in Radix Bupleuri for quality control. Summary of the invention

[0006] The technical problem to be solved by the present invention is to fill the gap in the prior art and provide a method for quantitative analysis of the effective ingredients of the medicinal material of Bupleurum chinense. The method is used to detect the content of the effective ingredients in the medicinal material of Bupleurum chinense with good precision, good repeatability and good stability, and can effectively control the quality of the medicinal material of Bupleurum chinense.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is to provide a method for quantitative analysis of the effective components of Radix Bupleuri medicinal materials, the method comprising the following steps:

[0008] a. Preparation of reference solution: accurately weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid reference substances, and add methanol to prepare a mixed reference solution;

[0009] b. Preparation of test solution: grind Radix Bupleuri, extract with methanol solution, and filter to obtain the solution;

[0010] c. Determination: Accurately aspirate the reference solution and the test solution respectively, inject them into the ultra-high performance liquid chromatography, determine, and calculate the content using the external standard method.

[0011] Furthermore, in step a, the preparation method of the reference solution is: accurately weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid reference substances, and add methanol to make a mixed reference solution containing 0.01 mg of neochlorogenic acid, 0.1 mg of chlorogenic acid, 0.01 mg of cryptochlorogenic acid, 0.1 mg of rutin, 0.01 mg of 3,5-O-dicaffeoylquinic acid, 0.1 mg of quercetin, and 0.01 mg of 4,5-O-dicaffeoylquinic acid per 1 mL.

[0012] Furthermore, in step b, the preparation method of the test solution is: grind the Bupleurum chinense medicinal material, pass it through a No. 3 sieve, take 0.5-2g and place it in a stoppered conical flask, add a methanol solution with a volume concentration of 30% to 100%, the solid-liquid ratio of the Bupleurum chinense medicinal material to the methanol solution is 1:25-100, weigh, ultrasonically extract for 0.5-2h, cool, and then add a methanol solution with a volume concentration of 30% to 100% to make up the weight, shake well, and filter to obtain.

[0013] Preferably, in step b, the preparation method of the test solution is: grind the Radix Bupleuri medicinal material, pass it through a No. 3 sieve, take 1 g, accurately weigh it, put it in a stoppered conical flask, add a methanol solution with a volume concentration of 70%, the solid-liquid ratio of the Radix Bupleuri medicinal material to the methanol solution is 1:50, weigh it, ultrasonically extract it for 1 hour, cool it, and then add a methanol solution with a volume concentration of 70% to make up the weight, shake it well, and filter it to obtain it.

[0014] Furthermore, in step c, the chromatographic conditions of the determination are: the chromatographic column is filled with octadecylsilane bonded silica gel; acetonitrile and 5-50mmol / L potassium dihydrogen phosphate solution are used as mobile phases for gradient elution; the detection wavelength is 320-330nm; the column temperature is 25-40°C; the flow rate is 0.2-0.5mL / min; and the theoretical plate number calculated based on the quercetin peak should be no less than 150,000.

[0015] Furthermore, in step c, the gradient elution procedure of the mobile phase is: 50 mmol / L potassium dihydrogen phosphate solution is used as mobile phase A, and acetonitrile is used as mobile phase B;

[0016] From 0 to 30 min, the volume fraction of mobile phase A decreased from 95% to 80%, and the volume fraction of mobile phase B increased from 5% to 20%;

[0017] From 30 to 31 min, the volume fraction of mobile phase A decreased from 80% to 40%, and the volume fraction of mobile phase B increased from 20% to 60%;

[0018] 31-35 min, maintain the volume fraction of mobile phase A at 40%, and the volume fraction of mobile phase B at 60%;

[0019] From 35 to 36 min, the volume fraction of mobile phase A increased from 40% to 95%, and the volume fraction of mobile phase B decreased from 60% to 5%;

[0020] From 36 to 45 min, the volume fraction of mobile phase A was maintained at 95%, and the volume fraction of mobile phase B was maintained at 5%.

[0021] Furthermore, the pH value of the potassium dihydrogen phosphate solution is 2.0-7.0.

[0022] Preferably, the pH value of the potassium dihydrogen phosphate solution is 3.0.

[0023] Preferably, the detection wavelength is 325 nm; the column temperature is 25° C.; and the flow rate is 0.3 mL / min.

[0024] Preferably, the chromatographic column is a Shimadzu Shim-pack HD-C18-80 column, 2.1×100 mm, 1.9 μm.

[0025] Furthermore, in step c, the external standard method is used to calculate the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid in each 1 g of Bupleurum chinense medicinal material.

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides a quantitative analysis method for the effective components of Radix Bupleuri medicinal materials by optimizing the chromatographic conditions and the preparation parameters of the test solution, which can simultaneously detect 7 effective components such as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin and 4,5-O-dicaffeoylquinic acid, and realizes the detection of multi-component content of Radix Bupleuri medicinal materials. Such components have multiple pharmacological activities and are related to the efficacy of the medicinal materials. The detection operation is simple and the results are stable and repeatable, so that it can be used to effectively control the quality control of Radix Bupleuri medicinal materials. The method of the present invention has good linearity, repeatability, stability and recovery rate, can well control the quality of Radix Bupleuri medicinal materials, provides a rapid and comprehensive detection means for the quality control of Radix Bupleuri medicinal materials, and provides a scientific experimental basis for the research of the quality evaluation system of Radix Bupleuri medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the chromatogram of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid in Bupleurum chinense measured in Example 1 (peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is rutin, peak 5 is 3,5-O-dicaffeoylquinic acid, peak 6 is quercetin, and peak 7 is 4,5-O-dicaffeoylquinic acid).

[0029] Figure 2 This is an investigation spectrum of the mobile phase composition of Example 2.

[0030] Figure 3 This is the investigation graph of the pH value of the mobile phase in Example 2.

[0031] Figure 4 This is the investigation spectrum of potassium dihydrogen phosphate concentration in Example 2.

[0032] Figure 5 This is the investigation spectrum of the chromatographic column of Example 2.

[0033] Figure 6 This is the investigation spectrum of the column temperature of Example 2.

[0034] Figure 7 This is the chromatographic peak positioning map of Example 2 (Note: from bottom to top, they are Radix Bupleuri medicinal material, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid reference substance).

[0035] Figure 8 A new chlorogenic acid standard curve was investigated for the linear relationship in Example 3.

[0036] Fig. 9 The linear relationship of Example 3 was investigated in the standard curve of chlorogenic acid.

[0037] Fig.10 The linear relationship of Example 3 was investigated in the standard curve of cryptochlorogenic acid.

[0038] Fig.11 The rutin standard curve is investigated for the linear relationship of Example 3.

[0039] Fig.12 The linear relationship of Example 3 was investigated in the standard curve of 3,5-O-dicaffeoylquinic acid.

[0040] Fig.13 The quercetin standard curve was investigated for the linear relationship in Example 3.

[0041] Fig.14 The linear relationship of Example 3 was investigated for the standard curve of 4,5-O-dicaffeoylquinic acid.

[0042] Fig.15 This is the specificity investigation spectrum of Example 3 (1-7 are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid, respectively). DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below through specific examples. The raw materials and reagents involved in the specific implementation methods are all commercially available.

[0044] Instruments: H-CLASS ultra-high performance liquid chromatograph from Waters, PDA detector, Empower3 chromatography workstation; XS105DU electronic analytical balance from Mettler-Toledo; KQ5200DB CNC ultrasonic cleaner from Kunshan Ultrasonic Instrument Co., Ltd.; TG16-W micro high-speed centrifuge from Changsha Xiangyi Centrifuge Co., Ltd.

[0045] Reference substance: Neochlorogenic acid, batch number 112110-202401, content 99.2%, China Food and Drug Inspection Institutes.

[0046] Chlorogenic acid, batch number 110753-202018, content 96.1%, China Food and Drug Inspection Institute.

[0047] Cryptochlorogenic acid, batch number 112111-202401, content 96.7%, China Food and Drug Inspection Institute.

[0048] Rutin, batch number 100080-202012, content 91.6%, China Food and Drug Inspection Institute.

[0049] 4,5-O-dicaffeoylquinic acid, batch number 111894-202205, content 94.9%, China Food and Drug Inspection Institute.

[0050] Quercetin, batch number 111538-202308, content 95.3%, China Food and Drug Inspection Institute.

[0051] 3,5-O-dicaffeoylquinic acid, batch number 111782-202208, content 95.9%, China Food and Drug Inspection Institutes.

[0052] New chlorogenic acid, batch number N2205002, content 99.24%, Shanghai Yuanye Biotechnology Co., Ltd.

[0053] Cryptochlorogenic acid, batch number C2205003, content 98.39%, Shanghai Yuanye Biotechnology Co., Ltd.

[0054] Reagents: chromatographic grade methanol and acetonitrile from Fisher Company, USA; water was distilled water from Watsons; other reagents were of analytical grade.

[0055] The medicinal material of Bupleurum chinense was provided by Yunnan Shengke Pharmaceutical Co., Ltd.

[0056] Example 1

[0057] Preparation of test solution: Accurately weigh 1 g of Bupleurum chinense powder (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 mL of 70% methanol solution by volume, weigh it, extract it by ultrasonic for 1 h, cool it, add 70% methanol solution to make up the weight, shake it well, filter it, and obtain the solution.

[0058] Preparation of reference solution: Accurately weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid reference substances, and add methanol to make a mixed reference solution containing 0.01 mg neochlorogenic acid, 0.1 mg chlorogenic acid, 0.01 mg cryptochlorogenic acid, 0.1 mg rutin, 0.01 mg 3,5-O-dicaffeoylquinic acid, 0.1 mg quercetin, and 0.01 mg 4,5-O-dicaffeoylquinic acid per 1 mL.

[0059] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as filler (2.1×100 mm, 1.9 μm) mobile phase: 50 mmol potassium dihydrogen phosphate solution (adjusted to pH=3.0) as mobile phase A, acetonitrile as mobile phase B, gradient elution according to Table 1, column temperature 25°C; flow rate 0.3 mL / min; detection wavelength 325 nm; injection volume 1 μL. The theoretical plate number calculated based on the quercetin peak should be no less than 150,000.

[0060] Table 1 Mobile phase gradient conditions used in Example 1

[0061] Time (minutes) Acetonitrile (%) 50mmol / L potassium dihydrogen phosphate (%) 0～30 5→20 95→80 30～31 20→60 80→40 31～35 60 40 35～36 60→5 40→95 36～45 5 95

[0062] Determination method: Accurately pipette 1 μL of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography, and determine. Figure 1 .

[0063] The external standard method was used to calculate the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin and 4,5-O-dicaffeoylquinic acid in Radix Bupleuri.

[0064] Example 2 Screening of Chromatographic Conditions and Preparation of Test Solution

[0065] 2.1 Screening of chromatographic conditions

[0066] 2.1.1 Investigation of mobile phase composition

[0067] The inventors investigated four mobile phases: acetonitrile-50mmol / L potassium dihydrogen phosphate solution, acetonitrile-0.1% phosphoric acid solution, acetonitrile-50mmol / L ammonium formate solution, and acetonitrile-0.1% trifluoroacetic acid solution. The results showed that when the mobile phase was acetonitrile-potassium dihydrogen phosphate solution (adjusted to pH 3.0), the target peaks in the medicinal sample were best separated without obvious interference, so the mobile phase was acetonitrile-potassium dihydrogen phosphate solution. Figure 2 .

[0068] 2.1.2 Investigation of mobile phase pH value

[0069] The inventors investigated the pH values ​​of potassium dihydrogen phosphate solution at 2.5, 3.0, 3.5 and 4.0 (all pH values ​​were adjusted with phosphoric acid). When the pH value was 3.0, the separation effect of each target peak in the medicinal material sample was the best, and there was no obvious interference. Therefore, the pH value of potassium dihydrogen phosphate solution was selected to be 3.0. Figure 3 .

[0070] 2.1.3 Investigation of potassium dihydrogen phosphate concentration

[0071] The inventors investigated potassium dihydrogen phosphate solutions with concentrations of 10mmol / L, 25mmol / L and 50mmol / L (adjusted to pH = 3.0). When the concentration of potassium dihydrogen phosphate solution was 50mmol / L, the target peaks in the medicinal sample were best separated without obvious interference, so the concentration of potassium dihydrogen phosphate solution was selected to be 50mmol / L. Figure 4 .

[0072] 2.1.4 Investigation of chromatographic columns

[0073] The inventors investigated a number of chromatographic columns from three manufacturers, Shimadzu, Thermo Fisher Scientific and Waters. The results showed that the Shimadzu chromatographic column Shim-packHD-C18-80, 2.1×100mm, 1.9μm, had the best separation of target peaks in the medicinal material samples without obvious interference. Figure 5 .

[0074] 2.1.5 Investigation of column temperature

[0075] The inventors investigated four column temperatures of 25°C, 30°C, 35°C, and 40°C. The results showed that when the column temperature was 25°C, the target peaks in the medicinal sample were best separated without obvious interference, so the column temperature was selected to be 25°C. Figure 6 .

[0076] 2.1.6 Positioning of chromatographic peaks

[0077] Use a single reference substance to locate each, and the peaks in the chromatogram of the test solution are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid. Figure 7 .

[0078] 2.2 Investigation of test solution preparation

[0079] 2.2.1 Investigation of extraction method and extraction solvent of test solution

[0080] The inventors investigated four extraction solvents with a volume concentration of 30% methanol, 50% methanol, 70% methanol, and methanol, as well as two extraction methods, ultrasonic and reflux. The results showed that 70% methanol extraction had the best effect, and reflux extraction was slightly higher than ultrasonic extraction, but the difference was not obvious. Considering that ultrasonic extraction is simpler, faster, and more environmentally friendly, the extraction method selected was 70% methanol ultrasonic extraction. The results are shown in Table 2.

[0081] Table 2: Extraction method and extraction solvent investigation (n=3, content unit: mg / g)

[0082]

[0083] 2.2.2 Investigation of extraction time

[0084] The inventors investigated the ultrasonic extraction time of 0.5h, 1h, and 1.5h, and found that there was no significant difference among the three. Ultrasonic extraction for 1h can ensure complete extraction, so the extraction time was selected as 1h. The results are shown in Table 3.

[0085] Table 3 Extraction time investigation (n=3, content unit: mg / g)

[0086]

[0087] 2.2.3 Investigation of material-liquid ratio

[0088] The inventors investigated the solid-liquid ratios of 1:25, 1:50 and 1:100 for extracting Radix Bupleuri with methanol solution, and found that there was no significant difference among the three. The solid-liquid ratio of 1:50 can ensure complete extraction, so the solid-liquid ratio of 1:50 was selected. The results are shown in Table 4.

[0089] Table 4 Investigation of extraction solid-liquid ratio (n=3, content unit: mg / g)

[0090]

[0091] Example 3 Methodology Verification

[0092] 3.1 System Applicability

[0093] The mixed reference solution prepared in Example 1 was injected 5 times continuously, the peak area was recorded, and the RSD value was calculated. The results are shown in Table 5, indicating that the injection precision was good, the theoretical plate number of the quercetin peak was greater than 150,000, and the separation degree of each peak was greater than 1.5, indicating that the system applicability was good.

[0094] Table 5 Injection precision investigation results (n=5)

[0095]

[0096] 3.2 Linearity

[0097] Preparation of reference solution: Accurately weigh appropriate amount of chlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid reference substances, and add methanol to prepare reference substance stock solution 1 containing 389.8224 μg of chlorogenic acid, 480.3504 μg of rutin, 180.52956 μg of 3,5-O-dicaffeoylquinic acid, 452.1032 μg of quercetin, and 169.3016 μg of 4,5-O-dicaffeoylquinic acid per 1 mL. Accurately weigh appropriate amount of neochlorogenic acid and cryptochlorogenic acid reference substances, and add methanol to prepare reference substance stock solution 2 containing 42.6732 μg of neochlorogenic acid and 36.99464 μg of cryptochlorogenic acid per 1 mL.

[0098] Accurately measure 1 mL, 1 mL, 1 mL, 1 mL, and 1 mL of the above-mentioned reference substance stock solution 1, respectively, and place them in 2 mL, 5 mL, 10 mL, 50 mL, and 100 mL volumetric flasks, dilute to the mark with methanol, shake well, filter, and take the filtrate as reference substance solutions 1, 2, 3, 4, and 5. Accurately measure 1 mL, 1 mL, 1 mL, 1 mL, and 1 mL of the above-mentioned reference substance stock solution 2, respectively, and place them in 2 mL, 5 mL, 10 mL, 25 mL, and 50 mL volumetric flasks, dilute to the mark with methanol, shake well, filter, and take the filtrate as reference substance solutions 6, 7, 8, 9, and 10.

[0099] 1 μL of the reference substance solution and reference substance stock solution were taken respectively and injected into the ultra-high performance liquid chromatograph to determine the peak area. The standard curve of each reference substance was drawn with the reference substance concentration as the abscissa and the peak area as the ordinate, and the linear regression equation, correlation coefficient and linear range were calculated. The results are shown in Tables 6 to 12. Figure 8 to Figure 14 .

[0100] Table 6 Investigation of linear relationship of new chlorogenic acid

[0101] Concentration μg / mL 0 0.853464 1.706928 4.26732 8.53464 21.3366 42.6732 Peak area 0 14275 30846 76490 142253 401589 827581

[0102] The linear regression equation of neochlorogenic acid is y=19416x-6865.3, and the correlation coefficient is R 2 =0.9992.

[0103] Table 7 Investigation of linear relationship of chlorogenic acid

[0104] Concentration μg / mL 0 3.898224 7.796448 38.98224 77.96448 194.9112 389.8224 Peak area 0 31817 74234 425379 846577 2155859 4131718

[0105] The linear regression equation of chlorogenic acid is y=10670x+7723.6, and the correlation coefficient is R 2 =0.9995.

[0106] Table 8 Investigation of linear relationship of chlorogenic acid

[0107] Concentration μg / mL 0 0.7398928 1.4797856 3.699464 7.398928 18.49732 36.99464 Peak area 0 9306 22007 54668 107576 287127 588478

[0108] The linear regression equation of chlorogenic acid is y=15929x-3841, and the correlation coefficient is R 2 =0.9997.

[0109] Table 9 Investigation of linear relationship of rutin

[0110] Concentration μg / mL 0 4.803504 9.607008 48.03504 96.07008 240.1752 480.3504 Peak area 0 16895 38639 216899 423398 1083496 2056993

[0111] The linear regression equation of rutin is y=4312.9x+6439.8, and the correlation coefficient is R 2 =0.9993.

[0112] Table 103,5-O-dicaffeoylquinic acid linear relationship investigation

[0113] Concentration μg / mL 0 1.8052956 3.6105912 18.052956 36.105912 90.26478 180.52956 Peak area 0 20665 45922 238607 490215 1183038 2296076

[0114] The linear regression equation of 3,5-O-dicaffeoylquinic acid is y=12756x+8615, and the correlation coefficient is R 2 =0.9997.

[0115] Table 11 Investigation of linear relationship of quercetin

[0116] Concentration μg / mL 0 4.521032 9.042064 45.21032 90.42064 226.0516 452.1032 Peak area 0 22284 49368 260244 498688 1334221 2518442

[0117] The linear regression equation of quercetin is y=5618.4x+4977.3, and the correlation coefficient is R 2 =0.9991.

[0118] Table 124,5-O-dicaffeoylquinic acid linear relationship investigation

[0119] Concentration μg / mL 0 1.693016 3.386032 16.93016 33.86032 84.6508 169.3016 Peak area 0 21622 47045 264228 508456 1231140 2392283

[0120] The linear regression equation of 4,5-O-dicaffeoylquinic acid is y=14154x+11382, and the correlation coefficient is R 2 =0.9996.

[0121] 3.3 Exclusivity

[0122] Take 70% methanol (solvent blank) as negative control solution, then take mixed reference solution and test solution, and measure according to the law. The result is that the retention time of the test sample chromatographic peak is consistent with that of the reference chromatographic peak, and there is no interference peak in the negative control, indicating that the method of the present invention has good specificity. Fig.15 .

[0123] 3.4 Stability study of test solution

[0124] Take Radix Bupleuri and prepare the test solution according to the test solution preparation method. Determine at 0h, 2h, 4h, 6h, 8h, 12h, and 24h, record the peak areas of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid, calculate the RSD value, and the results are shown in Table 13. It shows that the test solution has good stability within 24h.

[0125] Table 13 Stability study of test solution (unit: peak area)

[0126]

[0127] 3.5 Repeatability

[0128] Take Bupleurum chinense medicinal material, prepare 6 test solutions according to the test solution preparation method of the present invention, measure according to the law, calculate the content and RSD value of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, 4,5-O-dicaffeoylquinic acid, and the results are shown in Table 14. It shows that the method has good repeatability.

[0129] Table 14 Repeatability study (n=6, content unit: mg / g)

[0130]

[0131] 3.6 Recovery rate

[0132] Take 6 portions of Bupleurum chinense medicinal materials with known content, the sampling amount is half of the proposed method, add appropriate amounts of 7 reference substances including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid, respectively, prepare 6 test sample solutions according to the proposed method, inject them into ultra performance liquid chromatography, determine the content, and calculate the recovery rate according to the following formula.

[0133]

[0134] The average recovery rate of neochlorogenic acid was 96.2%, and the RSD value was 2.12%; the average recovery rate of chlorogenic acid was 97.2%, and the RSD value was 1.46%; the average recovery rate of cryptochlorogenic acid was 95.1%, and the RSD value was 2.28%; the average recovery rate of rutin was 96.3%, and the RSD value was 0.89%; the average recovery rate of 3,5-O-dicaffeoylquinic acid was 97.8%, and the RSD value was 1.65%; the average recovery rate of quercetin was 98.6%, and the RSD value was 0.94%; the average recovery rate of 4,5-O-dicaffeoylquinic acid was 97.0%, and the RSD value was 1.35%. This shows that the method has good accuracy. The results are shown in Tables 15 to 21.

[0135] Table 15 New chlorogenic acid sample recovery test results (n = 6)

[0136]

[0137] Table 16 Chlorogenic acid recovery test results (n = 6)

[0138]

[0139] Table 17 Chlorogenic acid recovery test results (n = 6)

[0140]

[0141] Table 18 Rutin sample recovery experimental results (n = 6)

[0142]

[0143] Table 19 3,5-O-dicaffeoylquinic acid recovery test results (n=6)

[0144]

[0145] Table 20 Quercetin sample recovery experimental results (n = 6)

[0146]

[0147] Table 21 4,5-O-dicaffeoylquinic acid recovery test results (n=6)

[0148]

[0149] 3.7 Content range

[0150] According to the method of the present invention, three batches of Radix Bupleuri were tested, and the total amount of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid was 19.99-23.75 mg / g. See Table 22.

[0151] Table 22 Determination results of different batches of medicinal materials content (n = 3, content unit: mg / g)

[0152]

[0153] In summary, the method of the present invention has good linearity, repeatability, stability and recovery rate, and can effectively control the quality of Radix Bupleuri. Compared with the existing standards, the present invention establishes a quantitative analysis method for 7 active ingredients in Radix Bupleuri, provides a rapid and reliable detection method for the quality control of Radix Bupleuri, and provides an important reference for the research of the quality evaluation system of Radix Bupleuri.

Claims

1. A quantitative analysis method for the effective components of Radix Bupleuri Radix, characterized in that: The steps include: a. Preparation of reference solution: accurately weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid reference substances, and add methanol to prepare a mixed reference solution; b. Preparation of test solution: grind Radix Bupleuri, extract with methanol solution, and filter to obtain the solution; c. Determination: Accurately aspirate the reference solution and the test solution respectively, inject them into the ultra-high performance liquid chromatography, determine, and calculate the content using the external standard method.

2. The method for quantitative analysis of effective components of Radix Bupleuri medicinal material according to claim 1, characterized in that: In step a, the preparation method of the test solution is: grind the Radix Bupleuri medicinal material, pass it through a No. 3 sieve, take 0.5-2g and place it in a stoppered conical flask, add a methanol solution with a volume concentration of 30%-100%, the solid-liquid ratio of the Radix Bupleuri medicinal material to the methanol solution is 1:25-100, weigh, ultrasonically extract for 0.5-2h, cool, and then add a methanol solution with a volume concentration of 30%-100% to make up the weight, shake well, filter, and take the filtrate to obtain.

3. The method for quantitative analysis of effective components of Radix Bupleuri medicinal material according to claim 2, characterized in that: In step a, the preparation method of the test solution is: grind the Radix Bupleuri medicinal material, pass it through a No. 3 sieve, place it in a stoppered conical flask, add a methanol solution with a volume concentration of 70%, the solid-liquid ratio of the Radix Bupleuri medicinal material to the methanol solution is 1:50mL, weigh it, ultrasonically extract it for 1h, cool it, add a methanol solution with a volume concentration of 70% to make up the weight, shake it well, and filter it to obtain it.

4. The method for quantitative analysis of effective components of Radix Bupleuri medicinal material according to claim 1, characterized in that: In step b, the preparation method of the reference solution is as follows: accurately weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercetin, and 4,5-O-dicaffeoylquinic acid reference substances, and add methanol to make a mixed reference solution containing 0.01 mg of neochlorogenic acid, 0.1 mg of chlorogenic acid, 0.01 mg of cryptochlorogenic acid, 0.1 mg of rutin, 0.01 mg of 3,5-O-dicaffeoylquinic acid, 0.1 mg of quercetin, and 0.01 mg of 4,5-O-dicaffeoylquinic acid per 1 mL.

5. The method for quantitative analysis of effective components of Radix Bupleuri Radix according to claim 1, characterized in that: In step c, the chromatographic conditions for the determination are: the chromatographic column is filled with octadecylsilane bonded silica gel; acetonitrile and 5-50mmol / L potassium dihydrogen phosphate solution are used as mobile phases for gradient elution; the detection wavelength is 320-330nm; the column temperature is 25-40°C; the flow rate is 0.2-0.5mL / min; and the theoretical plate number calculated based on the quercetin peak should be no less than 150,000.

6. The method for quantitative analysis of effective components of Radix Bupleuri Radix according to claim 5, characterized in that: In step c, the gradient elution procedure of the mobile phase is: 50 mmol / L potassium dihydrogen phosphate solution is used as mobile phase A, and acetonitrile is used as mobile phase B; From 0 to 30 min, the volume fraction of mobile phase A decreased from 95% to 80%, and the volume fraction of mobile phase B increased from 5% to 20%; From 30 to 31 min, the volume fraction of mobile phase A decreased from 80% to 40%, and the volume fraction of mobile phase B increased from 20% to 60%; 31-35 min, maintain the volume fraction of mobile phase A at 40%, and the volume fraction of mobile phase B at 60%; From 35 to 36 min, the volume fraction of mobile phase A increased from 40% to 95%, and the volume fraction of mobile phase B decreased from 60% to 5%; From 36 to 45 min, the volume fraction of mobile phase A was maintained at 95%, and the volume fraction of mobile phase B was maintained at 5%.

7. The method for quantitative analysis of effective components of Radix Bupleuri Radix according to claim 5 or 6, characterized in that: The pH value of the potassium dihydrogen phosphate solution is 2.0-7.

0.

8. The method for quantitative analysis of effective components of Radix Bupleuri Radix according to claim 7, characterized in that: The pH value of the potassium dihydrogen phosphate solution is 3.

0.

9. The method for quantitative analysis of effective components of Radix Bupleuri Radix according to claim 5, characterized in that: The detection wavelength was 325 nm; the column temperature was 25°C; and the flow rate was 0.3 mL / min.

10. The method for quantitative analysis of effective components of Radix Bupleuri Radix according to claim 5, characterized in that: The chromatographic column is a Shimadzu Shim-pack HD-C18-80 column, 2.1×100 mm, 1.9 μm.

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