Quantitative analysis method of effective components in radix bupleuri

Abstract

The present application belongs to the technical field of quality detection of traditional Chinese medicinal materials, and particularly relates to a method for quantitatively analyzing effective components of Xiaochaihu medicinal materials. The method comprises the following steps: a, preparation of a control solution: precisely take new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin and 4,5-O-dicaffeoylquinic acid control samples, and add methanol to prepare a mixed control solution; b, preparation of a test solution: crush Xiaochaihu medicinal materials, add a methanol solution for extraction, and filter to obtain the test solution; and c, determination: precisely take the control solution and the test solution, inject them into an ultra-high performance liquid chromatograph, and determine the content by using an external standard method. The method can simultaneously detect and analyze the content of seven chemical components in Xiaochaihu medicinal materials, has good linearity, repeatability, stability and recovery rate, and can effectively control the quality of Xiaochaihu medicinal materials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of quality detection of traditional Chinese medicinal materials, and particularly relates to a method for quantitatively analyzing effective components of Xiaochaihu medicinal materials. BACKGROUND

[0002] Bupleurum is a traditional Chinese medicinal material in China, which has been used for more than 2000 years. It was first recorded in Shennong Bencao Jing and listed as the top grade. It has the effects of dispersing heat, soothing the liver and relieving depression, and lifting Yang Qi. Plants of the genus Bupleurum in the family Umbelliferae are mainly distributed in the temperate and subtropical regions of the Northern Hemisphere, with about 200 genera and 2500 species, of which 36 species are found in China, and 25 species are used as medicine. Only two species, Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd., are recorded in the Chinese Pharmacopoeia 2020 edition. There are 590 compound preparations using Bupleurum as raw material in the existing drug standards in China. The market demand is increasing year by year, while the wild resources are greatly reduced, leading to a shortage of Bupleurum medicinal materials, which cannot meet the market demand.

[0003] At present, most other plants of the genus Bupleurum, such as Bupleurum falcatum, Bupleurum smithii and Bupleurum marcoangense, are used as Bupleurum in their producing areas. These plants have a long history of medicine use and rich resource reserves in their producing areas, which greatly alleviate the supply demand of Bupleurum chinense and Bupleurum scorzonerifolium. Previous research results have shown that the medicinal parts and effective components of other medicinal plants of the genus Bupleurum are different from those of the pharmacopoeia Bupleurum. Therefore, the study of the material basis of local medicinal materials, the exploration of new high-quality resources, and the establishment of effective quality control methods can better meet the market demand for medicine, and ensure the rational development and application of medicinal materials.

[0004] The Xiaochaihu medicinal material is recorded under the standard of “Dianchaihu” (Yun YNZYC-0343-2010) in Yunnan Province, which is the dried whole plant of Bupleurum hamiltonii N.P.Balakr. of the genus Bupleurum in the family Umbelliferae. It is distributed in Yunnan, Guizhou, Sichuan, Hubei and Guangxi provinces, and its aboveground part is rich in resources and easy to collect, which has a good utilization prospect.

[0005] The quality control of Xiaochaihu medicinal material in the existing standard only has simple thin layer identification, moisture and ash inspection, extract, no content determination method, which is difficult to effectively control the quality. At present, the research data of the chemical components and quantitative control method of Xiaochaihu medicinal material is very few. The test research finds that Xiaochaihu medicinal material mainly contains more organic acid and flavonoid components, and the components have many pharmacological activities such as antipyretic analgesic, anti-inflammatory, antiviral and immune enhancement. Therefore, it is necessary to develop a method for quantitatively detecting the content of the effective components in Xiaochaihu medicinal material for quality control. SUMMARY

[0006] The technical problem to be solved by the present application is to fill the blank of the prior art, and to provide an effective component quantitative analysis method of Xiaochaihu medicinal material. The effective component content in Xiaochaihu medicinal material is detected by the method, and the precision, repeatability and stability are good, so that the quality of Xiaochaihu medicinal material can be effectively controlled.

[0007] In order to solve the above technical problems, the technical scheme adopted by the present application is to provide an effective component quantitative analysis method of Xiaochaihu medicinal material, which comprises the following steps:

[0008] a. Preparation of reference solution: accurately weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin and 4,5-O-dicaffeoylquinic acid reference substances, and prepare a mixed reference solution by adding methanol;

[0009] b. Preparation of test solution: crush Xiaochaihu medicinal material, add methanol solution for extraction, and filter to obtain;

[0010] c. Determination: accurately pipette the reference solution and the test solution respectively, inject into the ultra-high performance liquid chromatograph, and determine the content by using the external standard method.

[0011] Further, in step a, the preparation method of the reference solution is as follows: accurately weigh new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin and 4,5-O-dicaffeoylquinic acid reference substances, and prepare a mixed reference solution containing 0.01 mg of new chlorogenic 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 quercitrin and 0.01 mg of 4,5-O-dicaffeoylquinic acid per 1 mL of methanol.

[0012] Further, in step b, the preparation method of the test solution is as follows: the Bupleurum Chinese DC. is crushed, passed through a No. 3 sieve, 0.5-2 g is taken and put into a conical flask with a stopper, 30%-100% methanol solution is added, the ratio of Bupleurum Chinese DC. to methanol solution is 1:25-100, weighed, ultrasonic extraction is conducted for 0.5-2 h, cooled, 30%-100% methanol solution is added to make up the weight, shaken uniformly, and filtered to obtain the test solution.

[0013] Preferably, in step b, the preparation method of the test solution is as follows: the Bupleurum Chinese DC. is crushed, passed through a No. 3 sieve, 1 g is taken and put into a conical flask with a stopper, 70% methanol solution is added, the ratio of Bupleurum Chinese DC. to methanol solution is 1:50, weighed, ultrasonic extraction is conducted for 1 h, cooled, 70% methanol solution is added to make up the weight, shaken uniformly, and filtered to obtain the test solution.

[0014] Further, in step c, the chromatographic conditions of the determination are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel; acetonitrile and 5-50 mmol / L potassium dihydrogen phosphate solution are used as the mobile phase for gradient elution; the detection wavelength is 320-330 nm; the column temperature is 25-40 °C; the flow rate is 0.2-0.5 mL / min; and the theoretical plate number calculated according to the quercitrin peak should be not less than 150000.

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

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

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

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

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

[0020] 36-45 min, the volume fraction of the mobile phase A is kept at 95% and the volume fraction of the mobile phase B is kept at 5%.

[0021] Further, 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 DEG C; and the flow rate is 0.3 mL / min.

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

[0025] Further, in step c, the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin and 4,5-O-dicaffeoylquinic acid in 1 g of the small Chaihu medicinal material are calculated by using an external standard method.

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

[0027] The present application provides an effective component quantitative analysis method for the small Chaihu medicinal material by optimizing chromatographic conditions and preparation parameters of the test sample solution, can simultaneously detect 7 kinds of effective components such as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin and 4,5-O-dicaffeoylquinic acid, realizes the multi-component content detection of the small Chaihu medicinal material, the components have various pharmacological activities and are related to the efficacy of the medicinal material, the detection operation is simple, the result is stable and repeatable, thereby the quality control of the small Chaihu medicinal material can be effectively controlled. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The chromatogram (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 quercitrin, and peak 7 is 4,5-O-dicaffeoylquinic acid) of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin and 4,5-O-dicaffeoylquinic acid in the small Chaihu medicinal material measured in Example 1.

[0029] Figure 2 The investigation atlas of the composition of the mobile phase in Example 2.

[0030] Figure 3 The investigation atlas of the pH value of the mobile phase in Example 2.

[0031] Figure 4 The investigation atlas of the concentration of potassium dihydrogen phosphate in Example 2.

[0032] Figure 5 The investigation atlas of the chromatographic column in Example 2.

[0033] Figure 6 The column temperature of Example 2 is shown in the following chart.

[0034] Figure 7 The peak positioning chart of Example 2 is shown in the following chart (Note: from bottom to top, they are Xiaochaihu medicinal material, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin, and 4,5-O-dicaffeoylquinic acid reference substance).

[0035] Figure 8 The standard curve of neochlorogenic acid for linear relationship investigation of Example 3 is shown in the following chart.

[0036] Figure 9 The standard curve of chlorogenic acid for linear relationship investigation of Example 3 is shown in the following chart.

[0037] Figure 10 The standard curve of cryptochlorogenic acid for linear relationship investigation of Example 3 is shown in the following chart.

[0038] Figure 11 The standard curve of rutin for linear relationship investigation of Example 3 is shown in the following chart.

[0039] Figure 12 The standard curve of 3,5-O-dicaffeoylquinic acid for linear relationship investigation of Example 3 is shown in the following chart.

[0040] Figure 13 The standard curve of quercitrin for linear relationship investigation of Example 3 is shown in the following chart.

[0041] Figure 14 The standard curve of 4,5-O-dicaffeoylquinic acid for linear relationship investigation of Example 3 is shown in the following chart.

[0042] Figure 15 The specificity investigation chart of Example 3 is shown in the following chart (1-7 are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin, and 4,5-O-dicaffeoylquinic acid, respectively). DETAILED DESCRIPTION

[0043] The present application will be further described in detail by specific examples. The raw materials and reagents involved in the specific embodiments are all commercially available.

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

[0045] Reference substance: neochlorogenic acid, batch number 112110-202401, content 99.2%, China Institute for Drug Control.

[0046] Chlorogenic acid, batch number 110753-202018, content 96.1%, China Institute for Drug Control.

[0047] Cryptochlorogenic acid, batch number 112111-202401, content 96.7%, China Institute for Drug Control.

[0048] Rutin, batch number 100080-202012, content 91.6%, China Institute for Drug Control.

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

[0050] Quercitrin, batch number 111538-202308, content 95.3%, China Institute for Drug Control.

[0051] 3,5-O-dicaffeoylquinic acid, batch number 111782-202208, content 95.9%, China Institute for Drug Control.

[0052] Neochlorogenic acid, batch number N2205002, content 99.24%, Shanghai Yuan Ye Biological Technology Co., Ltd.

[0053] Cryptochlorogenic acid, batch number C2205003, content 98.39%, Shanghai Yuan Ye Biological Technology Co., Ltd.

[0054] Reagents: chromatographic grade methanol and acetonitrile from Fisher Company, USA, water is distilled water from Watsons, and the rest of the reagents are analytical pure.

[0055] Xiaochaihu medicinal materials were provided by Yunnan Shengke Pharmaceutical Co., Ltd.

[0056] Example 1

[0057] Preparation of test sample solution: accurately weigh 1 g of Xiaochaihu medicinal material powder (pass through No. 3 sieve) into a stoppered conical flask, add 50 mL of 70% methanol solution, weigh, ultrasonic extraction for 1 h, cool, add 70% methanol solution to make up the weight, shake well, and filter to obtain the test sample solution.

[0058] Preparation of the control solution: precisely take the control samples of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin and 4,5-O-dicaffeoylquinic acid, and add methanol to prepare a mixed control 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 quercitrin and 0.01 mg of 4,5-O-dicaffeoylquinic acid per 1 mL.

[0059] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler (2.1x100mm, 1.9μm), mobile phase: 50mmol potassium dihydrogen phosphate solution (pH=3.0) as mobile phase A, acetonitrile as mobile phase B, gradient elution according to Table 1, column temperature 25℃; flow rate 0.3mL / min; detection wavelength 325nm; injection volume 1μL. The theoretical plate number calculated according to the quercitrin peak should not be less than 150000.

[0060] Table 1 Gradient conditions of the mobile phase used in Example 1

[0061] Time (min) Acetonitrile (%) 50 mmol / 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: precisely take 1μL of the control solution and the sample solution respectively, inject into the ultra-high performance liquid chromatograph, and determine, and then obtain. See Figure 1 .

[0063] The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin and 4,5-O-dicaffeoylquinic acid in the Xiaochaihu medicinal material are calculated by the external standard method.

[0064] Example 2 Screening of chromatographic conditions and investigation of preparation of the sample solution

[0065] 2.1 Screening of chromatographic conditions

[0066] 2.1.1 Investigation of the composition of the mobile phase

[0067] The inventors investigated four kinds of mobile phases, i.e. 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, and the results showed that when the mobile phase was acetonitrile-potassium dihydrogen phosphate solution (pH value adjusted to 3.0), the separation of each target peak in the medicinal material sample was the best, and there was no obvious interference, so the mobile phase was selected to be acetonitrile-potassium dihydrogen phosphate solution. See Figure 2 .

[0068] 2.1.2 Investigation of the pH value of the mobile phase

[0069] The inventors respectively investigated the pH value of potassium dihydrogen phosphate solution under four conditions of 2.5, 3.0, 3.5 and 4.0 (all using phosphoric acid to adjust pH), and 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, so the pH value of the potassium dihydrogen phosphate solution was selected as 3.0. See Figure 3 .

[0070] 2.1.3 Investigation of potassium dihydrogen phosphate concentration

[0071] The inventors respectively investigated the potassium dihydrogen phosphate solution with concentrations of 10 mmol / L, 25 mmol / L and 50 mmol / L (pH adjusted to 3.0), and when the concentration of the potassium dihydrogen phosphate solution was 50 mmol / L, the separation of each target peak in the medicinal material sample was the best, and there was no obvious interference, so the concentration of the potassium dihydrogen phosphate solution was selected as 50 mmol / L. See Figure 4 .

[0072] 2.1.4 Investigation of chromatographic column

[0073] The inventors respectively investigated multiple chromatographic columns from Shimadzu, Thermo and Waters, and the results showed that when the Shimadzu chromatographic column Shim-pack HD-C18-80, 2.1x100mm, 1.9μm was used, the separation of each target peak in the medicinal material sample was the best, and there was no obvious interference, see Figure 5 .

[0074] 2.1.5 Investigation of column temperature

[0075] The inventors respectively investigated four column temperatures of 25℃, 30℃, 35℃ and 40℃, and the results showed that when the column temperature was 25℃, the separation of each target peak in the medicinal material sample was the best, and there was no obvious interference, so the column temperature was selected as 25℃. See Figure 6 .

[0076] 2.1.6 Positioning of chromatographic peaks

[0077] A single control was used for positioning, and the peaks in the chromatogram of the test sample solution were new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin and 4,5-O-dicaffeoylquinic acid in turn. See Figure 7 .

[0078] 2.2 Investigation of preparation of test sample solution

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

[0080] The inventors respectively investigated four kinds of extraction solvents of 30% methanol, 50% methanol, 70% methanol and methanol, and two kinds of extraction methods of ultrasonic and reflux, and the results showed that the extraction effect of 70% methanol was the best, the reflux extraction was slightly higher than the ultrasonic extraction but the difference was not obvious, considering that the ultrasonic extraction operation was more simple and convenient, more environmental protection, therefore the extraction method was selected as 70% methanol ultrasonic extraction. The results are shown in Table 2.

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

[0082]

[0083] 2.2.2 Investigation of extraction time

[0084] The inventors respectively investigated the ultrasonic extraction time of 0.5h, 1h and 1.5h, and the results showed that there was no obvious difference among the three, and the ultrasonic extraction for 1h could ensure complete extraction, therefore the extraction time was selected as 1h. The results are shown in Table 3.

[0085] Table 3 Investigation of extraction time (n = 3, content unit: mg / g)

[0086]

[0087] 2.2.3 Investigation of solid-liquid ratio

[0088] The inventors respectively investigated the solid-liquid ratio of using methanol solution to extract Xiaochaihu medicinal materials, 1:25, 1:50 and 1:100, and the results showed that there was no obvious difference among the three, and the solid-liquid ratio of 1:50 could ensure complete extraction, therefore the solid-liquid ratio was selected as 1:50. The results are shown in Table 4.

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

[0090]

[0091] Methodology verification of Example 3

[0092] 3.1 System suitability

[0093] The mixed reference solution prepared in Example 1 was continuously injected for 5 times, the peak area was recorded, and the RSD value was calculated, and the results are shown in Table 5, which showed that the injection precision was good, the theoretical column plate number of quercitrin peak was greater than 150000, and the separation degree of each peak was greater than 1.5, which showed that the system suitability was good.

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

[0095]

[0096] 3.2 Linearity

[0097] Preparation of the control solution: accurately weigh green acid, rutin, 3, 5-O-dicaffeoyl quinic acid, quercitrin, 4, 5-O-dicaffeoyl quinic acid control substance, add methanol to prepare 1 mL of control solution containing green acid 389.8224 μg, rutin 480.3504 μg, 3, 5-O-dicaffeoyl quinic acid 180.52956 μg, quercitrin 452.1032 μg, 4, 5-O-dicaffeoyl quinic acid 169.3016 μg. Accurately weigh neochlorogenic acid, cryptochlorogenic acid control substance, add methanol to prepare 1 mL of control solution containing neochlorogenic acid 42.6732 μg, cryptochlorogenic acid 36.99464 μg.

[0098] Accurately take 1 mL, 1 mL, 1 mL, 1 mL, 1 mL of the above control solution 1, respectively, and place it in a 2 mL, 5 mL, 10 mL, 50 mL, 100 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and take the filtrate as the control solution 1, 2, 3, 4, 5. Accurately take 1 mL, 1 mL, 1 mL, 1 mL, 1 mL of the above control solution 2, respectively, and place it in a 2 mL, 5 mL, 10 mL, 25 mL, 50 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and take the filtrate as the control solution 6, 7, 8, 9, 10.

[0099] Take 1 μL of the above control solution and control solution, respectively, and inject it into the ultra-high performance liquid chromatograph, measure the peak area, take the control concentration as the abscissa and the peak area as the ordinate, draw the standard curve of each control, and calculate the linear regression equation, correlation coefficient and linear range. The results are shown in Tables 6-12, Figure 8 to Figure 14 .

[0100] Table 6 Linear relationship of neochlorogenic acid

[0101] Concentration pg / 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, the correlation coefficient R 2 = 0.9992.

[0103] Table 7 Linear relationship of green acid

[0104] Concentration pg / 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 green acid is y = 10670x + 7723.6, the correlation coefficient R 2 = 0.9995.

[0106] Table 8 Linear relationship of cryptochlorogenic acid

[0107] Concentration pg / 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 cryptochlorogenic acid is y = 15929x - 3841, and the correlation coefficient R = 0.9997. 2

[0109] Table 9 Rutin linear relationship investigation

[0110] Concentration pg / 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 R = 0.9993. 2

[0112] Table 10 3,5-O-dicaffeoylquinic acid linear relationship investigation

[0113] Concentration pg / 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 R = 0.9997. 2

[0115] Table 11 Quercitrin linear relationship investigation

[0116] Concentration pg / 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 quercitrin is y = 5618.4x + 4977.3, and the correlation coefficient R = 0.9991. 2

[0118] Table 12 4,5-O-dicaffeoylquinic acid linear relationship investigation

[0119] Concentration pg / 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 R = 0.9996. 2

[0121] 3.3 Specificity

[0122] Take 70% methanol (solvent blank) as a negative control solution, and then take the mixed control solution and the test solution, and determine according to the method. The results show that the retention time of the test solution chromatographic peak is consistent with the retention time of the control product chromatographic peak, and there is no interference peak in the negative control, indicating that the method has good specificity, as shown in Table 3. Figure 15 .

[0123] 3.4 Test solution stability investigation

[0124] ​​​​​Take the small Chaihu medicinal materials, according to the test solution preparation method for test solution, respectively in 0h, 2h, 4h, 6h, 8h, 12h, 24h determination, record the peak area of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3, 5-O-dicaffeoyl quinic acid, quercitrin, 4, 5-O-dicaffeoyl quinic acid, calculate the RSD value, the results are shown in table 13. The test solution is stable within 24h.

[0125] Table 13 test solution stability investigation (unit: peak area)

[0126]

[0127] 3.5 repeatability

[0128] Take the small Chaihu medicinal materials, according to the test solution preparation method of the application, 6 test solution was prepared, respectively, according to the law, the content of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3, 5-O-dicaffeoyl quinic acid, quercitrin, 4, 5-O-dicaffeoyl quinic acid and RSD value were calculated, and the results were shown in table 14. The method is good in repeatability.

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

[0130]

[0131] 3.6 recovery

[0132] Take the known content of small Chaihu medicinal materials 6, the sample amount is half of the amount of the proposed method, respectively, add neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3, 5-O-dicaffeoyl quinic acid, quercitrin, 4, 5-O-dicaffeoyl quinic acid 7 kinds of control product, according to the proposed method, 6 test solution was prepared, injected into the ultra performance liquid chromatograph, the content was determined, and the recovery was calculated according to the following formula.

[0133]

[0134] The average recovery of neochlorogenic acid was 96.2%, and the RSD value was 2.12%; the average recovery of chlorogenic acid was 97.2%, and the RSD value was 1.46%; the average recovery of cryptochlorogenic acid was 95.1%, and the RSD value was 2.28%; the average recovery of rutin was 96.3%, and the RSD value was 0.89%, the average recovery of 3, 5-O-dicaffeoyl quinic acid was 97.8%, and the RSD value was 1.65%; the average recovery of quercitrin was 98.6%, and the RSD value was 0.94%; the average recovery of 4, 5-O-dicaffeoyl quinic acid was 97.0%, and the RSD value was 1.35%. The method is good in accuracy. The results are shown in tables 15-21.

[0135] Table 15 New chlorogenic acid spiking recovery experiment results (n = 6)

[0136]

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

[0138]

[0139] Table 17 Cryptochlorogenic acid spiking recovery experiment results (n = 6)

[0140]

[0141] Table 18 Rutin spiking recovery experiment results (n = 6)

[0142]

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

[0144]

[0145] Table 20 Quercitrin spiking recovery experiment results (n = 6)

[0146]

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

[0148]

[0149] 3.7 Content range

[0150] According to the method of the present application, 3 batches of Xiaochaihu medicinal materials were determined, and the total amount of new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, 3,5-O-dicaffeoylquinic acid, quercitrin and 4,5-O-dicaffeoylquinic acid was 19.99-23.75 mg / g. See Table 22.

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

[0152]

[0153] In summary, the method of the present application has good linearity, repeatability, stability and recovery rate, and can effectively control the quality of Xiaochaihu. Compared with the existing standard, the present application establishes a quantitative analysis method for 7 kinds of effective components in Xiaochaihu medicinal materials, provides a fast and reliable detection means for the quality control of Xiaochaihu medicinal materials, and provides an important reference for the research of Xiaochaihu medicinal material quality evaluation system.

Claims

1. A method for quantitative analysis of the effective components of Bupleurum chinense, characterized in that: Includes the following steps: 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 standards, and add methanol to prepare a mixed reference solution; b. Preparation of the test solution: Take pulverized Bupleurum chinense, add methanol solution for extraction, and filter to obtain the test solution; take pulverized Bupleurum chinense, pass it through a No. 3 sieve, take 0.5-2g and place it in a stoppered conical flask, add methanol solution with a volume concentration of 30%-100%, the material-to-liquid ratio of Bupleurum chinense to methanol solution is 1:25-100, weigh, extract ultrasonically for 0.5-2h, cool, then add methanol solution with a volume concentration of 30%-100% to make up the weight, shake well, filter, and take the filtrate to obtain the test solution; c. Determination: Accurately pipette the reference solution and the test solution into an ultra-high performance liquid chromatograph (UHPLC) and determine the content using the external standard method. The chromatographic conditions for step c are as follows: the column is packed with octadecylsilane-bonded silica gel; gradient elution is performed using acetonitrile and 10–50 mmol / L potassium dihydrogen phosphate solution as the mobile phase; the detection wavelength is 320–330 nm; the column temperature is 25–40 °C; the flow rate is 0.2–0.5 mL / min; the theoretical plate number, calculated based on the quercetin peak, should not be less than 150,000; the gradient elution program is as follows: From 0 to 30 minutes, the volume fraction of mobile phase A decreased from 95% to 80%, while the volume fraction of mobile phase B increased from 5% to 20%. Over 30–31 minutes, the volume fraction of mobile phase A decreased from 80% to 40%, while the volume fraction of mobile phase B increased from 20% to 60%. 31–35 min, maintaining the volume fraction of mobile phase A at 40% and the volume fraction of mobile phase B at 60%; Over 35–36 minutes, the volume fraction of mobile phase A increased from 40% to 95%, while the volume fraction of mobile phase B decreased from 60% to 5%. For 36–45 min, maintain the volume fraction of mobile phase A at 95% and the volume fraction of mobile phase B at 5%. The pH value of the potassium dihydrogen phosphate solution is 2.0 to 4.

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2. The method for quantitative analysis of the effective components of Bupleurum chinense according to claim 1, characterized in that: In step a, the preparation method of the test solution is as follows: take the small bupleurum medicinal material, crush it, 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 material-to-liquid ratio of small bupleurum medicinal material to methanol solution is 1:50mL, weigh it, extract it by ultrasonication 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 the test solution.

3. The method for quantitative analysis of the effective components of Bupleurum chinense 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 standards, and add methanol to prepare 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.

4. The method for quantitative analysis of the effective components of Bupleurum chinense according to claim 1, characterized in that: The detection wavelength was 325 nm; the column temperature was 25 °C; and the flow rate was 0.3 mL / min.

5. The method for quantitative analysis of the effective components of Bupleurum chinense according to claim 1, characterized in that: The chromatographic column was a Shimadzu Shim-pack HD-C18-80 column, 2.1×100mm, 1.9μm.

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