Method for detecting contents of saikosides A, B2 and B1 in pediatric radix bupleuri-cinnamon antipyretic granules

Abstract

The invention provides a method for determining the content of saikosides A, B2 and B1 in pediatric radix bupleuri and cortex cinnamomi antipyretic granules. The preparation method comprises the following steps: adding medicinal powder of the pediatric Chaigui antipyretic granules into a methanol solution containing 4-6% of a concentrated ammonia test solution for ultrasonic treatment and extraction, or sequentially adding a proper amount of calcium hydroxide, water and absolute ethyl alcohol into the powder of the pediatric Chaigui antipyretic granules for ultrasonic treatment and extraction, and purifying an extracting solution through MCI small-pore adsorption resin to obtain a test solution; and finally, performing high performance liquid chromatography determination. The method solves the problem that accurate results are difficult to obtain because saikosides A, B2 and B1 are easily interfered by other medicines in a formula during content determination. The detection result of the sample prepared by the method shows that the chromatographic peak purity factors of saikosides A, B2 and B1 are all greater than 980, all peaks are completely separated, and accurate determination of the contents of saikosides A, B2 and B1 in the pediatric antipyretic granules can be realized by adopting a high performance liquid chromatography-diode array detector.

Description

Technical Field

[0001] The invention relates to the technical field of quality control of traditional Chinese medicines, and in particular to a method for determining saikosaponin components A, B2 and B1 in Xiaoer Chaigui Tuire granules. Background Art

[0002] The prescription of Xiaoer Chaigui Antipyretic Granules is composed of seven herbs: Bupleurum, Cinnamon Twig, Pueraria lobata, Duckweed, Scutellaria baicalensis, White Peony Root, and Periostracum Cicadae, which are mainly used for sweating and relieving exterior symptoms, clearing away heat and relieving interior symptoms. In the prescription, Bupleurum and Cinnamon Twig sweat and relieve exterior symptoms, and are the main herbs; Pueraria lobata and Duckweed relieve muscles and penetrate the exterior, and are the ministers; White Peony Root restrains Yin and harmonizes the Ying to prevent Bupleurum and Cinnamon Twig from sweating too much, Scutellaria baicalensis clears the heat of the exterior and interior, and Periostracum Cicadae disperses wind-heat, clears heat and relieves sore throat, and are the adjuvants. All the herbs work together to achieve the effects of sweating and relieving exterior symptoms, clearing away interior symptoms and relieving fever. Xiaoer Chaigui Antipyretic Granules had sales of 247 million in physical stores in 2020, ranking ninth among pediatric Chinese patent medicine brands, and is trusted by the general public.

[0003] Modern studies have shown that the content of saikosaponins in Bupleurum is closely related to its antipyretic and anti-inflammatory effects. Therefore, the content of saikosaponins is a key indicator for evaluating the quality of Bupleurum formula preparations. The standard for Xiaoer Chaigui Tuire Granules in the Chinese Pharmacopoeia (2020 edition) only determines the content of puerarin in the ministerial drug Pueraria root, but does not set a content limit for the monarch drug Bupleurum in the preparation, making it impossible to effectively evaluate the quality of the preparation to ensure its effectiveness. In order to more effectively evaluate the quality of Xiaoer Chaigui Tuire Granules and thus ensure the safety and effectiveness of the product in pediatric treatment, it is urgently necessary to establish an accurate and feasible method for determining the content of saikosaponins in Xiaoer Chaigui Tuire Granules.

[0004] At present, the method for determining the content of saikosaponins in Bupleurum is relatively simple. It is mainly based on the provisions of the Chinese Pharmacopoeia (2020 edition) for ultrasonic extraction and determination using a high-performance liquid chromatography-tandem ultraviolet detector. The 2020 edition of the Chinese Pharmacopoeia was consulted to collect single and compound preparations containing Bupleurum. It was found that there were 2 single preparations of Bupleurum and 99 compound preparations, involving 70 prescriptions, of which 31 prescriptions used Bupleurum as the main drug, and 40 preparations used thin-layer chromatography identification to control the quality of saikosaponin A. However, only the single preparation Bupleurum Drop Pills used saikosaponin A for content control, and there was no requirement for the determination of saikosaponins in Bupleurum compound preparations. Because saikosaponin A is determined at a wavelength of 210 nm, the end absorption at this wavelength is severe, which interferes with the content determination. The chemical composition of Bupleurum compound preparations is more complex than that of single Chinese herbal medicine Bupleurum. Other chemical components will interfere with the determination, resulting in substandard separation or reduced purity of index component peaks, making it difficult to accurately determine their content. Zhao Yiqing (Zhao Yiqing, Wang Yanwei, Yang Yuan, et al. Simultaneous determination of the contents of saikosaponin a, saikosaponin d, saikosaponin b1, saikosaponin b2 and saikosaponin k in Ruhe Nei Xiao Liquid by UPLC-MS / MS [J]. Journal of Drug Analysis, 2023, 43(10): 1702-1708) et al. used ultra-performance liquid chromatography-tandem mass spectrometry to simultaneously determine the contents of saikosaponin a, saikosaponin d, saikosaponin b1, saikosaponin b2 and saikosaponin k in Ruhe Nei Xiao Liquid (prescription: Bupleurum, Fritillaria thunbergii, Angelica sinensis, Paeonia lactiflora, Rhizoma Cyperi, Rubia cordifolia, Cyperus rotundus, Citrus aurantium, Prunella vulgaris, Luffa fructus, Curcuma aromatica, Licorice) in the main herbal medicine Bupleurum. The prior art uses a mass spectrometer. The mass spectrometer uses an ion source to convert the sample to be tested into ions, the analyzer separates the ions, and the detector measures the relative abundance of the ions for quantitative determination. As long as the precise molecular weight of the determined components is different, it can be accurately determined without using spectral technology to measure at relevant specific wavelengths. A Chinese patent with announcement number CN112526006A discloses a method for detecting saikosaponins in Xiao Chaihu granules (prescription is bupleurum, scutellaria, ginger pinellia, codonopsis, ginger, licorice, jujube), that is, a high performance liquid chromatograph combined with an electrospray detector is used to detect saikosaponins in the monarch drug bupleurum. The electrospray detector measures the charge of the solute saikosaponin component particles through a highly sensitive electrostatic detector, and the signal current generated is proportional to the content of the solute, which is obviously different from the ultraviolet absorption as the property of the substance itself, and the accuracy of the detection result is slightly inferior to that of the ultraviolet detector. At present, there is a lack of a simple and reliable determination method using a high performance liquid chromatograph in series with an ultraviolet detector. The technical gap in this area has greatly restricted the development of Bupleurum compound preparations in terms of quality control. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a method for detecting the contents of saikosaponin A, saikosaponin B2 and saikosaponin B1 in Xiaoer Chaigui Tuire Granules by using a high performance liquid chromatography-diode array detector.

[0006] The technical solution of the present invention is as follows: A method for determining the contents of saikosaponins A, B2 and B1 in Xiaoer Chaigui Tuire Granules, characterized in that it comprises the following steps: Step 1, preparation of the test solution: ① Take an appropriate amount of Xiaoer Chaigui Tuire Granules powder, accurately weigh it, add alkaline liquid or alkaline solid, weigh it again after the volumetric bottle is fixed to volume; ② Ultrasonic treatment extraction, cooling, make up the lost weight with methanol, filter, and obtain the subsequent filtrate; ③ Accurately measure 50 mL of the subsequent filtrate, dry it to residue, dissolve the residue in pure water to obtain an aqueous solution; ④ The aqueous solution is applied to the MCI small pore adsorption resin column, and eluted with pure water and 45~55% ethanol (mass fraction) in turn; ⑤ Then elute with 70% ethanol (mass fraction), collect the 70% ethanol eluate, evaporate to dryness, dissolve in methanol, fix the volume, filter with a microporous filter membrane, and take the subsequent filtrate to obtain the test solution; the alkaline liquid is diluted with methanol to an alkaline methanol solution; after the alkaline solid is added, 10 mL of purified water and anhydrous ethanol are added in turn; Step 2: Determination of the content of saikosaponin A, B2 and B1: ① Accurately weigh saikosaponin A, B2, and B1 reference substances, dissolve them in methanol, and prepare a mixed reference solution of saikosaponin A, B2, and B1; ② Accurately pipette 20 μL of the mixed reference solution and the test solution respectively, inject into the liquid chromatograph, detect with a diode array detector, and calculate the content of each index component by the external standard method; The liquid chromatography conditions were as follows: Chromatographic column: TC-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm); Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in the table below, the theoretical plate number calculated based on saikosaponin A peak should be no less than 10000; The gradient elution procedure includes:

[0007] Wavelength: Simultaneous detection at 210 nm and 254 nm (DAD detector); the detection wavelength of saikosaponin A is 210 nm, and the detection wavelength of saikosaponin B2 and saikosaponin B1 is 254 nm.

[0008] Temperature: 30℃; Flow rate: 1 mL / min.

[0009] Furthermore, the alkaline methanol solution is a methanol solution containing 4-6% (V / V) concentrated ammonia.

[0010] Furthermore, the alkaline solid is calcium hydroxide, and the amount of calcium hydroxide added is 12.5-37.5% of the mass of the Xiaoer Chaigui Antipyretic Granules powder.

[0011] Furthermore, the model of the MCI small pore adsorption resin is CHP20 / P120.

[0012] Furthermore, the ambient temperature during the preparation of the test solution should be maintained at 20-25° C. to prevent the adsorption capacity of the small pore adsorption resin from being affected by temperature.

[0013] Furthermore, the diameter-to-height ratio of the MCI small-pore adsorption resin column is 1:4-7.

[0014] Furthermore, after the pure water elution, 30% ethanol is added for elution, and the amount of the 30% ethanol is 30 mL. Adding 30% ethanol can effectively reduce the speed of bubble generation and make the experimental results more stable.

[0015] Furthermore, the amount of 45-55% ethanol is 30-50 mL. 55% ethanol has the best impurity removal ability, and part of saikosaponin A may be lost when the ethanol concentration is higher than 55%.

[0016] Furthermore, the power of the ultrasonic treatment is 300-400 W, and the frequency of the ultrasonic treatment is 50-60 kHz. The ultrasonic temperature should not be too high to prevent the decomposition of saikosaponin.

[0017] Furthermore, the concentration of the test solution is 1-2 g / mL, based on the weight of the drug powder.

[0018] Furthermore, the concentrations of saikosaponin A, B2 and B1 in the mixed reference solution are 0.0501 mg / mL, 0.0853 mg / mL and 0.0440 mg / mL, respectively. The concentration of the reference solution is similar to that of the test solution, and the measurement results are more consistent with the actual results.

[0019] Furthermore, the elution solvent used in the preparation process of the test solution needs to be ultrasonically removed from the air dissolved in the solvent, and the ethanol concentration used in the present invention is the mass concentration.

[0020] Beneficial technical effects of the present invention: 1. The present invention determines the contents of saikosaponins A, B2 and B1, the main active ingredients of the monarch drug Bupleurum, in the prescription of Xiaoer Chaigui Tuire Granules, which is beneficial to the improvement of the quality standard of Xiaoer Chaigui Tuire Granules.

[0021] 2. The present invention uses a methanol solution of concentrated ammonia or a combination of calcium hydroxide and ethanol solutions to extract multiple saponin active ingredients of the main herb Bupleurum in the Children's Chaigui Antipyretic Granules, which can effectively remove the interference of sugar substances in the Children's Chaigui Antipyretic Granules.

[0022] 3. The MCI small pore adsorption resin column was used to optimize the preparation conditions of the test solution of Xiaoer Chaigui Tuire Granules, which can remove most of the interfering components in the test sample, so that the separation of the chromatographic peaks of saikosaponin A, B2, and B1 is greater than 2, and the purity factor is above 980. The sample recovery rate of the method is between 95% and 105%, and the RSD value of the determination result is less than 3%. The methodological verification results show that the determination method of the present invention is accurate and reliable.

[0023] 4. The determination parameters of high performance liquid chromatography were optimized, and the commonly used ultraviolet detector-diode array detector was used, which broke through the dilemma that Bupleurum compound preparations could not be determined using high performance liquid chromatography tandem with ultraviolet detector. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a liquid chromatogram of the specificity inspection of Example 1 of the present invention at a detection wavelength of 210 nm.

[0025] Figure 2 This is a liquid chromatogram of Example 1 of the present invention with a specificity inspection at a detection wavelength of 254 nm.

[0026] Figure 3 This is a liquid chromatogram at a detection wavelength of 210 nm of the extract of Experimental Example 1 of the present invention before and after purification using a small-pore adsorption resin column.

[0027] Figure 4 This is a liquid chromatogram at a detection wavelength of 210 nm of the extract of Experimental Example 7 of the present invention before and after purification using a small-pore adsorption resin column.

[0028] In the figure: A. negative control, B. test sample, C. reference sample, D. test sample before purification in Example 1, E. test sample before purification in Example 7, 1. saikosaponin A, 2. saikosaponin B2, 3. saikosaponin B1. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the present invention and implement it, the present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to the scope of the embodiments. The test methods used in the embodiments are all conventional methods unless otherwise specified. Those skilled in the art are within the scope of protection of the present invention without making creative work.

[0030] Example 1 Preparation of the test solution of Xiaoer Chaigui Antipyretic Granules Step 1: Grind an appropriate amount of Xiaoer Chaigui Tuire Granules (pass through a No. 3 sieve) to obtain a powder. Take 3 g of the powder, weigh it accurately, place it in a stoppered conical flask, add 100 mL of a methanol solution containing 5% concentrated ammonia test solution, stopper it tightly, and weigh it. Ultrasonicate and extract for 30 min below 30°C, take it out, cool it, weigh it again, make up the lost weight with methanol, and filter it. Accurately measure 50 mL of the filtrate, place it in a recovery bottle, concentrate it to dryness under reduced pressure, and dissolve the residue in water to obtain an aqueous solution.

[0031] Step 2: The aqueous solution passes through an MCI small pore adsorption resin (CHP20 / P120) column (inner diameter 1.2 cm, column height 6 cm), eluted with 30 mL of water, discarded the aqueous solution, then eluted with 30 mL of 30% ethanol, discarded the 30% ethanol eluate, then eluted with 50 mL of 55% ethanol, discarded the 55% ethanol eluate, and finally eluted with 30 mL of 70% ethanol, collected the eluate, evaporated to dryness, dissolved the residue with methanol, transferred to a 2 mL volumetric flask, added methanol to the scale, shook well, filtered through a microporous filter membrane, and took the filtrate to obtain the product.

[0032] Example 2 Preparation of the test solution of Xiaoer Chaigui Antipyretic Granules On the basis of Example 1, the drug powder was adjusted from 3 g to 2 g, the elution with 55% ethanol 50 mL was adjusted to 55% ethanol 30 mL, and the rest remained unchanged.

[0033] Example 3 Preparation of the test solution of Xiaoer Chaigui Antipyretic Granules On the basis of Example 1, the 5% concentrated ammonia test solution in methanol was adjusted to a 4% concentrated ammonia test solution in methanol, and the other contents remained unchanged.

[0034] Example 4 Preparation of the test solution of Xiaoer Chaigui Antipyretic Granules On the basis of Example 1, the 5% concentrated ammonia test solution in methanol was adjusted to a 6% concentrated ammonia test solution in methanol, and the other contents remained unchanged.

[0035] Example 5 Preparation of the test solution of Xiaoer Chaigui Antipyretic Granules On the basis of Example 1, the inner diameter of the small pore adsorption resin was adjusted from 1.2 cm to 1.0 cm, the column height was adjusted from 6 cm to 7 cm, the elution with 30% ethanol 30 mL and the elution with 55% ethanol 50 mL was adjusted to elution with 45% ethanol 50 mL once, and the rest remained unchanged.

[0036] Example 6 Preparation of the test solution of Xiaoer Chaigui Antipyretic Granules On the basis of Example 1, the inner diameter of the small-pore adsorption resin was adjusted from 1.2 cm to 1.5 cm, and the other parameters remained unchanged.

[0037] Example 7 Preparation of the test solution of Xiaoer Chaigui Antipyretic Granules Step 1: Take an appropriate amount of Xiaoer Chaigui Antipyretic Granules, grind them finely (pass through a No. 3 sieve), take 4 g of the powder, weigh it accurately, and place it in a 100 mL volumetric flask. Add 1 g of calcium hydroxide powder, 10 mL of water and anhydrous ethanol in turn, make up to volume, seal it, and weigh it. Ultrasonic extraction below 30°C for 30 min, take it out, cool it, weigh it again, make up the lost weight with anhydrous ethanol, and filter it. Accurately measure 50 mL of the filtrate, place it in a flask, concentrate it to dryness under reduced pressure, and dissolve the residue in water to obtain an aqueous solution.

[0038] Step 2: The aqueous solution passes through an MCI small pore adsorption resin (CHP20 / P120) column (inner diameter 1.2 cm, column height 6 cm), eluted with 30 mL of water, discarded the aqueous solution, then eluted with 30 mL of 30% ethanol, discarded the 30% ethanol eluate, then eluted with 50 mL of 55% ethanol, discarded the 55% ethanol eluate, and finally eluted with 30 mL of 70% ethanol, collected the eluate, evaporated to dryness, dissolved the residue with methanol, transferred to a 2 mL volumetric flask, added methanol to the scale, shook well, filtered through a microporous filter membrane, and took the filtrate to obtain the product.

[0039] Example 8 Preparation of the test solution of Xiaoer Chaigui Antipyretic Granules On the basis of Example 7, the calcium hydroxide powder was changed from 1 g to 0.5 g, and the others remained unchanged.

[0040] Example 9 Preparation of the test solution of Xiaoer Chaigui Antipyretic Granules On the basis of Example 7, the calcium hydroxide powder was changed from 1 g to 1.5 g, and the others remained unchanged.

[0041] Example 10 Determination of the contents of saikosaponins A, B2 and B1 in Xiaoer Chaigui Tuire Granules (1) Accurately weigh appropriate amounts of saikosaponin A, B2, and B1 reference substances and dissolve them in methanol to prepare mixed reference substance solutions containing saikosaponin A, B2, and B1 at concentrations of 0.0501 mg / mL, 0.0853 mg / mL, and 0.0440 mg / mL, respectively, for later use.

[0042] (2) Accurately pipette 20 μL of the mixed reference solution and test solution prepared in step 1 respectively, inject them into the liquid chromatograph, and calculate the content of each index component by the external standard method.

[0043] (3) Liquid chromatography conditions: Chromatographic column: TC-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm); Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in the table below, the theoretical plate number calculated based on saikosaponin A peak should be no less than 10000; Gradient elution procedure

[0044] Wavelength: Simultaneous detection at 210 nm and 254 nm (DAD detector); the detection wavelength of saikosaponin A is 210 nm, and the detection wavelength of saikosaponin B2 and saikosaponin B1 is 254 nm; Column temperature: 30°C; Flow rate: 1.0 mL / min.

[0045] Example 11 Determination of the contents of saikosaponins A, B2 and B1 in Xiaoer Chaigui Tuire Granules Based on Example 10, the gradient elution procedure was changed to

[0046] Example 12 Determination of the contents of saikosaponins A, B2 and B1 in Xiaoer Chaigui Tuire Granules Based on Example 10, the gradient elution procedure was changed to

[0047] Example 13 Determination of the contents of saikosaponins A, B2 and B1 in Xiaoer Chaigui Tuire Granules Based on Example 10, the gradient elution procedure was changed to

[0048] Example 14 Study on the methodological determination of saikosaponins A, B2 and B1 in Xiaoer Chaigui Tuire Granules (1) Specificity test According to the method of Example 1, the test solution and the negative sample solution were prepared respectively, and the negative sample solution, the test solution and the reference solution were respectively aspirated, and the detection was carried out according to the detection conditions of Example 10. The results showed that at the corresponding retention times of saikosaponin A, saikosaponin B2 and saikosaponin B1, no corresponding chromatographic peaks appeared in the negative sample, and the negative sample had no interference. See the attached chromatogram for the Figure 1 and attached Figure 2 .

[0049] (2) System durability investigation The changes in the instrumental chromatographic behavior under different flow rates, different temperatures, different elution programs, and different chromatographic columns were investigated. The results are shown in Table 1.

[0050] Table 1 System durability investigation

[0051] (3) Linear relationship Mixed reference solutions of different concentrations were prepared, and each solution was injected into a liquid chromatograph and measured according to the method of Example 10. The concentrations of saikosaponin A, saikosaponin B2, and saikosaponin B1 were ( X ) and peak area ( Y ) The standard curve was drawn, and the linear regression equation and linear range are shown in Table 2.

[0052] Table 2 Linear regression equation and linear range

[0053] (4) Precision The mixed reference substance was sampled 6 times continuously and detected according to the method of Example 10. The RSD value of the peak area was no more than 0.49%. The results showed that the parallel injection precision of the instrument was good. The results are shown in Table 3.

[0054] Table 3 Peak area results of precision investigation

[0055] (5) Repeatability Weigh 6 portions of the same batch of Xiaoer Chaigui Tuire Granules, prepare the test solution according to the method in Example 1, inject and measure according to the detection conditions in Example 10, and the RSD values ​​of the contents of saikosaponin A, saikosaponin B2 and saikosaponin B1 are 1.86%, 1.15% and 1.19%, respectively, indicating that the method has good repeatability. The results are shown in Table 4.

[0056] Table 4 Results of repeatability test

[0057] (6) Stability The test solution prepared according to the method of Example 1 was taken and sampled and measured at 0, 2, 4, 6, 8, 10, 12 and 24 hours according to the detection conditions of Example 10. The RSDs of the peak areas of saikosaponin A, saikosaponin B2 and saikosaponin B1 were all less than 3%, indicating that the test solution prepared by this method had good stability within 24 hours. The results are shown in Table 5.

[0058] Table 5 Stability test results

[0059] (7) Sample recovery rate Six portions of Xiaoer Chaigui Tuire Granules with known contents were accurately weighed, each portion was 1.5 g, and 1 mL of the corresponding reference solution was added respectively. The samples were prepared according to the test sample preparation method, and the samples were measured according to the above chromatographic conditions. The recovery rate was calculated, and the results are shown in Table 6. The results showed that the recovery rate was between 95% and 105%, indicating that the method had good accuracy and met the requirements.

[0060] Recovery rate %=(C–A) / B×100%, where A is the amount of saikosaponin in the test sample, B is the amount of reference substance added, and C is the measured value.

[0061] Table 6 Sample recovery test results

[0062] (8) Intermediate precision experiment Different analysts, one day apart, used two different liquid chromatographs to determine the content of the samples at different locations, and calculated the average value. Three samples of the same batch were taken each time to calculate the content of saikosaponin A, saikosaponin B2, and saikosaponin B1, and to investigate their intermediate precision. The results are shown in Table 7.

[0063] Table 7 Intermediate precision test results

[0064] Example 15 Investigation of the preparation method and analysis method of the test solution The test solutions prepared in Examples 1 to 9 were taken and tested according to the chromatographic conditions of Examples 10 to 13 to determine the chromatographic peak purity factors and separations of saikosaponin A, saikosaponin B2, and saikosaponin B1 in Xiaoer Chaigui Tuire Granules. The results are shown in Table 8.

[0065] Table 8 Purity factors and separation of saikosaponin A, B2 and B1 peaks in the chromatograms of each method

[0066] Example 16: Extraction method of Xiaoer Chaigui antipyretic granules Take 4 g of the powder of Xiaoer Chaigui Tuire Granules and put it in a 100 mL volumetric flask, add 0.5 g of calcium hydroxide and 10 mL of water in sequence, mix well, slowly add ethanol to the scale, ultrasonically treat for 30 min, filter, take 50 mL of the filtrate, evaporate to dryness, and transfer the residue to a 2 mL volumetric flask with methanol.

[0067] Example 17: Extraction method of Xiaoer Chaigui antipyretic granules Take 4 g of the powder of Xiaoer Chaigui Tuire Granules and put it in a 100 mL volumetric flask, add 1 g of calcium hydroxide and 10 mL of water in sequence, mix well, slowly add ethanol to the scale, ultrasonically treat for 30 min, filter, take 50 mL of the filtrate, evaporate to dryness, and transfer the residue to a 2 mL volumetric flask with methanol.

[0068] Example 18: Extraction method of Xiaoer Chaigui antipyretic granules Take 4 g of Xiaoer Chaigui Tuire Granules powder and place it in a 100 mL conical flask, add 100 mL of methanol solution containing 5% concentrated ammonia test solution, ultrasonically treat for 30 min, filter, take 50 mL of the filtrate, evaporate to dryness, and transfer the residue to a 2 mL volumetric flask with methanol.

[0069] Comparative Example 1: Extraction method of Xiaoer Chaigui antipyretic granules Take 4 g of Xiaoer Chaigui Tuire Granules and place them in a 100 mL volumetric flask. Add 1 g of calcium hydroxide and 20 mL of water in sequence, mix well, slowly add ethanol to the scale, ultrasonically treat for 30 min, filter, take 50 mL of the filtrate, evaporate to dryness, and transfer the residue to a 2 mL volumetric flask with methanol.

[0070] Comparative Example 2: Extraction method of Xiaoer Chaigui antipyretic granules Take 4 g of Xiaoer Chaigui Tuire Granules and place them in a 100 mL volumetric flask. Add 1.5 g of calcium hydroxide and 20 mL of water in sequence, mix well, slowly add methanol to the scale, ultrasonically treat for 30 min, filter, take 50 mL of the filtrate, evaporate to dryness, and transfer the residue to a 2 mL volumetric flask with methanol.

[0071] Comparative Example 3: Extraction method of Xiaoer Chaigui antipyretic granules Take 4 g of Xiaoer Chaigui Tuire Granules and place them in a 100 mL volumetric flask. Add 1 g of calcium hydroxide and 20 mL of water in sequence, mix well, slowly add methanol to the scale, ultrasonically treat for 30 min, filter, take 50 mL of the filtrate, evaporate to dryness, and transfer the residue to a 2 mL volumetric flask with methanol.

[0072] Comparative Example 4: Extraction method of Xiaoer Chaigui antipyretic granules Take 4 g of Xiaoer Chaigui Tuire Granules and place them in a 100 mL volumetric flask. Add 1.5 g of calcium hydroxide and 10 mL of water in sequence, mix well, slowly add methanol to the scale, ultrasonically treat for 30 min, filter, take 50 mL of the filtrate, evaporate to dryness, and transfer the residue to a 2 mL volumetric flask with methanol.

[0073] Comparative Example 5: Purification method of extract of Xiaoer Chaigui Antipyretic Granules Preparation of test sample: Take the aqueous solution extracted in step 1 of Example 7, pass the aqueous solution through a D101 macroporous adsorption resin column (column height 6 cm, diameter 1.5 cm), and use 100 mL of water, 100 mL of 40% ethanol and 100 mL of 80% ethanol to elute in sequence, evaporate the 80% ethanol fraction to dryness, dissolve the residue with methanol, transfer to a 2 mL volumetric flask, add methanol to the scale, shake well, filter through a microporous filter membrane, and take the filtrate to obtain.

[0074] Comparative Example 6: Purification method of extract of Xiaoer Chaigui Antipyretic Granules Preparation of test sample: Take the aqueous solution extracted in step 1 of Example 1, dilute the aqueous solution to 30 mL, extract 5 times with water-saturated n-butanol at a ratio of 1:1 (volume ratio), combine the 5 extracts and evaporate to dryness, dissolve the residue with methanol, transfer to a 2 mL volumetric flask, add methanol to the scale, shake well, filter through a microporous filter membrane, and take the filtrate to obtain.

[0075] Comparative Example 7: Purification method of extract of Xiaoer Chaigui Antipyretic Granules The preparation was carried out according to Example 7, except that the amount of 70% ethanol was adjusted from 30 mL to 25 mL. The detection was carried out according to Example 10. As a result, a small amount of saikosaponin B1 remained in the column.

[0076] Comparative Example 8: Purification method of extract of Xiaoer Chaigui Antipyretic Granules The preparation was carried out according to Example 1, and 30% ethanol was removed, resulting in a large number of bubbles and fractures in the chromatographic column. The results could still be obtained but were unstable.

[0077] Comparative Example 9: Purification method of extract of Xiaoer Chaigui Antipyretic Granules The method was carried out according to Example 7, except that the concentration of 70% ethanol was adjusted to 65%. The method was tested according to Example 10. As a result, a small amount of saikosaponin B1 remained in the column.

[0078] Example 19: Investigation of different extraction methods of Xiaoer Chaigui Tuire Granules Take the sample solutions prepared in Examples 16-18 and Comparative Examples 1-4, detect according to the method of Example 10, and compare the peak areas of saikosaponin A, saikosaponin B2 and saikosaponin B1. The results are shown in Table 9. The extraction method of Comparative Examples 2-4 adopts the combination of "calcium hydroxide + methanol", and the total content of the three saponins in the samples is low, the difference is large, and it is unstable. The extraction methods of Examples 16, 17 and Comparative Example 1 adopt the combination of "calcium hydroxide + ethanol", and Example 17 extracts the highest content of saikosaponin A. Example 18 uses 5% concentrated ammonia test solution for extraction, and the total content of the three saponins is the highest. The powder used in Examples 16-18 and Comparative Examples 1-4 comes from the same batch of Xiaoer Chaigui Antipyretic Granules from the same manufacturer.

[0079] Table 9 Comparison of seven extraction methods

[0080] Example 20: Investigation of different purification methods for Xiaoer Chaigui Antipyretic Granules The test samples and test results of Example 1, Example 7 and Comparative Examples 5-9 are shown in Table 10.

[0081] Table 10 Effects of different purification methods

[0082] By comparing the elution capacity of methanol and ethanol, it was found that ethanol had a suitable elution effect, so ethanol was selected as the elution solvent; by comparing the effects of different concentrations of ethanol on the chromatographic column, it was found that 55% ethanol had the best impurity removal effect, so 55% ethanol was selected for impurity removal, and the 70% ethanol elution fraction was used as the target fraction.

[0083] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for determining the contents of saikosaponins A, B2 and B1 in Xiaoer Chaigui Tuire Granules, characterized in that: The following steps are involved: Step 1, preparation of the test solution: ① Take an appropriate amount of Xiaoer Chaigui Tuire Granules powder, accurately weigh it, add alkaline liquid or alkaline solid, weigh it again after the volumetric bottle is fixed to volume; ② Ultrasonic treatment extraction, cool, make up the lost weight with methanol, filter, and obtain the subsequent filtrate; ③ Accurately measure 50 mL of the subsequent filtrate, dry it to residue, dissolve the residue in pure water to obtain an aqueous solution; ④ The aqueous solution is applied to the MCI small pore adsorption resin column, and eluted with pure water and 45~55% ethanol (mass fraction) in turn; ⑤ Then elute with 70% ethanol (mass fraction), collect the 70% ethanol eluate, evaporate to dryness, dissolve in methanol, fix the volume, filter with a microporous filter membrane, and take the subsequent filtrate to obtain the test solution; the alkaline liquid is diluted with methanol to an alkaline methanol solution; after the alkaline solid is added, 10 mL of purified water and anhydrous ethanol are added in turn; Step 2: Determination of the content of saikosaponin A, B2 and B1: ① Accurately weigh saikosaponin A, B2, and B1 reference substances, dissolve them in methanol, and prepare a mixed reference solution of saikosaponin A, B2, and B1; ② Accurately pipette 20 μL of the mixed reference solution and the test solution respectively, inject into the liquid chromatograph, detect with a diode array detector, and calculate the content of each index component by the external standard method; The liquid chromatography conditions were as follows: Chromatographic column: TC-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm); Mobile phase: acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in the table below, the theoretical plate number calculated based on saikosaponin A peak should be no less than 10000; Gradient elution procedure include: Wavelength: Simultaneous detection at 210 nm and 254 nm (DAD detector); the detection wavelength of saikosaponin A is 210 nm, and the detection wavelength of saikosaponin B2 and saikosaponin B1 is 254 nm; Temperature: 30℃; Flow rate: 1 mL / min.

2. The content determination method according to claim 1, characterized in that: The alkaline methanol solution is a methanol solution containing 4-6% (V / V) concentrated ammonia.

3. The content determination method according to claim 1, characterized in that: The amount of calcium hydroxide added is 12.5-37.5% of the weight of the Xiaoer Chaigui Antipyretic Granules powder.

4. The content determination method according to claim 1, characterized in that: The model of the MCI small pore adsorption resin is CHP20 / P120.

5. The content determination method according to claim 1, characterized in that: The diameter-to-height ratio of the MCI small-pore adsorption resin column is 1:4-7.

6. The content determination method according to claim 1, characterized in that: After the pure water elution, 30% ethanol is added for elution, and the amount of the 30% ethanol used is 30 mL.

7. The content determination method according to claim 1, characterized in that: The amount of the 45-55% ethanol is 30-50 mL.

8. The content determination method according to claim 1, characterized in that: The power of the ultrasonic treatment is 300-400 W, and the frequency of the ultrasonic treatment is 50-60 kHz.

9. The content determination method according to claim 1, characterized in that: The concentration of the test solution is 1-2 g / mL, based on the weight of the drug powder.

10. The content determination method according to claim 1, characterized in that: The concentrations of saikosaponin A, B2 and B1 in the mixed reference solution are 0.0501 mg / mL, 0.0853 mg / mL and 0.0440 mg / mL respectively.

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