A quality detection method for schisandra chinensis or schisandra chinensis formula granules

Abstract

The present application relates to a kind of quality detection method of schisandra or schisandra formula granules, including characteristic spectrum, content determination and the like method;Characteristic spectrum determination method includes: (1) the preparation of schisandra reference solution of control medicinal material;(2) the preparation of reference solution of control sample;(3) by liquid chromatograph with acetonitrile as mobile phase A, 0.05% formic acid solution of volume concentration as mobile phase B is determined to obtain the characteristic spectrum of schisandra control medicinal material;(4) the preparation and determination of test sample solution schisandra or schisandra formula granules;(5) the quality of schisandra or schisandra formula granules is judged using characteristic spectrum;The present application can effectively control the quality of schisandra or schisandra formula granules by determining the characteristic spectrum of schisandra or schisandra formula granules.

Description

Technical Field

[0001] The invention belongs to a quality detection method for traditional Chinese medicine or its formula granules, and particularly relates to a quality detection method for acanthopanax acanthopanax cortex or acanthopanax acanthopanax cortex formula granules. Background Art

[0002] Acanthopanax acanthopanax was first recorded in the "Shennong Bencao Jing" and listed as a top-grade medicine. It has been recorded in all the herbal books of all dynasties. It is also known as Wuqia, and has other names such as Wuhua, Mugu, Zhuifengshi, Citong, and Baici. "Ming Yi Bie Lu" says: "The five-leaf ones are good. They grow in Hanzhong and Yuanju. The stems are collected in May and July, and the roots are collected in October. They are dried in the shade." Because of its effect of dispelling rheumatism and strengthening muscles and bones, manufacturers have developed a variety of products such as Acanthopanax acanthopanax health wine, which is an in-depth development and exploration beyond traditional medicine. According to the 2020 edition of the "Chinese Pharmacopoeia", the clinical application of this product has the effects of dispelling wind and dampness, nourishing the liver and kidneys, strengthening muscles and bones, and promoting diuresis and reducing swelling. It is mainly used for rheumatic arthritis, weakness of muscles and bones, delayed walking in children, physical weakness, edema, beriberi, etc.

[0003] There is considerable confusion regarding the variety of medicinal Acanthopanax bark. Other species collectively referred to as Acanthopanax bark include Acanthopanax senticosus, Acanthopanax rubra, and Tripterygium wilfordii. It is now clear that the root bark of Acanthopanax stenoticum (Araliaceae) is the authentic Acanthopanax bark. Perilla frutescens bark (Asclepiadaceae), also known as Northern Acanthopanax bark due to its predominant presence in northern my country and its distinctive aroma, is also known as Perilla frutescens bark (now known as Perilla frutescens bark or Northern Acanthopanax bark). Perilla frutescens bark is toxic and has cardiotonic and diuretic properties, so excessive use is advised. Numerous reports of poisoning from Perilla frutescens bark have been reported in modern clinical practice. Lycium bark, derived from the dried root bark of Lycium barbarum (Lycium chinense), is widely distributed and grows throughout much of my country. It is often mistaken for Acanthopanax bark in commercial products. The three root bark medicinal herbs, Acanthopanax bark, Perilla frutescens bark, and Lycium bark, are similar in appearance, making identification difficult and potentially confusing during clinical use. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a quality detection method for Acanthopanax Cortex or Acanthopanax Cortex Formula Granules, which overcomes the shortcomings of the existing technology and uses a characteristic spectrum to judge the quality of Acanthopanax Cortex or Acanthopanax Cortex Formula Granules, further effectively ensuring the quality of Acanthopanax Cortex or Acanthopanax Cortex Formula Granules.

[0005] A quality detection method for Acanthopanax acanthopanax or Acanthopanax acanthopanax formula granules, comprising properties, thin layer chromatography identification, characteristic spectrum, inspection, extract and content determination methods, wherein the characteristic spectrum determination method comprises:

[0006] (1) Preparation of a reference solution of Acanthopanax Cortex: Add water to the Acanthopanax Cortex, heat, filter, add methanol solution to the residue, and filter to obtain a reference solution of Acanthopanax Cortex;

[0007] (2) Preparation of reference substance solution: Take appropriate amount of chlorogenic acid and 3,5-di-O-caffeoylquinic acid, add methanol to make a mixed solution, which is used as the reference substance solution;

[0008] (3) Determination: Aspirate the Acanthopanax Cortex reference medicinal material solution and the reference substance solution, inject them into the liquid chromatograph, use acetonitrile as mobile phase A and 0.05% volume concentration formic acid solution as mobile phase B, and determine to obtain a characteristic spectrum;

[0009] (4) Determination of the test solution: add water to Acanthopanax acanthopanax or Acanthopanax acanthopanax granules, heat, filter, treat the residue with methanol solution, filter to obtain the test solution, draw the test solution, inject into liquid chromatography, and determine;

[0010] (5) Use the characteristic spectrum to judge the quality of Acanthopanax acanthopanax or Acanthopanax acanthopanax formula granules.

[0011] Furthermore, the preparation method of the acanthopanax cortex formula granules is as follows: take 5600g of acanthopanax cortex slices, add water to boil, filter, concentrate the filtrate into a clear paste (the dry extract paste rate is 9.0% to 16.0%), add an appropriate amount of auxiliary materials, dry (or dry and crush), add an appropriate amount of auxiliary materials, mix well, granulate, and make 1000g.

[0012] Furthermore, the quality judgment of Acanthopanax Cortex includes: judging whether the test sample is Acanthopanax Cortex, including similar products such as Cortex Acanthopanax and Cortex Lycii, and distinguishing different medicinal parts of Cortex Acanthopanax.

[0013] Furthermore, the quality of the Acanthopanax Cortex formula granules can be determined through the characteristic spectra of the Acanthopanax Cortex control medicinal material and chlorogenic acid and 3,5-di-O-caffeoylquinic acid, because the characteristic peaks of the control medicinal material represent the content of these substances. If there are no peaks of these substances in the test sample, it is inferred that these substances are also not present in the formula granules, thereby determining the quality of the Acanthopanax Cortex formula granules.

[0014] Furthermore, 12 characteristic peaks should be present in the chromatogram of the test sample, and the retention times of the 12 characteristic peaks in the chromatogram of the Acanthopanax Cortex reference medicinal material should correspond to each other, among which Peak 4 and Peak 10 should correspond to the retention times of the corresponding reference peaks respectively; the peak corresponding to the chlorogenic acid reference peak is the S1 peak, and the relative retention times of Peak 1, Peak 2, Peak 3, Peak 5, Peak 6 and S1 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, and the specified values ​​are: 0.55 (Peak 1), 0.63 (Peak 2), 0.77 (Peak 3), 1.05 (Peak 5), and 1.10 (Peak 6). The peak corresponding to the 3,5-O-dicaffeoylquinic acid reference substance is the S2 peak. Calculate the relative retention times of Peaks 7, 8, 9, 11, and 12 relative to the S2 peak. These relative retention times should be within ±10% of the specified values: 0.72 (Peak 7), 0.81 (Peak 8), 0.98 (Peak 9), 1.04 (Peak 11), and 1.06 (Peak 12). Furthermore, the ratio of the peak area of ​​Peak 7 to the peak area of ​​Peak 4 should be greater than 0.88.

[0015] Furthermore, the characteristic peak numbers of the Cortex Periplocae and Cortex Acanthopanacis are significantly different, making them easy to distinguish.

[0016] Furthermore, the characteristic peak numbers of the Cortex Lycii and Cortex Acanthopanacis are significantly different and can be easily distinguished.

[0017] Furthermore, the different medicinal parts of Acanthopanax Cortex are distinguished as follows: by comparing the characteristic spectra of different parts of Acanthopanax Cortex, the characteristic peaks of the root bark and other parts are significantly different, the stem bark part has no obvious characteristic peaks at peak 1, peak 2, and peak 12, and the response value of peak 7 (1,3-di-O-caffeoylquinic acid) is low, the root wood core has no obvious characteristic peaks at peak 1 and peak 9, and the response value of peak 7 (1,3-di-O-caffeoylquinic acid) is low, the stem wood core has no obvious characteristic peaks at peak 1 and peak 9, and the response value of peak 7 (1,3-di-O-caffeoylquinic acid) is low.

[0018] Furthermore, the content determination method includes:

[0019] (1) Preparation of reference solution: Chlorogenic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid were added to ethanol solution to prepare a mixed solution;

[0020] (2) Preparation of test solution: add methanol to Acanthopanax acanthopanax or Acanthopanax acanthopanax granules, treat, and filter to obtain the test solution;

[0021] (3) Determination: Aspirate the reference solution and the test solution separately, inject them into the liquid chromatograph, use acetonitrile as mobile phase A and 0.05% by volume formic acid solution as mobile phase B, and perform determination.

[0022] Furthermore, in step (1) of the characteristic spectrum determination method, the volume concentration of methanol in the preparation of the Acanthopanax Cortex reference medicinal material reference solution is 50%, and the concentrations of chlorogenic acid and 3,5-di-O-caffeoylquinic acid in the reference substance solution in step (2) are 50 μg / ml respectively.

[0023] Furthermore, in step (4) of the characteristic spectrum determination method, the volume concentration of methanol is 50%, and the treatment method is ultrasonic treatment.

[0024] Furthermore, in step (4) of the characteristic spectrum determination method, methanol with a volume concentration of 50% is used to make up for the lost weight after treatment.

[0025] Furthermore, the chromatographic conditions in step (3) of the characteristic spectrum determination method are as follows: octadecylsilane bonded silica gel is used as the filler, the flow rate is 0.8 ml per minute, the column temperature is 20° C., the detection wavelength is 280 nm, and the gradient elution is performed according to the following procedure:

[0026] 0-6 min, mobile phase A is 5%, mobile phase B is 95%;

[0027] 6-55 min, mobile phase A from 5% to 13%, mobile phase B from 95% to 87%;

[0028] 55-70 min, mobile phase A from 13% to 19%, mobile phase B from 87% to 81%;

[0029] 70-80 min, mobile phase A: 19%, mobile phase B: 81%;

[0030] 80-105 min, mobile phase A from 19% to 30%, mobile phase B from 81% to 70%;

[0031] 105-107 min, mobile phase A is 30%→5%, mobile phase B is 70%→95%.

[0032] Furthermore, in step (1) of the content determination method, the volume concentration of methanol is 75%, the concentration of chlorogenic acid in the reference solution is 50 μg / ml, the concentration of 3,5-di-O-caffeoylquinic acid is 10 μg / ml, and the concentration of 4,5-di-O-caffeoylquinic acid is 10 μg / ml.

[0033] Furthermore, in step (2) of the content determination method: the volume concentration of methanol is 75%, and the treatment method is ultrasonic treatment.

[0034] Furthermore, in step (2) of the content determination method, methanol with a volume concentration of 75% is used to make up for the lost weight after treatment.

[0035] Furthermore, the chromatographic conditions in step (3) of the content determination method are: octadecylsilane bonded silica gel as the filler, column temperature of 22° C., flow rate of 0.7 ml per minute, and detection wavelength of 327 nm.

[0036] Beneficial effects of the present invention:

[0037] (1) The present invention establishes a characteristic spectrum and content determination method for Acanthopanax acanthopanax or Acanthopanax acanthopanax granules. The characteristic spectrum determination method is simple and efficient. The chromatographic peaks in the chromatogram can be well separated and have good peak shapes. The quality of Acanthopanax acanthopanax or Acanthopanax acanthopanax granules can be effectively controlled, providing a guarantee for industrialization.

[0038] (2) The present invention distinguishes Acanthopanax Cortex, Periploca Cortex, and Lycium Bark Cortex through characteristic patterns, thereby avoiding the use of incorrect medicinal materials, which may lead to delayed treatment and have the opposite effect.

[0039] (3) The present invention can distinguish different medicinal parts of Acanthopanax Cortex by comparing the characteristic spectra of the medicinal pieces of different parts of Acanthopanax Cortex, prevent the use of other medicinal parts of Acanthopanax Cortex, and further ensure the quality of Acanthopanax Cortex and its formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the characteristic spectrum of the reference medicinal material of Acanthopanax acanthopanax; Figure 1 Peak 2: neochlorogenic acid; Peak 4 (S1): chlorogenic acid; Peak 5: cryptochlorogenic acid; Peak 6: caffeic acid; Peak 7: 1,3-O-dicaffeoylquinic acid; Peak 10 (S2): 3,5-di-O-caffeoylquinic acid; Peak 11: 4,5-O-dicaffeoylquinic acid;

[0041] Figure 2 It is the common peak superposition characteristic spectrum of chromatographic column (different manufacturers and different batches) tests;

[0042] Figure 3 It is the common peak superposition characteristic spectrum of different flow rate tests;

[0043] Figure 4 This is a comparison chart of similar HPLC products of Acanthopanax acanthopanax and Periploca odorata;

[0044] Figure 5 This is a comparison chart of HPLC anti-counterfeit products of Acanthopanax Cortex and Rehmanniae Radix;

[0045] Figure 6 This is a comparison chart of the characteristic spectra of different medicinal parts of Acanthopanax acanthopanax;

[0046] Figure 7 This is the characteristic spectrum of the reference medicinal material Acanthopanax Cortex obtained according to the original method, and Peak 2 is the green source peak. DETAILED DESCRIPTION

[0047] Example 1

[0048] 1. Instruments, reagents and test drugs

[0049] (1) Instruments: Shimadzu high performance liquid chromatograph (LC-2030 Plus, Shimadzu Co., Ltd., Japan), LabSolutions chromatography workstation, SPD-10A UV detector; Thermo Fisher Scientific (U3000, Thermo Fisher Scientific (China) Technology Co., Ltd.), Chromeleon 7.2 chromatographic workstation, VWD-3x00 UV detector; Thermo Fisher Scientific (U3000, Thermo Fisher Scientific (China) Technology Co., Ltd.), Chromeleon 7.2 chromatographic workstation, DAD-3x00 detector; Shimadzu Shim-pack GISTC18-AQ (4.6 mm × 250 mm, 5 μm), Medium Spectrum Blue XR-C18 (4.6 mm × 250 mm, 5 μm), Shimadzu Shim-pack GISTC18 (4.6 mm × 250 mm, 5 μm); constant temperature water bath (HMTD-7000, Beijing Yongguangming Medical Instrument Co., Ltd.); ultrasonic cleaner (KQ-300DE, Kunshan Ultrasonic Instrument Co., Ltd.); 1 / 10,000 balance (PX224ZH, Ohaus Instrument Co., Ltd.); 1 / 100,000 balance (AWU220D, Shimadzu Corporation, Japan).

[0050] (2) Reagents: Methanol (Tianjin Komeiou Chemical Reagent Co., Ltd.) of chromatographic grade; acetonitrile (Tianjin Komeiou Chemical Reagent Co., Ltd.) of chromatographic grade; ethanol (Tianjin Zhiyuan Chemical Reagent Co., Ltd.), formic acid (Tianjin Komeiou Chemical Reagent Co., Ltd.); and water of ultrapure grade (prepared in the laboratory).

[0051] (3) Reference substances and reference medicinal materials: chlorogenic acid (batch number: 110753-202119, content: 96.3%, China Food and Drug Inspection Institute), 3,5-O-dicaffeoylquinic acid (batch number: 111782-202208, content: 100%, China Food and Drug Inspection Institute), 4,5-di-O-caffeoylquinic acid (batch number: 111894-202103, content: 95.2%, China Food and Drug Inspection Institute), Acanthopanax cortex control medicinal materials (batch number: 121523-201703, China Food and Drug Inspection Institute), Perennial Croton cortex (batch number: 230201-230205, China Food and Drug Inspection Institute), and Rehmannia root bark (batch number: 230201-230205, China Food and Drug Inspection Institute).

[0052] 2. Characteristic spectrum determination method

[0053] Preparation of the reference solution of Acanthopanax Cortex: Take 1.0 g of Acanthopanax Cortex reference medicinal material, place it in a stoppered conical flask, add 50 ml of water, heat and reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 50% methanol solution by volume to the residue, treat it with ultrasound (power 300 W, frequency 40 kHz) for 30 minutes, cool, filter, and take the filtrate as the reference solution of the control medicinal material.

[0054] Preparation of reference substance solution: Take appropriate amount of chlorogenic acid reference substance and 3,5-di-O-caffeoylquinic acid reference substance, weigh them accurately, add methanol to make a mixed solution containing 50 μg of each per 1 ml, which is used as the reference substance solution.

[0055] Assay Method: Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as the packing (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile as mobile phase A, 0.2% by volume formic acid solution as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.8 ml / min; column temperature, 20°C; detection wavelength, 280 nm. The number of theoretical plates, calculated based on the chlorogenic acid peak, should be no less than 3000.

[0056] Table 1 Gradient elution program

[0057]

[0058] Accurately draw 10 μl of reference solution and test solution respectively, inject into liquid chromatography for determination, and the characteristic spectrum of the reference medicinal material of Acanthopanax Cortex is shown in Figure 1 .

[0059] Preparation of test solution: Take an appropriate amount of Acanthopanax Cortex granules, grind them into powder, take 1.0 g, place it in a stoppered conical flask, add 25 mL of methanol with a volume concentration of 50%, and ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes. Take it out, cool, filter, and take the filtrate to obtain the test solution. Take the test solution and inject it into the liquid chromatograph for determination.

[0060] There should be 12 characteristic peaks in the chromatogram of the test sample, and the retention times of the 12 characteristic peaks in the chromatogram of the Acanthopanax Cortex reference medicinal material should correspond to each other. Peak 4 and Peak 10 should correspond to the retention times of the corresponding reference peaks respectively; the peak corresponding to the chlorogenic acid reference peak is the S1 peak. The relative retention times of Peak 1, Peak 2, Peak 3, Peak 5, Peak 6 and S1 peak are calculated. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​are: 0.55 (Peak 1), 0.63 (Peak 2), 0.77 (Peak 3), 1.05 (Peak 5), and 1.10 (Peak 6). The peak corresponding to the 3,5-O-dicaffeoylquinic acid reference substance is the S2 peak. Calculate the relative retention times of Peaks 7, 8, 9, 11, and 12 relative to the S2 peak. These relative retention times should be within ±10% of the specified values: 0.72 (Peak 7), 0.81 (Peak 8), 0.98 (Peak 9), 1.04 (Peak 11), and 1.06 (Peak 12). Furthermore, the ratio of the peak area of ​​Peak 7 to the peak area of ​​Peak 4 should be greater than 0.88.

[0061] 3. Methodological Investigation of Characteristic Spectrum Determination

[0062] (1) Investigation of extraction methods: Test solutions were prepared using different extraction methods and measured according to "2. Characteristic spectrum determination method" in Example 1.

[0063] Table 2 Comparison of extraction methods

[0064]

[0065] The results showed that there was little difference between the different extraction methods, but ultrasonic extraction was convenient for subsequent experiments, so ultrasonic extraction was selected as the sample extraction method.

[0066] (2) Extraction time investigation: The test sample solutions were prepared at different extraction times and measured according to "2. Characteristic spectrum determination method" in Example 1.

[0067] Table 3 Comparison of extraction time

[0068]

[0069] The results showed that the difference between different extraction times was not obvious, so the 30 minutes with the highest response value was selected as the extraction time of the sample.

[0070] (3) Investigation of extraction solvents: Prepare test sample solutions using different extraction solvents and perform the measurement according to “2. Characteristic spectrum determination method” in Example 1.

[0071] Table 4 Comparison of extraction solvents

[0072]

[0073] The results showed that there was no significant difference in the test samples prepared with the three extraction solvents. To facilitate subsequent experiments, 50% methanol was selected as the extraction solvent for the test samples.

[0074] (4) Investigation of sampling amount: Prepare test sample solutions with different sampling amounts and perform the measurement according to “2. Characteristic spectrum measurement method” in Example 1.

[0075] Table 5 Comparison of sampling amounts

[0076]

[0077]

[0078] The results showed that the differences in the test solutions with different sampling amounts were small, but the response value was the highest when the sampling amount was 1.0 g, so the sampling amount of the test solution was selected as 1.0 g.

[0079] (5) Investigation of different chromatographic columns:

[0080] About 1.0 g of the Acanthopanax Cortex Granules were taken and chromatographically analyzed on three chromatographic columns (4.6 mm x 250 mm, 5 μm) of different manufacturers and models [CAPCELL PAK C18 AQ S5 (CX-143), Shimadzu GIST AQ-C18 (PF-151), AQ-C18 (YX-UAQ-2201)] was measured according to "2. Characteristic spectrum measurement method" in Example 1.

[0081] Table 6 Relative retention time of experimental characteristic spectra of chromatographic columns (different manufacturers and different batches)

[0082]

[0083] Table 7 Relative peak areas of experimental characteristic spectra of chromatographic columns (different manufacturers and different batches)

[0084]

[0085]

[0086] The results showed that the peak shape and number of peaks of the characteristic spectrum of the test sample were inconsistent (see Figure 2). There are 12 common peaks in the characteristic spectrum, among which peak 4 is chlorogenic acid. The peak corresponding to the chlorogenic acid reference peak is the S1 peak. The relative retention times and relative peak areas of peaks 1, 2, 3, 5, and 6 with respect to the S1 peak are calculated. Peak 10 is 3,5-di-O-caffeoylquinic acid. The peak corresponding to the 3,5-di-O-caffeoylquinic acid reference peak is the S2 peak. The relative retention times and relative peak areas of peaks 7, 8, 9, 11, and 12 with respect to the S2 peak are calculated, and the RSD% values ​​are calculated. The calculation results show that the relative retention time RSD% values ​​and the relative peak area RSD% values ​​are both outside the qualified range (see Tables 6 and 7). The experiment shows that different models of chromatographic columns from different manufacturers have a significant impact on the characteristic spectrum determination, that is, only chromatographic columns of specified models can be used for the characteristic spectrum determination.

[0087] (6) Investigation of different flow rates:

[0088] About 1.0 g of a sample of Acanthopanax Cortex granules was taken and measured according to "2. Characteristic Spectrum Determination Method" of Example 1 at flow rates of 0.78 ml / min, 0.80 ml / min, 0.82 ml / min, 1.0 ml / min, 1.02 ml / min, and 1.20 ml / min.

[0089] Table 8 Relative retention time of characteristic spectra at different flow rates

[0090]

[0091] Table 9 Relative peak areas of characteristic spectra at different flow rates

[0092]

[0093]

[0094] The results showed that the peak shape and peak number of the characteristic spectrum of the test samples were basically consistent (see Figure 3). There are 12 common peaks in the characteristic spectrum, among which peak 4 is chlorogenic acid. The peak corresponding to the chlorogenic acid reference peak is the S1 peak. The relative retention time and relative peak area of ​​peaks 1, 2, 3, 5, and 6 with the S1 peak are calculated. Peak 10 is 3,5-di-O-caffeoylquinic acid. The peak corresponding to the 3,5-di-O-caffeoylquinic acid reference peak is the S2 peak. The relative retention time and relative peak area of ​​peaks 7, 8, 9, 11, and 12 with the S2 peak are calculated, and the RSD% value is calculated. The calculation results show that the relative retention time RSD% value and the relative peak area RSD% value are both within the range of ±10% (see Table 8 and Table 9). The experiment shows that slight changes in flow rate have little effect on the characteristic spectrum measurement, that is, the durability of different flow rates is good. However, when the flow rate is ±0.2 ml / min, it has a greater impact on the separation and peak width of the characteristic peaks, and the RSD value of the relative retention time is also greater than the qualified range.

[0095] Example 2

[0096] Preparation of reference solution: Take appropriate amount of chlorogenic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid reference substances, accurately weigh them, and add methanol with a volume concentration of 75% to make a mixed solution containing 50 μg of chlorogenic acid, 10 μg of 3,5-di-O-caffeoylquinic acid, and 10 μg of 4,5-di-O-caffeoylquinic acid per 1 ml.

[0097] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 75% methanol by volume, stopper it tightly, weigh it, ultrasonically treat it (power 300W, frequency 40kHz) for 20 minutes, let it cool, weigh it again, make up the loss with 75% methanol by volume, shake it well, filter it, and take the filtrate to obtain it.

[0098] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph for determination.

[0099] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.05% formic acid solution was used as mobile phase B. Gradient elution was performed according to the requirements in Table 2 below. The column temperature was 22°C, the flow rate was 0.7 ml / min, and the detection wavelength was 327 nm. The number of theoretical plates calculated based on the chlorogenic acid peak should be no less than 10,000.

[0100] Table 10 Gradient elution program

[0101]

[0102] Results: Each 1g of this product contains phenolic acids, mainly chlorogenic acid (C 16 H18 O9), 3,5-di-O-caffeoylquinic acid (C 25 H 24 O 12 ) and 4,5-di-O-caffeoylquinic acid (C 25 H 24 O 12 ) should be 4.3mg~16.2mg.

[0103] Example 3

[0104] (1) Preparation of five batches of Perennial Cortex test solutions: Five batches of Perennial Cortex samples were taken and ground into powder. 1.0 g of each sample was placed in five stoppered conical flasks. 25 mL of 50% methanol was added to each sample. The mixture was ultrasonically treated (power 300 W, frequency 40 kHz) for 30 minutes. The mixture was removed, cooled, filtered, and the filtrate was collected to obtain the Perennial Cortex test solution.

[0105] (2) Preparation of five batches of Acanthopanax Cortex test solutions: Five batches of Acanthopanax Cortex samples were taken and ground into powder. 1.0 g of each sample was placed in five stoppered conical flasks. 25 mL of 50% methanol was added to each sample. The mixture was ultrasonically treated (power 300 W, frequency 40 kHz) for 30 minutes. The sample was removed, cooled, filtered, and the filtrate was collected to obtain five batches of Acanthopanax Cortex test solutions.

[0106] (3) According to the "2. Characteristic spectrum determination method" of Example 1, the results are as follows Figure 4 shown.

[0107] Results: The characteristic spectra of Acanthopanax acanthopanax and Cortex Periplocae were used to identify each other. In the characteristic spectra, the number of characteristic peaks of Cortex Periplocae and Acanthopanax acanthopanax were significantly different, making them easy to distinguish.

[0108] Example 4

[0109] (1) Preparation of five batches of Radix Lycii test solutions: Five batches of Radix Lycii samples were taken, ground into powder, and 1.0 g of each was taken and placed in five stoppered conical flasks. 25 mL of methanol with a volume concentration of 50% was added to each flask, and ultrasonic treatment (power 300 W, frequency 40 kHz) was performed for 30 minutes. The samples were taken out, cooled, filtered, and the filtrate was taken to obtain the Radix Lycii test solution;

[0110] (2) Preparation of five batches of Acanthopanax Cortex test solutions: Five batches of Acanthopanax Cortex samples were taken and ground into powder. 1.0 g of each sample was placed in five stoppered conical flasks. 25 mL of 50% methanol was added to each sample. The mixture was ultrasonically treated (power 300 W, frequency 40 kHz) for 30 minutes. The sample was removed, cooled, filtered, and the filtrate was collected to obtain the Acanthopanax Cortex test solution.

[0111] (3) According to the "2. Characteristic spectrum determination method" of Example 1, the results are as follows Figure 5shown.

[0112] Results: The characteristic spectrum was used to identify the anti-counterfeiting products of Acanthopanax Cortex and Radix Lycoris. It can be seen from the characteristic spectrum that Radix Lycoris does not have the corresponding characteristic peaks in the characteristic spectrum of Acanthopanax Cortex, and has obvious differences from Acanthopanax Cortex decoction pieces, making it easy to distinguish.

[0113] Example 5

[0114] (1) Prepare three batches of Acanthopanax acanthopanax root bark test solution using the same method as in Example 1.

[0115] (2) Prepare three batches of Acanthopanax acanthopanax stem bark test solution using the same method as in Example 1;

[0116] (3) Prepare three batches of test solution of the root core of Acanthopanax acanthopanax bark using the same method as that of Example 1;

[0117] (4) Prepare three batches of Acanthopanax acanthopanax stem bark and wood core test solution using the same method as in Example 1;

[0118] (5) According to the "2. Characteristic spectrum determination method" of Example 1, the results are as follows Figure 6 shown.

[0119] Results: The characteristic spectra of different parts of Acanthopanax acanthopanax were compared. The characteristic peaks of the root bark were significantly different from those of other parts. The stem bark had no obvious characteristic peaks at peak 1, peak 2, and peak 12, and the response value of peak 7 (1,3-di-O-caffeoylquinic acid) was low. The root pith had no obvious characteristic peaks at peak 1 and peak 9, and the response value of peak 7 (1,3-di-O-caffeoylquinic acid) was low. The stem pith had no obvious characteristic peaks at peak 1 and peak 9, and the response value of peak 7 (1,3-di-O-caffeoylquinic acid) was low.

[0120] Comparative Example 1

[0121] Preparation of the reference solution of Acanthopanax Cortex: Take 0.2 g of Acanthopanax Cortex reference medicinal material, place it in a stoppered conical flask, add 50 ml of a 50% methanol solution by volume, treat it with ultrasound (power 300 W, frequency 40 kHz) for 20 minutes, cool it, filter it, and take the filtrate as the reference solution of the control medicinal material.

[0122] Preparation of reference substance solution: Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add methanol to make a mixed solution containing 20 μg of each substance per 1 ml, which is used as the reference substance solution.

[0123] Preparation of the test solution: Grind an appropriate amount of the granules without the acanthopanax cortex into powder. Take about 0.2 g and put it into a stoppered conical flask. Add 50 ml of a 50% methanol solution by volume. Treat with ultrasound (power 300 W, frequency 40 kHz) for 20 minutes. Cool, filter, and take the filtrate to obtain the test solution.

[0124] Assay Method: Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as the packing (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile as mobile phase A, 0.2% by volume formic acid solution as mobile phase B, gradient elution as specified in Table 11 below; flow rate, 1.0 ml / min; column temperature, 20°C; detection wavelength, 280 nm. The number of theoretical plates, calculated based on the chlorogenic acid peak, should be no less than 1500.

[0125] Accurately draw 10 μl of reference solution and test solution respectively, inject into liquid chromatography for determination, the characteristic spectrum of the Acanthopanax Cortex reference medicinal material is as follows Figure 7 shown.

[0126] Table 11 Gradient elution program

[0127]

[0128] There are 5 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 5 characteristic peaks in the chromatogram of the control medicinal material reference. The peak corresponding to the chlorogenic acid reference peak is the S peak. Calculate the relative retention times of Peak 1, Peak 3, Peak 4, Peak 5 and the S peak. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​are: 0.80 (Peak 1), 1.28 (Peak 3), 1.91 (Peak 4), and 2.50 (Peak 5).

[0129] After optimizing the chromatographic conditions in Example 1, the results of Comparative Example 7 (before optimization) were compared. Figure 1 and Figure 7 The number of characteristic peaks increased from 5 to 12, and two reference peaks were selected. Based on the identification of the chlorogenic acid peak, a total of 7 peaks were identified: neochlorogenic acid, cryptochlorogenic acid, caffeic acid, 1,3-O-dicaffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid. The peak area ratio of Peak 7 to Peak 4 was increased to distinguish different medicinal parts.

[0130] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Within the scope of the present disclosure, the above embodiments or technical features in different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0131] The one or more embodiments of this application are intended to encompass all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of this application should be included within the scope of protection of this disclosure.

Claims

1. A quality detection method for Acanthopanax Cortex or Acanthopanax Cortex Formula Granules, characterized in that the characteristic spectrum Measurement methods include: (1) Preparation of a reference solution of Acanthopanax cortex medicinal materials: Add water to the Acanthopanax cortex medicinal materials, heat, filter, treat the residue with methanol solution, and filter to obtain a reference solution of Acanthopanax cortex medicinal materials; (2) Preparation of reference substance solution: Take appropriate amount of chlorogenic acid and 3,5-di-O-caffeoylquinic acid, add methanol to make a mixed solution, which is used as the reference substance solution; (3) Determination: The reference solution of Acanthopanax Cortex and the reference solution of the reference substance were respectively drawn and injected into a liquid chromatograph, with acetonitrile as mobile phase A and 0.05% volume concentration of formic acid solution as mobile phase B, and the characteristic spectrum was obtained; in the characteristic spectrum of the Acanthopanax Cortex reference medicinal material, peak 2 is neochlorogenic acid; peak 4 is chlorogenic acid; peak 5 is cryptochlorogenic acid; peak 6 is caffeic acid; peak 7 is 1,3-O-dicaffeoylquinic acid; peak 10 is 3,5-di-O-caffeoylquinic acid; peak 11 is 4,5-O-dicaffeoylquinic acid; (4) Determination of the test solution: add water to Acanthopanax acanthopanax or Acanthopanax acanthopanax granules, heat, filter, add methanol solution to the residue, filter to obtain the test solution, draw up the test solution, inject into liquid chromatography, and determine; (5) Use characteristic spectra to judge the quality of Acanthopanax acanthopanax or Acanthopanax acanthopanax granules; The chromatographic conditions in step (3) of the characteristic spectrum determination method are as follows: octadecylsilane bonded silica gel is used as the filler, the flow rate is 0.8 ml per minute, the column temperature is 20°C, the detection wavelength is 280 nm, and the gradient elution is performed according to the following procedure: 0-6 min, mobile phase A is 5%, mobile phase B is 95%; 6-55 min, mobile phase A from 5% to 13%, mobile phase B from 95% to 87%; 55-70 min, mobile phase A from 13% to 19%, mobile phase B from 87% to 81%; 70-80 min, mobile phase A: 19%, mobile phase B: 81%; 80-105 min, mobile phase A from 19% to 30%, mobile phase B from 81% to 70%; 105-107 min, mobile phase A is 30%→5%, mobile phase B is 70%→95%.

2. The quality detection method of Acanthopanax acanthopanax or Acanthopanax acanthopanax granules according to claim 1, characterized in that: In step (1) of the characteristic spectrum determination method, the volume concentration of methanol in the preparation of the Acanthopanax Cortex reference medicinal material reference solution is 50%, and the concentrations of chlorogenic acid and 3,5-di-O-caffeoylquinic acid in the reference substance solution in step (2) are 50 μg / ml respectively.

3. The quality detection method of Acanthopanax acanthopanax or Acanthopanax acanthopanax granules according to claim 1, characterized in that: In step (4) of the characteristic spectrum determination method, the volume concentration of methanol is 50%, and the treatment method is ultrasonic treatment.

4. The quality detection method of Acanthopanax acanthopanax or Acanthopanax acanthopanax granules according to claim 1, characterized in that: In step (4) of the characteristic spectrum determination method, the weight loss is supplemented with methanol having a volume concentration of 50%.

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