Quality detection method for a Sichuan parasitic medicinal material and its preparation

Abstract

The present invention relates to the technical field of detection of traditional Chinese medicine components, and particularly to a quality detection method for Sichuan parasitic herbs and their preparations, including: detecting the test solution and the reference solution by high performance liquid chromatography; the test solution contains Sichuan parasitic herbs; the reference solutions are gallic acid solution, protocatechuic acid solution, catechin solution, isoquercitrin solution and quercitrin solution respectively; the conditions for high performance liquid chromatography detection include: the mobile phase includes mobile phase A and mobile phase B, acetonitrile is used as mobile phase A, and glacial acetic acid solution is used as mobile phase B for gradient elution; the flow rate of the mobile phase is 0.8-1.2 mL / min. By establishing a new characteristic chromatogram method to control its quality, it can solve the problem that it is difficult to comprehensively evaluate and control the process from Sichuan parasitic herbs to the finished Sichuan parasitic formula granules in the prior art, and the quality detection of Sichuan parasitic herbs in production is comprehensive and reliable, and the detection time is relatively short.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of traditional Chinese medicine components, and particularly to a quality detection method for Sichuan Taxillus chinensis herbs and their preparations. Background Art

[0002] Sichuan Taxillus chinensis belongs to the Loranthaceae family in the plant kingdom, and its subclass belongs to the Archichlamydeae.

[0003] Existing literature on Sichuan Taxillus chinensis (such as: Sun Riqiang, Sui Changhui, Xu Chunquan, etc. Determination of quercetin content in Taxillus sutchuenensis and Taxillus rubiginosus [J]. Journal of Modern Applied Pharmacy, 1993, 3(10): 36-37; such as: Li Meirong, Li Liangqiong, Li Ping. Study on flavonoid components of Taxillus sutchuenensis and Taxillus sutchuenensis var. duclouxii [J]. Bulletin of Chinese Traditional Medicine, 1987, 12(12): 34-36; such as: Chen Jiangtao, Feng Feng. Study on the chemical constituents of Taxillus sutchuenensis [J]. Journal of Chinese Medicinal Materials, 2007, 11(30): 1393-1395) is limited to the determination of single chemical components, making it difficult to effectively compare and analyze the differences and changes in the characteristic fingerprints of Sichuan Taxillus chinensis herbs, cut pieces, standard decoctions, and formula granules, difficult to deeply understand the transfer of the effective substances of Sichuan Taxillus chinensis from herbs to formula granules during the technological process, and difficult to comprehensively evaluate and control the technological process from Sichuan Taxillus chinensis herbs to the finished Sichuan Taxillus chinensis formula granules. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a quality detection method for Sichuan Taxillus chinensis herbs and their preparations, and to control their quality by establishing a new characteristic fingerprint method.

[0005] The present invention provides a quality detection method for Sichuan Taxillus chinensis herbs and their preparations, including the following steps:

[0006] Detecting the test solution and the reference solution by high performance liquid chromatography;

[0007] The test solution contains Sichuan Taxillus chinensis;

[0008] The reference solutions are gallic acid solution, protocatechuic acid solution, catechin solution, isoquercitrin solution, and quercitrin solution respectively;

[0009] The conditions for the high performance liquid chromatography detection include:

[0010] The mobile phase includes mobile phase A and mobile phase B. Acetonitrile is used as mobile phase A, and glacial acetic acid solution is used as mobile phase B for gradient elution;

[0011] The flow rate of the mobile phase is 0.8 - 1.2 mL / min.

[0012] In certain embodiments of the present invention, the flow rate of the mobile phase is 1.0 mL / min.

[0013] In certain embodiments of the present invention, the volume ratio of mobile phase A to mobile phase B is 2 - 36:64 - 98;

[0014] In mobile phase B, the mass concentration of the glacial acetic acid solution is 0.2%.

[0015] In certain embodiments of the present invention, during the gradient elution process, the volume ratio of mobile phase A to mobile phase B is:

[0016]

[0017] In certain embodiments of the present invention, the chromatographic column used in the high performance liquid chromatography is a C18 column, such as DIKMA Diamonsil C18(2), Agilent ZORBAX SB - C18 or Waters XBridge C18; the column length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the particle size of the packing is 5 μm.

[0018] The detector used in the high performance liquid chromatography is a diode array detector.

[0019] In certain embodiments of the present invention, the conditions for detection by the high performance liquid chromatography further include: the column temperature is 20 - 30 °C, such as 30 °C; the detection wavelength is 250 - 310 nm, such as 250 nm.

[0020] In certain embodiments of the present invention, the method for preparing the test solution includes the following steps:

[0021] Mix the sample to be tested with a methanol solution, and perform ultrasonic treatment or reflux extraction to obtain the test solution;

[0022] The sample to be tested contains Taxillus sutchuenensis.

[0023] The form of the sample to be tested includes at least one of medicinal materials, cut pieces, standard decoctions, and formula granules.

[0024] In the method for preparing the test solution, the mass concentration of the methanol solution is 50%.

[0025] In certain embodiments of the present invention, the method for preparing the test solution includes the following steps:

[0026] For the case where Taxillus sutchuenensis is a medicinal material or cut pieces:

[0027] Mix the powder of the test sample with water, decoct, filter, evaporate the filtrate to dryness, mix the residue obtained with a methanol solution with a mass concentration of 50%, and perform ultrasonic treatment or reflux extraction to obtain a test solution.

[0028] For the case where Sichuan Mistletoe is the standard decoction:

[0029] Mix the test sample with a methanol solution with a mass concentration of 50%, and perform ultrasonic treatment or reflux extraction to obtain a test solution.

[0030] For the case where Sichuan Mistletoe is the formula granule:

[0031] Grind the test sample finely, mix it with a methanol solution with a mass concentration of 50%, and perform ultrasonic treatment or reflux extraction to obtain a test solution.

[0032] In some embodiments of the present invention, the power of the ultrasonic treatment is 500 - 700 W, such as 600 W; the frequency is 35 - 45 kHz, such as 40 kHz;

[0033] The time of the ultrasonic treatment or reflux extraction is 20 - 40 min, such as 30 min;

[0034] The concentration of the test solution is 4 - 40 mg / mL, such as 4 mg / mL, 8 mg / mL or 12 mg / mL.

[0035] In some embodiments of the present invention, the preparation method of the reference solution includes the following steps:

[0036] Mix the reference with a methanol solution to obtain a reference solution;

[0037] The reference includes gallic acid, protocatechuic acid, catechin, isoquercitrin or quercitrin;

[0038] The concentration of the reference solution is 70 μg / mL.

[0039] In some embodiments of the present invention, in the preparation method of the reference solution, the mass concentration of the methanol solution is 100%.

[0040] In some embodiments of the present invention, the theoretical plate number adopted by the high performance liquid chromatography method is not less than 5000 calculated according to the gallic acid peak.

[0041] In the quality inspection method provided by the present invention, for the case where Sichuan Mistletoe is in the form of formula granules, 7 characteristic peaks should appear in the test sample chromatogram. The peak corresponding to the reference peak of gallic acid is the S1 peak, and the relative retention times of peak 2, peak 3 and the S1 peak are calculated; the peak corresponding to the reference peak of quercitrin is the S2 peak, and the relative retention times of peak 4, peak 5, peak 6 and the S2 peak are calculated. Their relative retention times should all be within the range of ±10% of the specified values, and the specified values are: 1.83 (peak 2), 2.70 (peak 3), 0.86 (peak 4), 0.90 (peak 5), 0.98 (peak 6).

[0042] In the quality inspection method provided by the present invention, for the case where Sichuan Mistletoe is in the form of a standard decoction, 7 characteristic peaks should appear in the test sample chromatogram. The peak corresponding to the reference peak of gallic acid is the S1 peak, and the relative retention times of peak 2 and peak 3 and the S1 peak are calculated, and their relative retention times should be within the range of ±10% of the specified values. The specified values are: 1.620 (peak 2), 2.395 (peak 3); the peak corresponding to the reference peak of quercitrin is the S2 peak, and the relative retention times of peak 4, peak 5, peak 6 and the S2 peak are calculated, and their relative retention times should be within the range of ±10% of the specified values. The specified values are 0.894 (peak 4), 0.908 (peak 5), 0.983 (peak 6).

[0043] In the quality inspection method provided by the present invention, for the case where Sichuan Mistletoe is in the form of medicinal materials or cut pieces, 7 characteristic peaks should appear in the test sample chromatogram. The peak corresponding to the reference peak of gallic acid is the S1 peak, and the relative retention times of peak 2 and peak 3 and the S1 peak are calculated, and their relative retention times should be within the range of ±10% of the specified values. The specified values are: 1.634 (peak 2), 2.442 (peak 3); the peak corresponding to the reference peak of quercitrin is the S2 peak, and the relative retention times of peak 4, peak 5, peak 6, peak 7 and the S2 peak are calculated, and their relative retention times should be within the range of ±10% of the specified values. The specified values are 0.894 (peak 4), 0.908 (peak 5), 0.983 (peak 6).

[0044] The present invention can solve the problem in the prior art that it is difficult to comprehensively evaluate and control the process from Sichuan Mistletoe medicinal materials to Sichuan Mistletoe formula granule products, so as to fill the blank in the prior art, and the quality inspection of Sichuan Mistletoe in production is comprehensive and reliable. At the same time, the detection time is short, which is convenient for large-scale popularization and application. Description of the Drawings

[0045] Figure 1 It is the ultraviolet absorption spectrogram of gallic acid;

[0046] Figure 2 It is the ultraviolet absorption spectrogram of protocatechuic acid;

[0047] Figure 3It is the ultraviolet absorption spectrogram of catechin;

[0048] Figure 4 It is the ultraviolet absorption spectrogram of isoquercitrin;

[0049] Figure 5 It is the ultraviolet absorption spectrogram of quercitrin;

[0050] Figure 6 It is the chromatogram of Sichuan Taxillus chinensis formula granules at different wavelengths;

[0051] Figure 7 It is the 3D map of Sichuan Taxillus chinensis formula granules;

[0052] Figure 8 It is the chromatogram for investigating the column temperature of the present invention;

[0053] Figure 9 It is the chromatogram for investigating the flow rate of the present invention;

[0054] Figure 10 It is the chromatogram for investigating the extraction solvent of the present invention;

[0055] Figure 11 It is the chromatogram for investigating the extraction method of the test sample of the present invention;

[0056] Figure 12 It is the chromatogram for investigating the extraction time of the test sample of the present invention;

[0057] Figure 13 It is the chromatogram for investigating the concentration of the test sample solution of the present invention;

[0058] Figure 14 It is the chromatographic peak identification map of Sichuan Taxillus chinensis formula granules of the present invention;

[0059] Figure 15 It is the chromatogram for investigating different instruments of the present invention;

[0060] Figure 16 It is the chromatogram for investigating different chromatographic columns;

[0061] Figure 17 It is the verification map of the characteristic fingerprints of 3 batches of Sichuan Taxillus chinensis formula granules of the present invention;

[0062] Figure 18 It is the reference fingerprint of the Sichuan Taxillus chinensis formula granules of the present invention;

[0063] Figure 19 It is the reference fingerprint of the characteristic fingerprints of the Sichuan Taxillus chinensis standard decoction of the present invention;

[0064] Figure 20 It is the reference characteristic fingerprint of the Sichuan Taxillus chinensis medicinal materials of the present invention;

[0065] Figure 21 It is the reference fingerprint of the Sichuan Taxillus chinensis decoction pieces of the present invention;

[0066] Figure 22 This is the comparison chart of the characteristic chromatograms of Sichuan Taxillus chinensis herbs, standard decoctions, standard decoction pieces, and formula granules of the present invention. Detailed implementation manners

[0067] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0068] Experimental instruments and materials:

[0069] High-performance liquid chromatographs: Agilent 1260 high-performance liquid chromatograph, LC-20AD high-performance liquid chromatograph, Waters e 2695 high-performance liquid chromatograph;

[0070] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0071] Ultra-pure water machines: Cell type 1810A (Shanghai Moller Scientific Instruments Co., Ltd.);

[0072] Ultrasonic cleaners: KQ600DB type (600W, 40KHz; Kunshan Ultrasonic Instruments Co., Ltd.);

[0073] Chromatographic columns: Agilent ZORBAX SB-C18 250×4.6mm, 5μm; Waters XBridge C18 250×4.6mm, 5μm; DIKMA Diamonsil C18(2) 250×4.6mm, 5μm. Except for the subsequent 3-5), in the rest of the investigations, the chromatographic column used was Agilent ZORBAX SB-C18 250×4.6mm, 5μm.

[0074] Reagents and test drugs:

[0075] Gallic acid (National Institutes for Food and Drug Control, batch number: 110831-201605, mass content calculated as 90.8%);

[0076] Protocatechuic acid (National Institutes for Food and Drug Control, batch number: 110809-201205, mass content calculated as 99.9%);

[0077] Catechin (National Institutes for Food and Drug Control, batch number: 110877-201604, mass content calculated as 99.2%);

[0078] Isoquercitrin (Sichuan Victy Biotech Co., Ltd., batch number: wkq17060202, mass content calculated as 99.8%);

[0079] Quercitrin (National Institutes for Food and Drug Control, batch number: 111538 - 201606, mass content calculated as 90.6%);

[0080] Acetonitrile (SIGMA, chromatographic grade); Phosphoric acid (chromatographic grade); Water is ultrapure water; Other reagents are all of analytical grade;

[0081] Sichuan Taxillus parasiticus formula granules: JS - KL - 01, JS - KL - 02, JS - KL - 03, JS - KL - 04

[0082] Sichuan Taxillus parasiticus standard decoction: JS - BT - 01, JS - BT - 02, JS - BT - 03, JS - BT - 04, JS - BT - 05, JS - BT - 06, JS - BT - 07, JS - BT - 08, JS - BT - 09, JS - BT - 10, JS - BT - 11, JS - BT - 12, JS - BT - 13, JS - BT - 14, JS - BT - 15, JS - BT - 16, JS - BT - 17, JS - BT - 18, JS - BT - 19, JS - BT - 20, JS - BT - 21, JS - BT - 22;

[0083] Sichuan Taxillus parasiticus medicinal materials: YC - JS - 01, YC - JS - 02, YC - JS - 03, YC - JS - 04, YC - JS - 05, YC - JS - 06, YC - JS - 07, YC - JS - 08, YC - JS - 09, YC - JS - 10, YC - JS - 11, YC - JS - 12, YC - JS - 13, YC - JS - 14, YC - JS - 15, YC - JS - 16, YC - JS - 17, YC - JS - 18, YC - JS - 19, YC - JS - 20, YC - JS - 21, YC - JS - 22;

[0084] Sichuan Taxillus parasiticus cut crude drugs: YP - JS - 01, YP - JS - 02, YP - JS - 03, YP - JS - 04, YP - JS - 05, YP - JS - 06, YP - JS - 07, YP - JS - 08, YP - JS - 09, YP - JS - 10, YP - JS - 11, YP - JS - 12, YP - JS - 13, YP - JS - 14, YP - JS - 15, YP - JS - 16, YP - JS - 17, YP - JS - 18, YP - JS - 19, YP - JS - 20, YP - JS - 21, YP - JS - 22.

[0085] (1) Establishment of the characteristic chromatogram of Sichuan Mistletoe Formula Granules

[0086] Selection of chromatographic conditions:

[0087] Using octadecylsilane chemically bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); using acetonitrile as mobile phase A; using glacial acetic acid with a mass concentration of 0.2% as mobile phase B, and performing gradient elution; during the gradient elution process, the volume ratio of mobile phase A to mobile phase B is:

[0088]

[0089]

[0090] The flow rate of the mobile phase is: 1.0 mL / min, the column temperature is 30 °C; the detection wavelength is 250 nm; the number of theoretical plates calculated by the kynurenic acid peak is not less than 5000.

[0091] Preparation of the reference solution:

[0092] Accurately weigh appropriate amounts of gallic acid, protocatechuic acid, catechin, isoquercitrin, and quercitrin reference substances, and separately dissolve them in methanol solution with a mass concentration of 100% to prepare solutions containing 70 μg per 1 mL, respectively.

[0093] Preparation of the test solution:

[0094] Take an appropriate amount of Sichuan Mistletoe Formula Granules, grind them finely, take 0.2 g, accurately weigh, place it in a stoppered conical flask, add 25 mL of methanol solution with a mass concentration of 50%, tightly stopper, ultrasonically treat (power: 600 W, frequency: 40 kHz) for 30 min, let it cool, shake well, filter, and take the subsequent filtrate, that is, obtain.

[0095] Determination method:

[0096] Precisely pipette 10 μL of the reference solution and the test solution respectively, inject them into the high-performance liquid chromatograph, and determine, that is, obtain.

[0097] 1) Chromatographic conditions and system suitability test:

[0098] 1-1) Wavelength selection

[0099] On the basis of the above-mentioned experimental conditions, use a diode array detector to perform full-wavelength scanning on gallic acid, protocatechuic acid, catechin, isoquercitrin, quercitrin, and the test solution respectively, and extract the chromatograms of the test solution at wavelengths of 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, and ..... Figures 1 to 7 as shown. Figure 1 is the ultraviolet absorption spectrum of gallic acid,Figure 2 This is the ultraviolet absorption spectrum of protocatechuic acid, Figure 3 This is the ultraviolet absorption spectrum of catechin, Figure 4 This is the ultraviolet absorption spectrum of isoquercitrin, Figure 5 This is the ultraviolet absorption spectrum of quercitrin, Figure 6 This is the chromatogram of Sichuan Taxillus parasiticus formula granules at different wavelengths, Figure 7 This is the 3D map of Sichuan Taxillus parasiticus formula granules.

[0100] The results show that when the detection wavelength is 250 nm, the chromatographic peak information is larger and the chromatogram baseline is more stable. Therefore, the detection wavelength is determined to be 250 nm.

[0101] 1-2) Column temperature investigation

[0102] On the basis of the above-determined experimental conditions, the column temperature was investigated at 20 °C, 25 °C, and 30 °C respectively. The results are as Figure 8 shown. Figure 8 This is the chromatogram for investigating the column temperature of the present invention.

[0103] The results show that when the column temperature is 30 °C, the chromatogram peak shapes are relatively symmetrical and the resolutions are good. Therefore, 30 °C is finally determined as the column temperature for the characteristic chromatogram method of Taxillus parasiticus formula granules.

[0104] 1-3) Flow rate investigation

[0105] On the basis of the above-determined experimental conditions, the flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min were investigated respectively. The results are as Figure 9 shown. Figure 9 This is the chromatogram for investigating the flow rate of the present invention.

[0106] The results show that when the flow rates are 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min respectively, the relative retention time RSD of each characteristic peak is 0.10% - 5.62%. When the flow rate is 1.0 mL / min, the chromatogram peak shape is good and the resolution is moderate. Therefore, the flow rate is determined to be 1.0 mL / min.

[0107] 2) Investigation on the preparation of the test solution:

[0108] 2-1) Investigation on the extraction solvent

[0109] Take an appropriate amount of this product (JS-KL-01), grind it finely, take 0.25 g, place it in a stoppered conical flask, and examine it when the extraction solvents for the test sample are methanol solution with a mass concentration of 100% (which can be denoted as methanol), ethanol solution with a mass concentration of 100% (which can be denoted as ethanol), methanol solution with a mass concentration of 70%, ethanol solution with a mass concentration of 70%, methanol solution with a mass concentration of 50%, ethanol solution with a mass concentration of 50%, methanol solution with a mass concentration of 30%, ethanol solution with a mass concentration of 30%, and water, 25 mL each. Stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 min, let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample. As Figure 10 shown. Figure 10 This is the chromatogram for the investigation of the extraction solvent of the present invention.

[0110] The results show that when the extraction solvent is methanol solution with a mass concentration of 50%, the chromatographic peak information is large, the resolution is good, and the overall is more harmonious. Therefore, the extraction solvent for the test sample is determined to be methanol solution with a mass concentration of 50%.

[0111] 2-2) Investigation of extraction method

[0112] Take an appropriate amount of this product (JS-KL-01), grind it finely, take 0.2 g, place it in a stoppered conical flask, add 25 mL of methanol with a mass concentration of 50%, stopper tightly, and examine it when the extraction methods for the test sample are reflux and ultrasonic extraction respectively. The extraction time is 30 min, let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample. The results are as Figure 11 shown. Figure 11 This is the chromatogram for the investigation of the extraction method of the test sample of the present invention.

[0113] The results show that the effects are the same when ultrasonic extraction and reflux extraction are carried out on the test sample respectively. Since the ultrasonic extraction operation is more convenient, the extraction method for the test sample is determined to be ultrasonic extraction.

[0114] 2-3) Investigation of extraction time

[0115] Take an appropriate amount of this product (JS-KL-01), grind it finely, take 0.2 g, place it in a stoppered conical flask, add 25 mL of methanol with a mass concentration of 50%, stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz), and examine it when the extraction times for the test sample are 20 min, 30 min, and 40 min respectively. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample. The results are as Figure 12 shown. Figure 12 This is the chromatogram for the investigation of the extraction time of the test sample of the present invention.

[0116] The results show that the chromatographic peak information at different extraction times is roughly the same. Considering comprehensively, the extraction time for the test sample of the parasitic formula granules is determined to be ultrasonic for 30 min.

[0117] 2-4) Investigation of sampling amount

[0118] Take an appropriate amount of this product (JS-KL-01), grind it finely, and take 0.1 g, 0.2 g, and 0.3 g respectively, place them in a stoppered conical flask, add 25 mL of methanol with a mass concentration of 50%, stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 min, let it cool, shake well, filter, and take the continuous filtrate to obtain the solution. The results are as Figure 13 shown. Figure 13 This is the chromatogram for investigating the concentration of the test solution of the present invention. The results show that the chromatographic peak information of different sampling amounts is roughly the same. Considering comprehensively, the sampling amount of the test solution of the parasitic formula granules is determined to be 0.2 g for ultrasonic treatment.

[0119] 3) Methodology investigation:

[0120] 3-1) Chromatographic peak identification

[0121] Preparation of the test solution: Prepare the test solution of Sichuan parasitic formula granules according to the above-determined experimental conditions.

[0122] Preparation of the reference solution: Take appropriate amounts of gallic acid, protocatechuic acid, catechin, isoquercitrin, and quercitrin reference substances, weigh them accurately, and prepare solutions containing 70 μg per 1 mL with methanol respectively to obtain the solution.

[0123] Preparation of the negative control solution: Prepare the negative control solution lacking Sichuan parasitic formula granules according to the above-determined experimental conditions.

[0124] Locate the characteristic chromatogram peaks of Sichuan parasitic formula granules. The results are as Figure 14 shown. Figure 14 This is the chromatographic peak identification diagram of Sichuan parasitic formula granules of the present invention.

[0125] 3-2) Precision test

[0126] Take the test solution of Sichuan parasitic formula granules (JS-KL-01), inject 10 μL each time continuously for 6 times according to the determined experimental method, and calculate the relative retention time of each characteristic peak. As shown in Table 1.

[0127] Table 1 Precision investigation - retention time

[0128]

[0129]

[0130] The results show that the precision of this instrument is good.

[0131] 3-3) Reproducibility investigation

[0132] Accurately weigh 6 portions of Sichuan parasitic formula granules (JS-KL-01), prepare and determine them according to the determined experimental method. See Table 2.

[0133] Table 2 Repeatability Investigation - Relative Retention Time Ratio

[0134]

[0135] The results show that the method has good repeatability.

[0136] 3 - 4) Intermediate Precision Investigation

[0137] 3 - 4 - 1) Investigation with Different Instruments

[0138] On the basis of the above - established experimental conditions, accurately weigh Sichuan Taxillus chinensis formula granules (JS - KL - 01), prepare the test solution, and determine them on Agilent 1260, Shimadzu LC - 20AD, and Waters e2695 high - performance liquid chromatographs respectively. As Figure 15 shown in Table 3. Figure 15 This is the chromatogram for the investigation with different instruments of the present invention.

[0139] Table 3 Instrument Durability Investigation - Relative Retention Time Ratio

[0140]

[0141]

[0142] The results show that when the test samples are detected with the above three instruments, the RSD range of the relative retention time of each characteristic peak is 0.17% - 2.89%.

[0143] 3 - 4 - 1) Investigation with Different Persons and at Different Times

[0144] On the basis of the above - established experimental conditions, different persons (A, B) accurately weigh two portions of Sichuan Taxillus chinensis formula granules (JS - KL - 01) respectively at different times (T1, T2), prepare the test samples, and conduct the determination. See Table 4.

[0145] Table 4 Investigation with Persons and at Different Times - Relative Retention Time Ratio

[0146]

[0147] The results show that when the same sample is determined by different persons at different times, the method has good stability.

[0148] 3 - 5) Durability Investigation

[0149] On the basis of the above - determined experimental conditions, the following chromatographic columns were investigated respectively: Agilent ZORBAX SB - C18 250×4.6mm, 5μm; Waters XBridge C18 250×4.6mm, 5μm; DIKMA Diamonsil C18(2) 250×4.6mm, 5μm. See Figure 16 , Table 5. Figure 16 It is the investigation diagram of different chromatographic columns.

[0150] Table 5 Chromatographic column durability investigation - relative retention time ratio

[0151]

[0152] The results showed that when the samples were detected with the above - mentioned 3 chromatographic columns, the RSD of the relative retention time of the characteristic peaks was between 0.16% and 4.29%.

[0153] 3 - 6) Stability investigation

[0154] On the basis of the above - determined experimental conditions, the same test solution was taken and determined at 0h, 3h, 6h, 9h, 12h, and 24h respectively. See Table 6.

[0155] Table 6 Stability investigation - retention time

[0156]

[0157] The results showed that the sample solution was stable within 24 hours.

[0158] To sum up, the RSD of the relative retention time of each characteristic peak met the requirements in the above - mentioned various investigations, and this method was good. The above - mentioned 7 characteristic peaks were included in the subsequent investigations.

[0159] 3 - 7) Determination of characteristic peaks and establishment of reference chromatogram

[0160] 3 - 7 - 1) Verification results of 3 batches of Sichuan Taxillus formula granules

[0161] The characteristic chromatograms of 3 batches of samples of this product were determined by the proposed method, and the relative retention time was calculated. See Figure 17 and Table 7. Figure 17 It is the verification diagram of the characteristic chromatograms of 3 batches of Sichuan Taxillus formula granules of the present invention.

[0162] Table 7 Relative retention time of 3 batches of Sichuan Taxillus formula granules

[0163]

[0164] According to the principle of stable relative retention time, detectable in each batch of samples, and relatively high peaks, a total of 7 peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 1 was used as S1 peak and peak 7 was used as S2 peak, the relative standard deviation (RSD) of the relative retention times of the 7 characteristic peaks in 3 batches of parasitic formula granules was less than 2%.

[0165] 3-7-2) Establishment of the specified limit of relative retention time

[0166] The summary of the investigation items and verification results of the methodology is shown in Table 8.

[0167] Table 8 Summary of the RSD% results of each item of the methodology - relative retention time

[0168]

[0169] Final regulation: Seven characteristic peaks should be presented in the chromatogram of the test sample. The peak corresponding to the reference peak of gallic acid reference substance is S1 peak, and the relative retention times of peak 2 and peak 3 with respect to S1 peak are calculated; the peak corresponding to the reference peak of quercitrin reference substance is S2 peak, and the relative retention times of peak 4, peak 5, and peak 6 with respect to S2 peak are calculated. Their relative retention times should all be within the range of ±10% of the specified value, and the specified values are: 1.83 (peak 2), 2.70 (peak 3), 0.86 (peak 4), 0.90 (peak 5), 0.98 (peak 6).

[0170] Using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition) to synthesize the above-mentioned 3 batches of Sichuan parasitic formula granules, a reference chromatogram of the characteristic chromatogram of Sichuan parasitic formula granules was established. See Figure 18 . Figure 18 This is the reference chromatogram of the characteristic chromatogram of the Sichuan parasitic formula granules of the present invention; among them, peak 1 (S1): gallic acid; peak 2: protocatechuic acid; peak 3: catechin; peak 5: isoquercitrin; peak 7 (S2): quercitrin.

[0171] (II) Establishment of the characteristic chromatogram of the standard decoction of Sichuan parasitica

[0172] Method for formulating the characteristic chromatogram of the standard decoction

[0173] The chromatographic conditions and system suitability test are the same as those in the [Characteristic Chromatogram] item of the formula granules.

[0174] The preparation of the reference substance solution is the same as that in the [Characteristic Chromatogram] item of the formula granules.

[0175] Preparation of the test sample solution: Take 0.2 g of this product, add 25 mL of methanol solution with a mass concentration of 50%, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 min, let it cool, shake well, filter, and take the subsequent filtrate as the test sample solution.

[0176] The determination method is the same as that of the [characteristic spectrum] item of the formula granules.

[0177] Verification of characteristic spectrum of standard decoction

[0178] The proposed method was used to determine the characteristic spectra of 22 batches of samples of this product, and the relative retention times were calculated, see Table 9.

[0179] Table 9 Relative retention time of 22 batches of parasitic standard decoction

[0180]

[0181]

[0182] Based on the principle of stable relative retention time and that all batches of samples can be detected and the peaks are relatively high, a total of 7 peaks with good repeatability were selected as characteristic peaks. The final regulations are: 7 characteristic peaks should appear in the chromatogram of the test sample. The peak corresponding to the gallic acid reference peak is the S1 peak. The relative retention times of peaks 2 and 3 to the S1 peak are calculated, and their relative retention times should be within the ±10% range of the specified value. The specified values are: 1.620 (peak 2), 2.395 (peak 3); the peak corresponding to the quercetin reference peak is the S2 peak. The relative retention times of peaks 4, 5, and 6 to the S2 peak are calculated, and their relative retention times should be within the ±10% range of the specified value. The specified values are 0.894 (peak 4), 0.908 (peak 5), and 0.983 (peak 6).

[0183] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 22 batches of standard decoctions of parasitic plants, and a comparison spectrum of the characteristic spectrum of Sichuan standard decoctions of parasitic plants was established. Figure 19 . Figure 19 It is a reference spectrum of the characteristic spectrum of the Sichuan parasitic standard decoction of the present invention; wherein, peak 1 (S1): gallic acid; peak 2: protocatechuic acid; peak 3: catechin; peak 5: isoquercetin; peak 7 (S2): quercetin.

[0184] (III) Establishment of characteristic maps of Sichuan parasitic medicinal materials

[0185] Method for developing characteristic maps of medicinal materials

[0186] The chromatographic conditions and system suitability test are the same as those in the [characteristic spectrum] item of the formula particles.

[0187] The preparation of the reference solution is the same as that of the formula particles [characteristic spectrum].

[0188] Preparation of the test solution: Weigh 1.0 g of the powder of this product (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 25 mL of methanol with a mass concentration of 50%, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0189] Determination method: The same as the [Characteristic Chromatogram] item of the formula granules.

[0190] Verification of the characteristic chromatogram of the medicinal material

[0191] Determine 22 batches of samples according to the above method, and calculate the relative retention time ratio. The results are shown in Table 10.

[0192] Table 10 Relative retention time ratio of the characteristic chromatogram of 22 batches of Sichuan Taxillus chinensis medicinal materials

[0193]

[0194]

[0195] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 7 peaks with good repeatability were selected as characteristic peaks. The relative standard deviation (RSD) of the relative retention time of the 7 characteristic peaks of 22 batches of Taxillus chinensis medicinal materials is less than 2.0%. Finally, it is stipulated that: 7 characteristic peaks should be presented in the chromatogram of the test solution. The peak corresponding to the gallic acid reference peak is the S1 peak. Calculate the relative retention time of peak 2 and peak 3 with respect to the S1 peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: 1.634 (peak 2), 2.442 (peak 3); the peak corresponding to the quercitrin reference peak is the S2 peak. Calculate the relative retention time of peak 4, peak 5, and peak 6 with respect to the S2 peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are 0.894 (peak 4), 0.908 (peak 5), 0.983 (peak 6).

[0196] The similarity evaluation system for traditional Chinese medicine chromatographic fingerprints (2012 version) was used to synthesize 22 batches of Taxillus chinensis medicinal materials, and a reference characteristic chromatogram of the characteristic chromatogram of Taxillus chinensis medicinal materials was established. See Figure 20 . Figure 20 This is the reference characteristic chromatogram of Sichuan Taxillus chinensis medicinal materials of the present invention. Among them, peak 1 (S1): gallic acid; peak 2: protocatechuic acid; peak 3: catechin; peak 5: isoquercitrin; peak 7 (S2): quercitrin.

[0197] (IV) Establishment of the characteristic chromatogram of Sichuan Taxillus chinensis cut pieces

[0198] Method for formulating the characteristic chromatogram of cut pieces

[0199] Chromatographic conditions and system suitability test: The same as the [Characteristic Chromatogram] item of the formula granules.

[0200] The preparation of the reference solution is the same as that in the [Characteristic Spectrum] item of the formula granules.

[0201] The preparation of the test solution is the same as that in the [Characteristic Spectrum] item of the medicinal materials.

[0202] The determination method is the same as that in the [Characteristic Spectrum] item of the formula granules.

[0203] Verification of the characteristic spectrum of the cut crude drugs

[0204] Determine 20 batches of samples according to the above method, and calculate the relative retention time ratio. The results are shown in Table 11.

[0205] Table 11 Relative retention time ratio of the characteristic spectrum of 22 batches of Sichuan Taxillus parasiticus cut crude drugs

[0206]

[0207]

[0208] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 7 peaks with good repeatability were selected as characteristic peaks. The results show that the RSD of the relative retention time of the 7 characteristic peaks of 22 batches of Taxillus parasiticus cut crude drugs is less than 2.0%. Considering the RSD value range of the methodology investigation of Taxillus parasiticus cut crude drugs, it is finally stipulated that: 7 characteristic peaks should be presented in the chromatogram of the test solution, and the peak corresponding to the gallic acid reference peak is the S1 peak. Calculate the relative retention time of peak 2, peak 3 and the S1 peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: 1.634 (peak 2), 2.442 (peak 3); the peak corresponding to the quercitrin reference peak is the S2 peak. Calculate the relative retention time of peak 4, peak 5, peak 6 and the S2 peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are 0.894 (peak 4), 0.908 (peak 5), 0.983 (peak 6).

[0209] The 22 batches of Taxillus parasiticus cut crude drugs were synthesized using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition) to establish a reference chromatogram of the characteristic spectrum of Taxillus parasiticus cut crude drugs. See Figure 21 。 Figure 21 This is the reference chromatogram of Sichuan Taxillus parasiticus cut crude drugs of the present invention; among them, peak 1 (S1): gallic acid; peak 2: protocatechuic acid; peak 3: catechin; peak 5: isoquercitrin; peak 7 (S2): quercitrin.

[0210] (V) Correlation results of Sichuan Taxillus parasiticus medicinal materials, cut crude drugs, standard decoctions and formula granules

[0211] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), the characteristic fingerprints of 22 batches of Sichuan Taxillus chinensis herbs were synthesized into a single herb control fingerprint, the characteristic fingerprints of 22 batches of Sichuan Taxillus chinensis decoction pieces were synthesized into a single decoction piece control fingerprint, the characteristic fingerprints of 22 batches of Sichuan Taxillus chinensis standard decoctions were synthesized into a single standard decoction control fingerprint, and the characteristic fingerprints of 3 batches of Sichuan Taxillus chinensis formula granules were synthesized into a single formula granule control fingerprint. Comparing the above-mentioned Sichuan Taxillus chinensis herb, decoction piece, standard decoction, and formula granule control fingerprints, the results are shown in Figure 22 . Figure 22 This is the comparison chart of the control characteristic fingerprints of Sichuan Taxillus chinensis herbs, standard decoctions, standard decoctions, and formula granules of the present invention; among them, peak 1 (S1): gallic acid; peak 2: protocatechuic acid; peak 3: catechin; peak 5: isoquercitrin; peak 7 (S2): quercitrin.

[0212] The results show that 7 characteristic peaks can be detected in Sichuan Taxillus chinensis herbs, decoction pieces, standard decoctions, and formula granules, and their material bases are the same. The method of the present invention can accurately and efficiently detect the characteristic components in Sichuan Taxillus chinensis herbs with petioles, decoction pieces, standard decoctions, and formula granules, and achieve the purpose of overall controlling the quality of Sichuan Taxillus chinensis herbs, decoction pieces, standard decoctions, and formula granules.

[0213] In summary, the detection method applicable to the high-performance liquid chromatography characteristic fingerprints of Sichuan Taxillus chinensis herbs, decoction pieces, standard decoctions, and formula granules can overall control the characteristic components in Sichuan Taxillus chinensis herbs, decoction pieces, standard decoctions, and formula granules, ensure the overall stability of the quality of Sichuan Taxillus chinensis herbs, decoction pieces, standard decoctions, and formula granules, and the method is simple to operate, has high precision, good stability, good repeatability, and high accuracy.

[0214] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A quality detection method for a Sichuan parasitic medicinal material and its preparations, comprising the following steps: Detecting the test solution and the reference solution by high performance liquid chromatography; The preparation method of the test solution comprises the following steps: Mixing the test sample with a methanol solution, and performing ultrasonic treatment or reflux extraction to obtain the test solution; the test sample contains Sichuan parasitic; The reference solutions are respectively gallic acid solution, protocatechuic acid solution, catechin solution, isoquercitrin solution and quercitrin solution; The conditions for the high performance liquid chromatography detection include: The mobile phase comprises mobile phase A and mobile phase B. Using acetonitrile as mobile phase A and acetic acid solution as mobile phase B, gradient elution is carried out; During the gradient elution process, the volume ratio of mobile phase A to mobile phase B is: ； The flow rate of the mobile phase is 0.8 - 1.2 mL / min, and the detection wavelength is 250 nm.

2. The quality inspection method according to claim 1, wherein In the mobile phase B, the mass concentration of the acetic acid solution is 0.2%.

3. The quality inspection method according to claim 1, characterized in that The conditions for the high performance liquid chromatography detection also include: the column temperature is 20 - 30 °C.

4. The quality inspection method according to claim 1, wherein The form of the test sample includes at least one of medicinal materials, cut pieces, standard decoctions and formula granules.

5. The quality inspection method according to claim 1, characterized in that, The power of the ultrasonic treatment is 600 W, and the frequency is 40 kHz; The time for the ultrasonic treatment or reflux extraction is 20 - 40 min.

6. The quality inspection method according to claim 1, characterized in that, The solvent of the reference solution is methanol solution; The preparation method of the reference solution comprises the following steps: Mixing the reference with a methanol solution to obtain the reference solution; The reference includes gallic acid, protocatechuic acid, catechin, isoquercitrin or quercitrin.

7. The quality inspection method according to claim 1, wherein, The theoretical plate number adopted by the high performance liquid chromatography is not less than 5000 calculated by the gallic acid peak.

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