HPLC (High Performance Liquid Chromatography) fingerprint spectrum of compound dendrobium granule for quenching thirst and method for determining contents of 21 components

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of compound dendrobium granules for quenching thirst and a method for determining the contents of 21 components, which are characterized in that through sample extraction and HPLC detection, the fingerprint spectrum of 36 chemical component characteristic peaks in the compound dendrobium granules for quenching thirst can be obtained; the method also can be used for detecting gallic acid, tanshinol, protocatechuic acid, protocatechualdehyde, 3 '-hydroxy puerarin, caffeic acid, puerarin, mangiferin, 3'-methoxy puerarin, puerarin-6 ''-O-glucoside, puerarin apigenin, daidzin, daidzein, calycosin-7-glucoside, genistin, rosmarinic acid, alkannic acid, daidzein and isoflavones in the compound dendrobium nobile thirst quenching granules. According to the method, the contents of 21 components including salvianolic acid A, salvianolic acid B, calycosin and salvianolic acid A are measured, so that the quality of the compound dendrobium Xiaoke granule preparation can be comprehensively and effectively controlled.

Description

Technical Field

[0001] The present invention belongs to the field of quality control of traditional Chinese medicine preparations, and particularly relates to a method for determining the HPLC fingerprint and the contents of 21 components of Compound Shihu Xiaoke Granules. Background Art

[0002] Compound Shihu Xiaoke Granules are composed of twelve medicinal materials such as Pueraria lobata, Salvia miltiorrhiza, Dendrobium officinale, Astragalus membranaceus, and Schisandra chinensis, and have the effects of nourishing yin and clearing heat, and supplementing qi and activating blood circulation. It is mainly used for treating consumptive thirst caused by deficiency of both qi and yin. This medicine is a hospital preparation and has been used for many years, and has a good effect in treating consumptive thirst, especially type II diabetes. During the process of quality supervision and inspection, it is found that the current standard is relatively simple, and only thin-layer chromatography identification is carried out on astragaloside in Astragalus membranaceus, schisandrin A and Schisandra chinensis medicinal materials in Schisandra chinensis. However, the components of traditional Chinese medicine are complex and diverse, and the exertion of its medicinal effects is often the result of the combined action of multiple components. Therefore, it is very necessary to comprehensively determine its components. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for determining the HPLC fingerprint and the contents of 21 components of Compound Shihu Xiaoke Granules, which can realize the comprehensive and effective control of the quality of Compound Shihu Xiaoke Granules.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions: A method for determining the HPLC fingerprint and the contents of 21 components of Compound Shihu Xiaoke Granules, which comprises the following steps: 1) Preparation of reference substance solution: Weigh accurately appropriate amounts of reference substance powders of gallic acid, danshensu, protocatechuic acid, protocatechualdehyde, 3 ' -hydroxy puerarin, caffeic acid, puerarin, mangiferin, 3 ' -methoxy puerarin, puerarin-6 ''-O -glucoside, puerarin apigenin glycoside, daidzin, glycitin, calycosin-7-O-β-D-glucoside, genistin, rosmarinic acid, lithospermic acid, daidzein, salvianolic acid B, calycosin, salvianolic acid A, place them in a volumetric flask, dissolve with methanol and make up the volume to obtain a single reference substance stock solution. Then, accurately measure appropriate amounts of each reference substance stock solution, place them in the same volumetric flask, dilute with methanol and make up the volume to obtain a mixed reference substance mother liquor; then accurately measure an appropriate amount of the mixed reference substance mother liquor, and dilute it with a methanol solution with a volume concentration of 50% to obtain a reference substance solution with a series of concentrations; 2) Preparation of the test solution: Grind the sample of compound Dendrobium and Xiaoke Granules to be tested into powder. Weigh 2 g of the powder precisely, add 20 mL of methanol solution with a volume concentration of 50% precisely, weigh and record. Then, perform ultrasonic extraction for 30 min. After cooling to room temperature, make up the weight loss with methanol solution with a volume concentration of 50%. Shake well, filter, take 10 mL of the filtrate and place it in an evaporating dish, evaporate the solvent in a water bath, then redissolve the residue with 2 mL of methanol, filter through a 0.22 μm microporous filter membrane, and take the subsequent filtrate as the test solution; 3) Determination of the sample: Precisely pipette the prepared mixed reference substance stock solution, reference substance solutions with a series of concentrations, and the test solution respectively, and perform HPLC detection to obtain the corresponding chromatograms; 4) Establishment of the fingerprint: Import the chromatogram of the test solution into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" for processing to obtain the corresponding fingerprint, and assign 21 characteristic components using the chromatogram of the mixed reference substance stock solution; 5) Determination of the contents of 21 characteristic components in the sample: Perform linear regression with the peak areas of the chromatographic peaks of these 21 characteristic components in the chromatograms of the reference substance solutions with a series of concentrations as the ordinate and the concentrations of these 21 characteristic components in the reference substance solutions as the abscissa to obtain the standard curves and linear equations of the 21 characteristic components respectively. Then, calculate the contents of these 21 characteristic components in the test solution using the obtained linear equations and the chromatogram of the test solution.

[0005] Among them, the concentrations of gallic acid, danshensu, protocatechuic acid, protocatechualdehyde, 3 ' -hydroxy puerarin, caffeic acid, puerarin, mangiferin, 3 ' -methoxy puerarin, puerarin-6 ''-O -glucoside, puerarin apioglucoside, daidzin, glycitin, calycosin-7-O-β-D-glucoside, genistin, rosmarinic acid, lithospermic acid B, daidzein, salvianolic acid B, calycosin, salvianolic acid A in the mixed reference substance stock solution obtained in step 1) are 23.60, 227.00, 34.60, 12.85, 380.00, 37.30, 1012.50, 302.00, 236.00, 98.40, 204.40, 178.00, 23.80, 80.00, 27.20, 238.00, 198.00, 30.40, 1215.00, 51.20, 110.40 μg / mL respectively.

[0006] The chromatographic conditions for the HPLC detection in step 3) are: Thermo Scientific Accucore TM XL C 18Chromatographic column, with a specification of 4.6 mm×250 mm, 4 μm; mobile phase A is acetonitrile, mobile phase B is an aqueous phosphoric acid solution with a volume concentration of 0.2%, the elution method is gradient elution, and its elution program is 0 - 5 min, 3% - 5%A; 5 - 10 min, 5% - 9%A; 10 - 23 min, 9% - 9%A; 23 - 34 min, 9% - 19%A; 34 - 50 min, 19% - 29%A; 50 - 53 min, 29% - 60%A; the column temperature is 30°C, the flow rate is 1.0 mL / min, the detection wavelength is 265 nm, and the injection volume is 10 μL.

[0007] The significant advantages of the present invention are as follows: The existing method for determining the components in Compound Dendrobium and Schisandra Granules by HPLC has the problem of low analysis efficiency. The established fingerprint of Compound Dendrobium and Schisandra Granules only has 7 calibrated common characteristic peaks, the number of detection indicators is small, and the content of only 2 components can be determined, and it is impossible to effectively control the overall quality of Compound Dendrobium and Schisandra Granules. However, the method established by the present invention through the research on the sample extraction conditions and HPLC chromatographic conditions can calibrate 36 common characteristic peaks in the fingerprint of Compound Dendrobium and Schisandra Granules in only 53 minutes. At the same time, based on single-wavelength (265 nm) detection, the content of 21 components can be determined, enabling the quantification of the content of common components, better reflecting the internal quality of the preparation, and achieving comprehensive and effective control of the quality of Compound Dendrobium and Schisandra Granule preparations. Therefore, compared with the prior art, the present invention has high analysis efficiency, is simple and easy to operate, has strong specificity, and can provide a basis for improving the quality control level of existing Compound Dendrobium and Schisandra Granules. Description of the Drawings

[0008] Figure 1 It is the HPLC chromatogram obtained by extracting Compound Dendrobium and Schisandra Granules by different extraction methods.

[0009] Figure 2 It is the HPLC chromatogram obtained by extracting Compound Dendrobium and Schisandra Granules with different extraction times.

[0010] Figure 3 It is the HPLC chromatogram obtained by extracting Compound Dendrobium and Schisandra Granules with different extraction solvents.

[0011] Figure 4 It is the HPLC chromatogram obtained by extracting Compound Dendrobium and Schisandra Granules with different solid-to-liquid ratios.

[0012] Figure 5 It is the chromatogram obtained by HPLC detection of Compound Dendrobium and Schisandra Granules with different chromatographic columns.

[0013] Figure 6Chromatograms obtained by HPLC detection of Compound Shihu Xiaoke Granules using different detection wavelengths.

[0014] Figure 7 Chromatograms obtained by HPLC detection of Compound Shihu Xiaoke Granules using different mobile phase systems.

[0015] Figure 8 Chromatograms obtained by HPLC detection of Compound Shihu Xiaoke Granules with different flow rates of the mobile phase.

[0016] Figure 9 Chromatograms obtained by HPLC detection of Compound Shihu Xiaoke Granules using different column temperatures.

[0017] Figure 10 HPLC chromatograms (S1 - S15) of 15 batches of Compound Shihu Xiaoke Granule samples measured and the generated reference fingerprint chromatogram (R).

[0018] Figure 11 HPLC chromatograms of each single herb in Compound Shihu Xiaoke Granules (where A - Dendrobium officinale, B - Astragalus membranaceus, C - Schisandra chinensis, D - Pueraria lobata, E - Rehmannia glutinosa, F - Scrophularia ningpoensis, G - Anemarrhena asphodeloides, H - Salvia miltiorrhiza, I - Dioscorea opposita, J - Poria cocos, K - Cyathula officinalis, L - Turtle shell vinegar, M - Compound Shihu Xiaoke Granule sample).

[0019] Figure 12 HPLC chromatogram comparison of Compound Shihu Xiaoke Granule sample and mixed reference substances (where A - Compound Shihu Xiaoke Granule sample, B - mixed reference substances).

[0020] Figure 13 HPLC chromatogram comparison of mixed reference substances, Compound Shihu Xiaoke Granule sample and negative sample (where A - lacking Astragalus membranaceus, B - lacking Salvia miltiorrhiza, C - lacking Pueraria lobata, D - lacking Scrophularia ningpoensis, E - lacking Anemarrhena asphodeloides, F - lacking Schisandra chinensis, G - Compound Shihu Xiaoke Granule sample, H - mixed reference substances).

[0021] Figure 14 Cluster heat map analysis of the contents of 21 components in 15 batches of Compound Shihu Xiaoke Granules. Detailed implementation methods

[0022] To make the content of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0023] 1 Materials 1.1 Instruments U3000 high performance liquid chromatograph equipped with diode array detector (Thermo Fisher Scientific, USA); CLEAE01 sonicator (Ningbo Xinzhi Biological Co., Ltd.); EX125ZH electronic balance (Ohaus Instrument (Changzhou) Co., Ltd.); BHS-2 water bath (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); ultrapure water machine (Sichuan Excellence Water Treatment Equipment Co., Ltd.); H17.5 high-speed centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.).

[0024] 1.2 Drugs and reagents Reference substances: Gallic acid (Batch No.: 110831-201906), caffeic acid (Batch No.: 110885-201703), daidzein (Batch No.: 111738-202305), calycosin glucoside (Batch No.: 111920-202308), genistein (Batch No.: 111709-202303), rosmarinic acid (Batch No.: 11187 1-202007), daidzein (batch number: 111502-202304), and salvianolic acid B (batch number: 111562-201917) were purchased from the China Food and Drug Administration; danshensu (batch number: H09N10S102463); protocatechuic acid (batch number: M11GB140672), protocatechuic aldehyde (batch number: L17A9D59027), 3 ' -Hydroxypuerarin (Batch No.: A10HB191367), 3 ' -Methoxypuerarin (Batch No.: J12HB173942), Puerarin-6 '' - O -Glucoside (batch number: P14D9S77617), glycitin (batch number: O20IB229083), and lithospermic acid (batch number: Y30S7H22269) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; puerarin (batch number: 3681-99-0) was purchased from Beijing Beina Chuanglian Biotechnology Research Institute, and puerarin apiglycoside (batch number: PS000386) were all purchased from Chengdu Pusi Biotechnology Co., Ltd.; mangiferin (batch number: 1223-RF-0086), calycosin (batch number: 1215-RD-0018), and salvianolic acid A (batch number: 0305-RF-0102) were all purchased from Guangzhou Jiatu Technology Co., Ltd.; the purity of the above reference substances was ≥98%.

[0025] Single herbal medicine: Astragalus (batch number: X23070502D) and Scrophularia (batch number: X24060401B) were purchased from Tianjin Shengshi Pharmaceutical Co., Ltd., Schisandra (batch number: 240601) was purchased from Anhui Shouxin Chinese Medicine Technology Co., Ltd.; Pueraria (batch number: 240722), Radix Rehmanniae (batch number: 241101), Rhizoma Anemarrhenae (batch number: 240829) were purchased from Xiamen Yanlaifu Pharmaceutical Co., Ltd.; Dendrobium (batch number: 240301) was purchased from from Anhui Xintai Pharmaceutical Co., Ltd.; Salvia miltiorrhiza (batch number: 2207251) was purchased from Anhui Puren Chinese Medicine Pieces Co., Ltd.; Dioscorea opposita (batch number: A230923) was purchased from Haozhou Yonggang Pieces Factory Co., Ltd.; Vinegar turtle shell (batch number: 2300220101) was purchased from Guilu Pharmaceutical Group Co., Ltd.; Cyathula officinalis (batch number: 240401) was purchased from Guangdong Rongxiang Pharmaceutical Co., Ltd.; Poria cocos (batch number: A230402) was purchased from Hubei Jingui Chinese Medicine Pieces Co., Ltd.

[0026] Samples to be tested: 15 batches of Compound Dendrobium Xiaoke Granules, batch numbers 202407001, 202312010, 202408002, 202402005, 202410008, 202308004, 202310007, 202312010, 202306008, 202407005, 202401002, 2023090003, 202302004, 202305007, 202307008, numbered S1 to S15, specification 10g / bag, all provided by the Preparation Center of the Second People's Hospital Affiliated to Fujian University of Traditional Chinese Medicine.

[0027] Methanol and acetonitrile were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade.

[0028] 2. Investigation of extraction and chromatographic conditions 2.1 Selection of extraction method Accurately weigh 3 portions of the same batch of Compound Dendrobium Xiaoke Granules powder, 2.0 g each, accurately add 20 mL of 50% methanol solution respectively, weigh and record, and extract by heating reflux for 30 min, ultrasonic extraction for 30 min, and soaking overnight. After cooling to room temperature, add 50% methanol solution to make up the weight, shake well, filter, take 10 mL of the filtrate and place it in an evaporating dish in a water bath to evaporate the solvent, add 2 mL of methanol to dissolve, pass through a 0.22 μm microporous filter membrane, take the filtrate, and inject it for HPLC detection. The total number of chromatographic peaks and peak area were used as indicators to determine the optimal extraction method.

[0029] Depend on Figure 1 It can be seen that the number of chromatographic peaks obtained by ultrasonic extraction is large and the peak area is large, so ultrasonic extraction is selected as a suitable extraction method.

[0030] 2.2 Selection of extraction time Accurately weigh 4 portions of the same batch of Fufang Shihu Xiaoke Granule powder, 2.0 g for each portion, place them in conical flasks, accurately add 20 mL of 50% methanol solution to each, weigh and record. Ultrasonically extract for 15 min, 30 min, 45 min, and 60 min respectively. After cooling to room temperature, make up the weight with 50% methanol solution, shake well, filter, take 10 mL of the filtrate and place it in an evaporating dish, evaporate the solvent in a water bath until dry, redissolve with 2 mL of methanol, filter through a 0.22 μm microporous membrane, take the subsequent filtrate, inject the sample for HPLC detection, and determine the optimal extraction time based on the total number of chromatographic peaks and peak area.

[0031] From Figure 2 It can be seen that after the ultrasonic time exceeds 30 min, there is no obvious change in the number of chromatographic peaks and peak area obtained. Therefore, ultrasonic extraction for 30 min is selected as the appropriate extraction time.

[0032] 2.3 Selection of extraction solvent Accurately weigh 4 portions of the same batch of Fufang Shihu Xiaoke Granule powder, 2.0 g for each portion, place them in conical flasks, accurately add 20 mL of 20% methanol solution, 50% methanol solution, 80% methanol solution, and pure methanol respectively, weigh and record. Ultrasonically extract for 30 min, after cooling to room temperature, make up the weight with 50% methanol solution, shake well, filter, take 10 mL of the filtrate and place it in an evaporating dish, evaporate the solvent in a water bath until dry, redissolve with 2 mL of methanol, filter through a 0.22 μm microporous membrane, take the subsequent filtrate, inject the sample for HPLC detection, and determine the optimal extraction solvent based on the total number of chromatographic peaks and peak area.

[0033] From Figure 3 It can be seen that when extracting with 50% methanol, the number of chromatographic peaks of the obtained sample is relatively large and the peak area is also large. Therefore, 50% methanol is selected as the optimal extraction solvent.

[0034] 2.4 Selection of extraction material-liquid ratio Accurately weigh 4 portions of the same batch of Fufang Shihu Xiaoke Granule powder, 2.0 g for each portion, accurately add 10 mL, 20 mL, 40 mL, and 100 mL of 50% methanol solution respectively, weigh and record. Ultrasonically extract for 30 min, after cooling to room temperature, make up the weight with 50% methanol solution, shake well, filter, take 10 mL of the filtrate and place it in an evaporating dish, evaporate the solvent in a water bath until dry, redissolve with 2 mL of methanol, filter through a 0.22 μm microporous membrane, take the subsequent filtrate, inject the sample for HPLC detection, and determine the optimal extraction material-liquid ratio based on the total number of chromatographic peaks and peak area.

[0035] From Figure 4 It can be seen that when the material-liquid ratio is 1:10, that is, when the amount of 50% methanol used is 20 mL, the number of chromatographic peaks of the obtained sample is relatively large and the peak area is also large. Therefore, the material-liquid ratio of 1:10 is selected as the optimal extraction material-liquid ratio.

[0036] 2.5 Selection of Different Chromatographic Columns Accurately weigh 2.0 g of the powder of the same batch of Compound Dendrobium Diabetes Granules, place it in a conical flask, accurately add 20 mL of 50% methanol solution, weigh and record. Ultrasonically extract for 30 min, after cooling to room temperature, make up the weight with 50% methanol solution, shake well, filter, take 10 mL of the filtrate and place it in an evaporating dish, evaporate the solvent in a water bath, redissolve with 2 mL of methanol, filter through a 0.22 μm microporous filter membrane, take the subsequent filtrate, inject the sample, and perform HPLC detection using different brand C18 chromatographic columns such as Thermo (4.6 mm×250 mm, 4 μm), Waters (4.6 mm×250 mm, 5 μm), Nano (4.6 mm×250 mm, 5 μm), and Yilite (4.6 mm×250 mm, 5 μm). Take the total number of chromatographic peaks and the resolution as indicators to determine the best chromatographic column.

[0037] From Figure 5 It can be seen that when using the Thermo chromatographic column for HPLC detection, the number of chromatographic peaks of the obtained sample is relatively large and the resolution is good. Therefore, the Thermo chromatographic column is selected for HPLC detection.

[0038] 2.6 Determination of Detection Wavelength Accurately weigh 2.0 g of the powder of the same batch of Compound Dendrobium Diabetes Granules, place it in a conical flask, accurately add 20 mL of 50% methanol solution, weigh and record. Ultrasonically extract for 30 min, after cooling to room temperature, make up the weight with 50% methanol solution, shake well, filter, take 10 mL of the filtrate and place it in an evaporating dish, evaporate the solvent in a water bath, redissolve with 2 mL of methanol, filter through a 0.22 μm microporous filter membrane, take the subsequent filtrate, inject the sample, and perform HPLC detection at wavelengths of 210, 230, 250, 265, 280, and 320 nm respectively. Take the total number of chromatographic peaks and the resolution as indicators to determine the best detection wavelength.

[0039] From Figure 6 It can be seen that in the chromatogram obtained at a wavelength of 265 nm, the number of chromatographic peaks is relatively large and the resolution is good. Therefore, 265 nm is selected as the best wavelength for HPLC detection.

[0040] 2.7 Selection of Mobile Phase System Accurately weigh 2.0 g of the powder of the same batch of Compound Dendrobium Granules for Treating Thirst, place it in a conical flask, accurately add 20 mL of 50% methanol solution, weigh and record. Ultrasonically extract for 30 min, after cooling to room temperature, make up the weight with 50% methanol solution, shake well, filter, take 10 mL of the filtrate and place it in an evaporating dish, evaporate the solvent in a water bath, redissolve with 2 mL of methanol, filter through a 0.22 μm microporous filter membrane, take the subsequent filtrate, inject the sample, and perform HPLC detection under the mobile phase systems of methanol - 0.1% formic acid water, methanol - 0.1% phosphoric acid water, acetonitrile - 0.1% formic acid water, acetonitrile - 0.1% phosphoric acid water, acetonitrile - 0.2% phosphoric acid water, and acetonitrile - 0.3% phosphoric acid water. Determine the optimal mobile phase system with the total number of chromatographic peaks and resolution as the indicators.

[0041] It can be seen from Figure 7 that when the mobile phase system is acetonitrile - 0.2% phosphoric acid water, the number of chromatographic peaks in the obtained chromatogram is relatively large and the resolution is good. Therefore, acetonitrile - 0.2% phosphoric acid water is selected as the mobile phase system for HPLC detection.

[0042] 2.8 Selection of Different Flow Rates Accurately weigh 2.0 g of the powder of the same batch of Compound Dendrobium Granules for Treating Thirst, place it in a conical flask, accurately add 20 mL of 50% methanol solution, weigh and record. Ultrasonically extract for 30 min, after cooling to room temperature, make up the weight with 50% methanol solution, shake well, filter, take 10 mL of the filtrate and place it in an evaporating dish, evaporate the solvent in a water bath, redissolve with 2 mL of methanol, filter through a 0.22 μm microporous filter membrane, take the subsequent filtrate, inject the sample, and perform HPLC detection at the mobile phase flow rates of 0.8, 1.0, and 1.2 mL / min. Determine the optimal flow rate with the total number of chromatographic peaks and resolution as the indicators.

[0043] It can be seen from Figure 8 that when the mobile phase flow rate is 1.0 mL / min, the number of chromatographic peaks in the obtained chromatogram is relatively large and the resolution is good. Therefore, a flow rate of 1.0 mL / min is selected as the optimal flow rate for HPLC detection.

[0044] 2.9 Selection of Different Column Temperatures Accurately weigh 2.0 g of the powder of the same batch of Compound Dendrobium Granules for Treating Thirst, place it in a conical flask, accurately add 20 mL of 50% methanol solution, weigh and record. Ultrasonically extract for 30 min, after cooling to room temperature, make up the weight with 50% methanol solution, shake well, filter, take 10 mL of the filtrate and place it in an evaporating dish, evaporate the solvent in a water bath, redissolve with 2 mL of methanol, filter through a 0.22 μm microporous filter membrane, take the subsequent filtrate, inject the sample, and perform HPLC detection at the column temperatures of 25, 30, and 35 °C. Determine the optimal column temperature with the total number of chromatographic peaks and resolution as the indicators.

[0045] It can be seen from Figure 9It can be seen that in the chromatogram obtained at a column temperature of 30 °C, the number of chromatographic peaks is relatively large and the resolution is good. Therefore, 30 °C is selected as the optimal column temperature for HPLC detection.

[0046] 3 Methods and Results 3.1 Chromatographic Conditions The chromatographic column used was a Thermo Scientific Accucore TM XL C 18 chromatographic column (4.6 mm × 250 mm, 4 μm). The mobile phase was acetonitrile (A) - 0.2% phosphoric acid in water (B), and the gradient elution was as follows: 0 - 5 min, 3% - 5% A; 5 - 10 min, 5% - 9% A; 10 - 23 min, 9% - 9% A; 23 - 34 min, 9% - 19% A; 34 - 50 min, 19% - 29% A; 50 - 53 min, 29% - 60% A; the column temperature was 30 °C, the flow rate was 1.0 mL / min, the detection wavelength was 265 nm, and the injection volume was 10 μL.

[0047] 3.2 Preparation of Stock Solution of Reference Substances Appropriate amounts of reference substance powders of gallic acid, danshensu, protocatechuic acid, protocatechuic aldehyde, 3 ' -hydroxy puerarin, caffeic acid, puerarin, mangiferin, 3 ' -methoxy puerarin, puerarin-6 ''-O -glucoside, puerarin apiin, daidzin, glycitin, calycosin-7-O-glucoside, genistin, rosmarinic acid, lithospermic acid, daidzein, salvianolic acid B, calycosin, salvianolic acid A were accurately weighed and placed in a volumetric flask. They were dissolved with methanol and made up to the mark to prepare single stock solutions of reference substances, with their mass concentrations being 2.360, 2.270, 3.460, 2.570, 7.600, 7.460, 10.125, 3.020, 2.360, 2.460, 4.088, 4.450, 2.380, 4.000, 2.720, 2.380, 3.960, 3.040, 12.150, 5.120, 5.520 mg / mL respectively. Then, appropriate amounts of each stock solution of reference substance were accurately measured and placed in the same volumetric flask, diluted with methanol and made up to the mark to obtain the mixed stock solution of reference substances. Among them, gallic acid, danshensu, protocatechuic acid, protocatechuic aldehyde, 3 ' -hydroxy puerarin, caffeic acid, puerarin, mangiferin, 3 ' -methoxy puerarin, puerarin-6 ''-OThe concentrations of β-glucoside, puerarin apioglucoside, daidzin, glycitin, calycosin-7-O-β-D-glucoside, genistin, rosmarinic acid, lithospermic acid, daidzein, salvianolic acid B, calycosin, salvianolic acid A were 23.60, 227.00, 34.60, 12.85, 380.00, 37.30, 1012.50, 302.00, 236.00, 98.40, 204.40, 178.00, 23.80, 80.00, 27.20, 238.00, 198.00, 30.40, 1215.00, 51.20, 110.40 μg / mL, respectively.

[0048] 3.3 Preparation of the test solution The compound Shihu Xiaoke granules were ground into powder. 2 g of the powder was precisely weighed and placed in a stoppered flask. 20 mL of 50% methanol was precisely added, weighed, sonicated (power 250 W, frequency 50 kHz) for 30 min, allowed to cool to room temperature and weighed again, and the lost weight was made up with 50% methanol. After shaking well, it was filtered. 10 mL of the filtrate was placed in an evaporating dish and the solvent was evaporated in a water bath. Then 2 mL of methanol was added for re-dissolution, and it was filtered through a 0.22 μm microporous filter membrane. The subsequent filtrate was taken to obtain the test solution.

[0049] 3.4 Preparation of the solutions of single herbs and negative samples Each single herb was weighed according to the prescription ratio of the compound Shihu Xiaoke granules, and a negative sample (lacking a certain single herb in the prescription) was prepared. They were prepared according to the method under item "3.3" respectively to obtain the solutions of single herbs and negative samples.

[0050] 3.5 Methodology investigation 3.5.1 Precision test 2 g of the compound Shihu Xiaoke granule sample (S1) was precisely weighed, prepared according to the method under item "3.3", and detected by HPLC under the chromatographic conditions in item "3.1". It was injected continuously for 6 times, and the chromatogram was recorded. Taking the 16th peak (puerarin) as the reference peak, the RSD values of the peak areas and relative retention times of each chromatographic peak in the obtained chromatogram were less than 2.46% and 2.73% respectively, indicating that the method had good precision.

[0051] 3.5.2 Repeatability test 2 g of the compound Shihu Xiaoke granule sample (S1) was precisely weighed, prepared according to the method under item "3.3", and 6 sample solutions were prepared in parallel. They were injected and detected by HPLC under the chromatographic conditions in item "3.1", and the chromatogram was recorded. Taking the 16th peak (puerarin) as the reference peak, the RSD values of the peak areas and relative retention times of each chromatographic peak in the chromatograms of the 6 samples were less than 0.85% and 2.71% respectively, indicating that the method had good repeatability.

[0052] 3.5.3 Stability Test Accurately weigh 2 g of the compound Shihu Xiaoke Granules sample (S1), prepare it according to the method under item "3.3", and inject samples for HPLC determination according to the chromatographic conditions under item "3.1" at 0, 2, 4, 8, 12, 16, and 24 h at room temperature, and record the chromatogram. Taking the 16th peak (puerarin) as the reference peak, calculate the RSD of the areas and relative retention times of each chromatographic peak in the obtained chromatogram, which are less than 1.92% and 2.28% respectively, indicating that the solution has good stability within 24 h.

[0053] 3.5.4 Establishment and Similarity Analysis of Fingerprint Take 15 batches of compound Shihu Xiaoke Granules samples (S1 - S15), prepare them according to the method under item "3.3", and inject samples for HPLC determination according to the chromatographic conditions under item "3.1", and record the generated chromatogram. Use the software "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" to process and analyze the above chromatograms. Taking the S1 batch as the reference chromatogram, using the median method, with a time window of 0.1 min, calibrate the common peaks with strong signals, obvious peak shapes, and good stability in the chromatogram. A total of 36 common peaks are calibrated, and after multi-point calibration, a reference fingerprint (R) is generated by fitting. Then, match the chromatograms of 15 batches of compound Shihu Xiaoke Granules samples (S1 - S15) with the reference fingerprint and calculate the similarity, which are 0.999, 0.999, 0.998, 0.999, 0.993, 0.994, 0.993, 0.993, 0.989, 0.989, 0.986, 0.985, 0.993, 0.982, 0.994 respectively, all greater than 0.980, indicating that the quality of each batch of compound Shihu Xiaoke Granules samples is relatively stable.

[0054] 3.5.5 Attribution and Identification of Common Peaks Take the mixed reference substance stock solution, test solution, and single-herb solutions prepared under items "3.2", "3.3", and "3.4", inject samples for HPLC detection according to the chromatographic conditions under item "3.1", and record the chromatogram. Compare the relative retention times and spectrograms of each chromatographic peak of the test solution and the single-herb solutions to determine the attribution of the 36 common peaks of compound Shihu Xiaoke Granules in 12 herbs. The results are shown in Figure 11 .

[0055] Figure 11The results showed that in the chromatogram of the compound Dendrobium and Anemarrhena Granules, peak 1 was from Schisandra chinensis, peaks 2 - 5 were from Rehmannia glutinosa, peaks 6, 28, 30, 33, 35 were from Salvia miltiorrhiza, peaks 7, 8, 25, 34 were from Astragalus membranaceus, peaks 10, 14, 17 were from Anemarrhena asphodeloides, peaks 11 - 13, 15, 16, 18 - 24, 26, 27, 32 were from Pueraria lobata, peak 36 was from Scrophularia ningpoensis, peak 9 was common to Scrophularia ningpoensis and Anemarrhena asphodeloides, peak 29 was common to Salvia miltiorrhiza and Scrophularia ningpoensis, and peak 31 was common to Astragalus membranaceus and Pueraria lobata.

[0056] By comparing with the chromatogram of the mixed reference substance stock solution, 21 components in the chromatogram of the compound Dendrobium and Anemarrhena Granules samples were determined (such as Figure 12 ), namely peaks 1 (gallic acid), 6 (danshensu), 7 (protocatechuic acid), 10 (protocatechualdehyde), 13 (3 ' -hydroxy puerarin), 14 (caffeic acid), 16 (puerarin), 17 (mangiferin), 18 (3 ' -methoxy puerarin), 19 (puerarin-6 '' - O -glucoside), 20 (puerarin apiin), 21 (daidzin), 22 (glycitin), 25 (calycosin-7-O-β-D-glucoside), 26 (genistin), 29 (rosmarinic acid), 30 (lithospermic acid), 32 (daidzein), 33 (salvianolic acid B), 34 (calycosin), 35 (salvianolic acid A).

[0057] 3.6 Determination of the Contents of 21 Components in Compound Dendrobium and Anemarrhena Granules 3.6.1 Specificity Test Take the mixed reference substance stock solution, the test solution, and the negative sample solution prepared under items "3.2", "3.3", and "3.4", and inject them for HPLC detection according to the chromatographic conditions under item "3.1", and record the chromatogram.

[0058] Figure 13 The results showed that the resolution of the chromatographic peaks of the 21 components in the test solution was good, and the chromatographic peaks of the negative sample showed no interfering peaks, indicating that the quantitative method had good specificity.

[0059] 3.6.2 Investigation of Linear Relationship Take the mixed reference substance stock solution prepared under item "3.2", dilute it with 50% methanol to prepare a series of gradient concentration reference solutions, inject them for HPLC detection according to the chromatographic conditions under item "3.1", and record the peak areas of the 21 components. Then, with the concentration of the reference solution as the abscissa ( X ), and the peak area as the ordinate ( Y ), perform linear regression to obtain the standard curves, regression equations, correlation coefficients, and linear ranges of each component. The results are shown in Table 1.

[0060] Table 1 Linear relationship of each component

[0061] 3.6.3 Precision investigation Take about 2 g of the compound Dendrobium officinale granules for relieving thirst (S1), prepare according to the method under item "3.3", and under the chromatographic conditions under item "3.1", inject samples continuously 6 times within 1 day, and repeat the injection continuously for 3 days (inject 3 times a day), record the peak areas of 21 components, and calculate the RSD values of inter-day and intra-day precision. The results show that the RSDs are all less than 2.84%, indicating good precision of the instrument.

[0062] 3.6.4 Stability investigation Take about 2 g of the compound Dendrobium officinale granules for relieving thirst (S1), prepare according to the method under item "3.3", and after standing at room temperature for 0, 2, 4, 8, 12, and 24 h, inject samples for HPLC detection respectively under the chromatographic conditions under item "3.1", obtain the peak areas of 21 components, and calculate the RSD values of 21 components. The results show that the RSDs are all less than 2.64%, indicating good stability of the test solution within 24 h.

[0063] 3.6.5 Repeatability investigation Take 6 portions of the compound Dendrobium officinale granules for relieving thirst (S1) of the same batch, each about 2 g, prepare in parallel according to the method under item "3.3", and inject samples for HPLC detection respectively under the chromatographic conditions under item "3.1", record the peak areas of 21 components, and then substitute them into the regression equation under item "3.6.2" to calculate the contents and RSD values of each component. The results show that the RSDs of 21 components are all less than 2.89%, indicating good repeatability of the method.

[0064] Table 2 Results of methodology investigation for 21 components (%)

[0065] 3.6.6 Test for recovery of added samples Take 6 portions of the compound Dendrobium officinale granules for relieving thirst (S1) of the same batch with known contents, each about 1.0 g, weigh accurately, add each reference substance approximately according to the ratio of the content in the sample to the amount of the reference substance being about 1:1, then prepare according to the method under item "3.3", and inject samples for HPLC detection under the chromatographic conditions under item "3.1", record the peak areas of 21 components, and then substitute them into the regression equation under item "3.6.2" to calculate the contents and recoveries of added samples of each component. The results are shown in Table 3. The results indicate high accuracy of this method.

[0066] Table 3 Results of test for recovery of added samples of 21 components

[0067]

[0068]

[0069] 3.6.7 Determination of Sample Content Take 15 batches of compound Dendrobium officinale granules for relieving thirst samples (S1~S15), prepare them in parallel according to the method under "3.3", and inject samples separately under the chromatographic conditions under "3.1" for HPLC detection. Record the peak areas of 21 components, and then substitute them into the regression equation under "3.6.2" to calculate the contents of each component. The results are shown in Table 4.

[0070] Then import the content data of 21 components of 15 batches of samples into Origin 2024 software, and use the Heat MapDendrogram.opx plug-in to perform heat map clustering analysis on them. According to the color difference of the heat map squares, the content differences of each component among different batches can be visually found.

[0071] Table 4 Determination results of 21 components in 15 batches of compound Dendrobium officinale granules for relieving thirst ( n =3, mg / g)

[0072] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. A method for HPLC fingerprint of compound Shihu Xiaoke granules and determination of 21 components, characterized in that: The following steps are involved: 1) Preparation of reference solution: accurately weigh gallic acid, danshensu, protocatechuic acid, protocatechuic aldehyde, 3 ' -Hydroxypuerarin, caffeic acid, puerarin, mangiferin, 3 ' -Methoxypuerarin, Puerarin-6 ''-O - glucoside, puerarin apiglycoside, daidzein, glycitin, calycosin glucoside, genistin, rosmarinic acid, lithospermic acid, daidzein, salvianolic acid B, calycosin, salvianolic acid A reference substance powders in appropriate amounts are dissolved in methanol and fixed to volume to prepare single reference substance stock solutions, then each reference substance stock solution is accurately measured, placed in the same volumetric flask, diluted with methanol, fixed to volume, and a mixed reference substance mother solution is obtained; then the mixed reference substance mother solution is accurately measured, and diluted with a methanol solution with a volume concentration of 50% to obtain reference substance solutions with a series of concentrations; 2) Preparation of test solution: Grind the compound Shihu Xiaoke granules sample to be tested into powder, accurately weigh the powder and accurately add a 50% volume concentration of methanol solution, make up for the weight loss after ultrasonic extraction, shake well, filter, take the filtrate and place it in an evaporating dish in a water bath to evaporate the solvent, add methanol to the residue and filter it through a 0.22 μm microporous filter membrane, and take the filtrate as the test solution; 3) Sample determination: Accurately pipette the prepared mixed reference substance mother solution, reference substance solution with a series of concentrations and test substance solution, respectively, and obtain the corresponding chromatogram by HPLC detection; 4) Establishment of fingerprint: The chromatogram of the test sample was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for processing to obtain the corresponding fingerprint, and the chromatogram of the mixed reference substance mother solution was used to attribute the 21 characteristic components; 5) Determination of the content of 21 characteristic components in the sample: Linear regression is performed with the peak areas of the chromatographic peaks of the 21 characteristic components in the chromatogram of the reference solution with a series of concentrations as the ordinate and the concentrations of the 21 characteristic components in the reference solution as the abscissa to obtain the standard curves and linear equations of the 21 characteristic components, respectively. The obtained linear equations and the chromatogram of the test sample are then used to calculate the contents of the 21 characteristic components in the test solution.

2. The method according to claim 1, characterized in that Step 2) The solid-liquid ratio of the extraction is 1 g:10 mL, and the extraction time is 30 min.

3. The method according to claim 1, characterized in that Step 3) The chromatographic conditions of the HPLC detection are: Thermo Scientific Accucore TM XL C 18 The chromatographic column had a specification of 4.6 mm×250 mm and 4 μm. The mobile phase A was acetonitrile, and the mobile phase B was a 0.2% volume concentration of phosphoric acid aqueous solution. The elution method was gradient elution, and the elution program was as follows: 0-5 min, the volume of mobile phase A increased from 3% to 5%; 5-10 min, the volume of mobile phase A increased from 5% to 9%A; 10-23 min, the volume of mobile phase A remained at 9%; 23-34 min, the volume of mobile phase A increased from 9% to 19%A; 34-50 min, the volume of mobile phase A increased from 19% to 29%A; 50-53 min, the volume of mobile phase A increased from 29% to 60%A; the column temperature was 30°C, the flow rate was 1.0 mL / min, the detection wavelength was 265 nm, and the injection volume was 10 μL.

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