Construction method of gallnut aqueous extract characteristic spectrum and contrast spectrum and distinguishing method of gallnuts with different commodity specifications

Abstract

The invention discloses a construction method of a gallnut aqueous extract characteristic chromatogram and a control chromatogram and a distinguishing method of gallnuts with different commodity specifications. The construction method comprises the steps of preparation of a test solution, preparation of a reference solution, establishment of a chromatographic method, determination and comparison of gallnut samples with different specifications, material basic research, identification of samples to be detected and the like. According to the invention, a chromatographic peak method is carried out by comparing and researching component difference of gallnuts with different commodity specifications, an ultra-high performance liquid chromatography is adopted, standard decoction with different commodity specifications is compared and researched, the internal quality of the gallnut standard decoction is controlled, the characteristics of the gallnut standard decoction can be comprehensively reflected, and the method has a wide application prospect. The method can provide a new analysis means for quality control of gallnut standard decoction and formula granules, and achieves the purpose of identifying the gallnut standard decoction and the gallnut standard formula granules.

Description

Technical Field

[0001] The invention relates to a method for constructing a characteristic spectrum and a reference spectrum of a gallnut water extract and a method for distinguishing gallnuts of different commercial specifications, and belongs to the technical field of traditional Chinese medicine analysis. Background Art

[0002] Gallnut is a plant of Anacardiaceae Rhus chinensis Mill. Rhus potaninii Maxim. or Red Bran Poplar Rhus punjabensis Galls on the leaves of Stew.var.sinica (Diels)Rehd.et Wils., mainly caused by Galla aphid Melaphis chinensis (Bell) Baker parasites. It has the effects of astringing the lungs and reducing fire, astringing the intestines and stopping diarrhea, astringing sweat, stopping bleeding, and astringing moisture. It is used for chronic cough due to lung deficiency, cough with phlegm caused by lung heat, chronic diarrhea and dysentery, spontaneous sweating, night sweats, thirst, blood in stool and hemorrhoids, bleeding from trauma, carbuncle, sore, and wet and rotten skin.

[0003] Modern research shows that the chemical components of gallnut include tannin (also known as tannic acid, tannic acid), gallic acid, gallnut oil, etc. Among them, tannin has the highest content, which can reach 70%. Gallnut has pharmacological effects such as antidiarrhea, antibacterial, anti-inflammatory, antioxidant, antiviral, anti-aging and hypoglycemic, in addition to anti-cancer, anti-fertility, and free radical scavenging effects.

[0004] The quality of gallnut is judged by its medicinal shape, and it is divided into two specifications: "Jiaobei" and "Dubei" based on the difference in shape. The Identification of Chinese Medicinal Materials Commodity Specifications and Quality (1995) records: "The best ones are those that are large, complete, thick-walled and gray-brown in color"; the Chinese Materia Medica (1996) records: The appearance of medicinal materials is divided into "horn-shaped" and "belly-shaped", "The best ones are those that are large, complete, thick-walled, gray-brown and pure"; the Chinese Materia Medica (1999) records: The appearance of medicinal materials is divided into "horn-shaped" and "belly-shaped", "The best ones are those that are large, complete, thick-walled, gray-brown and pure"; the Modern Chinese Medicinal Materials Commodity History (2001) records: According to the appearance, they are divided into "horn-shaped" and "belly-shaped", and the "belly-shaped" is of high quality, while the horn-shaped is of low quality; the Experience Identification of 500 Commonly Used Chinese Medicinal Materials (2002) evaluates the quality as "the best ones are those that are large, complete, thick-walled and gray-brown in color"; and according to the appearance, they are divided into two specifications, "horn-shaped" and "belly-shaped", and divided into two grades according to size and individual quality. The Illustrated Dictionary of Authentic Medicinal Materials (Southwest Volume) (2003) states: According to their appearance, they are divided into "horn-shaped" and "belly-shaped" types, and "the ones that are large, complete, thick-walled, gray-brown in color and pure are the best."

[0005] At present, the comparative research on different commodity specifications of Dubei mainly focuses on the research on properties and content. Liang Qing et al. studied the quality of different commodity specifications of gallnut. The quality evaluation results showed that the gallic acid content of each grade of Dubei was higher than that of Jiaobei, which is consistent with the traditional belief that "Dubei is of high quality and Jiaobei is of low quality"; the wall thickness measurement results found that the wall thickness of Dubei was significantly greater than that of Jiaobei, making Dubei look more solid, while Jiaobei is fragile, so most of the broken samples on the market are Jiaobei, which may be the main reason why traditional Dubei is better than Jiaobei. After decocting, the gallnut slices are made into standard decoctions, which lose the shape of the slices and cannot distinguish the gallnut standard decoctions of different commodity specifications. Therefore, it is necessary to establish a more specific characteristic map for distinction. The patent (application number: 2019108219503, a method for constructing a UPLC fingerprint of Chinese gallnut medicinal materials, a fingerprint constructed by the method and its application) discloses that the characteristic spectra of Chinese gallnut (Dubei) and Chinese gallnut (Jiaobei) medicinal materials were established respectively, and the similarity was used to distinguish the two. The characteristic peaks that differed between the two were not clearly defined, and the distinguishing points were not quantified, so they cannot be used to distinguish the water extracts of Chinese gallnut (Dubei) and Chinese gallnut (Jiaobei). Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is a method for constructing a characteristic spectrum and a reference spectrum of gallnut water extract, and a method for distinguishing gallnuts of different commercial specifications.

[0007] Technical solution: To solve the above technical problems, the present invention provides a method for constructing a characteristic map of Galla chinensis (Galax chinensis), comprising the following steps: (1) Preparation of test solution: Take the standard decoction of Galla chinensis (Galla chinensis), extract it with organic solvent, shake well, filter, and take the filtrate to obtain the test solution; (2) Preparation of reference solution: Take gallic acid reference substance, 1,2,3,4,6- O -Pentagalloylglucose reference substance, respectively, add solvent to dissolve, and obtain reference substance solution; take gallnut (horn or belly) reference medicinal material, add methanol, ultrasonically treat, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution; (3) Establishment of characteristic spectrum: Take the standard decoction of Galla chinensis (Dubei) and the reference solution, inject them into the high performance liquid chromatography respectively, and measure to obtain the characteristic spectrum; the characteristic spectrum has 10 peaks in total, take gallic acid as the reference, peak 1 is the chromatographic peak of gallic acid, the peak corresponding to the gallic acid reference is the S1 peak, calculate the relative retention time of peaks 2 to 4 and S1 peak, and the relative retention time is within the range of ±10% of the specified value. The specified values ​​of peaks 2 to 4 are 2.85, 3.18, and 3.86 respectively; take 1, 2, 3, 4, 6- O -pentagalloylglucose as the reference substance, peak 8 is 1,2,3,4,6-O -pentagalloylglucose; and 1,2,3,4,6- O The peak corresponding to -pentagalloylglucose is S2 peak. The relative retention times of peaks 5 to 7, 9, 10 and S2 peak are calculated, and their relative retention times are within ±10% of the specified values. The specified values ​​of peaks 5 to 7, 9, and 10 are 0.49, 0.73, 0.89, 1.11, and 1.24, respectively.

[0008] The chromatographic conditions of the high performance liquid chromatography in step (3) are as follows: chromatographic column: Agilent Aq-C18; injection volume: 1 μl; flow rate: 0.25-0.35 ml / min; column temperature: 25-35°C; detection wavelength: 273 or 280 nm; acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution.

[0009] Wherein, the gradient elution in step (3) is as follows: 0-5 min, the volume fraction of mobile phase A is 8%, and the volume fraction of mobile phase B is 92%; 5-6 min, the volume fraction of mobile phase A is 8→14%, and the volume fraction of mobile phase B is 92→86%; 6-13 min, the volume fraction of mobile phase A is 14→16%, and the volume fraction of mobile phase B is 86→84%; 13-17 min, the volume fraction of mobile phase A is 16→17%, and the volume fraction of mobile phase B is 84→83%; 17-20 min, the volume fraction of mobile phase A is 17→19%, and the volume fraction of mobile phase B is 83→81%; 20-25 min, the volume fraction of mobile phase A is 19→23%, and the volume fraction of mobile phase B is 81→77%; 25-30 min, the volume fraction of mobile phase A is 23%, and the volume fraction of mobile phase B is 77%.

[0010] Wherein, in step (1), take the slices of Galla chinensis (Dubei), soak them in water, decoct them, filter them, concentrate the filtrate under reduced pressure, and freeze-dry them to obtain the standard decoction of Galla chinensis (Dubei).

[0011] The present invention also provides a method for establishing a comparison map of Galla chinensis (Galla chinensis), comprising the following steps: (1) Prepare multiple batches of Galla chinensis (Dubei) standard decoction test sample solutions; (2) According to the method for constructing the characteristic spectrum of Galla chinensis (Dubei) as described in claim 1, the test solution is tested respectively to obtain the characteristic spectrum of the standard decoction of Galla chinensis; (3) The obtained characteristic spectrum was imported into the Chinese medicine chromatographic fingerprint similarity evaluation system to establish the reference spectrum of the standard decoction of Galla chinensis.

[0012] The present invention also provides a method for distinguishing gallnut (Dubei) and gallnut (Jiaobei), comprising the following steps: (1) Preparation of test solution: extract the standard decoction of Galla chinensis with organic solvent, shake well, filter, and take the filtrate to obtain the test solution; (2) Preparation of reference solution: Weigh reference gallic acid and 1,2,3,4,6-O-pentagalloylglucose respectively, and add methanol solution as reference solution; weigh reference Chinese medicinal material Galla chinensis, add methanol to extract, shake well, filter, and take the filtrate as reference solution.

[0013] (3) HPLC chromatogram detection: Take the test solution or the reference solution respectively, inject it into the HPLC, perform the test, and record the chromatogram; take gallic acid as the reference, peak 1 is the chromatographic peak of gallic acid, and the peak corresponding to the gallic acid reference is the S1 peak; take 1,2,3,4,6- O -pentagalloylglucose as the reference substance, peak 8 is 1,2,3,4,6- O -pentagalloylglucose; and 1,2,3,4,6- O -The peak corresponding to pentagalloylglucose is S2 peak; the chromatographic conditions of the high performance liquid chromatograph are: chromatographic column: Agilent Aq-C18; injection volume: 1 μl; flow rate: 0.25~0.35 ml / min; column temperature: 25~35°C; detection wavelength: 273 or 280 nm; acetonitrile is used as mobile phase A, and 0.1% phosphoric acid aqueous solution is used as mobile phase B for gradient elution; the gradient elution is: 0~5 min, the volume fraction of mobile phase A is 8%, and the volume fraction of mobile phase B is 92%; 5~6 min, the volume fraction of mobile phase A is 8→14%, and the volume fraction of mobile phase B is 92→86%; 6~13 min, the volume fraction of mobile phase A is 1 4→16%, the volume fraction of mobile phase B is 86→84%; 13~17min, the volume fraction of mobile phase A is 16→17%, the volume fraction of mobile phase B is 84→83%; 17~20min, the volume fraction of mobile phase A is 17→19%, the volume fraction of mobile phase B is 83→81%; 20~25min, the volume fraction of mobile phase A is 19→23%, the volume fraction of mobile phase B is 81→77%; 25~30min, the volume fraction of mobile phase A is 23%, the volume fraction of mobile phase B is 77%; (4) Analysis: Select the peak with stable relative retention time in the chromatogram as the characteristic peak, and then compare it with the peak of the reference solution spectrum; (5) Discrimination: Compare the chromatogram of the test solution with that of the reference solution; when the chromatogram of the test solution has peaks 1 to 10, the test sample is Galla chinensis (Galax chinensis); when the chromatogram of the test solution has peaks 1, 2 to 4, 7, 8, and 10, the test sample is Galla chinensis (Galax chinensis).

[0014] Among them, when the gallnut standard decoction is adulterated, the relative peak area of ​​peak 7 is used as the reference peak to distinguish the adulterated samples in step (5), and 0.15 is used as the specified value. Finally, gallic acid is used as the reference peak. If the sum of the relative peak areas of peaks 5 and 6 is greater than 0.15, the sample being tested is gallnut (galnut); the peak corresponding to the peak of the gallic acid reference substance is the S1 peak, and the relative peak area of ​​the sum of the peak areas of peaks 5 and 6 and the S1 peak is calculated. If the relative peak area is less than 0.15, it is gallnut.

[0015] The relative retention time in step (4) is the average value of several groups of experimental results.

[0016] The characteristic spectrum of Galla chinensis (Galla chinensis) in step (4) has 10 characteristic peaks, gallic acid is taken as the reference substance, and the S1 peak is the chromatographic peak of gallic acid; 1,2,3,4,6-O-pentagalloylglucose is taken as the reference substance, and the S2 peak is 1,2,3,4,6-O-pentagalloylglucose; the relative retention times of peaks 5, 6 and S2 are calculated, and the relative retention times should be within the range of ±10% of the specified value, the specified value of peak 5 is 0.49, and the specified value of peak 6 is 0.73; the peak corresponding to the peak of the gallic acid reference substance is the S1 peak, and the relative peak area of ​​the sum of the peak areas of peaks 5 and 6 and the S1 peak is calculated, and the relative peak area is greater than 0.15.

[0017] Wherein, the characteristic spectrum of gallnut (Galnut) in step (4) has 7 characteristic peaks, gallic acid is taken as the reference substance, and the S1 peak is the chromatographic peak of gallic acid; 1,2,3,4,6-O-pentagalloylglucose is taken as the reference substance, and the S2 peak is 1,2,3,4,6-O-pentagalloylglucose; the relative retention times of peaks 5, 6 and S2 are calculated, and the relative retention times should be within the range of ±10% of the specified value, the specified value of peak 5 is 0.49, and the specified value of peak 6 is 0.73; the peak corresponding to the peak of the gallic acid reference substance is the S1 peak, and the relative peak area of ​​the sum of the peak areas of peaks 5 and 6 and the S1 peak is calculated, and the relative peak area is less than 0.15.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The method is simple, reproducible, accurate and reliable, easy to operate, short time, low solvent consumption and little environmental pollution; 2. The present invention adopts ultra-high performance liquid chromatography through a chromatographic method for the difference in the components of different commercial specifications of gallnut (doubei, jiaobei), and reasonably controls the chromatographic method, using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B for gradient elution, and establishing 10 common peaks of gallnut (doubei); 3. The characteristic spectrum of the standard decoction of gallnut (doubei) is constructed, and the gallnut (doubei, jiaobei) of different commercial specifications is compared and studied, and the intrinsic quality of the entire standard decoction of gallnut is controlled, which can more comprehensively reflect the characteristics of the standard decoction of gallnut (doubei, jiaobei) of different commercial specifications, and can provide a new analytical means for the intrinsic quality control of the standard decoction of gallnut (doubei) and the formula granules, and achieve the purpose of identifying the gallnut (doubei, jiaobei) of different commercial specifications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 1; Figure 2 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 2; Figure 3 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 3; Figure 4 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 4; Figure 5 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 5; Figure 6 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 6; Figure 7 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 7; Figure 8 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 8; Fig. 9 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 9; Fig.10 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 10; Fig.11 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 11; Fig.12 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 12; Fig.13 The characteristic spectrum of the standard decoction of Galla chinensis in chromatographic condition 13; Fig.14The chromatograms of standard decoction of Galla chinensis (Dubei) under different chromatographic columns; Fig.15 The chromatograms of the standard decoction of Galla chinensis (Dubei) under different column temperature conditions; Fig.16 The chromatograms of the standard decoction of Galla chinensis (Dubei) under different flow rate conditions; Fig.17 The chromatograms of the standard decoction of Galla chinensis (Dubei) under the conditions of different brands of instruments; Fig.18 Overlay of 17 batches of standard decoctions of Galla chinensis (Dubei) Fig.19 This is the reference characteristic spectrum of the standard decoction of Galla chinensis (Dubei); Fig. 20 This is a comparison chart of the standard decoction of Gallic Acid (Dubei) and the standard decoction of Gallic Acid (Jiaobei); Fig.21 This is the identification chart of the reference material of Galla chinensis (Dubei); Fig. 22 These are the chromatograms of the reference medicinal materials of Gallic Acid (Dubei) and Gallic Acid (Jiaobei). DETAILED DESCRIPTION

[0020] In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0021] Unless otherwise specified, the term "test article" as used herein refers to an experimental sample used for detection or identification.

[0022] Unless otherwise specified, the term "reference material" as used herein refers to a standard substance used for identification, inspection, content determination and calibration of instrument performance.

[0023] Unless otherwise specified, the term "precision weighing" used in this article means that the weight should be weighed accurately to one thousandth of the weight taken, the term "weighing" means that the weight should be weighed accurately to one hundredth of the weight taken, the term "precision measuring" means that the volume should be measured accurately to one thousandth of the volume taken, and the term "precision pipetting" refers to the operating method of accurately measuring samples through a microinjector.

[0024] Unless otherwise specified, the term "filler" as used herein refers to a substance used as a stationary phase in chromatography, the term "stationary phase" refers to a phase that is fixed in position in chromatography and has an adsorption effect on the sample, and the term "mobile phase" refers to a phase that can flow freely in chromatography and has a desorption effect on the sample.

[0025] Unless otherwise specified, the term "gradient elution" used herein refers to an elution method in which the composition and proportion of the mobile phase change according to a certain procedure during the chromatographic analysis cycle of the sample.

[0026] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0027] Instruments, reagents and samples Instruments: Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatography system (Waters); Waters Quaternary Solvent Manager quaternary pump (Waters); Sample Manager-FTN autosampler (Waters); Waters UPLC PDA detector (Waters); Empower 3 chromatography workstation (Waters); Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent); KQ-250E ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); pure water system (Sartorius); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory); AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.).

[0028] Reagents: acetonitrile (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, Aladdin); water is ultrapure water; other reagents are of analytical grade.

[0029] References: 1, 2, 3, 4, 6- O-Pentagalloylglucose reference substance was purchased from Shanghai Shidande Biotechnology Co., Ltd. with batch number 3367. Gallic acid reference substance was purchased from China Food and Drug Inspection Institute with batch number 110831-201906.

[0030] Reference medicinal materials: Galla chinensis (Dubei) reference medicinal material (batch number: 121078-201103); Galla chinensis (Jiaobei) reference medicinal material (batch number: 121078-201504), both purchased from China Food and Drug Inspection Institutes.

[0031] Samples (Table 1): 17 batches of Galla chinensis (Dubei) decoction pieces were collected; 3 batches of Galla chinensis (Jiaobei) decoction pieces. All of the above Galla chinensis decoction pieces comply with the various provisions under the Galla chinensis item of the 2020 edition of the "Chinese Pharmacopoeia".

[0032] Table 1 Origin information of different specifications of Galla chinensis slices

[0033] Preparation of standard decoction: take 100g of Galla chinensis (Dubei) slices (hereinafter referred to as Dubei slices), weigh them, put them in a casserole, add 800ml of water and soak them for 30min, use a YMW mechanical split decoction pot, first boil over high heat, then simmer over low heat for 30min, select a 200-mesh standard sieve to filter while hot, and cool the filtrate in a cold water bath; add 600ml of water for the second decoction, boil over high heat, then simmer over low heat for 25min, filter through a 200-mesh standard sieve while hot, and cool the filtrate in a cold water bath; combine the two filtrates and concentrate them under reduced pressure at 65℃; concentrate to a relative density of 1.06~1.07 (65℃), divide the concentrate into vials, place them in a freeze dryer, and freeze-dry them to obtain the standard decoction of Galla chinensis.

[0034] Preparation of reference solution: Take gallic acid reference substance, 1,2,3,4,6- O -Pentagalloylglucose reference substance was weighed accurately and methanol was added to prepare solutions containing 20µg gallic acid per 1ml and 1,2,3,4,6- O -Pentagalloylglucose 200µg solution was used as the reference solution. Take 0.2g of the Chinese gallnut reference medicinal material, place it in a stoppered conical bottle, add 25ml of methanol, and ultrasonically treat (power 250W, frequency 40kHz) for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material.

[0035] Preparation of test solution: Take an appropriate amount of standard decoction of Gallic chinensis, grind it into powder, take 0.1g, weigh it accurately, put it in a stoppered conical flask, add 25ml of methanol accurately, stopper it tightly, weigh it, treat it with ultrasound (power 250W, frequency 40kHz) for 60 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0036] Example 1 Determination of chromatographic conditions Chromatographic conditions 1: Chromatographic conditions used ZORBAX SB C18 RRHD (2.1 mm × 150 mm, 1.8 µm); methanol was used as mobile phase A, 0.1% phosphoric acid was used as mobile phase B, and gradient elution was performed according to the provisions in Table 2; the flow rate was 0.3 ml per minute; the column temperature was 40 °C, and the detection wavelength was 273 nm.

[0037] Table 2 Gradient elution table

[0038] The results show that ( Figure 1 ), when the organic phase in chromatographic condition 1 is methanol, the separation of the chromatographic peaks in the 9-10 minute spectrum is poor. The chromatographic conditions were further optimized to improve the separation of the chromatographic peaks in this section.

[0039] Chromatographic condition 2: Chromatographic condition: ZORBAX SB C18 RRHD (2.1 mm × 150 mm, 1.8 µm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 3; flow rate 0.3 ml per minute; column temperature 30 °C, detection wavelength 273 nm.

[0040] Table 3 Gradient elution table

[0041] The results show that ( Figure 2 ), after adjusting the elution gradient of chromatographic condition 2, the separation of the chromatographic peaks in the 17-18 minute spectrum was poor. The chromatographic conditions were further optimized to improve the separation of the chromatographic peaks in this section.

[0042] Chromatographic condition 3: Use chromatographic condition ZORBAX SB C18 RRHD (2.1 mm × 150 mm, 1.8 µm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 4; flow rate 0.3 ml per minute; column temperature 35 °C, detection wavelength 273 nm.

[0043] Table 4 Gradient elution table

[0044] The results show that ( Figure 3), chromatographic condition 3. Under the adjustment of this method, the peak coverage has been improved, but the chromatographic peak separation in the 18-19 minute spectrum is still poor, and the 3-8 minute blank section is long. Therefore, the separation of each peak needs to be further optimized.

[0045] Chromatographic condition 4: Use chromatographic condition ZORBAX SB C18 RRHD (2.1 mm × 150 mm, 1.8 µm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 5; flow rate 0.3 ml per minute; column temperature 35 °C, detection wavelength 273 nm.

[0046] Table 5 Gradient elution table

[0047] The results show that ( Figure 4 ), chromatographic condition 4. Under the adjustment of this method, the peak wrapping situation has been improved, but the chromatographic peaks presented in the 18-19 minute spectrum still have peak wrapping. Therefore, the separation degree of each peak needs to be further optimized.

[0048] Chromatographic condition 5: Use chromatographic condition ZORBAX SB C18 RRHD (2.1 mm × 150 mm, 1.8 µm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 6; flow rate 0.3 ml per minute; column temperature 30 °C, detection wavelength 273 nm.

[0049] Table 6 Gradient elution table

[0050] The results show that ( Figure 5 ), the gradient change in chromatographic condition 5 did not improve the separation much, and other types of chromatographic columns were tried for optimization in the future.

[0051] Chromatographic condition 6: Use chromatographic condition Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 7; flow rate 0.3 ml per minute; column temperature 30 ° C, detection wavelength 273 nm.

[0052] Table 7 Gradient elution table

[0053] The results show that ( Figure 6 ), chromatographic condition 6 This method can basically reproduce the method of the previous chromatographic column, so the separation degree of each peak will be further optimized based on this method.

[0054] Chromatographic condition 7: Use chromatographic condition Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 8; flow rate 0.3 ml per minute; column temperature 30 ° C, detection wavelength 273 nm.

[0055] Table 8 Gradient elution table

[0056] The results show that ( Figure 7 ), chromatographic condition 7. With the adjustment of this method, the peak coverage was improved and the time was shortened. Therefore, based on this method, the separation of each peak in 12-15 minutes was further optimized.

[0057] Chromatographic conditions 8: Use chromatographic conditions Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 9; flow rate 0.3 ml per minute; column temperature 30 ° C, detection wavelength 273 nm.

[0058] Table 9 Gradient elution table

[0059] The results show that ( Figure 8 ), chromatographic condition 8. With the adjustment of this method, the peak wrapping situation has been improved, but the chromatographic peak at 17 minutes still has the peak wrapping phenomenon, so the separation degree of each peak will be further optimized based on this method.

[0060] Chromatographic conditions 9: Use chromatographic conditions Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm); methanol as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 10; flow rate 0.3 ml per minute; column temperature 25 ° C, detection wavelength 273 nm.

[0061] Table 10 Gradient elution table

[0062] The results show that ( Fig. 9 ), the gradient change of chromatographic condition 9 did not improve the separation much, and other mobile phases were tried for optimization in the future.

[0063] Chromatographic condition 10: Use chromatographic condition Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm); use acetonitrile as mobile phase A, use 0.1% phosphoric acid as mobile phase B, and perform gradient elution as specified in Table 11; the flow rate is 0.3 ml per minute; the column temperature is 25°C, and the detection wavelength is 273 nm.

[0064] Table 11 Gradient elution table

[0065] The results show that ( Fig.10 ), chromatographic condition 10. Under the adjustment of this method, the chromatographic peak information is richer, but the chromatographic peaks in the first 10 minutes are too dispersed, and the chromatographic peak at 16 minutes still has a peak-wrapping phenomenon. Therefore, the separation degree of each peak is further optimized based on this method.

[0066] Chromatographic condition 11: Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm) was used; acetonitrile was used as mobile phase A, 0.1% phosphoric acid was used as mobile phase B, and gradient elution was performed according to the provisions in Table 12; the flow rate was 0.3 ml per minute; the column temperature was 25°C, and the detection wavelength was 273 nm.

[0067] Table 12 Gradient elution table

[0068] The results show that ( Fig.11 ), chromatographic condition 11. Under the adjustment of this method, the peak wrapping was improved, but the chromatographic peaks at 6 to 7 minutes showed a peak wrapping phenomenon. Therefore, the separation degree of each peak was further optimized based on this method.

[0069] Chromatographic condition 12: Use Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm); use acetonitrile as mobile phase A, use 0.1% phosphoric acid as mobile phase B, and perform gradient elution as specified in Table 13; the flow rate is 0.3 ml per minute; the column temperature is 30°C, and the detection wavelength is 273 nm.

[0070] Table 13 Gradient elution table

[0071] The results show that ( Fig.12 ), chromatographic condition 12. Under the adjustment of this method, the peak wrapping was improved, but the chromatographic peak around 14 minutes showed a peak wrapping phenomenon. Therefore, the separation degree of each peak was further optimized based on this method.

[0072] Chromatographic condition 13: Use Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm); use acetonitrile as mobile phase A, use 0.1% phosphoric acid as mobile phase B, and perform gradient elution as specified in Table 14; the flow rate is 0.3 ml per minute; the column temperature is 35°C, and the detection wavelength is 280 nm.

[0073] Table 14 Gradient elution table

[0074] The results show that ( Fig.13 ), under chromatographic condition 13, the separation of some characteristic peaks was good, basically meeting the requirements of the characteristic spectrum. There was little difference between 273 nm and 280 nm. Considering that the maximum absorption wavelength of most peaks was around 280 nm and 1,2,3,4,6- O -The maximum absorption wavelength of pentagalloylglucose is 280 nm, and the spectrum of gallnut medicinal materials, standard soup, and granules is also consistent. The final chromatographic conditions are as follows: Chromatographic conditions and system suitability test Agilent Aq-C18 (2.1mm×150mm, 2.7μm) was used; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 15; the flow rate was 0.30ml per minute; the column temperature was 35℃; the detection wavelength was 280nm. The theoretical plate number calculated based on the gallic acid peak should not be less than 5000.

[0075] Table 15 Gradient elution table

[0076] Example 2 Methodological verification of the characteristic spectrum detection method of the standard decoction of Galla chinensis (Dubei) 1. Precision inspection Take the standard decoction of Galla chinensis (batch number: WBZ-DB-DG-1, prepared from Galla chinensis (Dubei) decoction piece WBZ-DB-YP-1), prepare the test solution according to the test solution preparation method, and inject 6 times continuously under the chromatographic conditions determined in Example 1, 1 μl each time, to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peak. The results are shown in Tables 16 and 17.

[0077] Table 16 Precision test results (relative retention time)

[0078] Table 17 Precision test results (relative peak area)

[0079] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, and the precision was good.

[0080] 2. Stability test Take this product (batch number: WBZ-DB-DG-1) and prepare a test solution according to the test solution preparation method. Inject 1 μl of the sample every 4 hours for a total of 24 hours. Under the chromatographic conditions determined in Example 1, examine the consistency of the relative retention time and relative peak area of ​​the characteristic peak. The results are shown in Tables 18 and 19.

[0081] Table 18 Stability test results (relative retention time)

[0082] Table 19 Stability test results (relative peak area)

[0083] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, indicating good stability.

[0084] 3. Repeatability Investigation Take an appropriate amount of this product (batch number: WBZ-DB-DG-1), grind it into powder, take about 0.1g, and prepare 6 parallel portions into test solutions according to the test solution preparation method. Under the chromatographic conditions determined in Example 1, inject and analyze, calculate the relative retention time and relative peak area, and examine the consistency of the relative retention time and relative peak area of ​​the characteristic peak. The results are shown in Table 20 and Table 21.

[0085] Table 20 Repeatability results of characteristic spectra of Galla chinensis formula granules (relative retention time)

[0086] Table 21 Repeatability results of characteristic spectrum of Galla chinensis formula granules (relative peak area)

[0087] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, and the method had good repeatability.

[0088] 4. Durability inspection 4.1 Investigation of different chromatographic columns In this example, the sample separation effect of different batches of Agilent Aq-C18 columns, S / N numbers: 115-ENGR3018, 116-ENGR3026, 118-ENGR3026, Waters H class ultra-high performance liquid chromatograph, flow rate of 0.30 ml / min, column temperature of 35 ° C was investigated. The results are shown in Fig.14 And Table 22.

[0089] Table 22 Effect of chromatographic column on separation effect (relative retention time)

[0090] The results showed that the characteristic peaks in the three chromatographic columns could be well separated. In this experiment, Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm) was used for subsequent research.

[0091] 4.2 Investigation of different column temperatures A Waters H class ultra-high performance liquid chromatograph and an Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm) column were used. Acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, the flow rate was 0.30 ml / min, the column temperature was 35 °C, the detection wavelength was 280 nm, and gradient elution was performed according to the regulations in Table 15. The sample separation effects were investigated when the column temperatures were 30 °C, 35 °C, and 40 °C. The results are shown in Table 15. Fig.15 And Table 23.

[0092] Table 23 Effect of column temperature on separation effect (relative retention time)

[0093] The results showed that the analytical method had good separation of characteristic peaks when the column temperature was 30℃~40℃, and could meet the system suitability requirements with small column temperature changes.

[0094] 4.3 Investigation of flow rate A Waters H class ultra-high performance liquid chromatograph and an Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm) column were used. Acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B. The column temperature was 35 °C and the detection wavelength was 280 nm. Gradient elution was performed according to the requirements in Table 15. The sample separation effects at flow rates of 0.25 ml / min, 0.30 ml / min, and 0.35 ml / min were investigated. The results are shown in Tables 24 and Fig.16 .

[0095] Table 24 Effect of flow rate on separation effect (relative retention time)

[0096] The results show that when the flow rate is in the range of 0.25ml / min~0.35ml / min, the peak shapes of each characteristic peak are relatively small, and slight changes in the flow rate can meet the system suitability requirements.

[0097] 4.4 Investigation of different brands of liquid chromatographs Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm) chromatographic column was used, acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, the flow rate was 0.30 ml / min, the column temperature was 35 °C, the detection wavelength was 280 nm, and gradient elution was performed according to the provisions in Table 15. The sample separation effects under the conditions of Waters H class, Agilent 1290, and Thermo Vanquish ultra-high performance liquid chromatography were investigated respectively. The results are shown in Table 25 and Fig.17 .

[0098] Table 25 Determination results of different instruments (relative retention time)

[0099] Due to the differences in residence volume between different instruments, the relative retention times differed slightly, and this method was reproducible on different brands of HPLC instruments, indicating good robustness.

[0100] Example 3: Determination of the characteristic spectrum of the standard decoction of Galla chinensis (Dubei) 1. Chromatographic conditions: Agilent Aq-C18 (2.1 mm × 150 mm, 2.7 μm) column; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 15; flow rate 0.30 ml per minute; column temperature 35 °C; detection wavelength 280 nm. The theoretical plate number calculated based on the gallic acid peak should be no less than 5000.

[0101] 2. Preparation of reference solution: Take gallic acid reference substance, 1,2,3,4,6- O -Pentagalloylglucose reference substance was weighed accurately and methanol was added to prepare solutions containing 20µg gallic acid per 1ml and 1,2,3,4,6- O -Pentagalloylglucose 200µg solution was used as the reference solution. Take 0.2g of the reference medicinal material of Galla chinensis (Dubei), place it in a stoppered conical bottle, add 25ml of methanol, ultrasonically treat (power 250W, frequency 40kHz) for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material.

[0102] 3. Preparation of test solution: Take an appropriate amount of 17 batches of Galla chinensis (Dubei) standard decoction (WBZ-DB-DG-1~17, prepared from WBZ-DB-YP-1~17 respectively), grind it into powder, take 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of methanol, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 60 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0103] 4. Accurately pipette 1µl of the reference solution and the test solution respectively and inject them into the ultra-high performance liquid chromatography.

[0104] 5. Establishment of the characteristic spectrum of the standard decoction of Galla chinensis (Dubei) The “Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)” issued by the Chinese Pharmacopoeia Committee was used to superimpose the characteristic spectra of 17 batches of gallnut (Dubei) standard decoction samples to generate a reference spectrum. Fig.18 , generate the control map see Fig.19 , select the common peaks with relatively stable relative retention time and relative peak area RSD as characteristic peaks (peak 1 to peak 10).

[0105] Table 27 Sample measurement results (relative retention time)

[0106] Table 28 Sample measurement results (relative peak area)

[0107] As shown in Tables 27 and 28, there are 10 characteristic peaks in the chromatogram of the test sample, and the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other, and 2 of the peaks should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the peak of the gallic acid reference is the S1 peak, and the relative retention times of peaks 2 to 4 and S1 are calculated; O -The peak corresponding to the reference peak of the pentagallocetanoyl glucose reference substance is the S2 peak. The relative retention times of peaks 5 to 7, 9, and 10 with the S2 peak are calculated, and their relative retention times should be within the range of ±10% of the specified value. The specified values ​​are: 2.85 (peak 2), 3.18 (peak 3), 3.86 (peak 4), 0.49 (peak 5), 0.73 (peak 6), 0.89 (peak 7), 1.11 (peak 9), and 1.24 (peak 10). The relative retention times of the characteristic peaks are all within ±10% of the specified values, which meets the standard requirements of the characteristic spectrum of the standard decoction.

[0108] Example 4 Comparison of different commercial specifications of gallnut (belly-sized and horn-sized) 1 Comparison of characteristic spectra of Gallic chinensis (Dubei and Jiaobei) The "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)" promulgated by the Chinese Pharmacopoeia Committee was used to measure three batches of standard decoctions of Gallic (Jiaobei) (WBZ-JB-DG-1~3, prepared from WBZ-JB-YP-1~3) according to the chromatographic conditions determined in Example 1. The results are shown in Tables 29 and 30. The comparison of standard decoctions of Gallic (Dubei, Jiaobei) is shown in Tables 31 and 32. Fig. 20 .

[0109] Table 29 Relative retention time results of 3 batches of standard decoction of Galla chinensis (Gala chinensis)

[0110] Table 30 Relative peak area results of 3 batches of standard decoction of Galla chinensis (Gala chinensis)

[0111] The results showed that there were significant differences in the characteristic spectra of decoctions of Galla chinensis (Dubei) and Galla chinensis (Jiaobei).

[0112] 2. Research on the material basis and identification of characteristic peaks of Galla chinensis (Dubei) Liquid chromatography-mass spectrometry (LC-MS) was used to collect high-resolution mass spectrometry data according to the liquid chromatography conditions of the characteristic spectrum of the standard decoction of Galla chinensis (Dubei), and the molecular formula of the compounds in the standard decoction of Galla chinensis (Dubei) was inferred by combining isotope analysis. The common peaks were attributed by comparing with references and related reference substances. The retention time of peak 1 was consistent with that of the gallic acid reference substance in the reference substance, so peak 1 in the characteristic spectrum of the test sample was identified as gallic acid, see Fig.21 The retention time of peak 8 is similar to that of 1,2,3,4,6- O -pentagalloylglucose reference substance has the same retention time, so peak 8 in the characteristic spectrum of the test sample was identified as 1, 2, 3, 4, 6- O - Pentagalloylglucose, the retention time of 10 characteristic peaks in the standard decoction of Gallic chinensis (Dubei) is consistent with that of 10 characteristic peaks in the reference medicinal material of Gallic chinensis (Dubei), and the retention time of 7 characteristic peaks in the standard decoction of Gallic chinensis (Jiaobei) is consistent with that of 7 characteristic peaks in the reference medicinal material of Gallic chinensis (Jiaobei). Fig. 22 .

[0113] Example 4 Determination of characteristic peaks for distinguishing different commercial specifications of gallnuts (Galnuts with different diameters and different horns) Through mass spectrometry and reference substances, the differential components were determined, and the components were screened to distinguish the standard decoction of Gallic Acid (Dubei, Jiaobei). Dubei and Jiaobei can be distinguished by Peak 5 and Peak 6. Due to the phenomenon of partial adulteration in actual production, the relative peak area with Peak 7 as the reference peak was further used to distinguish the adulterated samples, and 0.15 was used as the specified value. Finally, it was stipulated that gallic acid was used as the reference peak. If the sum of the relative peak areas of Peak 5 and Peak 6 was greater than 0.15, the sample being tested was Gallic Acid (Dubei).

[0114] Table 31 The sum of the relative peak areas of peak 5 and peak 6 of different commercial specifications of gallnut standard decoction

[0115] In summary: a. If the retention time of the chromatogram of the sample to be tested corresponds to the retention time of the 10 characteristic peaks in the chromatogram of the reference material of the gallic acid reference material and the 1,2,3,4,6-O-pentagalloylglucose reference material, two of which should correspond to the retention time of the reference material peaks corresponding to the gallic acid reference material and the 1,2,3,4,6-O-pentagalloylglucose reference material. The peak corresponding to the peak of the 1,2,3,4,6-O-pentagalloylglucose reference material is the S2 peak. The relative retention time of peak 5, peak 6 and S2 peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.49 (peak 5), 0.73 (peak 6), and the peak corresponding to the peak of the gallic acid reference material is the S1 peak. The relative peak area of ​​the sum of the peak areas of peak 5 and peak 6 and the S1 peak is calculated. If the relative peak area is greater than 0.15, it is the gallic acid reference material.

[0116] b. If the retention time of the chromatogram of the sample to be tested corresponds to the retention time of the 7 characteristic peaks in the chromatogram of the reference material of the Chinese gallic acid reference material (Jiaobei), 2 of the peaks should correspond to the retention time of the reference material peaks corresponding to the gallic acid reference material and the 1,2,3,4,6-O-pentagalloylglucose reference material. The peak corresponding to the peak of the 1,2,3,4,6-O-pentagalloylglucose reference material is the S2 peak. The relative retention time of peak 5, peak 6 and S2 peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.49 (peak 5), 0.73 (peak 6), and the peak corresponding to the peak of the gallic acid reference material is the S1 peak. The relative peak area of ​​the sum of the peak areas of peak 5 and peak 6 and the S1 peak is calculated. If the relative peak area is less than 0.15, it is Jiaobei.

Claims

1. A method for constructing a characteristic map of Galla chinensis (Galax chinensis), characterized in that: The following steps are involved: (1) Preparation of test solution: Take the standard decoction of Galla chinensis (Galla chinensis), extract it with organic solvent, shake well, filter, and take the filtrate to obtain the test solution; (2) Preparation of reference solution: Take gallic acid reference substance, 1,2,3,4,6- O - Pentagalloylglucose reference substance, respectively, add solvent to dissolve, and obtain reference substance solution; take Galla chinensis (Dubei) reference medicinal material, add methanol to extract, shake well, filter, and take the filtrate as the reference medicinal material solution; (3) Establishment of characteristic spectrum: Take the standard decoction of Galla chinensis (Dubei) and the reference solution, inject them into the high performance liquid chromatography respectively, and measure to obtain the characteristic spectrum; the characteristic spectrum has 10 peaks in total, take gallic acid as the reference, peak 1 is the chromatographic peak of gallic acid, the peak corresponding to the gallic acid reference is the S1 peak, calculate the relative retention time of peaks 2 to 4 and S1 peak, and the relative retention time is within the range of ±10% of the specified value. The specified values ​​of peaks 2 to 4 are 2.85, 3.18, and 3.86 respectively; take 1, 2, 3, 4, 6- O -pentagalloylglucose as the reference substance, peak 8 is 1,2,3,4,6- O -pentagalloylglucose; and 1,2,3,4,6- O The peak corresponding to -pentagalloylglucose is S2 peak. The relative retention times of peaks 5 to 7, 9, 10 and S2 peak are calculated, and their relative retention times are within ±10% of the specified values. The specified values ​​of peaks 5 to 7, 9, and 10 are 0.49, 0.73, 0.89, 1.11, and 1.24, respectively.

2. The method for constructing the characteristic spectrum of Galla chinensis (Galla chinensis) according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography described in step (3) are: chromatographic column: Agilent Aq-C18; injection volume: 1 μl; flow rate: 0.25~0.35 ml / min; column temperature: 25~35°C; detection wavelength: 273 or 280 nm; acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution.

3. The method for constructing the characteristic spectrum of Galla chinensis (Galla chinensis) according to claim 1, characterized in that: The gradient elution in step (3) is as follows: 0-5 min, the volume fraction of mobile phase A is 8%, and the volume fraction of mobile phase B is 92%; 5-6 min, the volume fraction of mobile phase A is 8→14%, and the volume fraction of mobile phase B is 92→86%; 6-13 min, the volume fraction of mobile phase A is 14→16%, and the volume fraction of mobile phase B is 86→84%; 13-17 min, the volume fraction of mobile phase A is 16→17%, and the volume fraction of mobile phase B is 84→83%; 17-20 min, the volume fraction of mobile phase A is 17→19%, and the volume fraction of mobile phase B is 83→81%; 20-25 min, the volume fraction of mobile phase A is 19→23%, and the volume fraction of mobile phase B is 81→77%; 25-30 min, the volume fraction of mobile phase A is 23%, and the volume fraction of mobile phase B is 77%.

4. The method for constructing the characteristic spectrum of Galla chinensis (Galla chinensis) according to claim 1, characterized in that: In step (1), the Galla chinensis (Dubei) decoction pieces are taken, soaked in water, decocted, filtered, the filtrate is concentrated under reduced pressure, and freeze-dried to obtain the standard decoction of Galla chinensis (Dubei).

5. A method for establishing a reference map of Galla chinensis (Galla chinensis), characterized in that: The following steps are involved: (1) Prepare multiple batches of Galla chinensis (Dubei) standard decoction test sample solutions; (2) According to the method for constructing the characteristic spectrum of Galla chinensis (Dubei) as described in claim 1, the test solution is tested respectively to obtain the characteristic spectrum of the standard decoction of Galla chinensis; (3) The obtained characteristic spectrum was imported into the Chinese medicine chromatographic fingerprint similarity evaluation system to establish the reference spectrum of the standard decoction of Galla chinensis.

6. A method for distinguishing gallnut (Dubei) and gallnut (Jiaobei), characterized in that: The following steps are involved: (1) Preparation of test solution: extract the standard decoction of Galla chinensis with organic solvent, shake well, filter, and take the filtrate to obtain the test solution; (2) Preparation of reference solution: Weigh reference gallic acid and 1,2,3,4,6-O-pentagalloylglucose respectively, and add methanol solution as reference solution; weigh reference Chinese medicinal material Galla chinensis, add methanol to extract, shake well, filter, and take the filtrate as reference solution; (3) HPLC chromatogram detection: Take the test solution or the reference solution respectively, inject it into the HPLC, perform the test, and record the chromatogram; take gallic acid as the reference, peak 1 is the chromatographic peak of gallic acid, and the peak corresponding to the gallic acid reference is the S1 peak; take 1,2,3,4,6- O -pentagalloylglucose as the reference substance, peak 8 is 1,2,3,4,6- O -pentagalloylglucose; and 1,2,3,4,6- O -The peak corresponding to pentagalloylglucose is S2 peak; the chromatographic conditions of the high performance liquid chromatograph are: chromatographic column: Agilent Aq-C18; injection volume: 1 μl; flow rate: 0.25~0.35 ml / min; column temperature: 25~35°C; detection wavelength: 273 or 280 nm; acetonitrile is used as mobile phase A, and 0.1% phosphoric acid aqueous solution is used as mobile phase B for gradient elution; the gradient elution is: 0~5 min, the volume fraction of mobile phase A is 8%, and the volume fraction of mobile phase B is 92%; 5~6 min, the volume fraction of mobile phase A is 8→14%, and the volume fraction of mobile phase B is 92→86%; 6~13 min, the volume fraction of mobile phase A is 1 4→16%, the volume fraction of mobile phase B is 86→84%; 13~17min, the volume fraction of mobile phase A is 16→17%, the volume fraction of mobile phase B is 84→83%; 17~20min, the volume fraction of mobile phase A is 17→19%, the volume fraction of mobile phase B is 83→81%; 20~25min, the volume fraction of mobile phase A is 19→23%, the volume fraction of mobile phase B is 81→77%; 25~30min, the volume fraction of mobile phase A is 23%, the volume fraction of mobile phase B is 77%; (4) Analysis: Select the peak with stable relative retention time in the chromatogram as the characteristic peak, and then compare it with the peak of the reference solution spectrum; (5) Discrimination: Compare the chromatogram of the test solution with that of the reference solution; when the chromatogram of the test solution has peaks 1 to 10, the test sample is Galla chinensis (Galax chinensis); when the chromatogram of the test solution has peaks 1, 2 to 4, 7, 8, and 10, the test sample is Galla chinensis (Galax chinensis).

7. The method according to claim 6, characterized in that: When the gallic acid standard decoction is adulterated, the relative peak area of ​​peak 7 as the reference peak is used to distinguish the adulterated samples in step (5), and 0.15 is used as the specified value. Finally, gallic acid is used as the reference peak. If the sum of the relative peak areas of peak 5 and peak 6 is greater than 0.15, the sample being tested is gallic acid (gallic acid); the peak corresponding to the peak of the gallic acid reference substance is the S1 peak. The relative peak area of ​​the sum of the peak areas of peak 5 and peak 6 and the S1 peak is calculated. If the relative peak area is less than 0.15, it is gallic acid.

8. The method according to claim 6, characterized in that: The relative retention time described in step (4) is the average value of several groups of experimental results.

9. The method according to claim 6, characterized in that: The characteristic spectrum of Galla chinensis (Galla chinensis) described in step (4) has 10 characteristic peaks, gallic acid is taken as the reference substance, and the S1 peak is the chromatographic peak of gallic acid; 1,2,3,4,6-O-pentagalloylglucose is taken as the reference substance, and the S2 peak is 1,2,3,4,6-O-pentagalloylglucose; the relative retention times of peaks 5, 6 and S2 are calculated, and the relative retention times should be within the range of ±10% of the specified value, the specified value of peak 5 is 0.49, and the specified value of peak 6 is 0.73; the peak corresponding to the peak of the gallic acid reference substance is the S1 peak, and the relative peak area of ​​the sum of the peak areas of peaks 5 and 6 and the S1 peak is calculated, and the relative peak area is greater than 0.

15.

10. The method according to claim 6, characterized in that: In step (4), the characteristic spectrum of Galla chinensis (Gala chinensis) has 7 characteristic peaks, gallic acid is taken as the reference substance, and the S1 peak is the chromatographic peak of gallic acid; 1,2,3,4,6-O-pentagalloylglucose is taken as the reference substance, and the S2 peak is 1,2,3,4,6-O-pentagalloylglucose; the relative retention times of peaks 5, 6 and S2 are calculated, and the relative retention times should be within the range of ±10% of the specified value, the specified value of peak 5 is 0.49, and the specified value of peak 6 is 0.73; the peak corresponding to the peak of the gallic acid reference substance is the S1 peak, and the relative peak area of ​​the sum of the peak areas of peaks 5 and 6 and the S1 peak is calculated, and the relative peak area is less than 0.15.

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