Quality detection method of Qianzhida preparation for treating depression

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for detecting the quality of a Dezhihua preparation for treating depression, and further relates to a specific chromatogram of the Dezhihua preparation, an establishment method of the specific chromatogram and a method for detecting alpinetin and pinocembrin in the Dezhihua preparation. And performing chromatographic detection on the reference substance solution and the Dezhihua preparation test solution through high performance liquid chromatography to determine a common characteristic peak, thereby establishing a characteristic spectrum and detecting the content of alpinetin and pinocembrin. According to the specific chromatogram established by the method for establishing the specific chromatogram of the Xuezhida tablet and the method for determining the contents of the alpinetin and the pinocembrin, positive significance can be achieved for quality control of the Xuezhida preparation, and the method plays an important role in treatment of depression diseases.

Description

Technical Field

[0001] This invention relates to the field of detection and analysis technology of traditional Chinese medicine, specifically to the field of analysis of Yushuda preparations. Background Technology

[0002] Depression is a common mental disorder and one of the leading causes of illness with a high incidence rate in humans. Yushuda preparations are currently known on the market as traditional Chinese medicine preparations mainly made from herbs such as patchouli, tangerine peel, cardamom, amomum villosum, and atractylodes lancea. Their main functions are to invigorate the spleen and resolve dampness, regulate qi and relieve depression, and are used for mood swings, low mood, slow thinking, and poor appetite caused by qi stagnation. They are particularly effective in treating mild early symptoms of depressive disorders. However, to better understand the mechanism of action of Yushuda tablets, quality control of the components of Yushuda preparations is crucial. Summary of the Invention

[0003] Although the existing technology CN100464188C discloses the quality control method of Yushuda preparation, the control method mainly focuses on the identification of the properties of Chinese medicine and the raw materials of Chinese medicine (patchouli, atractylodes, cardamom, amomum villosum, tangerine peel). Regarding the determination of the content of the components, it only measures the content of hesperidin in the preparation. It has not truly and completely established the characteristic spectrum of each chemical component of Yushuda preparation, nor does it mention the determination of the content of sorbitol and pinosin in Yushuda preparation.

[0004] This invention addresses the technical problem of the current lack of methods for establishing fingerprint profiles of Yushuda preparations and accurately detecting the content of sorbitol and pinocembrin for quality control. The inventors have provided a method for establishing characteristic profiles of Yushuda preparations through research.

[0005] To address the aforementioned technical problems, the applicant has provided the following technical solutions.

[0006] A quality testing method for a Yushuda preparation for treating depression, wherein the Yushuda preparation is... The following steps are included in the preparation of the following traditional Chinese medicine mixture: 700-1200 parts by weight of patchouli, 200-400 parts by weight of dried tangerine peel, 300-700 parts by weight of cardamom, 300-700 parts by weight of amomum villosum, and 400-750 parts by weight of stir-fried atractylodes lancea are mixed to obtain a mixture of traditional Chinese medicine materials. The mixture of traditional Chinese medicine materials is decocted with water, and the filtrate is concentrated to a clear extract at a temperature of 48-52°C and a relative density of 1.04-1.05. Ethanol is added to concentrate the filtrate to an extract at a temperature of 60-70°C and a relative density of 1.14-1.17. Excipients are added to prepare the desired formulation. The cardamom and amomum villosum are preferably mixed with patchouli, dried tangerine peel, and stir-fried atractylodes lancea in a crushed powder state to obtain the mixture of traditional Chinese medicine materials.

[0007] The quality of the formulation was determined by high performance liquid chromatography and mass spectrometry.

[0008] Preferably, the quality detection method for the Yushuda preparation is characterized in that: the quality detection method includes a method for establishing a characteristic chromatogram and / or content determination, wherein the method for establishing the characteristic chromatogram uses hesperidin, sorbitol, pinosin, cardamom, alder ketone, and patchouli ketone as reference standards, and employs high-phase liquid chromatography with acetonitrile and phosphoric acid solution as the mobile phase; the content determination uses hesperidin, sorbitol, and pinosin as reference standards, and employs high-phase liquid chromatography with methanol and phosphoric acid solution as the mobile phase.

[0009] Specifically, the present invention provides a quality testing method for a Yushuda preparation for treating depression, wherein the Yushuda preparation is prepared by a method comprising the following steps: mixing 700-1200 parts by weight of patchouli, 200-400 parts by weight of dried tangerine peel, 300-700 parts by weight of cardamom, 300-700 parts by weight of amomum villosum, and 400-750 parts by weight of stir-fried Atractylodes lancea to obtain a mixture of traditional Chinese medicinal materials; decocting the mixture of medicinal materials with water and concentrating the filtrate to a clear extract at a temperature of 48-52°C and a relative density of 1.04-1.05; adding ethanol to concentrate the filtrate to an extract at a temperature of 60-70°C and a relative density of 1.14-1.17; and adding excipients to prepare the desired preparation. The quality of the formulation was determined by high performance liquid chromatography and mass spectrometry.

[0010] Preferably, the quality detection method for the above-mentioned Yushuda preparation is characterized in that: the quality detection method includes a method for establishing characteristic chromatograms and / or content determination, wherein the method for establishing characteristic chromatograms uses hesperidin, sorbitol, pinosin, cardamom, alder ketone, and patchouli ketone as reference standards, and employs high-phase liquid chromatography with acetonitrile and phosphoric acid solution as mobile phases; the content determination uses hesperidin, sorbitol, and pinosin as reference standards, and employs high-phase liquid chromatography with methanol and phosphoric acid solution as mobile phases.

[0011] This invention also provides a method for establishing the characteristic chromatogram of Yushuda preparation, characterized by using high performance liquid chromatography to analyze and detect Yushuda preparation, the method comprising the following steps: 1) Prepare reference solutions: Mix hesperidin reference standard, sorbitol reference standard, ginsenoside reference standard, cardamomin reference standard, alder ketone reference standard and patchouli ketone reference standard to prepare mixed reference solutions; 2) Prepare the test solution of Yushuda preparation; add solvent to Yushuda preparation to prepare the test solution; 3) The chromatograms of the mixed reference solution and the test solution were detected by high performance liquid chromatography (HPLC). The chromatograms of the test solution and the reference solution were compared to determine the common peaks and establish characteristic chromatograms. The common peaks include 6 peaks: hesperidin, sorbitol, pinocembrin, cardamomin, alder ketone and patchouli ketone. The high-performance liquid chromatography (HPLC) conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material, and the mobile phase includes phase A and phase B, wherein phase A is acetonitrile and phase B is a 0.05-0.15% by mass phosphoric acid solution, and gradient elution is performed.

[0012] Preferably, in the method for establishing the characteristic chromatogram of the Yushuda preparation, the chromatogram of the test solution has at least 13 common characteristic peaks.

[0013] Preferably, in the method for establishing the feature map, the gradient elution method of the mobile phase is as follows: Within 0-4 minutes, the volume of phase A increased from 5% to 10%, and the volume of phase B decreased from 95%. As low as 90% by volume; Within 4-10 minutes, the volume of phase A increased from 10% to 15%, and the volume of phase B increased from 90%. Reduced to 85% of volume; Within 10-35 minutes, the volume of phase A increased from 15% to 30% and the volume of phase B increased from 85% to 10%. Reduced to 70% of volume; Within 35-40 minutes, the volume of phase A increased from 30% to 38%, and the volume of phase B increased from 70% to... Reduced to 62% of volume; Within 40-45 minutes, the volume of phase A increased from 38% to 48% and the volume of phase B increased from 62% to 48%. Reduced to 52% of volume; Within 45-55 minutes, the volume of phase A increased from 48% to 60%, and the volume of phase B increased from 52%. Reduced to 40% of volume; Within 55-60 minutes, the volume of phase A increased from 60% to 76%, while the volume of phase B decreased from 40%. Up to 24% by volume Over 60-62 minutes, the volume of phase A decreased from 76% to 5%, while the volume of phase B increased from 24% to [missing value]. 95% by volume.

[0014] Preferably, in the detection method described above, the chromatographic column has an inner diameter of 2.1 mm and a column length of 100-150 mm. The inner diameter is 1.8 μm; preferably, the chromatographic column is a Waters ACQUITY UPLC® HSS T3 C18 or AmethSepC18 Aq.

[0015] Preferably, in the method for establishing the characteristic spectrum described in any of the preceding claims, the chromatographic detection... The measurement wavelength is 190-400 nm, preferably 315 nm; And / or, the column temperature of the chromatographic column is 25-35℃, preferably 28-32℃, more preferably 30℃; And / or, the flow rate of the mobile phase is 0.20-0.40 ml / min, preferably 0.28-0.32 ml / min.

[0016] Preferably, in the method for establishing the feature map described in any of the preceding items, in step 3), When detecting the test solution by chromatography, the injection volume of the test solution is 1-3 µl, preferably 2-3 µl, and more preferably 2 µl.

[0017] Preferably, in the method for establishing the characteristic chromatogram described in any of the preceding claims, the chromatogram of the test sample solution has at least 13 common characteristic peaks, wherein the 13 characteristic peaks are numbered 1-13 according to the retention time from smallest to largest, then peak 4 is the standard hesperidin peak, peak 7 is the standard sorbitol peak, the relative retention times of peaks 1, 2, 3, 5, and 6 relative to peak 4 are within ±10% of a specified value; the relative retention times of peaks 8 and 10 relative to peak 2 are within ±10% of a specified value.

[0018] Preferably, the substances corresponding to the six characteristic peaks in the common characteristic peaks are: peak 4 is hesperidin; peak 7 is sorbitol; peak 9 is pinoside; peak 11 is cardamomin; peak 12 is patchoulione; and peak 13 is alderne.

[0019] Preferably, the Yushuda preparation is prepared using the method for establishing the characteristic spectrum described in any of the preceding claims. The feature map.

[0020] This invention also provides a method for detecting galangin and pine resin in Yushuda preparations, characterized in that, The analysis and detection of Yushuda preparations were performed using high-performance liquid chromatography (HPLC). The method includes the following steps: 1) Prepare reference solution: Mix sorbitan reference standard and ginsenoside reference standard separately to prepare a mixed reference solution; 2) Prepare the test solution of Yushuda preparation: Add solvent to Yushuda preparation to prepare the test solution; 3) High-performance liquid chromatography (HPLC) was used to detect the mixed reference standard solution and the test solution separately; The high-performance liquid chromatography (HPLC) conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material, and the mobile phase includes phase A and phase B, wherein phase A is acetonitrile and phase B is a 0.05-0.15% by mass phosphoric acid solution.

[0021] Preferably, in the detection method of galangin and galangin in the Yushuda preparation described above, the mass ratio of phase A and phase B as the mobile phase is (55-68):(35-42), preferably 62:38.

[0022] Preferably, in the detection method of galangin and pine resin in the Yushuda preparation described in any of the preceding claims, the detection wavelength of the chromatography is 190-400 nm, preferably 290 nm; And / or, the chromatographic column is a column with an inner diameter of 4.6 mm, a column length of 250 mm, and a particle size of 5 μm, preferably selected from Xtimate. ® C18, Waters XBridge ® C18, Thermo Accliam TM 120 A 。 C18, GL Sciences Inertsil TM ODS-HL, JADE-PAK ® KP-C18, any of the Agilent ZORBAX Eclipse Plus C18; And / or, the column temperature of the chromatographic column is 25-35℃, preferably 28-32℃, more preferably 30℃; And / or, the flow rate of the mobile phase is 0.10-1.5 ml / min, preferably 0.8-1.2 ml / min.

[0023] Preferably, in the detection method of galangin and pine resin in the Yushuda preparation described in any of the preceding claims, or in the method for establishing the characteristic spectrum described in any of the preceding claims, in step 2), the solvent is selected from any one or more of 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water, preferably selected from any one or more of methanol, 75% methanol, 50% methanol, ethanol, 75% ethanol and water; more preferably selected from any one or more of methanol, 75% methanol, 50% methanol, 75% ethanol and water.

[0024] Preferably, in the detection method of galangin and pine resin in the Yushuda preparation described in any of the preceding claims, or in the method for establishing the characteristic spectrum described in any of the preceding claims, step 2) further includes extracting the chemical components of the test sample solution by ultrasonic treatment, reflux treatment, or shaking treatment. It is preferred to extract the chemical components by ultrasonic treatment, and more preferably, the ultrasonic treatment is carried out under the conditions of power of 100-200W and frequency of 15-30kHz. And / or, in step 2), the chemical components of the test sample solution are extracted using a solvent, and the extraction time is... The extraction time is 30-90 minutes, with an optimal extraction time of 45-50 minutes. And / or, in step 2), the chemical components of the test solution are extracted by solvent extraction relative to 1g. The solvent volume used in the Yushuda preparation is 30-100ml, preferably 30-60ml; more preferably 40-60ml.

[0025] Preferably, in the method for detecting galangin and galangin in the Yushuda preparation described in any of the preceding claims, when detecting the chromatogram of the test solution, the injection volume of the test solution is 7-15 µl, preferably 8-11 µl.

[0026] Preferably, the detection of galangin and pine resin in the Yushuda preparation described in any of the preceding items is... The method, wherein, relative to 0.5g of Yushuda preparation, the total content of sorbitol and pine resin is 1.53-3.09mg, preferably 2.10-2.50mg.

[0027] Preferably, the detection of galangin and pine resin in the Yushuda preparation described in any of the preceding items is... The method is used to control the quality of Yushuda preparations.

[0028] Preferably, the aforementioned characteristic chromatograms are used for quality control of the Yushuda preparation.

[0029] This invention establishes a method for creating characteristic spectra of hesperidin, sorbitol, pinoresin, cardamomin, alpinone, and patchouli in Yushuda preparations, and a method for determining the content of sorbitol and pinoresin. This can play a positive role in the quality control of Yushuda preparations. Through qualitative and quantitative detection and analysis of the components, the quality stability of different batches of the drug can be ensured, which plays an important role in ensuring the efficacy of Yushuda preparations in treating depression.

[0030] The quality detection method established in this invention employs ultra-high performance liquid chromatography (UHPLC), which is simple, stable, highly precise, and reproducible, providing an effective control method for the quality evaluation of Yushuda preparations. The qualitative method can simultaneously identify fingerprint peaks with good peak shapes, making them easy to identify and accurate and reliable; the quantitative detection method simultaneously detects the content of sorbitol and pinocembrin, improving detection efficiency. Attached Figure Description

[0032] Figure 1 This is a superimposed image of the characteristic spectrum of the control group in Example 1 and the common peak of Yushuda tablets in Example 1; Figure 2 is a chromatogram for the specificity investigation in Example 1; Figure 3 shows the chromatograms of different chromatographic columns used in Example 1; Figure 4 shows the durability test at different column temperatures in Example 1; Figure 5 shows the chromatographic results of different flow rates investigated in Example 1; Figure 6 shows the peak area / sample weight results of different extraction solvents in Example 1. Figure 7 shows the chromatograms of characteristic peaks under different extraction solvents in Example 1; Figure 8 shows the results of examining the peak area / sample quantity of each characteristic peak using different extraction methods in Example 1. Figure 9 shows the chromatograms of characteristic peaks for solvent analysis under different extraction methods in Example 1; Figure 10 shows the results of peak area / sample weight for each characteristic peak at different extraction times in Example 1. Figure 11 shows the chromatograms of characteristic peaks under different ultrasonic times in Example 1; Figure 12 is a graph showing the peak area / sample volume of each characteristic peak under different solvent dosages in Example 1. Figure 13 shows the chromatograms of various characteristic peaks in Example 1 under different amounts of extraction solvent. Figure 14 is a graph showing the peak area / sample volume of each characteristic peak under different injection volumes in Example 1. Figure 15 shows the chromatograms of various characteristic peaks under different injection volumes in Example 1; Figure 16 is a chromatographic overlay of 10 batches of Yushuda tablets in Example 1; Figure 17 shows the common peak pattern and control spectrum of Yushuda tablets in Example 1; Figure 18 This is the chromatogram used in Example 2 to investigate specificity; Figure 19 is a standard curve of galangin in Example 2; Figure 20 is a standard curve of galactopyrine in Example 2; Figure 21A and Figure 21BThese are the chromatograms of different flow rates examined in Example 2; Figure 22A and Figure 22B These are the chromatograms obtained from different column temperatures in Example 2; Figure 23A Figure 23B , Figure 23C and Figure 23D These are chromatograms from different chromatographic columns examined in Example 2.

[0033] in, Figure 21A and Figure 21B The retention times of the chromatographic peaks at a flow rate of 0.8 ml / min were 4.620, 5.148, 5.935, 7.889, 9.573, 10.130, 15.121, and 16.312, respectively; the retention times of the chromatographic peaks at a flow rate of 1.0 ml / min were 4.136, 4.769, 6.330, 8.145, 12.162, and 13.101, respectively; and the retention times of the chromatographic peaks at a flow rate of 1.2 ml / min were 5.284, 6.801, 10.155, 10.923, and 13.625, respectively. Figure 22A and Figure 22B The retention times of the chromatographic peaks at 25℃ were 4.286, 4.985, 6.613, 8.691, 11.596, 13.148, 14.280, and 15.139, respectively; those at 30℃ were 4.136, 4.769, 6.330, 8.145, 12.162, and 13.101, respectively; and those at 35℃ were 4.568, 6.047, 7.617, 11.209, and 11.984, respectively. Figure 23A , Figure 23B , Figure 23C and Figure 23D In the chromatography, the retention times of the chromatographic peaks were 4.136, 4.769, 6.330, 8.145, 12.162, and 13.101 when using the Ultimate® C18 column; and 6.733, 9.782, and 10.699 when using the Waters XBridge® C18 column; and 6.733, 9.782, and 10.699 when using the Thermo Accliam column. TM The retention times of the chromatographic peaks at 120 A°C 18 column were 9.866, 15.471, and 16.677, respectively; using GL Sciences Inertsil TMThe retention times of the chromatographic peaks were 12.783 and 23.949 when using the ODS-HL column, 10.871 and 19.191 when using the JADE-PAK® KP-C18 column, and 8.066, 11.953 and 13.127 when using the Agilent ZORBAX Eclipse Plus C18 column. Detailed Implementation

[0035] This invention addresses the technical problem of the current lack of methods for establishing fingerprint chromatograms of Yushuda tablets and accurately detecting the content of sorbitol and pinoresin in them for quality control. It provides a method for establishing characteristic chromatograms of Yushuda tablets and a method for detecting the content of sorbitol and pinoresin.

[0036] Specifically, a quality testing method for a Yushuda preparation for treating depression, wherein the Yushuda... The preparation is obtained by the following steps: mixing 700-1200 parts by weight of patchouli, 200-400 parts by weight of dried tangerine peel, 300-700 parts by weight of cardamom, 300-700 parts by weight of amomum villosum, and 400-750 parts by weight of stir-fried atractylodes lancea to obtain a mixture of Chinese medicinal materials, wherein the cardamom and amomum villosum are preferably mixed with patchouli, dried tangerine peel and stir-fried atractylodes lancea in the form of crushed powder; decocting the mixture of medicinal materials with water, concentrating the filtrate to a clear extract with a temperature of 48-52℃ and a relative density of 1.04-1.05; adding ethanol to concentrate the filtrate to an extract with a temperature of 60-70℃ and a relative density of 1.14-1.17; and adding excipients to prepare the desired formulation. The quality of the formulation was determined by high performance liquid chromatography and mass spectrometry.

[0037] In addition, the present invention also provides a method for establishing the characteristic chromatogram of Yushuda preparation, characterized in that the Yushuda preparation is analyzed and detected by high performance liquid chromatography, and the method includes the following steps: 1) Prepare reference solutions: Mix hesperidin reference standard, sorbitol reference standard, ginsenoside reference standard, cardamomin reference standard, alder ketone reference standard and patchouli ketone reference standard to prepare mixed reference solutions; 2) Prepare the test solution of Yushuda preparation; add solvent to Yushuda preparation to prepare the test solution; 3) The chromatograms of the mixed reference solution and the test solution were detected by high performance liquid chromatography (HPLC). The chromatograms of the test solution and the reference solution were compared to determine the common peaks and establish characteristic chromatograms. The common peaks include 6 peaks: hesperidin, sorbitol, pinocembrin, cardamomin, alder ketone and patchouli ketone. The high-performance liquid chromatography (HPLC) conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material, and the mobile phase includes phase A and phase B, wherein phase A is acetonitrile and phase B is a 0.05-0.15% by mass phosphoric acid solution, and gradient elution is performed.

[0038] The present invention establishes the characteristic spectrum of Yushuda preparation by means of the characteristic spectrum establishment method described above.

[0039] This invention also provides a method for detecting the content of sorbitol and pine resin in Yushuda preparations, characterized in that... The method involves analyzing and detecting Yushuda preparations using high-performance liquid chromatography (HPLC), which includes the following steps: 1) Prepare reference solution: Mix sorbitan reference standard and ginsenoside reference standard separately to prepare a mixed reference solution; 2) Prepare the test solution of Yushuda preparation; add solvent to Yushuda preparation to prepare the test solution; 3) High-performance liquid chromatography (HPLC) was used to detect the mixed reference standard solution and the test solution separately; The high-performance liquid chromatography (HPLC) conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material, and the mobile phase includes phase A and phase B, wherein phase A is acetonitrile and phase B is a 0.05-0.15% by mass phosphoric acid solution.

[0040] The Yushuda tablets used in this invention can be any commercially available known Yushuda tablets. Specifically, the Yushuda tablets used are a traditional Chinese medicine preparation made from the medicinal herbs of patchouli, tangerine peel, cardamom, and atractylodes. Preferably, the preparation is made by mixing 700-1200 parts by weight of patchouli, 200-400 parts by weight of tangerine peel, 300-700 parts by weight of cardamom, 300-700 parts by weight of cardamom, and 400-750 parts by weight of stir-fried atractylodes, decocting with water, extracting with ethanol, and adding conventional excipients. The excipients can be selected from one or more of lactose, silicon dioxide, dextrin, hydroxypropyl cellulose, microcrystalline cellulose, microcrystalline silica, magnesium stearate, etc.; specifically, various preparations are prepared with reference to the excipients described in patent application CN201610811277.1. For example, regarding Yushuda tablets, based on weight, relative to 100 parts of patchouli, add 340 parts of lactose, 104 parts of dextrin, 40 parts of hydroxypropyl cellulose, 8 parts of microcrystalline cellulose, 4.8 parts of micronized silica gel, and 3.2 parts of magnesium stearate, mix well, add an appropriate amount of ethanol to granulate, and dry.

[0041] Specifically, the Yushuda tablets used in this invention can be commercially available Yushuda tablets or prepared using conventional methods for preparing traditional Chinese medicine preparations.

[0042] In this invention, the raw materials used as traditional Chinese medicine are the dried aerial parts of *Pogostemon cablin* (Blanco) Benth., a plant of the Lamiaceae family. Dried tangerine peel is the dried mature pericarp of *Citrus reticulata* Blanco, a plant of the Rutaceae family, and its cultivated varieties. Cardamom is the dried mature fruit of *Amomum javanica*, a plant of the Zingiberaceae family, and is a multi-origin variety. Amomum villosum is the dried nearly mature seed of *Amomum villosum*, a plant of the Zingiberaceae family, and is listed as a single-origin variety under the *Amomum villosum* entry in various editions of the *Chinese Pharmacopoeia*. Stir-fried Atractylodes lancea refers to Atractylodes lancea processed by stir-frying with wheat bran. In this invention, stir-fried Atractylodes lancea is prepared by mixing wheat bran and Atractylodes lancea in a mass ratio of (13-18):100, preferably 15:18. Wheat bran is sprinkled into Atractylodes lancea (cut into 2-4 mm thick slices) and stir-fried until the surface of the Atractylodes lancea turns deep yellow. The wheat bran is then sieved out to obtain stir-fried Atractylodes lancea. In this invention, it is preferably prepared according to the "Processing Procedure of Atractylodes lancea stir-fried with wheat bran" specified in the 2020 edition of the Chinese Pharmacopoeia. Specifically, the processing procedure specified in the pharmacopoeia is as follows: Take Atractylodes lancea medicinal material, remove impurities and non-medicinal parts; wash the cleaned Atractylodes lancea medicinal material with water, and soak it for 4-6 hours until there is no dry core inside. Cut the prepared Atractylodes lancea into 2-4mm thick slices. Heat the slices in an oven (55±5℃) until the moisture content does not exceed 10.0%, then remove and let cool. Heat a wok over medium heat (160-190℃), add wheat bran and stir-fry until it smokes, then add the prepared Atractylodes lancea slices (15kg wheat bran for every 100kg of prepared Atractylodes lancea slices). Control the stir-frying temperature at 160-190℃ (wok wall temperature) for 10-40 minutes, until the surface turns a deep yellow. Stop heating, remove, sift out the wheat bran, and let cool. Place the sifted, bran-fried Atractylodes lancea slices on a sorting table to remove any mushy slices. After passing inspection, transfer the bran-fried Atractylodes lancea to the packaging room, package, and label.

[0043] In one specific embodiment of the present invention, for example, a mixture of patchouli, tangerine peel, cardamom and atractylodes can be used to obtain Chinese herbal raw materials. These raw materials are then decocted with water to obtain a clear extract. Ethanol is then added and the mixture is filtered to obtain a clear extract, which is Yushuda extract. Then, excipients are added and the mixture is dried under reduced pressure to obtain Yushuda tablets.

[0044] In a preferred embodiment, the Yushuda preparation used in this invention is prepared by a method comprising the following steps: mixing 700-1200 parts by weight of patchouli, 200-400 parts by weight of dried tangerine peel, 300-700 parts by weight of cardamom, 300-700 parts by weight of amomum villosum, and 400-750 parts by weight of stir-fried atractylodes lancea to obtain a mixture of traditional Chinese medicinal materials, wherein the cardamom and amomum villosum are preferably mixed with patchouli, dried tangerine peel, and stir-fried atractylodes lancea in the form of crushed powder; decocting the mixture of medicinal materials with water and concentrating the filtrate to a clear extract with a relative density of 1.04-1.05 (48-52℃); adding ethanol to concentrate the filtrate to an extract with a relative density of 1.14-1.17 (60-70℃); and adding excipients to prepare various preparations as required.

[0045] In a preferred embodiment, a medicinal mixture is prepared from 1000g of patchouli, 334g of dried tangerine peel, 500g of cardamom, 500g of amomum villosum, and 666g of stir-fried atractylodes lancea. Preferably, the cardamom and amomum villosum are mixed with patchouli, dried tangerine peel, and stir-fried atractylodes lancea in a crushed powder state to obtain the medicinal mixture. The medicinal mixture is decocted with water 2-4 times, 1-3 hours each time. The decoction is filtered, and the filtrate is concentrated to a clear extract with a relative density of 1.04-1.05 (48-52℃). Ethanol is added to make the alcohol content reach 70%, mixed well, refrigerated (5-10℃) overnight, filtered, and the filtrate is reserved. The filtered suspension was washed with 70% ethanol by stirring, allowed to stand, filtered, and all the filtrates were used to recover ethanol and concentrated to an extract with a relative density of 1.14-1.17 (60-70℃). Excipients were added to prepare various preparations as required, including capsules, tablets, granules, etc.

[0046] The characteristic spectrum established by the method of Yushuda tablets and the method for determining the content of sorbitol and pinocembrin of the present invention can play a positive role in the quality control of Yushuda tablets and play an important role in the treatment of depression.

[0047] The following examples illustrate the characteristic chromatogram of Yushuda tablets of the present invention and the method for determining the content of sorbitol and pinosin. Example

[0048] Example 1: Establishment of the characteristic spectrum of Yushuda tablets Based on the General Chapter 0512 High Performance Liquid Chromatography (HPLC) and the 9101 Analytical Method Validation Guidelines of the 2020 Edition of the Chinese Pharmacopoeia, Part IV, this invention demonstrates that the analytical method established by the inventors is suitable for the corresponding detection requirements. The results of the analytical method validation should comply with the provisions of the Pharmacopoeia Analytical Method Validation Guidelines.

[0049] The methodology validation content and results for Yushuda tablets are summarized in Table A below.

[0050] The method of using the characteristic spectrum analysis of Yushuda tablets was validated, and the results met the judgment criteria. This proves that the established and validated characteristic spectrum analysis method for Yushuda tablets is capable of successfully operating the verification method for the characteristic spectrum of Yushuda tablets.

[0051] The feature mapping method for Yushuda tablets in this embodiment is described in detail below.

[0052] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0053] 1. Chromatographic conditions and system suitability test for characteristic chromatograms: Octadecylsilane-bonded silica gel was used as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 µm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 0.30 ml / min; the column temperature was 30℃; and the detection wavelength was 315 nm. The theoretical plate number, calculated based on the sorbitol peak, should not be less than 7000.

[0054]

[0055] 2. Preparation of reference solutions: Take appropriate amounts of hesperidin reference standard, sorbitol reference standard, pinocembrin reference standard, cardamom reference standard, alder ketone reference standard, and patchouli reference standard, respectively, accurately weigh them, and add methanol to prepare a solution containing 80 µg of hesperidin, 40 µg of sorbitol, 40 µg of pinocembrin, 40 µg of cardamom, 80 µg of alder ketone, and 40 µg of patchouli per ml, as the mixed reference solution.

[0056] 3. Preparation of the test solution: Take 10 Yushuda tablets, remove the film coating, weigh accurately, grind finely, take about 0.5g, weigh accurately, place in a stoppered conical flask, accurately add 25ml of methanol, stopper tightly, weigh, sonicate (power 150W, frequency 20kHz) for 45 minutes, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0057] 4. Assay: Accurately pipette 2 μl each of the reference solution and the test solution into the liquid chromatograph and measure to obtain the result.

[0058] The chromatogram of the test sample should show 13 characteristic peaks. Peaks 4, 7, 9, 11, 12, and 13 correspond to the retention times of the reference peaks of hesperidin, sorbitol, pinosin, cardamomin, patchouli, and alder, respectively. The peak corresponding to the hesperidin reference peak is peak S1. The relative retention times of peaks 1, 2, 3, 5, and 6 with peak S1 should be calculated. The relative retention times should be within ±10% of the specified values, which are: 0.14 (peak 1), 0.51 (peak 2), 0.60 (peak 3), 1.07 (peak 5), and 1.11 (peak 6). The peak corresponding to the reference peak of sorbitol is peak S2. Calculate the relative retention times of peaks 8 and 10 with peak S2. The relative retention times should be within ±10% of the specified values, which are 1.16 (peak 8) and 1.21 (peak 10).

[0059] in, Figure 1 In this diagram, A represents the reference feature map. The relevant label information in this map is explained as follows: Peak 4 (S1): hesperidin; Peak 7 (S2): sorbitin; Peak 9: ginsenoside; Peak 11: Cardamom; Peak 12: Patchouli; Peak 13: Alder.

[0060] The chromatographic column was an ACQUITY UPLC® HSS T3 C18, 2.1 × 150 mm, 1.8 μm.

[0061] The experimental equipment and chromatographic columns used in the chromatographic detection process are shown in Tables 2 and 3.

[0062]

[0063] The experimental reagents, reference standards, test samples (Yushuda tablets), and excipients used are shown in Tables 4 and 5 below.

[0064]

[0065]

[0066] 5. Determination and establishment of characteristic spectra 5.1 Determination of Common Peaks UPLC chromatograms of different batches (220401, 220501, 220502, 220701, 220702, 220703, 220704, 231101, 231102, 231103) of Yushuda tablets were determined. The results were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission, and common peaks were selected. The overlay diagrams of characteristic peaks from the 10 batches of Yushuda tablets are shown below. Figure 1 B in the diagram is the superposition of the common peaks of the Yushuda film.

[0067] 5.2 Methodological Validation 5.2.1 Specificity Examination Preparation of the test solution: Take about 0.5g of finely ground Yushuda tablets (batch number: 220502), accurately weigh it, and prepare the test solution according to the method described in item 3 above, "Preparation of the test solution".

[0068] Preparation of blank solution of mixed excipients: Take about 0.5g of mixed excipient (lactose, dextrin, magnesium stearate, silicon dioxide, microcrystalline cellulose, low-substituted hydroxypropyl cellulose) powder, weigh accurately, and prepare the test solution according to the method described in item 3 "Preparation of test solution" above.

[0069] Preparation of blank solution of excipient for gastric-soluble film-coating premix: Take about 0.5g of gastric-soluble film-coating premix powder accurately, and prepare the test solution according to the method described in item 3 above, "Preparation of test solution".

[0070] Accurately pipette 2 μl each of the following: the test solution of Yushuda tablets (220502); the mixed reference solution (hesperidin, sorbitol, pinocembrin, cardamomin, alderne, patchouli ketone); the blank solution of mixed excipients (lactose, dextrin, magnesium stearate, silicon dioxide, microcrystalline cellulose, low-substituted hydroxypropyl cellulose); the blank solution of gastric-soluble film-coating premix; and the methanol blank. Inject these solutions according to the conditions outlined in Section 1 above, "Chromatographic conditions for characteristic chromatograms and system suitability test," and record the chromatograms. See below for results. Figure 2 .

[0071] in Figure 2 In the table, numbers 1-6 represent the following substances: 1-hesperidin; 2-izin; 3-pinene; 4-cardin; 5-patchouli; 6-alderne. The sample information represented by A and D is as follows: A-methanol blank, B-mixed excipient blank solution, C-gastric-soluble film-coating premix blank solution, D-mixed reference standard, E-Yushuda tablets 220502.

[0072] Depend on Figure 2It can be seen that the solvent and excipients did not interfere with the characteristic peaks in the spectrum of Yushuda tablets. This indicates that the method has good specificity.

[0073] 5.2.2 Instrument Precision Assessment Take approximately 0.5 g of finely ground Yushuda tablets (batch number: 220502), accurately weigh it, and prepare the test solution according to the method described in Section 3, "Preparation of Test Solution". Inject the solution 6 times under the conditions described in Section 1, "Chromatographic Conditions and System Suitability Test of Characteristic Chromatograms". Accurately pipette the test solution prepared from the same Yushuda tablet (220502) and inject it 6 times under the above chromatographic conditions. Record the retention times of each characteristic peak. Peaks 4 (S1), 7 (S2), 9, 11, 12, and 13 correspond to the retention times of the reference peaks of hesperidin, sorbitol, pinosin, cardamomin, patchouli, and alder, respectively. Calculate the relative retention times of peaks 1, 2, 3, 5, and 6 using the hesperidin peak as reference S1, and calculate the relative retention times of peaks 8 and 10 using the sorbitol peak as reference S2. The results are shown in Table 6.

[0074]

[0075] Experimental results show that the RSD of the relative retention time of each chromatographic peak is less than 2%, indicating that the instrument has good precision.

[0076] 5.2.3 Repeatability Test Take approximately 0.5 g of finely ground Yushuda tablets (220502), accurately weigh 6 portions, and prepare the test solution according to the method described in Section 3 "Preparation of Test Solution" above. Inject the sample according to the conditions in Section 1 "Chromatographic Conditions and System Suitability Test of Characteristic Chromatograms" above. Record the retention times of each characteristic peak. Peaks 4 (S1), 7 (S2), 9, 11, 12, and 13 correspond to the retention times of the reference peaks of hesperidin, sorbitol, pinocembrin, cardamomin, patchouli, and alder, respectively. Using the hesperidin peak as reference S1, calculate the relative retention times of peaks 1, 2, 3, 5, and 6. Using the sorbitol peak as reference S2, calculate the relative retention times of peaks 8 and 10. The results are shown in Table 7.

[0077] Experimental results show that the relative retention time (RSD) of each chromatographic peak is less than 2%, indicating that the method has good repeatability.

[0078] 5.2.4 Stability Study of the Test Sample Take 2 µl of the same test sample, Yushuda tablets (220502), under the repeatability test, and inject it at 0, 2, 4, 6, 8, 12, and 24 hours according to the conditions in Section 1 above, "Chromatographic conditions and system suitability test of characteristic chromatograms." Record the retention times of each characteristic peak. Peaks 4 (S1), 7 (S2), 9, 11, 12, and 13 correspond to the retention times of the reference peaks of hesperidin, sorbitol, pinosin, cardamomin, patchouli, and alder, respectively. Using the hesperidin peak as reference S1, calculate the relative retention times of peaks 1, 2, 3, 5, and 6. Using the sorbitol peak as reference S2, calculate the relative retention times of peaks 8 and 10. The results are shown in Table 8.

[0079]

[0080] The experimental results show that the relative retention time (RSD) of each chromatographic peak is less than 2%, indicating that the test solution has good stability within 24 hours.

[0081] 5.2.5 Durability Test The main focus was on the durability of the chromatographic column, chromatograph, column temperature, and flow rate on the characteristic chromatogram of Yushuda tablets.

[0082] (1) Chromatographic column investigation The effects of different manufacturers and models of chromatographic columns on each characteristic peak were investigated.

[0083] The chromatographic columns used were: Column 1 - Waters ACQUITY UPLC® HSS T3 (2.1×150mm, 1.8μm); Column 2 - AmethSep C18 Aq (2.1×100mm, 1.8μm); Column 3 - ZORBAX Eclipse Plus C18 (2.1×150mm, 1.8μm); Column 4 - ZORBAX SB-AQ (2.1×150mm, 1.8μm). The effect of the chromatographic column on the peak characteristics of Yushuda tablets was investigated.

[0084] Take approximately 0.5 g of finely ground Yushuda tablets (220502), accurately weigh it, and prepare the test solution according to the method described in Section 3, "Preparation of Test Solution". Inject the sample according to the conditions in Section 1, "Chromatographic Conditions and System Suitability Test of Characteristic Chromatograms" (using chromatographic columns 1, 2, 3, and 4 respectively). Record the retention times of each characteristic peak. Peaks 4 (S1), 7 (S2), 9, 11, 12, and 13 correspond to the retention times of the reference peaks of hesperidin, sorbitol, pinosin, cardamom, patchouli, and alder, respectively. Calculate the relative retention times of peaks 1, 2, 3, 5, and 6 using the hesperidin peak as reference S1, and calculate the relative retention times of peaks 8 and 10 using the sorbitol peak as reference S2. The results are shown in Table 9. Figure 3 ;

[0085] Figure 3 These are chromatograms of results obtained by examining different chromatographic columns. Figure 3 As can be seen from the chromatogram, the characteristic peaks of Yushuda tablets do not have ideal durability on the chromatographic column. According to the comparison of chromatographic columns from different manufacturers, Waters ACQUITY UPLC® HSS T3 (chromatographic column 1) has better resolution and peak shape for the 13 characteristic peaks. Therefore, this chromatographic column was selected for the experiment in this study.

[0086] (2) Column temperature investigation The effects of different column temperatures (25℃, 30℃, and 35℃) on the peak shape and resolution of each characteristic peak in the characteristic spectrum of Yushuda tablets were compared.

[0087] Take approximately 0.5 g of finely ground Yushuda tablets (220502), accurately weigh it, and prepare the test solution according to the method described in Section 3, "Preparation of Test Solution". Inject the sample according to the conditions in Section 1, "Chromatographic Conditions and System Suitability Test of Characteristic Chromatograms" (column temperatures of 25℃, 30℃, and 35℃). Record the retention times of each characteristic peak. Peaks 4 (S1), 7 (S2), 9, 11, 12, and 13 correspond to the retention times of the reference peaks of hesperidin, sorbitol, pinosin, cardamom, patchouli, and alder, respectively. Calculate the relative retention times of peaks 1, 2, 3, 5, and 6 using the hesperidin peak as reference S1, and calculate the relative retention times of peaks 8 and 10 using the sorbitol peak as reference S2. The results are shown in Table 10. Figure 4 .

[0088]

[0089] Figure 4 This is a graph showing the results of durability testing at different column temperatures; from this... Figure 4 The separation of peaks in the chromatogram shows that the peak separation is best at a column temperature of 30℃, less ideal at 35℃, and unsatisfactory at 25℃. This indicates that the column temperature is not very robust to characteristic peaks. Therefore, the column temperature should be fixed at 30℃. Thus, 30℃ was chosen as the column temperature for the characteristic chromatogram of Yushuda tablets.

[0090] (3) Flow velocity investigation The effects of different flow rates (0.28 ml / min, 0.30 ml / min, and 0.32 ml / min) on the peak shape and resolution of each characteristic peak in the characteristic spectrum of Yushuda tablets were compared.

[0091] Take approximately 0.5 g of finely ground Yushuda tablets (220502), accurately weigh it, and prepare the test solution according to the method described in Section 3, "Preparation of Test Solution". Inject the sample according to the conditions in Section 1, "Chromatographic Conditions and System Suitability Test of Characteristic Chromatograms" (flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min). Record the retention times of each characteristic peak. Peaks 4 (S1), 7 (S2), 9, 11, 12, and 13 correspond to the retention times of the reference peaks of hesperidin, sorbitol, pinosin, cardamom, patchouli, and alder, respectively. Calculate the relative retention times of peaks 1, 2, 3, 5, and 6 using the hesperidin peak as reference S1, and calculate the relative retention times of peaks 8 and 10 using the sorbitol peak as reference S2. The results are shown in Table 11 and... Figure 5 .

[0092]

[0093] Figure 5 These are chromatograms of the results obtained at different flow rates. Figure 5 The separation of peaks and their relative retention times in the chromatograms show that flow rates of 0.25 ml / min and 0.35 ml / min do not provide as good a resolution for the characteristic peaks as 0.30 ml / min, indicating that 0.30 ml / min is the optimal flow rate. Therefore, this study selected 0.30 ml / min as the flow rate for the chromatographic conditions used to obtain the characteristic chromatogram of Yushuda tablets.

[0094] (4) Investigation of the preparation conditions of the test solution The effects of different extraction solvents, extraction times, and extraction methods on the extraction rates of each characteristic peak were investigated to further determine the method for preparing the test solution of Yushuda tablets with characteristic chromatograms.

[0095] (4.1) Investigation of extraction solvent The effects of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and water on each characteristic peak were investigated to determine the optimal extraction solvent for preparing the test solution of Yushuda tablets characteristic chromatogram.

[0096] Take approximately 0.5g of finely ground Yushuda tablets (220502), accurately weigh 9 groups, and place them in stoppered conical flasks. Accurately add methanol, 5% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and 25ml of water respectively. Seal tightly, weigh, and sonicate (150W power, 20kHz frequency) for 45 minutes. Remove, cool, and weigh again. Make up the lost weight with the corresponding solvent, shake well, filter, and collect the filtrate. Inject 2µl of the sample under the conditions described in section 1 above, "Chromatographic conditions and system suitability test for characteristic chromatograms," measure and record the peak area of ​​each characteristic peak, calculate the ratio of the peak area to the sample weight, and plot a column chart. The results are shown in Table 12 and... Figure 6 , Figure 7 .

[0097] Figure 6 This is a graph showing the peak area / sample weight results for different extraction solvents. Figure 7 The chromatograms of characteristic peaks were examined using different extraction solvents. Based on the calculated peak area values, column charts, and comprehensive analysis of the chromatograms, methanol was found to have the highest peak areas when used as the extraction solvent. Among these, methanol, 75% methanol, and 50% methanol were preferred. Furthermore, it was found that 75% ethanol had the most prominent peak area when used as the solvent, with hesperidin showing a relatively prominent peak area and good separation of each peak. Therefore, methanol was selected as the extraction solvent for preparing the test solution of Yushuda tablets.

[0098] (4.2) Examination of extraction methods The effects of ultrasonic extraction, reflux extraction, and shaking extraction on each characteristic peak were investigated to determine the optimal extraction method for preparing the test solution of Yushuda tablets with characteristic chromatograms.

[0099] Take approximately 0.5g of finely ground Yushuda tablets (220502), accurately weigh 3 groups, place them in stoppered conical flasks, accurately add 25ml of methanol to each, seal tightly, weigh, and respectively use ultrasonic treatment (power 150W, frequency 20kHz) for 45 minutes, reflux extraction for 45 minutes, and shaking extraction for 45 minutes. Remove, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate. Inject 2µl of the sample according to the conditions in section 1 above, "Chromatographic conditions and system suitability test of characteristic chromatograms," measure and record the peak area of ​​each characteristic peak, calculate the ratio of the peak area of ​​each characteristic peak to the sample weight, and plot a column chart. The results are shown in Table 13. Figure 8 , Figure 9 .

[0100]

[0101] Figure 8 This is a graph showing the results of different extraction methods examining the peak area / sample size of each characteristic peak; Figure 9 The chromatograms of characteristic peaks were examined using different extraction methods and solvents. Based on... Figure 8 and Figure 9 Based on the comprehensive analysis of the peak area values, column charts, and chromatograms, ultrasonic extraction, reflux extraction, and shaking extraction showed no significant difference in the extraction rates of each characteristic peak. For ease of extraction operation, and based on the results of content determination, ultrasonic extraction can be considered as the extraction method for preparing the test solution of Yushuda tablets.

[0102] (4.3) Examination of extraction time The effects of ultrasonic extraction time (30 min, 45 min, 60 min, 90 min) on each characteristic peak were investigated to determine the optimal extraction time for preparing the test solution of Yushuda tablets characteristic chromatogram.

[0103] Take approximately 0.5g of finely ground Yushuda tablets (220502), accurately weigh four portions, place them in stoppered conical flasks, accurately add 25ml of methanol to each, seal tightly, weigh, and sonicate (power 150W, frequency 20kHz) for 30 minutes, 45 minutes, 60 minutes, and 90 minutes respectively. Remove, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate. Inject 2µl of the sample under the conditions described in section 1 above, "Chromatographic conditions and system suitability test for characteristic chromatograms," measure and record the peak area of ​​each characteristic peak, calculate the ratio of the peak area to the sample weight, and plot a column chart. The results are shown in Table 14. Figure 10 , Figure 11 .

[0104]

[0105] Figure 10 Figure 11 shows the chromatograms of each characteristic peak under different extraction times, calculated as peak area / sample weight. Figure 12 shows the chromatograms of each characteristic peak under different ultrasonic times. According to... Figure 10 and Figure 11 Based on the comprehensive analysis of the peak area values, column charts, and chromatograms, it was found that there was no significant difference in the extraction rate of each characteristic peak due to different ultrasonic extraction times. However, considering the results of the content determination indicators hesperidin, sorbitol, and arbutin, ultrasonic extraction for 45 minutes was selected as the extraction time for preparing the test solution of Yushuda tablets.

[0106] (4.4) Investigation of the amount of extraction solvent The effect of methanol volume (15 ml, 25 ml, 50 ml) on each characteristic peak during ultrasonic extraction was investigated to determine the optimal extraction solvent volume for preparing the test solution of Yushuda tablets characteristic chromatogram.

[0107] Take approximately 0.5g of finely ground Yushuda tablets (220502), accurately weigh three portions, and place them in stoppered conical flasks. Accurately add 15ml, 25ml, and 50ml of methanol to each flask, respectively. Seal the flasks tightly, weigh them, and sonicate each portion (150W power, 20kHz frequency) for 45 minutes. Remove the flasks, cool them, and weigh them again. Make up the lost weight with methanol, shake well, filter, and collect the filtrate. Inject 2µl of the sample under the conditions described in section 1 above, "Chromatographic conditions and system suitability test for characteristic chromatograms." Measure and record the peak area of ​​each characteristic peak, calculate the ratio of the peak area to the sample weight (using 25ml as a reference), and plot a column chart. The results are shown in Table 15. Figure 12 , Figure 13 .

[0108]

[0109] Figure 12 shows the peak area / sample volume results for different solvent dosages; Figure 13 shows the chromatograms of different characteristic peaks for different extraction solvent dosages. According to... Figure 12 and Figure 13 The results, column charts, and chromatograms were analyzed after converting the peak area ratio to the sample weight using the equivalent of 25 ml of extraction solvent. Different amounts of extraction solvent did not show significant differences in the extraction rates of each characteristic peak. Therefore, to ensure sufficient extraction of the test sample while also considering solvent conservation, the middle amount of 25 ml of methanol was selected as the extraction solvent for preparing the Yushuda tablet test sample solution.

[0110] (4.5) Investigation of the sample injection volume The effect of the injection volume (1µl, 2µl, 3µl) of the test solution of Yushuda tablets on each characteristic peak was investigated in order to determine the injection volume of the test solution for the characteristic spectrum of Yushuda tablets.

[0111] Take approximately 0.5g of finely ground Yushuda tablets (220502), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of methanol, seal tightly, weigh, and sonicate (150W power, 20kHz frequency) for 45 minutes. Remove, cool, and weigh again. Make up the lost weight with methanol, shake well, filter, and collect the filtrate. Inject 1µl, 2µl, and 3µl of the sample respectively under the conditions of Section 1 above, "Chromatographic conditions and system suitability test of characteristic chromatograms," measure and record the peak area of ​​each characteristic peak, calculate the ratio of the peak area of ​​each characteristic peak to the sample weight, and plot a column chart. The results are shown in Table 16. Figure 14 , Figure 15 .

[0112]

[0113] Figure 14 shows the peak area / sample volume ratio of each characteristic peak under different injection volumes; Figure 15 shows the chromatograms of each characteristic peak under different injection volumes. Based on the results of peak area ratio after sample volume weighing, column chart, and chromatogram analysis equivalent to a 2µl injection volume, there was no significant difference in the characteristic peaks under different injection volumes. Therefore, to ensure that the peak area of ​​the characteristic peaks is not too small, and to avoid overloading the injection volume, the middle value of 2µl was selected as the injection volume of the Yushuda tablet test solution.

[0114] 5.3 Determination of the preparation method for the test solution Based on the above research results and the findings of content determination, the preparation method for the test solution of Yushuda tablets is determined as follows: Take 10 tablets of this product, remove the film coating, weigh accurately, grind finely, take about 0.5g, weigh accurately, place in a stoppered conical flask, accurately add 25ml of methanol, stopper tightly, weigh, sonicate (power 150W, frequency 20kHz) for 45 minutes, remove, cool, weigh again, make up the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the product.

[0115] 5.4 Determination of Characteristic Spectra of Multiple Batch Samples Take 10 tablets of each of the following batches (batch numbers: 220401, 220501, 220502, 220701, 220702, 220703, 220704, 231101, 231102, and 231103): remove the film coating, weigh accurately, grind finely, take about 0.5g, weigh accurately into 3 portions, place in stoppered conical flasks, add 25ml of methanol to each, seal tightly, weigh, and sonicate (150W power, 20kHz frequency) for 45 minutes. Remove, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the final product. Inject 2 µl of the sample under the above chromatographic conditions and record the retention times of each characteristic peak. Peaks 4 (S1), 7 (S2), 9, 11, 12, and 13 correspond to the retention times of the reference peaks of hesperidin, sorbitol, pinosin, cardamomin, patchouli, and alder, respectively. The relative retention times of peaks 1, 2, 3, 5, and 6 are calculated using the hesperidin peak as reference S1, and the relative retention times of peaks 8 and 10 are calculated using the sorbitol peak as reference S2.

[0116] Sample information for 10 batches of Yushuda tablets is shown in Table 17. The relative retention times and overlay results of each characteristic peak in the characteristic chromatograms of the 10 batches of Yushuda tablets are shown in Table 18 and... Figure 16 .

[0117]

[0118]

[0119] According to the similarity evaluation results of the feature maps of 10 batches of Yushuda tablets, there are 13 common peaks in Yushuda tablets.

[0120] 5.5 Formulation of Feature Maps The UPLC characteristic chromatograms of 10 batches of Yushuda tablets were matched using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine". A control chromatogram was generated using the median method to establish a control characteristic chromatogram for Yushuda tablets. (See attached image.) Figure 16 and Figure 17 .

[0121] Figure 16 This is a superimposed chromatogram of 10 batches of Yushuda tablets. The chromatographic column type is: Waters ACQUITY UPLC® HSS T3, 2.1×150mm, 1.8μm; R is the reference chromatogram. The 10 batches of Yushuda tablets are numbered S1-S10. The results were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission, and a total of 6 common peaks were identified. Figure 17 shows the common peak pattern and reference chromatogram of Yushuda tablets. Based on the... Figure 17 The analysis of the characteristic chromatograms of the 10 batches of Yushuda tablets shown included peaks and reference peaks for hesperidin, sorbitol, cardamom, amylopectin, and patchouli. The final standard for the characteristic chromatograms of Yushuda tablets was determined: the test sample's characteristic chromatogram should show 13 characteristic peaks, among which peaks 4, 7, 9, 11, 12, and 13 correspond to the retention times of the reference peaks for hesperidin, sorbitol, cardamom, amylopectin, patchouli, and amylopectin, respectively. The peak corresponding to the hesperidin reference peak is peak S1. Calculate the relative retention times of peaks 1, 2, 3, 5, and 6 with peak S1. These relative retention times should be within ±10% of the specified values, which are: 0.14 (peak 1), 0.51 (peak 2), 0.60 (peak 3), 1.07 (peak 5), and 1.11 (peak 6). The peak corresponding to the sorbitol reference peak is peak S2. Calculate the relative retention times of peaks 8 and 10 with peak S2. These relative retention times should be within ±10% of the specified values, which are: 1.16 (peak 8) and 1.21 (peak 10).

[0122] The graph used for validation of the feature mapping methodology is... Figure 17 The spectrum contains 13 peaks.

[0123] Example 2: Determination and Analysis of the Content of Gingerol and Pinocene in Yushuda Tablets Based on the General Chapter 0512 High Performance Liquid Chromatography (HPLC) and the 9101 Analytical Method Validation Guidelines of the 2020 Edition of the Chinese Pharmacopoeia, Part IV, it is demonstrated that the analytical methods established by the R&D technicians are suitable for the corresponding testing requirements. The results of the analytical method validation should comply with the provisions of the Pharmacopoeia Analytical Method Validation Guidelines.

[0124] The verification results of the analytical method for determining the content of sorbitol and pine resin in Yushuda tablets are summarized in Table II-1 below.

[0125]

[0126] The method for determining the content of sorbitan and pinoresin in Yushuda tablets was validated, and the results met the judgment criteria. This demonstrates that the established and validated method for determining the content of sorbitan and pinoresin in Yushuda tablets is capable of successfully operating the test method for determining the content of sorbitan and pinoresin described in the "[Content Determination] Cardamom" section of Yushuda tablets. Specifically, the method for determining the content of sorbitan and pinoresin in Yushuda tablets is as follows: The following describes in detail the analytical method for determining the content of sorbitol and pinocembrin in this embodiment, and verifies the method.

[0127] [Content Determination] Cardamom was determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0128] 1. Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material; methanol-0.2% phosphoric acid solution (62:38) was used as the mobile phase; the detection wavelength was 290 nm; the theoretical plate number calculated based on the sorbitol peak should not be less than 3000.

[0129] 2. Preparation of reference solution: Take appropriate amounts of sorbitol and pinoresin reference standards, accurately weigh them, and add methanol to prepare a mixed reference solution containing 40 μg of sorbitol and 40 μg of pinoresin per 1 ml.

[0130] 3. Preparation of the test solution: Take 10 tablets of this product, remove the film coating, weigh accurately, grind finely, take about 0.5g, weigh accurately, place in a stoppered conical flask, accurately add 25ml of methanol, stopper tightly, weigh, sonicate (power 150W, frequency 20kHz) for 45 minutes, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0131] 4. Assay: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0132] Each tablet of this product contains cardamom and gingerol (C). 16 H 14 O4) and germinol (C 15 H12 The total amount of O4 is 1.53-3.09 mg / tablet.

[0133] The experimental equipment and chromatographic columns used in the chromatographic detection process are shown in Tables II-2 and II-3.

[0134]

[0135]

[0136] The experimental reagents, reference standards, test samples (Yushuda tablets), and excipients used are shown in Tables II-4 and II-5 below.

[0137]

[0138]

[0139] 5. Methodological validation for the determination of sorbitol and arbutin 5.1 Specificity Examination Take approximately 0.5 g of Yushuda tablets (batch number: 220502), Yushuda negative sample (lacking cardamom, batch number: 220725), and mixed excipients (lactose, dextrin, magnesium stearate, silicon dioxide, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, and gastric-soluble film-coating premix) powder, accurately weigh them, and prepare different solutions according to the method described in "3. Preparation of test solution" of cardamom in the "Content Determination" section above.

[0140] Accurately pipette 10 μl each of the following solutions: Yushuda tablets test solution (B), sorbitol and pinocembrin mixed reference solution (A), mixed excipient blank solution (D), Yushuda tablets negative solution (without cardamom) (E), and methanol blank. Inject each solution under the chromatographic conditions described above and record the chromatograms. Results are shown below. Figure 18 . Figure 18 It is a specific chromatogram for investigation, by Figure 18 The experimental results showed that no chromatographic peaks corresponding to sorbitol and pinoresin were found in the chromatograms of methanol blank (C), mixed excipient blank (D), and Yushuda tablet negative solution (without cardamom) (E). In summary, methanol blank, mixed excipient blank, and Yushuda tablet negative solution (without cardamom) do not interfere with the determination of sorbitol and pinoresin content. Therefore, the method for determining the content of sorbitol and pinoresin in Yushuda tablets is specific.

[0141] 5.2 Examination of Linear Relationships Accurately weigh 4.049 mg of sorbitol reference standard and 4.037 mg of pineapple reference standard, place them in a 50 ml volumetric flask, add methanol to dissolve and dilute to the mark to prepare a mixed reference solution containing 0.080332 mg of sorbitol and 0.080336 mg of pineapple per ml, which is used as the mother liquor.

[0142] Using the same pipette, accurately pipette 1, 2, 3, 4, 5, and 6 ml of the mixed reference solution of sorbitol and pinocembrin was placed into 10 ml volumetric flasks, diluted to the mark with methanol, and shaken well to obtain reference solutions of different concentrations of sorbitol and pinocembrin. Accurately pipette 10 μl of each solution was injected into the liquid chromatograph, and the chromatographic conditions were determined according to the conditions described in "1. Chromatographic Conditions and System Suitability Test" above. Standard curves and linear regression equations were plotted with the peak areas of sorbitol and pinocembrin as the ordinate and the concentrations (μg / ml) of sorbitol and pinocembrin as the abscissa. The results are shown in Tables II-6 and II-7. Figure 19 and Figure 20 .

[0143]

[0144]

[0145] Plotting the results from Tables II-6 and II-7 yields the following results: Figure 19 and Figure 20 The standard curve shown is as follows, where, Figure 19 This is the standard curve of sorbitol. Figure 20 This is the standard curve of galangin. The result shows the linear regression equation for galangin as follows: Y =33.724 X +6.7163, correlation coefficient R 2 The correlation coefficient was 0.9999. Experimental results showed that within the concentration range of 8.0332 μg / ml to 48.1992 μg / ml, the concentration of sorbitol reference standard showed a good linear relationship with the peak area; the linear regression equation for sorbitol was Y = 36.347X + 6.011, with a correlation coefficient of 0.9999. R 2 The value was 0.9999, and the experimental results showed that the concentration of berberine reference standard and the peak area had a good linear relationship in the range of 8.0336 μg / ml to 48.2016 μg / ml.

[0146] 5.3 Precision Test Take Yushuda tablets (220502), remove the film coating, weigh accurately, grind finely, and prepare the test solution according to the method described in "3. Preparation of test solution" of [Content Determination] of Amomum villosum. Repeat the injection 6 times according to the chromatographic conditions in "1. Chromatographic conditions and system suitability test" above, with an injection volume of 10 μl. Record the peak areas of sorbitol and pinocembrin in the test solution respectively, calculate the peak area RSD (%) value, and the determination results are shown in Table II-8.

[0147]

[0148] The experimental results showed that the RSD values ​​of the peak areas of sorbitol and arbutin were both less than 2.0%. Therefore, the results indicate that the instrument has good precision.

[0149] 5.4 Repeatability Test Take Yushuda tablets (220502), remove the film coating, weigh accurately, grind finely, and prepare 8 test solutions according to the method described in "3. Preparation of test solution" of [Content Determination] Cardamom. Determine the chromatographic conditions in "1. Chromatographic conditions and system suitability test" above, with an injection volume of 10 μl, and determine the content (mg / g) of sorbitol and pinocembrin in the test solution, and calculate the RSD (%) value of the content. The determination results are shown in Table II-9.

[0150] The experimental results showed that the RSD values ​​of both sorbitol and pinocembrin were less than 2%, indicating that the method had good repeatability.

[0151] 5.5 Stability Study of the Test Sample Take Yushuda tablets (220502), remove the film coating, accurately weigh, grind finely, and prepare the test solution according to the method described in "3. Preparation of test solution" of the content determination section of cardamom. Determine the chromatographic conditions according to "1. Chromatographic conditions and system suitability test" above, and inject at 0, 2, 4, 6, 8, 12 and 24 hours respectively, with an injection volume of 10 μl. Determine the content (mg / g) of sorbitol and pinocembrin in the test solution, and calculate the RSD (%) value of the content. The determination results are shown in Table II-10.

[0152] The experimental results showed that the RSD values ​​of sorbitol and pinocembrin were both less than 2%, indicating that the test solution was stable within 24 hours.

[0153] 5.6 Accuracy Test Take 0.25g of finely ground Yushuda tablets (220502, sorbitol content 1.90mg / g, pineapple content 0.57mg / g) with known content, accurately weigh 6 portions, and accurately add 5ml of sorbitol and pineapple reference solutions (sorbitol concentration 0.09492mg / ml, pineapple concentration 0.02932mg / ml) to each portion. Then accurately add 20ml of methanol solution, seal tightly, weigh, and sonicate (power 150W, frequency 20kHz) for 45 minutes. After cooling, weigh again, make up the weight loss with methanol solvent, shake well, filter, and collect the filtrate. Inject 10μl of each sample, determine the peak area of ​​sorbitol and pineapple, and calculate the content (mg) of sorbitol and pineapple respectively. The results are shown in Tables II-11 and II-12.

[0154]

[0155]

[0156] According to the "Guiding Principles for Analytical Method Validation" in Part IV of the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is 1mg-10mg, the recovery limit is 92%-105%. The results show that the recovery rate of sorbitol is 98%, which is within the specified range. Therefore, the results indicate that the accuracy of the assay method is good.

[0157]

[0158] According to the "Guiding Principles for Analytical Method Validation" in Part IV of the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is 0.1 mg-1 mg, the recovery limit is 90%-108%. The results show that the recovery rate of ginsenoside is 96%, which is within the specified range. Therefore, the results indicate that the accuracy of the assay method is good.

[0159] 5.7 Durability Test (1) Investigation of different flow velocities The effects of different flow rates (0.8 ml / min, 1.0 ml / min, and 1.2 ml / min) on the determination of sorbitol and pinocembrin in Yushuda tablets were compared.

[0160] Take Yushuda tablets (batch number: 220502) and prepare the test solution according to the method described in "3. Preparation of the test solution" of Cardamom [Content Determination]. Accurately inject 10 μl into the high-performance liquid chromatograph (HPLC). Perform the determination according to the chromatographic conditions in "1. Chromatographic conditions and system suitability test" above, changing the flow rate to 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min respectively. Record the relative retention time, symmetry, resolution, and plate number of sorbitol and pinocembrin, and calculate the content (mg / g). Experimental results are shown in Tables II-13 and II-14. Figure 21A and Figure 21B .

[0161]

[0162]

[0163] Figure 21A and Figure 21B The chromatograms were obtained at different flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min. The chromatograms and parameters show that the parameters meet the requirements at different flow rates, indicating that the method is robust to different flow rates.

[0164] (2) Investigation at different column temperatures The effects of different column temperatures (25℃, 30℃, and 35℃) on the determination of sorbitol and pinocembrin in Yushuda tablets were compared.

[0165] Take Yushuda tablets (220502) and prepare the test solution according to the method described in "3. Preparation of the test solution" of Cardamom in the section on "Content Determination". Accurately inject 10 μl into the high-performance liquid chromatograph. According to the chromatographic conditions in "1. Chromatographic conditions and system suitability test" above, change the column temperature to 25℃, 30℃ and 35℃ respectively, and record the relative retention time, symmetry, resolution and plate number of sorbitol and pinocembrin, and calculate the content (mg / g). Experimental results are shown in Tables II-15 and II-16. Figure 22A and Figure 22B .

[0166]

[0167] Figure 22A and Figure 22B The chromatograms were obtained at different column temperatures. As can be seen from the chromatograms and parameters, the parameters met the requirements at different column temperatures, indicating that the method has good robustness to column temperature.

[0168] (3) Investigation of different chromatographic columns The effects of different brands of chromatographic columns on the determination of sorbitol and pinosin in Yushuda tablets were investigated. Column 1: Xtimate ® C18 (4.6×250mm, 5μm), Column 2: Waters XBridge ® C18 (4.6×250mm, 5μm), column 3: Thermo Accliam TM 120 A ° C18 (4.6×250mm, 5μm), column 4: GL Sciences Inertsil TMODS-HL (4.6×250mm, 5μm), column 5: JADE-PAK ® KP-C18 (4.6×250mm, 5μm), column 6: Agilent ZORBAXEclipse Plus C18 (4.6×250mm, 5μm).

[0169] Take Yushuda tablets (220502) and prepare the test solution according to the method described in "3. Preparation of the test solution" of Cardamom in the section on "Content Determination". Accurately inject 10 μl into the high-performance liquid chromatograph. According to the chromatographic conditions in "1. Chromatographic conditions and system suitability test" above, change the chromatographic column to different brands of columns. Record the relative retention time, symmetry, resolution, and plate number of sorbitol and pinocembrin, and calculate the content (mg / g). The experimental results are shown in Tables II-17 and II-18. Figure 23A , Figure 23B , Figure 23C and Figure 23D .

[0170]

[0171]

[0172] Figure 23 A Figure 23B , Figure 23C and Figure 23D The chromatograms were examined using different chromatographic columns. Based on the above experimental results, it can be seen that there are certain differences in parameters between different chromatographic columns, but the differences in the contents of sorbitol and pinoresin are small. This indicates that using octadecylsilane-bonded silica gel as the packing material meets the requirements for the determination of sorbitol and pinoresin contents; therefore, no specific requirements are placed on the chromatographic column.

[0173] 6. Determination of sorbitan and pinocembrin content in samples Take 10 tablets each of Yushuda tablets (220701, 220702, 220703), remove the film coating, accurately weigh, grind into a fine powder, take about 0.5g, accurately weigh, place in a stoppered conical flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate (power 150W, frequency 20kHz) for 45 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and collect the filtrate. Accurately pipette 10μl each of the reference solution and the test solution, inject into the liquid chromatograph, and determine the content and total amount (mg / g) of sorbitol and pinocembrin according to the chromatographic conditions determined above. Calculate the total amount (mg / tablet) per tablet based on the average tablet weight. The calculation results are shown in Table II-19.

[0174]

[0175] According to the test results, the total amounts of scutellarin and galangin in the three batches of Yushuda tablets were 2.36 mg / tablet, 2.11 mg / tablet, and 2.25 mg / tablet, respectively.

[0176] The test results showed that each tablet in each batch of Yushuda tablets contained cardamom and galangin (C 16 H 14 O4) and ginsenosides (C 15 H 12 Total O4 content: 1.53-3.09 mg / tablet. Meets standard requirements.

Claims

1. A quality testing method for a Yushuda preparation for treating depression, wherein, The Yushuda preparation is prepared by a method including the following steps: mixing 700-1200 parts by weight of patchouli, 200-400 parts by weight of dried tangerine peel, 300-700 parts by weight of cardamom, 300-700 parts by weight of amomum villosum, and 400-750 parts by weight of stir-fried Atractylodes lancea to obtain a mixture of Chinese medicinal materials; decocting the mixture of medicinal materials with water and concentrating the filtrate to a clear extract with a temperature of 48-52℃ and a relative density of 1.04-1.05; adding ethanol to concentrate the filtrate to an extract with a temperature of 60-70℃ and a relative density of 1.14-1.17; and adding excipients to prepare the desired preparation. The quality of the formulation was determined by high performance liquid chromatography and mass spectrometry.

2. The quality testing method for Yushuda preparation according to claim 1, characterized in that: The quality detection method includes a method for establishing characteristic chromatograms and / or content determination. The method for establishing characteristic chromatograms uses hesperidin, sorbitol, pinocembrin, cardamom, alder ketone, and patchouli ketone as reference standards and employs high-phase liquid chromatography with acetonitrile and phosphoric acid solution as the mobile phase. The content determination uses hesperidin, sorbitol, and pinocembrin as reference standards and employs high-phase liquid chromatography with methanol and phosphoric acid solution as the mobile phase.

3. A method for establishing a characteristic spectrum of a Yushuda preparation, characterized in that, The analysis and detection of Yushuda preparations were performed using high-performance liquid chromatography (HPLC). The method includes the following steps: 1) Prepare reference solutions: Mix hesperidin reference standard, sorbitol reference standard, pinosin reference standard, cardamomin reference standard, alder ketone reference standard and patchouli ketone reference standard to prepare mixed reference solutions; 2) Prepare the test solution of Yushuda preparation; add solvent to Yushuda preparation to prepare the test solution; 3) The chromatograms of the mixed reference solution and the test solution were detected by high performance liquid chromatography (HPLC). The chromatograms of the test solution and the reference solution were compared to determine the common peaks and establish characteristic chromatograms. The common peaks include 6 peaks: hesperidin, sorbitol, pinocembrin, cardamomin, alder ketone and patchouli ketone. The high-performance liquid chromatography (HPLC) conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material, and the mobile phase includes phase A and phase B, wherein phase A is acetonitrile and phase B is a 0.05-0.15% by mass phosphoric acid solution, and gradient elution is performed.

4. The method for establishing the characteristic spectrum of the Yushuda preparation according to claim 1, wherein, The chromatogram of the test solution has at least 13 common characteristic peaks.

5. The method for establishing the feature map according to claim 1 or 2, wherein the gradient elution method of the mobile phase is as follows: Within 0-4 minutes, the volume of phase A increased from 5% to 10%, while the volume of phase B decreased from 95% to 90%. Within 4-10 minutes, the volume of phase A increased from 10% to 15%, while the volume of phase B decreased from 90% to 85%. Within 10–35 minutes, the volume of phase A increased from 15% to 30%, while the volume of phase B decreased from 85% to 70%. Within 35-40 minutes, the volume of phase A increased from 30% to 38%, while the volume of phase B decreased from 70% to 62%. Over 40-45 minutes, the volume of phase A increased from 38% to 48%, while the volume of phase B decreased from 62% to 52%. Within 45-55 minutes, the volume of phase A increased from 48% to 60%, while the volume of phase B decreased from 52% to 40%. Within 55-60 minutes, the volume of phase A increased from 60% to 76%, while the volume of phase B decreased from 40% to 24%. In 60-62 minutes, the volume of phase A decreased from 76% to 5%, while the volume of phase B increased from 24% to 95%.

6. The detection method according to claim 1 or 2, wherein, The chromatographic column has an inner diameter of 2.1 mm, a length of 100-150 mm, and an inner diameter of 1.8 μm; preferably, the chromatographic column is Waters ACQUITY UPLC® HSS T3 C18 or AmethSep C18 Aq.

7. The method for establishing a feature map according to any one of claims 1-3, wherein, The detection wavelength of the chromatogram is 190-400 nm, preferably 315 nm; And / or, the column temperature of the chromatographic column is 25-35℃, preferably 28-32℃, more preferably 30℃; And / or, the flow rate of the mobile phase is 0.20-0.40 ml / min, preferably 0.28-0.32 ml / min.

8. The method for establishing a feature map according to any one of claims 1-4, wherein, In step 3), When detecting the test solution by chromatography, the injection volume of the test solution is 1-3 µl, preferably 2-3 µl, and more preferably 2 µl.

9. The method for establishing a feature map according to any one of claims 1-5, wherein, The chromatogram of the test solution has at least 13 common characteristic peaks, which are numbered 1-13 according to their retention times from smallest to largest. Peak 4 is the standard hesperidin peak, peak 7 is the standard sorbitol peak, and the relative retention times of peaks 1, 2, 3, 5, and 6 relative to peak 4 are within ±10% of the specified value; the relative retention times of peaks 8 and 10 relative to peak 2 are within ±10% of the specified value. Preferably, the substances corresponding to the six characteristic peaks in the common characteristic peaks are: peak 4 is hesperidin; peak 7 is sorbitol; peak 9 is pinoside; peak 11 is cardamomin; peak 12 is patchoulione; and peak 13 is alderne.

10. A characteristic spectrum of the Yushuda preparation established by the method for establishing characteristic spectra according to any one of claims 1-6.

11. A method for detecting galangin and pine resin in a Yushuda preparation, characterized in that, The analysis and detection of Yushuda preparations were performed using high-performance liquid chromatography (HPLC). The method includes the following steps: 1) Prepare reference solution: Mix sorbitan reference standard and ginsenoside reference standard separately to prepare a mixed reference solution; 2) Prepare the test solution of Yushuda preparation: Add solvent to Yushuda preparation to prepare the test solution; 3) High-performance liquid chromatography (HPLC) was used to detect the mixed reference standard solution and the test solution separately; The high-performance liquid chromatography (HPLC) conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material, and the mobile phase includes phase A and phase B, wherein phase A is acetonitrile and phase B is a 0.05-0.15% by mass phosphoric acid solution.

12. The method for detecting galangin and pine resin in the Yushuda preparation according to claim 11, wherein, The mass ratio of phase A and phase B, which are used as the mobile phase, is (55-68):(35-42), preferably 62:

38.

13. The method for detecting galangin and pine resin in the Yushuda preparation according to claim 11 or 12, wherein, The detection wavelength of the chromatogram is 190-400 nm, preferably 290 nm; And / or, the chromatographic column is a column with an inner diameter of 4.6 mm, a column length of 250 mm, and a particle size of 5 μm, preferably selected from Xtimate. ® C18, Waters XBridge ® C18, Thermo Accliam TM 120 A°C18, GL Sciences Inertsil TM ODS-HL, JADE-PAK ® KP-C18, any of the Agilent ZORBAX Eclipse Plus C18; And / or, the column temperature of the chromatographic column is 25-35℃, preferably 28-32℃, more preferably 30℃; And / or, the flow rate of the mobile phase is 0.10-1.5 ml / min, preferably 0.8-1.2 ml / min.

14. The method for detecting sorbitol and pinosin in the Yushuda preparation according to any one of claims 8-10, or the method for establishing characteristic chromatograms according to any one of claims 1-6, wherein, In step 2), the solvent is selected from any one or more of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water, preferably from any one or more of methanol, 75% methanol, 50% methanol, ethanol, 75% ethanol and water; more preferably from any one or more of methanol, 75% methanol, 50% methanol, 75% ethanol and water.

15. The method for detecting sorbitol and pinocembrin in the Yushuda preparation according to any one of claims 11-14, or the method for establishing characteristic chromatograms according to any one of claims 1-6, wherein, When performing chromatographic analysis on the test solution, the injection volume of the test solution is 7-15 µl, preferably 8-11 µl.

16. The method for detecting galangin and pine resin in the Yushuda preparation according to any one of claims 11-15, wherein, Compared to the 0.5g Yushuda preparation, the total content of sorbitol and pine resin is 1.53-3.09mg, preferably 2.10-2.50mg.

17. The quality control of Yushuda preparation is carried out by using the detection method of sorbitol and pine resin in the Yushuda preparation according to any one of claims 11-15.

18. The characteristic spectrum of claim 10 is used for quality control of Yushuda preparation.

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