Quality detection method for all medicines in Pingwei pill

Abstract

The invention relates to the technical field of medicine quality detection, in particular to a quality detection method for all medicines in Pingwei pills, which comprises the following steps: qualitatively identifying rhizoma atractylodis, mangnolia officinalis, pericarpium citri reticulatae and liquorice by adopting a thin layer chromatography (TLC), and simultaneously determining the contents of hesperidin, honokiol, magnolol and atractydin by adopting a high performance liquid chromatography (HPLC). The established method is easy and convenient to operate, good in repeatability and high in accuracy, qualitative identification of the whole medicine in the Pingwei pill and simultaneous determination of the content of four active ingredients in the monarch medicine, the minister medicine and the adjuvant medicine are achieved, and the quality control level of the Pingwei pill is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical quality detection, and particularly relates to a quality detection method for all medicinal ingredients in Pingwei pills. BACKGROUND

[0002] The Pingwei pill is composed of atractylodes (fried), magnolia officinalis (processed), dried tangerine or orange peel and licorice root (roasted), has the effects of drying dampness, invigorating the spleen, and expanding the chest and relieving distension, and is often used in clinic for treating loss of appetite, abdominal fullness, nausea and vomiting caused by excessive dampness in the spleen and stomach. In the prescription, atractylodes is the monarch herb with the effects of drying dampness and invigorating the spleen; magnolia officinalis is the ministerial herb with the effects of bitter-warm descending, promoting qi and relieving fullness, and drying dampness and relieving distension; dried tangerine or orange peel is the auxiliary herb with the effects of regulating qi, harmonizing the center, drying dampness and invigorating the spleen; and licorice root (roasted) is the ministerial herb with the effects of regulating the other herbs, benefiting qi and harmonizing the center. The four herbs are used together to achieve the effects of drying dampness, invigorating the spleen, promoting qi and relieving distension.

[0003] At present, the quality standard for the Pingwei pill is the Tenth Volume of Chinese Patent Medicine Standards of the Ministry of Public Health, but the standard only collects the general inspection items of the pill, does not perform qualitative identification or content determination on any medicinal ingredient in the prescription, the quality standard is relatively simple, and it is difficult to comprehensively reflect the internal quality of the preparation. In recent years, although some studies have attempted to improve the quality control level, such as the study of Xuanfengguo et al. which adopts high performance liquid chromatography (HPLC) to determine the content of hesperidin in the Pingwei pill, and the study of Tanshengjian which establishes an HPLC method for simultaneously determining the contents of magnolol and honokiol, the above studies are only directed to a single component or a single medicinal ingredient, the quality control index is relatively one-sided, and the overall quality of the preparation cannot be comprehensively evaluated. SUMMARY

[0004] One of the purposes of the present application is to avoid the shortcomings in the prior art and provide a quality detection method for all medicinal ingredients in Pingwei pills, which can comprehensively detect the quality of all medicinal ingredients in the Pingwei pill, and has the characteristics of high efficiency and high accuracy.

[0005] To achieve one of the above purposes, the present application provides the following technical solutions: A quality detection method for all medicinal ingredients in Pingwei pills is provided, and atractylodes is identified, including the following steps: Preparation of a first test sample solution: finely grind the Pingwei pill, take 2 g, add 10 mL of ethyl acetate, ultrasonically treat for 5 minutes, filter, evaporate the filtrate, add 1 mL of ethyl acetate to the residue to dissolve, and take the residue as the first test sample solution, Preparation of a first control medicinal ingredient solution: take 0.5 g of atractylodes control medicinal ingredient, add 10 mL of ethyl acetate, ultrasonically treat for 5 minutes, filter, evaporate the filtrate, add 1 mL of ethyl acetate to the residue to dissolve, and take the residue as the first control medicinal ingredient solution, Preparation of a first control medicinal ingredient solution: take atractylodesin, and prepare a solution containing 1 mg per 1 mL of methanol as the first control sample solution; Preparation of the first negative sample solution: the negative sample without atractylodes rhizome was prepared according to the prescription and process of Pingwei pills, and the first negative sample solution was prepared according to the preparation method of the first test sample solution, Identification was carried out according to the thin layer chromatography method (Chinese Pharmacopoeia 2025 edition four general rules 0502): 5 μL of the first test sample solution, the first control medicinal material solution, the first control solution and the first negative sample solution were taken respectively and spotted on the same silica gel G thin layer plate; a mixed solution of petroleum ether-cyclohexane with a volume ratio of 9:1 at 60-90 ℃ was used as the developing agent, and after development, removal and air drying, 5% vanillin sulfuric acid solution was sprayed, and heating was carried out at 105 ℃ until the spots were clearly colored; when the first test sample chromatogram showed the same color spots at the corresponding positions of the first control medicinal material chromatogram and the first control solution chromatogram, and the first negative sample had no interference, it indicated that Pingwei pills contained atractylodes rhizome; Identification of Magnolia officinalis, including the following steps: Preparation of the second test sample solution: an appropriate amount of Pingwei pill sample was finely ground, 3 g was weighed, 50 mL of ethyl acetate was added, and ultrasonic extraction was carried out for 30 minutes, then it was cooled, filtered, and the filtrate was extracted with 2% sodium hydroxide solution for 3 times, 20 mL each time, the alkali solution was combined, and the pH was adjusted to 1-2 with hydrochloric acid, then it was extracted with petroleum ether at 30-60 ℃ for 3 times, 20 mL each time, the petroleum ether liquid was combined, and evaporated to dryness, then 1 mL of methanol was added to dissolve the residue, which was used as the second test sample solution; Preparation of the second control solution: another magnolol and honokiol control sample was taken, and methanol was added to prepare a mixed solution containing 1 mg of magnolol and 0.5 mg of honokiol per 1 mL, which was used as the second control solution; Preparation of the second negative sample solution: the negative sample without Magnolia officinalis was prepared according to the prescription and process of Pingwei pills, and the second negative sample solution was prepared according to the preparation method of the test sample solution; Identification was carried out according to the thin layer chromatography method (Chinese Pharmacopoeia 2025 edition four general rules 0502): 10 μL of the second test sample solution, the second control solution and the second negative sample solution were taken and spotted on the same silica gel G thin layer plate; a mixed solution of toluene-methanol with a volume ratio of 12:1 was used as the developing agent, and after development, removal and air drying, 5% vanillin sulfuric acid solution was sprayed, and heating was carried out at 105 ℃ until the spots were clearly colored; when the second test sample chromatogram showed the same color spots at the corresponding positions of the second control solution chromatogram, and the negative sample had no interference, it indicated that Pingwei pills contained Magnolia officinalis; Identification of Pericarpium Citri Reticulatae, including the following steps: Preparation of the third test sample solution: an appropriate amount of Pingwei pill sample was finely ground, 3 g was weighed, 50 mL of 0.2% calcium hydroxide solution was added, and ultrasonic extraction was carried out for 30 minutes, then it was filtered, and the filtrate was extracted with 1 mol·L -1The pH was adjusted to 3 with hydrochloric acid, and extracted with 20 mL of ethyl acetate three times, and the ethyl acetate was combined and evaporated to dryness. The residue was dissolved in 1 mL of methanol to serve as the third test solution, Preparation of the third control solution: another hynodulcin control was prepared into a saturated solution with methanol as the third control solution, Preparation of the third negative sample solution: a negative sample without Chenpi was prepared according to the prescription and process of Pingwei Pill, and the third negative sample solution was prepared according to the test solution preparation method, Identification according to the thin layer chromatography of the Pharmacopoeia of the People's Republic of China 2025 edition four general rules 0502: 10 μL of the third test solution, 10 μL of the third negative sample solution, and 5 μL of the third control solution were spotted on the same silica gel G thin layer plate. The lower layer solution of the mixture of chloroform, ethyl acetate, methanol and water (8:20:11:5 by volume) was placed at 10 ℃ for 12 hours or more, and then developed, taken out, dried, sprayed with aluminum chloride test solution, and heated at 105 ℃ for 2 minutes. Under the wavelength of 365 nm, the ultraviolet lamp was observed. When the third test chromatogram showed the same color spots at the corresponding positions of the third control chromatogram, and the negative sample solution had no interference, it indicated that Pingwei Pill contained Chenpi; Identification of licorice includes the following steps: Preparation of the fourth test solution: a certain amount of Pingwei Pill sample was finely ground, 6 g was weighed, and 50 ml of ethanol was added. Ultrasonic was performed for 30 minutes, and then filtered. The filtrate was evaporated to dryness. The residue was dissolved in 20 mL of water, and then extracted with 20 mL of n-butanol saturated with water three times. The n-butanol extract was combined, washed with 20 mL of water saturated with n-butanol twice, and then collected. The n-butanol liquid was evaporated to dryness, and then dissolved in 10% ethanol 20 mL. The residue was added to a D101 macroporous adsorption resin column with an inner diameter of 1.5 cm and a column height of 13 cm. The column was eluted with 50 mL of 10%, 30%, and 50% ethanol, respectively. The 50% ethanol eluate was collected, evaporated to dryness, and then dissolved in 1 mL of methanol to serve as the fourth test solution. Preparation of the fourth control medicinal material solution: another 1 g of licorice control medicinal material was prepared into a control medicinal material solution.

[0006] Preparation of the fourth negative sample solution: a negative sample without licorice was prepared according to the prescription and process of Pingwei Pill, and the fourth negative sample solution was prepared according to the test solution preparation method; According to the thin layer chromatography of the Pharmacopoeia of the People's Republic of China 2025 edition four general rules 0502, take 10 μL of the fourth test solution, the fourth control drug solution and the fourth negative sample solution respectively, and point them on the same silica gel G thin layer plate. Use toluene-ethyl acetate-water (15:4:0.5 by volume) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 ℃ until the spots develop clear color. When the fourth test chromatogram shows the same color spots at the corresponding positions of the fourth control drug chromatogram, and the negative sample solution has no interference, it indicates that the Pingwei Pill contains licorice; In some embodiments, the parameter of the ultrasound is 500 W, 45 kHz.

[0007] In some embodiments, the step of simultaneously measuring the contents of hesperidin, honokiol, magnolol and atractylodin in the Pingwei Pill comprises: Step one, use Shimadzu VP-ODS-C 18 chromatographic column; acetonitrile (A)-methanol (B)-water (C) as the mobile phase, gradient elution; flow rate is 1.0 mL·min –1 ; column temperature is 30 ℃; detection wavelength is 290 nm, 336 nm; injection volume: 10 μL; gradient elution program is shown in the table, ; Step two, preparation of mixed control solution: accurately weigh 7.52, 8.26, 7.59 mg of hesperidin, honokiol and magnolol control substances respectively into 20 mL volumetric flasks, and 6.06 mg of atractylodin control substance into a 50 mL volumetric flask. Dissolve and dilute with methanol to serve as reference stock solutions A, B, C and D. Accurately measure 7.0, 3.0, 7.5 and 7.0 mL of A, B, C and D control stock solutions respectively into the same 25 mL volumetric flask, and add methanol to the mark to obtain the mixed control stock solution. Step three, fifth test solution: take an appropriate amount of Pingwei Pill sample, grind it finely, take about 0.8 g, accurately weigh it, put it in a conical flask, accurately add 50 mL of methanol, weigh it, ultrasonic (500 W, 45 kHz) for 60 minutes, cool it down, weigh it again, add methanol to make up for the weight loss, shake it well, filter it, and filter the filtrate with a 0.45 μm filter membrane to obtain it. Step four, fifth negative sample solution: prepare negative samples without pericarpium citri reticulatae, without magnolia officinalis and without atractylodes according to the prescription proportion and process of Pingwei Pill, and prepare negative sample solutions without pericarpium citri reticulatae, without magnolia officinalis and without atractylodes according to the method of step three. Step five, specificity experiment: take mixed control solution, the fifth sample solution, the fifth negative sample solution respectively, according to the chromatographic condition of step one, sample determination, record chromatogram, when the chromatogram of the fifth sample solution is consistent with the chromatogram of the mixed control solution, and the separation degree between adjacent components is greater than 1.5, and the chromatogram of the negative sample solution is the same as the chromatogram of the four components, and no chromatographic peak appears, and the negative sample has no interference, then the chromatogram results of hesperidin, honokiol, magnolol and atractylodin can be read; Step six, linear relationship and quantitative limit investigation: precisely take 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 mL of mixed control stock solution into a 5 mL capacity bottle, add methanol to the mark, and obtain a series of mixed control solutions. Each takes 10 μL according to the chromatographic conditions of step one, sample determination, with the sample concentration as the abscissa, and the corresponding peak area as the ordinate for linear regression, to obtain the standard curve of hesperidin, honokiol, magnolol and atractylodin, r is greater than 0.999, the linear relationship is good, and the results are shown in the following table, take the mixed control solution, dilute it with methanol step by step, sample determination, and the sample concentration when the signal-to-noise ratio is 10:1 is taken as the quantitative limit, ; In some embodiments, The preparation method of the stomach flatting pill comprises atractylodes, magnolia officinalis, dried tangerine or orange peel, licorice, The weight ratio of atractylodes, magnolia officinalis, dried tangerine or orange peel and licorice is 160:100:20:20. The above four ingredients are crushed into fine powder, sieved, mixed, 4g of jujube and 3g of ginger are taken, the juice is extracted, the pill is coated with the juice, and dried to obtain the stomach flatting pill.

[0008] The stomach flatting pill has the following advantages: The application establishes a quality control method covering four medicinal materials in the prescription: TLC is used for qualitative identification of atractylodes, magnolia officinalis, dried tangerine or orange peel and licorice, and HPLC is used for simultaneous determination of the contents of hesperidin, honokiol, magnolol and atractylodin. The method is simple, reproducible, accurate, realizes the qualitative identification of all medicinal materials in the stomach flatting pill and the simultaneous determination of the contents of four active ingredients in three medicinal materials of monarch, minister and assistant, and improves the quality control level of the stomach flatting pill. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 The TLC diagram of atractylodes in the stomach flatting pill in Example 1 is shown in the figure, wherein 1-3. Stomach flatting pill sample 4. Atractylodes control drug 5. Negative sample 6. Atractylodin control.

[0010] Figure 2TLC of Atractylodes Rhizome in Pingwei Pills in Example 1 for Tolerance Test.

[0011] Figure 3 TLC of Magnoliae Officinalis Cortex in Pingwei Pills in Example 1, where 1-3. Pingwei pill sample 4. Negative sample 5. Mixed reference substance.

[0012] Figure 4 TLC of Magnoliae Officinalis Cortex in Pingwei Pills in Example 1 for Tolerance Test.

[0013] Figure 5 TLC of Citri Reticulatae Pericarpium in Pingwei Pills in Example 1, where 1-3. Pingwei pill sample 4. Negative sample 5. Reference substance of hesperidin.

[0014] Figure 6 TLC of Citri Reticulatae Pericarpium in Pingwei Pills in Example 1 for Tolerance Test.

[0015] Figure 7 TLC of Glycyrrhizae Radix et Rhizoma in Pingwei Pills in Example 1, where 1-3. Pingwei pill sample 4. Negative sample 5. Reference substance of Glycyrrhizae Radix et Rhizoma.

[0016] Figure 8 TLC of Glycyrrhizae Radix et Rhizoma in Pingwei Pills in Example 1 for Tolerance Test.

[0017] Figure 9 HPLC chromatogram of Pingwei Pills in Example 1, where 1. Hesperidin 2. and 3. Magnolol 4. Atractylodin.

[0018] A. Mixed reference substance B. Sample C. Negative sample without Citri Reticulatae Pericarpium D. Negative sample without Magnoliae Officinalis Cortex E. Negative sample without Atractylodes Rhizome.

[0019] Figure 10 HPLC chromatogram of Pingwei Pills in Example 1 on different instruments.

[0020] Figure 11 HPLC chromatogram of Pingwei Pills in Example 1 on different chromatographic columns.

[0021] Figure 12 HPLC chromatogram of Pingwei Pills in Example 1 at different temperatures.

[0022] Figure 13 HPLC chromatogram of Pingwei Pills in Example 1 at different flow rates. DETAILED DESCRIPTION

[0023] Preferred embodiments of the present application will be described in more detail below. While preferred embodiments of the application are shown, it should be understood that the application can be practiced in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will convey the full scope of the application to others skilled in the art. Embodiments of the present application are described herein with reference to the following drawings.

[0024] Example 1 1. Instruments and materials 1.1 Instruments Waters Arc high performance liquid chromatograph (equipped with 2998 PDA diode array detector, USA, Waters Corporation), Agilent 1260 high performance liquid chromatograph (equipped with G1315D diode array detector, USA, Agilent Corporation), Shimadzu LC-20AD high performance liquid chromatograph (equipped with SPD-M40 diode array detector, Japan, Shimadzu Corporation), LTH-250 constant temperature and humidity chamber (Shanghai Long Yue Instrument and Equipment Co., Ltd.), XSE-205 millionth electronic balance (Mettler-Toledo, Switzerland), KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), TLC Visualizer3 thin layer chromatography imaging system (CAMAG), ZOOMWOL ultrapure water instrument (Hunan Zhongwo Water Environmental Protection Technology Co., Ltd.), DZKW-D-2 electric heating constant temperature water bath (Beijing Yongguangming Instrument Co., Ltd.).

[0025] 1.2 Materials The reference standards hesperidin (batch number: 110721-202220, purity: 97.2%), magnolol (batch number: 110730-201915, purity: 99.8%), honokiol (batch number: 110729-202316, purity: 99.2%), and atractylodesin (batch number: 111924-201806, purity: 99.5%), and the reference medicinal materials Atractylodes lancea (batch number: 120983-202106) and licorice (batch number: 120904-202021) were purchased from the National Institutes for Food and Drug Control. Silica gel G plates were purchased from Qingdao Ocean Chemical Co., Ltd. (batch number: 20240812), Yantai Chemical Industry Research Institute (batch number: 20230919), and Merck GmbH (batch number: HX03164126), respectively. Chromatographic grade methanol and acetonitrile were from Tedia, USA. All other reagents were of analytical grade, and the water was ultrapure water. Ten batches of samples were purchased from Beijing Tongrentang Natural Medicines (Tangshan) Co., Ltd.: 24080041 (S1), 24080053 (S2), 23080156 (S3), 23080133 (S4), 23050146 (S5), 23080111 (S6), 23080094 (S7), 23080058 (S8), 23080020 (S9), and 23080022 (S10). Each batch contained 19 tablets, weighing 1 g, with a shelf life of 3 years.

[0026] 2. Methods and Results 2.1 TLC Identification 2.1.1 Atractylodes lancea Preparation of the test solution: Take an appropriate amount of Pingwei Pill sample, grind it finely, weigh 2 g, add 10 mL of ethyl acetate, sonicate (500 W, 45 kHz) for 5 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethyl acetate to obtain the test solution. Preparation of the reference medicinal material and reference solution: Take 0.5 g of Atractylodes lancea reference medicinal material and prepare the reference medicinal material solution using the same method. Separately, take Atractylodes lancea reference standard and add methanol to prepare a solution containing 1 mg per 1 mL to obtain the reference solution.

[0027] The negative sample solution was prepared according to the prescription and process of Pingwei Pill, and the negative sample solution was prepared according to the test sample solution preparation method.

[0028] Thin layer chromatography by thin layer chromatography (Chinese Pharmacopoeia 2025 edition four general rules 0502), 5 μL of the above four solutions were taken and spotted on the same silica gel G thin layer plate, with petroleum ether (60-90 ℃)-cyclohexane (9:1) as the developing agent, and developed. The test sample spots were clearly separated when developed with petroleum ether (60-90 ℃)-cyclohexane, and the separation degree was good. The ratio of the two was optimized, and the main spot developed under the condition of 9:1 was moderate, which was convenient for observation and aesthetics. Take out, air dry, and spray with 5% vanillin sulfuric acid solution. Atractylin is an olefin compound, and 5% vanillin sulfuric acid can cause the double bond of atractylin molecules to shift and double molecules to condense, forming a conjugated double bond system. Finally, under the action of sulfuric acid, a carbonium ion is formed to develop color. They only develop color at 105 ℃, so heat to 105 ℃ until the spots develop clear color. As a result, in the test sample chromatogram, spots of the same color appeared at the corresponding positions of the control drug and control substance chromatogram, the negative sample solution had no interference, and Figure 1 .

[0029] Tolerance investigation The effects of different thin layer plate manufacturers (Qingdao Haoyang Chemical Co., Ltd., Yantai Chemical Industry Research Institute, and Germany Merck Company), different temperatures (10-35 ℃), and different humidities (30%-75%) on the identification of atractylodes in Pingwei pills were investigated. The results showed that the test sample TLC chromatograms under each condition had good separation effect, and the negative sample had no interference. The method had good tolerance, and is shown in Figure 2 .

[0030] 2.1.2 Magnolia officinalis Preparation of test sample solution Take a suitable amount of Pingwei pill sample, grind it finely, weigh 3 g, add 50 mL of ethyl acetate, ultrasonic (500 W, 45 kHz) for 30 minutes, cool, filter, and extract the filtrate with 2% sodium hydroxide solution for 3 times. Honokiol and magnolol are easily soluble in alkaline solution, and their solubility is greater than that in ethyl acetate. Therefore, they are extracted with 2% sodium hydroxide solution. At the same time, it also plays a purifying role. Each time 20 mL, combine the alkali, adjust the pH value to 1-2 with hydrochloric acid, and the solubility of honokiol and magnolol is the smallest at pH 1-2. At this time, the solubility in petroleum ether (30-60 ℃) is much larger, so petroleum ether (30-60 ℃) is used to extract honokiol and magnolol. Changing different extraction reagents is equivalent to purifying honokiol and magnolol, reducing the interference of impurities. Use petroleum ether (30-60 ℃) to shake and extract 3 times, each time 20 mL, combine the petroleum ether liquid, and evaporate to dryness. Add 1 mL of methanol to dissolve the residue as the test sample solution.

[0031] Preparation of control solution Take another honokiol and magnolol control solution, add methanol to prepare a mixed control solution containing 1, 0.5 mg per 1 mL, respectively, as the control solution.

[0032] Preparation of negative sample solution The negative sample without Magnolia officinalis was prepared according to the prescription and process of Pingweiwan, and the negative sample solution was prepared according to the preparation method of test sample solution.

[0033] Thin layer chromatography According to the thin layer chromatography method (Chinese Pharmacopoeia 2025 edition four general rules 0502), 10 μL of each of the above three solutions was spotted on the same silica gel G thin layer plate, toluene-methanol (12:1) was used as the developing agent, and the plate was developed, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, and heated at 105 ℃ until the spots were clearly colored. Results: In the test sample chromatogram, the same colored spots appeared at the positions corresponding to the control sample chromatogram, and the negative sample solution had no interference, see Figure 3 .

[0034] Tolerance investigation The effects of different thin layer plate manufacturers (Qingdao Haoyang Chemical Co., Ltd., Yantai Chemical Industry Research Institute, and Germany Merck Company), different temperatures (10-35 ℃), and different humidities (30%-75%) on the identification of Magnolia officinalis in Pingweiwan by thin layer chromatography were investigated. The results showed that the test sample TLC chromatogram under each condition had good separation effect, and the negative sample had no interference, and the method had good tolerance, see Figure 4 .

[0035] 2.1.3 Pericarpium Citri Reticulatae Preparation of test sample solution Take Pingweiwan sample, grind it finely, and weigh 3 g, add 0.2% calcium hydroxide solution 50 mL, orange peel glycoside is better dissolved in 0.2% calcium hydroxide, use it to completely extract orange peel glycoside, adjust pH to 3, make orange peel glycoside crystallize, equivalent to purification, reduce the interference of impurities. Shake for 30 minutes, filter, and use 1 mol·L -1 -1 to adjust the pH of the filtrate to 3, and extract it with ethyl acetate for 3 times, 20 mL each time, and combine the ethyl acetate liquid. Under the condition of pH 3, orange peel glycoside is crystallized and basically insoluble, which is extracted with ethyl acetate for further purification to reduce the interference of impurities; then evaporate to dryness, dissolve the residue in 1 mL of methanol as the test sample solution.

[0036] Preparation of control sample solution Take another orange peel glycoside control sample and add methanol to prepare a saturated solution as the control sample solution.

[0037] Preparation of negative sample solution The negative sample without Pericarpium Citri Reticulatae was prepared according to the prescription and process of Pingweiwan, and the negative sample solution was prepared according to the preparation method of test sample solution.

[0038] Thin layer chromatography (ChP 2025, Vol. 4, General Rules 0502) 10 μL of the test solution, 10 μL of the negative sample solution and 5 μL of the reference solution were spotted on the same silica gel G thin layer plate. The lower solution of chloroform-ethyl acetate-methanol-water (8:20:11:5) was used as the developing agent and was placed at 10 ℃ for 12 hours. The sample was developed, removed, dried, sprayed with aluminum chloride solution, heated at 105 ℃ for 2 minutes, and observed under ultraviolet light (365 nm). The test chromatogram showed the same color spots as the reference chromatogram at the corresponding positions, and the negative sample solution had no interference. Figure 5 .

[0039] Tolerance test The effects of different thin layer plate manufacturers (Qingdao Haoyang Chemical Co., Ltd., Yantai Chemical Industry Research Institute, and Merck, Germany), different temperatures (10-35 ℃), and different humidities (30%-75%) on the identification of pericarpium citri reticulatae in Pingwei pills were investigated. The results showed that the test TLC chromatogram under each condition had good separation effect, and the negative sample had no interference. The method had good tolerance, as shown in Figure 6 .

[0040] 2.1.4 Glycyrrhiza Preparation of the test solution A certain amount of Pingwei pill sample was finely ground, 6 g was weighed, and 50 mL of ethanol was added. Glycyrrhiza flavonoids were easily soluble in ethanol, and the flavonoids were extracted by adding ethanol. Ultrasonic extraction (500 W, 45 kHz) was performed for 30 minutes, and the filtrate was evaporated to dryness. The residue was dissolved in 20 mL of water, and the water-saturated n-butanol was extracted three times. Glycyrrhiza flavonoids had low solubility in water but high solubility in n-butanol, and water and n-butanol were mutually insoluble, which extracted the flavonoids and removed impurities. Each time 20 mL, the n-butanol extract was washed with water saturated with n-butanol twice, each time 20 mL, the n-butanol solution was collected, evaporated to dryness, and the residue was dissolved in 10% ethanol 20 mL. The solution was added to a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 13 cm), and eluted with 10%, 30%, and 50% ethanol (50 mL each). The 50% ethanol eluate was collected, evaporated to dryness, and the residue was dissolved in 1 mL of methanol as the test solution.

[0041] Preparation of the reference medicinal material solution Another 1 g of glycyrrhiza reference medicinal material was prepared into a reference medicinal material solution by the same method.

[0042] Preparation of the negative sample solution A negative sample without glycyrrhiza was prepared according to the prescription and process of Pingwei pills, and a negative sample solution was prepared according to the preparation method of the test solution.

[0043] Thin layer chromatography (ChP 2025 edition four general rules 0502), 10 μL of each of the above three solutions was spotted on the same silica gel G thin layer plate, developed with toluene-ethyl acetate-water (15:4:0.5) as the developing agent, removed, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105 ℃ until the spots were clearly colored. The results showed that the test sample chromatogram had the same color spots as the control drug chromatogram, the negative sample solution had no interference, and the Figure 7 .

[0044] Tolerance investigation The effects of different thin layer plate manufacturers (Qingdao Haoyang Chemical Co., Ltd., Yantai Chemical Industry Research Institute, Germany Merck Company), different temperatures (10-35 ℃), and different humidities (30%-75%) on the identification of licorice in Pingwei pills were investigated. The results showed that the test sample TLC chromatogram under each condition had good separation effect, the negative sample had no interference, and the method had good tolerance, as shown in Figure 8 .

[0045] 2.2 Content determination of hesperidin, honokiol, magnolol, and atractylenolide 2.2.1 Chromatographic conditions Shimadzu VP-ODS-C 18 chromatographic column (250 mm x 4.6 mm, 5 μm) was used; acetonitrile (A)-methanol (B)-water (C) was used as the mobile phase for gradient elution; the flow rate was 1.0 mL·min –1 ; the column temperature was 30 ℃; the detection wavelength was 290 nm and 336 nm; the injection volume was 10 μL; the gradient elution program is shown in Table 1.

[0046] Table 1 Gradient elution program ; 2.2.2 Preparation of mixed reference solution Precisely weigh 7.52, 8.26, and 7.59 mg of hesperidin, honokiol, and magnolol reference substances, respectively, into 20 mL volumetric flasks, and 6.06 mg of atractylenolide reference substance into a 50 mL volumetric flask, dissolve in methanol and dilute to the mark to serve as reference stock solutions A, B, C, and D. Precisely take 7.0, 3.0, 7.5, and 7.0 mL of reference stock solutions A, B, C, and D, respectively, into the same 25 mL volumetric flask, add methanol to the mark to obtain the mixed reference solution.

[0047] 2.2.3 Test sample solution Take a suitable amount of Pingwei pill sample, grind it finely, accurately weigh about 0.8 g, accurately weigh, add 50 mL of methanol into a conical flask, weigh, ultrasonic (500 W, 45 kHz) for 60 minutes, cool, weigh again, add methanol to make up for the weight loss, shake well, filter, and filter the filtrate with a 0.45 μm filter membrane to obtain the test sample solution.

[0048] 2.2.4 Negative sample solution According to the prescription proportion and process of flat pills, negative samples without pericarpium citri reticulatae, without magnolia officinalis and without atractylodes rhizome were prepared respectively, and negative sample solutions without pericarpium citri reticulatae, without magnolia officinalis and without atractylodes rhizome were prepared according to the method under item 2.2.3.

[0049] 2.2.5 Specificity experiment Take appropriate amount of solution under 2.2.2-2.2.4 respectively, and determine by sample injection under the chromatographic conditions of item 2.2.1. Record the chromatogram, see Figure 9 . The results showed that the retention time of hesperidin, magnolol, honokiol and atractylodin in the test sample chromatogram was consistent with that of the mixed reference substance, and the separation degree between adjacent components was greater than 1.5. The retention time of the four components in the negative sample solution chromatogram was the same, and no chromatographic peak appeared, and the negative sample had no interference.

[0050] 2.2.6 Linear relationship and limit of quantification investigation Precisely take 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 mL of mixed reference substance stock solution into a 5 mL volumetric flask, add methanol to the mark, and obtain a series of mixed reference substance solutions. Take 10 μL of each according to the chromatographic conditions under item 2.2.1. The mass concentration (μg·mL -1 ) of the sample injection was taken as the abscissa (X), and the corresponding peak area was taken as the ordinate (Y) to perform linear regression to obtain the standard curve of hesperidin, magnolol, honokiol and atractylodin, and r was greater than 0.999, indicating good linear relationship. The results are shown in Table 2. Take an appropriate amount of mixed reference substance solution and dilute it with methanol step by step. The injection concentration at a signal-to-noise ratio of 10:1 was taken as the limit of quantification, and the results are shown in Table 2.

[0051] Table 2 Linear relationship investigation and limit of quantification of 4 active ingredients ; 2.2.7 Precision test Take an appropriate amount of the third point of the series of mixed reference substance solutions, and inject 10 μL continuously according to the chromatographic conditions under item 2.2.1 for 6 times. The RSD of the peak area of hesperidin, magnolol, honokiol and atractylodin was 1.3%, 1.4%, 1.2% and 0.54% respectively, indicating good instrument precision.

[0052] 2.2.8 Stability test Take the same test sample solution (No. S4) and determine by sample injection under the chromatographic conditions of item 2.2.1 at 0, 3, 9, 15, 24 and 30 h after preparation, and record the chromatogram. The RSD of the peak area of hesperidin, magnolol, honokiol and atractylodin was 1.1%, 1.3%, 0.67% and 1.3% respectively, indicating good stability of the test sample solution within 30 h.

[0053] 2.2.9 Reproducibility test Take the same batch (No. S4) of samples, prepare 6 test sample solutions according to the method under item “2.2.3”, and then inject them under the chromatographic conditions in item “2.2.1” to determine the content. The average content of hesperidin, honokiol, magnolol, and atractylin is 4.415, 1.866, 5.001, and 1.358 mg·g -1 , respectively, and the RSD is 2.0%, 2.0%, 1.0%, and 2.6%, respectively, indicating that the reproducibility of the method is good.

[0054] 2.2.10 Spiking recovery test Take the same batch of samples (No. S4) with known content, grind them finely, take about 0.4 g, accurately weigh 6 portions, and add 0.3507 mg·mL -1 of hesperidin, 0.3649 mg·mL -1 of honokiol, 0.3992 mg·mL -1 of magnolol, and 0.1307 mg·mL -1 of atractylin, respectively, according to 100% of the content of hesperidin, honokiol, magnolol, and atractylin, prepare the test sample solution according to the method under item “2.2.3”, and then determine it according to the method under item “2.2.1” to calculate the recovery rate. The results are shown in Table 3.

[0055] Table 3 Results of spiking recovery test of 4 components in Pingwan pills (n = 6) ; 2.2.11 Intermediate precision test Take the same batch of samples (No. S10), and 2 experimenters prepare test sample solutions according to the method under item “2.2.3” and determine them using 3 instruments of different brands according to the chromatographic conditions under item “2.2.1”. The results show that the resolution of hesperidin, honokiol, magnolol, and atractylin is greater than 1.5 (see Table 4) on different instruments, and the RSD of their content is 1.6%, 2.9%, 2.8%, and 2.9%, respectively (see Table 4), indicating that the intermediate precision of the method is good. Figure 10

[0056] Table 4 Results of intermediate precision test of content of hesperidin, honokiol, magnolol, and atractylin ; 2.2.12 Durability test Take the same batch of samples (No. S10), prepare test sample solutions according to the method under item “2.2.3”, and investigate the use of different chromatographic columns [Pntulips® NT-C 18 , phenomenex Luna-C 18 , and Shimadzu VP-ODS-C 18 ​[Shim-pack XR-ODSⅡ (250 mm x 4.6 mm, 5 μm)], different temperatures (25, 30, 35 ℃), different flow rates (0.9, 1.0, 1.1 mL·min -1 ) on the determination of hesperidin, honokiol, magnolol, and atractylenolide. The results showed that the separation degree of each component was greater than 1.5 (see Figures 11-13 ), the RSD of the content was less than 3.0% (see Table 5), and the method had good durability.

[0057] Table 5 Durability test results of hesperidin, honokiol, magnolol, and atractylenolide ; 2.2.13 Sample content determination Ten batches of Pingwei pills were prepared according to the method in "2.2.3", and two test sample solutions were prepared. The content of hesperidin, honokiol, magnolol, and atractylenolide in the test sample was determined by the method in "2.2.1", and the content was calculated by the external standard method. The results are shown in Table 6.

[0058] Table 6 Determination results of the contents of four components in Pingwei pill samples (mg·g -1 , n = 2) ; 3 Optimization of TLC identification 3.1 Atractylodes rhizome The effects of ethyl acetate and diethyl ether as extraction solvents on TLC of atractylodes were investigated, and it was found that the extraction effects of the two were consistent, but diethyl ether was a drug precursor, so ethyl acetate was selected as the extraction solvent in this test. The effects of petroleum ether (60-90 ℃), petroleum ether (60-90 ℃)-n-hexane (7:3), petroleum ether (60-90 ℃)-cyclohexane (9:1), and n-hexane-ethyl acetate (10:0.8) as developing agents on TLC of atractylodes were investigated. The spots of atractylodes test sample could be separated in the above developing systems, but petroleum ether (60-90 ℃)-cyclohexane (9:1) was the best.

[0059] 3.2 Magnolia bark Honokiol and magnolol are easily soluble in alkaline solution, ethyl acetate, and methanol. The spots of test samples extracted by ethyl acetate and methanol were more and the interference was greater. Subsequently, the residues extracted by ethyl acetate and methanol were dissolved in water and extracted by n-butanol, and the ethyl acetate extract was directly extracted by 2% sodium hydroxide. It was found that the spots of honokiol and magnolol were more obvious and the impurities were the least when 2% sodium hydroxide was used as the extractant. The effects of toluene-methanol (17:1), toluene-ethyl acetate-methanol (17:3:3), and petroleum ether (60-90 ℃)-ethyl acetate-formic acid (85:25:2) on TLC of magnolia bark were investigated. Toluene-methanol (17:1) had the best separation effect, but the spot distance was not high, so the ratio of the two was further optimized, and 12:1 was the most appropriate.

[0060] 3.3 Pericarpium Citri Reticulatae Hesperidin in Pericarpium Citri Reticulatae is easily soluble in alkaline solution and slightly soluble in methanol. The pre-test investigated methanol and 1% sodium hydroxide saturated ethyl acetate as extraction solvent. The results showed that the hesperidin spots in the test sample were not obvious, and the negative sample was interfered. The influence of lower layer solution placed below 10 ℃ with ethyl acetate-methanol-water (100:17:13), chloroform-methanol-water (28:10:1), ethyl acetate-methanol-chloroform-water (20:11:8:5) as developing agent on the development of hesperidin thin layer was investigated. The lower layer solution placed below 10 ℃ with ethyl acetate-methanol-chloroform-water (20:11:8:5) was the best for the separation of hesperidin. It was suggested that the developing system should be placed below 10 ℃ for more than 12 hours to avoid the occurrence of solubility phenomenon.

[0061] 3.4 Glycyrrhiza Glycyrrhiza mainly contains flavonoids. The amount of Glycyrrhiza in Pingwei Pills is small, and the sample size is large when preparing the test sample. The impurities are more when extracted with methanol or ethanol, which affects spotting and development. Therefore, the residue after ethanol extraction and evaporation to dryness needs to be purified. After dissolving with water, the sample is first extracted with n-butanol, and then purified with a purification column. The macroporous adsorption resin D101 and neutral alumina column were compared, and it was found that the macroporous adsorption resin D101 had better purification effect. The concentration and order of the elution solvent were also optimized. When developing, ethyl acetate-formic acid-glacial acetic acid-water, butyl acetate-formic acid-water, and toluene-ethyl acetate-water were compared as developing systems, and it was found that toluene-ethyl acetate-water as the developing system had the best developing effect. The ratio of the three was optimized to 15:4:0.5.

[0062] 4 Screening of HPLC determination conditions 4.1 Selection of detected components In order to comprehensively control the medicinal ingredients in Pingwei Pills, the pre-test investigated the index components of atractylin in Atractylodes, and the index components of magnolol and honokiol in Magnolia officinalis, hesperidin in Pericarpium Citri Reticulatae, and glycyrrhizic acid in Glycyrrhiza. It was found that glycyrrhizic acid had strong water solubility, and the extraction rate was not high when methanol was used as the extraction solvent. Hesperidin, magnolol, honokiol, and atractylin had good solubility in methanol. Therefore, hesperidin, magnolol, honokiol, and atractylin were selected as the index components for content determination.

[0063] 4.2 Optimization of chromatographic conditions The four components were scanned by a diode array detector, and the maximum absorption wavelengths of hesperidin, honokiol, magnolol and artactin were found to be 284, 292, 290 and 336 nm, respectively. Considering comprehensively, 290 nm was used to detect hesperidin, honokiol and magnolol, and 336 nm was used to detect artactin. When investigating the mobile phase, it was found that the separation of honokiol and magnolol was not good when using methanol or acetonitrile or their mixture as the organic phase. However, good separation was achieved by using the combination of the two. When adding formic acid, acetic acid or phosphoric acid to the water phase, the peak shapes of the four components were consistent. Therefore, water was selected as the inorganic phase.

[0064] 4.3 Screening of test sample preparation conditions The extraction solvent of hesperidin, honokiol, magnolol and artactin in the first volume of the Pharmacopoeia of the People's Republic of China 2025 is methanol. Therefore, methanol was directly selected as the extraction solvent in this experiment. Then, the extraction effects of the four components under different extraction methods (ultrasonic, heating reflux), different sampling amounts (0.6, 0.8, 1.0, 1.2 g), different extraction times (30, 40, 50, 60, 90 min) and different solvent volumes (25, 50, 100 mL) were investigated. Finally, the test sample preparation method used in this paper was determined according to the difficulty of test sample preparation and the completeness of the extraction of the four components.

[0065] 4.4 Suggestions on the content limit The content determination results of 10 batches of Pingwei pills showed that the content of hesperidin was in the range of 2.619-4.775 mg·g -1 , the average value was 3.869 mg·g -1 , the total amount of magnolol and honokiol was in the range of 5.964-8.692 mg·g -1 , the average value was 6.791 mg·g -1 , the content of artactin was in the range of 1.160-1.795 mg·g -1 , the average value was 1.413 mg·g -1 . Considering comprehensively, the limit was set as follows: the content of hesperidin in Pingwei pills was temporarily set to not less than 2.6 mg·g -1 , the total amount of magnolol and honokiol in Magnolia officinalis was temporarily set to not less than 4.7 mg·g -1 , and the content of artactin in Atractylodes lancea was temporarily set to not less than 0.9 mg·g -1 .

[0066] In summary, the study successfully established the thin-layer identification method of atractylodes, magnolia officinalis, pericarpium citri reticulatae, and glycyrrhiza in Pingwei Pills, as well as the content determination method of hesperidin, honokiol, magnolol, and atractylodin, covering all medicinal materials in the prescription, effectively improving the quality control standards of the preparation, and enabling comprehensive control of the quality of the preparation.

[0067] Example 2 Prescription of Pingwei Pills: Atractylodes (fried) 160 g, Magnolia officinalis (processed) 100 g, Pericarpium Citri Reticulatae 20 g, Glycyrrhiza 20 g Method of preparation: The above four ingredients are ground into fine powder, sieved, and mixed. Take 4 g of jujube and 3 g of ginger, decoct the juice, and coat the pills with the juice. Dry them to obtain the Pingwei Pills.

[0068]

Characteristics

[0069] Functions and indications of Pingwei Pills: Dry dampness, invigorate the spleen, and relieve chest distension. It is used for dampness accumulation in the spleen and stomach, loss of appetite, abdominal fullness, nausea, vomiting, and acid regurgitation.

[0070] Usage and dosage: Oral, 6 grams (1 bag) at a time, twice a day, taken before meals.

[0071] Specifications: 19 pills weighing 1 gram Storage: Keep airtight and moisture-proof.

[0072]

Identification

[0073] (3) Take 3 g of the product, grind finely, add 50 mL of 0.2% calcium hydroxide solution, and shake every 30 minutes. Filter, and extract the filtrate with 1 mol·L -1 -1 hydrochloric acid to adjust the pH to 3, and extract with ethyl acetate three times, 20 mL each time. Combine the ethyl acetate solution, evaporate to dryness, and dissolve the residue in 1 mL of methanol to obtain the test solution. Separately, prepare a saturated solution of hesperidin control solution in methanol, and use as the control solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2025, General Rules 0502), take 10 μL of the test solution and 5 μL of the control solution, spot on the same silica gel G thin layer plate, develop with the lower layer solution of chloroform-ethyl acetate-methanol-water (8:20:11:5) that has been stored at 10 ℃ for 12 hours or more, take out, air dry, spray with aluminum chloride reagent, heat at 105 ℃ for 2 minutes, and observe under a UV lamp (365 nm). The test chromatogram shows the same colored spots at the positions corresponding to the control chromatogram.

[0074] (4) Take the product 6 g, grind finely, add ethanol 50 mL, ultrasonic for 30 minutes, filter, evaporate the filtrate to dryness, add water 20 mL to dissolve, extract with water-saturated n-butanol for 3 times, 20 mL each time, combine the n-butanol extract, wash with n-butanol-saturated water for 2 times, 20 mL each time, collect the n-butanol liquid, evaporate to dryness, add 10% ethanol 20 mL to dissolve, add to a macroporous adsorption resin D101 column (inner diameter 1.5 cm, column height 13 cm), elute with 10%, 30%, 50% ethanol 50 mL respectively, collect the 50% ethanol eluate, evaporate to dryness, add methanol 1 mL to dissolve as the test solution. Take another 1 g of licorice reference material, prepare a reference material solution by the same method. According to the thin layer chromatography method (《People's Republic of China Pharmacopoeia》2020 edition volume four general rules 0502), take 10 μL of the above two solutions respectively, spot on the same silica gel G thin layer plate, develop with toluene-ethyl acetate-water (15:4:0.5) as the developing agent, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 ℃ until the spots develop clearly. The results show that the test sample chromatogram shows the same colored spots at the corresponding positions of the reference material chromatogram.

[0075]

Inspection

[0076]

Content Determination

[0077] Chromatographic conditions and system suitability test Use octadecylsilane-bonded silica gel as the filler; use acetonitrile (A)-methanol (B)-water (C) as the mobile phase, gradient elution according to the following table; the detection wavelength of aurantiamarin, honokiol, magnolol and atractylin is 290 nm, and the detection wavelength of atractylin is 336 nm. The theoretical plate number should not be less than 5000 calculated by aurantiamarin.

[0078] Gradient elution program ; Preparation of the reference solution Take the aurantiamarin, honokiol, magnolol and atractylin reference substances, accurately weigh, add methanol to prepare a mixed solution containing 50 μg of aurantiamarin, 25 μg of honokiol, 80 μg of magnolol and 20 μg of atractylin per 1 mL, which is the reference solution.

[0079] Preparation of test solution: accurately weigh about 0.8 g of the sample of Pingwei pills, finely grind, precisely add into a conical flask, precisely add 50 mL of methanol, weigh, ultrasonic (500 W, 45 kHz) for 60 minutes, cool, re-weigh, add the lost weight with methanol, shake well, filter, filter the filtrate with 0.45 μm filter membrane, and obtain.

[0080] Determination method: precisely take 10 μL of the control solution and the test solution respectively, inject into the liquid chromatograph, determine, and obtain.

[0081] Each 1 g of the product contains Chenpi calculated as hesperidin (C 28 H 34 O 15 ) not less than 2.6 mg, contains Hupu calculated as magnolol (C 18 H 18 O2) and honokiol (C 18 H 18 O2) not less than 4.7 mg, contains Cangzhu calculated as atractylenolide (C 13 H 10 O) not less than 0.9 mg.

[0082] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Various modifications and changes can be made by those skilled in the art to the present application without departing from the spirit and principle of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for quality testing of all medicinal ingredients in Pingwei Pills, characterized in that, Identifying Atractylodes lancea involves the following steps: Preparation of the first test solution: Grind Pingwei Pills into a fine powder, weigh 2 g, add 10 mL of ethyl acetate, sonicate for 5 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethyl acetate to obtain the first test solution. Preparation of the first control herbal solution: Take 0.5 g of Atractylodes lancea reference material, add 10 mL of ethyl acetate, sonicate for 5 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethyl acetate to obtain the first control herbal solution. Preparation of the first reference drug solution: Take atractylodes lancea and add methanol to prepare a solution containing 1 mg per 1 mL, which is used as the first reference solution; Preparation of the first negative sample solution: Prepare a negative sample of Atractylodes lancea without the required amount according to the prescription and process of Pingwei Pill, and prepare the first negative sample solution according to the preparation method of the first test sample solution. Identification was performed according to the thin-layer chromatography method in General Chapter 0502 of Part IV of the 2025 edition of the Pharmacopoeia of the People's Republic of China: Take 5 μL each of the first test sample solution, the first reference herb solution, the first reference solution, and the first negative sample solution, and spot them on the same silica gel G thin-layer plate; use a petroleum ether-cyclohexane mixture with a volume ratio of 9:1 at 60-90 °C as the developing solvent, develop, remove, air dry, spray with 5% vanillin-sulfuric acid solution, and heat at 105 °C until the spots are clearly visible. When the chromatogram of the first test sample shows a spot of the same color at the corresponding position as the chromatograms of the first reference herb and the first reference solution, and there is no interference from the first negative sample, it indicates that Pingwei Pill contains Atractylodes lancea. Identifying Magnolia officinalis involves the following steps: Preparation of the second test solution: Take an appropriate amount of Pingwei Pill sample, grind it into a fine powder, weigh 3 g, add 50 mL of ethyl acetate, sonicate for 30 minutes, cool, filter, and extract the filtrate with 2% sodium hydroxide solution by shaking 3 times, 20 mL each time. Combine the alkaline solutions, add hydrochloric acid to adjust the pH to 1-2, and extract with petroleum ether at 30-60 ℃ by shaking 3 times, 20 mL each time. Combine the petroleum ether solutions, evaporate to dryness, and dissolve the residue in 1 mL of methanol to obtain the second test solution. Preparation of the second reference solution: Take magnolol and honokiol reference standards separately, add methanol to prepare a mixed solution containing 1 mg of magnolol and 0.5 mg of honokiol per 1 mL, as the second reference solution; Preparation of the second negative sample solution: Prepare a negative sample lacking Magnolia officinalis according to the prescription and process of Pingwei Pill, and prepare the second negative sample solution according to the test sample solution preparation method; Identification was performed according to thin-layer chromatography (General Chapter 0502, Part IV, Chinese Pharmacopoeia 2025 Edition): 10 μL each of the second test sample solution, the second reference solution, and the second negative sample solution were spotted onto the same silica gel G thin-layer plate; a toluene-methanol mixture (12:1 v / v) was used as the developing solvent, the plate was developed, removed, and air-dried; 5% vanillin-sulfuric acid solution was sprayed on the plate, and the plate was heated at 105℃ until the spots were clearly visible. If the second test sample chromatogram showed spots of the same color at the corresponding positions as the second reference sample chromatogram, and there was no interference from the negative sample, it indicated that Pingwei Pill contained Magnolia officinalis. Identifying dried tangerine peel involves the following steps: Preparation of the third test solution: Take an appropriate amount of Pingwei Pill sample, grind it finely, weigh 3 g, add 50 mL of 0.2% calcium hydroxide solution, shake frequently for 30 minutes, filter, and use 1 mol·L⁻¹ water to extract the filtrate. -1 Adjust the pH to 3 with hydrochloric acid, extract three times with 20 mL of ethyl acetate each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 mL of methanol, and use this as the third test solution. Preparation of the third reference solution: Separately, prepare a saturated solution of hesperidin reference standard in methanol, which will serve as the third reference solution. Preparation of the third negative sample solution: Prepare a negative sample lacking tangerine peel according to the Pingwei Pill prescription and process, and prepare the third negative sample solution according to the test sample solution preparation method. Identification was performed according to the thin-layer chromatography method in General Chapter 0502 of Part IV of the 2025 edition of the Pharmacopoeia of the People's Republic of China: 10 μL of the third test sample solution, 10 μL of the third negative sample solution, and 5 μL of the third reference solution were spotted separately onto the same silica gel G thin-layer plate. The lower layer of a mixed solution of chloroform-ethyl acetate-methanol-water (volume ratio 8:20:11:5) was used as the developing solvent and left at below 10 ℃ for at least 12 hours. After development, the plate was removed, dried, sprayed with aluminum trichloride test solution, heated at 105 ℃ for 2 minutes, and examined under a 365 nm ultraviolet lamp. If the third test sample chromatogram showed a spot of the same color at the corresponding position as the third reference chromatogram, and the negative sample solution showed no interference, it indicated that Pingwei Pills contained tangerine peel. Identifying licorice involves the following steps: Preparation of the fourth test solution: Take an appropriate amount of Pingwei Pill sample, grind it finely, weigh 6 g, add 50 mL of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 mL of water, extract three times with 20 mL of water-saturated n-butanol each time, combine the n-butanol extracts, wash twice with 20 mL of water-saturated n-butanol each time, collect the n-butanol solution, evaporate to dryness, dissolve the residue in 20 mL of 10% ethanol, and add it to a macroporous adsorption resin D101 column, the inner diameter of which is [missing information]. The column was 1.5 cm high and 13 cm long. It was eluted with 50 mL each of 10%, 30%, and 50% ethanol. The 50% ethanol eluent was collected, evaporated to dryness, and the residue was dissolved in 1 mL of methanol to obtain the fourth test solution. Preparation of the fourth reference herb solution: Take another 1 g of licorice reference herb and prepare the reference herb solution using the same method; Preparation of the fourth negative sample solution: Prepare a negative sample lacking licorice according to the prescription and process of Pingwei Pill, and prepare the fourth negative sample solution according to the preparation method of the test sample solution; According to the thin-layer chromatography method in General Chapter 0502 of Part IV of the 2025 edition of the Pharmacopoeia of the People's Republic of China, 10 μL each of the fourth test sample solution, the fourth reference medicinal material solution, and the fourth negative sample solution were spotted separately on the same silica gel G thin-layer plate. A toluene-ethyl acetate-water solution (v / v) with a volume ratio of 15:4:0.5 was used as the developing solvent. After development, the plate was removed, dried, and sprayed with 10% sulfuric acid ethanol solution. The plate was heated to 105℃ until the spots were clearly visible. When the fourth test sample chromatogram showed a spot of the same color at the corresponding position as the fourth reference medicinal material chromatogram, and the negative sample solution did not interfere, it indicated that Pingwei Pill contained licorice.

2. The method for quality testing of all medicinal ingredients in Pingwei Pills according to claim 1, characterized in that, The ultrasound parameters are 500 W and 45 kHz.

3. The method for quality testing of all medicinal ingredients in Pingwei Pills according to claim 1, characterized in that, The steps for simultaneously measuring the content of hesperidin, magnolol, honokiol, and atractylodesin in Pingwei Pills include: Step 1: Use Shimadzu VP-ODS-C 18 Chromatographic column; acetonitrile (A)-methanol (B)-water (C) as mobile phase, gradient elution; flow rate 1.0 mL·min –1 Column temperature: 30 ℃; Detection wavelengths: 290 nm, 336 nm; Injection volume: 10 μL; Gradient elution program is shown in the table below. ； Step 2: Preparation of mixed reference solutions: Accurately weigh 7.52, 8.26, and 7.59 mg of hesperidin, magnolol, and honokiol reference standards into 20 mL volumetric flasks, and 6.06 mg of atractylodesin reference standard into a 50 mL volumetric flask. Dissolve in methanol and dilute to volume to obtain reference stock solutions A, B, C, and D. Accurately measure 7.0, 3.0, 7.5, and 7.0 mL of reference stock solutions A, B, C, and D into the same 25 mL volumetric flask, and add methanol to the mark to obtain mixed reference stock solutions. Step 3: Preparation of the fifth test solution: Take an appropriate amount of Pingwei Pill sample, grind it into a fine powder, take about 0.8 g, weigh it accurately, place it in an Erlenmeyer flask, add 50 mL of methanol accurately, weigh it, sonicate (500 W, 45 kHz) for 60 min, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, and filter the filtrate through a 0.45 μm filter membrane to obtain the solution. Step 4, Preparation of the fifth negative sample solution: According to the prescription ratio and process of Pingwei Pill, prepare negative samples without tangerine peel, magnolia bark and atractylodes rhizome respectively, and prepare negative sample solutions without tangerine peel, magnolia bark and atractylodes rhizome according to the method in step 3; Step 5, Specificity Experiment: Take appropriate amounts of the mixed reference solution, the fifth test solution, and the fifth negative sample solution, respectively, and inject them according to the chromatographic conditions in Step 1. Record the chromatograms. When the retention times of the peaks of hesperidin, honokiol, magnolol, and atractylodes in the chromatogram of the fifth test solution are consistent with the retention times of the peaks of the mixed reference solution, and the resolution between them and adjacent components is greater than 1.5, and no chromatographic peaks appear at the same retention times of the four components in the chromatograms of the negative sample solutions, and there is no interference from the negative samples, then the chromatographic results of hesperidin, honokiol, magnolol, and atractylodes can be read. Step Six: Linearity and Limit of Quantification (LOQ) Assessment: Accurately measure 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the mixed reference stock solution into 5 mL volumetric flasks, and add methanol to the mark to obtain a series of mixed reference solutions. Inject 10 μL of each solution under the chromatographic conditions described in Step One. Perform linear regression with the injection concentration as the abscissa and the corresponding peak area as the ordinate to obtain the standard curves for hesperidin, honokiol, magnolol, and atractylodesin. All values ​​showed r > 0.999, indicating good linearity. The results are shown in the table below. Take an appropriate amount of the mixed reference solution, dilute it stepwise with methanol, and inject it for analysis. The injection concentration at a signal-to-noise ratio of 10:1 is taken as the limit of quantification.

4. The method for quality testing of all medicinal ingredients in Pingwei Pills according to any one of claims 1 to 3, characterized in that, The preparation method of the Pingwei Pill includes: Atractylodes lancea, Magnolia officinalis, Citrus reticulata peel, and Glycyrrhiza uralensis. The weight ratio of Atractylodes lancea, Magnolia officinalis, Citrus reticulata peel, and Glycyrrhiza uralensis is 160:100:20:

20. The above four ingredients are ground into fine powder, sieved, and mixed evenly. Separately, 4g of jujube and 3g of ginger are decocted to extract the juice. The juice is used to make pills, which are then dried.

Images