Quality control method for peony leaves
A quality control method for peony leaves was established by combining thin-layer chromatography, spectrophotometry, and high-performance liquid chromatography, which solved the problems of quality standardization and stability. This method is applicable to the treatment of hyperuricemia and enriches the resources of traditional Chinese medicine.
Patent Information
- Application Number
- CN202511104607.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-07
- Publication Date
- 2025-11-04
AI Technical Summary
In the existing technology, the quality control standardization of peony leaves, drug stability, and shelf life are unclear, making it difficult to meet the treatment needs of hyperuricemia.
Thin-layer chromatography was used to identify paeoniflorin in peony leaves, total flavonoids were determined by spectrophotometry, and the contents of paeoniflorin and apigenin 7-O-glucoside were determined by high-performance liquid chromatography. Based on the combination of the three techniques, a quality control standard for peony leaves was established.
This achievement ensures the precision and stability of peony leaf quality, guaranteeing the reliability and effectiveness of the medicine, which is indicated for the treatment of hyperuricemia, thus enriching the application of traditional Chinese medicine resources.
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Figure CN120891106A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of traditional Chinese medicine identification, and particularly relates to a quality control method of peony leaf tablets. BACKGROUND
[0002] Peony leaf is the dried leaf of Paeonia suffruticosa Andr. of Ranunculaceae, which is collected in autumn and dried. Peony is widely distributed in China and has very rich resources. The chemical components of peony leaf mainly include total glycosides, flavonoids, polyphenols and other active ingredients, such as paeoniflorin, apigenin-7-O-glucoside and gallic acid. In addition to the effects of lowering uric acid, anti-inflammatory and analgesic, paeoniflorin, apigenin-7-O-glucoside and gallic acid also have the effects of antioxidant, antitumor and immunomodulation.
[0003] The peony leaf tablet is composed of peony leaf single herb. The preparation has the effects of clearing heat and resolving toxicity, removing dampness and phlegm, relieving inflammation and pain and lowering uric acid, and is used for treating hyperuricemia. The preparation is prepared by modern pharmaceutical technologies such as water extraction, spray drying and film coating. In order to better control the quality of the preparation, the research group establishes the quality standard of peony leaf tablet and conducts the accelerated and long-term stability research on the preparation. SUMMARY
[0004] In view of the above problems in the prior art, the present application provides a quality control method of peony leaf tablet, which can be used for the quality control of peony leaf tablet, has good precision, sensitivity and stability, and effectively solves the problems of quality control standardization, drug stability, unclear effective period and the treatment demand of hyperuricemia.
[0005] To achieve the above object, the technical scheme adopted by the present application to solve its technical problems is to provide a quality control method of peony leaf tablet, comprising the following steps: S1, identifying paeoniflorin in the peony leaf tablet by thin layer chromatography; S2, determining total flavonoids in the peony leaf tablet by spectrophotometry; S3, determining the content of paeoniflorin in the peony leaf tablet by high performance liquid chromatography; S4, determining the content of apigenin-7-O-glucoside in the peony leaf tablet by high performance liquid chromatography.
[0006] Further, the above step S1, step S2, step S3 and step S4 can exchange the order.
[0007] The preparation method of the peony leaf tablet comprises the following steps: decocting peony leaf decoction pieces with water, filtering, collecting the filtrate, adding water to the residue and decocting, filtering, collecting the filtrate, combining the filtrates, reducing pressure and concentrating, collecting the extract and drying, obtaining peony leaf extract, granulating by adding ethanol, drying, compressing into tablets, and coating with film to obtain the peony leaf tablet.
[0008] Further, the peony leaf decoction pieces are decocted with 9-11 times the amount of water for 1-1.5 h.
[0009] Further, the peony leaf decoction pieces are decocted with 10 times the amount of water for 1 h.
[0010] Further, the residue is decocted with 7-9 times the amount of water for 35-45 min.
[0011] Further, the residue is decocted with 8 times the amount of water for 40 min.
[0012] Further, the concentration is performed at 70-80 ℃ under reduced pressure.
[0013] Further, the concentration is performed at 75 ℃ under reduced pressure.
[0014] Further, the extract is collected at 55-65 ℃.
[0015] Further, the extract is collected at 60 ℃.
[0016] Further, the relative density of the extract is 1.05-1.15.
[0017] Further, the extract is collected and spray dried, the temperature of the air inlet is 155-160 ℃, and the temperature of the air outlet is 80-85 ℃.
[0018] Further, the mass-volume ratio of the peony leaf extract and ethanol is 450-550 g:5-20 mL.
[0019] Further, the mass-volume ratio of the peony leaf extract and ethanol is 500 g:10 mL.
[0020] Further, the concentration of the ethanol is 70-80 vt%.
[0021] Further, the ethanol is added in the form of spray.
[0022] Further, the drying is performed at 40-60 ℃.
[0023] Further, the drying is performed at 55 ℃.
[0024] The method for identifying paeoniflorin in the peony leaf tablet by thin layer chromatography comprises the following steps: S101. Take peony leaves, remove the thin film coating, grind them into a fine powder, add acetone to the peony leaf powder, sonicate and filter, evaporate the residue, add acetone again, sonicate and filter, and allow the filtrate to evaporate to obtain the test solution. S102. Take paeoniflorin as a reference standard, add acetone to obtain the reference standard solution; S103. Take the test solution obtained in step S101 and the reference solution obtained in step S102 respectively, and spot them on the same silica gel G thin layer plate. Use chloroform-ethyl acetate-methanol-formic acid as the developing solvent, develop, remove, air dry, spray with vanillin sulfuric acid solution, and heat until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.
[0025] Furthermore, in step S101, the mass-to-volume ratio of peony leaf powder to acetone is 0.9-1.1 g: 45-55 mL.
[0026] Furthermore, in step S101, the mass-to-volume ratio of peony leaf powder to acetone is 1 g: 50 mL.
[0027] Furthermore, in step S101, the ultrasonic treatment lasts for 25-35 minutes.
[0028] Furthermore, in step S101, the ultrasonic treatment lasts for 30 minutes.
[0029] Furthermore, in step S101, the mass-to-volume ratio of peony leaf powder to acetone added to the residue is 0.9-1.1 g: 45-55 mL.
[0030] Furthermore, in step S101, the mass-to-volume ratio of peony leaf powder to acetone added to the residue is 1 g: 50 mL.
[0031] Furthermore, in step S101, acetone is added and ultrasonically treated for 25-35 minutes.
[0032] Furthermore, in step S101, acetone is added and ultrasonically treated for 30 minutes.
[0033] Further, in step S101, the filtrate is evaporated to 1.5-2.5 mL.
[0034] Further, in step S101, the filtrate is evaporated to 2 mL.
[0035] Furthermore, in step S102, the mass-to-volume ratio of paeoniflorin to acetone is 0.9-1.1 mg:0.9-1.1 mL.
[0036] Furthermore, in step S102, the mass-to-volume ratio of paeoniflorin to acetone is 1 mg:1 mL.
[0037] Further, in step S103, 9-11 μL of the test sample solution and the control sample solution respectively.
[0038] Further, in step S103, 10 μL of the test sample solution and the control sample solution respectively.
[0039] Further, in step S103, the volume ratio of chloroform, ethyl acetate, methanol and formic acid is 35-45:9-11:18-22:0.1-0.3.
[0040] Further, in step S103, the volume ratio of chloroform, ethyl acetate, methanol and formic acid is 40:10:20:0.2.
[0041] Further, in step S103, the concentration of vanillin sulfuric acid solution is 4-6 vt%.
[0042] Further, in step S103, the concentration of vanillin sulfuric acid solution is 5 vt%.
[0043] Further, in step S103, heating is performed at 104-106 ℃.
[0044] Further, in step S103, heating is performed at 105 ℃.
[0045] The above determination of total flavonoids in peony leaves by spectrophotometry comprises the following steps: S201, dissolve apigenin control sample in ethanol to obtain a control sample solution; S202, take different volumes of the control sample solution, add ethanol and triethylamine solution and mix well, take the corresponding reagent as blank, measure the absorbance by ultraviolet-visible spectrophotometry, and draw a standard curve with absorbance as vertical coordinate and concentration as horizontal coordinate; S203, grind the film-coated peony leaves to obtain peony leaf powder, then add ethanol, cool after ultrasonic treatment, make up the weight loss with ethanol, filter, take the filtrate, add ethanol to obtain a test sample solution, measure the absorbance according to the method, read the weight of apigenin in the test sample solution from the standard curve, and calculate the total flavonoid content. The total flavonoid content in every 0.5 g of peony leaves should not be less than 11.0 mg.
[0046] Further, in step S201, 13-14 mg of apigenin control sample is taken in a 50 mL volumetric flask, and a 55-65 vt% ethanol solution is added to the mark to obtain a dissolved mixed solution. 0.9-1.1 mL of the mixed solution is taken in a 20 mL volumetric flask, and a 55-65 vt% ethanol solution is added to the mark.
[0047] Further, in step S201, 13.95 mg of the apigenin reference substance is taken into a 50 mL volumetric flask, and 60% (v / v) ethanol solution is added to the calibration mark to obtain a mixed solution after dissolution. 1 mL of the mixed solution is taken into a 20 mL volumetric flask, and then 60% (v / v) ethanol solution is added to the calibration mark.
[0048] Further, in step S202, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of the reference substance solution are respectively taken into 10 mL volumetric flasks, and then 55-65% (v / v) ethanol solution is added to 5 mL, and 0.9-1.1% (v / v) triethylamine solution is added to the calibration mark and mixed.
[0049] Further, in step S202, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of the reference substance solution are respectively taken into 10 mL volumetric flasks, and then 60% (v / v) ethanol solution is added to 5 mL, and 1% (v / v) triethylamine solution is added to the calibration mark and mixed.
[0050] Further, in step S202, the absorbance is measured at a wavelength of 400 nm.
[0051] Further, in step S203, 0.1-0.2 g of peony leaf powder is added to 90-110 mL of 55-65% (v / v) ethanol solution.
[0052] Further, in step S203, 0.1 g of peony leaf powder is added to 100 mL of 60% (v / v) ethanol solution.
[0053] Further, in step S203, ultrasonic treatment is performed for 40-50 min.
[0054] Further, in step S203, ultrasonic treatment is performed for 45 min.
[0055] Further, in step S203, the ultrasonic power is 230-250 W, and the frequency is 40-50 kHz.
[0056] Further, in step S203, the ultrasonic power is 250 W, and the frequency is 40 kHz.
[0057] Further, in step S203, the weight loss is made up with 55-65% ethanol.
[0058] Further, in step S203, the weight loss is made up with 60% ethanol.
[0059] Further, in step S203, 0.9-1.1 mL of the filtrate is taken into a 10 mL volumetric flask, and 55-65% (v / v) ethanol solution is added to 5 mL.
[0060] Further, in step S203, 1 mL of the filtrate is taken in a 10 mL volumetric flask, and an ethanol solution with a concentration of 60 vt% is added to 5 mL.
[0061] The method for determining paeoniflorin in peony tablets by high performance liquid chromatography, comprises the following steps: S301, adding methanol to the paeoniflorin reference substance, dissolving to obtain a reference solution; S302, removing the film coating of the peony leaf and grinding to obtain peony leaf powder, adding ethanol to the peony leaf, ultrasonic treatment, cooling, adding an ethanol solution, shaking, filtering, taking the filtrate to obtain a test solution; S303, determining the blank control, the reference solution and the test solution respectively, and the content of paeoniflorin in each 0.5 g of peony leaf should be not less than 5.8 mg.
[0062] Further, in step S301, 17-18 mg of paeoniflorin reference substance is taken in a 90-110 mL volumetric flask, and methanol is added to the scale, and dissolved.
[0063] Further, in step S301, 17.95 mg of paeoniflorin reference substance is taken in a 100 mL volumetric flask, and methanol is added to the scale, and dissolved.
[0064] Further, in step S302, 0.1-0.3 g of peony leaf powder is taken in a 50 mL volumetric flask.
[0065] Further, in step S302, 0.5 g of peony leaf powder is taken in a 50 mL volumetric flask.
[0066] Further, in step S302, an ethanol solution with a concentration of 49.5-50.5 vt% is added.
[0067] Further, in step S302, ultrasonic treatment is performed for 14-16 min.
[0068] Further, in step S302, ultrasonic treatment is performed for 15 min.
[0069] Further, in step S302, the power of ultrasonic treatment is 230-250 W, and the frequency is 40-50 kHz.
[0070] Further, in step S302, the power of ultrasonic treatment is 240 W, and the frequency is 45 kHz.
[0071] Further, in step S302, an ethanol solution with a concentration of 65-75 vt% is added to the scale.
[0072] Further, in step S302, 70% by volume of ethanol solution is added to the scale.
[0073] Further, in step S303, high performance liquid chromatography is performed using ShimPack Scepter C 18 a chromatographic column, a mobile phase of acetonitrile-0.085wt% phosphoric acid aqueous solution = 13-15:85-87 (V / V), a flow rate of 0.9-1.1 mL / min, a column temperature of 25-35°C, a detection wavelength of 230 nm, and an injection volume of 9-11 μL.
[0074] Further, in step S303, high performance liquid chromatography is performed using ShimPack Scepter C 18 a chromatographic column, a mobile phase of acetonitrile-0.085wt% phosphoric acid aqueous solution = 14:86 (V / V), a flow rate of 1 mL / min, a column temperature of 30°C, a detection wavelength of 230 nm, and an injection volume of 10 μL.
[0075] The method for determining apigenin 7-O-glucoside in peony tablets by high performance liquid chromatography includes the following steps: S401, adding methanol to the apigenin 7-O-glucoside reference substance, dissolving to obtain a reference solution; S402, removing the film coating of the peony leaf and grinding to obtain peony leaf powder, adding ethanol to the peony leaf and weighing, ultrasonic treatment, cooling, supplementing the weight loss with ethanol, shaking and filtering to obtain a test solution; S403, determining the blank control, the reference solution and the test solution, respectively, and the content of apigenin 7-O-glucoside in each 0.5 g of peony leaf should be not less than 0.95 mg.
[0076] Further, in step S401, 14-15 mg of apigenin 7-O-glucoside reference substance is taken in a 45-55 mL volumetric flask, and methanol is added to the scale and dissolved.
[0077] Further, in step S401, 14.45 mg of apigenin 7-O-glucoside reference substance is taken in a 50 mL volumetric flask, and methanol is added to the scale and dissolved.
[0078] Further, in step S402, 90-110 mL of ethanol solution with a concentration of 65-75% by volume is added to 0.4-0.6 g of peony leaf.
[0079] Further, in step S402, 100 mL of ethanol solution with a concentration of 70% by volume is added to 0.5 g of peony leaf.
[0080] Further, in step S402, ultrasonic treatment is performed for 25-35 min.
[0081] Further, in step S402, ultrasonic treatment is performed for 30 min.
[0082] Further, in step S402, the weight loss is made up with ethanol with a concentration of 70 vt%.
[0083] Further, high performance liquid chromatography is performed by using ShimPack Scepter C 18 As a chromatographic column, acetonitrile is used as mobile phase A, 0.1 vt% formic acid aqueous solution is used as mobile phase B, the detection wavelength is 340 nm, the flow rate is 0.7-0.9 mL / min, the column temperature is 20-30℃, and the injection amount is 4-6 μL.
[0084] Further, high performance liquid chromatography is performed by using ShimPack Scepter C 18 As a chromatographic column, acetonitrile is used as mobile phase A, 0.1 vt% formic acid aqueous solution is used as mobile phase B, the detection wavelength is 340 nm, the flow rate is 0.8 mL / min, the column temperature is 25℃, and the injection amount is 5 μL.
[0085] Further, the specification of the chromatographic column is 4-5 mm×240-260 mm, 4-6 μm.
[0086] Further, the specification of the chromatographic column is 4.6 mm×250 mm, 5 μm.
[0087] Further, the elution procedure of high performance liquid chromatography is as follows: 0-5 min, mobile phase A: 20-22%, mobile phase B: 80-78%; 5-40 min, mobile phase A: 22-23%, mobile phase B: 78-77%.
[0088] The present application has the following beneficial effects: 1、The present application combines thin layer chromatography (TLC), spectrophotometry (UV) and high performance liquid chromatography (HPLC) three kinds of technologies, respectively qualitative identification of paeoniflorin, quantitative determination of total flavonoids and apigenin 7-O-glucoside / paeoniflorin content, covers the detection requirement quality standard research of main active ingredients, through 15 batches of peony leaf paeoniflorin TLC qualitative research, ultraviolet spectrophotometer quantitative total flavonoids, HPLC method for determining the content of paeoniflorin and apigenin 7-O-glucoside. These qualitative and quantitative methods have strong specificity, repeatability and stability, and the quality of peony leaf products is well controlled.
[0089] 2、In the application, TLC spots are clear without interference, HPLC peaks are well separated (theoretical plate number is greater than or equal to 5000), method verification shows that the precision (RSD is less than or equal to 1.94%) and the recovery rate (97.86% to 99.53%) meet the requirements of the pharmacopoeia, ensuring the reliability of the data.
[0090] 3、The peony leaf provided by the application is suitable for hyperuricemia, and hyperuricemia (HUA) is a metabolic disease caused by purine metabolism disorder and / or reduced uric acid excretion. The peony leaf resource is extremely rich in China, and it is of great significance to develop the peony leaf into a new uric acid-lowering traditional Chinese medicine drug. BRIEF DESCRIPTION OF DRAWINGS
[0091] Figure 1 is a TLC identification diagram of paeoniflorin in peony leaf tablets; Figure 2 is a TLC identification diagram of paeoniflorin in peony leaf tablets; Figure 3 is a TLC identification diagram of paeoniflorin in peony leaf tablets; Figure 4 is a TLC identification diagram of paeoniflorin in peony leaf tablets; Figure 5 is a high performance liquid chromatogram; Figure 6 is a high performance liquid chromatogram; Figure 7 is a total flavone content acceleration test result diagram; Figure 8 is an apigenin 7-O-glucoside acceleration test result diagram; Figure 9 is a paeoniflorin content acceleration test result diagram; Figure 10 is a total flavone content long-term stability test result diagram; Figure 11 is an apigenin 7-O-glucoside content long-term stability test result diagram; Figure 12 is a paeoniflorin content long-term stability test result diagram. DETAILED DESCRIPTION
[0092] The principles and characteristics of the application are described below, and the examples are only used to explain the application and are not used to limit the scope of the application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.
[0093] The peony leaf used in the application: specification 0.5 g / tablet, 1000 pieces of prescription sample (number A1, A2, A3), 5 times magnification sample (number B1, B2, B3), 10 times magnification adaptive sample (number C1, C2, C3), 20 times magnification adaptive sample (number D1, D2, D3), pilot sample (batch number: 20221201, 20221202, 20221203), provided by Shaanxi Fengdan Zhengyuan Biological Technology Co., Ltd.
[0094] Example 1 The preparation method of peony leaf tablet comprises the following steps: decocting peony leaf decoction pieces with 10 times the amount of water for 1 h, filtering, collecting the filtrate, decocting the residues with 8 times the amount of water for 40 min, filtering, collecting the filtrate, combining the filtrates, concentrating under reduced pressure at 75 ℃, collecting the extract at 60 ℃, and then spray drying, the inlet temperature is 155-160 ℃, and the exhaust outlet temperature is 80-85 ℃, to obtain peony leaf extract, then adding 10 mL of 75 vt% ethanol to 500 g of peony leaf extract in the form of spraying, drying, pressing into tablets, drying at 55 ℃, and coating with film to obtain peony leaf tablets.
[0095] The TLC method is used to identify paeoniflorin in peony leaf tablets, which comprises the following steps: S101, take peony leaf tablets, remove the film coating, grind finely, add 50 mL of acetone to 1 g of peony leaf tablet powder, ultrasonic treat for 30 min, filter, evaporate the residues, then add 50 mL of acetone, ultrasonic treat for 30 min, filter, and evaporate the filtrate to 2 mL to obtain a test solution; S102, take paeoniflorin as a control, add acetone to obtain a control solution containing 1 mg per 1 mL; S103, respectively, 10 μL of the test solution obtained in step S101 and the control solution obtained in step S102 are spotted on the same silica gel G thin layer plate, chloroform-ethyl acetate-methanol-formic acid (40:10:20:0.2) is used as the developing agent, develop, take out, dry, spray with 5 vt% vanillin sulfuric acid solution, heat at 105 ℃ until the spots develop clear, the test chromatogram shows the same color spots at the corresponding positions of the control chromatogram. Through negative control, peony leaf tablet samples (pilot sample, adaptive pilot sample and 3 batches of pilot sample), the results are shown in Figures 1-4 . Figure 1 In the figure, 1 and 2 are paeoniflorin, 3 and 4 are A1, 5 is A2, 6 is A3, and 7 is blank. Figure 2 In the figure, 1 and 6 are paeoniflorin, 2 is B1, 3 is B2, 4 is B3, and 5 is C1. Figure 3 In the figure, 1 and 6 are paeoniflorin, 2 is C2, 3 is C3, 4 is D1, and 5 is D2.Figure 4 In particular, 1 and 6 are paeoniflorin, 2 is D3, 3 is 20221201 batch, 4 is 20221202 batch, and 5 is 20221203 batch.
[0096] By Figures 1-4 It can be seen that the negative sample has no interference, the spot is obvious, the reproducibility is good, the method is simple and sensitive, and therefore is included in the standard.
[0097] Example 2 The total flavonoids in peony leaves are determined by spectrophotometry, including the following steps: S201, 13.95 mg of apigenin reference substance is taken in a 50 mL volumetric flask, and 60 vt% ethanol solution is added to the calibration mark to obtain a dissolved mixed solution. 1 mL of the mixed solution is taken in a 20 mL volumetric flask, and then 60 vt% ethanol solution is added to the calibration mark to obtain a reference solution; S202, 1 mL, 2 mL, 3 mL, 4 mL and 5 mL of the reference solution are respectively taken in 10 mL volumetric flasks, and then 60 vt% ethanol solution is added to 5 mL, and 1 vt% triethylamine solution is added to the calibration mark and mixed, taking the corresponding reagent as the blank. The absorbance is determined at a wavelength of 400 nm by ultraviolet-visible spectrophotometry. The results are shown in Table 1. The absorbance is taken as the vertical coordinate (y), and the concentration is taken as the horizontal coordinate (x, μg / mL) for linear regression. y=0.1101 x-0.0119, r=0.9991, and the linear range is 0.135-0.749 μg / mL; Table 1: Absorbance value results of different concentrations of reference substance
[0098] S203, the film coating of peony leaves is finely ground to obtain peony leaf powder. Then 100 mL of 60 vt% ethanol solution is added to 0.1 g of peony leaf powder, and ultrasonic treatment is performed for 45 min. After cooling, the weight loss is made up with 60% ethanol, and then filtered. 1 mL of the filtrate is taken in a 10 mL volumetric flask, and 60 vt% ethanol solution is added to 5 mL to obtain a test solution. The absorbance is determined at a wavelength of 400 nm according to the method, and the weight of apigenin in the test solution is read from the standard curve. The total flavonoid content is calculated to be 14.44 mg / piece.
[0099] Example 3 The above method for determining paeoniflorin in peony tablets by high performance liquid chromatography includes the following steps: S301, 17.95 mg of paeoniflorin reference substance is taken in a 100 mL volumetric flask, and methanol is added to the calibration mark to obtain a reference solution after dissolution; S302, the peony leaf film was removed and finely ground to obtain peony leaf powder, 0.5 g of peony leaf powder was taken in a 50 mL volumetric flask, 49.5-50.5 vt% ethanol solution was added, and after ultrasonic treatment for 15 min, the power was 240W, the frequency was 45 kHz, 70 vt% ethanol solution was added to the mark, shaken, filtered, and the filtrate was taken to obtain the test solution; S303, the blank control, the control solution and the test solution were determined respectively, the high performance liquid chromatography used ShimPack Scepter C 18 chromatographic column, the mobile phase was acetonitrile-0.085wt% phosphoric acid aqueous solution=14:86 (V / V), the flow rate was 1 mL / min, the column temperature was 30℃, the detection wavelength was 230 nm, the injection amount was 10 μL, and the results were as shown in Figure 5
[0100] It can be seen from Figure 5 that the result of the blank control has no interference, indicating that the method has strong specificity.
[0101] Example 4 The method for determining apigenin 7-O-glucoside in peony tablets by high performance liquid chromatography, comprising the following steps: S401, 14.45 mg of apigenin 7-O-glucoside control was taken in a 50 mL volumetric flask, and methanol was added to the mark. After dissolution, the control solution was obtained; S402, the peony leaf film was removed and finely ground to obtain peony leaf powder, 0.5 g of peony leaf powder was taken in a 50 mL volumetric flask, 49.5-50.5 vt% ethanol solution was added, and after ultrasonic treatment for 15 min, the power was 240W, the frequency was 45 kHz, 70 vt% ethanol solution was added to the mark, shaken, filtered, and the filtrate was taken to obtain the test solution; S403, the blank control, the control solution and the test solution were determined respectively, the high performance liquid chromatography used ShimPack Scepter C 18 as the chromatographic column, acetonitrile was used as the mobile phase A, 0.1 vt% formic acid aqueous solution was used as the mobile phase B, the detection wavelength was 340 nm, the flow rate was 0.8 mL / min, the column temperature was 25℃, the injection amount was 5 μL, the specification of the chromatographic column was 4.6mm×250 mm, 5 μm, and the elution program of the high performance liquid chromatography was: 0-5 min, mobile phase A: 20-22%, mobile phase B: 80-78%; 5-40 min, mobile phase A: 22-23%, mobile phase B: 78-77%. The results are shown in Figure 6
[0102] It can be seen from Figure 6 that the result of the blank control has no interference, indicating that the method has strong specificity.
[0103] Test Example 1 The performance in Test Example 2 (determination of total flavones in peony leaves by spectrophotometry) was tested.
[0104] (1) Precision test The control solution (concentration 4.16 μg / mL) was continuously determined for 6 times, and the results of absorbance values were 0.460, 0.459, 30.459, 40.459, 50.458, 60.458, respectively, and the RSD was 0.17%, indicating that the precision of the instrument was good.
[0105] (2) Stability test The absorbance of the test sample solution (A1 sample) was determined at 0, 0.5, 1, 2, 4, 8, 12, 24 h, and the results were 0.460, 0.459, 0.458, 0.458, 0.458, 0.457, 0.457, 0.457, respectively, and the RSD of the results of absorbance values was 0.12%, indicating that the stability of the detection sample was good within 24 h.
[0106] (3) Reproducibility test The sample (A1 sample) was prepared into 6 test sample solutions according to the test sample preparation method, and determined according to the determination method, and the results were 18.01, 18.50, 18.63, 18.69, 18.41, 18.44, respectively, and the average content of total flavones in each piece was 18.44449 mg / piece, and the RSD was 1.31%, indicating that the sample reproducibility was good.
[0107] (4) Sample recovery rate test 0.05 g of sample (A1 sample, content 18.44449 mg / piece, i.e. 36.88898 mg / g) was precisely weighed, and 1.90 mg was added according to the test sample preparation method to prepare 6 test sample solutions, and determined according to the determination method, and the recoveries were 96.12%, 97.20%, 97.60%, 96.32%, 95.37%, 96.40%, respectively, and the average sample recovery rate was 96.50%, and the RSD was 0.83%, indicating that the sample recovery rate of the method was good.
[0108] (5) Sample content determination Different samples were determined according to the method provided by the application, and the determination results of total flavones in the small test sample of peony leaves were shown in Table 2, and the determination results of total flavones in the pilot sample of 6 batches were shown in Table 3.
[0109] Table 2 Determination results of total flavones in the small test sample of peony leaves
[0110] Table 3 Total flavone determination results of 6 batches of pilot samples
[0111] From Table 2 and Table 3, according to the total flavone detection results of 15 batches of peony leaves, the provisional standard is that the total flavone content of peony leaf medicinal materials should not be less than 1.1 % (provisional standard of declaration materials); according to the small test and pilot test results, the total flavone extraction transfer rate is not less than 75 %, and the total flavone content of each peony leaf is not less than 11.0 mg, which is included in the quality standard.
[0112] Test Example 2 The performance of Example 3 (determination of paeoniflorin in peony tablets by high performance liquid chromatography) was tested.
[0113] (1) Preparation of standard curve Take 0.100, 0.250, 0.500, 1.250, 2.500, 5.000, 10.000 mL of control solution, respectively, in 10 mL volumetric flask, add methanol to the mark, and obtain a series of concentration control solution, and the sample is measured for peak area. Take the peak area (y) as the vertical coordinate and the concentration (x, μg / mL) as the horizontal coordinate, and draw the standard curve. The standard curve is y = 13164.59 x + 11886.87, R² = 0.9994, which shows that paeoniflorin has good linear relationship in the concentration range of 1.74-173.76 μg / mL.
[0114] (2) Precision test Take the control solution (concentration 86.88 μg / mL) and continuously measure 6 times, and measure the peak area. The results are shown in Table 4.
[0115] Table 4 Precision test results
[0116] From Table 4, the RSD of peak area is 1.47 %, which shows that the instrument precision is good.
[0117] (3) Stability test Take the test sample solution (20221201 sample), and measure the peak area at 0, 1, 2, 4, 8, 12, 24 h, respectively. The results are shown in Table 5.
[0118] Table 5 Stability determination results
[0119] From Table 5, the RSD of peak area is 1.12 %, which shows that the detection sample has good stability within 24 h.
[0120] (4) Repetitive test The sample (20221201 sample) was prepared into 6 sample solutions according to the sample preparation method, and the determination was performed according to the determination method, and the results are shown in Table 6.
[0121] Table 6 Determination results of repetitive test
[0122] As shown in Table 6, the average content of paeoniflorin in the sample is 24.37714 mg / g, and the RSD is 1.23 %, indicating that the sample has good repeatability.
[0123] (5) Spiking recovery rate test The sample (20221201 sample, paeoniflorin content is 24.37714 mg / g) was prepared into 6 sample solutions according to the sample preparation method, and the amount of addition is shown in Table 7, and the determination was performed according to the determination method. The results are shown in Table 7.
[0124] Table 7 Determination results of spiking recovery rate
[0125] As shown in Table 7, the average spiking recovery rate is 99.53 %, and the RSD is 1.01 %, indicating that the method has good spiking recovery rate.
[0126] (6) Determination of paeoniflorin content in sample Different samples were taken and determined according to the method provided by the present application. The results are shown in Table 8.
[0127] Table 8 Content of paeoniflorin in different samples
[0128] As shown in Table 8, the determination results of 15 batches of samples show that the minimum average value of paeoniflorin in the sample is 7.74 mg / piece, and the RSD values of small test, adaptive pilot test and pilot test are all less than 2 %, indicating that the process is stable. Based on the paeoniflorin transfer rate of more than 75 % and the content standard of peony leaf medicinal material not less than 0.50 % (provisional standard of declaration material), the content limit of paeoniflorin in peony leaf is temporarily set as: each piece (0.5 g) of paeoniflorin (C 23 H 28 O 11 ) should not be less than 5.8 mg, and is included in the quality standard.
[0129] Test example 3 The performances in example 4 (determination of apioin 7-O-glucoside content) were tested.
[0130] (1) Standard curve and linear range Take 0.10, 0.35, 0.50, 1.25, 2.50, 6.00, 10.00 of the control solution respectively into 10 mL volumetric flask, add methanol to constant volume, get a series of concentrations respectively, determine the peak area under the above conditions, the results are shown in Table 9. Take the control peak area value (y) as the vertical coordinate, and the apigenin-7-O-glucoside concentration (x) as the horizontal coordinate to carry out linear regression, the regression equation is y = 19847 x + 22845, r = 0.9997, and the linear range is 2.83-283.22 μg / mL.
[0131] Table 9 Relationship between apigenin-7-O-glucoside control concentration and peak area
[0132] (2) Precision test Take the linear sample solution (concentration is 14.16 μg / mL), and inject 6 times repeatedly under the above chromatographic conditions, and the apigenin-7-O-glucoside peak area values are 298473, 299138, 297407, 298516, 298575, 299081 respectively, and the RSD % is 0.21 %, which shows that the precision is good.
[0133] (3) Stability test Precisely take 1 portion of sample (20221201 batch, 0.45575 g), and operate according to the preparation method of test solution, and inject under the above chromatographic conditions at 0, 2, 4, 6, 8, 12, 24, 36 hours, and the apigenin-7-O-glucoside peak area values are 236478, 237634, 236716, 236545, 236958, 236568, 236357, 235524 respectively, and the RSD of area value is 0.25 %, which shows that the apigenin-7-O-glucoside has good stability within 36 h.
[0134] (4) Reproducibility test Precisely take 6 portions of sample (20221201 batch), and operate according to the preparation method of test solution, and determine under the above chromatographic conditions, and the apigenin-7-O-glucoside content in each tablet of the test sample is 1.30537, 1.36710, 1.34116, 1.31880, 1.35385, 1.33342 mg / tablet respectively, and the average content is 1.33662 mg / tablet, and the RSD of content is 1.69 %, which shows good reproducibility.
[0135] (5) Recovery rate test Precisely take the sample (20221201 batch, the content of apigenin 7-O-glucoside 2.67323 mg / g) 6 parts, 0.25 g each, precisely add apigenin 7-O-glucoside reference substance, operate according to the preparation method of test sample solution, and determine according to the chromatographic condition. The results of recovery rate are 96.72069%, 99.12587%, 98.88112%, 99.28630%, 96.15428%, and 97.00281%. The average recovery rate of sample addition is 97.86%, and the RSD is 1.42%, indicating that the recovery rate of sample addition is good.
[0136] (6) Sample determination Precisely take 5 μL of the reference solution and the test sample solution respectively, inject into the liquid chromatograph, and determine. The results are shown in Table 10.
[0137] Table 10 Determination results of apigenin 7-O-glucoside in peony leaf
[0138] From Table 10, it can be seen that the determination results of 15 batches of samples show that the minimum average value of apigenin 7-O-glucoside in the samples is 1.36 mg / piece. The RSD values of small test, adaptive pilot test, and pilot test are all less than 2%, indicating that the process is stable. Based on the apigenin 7-O-glucoside transfer rate of more than 70% and the content standard of peony leaf medicinal material not less than 0.13% (provisional standard in the declaration material), the content limit of apigenin 7-O-glucoside in peony leaf is temporarily set as: each piece (0.5 g) of peony leaf contains apigenin 7-O-glucoside (C 21 H 20 O 10 ) should not be less than 0.95 mg, which is included in the quality standard.
[0139] Test example 4 stability study (1) Accelerated stability test Take three batches of peony leaf tablet samples with batch numbers 20221201, 20221202, and 20221203, and perform an accelerated stability test (set temperature and humidity: 40℃±2℃, RH 75%±5%). Take samples at the end of the 0th, 1st, 2nd, 3rd, and 6th months during the test period, and perform inspection according to the draft quality standard of peony leaf tablet (full inspection including appearance, tablet weight difference, moisture, disintegration time limit, microbial limit test, TLC identification, and content determination). The total flavone content results are shown in Table 11, the paeoniflorin content is shown in Table 12, and the apigenin 7-O-glucoside content is shown in Table 13. Figure 6 Figure 7 Figure 8
[0140] As shown in Table 1, the three batches of trial samples of peony leaves all meet the quality standard draft of the product, and the stability is good compared with the 0-month accelerated test results. Figures 6-8 As shown in Table 1, the three batches of trial samples of peony leaves all meet the quality standard draft of the product, and the stability is good compared with the 0-month accelerated test results.
[0141] (2) Long-term stability test Three batches of trial samples of peony leaves with batch numbers 20221201, 20221202 and 20221203 were taken for long-term stability test (set temperature and humidity: 25℃±2℃, RH 60%±5%, samples were taken for detection at the end of 0, 3, 6, 9, 12, 18 and 24 months), and were tested according to the quality standard draft of peony leaves (full inspection including appearance, tablet weight difference, moisture, disintegration time limit, microbial limit test, TLC identification and content determination). The results are shown in Table 2. Among them, the accelerated time, "1" is 0 month, "2" is 3 months, "3" is 6 months, "4" is 9 months, "5" is 12 months, "6" is 18 months, and "7" is 24 months. Figures 9-11
[0142] As shown in Table 1, the three batches of trial samples of peony leaves all meet the quality standard draft of the product, and the stability is good compared with the 0-month accelerated test results. Figures 9-11 As shown in Table 1, the three batches of trial samples of peony leaves all meet the quality standard draft of the product, and the stability is good compared with the 0-month accelerated test results.
[0143] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.
Claims
1. A method for quality control of peony leaves, characterized in that, Includes the following steps: S1. Thin-layer chromatography was used to identify paeoniflorin in peony leaves; S2. Total flavonoids in peony leaves were determined by spectrophotometry. S3. The content of paeoniflorin in peony leaves was determined by high performance liquid chromatography. S4. The content of apigenin 7-O-glucoside in peony leaves was determined by high performance liquid chromatography.
2. The quality control method for peony leaves as described in claim 1, characterized in that, The method for preparing peony leaves includes the following steps: decocting peony leaf slices with water and filtering, collecting the filtrate, adding water to the residue and decocting again, filtering and collecting the filtrate, combining the filtrates and concentrating under reduced pressure, collecting the extract and drying it to obtain peony leaf extract, then adding ethanol to granulate, drying and pressing it into tablets, coating it with a film to obtain peony leaves.
3. The quality control method for peony leaves as described in claim 1, characterized in that, The method for identifying paeoniflorin in peony leaves using thin-layer chromatography includes the following steps: S101. Take peony leaves, remove the thin film coating, grind them into a fine powder, add acetone to the peony leaf powder, sonicate and filter, evaporate the residue, add acetone again, sonicate and filter, and allow the filtrate to evaporate to obtain the test solution. S102. Take paeoniflorin as a reference standard, add acetone to obtain the reference standard solution; S103. Take the test solution obtained in step S101 and the reference solution obtained in step S102 respectively, and spot them on the same silica gel G thin layer plate. Use chloroform-ethyl acetate-methanol-formic acid as the developing solvent, develop, remove, air dry, spray with vanillin sulfuric acid solution, and heat until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.
4. The quality control method for peony leaves as described in claim 3, characterized in that, In step S3, the volume ratio of chloroform, ethyl acetate, methanol and formic acid is 35-45:9-11:18-22:0.1-0.
3.
5. The quality control method for peony leaves as described in claim 1, characterized in that, The method for determining total flavonoids in peony leaves using spectrophotometry includes the following steps: S201. Dissolve apigenin reference standard in ethanol to obtain reference standard solution; S202. Take different volumes of reference solutions, add ethanol and triethylamine solution and mix well. Using the corresponding reagents as blanks, measure the absorbance by ultraviolet-visible spectrophotometry. Plot a standard curve with absorbance as the ordinate and concentration as the abscissa. S203. Peony leaves, after removing the thin film coating, are finely ground to obtain peony leaf powder. Ethanol is then added, and the mixture is sonicated and cooled. The weight loss is made up with ethanol, and the mixture is filtered. The filtrate is then added to ethanol to obtain the test solution. The absorbance is determined according to the method, and the weight of apigenin in the test solution is read from the standard curve. The total flavonoid content is calculated, and the total flavonoid content in each 0.5 g of peony leaves should not be less than 11.0 mg.
6. The quality control method for peony leaves as described in claim 5, characterized in that, In step S3, the absorbance is measured at a wavelength of 400 nm.
7. The quality control method for peony leaves as described in claim 1, characterized in that, The method for determining the paeoniflorin content in peony leaves using high performance liquid chromatography includes the following steps: S301. Add methanol to paeoniflorin reference standard, dissolve, and obtain reference standard solution; S302. Peony leaves are ground to remove the thin film coating and obtained as peony leaf powder. Ethanol is added to the peony leaves, ultrasonic treatment is performed, and then the mixture is cooled. Ethanol solution is added, shaken well, filtered, and the filtrate is collected to obtain the test solution. S303. Take blank control, reference solution and test solution respectively for determination. The paeoniflorin content in each 0.5g peony leaf should not be less than 5.8 mg.
8. The quality control method for peony leaves as described in claim 7, characterized in that, In step S3, high-performance liquid chromatography was performed using ShimPack Scepter C 18 The chromatographic column was used with a mobile phase of acetonitrile-0.085wt% phosphoric acid aqueous solution = 13-15∶85-87, a flow rate of 0.9-1.1 mL / min, a column temperature of 25-35℃, a detection wavelength of 230 nm, and an injection volume of 9-11 μL.
9. The quality control method for peony leaves as described in claim 1, characterized in that, The method for determining the content of apigenin 7-O-glucoside in peony leaves using high performance liquid chromatography includes the following steps: S401. Add methanol to apigenin 7-O-glucoside reference standard, dissolve, and obtain reference standard solution; S402. Peony leaves are ground to remove the thin film coating and then ground into peony leaf powder. Ethanol is added to the peony leaves and weighed. After ultrasonic treatment and cooling, the weight loss is made up with ethanol, shaken well and filtered. The filtrate is collected to obtain the test solution. S403. Take blank control, reference solution and test solution respectively for determination. The content of apigenin 7-O-glucoside in each 0.5g peony leaf should not be less than 0.95 mg.
10. The quality control method for peony leaves as described in claim 9, characterized in that, The high-performance liquid chromatography was performed using ShimPack Scepter C 18 The chromatographic column used acetonitrile as mobile phase A and 0.1 vt % formic acid aqueous solution as mobile phase B. The detection wavelength was 340 nm, the flow rate was 0.7-0.9 mL / min, the column temperature was 20-30℃, and the injection volume was 4-6 μL. The elution program for the high performance liquid chromatography was as follows: 0-5 min, mobile phase A: 20-22%, mobile phase B: 80-78%; 5-40 min, mobile phase A: 22-23%, mobile phase B: 78-77%.