Quality detection method of poria cocos and coix seed paste for children

Abstract

The invention discloses a quality detection method of an infantile poria cocos and coix seed paste, the detection method sequentially comprises the steps of preparation of the infantile poria cocos and coix seed paste, thin-layer identification, finished product inspection and content determination, and according to inspection items (general rule 0183) under oral thick paste items of four parts of Chinese Pharmacopoeia of 2020 edition, inspection items of characters, relative density, insoluble substances and water content are studied; identifying poria cocos, semen euryales and semen coicis in the root-benefiting conditioning paste by adopting a thin layer chromatography (TLC); according to the method, the content of pachymic acid serving as an effective component in the poria cocos and coix seed paste for children is measured and researched by adopting an HPLC (High Performance Liquid Chromatography) method, so that a basis is provided for quality control of the product, and the method is good in reproducibility, good in durability, simple and convenient, and capable of effectively controlling the quality of the poria cocos and coix seed paste for children.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a quality detection method for pediatric Lingyi Gao. Background Art

[0002] Xiaoer Lingyi Gao is a traditional Chinese medicine paste preparation used to treat diarrhea caused by weak spleen and stomach in children. It is composed of three herbs: Poria cocos, Euryale ferox, and Coix seed. According to literature research, the main chemical components of Poria cocos are polysaccharides and triterpenes. Its polysaccharides have the function of regulating intestinal flora and enhancing immunity. Poria cocos polysaccharides are soluble in water and insoluble in organic solvents such as high concentrations of ethanol. Euryale ferox is rich in starch, protein, multiple vitamins and minerals, etc. It has the effects of benefiting the kidney and consolidating essence, tonifying the spleen and stopping diarrhea. Its starch component helps to strengthen the spleen and stomach and provide gentle nourishment for the intestines. Coix seed contains active ingredients such as coix ester, coix seed oil, sterols, amino acids, vitamin B, etc. Its aqueous extract has anti-inflammatory and immune-regulating functions. Coix ester has a positive effect on improving digestive dysfunction caused by weak spleen and stomach, and can effectively relieve the symptoms of diarrhea in children and help restore the normal transportation and transformation function of the spleen and stomach.

[0003] The main active ingredients of pediatric Lingyi Gao come from Poria cocos, Euryale ferox and Coix seed. In order to study the quality level of pediatric Lingyi Gao, it is necessary to establish an efficient detection method to ensure that the pediatric Lingyi Gao with the best efficacy can be screened out. Summary of the invention

[0004] The purpose of the invention is to design a quality detection method for Xiaoer Lingyi Gao.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A quality detection method for pediatric Lingyi Gao, characterized in that: the detection method is the preparation of pediatric Lingyi Gao, thin layer identification, finished product inspection and content determination in sequence;

[0007] The thin layer identification method is as follows:

[0008] S1: Accurately pipette 10 mL of Xiaoer Lingyi Gao sample, treat with ultrasound and extraction, evaporate to dryness in a water bath at 80°C, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution;

[0009] S2: Accurately pipette 10 mL of the sample made with missing medicinal materials, and prepare the negative control solution using the same method as the test solution in S1;

[0010] S3: Accurately weigh the reference medicinal material and prepare the reference medicinal material solution in the same manner as the test solution preparation method in S1;

[0011] S4: Take the reference substance and add methanol to make a solution containing 1 mg per 1 mL as the reference substance solution;

[0012] S5: 10 μL of the test solution, negative control solution, and control medicinal material solution, i.e., the reference solution, are taken separately and spotted on the same silica gel G thin layer plate, using dot-shaped spots and a mixed solution of n-hexane-ethyl acetate-formic acid as a developing agent. The plate is developed, pre-saturated for 30 minutes, with a development distance of 8 cm, taken out, dried, sprayed with 2% vanillin sulfuric acid solution, heated at 105°C until the spots are clearly colored, and placed under sunlight for inspection; in the chromatogram of the test sample, the same fluorescent spots are shown at the corresponding positions of the chromatogram of the control medicinal material, and there is no interference with the negative control; the same method is used to conduct qualitative analysis on Euryale ferox and Coix seeds in turn;

[0013] The finished product inspection method is to check the properties, relative density, insoluble matter and water content of Xiaoer Lingyi Gao one by one;

[0014] Content determination method: The first step is the chromatographic conditions and system suitability test; the second step is to prepare the test solution; the third step is to prepare the reference solution; the fourth step is to determine the results.

[0015] Further, the preparation steps of Xiaoer Lingyi Gao are as follows:

[0016] (1) Weigh the following medicinal materials by weight: 6 parts of Poria cocos, 6 parts of Euryale ferox, and 6 parts of Coix seeds;

[0017] (2) Mix Poria cocos, Euryale ferox and Coix seeds evenly, add water equivalent to 10 times the weight of the mixed medicinal materials, extract for 100 min, extract 3 times, filter and concentrate;

[0018] (3) Combine the three concentrated solutions and concentrate to a relative density of 1.1-1.2, then add a certain amount of syrup; stir and mix continuously, and divide into packages to obtain the Xiaoer Lingyi Gao.

[0019] Furthermore, the volume ratio of n-hexane-ethyl acetate-formic acid mixture used for Poria cocos is 7:4:0.5; the volume ratio of n-hexane-acetone mixture used for Euryale ferox is 5:1; and the volume ratio of petroleum ether-ethyl ether-formic acid mixture used for Coix seed is 8.8:3.6:0.4.

[0020] Furthermore, the content was determined by high performance liquid chromatography, and the chromatographic column was Waters C18 (4.6×250mm×5μm), with acetonitrile:phosphoric acid water in a ratio of 75:25 as the mobile phase, the detection wavelength was 210nm; the flow rate was 0.6mL / min, the column temperature was 30℃, the injection volume was 10μL, and isocratic elution was performed.

[0021] The following beneficial effects can be obtained through the above technical solution:

[0022] The present invention studies the properties, relative density, insoluble matter and water content inspection items according to the inspection items under the four ointments of the 2020 edition of the "Chinese Pharmacopoeia" (General Rule 0183); uses thin layer chromatography (TLC) to identify the Poria cocos, Euryale ferox and Coix seeds in the Yigen conditioning ointment; and uses HPLC to determine the content of pachymic acid, an effective ingredient in the children's Lingyi ointment, in order to provide a basis for the quality control of the product. The method has good reproducibility, good durability and simplicity, and can effectively control the quality of the children's Lingyi ointment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the identification chromatogram of Poria cocos thin layer chromatography, where 1 is sample (20240901); 2 is sample (20240902); 3 is sample (20240903); 4 is Poria cocos reference material; 5 is negative control; 6 is pachymic acid reference.

[0024] Figure 2 This is the identification chromatogram of Gorgon Fruit Thin Layer Poria, where 1 is sample (20240901); 2 is sample (20240902); 3 is sample (20240903); 4 is Gorgon Fruit reference medicinal material; 5 is the negative control.

[0025] Figure 3 This is the identification chromatogram of coix seed and Poria cocos thin layer, where 1 is sample (20240901); 2 is sample (20240902); 3 is sample (20240903); 4 is coix seed control medicinal material; 5 is the negative control.

[0026] Figure 4 It is the HPLC chromatogram of blank solution, reference solution and test solution (the peak 1 in the figure represents Pachycolic acid). DETAILED DESCRIPTION

[0027] The following is combined with Figure 1-4 The present invention is further described as follows:

[0028] The present invention provides a method for detecting the quality standard of Xiaoer Lingyi Gao, and the method is as follows

[0029] Step 1: Preparation of Lingyi Gao for Children

[0030] Weigh the following medicinal materials by weight: 6 parts of Poria cocos, 6 parts of Euryale ferox, and 6 parts of Coix seeds, mix them evenly, add water equivalent to 10 times the weight of the mixed medicinal materials, extract 3 times, 100 minutes each time, filter, and concentrate; combine the 3 concentrates, concentrate to a relative density of 1.1-1.2, and add a certain amount of syrup. Boil for 30 minutes, stir and mix continuously, and divide into packages to obtain Xiaoer Lingyi Gao. The amount of syrup added is 1:2 of clear paste to syrup.

[0031] Step 1: Thin layer identification of Lingyi Gao for children

[0032] TLC was used to identify the three components of Poria cocos, Euryale ferox and Coix seeds in Xiaoer Lingyi Gao, and qualitative analysis was performed, including the following contents:

[0033] (1) TLC Identification of Poria

[0034] ① Preparation of test solution

[0035] Accurately weigh 10 g of Xiaoer Lingyi Gao, add 50 mL of ether, ultrasonically treat for 15 min, filter, evaporate to dryness in a water bath at 80°C, add 1 mL of methanol to the residue to dissolve, and obtain the test solution. Prepare 3 copies in parallel.

[0036] ②Preparation of negative control solution

[0037] Take the prescription medicinal material lacking Poria cocos, and prepare the Poria cocos paste prescription according to the preparation process. Prepare the negative control solution in the same way as the test sample preparation method.

[0038] ③ Preparation of reference medicinal material solution

[0039] Take 1 g of Poria cocos control medicinal material, add 50 mL of ether, and prepare the control medicinal material solution in the same way as the test sample preparation method.

[0040] ④ Preparation of reference solution

[0041] Take 1 mg of pachymic acid reference substance and add methanol to prepare a 1 mg / mL pachymic acid solution as the reference substance solution.

[0042] ⑤Methods

[0043] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 10 μL of each of the above solutions were taken and spotted on the same silica gel G thin layer plate, using n-hexane-ethyl acetate-formic acid (7:4:0.5) as the developing solvent, pre-saturated for 30 minutes, developed, with a development distance of 8 cm, taken out, dried, sprayed with 2% vanillin sulfuric acid solution, heated at 105°C until the spots were clearly colored, and placed under sunlight for inspection. In the chromatogram of the test sample, the same fluorescent spots appeared at the corresponding position of the chromatogram of the control medicinal material, and there was no interference with the negative control. See the results. Figure 1 .

[0044] (2) TLC Identification of Gorgon Fruit

[0045] ① Preparation of test solution

[0046] Accurately weigh 10 g of Xiaoer Lingyi Gao, add 30 mL of dichloromethane, extract twice, combine the chloroform extracts, evaporate to dryness in a water bath at 80°C, dissolve the residue in 1 mL of ethyl acetate to obtain the test solution, and prepare 3 copies in parallel.

[0047] ②Preparation of negative control solution

[0048] Take the prescription medicinal materials without Gorgon Fruit, prepare the Gorgon Fruit paste prescription according to the preparation process, and prepare the negative control solution with the same preparation method as the test sample.

[0049] ③ Preparation of reference medicinal material solution

[0050] Take 0.5 g of Euryale ferox reference medicinal material, add 15 mL of dichloromethane, ultrasonically treat for 30 min, filter, evaporate to dryness in a water bath at 80°C, add 1 mL of ethyl acetate to the residue to dissolve it, and obtain a reference medicinal material solution.

[0051] ④Methods

[0052] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 10 μL of each of the above solutions was taken and spotted on the same silica gel G thin layer plate, using n-hexane-acetone (5:1) as the developing agent, pre-saturated for 30 minutes, developed, with a development distance of 8 cm, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and placed under ultraviolet light (365nm) for inspection. In the chromatogram of the test sample, the same fluorescent spots appeared at the corresponding position of the chromatogram of the control medicinal material, and there was no interference with the negative control. See the results. Figure 2 .

[0053] (3) TLC Identification of Coix Seed

[0054] ① Preparation of test solution

[0055] Accurately weigh 10 g of Xiaoer Lingyi Gao, add 30 mL of petroleum ether, ultrasonically treat for 30 min, filter, evaporate to dryness in a water bath at 80°C, add 1 mL of petroleum ether to the residue to dissolve it to obtain the test solution, and prepare 3 copies in parallel.

[0056] ②Preparation of negative control solution

[0057] Take the prescription medicinal materials lacking coix seed, and prepare the paste recipe lacking coix seed according to the preparation process, and prepare the negative control solution with the same preparation method as the test sample.

[0058] ③ Preparation of reference medicinal material solution

[0059] Take 1 g of Euryale ferox reference medicinal material, add 30 mL of petroleum ether, ultrasonically treat for 30 min, filter, evaporate to dryness in a water bath at 80°C, add 1 mL of petroleum ether to the residue and dissolve it to obtain a reference medicinal material solution.

[0060] ④Methods

[0061] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 10 μL of each of the above solutions were taken and spotted on the same silica gel G thin layer plate, with petroleum ether-ether-formic acid (8.8:3.6:0.4) as the developing agent, pre-saturated for 30 minutes, developed, with a development distance of 8 cm, taken out, dried, sprayed with 2% vanillin sulfuric acid solution, heated at 105°C until the spots were clearly colored, and placed under sunlight for inspection. In the chromatogram of the test sample, the same fluorescent spots appeared at the corresponding position of the chromatogram of the control medicinal material, and there was no interference with the negative control. See the results. Figure 3 .

[0062] Step 3: Finished product inspection of Lingyi Gao for children

[0063] ①Characteristics

[0064] The appearance, shape, color, smell and taste of three batches of Xiaoer Lingyi Gao were observed, and the results are shown in Table 1. The product is dark brown semi-fluid with uniform color and sweet smell.

[0065] Table 1 Properties of three batches of samples

[0066]

[0067] ②Relative density

[0068] The relative density was determined according to the general rules of relative density method (0601) in Part IV of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 2. The relative density of the six batches of Xiaoer Lingyi Gao was stable and fluctuated within a certain range. According to the maximum relative density of Xiaoer Lingyi Gao was 1.29 and the minimum was 1.26, considering the impact of the environment on the relative density, the relative density of this product was tentatively determined to be no less than 1.25.

[0069] Table 2 Relative density measurement results (n=6)

[0070]

[0071] ③Insoluble matter

[0072] Take 3 batches of Lingyi Gao for children. According to the fourth ointment item of the 2020 edition of the Chinese Pharmacopoeia (General Rule 0183), take 5g of Lingyi Gao for children respectively, add 200mL of hot water, stir to dissolve, and observe after standing for 3 minutes. There is no burnt debris or foreign matter in the three batches of Lingyi Gao for children. The results are shown in Table 3.

[0073] Insoluble matter determination results (n=3)

[0074]

[0075] ④Water content

[0076] Six batches of pediatric Lingyi Gao were taken and measured according to the drying method under the moisture determination method of Part IV of the 2020 edition of the "Chinese Pharmacopoeia" (General Rule 0832). The results are shown in Table 4. The results show that the maximum water content is 21.11% and the minimum is 18.89%. The water content is tentatively set at 18%-22% based on appropriate adjustments according to the situation.

[0077] Table 4 Water content measurement results (n = 6)

[0078]

[0079] Step 4: Determination of the content of Xiaoer Lingyi Gao

[0080] The content of pachymic acid in Xiaoer Lingyi Gao was detected by high performance liquid chromatography and quantitative analysis was performed, including the following contents:

[0081] ①Chromatographic conditions: Shimadzu high performance liquid chromatograph LC-20ADxr. Chromatographic column: Waters C18 5μm (4.6×250mm). Mobile phase: acetonitrile: phosphoric acid water (75:25). Flow rate: 0.6mL / min, column temperature: 30℃, detection wavelength: 210nm.

[0082] ② Preparation of test solution: Take 1 g of Xiaoer Lingyi Gao, weigh it accurately, put it in a stoppered conical flask, add 10 mL of methanol solution accurately, weigh it, treat it with ultrasound for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, filter it, and take the filtrate to obtain the solution.

[0083] ③ Preparation of reference solution: Accurately weigh 2 mg of pachymic acid reference substance, place it in a 10 mL volumetric flask, accurately add 10 mL of methanol solution to obtain pachymic acid reference substance solution (200 μg / mL).

[0084] ④ Drawing of standard curve:

[0085] Accurately measure 50%, 80%, 100%, 120%, and 150% of the reference solution in step ③ and place them in injection vials. Then inject 0.6 μL of the sample according to the chromatographic conditions described in step 1.1, and draw a standard curve with the reference concentration as the abscissa (X) and the peak area as the ordinate (Y). The standard curve of Pachycolic acid is y=8005.2x-61340 (r=0.9997), and the linear range is: 54-162 μg / mL.

[0086] ⑤Stability test: Take the same test solution and inject it at 0, 4, 8, 12, 16, and 24 hours, record the peak area and calculate the RSD. The results show that the RSD of the pachymic acid peak area is 0.69%, and the sample solution is stable within 24 hours.

[0087] Table 5 Stability test results (n=6)

[0088]

[0089] ⑥Precision test: Take the same reference solution, inject it 6 times continuously, record the peak area and calculate the RSD. The results show that the RSD of the peak area of ​​pachymic acid is 1.81%, indicating that the precision of this method is good.

[0090] Table 6 Precision test results (n = 6)

[0091]

[0092] ⑦ Repeatability test: Prepare 6 test solutions according to the preparation method of the test solution in step ②, inject and measure them respectively, record the peak area and calculate the content of pachymic acid and RSD. The results show that the RSD of pachymic acid is 0.76%, indicating that the method has good repeatability.

[0093] Table 7 Repeatability test results (n=6)

[0094]

[0095]

[0096] ⑧ Sample recovery test: Accurately measure 0.5mL of the test solution with known content, add 0.5mL of the reference solution to make a 100% spiked test solution, repeat 6 times, continuously inject and analyze, calculate the recovery rate and average recovery rate. The results show that the average recovery rate of pachymic acid is 104.33%, and the RSD is 3.52%, indicating that this method is feasible.

[0097] Table 8 Sample recovery test results (n = 6)

[0098]

[0099] ⑨Content determination

[0100] Take 3 batches of pediatric ointment and prepare 3 test solutions according to the preparation method of test solution in step ②. Operate according to the chromatographic conditions in step ①, record the peak area, calculate RSD, and the results are shown in Table 9.

[0101] Table 9 Content determination results (n=3)

[0102]

[0103] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention belong to the protection scope of the claims attached to this application.

Claims

1. A quality detection method for Xiaoer Lingyi Gao, characterized in that: The detection methods are preparation, thin layer identification, finished product inspection and content determination of Xiaoer Lingyi Gao; The thin layer identification method is as follows: S1: Accurately pipette 10 mL of Xiaoer Lingyi Gao sample, treat with ultrasound and extraction, evaporate to dryness in a water bath at 80°C, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution; S2: Accurately pipette 10 mL of the sample made with missing medicinal materials, and prepare the negative control solution using the same method as the test solution in S1; S3: Accurately weigh the reference medicinal material and prepare the reference medicinal material solution in the same manner as the test solution preparation method in S1; S4: Take the reference substance and add methanol to make a solution containing 1 mg per 1 mL as the reference substance solution; S5: 10 μL of the test solution, negative control solution, and control medicinal material solution (i.e., reference solution) were taken respectively, and spotted on the same silica gel G thin layer plate, using dot pattern, a mixed solution of n-hexane-ethyl acetate-formic acid as the developing agent, developed, pre-saturated for 30 minutes, with a development distance of 8 cm, taken out, dried, sprayed with 2% vanillin sulfuric acid solution, heated at 105°C until the spots were clearly colored, and placed under sunlight for inspection; in the chromatogram of the test sample, the same fluorescent spots were shown at the corresponding positions of the chromatogram of the control medicinal material, and there was no interference with the negative control; the same method was used to conduct qualitative analysis on Euryale ferox and Coix seeds in turn; The finished product inspection method is to check the properties, relative density, insoluble matter and water content of Xiaoer Lingyi Gao one by one; Content determination method: The first step is the chromatographic conditions and system suitability test; the second step is to prepare the test solution; the third step is to prepare the reference solution; the fourth step is to determine the results.

2. The quality detection method of Xiaoer Lingyi Gao according to claim 1, characterized in that: The preparation steps of Xiaoer Lingyi Gao are as follows: (1) Weigh the following medicinal materials by weight: 6 parts of Poria cocos, 6 parts of Euryale ferox, and 6 parts of Coix seeds; (2) Mix Poria cocos, Euryale ferox and Coix seeds evenly, add water equivalent to 10 times the weight of the mixed medicinal materials, extract for 100 min, extract 3 times, filter and concentrate; (3) Combine the three concentrated solutions and concentrate to a relative density of 1.1-1.2, then add a certain amount of syrup; stir and mix continuously, and divide into packages to obtain the Xiaoer Lingyi Gao.

3. The quality detection method of Xiaoer Lingyi Gao according to claim 1, characterized in that: The volume ratio of n-hexane-ethyl acetate-formic acid mixture used for the Poria cocos is 7:4:0.5; the volume ratio of n-hexane-acetone mixture used for the Euryale ferox is 5:1; the volume ratio of petroleum ether-ethyl ether-formic acid mixture used for the Coix seeds is 8.8:3.6:0.

4.

4. The quality detection method of Xiaoer Lingyi Gao according to claim 1, characterized in that: The content was determined by high performance liquid chromatography with a Waters Symmetry ® C18 column (4.6×250mm×5μm), acetonitrile:phosphoric acid water in a ratio of 75:25 as the mobile phase, a detection wavelength of 210nm, a flow rate of 0.6mL / min, a column temperature of 30°C, an injection volume of 10μL, and isocratic elution.

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