A thin layer chromatography quality control method for bran-fried gorgon fruit formula granules and its application

Abstract

The present invention provides a thin layer chromatography quality control method for bran-fried gorgon fruit granules and its application. The thin layer chromatography quality control method comprises the following steps: (1) mixing a sample to be tested with alcohol and refluxing to obtain a test solution; (2) mixing a bran-fried gorgon fruit reference medicinal material with water and refluxing to obtain a control medicinal material solution; (3) mixing a reference substance with alcohol to obtain a reference substance solution; (4) subjecting the test solution, the control medicinal material solution, and the reference substance solution to thin layer chromatography testing, and judging the quality of the sample to be tested based on the test results. The method provided by the present invention discards starch components, effectively reduces the interference of starch components, and improves the accuracy of the test results.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine quality control, and specifically relates to a thin layer chromatography quality control method for bran-fried gorgon fruit formula granules and an application thereof, and especially relates to a thin layer chromatography quality control method for bran-fried gorgon fruit formula granules with low interference and an application thereof. Background Art

[0002] Gorgon fruit is the dried, mature kernel of the Euryale ferox (Euryale ferox) plant of the Nymphaeaceae family. Gorgon fruit has a long history of medicinal use in my country and is a renowned traditional herbal medicine. It is mild in nature, sweet, and astringent in flavor, and enters the spleen and kidney meridians. It is a tonic Chinese herbal medicine, known for its benefits of strengthening the kidneys and consolidating essence, tonifying the spleen and stopping diarrhea, and removing dampness and stopping leukorrhea. It is used to treat nocturnal emission, spermatorrhea, enuresis, frequent urination, chronic diarrhea due to spleen deficiency, leukorrhea, and leukorrhea. The 2020 edition of the Pharmacopoeia specifies testing items including herbal properties, identification, and inspection.

[0003] Fried gorgon fruit with bran is a processed gorgon fruit with a bright yellow or yellowish surface and a slight aroma. Fried gorgon fruit with bran has a warming nature, enhancing its spleen-tonifying and astringent properties. It is suitable for spleen deficiency and those with more deficiency than excess. Fried gorgon fruit with bran has similar efficacy to stir-fried gorgon fruit, both emphasizing spleen-tonifying and astringent properties. It is primarily used for diarrhea caused by spleen deficiency and spermatorrhea caused by kidney deficiency and spermatorrhea. It can also be used for leukorrhea caused by spleen deficiency.

[0004] Currently, thin-layer chromatography (TLC) methods for analyzing gorgon fruit (Euphorbia glutinosa) are generally based on pharmacopoeial methods. The components of the aqueous extract of gorgon fruit tested in these methods are primarily starch. However, during the preparation of gorgon fruit granules, starch-containing excipients such as maltodextrin are artificially added, which interferes with the detection and makes it difficult to authenticate the granules. Therefore, finding a TLC method for analyzing gorgon fruit granules that can effectively reduce starch interference has become an urgent problem to be solved. Summary of the Invention

[0005] In response to the shortcomings of the prior art, the present invention aims to provide a thin-layer chromatography quality control method for bran-fried gorgon fruit granules and its application, particularly to provide a thin-layer chromatography quality control method for bran-fried gorgon fruit granules with low interference and its application. The method provided by the present invention eliminates starch components, effectively reducing the interference of starch components and improving the accuracy of the test results.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides a thin layer chromatography quality control method for bran-fried gorgon fruit formula granules, the thin layer chromatography quality control method comprising the following steps:

[0008] (1) Mixing the sample to be tested with alcohol and refluxing to obtain a test solution;

[0009] (2) mixing the bran-fried Euryale ferox seed control medicinal material with water and refluxing to obtain a control medicinal material solution;

[0010] (3) mixing the reference substance with alcohol to obtain a reference substance solution;

[0011] (4) The test sample solution, the control medicinal material solution and the reference substance solution are subjected to thin layer chromatography, and the quality of the test sample is determined based on the test results.

[0012] Steps (1), (2), and (3) are performed in no particular order.

[0013] The above method processes the sample and then performs thin layer chromatography detection in combination with reference substances and reference medicinal materials, discarding starch components in the detection, effectively reducing the interference of starch components and improving the accuracy of the detection results.

[0014] Preferably, the alcohol comprises ethanol.

[0015] Preferably, the material-liquid ratio of the sample to be tested to the alcohol solution in step (1) is 1:(10-20) g / mL, for example 1:10 g / mL, 1:11 g / mL, 1:12 g / mL, 1:13 g / mL, 1:14 g / mL, 1:15 g / mL, 1:16 g / mL, 1:17 g / mL, 1:18 g / mL, 1:19 g / mL or 1:20 g / mL, etc., but is not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are also applicable.

[0016] Preferably, the material-liquid ratio of the bran-fried Euryale ferox control medicinal material to water in step (2) is 1: (10-20) g / mL, for example 1: 10 g / mL, 1: 11 g / mL, 1: 12 g / mL, 1: 13 g / mL, 1: 14 g / mL, 1: 15 g / mL, 1: 16 g / mL, 1: 17 g / mL, 1: 18 g / mL, 1: 19 g / mL or 1: 20 g / mL, etc., but is not limited to the above-listed values, and other unlisted values ​​within the above numerical range are equally applicable.

[0017] Preferably, the reference substance in step (3) comprises gallic acid and / or linoleic acid.

[0018] Preferably, the reference substance in step (3) comprises gallic acid and linoleic acid.

[0019] Preferably, in the thin layer chromatography detection in step (4), the developing solvent is a mixed solution of chloroform, ethyl formate and formic acid.

[0020] The above-mentioned specific developing agent combination can effectively analyze the characteristic spots in the test samples and reference substances, reduce the interference of starch components, and improve the accuracy of the test results.

[0021] Preferably, in the mixed solution of chloroform, ethyl formate and formic acid, the volume ratio of chloroform, ethyl formate and formic acid is (4-6):(4-6):(0.8-1.2), wherein the number of parts of chloroform can be 4, 4.5, 5, 5.5 or 6, the number of parts of ethyl formate can be 4, 4.5, 5, 5.5 or 6, and the number of parts of formic acid can be 0.8, 0.9, 1, 1.1 or 1.2, but are not limited to the values ​​listed above. Other values ​​not listed within the above numerical range are also applicable.

[0022] On the other hand, the present invention also provides the application of the thin layer chromatography quality control method described above in the detection of bran-fried gorgon fruit formula granules.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a thin-layer chromatography quality control method for bran-fried gorgon fruit formula granules. The method comprises the following steps: processing a sample and then performing thin-layer chromatography detection in combination with a reference substance and a reference medicinal material; discarding starch components in the detection; effectively reducing the interference of starch auxiliary materials such as maltodextrin artificially added in the prior art on the detection; effectively distinguishing bran-fried gorgon fruit formula granules from other formula granules added with starch auxiliary materials such as maltodextrin, avoiding the false negative problem in the prior method, and improving the accuracy of the detection result; and adopting reference substances and reference medicinal materials for multi-faceted and multi-angle comparison, which can more comprehensively and effectively detect the bran-fried gorgon fruit formula granules, thereby ensuring the comprehensiveness and accuracy of quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a thin layer identification chromatogram of the bran-fried Euryale ferox reference medicinal material and the bran-fried Euryale ferox formula granules in the durability and specificity investigation in Example 1;

[0026] Figure 2 This is a thin layer identification chromatogram of the reference substance, the bran-fried gorgon fruit reference medicinal material, and the bran-fried gorgon fruit formula granules in the durability and specificity investigation in Example 1;

[0027] Figure 3 This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granules at room temperature (26° C.) in Example 1;

[0028] Figure 4 This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granules at low temperature (6° C.) in Example 1;

[0029] Figure 5 This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granules under high humidity (relative humidity 88%) in Example 1;

[0030] Figure 6This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granules under low humidity (relative humidity 23%) in Example 1;

[0031] Figure 7 This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granules using Merck silica gel G plate in Example 1;

[0032] Figure 8 This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granules using the silica gel G plate produced by Tianjin Silida Technology Co., Ltd. in Example 1;

[0033] Figure 9 This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granules using the silica gel G plate of Yantai Huayang New Materials Technology Co., Ltd. in Example 1;

[0034] Figure 10 1 is a thin layer identification chromatogram of three batches of bran-fried gorgon fruit formula granule samples in Example 1;

[0035] Figure 11 1 is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granule sample in Example 2;

[0036] Figure 12 This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granule sample in Example 3;

[0037] Figure 13 This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granule sample in Example 4;

[0038] Figure 14 This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granule sample in Example 5;

[0039] Figure 15 This is a thin layer identification chromatogram of the bran-fried gorgon fruit formula granule sample in Example 6;

[0040] Figure 16 This is the thin layer identification chromatogram in Comparative Example 1;

[0041] Figure 17 This is the thin layer identification chromatogram in Comparative Example 2. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0043] In the following example, the sources of raw materials and consumables are as follows:

[0044] Test drugs: reference medicinal materials of fried gorgon fruit with bran (batch number: K145YC01, homemade), fried gorgon fruit formula granules with bran (batch number: K145CP01, K145CP06, K145CP11), linoleic acid (batch number: 111622-202105, China Food and Drug Inspection Institute); gallic acid reference substance (batch number: 110831-201906, China Food and Drug Inspection Institute).

[0045] Reagents: Ethyl formate, formic acid, chloroform, and anhydrous ethanol were of analytical grade, and water was ultrapure water.

[0046] Consumables: Merck silicone G plate (specification: 200 × 200 mm; batch number: 1.05721.0001), Yantai Huayang New Material Technology Co., Ltd. silicone G plate (model: HSG-G, specification: 100 × 200 mm, batch number: 20210309), Tianjin Silida Technology Co., Ltd. silicone G plate (model: HSG-G, specification: 100 × 200 mm, batch number: 20210707).

[0047] Example 1

[0048] This embodiment provides a thin layer chromatography quality control method for bran-fried gorgon fruit formula granules, and the specific steps are as follows:

[0049] Preparation of the test solution: Take 2.0 g of the granules of fried gorgon fruit with bran, grind them into powder, add 50 mL of anhydrous ethanol, heat and reflux for 30 minutes, filter, and concentrate the filtrate to 2 mL as the test solution.

[0050] Preparation of control medicinal material solution: Take 12.0 g of bran-fried Euryale ferox seed control medicinal material, add 200 mL of water, reflux for 30 minutes, centrifuge (4000 rpm, 20 minutes), take the supernatant to a 100 mL conical flask and concentrate to dryness under reduced pressure, add 60 mL of anhydrous ethanol to the residue, heat and reflux for 30 minutes, filter, and concentrate the filtrate to 2 mL as the control medicinal material solution.

[0051] Preparation of reference solution: Take linoleic acid reference substance and add anhydrous ethanol to make a solution containing 0.5 mg per 1 mL as the reference solution.

[0052] Take gallic acid reference substance and add anhydrous ethanol to make a solution containing 0.2 mg per 1 mL as the reference solution.

[0053] Preparation of negative control solution: According to the proportion of excipients in the formula of fried gorgon fruit granules with bran, take an appropriate amount of maltodextrin and prepare it in the same way as the test solution as the negative control solution.

[0054] Durability and specificity inspection

[0055] Investigation on different spot amounts and specificity of test and control medicinal material solutions

[0056] 15 μL, 20 μL, 25 μL, and 30 μL of the bran-fried Euryale Root control medicinal material solution, 10 μL, 15 μL, 20 μL, and 25 μL of the bran-fried Euryale Root formula granules (batch number: K145CP01) test sample solution, 3 μL, 4 μL, and 5 μL of the linoleic acid reference solution, and 3 μL, 4 μL, and 5 μL of the gallic acid reference solution were respectively aspirated and spotted onto a silica gel G plate (Silica G plate, Yantai Huayang New Materials Technology Co., Ltd.) using chloroformane-ethyl formate-formic acid (volume ratio 5:5:1) as the developing solvent. The plate was developed, removed, air-dried, sprayed with phosphomolybdic acid test solution, and heated at 105°C for 3 min until the spots were clearly colored. The plates were then examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions in the chromatograms of the reference sample and the control medicinal material.

[0057] The experimental results are as follows Figure 1 (1. Fried gorgon fruit with bran as control material (15μL); 2. Fried gorgon fruit with bran as control material (20μL); 3. Fried gorgon fruit with bran as control material (25μL); 4. Fried gorgon fruit with bran as control material (30μL); 5. Fried gorgon fruit formula granules (K145CP01) test sample (10μL); 6. Fried gorgon fruit formula granules (K145CP01) test sample (15μL); 7. Fried gorgon fruit formula granules (K145CP01) test sample (20μL); 8. Fried gorgon fruit formula granules (K145CP01) test sample (25μL)) and Figure 2 (1. Linoleic acid reference substance (3μL); 2. Linoleic acid reference substance (4μL); 3. Linoleic acid reference substance (5μL); 4. Gallic acid reference substance (3μL); 5. Gallic acid reference substance (4μL); 6. Gallic acid reference substance (5μL); 7. Fried gorgon fruit with bran reference medicinal material (20μL); 8. Fried gorgon fruit formula granules (K145CP01) test sample (15μL)).

[0058] Depend on Figure 1-2 It can be seen that the thin layer chromatographic spots of the test samples with different spot amounts are all clear, without tailing phenomenon, the spot separation is good, all can present relatively clear spots, and the number does not change, indicating that fine-tuning of the spot amount has no effect on the detection; the thin layer chromatographic spots of the control medicinal materials with different spot amounts are all clear, without tailing phenomenon, the spot separation is good, all can present relatively clear spots, and the number does not change, indicating that fine-tuning of the spot amount has no effect on the detection.

[0059] Investigation at different temperatures

[0060] ①Normal temperature inspection

[0061] Experimental Conditions: Normal temperature refers to 10-30°C, relative humidity is controlled at 45%-75%. Place the spotted thin-layer plate in a developing chamber, then add the developing agent. Seal and place for 20 minutes (saturation). Develop, remove, air dry, spray with phosphomolybdic acid solution, and heat at 105°C for 3 minutes until the spots are clearly colored. Inspect under sunlight. The test sample chromatogram should show spots of the same color at the corresponding positions in the chromatograms of the reference sample and reference medicinal material.

[0062] Experimental operation method: 15 μL of maltodextrin negative solution, 15 μL of the test solution of bran-fried gorgon fruit formula granules (batch number: K145CP01), 20 μL of bran-fried gorgon fruit control medicinal material solution, 4 μL of linoleic acid reference substance, and 4 μL of gallic acid reference substance were respectively aspirated and spotted on the same silica gel G plate. Chloroformane-ethyl formate-formic acid (volume ratio 5:5:1) was used as the developing agent. The plate was developed at room temperature (temperature: 26°C, relative humidity: 60%). The plate was taken out, dried, sprayed with phosphomolybdic acid test solution, heated at 105°C for 3 minutes until the spots were clearly colored, and inspected under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference sample and the control medicinal material. The experimental results are shown in Figure 3 (1. Linoleic acid reference substance (4 μL); 2. Gallic acid reference substance (4 μL); 3. Fried gorgon fruit with bran reference medicinal material (20 μL); 4. Fried gorgon fruit with bran formula granules (K145CP01) test sample (15 μL); 5. Maltodextrin negative solution (15 μL)).

[0063] ② Low temperature (4-10℃) inspection

[0064] Experimental Conditions: Use a refrigerator set to 4°C, maintaining the temperature between 4 and 10°C. Place the spotted thin-layer plate in a developing chamber, add the developing agent, and seal for 20 minutes (saturation). Develop the plate, remove it, air-dry it, spray it with phosphomolybdic acid solution, and heat it at 105°C for 3 minutes until the spots are clearly colored. Inspect the plate under sunlight. The test sample chromatogram should display spots of the same color at the corresponding positions on the chromatograms of the reference sample and reference medicinal material.

[0065] Experimental operation method: 15 μL of maltodextrin negative solution, 15 μL of the test solution of bran-fried gorgon fruit formula granules (batch number: K145CP01), 20 μL of bran-fried gorgon fruit control medicinal material solution, 4 μL of linoleic acid reference substance, and 4 μL of gallic acid reference substance were respectively aspirated and spotted on the same silica gel G plate. Chloroformane-ethyl formate-formic acid (volume ratio 5:5:1) was used as the developing agent. The plate was developed at low temperature (temperature: 6°C, relative humidity: 70%). The plate was taken out, dried, sprayed with phosphomolybdic acid solution, heated at 105°C for 3 minutes until the spots were clearly colored, and inspected under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference sample and the control medicinal material. The experimental results are shown in Figure 4(1. Linoleic acid reference substance (4 μL); 2. Gallic acid reference substance (4 μL); 3. Fried gorgon fruit with bran reference medicinal material (20 μL); 4. Fried gorgon fruit with bran formula granules (K145CP01) test sample (15 μL); 5. Maltodextrin negative solution (15 μL)).

[0066] Depend on Figure 3-4 It can be seen that the experimental results show that the spots of the same color can be seen in the chromatogram of the test sample of the glutinous rice flour formula granules at the corresponding positions in the chromatogram of the glutinous rice flour control medicinal material, and the chromatographic spot separation effect is good.

[0067] Investigation of different humidity (high humidity, low humidity)

[0068] High Humidity (88% Relative Humidity) Experimental Condition Control Method: This experiment examines the effect of 88% relative humidity on thin-layer identification. A solution of 108 mL of sulfuric acid, 1000 mL of water, and a uniform mixture is prepared (placed in a desiccator). The spotted thin-layer plate is placed in a developing cylinder in the desiccator, and then the developing agent is added. The plate is sealed and allowed to stand for 20 minutes (saturation). After development, the plate is removed and sprayed with phosphomolybdic acid solution. Heat at 105°C for 3 minutes until the spots are clearly colored. The plate is then examined under sunlight. The test sample chromatogram should display spots of the same color at the corresponding positions in the chromatograms of the reference sample and reference medicinal material.

[0069] Experimental operation method: 15 μL of maltodextrin negative solution, 15 μL of the test solution of bran-fried gorgon fruit formula granules (batch number: K145CP01), 20 μL of bran-fried gorgon fruit control medicinal material solution, 4 μL of linoleic acid reference substance, and 4 μL of gallic acid reference substance were respectively absorbed and spotted on the same silica gel G plate. Chloroformane-ethyl formate-formic acid (volume ratio 5:5:1) was used as the developing agent. The plate was developed in high humidity (temperature: 26°C, relative humidity: 88%). The plate was taken out, dried, sprayed with phosphomolybdic acid test solution, heated at 105°C for 3 minutes until the spots were clearly colored, and inspected under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference sample and the control medicinal material. The experimental results are shown in Figure 5 (1. Linoleic acid reference substance (4 μL); 2. Gallic acid reference substance (4 μL); 3. Fried gorgon fruit with bran reference medicinal material (20 μL); 4. Fried gorgon fruit with bran formula granules (K145CP01) test sample (15 μL); 5. Maltodextrin negative solution (15 μL)).

[0070] Low Humidity (23% Relative Humidity) Experimental Condition Control Method: This experiment examines the effect of 23% relative humidity on thin-layer identification. Dry beads were placed in a desiccator. The spotted thin-layer plate was placed in a developing jar. The developing agent was then added and sealed for 20 minutes (saturation). The plate was then developed, removed, air-dried, and sprayed with phosphomolybdic acid solution. The plate was heated at 105°C for 3 minutes until the spots were clearly colored. The plates were then examined under sunlight. The test sample chromatogram should show spots of the same color at the corresponding positions on the chromatograms of the reference sample and reference medicinal material.

[0071] Experimental operation method: 15 μL of maltodextrin negative solution, 15 μL of the test solution of bran-fried gorgon fruit formula granules (batch number: K145CP01), 20 μL of bran-fried gorgon fruit control medicinal material solution, 4 μL of linoleic acid reference substance, and 4 μL of gallic acid reference substance were respectively absorbed and spotted on the same silica gel G plate. Chloroformane-ethyl formate-formic acid (volume ratio 5:5:1) was used as the developing agent. The plate was developed in low humidity (temperature: 26°C, relative humidity: 23%). The plate was taken out, dried, sprayed with phosphomolybdic acid solution, heated at 105°C for 3 minutes until the spots were clearly colored, and inspected under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference sample and the control medicinal material. The experimental results are shown in Figure 6 (1. Linoleic acid reference substance (4 μL); 2. Gallic acid reference substance (4 μL); 3. Fried gorgon fruit with bran reference medicinal material (20 μL); 4. Fried gorgon fruit with bran formula granules (K145CP01) test sample (15 μL); 5. Maltodextrin negative solution (15 μL)).

[0072] Depend on Figure 5-6 As can be seen, under both high and low humidity conditions, the chromatograms of the test sample of the bran-fried gorgon fruit granules showed spots of the same color at the corresponding positions in the chromatograms of the bran-fried gorgon fruit control medicinal material, and the chromatographic spot separation was good in both cases. The experimental results show that humidity has no significant effect on the thin-layer chromatography identification of the bran-fried gorgon fruit granules, and the thin-layer chromatography identification method has good humidity resistance.

[0073] Investigation of thin layer boards from different manufacturers

[0074] (1) Merck silica gel G plate (specification: 200 × 200 mm; batch number: 1.05721.0001)

[0075] (2) Silica gel G plate (model: HSG-G, specification: 100×200 mm, batch number: 20210309) produced by Yantai Huayang New Materials Technology Co., Ltd.

[0076] (3) Tianjin Silida Technology Co., Ltd. Silicone G plate (model: HSG-G, specification: 100×200mm, batch number: 20210707)

[0077] Take one piece of each of the above brands of prefabricated plates and operate according to the above method. Separately aspirate 15μL of maltodextrin negative solution, 15μL of the test solution of bran-fried gorgon fruit formula granules (batch number: K145CP01), 20μL of bran-fried gorgon fruit control medicinal material solution, 4μL of linoleic acid reference substance, and 4μL of gallic acid reference substance, and spot them on the same silica gel G plate. Use chloroform-ethyl formate-formic acid (volume ratio 5:5:1) as the developing agent, develop, take out, dry, spray with phosphomolybdic acid test solution, heat at 105°C for 3 minutes until the spots are clearly colored, and inspect under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference sample and the control medicinal material. See the experimental results. Figure 7-9 (1. Linoleic acid reference substance (4 μL); 2. Gallic acid reference substance (4 μL); 3. Fried gorgon fruit with bran reference medicinal material (20 μL); 4. Fried gorgon fruit with bran formula granules (K145CP01) test sample (15 μL); 5. Maltodextrin negative solution (15 μL)).

[0078] Depend on Figure 7-9 It can be seen that the separation effects of the three brands of silica gel G plates are similar. The chromatogram of the test sample of the glutinous rice flour granules shows spots of the same color at the corresponding positions of the chromatograms of the glutinous rice flour reference medicinal materials and reference substances. The experimental results show that thin layer plates from different manufacturers have no significant effect on the thin layer identification of the glutinous rice flour granules. The thin layer identification method has good durability and is suitable for the experimental conditions.

[0079] The above method was used to perform thin layer chromatography identification on three batches of bran-fried gorgon fruit granule samples. The results were as follows: Figure 10 As shown (1. linoleic acid reference substance (4 μL); 2. gallic acid reference substance (4 μL); 3. bran-fried gorgon fruit reference medicinal material (20 μL); 4. bran-fried gorgon fruit formula granules (K145CP01) test sample (15 μL); 5. bran-fried gorgon fruit formula granules (K145CP06) test sample (15 μL); 6. bran-fried gorgon fruit formula granules (K145CP11) test sample (15 μL)).

[0080] from Figure 10 It can be found that this method can effectively identify the granules of fried gorgon fruit with bran, with good durability and specificity, clear spots and good separation effect.

[0081] Example 2

[0082] This embodiment provides a thin layer chromatography quality control method for bran-fried gorgon fruit formula granules. The specific steps are consistent with those in Example 1 except that the developing solvent is a chloroform-ethyl formate mixed solution (volume ratio of 5:5).

[0083] See the results Figure 11(1. Gallic acid reference substance (4μL); 2. Linoleic acid reference substance (4μL); 3. Fried gorgon fruit with bran and bran reference medicinal material (20μL); 4. Fried gorgon fruit with bran and bran formula granules (K145CP01) test sample (15μL); 5. Maltodextrin negative solution (15μL)). Among them, the Rf value of the gallic acid spot is low, the development system is not suitable, the development effect is poor, and the developing agent needs to be replaced.

[0084] Example 3

[0085] This embodiment provides a thin layer chromatography quality control method for bran-fried gorgon fruit formula granules. The specific steps are consistent with those in Example 1 except that the developing solvent is a chloroform-formic acid mixed solution (volume ratio of 5:1).

[0086] See the results Figure 12 (1. Gallic acid reference substance (4μL); 2. Linoleic acid reference substance (4μL); 3. Fried gorgon fruit with bran and bran reference medicinal material (20μL); 4. Fried gorgon fruit with bran and bran formula granules (K145CP01) test sample (15μL); 5. Maltodextrin negative solution (15μL)). Among them, the gallic acid spots and the linoleic acid spots did not achieve the development effect, the development effect was poor, and the developing agent needed to be replaced.

[0087] Example 4

[0088] This embodiment provides a thin layer chromatography quality control method for bran-fried gorgon fruit formula granules. The specific steps are consistent with those in Example 1 except that the developing solvent is a mixed solution of ethyl formate and formic acid (volume ratio of 5:1).

[0089] See the results Figure 13 (1. Gallic acid reference substance (4μL); 2. Linoleic acid reference substance (4μL); 3. Fried gorgon fruit with bran (20μL); 4. Fried gorgon fruit formula granules (K145CP01) test sample (15μL); 5. Maltodextrin negative solution (15μL)). Among them, the Rf value of the gallic acid spot is higher, and the linoleic acid spot reaches the front of the solvent. The developing effect is poor, and the developing agent needs to be replaced.

[0090] Example 5

[0091] This embodiment provides a thin layer chromatography quality control method for bran-fried gorgon fruit formula granules. The specific steps are consistent with those in Example 1 except that the developing solvent is a mixed solution of chloroform-ethyl formate-formic acid (volume ratio of 5:10:1).

[0092] See the results Figure 14(1. Gallic acid reference substance (4μL); 2. Linoleic acid reference substance (4μL); 3. Fried gorgon fruit with bran (20μL); 4. Fried gorgon fruit with bran formula granules (K145CP01) test sample (15μL); 5. Maltodextrin negative solution (15μL)). The linoleic acid spots dispersed during the development process. The development system was not suitable, the development effect was poor, and the developing agent needed to be replaced.

[0093] Example 6

[0094] This embodiment provides a thin layer chromatography quality control method for bran-fried gorgon fruit formula granules. The specific steps are consistent with those in Example 1 except that the developing solvent is a mixed solution of chloroform-ethyl formate-formic acid (volume ratio of 10:5:1).

[0095] See the results Figure 15 (1. Gallic acid reference substance (4μL); 2. Linoleic acid reference substance (4μL); 3. Fried gorgon fruit with bran and bran reference medicinal material (20μL); 4. Fried gorgon fruit with bran and bran formula granules (K145CP01) test sample (15μL); 5. Maltodextrin negative solution (15μL)). Among them, the Rf value of the gallic acid spot is low, the development system is not suitable, the development effect is poor, and the developing agent needs to be replaced.

[0096] Comparative Example 1

[0097] This comparative example provides a thin layer chromatography quality control method for bran-fried gorgon fruit granules, and the specific steps are as follows:

[0098] Take 2g of gorgon fruit powder, add 30mL of dichloromethane, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, and add 2mL of ethyl acetate to the residue to dissolve it as the test solution. Take another 2g of gorgon fruit control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 10μL of each of the above two solutions were taken and spotted on the same silica gel G thin layer plate, and n-hexane-acetone (volume ratio 5:1) was used as the developing agent. Develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored. In the test sample chromatogram, spots of the same color appear at the corresponding position of the control medicinal material chromatogram. See the results. Figure 16 (1. Linoleic acid reference substance (4μL); 2. Fried gorgon fruit medicinal material with bran (15μL); 3. Fried gorgon fruit formula granules with bran (15μL); 4. Maltodextrin negative test substance (15μL). The left picture is the picture under sunlight, and the right picture is the picture under ultraviolet light).

[0099] Using the pharmacopoeia method, it is clear that after water extraction, the formulated granules become lighter in color than the medicinal material spots, and some spots are lost. However, the linoleic acid spots are still present when observed at 365nm, proving that linoleic acid spots still exist in the formulated granules. However, the spots of maltodextrin and the formulated granules in this development system are basically the same. Subsequent testing cannot prove whether the sample is a bran-fried gorgon fruit granule or another formulated granule with added excipients (maltodextrin). Therefore, it is impossible to perform quality control on the bran-fried gorgon fruit granules. It is necessary to change the development system and discard the maltodextrin interference spots to ensure quality control.

[0100] Comparative Example 2

[0101] This comparative example provides a thin layer chromatography quality control method for bran-fried gorgon fruit granules, and the specific steps are as follows:

[0102] Take an appropriate amount of bran-fried gorgon fruit granules, grind them finely, take 1g, add 30mL of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20mL of water, extract with ethyl acetate twice, shake and extract 20mL each time, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1mL of methanol to prepare the test solution. Separately, take 10g of bran-fried gorgon fruit control medicinal material, add 100mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, and prepare the control medicinal material solution from "add 30mL of methanol..." by the same method. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition General Chapter 0502), take 20μL of each of the above two solutions and spot them on the same silica gel G thin layer plate with toluene-ethyl formate-formic acid (10:8:1) as the developing solvent. Develop, remove, dry, spray with 5% phosphomolybdic acid ethanol test solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material. Figure 17 (1. Linoleic acid; 2. Gallic acid; 3. Formula granules of fried gorgon fruit with bran).

[0103] The developing solvent in this method contains toluene, which is too toxic, and the other spots are blurred and the separation is poor.

[0104] The applicant declares that the present invention illustrates the thin-layer chromatography quality control method and application of the bran-fried gorgon fruit formula granules of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

[0105] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0106] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A thin layer chromatography quality control method for bran-fried gorgon fruit formula granules, characterized in that: The thin layer chromatography quality control method comprises the following steps: (1) Mix the sample to be tested with alcohol and reflux to obtain a test solution; (2) Mixing the bran-fried Euryale ferox seed control medicinal material with water and refluxing to obtain a control medicinal material solution; (3) Mixing the reference substance with alcohol to obtain a reference solution; (4) Perform thin layer chromatography on the test sample solution, control medicinal material solution, and reference substance solution, and judge the quality of the test sample based on the test results; Steps (1), (2), and (3) are not sequential; The alcohol includes ethanol; The reference substances in step (3) include gallic acid and linoleic acid; In the thin layer chromatography test in step (4), the developing solvent is a mixed solution of chloroform, ethyl formate, and formic acid, and the volume ratio of chloroform, ethyl formate, and formic acid is 5:5:1; The thin layer chromatography detection in step (4) is performed using a silica gel G plate.

2. The thin layer chromatography quality control method according to claim 1, characterized in that: In step (1), the material-liquid ratio of the sample to be tested to the alcohol solution is 1:(10-20) g / mL.

3. The thin layer chromatography quality control method according to claim 1, characterized in that: The material-liquid ratio of the bran-fried Euryale ferox reference medicinal material to water in step (2) is 1:(10-20) g / mL.

4. An application of the thin layer chromatography quality control method according to any one of claims 1 to 3 in the detection of bran-fried gorgon fruit formula granules.

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