Quality control method of compound radix biondiellae stomachache capsule

Abstract

The application discloses a quality control method of compound radix bidentatae stomachache capsules, which comprises the following steps: (1) preparing a test sample solution of the compound radix bidentatae stomachache capsules; (2) preparing a control sample solution containing ginsenoside and panax notoginseng saponin; (3) using a thin layer chromatography method, spotting the test sample and the control sample on the same thin layer plate, using a mixed solution of chloroform-methanol-water-ammonia water as a developing agent, developing, taking out, airing, developing color and observing, and judging the quality of the compound radix bidentatae stomachache capsules according to the observation result; and the volume ratio of chloroform-methanol-water-ammonia water in the developing agent is 13:9:2:2. The method has the advantages of simple processing process, clear spots, less interference, high detection specificity of panax notoginseng in the compound radix bidentatae stomachache capsules, good reproducibility, improved standard level, and is more favorable for the quality control of the compound radix bidentatae stomachache capsules.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical quality control, and particularly relates to a quality control method of compound radix notoginseng stomachache capsules. BACKGROUND

[0002] Chronic gastritis, gastric ulcer and duodenal ulcer are a kind of frequently-occurring and common digestive system diseases. Men are more than women. They are often induced by factors such as emotional fluctuation, overwork, diet disorder, smoking, alcoholism, adverse effects of some drugs, etc. The disease course is long, and it is recurrent. Chronic disease can make the pathogenesis more complex.

[0003] The compound radix notoginseng stomachache capsules are made of radix notoginseng, rhizoma corydalis (vinegar fried), rhizoma cyperi (vinegar fried), fructus evodiae (vinegar fried), concha arcae (calcined), vitriol, radix glycyrrhizae, radix paeoniae alba, rhizoma bletillae, fructus aurantii, magnesium oxide, sodium bicarbonate and belladonna extract. The function is to regulate qi and remove blood stasis, warm the middle and invigorate the spleen, and astringe and stop bleeding. It is used for hyperacidity, stomachache, gastric ulcer, duodenal bulb ulcer and chronic gastritis, etc.

[0004] The compound radix notoginseng stomachache capsules are recorded in the Ministry of Public Health Pharmaceutical Standard Traditional Chinese Prescription Preparation 16 (Protected Variety Volume 1), WS3-B-3078-98. The standard detection content is less, only microscopic and test tube reaction, which is not conducive to the quality control of the product.

[0005] In the research of quality control method of compound radix notoginseng stomachache capsules, Chen Yong reports that the radix notoginseng in the compound radix notoginseng stomachache capsules is identified by thin layer chromatography. The reference substance is radix notoginseng reference material. The test sample, reference substance and thin layer chromatography method all refer to the 1995 edition of the Pharmacopoeia. The specificity of the method for identifying the radix notoginseng in the compound radix notoginseng stomachache capsules is poor.

[0006] In order to improve the quality standard control level of the compound radix notoginseng stomachache capsules, we have carried out systematic standard research, and established a radix notoginseng thin layer identification method for the compound radix notoginseng stomachache capsules.

[0007] Therefore, the present application is proposed. SUMMARY

[0008] The technical problem solved by the present application is to overcome the deficiencies of the prior art, and to provide a quality control method of compound radix notoginseng stomachache capsules. The processing process of the method is simple, the spot is clear and the interference is less. The detection specificity of the method for the radix notoginseng in the compound radix notoginseng stomachache capsules is high, and the reproducibility is good. The standard level is improved, and it is more conducive to the quality control of the compound radix notoginseng stomachache capsules.

[0009] To solve the above technical problems, the basic idea of the technical scheme of the present application is:

[0010] The purpose of the present application is to provide a quality control method of compound radix notoginseng stomachache capsules. The quality control method comprises:

[0011] (1) Preparation of the test solution of Compound Gouteng Capsules;

[0012] (2) Preparation of the reference solution containing Panax notoginseng saponins and ginsenosides;

[0013] (3) The test solution and the reference solution are spotted on the same thin layer plate by using thin layer chromatography, a mixed solution of chloroform-methanol-water-ammonia water is used as the developing agent, and after development, taking out, drying, color development and inspection, the quality of Compound Gouteng Capsules is determined according to the inspection results.

[0014] The existing Panax notoginseng thin layer identification method for detecting Compound Gouteng Capsules has the problems of complicated processing process, many interferences, poor specificity and poor reproducibility. The thin layer identification method for detecting Panax notoginseng components in Compound Gouteng Capsules is found in the present application, which has the advantages of simple processing process, clear spots, few interferences, high specificity for detecting Panax notoginseng in Compound Gouteng Capsules and good reproducibility. The standard level of the existing Compound Gouteng Capsules can be improved, and the quality control of Compound Gouteng Capsules is more beneficial.

[0015] In a further scheme, in step (3), the volume ratio of chloroform-methanol-water-ammonia water in the developing agent is 13:9:2:2.

[0016] The inventors found in the exploration process that when the mixed solution of chloroform-methanol-water-ammonia water is used as the developing agent, and the volume ratio is controlled at 13:9:2:2, the separation degree is good, the spots are clear and obvious, there are few interferences, and the specificity is good.

[0017] In a further scheme, in step (1), the contents of Compound Gouteng Capsules are taken, finely ground, added with water, ultrasonically treated, filtered, and the filtrate is extracted with water-saturated n-butanol, and the n-butanol liquid is separated, washed with water saturated with n-butanol, and the n-butanol layer is taken, evaporated to dryness, and the residue is dissolved with methanol as the test solution.

[0018] In a further scheme, the mass-volume ratio of the contents to water is 1:4.

[0019] In a further scheme, the volume of water-saturated n-butanol used for shaking extraction is equal to the volume of water added.

[0020] In a further scheme, the volume of water saturated with n-butanol used for washing is 75% of the volume of water-saturated n-butanol used for shaking extraction.

[0021] As a specific embodiment, 5 g of the contents of Compound Gouteng Capsules are taken, finely ground, added with 20 ml of water, the volume of water-saturated n-butanol used for shaking extraction is 20 ml, and the volume of water saturated with n-butanol used for washing is 15 ml.

[0022] By controlling the mass-volume ratio of the content, the extraction solvent, the shaking extraction solvent and the washing solvent, clear spots can be obtained, and background interference can be greatly reduced, and specificity can be improved.

[0023] In a further aspect, the ultrasonic treatment is performed for 10-30 minutes, preferably 20 minutes.

[0024] In a further aspect, the filtrate is extracted with water-saturated n-butanol twice, and the volume of the water-saturated n-butanol used each time is equal to the volume of the water added.

[0025] In a further aspect, the filtrate is washed with water saturated with n-butanol twice, and the volume of the water saturated with n-butanol used each time is 75% of the volume of the water-saturated n-butanol used for shaking extraction.

[0026] The inventors found that shaking extraction with water-saturated n-butanol twice combined with washing with water saturated with n-butanol twice showed the clearest spots and the least background interference, and therefore selected shaking extraction with water-saturated n-butanol twice and washing with water saturated with n-butanol twice as the shaking extraction method.

[0027] In a further aspect, in step (2), Panax notoginseng saponin R1 and ginsenoside Rg1 reference substances are taken to prepare a mixed solution with methanol as a reference solution;

[0028] In a further aspect, in step (2), the mass concentration of Panax notoginseng saponin R1 and ginsenoside Rg1 in the reference solution is 0.4-1 mg / ml respectively.

[0029] Preferably, the mass concentration of Panax notoginseng saponin R1 and ginsenoside Rg1 in the mixed solution is 1 mg / ml respectively.

[0030] In a further aspect, in step (3), the conditions of the thin layer chromatography include: the sample application amount of the test sample is 10-15 μl, the sample application amount of the reference sample is 3-8 μl, and development is performed below 10℃ with a development distance of 15 cm.

[0031] By controlling the sample application amount of the test sample and the reference sample, background interference can be reduced, and the detectability of samples with different contents can be ensured. The method of the present application is developed below 10℃, has good separation degree, and has no negative interference.

[0032] In a further aspect, in step (3), 10% sulfuric acid ethanol solution is sprayed, and heating is performed at 105℃ until the spots are clearly colored, and observation is performed under sunlight and / or ultraviolet light 365 nm respectively.

[0033] As a specific embodiment, the quality control method of the compound notoginseng stomachache capsule of the present application includes:

[0034] (1) Take 5g of the contents of this product, grind it into a fine powder, add 20ml of water, sonicate for 20 minutes, filter, extract the filtrate by shaking with water-saturated n-butanol twice, 20ml each time, separate the n-butanol solution, wash it twice with water saturated with n-butanol, 15ml each time, take the n-butanol layer, evaporate to dryness, add 0.5ml of methanol to dissolve the residue, and use it as the test solution.

[0035] (2) Take the reference standards of Panax notoginseng saponin R1 and Ginsenoside Rg1, add methanol to prepare a mixed solution containing 1 mg of each per 1 ml, and use it as the reference solution.

[0036] (3) Perform thin-layer chromatography (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia). Apply 10–15 μl of the test solution and 3 μl of the reference solution separately to the same silica gel G thin-layer plate. Use the lower layer of a chloroform-methanol-water-ammonia solution (13:9:2:2) stored below 10°C as the developing solvent. Develop the plate below 10°C. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly visible. Examine the plate under sunlight and ultraviolet light (365 nm). In the chromatogram of the test sample, spots or fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference sample.

[0037] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0038] 1. The original standard for Compound Panax notoginseng Stomach Pain Capsules has limited testing content. The quality control method for Compound Panax notoginseng Stomach Pain Capsules provided by this invention adds thin-layer chromatography identification of Panax notoginseng to the original standard, thereby improving its standard level and making it more conducive to the quality control of Compound Panax notoginseng Stomach Pain Capsules.

[0039] 2. The quality control method of Compound Panax notoginseng Stomach Pain Capsules of the present invention has a simple processing procedure, clear spots with little interference, high specificity for the detection of Panax notoginseng in Compound Panax notoginseng Stomach Pain Capsules, and good reproducibility.

[0040] 3. The quality control method for Compound Tianqi Stomach Pain Capsules of the present invention uses a chloroform-methanol-water-ammonia mixed solution as the developing solvent, and it was found that the chloroform-methanol-water-ammonia (13:9:2:2) solution is the most effective. Furthermore, various conditions such as extraction solvent, shaking extraction solvent, number of shaking extractions, number of washings, sample volume, and spotting volume were explored to obtain the optimal thin-layer chromatography detection method, which has high specificity, low interference, and good reproducibility.

[0041] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0042] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the application and together with the description, serve to explain the application. Obviously, the drawings shown are only a few embodiments and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings. In the drawings:

[0043] Figure 1 is a chromatogram of solvent selection of thin-layer identification and shaking extraction of Panax notoginseng of the present application;

[0044] Figure 2 is a chromatogram of shaking extraction and washing times investigation of thin-layer identification of Panax notoginseng of the present application;

[0045] Figure 3 is a chromatogram of different sampling amount investigation of thin-layer identification of Panax notoginseng of the present application;

[0046] Figure 4 is a chromatogram of different sample amount investigation of thin-layer identification of Panax notoginseng of the present application;

[0047] Figures 5-9 is a chromatogram of different thin-layer silica gel precast plates with chloroform-methanol-water (13:7:2) as developing agent;

[0048] Figures 10-12 is a chromatogram of different thin-layer silica gel precast plates with chloroform-ethyl acetate-methanol-water (15:40:22:10) as developing agent at 10℃ or below;

[0049] Figures 13-15 is a chromatogram of different thin-layer silica gel precast plates with chloroform-ethyl acetate-methanol-water (15:40:22:10) as developing agent at room temperature;

[0050] Figures 16-17 is a chromatogram of different thin-layer silica gel precast plates with chloroform-methanol-water-formic acid (13:7:2:0.5) as developing agent;

[0051] Figures 18-19 is a chromatogram of different thin-layer silica gel precast plates with chloroform-methanol-water-ammonia (13:7:2:1) as developing agent;

[0052] Figures 20-21 is a chromatogram of different thin-layer silica gel precast plates with chloroform-methanol-water-ammonia (11.5:7:2:1) as developing agent;

[0053] Figure 22 is a chromatogram of chloroform-methanol-ammonia (13:7:2) as developing agent;

[0054] Figure 23 is a chromatogram with chloroform-methanol-water-ammonia (13:9:2:2) as developing agent, using different pre-coated thin layer silica gel plates;

[0055] Figures 24-26 is a chromatogram with chloroform-methanol-water-ammonia (13:9:2:2) as developing agent, using different pre-coated thin layer silica gel plates;

[0056] Figure 27 is a chromatogram with chloroform-methanol-water-ammonia (13:9:2:2) as developing agent, using different pre-coated thin layer silica gel plates;

[0057] Figure 28 is a chromatogram of the specificity investigation of the Panax notoginseng thin layer identification;

[0058] Figure 29 is a chromatogram of the Panax notoginseng thin layer identification developed at room temperature;

[0059] Figure 30 is a chromatogram of the Panax notoginseng thin layer identification of different pre-coated thin layer plates;

[0060] Figure 31 is a chromatogram of the Panax notoginseng thin layer identification of different batches of samples.

[0061] It should be noted that these drawings and written descriptions are not intended to limit the scope of the inventive concept in any way, but to illustrate the inventive concept to those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments will be described clearly and completely below with reference to the drawings of the embodiments of the present application. The following embodiments are used to illustrate the present application, but not to limit the scope of the present application.

[0063] The compound Panax notoginseng stomach pain capsules used in the present application are all produced by Guilin Sanjin Pharmaceutical Co., Ltd., with batch number 200102.

[0064] Example 1

[0065] (1) Take 5 g of the content of the product, grind finely, add 20 ml of water, and ultrasonically treat for 20 minutes. Filter, and extract the filtrate with water-saturated n-butanol twice, 20 ml each time. Separate the n-butanol liquid, wash with water saturated with n-butanol twice, 15 ml each time, take the n-butanol layer, evaporate to dryness, and add 0.5 ml of methanol to dissolve, as the test solution.

[0066] (2) Take another Panax notoginseng saponin R1 and ginsenoside Rg1 reference substances, add methanol to prepare a mixed solution containing 1 mg of each per 1 ml, as the reference solution.

[0067] (3) Take 10-15 μl of the test solution and 3 μl of the reference solution and apply separately to the same silica gel G plate. Use the lower layer solution of chloroform-methanol-water-ammonia (13:9:2:2) kept at 10 ℃ as the developing agent, develop at 10 ℃, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 ℃ until the spots are clearly colored, and observe under daylight and ultraviolet light (365 nm). The same colored spots or fluorescent spots appear in the test solution chromatogram at the corresponding positions of the reference solution chromatogram.

[0068] Test Example 1: Investigation of extraction solvents and shaking extraction solvents

[0069] 1.1 Take 5 g of the contents of the product, finely grind, add 20 ml of water, ultrasonically treat for 20 minutes, filter, and extract the filtrate with water-saturated n-butanol twice, 20 ml each time. Separate the n-butanol solution, wash with water saturated with n-butanol twice, 15 ml each time, take the n-butanol layer, evaporate to dryness, add 0.5 ml of methanol to the residue to dissolve, and use as the test solution.

[0070] 1.2 Take 5 g of the contents of the product, finely grind, add 20 ml of dilute ethanol, ultrasonically treat for 20 minutes, filter, evaporate the filtrate to dryness, dissolve with 20 ml of water, extract with water-saturated n-butanol three times, 20 ml each time, separate the n-butanol solution, wash with water saturated with n-butanol twice, 15 ml each time, take the n-butanol layer, evaporate to dryness, add 0.5 ml of methanol to the residue to dissolve, and use as the test solution.

[0071] 1.3 Take 5 g of the contents of the product, finely grind, add 20 ml of water, ultrasonically treat for 20 minutes, filter, and extract the filtrate with ethyl acetate three times, 20 ml each time. Evaporate to dryness, add 0.5 ml of methanol to the residue to dissolve, and use as the test solution.

[0072] 1.4 Preparation of the reference solution: Take the ginsenoside R1 and ginsenoside Rg1 reference solutions, and add methanol to prepare a solution containing 0.4 mg of each per 1 ml.

[0073] Take 15 μl of the test solution and 8 μl of the reference solution, apply to the same silica gel G plate (Qingdao Marine Silica Gel G plate), use the lower layer solution of chloroform-methanol-water-ammonia (13:9:2:2) kept at 10 ℃ as the developing agent, develop at 10 ℃, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 ℃ until the spots are clearly colored, observe under daylight and at 365 nm under ultraviolet light. The same colored spots appear in the test solution chromatogram at the corresponding positions of the reference solution chromatogram.

[0074] The chromatogram of this test example is shown in Figure 1Lane 1: dilute ethanol extraction water-saturated n-butanol shaking extraction; Lane 2: Panax notoginseng saponin Rl control; Lane 3: ginsenoside Rgl control; Lane 4: water extraction water-saturated n-butanol shaking extraction; Lane 5: water extraction ethyl acetate shaking extraction. Chromatographic conditions: temperature: 8°C, distance: 15 cm;

[0075] The results show that the sample obtained by dilute ethanol extraction has many impurities and large background interference, so water is selected as the extraction solvent. The shaking extraction solvent ethyl acetate has low extraction rate, and Panax notoginseng saponin Rl is not detected, so n-butanol is selected as the shaking extraction solvent.

[0076] Test Example 2: Shaking extraction and washing times investigation

[0077] 2.1 Take 5 g of the content of the product, grind finely, add 20 ml of water, and ultrasonically treat for 20 minutes. Filter, and extract the filtrate with water-saturated n-butanol by shaking extraction twice, 20 ml each time. Separate the n-butanol liquid, wash with water saturated with n-butanol twice, 15 ml each time, take the n-butanol layer, evaporate to dryness, add 0.5 ml of methanol to dissolve, and use as the test sample solution.

[0078] 2.2 Take 5 g of the content of the product, grind finely, add 20 ml of water, and ultrasonically treat for 20 minutes. Filter, and extract the filtrate with water-saturated n-butanol by shaking extraction twice, 20 ml each time. Separate the n-butanol liquid, wash with 15 ml of water saturated with n-butanol once, take the n-butanol layer, evaporate to dryness, add 0.5 ml of methanol to dissolve, and use as the test sample solution.

[0079] 2.3 Take 5 g of the content of the product, grind finely, add 20 ml of water, and ultrasonically treat for 20 minutes. Filter, and extract the filtrate with water-saturated n-butanol by shaking extraction three times, 20 ml each time. Separate the n-butanol liquid, wash with water saturated with n-butanol twice, 15 ml each time, take the n-butanol layer, evaporate to dryness, add 0.5 ml of methanol to dissolve, and use as the test sample solution.

[0080] 2.4 Preparation of control solution: take Panax notoginseng saponin Rl and ginsenoside Rgl control, and add methanol to prepare a solution containing 0.4 mg per 1 ml.

[0081] Take 15 μl of the test sample solution and 8 μl of the control solution each, and spot on the same plate of silica gel G plate (Qingdao Marine silica gel G plate). Use the lower layer solution of chloroform-methanol-water-ammonia (13:9:2:2) placed below 10°C as the developing agent, develop at 10°C, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, observe under sunlight, and observe under ultraviolet light at 365 nm. In the test sample chromatogram, the same color spots appear at the positions corresponding to the control sample chromatogram.

[0082] The chromatogram of this test example is shown in Figure 2Wherein, each lane of the sample is: 1. water saturated n-butanol shaking extraction twice n-butanol saturated water washing once; 2. water saturated n-butanol shaking extraction twice n-butanol saturated water washing twice; 3. Panax notoginseng saponins R1 control; 4. ginsenoside Rg1 control; 5. water saturated n-butanol shaking extraction three times n-butanol saturated water washing twice.

[0083] Chromatographic conditions: temperature: 8 ℃, distance: 15 cm.

[0084] The results show that the shaking extraction 2 times washing 2 times the spot clear, the background interference is least, so select water saturated n-butanol shaking extraction 2 times n-butanol saturated water washing 2 times as shaking extraction method.

[0085] Test example 3 sample size investigation

[0086] 3.1 respectively take the content of the product after fine grinding 2.5g, 5g, 7.5g, add water 20ml, ultrasonic treatment 20 minutes, filter, filter with water saturated n-butanol shaking extraction 2 times, 20ml each time, separate n-butanol liquid, with n-butanol saturated water washing 2 times, 15ml each time, take n-butanol layer, evaporate to dryness, add methanol 0.5ml to make dissolution, as test solution.

[0087] 3.2 preparation of control solution: take Panax notoginseng saponins R1 and ginsenoside Rg1 control, add methanol to prepare each containing 0.4mg per 1ml solution.

[0088] Take 15μl test solution, 8μl control solution respectively, point on the same plate silica gel G plate (Qingdao marine silica gel G plate), with chloroform-methanol-water-ammonia (13:9:2:2) 10℃ below the lower solution as developing agent, develop below 10℃, take out, dry, spray with 10% sulfuric acid ethanol solution, heat to 105℃ until the spot color clear, observe under sunlight, and observe under ultraviolet 365nm. In the test chromatogram, the same color spot appears in the corresponding position of the control chromatogram.

[0089] The chromatogram of this test example is shown in Figure 3 Wherein, each lane of the sample is: 1. 2.5g sample size; 2. 5g sample size; 3. Panax notoginseng saponins R1 control; 4. ginsenoside Rg1 control; 5. 7.5g sample size;

[0090] Chromatographic conditions: temperature: 8 ℃, distance: 15 cm.

[0091] The results show that the sample size is 2.5, 5g, the spot color is relatively obvious, the background interference is small, 7.5g under sunlight, the background color is deep, considering the difference of sample content, in order to ensure the detectability of different content samples, so select 5g as the sample size.

[0092] Investigation of sample application amount of Test Example 4

[0093] 4.1 Take 5 g of the contents of the product, finely grind, add 20 ml of water, and ultrasonically treat for 20 minutes. Filter, extract the filtrate twice with n-butanol saturated with water, each time with 20 ml of n-butanol, separate the n-butanol solution, wash twice with water saturated with n-butanol, each time with 15 ml, take the n-butanol layer, evaporate to dryness, add 0.5 ml of methanol to dissolve, and use as the test sample solution.

[0094] 4.2 Preparation of the control solution: take the Panax notoginseng saponin Rl and ginsenoside Rgl control samples, add methanol to make a solution containing 0.4 mg per 1 ml.

[0095] Take 10, 15, and 20 μl of the test sample solution and 8 μl of the control solution each, apply to the same plate of silica gel G (Qingdao Marine silica gel G plate), use the lower layer solution of chloroform-methanol-water-ammonia (13:9:2:2) that has been kept below 10°C as the developing agent, develop at below 10°C, take out, air dry, spray with 10% sulfuric acid in ethanol, and heat at 105°C until the spots develop clearly. In the test sample chromatogram, the same colored spots appear at the positions corresponding to the control sample chromatogram, observe under daylight and under ultraviolet light at 365 nm.

[0096] The chromatogram of this test example is shown in Figure 4 wherein the samples of each lane are: 1. 10 μl; 2. 15 μl; 3. Panax notoginseng saponin Rl control; 4. ginsenoside Rgl control; 5. 20 μl.

[0097] Chromatographic conditions: temperature: 8°C; developing distance: 15 cm.

[0098] The results show that when the sample application amount is 10, 15, and 20 μl, the spot color is relatively obvious and the background interference is small. Considering the diversity of samples, 10-15 μl is selected as the sample application amount.

[0099] Investigation of different developing agents of Test Example 5

[0100] The following thin layer silica gel precast plates were investigated: Qingdao Yumin Source (2019.8.20), Merck (1.05626.0001), Qingdao Ocean (2020.11.9), MN (HPTLC 605130), and Yinlong (HSG 20210305, Yantai).

[0101] 1. Developing agent: chloroform-methanol-water (13:7:2) lower layer solution placed below 10℃

[0102] The following thin layer silica gel precast plates were investigated: Qingdao Yumin Source (2019.8.20), Merck (1.05626.0001), Qingdao Ocean (2020.11.9), MN (HPTLC 605130), and Yinlong (HSG 20210305, Yantai).

[0103] Sample 1. 200102 batch sample, 2. 200103 batch sample, 3. Sanchi saponin R1 control, 4. Ginsenoside Rg1 control, 5. Sanchi negative sample, 6. 200302 batch sample

[0104] Chromatographic conditions: temperature: 8℃, developing distance: 15cm, control sample sample amount: 8μl, test sample sample amount: 15μl.

[0105] Coloration and observation: spray with 10% sulfuric acid ethanol solution, heat to 105℃ until the spots are clearly colored, observe under daylight, and observe under ultraviolet 365nm.

[0106] It was found that under daylight observation, the spots of the five brands of thin layer plates were clear and had no interference, but under ultraviolet 365nm observation, except for the Merck plate, the other four brands of thin layer chromatography plates had interference. The thin layer chromatogram is shown in Figures 5-9 .

[0107] 2. Developing agent: chloroform-ethyl acetate-methanol-water (15:40:22:10) lower layer solution placed below 10℃

[0108] The following thin layer silica gel precast plates were investigated: Qingdao Yumin Source (2019.8.20), Merck (1.05626.0001), Qingdao Ocean (2020.11.9)

[0109] Sample 1. 200102 batch sample, 2. 200103 batch sample, 3. Panax notoginseng saponin R1 control, 4. ginsenoside Rg1 control, 5. Panax notoginseng negative sample, 200302 batch sample

[0110] Chromatographic conditions: temperature: 8℃, development distance: 15cm, control sample sample amount: 8μl, test sample sample amount: 15μl.

[0111] Color development and observation: spray with 10% sulfuric acid ethanol solution, heat to 105℃ until the spot color is clear, observe under daylight, and observe under ultraviolet 365nm.

[0112] The results show that the Qingdao marine thin layer plate spot is clear without interference under daylight observation, and the other two brands of thin layer plate have interference under daylight. Under ultraviolet 365nm observation, the negative sample of the three brands of thin layer plate has interference, and the Rf value is very low. The thin layer chromatogram is shown in Figures 10-12 .

[0113] Because the Rf value of the refrigerator development is low, the same development system is developed at room temperature. The following several thin layer silica gel precast plates are investigated: Merck (1.05626.0001), MN (HPTLC 605130), Silver Dragon (HSG 20210305, Yantai).

[0114] Sample 1. 200102 batch sample, 2. 200103 batch sample, 3. Panax notoginseng saponin R1 control, 4. ginsenoside Rg1 control, 5. Panax notoginseng negative sample, 200302 batch sample

[0115] Chromatographic conditions: temperature: 25℃, development distance: 15cm, control sample sample amount: 8μl, test sample sample amount: 15μl.

[0116] Color development and observation: spray with 10% sulfuric acid ethanol solution, heat to 105℃ until the spot color is clear, observe under daylight, and observe under ultraviolet 365nm.

[0117] The results show that there is interference under daylight observation except for Silver Dragon, and under ultraviolet 365nm observation, the negative sample of the three brands of thin layer chromatography plate has interference. The thin layer chromatogram is shown in Figures 13-15 .

[0118] 3, Developing agent: chloroform-methanol-water-formic acid (13:7:2:0.5) lower solution placed below 10℃

[0119] The following two thin layer silica gel precast plates are investigated: MN (HPTLC 605130), Silver Dragon (HSG 20210305, Yantai).

[0120] Sample 1. 200102 batch sample, 2. 200103 batch sample, 3. Panax notoginseng saponin R1 control, 4. Ginsenoside Rg1 control, 5. Panax notoginseng negative sample, 200302 batch sample

[0121] Chromatographic conditions: Temperature: 8℃, Developing distance: 15cm, Control sample spotting amount: 8μl, Test sample spotting amount: 15μl;

[0122] Color development and observation: Spray with 10% sulfuric acid ethanol solution, heat to 105℃ until the spots are clear, observe under daylight, and observe under ultraviolet 365nm.

[0123] The results show that the spots of the two brands of thin layer plates are clear without interference under daylight observation, but the negative samples have interference under ultraviolet 365nm observation. The thin layer chromatogram is shown in Figures 16-17 .

[0124] 4, Developing agent: chloroform-methanol-water-ammonia water (13:7:2:1) lower solution placed below 10℃

[0125] The following two thin layer silica gel pre-coated plates were investigated: MN (HPTLC 605130), Yinlong (HSG 20210305, Yantai).

[0126] Sample 1. 200102 batch sample, 2. 200103 batch sample, 3. Panax notoginseng saponin R1 control, 4. Ginsenoside Rg1 control, 5. Panax notoginseng negative sample, 200302 batch sample

[0127] Chromatographic conditions: Temperature: 8℃, Developing distance: 15cm, Control sample spotting amount: 8μl, Test sample spotting amount: 15μl.

[0128] Color development and observation: Spray with 10% sulfuric acid ethanol solution, heat to 105℃ until the spots are clear, observe under daylight, and observe under ultraviolet 365nm.

[0129] The results show that the spots are clear and the negative sample has no interference, but the Panax notoginseng saponin R1 spot Rf value is too low, and the spots are close. Therefore, the developing system is fine-tuned based on this chromatographic condition. The thin layer chromatogram is shown in Figures 18-19 .

[0130] 5, Developing agent: chloroform-methanol-water-ammonia water (11.5:7:2:1) lower solution placed below 10℃

[0131] The following two thin layer silica gel pre-coated plates were investigated: Yinlong (HSG 20210305, Yantai), Qingdao Marine (2020.11.9)

[0132] Sample: 1. 200102 batch sample, 2. 200103 batch sample, 3. Panax notoginseng saponin R1 control, 4. ginsenoside Rg1 control, 5. Panax notoginseng negative sample, 200302 batch sample

[0133] Chromatographic conditions: temperature: 8℃, development distance: 15cm, control sample sample amount: 8μl, test sample sample amount: 15μl.

[0134] Color development and observation: spray with 10% sulfuric acid ethanol solution, heat to 105℃ until the spot color is clear, observe under daylight, and observe under ultraviolet 365nm.

[0135] The results show that the Rf value of Panax notoginseng saponin R1 spot is still low, and the spots are close. The thin layer chromatogram is shown in Figures 20-21 .

[0136] 6, Developing agent: chloroform-methanol-ammonia water (13:7:2) lower solution placed below 10℃

[0137] Qingdao Marine (2020.11.9) thin layer silica gel pre-coated plate was investigated

[0138] Sample: 1. 200102 batch sample, 2. 200103 batch sample, 3. Panax notoginseng saponin R1 control, 4. ginsenoside Rg1 control, 5. Panax notoginseng negative sample, 200302 batch sample

[0139] Chromatographic conditions: temperature: 8℃, development distance: 15cm, control sample sample amount: 8μl, test sample sample amount: 15μl.

[0140] Color development and observation: spray with 10% sulfuric acid ethanol solution, heat to 105℃ until the spot color is clear, observe under daylight, and observe under ultraviolet 365nm.

[0141] The results show that the negative sample has interference under ultraviolet 365nm. The thin layer chromatogram is shown in Figure 22 .

[0142] 7, Developing agent: chloroform-methanol-water-ammonia water (13:8:2:2) lower solution placed below 10℃

[0143] Qingdao Marine (2020.11.9) thin layer silica gel pre-coated plate was investigated

[0144] Sample 1. 200102 batch sample, 2. 200103 batch sample, 3. Panax notoginseng saponin R1 control, 4. ginsenoside Rg1 control, 5. Panax notoginseng negative sample, 200302 batch sample

[0145] Chromatographic conditions: temperature: 8℃, development distance: 15cm, control sample sample amount: 8μl, test sample sample amount: 15μl;

[0146] Color development and inspection: spray with 10% sulfuric acid ethanol solution, heat to 105 ℃ until the spot color is clear, observe under daylight, and observe under ultraviolet 365 nm.

[0147] It was found that the Rf value of notoginsenoside R1 spot was still low, and the spots were close. The thin layer chromatogram is shown in Figure 23 .

[0148] 8, Developing agent: chloroform-methanol-water-ammonia water (13:9:2:2) lower solution placed below 10 ℃

[0149] The following thin layer silica gel precast plates were investigated: Qingdao Yumin Source (2019.8.20), Merck (1.05626.0001), Qingdao Ocean (2020.11.9).

[0150] Sample: 1. 200102 batch sample, 2. 200103 batch sample, 3. notoginsenoside R1 reference substance, 4. ginsenoside Rg1 reference substance, 5. negative sample without notoginseng, 200302 batch sample

[0151] Chromatographic conditions: temperature: 8 ℃, development distance: 15 cm, reference substance sample amount: 8 μl, test sample sample amount: 15 μl.

[0152] Color development and inspection: spray with 10% sulfuric acid ethanol solution, heat to 105 ℃ until the spot color is clear, observe under daylight, and observe under ultraviolet 365 nm.

[0153] It was found that the spots on the three brands of thin layer plates were clear under daylight and ultraviolet 365 nm, and the negative sample had no interference. The thin layer chromatogram is shown in Figures 24-26 .

[0154] 9, Developing agent: chloroform-methanol-water-ammonia water (13:10:2:2) lower solution placed below 10 ℃

[0155] Qingdao Ocean (2020.11.9) thin layer silica gel precast plate was investigated

[0156] Sample: 1. 200102 batch sample, 2. 200103 batch sample, 3. notoginsenoside R1 reference substance, 4. ginsenoside Rg1 reference substance, 5. negative sample without notoginseng, 200302 batch sample

[0157] Chromatographic conditions: temperature: 8 ℃, development distance: 15 cm, reference substance sample amount: 8 μl, test sample sample amount: 15 μl.

[0158] Color development and inspection: spray with 10% sulfuric acid ethanol solution, heat to 105 ℃ until the spot color is clear, observe under daylight, and observe under ultraviolet 365 nm.

[0159] The result showed that the spot of ginsenoside Rg1 was close to the previous spot under UV 365 nm. The TLC chromatogram was shown in Figure 1. Figure 27 .

[0160] Summary: In summary, the developing system of chloroform-methanol-water-ammonia water (13:9:2:2) had the best developing effect, the spot was clear and the negative sample had no interference, so it was finally selected as the developing agent for TLC identification of Panax notoginseng in Compound Notoginseng Stomachache Capsule.

[0161] Test Example 6: Specificity Investigation

[0162] Different batches of products and negative samples were taken and operated according to the method of Example 1, and the chromatogram was shown in Figure 2. Figure 28 .

[0163] Among them, the samples of each lane were: sample 1. 200102 batch sample; 2. 200103 batch sample; 3. Panax notoginseng saponin R1 reference substance; 4. Ginsenoside Rg1 reference substance; 5. Negative sample without Panax notoginseng; 200302 batch sample.

[0164] Chromatographic conditions: temperature: 8℃, developing distance: 15cm, reference substance sample amount: 8μl, test sample sample amount: 15μl;

[0165] Developing agent: chloroform-methanol-water-ammonia water (13:9:2:2) lower solution placed below 10℃

[0166] TLC plate: Qingdao Marine Silica Gel G plate

[0167] Coloration and observation: sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ until the spot was clear, observed under daylight and under UV 365nm.

[0168] Appendix: Preparation of negative sample without Panax notoginseng: about 1 prescription amount of medicinal materials of the product was taken without Panax notoginseng, and prepared according to the process method of Example 1, to obtain the blank sample of Panax notoginseng.

[0169] The result showed that the negative control had no interference, the sample reproducibility was good, the operation was simpler than the original method, and the spot was clearer.

[0170] Test Example 7: The related research on TLC condition durability was carried out by using the text method

[0171] 7.1 Investigation of different temperature and humidity: developed at room temperature (27.9℃, relative humidity 82%), and the chromatogram was shown in Figure 3. Figure 29 .

[0172] The samples in each lane are: 1. 200102 batch sample; 2. 200103 batch sample; 3. Sanchi saponin Rl control; 4. Ginseng saponin Rg1 control; 5. Sanchi negative sample; 6. 200302 batch sample.

[0173] Chromatographic conditions: Temperature: 8℃; development distance: 15cm; control sample spotting amount: 8μl; test sample spotting amount: 15μl;

[0174] Chromatographic conditions: Temperature: 8℃; development distance: 15cm; control sample spotting amount: 8μl; test sample spotting amount: 15μl;

[0175] Developing agent: chloroform-methanol-water-ammonia water (13:9:2:2) lower layer solution placed below 10℃

[0176] Thin layer plate: Qingdao Haoyuan silica gel G plate

[0177] Coloration and observation: sprayed with 10% sulfuric acid ethanol solution, heated to 105℃ until the spots color clearly, observed under sunlight, and observed under ultraviolet 365nm.

[0178] 7.2 Investigation of different brands of thin layer plate: the test sample was spotted on different brands of silica gel precast plate for development, and the chromatogram is shown in Figure 7.2. Figure 30 .

[0179] Figure 7 The samples in each lane are: 1. 200102 batch sample; 2. 200103 batch sample; 3. Sanchi saponin Rl control; 4. Ginseng saponin Rg1 control; 5. Sanchi negative sample; 6. 200302 batch sample.

[0180] Chromatographic conditions: Temperature: 8℃; development distance: 15cm; control sample spotting amount: 8μl; test sample spotting amount: 15μl;

[0181] Chromatographic conditions: Temperature: 8℃; development distance: 15cm; control sample spotting amount: 8μl; test sample spotting amount: 15μl;

[0182] Thin layer plate: Qingdao Yumin source silica gel G plate, Merck silica gel G plate, Qingdao Haoyuan silica gel G plate

[0183] Coloration and observation: sprayed with 10% sulfuric acid ethanol solution, heated to 105℃ until the spots color clearly, observed under sunlight, and observed under ultraviolet 365nm.

[0184] The results show that at room temperature, the target spots cannot be separated under ultraviolet and the negative sample has interference, so this method is only suitable for development below 10℃. Different brands of silica gel precast plate are separated well, and the durability meets the requirements.

[0185] Test example 8 determination results of different batch samples

[0186] The method of Example 1 was used to test 10 batches of the test sample, and the chromatogram is shown in Figure 1. Figure 28 , Figure 1 Figure 31 .

[0187] Figure 8 In the figure, the samples in each lane are: 1. 200402 batch sample; 2. 200603 batch sample; 3. Panax notoginseng saponin R1 reference substance; 4. Ginsenoside Rg1 reference substance; 5. 200902 batch sample; 6. 201102 batch sample; 7. 210102 batch sample; 8. 210403 batch sample; 9. 210602 batch sample.

[0188] Chromatographic conditions: temperature: 8℃, distance: 15cm

[0189] Reference substance sample amount: 8μl Test sample amount: 15μl

[0190] Developing agent: chloroform-methanol-water-ammonia water (13:9:2:2) lower solution placed below 10℃

[0191] Thin layer plate: Qingdao marine silica gel G plate

[0192] Color development and observation: spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, observe under daylight, and observe under ultraviolet light at 365nm.

[0193] The results show that the 10 batch samples can be determined under the method conditions, and the spots are clear and without interference.

[0194] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiment, it is not intended to limit the present application, and any skilled person in the art can make some changes or modifications to the above-mentioned technical content without departing from the technical solution of the present application, and any simple modification, equivalent change and modification of the above-mentioned embodiment, which does not depart from the technical solution of the present application, are still within the scope of the present application.

Claims

1. A quality control method for compound Radix Bidentis stomachache capsules, characterized in that, The quality control method comprises: (1) preparing a test sample solution of the compound notopterygium stomach pain capsule, including: taking the content of the compound notopterygium stomach pain capsule, grinding, adding water, ultrasonic treatment, filtering, extracting with water-saturated n-butanol, separating the n-butanol liquid, washing with water saturated with n-butanol, taking the n-butanol layer, evaporating to dryness, adding methanol to dissolve the residue as a test sample solution; (2) preparing a control sample solution containing panax notoginseng saponin R1 and ginsenoside Rg1; (3) using thin layer chromatography, spotting the test sample and the control sample on the same silica gel G thin layer plate, using a mixed solution of chloroform-methanol-water-ammonia water as the developing agent, the volume ratio of chloroform-methanol-water-ammonia water in the developing agent being 13:9:2:2; developing, taking out, drying, developing color and observing, the developing color agent being 10% sulfuric acid ethanol solution, and judging the quality of the compound notopterygium stomach pain capsule according to the observation results.

2. The quality control method according to claim 1, characterized by, In step (1), the mass-volume ratio of the content to water is 1:4 g / ml.

3. The quality control method according to claim 1, characterized by, In step (1), the volume of water-saturated n-butanol used for shaking extraction is equal to the volume of water added.

4. The quality control method according to claim 1, characterized by, In step (1), the volume of water saturated with n-butanol used for washing is 75% of the volume of water-saturated n-butanol used for shaking extraction.

5. The quality control method of claim 1, wherein, In step (1), the water-saturated n-butanol used for shaking extraction is shaken twice, and the volume of water-saturated n-butanol each time is equal to the volume of water added.

6. The quality control method of claim 1, wherein, In step (1), the water saturated with n-butanol is washed twice, and the volume of water saturated with n-butanol each time is 75% of the volume of water-saturated n-butanol used for shaking extraction.

7. The method of quality control according to any one of claims 1 to 6, characterized in that, In step (2), take panax notoginseng saponin R1 and ginsenoside Rg1 control samples, add methanol to prepare a mixed solution as a control sample solution.

8. The quality control method according to claim 7, characterized in that, In step (2), the mass concentration of panax notoginseng saponin R1 and ginsenoside Rg1 in the control sample solution is 0.4-1 mg / ml respectively.

9. The quality control method according to claim 7, characterized by, The mass concentration of panax notoginseng saponin R1 and ginsenoside Rg1 in the control sample solution is 1 mg / ml respectively.

10. The method of quality control according to any one of claims 1 to 6, wherein In step (3), the conditions of the thin layer chromatography include: the spotting amount of the test sample solution is 10-15 μl, the spotting amount of the control sample solution is 3-8 μl, and the developing distance is 15 cm below 10℃.

11. The method of quality control according to any one of claims 1 to 6, wherein In step (3), spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots develop color clearly, and observe under sunlight and / or ultraviolet light 365 nm respectively.

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