CN117288882A - Quality control method of compound pseudo-ginseng stomach-ache capsule - Google Patents
Quality control method of compound pseudo-ginseng stomach-ache capsule Download PDFInfo
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- CN117288882A CN117288882A CN202210690386.8A CN202210690386A CN117288882A CN 117288882 A CN117288882 A CN 117288882A CN 202210690386 A CN202210690386 A CN 202210690386A CN 117288882 A CN117288882 A CN 117288882A
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- 244000131316 Panax pseudoginseng Species 0.000 title claims abstract description 65
- 235000003181 Panax pseudoginseng Nutrition 0.000 title claims abstract description 65
- 239000002775 capsule Substances 0.000 title claims abstract description 43
- 150000001875 compounds Chemical class 0.000 title claims abstract description 43
- 206010000087 Abdominal pain upper Diseases 0.000 title claims abstract description 41
- 238000003908 quality control method Methods 0.000 title claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 86
- 239000000243 solution Substances 0.000 claims abstract description 66
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 43
- PKSROMPNLONTJT-UHFFFAOYSA-N azanium;chloroform;methanol;hydroxide Chemical compound N.O.OC.ClC(Cl)Cl PKSROMPNLONTJT-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000013558 reference substance Substances 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 26
- 235000003143 Panax notoginseng Nutrition 0.000 claims abstract description 25
- 241000180649 Panax notoginseng Species 0.000 claims abstract description 25
- 238000007689 inspection Methods 0.000 claims abstract description 20
- 230000007480 spreading Effects 0.000 claims abstract description 20
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 12
- 239000011259 mixed solution Substances 0.000 claims abstract description 11
- 238000007605 air drying Methods 0.000 claims abstract description 9
- 239000012085 test solution Substances 0.000 claims abstract description 6
- 229930189092 Notoginsenoside Natural products 0.000 claims abstract description 3
- 229930182494 ginsenoside Natural products 0.000 claims abstract description 3
- 229940089161 ginsenoside Drugs 0.000 claims abstract description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 116
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 60
- YURJSTAIMNSZAE-HHNZYBFYSA-N ginsenoside Rg1 Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O YURJSTAIMNSZAE-HHNZYBFYSA-N 0.000 claims description 31
- LLPWNQMSUYAGQI-OOSPGMBYSA-N notoginsenoside R1 Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)CO2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O LLPWNQMSUYAGQI-OOSPGMBYSA-N 0.000 claims description 26
- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 claims description 22
- 238000000605 extraction Methods 0.000 claims description 22
- 238000005406 washing Methods 0.000 claims description 20
- 239000000706 filtrate Substances 0.000 claims description 15
- 229920006395 saturated elastomer Polymers 0.000 claims description 14
- 238000009210 therapy by ultrasound Methods 0.000 claims description 13
- LLPWNQMSUYAGQI-QBQUQATFSA-N Ginsenoside R1 Natural products O([C@](CC/C=C(\C)/C)(C)[C@@H]1[C@H]2[C@@H](O)C[C@H]3[C@](C)([C@]2(C)CC1)C[C@H](O[C@@H]1[C@H](O[C@@H]2[C@@H](O)[C@@H](O)[C@@H](O)CO2)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@H]1C(C)(C)[C@@H](O)CC[C@]31C)[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1 LLPWNQMSUYAGQI-QBQUQATFSA-N 0.000 claims description 7
- YURJSTAIMNSZAE-UHFFFAOYSA-N UNPD89172 Natural products C1CC(C2(CC(C3C(C)(C)C(O)CCC3(C)C2CC2O)OC3C(C(O)C(O)C(CO)O3)O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O YURJSTAIMNSZAE-UHFFFAOYSA-N 0.000 claims description 7
- CBEHEBUBNAGGKC-UHFFFAOYSA-N ginsenoside Rg1 Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5CC(OC6OC(CO)C(O)C(O)C6O)C34C)C CBEHEBUBNAGGKC-UHFFFAOYSA-N 0.000 claims description 7
- JURZHOVRCOWZFN-UHFFFAOYSA-N notoginsenoside R1 Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5C(CC34C)OC6OC(COC7OCC(O)C(O)C7O)C(O)C(O)C6O)C JURZHOVRCOWZFN-UHFFFAOYSA-N 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 4
- 239000012088 reference solution Substances 0.000 claims description 4
- 230000008569 process Effects 0.000 abstract description 7
- 230000009286 beneficial effect Effects 0.000 abstract description 5
- 239000000523 sample Substances 0.000 description 57
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 35
- 238000010438 heat treatment Methods 0.000 description 20
- 238000005507 spraying Methods 0.000 description 20
- 239000000741 silica gel Substances 0.000 description 19
- 229910002027 silica gel Inorganic materials 0.000 description 19
- 238000012360 testing method Methods 0.000 description 19
- 238000011161 development Methods 0.000 description 17
- 239000013068 control sample Substances 0.000 description 15
- 239000012488 sample solution Substances 0.000 description 15
- 239000000047 product Substances 0.000 description 13
- 238000001704 evaporation Methods 0.000 description 11
- 239000002904 solvent Substances 0.000 description 11
- 238000001914 filtration Methods 0.000 description 10
- 238000000227 grinding Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 8
- 229930182490 saponin Natural products 0.000 description 8
- 150000007949 saponins Chemical class 0.000 description 8
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 6
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- 229910052709 silver Inorganic materials 0.000 description 6
- 239000004332 silver Substances 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 239000000779 smoke Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- YEZWWUMWIFKEQM-UHFFFAOYSA-N O.OC.ClC(Cl)Cl.CCOC(C)=O Chemical compound O.OC.ClC(Cl)Cl.CCOC(C)=O YEZWWUMWIFKEQM-UHFFFAOYSA-N 0.000 description 4
- 230000007812 deficiency Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- WIYUJTDPESFNJV-UHFFFAOYSA-N chloroform formic acid methanol hydrate Chemical compound O.OC.OC=O.ClC(Cl)Cl WIYUJTDPESFNJV-UHFFFAOYSA-N 0.000 description 3
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000013074 reference sample Substances 0.000 description 3
- 235000021419 vinegar Nutrition 0.000 description 3
- 239000000052 vinegar Substances 0.000 description 3
- 208000007882 Gastritis Diseases 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 208000007107 Stomach Ulcer Diseases 0.000 description 2
- OJHAHQJRQIOCFK-UHFFFAOYSA-N azane;chloroform;methanol Chemical compound N.OC.ClC(Cl)Cl OJHAHQJRQIOCFK-UHFFFAOYSA-N 0.000 description 2
- 208000023652 chronic gastritis Diseases 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 201000005917 gastric ulcer Diseases 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 208000007848 Alcoholism Diseases 0.000 description 1
- 241001106067 Atropa Species 0.000 description 1
- 241001313855 Bletilla Species 0.000 description 1
- 241000218176 Corydalis Species 0.000 description 1
- 244000150195 Cyperus longus Species 0.000 description 1
- 235000018109 Cyperus longus Nutrition 0.000 description 1
- 244000075634 Cyperus rotundus Species 0.000 description 1
- 208000030814 Eating disease Diseases 0.000 description 1
- 208000019454 Feeding and Eating disease Diseases 0.000 description 1
- 206010017472 Fumbling Diseases 0.000 description 1
- 244000303040 Glycyrrhiza glabra Species 0.000 description 1
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 description 1
- 206010020601 Hyperchlorhydria Diseases 0.000 description 1
- 240000000233 Melia azedarach Species 0.000 description 1
- 206010027951 Mood swings Diseases 0.000 description 1
- 244000236658 Paeonia lactiflora Species 0.000 description 1
- 235000008598 Paeonia lactiflora Nutrition 0.000 description 1
- 235000005010 Scirpus paludosus Nutrition 0.000 description 1
- 241001078983 Tetradium ruticarpum Species 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
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- 229940037003 alum Drugs 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 235000014632 disordered eating Nutrition 0.000 description 1
- 230000002183 duodenal effect Effects 0.000 description 1
- 208000000718 duodenal ulcer Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 235000011477 liquorice Nutrition 0.000 description 1
- 239000000395 magnesium oxide Substances 0.000 description 1
- CPLXHLVBOLITMK-UHFFFAOYSA-N magnesium oxide Inorganic materials [Mg]=O CPLXHLVBOLITMK-UHFFFAOYSA-N 0.000 description 1
- 235000012245 magnesium oxide Nutrition 0.000 description 1
- AXZKOIWUVFPNLO-UHFFFAOYSA-N magnesium;oxygen(2-) Chemical compound [O-2].[Mg+2] AXZKOIWUVFPNLO-UHFFFAOYSA-N 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 230000001105 regulatory effect Effects 0.000 description 1
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
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- 210000000952 spleen Anatomy 0.000 description 1
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- 231100000397 ulcer Toxicity 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/94—Development
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a quality control method of a compound pseudo-ginseng stomach-ache capsule, which comprises the following steps: (1) preparing a test solution of a compound pseudo-ginseng stomach-ache capsule; (2) Preparing reference substance solution containing notoginsenoside and ginsenoside; (3) Sample the sample and reference substance on the same thin layer plate by thin layer chromatography, spreading with mixed solution of chloroform-methanol-water-ammonia water as developing agent, taking out, air drying, developing and inspecting, and judging quality of the compound Notoginseng radix stomach ache capsule according to inspection result. 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 treatment process, clear spots, less interference, high specificity for detecting the pseudo-ginseng in the compound pseudo-ginseng stomach-ache capsule and good reproducibility; improves the standard level of the compound pseudo-ginseng capsules and is more beneficial to the quality control of the compound pseudo-ginseng capsules for treating the stomach ache.
Description
Technical Field
The invention belongs to the field of medicine quality control, and in particular relates to a quality control method of a compound pseudo-ginseng stomach-ache capsule.
Background
Chronic gastritis, gastric ulcer and duodenal ulcer are a common digestive system disease. Men are more than women. Often induced by mood swings, overfatigue, eating disorders, smoking, alcoholism, adverse effects of certain drugs, and the like. Longer course, repeated attacks, and longer illness can lead to more complex pathogenesis.
The compound pseudo-ginseng stomach ache capsule is prepared from pseudo-ginseng, rhizoma corydalis (stir-baked with vinegar), nutgrass galingale rhizome (stir-baked with vinegar), evodia rutaecarpa (stir-baked with vinegar), concha arcae (calcined), dried alum, liquorice, white peony root, common bletilla pseudobulb, szechwan chinaberry fruit, magnesium oxide, sodium bicarbonate and belladonna fluid extract, and has the functions of regulating qi, removing blood stasis, warming spleen and stomach, astringing and stopping bleeding. Can be used for treating gastric hyperacidity, gastralgia, gastric ulcer, duodenal bulbar ulcer, and chronic gastritis.
The compound pseudo-ginseng stomach-ache capsule is loaded in a 16-book (protection variety division I) of a standard Chinese medicine prescription preparation of a medicine of a ministry of health, WS3-B-3078-98, has less standard detection content, only has microscopic and test tube reactions, and is not beneficial to quality control of products.
Chen Yong in "study of quality control method of Compound Notoginseng radix and stomach ache Capsule", it is reported that thin-layer identification is performed on Notoginseng radix in Compound Notoginseng radix and stomach ache Capsule, and Notoginseng radix control drug is used as control, and the test sample, control and thin-layer chromatography are all referred to pharmacopoeia of 1995 edition. The method has poor specificity for identifying Notoginseng radix in the compound Notoginseng radix stomach ache capsule.
In order to improve the quality standard control level of the compound pseudo-ginseng stomach-ache capsule, a standard research of a system is developed, and a pseudo-ginseng thin layer identification method aiming at the compound pseudo-ginseng stomach-ache capsule is established.
The present invention has been made in view of this.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a quality control method of a compound pseudo-ginseng stomach-ache capsule; the method has simple treatment process, clear spots and less interference, and has high specificity and good reproducibility for detecting Notoginseng radix in the compound Notoginseng radix stomach ache capsule; improves the standard level of the compound pseudo-ginseng capsules and is more beneficial to the quality control of the compound pseudo-ginseng capsules for treating the stomach ache.
In order to solve the technical problems, the invention adopts the basic conception of the technical scheme that:
the invention aims to provide a quality control method of a compound pseudo-ginseng stomach-ache capsule, which comprises the following steps:
(1) Preparing a test solution of the compound pseudo-ginseng stomach-ache capsule;
(2) Preparing reference substance solution containing notoginsenoside and ginsenoside;
(3) Sample the sample and reference substance on the same thin layer plate by thin layer chromatography, spreading with mixed solution of chloroform-methanol-water-ammonia water as developing agent, taking out, air drying, developing and inspecting, and judging quality of the compound Notoginseng radix stomach ache capsule according to inspection result.
The compound pseudo-ginseng stomach-ache capsule is detected by the existing pseudo-ginseng thin layer identification method, and the problems of complex treatment process, more interference, poor specificity and poor reproducibility exist. The invention discloses a thin-layer identification method for detecting the components of pseudo-ginseng in the compound pseudo-ginseng stomach-ache capsule, which is simple in treatment process, clear in spots and less in interference, and has high specificity and good reproducibility in detecting pseudo-ginseng in the compound pseudo-ginseng stomach-ache capsule; can improve the standard level of the existing compound pseudo-ginseng stomach-ache capsule and is more beneficial to the quality control of the compound pseudo-ginseng stomach-ache capsule.
In a further scheme, in the step (3), the volume ratio of chloroform-methanol-water-ammonia water in the developing agent is 13:9:2:2.
The inventor finds that the developing agent adopts the mixed solution of chloroform-methanol-water-ammonia water in the fumbling process, and the volume ratio is controlled at 13:9:2:2, so that the separating degree is good, the spots are clear and obvious, the interference is less, and the specificity is good.
In a further scheme, in the step (1), the content of the compound pseudo-ginseng stomach-ache capsule is taken, ground, added with water, subjected to ultrasonic treatment, filtered, and the filtrate is extracted by shaking with water-saturated n-butanol, the n-butanol liquid is separated, the n-butanol liquid is washed with water saturated with n-butanol, the n-butanol layer is taken, evaporated to dryness, and the residue is added with methanol to be dissolved to be used as a test solution.
Further, the mass-to-volume ratio of the content to the water is 1:4;
further, the volume of the water saturated n-butanol used for the shaking extraction is equal to the volume of the added water;
in a further embodiment, the volume of water saturated with n-butanol used for washing is 75% of the volume of water saturated with n-butanol used for shaking extraction.
As a specific implementation mode, taking 5g of the content of the compound pseudo-ginseng stomach-ache capsule, grinding, and adding 20ml of water; the volume of water-saturated n-butanol used for the shaking extraction was 20ml, and the volume of water-saturated n-butanol used for the washing was 15ml.
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, background interference can be greatly reduced, and the specificity can be improved.
Further, the time of the ultrasonic treatment is 10 to 30 minutes, preferably 20 minutes.
In a further scheme, the filtrate is extracted for 2 times by shaking with water-saturated n-butanol, and the volume of water-saturated n-butanol is equal to the volume of water added each time.
Further, the method is characterized in that the method is carried out by washing the mixture with water saturated by n-butanol for 2 times, wherein the volume of water saturated by n-butanol is 75% of the volume of water saturated by n-butanol used for shaking extraction.
The inventor finds that the water saturated n-butanol is extracted by shaking for 2 times and is matched with the water saturated n-butanol for 2 times, the obtained spots are the most clear, and the background interference is the least, so the water saturated n-butanol is extracted by shaking for 2 times and is extracted by shaking for 2 times.
In a further scheme, in the step (2), taking reference substances of notoginsenoside R1 and ginsenoside Rg1, and adding methanol to prepare a mixed solution serving as a reference substance solution;
in a further scheme, in the step (2), the mass concentration of the notoginsenoside R1 and the mass concentration of the ginsenoside Rg1 in the reference substance solution are respectively 0.4-1mg/ml;
preferably, in the mixed solution, the mass concentration of the notoginsenoside R1 and the mass concentration of the ginsenoside Rg1 are respectively 1mg/ml.
Further, in step (3), the conditions of the thin layer chromatography include: the sample application amount of the sample is 10-15 μl, the sample application amount of the control is 3-8 μl, and the sample application range is 15cm below 10deg.C.
By controlling the sample application amount of the sample to be tested and the sample application amount of the reference sample, the background interference can be reduced, the difference of the sample contents is also considered, and the detectability of the samples with different contents is ensured. The method of the invention is developed below 10 ℃, has good separation degree and no negative interference.
In a further scheme, in the step (3), 10% sulfuric acid ethanol solution is sprayed, and the mixture is heated at 105 ℃ until the color of spots is clear, and is respectively inspected under sunlight and/or ultraviolet light at 365 nm.
As a specific implementation mode, the quality control method of the compound pseudo-ginseng stomach-ache capsule comprises the following steps:
(1) Taking 5g of the content of the product, grinding, adding 20ml of water, carrying out ultrasonic treatment for 20 minutes, filtering, shaking and extracting the filtrate with water saturated n-butanol for 2 times, respectively taking 20ml of n-butanol liquid each time, washing with water saturated with n-butanol for 2 times, respectively taking 15ml of n-butanol layer each time, evaporating to dryness, and adding 0.5ml of methanol into residues to dissolve the residues to obtain a sample solution.
(2) Taking notoginsenoside R 1 Ginsenoside Rg 1 The reference substance was prepared by adding methanol to prepare a mixed solution containing 1mg of each 1ml of the reference substance as a reference substance solution.
(3) According to thin layer chromatography (rule 0502 of the general rule of 2020 of Chinese pharmacopoeia), sucking 10-15 μl of sample solution and 3 μl of reference solution, respectively spotting on the same silica gel G thin layer plate, spreading with lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) placed below 10deg.C as spreading agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until spot color is clear, and respectively placing under sunlight and ultraviolet lamp (365 nm) for inspection. In the chromatogram of the test sample, spots or fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the reference sample.
By adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects.
1. The original standard detection content of the compound pseudo-ginseng stomach-ache capsule is less, and the quality control method of the compound pseudo-ginseng stomach-ache capsule provided by the invention has the advantages that the thin layer identification of pseudo-ginseng is added on the basis of the original standard, the standard level of the pseudo-ginseng is improved, and the quality control of the compound pseudo-ginseng stomach-ache capsule is more facilitated.
2. The quality control method of the compound pseudo-ginseng stomach-ache capsule has the advantages of simple treatment process, clear spots, less interference, high specificity for detecting pseudo-ginseng in the compound pseudo-ginseng stomach-ache capsule and good reproducibility.
3. The quality control method of the compound pseudo-ginseng stomach-ache capsule adopts the mixed solution of chloroform-methanol-water-ammonia water as a developing agent, and the research shows that the effect of the chloroform-methanol-water-ammonia water (13:9:2:2) is optimal. In addition, various conditions such as an extraction solvent, a shaking extraction solvent, shaking extraction times, washing times, sampling amount, sample application amount and the like are also searched, and the optimal thin layer detection method is obtained, so that the specificity is high, the interference is less, and the repeatability is good.
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. It is evident that the drawings in the following description are only examples, from which other drawings can be obtained by a person skilled in the art without the inventive effort. In the drawings:
FIG. 1 is a selective chromatogram of a thin-layer differential extraction and shake extraction solvent of Notoginseng radix of the present invention;
FIG. 2 is a chromatogram of the invention for identifying shaking extraction and washing times of pseudo-ginseng thin layer;
FIG. 3 is a chart of a thin layer chromatography of pseudo-ginseng for identifying different sampling amounts;
FIG. 4 is a chart of the thin-layer identification of the pseudo-ginseng of the present invention for different sample application amounts;
FIGS. 5-9 are chromatograms of preformed plates using chloroform-methanol-water (13:7:2) as developing agent and different thin silica gel layers;
FIGS. 10-12 are chromatograms of different thin silica gel preformed plates at below 10deg.C using chloroform-ethyl acetate-methanol-water (15:40:22:10) as developing agent;
FIGS. 13-15 are chromatograms of different thin silica gel preformed plates using chloroform-ethyl acetate-methanol-water (15:40:22:10) as developing agent at room temperature;
FIGS. 16-17 are chromatograms of different thin silica gel preformed plates using chloroform-methanol-water-formic acid (13:7:2:0.5) as developing agent;
FIGS. 18-19 are chromatograms of different thin silica gel preformed plates using chloroform-methanol-water-ammonia (13:7:2:1) as developing agent;
FIGS. 20-21 are chromatograms of different thin silica gel preformed plates using chloroform-methanol-water-ammonia (11.5:7:2:1) as developing agent;
FIG. 22 is a chromatogram with chloroform-methanol-ammonia (13:7:2) as a developing agent;
FIG. 23 is a chromatogram with chloroform-methanol-water-ammonia (13:8:2:2) as a developing agent;
FIGS. 24-26 are chromatograms of different thin-layer silica gel prefabricated panels using chloroform-methanol-water-ammonia (13:9:2:2) as developing agent;
FIG. 27 is a chromatogram with chloroform-methanol-water-ammonia (13:10:2:2) as a developing agent;
FIG. 28 is a thin-layer differential specificity-looking chromatogram of Notoginseng radix;
FIG. 29 is a developed chromatogram of Notoginseng radix at room temperature for thin-layer identification;
FIG. 30 is a survey chromatogram of a thin-layer panel of Notoginseng identifying different thin-layer panels;
fig. 31 is a survey chromatogram of a thin layer identification of Notoginseng radix samples of different lot numbers.
It should be noted that these drawings and the written description 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 the specific embodiments.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments will be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention, and the following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
The compound pseudo-ginseng stomach-ache capsules adopted in the invention are all manufactured by Gui Linsan golden pharmaceutical industry Co., ltd., batch No. 200102.
Example 1
(1) Taking 5g of the content of the product, grinding, adding 20ml of water, carrying out ultrasonic treatment for 20 minutes, filtering, shaking and extracting the filtrate with water saturated n-butanol for 2 times, respectively taking 20ml of n-butanol liquid each time, washing with water saturated with n-butanol for 2 times, respectively taking 15ml of n-butanol layer each time, evaporating to dryness, and adding 0.5ml of methanol into residues to dissolve the residues to obtain a sample solution.
(2) Taking notoginsenoside R 1 Ginsenoside Rg 1 The reference substance was prepared by adding methanol to prepare a mixed solution containing 1mg of each 1ml of the reference substance as a reference substance solution.
(3) According to thin layer chromatography (rule 0502 of the general rule of 2020 of Chinese pharmacopoeia), sucking 10-15 μl of sample solution and 3 μl of reference solution, respectively spotting on the same silica gel G thin layer plate, spreading with lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) placed below 10deg.C as spreading agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until spot color is clear, and respectively placing under sunlight and ultraviolet lamp (365 nm) for inspection. In the chromatogram of the test sample, spots or fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the reference sample.
Test example 1 investigation of extraction solvent and shaking extraction solvent
1.1 taking 5g of the content of the product, grinding, adding 20ml of water, carrying out ultrasonic treatment for 20 minutes, filtering, shaking and extracting the filtrate with water saturated n-butanol for 2 times, respectively taking 20ml of n-butanol liquid each time, washing with water saturated n-butanol for 2 times, respectively taking 15ml of n-butanol layer each time, evaporating to dryness, and adding 0.5ml of methanol into residues to dissolve the residues to obtain a sample solution.
1.2 taking 5g of the content of the product, grinding, adding 20ml of diluted ethanol, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating the filtrate to dryness, dissolving with 20ml of water, shaking and extracting with water saturated n-butanol for 3 times, 20ml each time, separating n-butanol liquid, washing with water saturated n-butanol for 2 times, 15ml each time, taking n-butanol layer, evaporating to dryness, and adding 0.5ml of methanol into residues to dissolve, thereby obtaining a sample solution.
1.3 taking 5g of the content of the product, grinding, adding 20ml of water, carrying out ultrasonic treatment for 20 minutes, filtering, extracting the filtrate with ethyl acetate for 3 times by shaking, evaporating 20ml each time, and adding 0.5ml of methanol into the residue to dissolve the residue to obtain a sample solution.
1.4 preparation of control solution: taking notoginsenoside R 1 Ginsenoside Rg 1 The reference substance was added with methanol to prepare solutions each containing 0.4mg per 1 ml.
Sucking 15 μl of the sample solution and 8 μl of the control solution, spotting on the same plate of silica gel G plate (Qingdao ocean silica gel G plate), spreading with lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) below 10deg.C as spreading agent, spreading at 10deg.C, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm. Spots of the same color appear in the sample chromatogram at positions corresponding to those of the control chromatogram.
The chromatogram of this test example is shown in FIG. 1, wherein the samples of each lane are: 1. extracting water saturated n-butanol with diluted ethanol, and shaking; 2. notoginseng radix saponin R 1 A reference; 3. ginsenoside Rg 1 A reference; 4. extracting water-saturated n-butanol with shaking; 5. extracting ethyl acetate with water, and shaking. Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm;
the results show that the samples obtained by dilute ethanol extraction have more impurities and large background interference, so water is selected as an extraction solvent. The shaking extraction solvent ethyl acetate has low shaking extraction rate, and notoginsenoside R 1 N-butanol was used as the shake extraction solvent, as it was not detected.
Test example 2 examination of shaking extraction and washing times
2.1 taking 5g of the content of the product, grinding, adding 20ml of water, carrying out ultrasonic treatment for 20 minutes, filtering, shaking and extracting the filtrate with water saturated n-butanol for 2 times, respectively taking 20ml of n-butanol liquid each time, washing with water saturated n-butanol for 2 times, respectively taking 15ml of n-butanol layer each time, evaporating to dryness, and adding 0.5ml of methanol into residues to dissolve the residues to obtain a sample solution.
2.2 taking 5g of the content of the product, grinding, adding 20ml of water, carrying out ultrasonic treatment for 20 minutes, filtering, shaking and extracting the filtrate with water saturated n-butanol for 2 times, 20ml each time, separating n-butanol liquid, washing with 15ml of water saturated n-butanol for 1 time, taking n-butanol layer, evaporating to dryness, and adding 0.5ml of methanol into residues to dissolve the residues to obtain a sample solution.
2.3 taking 5g of the content of the product, grinding, adding 20ml of water, carrying out ultrasonic treatment for 20 minutes, filtering, shaking and extracting the filtrate with water saturated n-butanol for 3 times, respectively taking 20ml of the filtrate, respectively taking n-butanol liquid, washing with water saturated n-butanol for 2 times, respectively taking 15ml of the filtrate, taking n-butanol layer, evaporating to dryness, and adding 0.5ml of methanol into the residue to dissolve the residue to obtain a sample solution.
2.4 preparation of control solution: taking notoginsenoside R 1 Ginsenoside Rg 1 The reference substance was added with methanol to prepare solutions each containing 0.4mg per 1 ml.
Sucking 15 μl of the sample solution and 8 μl of the control solution, spotting on the same plate of silica gel G plate (Qingdao ocean silica gel G plate), spreading with lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) below 10deg.C as spreading agent, spreading at 10deg.C, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm. . Spots of the same color appear in the sample chromatogram at positions corresponding to those of the control chromatogram.
The chromatogram of this test example is shown in FIG. 2, wherein the samples of each lane are: 1. extracting the secondary n-butanol by shaking the water-saturated n-butanol, and washing the secondary n-butanol with water once; 2. extracting secondary n-butanol saturated water by shaking the water saturated n-butanol, and washing twice; 3. notoginseng radix saponin R 1 A reference; 4. ginsenoside Rg 1 A reference; 5. extracting with water saturated n-butanol by shaking for three times, and washing with water saturated n-butanol twice.
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm.
The results show that the spots are clear after 2 times of shaking and the background interference is the least, so that the method for extracting the water-saturated n-butanol by 2 times of shaking and extracting the water-saturated n-butanol by 2 times is selected.
Test example 3 investigation of sample size
3.1 respectively taking 2.5g, 5g and 7.5g of ground product content, adding 20ml of water, carrying out ultrasonic treatment for 20 minutes, filtering, shaking and extracting filtrate with water saturated n-butanol for 2 times, 20ml each time, separating n-butanol liquid, washing with water saturated n-butanol for 2 times, 15ml each time, taking n-butanol layer, evaporating to dryness, and adding 0.5ml of methanol into residues to dissolve the residues to obtain a sample solution.
3.2 preparation of control solution: taking notoginsenoside R 1 Ginsenoside Rg 1 The reference substance was added with methanol to prepare solutions each containing 0.4mg per 1 ml.
Sucking 15 μl of the sample solution and 8 μl of the control solution, spotting on the same plate of silica gel G plate (Qingdao ocean silica gel G plate), spreading with lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) below 10deg.C as spreading agent, spreading at 10deg.C, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm. . Spots of the same color appear in the sample chromatogram at positions corresponding to those of the control chromatogram.
The chromatogram of this test example is shown in FIG. 3, wherein the samples of each lane are: 1.2.5g sample; a sample of 2.5 g; 3. notoginseng radix saponin R 1 A reference; 4. ginsenoside Rg 1 A reference; a sample of 5.7.5 g;
chromatographic conditions: temperature: 8 ℃, spread distance: 15cm.
The results show that when the sampling amount is 2.5g and 5g, the spot color is obvious, the background interference is small, the inspection background color is deeper under 7.5g sunlight, and the 5g is selected as the sampling amount in order to ensure the detectability of samples with different contents by considering the difference of the sample contents.
Test example 4 investigation of sample application amount
4.1 collecting 5g of the product, grinding, adding 20ml of water, performing ultrasonic treatment for 20 min, filtering, extracting the filtrate with water saturated n-butanol for 2 times, each time 20ml, separating n-butanol solution, each time 15ml, collecting n-butanol layer, evaporating, and dissolving the residue with 0.5ml of methanol to obtain test solution.
4.2 preparation of control solution: taking notoginsenoside R 1 Ginsenoside Rg 1 The reference substance was added with methanol to prepare solutions each containing 0.4mg per 1 ml.
Respectively sucking 10, 15, 20 μl of sample solution and 8 μl of control solution, spotting on the same plate of silica gel G plate (Qingdao ocean silica gel G plate), spreading with lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) below 10deg.C as developing agent, spreading below 10deg.C, taking out, air drying, spraying 10% sulfuric acid ethanol solution, and heating at 105deg.C until the color of spots is clear. In the chromatogram of the test sample, spots of the same color appear at the positions corresponding to those of the chromatogram of the control sample, and the spots are observed under sunlight and at 365nm of ultraviolet.
The chromatogram of this test example is shown in FIG. 4, wherein the samples of each lane are: 1.10 μl;2.15 μl;3. notoginseng radix saponin R 1 A reference; 4. ginsenoside Rg 1 A reference; 5.20 μl.
Chromatographic conditions: temperature: 8 ℃; spreading: 15cm.
The results show that when the sample application amounts are 10, 15 and 20 mu l, the spot colors are obvious, the background interference is small, and 10-15 mu l is selected as the sample application amount in consideration of the diversity of the samples.
Test example 5 examination of different developing Agents
Chloroform-methanol-water (13:7:2), chloroform-ethyl acetate-methanol-water (15:40:22:10), chloroform-methanol-water-formic acid (13:7:2:0.5), chloroform-methanol-water-ammonia (13:7:2:1), chloroform-methanol-water-ammonia (11.5:7:2:1), chloroform-methanol-ammonia (13:7:2), chloroform-methanol-water-ammonia (13:8:2), and chloroform-methanol-water-ammonia (13:9:2:2) were examined, and the above developing agents were all lower solutions placed at 10℃or lower and placed in a refrigerator to develop. As a result, it was found that chloroform-methanol-water-ammonia (13:9:2:2) developing agent developed with the best effect, clear spots and no interference with the negative sample.
1. Developing agent: lower layer solution of chloroform-methanol-water (13:7:2) placed below 10deg.C
The following thin-layer silica gel precast slabs were examined: qingda Yumin source (2019.8.20), merck (1.05626.0001), qingda ocean (2020.11.9), MN (HPTLC 605130), silver dragon (HSG 20210305, smoke table).
Sample 1.200102 samples, 2.200103 samples, 3. Notoginsenoside R1 reference, 4. Ginsenoside Rg1 reference, 5. Negative sample of Notoginseng radix, 6.200302 samples
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl.
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
As a result, it was found that the spots of all five brands of thin layer plates were clear and undisturbed when observed under sunlight, but the four brands of thin layer chromatography plates were disturbed when observed under ultraviolet 365nm, except for the Merck plate. The thin layer chromatograms are shown in figures 5-9.
2. Developing agent: lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) placed below 10 DEG C
For the unfolding system used by the pseudo-ginseng medicinal material pharmacopoeia standard, the following thin-layer silica gel precast slabs are examined: yumin Yuyuan Qingdao (2019.8.20), merck (1.05626.0001) and Qingdao ocean (2020.11.9)
Sample 1.200102 samples, 2.200103 samples, 3. Notoginsenoside R 1 Reference substance, 4. Ginsenoside Rg 1 Control, 5. Negative samples of pseudo-ginseng lack, 200302 batches of samples
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl.
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
As a result, the blue island ocean thin-layer plate spots observed under the sunlight are clear and have no interference, and the other two brands of thin-layer plates have interference under the sunlight. Three brands of lamina negative samples all interfere with uv 365nm observation and have very low Rf values. The thin layer chromatograms are shown in figures 10-12.
Since the refrigerator is low in the Rf value, it is developed at room temperature with the same development system. The following thin-layer silica gel precast slabs were examined: merck (1.05626.0001), MN (HPTLC 605130), silver dragon (HSG 20210305, smoke table).
Sample 1.200102 samples, 2.200103 samples, 3. Notoginsenoside R 1 Reference substance, 4. Ginsenoside Rg 1 Control, 5. Negative samples of pseudo-ginseng lack, 200302 batches of samples
Chromatographic conditions: temperature: 25 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl.
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
As a result, it was found that the observation under sunlight was interfered with except for silver dragon, and that the observation under ultraviolet 365nm was interfered with for all three brands of thin layer chromatography plate negative samples. The thin layer chromatograms are shown in figures 13-15.
3. Developing agent: lower solution of chloroform-methanol-water-formic acid (13:7:2:0.5) placed below 10 DEG C
The following two thin-layer silica gel precast slabs were examined: MN (HPTLC 605130), silver dragon (HSG 20210305, smoke table).
Sample 1.200102 samples, 2.200103 samples, 3. Notoginsenoside R 1 Reference substance, 4. Ginsenoside Rg 1 Control, 5. Negative samples of pseudo-ginseng lack, 200302 batches of samples
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl;
color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
As a result, it was found that both brands of lamina plates were clear and undisturbed when viewed in daylight, but that negative samples were disturbed when viewed at 365nm in UV. The thin layer chromatograms are shown in figures 16-17.
4. Developing agent: lower layer solution of chloroform-methanol-water-ammonia water (13:7:2:1) placed below 10 DEG C
The following two thin-layer silica gel precast slabs were examined: MN (HPTLC 605130), silver dragon (HSG 20210305, smoke table).
Sample: 1.200102 and 2.200103 samples, 3. Notoginsenoside R 1 Reference substance, 4. Ginsenoside Rg 1 Control, 5. Negative samples of pseudo-ginseng lack, 200302 batches of samples
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl.
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
As a result, it was found that the spots were clear and that the negative samples were undisturbed, but that notoginsenoside R 1 The spot Rf value is too low and the spots are closely spaced. The development system is thus fine-tuned based on the chromatographic conditions. The thin layer chromatograms are shown in figures 18-19.
5. Developing agent: lower layer solution of chloroform-methanol-water-ammonia water (11.5:7:2:1) placed below 10 DEG C
The following two thin-layer silica gel precast slabs were examined: silver dragon (HSG 20210305, smoke table), qingdao ocean (2020.11.9)
Sample: 1.200102 and 2.200103 samples, 3. Notoginsenoside R 1 Reference substance, 4. Ginsenoside Rg 1 Control, 5. Negative samples of pseudo-ginseng lack, 200302 batches of samples
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl.
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
As a result, it was found that notoginsenoside R 1 The spot Rf value is still low and the spots are closely spaced. The thin-layer chromatograms are shown in figures 20-21.
6. Developing agent: lower layer solution of chloroform-methanol-ammonia water (13:7:2) placed below 10 DEG C
Thin-layer silica gel precast slab for examining Qingdao ocean (2020.11.9)
Sample: 1.200102 and 2.200103 samples, 3. Notoginsenoside R 1 Reference substance, 4. Ginsenoside Rg 1 Control, 5. Negative samples of pseudo-ginseng lack, 200302 batches of samples
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl.
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
As a result, it was found that the negative sample was observed to have an interference at 365nm in ultraviolet. The thin layer chromatogram is shown in FIG. 22.
7. Developing agent: lower layer solution of chloroform-methanol-water-ammonia water (13:8:2:2) placed below 10 DEG C
Thin-layer silica gel precast slab for examining Qingdao ocean (2020.11.9)
Sample 1.200102 samples, 2.200103 samples, 3. Notoginsenoside R 1 Reference substance, 4. Ginsenoside Rg 1 Control, 5. Negative samples of pseudo-ginseng lack, 200302 batches of samples
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl;
color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
As a result, it was found that notoginsenoside R 1 The spot Rf value is still low and the spots are closely spaced. The thin layer chromatogram is shown in FIG. 23.
8. Developing agent: lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) placed below 10 DEG C
The following thin-layer silica gel precast slabs were examined: qingdao Yumin source (2019.8.20), merck (1.05626.0001), qingdao ocean (2020.11.9).
Sample: 1.200102 and 2.200103 samples, 3. Notoginsenoside R 1 Reference substance, 4. Ginsenoside Rg 1 Control, 5. Negative samples of pseudo-ginseng lack, 200302 batches of samples
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl.
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
As a result, it was found that the spots on all three brands of thin layer plates were clear and the negative samples were undisturbed when observed at 365nm in sunlight and ultraviolet. The thin layer chromatograms are shown in FIGS. 24-26.
9. Developing agent: lower layer solution of chloroform-methanol-water-ammonia water (13:10:2:2) placed below 10 DEG C
Thin-layer silica gel precast slab for examining Qingdao ocean (2020.11.9)
Sample: 1.200102 and 2.200103 samples, 3. Notoginsenoside R 1 Reference substance, 4. Ginsenoside Rg 1 Control, 5. Negative samples of pseudo-ginseng lack, 200302 batches of samples
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl.
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
As a result, it was found that ginsenoside Rg 1 The spot was located closer to the last spot at 365nm in uv. The thin layer chromatogram is shown in FIG. 27.
Summarizing: in conclusion, the chloroform-methanol-water-ammonia water (13:9:2:2) developing agent in the developing system has the best developing effect, clear spots and no interference to negative samples, so that the developing agent is finally selected as the compound pseudo-ginseng capsule pseudo-ginseng thin layer identification developing agent.
Test example 6 specificity investigation
Different batches of products and negative samples were taken and run as in example 1, with a chromatogram as shown in FIG. 28.
Wherein, the samples of each lane are: sample 1.200102 batches of samples; 2.200103 samples; 3. notoginseng radix saponin R 1 A reference; 4. ginsenoside Rg 1 A reference; 5. negative samples of pseudo-ginseng deficiency; 200302 samples.
Chromatographic conditions: temperature: 8 ℃, spread distance: 15cm, control sample application amount: sample application amount of 8. Mu.l: 15 μl;
developing agent: lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) at below 10deg.C
Thin layer plate: qingdao ocean silica gel G plate
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
The method comprises the following steps: preparation of negative samples of pseudo-ginseng deficiency: taking about 1 medicinal material with prescription amount of the product, preparing the medicinal material without adding pseudo-ginseng according to the process method of the embodiment 1, and obtaining a blank sample of pseudo-ginseng.
The result shows that the negative control is free from interference, the sample reproducibility is better, the operation is simpler than the original method, and the spots are clearer.
Experimental example 7 related studies on TLC condition durability were performed using text method
7.1 investigation of different humiture: the development was carried out at room temperature (27.9 ℃ C., 82% relative humidity) and the chromatogram was shown in FIG. 29.
Wherein, the samples of each lane are: 1.200102 samples; 2.200103 samples; 3. notoginseng radix saponin R 1 A reference; 4. ginsenoside Rg 1 A reference; 5. negative samples of pseudo-ginseng deficiency; 6.200302 samples.
Chromatographic conditions: temperature: relative humidity at 27.9 ℃): 82% span: 15cm
Control sample application amount: sample application amount of 8. Mu.l test article: 15 μl
Developing agent: lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) at below 10deg.C
Thin layer plate: qingdao ocean silica gel G plate
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
7.2 investigation of different brands of lamina plates: the sample is respectively dotted on silica gel precast slabs of different brands for unfolding, and the chromatogram is shown in figure 30.
In fig. 7, samples for each lane are: 1.200102 samples; 2.200103 samples; 3. notoginseng radix saponin R 1 A reference; 4. ginsenoside Rg 1 A reference; 5. negative samples of pseudo-ginseng deficiency; 6.200302 samples.
Chromatographic conditions: temperature: 8 ℃; spreading: 15cm; control sample application amount: 8 μl; sample application amount of test article: 15 μl;
developing agent: lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) at below 10deg.C
Thin layer plate: qingdao Yumin source silica gel G plate, merck silica gel G plate and Qingdao ocean silica gel G plate
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
The results show that the target spots cannot be separated under ultraviolet and are negative and interfered when the target spots are unfolded at room temperature, so that the method is only applicable to the unfolding at the temperature of below 10 ℃. The silica gel precast slabs of different brands are better in separation, and the durability meets the requirements.
Test example 8 measurement results of different lot number samples
10 batches of samples were tested by the method of example 1, and the chromatograms are shown in fig. 28 and 31.
In fig. 8, samples for each lane are: 1.200402 samples; 2.200603 samples; 3. notoginseng radix saponin R 1 A reference; 4. ginsenoside Rg 1 A reference; 5.200902 samples; 6.201102 samples; 7.210102 samples 8.210403 samples; 9.210602 samples.
Chromatographic conditions: temperature: spread at 8 ℃): 15cm
Control sample application amount: sample application amount of 8. Mu.l test article: 15 μl
Developing agent: lower layer solution of chloroform-methanol-water-ammonia water (13:9:2:2) at below 10deg.C
Thin layer plate: qingdao ocean silica gel G plate
Color development and inspection: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, observing under sunlight, and observing under ultraviolet 365 nm.
The results show that 10 lot number samples can be measured under the conditions of the method, and the spots are clear and have no interference.
The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.
Claims (10)
1. A quality control method of a compound pseudo-ginseng stomach-ache capsule is characterized by comprising the following steps:
(1) Preparing a test solution of the compound pseudo-ginseng stomach-ache capsule;
(2) Preparing reference substance solution containing notoginsenoside and ginsenoside;
(3) Sample the sample and reference substance on the same thin layer plate by thin layer chromatography, spreading with mixed solution of chloroform-methanol-water-ammonia water as developing agent, taking out, air drying, developing and inspecting, and judging quality of the compound Notoginseng radix stomach ache capsule according to inspection result.
2. The quality control method according to claim 1, wherein in the step (3), the volume ratio of chloroform-methanol-water-ammonia water in the developing agent is 13:9:2:2.
3. The quality control method according to claim 1 or 2, wherein in the step (1), the content of the compound pseudo-ginseng stomach-ache capsule is taken, ground, added with water, subjected to ultrasonic treatment, filtered, extracted by shaking with water saturated n-butanol, the n-butanol solution is separated, the n-butanol solution is taken and washed with water saturated n-butanol, the n-butanol layer is taken, evaporated to dryness, and the residue is dissolved by adding methanol to be used as a test solution.
4. A quality control method according to claim 3, wherein the mass to volume ratio of the content to water is 1:4;
preferably, the volume of water-saturated n-butanol used for the shaking extraction is equal to the volume of water added;
preferably, the volume of water saturated with n-butanol used for washing is 75% of the volume of water saturated with n-butanol used for shaking extraction.
5. A quality control method according to claim 3, wherein the filtrate is extracted 2 times with water-saturated n-butanol by shaking, each time the volume of water-saturated n-butanol is equal to the volume of water added.
6. A quality control method according to claim 3, characterized in that the washing with n-butanol-saturated water is performed 2 times, the volume of water saturated with n-butanol each time being 75% of the volume of water saturated with n-butanol used for the shaking extraction.
7. The method according to any one of claims 1 to 6, wherein in the step (2), a reference substance of notoginsenoside R1 and ginsenoside Rg1 is prepared by adding methanol to prepare a mixed solution as a reference solution.
8. The quality control method according to claim 7, wherein in the step (2), the mass concentrations of the notoginsenoside R1 and the ginsenoside Rg1 in the reference solution are respectively 0.4-1mg/ml;
preferably, in the mixed solution, the mass concentration of the notoginsenoside R1 and the mass concentration of the ginsenoside Rg1 are respectively 1mg/ml.
9. The quality control method according to any one of claims 1 to 6, wherein in the step (3), the conditions of the thin layer chromatography include: the sample application amount of the sample is 10-15 μl, the sample application amount of the control is 3-8 μl, and the sample application range is 15cm below 10deg.C.
10. The method according to any one of claims 1 to 6, wherein in the step (3), a 10% sulfuric acid ethanol solution is sprayed, and the mixture is heated at 105 ℃ until the color of the spots becomes clear, and the mixture is inspected under sunlight and/or ultraviolet light at 365 nm.
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