CN103245754B - Improved radix sophorae flavescentis thin-layer chromatography identification method - Google Patents
Improved radix sophorae flavescentis thin-layer chromatography identification method Download PDFInfo
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Abstract
The invention discloses an improved radix sophorae flavescentis thin-layer chromatography identification method. In comparison with the thin-layer chromatography identification method specified under a radix sophorae flavescentis item of Chinese Pharmacopoeia 2010, the method has been improved in the following aspects: (1) the test solution is prepared by an ultrasonic method from a soaking method; the amount of thin layer plates is reduced to be one from two, and three components of sophocarpidine, oxymatrine and sophoridine are unfolded on the same thin layer plate; (3) the amount of developing solvents is reduced to be two from three, that is methylbenzene-acetone-ethanol-ammonia water (6:8:2:0.28), methanol-chloroform (1.6:10); and (4) developing by a single color agent: bismuth potassium iodide-0.6mol/L hydrochloric acid (1:1). The method is simple to operate, time-saving, low in cost and good in separation effect and developing effect, the result can be easily judged and the color agent can be stored for a long time. A system suitability study proves that the method has favorable repeatability, sensitivity and developing stability, and is suitable for radix sophorae flavescentis thin-layer chromatography identification.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical field, relate to a kind of distinguishing method between true and false of medicine.
Background technology
Kuh-seng is the dry root of legume kuh-seng Sophora flavescens Ait.; Bitter, cold in nature; The thoughts of returning home, liver, stomach, large intestine, urinary bladder channel; There is heat-clearing and damp-drying drug, desinsection, effect of diuresis; For hot dysentery, have blood in stool, jaundice renal shutdown, leukorrhea with reddish discharge, swelling of vulva pruritus vulvae, eczema, wet sore, pruitus, mange leprosy, controls trichomonas vaginitis outward.In recent years, along with the increasing gradually of kuh-seng market consumption, its price continues soaring, has occurred the phenomenon of much adulterating, mixing the spurious with the genuine not only having reduced drug quality, also causes great negative effect to Chinese Medicinal Materials Markets.
Version Chinese Pharmacopoeia regulation in 2010, the discriminating of kuh-seng adopts thin-layered chromatography, and concrete grammar is as follows: (1) gets this product powder 0.5g, add strong ammonia solution 0.3ml, methenyl choloride 25mI, placement is spent the night, and filters, filtrate evaporate to dryness, residue adds methenyl choloride 0.5ml to be made to dissolve, as need testing solution.Separately get matrine reference substance and Sophoridine reference substance, add ethanol and make the mixed solution that every 1ml respectively contains 0.2mg, product solution in contrast.According to thin-layered chromatography test, draw the each 4 μ l of above-mentioned two kinds of solution, put respectively on the silica gel g thin-layer plate of preparing in same use 2% sodium hydroxide solution, taking toluene-acetone-methyl alcohol (8:3:0.5) as developping agent, launch, exhibition is apart from 8cm, take out, dry, then taking 10 DEG C of following upper solution of placing of toluene-ethyl acetate-methanol-water (2:4:2:1) as developping agent, launch, exhibition is apart from the same, take out, dry, spray successively with bismuth potassium iodide test solution and sodium nitrite ethanol test solution.In test sample chromatogram, with the corresponding position of reference substance chromatogram on, aobvious two identical orange spots.(2) separately get oxymatrine reference substance, add ethanol and make the solution of every 1ml containing 0.2mg, product solution in contrast.Draw above-mentioned need testing solution and the each 4 μ l of this reference substance solution, put respectively on the silica gel g thin-layer plate of preparing in same use 2% sodium hydroxide solution, taking 10 DEG C of following lower floor's solution of placing of methenyl choloride-methyl alcohol-strong ammonia solution (5:0.6:0.3) as developping agent, launch, take out, dry, spray successively with bismuth potassium iodide test solution and sodium nitrite ethanol test solution.In test sample chromatogram, with the corresponding position of reference substance chromatogram on, aobvious identical orange spot.
Above-mentioned TLC Identification judges the true and false of kuh-seng by differentiating index components matrine, oxymatrine and the Sophoridine of kuh-seng, there is following problem: the preparation of (1) need testing solution adopts infusion method, consuming timely grow (approximately needing 12 hours), index components dissolution rate is lower; (2) thin-layer chromatography is differentiated and need be used two blocks of thin layer plates, three kinds of developping agents, two kinds of developers, carry out three times and launch operation and four color operations, and the preparation of two kinds of developping agents need to be 10 DEG C of following placements, developer bismuth potassium iodide test solution is preserved easy generation precipitation at normal temperatures, need be now with the current, and whole process is loaded down with trivial details, consuming time; (3) the colour developing principle of the method is actual is first to develop the color with bismuth potassium iodide test solution, then decolours with sodium nitrite ethanol test solution, and the more difficult control of dosage in the time of colour developing and decolouring easily causes and develop the color dark or decolouring is excessive, affects the judgement to result.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of improved kuh-seng TLC Identification, the preparation of need testing solution is simple, save time; Matrine, oxymatrine and three kinds of components of Sophoridine can be launched on same thin laminate; Single Reagent colour developing, colour developing clear spot, retention time is long, and result is easy to judgement, and developer can be preserved use for a long time.
For achieving the above object, after deliberation, the invention provides following technical scheme:
Improved kuh-seng TLC Identification, comprises the following steps:
A. the preparation of reference substance solution: get matrine reference substance, oxymatrine reference substance and Sophoridine reference substance, add respectively ethanol and make the solution of every 1ml containing 0.2mg, product solution in contrast;
B. the preparation of need testing solution: get kuh-seng test sample powder 1g, add strong ammonia solution 0.3ml, methenyl choloride 25ml, ultrasonic 20 minutes of 250W, filters, filtrate evaporate to dryness, residue adds methenyl choloride 1ml to be made to dissolve, as need testing solution;
C. launch and develop the color: the each 4 μ l of need testing solution that three kinds of reference substance solution drawing that step a makes and step b make, put respectively on the silica gel g thin-layer plate of preparing in same use 2% sodium hydroxide solution, launch with developping agent A, exhibition, apart from 8cm, is taken out, dry, launch with developping agent B, exhibition, apart from 8cm, is taken out again, dry spray chromogenic reagent; Described developping agent A is that 6:8:2:0.28 mixes by toluene, acetone, ethanol and ammoniacal liquor according to volume ratio; Described developping agent B is that 1.6:10 mixes by methyl alcohol and chloroform according to volume ratio; Described developer is that 1:1 mixes by bismuth potassium iodide test solution and 0.6mol/L hydrochloric acid according to volume ratio;
D. result judgement: in test sample chromatogram, with the corresponding position of reference substance chromatogram on, aobvious identical orange spot, judges that kuh-seng test sample is genuine piece, otherwise is adulterant.
Beneficial effect of the present invention is: the present invention has set up a kind of improved kuh-seng TLC Identification, compared with the TLC Identification specifying under kuh-seng item of version Chinese Pharmacopoeia in 2010, (1) preparation of need testing solution changes ultrasonic method into by infusion method, operate easylier, expend time in greatly and to shorten; (2) thin layer plate reduces to one by two, and matrine, oxymatrine and three kinds of components of Sophoridine can be launched on same thin laminate, operates easylier, and duration of run shortens greatly, cost; (3) developping agent is reduced to two kinds by three kinds, and preparation time do not need to carry out the special processings such as 10 DEG C of following placements, operate easier, save time, cost; Meanwhile, the degree of separation of matrine, oxymatrine and three kinds of components of Sophoridine increases, and result is easier to observe; (4) Single Reagent colour developing, spot colors is evenly clear, and with distinct contrast with background colour, spot is round, and without obvious conditions of streaking, latter 30 minutes of colour developing, without the phenomenon of obviously fading, result was easy to judgement; (5) developer can be preserved use for a long time, does not need matching while using.Investigate and confirm through system suitability, the inventive method has good repeatability, sensitivity and color stability, and the thin-layer chromatography that is applicable to kuh-seng is differentiated.
Brief description of the drawings
In order to make object of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing and describe:
Fig. 1 is the thin-layer chromatogram that adopts version Chinese Pharmacopoeia prescriptive procedure discriminating kuh-seng in 2010, and wherein 1,4 is oxymatrine reference substance, and 2,3,5,8 is kuh-seng test sample, and 6 is matrine reference substance, and 7 is Sophoridine reference substance.
Fig. 2 adopts the present invention to improve one's methods to differentiate the thin-layer chromatogram of kuh-seng, and wherein 1 is Sophoridine reference substance, and 2 is oxymatrine reference substance, and 3 is matrine reference substance, and 4 is kuh-seng test sample.
Fig. 3 adopts the present invention to improve one's methods to differentiate the detectability test findings of kuh-seng, wherein 1 is Sophoridine reference substance, 2 is oxymatrine reference substance, and 3 is matrine reference substance, and 4～11 to be respectively extension rate be the kuh-seng need testing solution of 7,6,5,4,3,2,1,0 times.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
The main material using in preferred embodiment: matrine reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, 110805-200508); Oxymatrine reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, 110780-200506); Sophoridine reference substance (Yuan Ye bio tech ltd, Shanghai); Sophora flavescens, purchased from Chinese crude drug company, differentiates by the 188th page of kuh-seng of version Chinese Pharmacopoeia in 2010 a lower prescriptive procedure differentiates to be genuine piece; Silica G (Qingdao wave silica-gel desiccant factory); It is pure that all the other reagent are analysis; Bismuth potassium iodide test solution is prepared according to prescriptive procedure under annex XVB item of version Chinese Pharmacopoeia in 2010: get basic bismuth nitrate 0.85g, add after glacial acetic acid 10ml and water 40ml dissolving, add liquor kalii iodide (4 → 10) 20ml, shake up, to obtain final product.
The kuh-seng TLC Identification discriminating kuh-seng that comparative example 1, application version Chinese Pharmacopoeia in 2010 specifys
Differentiate that according to the 188th page of kuh-seng of version Chinese Pharmacopoeia in 2010 lower (3), (4) event prescriptive procedure carries out.Test findings as shown in Figure 1, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, aobvious identical orange spot, and spot colors is evenly clear, with distinct contrast with background colour, without obvious conditions of streaking.Wherein, the Rf value of matrine component, Oxymatrine alkaline constituents, Sophoridine component is respectively: 0.361 ± 0.052,0.394 ± 0.028,0.127 ± 0.015(n=3), degree of separation is respectively: 1.264 ± 0.046,1.315 ± 0.024,1.893 ± 0.031(n=3).
Embodiment 1, the improved kuh-seng TLC Identification of application the present invention are differentiated kuh-seng
The improved kuh-seng TLC Identification of the present invention, comprises the following steps:
A. the preparation of reference substance solution: get matrine reference substance, oxymatrine reference substance and Sophoridine reference substance, add respectively ethanol and make the solution of every 1ml containing 0.2mg, product solution in contrast;
B. the preparation of need testing solution: get kuh-seng test sample powder 1g, add strong ammonia solution 0.3ml, methenyl choloride 25ml, ultrasonic 20 minutes of 250W, filters, filtrate evaporate to dryness, residue adds methenyl choloride 1ml to be made to dissolve, as need testing solution;
C. launch and develop the color: the each 4 μ l of need testing solution that three kinds of reference substance solution drawing that step a makes and step b make, put respectively on the silica gel g thin-layer plate of preparing in same use 2% sodium hydroxide solution, launch with developping agent A, exhibition, apart from 8cm, is taken out, dry, launch with developping agent B, exhibition, apart from 8cm, is taken out again, dry spray chromogenic reagent; Described developping agent A is that 6:8:2:0.28 mixes by toluene, acetone, ethanol and ammoniacal liquor according to volume ratio; Described developping agent B is that 1.6:10 mixes by methyl alcohol and chloroform according to volume ratio; Described developer is that 1:1 mixes by bismuth potassium iodide test solution and 0.6mol/L hydrochloric acid according to volume ratio;
D. result judgement: in test sample chromatogram, with the corresponding position of reference substance chromatogram on, aobvious identical orange spot, judges that kuh-seng test sample is genuine piece, otherwise is adulterant.
Test findings as shown in Figure 2, in test sample chromatogram, with the corresponding position of reference substance chromatogram on, all aobvious identical orange spots, and spot colors is evenly clear, and with distinct contrast with background colour, spot is round, without obvious conditions of streaking, latter 30 minutes of colour developing without the phenomenon of obviously fading.Wherein, the Rf value (Rf) of matrine component, Oxymatrine alkaline constituents, Sophoridine component is respectively 0.721 ± 0.014,0.304 ± 0.018,0.580 ± 0.025(n=3), degree of separation (R) is respectively 1.719 ± 0.106,4.348 ± 0.175,3.125 ± 0.162(n=3), all meet thin-layered chromatography to 0.2 < Rf < 0.8, the requirement of R > 1.And compared with comparative example 1 test findings, the degree of separation of three kinds of target components is larger, result is easier to observe.
The system suitability of embodiment 2, the improved kuh-seng TLC Identification of the present invention is investigated
1, repeatability
Get the kuh-seng test sample of three parts of different batches, test by improved kuh-seng TLC Identification described in embodiment 1, every part of kuh-seng test sample does revision test three times, 9 test findings is evaluated to the repeatability of investigation method.
Test findings shows, in 9 test sample chromatograms, with the corresponding position of reference substance chromatogram on, all aobvious identical orange spots, and spot colors is evenly clear, with distinct contrast with background colour, without obvious conditions of streaking.In test sample chromatogram, Rf value and the degree of separation of matrine component, Oxymatrine alkaline constituents, Sophoridine component refer to table 1.Can find out from above-mentioned test findings, the improved kuh-seng TLC Identification of the present invention has good repeatability.
Table 1 repeatability is investigated test findings (n=9)
2, detectability
Prepare kuh-seng need testing solution by method described in embodiment 1, using this solution as mother liquor, carry out respectively 0,1,2,3,4,5,6,7 times of dilution with methenyl choloride, the need testing solution of testing as detectability using the dilution of gained variable concentrations again, launch and develop the color according to method described in embodiment 1, using the visible minimum test sample concentration of target components clear spot after developing the color as lowest detectable limit.
Test findings as shown in Figure 3, matrine component in kuh-seng need testing solution, Oxymatrine alkaline constituents, Sophoridine component respectively in the time of 3 times, 4 times, 4 times of dilutions spot colour developing clear, after continuing to strengthen extension rate, spot colour developing is unintelligible, therefore, determine the lowest detectable limit that the concentration of the capable 3 times of dilutions of kuh-seng need testing solution is the inventive method.Can find out from above-mentioned test findings, the improved kuh-seng TLC Identification of the present invention has higher sensitivity.
3, color stability
Press method preparation developer described in embodiment 1, under normal temperature, preserve, respectively at after preparation 1,5,15,30,45,60 day, carry out discrimination test according to method described in embodiment 1, whether stable the fading time of the each test colour developing of record spot, observe developer.
Test findings demonstration, the improved developer of the present invention is preserved 60 days at normal temperatures, and colour developing spot is still high-visible, and after each colour developing, retention time can reach more than 25 minutes.
Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can make various changes to it in the form and details, and not depart from the claims in the present invention book limited range.
Claims (1)
1. improved kuh-seng TLC Identification, is characterized in that: comprise the following steps:
A. the preparation of reference substance solution: get matrine reference substance, oxymatrine reference substance and Sophoridine reference substance, add respectively ethanol and make the solution of every 1ml containing 0.2mg, product solution in contrast;
B. the preparation of need testing solution: get kuh-seng test sample powder 1g, add strong ammonia solution 0.3ml, methenyl choloride 25ml, ultrasonic 20 minutes of 250W, filters, filtrate evaporate to dryness, residue adds methenyl choloride 1ml to be made to dissolve, as need testing solution;
C. launch and develop the color: the each 4 μ l of need testing solution that three kinds of reference substance solution drawing that step a makes and step b make, put respectively on the silica gel g thin-layer plate of preparing in same use 2% sodium hydroxide solution, launch with developping agent A, exhibition, apart from 8cm, is taken out, dry, launch with developping agent B, exhibition, apart from 8cm, is taken out again, dry spray chromogenic reagent; Described developping agent A is that 6:8:2:0.28 mixes by toluene, acetone, ethanol and ammoniacal liquor according to volume ratio; Described developping agent B is that 1.6:10 mixes by methyl alcohol and chloroform according to volume ratio; Described developer is that 1:1 mixes by bismuth potassium iodide test solution and 0.6mol/L hydrochloric acid according to volume ratio;
D. result judgement: comparison test sample chromatogram and the orange spot of reference substance chromatogram on relevant position.
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| CN104730169A (en) * | 2015-04-03 | 2015-06-24 | 宁夏职业技术学院 | Quality detection method of total alkaloid of sophora alopecuroide |
| CN104833764B (en) * | 2015-05-07 | 2016-08-24 | 河南省康星药业股份有限公司 | Matrine and the tlc identification method of oxymatrine in a kind of Sophora flavescens |
| CN112180024A (en) * | 2020-09-30 | 2021-01-05 | 西安雨田农业科技股份有限公司 | Quality control method of traditional Chinese medicine compound preparation for treating piglet diarrhea |
| CN113125628A (en) * | 2021-03-12 | 2021-07-16 | 河北锦坤动物药业有限公司 | Thin-layer chromatography identification method for radix sophorae flavescentis and radix sophorae flavescentis in three-flavor bistort oral liquid |
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