CN104730169A - Quality detection method of total alkaloid of sophora alopecuroide - Google Patents
Quality detection method of total alkaloid of sophora alopecuroide Download PDFInfo
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Abstract
The invention relates to a quality detection method of total alkaloid of sophora alopecuroide. The method comprises the steps of content measurement of effective components and verification and detection of main components, wherein the content measurement of effective components refers to measurement on the sum of oxymatrine and sophocarpidine or the sum of n-oxysophocarpine and sophocarpine in the total alkaloid of sophora alopecuroide by using high performance liquid chromatography; the verification and detection of main components refers to verification on sophoridine, oxymatrine, n-oxysophocarpine and sophocarpine in the total alkaloid of sophora alopecuroide by using high performance liquid chromatography or thin layer chromatography. The method provided by the invention is high in accuracy, strong in specificity and good in reproducibility and is conducive to quality detection of the total alkaloid of sophora alopecuroide.
Description
Technical field
The present invention relates to Chinese medicine detection technique field, particularly relate to a kind of sophora alapecuroides quality determining method.
Background technology
Sophora alapecuroides is the total alkaloids extracted from cassia leguminous plant Sophora alopecuroide, and sophora alapecuroides contains more than 20 kind of alkaloid, and Sophoridine, matrine, oxymatrine, sophocarpine, N-Oxysophocarpine are its principal ingredients.In Sophora alopecuroide plant, the content of oxymatrine, N-Oxysophocarpine is higher than the content of Sophoridine, matrine, sophocarpine, show to should be more stable natural existence form at plant internal oxidition matrine, N-Oxysophocarpine, and matrine and oxymatrine, sophocarpine and N-Oxysophocarpine also exist mutual conversion in leaching process, along with the increase of extraction time, oxidized form alkaloid is converted into the alkaloidal amount of reduced form to be increased.Office of State Food and Drug Administration issues in the quality standard of standard sophora alapecuroides (WS-10001 (HD-1391)-2003) method adopting HPLC to measure Sophoridine, do not adopt the method measuring matrine, oxymatrine or N-Oxysophocarpine, sophocarpine, have ignored the factor that Sophora alopecuroide leaching process oxidized form alkaloid and reduced form alkaloid transform mutually.The content only measuring single component Sophoridine is difficult to alkaloidal actual content situation in reflection Sophora alopecuroide, therefore sets up with matrine and oxymatrine sum or has very important significance for sophora alapecuroides quality control as quality control index using N-Oxysophocarpine and sophocarpine sum.
Traditional Chinese medicine ingredients is complicated, sets up the quality control of many indexes composition, and many-sided control product quality, improves the quality control level of modern Chinese herbal medicine, is the direction of development.
Summary of the invention
The object of this invention is to provide a kind of to set up many indexes composition to carry out the quality determining method of the sophora alapecuroides that production quality control is target.
The object of the invention is to realize according to following proposal:
A kind of quality determining method of sophora alapecuroides, the discriminating of the assay and principal ingredient that it is characterized in that comprising effective constituent detects, and wherein the assay of effective constituent refers to and adopts high performance liquid chromatography to measure the oxymatrine in sophora alapecuroides and matrine sum or N-Oxysophocarpine and sophocarpine sum; The discriminating of principal ingredient detects and refers to and adopt high performance liquid chromatography or thin-layered chromatography to differentiate alkaloid contained by sophora alapecuroides.
During described employing high performance liquid chromatography measures the oxymatrine in sophora alapecuroides and matrine sum or N-Oxysophocarpine and sophocarpine sum, chromatographic condition: with the strong silica gel that closes of octadecylsilane for filling agent; Take acetonitrile as mobile phase A, with the potassium dihydrogen phosphate of the 0.05moL/L containing 2.0mL/L triethylamine for Mobile phase B, by 0 ~ 12min mobile phase A-Mobile phase B (6:94), 12 ~ 60min mobile phase A-Mobile phase B (10:90) carries out gradient elution; Determined wavelength is 205nm, and theoretical cam curve calculates should be not less than 5000 by oxymatrine peak or N-Oxysophocarpine peak.
Described employing high performance liquid chromatography is carried out in mensuration process to the oxymatrine in sophora alapecuroides and matrine sum or N-Oxysophocarpine and sophocarpine sum,
The preparation of reference substance solution: get matrine reference substance, oxymatrine reference substance is appropriate, accurately weighed, add methyl alcohol make every 1mL containing matrine 0.10mg, oxymatrine 0.15mg mixed solution or get sophocarpine reference substance, N-Oxysophocarpine reference substance is appropriate, accurately weighed, add methyl alcohol and make the mixed solution of every 1mL containing sophocarpine 0.10mg, N-Oxysophocarpine 0.15mg;
The preparation of need testing solution: get sophora alapecuroides test sample and be about 0.2g, accurately weighed, add methyl alcohol dissolving and be settled in 100mL measuring bottle, mixing, to obtain final product;
Determination method: accurate absorption matrine and oxymatrine mix reference substance solution or sophocarpine and N-Oxysophocarpine and mix reference substance solution 0 μ L respectively, separately get need testing solution 10 μ L, injection liquid chromatography, measure, the amount of matrine and oxymatrine or the amount of sophocarpine and N-Oxysophocarpine is calculated respectively by external standard method, both are added and, to obtain final product.
During described high performance liquid chromatography detects the discriminating of effective constituent, chromatographic condition is: with the strong silica gel that closes of octadecylsilane for filling agent; Take acetonitrile as mobile phase A, with the potassium dihydrogen phosphate of the 0.05moL/L containing 2.0mL/L triethylamine for Mobile phase B, by 0 ~ 12min mobile phase A-Mobile phase B (6:94), 12 ~ 60min mobile phase A-Mobile phase B (10:90) carries out gradient elution; Determined wavelength is 205nm, and number of theoretical plate calculates should be not less than 5000 by oxymatrine peak.
During described high performance liquid chromatography detects the discriminating of effective constituent,
The preparation of object of reference solution: get Sophoridine reference substance, oxymatrine reference substance, N-Oxysophocarpine reference substance and matrine reference substance appropriate, adds methyl alcohol respectively and makes every 1mL respectively containing the solution of 0.10mg, as object of reference solution;
The preparation of need testing solution: get sophora alapecuroides test sample and be about 0.1g, accurately weighed, add methyl alcohol and dissolve, and make the solution of every mL containing 4mg, as need testing solution with methyl alcohol;
Determination method: get need testing solution and each 10 μ L of object of reference solution, injection liquid chromatography, measure test sample chromatogram, test sample chromatogram should present four characteristic peaks corresponding with contrast characteristic spectrum, its retention time should be corresponding with object of reference peak retention time, and the peak corresponding with oxymatrine peak retention time is S peak; The relative retention time setting of each characteristic peak is respectively 0.72 (peak 1), 1.00 (peaks 2), 1.13 (peaks 3), 1.57 (peaks 4), 2.06 (peaks 5), in test sample chromatogram, the relative retention time of each characteristic peak should its setting ± 5% within.
During described thin-layered chromatography detects the discriminating of effective constituent, need testing solution and reference substance solution are put respectively on same silica gel g thin-layer plate, take volume ratio as the methenyl choloride-methanol-conc. aq. ammonia of 5:0.6:0.3 be developping agent, ascending development half, takes out airing, then be 7:3:3 toluene-acetone-ethyl acetate with volume ratio is developping agent, second outspread, take out, dry, spray to improve bismuth potassium iodide test solution.
The present invention by chromatographic condition preferably and test sample, reference substance preparation method test, to establish in sophora alapecuroides principal ingredient matrine and oxymatrine sum or using N-Oxysophocarpine and sophocarpine sum as the HPLC analytical method of assay index; Set up using Sophoridine reference substance, oxymatrine reference substance, N-Oxysophocarpine reference substance and matrine reference substance as object of reference solution, be need testing solution with sophora alapecuroides, calculate the high performance liquid chromatography of the discriminating sophora alapecuroides of the relative retention time at each characteristic peak and oxymatrine object of reference peak; By the optimization to chromatographic fractionation system, the adjustment of developping agent proportioning, establishes the TLC Identification of sophora alapecuroides.By showing methodology experimental study: the method accuracy of setting up is high, specificity is strong, reappearance is good, is conducive to the quality control of sophora alapecuroides.
Embodiment
Sophora alapecuroides quality determining method, the discriminating of the assay and principal ingredient that comprise effective constituent detects.Wherein the assay of effective constituent refers to and adopts high performance liquid chromatography to measure the oxymatrine in sophora alapecuroides and matrine sum or N-Oxysophocarpine and sophocarpine sum.The discriminating of principal ingredient detects and refers to that employing high performance liquid chromatography or thin-layered chromatography are differentiated the Sophoridine in sophora alapecuroides, oxymatrine, N-Oxysophocarpine and matrine.
Embodiment 1: the assay of effective constituent
1, chromatographic condition: with the strong silica gel that closes of octadecylsilane for filling agent; Take acetonitrile as mobile phase A, with the potassium dihydrogen phosphate of 0.05moL/L (including 2.0mL/L triethylamine) for Mobile phase B, the regulation according to the form below carries out gradient elution; Determined wavelength is 205nm.Theoretical cam curve calculates should be not less than 5000 by oxymatrine peak or with N-Oxysophocarpine peak.
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0～12 | 6 | 94 |
| 12～60 | 10 | 90 |
2, the preparation method of need testing solution: get sophora alapecuroides and be about 0.2g, accurately weighed, add methyl alcohol dissolving and be settled in 100mL measuring bottle, mixing, to obtain final product.
3, the preparation method of reference substance solution: get matrine reference substance, oxymatrine reference substance is appropriate, accurately weighed, add methyl alcohol or mobile phase makes the mixed solution of every 1mL containing matrine 0.10mg, oxymatrine 0.15mg, to obtain final product.
4, the above-mentioned need testing solution of accurate absorption and each 10 μ L of reference substance solution, injection liquid chromatography, records the chromatogram of 60 minutes, calculates the content of matrine and oxymatrine in test sample, and sum up by external standard method.
Embodiment 2: the assay of effective constituent
1, chromatographic condition: with the strong silica gel that closes of octadecylsilane for filling agent; Take acetonitrile as mobile phase A, with the potassium dihydrogen phosphate of 0.05moL/L (including 2.0mL/L triethylamine) for Mobile phase B, the regulation according to the form below carries out gradient elution; Determined wavelength is 205nm.Theoretical cam curve calculates should be not less than 5000 by oxymatrine peak or with N-Oxysophocarpine peak.
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0～12 | 6 | 94 |
| 12～60 | 10 | 90 |
2, the preparation method of need testing solution: get sophora alapecuroides and be about 0.2g, accurately weighed, add methyl alcohol dissolving and be settled in 100mL measuring bottle, mixing, to obtain final product.
3, the preparation method of reference substance solution: get sophocarpine reference substance, N-Oxysophocarpine reference substance is appropriate, accurately weighed, add methyl alcohol or mobile phase makes the mixed solution of every 1mL containing sophocarpine 0.10mg, N-Oxysophocarpine 0.15mg, to obtain final product.
4, the above-mentioned need testing solution of accurate absorption and each 10 μ L of reference substance solution, injection liquid chromatography, records the chromatogram of 60 minutes, calculates the content of sophocarpine and N-Oxysophocarpine in test sample, and sum up by external standard method.
Embodiment 3: the discriminating (high performance liquid chromatography) of principal ingredient
1, the preparation of need testing solution: get sophora alapecuroides and be about 0.1g, accurately weighed, add methyl alcohol and dissolve, make the solution of every 1mL containing 4mg.
2, the preparation of object of reference solution: get Sophoridine reference substance, oxymatrine reference substance, N-Oxysophocarpine reference substance and matrine reference substance respectively appropriate, adds methyl alcohol respectively or mobile phase makes every 1mL respectively containing the solution of 0.10mg.
3, chromatographic condition: with the strong silica gel that closes of octadecylsilane for filling agent; Take acetonitrile as mobile phase A, with the potassium dihydrogen phosphate of 0.05moL/L (including 2.0mL/L triethylamine) for Mobile phase B, the regulation according to the form below carries out gradient elution; Determined wavelength is 205nm.Number of theoretical plate calculates should be not less than 5000 by oxymatrine peak.
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0～12 | 6 | 94 |
| 12～60 | 10 | 90 |
4, accurate Sophoridine, oxymatrine, N-Oxysophocarpine and matrine four kinds of object of reference solution and each 10 μ L of need testing solution, injection liquid chromatography, record the chromatogram of 60 minutes, calculate the relative retention time at each characteristic peak and oxymatrine object of reference peak, to obtain final product.
Test sample chromatogram should present four characteristic peaks corresponding with contrast characteristic spectrum, and retention time should be corresponding with object of reference peak retention time, and the peak corresponding with oxymatrine peak retention time is S peak; The relative retention time setting of each characteristic peak is respectively 0.72 (peak 1), 1.00 (peaks 2), 1.13 (peaks 3), 1.57 (peaks 4), 2.06 (peaks 5).In test sample chromatogram, the relative retention time of each characteristic peak should its setting ± 5% within.
Embodiment 4: (tlc identification method, first prepares test sample and reference substance solution, and two kinds of solution are put respectively on same silica gel g thin-layer plate, comparison colour developing situation in the discriminating of principal ingredient.)
1, the preparation of need testing solution: get sophora alapecuroides appropriate, adds methyl alcohol and makes the solution of every 1mL containing 2.5mg, as need testing solution.
2, the preparation of reference substance solution: get Sophoridine, oxymatrine, N-Oxysophocarpine and matrine reference substance, adds methyl alcohol and makes every 1mL respectively respectively containing the solution of 1mg, product solution in contrast.
3, Sophoridine, oxymatrine, N-Oxysophocarpine and matrine four kinds of reference substance solution and each 10 μ L of need testing solution are drawn, put respectively on same silica gel g thin-layer plate, with methenyl choloride-methanol-conc. aq. ammonia (5:0.6:0.3) for developping agent, ascending development half, takes out airing, then with toluene-acetone-ethyl acetate (7:3:3) for developping agent, second outspread, take out, dry, spray to improve bismuth potassium iodide test solution.In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
Claims (6)
1. the quality determining method of a sophora alapecuroides, the discriminating of the assay and principal ingredient that it is characterized in that comprising effective constituent detects, and wherein the assay of effective constituent refers to and adopts high performance liquid chromatography to measure the oxymatrine in sophora alapecuroides and matrine sum or N-Oxysophocarpine and sophocarpine sum; The discriminating of principal ingredient detects and refers to and adopt high performance liquid chromatography or thin-layered chromatography to differentiate alkaloid contained by sophora alapecuroides.
2. the quality determining method of sophora alapecuroides as claimed in claim 1, in it is characterized in that described employing high performance liquid chromatography measures the oxymatrine in sophora alapecuroides and matrine sum or N-Oxysophocarpine and sophocarpine sum, chromatographic condition: with the strong silica gel that closes of octadecylsilane for filling agent; Take acetonitrile as mobile phase A, with the potassium dihydrogen phosphate of the 0.05moL/L containing 2.0mL/L triethylamine for Mobile phase B, by 0 ~ 12 min mobile phase A-Mobile phase B (6:94), 12 ~ 60 min mobile phase A-Mobile phase B (10:90) carry out gradient elution; Determined wavelength is 205nm, and theoretical cam curve calculates should be not less than 5000 by oxymatrine peak or N-Oxysophocarpine peak.
3. the quality determining method of sophora alapecuroides as claimed in claim 2, is characterized in that described employing high performance liquid chromatography is carried out in mensuration process to the oxymatrine in sophora alapecuroides and matrine sum or N-Oxysophocarpine and sophocarpine sum,
The preparation of reference substance solution: get matrine reference substance, oxymatrine reference substance is appropriate, accurately weighed, add methyl alcohol make every 1mL containing matrine 0.10mg, oxymatrine 0.15mg mixed solution or get sophocarpine reference substance, N-Oxysophocarpine reference substance is appropriate, accurately weighed, add methyl alcohol and make the mixed solution of every 1mL containing sophocarpine 0.10mg, N-Oxysophocarpine 0.15mg;
the preparation of need testing solution: get sophora alapecuroides test sample and be about 0.2g, accurately weighed, add methyl alcohol dissolving and be settled in 100mL measuring bottle, mixing, to obtain final product;
determination method: accurate absorption matrine and oxymatrine mix reference substance solution or sophocarpine and N-Oxysophocarpine and mix reference substance solution 0 μ L respectively, separately get need testing solution 10 μ L, injection liquid chromatography, measure, the amount of matrine and oxymatrine or the amount of sophocarpine and N-Oxysophocarpine is calculated respectively by external standard method, both are added and, to obtain final product.
4. the quality determining method of sophora alapecuroides as claimed in claim 1, in it is characterized in that described high performance liquid chromatography detects the discriminating of effective constituent, chromatographic condition is: with the strong silica gel that closes of octadecylsilane for filling agent; Take acetonitrile as mobile phase A, with the potassium dihydrogen phosphate of the 0.05moL/L containing 2.0mL/L triethylamine for Mobile phase B, by 0 ~ 12 min mobile phase A-Mobile phase B (6:94), 12 ~ 60 min mobile phase A-Mobile phase B (10:90) carry out gradient elution; Determined wavelength is 205nm, and number of theoretical plate calculates should be not less than 5000 by oxymatrine peak.
5. the quality determining method of sophora alapecuroides as claimed in claim 5, in it is characterized in that described high performance liquid chromatography detects the discriminating of effective constituent,
The preparation of object of reference solution: get Sophoridine reference substance, oxymatrine reference substance, N-Oxysophocarpine reference substance and matrine reference substance appropriate, adds methyl alcohol respectively and makes every 1mL respectively containing the solution of 0.10mg, as object of reference solution;
The preparation of need testing solution: get sophora alapecuroides test sample and be about 0.1g, accurately weighed, add methyl alcohol and dissolve, and make the solution of every mL containing 4mg, as need testing solution with methyl alcohol;
Determination method: get need testing solution and each 10 μ L of object of reference solution, injection liquid chromatography, measure test sample chromatogram, test sample chromatogram should present four characteristic peaks corresponding with contrast characteristic spectrum, its retention time should be corresponding with object of reference peak retention time, and the peak corresponding with oxymatrine peak retention time is S peak; The relative retention time setting of each characteristic peak is respectively 0.72(peak 1), 1.00(peak 2), 1.13(peak 3), 1.57(peak 4), 2.06(peak 5), in test sample chromatogram, the relative retention time of each characteristic peak should its setting ± 5% within.
6. the quality determining method of sophora alapecuroides as claimed in claim 1, in it is characterized in that described thin-layered chromatography detects the discriminating of effective constituent, need testing solution and reference substance solution being put respectively on same silica gel g thin-layer plate, is that the methenyl choloride-methanol-conc. aq. ammonia of 5:0.6:0.3 is developping agent with volume ratio, ascending development half, take out airing, be 7:3:3 toluene-acetone-ethyl acetate again with volume ratio be developping agent, second outspread, takes out, dry, spray to improve bismuth potassium iodide test solution.
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| CN106198802A (en) * | 2016-07-11 | 2016-12-07 | 广东医科大学 | A kind of method of quality control of Herba Sophorae alopecuroidis total alkaloids |
| CN109856287A (en) * | 2019-03-25 | 2019-06-07 | 广西壮族自治区药用植物园 | The detection method of alkaloid in toothpaste |
| 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 |
| CN113358772A (en) * | 2021-05-21 | 2021-09-07 | 河北化工医药职业技术学院 | Establishment and application of HPLC fingerprint spectrum series method based on different separation mechanisms |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106198802A (en) * | 2016-07-11 | 2016-12-07 | 广东医科大学 | A kind of method of quality control of Herba Sophorae alopecuroidis total alkaloids |
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| CN109856287A (en) * | 2019-03-25 | 2019-06-07 | 广西壮族自治区药用植物园 | The detection method of alkaloid in toothpaste |
| WO2022116971A1 (en) * | 2020-12-02 | 2022-06-09 | 北京振东光明药物研究院有限公司 | Method for detecting content of active ingredients of compound sophorae flavescentis radix injection and fingerprint spectrum thereof |
| 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 |
| CN113358772A (en) * | 2021-05-21 | 2021-09-07 | 河北化工医药职业技术学院 | Establishment and application of HPLC fingerprint spectrum series method based on different separation mechanisms |
| CN113358772B (en) * | 2021-05-21 | 2022-09-20 | 河北化工医药职业技术学院 | Establishment and application of HPLC fingerprint spectrum series method based on different separation mechanisms |
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Application publication date: 20150624 |