CN107505426A - A kind of method for quick identification of ingenol - Google Patents
A kind of method for quick identification of ingenol Download PDFInfo
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- CN107505426A CN107505426A CN201710611366.6A CN201710611366A CN107505426A CN 107505426 A CN107505426 A CN 107505426A CN 201710611366 A CN201710611366 A CN 201710611366A CN 107505426 A CN107505426 A CN 107505426A
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- ingenol
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- petroleum ether
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- 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
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Abstract
The invention belongs to Natural Medicine Chemistry technical field, and in particular to a kind of ingenol method for quick identification；In turn include the following steps：1）Top section after lathyrol extract recycling design is subjected to basic hydrolysis, neutralization, extraction, adds methanol dissolving after volatilizing solvent；2）Supernatant is taken to be tested according to thin-layered chromatography, with petroleum ether（60℃~90℃）Ethyl acetate is solvent, expansion；3）Lamellae is taken out, drying, sprays with chromogenic reagent, it is clear to be heated to band；4）Lamellae is placed under 365nm and observed, it is seen that ingenol is light blue spot, and other materials are orange-yellow, cyan, pink spot.The present invention has the advantages of efficient, easy, accurate, reliable, has filled up the blank of ingenol specificity quick detection discrimination method, the quick detection available for ingenol.
Description
Technical field
The invention belongs to Natural Medicine Chemistry authentication technique field, and in particular to the high performance thin layer chromatography of ingenol is quick
Discrimination method.
Background technology
Nineteen sixty-eight ingenol（Ingenol）First from euphorbia plantEIt is isolated in ingens, there is uniqueness
The Fourth Ring parent nucleus of high-tension 5/7/7/3, by being interacted with protein kinase C, produce the pharmacological activity such as anticancer, anti AIDS virus.
2012, the ingenol methylcrotonic acid gels researched and developed by LEO drugmakers（Ingenol mebutate,
Medicine name Picato）Obtain in U.S. FDA approval, turn into first and be used to treat solar keratosis（actinic keratosis）
Gel preparation.In addition ingenol methyl butene acid esters can be additionally used in basal-cell carcinoma treatment, carry out the clinical examination of II phase
Test.Ingenol methyl butene acid esters is obtained using ingenol as raw material is semi-synthetic in production, yield 62%.It is however, huge
Halberd alcohol is only in Euphorbia lathyris（Euphorbia lathryis）Etc. being distributed in a small number of euphorbias, separated from Euphorbia lathyris seed
Yield is 275 mg, than the yield that qinghaosu is separated directly from plant（5000mg）Low nearly 20 times.LEO drugmakers in 2013
The Scripps Research Inst. of the joint U.S.（The Scripps Research Institute）Phil S. Baran research teams
Develop the step total synthesis method of ingenol 14.Therefore, establish a kind of method of quick discriminating ingenol and can be applied to
In the industrialized productions such as ingenol separation, purifying and synthesis, be advantageous to the quick identification and detection of ingenol.
The detection of ingenol at present is mainly using high performance liquid chromatography combination EISD to naboom
Alcohol carries out qualitative and quantitative analysis.Because composition is various and complicated in Euphorbia lathyris, different compounds may have identical
Or similar keeping characteristics, easily cause qualitative results mistake.In addition, the liquid-phase chromatographic analysis time is longer, organic phase flow consumption
Amount is big, easily causes testing cost height, is not suitable for ingenol quick detection and discriminating.Therefore develop efficiently, it is easy, accurate, can
The ingenol method for quick identification leaned on is significant.
The content of the invention
In order to solve the above problems, it is an object of the invention to provide a kind of discriminating of the high performance thin layer chromatography of ingenol
Method.
Invention is achieved by the following technical solution：
A kind of ingenol method for quick identification, the assay method comprise the following steps：
1）Ingenol standard items are taken in volumetric flask, adding solvent makes its dissolving, constant volume.As reference substance solution.
2）Euphorbia lathyris seed is taken to crush, alcohol reflux extracts, and recycling design and stratification, takes a small amount of upper strata material to use
Petroleum ether（60℃~90℃）Dissolving, acetonitrile extraction 2 times, merges and reclaims acetonitrile, add appropriate methanol and dissolve and adjust pH value
To alkalescence, after being stirred at room temperature, pH value is adjusted recycling design, then to be extracted with ethyl acetate to neutrality, merge acetic acid second with acid
Ester layer, ethyl acetate is reclaimed, adding solvent is uniformly dissolved it, as need testing solution.
3）Above-mentioned reference substance and need testing solution are drawn, is put on same GF254 silica gel plates, with petroleum ether（60℃~90
℃）- ethyl acetate is solvent, expansion, takes out, dries up, spray developer, being heated to clear spot, be placed under 365nm and observe,
In reference substance and test sample thin-layer chromatography, the visible bright light blue spot of ingenol, other materials show orange-yellow, cyan and pink
Color spot point.
Wherein, the step 1）In solvent be methanol.
It is preferred that described solvent is petroleum ether（60℃~90℃）, ethyl acetate mixture, the body between them
Product ratio is petroleum ether（60℃~90℃）:Ethyl acetate is 3:7.
It is preferred that described developer is 5% vanillic aldehyde-concentrated sulfuric acid.
It is preferred that adjust methanol solution of the reagent that pH value uses to alkalescence for 3% sodium hydroxide.
It is preferred that the reagent that pH value to neutrality uses is adjusted as 1 N hydrochloric acid.
The high performance thin layer chromatography quick discriminating ingenol method of the present invention, it has efficient, easy, accurate, reliable
The advantages of, fill up the blank of ingenol method for quick identification.
4th, illustrate
The inventive method of accompanying drawing 1 detects the HPTLC chromatograms of ingenol；
Wherein 1 is ingenol standard items, and 2-6 is test sample；
Accompanying drawing 2 is the structural formula of ingenol monomeric compound, and accompanying drawing 2 is Figure of abstract；
Embodiment
With reference to embodiment, the present invention is described in further detail, to help skilled artisan understands that hair
It is bright.
If without specified otherwise, test method used is conventional methods；Agents useful for same material can be obtained by commercial sources
.
The analyzing detecting method for the preferred embodiment implemented according to the present invention is as follows
Embodiment 1
The mg of ingenol reference substance 1 is taken in volumetric flask, adding 1 mL methanol is completely dissolved it, as reference substance solution.Separately
Lathyrol extract recycling design upper strata is taken, adjusts pH value to be made into need testing solution by [0009] to 12-14, Hydrolysis At Room Temperature 0.5h；
Above-mentioned reference substance solution and each 5 uL of need testing solution are drawn according to thin-layered chromatography experiment, is put respectively in same GF254 silica gel
On plate, expansion（Using petroleum ether and ethyl acetate mixture as solvent, the volume ratio petroleum ether:Ethyl acetate=3:7）, take
Go out drying, spray with 5% vanillic aldehyde-concentrated sulfuric acid solution（Vanillic aldehyde 5g, the mL of 10% ethanol solution of sulfuric acid 100）
Colour developing, is heated to clear spot, is placed in being observed under 365nm；In reference substance and test sample thin-layer chromatography, naboom
Alcohol shows light blue, and other spots show orange-yellow, cyan or pink, specifically see No. 1 point and No. 2 points in Fig. 1.
Embodiment 2
Lathyrol extract recycling design upper strata is taken to adjust pH value that it is molten to be made into test sample according to [0009] to 12-14, Hydrolysis At Room Temperature 1h
Liquid；Above-mentioned each 5 uL of need testing solution is drawn according to thin-layered chromatography experiment, and reference substance solution point is in same GF254 silica gel plates
On, expansion（Using petroleum ether and ethyl acetate mixture as solvent, the volume ratio petroleum ether:Ethyl acetate=3:7）, take out
Drying, sprays with 5% vanillic aldehyde-concentrated sulfuric acid solution（Vanillic aldehyde 5g, the mL of 10% ethanol solution of sulfuric acid 100）Colour developing, it is clear to be heated to spot
It is clear, it is placed in being observed under 365nm；In reference substance and test sample thin-layer chromatography, ingenol shows light blue, and other spots show
Orange-yellow, cyan or pink, specifically see No. 3 points in Fig. 1.
Embodiment 3
Lathyrol extract recycling design upper strata is taken to adjust pH value that it is molten to be made into test sample according to [0009] to 12-14, Hydrolysis At Room Temperature 2h
Liquid；Above-mentioned each 5 uL of need testing solution is drawn according to thin-layered chromatography experiment, and reference substance solution point is in same GF254 silica gel plates
On, expansion（Using petroleum ether and ethyl acetate mixture as solvent, the volume ratio petroleum ether:Ethyl acetate=3:7）, take out
Drying, sprays with 5% vanillic aldehyde-concentrated sulfuric acid solution（Vanillic aldehyde 5g, the mL of 10% ethanol solution of sulfuric acid 100）Colour developing, it is clear to be heated to spot
It is clear, it is placed in being observed under 365nm；In reference substance and test sample thin-layer chromatography, ingenol shows light blue, and other spots show
Orange-yellow, cyan or pink, specifically see No. 4 points in Fig. 1.
Embodiment 4
Lathyrol extract recycling design upper strata is taken to adjust pH value that it is molten to be made into test sample according to [0009] to 12-14, Hydrolysis At Room Temperature 3h
Liquid；Above-mentioned each 5 uL of need testing solution is drawn according to thin-layered chromatography experiment, and reference substance solution point is in same GF254 silica gel plates
On, expansion（Using petroleum ether and ethyl acetate mixture as solvent, the volume ratio petroleum ether:Ethyl acetate=3:7）, take out
Drying, sprays with 5% vanillic aldehyde-concentrated sulfuric acid solution（Vanillic aldehyde 5g, the mL of 10% ethanol solution of sulfuric acid 100）Colour developing, it is clear to be heated to spot
It is clear, it is placed in being observed under 365nm；In reference substance and test sample thin-layer chromatography, ingenol shows light blue, and other spots show
Orange-yellow, cyan or pink, No. 5 points being specifically shown in Fig. 1.
Embodiment 5
Lathyrol extract recycling design upper strata is taken to adjust pH value that it is molten to be made into test sample according to [0009] to 12-14, Hydrolysis At Room Temperature 4h
Liquid；Above-mentioned each 5 uL of need testing solution is drawn according to thin-layered chromatography experiment, and reference substance solution point is in same GF254 silica gel plates
On, expansion（Using petroleum ether and ethyl acetate mixture as solvent, the volume ratio petroleum ether:Ethyl acetate=3:7）, take out
Drying, sprays with 5% vanillic aldehyde-concentrated sulfuric acid solution（Vanillic aldehyde 5g, 10% ethanol solution of sulfuric acid）Colour developing, is heated to clear spot, and
It is placed under 365nm and is observed；In reference substance and test sample thin-layer chromatography, ingenol shows light blue, and other spots show orange
Color, cyan or pink, specifically see No. 6 points in Fig. 1.
Claims (6)
1. a kind of method for quick identification of ingenol, it is characterised in that the discrimination method comprises the following steps：
1）Ingenol standard items are taken in volumetric flask, add solvent as reference substance solution,
2）The upper strata material after lathyrol extract recycling design separately is taken, through basic hydrolysis, neutralization, extraction processing, adding solvent makes
It is uniformly dissolved, as need testing solution,
3）Above-mentioned reference substance and need testing solution are drawn, is put on same GF254 silica gel plates, with petroleum ether（60℃~90℃）- second
Acetoacetic ester is solvent, expansion, take out, drying, spray chromogenic reagent, it is clear to be heated to band, is positioned under 365nm and observes,
In reference substance and test sample thin-layer chromatography, ingenol is light blue spot, and other materials show orange-yellow, cyan and pink color spot
Point.
2. ingenol method for quick identification according to claim 1, it is characterised in that the step 1）In solvent be
Methanol.
3. ingenol method for quick identification according to claim 1, it is characterised in that the step 2）In solvent be
Methanol.
4. ingenol method for quick identification according to claim 1, it is characterised in that the step 1）In, take naboom
The mg of alcohol reference substance 1 adds methanol dissolve, shake up, constant volume is as reference substance solution in 1 mL volumetric flasks.
5. ingenol method for quick identification according to claim 1, it is characterised in that：Described solvent is petroleum ether
（60℃~90℃）, ethyl acetate mixture, the volume ratio between them is petroleum ether（60℃~90℃）:Ethyl acetate=3:
7。
6. ingenol method for quick identification according to claim 1, it is characterised in that：Described developer is 5% vanilla
Aldehyde-the concentrated sulfuric acid.
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| CN201710611366.6A CN107505426A (en) | 2017-07-25 | 2017-07-25 | A kind of method for quick identification of ingenol |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694096A (en) * | 2013-12-23 | 2014-04-02 | 成都普思生物科技有限公司 | Separation and purification method for ingenol monomers |
| CN105061169A (en) * | 2015-08-03 | 2015-11-18 | 江苏红豆杉药业有限公司 | Ingenol and preparation method therefor |
| CN105272838A (en) * | 2014-06-11 | 2016-01-27 | 成都普瑞法科技开发有限公司 | Separating purifying method for ingenol extract |
| US20160107977A1 (en) * | 2013-05-31 | 2016-04-21 | Leo Laboratories Limited | Methods of synthesis of ingenol and intermediates thereof |
| CN106083555A (en) * | 2016-06-14 | 2016-11-09 | 宁波绿之健药业有限公司 | The extracting method of ingenol |
-
2017
- 2017-07-25 CN CN201710611366.6A patent/CN107505426A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160107977A1 (en) * | 2013-05-31 | 2016-04-21 | Leo Laboratories Limited | Methods of synthesis of ingenol and intermediates thereof |
| CN103694096A (en) * | 2013-12-23 | 2014-04-02 | 成都普思生物科技有限公司 | Separation and purification method for ingenol monomers |
| CN105272838A (en) * | 2014-06-11 | 2016-01-27 | 成都普瑞法科技开发有限公司 | Separating purifying method for ingenol extract |
| CN105061169A (en) * | 2015-08-03 | 2015-11-18 | 江苏红豆杉药业有限公司 | Ingenol and preparation method therefor |
| CN106083555A (en) * | 2016-06-14 | 2016-11-09 | 宁波绿之健药业有限公司 | The extracting method of ingenol |
Non-Patent Citations (4)
| Title |
|---|
| 徐东翔: "《植物资源化学》", 30 September 2004, 湖南科学技术出版社 * |
| 杜海燕 译: "从续随子种子中快速分离巨大戟二萜醇的方法研究", 《国外医学中医中药分册》 * |
| 沈彤 等: "《天然药物化学》", 30 April 2010, 甘肃科学技术出版社 * |
| 焦威 等: "千金子化学成分的研究", 《中草药》 * |
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