CN107505426A - A kind of method for quick identification of ingenol - Google Patents

A kind of method for quick identification of ingenol Download PDF

Info

Publication number
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
Authority
CN
China
Prior art keywords
ingenol
solvent
quick identification
reference substance
petroleum ether
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710611366.6A
Other languages
Chinese (zh)
Inventor
姜杉
陈雨
赵兴增
刘飞
吕慧
王鸣
冯煦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Botany of CAS
Original Assignee
Institute of Botany of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Botany of CAS filed Critical Institute of Botany of CAS
Priority to CN201710611366.6A priority Critical patent/CN107505426A/en
Publication of CN107505426A publication Critical patent/CN107505426A/en
Pending legal-status Critical Current

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90Plate chromatography, e.g. thin layer or paper chromatography

Landscapes

  • 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)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

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

A kind of method for quick identification of ingenol
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.
CN201710611366.6A 2017-07-25 2017-07-25 A kind of method for quick identification of ingenol Pending CN107505426A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710611366.6A CN107505426A (en) 2017-07-25 2017-07-25 A kind of method for quick identification of ingenol

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710611366.6A CN107505426A (en) 2017-07-25 2017-07-25 A kind of method for quick identification of ingenol

Publications (1)

Publication Number Publication Date
CN107505426A true CN107505426A (en) 2017-12-22

Family

ID=60688796

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710611366.6A Pending CN107505426A (en) 2017-07-25 2017-07-25 A kind of method for quick identification of ingenol

Country Status (1)

Country Link
CN (1) CN107505426A (en)

Citations (5)

* Cited by examiner, † Cited by third party
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

Patent Citations (5)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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》 *

Similar Documents

Publication Publication Date Title
Wei et al. Pharmaceutical applications of affinity-ultrafiltration mass spectrometry: Recent advances and future prospects
CN109470791A (en) A kind of method and kit of high performance liquid chromatography-tandem mass detection serum estradiol
Lovett et al. Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard
CN107179404B (en) A kind of furans metabolite derivatization reagent and its rapid detection card
CN107505426A (en) A kind of method for quick identification of ingenol
CN101008634A (en) Method for identifying hawthorn of hawthorn extract lipid-lowering dispersion tablet
CN102539592A (en) Method for detecting content of VLCFAs (very long chain fatty acids) in body fluid
EP1456657A1 (en) Tagged microparticle compositions and methods
Pang et al. Simultaneous determination of main bioactive components in Rosa multiflora Thunb. and their fragmentation study by LC–MS
CN103102386B (en) The preparation method of tigogenin
CN101658546B (en) TLC identification method for Naoxinqing tablet and persimmon leaf extract
CN109085284A (en) A kind of quick thin-layer identification method of multiple medicine taste of Ershiwuwei zhenzhu wan
CN101781345B (en) Method for preparing tilianin
CN113533611A (en) Thin-layer chromatography identification method of agastache rugosus wanying powder
CN104402992A (en) Synthetic method for synthesizing cannabis sativa antigen and application of synthesized cannabis sativa antigen
CN104713979A (en) Thin-layer identification method for Dendrobium officinale
CN111004302A (en) A method for preparing 5 ferulic acid esters with soapstock containing oryzanol as raw material
Aslam et al. Phytochemical study of Ariel parts of Ranunculus muricatus for the pharmacological active compounds
Parker et al. Thin layer chromatography of marihuana
CN108623551B (en) Pentacene fluorescence ratio probe and preparation method and application thereof
CN103926368A (en) Method for extracting biotin from corn steep liquor and thin layer chromatography (TLC) scanning detection method of biotin
CN114814002B (en) Quick identification method for semen trichosanthis and modified semen trichosanthis
CN202372504U (en) Estradiol enzyme linked immunosorbent assay kit
CN107462658B (en) A kind of thin-layer chromatography qualitative identification method of Pueraria lobota ' Yanming ' capsules for clearing
CN110229169A (en) A kind of method Neo-garcinolic acid separation and its prepare the new gamboge amide of N- aryl

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20171222

WD01 Invention patent application deemed withdrawn after publication