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CONTENT
Defination of coumarin Classification Extraction methods Isolation Identification methods Structure elucidation
The name coumarin is derived from the carribbean word coumarou for the tonka tree from which coumarin with the characteristics aroma of newmown hay was isolated.
coumarins along with its derivative occurs abundantly in plant families such as umbelliferae,rutaceae,leguminaceae , orchidaceae,asteraceae,guttiferae, thymelaceae and solanaceae.
Coumarins play role in plant protection and are biosynthesized de novo in stress conditions as phytoalexins.
In low concentration they show synergistic activity with plant growth promoting.
Oldest medicine from this group is dicoumarol ,isolated from Melilotus alba linn. have antithrombotic and anticoagulative properties.
Solubility of coumarins depend upon phenolic hydroxyl group and the glycosidic bond.
Aglycone are soluble in petrol,benzene,ether,chloroform, diethylether,alcohol(nonpolar solvent) Glycosides are soluble in water and alcohols(polar solvent)
1--simple coumarins with substituents both in benzene ring and pyrone ring
2--furanocoumarins with substituents on benzene nucleus or pyrone ring
SIMPLE COUMARINS
HO HO
AESCULIN
HO
UMBELLIFERONE
FUROANOCOUMARINS
Furane ring is condensed with coumarin structure. --Psoralen type --Angelicin type
ANGELICIN
PSORALEN
FURANOCOUMARINS
Solubility-- lipid soluble Isolation-- by extraction with ether/light petroleum SeparationTLC Adsorbentsilica gel Solventether-benzene(1:1) Development time1 to 2hours Detection under UV light
PYRANOCOUMARIN(linear)
H3C H3C
XANTHYLETIN
PYRANOCOUMARINS(angul ar)
OH H3C H3C O O O
LOMATIN
COUMARIN GLYCOSIDE
Acsulin- bark of Aesculus hippocastanum family-Hippocastanaceae Cichorin-flowers of Cichorium intybus linn family-Compositae Daphnin-bark of Daphne mezerium family-Thymelaceae Fraxin-bark of Fraxinus excelsir linn. family-Oleaceae
FURANOCOUMARIN GLYCOSIDE
Dried sample, ground in to small particles and placed in a porous cellulose thimble Thimble placed in extracted chamber followed by flask heated with solvent and a condenser
MODERN TECHINQUE
supercritical fluid extraction ultrasonification aided extraction microwave-assisted solvent extraction pressurised liquid extraction medium pressure solid liquid extraction accelerated solvent extraction
isolation
Based on lactone type of coumarin structure PROCEDUREalcoholic solution of pottassium hydroxide crashes the lactone ring in coumarin result in coumaric acid After acidification these acid cyclize to coumarin again
3 volume propylene glycol 5 volume acetic acid 43 volume water and shake
DRUG
NaOH solution
TLC IS used for identification of compounds presented in plant extract by retention parameters as well as UV spectra taken directly from the layer by densitometry.
TLC of coumarins
Adsorbent silica,florosil,polyamide,alumina (normal phase) Silanized silica(reverse phase)
Adsorbentsilica, florisil, polyamide, alumina Eluentweak polar solvent: petrol+diethyl ether toluene+ethyl acetate n-hexane+ethyl acetate
Rf VALUE OF FURANOCOUMARIN
FURANOCOUMARI NS Bergapten Isobergapten Pimpinellin Isopinellin sphondin ETHER:BENZENE CHLOROFORM
100 167 86 43 90
2-D TLC
Adsorbent-diol silica(polar bonded) 1st direction eluent-10% methanol in water 2nd direction eluent-100% diisopropyl ether
PREPARATIVE TLC
PRINCIPLE-Components are applied in the form of a band and rechromatography of partly separated fractions. For separation of coumarins from Heracleum sosnowskyj fruits extract is applied as band on silanized silica layer eluted with methanol:water(6:4).
UV-SPECTROSCOPY
-pyrone-300 nm Unsubstituted coumarins-274nm and 311nm 7-hydroxycoumarins-217nm and 315330nm Linear furanocoumarins-205-225nm and 240-255nm Angular furanocoumarins-240-255nm and 260-270nm are abssent
EtOH max
WATER
HOAc
UV light
BAW
aesculin scopolin
224,252,293,338 227,250,288,339
56 64
13 44
53 53
STRUCTURE ELUCIDATION
INFRARED SPECTROSCOPY
H-NMR SPECTROSCOPY
NMR spectra of coumarins H-3 and H-4 protons exhibits characterstics chemical shift which distinguish different coumarin
Upfield shift of 0.17 ppm of H-3 Proton in 7oxygenated coumarins as compared with coumarins is due to electron release resulting in electron density at C-3 An oxygen at C-5 shift the resonance of H-4 downfield by 0-3 ppm due to peri effect
MASS SPECTROMETRY
Coumarin on electron impact gives a strong molecular ion peak (M) at m/e 146 (76%) and a base peak at m/z 118(100%) by the loss of 28 mass units equivalent to carbon monoxide
7-hydroxy coumarin show a strong Mion at m/e 162(80%) and base peak at m/z 134 due to loss of carbon monoxide
coumarins
O O
m/e 146(76%)
O
m/e 118(100%)
7 hydroxycoumarins
HO O O
m/e 162(80%)
HO
O
m/e 134(100%)
REFERENCES
Harborne J.B,Phenylpropanoid,A guide to modern techniques of plant analysis 2nd,champan and hall,New york 44-47 Kar Ashutosh ,coumarin glycoside,pharmacognosy and pharmacobiotechnology,new age Ltd. New delhi 512-515 Kowalska teresa,application of tlc in analysis of coumarin,TLC in phytochemistry,CRC press, London 7884 Bhatt S.V chemistry of natural products norosa pulisher 412-416