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this ppt. describe the coumarin present in herbal drugs and plant sources.
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GURU JAMBHESHWAR UNIVERSITY OF SCIENCE &TECHNOLOGYHISAR “ COUMARINS”
Submitted by shiva(11124002) M.Pharm 2nd sem (pharmacognosy)
Extraction,isolation and characterization of coumarins
CONTENTDefination of coumarinClassificationExtraction methodsIsolationIdentification methodsStructure elucidation
Coumarin is 5,6-benzo-2-pyrone derivative.
O O
The name coumarin is derived from the carribbean word “coumarou” for the tonka tree from which coumarin with the characteristics aroma of new-mown hay was isolated.
Unsubstituted coumarin is widespread in nature in free form or as glycoside.
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.
Biological activity of coumarin is diverse and depend on their chemical structure.
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.
Coumarins occur in plants in free form or as glycosides.
Aglycone are soluble in petrol,benzene,ether,chloroform,diethylether,alcohol(nonpolar solvent)
Glycosides are soluble in water and alcohols(polar solvent)
Coumarins are classified based on substitution in benzene and pyrone rings.
1--simple coumarins with substituents both in benzene ring and pyrone ring
2--furanocoumarins with substituents on benzene nucleus or pyrone ring
3--pyranocoumarins with substituents on benzene and pyrone rings
4--bis and tri-coumarins
SIMPLE COUMARINS
OOH O
OOH
OH
O
AESCULIN
UMBELLIFERONE
FUROANOCOUMARINS
Furane ring is condensed with coumarin structure.--Psoralen type--Angelicin type
ANGELICIN
O O O
PSORALEN
O O O
FURANOCOUMARINSSolubility-- lipid solubleIsolation-- by extraction with
ether/light petroleumSeparation—TLCAdsorbent—silica gelSolvent—ether-benzene(1:1)Development time—1 to 2hoursDetection– under UV light
PYRANOCOUMARIN(linear)
O O OCH3CH3
XANTHYLETIN
PYRANOCOUMARINS(angular)
O OO
OHCH3
CH3
LOMATIN
COUMARIN GLYCOSIDEAcsulin- 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
Khellol-seeds of Eranthis hyemalis linn
family-Ranunculaceae
Visnagin-seeds of Ammi visnaga family-Umbelliferae
Psoralea-dried ripe fruit of Psoralea corylifolia linn family-Leguminaceae
CLASSICAL EXTRACTION METHODS
continous hot percolation
maceration
percolation
ultrasonic-aided extraction
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
Continous hot percolation n
MODERN TECHINQUE
supercritical fluid extractionultrasonification aided extractionmicrowave-assisted solvent extractionpressurised liquid extractionmedium pressure solid liquid extractionaccelerated solvent extraction
SUPERCRITICAL FLUID EXTRACTIONPrinciple- supercritical fluid is applied
as an extractantAdvantages- high process speed lack of organic solvent possibility of coupling withOther methods like GC,HPLC
Used for separation of furanocoumarins from Angelica archangelica.
isolationBased on lactone type of
coumarin structure
PROCEDURE—alcoholic solution of pottassium hydroxide crashes the lactone ring in coumarin result in coumaric acid
After acidification these acid cyclize to coumarin again
NEW METHODS OF ISOLATION
Sublimation and fractionating distillation in high vaccum
Crystallization from organic solvents
Distillation with water vapours
GENERAL CHEMICAL TEST
3 volume propylene glycol 5 volume acetic acid 43 volume water and shake
blue fluorescence under UV light
NaOH solution yellow fluorescence under UV light
DRUG
DRUG
THIN LAYER CHROMATOGRAPHY
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 coumarinsAdsorbent –
silica,florosil,polyamide,alumina (normal phase)
Silanized silica(reverse phase)
Eluent---medium polar solvent like dichloromethane(normal phase)
aqueous solvent(reverse phase)
Development chamber-rectangular shaped
TLC of furanocoumarins and pyranocoumarins
Adsorbent—silica, florisil, polyamide,
alumina
Eluent—weak polar solvent: petrol+diethyl ether toluene+ethyl acetate n-hexane+ethyl acetate
Rf VALUE OF FURANOCOUMARIN
FURANOCOUMARINS
ETHER:BENZENE CHLOROFORM
Bergapten 100 100
Isobergapten 112 167
Pimpinellin 108 86
Isopinellin 97 43
sphondin 92 90
2-D TLC Adsorbent-diol silica(polar bonded)
1st direction eluent-10% methanol in water
2nd direction eluent-100% diisopropyl ether
PRINCIPLE-coumarins are identified by comparing retardation factors in both directions.
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-SPECTROSCOPYὰ-pyrone-300 nm Unsubstituted coumarins-274nm and
311nm7-hydroxycoumarins-217nm and 315-
330nmLinear furanocoumarins-205-225nm and
240-255nmAngular furanocoumarins-240-255nm
and 260-270nm are abssent
SPECTRAL DATA OF HYDROXYCOUMARINS
COUMARIN EtOH ʎmax WATER HOAc UV light BAW
AGLYCONE
coumarin 212,274,282,312
67 76 None 92
umbelliferone 210,240,325 57 60 Bright blue
89
aesculetin 230,260,303,351
28 45 Blue 79
scopoletin 229,253,300,346
29 51 Blue violet 83
daphnetin 215,263,328 61 54 Pale yellow
81
GLYCOSIDE Water BN BAW
aesculin 224,252,293,338
56 13 Clear blue 53
scopolin 227,250,288,339
64 44 mauve 53
STRUCTURE ELUCIDATIONIR Spectroscopy
NMR
Mass spectoscopy
INFRARED SPECTROSCOPYStretching frequency -1700-
1750cm⁻₁(c=o)
Skeletal vibrations-1600-1660cm⁻₁(c=c)
Stretching frequency-1500cm⁻₁(aromatic ring)
₁H-NMR SPECTROSCOPYNMR 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 7-oxygenated 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
₁H NMR chemical shift of coumarinsCoumarin derivatives
H₃-ᵹ(ppm) H₄-ᵹ(ppm) ᴊ(Hz)
Coumarins5-oxygenated7-oxygenated8-substituted5,7-disubstituted6-substitutedAngular furanocoumarins
6.1-6.46.1-6.46.2-6.56.6-6.96.7
6.7-7.2
7.5-7.7
7.5-7.97.9-8.27.6-7.87.1-7.56-7
7.5-7.7
6.7-7.2
9.59.59.58.52.5
2.5
2.5
MASS SPECTROMETRYCoumarin 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 M⁺ion at
m/e 162(80%) and base peak at m/z 134 due to loss of carbon monoxide
MASS FRAGMENTATION PATTERN OF COUMARIN AND FURANOCOUMARINS.
coumarinsO O
O
m/e 146(76%)
m/e 118(100%)
7 hydroxycoumarinsO OOH
OH O
m/e 162(80%)
m/e 134(100%)
REFERENCESHarborne 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 78-84
Bhatt S.V chemistry of natural products norosa pulisher 412-416