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