GURU JAMBHESHWAR UNIVERSITY OF SCIENCE & TECHNOLOGY HISAR COUMARINS Submitted by shiva(11124002) M.

Pharm 2nd sem (pharmacognosy)

Extraction,isolation and characterization of coumarins

CONTENT
Defination of coumarin Classification Extraction methods Isolation Identification methods Structure elucidation

Coumarin is 5,6-benzo-2-pyrone derivative.

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.

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

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

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

Continous hot percolation n

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

SUPERCRITICAL FLUID EXTRACTION

Principle- supercritical fluid is applied as an extractant Advantages- high process speed lack of organic solvent possibility of coupling with Other methods like GC,HPLC

Used for separation of furanocoumarins from Angelica archangelica.

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

NEW METHODS OF ISOLATION

Sublimation and fractionating distillation in high vaccum

Crystallization from organic solvents

Distillation with water vapours

GENERAL CHEMICAL TEST

DRUG

3 volume propylene glycol 5 volume acetic acid 43 volume water and shake

blue fluorescence under UV light

DRUG

NaOH solution

yellow fluorescence under UV light

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 coumarins
Adsorbent silica,florosil,polyamide,alumina (normal phase) Silanized silica(reverse phase)

Eluent---medium polar solvent like dichloromethane(normal phase) aqueous solvent(reverse phase)

TLC of furanocoumarins and pyranocoumarins

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

100 112 108 97 92

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

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-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

SPECTRAL DATA OF HYDROXYCOUMARINS

COUMARIN AGLYCONE
coumarin umbelliferone aesculetin scopoletin daphnetin GLYCOSIDE 212,274,282,312 210,240,325 230,260,303,351 229,253,300,346 215,263,328 67 57 28 29 61 Water 76 60 45 51 54 BN None Bright blue Blue Blue violet Pale yellow 92 89 79 83 81 BAW

EtOH max

WATER

HOAc

UV light

BAW

aesculin scopolin

224,252,293,338 227,250,288,339

56 64

13 44

Clear blue mauve

53 53

STRUCTURE ELUCIDATION

IR Spectroscopy NMR Mass spectoscopy

INFRARED SPECTROSCOPY

Stretching frequency -17001750cm(c=o) Skeletal vibrations-1600-1660cm(c=c) Stretching frequency-1500cm(aromatic ring)

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

H NMR chemical shift of coumarins

Coumarin derivatives Coumarins 5-oxygenated 7-oxygenated 8-substituted 5,7disubstituted 6-substituted Angular furanocoumar ins H-(ppm) 6.1-6.4 6.1-6.4 6.2-6.5 6.6-6.9 6.7 6.7-7.2 7.5-7.7 H-(ppm) 7.5-7.9 7.9-8.2 7.6-7.8 7.1-7.5 6-7 7.5-7.7 6.7-7.2 (Hz) 9.5 9.5 9.5 8.5 2.5 2.5 2.5

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

MASS FRAGMENTATION PATTERN OF COUMARIN AND FURANOCOUMARINS.

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