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Isolation of coumarin from Mellilotus officinalis of Iraq

Working Paper · December 2014

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Available online at www.pharmacie-globale.info ISSN 0976-8157

Research Article
PHARMACIE GLOBALE
INTERNATIONAL JOURNAL OF COMPREHENSIVE PHARMACY

ISOLATION OF COUMARIN FROM Mellilotus officinalis OF IRAQ

Noor Mohsen Nasser and Widad M K Al-Ani*
Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, Al-Mustansiriya University, Baghdad, Iraq.
Received: 14 May 2014; Revised: 21 May 2014; Accepted: 19 June 2014; Available online: 24 June 2014

ABSTRACT
Melilotus officinalis, a plant belong to the family fabaceae. It is characterized by a particularly wide spectrum of
different plant constituents. Many compounds have been isolated and identified from the plant. These included
coumarins, flavonoids, essential oils, vitamin C, allantoin, and tannins. Coumarins are the major constituent of
melilot they are blood thinner used to keep blood flowing smoothly and prevent the formation of blood clots. In this
study coumarin was isolated from melilotus officinalis widely grown in Iraq by column chromatography. The
isolated coumarin was identified by measuring its melting point, GC/MS, TLC, HPTLC and UV spectroscopy.
Preparative TLC was performed to measure the percentage of coumarin in the Iraqi plant.
Keywords: Melilot; Coumarin; Isolation.

INTRODUCTION
Melilotus officinalis, known as yellow sweet clover, yellow
melilot and common melilot, a plant belong to the family
fabaceae, Melilotus officinalis is biennial plant. Sweet
clover can grow 2 m tall but reaches usually less than 1 m.1
The plant has a bitter taste. Many compounds have been
isolated and indentified, these belong to major classes of
substances namely coumarins like dicoumarol,
dihydrocoumarin, flavonoids like Kaempferol glycosides,
Quercetin glycosides2 The plant also contains vitamin C,
allantoin, tannins and mineral salts for this reason
melilotus officinalis is valuable as medicinal plant.3 Its
characteristic sweet odor is intensified by drying4. Melilot
contain up to 0.9 % of coumarin and the minimum amount
of the official drug is 0.3 % Ph. Eur).5
The herb was used in traditional medicine in treatment of
problems related to varicose veins such as painful and
heavy legs, cramps in the legs and itching.
The herb is marketed in Germany in form of dry or fluid
extract with different concentration to treat several
inflammatory conditions.6
In Iran the dry extract of mellilotus officinalis is marketed
under the name Semelil (ANGIPARS™) which is used as
herbal formulation for treatment of chronic wounds,
particularly diabetic foot ulcers. Clinical trials had proven
the efficacy of this herb in healing of diabetes ulcer.7
Coumarin is a natural compound derived from shikimate
subunit. It is formed when the amino acid phenylalanine is
deaminated and then hydroxylated to trans-
hydroxycinnamic acid. UV light converts the trans alkene
to the cis configuration. This isomerisation leads to the
close proximity of the phenolic hydroxyl group with the
carboxyl group of the cis acid. Intramolecular lactonisation
*Corresponding Author:
Widad M K Al-Ani
Department of Pharmacognosy and Medicinal Plants,
College of Pharmacy, Al-Mustansiriya University, Baghdad, Iraq.
Contact no: +96-47901594250; Email: wmkalani@gmail.com
reaction resulted in the formation of the natural
compound coumarin which contributes to the smell of
newly mown hay (Figure 1).8
Figure 1. Biosynthesis of coumarin from cis-cinnamic
acid
MATERIAL & METHODS
GC-MS (Shimadzu), preparative thin layer
chromotagraphy HPTLC (CAMAG), IR, UV spectroscopy. All
chemicals and solvents are of analytical grade. Standard
coumarin was obtained from Sigma Aldrich Company.
Plant material
The aerial parts of Melilotus officinalis were collected from
area Abu-Graib in Baghdad and authenticated by the
National Herbarium at Abu-Graib, they were dried in
shade for several days at room temperature and then
grinded as powder.
Extraction and Isolation of Coumarin
Powdered plant aerial part (50g) was extracted by Soxhlet
apparatus with ethanol (80%, 250mL) till exhaustion. The
extract was concentrated by evaporation under vacuum.
Water (100g) was added and the suspension was
partitioned with petroleum ether (2x 100ml). The pet
ether layer was discarded and the aqueous layer was
extracted with ether (3x100ml). The combined ether
layers were dried with anhydrous sodium sulphate,
filtered and evaporated under vacuum. The residue
purified by column chromatography (silica gel, DCM

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mobile phase). The collected fractions were analysed by Figure 5. UV spectra

TLC comparing with standard coumarin.
Thinlayer Chromatography
The Rf value of isolated coumarin was compared with
standard coumarin in three solvents system. Preparative
TLC was performed using 0.5 mm thickness silica gel.
Elution of the isolated band was conducted with AR grade
acetone.
RESULTS AND DISCUSSION
Isolated coumarin was identified by melting oint. 71°C
which was identical with that reported in the literature.9
TLC of isolated comarin was compared with standard in
three solvent systems (table 1).
Table 1. Rf of standard and isolated coumarin in three
solvent system
Solvent system Rf of standard Rf of isolated
coumarin coumarin
Toulen:acetone: water
0.65 0.64
(4: 5:1)
Tolune: acetone: chloroform
0.55 0.54
(45:55:5)
Dichloromethane 0.3 0.29 HPTLC also support the occurrence of coumarin in
The occurrence of coumarin in the plant was confirmed by melilotus officinalis of Iraq. (Figure 6)
GC/MS (Figure 2). Figure 6. Track at 204 nm
Figure 2. GC/MS analysis of melilotus officinalis

Retention time of coumarin was 14.7 mjn. Fragmentation

pattern of isolated coumarin was identical with that
reported in the literature (Figure 3)10
Figure 3. Fragmentation pattern of isolated coumarin

Isolated coumarin data UV and IR are shown in Figure 4

and 5 respectively.
Figure 4. IR of isolated and standard coumarin

Preparative TLC
TLC with 0.5 mm thickness was conducted on 1.5 g of
extract corresponding to 13 g of the plant to give 60 mg of
coumarin (0.46%). This percentage was within the range
of the official drug according to Eu monograph.6
CONCLUSION
The valuable Iraqi medicinal plant Melilotus officinalis,
contains coumarin as a major active constituents.
Coumarin is responsible for the use of this plant in
treatment of problems related to varicose veins such as
painful and heavy legs. The percentage of coumarin in
melilot of Iraq was 0.46 %. This percentage indicates that
melilotus officinalis of Iraq is official herb (BP) since the
percentage is more than 0.3% and less than 0.9%.
REFERENCES
1. Maria Krzakowa and Ewa Grzywacz,; Phenolic officinalis) vs white clover (M. alba) revealed by 2D TLC
compounds pattern in sweet clover (Melilotus (two-dimentional thin-layer chromatography) and its
2 Pharmacie Globale© (IJCP), Vol. 05, Issue 02
 Al-Ani W M K et al. / Pharmacie Globale (IJCP) 2014, 02 (06)

taxonomic significance. Keria polonica. 2010; 56(3):53.
2. Clara E Quijano-Celis, Jorge A Pino & Gustavo Morales;
Chemical Composition of the Leaves Essential Oil of
Melilotus officinalis (L.) Pallas from Colombia. Journal of
Essential Oil Bearing Plants. 2010; 13(3):313.
3. American Diabetes Association: Poistion Statement:
Consensus Development Conference on Diabetic Foot
Wound Care. Diabetes Care. 1999; 22(8):1354-60.
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Effects of Melilotus officinalis on acute inflammation.
Phytotherapy Res. 2002; 16:316-19.
5. British herbal compendium. Bradley, 2006.
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for Human Use, London, 3 July 2008.
7. Masoompour S M, Bagheri M H, Borhani Haghighi A,
Novitsky Y A, Sadeghi B, Gharibdoust F, Larijani B,
Ranjbar Omrani G, Effect of ANGIPARS™, a new herbal
drug on diabetic foot ulcer: A phase 2 clinical study,
DARU. 2008; 16:1.
8. Michael Hienrich, Joanne Barnes, Simon gibbons and
Elizabeth M Williamson, Fundamentals of
Pharmacognosy and phytotherapy. 2012; 73.
9. Schäfer, F P (Ed.). Dye Lasers, 3rd Ed. Berlin, Springer-
Verlag. 1990.
10. Viorica Lopez-Avila and George Yefchak; Mass Spectral
Fragmentation Studies of Coumarin-Type Compounds
Using GC High-Resolution MS. The Open Analytical
Chemistry Journal. 2011; 5, 27.

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