Hamad M N et al.

/ Pharmacie Globale (IJCP) 2012, 4 (01)

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

Research Article
PHARMACIE GLOBALE
INTERNATIONAL JOURNAL OF COMPREHENSIVE PHARMACY

ISOLATION OF RUTIN FROM Ruta graveolens (RUTACEAE) CULTIVATED IN IRAQ BY

PRECIPITATION AND FRACTIONAL SOLUBILIZATION
Maha Noori Hamad
Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, University of Baghdad, Baghdad, Iraq.
Received: 17 February 2012; Revised: 15 March 2012; Accepted: 28 March 2012; Available online: 5 April 2012

ABSTRACT
Ruta graveolens L., a plant belongs to the family Rutaceae, is characterized by a particularly wide spectrum of
different plant constituents. More than 100 compounds have been indentified, these belong to four major classes
of substances namely alkaloids, coumarins, flavonoids, and essential oils. Rutin is the major flavonoid glycoside
found in Ruta graveolens is the rhamnoglucoside of the flavonoid quercetin has been called vitamin P or the
permeability factor. Several studies had been done for the isolation of rutin by different chromatographic method.
In this study rutin was isolated from Ruta graveolens by precipitation and fractional solubilization without the use
of any chromatographic technique. The isolated rutin was identified by measuring its melting point, UV absorption,
FTIR spectra, HPLC and TLC.
Keywords: Rue; flavonoids; rutin; isolation.

INTRODUCTION
Ruta graveolens L., synonyms bitter herb or garden rue is a
plant of the family Rutaceae. It is characterized by a
particularly wide spectrum of different plant constituents.
More than 100 compounds have been identified and
belong to four major chemical classes which may be
requires a minimum content of 4.0% flavonoids
determined by liquid chromatography with absorbance at
350 nm.7
Figure 1. Chemical structure of rutin
HO

present in variable amounts in the aerial parts of the plant, OH

namely alkaloids of quinoline and quinolinone
(graveoline), furoquinoline (dictamine, skimmianine), HO
O

pyrano-quinoline (rutalinium) and acridone

(funacridone); coumarins, like dicoumarins,
furacoumarins (bergaptene), pyranocoumarins; flavonoids
O
O
(rutin) and essential oils.1 It is an evergreen shrub that OH O O
O
OH
grows up to one meter tall and well known for its offensive OH
OH
HO
odor.2,3 Rue was used by the ancients as a medicinal plant. HO HO

In Iraq, it was given to soldiers to instill courage.4
Flavonoids are among the most widely produced natural
products in plants with over 2000 different compounds
present both as free aglycons and glycosides.5 Recently,
naturally occurring and synthetic flavonoids are used in
modern medicine for various therapeutic effects,
especially in USA, western and central Europe. They are
used for treatment of various conditions related to
capillary bleeding and increased capillary fragility and
permeability.1,5 Rutin is the rhamnoglucoside of the
flavonoid quercetin, and found in many plants1 and used
for treatment of various diseases related to the vascular.6
It is quercetin-3-rutinoside or 3,3',4', 5,7-pentahydroxy
flavones-3-rutinoside,7 and has a chemical formula
C27H30O16. Major commercial sources of rutin include
Saphora japonica, Eucalyptus spp., Fagopyrum esculatum
and Ruta graveolens. As a constituent of Fagopyrum
esculatum, the British and European pharmacopoeias
*Corresponding Author:
Maha Noori Hamad
Department of Pharmacognosy and Medicinal Plants,
College of Pharmacy, University of Baghdad, Baghdad, Iraq.
Email: manoha_1957@yahoo.com
Rutin (Figure 1) and quercetin were isolated from tobacco
leaves by column chromatography and preperative TLC8
and from Ruta graveative and other plants using
chromatographic techniques and ultrasonic methods.9,10
The present study was designed to isolate rutin from Iraqi
Ruta graveolens based on its solubility in different solvents
without any other separation techniques.
MATERIAL AND METHODS
HPLC (waters 1525), UV-visible spectrophotometer (Cary
100 conc), FT/IR (Jasco-4200). All chemicals and solvents
are of analytical grade. Standard rutin was obtained from
Sigma chemical company.
Plant material
The aerial parts of Ruta graveolens were collected from Al-
Graiaat area 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
Twenty grams of the powdered aerial part was extracted
by Soxhlet apparatus with 250 ml of 80% ethanol till

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exhaustion. The extract was filtered and concentrated by Figure 3. HPLC chromatogram of the isolated rutin
evaporation under vacuum to about 10ml then mixed with
25ml distilled water, and extracted with petroleum ether
(50ml x 3), then with chloroform (50 ml x 3).
After extraction, the aqueous layer was collected and left
to stand in a cold place for 72 hours; a yellow precipitate
separated out of the solution. The precipitate was filtered
and washed with a mixture of chloroform: ethylacetate:
ethanol (50:25:25). The un-dissolved part of the
precipitate was dissolved in hot methanol and filtered, the
filtrate was evaporated to dryness to give 115 mg yellow
powder (rutin), and its melting point was measured.
Identification of isolated rutin Figure 4. HPLC chromatogram of standard rutin
The isolated rutin was identified by HPLC method and
compared with standard rutin using hyperclone ODCC C18
V-25cm column and a mixture of methanol: water (70:30
ratio) as a mobile phase with a flow rate of 0.5ml/min, and
detected at 280 nm.
TLC and paper chromatography
Isolated rutin was also compared with standard rutin
using TLC method; a pre-coated aluminum sheet with
silica gel GF254 with the following mobile phases: ethyl
acetate: butanone: formic acid: water (50:30:10:10), ethyl
acetate: formic acid: acetic acid: water (100: 11: 11:27). In
paper chromatography, Watman No.1 filter paper was
used as a stationary phase and mobile phases of: Acetic The infrared spectrum is shown in figure 5, while the UV
acid: water (15:85) and isopropyl alcohol: water (60:40).12 spectra of isolated and standard rutin are shown in figure
6. The spectrum of rutin showed two major absorption
Spectrophotometric analysis
bands at 359 and 370nm which indicated the presence of
The isolated rutin was dissolved in methanol and its UV
flavonol structure. The first absorption maximum can be
absorption peaks were determined and compared with
considered as originating from π-π* transitions in the ring
standard rutin. Infrared spectrum of the isolated rutin was
A (aromatic system) and the second absorption maximum
determined using KBr disk method.
observed around 370nm, which may be assigned to
RESULTS AND DISCUSSION transitions in ring B (cinnamayl system); this band
Isolated rutin showed a melting point at 194-196°C which appeared broad as a result of overlapping with LMCT
is identical with that reported for rutin;7 the HPLC band.13,14 The principle of this method depends on the
chromatograms of the aqueous fraction after extraction differences in solubility between the glycoside and its
and isolation with chloroform and standard rutin are aglycone.7
shown in figure 2, 3 and 4 respectively, while the Rf values Figure 5. IR spectrum of the isolated rutin
of isolated and standard rutin in different mobile phases
are shown in table 1.
Table 1. Comparison between the Rf values of isolated
and standard rutin in different mobile phases
Rf value of Rf value
Solvent systems in TLC and Paper
isolated of standard
Chromatography
rutin rutin
Ethyl acetate: butanone: formic acid: water 0.31 0.28
Ethyl acetate: formic acid: acetic acid: water 0.38 0.35
Acetic acid: water 0.57 0.60
Isopropyl alcohol: water 0.78 0.80

Figure 2. HPLC chromatogram of the aqueous fraction

Figure 6. UV spectrum of rutin. Lower spectrum:
isolated rutin; upper spectrum: standard rutin

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After concentrating the aqueous-alcoholic extract the
solubility of the glycoside and the aglycones decrease and
therefore they precipitate out of the solution and the
mixture of precipitates is further fractionated by
dissolving the aglycone in the chloroform: ethylacetate:
ethanol mixture leaving the glycoside which was later on
dissolved in hot methanol, and so this method of isolation
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CONCLUSION
The flavonoid rutin can be isolated & purified from natural
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