in vitro antioxidant and free radical scavenging

Journal of Herbal Medicine and Toxicology 4 (1) 97-102 (2010)
ISSN : 0973-4643
Original Article
Bhawya Dayananda, Anilakumar Kandangath Raghavan*, Farhath Khanum and
Bawa Amarinder Sngh
Biochemistry & Nutrition Discipline, Defence Food Research Laboratory, Mysore-570011, India.
E-mail: anilakumarkr
Received- 21st Oct. 2009, Revised -12th Dec. 2009, Accepted- 30th Dec. 2009
Abstract: Glycyrrhiza glabra (Fabaceae) commonly called as licorice is one of the
oldest and most widely used herbs in Eastern traditional medicine. The powdered
extracts of Glycyrrhiza glabra were prepared using solvents based on polarity and
their antioxidant properties were evaluated employing various established in vitro
systems viz. 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion radicals
scavenging, hydroxyl radicals scavenging, metal ion chelation, reducing power along
with polyphenols and flavonoids. Proximate composition and mineral content analysis
was carried out along with vitamins and tannins in the powdered root. The study
revealed a notable inhibitory activity of ethanol extract, followed by methanol and
water extracts.
Key words: Glycyrrhiza glabra, free radicals, superoxide anion, metal ion chelation,
reducing power.
Many of the diseases are mainly linked to oxidative
stress due to free radicals[1] Antioxidants have been
reported to prevent oxidative damage caused by free
radicals and may prevent the occurrence of diseases.
It can interfere with the oxidation process by reacting
with free radicals, chelating, and catalytic metals and
also by acting as oxygen scavengers [2]. Currently
available synthetic antioxidants have been suspected
to cause or prompt negative health effects. Hence
strong restrictions have been placed on their
application and there is a trend to substitute them with
naturally occurring antioxidants. Recently there has
been an upsurge of interest in the therapeutic
potentials of medicinal plants as antioxidants in
reducing such free radical induced tissue injury.
Glycyrrhiza glabra (Licorice) root is one of the
common traditional Chinese medicines and used as
flavoring and sweetening agents for tobaccos, chewing
gums, candies, toothpaste and beverages.
Polysaccharides from Glycyrrhizia glabra are
enriched with glucuronic acid which showed strong
anti-adhesive properties against H. pylori and P.
gingivitis, [3].The main components (glycyrrhizin, 18beta-glycyrrhetinic acid, isoliquiritin, and liquiritigenin)
isolated from Glycyrrhizia glabra possess antiallergic effects, such as antiscratching behaviour and
IgE production-inhibitory activity [4]. Glabridin, a major
active constituent of Glycyrrhiza glabra is commonly
used in the treatment of cardiovascular and central
nervous system (CNS) diseases [5]. It could be an
effective agent for treatment of atopic dermatitis and
a promising memory enhancer in both exteroceptive
and interoceptive behavioural models of memory [6].
The extract possesses an antidepressant-like effect
[7].The main bioactive constituents of licorice are
triterpene saponins and various types of flavonoids .
Here we report the antioxidant ability in sequential
extracts based on polarity, in order to find a better
application because; free radicals are of great concern
in the food industry and among consumers of
processed food.
Journal of Herbal Medicine & Toxicology
Plant materials
The roots of Glycyrrhiza glabra were procured from
the reliable local herbal suppliers of Mysore,
Karnataka, India.
Chemicals and instruments
Butylated hydroxyanisole (BHA), nitroblue tetrazolium
(NBT), 1,1-diphenyl-2-picrylhydrazyl (DPPH),
Phenazine methosulphate (PMS), Nicotinamide
adenine dinucleotide reduced (NADH), deoxyribose
4-dimethyl amino cinnamaldehyde, Catechin were
purchased from M/s Sigma Chemicals Co. (St. Louis,
MO). Thiobarbituric acid (TBA), gallic acid,
trichloroacetic acid (TCA), Ethylenediamine tetraacetic acid (TCA), Ascorbic acid and other chemicals
were purchased from M/s Sisco Research
Laboratories, Mumbai, India. All reagents procured
were analytical grade. Absorbance was measured
using Shimadzu U V spectrophotometer, atomic
absorption spectrophotometer (model AAS Vario 6,
Shimadzu, Japan).
Extraction of plant samples
For antioxidant analysis, Glycyrrhiza glabra roots
were washed, dried in hot air oven at 400C and then
finely powdered. The powder was used for extraction.
Sequential extraction of the powder was done with
different solvents with increasing polarity i.e. hexane,
chloroform, ethylacetate, acetone, methanol, ethanol
and water. 30g of the powder were extracted in 300ml
of the solvent in glass conical flask on a mechanical
shaker for 24h at room temperature. The extract was
filtered with Whatmann filter paper no. 1 and dried
by flash evaporation/lyophilisation.
Proximate Composition
The proximate composition of Glycyrrhiza glabra
was determined using the Association of Official
Chemists (AOAC) method [8].The mineral content
of the stem was analyzed by an atomic absorption
spectrophotometer. The vitamin C content was
estimated by titrimetric method using 1, 2dicholorophenol indophenol dye. Vitamin E and total
carotenoids in the powdered extract of Glycyrrhiza
glabra were estimated as prescribed by Sadasivam
and Manickam [9].
Antioxidant activity by free radical scavenging method
The 1, 1-diphenyl 2-picrylhydrazyl (DPPH) radicalscavenging assay reported by Hatano et al., [10] was
adopted for the determination of the antioxidant
activity of Glycyrrhiza glabra extract with
modifications. Briefly, 3ml of the reaction mixture
contained 0.1ml of different concentration of the
extract (1-10mg/ml) and aliquot of DPPH solution
(0.5ml, 50mg/100ml) in methanol. Reaction mixture
without the extract was used as control. The mixture
was shaken vigorously and allowed to stand in dark
for 45min. The antioxidant scavenging activity was
measured as the decrease in absorbance of DPPH
at 515nm against a blank and calculated using the
following formula.
Scavenging Effect (%) = [(A0 - A1) / A0 ] X 100
Where, A0 is the absorbance of control and A1 is the
absorbance of sample. All the values expressed are
the mean values carried out in triplicates. BHA was
used as a positive control.
Superoxide anion scavenging activity
Measurement of superoxide radical scavenging
activity of the Glycyrrhiza glabra extracts was based
on the method reported by Liu et al. [11] with
modifications .The superoxide is generated in 3ml of
Tris HCl (16mM pH8.0) containing 1ml of NBT
(50µm) solution, 1ml NADH (78µM) solution and
sample solution of different extracts in a concentration
of 1-10µg/ml .The reaction was started by adding 1ml
of PMS solution (10um) to the mixture. The reaction
mixture was incubated at 25oC for 5 min. and the
absorbance at 560nm was measured against the blank
samples. L-Ascorbic acid was used as a control. BHA
was used as positive control.
% Inhibition = [( Acontrol - Asample ) / Acontrol ) x100]
Where Acontrol is the absorbance of the L-Ascorbic
acid and Asample is the absorbance of standards. The
data at each point were the average of three
Hydroxyl radical scavenging activity
1. Non-site specific hydroxyl radical mediated 2deoxy-D-ribose degradation
The assay was performed as reported by Halliwell et
al., [12] with modifications. The reaction mixture
contained 100µl of 28mM 2-deoxy D ribose (dissolved
in KH2PO4- K2HPO4) phosphate buffer pH 7.4, 500µl
solution of various concentration of the G.g extracts
(100µg-1000µg/ml), 200µl of 200µM FeCl3 and
1.04mM EDTA (1:1 v/v), 100µl of H2O2 (1.0 mM)
and 100µl ascorbic acid (1.0mM). After an incubation
Raghavan et al.
period of 1hr. at 37oC the extent of deoxy ribose
degradation was measured by the TBA reaction. 1ml
of TBA (1% in 50mM NaOH) and 1ml of TCA were
added to the reaction mixture, tubes were heated at
100o C for 20 min. After cooling the absorbance was
read at 532 nm against a blank (containing only buffer
and deoxy ribose). The % inhibition was calculated
by the formula
Inhibition (%) = [(A0 - A1) / A0 ] X 100
Where, A0 is the absorbance of control and A1 is the
absorbance of sample. All the values expressed are the
mean values carried out in triplicates. BHA was used
as a positive control.
2. Site-specific hydroxyl radical mediated 2deoxy-D-ribose degradation
The ability of the Glycyrrhiza glabra extracts to inhibit
site-specific hydroxyl radical mediated degradation was
also carried out to understand its role as a metal chelator.
The method was the same as described above except
that buffer replaced EDTA.
Reducing Power
The reducing power of the prepared Glycyrrhiza
glabra extract was determined by the method of
Oyaizu [13]. Briefly various concentration of the
extract (0-5mg/ml) and the standard compound were
mixed with phosphate buffer (2.5ml, 0.2mol/L, pH 6.6)
and potassium ferricyanide [K3 Fe(CN)6], (2.5ml, 1%).
The mixture was incubated at 50oCfor 20min. To the
mixture 10% TCA was added to the mixture, which
was then centrifuged at 3000rpm for 10min. The upper
layer of solution (2.5ml) was mixed with distilled water
(2.5ml), FeCl3 (0.5ml, 0.1%) and the absorbance was
measured at 700nm.Increased absorbance of the
reaction mixture indicated increased reducing power.
α- tocopherol was used as a positive control.
Ferrous ion chelating activity
The assay was determined as described by Dinis et
al., [14]. Briefly the extracts were added to a solution
of 2mmol/L FeCl2 (.05ml). The reaction mixture was
initiated by the addition of 5mmol/L ferrozine (0.2ml),
the mixture was shaken vigorously and left at room
temperature for 10 min. Absorbance of the solution
was then measured spectrophotometrically at 562nm.
The % of Inhibition of ferrozine-Fe2+ complex was
given by
% Inhibition = [(A0 - A1 ) / A0 ] X 100
Moisture %
06.50 ± 0.70
Where, A0 was the absorbance of the control & A1
was the absorbance in the presence of the samples
of G.G extracts and standards. The ability of the
extract to chelate ferrous ion was compared with the
standard BHA.
Protein %
07.17 ± 0.74
Total phenolic contents
Fat %
03.50 ± 0.70
Carbohydrates %
63.6 ± 1.69
Ash %
05.05 ± 0.07
Crude Fiber %
21.75 ± 1.06
Vitamin C mg/100g
27.65 ± 1.20
Vitamin E mg/100g
98.21 ± 1.22
Total carotenoids g/L
72.22 ± 3.72
Iron mg/100g
08.08 ± 0.02
Zinc mg/100g
00.96 ± 0.01
Sodium mg/100g
63.20 ± 0.56
Total phenolic content was estimated
spectrophotometrically by Folin-Ciocalteau method
carried out according to Singleton and Rossi [15] with
some modifications. The 0.1ml Glycyrrhiza glabra
extract concentration was made up to 3ml with double
distilled water, to this 0.5ml of folin-Ciocalteau reagent
was added and allowed to stand at room temperature
for 10min, to the mixture 2ml of 7% sodium carbonate
was added and kept in boiling water bath for 1min,
cooled, after which the absorbance was read at 650nm
against blank. The concentration was calculated using
gallic acid as standard, and the results were expressed
as milligram gallic acid equivalents per gram extract.
Potassium mg/100g
180.48 ± 0.12
Polyphenols mg/100g
28.62 ± 1.05
Flavonoids mg/100g
08.63 ± 0.98
Tannins mg/100g
06.82 ± 1.02
The determination of flavanoids was carried out
according to Delcour and Varebeke [16] .To 1ml of
Glycyrrhiza glabra extract, 5ml chromogen reagent
(1.0 g 4-dimethyl amino cinnamaldehyde dissolved in
Table 1: Proximate composition and phytoconsttuents of Glycyrrhiza glabra
Journal of Herbal Medicine & Toxicology
a cooled mixture of 250ml of concentrated HCl and
750ml of methanol, made upto 1 L methanol) was
added. After 10min, the absorbance was measured
at 640nm against a blank consisting of water instead
of extract and the flavanoids content was calculated
with (±) catechin and the concentration was expressed
as (±) catechin equivalents.
Raghavan et al.
A 0.5g sample was weighed and boiled with 75ml of
water for 30min. The contents were centrifuged at
800 x g for 20min and the supernatant was made
upto 100ml in a volumetric flask using water. Aliquots
were treated with Folin-Denis reagent and absorbance
was measured at 700nm as described by Sadasivam
and Manickam [9].
Statistical analysis
The statistical processing of the data obtained from
all studies is expressed as mean ± standard deviation
(SD) of three separate experiments using the
computer programme Excel.
DPPH free radical scavenging activity
Scavenging the stable DPPH radical is widely used a
method to evaluate the free radical scavenging ability
of various samples [17]. Antioxidants act mainly on
DPPH radical by their hydrogen-donating ability and
hence its scavenging activities by the extract are most
widely known to assess the antioxidant potential
antioxidants, where they reduce the stable radical
DPPH to the yellow-coloured diphenylpicrylhydrazine. The reduction capability of DPPH radical
is determined by the decrease in absorbance at 517nm.
The scavenging effect of the extracts and standards
with the DPPH radical is presented in Fig.1. The
methanol extract showed better radical scavenging
activity than the other extracts.
Superoxide radical (O2.-) scavenging activity
Superoxide radical is known to be produced in vivo
and can result in the formation of H2O2 via dismutation
reaction and is known to be a very harmful species to
cellular components [18]. Fig 2 shows the inhibitory
effect of methanol on superoxide radical generation.
The scavenging activity of methanol extracts on
superoxide radicals was increased with increase in
concentration. The data showed that the extract was
markedly a more potent scavenger of superoxide
anion than other solvent extracts.
Hydroxyl (OH.) radical-scavenging activity
Of all the oxygen free radicals, the hydroxyl radical
is the most reactive which severely damages adjacent
bimolecules such as proteins, DNA, PUFA and nucleic
acid causing aging, cancer and several diseases [19],
and also known to be one of the quick initiators of the
lipid oxidation process, abstracting hydrogen atoms
from unsaturated fatty acids [20]. Therefore, the
removal of hydroxyl radical is probably one of the
most effective defences of a living body against various
diseases. Fig 3 and 4 show the effect of various
Glycyrrhiza glabra extracts in deoxyribose
scavenging assays (both non-site and site-specific).
It was observed that all the extracts were effective
in scavenging the hydroxyl radicals in non site-specific
assay as well as in site-specific assay. However, the
change was comparatively greater in the non site
specific than in the site-specific assay indicating their
radical scavenging activity.
Metal chelating activity
Metal chelation indicates the effective capacity for
iron binding, suggesting its action as an antioxidant
relating to its iron binding capacity wherein ferrozine
can quantitatively form complexes with Fe2+. In the
presence of other chelating agents, the complex
formation is disrupted with the result that the red
colour of the complex is decreased. Measurement of
the rate of colour of reduction therefore allows
estimation of the chelating activity of the coexisting
chelator [21]. Fig.5 shows that the formation of the
ferrozine- Fe 2+ complex is not complete in the
presence of methanol, ethanol and water extracts
indicating their capacity in chelating iron. However,
the chelating ability was relatively lower than that of
EDTA. In this assay the extracts and standard
compounds interfered with the formation of ferrous
complex with the reagent ferrozine, indicating their
metal chelating activity.
Reducing power
In reducing power assay, the presence of antioxidants
in the sample result in the reducing of Fe3+ to Fe2+ by
donating an electron. The reducing properties are
generally associated with the presence of reductones
[22](Duh, 1998), which have been shown to exert
antioxidant action by breaking the free radical chain
by donating hydrogen atom [23]. Measuring the
formation of Perl’s blue at 700nm can then monitor
amount of Fe2+ complex. Fig. 6 shows the reducing
power of methanol, ethanol and water extracts in
increasing order in a dose dependent manner in
comparison with the reference compound átocopherol. Here, the methanol extract was shown
more reducing power than ethanol and water extracts.
Journal of Herbal Medicine & Toxicology
Composition and phytoconstituents
The Table 1 shows that the root is rich in fibre, vitamin
E, potassium, polyphenols and flavonoids . It is well
known that plant phenolics, in general, are highly
effective free radical scavengers and antioxidants .It
is known that hydrogen-donating property of the
polyphenolic compounds is responsible for the
inhibition of free radical induced lipid peroxidation [24].
Consequently the antioxidant activities of the
Glycyrrhiza glabra extracts may be explained with
respect to their total phenolics and flavonoid contents,
with good correlation.
This work shows the effective antioxidative activity
of methanol extract in scavenging free radical in
comparison to other solvents. Nowadays, there is an
increasing interest in detecting the natural antioxidants,
which are safe and effective in order to replace the
commercial synthetic antioxidants. Hence
Glycyrrhiza glabra extracts can be a source of
natural antioxidants in replacing the synthetic ones.
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