Natural Product Sciences

13(3) : 199-206 (2007)

Antioxidant and Free Radical Scavenging Potential

of Burm. Leaves
Justicia gendarussa in vitro.

K. Mruthunjaya and V. I. Hukkeri *

1 2

1
Department of Pharmacognosy and Phytochemistry, J.S.S. College of Pharmacy, Vidyanagar,
S. S. Nagar, Mysore-570 015 Karnatakata, India
2
Director, Herbals Research Division, Dr. Nargund Research Foundation, Dattatreya Nagar, BSK III Stage,
100 feet Ring Road, Bangalore-560 085, Karnataka, India.

Abstract − Antioxidant activity of 70% aqueous ethanolic extract of leaves of Justicia gendarussa (EJ) was
evaluated. EJ was prepared by cold maceration method. The antioxidant potency of EJ was investigated
employing various established in vitro systems, such as DPPH radical scavenging, nitric oxide (NO) scavenging,
β-carotene linoleic acid module system (β CLAMS), hydroxyl (OH) radical scavenging, anti lipid peroxidation.
IC values were determined in each experiment. Also, ferric ion reduction capacity of extracts in presence and
50

absence of chelating agent (EDTA) and total antioxidant capacity were determined. Preliminary phytochemical
investigation was carried out to know the nature of constituents present in the leaves and correlate it with
antioxidant activity. Further total phenolic content was determined in EJ. IC values of EJ were 123.09 ± 3.01,
50

643.0 ± 61.10, 132.3 ± 6.03, 68.5 ± 11.5 and 68.13 ± 1.38 µg/mL in DPPH radical scavenging, NO scavenging, β
CLAMS, OH radical scavenging and anti lipid peroxidation activity respectively. In total antioxidant capacity assay,
ascorbic acid equivalent value was found to be 205.56 ± 4.69 µg/mg of extract. Total phenolic content was found to
be 43.76 ± 4.27 µg equivalent of gallic acid per mg of extract. Phytochemical investigation reveals the presence of
flavonoids. The results indicate that EJ possess antioxidant activity and flavonoids are responsible for this activity.
Keywords − Justicia gendarussa, antioxidant, DPPH, free radical scavenging, hydroxyl radical scavenging, lignans

Introduction biological activities including antioxidant (Jyotishi and

Bagavant, 1992a; 1992b). It is also reported to contain β-
Reactive oxygen species (ROS) can contribute to the sitosterol, friedelin, lupeol and four simple -substituted
0

etiology of disorders such as cancer, liver diseases, aromatic amines (Chakravarty ., 1982). Its medicinal
et al

atherosclecrosis, respiratory diseases and inflammatory properties and presence of lignans inspired us to take up
response syndrome. In recent years there is great deal of the particular study.
interest in developing agents to control damage induced
by ROS in biological systems. As plants produce a lot of Experimental
antioxidants to control the oxidative stress caused by
sunbeams and oxygen, they can represent a source of new Chemicals − α,α-diphenyl-β-picryl hydrazyl (DPPH),
compounds with antioxidant activity. egg phospatidylcholine, β-carotene and γ-linoleic acid
Justicia gendarussa belonging to family Acanthaceae is were obtained from Sigma Chemical Co.(St. Louis USA),
known for its medicinal properties in Ayurveda, an Indian butylated hydroxy toluene (BHT), ascorbic acid, ethylene-
system of medicine in inflammations, bronchitis, vaginal diaminetetraacetic acid (EDTA), Tween-40, deoxy- - d

discharges, dyspepsia, eye diseases and fever (Kirthikar ribose, trichloroacetic acid (TCA) and thiobarbituric acid
and Basu, 2001). Decoction of leaves is used to treat (TBA) were obtained by Hi-Media Labs (Mumbai, India),
chronic rheumatism (Anonymus 1959). species
Justicia 1,10- -phenanthroline, ferric chloride (FeCl3), hydrogen
O

found to contain lignans, naturally occurring phenolic peroxide, ammonium molybdate, sodium dithionite were
dimers. Lignans reported to have various significant obtained from Ranbaxy Fine Chemicals (New Delhi,
India), phenyl hydrazine, folin-ciocalteau phenol reagent
*Author for correspondence were obtained from BDH Products (UK). Silymarin was
Fax: +91-80-26421903; E-mail: [email protected] kind gift from Dr. Chidambaramurthy K.N., CFTRI,
199
200 Natural Product Sciences

Mysore, India. All other chemicals used were of from A : B-30 : 70, 50 : 50, 70 : 30, 90 : 10 and finally
analytical grade. The solvents used for extraction were 100% A for 27 min. The flow rate was maintained at 1
from Ranbaxy Fine Chemicals (New Delhi, India). The mL/min. and the ratio changed at every 5 min. The
UV-visible spectrophotometric values were recorded in chromatogram of the EJ was recorded at 275 nm.
JASCO UV-500 spectrophotometer. DPPH radical scavenging activity (Singh et .,
al

Plant material and extraction − The leaves of the 2002) − Free radical scavenging potentials of the extracts
plant were collected during September 2004 in Wynadu was tested against a methanolic solution of DPPH.
district of Kerala State, India, authenticated, by Dr. Different concentrations of EJ, BHT and ascorbic acid in
B.D.Huddar, Professor and Head, Department of Botany, 500 µL ethanol were taken and added with 5 mL of
HSK Science Institute, Hubli, India. The voucher specimen 100 µM DPPH in methanol to give final concentration of
(KMJG01) of the plant is preserved in Department of 9.09 to 181.82 µg/mL of EJ, 4.54 to 27.27 µg/mL of BHT
Pharmacognosy, KLES’ College of Pharmacy, Hubli, and ascorbic acid. The mixtures were allowed to stand at
India. Leaves dried under shed, powdered and extracted room temperature for 20 minutes. The control was
with 70% aqueous ethanol by cold maceration. The prepared as above without extract. The readings were read
extraction was done for 72 hours. After extraction the at 517 nm using methanol as blank. The absorbance of
extract was separated from marc by filtration through control was first noted at 517 nm. The changes in
filter paper. The marc was pressed in muslin cloth to absorbance of the samples were measured. Scavenging
remove the solvent which is left in the marc after activity was expressed as the inhibition percentage
filtration. Filtrate was preserved in a well closed container. calculated using the formula % anti radical activity =
Marc left after extraction was extracted by cold maceration [(Control Abs. − Sample Abs.) / Control Abs.] × 100.
for 3 more days with same amount of fresh solvent and the Each experiment was carried out in triplicate and results
process was repeated for one more time. i.e. the drug was were expressed as mean % antiradical activity ± SD.
extracted 3 times with a gap of 3 days each. The 10th day Nitric oxide scavenging activity (Govindarajan et

the filtrates were pooled and concentrated to syrupy liquid ., 2003) − Scavenging of NO was determined using
al

under reduced pressure using superfit rotary vacuum sodium nitroprusside (SNP) as NO donor. SNP (10 mM)
evaporator, dried and stored in dessicator. Same is used for in phosphate buffered saline was mixed with different
below mentioned experiments. concentrations of extract (100 to 1000 µg/ml) in ethanol,
Preliminary phytochemical investigations − The ascorbic acid as standard (25 µg/mL to 125 µg/mL) and
preliminary phytochemical screening of the extract was incubated at 25 oC for 180 min, then Griess reagent (1%
carried out to know the different constituents present in it sulfanilamide, 0.1% naphylethylenediamine dihydrochloride
as per the standard procedures. The extract was tested for and 3% phosphoric acid) volume equal to incubated
alkaloids (Kokate et al ., 1996), sterols and triterpenes solution was added. The absorbance was immediately
(Peach et al ., 1955a), saponins (Peach et ., 1955b),
al measured at 546 nm. The NO scavenging activity was
flavonoids (Geinssman, 1955; Trease et al., 1989), tannins calculated from the formula, percentage NO scavenging
(Kokate et al ., 1996; Trease
et al ., 1989), carbohydrates activity = [(Abs of Control − Abs of Sample) / Abs of
(Hawks, 1971), cardiac glycosides, lactones and amino Control] × 100. Each experiment was carried out in
acids (Stevens, 1986). triplicate and results were expressed as mean % NO
HPLC fingerprinting of the extract − The high scavenging activity ± SD.
pressure liquid chromatography (HPLC) finger printing Antioxidant assay using β-Carotene Linoleate Model
analysis was performed on a Shimadzu (Japan) chromato- System (β CLAMS) (Hidalgo 1994; Singh .,
et al., et al

graphic system, which included a binary pump model LC 2002) − The antioxidant activity of EJ was evaluated by
10AT, a rheodyne injector with a 20 µl sample loop and a slightly modified method of Hidalgo 1994. Briefly
et al.,

UV-Vis detector model SPD 10AVP. Spinchrom computer 5 mg β-carotene, 40 mg γ-linoleic acid and 400 mg of
software was used to control the system and collect the Tween-40 were mixed in 1 mL chloroform. Chloroform
data. The separation was performed on reverse phase was removed under vacuum using the flash rotary
packed column (RP C-18; Phenomenex, USA; 250 mm × evaporator at 40 oC. The resulting mixture was added
4.6 mm; particle size 5 µm). 3 mg/ml solution of EJ was with 20 mL water and emulsion was prepared. The
prepared in methanol : water 7 : 3 and filtered through emulsion was further diluted with 80 mL of oxygenated
0.45 µm membrane filter. The elution was performed water. 100 to 600 µg of extract and BHT were added in
using methanol (A) and water (B), employing a program separate test tubes and volume was made up to 0.4 mL
Vol. 13, No. 3, 2007 201

with ethanol. 0.6 mL of water and 3 mL of emulsion was containing 20% w/v TCA, 0.4% w/v of TBA and 0.05%
added to each test tube. Absorbances of all samples were w/v BHT. After keeping in boiling water bath for 20 min,
taken at 470 nm at zero time and test tubes were placed at the samples were cooled. The pink chromogen was
50 oC in water bath. Measurement of absorbance was extracted with a constant amount of -butanol, and the
n

continued at an interval of 30 minutes, till the color of β- absorbance of the upper organic layer was measured at
carotene disappeared in the control reaction (t = 180 min). 532 nm. % Anti lipid peroxidation activity was calculated
A mixture prepared as above without β-carotene emulsion by the formula, % Anti lipid peroxidation activity = [(C −
served as blank and mixture without extract served as S) / C] × 100, where C is the absorbance of the control
control. Dose response of antioxidant activity of EJ was and S is the absorbance of the sample. Each experiment
determined at different concentrations. The antioxidant was carried out in triplicate and results were expressed as
activity (%AA) of EJ was evaluated in terms of bleaching mean % Anti lipid peroxidation activity ± SD.
of β-carotene using the following formula. % AA = 100 Reduction of Ferric Ions (Rajkumar and Rao, 1993) −
[1 − (Ao – At) / Ao 0 – At0)], where % AA = Antioxidant The reaction mixture containing -phenanthroline (0.5
O

activity, Ao = absorbance of sample at zero time, A00 = mg), ferric chloride (0.2 mM) and extracts of different
zero time absorbance of control, At = absorbance of concentrations, 100 to 1000 µg or ascorbic acid 10 to 50
sample after incubation for 180 min, At0 = absorbance of µg dissolved in ethanol, in a final volume of 5 mL and
control after incubation for 180 min. Each experiment was incubated for 15 - 20 min. at ambient temperature.
was carried out in triplicate and results were expressed as The absorbance at 510 nm was measured. In another set,
mean % antioxidant activity ± SD. sodium dithionite (0.3 mM) was added instead of the
Hydroxyl radical scavenging activity (Chakrabarti extract and the absorbance was taken as equivalent to
et al., 1995) − The reaction volume contains different 100% reduction of all the ferric ions present. Each
concentrations of EJ (from 5 to 80 µg) or standard gallic experiment was carried out in triplicate and results were
acid (0.5 to 4 µg), 2.8 mM deoxy- -ribose andd expressed as mean % reduction of ferric ions ± SD.
phenylhydrazine 0.2 mM incubated for 2 hours at 37 oC in Reduction of Ferric Ions in presence of EDTA
incubator and hydroxyl radical scavenging was measured (Kunchandy and Rao, 1989) − The reaction mixture
by thiobarbituric acid reactive substances (TBARS) containing -phenanthroline (0.5 mg), ferric chloride (0.2
O

method (Ohkawa ., 1979). To each test tube was

et al mM), EDTA (0.2 mM) and extracts of different concen-
added 1 mL of 2.8% TCA containing 1% TBA and trations, 100 to 600 µg ascorbic acid 10 to 50 µg
heated in boiling water bath for 20 min., cooled and (dissolved in ethanol) in a final volume of 5 mL and was
absorbance was read at 532 nm. Percentage hydroxyl incubated for 15 - 20 min. at ambient temperature. The
radical scavenging activity was calculated by the formula, absorbance at 510 nm was measured. In another set,
percentage hydroxyl radical scavenging activity = [(C − sodium dithionite (0.3 mM) was added instead of the
S) / C] × 100, where C is the absorbance of the control extract and the absorbance was taken as equivalent to
and S is the absorbance of the sample. Each experiment 100% reduction of all the ferric ions present.
was carried out in triplicate and results were expressed as Hydrogen peroxide scavenging activity (Ilhami,
mean % OH radical scavenging activity ± SD. 2006) − The hydrogen peroxide scavenging ability of EJ
Lipid peroxidation assay (Sudheerkumar et., al was determined by simple UV spectroscopic method.
2003) − Egg phospatidylcholine (20 mg) in chloroform (2 Different concentrations of EJ and BHT from 5 µg to 50
mL) was dried under vacuum in a rotary evaporator to µg were taken, volume adjusted to 3 mL with phosphate
give a thin homogenous film and further dispersed in buffer and 1 mL of 30 mM H2O2 was added. After 10
normal saline (5 mL) with a vortex mixer. The mixture min. the absorbance of the reaction mixture was recorded
was sonicated to get a homogeneous suspension of at 230 nm. Blank solution was containing the phosphate
liposomes. Lipid peroxidation was initiated by adding buffer without H2O2. Percentage scavenged [H2O2] was
0.05 mM ascorbic acid to a mixture containing liposome calculated using the formula. Percentage scavenged
(0.1 mL), 150 mM potassium chloride, 0.2 mM ferric [H2O2]= [(A0 − A1) / A0] × 100, where A0 is the absorbance
chloride, EJ (20 to 80 µg/mL) or standard Silymarin (1 to of the control and A1 is the absorbance of the sample.
10 µg) were added separately, in a total volume of 1 mL. Each experiment was carried out in triplicate and results
The reaction mixture was incubated for 40 minutes at were expressed as percentage scavenged [H2O2] ± SD.
37 oC. After incubation, the reaction was terminated by Total antioxidant capacity (Prieto ., 1999;
et al

adding 1 ml of ice cold 0.25 M Sodium hydroxide Govindarajan ., 2003) − Total antioxidant capacity
et al
202 Natural Product Sciences

was measured according to slightly modified method of gallic acid. The experiment was conducted in triplicate
Prieto ., 1999. 100 µg of EJ, BHT and silymarin were
et al and values are expressed in mean ± SD.
taken in 0.1 mL of alcohol, combined separately in an
eppendroff tube with 1.9 mL of reagent solution (0.6 M Results and discussion
sulfuric acid, 28 mM sodium phosphate, and 4 mM
ammonium molybdate). The tubes were capped and Preliminary phytochemical screening of EJ showed the
incubated in a thermal block at 95 oC for 90 min. After presence of sterols, flavonoids, carbohydrates and
the samples were cooled to room temperature, the glycosides.
absorbance of the aqueous solution of each was measured The HPLC chromatogram of EJ measured at 275 nm is
at 695 nm against a blank. A typical blank solution represented in Fig. 1. at a chromatogram of EJ at the
contained 1.9 mL of reagent solution and the appropriate concentration of 5 mg/mL showed that, it contains
volume of the same solvent used for the sample, and it constituents eluting between 2 - 25 min with major peaks
was incubated under the same conditions as the rest of the at 2.68, 2.833, 12.317, 23.37 and 23.46 min. The solvent
samples. Ascorbic acid equivalents were calculated using system, concentration of sample and wavelength for
standard graph of ascorbic acid. The experiment was detection (275 nm) was selected after trying different
conducted in triplicate and values are expressed as combinations. The method which is adopted was found
ascorbic acid equivalents in µg per mg of extract the most suitable one with highest reproducibility.
(mean ± SD). As shown in Table 1, EJ strongly scavenged DPPH
Total Phenolic Content (TPC) (Mathew and radical with the IC50 value of 123.09 ± 3.01 µg/mL. The
Abraham, 2006) − EJ was diluted with the alcohol 95% scavenging was found to be dose dependent. Where as
to a suitable concentration for analysis. TPC of EJ was ascorbic acid and BHT used as standards were shown
assessed approximately by using the Folin-Ciocalteau IC50 value of 4.91 ± 0.36 and 21.88 ± 2.12 µg/mL. DPPH
phenol reagent method. To 100 µL of the extract (200 µg) radicals react with suitable reducing agents then losing
was added 0.5 mL of Folin–Ciocalteau reagent and 1 mL colour stiochiometrically with the number of electrons
of sodium carbonate (4% w/v) and the volume made upto consumed which is measured spectrophotometrically at
to 2 mL, contents were mixed and allowed to stand for 30 517 nm. Ascorbic acid is a potent free radical scavenger
min. Absorption at 765 nm was measured in a UV-Vis. and BHT is known antioxidant and is used as preservative
spectrophotometer. The total phenolic content was (Singh et al., 2002; Mathew and Abraham, 2006). So
expressed as gallic acid equivalents (GAE) in micrograms when compared to such pure components, IC50 value of
per mg of sample, using a standard curve generated with 123.09 ± 3.01 µg/mL of EJ is quiet high, and shows that

HPLC finger printing. HPLC chromatogram of EJ detected at 275 nm separated on a RP-C18 column, Phenomenex, USA (250 ×
Fig. 1.

4.6 mm; particle size 5 µm) using methanol: water in different ratio for 27 min.
Vol. 13, No. 3, 2007 203

Table 1. Percentage Free radical scavenging activity of EJ, BHT Table 3.Percentage Antioxidant property of EJ and BHT in β-
and Ascorbic acid in DPPH method CLAMS method
Conc. EJ Conc. BHT Ascorbic acid Conc. (µg/mL) EJ BHT
(µg/mL) (µg/mL)
25 3.64 ± 1.58 20.71 ± 3.34
09.09 06.34 ± 1.60 4.54 32.05 ± 2.62 49.19 ± 1.52 50 27.41 ± 0.61 36.17 ± 2.39
18.18 14.13 ± 1.57 9.09 36.96 ± 1.56 76.30 ± 2.65 75 --- 54.73 ± 0.39
36.36 22.97 ± 2.63 13.63 40.06 ± 0.95 80.01 ± 1.98
100 43.91 ± 0.65 61.58 ± 1.43
54.54 31.87 ± 1.03 18.18 47.03 ± 0.89 86.03 ± 1.21
72.73 38.29 ± 1.18 22.73 51.19 ± 3.21 93.90 ± 2.36 150 55.79 ± 0.73 71.47 ± 0.33
109.09 45.08 ± 1.30 27.27 59.98 ± 1.65 99.38 ± 1.87 IC 50 132.3 ± 6.03 68.51 ± 2.5
145.45 51.85 ± 1.60 ---
181.82 63.49 ± 1.11 --- of EJ and BHT were found to be 132.3 ± 6.03 and 68.51 ±
IC 50 123.09 ± 3.01 21.88 ± 2.12 4.91 ± 0.36 2.5 µg/mL respectively. The mechanism of bleaching β-
carotene is a free radical mediated phenomenon resulting
from hydroperoxides formed from linoleic acid. β-
Nitric Oxide scavenging activity of EJ and Ascorbic acid
Table 2.

in percentage carotene in this model system undergoes rapid

discoloration in the absence of an antioxidant. The
Conc. Conc.
(µg/mL) EJ (µg/mL) Ascorbic acid linoleic acid free radicals, upon the abstraction of
hydrogen from one of its diallylic methylene group
100 17.09 ± 2.78 25 33.53 ± 2.17 attacks the highly unsaturated β-carotene molecules. As
200 26.65 ± 4.55 50 42.56 ± 2.58 β-carotene molecules loose their double bonds by
300 34.21 ± 3.89 75 46.23 ± 2.10 oxidation, the compound looses its chromophore and
400 41.98 ± 3.66 100 59.76 ± 4.16 characteristic orange color, which can be monitored
600 48.79 ± 3.42 125 69.56 ± 0.53 spectrophotometrically at 470 nm. So in presence of
800 53.61 ± 2.37 antioxidants, β-carotene retains its color. Because
1000 64.18 ± 0.98 antioxidants prevent abstract of hydrogen from linoleic
IC 50 4643.0 ± 61.10 83.80 ± 3.75 acid from its diallylic methylene group by donating
hydrogen from itself. Thus prevents the oxidation of β-
EJ is potent DPPH free radical scavenger. carotene. Here the IC50 values of BHT which is used as
EJ also strongly inhibited NO in dose dependent standard and EJ are having near values. This shows that
manner (Table 2) with the IC50 being 643.0 ± 61.10 µg/ EJ can donate hydrogen to linoleic acid free radicals as
mL. In the same manner ascorbic acid also scavenged NO much as BHT can. EJ can be used as natural antioxidant
and IC50 value being 83.80 ± 3.75 µg/mL. Nitric oxide is instead of BHT which is synthetic and use of BHT is said
a potent pleiotropic mediator of physiological process to unsafe and their toxicity is a problem of concern. (Kaur
such as smooth muscle relaxation, neuronal signaling, et al., 2006; Madhavi and Salunkhe., 1995).
initiation of platelet aggregation and regulation of cell Hydroxyl radical scavenging activity was estimated by
mediated toxicity. It is a diffusible free radical which generating the hydroxyl radicals using phenylhydrazine
plays many roles as an effector molecule in diverse (Chakrabarti ., 1995). Hydroxyl radical scavenging
et al

biological systems including neuronal messenger, was measured by studying the competition between
vasodialtion and antimicrobial and antitumor activities. deoxy- -ribose and sample extracts for hydroxyl radicals
d

Studies in animal models have suggested a role for NO in produced by phenyl hydrazine. The extent of deoxy- - d

the pathogenesis of inflammation and pain and NOS ribose degradation is measured as TBARS method of
inhibitors have been shown to have beneficial effects on Ohkawa ., 1979. EJ scavenged the hydroxyl radicals
et al

some aspects of the inflammation and tissue changes seen strongly with an IC50 value of 68.5 ± 11.5 µg/mL. IC50
in models of inflammatory bowel disease (Govindarajan value of GA is 3.3 ± 0.2 µg/mL (Table 4). The hydroxyl
et al ., 2003). Thus establishing the usage of the plant in radical is an extremely reactive free radical formed in
the Indian indigenous system as an anti-inflammatory biological systems and has been implicated as a highly
agent (Chopra et al., 1956 anonymus, 1959). damaging species in free radical pathology, capable of
The antioxidant activity of EJ as measured by the damaging almost every molecule found in living cell.
bleaching of β-carotene is presented in Table 3. IC50 value This radical has the capacity to join nucleotide in DNA
204 Natural Product Sciences

Table 4. Hydroxyl radical scavenging activity of EJ and Gallic Table 5. Anti Lipid Peroxidation activity of EJ and silymarin in
acid (GA) in percentage percentage
Conc. (µg/mL) EJ Conc. (µg/mL) GA Conc. (µg/mL) EJ Conc. (µg/mL) Silymarin
5 8.167 ± 4.06 0.5 20.91 ± 6.51 20 3.62 ± 1.54 1.0 3.13 ± 3.65
10 29.34 ± 4.08 1 25.81 ± 5.65 40 18.09 ± 0.44 2.0 29.66 ± 1.00
20 40.20 ± 4.51 2 36.54 ± 3.97 60 38.26 ± 1.54 5.0 43.34 ± 0.48
50 44.14 ± 3.61 3 46.41 ± 2.81 80 68.49 ± 1.86 7.5 59.44 ± 1.85
80 53.68 ± 3.58 4 58.39 ± 2.62 --- --- 10.0 89.84 ± 1.48
IC 68.5 ± 11.5 3.3 ± 0.2 IC 50 68.13 ± 1.38 IC50 6.0 ± 0.2
50

and cause strand breakage which contributes to al., 1996). Determination of lipid peroxidase content was
carcinogenesis, mutagenesis, and cytotoxicity. In addition, carried out indirectly by means of derivatizing MDA with
this species is considered to be one of the quick initiator TBA at high temperature and acidic condition (Halliwell
of lipid peroxidation process, abstracting hydrogen atom and Guttridge, 1989). EJ has strongly inhibited the lipid
form the unsaturated fatty acids (Singh ., 2002; Lee
et al et peroxidation a shown in the Table 5, IC50 value of EJ is
al., 2004). EJ has found to scavenge OH radicals but not 68.13 ± 1.38 µg/mL and that of silymarin is 6.0 ± 0.2 µg/
as strongly as gallic acid. Because GA is very potent OH mL. this indicates that silymarin is more than ten times
radical scavenger (Yen et ., 2002) and is a pure
al potent than EJ. Silymarin is a mixture of flavonolignan
compound. Thus EJ may be useful as an antioxidant and and is proven potent hepatoprotective and antioxidant
may prevent damages that arise from OH radicals by agent (Varga et al., 2006; De ., 1993). So when
et al

scavenging them. This OH radical capacity of EJ may be compared to such potent agent, the values obtained
due lignans and flavonoids as found present in EJ by indicate that EJ is good antilipid peroxidation agent.
qualitative chemical tests. It is found that flavonoids are Fe2+ reacts rapidly with 1, 10- -phenanthroline and
O

potent OH radical scavenging agents (Amic ., 2003;

et al forms red colored complex (λmax 510) which is
Dai ., 2006).
et al exceptionally stable. Extracts reacts with Fe3+ to reduce
EJ prevented lipid peroxidation strongly but less than and convert it to Fe2+. The degree of coloration measured
the standard (silymarin). IC50 values of antilipid peroxida- at 510 nm indicates the reduction potential of the extracts
tion of EJ and silymarin were 68.13 ± 1.38 and 6.0 ± 0.2 (Rajkumar and Rao, 1993; Kunchady and Rao, 1989). As
µg/mL respectively. The effect of EJ to prevent lipid shown in the Table 6, in absence of EDTA, EJ has shown
peroxidation is shown in Table 5. In biological systems, 41.82 ± 0.41% activity at 1000 µg concentration. IC50 of
malondialdehye (MDA) is very reactive species and takes ascorbic acid is 39.15 ± 3.65 µg. But in presence of EDTA
part in the cross linking of DNA, with protein and also both EJ and ascorbic acid failed to show ferric ion
damaging the liver cells. The lipid peroxidation has been reduction activity. Both EJ and ascorbic acid have
broadly defined as the oxidation deterioration of reduced the Fe3+ to Fe2+ moderately. But in presence of
polyunsaturated lipids. The initiation of peroxidation EDTA (strong chelating agent), ferric ion reduction
sequence in membrane or polyunsaturated fatty acids is activity of EJ and ascorbic acid were nil. This is because
due to abstraction of a hydrogen atom from the double of EDTA being strong chelating agent, complexed with
bond in the fatty acids. The free radicals tends to be ferric ions. EJ and ascorbic acid were not able to reverse
stabilized by a molecular rearrangement to produce a this complexation. So there is no reduction of ferric ions.
conjugated dienes, which then easily react with an oxygen The reducing properties are generally associated with the
molecule to give a peroxy radical. Peroxy radical can presence of reductones. Antioxidant action of the
abstract a hydrogen atom from another molecule or they reductones is based on the breaking of free radicals chain
can abstract hydrogen atom to give lipid hydroperoxide, by the donation of a hydrogen atom. The reductones also
R-OOH. A probable alternative fat of peroxy radicals is to react with certain precursors of peroxide, thus preventing
form cyclic peroxidase; these cyclic peroxidase, lipid formation of peroxide (Gordon, 1991).
peroxidase and cyclic endoperoxidase fragment to H2O2 in phosphate buffer has the λmax of 230 nm. In
aldehyde including MDA and polymerization product. presence of extracts the reduction of absorbance at 230
MDA is the major product of lipid peroxidation and is nm indicates scavenging or breakdown of H2O2 (Ilhami,
used to study the lipid peroxidation process. (Jadhav et 2006). When breakdown of H2O2 occurs, there will be
Vol. 13, No. 3, 2007 205

Table 6. Ferric ion reduction activity of EJ and Ascorbic acid in percentage

EJ Ascorbic acid
Conc. In absence In presence Conc. In absence In presence
(µgs) of EDTA of EDTA (µgs) of EDTA of EDTA
100 22.14 ± 0.84 ND 10 18.87 ± 0.64 ND
200 27.24 ± 0.90 ND 20 26.63 ± 2.06 ND
300 30.03 ± 1.36 ND 30 38.72 ± 5.62 ND
400 31.01 ± 1.84 ND 40 48.60 ± 5.09 ND
600 33.85 ± 1.40 ND 50 60.95 ± 1.43 ND
800 38.34 ± 0.84 ND -- -- --
1000 41.82 ± 0.41 ND -- -- --
IC 50 ND 39.15 ± 3.65
Concentration is the total extract present in reaction mixture in µgs.
ND - Not Detected at tested concentration.
ascorbic acid respectively. In the same manner, in total
Percentage H O scavenging activity of EJ and BHT
Table 7. 2 2
phenolic content assay, it was found that 1 mg of EJ and 1
Conc. (µg/mL) EJ BHT mg of silymarin were equivalent to 43.76 ± 4.27 and
10 5.93 ± 0.29 15.34 ± 0.25 42.49 ± 3.84 µg of gallic acid respectively. Total antioxidant
20 12.46 ± 0.58 22.16 ± 0.70 capacity and total phenolic content were found to be more
40 22.74 ± 0.25 30.66 ± 1.92 in EJ than the silymarin which is used as a standard. But
50 37.97 ± 0.43 33.86 ± 1.47 total antioxidant capacity of EJ is less than the BHT.
Silymarin was used as a standard because it is a mixture
of flavonolignans and is potent well known hepatopro-
reduction of absorbance at 230 nm. As shown in Table 7, tective agent (Varga et al., 2006; De et al., 1993). The
EJ and BHT scavenged the H2O2 to the extent of 37.97 ± genus Justiciaalso found to contain lignans (Jyotishi and
0.43% and 30.66 ± 1.92 at 50 µg/mL concentration. Both Bagavant, 1992a; 1992b) and also preliminary tests
EJ and BHT both caused weak decomposition of H2O2 in showed that EJ contains flavonoids. Hence total phenolic
a dose dependent manner as shown in Table 4. H2O2 content of EJ is found to be more than the silymarin and
scavenging capacity of EJ is found to be little higher than because of this total antioxidant capacity of EJ is also
the BHT. Hydrogen peroxide is a weak oxidizing agent more than the silymarin.
and can inactivate a few enzymes directly, usually by The data presented here indicate that the marked
oxidation of essential thiol (–SH) groups. Hydrogen antioxidant activity of EJ extracts seems to be due to
peroxide can cross cell membranes rapidly. Once enter presence of flavonoids and lignans, which may act in a
inside the cell, H2O2 can probably react with Fe2+, and similar fashion as reductones by donating the electrons
possibly Cu2+ ions to form hydroxyl radical and this may and reacting with free radicals to convert them to more
be the origin of many of its toxic effects. It is therefore stable product and terminate free radical chain reaction.
biologically advantageous for cells to control the amount Free radicals and reactive oxygen species are involved in
of hydrogen peroxide that is allowed to accumulate. Two a variety of pathological events such as aging,
types of enzymes exist to remove hydrogen peroxide inflammation, cancer, atherosclerosis and diabetes (Lee et

within cells. They are catalase and the peroxidase. The al., 2004). The plant would be useful for the treatment of
extract has shown to decompose H2O2 and thus may help various diseases mediated by free radicals. The flavonids
in preventing the imbalance in oxidative stress and and other components present in the extract found to
antioxidants including antioxidant enzymes like catalase suppress lipid peroxidation, hydroxyl radical formation
and peroxidase in the biological system (Ilhami, 2006; Le through different chemical mechanisms, including free
et al, 2007; Kaur, 2006). radical quenching, electron transfer, radical addition or
In total antioxidant capacity assay, it was found that 1 radical recombination. Overall, the plant would be useful
mg of EJ is equivalent to 205.56 ± 34.69 µg of ascorbic as an antioxidant and free radical scavenging agent and
acid. Similarly 1 mg of slymarin and BHT were found thus help in treatment of many diseases mediated by
equivalent to 197.22 ± 4.81 and 400.00 ± 22.05 µg of ROS.
206 Natural Product Sciences

Acknowledgement and Occurrence in Justicia-I. Indian J. Nat. Prod , 3-9 (1992a).

. 8

Jyotishi, S.G. and Bagavant, G., Lignans: their Pharmacological Activity

and Occurrence in Justicia-I. Indian J. Nat. Prod. , 3-17 (1992b).
One of the authors, K.Mruthunjaya is highly thankful
8

Kaur, G., Jabbar, Z., Athar, M., and Alam, M.S., Punica granatum
to JSS College of Pharmacy, and JSS Mahavidyapeetha, (pomegranate) flower extract possesses potent antioxidant activity and
Mysore, India for providing financial assistance during abrogates Fe-NTA induced hepatotoxicity in mice, Food Chem.
Toxicol. , 984-993 (2006).
the research work. And also thankful to Dr. 44

Kirthikar, K.R. and Basu, B.D., Indian medicinal plants vol. 8, Oriental
K.N.Chidambara Murthy, CFTRI, Mysore, India, for enterprises, Dehradun, 2001.
providing pure chemicals that were required to carryout Kokate, C.K., Purohit, A.P., and Gokhale, S.B., Pharmacognosy, Nirali
few experiments in the present study and Dr.L.V.G Prakashan, Pune, 1996.
Kunchandy, E. and Rao, M.N.A., Effect of curcumin on hydroxyl radical
Nargund, Professor & Director, Drug Discovery Division, generation through Fenton reaction, Int. J. Pharm. , 173-176 (1989).
Dr. Nargund Research Foundation, Bangalore, India for
57

Lee, J., Koo, N., and Min, D.B., Reactive Oxygen Species, Aging, and
his kind permission to utilize the research facilities Antioxidative Nutraceuticals, Comp. Rev. Food Sci. Safety. , 21-333

available in their college. (2004).

Li, Xin-Ming., Shi, Yong-Hui., Wang, Fang., Wang, Hai-Song., and Le,
Guo-Wei., In vitro free radical scavenging activities and effect of
References synthetic oligosaccharides on antioxidant enzymes and lipid
peroxidation in aged mice, J. Pharm. Biomed. Anal. , 364-370 43

Amic, D., Davidovic-Amic, D., Beslo, D., and Trinajstic, N., Structure- (2007).
Radical Scavenging Activity Relationships of Flavonoids, Croat. Madhavi, D.L., and Salunkhe, D.K., Toxicological aspects of food
Chem. Acta , 55-61 (2003).
76
antioxidants, in Madhavi, D.L., Deshpande, S.S., Salunkhe, D.K.,
Anonymous, The Wealth of India: Raw Materials-Vol V, Publication & (eds.), Food Antioxidants. Dekker, New York, 1995, pp. 267.
Information Directorate (CSIR), New Delhi, 1959. Mathew, S. and Abraham, T.E., In vitro antioxidant activity and
Chakrabarti, S., Sonaye, B., Naik, A.A., and Nadkarni, P.P., Erythrocyte scavenging effects of Cinnamomum verum leaf extract assayed by
membrane protein damage by oxidation products of phenylhydrazine, different methodologies, Food Chem.l Toxicol. , 198-206 (2006).
44

Biochem. Mol. Biol. Int. , 255-263 (1995).

35
Ohkawa, H., Ohishi, N., and Yagi, K., Assay for lipid peroxides in animal
Chakravarty, A.K., Dastidar, P.G., and Pakrash, S.C., Simple aromatic tissues by thiobarbituric acid reaction, Anal. Biochem. , 351-358,
95

amines from Justicia gendarussa-13C NMR spectra of the bases and (1979).
their analogues. Tetrahedron , 1797-1802 (1982).
38
Peach, K. and Tracey, M.V., Modern Methods of Plant Analysis Vol. 3.,
Chopra, R.N., Nayar, S.L. and Chopra, I.C., Glossary of Indian medicinal Springer Verlag, Berlin, 1955a.
plants, CSIR, New Delhi, 1956. Peach, K. and Tracey, M.V., Modern Methods of Plant Analysis Vol. 4.,
Dai, F., Miao, Q., Zhou, B., Yang, L, Liu, and Zhong-Li., Protective Springer Verlag, Berlin, 1955b.
effects of flvonols and their glycosides against free radical-induced Prieto, P., Pineda, M., and Aguilar, M., Spectrophotometric Quantitation
oxidative hemolysis of red blood cells, Life Sci. , 2488-2493 (2006).
78
of Antioxidant Capacity through the Formation of a Phospho-
De, S., Ravishankar, B., and Bhavsar, G.C., Plants with hepatoprotective molybdenum Complex: Specific Application to the Determination of
activity-A Review, Indian Drugs , 363 (1993).
38
Vitamin E1, Anal. Biochem. , 337-341(1999).
269

Geinssman, T.A., Flavonoids, in Peach, K., and Tracey, M.V., (eds.), Rajkumar, D.V. and Rao, M.N.A., Dehydrozingerone and isougenol as
Modern Methods of Plant Analysis Vol. 3, Springer Verlag, Berlin, inhibitors of lipid peroxidation and as free radical scavengers,
1955, pp. 467-474. Biochem. Pharmaco. , 2067-2072 (1993).
46

Gordon, M.F., The Mechanism of antioxidant action in vitro, in Hudson, Singh, R.P., Murthy, K.N.C., and Jayaprakash, G.K., Studies on the
B.J.F., (ed.), Food antioxidants, Elsevier Apllied Science, London, Antioxidant activity of Pomegranate (Punica granatum) peel and seed
1991, pp. 1-88. extracts using in vitro models, Journal of Agricult. Food Chem. , 50

Govindarajan, R., Rastogi, S., Vijayakumar, M., Shirwaikar, A., Rawat, 81-86 (2002).
A.K.S., Mehrotra, S., and Palpu, P., Studies on the Antioxidant Stevens, H.M., Analytical techniques, in Moffat, A.C., Jackson, J.V.,
Activities of Desmodium gangeticum, Biol. Pharm. Bull. , 1424-
26
Moss, M.S., and Widdop, B., (eds.), Clarke’s isolation and
1427 (2003). Identification of drugs in Pharmaceuticals, Body fluids and post-
Halliwell, B., and Guttridge, J.M.C., Free radicals in Biology and mortem material, The pharmaceutical Press, London, 1986, pp. 457-
Medicine, Japan Scientific Press, Tokyo, 1989. 472.
Hawks, P.B., Hawk's Physiological chemistry, Tata Macgraw Hill Sudheerkumar, M., Jagadish, P.C., Sridhar, R.B., Kiran, B.S., and
Publishing Company Ltd., New Delhi 1971. Unnikrishnan, M.K., In-vitro evaluation of antioxidant properties of
Hidalgo, M.E., Fernandez, E., Quilhot, W., and Lissi, E., Antioxidant Cocos nucifera Linn. Water, Nahrung/Food. , 126-131 (2003).
47

activity of depsides and desidones, Phytochemistry , 1585-1587

37
Trease, G.E. and Evans, W.C., Pharmacognosy, ELBS, London, 1989.
(1994). Varga, Zs., Seres, I., Nagy, E., Ujhelyi, L., Balla, G., Balla, J., and Antus,
Ilhami, Gulcin., Antioxidant and antiradical activities of l-carnitine, Life S., Structure prerequisite for antioxidant activity of silybin in different
Sci. , 803-811 (2006).
78
biochemical systems in vitro, Phytomedicine , 85-93 (2006).
13

Jadhav, S.J., Nimbalkar, Kulkarni, A.D., and Madhavi, D.L., Lipid Yen, Gow-Chin., Duh, Pin-Der., and Tsai, Hui-Ling., Antioxidant and
oxidation in biological and food systems, in Madhavi, D.L., pro-oxidant properties of ascorbic acid and gallic acid. Food Chem.
Deshpande S.S., Salunkhe, D.S., (eds.), Food antioxidants, Dekker, 79 , 307-313 (2002).
New york, 1996, pp. 5-63.
Jyotishi, S.G. and Bagavant, G., Lignans: their Pharmacological Activity (Accepted June 18, 2007)