Global Veterinaria 14 (4): 595-602, 2015

ISSN 1992-6197
© IDOSI Publications, 2015
DOI: 10.5829/[Link].2015.14.04.9427

In vitro Antioxidant Activity of Methanol Extract of Lantana camara Leaves

C.L. Asadu, C.A. Anosike, P.N. Uzoegwu, O. Abonyi, A.L. Ezugwu and R.I. Uroko

Department of Biochemistry, University of Nigeria, Nsukka, Nigeria

Abstract: The plant kingdom offers a wide range of natural antioxidant and medicinal values. These are made
possible as a result of plethora of different chemical constituents of these plants. The study was carried out
to ascertain the antioxidant status of Lantana camara leaves and its potential for scavenging free radical
species in the body. The quantitative phytochemical screening of Lantana camara showed that the leaves
contain flavonoids (11.08±0.05 mg/g), tannins (9.0±0.03 mg/g), alkaloids (9.76±0.02 mg/g), saponin (6.07±0.06
mg/g), reducing sugar (4.86 ±0.05 mg/g) and carbohydrate (5.08± 0.03 mg/g). Micronutrients analysis showed
vitamin A 0.50 mg/100g, vitamins C 6.5 mg/100g and vitamin E 1.6 mg/100g and total phenolic compounds 2.36
GAE (Gallic Acid Equivalent). The antioxidant activity of the extract was determined on 1,1-diphenyl-2-
picrylhydrazyl radical (DPPH),.
superoxide (O2.-), hydroxyl (OH.) and nitric oxide (NO.) radicals. The percentage
inhibition of methanol extract of Lantana camara leaves extracts on DPPH radical was concentration dependent
with an effective concentration at fifty percent (EC50) of 27.56±0.02µg/ml compared to standard (ascorbic acid)
with EC50 of 11.07±0.03µg/ml. The extract inhibited hydroxyl radical-induced 2-deoxy-ribose degradation, EC50
(22.18 ± 0.02 µg/ml) compared to the standard (á-tocopherol) EC50 (18.60 ± 0.02 µg/ml). The superoxide anion
radical was inhibited in a concentration dependent manner. The extract had a significant 0 2.- anion radical
scavenging ability, EC50 was 27.94 ± 0.03 µg/ml compared to ascorbic acid standard EC50 of 62.47 ± 0.02 µg/ml.
The overall scavenging activity of the extract on nitric oxide radical showed that the extract at 500 µg/ml was
most potent in scavenging nitric oxide radical compared to ascorbic acid standard at 500 µg/ml. Hydroxyl radical
showed the highest anti-radical power (ARP) compared to DPPH and superoxide. Superoxide free radical
recorded the lowest anti-radical power. The extract was found to be effective scavengers of the above-
mentioned free radicals. These results show that the extract possesses antioxidant potential, which is very
beneficial in tackling the damages caused by free radicals.

Key words: DPPH Radical Hydroxyl Radical Nitric Oxide Radical Superoxide Radical Ascorbic Acid
Antioxidant Activity Scavenging

INTRODUCTION been extensively studied by advanced scientific

techniques and reported for various medicinal properties
Nature has provided an excellent source of remedies which include: anticancer activity, antioxidant activity,
to cure many of the ailments of mankind. In ancient days, anti-inflammatory, antidiabetic, antibacterial, antifungal
almost all the medicine used were from natural sources, and hepato-protective activities [3].
particularly from plants. Plants continue to be important Free radical reactions have been implicated in the
sources of new drugs even now. The importance of pathology of many human diseases such as
biological, chemical and pharmacological evaluation of atherosclerosis, ischemic heart disease, diabetes,
plant derived agents used in the treatment of human neurodegenerative diseases and disease conditions such
ailments has been increasingly recognized in the last as ageing process, inflammation and immune-suppression.
decades [1]. World health organisation (WHO) currently A number of plants and plant products have been
encourages, recommends and promotes traditional reported to protect against free radical induced damages
herbal medicines due to their case of availability, low cost, in various experimental models [4]. Exposure to ionizing
safety and people’s faith in such remedies [2]. In the last radiation, smoking, herbicides, pesticides and fried foods
few decades, many of traditionally known plants have generate free radicals. Reactive oxygen species (ROS) are

Corresponding Author: C.L. Asadu, Department of Biochemistry, University of Nigeria, Nsukka, Nigeria .
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chemically reactive molecules containing oxygen which be dangerous for human health [14]. Both enzymatic and
includes superoxide (O2•-), hydroxyl (OH•), peroxyl (ROO ). non-enzymatic antioxidants exist in the intracellular and
Nitric oxide (NO•) and nitrogen dioxide ( NO2) are two extracellular environment to detoxify free radicals. An
nitrogen free radicals. Oxygen and nitrogen free radicals imbalance between antioxidants and reactive oxygen
can be converted to other non-radical reactive species, species results in oxidative stress, leading to cellular
such as hydrogen peroxide (H2O2), hypochlorous acid damage. Examples of antioxidants are vitamin C, vitamin E,
(HOCl), hypobromous acid (HOBr) and peroxynitrite selenium and carotenoids [15]. Natural antioxidants such
(ONOO•) [5]. Reactive oxygen species (ROS) and reactive as phenols, flavonoids and tannins are increasingly
nitrogen species (RNS) are produced in animals and attracting attention because they are natural disease
humans under physiologic and pathologic conditions. preventing, health promoting and anti-ageing substances
During energy transduction, a small number of electrons [16]. Antioxidants may serve the task of reducing
“leak” to oxygen prematurely, forming the oxygen free oxidative damage in human induced by free radicals and
radical superoxide, which has been implicated in the reactive oxygen species under oxidative stress conditions.
patho-physiology of a variety of diseases [6]. Superoxide Lantana camara Linn, (Verbenaceae) is an
anion, O2- can capture further electron to form hydrogen ornamental weed with aromatic leaves, orange, blue,
peroxide. Hydrogen peroxide (H2O2) is toxic, injurious and yellow and bright red flowers with dark blue and black
can further react with “superoxide” anion, in the presence fruits (drupes). It is a low, erect vigorous shrub which can
of ferrous ion or copper to form “hydroxyl” radical and grow up to 2 - 4 meters in height [17]. Lantana camara
“singlet oxygen”. Whenever superoxide anion, O2-• is has been used in many parts of the world to treat a wide
formed in the tissues, it will lead to the formation of variety of disorders [18]. Fevers, colds, rheumatism,
another free radical and hydrogen peroxide [7]. Nitric asthma and high blood pressure were treated with
oxide (NO•) has also been involved in a variety of preparations from the plant [19]. The leaves are also used
biological functions, including neurotransmission, to treat cuts, rheumatism, ulcers and intestinal worms.
vascular homeostasis, antimicrobial and antitumor Leaf extracts of Lantana exhibit antioxidant,
activities [8]. When antioxidants react with DPPH•, which antihypertensive, antimicrobial, fungicidal, insecticidal
is a stable free radical, the radical becomes paired off in and nematicidal properties [20- 24]. Lantana camara oil is
the presence of a hydrogen donor and is reduced to the sometimes used for the treatment of skin itches, as an
DPPHH and as consequence the absorbance decreases antiseptic for wounds and externally for leprosy and
from the DPPH (hydrazyl radical) to the DPPH-H scabies [25]. Pharmacological investigations indicated
(hydrazine) form [9]. The hydroxyl radical has a high that extracts of leaves of Lantana camara exhibited
reactivity, making it a very dangerous radical [10]. ROS strong antioxidant activities [26]. Thus, the search for
induced cell death can result from oxidative processes effective, non-toxic natural compounds with antioxidant
such as membrane lipid peroxidation, protein oxidation, activity has been intensified in recent years- hence the
enzyme inhibition, DNA and RNA damage [11]. rationale of the study.
Oxidative stress represents an imbalance between the
production of reactive oxygen species and a biological MATERIALS AND METHODS
system’s ability to readily detoxify the reactive
intermediates or to repair the resulting damage. Plant Material: The leaves of Lantana camara were used
Disturbances in the normal redox state of tissues can for the study. They were collected from Amokwe village
cause toxic effects through the production of peroxides Nsukka and were identified by Mr. Alfred Ozioko of the
and free radicals that damage all components of the cell, Bioresources Development and Conservation Programme
including protein lipids and DNA. Chemically, oxidative (BDCP) Research Centre Nsukka, Enugu State.
stress is associated with increased production of oxidizing
species or significant decrease in the capability of Equipment: The equipment used were obtained from the
oxidants defences, such as Gluthathione [12]. Department of Biochemistry, University of Nigeria and
An antioxidant is a molecule capable of preventing or other scientific shops in Nsukka.
inhibiting the oxidation of other molecules [13]. Synthetic
antioxidants such as butylated hydroxyl-toluene and Chemicals and Reagents: The chemicals and reagents
butylated hydroxyl-anisole have recently been reported to used were of analytical grade.

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Preparation of Plant Material: The fresh leaves of

Lantana camara were collected, washed with clean
water to remove dirt and drained. They were dried The EC50 value was calculated using a plot of %
under shade for several days and then pulverised into inhibition against different concentrations of the extract
powder. and it represented the concentration of the sample leading
to 50% reduction of the initial DPPH concentration.
Extraction of Plant Material: Powdered leaves (500 g) of
Lantana camara was macerated in one litre of methanol Hydroxyl Radical (OH.) Scavenging Assay: The 2-
for 24 h. The solution was filtered with Whatman no.1 deoxyribose assay was used to determine the scavenging
filter paper and the filtrate was concentrated to a effect of the extract on the hydroxyl (OH) radical as
semi-solid residue using rotary evaporator. reported by Halliwell et al. [29]. Each reaction mixture
contained the following concentration of reagents in a
Determination of Total Phenolic Contents: Total volume of 1.0ml: 2-deoxyribose (2.5µM) potassium
phenolics were determined using Folin-ciocalteu reagent phosphate buffer (pH 7.4, 20mM,), FeCl3 (100µM), EDTA
(FCR) as described by Velioglu et al. [27]. Folin-ciocalteu (104µM), H2O2 (1mM) and L-ascorbic acid. These were
reagent (FCR) consists of a yellow acidic solution prepared immediately before using in distilled water.
containing complex polymeric ions formed from The mixtures were incubated for 1hour at 37°C, followed
phosphomolybdic and phosphotungsticheteropoly acids. by addition of 1.0ml of 1% (w/v) thiobarbituric acid (TBA)
Dissociation of a phenolic proton in a basic medium leads in 0.05M NaOH and 1.0ml of 2.8%(w/v) trichloroacetic
to a phenolate anion, which reduces FCR forming blue acid(TCA). The resulting mixture was heated for 15min at
coloured molybdenum oxide. The colour intensity is 100°C. After cooling on ice, absorbance was measured at
532 nm. The inhibition of 2-deoxyribose degradation
directly proportional to the phenolic contents. The extract
expressed in percentage was calculated using the
(100µl) dissolved in methanol (1mg/ml) was mixed with
equation:
750µl of Folin-Ciocalteu reagent (diluted 10-fold in dH2O),
shaken slightly and allowed to stand at 22°C for 5mins;
750µl of Na2CO3 (60g/l) solution was then added to the
mixture. After 90 min the absorbance was measured at
IC50 value represented the concentration of the
725nm. Results were expressed as gallic acid equivalents.
extract that caused 50% inhibition of 2-
For the standard; 100µl of diluted gallic acid (0.1g gallic
deoxyribosedegradation. All determinations were carried
acid salt in 10ml 0f 80% methanol) was mixed with 750µl of
out in triplicate.
FCR as was with the extract. The blank was made up of
1ml of FCR and 1ml of Na2CO3.
Superoxide Radical Scavenging Assay: The methodof
Martinez et al. [30] was used to determine superoxide
Quantitative Scavenging Assay of 1, 1-diphenyl-2- dismutase in superoxide radical- scavenging assay.
Picrylhydrazyl (DPPH•) Radical: Scavenging activity of The assay based on the capacity of the extract to inhibit
DPPH free radicals by the extract was assessed according the photochemical reduction of nitro blue tetrazolium
to the method of Gyamfi et al. [28]. A solution of the (NBT). Each 3ml reaction mixture contained 0.05M
extract (1.0ml) at different concentrations (2.5-80µg/ml) in phosphate – buffered saline (PBS) (pH 7.8), 13mM
80% methanol was mixed with 1.0ml of 0.3mM DPPH in methionine, 2µM riboflavin, 100µM EDTA, NBT (75µM)
methanol. The mixture was shaken vigorously and and 1.0ml of test sample solutions (10-250 µg/ml).
allowed to stand at room temperature in the dark for The tubes were kept in front of a fluorescent light for
25 min. The negative control was 1.0ml of 0.3mM DPPH 20 minutes and absorbance was read at 560 nm. The entire
solution plus 2.0ml of methanol. L-Ascorbic acid was used reaction assembly was enclosed in a box lined with
as positive control. Thereafter, the absorbance of the aluminium foil. Identical tubes containing reaction mixture
assay mixture was measured at 518 nm against each blank were kept in the dark and served as blanks. The
with UV/Vis spectrophotometer. Lower absorbance of the percentage inhibition of superoxide generation was
reaction mixture indicated higher radical scavenging estimated by comparing the absorbance of the control and
activity. DPPH radical scavenging activity was calculated those of the reaction mixture containing test sample using
using the equation: the equation:

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Table 1: Percentage Inhibition of DPPH. Radical by Methanol Extracts of

Lantana camara Leaves (MELC)
Concentration (µg/ml) Percentage (%) Inhibition
In vitro Nitric Oxide Radical (NO.) Scavenging Assay
2.5 32.49±0.01
Nitric oxide generated from sodium nitroprusside (SNP) 5 40.61±0.03
was measured according to the method of [31]. The 10 70.00±0.03
reaction mixture (5.0ml) containing SNP (5 mM) in 20 50.79±0.02
phosphate buffered saline (pH 7.3), with plant extract at 40 58.38±0.01
different concentrations, was incubated at 25°C for 180min 80 68.02±0.02
EC50 (µg/ml) 27.56±0.02 (MELC) 11.07±0.03
in front of a visible polychromatic light source. The NO.
(ascorbic acid standard)
radical thus generated interacted with oxygen to produce
Values are represented as mean ± SEM (n=3)
the nitric ion (NO2-) which was assayed at 30 min intervals
by mixing 1 ml of incubation mixture with an equal amount Table 2: Hydroxyl Radical Scavenging Activity of MELC
of Griess reagent (1% sulphanilamide in 5% phosphoric Concentrations (µg/ml) Percentage (%) Inhibition
acid and 0.1% naphthyl-ethylene-dihydrochloride). The 2.5 30.24±0.02
absorbance of the chromophore (purple azo dye) formed 5 38.05±0.05
10 43.90±0.04
during the diazotisation of nitrite ions with sulphanilamide
20 54.15±0.02
and subsequent coupling with naphthyl-ethylene-
40 69.27 ±0.03
diamine-dihyhrochloride was measured at 546 nm. Each 80 79.02± 0.02
experiment was carried out in triplicates. EC50 (µg/ml) 22.18±0.02 (MELC)
standard’s EC50 18.60 ± 0.02
RESULTS Values are represented as mean ± SEM (n=3)

Table 3: Superoxide Radical Scavenging Activity of MELC

Effect of Radical Scavenging Activity of MELC Leaves on
Concentrations (µg/ml) Percentage (%) Inhibition
DPPH: The ability of the extract to scavenge DPPH
2.5 40.82 ± 0.04
radicals was investigated at various concentrations of the 5 43.83 ± 0.02
extract. The addition of MELC to the DPPH solution 10 48.58 ±0.02
caused a rapid decrease in absorbance at 518 nm 20 52.06 ± 0.03
indicating the good scavenging capacity of the extract as 40 54.27 ± 0.05
shown in Table 1, the extract possessed substantial 80 58.54 ± 0.01
EC50 (µg/ml) 27.94 ± 0.03(MELC)
dose-dependent antioxidant activity and the activity was
standard’s EC50 62.47 ± 0.02
compared to that of L-ascorbic acid, which was used as a Values are represented as mean ± SEM (n=3)
control antioxidant. The percentage inhibition of extract of
Lantana camara leaves was concentration dependent Scavenging Capacity of Methanol Extracts of Lantana
with an effective concentration at fifty percent (EC50) of
camara Leaves (MELC) on Superoxide Radical: Table 3
27.56±0.02µg/ml compared to that of standard (ascorbic
showed the radical scavenging activity of different
acid) with EC50 of 11.07±0.03µg/ml.
concentrations of the extract against superoxide radical.
The superoxide anion radical was inhibited in
Scavenging Activity of Methanol Extracts of Lantana
concentration dependent manner. The extract had a
camara Leaves (MELC) on Hydroxyl Radical (OH.): The significant 02.- anion radical scavenging ability EC 50= of
radical scavenging potential of the extract was further 27.94 ± 0.03 µg/ml compared to ascorbic acid EC50 of
assessed by investigating its ability to scavenge •OH 62.47 ± 0.02 µg/ml. The lower EC50 of the extract implies
radical using a Fe3+ dependent hydroxyl radical generation that the extract was more effective in scavenging
assay. The effect of Lantana camara extract on •OH superoxide radicals than the standard.
radicals generated by Fe3+ ions was measured by
determining the degree of deoxyribose degradation, an Effect of Methanol Extract of Lantana camara
indicator of thiobarbituric acid-malonaldehyde Leaves (MELC) on Nitric Oxide (NO.) Radical: Sodium
(TBA-MDA) adduct formation. The extract was observed nitro-prusside (SNP) in aqueous solution at physiological
to inhibit hydroxyl radical-induced deoxyribose pH spontaneously generates nitric oxide which interacts
degradation, EC50 (22.18 ± 0.02) compared to that of the with oxygen to produce nitrite ions (NO2-). At the first
standard ( -tocopherol) EC50 (18.60 ± 0.02) (Table 2). 90 minutes there was no significant reduction in nitrite

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Fig. 1: Nitric Oxide Radical Scavenging Activity of MELC

Radical Scavenging Activity: Fig. 2 shows the anti-radical

power exhibited by the extract against hydroxyl radical,
DPPH and superoxide. The extract showed highest ARP
agains thydroxyl radical, followed by DPPH radical and
finally superoxide [Link]-Radical Power (ARP) is the
inverse of the effective concentration (EC50) and it shows
the strength of the extracts in reducing the free radicals
(DPPH•, O2. and OH.). The higher the ARP the more
powerful the extract was in reducing the free radical with
respect to the method used.
Fig. 2: Anti Radical Power (ARP) of the MELC against the
various Free Radicals Antioxidant Vitamins and Total Phenolic Contents of
Methanol Extracts of Lantana Camara Leaves (MELC):
Table 4: Antioxidant Vitamins and Total Phenolic Contents of Methanol
Extract of Lantana Camara Leaves
Table 4 shows that vitamin A (fat soluble antioxidant
Vitamins Concentrations
vitamin) has the lowest concentration (0.50 mg/100g)
A 0.50 mg/100g compared to those of vitamins C and E with 6.5 and
E 1.64mg/100g 1.6mg/100g respectively. The total phenolic compound
C 6.585mg/100g detected was 2.36 GAE (gallic acid equivalent).
Total Phenolics 2.36±0.01 GAE

ions showing little scavenging activity of nitric oxide by DISCUSSION

both the extract and ascorbic acid standard. At 90 - 180
minutes, 250, 500 µg/ml of the extract and 500 µg/ml of Free radicals are involved in many disorders such as
ascorbic acid scavenged the nitric oxide which is neurodegenerative diseases, cancer and rheumatoid
shown as a reduction in the concentration of nitrite ions. arthritis. Antioxidants, through their scavenging power
The overall scavenging activity of the extract on nitric are useful for the management of these ailments. In the
oxide radical showed that the extract at 500 µg/ml was present study, leaves of Lantana camara, traditionally
most potent in scavenging nitric oxide radical compared used for the treatment of various disorders, were studied
to ascorbic acid standard at 500 µg/ml with respect to for their free radical scavenging activities on such radicals
other concentrations of the extract as shown in Fig. 1. as DPPH, superoxide, hydroxyl and nitric oxide radicals.
Natural antioxidants which could be secondary
Comparison of the Anti-Radical-Power (ARP) of the metabolites make plants to be beneficial medicinally [32].
Extract against DPPH., Superoxide Radical and Hydroxyl In the present study, the extract was found to be effective

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scavenger against DPPH. radical. It was observed that the beneficial effects of nitric oxide (NO•), its contribution to
more the decolourization of DPPH., the more its reducing oxidative damage is increasingly becoming evident since
ability [33, 34], suggesting that the extract is capable of NO• can react with superoxide ion to form peroxynitrite
donating a hydrogen atom to the stable free radical anion, which is a potential strong oxidant that can
(DPPH.). Quantitative DPPH radical scavenging assay decompose to produce •OH and NO2 [39]. The extract
also revealed the ability of the extract to donate hydrogen exhibited strong NO • radical scavenging activity leading
atom to the stable free radical, the activity increasing in a to the reduction of the nitrite ion concentration in the
concentration dependent manner. The EC50 value for assay medium, a possible protective effect against
DPPH scavenging activity of the extract was found to be oxidative damage. The NO• scavenging capacity was
27.56 ± 0.02µg/ml compared to a known strong antioxidant concentration dependent with 500µg/ml of the extract
(Ascorbic acid) with a value of 11.07±0.02µg/ml, thereby scavenging most efficiently as shown in Fig. 1 in the
suggesting a moderate scavenging activity of the extract. result. It has also been observed that antioxidant activity
Hydroxyl radical is a potent extremely reactive oxidising of plant extracts is not limited to phenolics. Activity may
radical that reacts with most bio-molecules in diffusion- also come from the presence of other antioxidant
controlled rates [35]. This radical has the capacity to join secondary metabolites such as volatile oils, carotenoids
nucleotides in DNA and as well cause strand breakage, and vitamins [40]. The presence of these vitamins in the
which contributes to condition such carcinogenesis, plant may suggest their possible role in curbing the
mutagenesis and cytotoxicity. The extract showed incidence of oxidative stress in humans [22]. Vitamin C is
hydroxyl radical scavenging activity with an effective considered the most important water-soluble antioxidant
concentration at fifty percent (EC50) of 22.18±0.02µg/ml in extracellular fluids [17]. It is capable of neutralizing
compared to that of the standard á-tocopherol’s EC50 of reactive oxygen species (ROS) in the aqueous phase
18.60±0.01 µg/ml thereby suggesting that the extract is a before lipid peroxidation is initiated. Vitamin E, a major
potentially high radical scavenger. The extract was found lipid-soluble antioxidant, is the most effective chain-
to inhibit the degradation of 2- deoxy-ribose generated breaking antioxidant within the cell membrane where it
from the Fe3+-dependent system. The extract could be protects membrane fatty acids from lipid peroxidation [25].
acting as a chelator of the Fe3+ ions in the system, thereby Beta carotene and other carotenoids are also believed to
preventing them from complexing with the deoxyribose, or provide antioxidant protection to lipid-rich tissues [20].
donating hydrogen atoms and accelerating the Anti-Radical Power (ARP) is the inverse of the effective
conversion of H2O2 to H2O [36]. The observed ability of concentration (EC50) and it showed the strength of the
the extract to scavenge or inhibit the•OH radical indicates extract in reducing the free radicals (OH ., DPPH • and O 2.).
that the extract could significantly inhibit lipid The higher the ARP the more powerful the extract was in
peroxidation since •OH radical is highly implicated in reducing the free radical with respect to the method used.
peroxidation reaction. The extract exhibited scavenging Lantana camara leaf extract contained vitamins A, C and
ability for superoxide anion radicals generated from the E, with Vitamin C having the highest concentration.
photochemical reduction of the nitro-blue tetrazolium The presence of these vitamins and phenolic compounds
(NBT) resulting in the formation of blue formazan could make Lantana camara leaf a good source of
solution. The plant extract inhibited the formation of antioxidant.
reduced nitro-blue tetrazolium (NBT) in a concentration-
dependent manner and had a significant O2•- anion radical CONCLUSION
scavenging ability of EC50 of 27.94 ± 0.03 µg/ml compared
to ascorbic acid with EC50 of 62.47± 0.02 µg/ml. Flavonoids Lantana camara exhibited antioxidant property
and catechins have been reported to be effective within the test systems used. Since this study used
scavengers of superoxide anion radical [37]. The presence in vitro approach, there is therefore a compelling
of flavonoids in the extract, therefore could be responsible need to check the in vivo antioxidant efficacy of the plant
for the extract’s antioxidant potency. The presence of for a better biochemical knowledge and utilization of the
tannin was reported to inhibit the generation of plant.
superoxide [38]. This further could confirm the antioxidant
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