American Journal of Plant Sciences, 2013, 4, 1709-1712 Published Online September 2013 (
Evaluation of Antioxidant and Cy totoxic Capacity of
Croton bonplandianum. Baill
Muhammad Naeem Qaisar*, Bashir Ahmad Chaudary, Muhammad Uzair, Sajid Nawaz Hussain
Faculty of Pharmacy, Bahauddun Zakariya University, Multan, Pakistan.
Email: *
Received June 3rd, 2013; revised July 3rd, 2013; accepted July 31st, 2013
Copyright © 2013 Muhammad Naeem Qaisar et al. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
The antioxidant and cytotoxic activities of Methanolic and Dichloromethane extracts of Croton bonplandianum Baill
were evaluated in a bid to provide better scientific basis for the isolation of bioactive compounds from the plant. The
DPPH and hydroxyl radical scavenging activities o f the Methanolic extract were 59.62%, and Dichloromethane extract
was not toxic. Gallic acid used as reference compounds sho wed higher antioxidant activities to the plant ex tracts. Both
methanolic and dichloromethane extracts were evaluated for their brine shrimp lethality. Methanolic extract showed
cytotoxic activity in vitro with a LD50 value of 115.76 (0.0048 - 13.76) µg/ml. Etoposide was used as standard drug. Put
together, these results confirm that Croton bonplandianum extracts possess appreciable natural antioxidant and cyto-
toxic potentials, thereby providing good justification for the isolation of pure bioactive compounds.
Keywords: Antioxidant Activity; Scavenging Activity; Cytotoxic Activity; Brine Shrimp; Croton bonplandianum
1. Introduction
Plants are serving human needs in the forms of foods and
fruits. Plants also provide the important components of
medicines, cosmetics and beverages. The most recent
study by the World Health Organization (WHO) approxi-
mated that for the primary health care needs, four fifths
of the total population still put confidence on the plant
medicine [1], and most of these plant extracts or their
bioactive comp onents. Still tod ay, a huge public is taking
interest to use the herbal remedies.
Croton bonplan dianum Baill (syn. Croton sparsiflorus
Morong ) is a monoecious wood y shrub, which is 1 - 5 m
in height, but more usually c. 30 - 40 cm, with whorled
branches. The plant grows in sandy clay soil along road-
side, irrigation canal banks, in plantations and on waste
ground [2]. It has been reported that this plant grows in S.
Balivia, Paraguay, S. W. Brazil, N. Argentina, Bangla-
desh, South America, South India and Pakistan [3,4]. In
Pakistan, this plant is found near Khyber, Attock, Wah,
Rawalpindi, Sargodha, Gujarat, Sialkot, Lahore and Ka-
Among the medicinal benefits of plants, antioxidant
properties have received increasing attention due to their
role in preventing or down-regulating myriads of oxida-
tive damages caused by free radicals in the body [5].
Oxidative stress is initiated by free radicals, which seek
stability through electron pairing with biological macro-
molecules such as proteins, lipids and DNA in healthy
human cells and cause protein and DNA damage along
with lipid peroxid ation. These changes contribute to can-
cer, atherosclerosis, cardiovascular diseases, ageing and
inflammatory diseases [6,7].
The antioxidant capacities of Croton bonplandianum
Baill consumed locally in Pakistan have not been clearly
presented. Such information, if provided, will not only
possibly introduce the plants as cost effective and acces-
sible sources of natural antioxidants but also justify the
need for renewed domestication effor ts on them. Against
this back-drop, th is study is aimed at evaluating the anti-
oxidant activities of Croton bonplandianum Baill in se-
lected in vitro assay systems.
Apart from already documented traditional uses, tradi-
tional healers claim to be using the leave extracts for
treating cancer, although it was difficult to establish the
type of cancer being treated. This indicated that the plant
might contain potentially useful anticancer compounds.
However, up to now there is little phyto chemical or phar-
macological work done on this. Therefore in this study
*Corresponding a uthor.
Copyright © 2013 SciRes. AJPS
Evaluation of Antioxidant and Cytotoxic Capacity of Croton bonplandianum. Baill
we present results of the brine sh rimp lethality test (BST)
of Methanolic and Dichloromethane extracts of Croton
2. Materials and Methods
2.1. Collection and Extraction of Plant Material
The plant material was collected from surroundings of
Sargodha district. The plant was identified as Croton
bonplandianum by Professor Dr. Altaf Hussain Dasti,
Institute of pure and applied Biology, Bahauddin Zaka-
riya University, Multan. For the purpose of effective ex-
traction, whole plant material was shade dried for 15
days. Then dried plant material was ground in blender
and weighed. The extraction of this finely ground mate-
rial was affected by simple maceration. The weighed
amount of plant material was taken in extraction bottle
and measured volume of dichloromethane was added to
it. Ultrasonication of this mixture was carried out occa-
sionally in order to achieve maximum possible extraction.
Filtration was carried out after 24 hours of addition of
solvent. The process was repeated three times with di-
chloromethane. The extraction of the marc was done by
methanol in the same manner. The Dichloromethane and
methanol extracts were concentrated separately under re-
duced pressure by using rotary evaporator. The dichloro-
methane (20.2 g) and methanol (48.9 g) extracts were
collected in separate sample bottles and designated with
codes as CBD and CBM respectively.
2.2. Chemicals
DPPH (1,1-diphenyl-2-picryl hydrazyl) radical and Rutin
were purchased from Sigma Aldrich Chemical Company,
USA; Folin and Ciocalteau’s Phenol reagent and Tri-
chloroacetic acid (TCA) from Qualikems Fine Chemical
Pvt. Ltd., New Delhi, India; Gallic acid monohydrate
from Kem Light Laboratories Pvt. Ltd., Mumbai, India.
Solvents and other chemicals used for this study were of
analytical grade, while water was glass distilled.
2.3. DPPH (1,1-Diphenyl-2-Picryl-Hydrazyl)
Radical Scavenging Assay
Antioxidant activity of the extracts of Croton bonplan-
dianum were measured in this assay as ability to scav-
enge stable DPPH radicals according to [8]. A con centr a-
tions (0.5 µg/ml) of the test extracts were prepared in
methanol. To 2.5 ml solution of each extract concentra-
tion was added 1 ml of 0.3 mM of freshly prepared
DPPH solution in methanol and allowed to react in the
dark at room temperature for 30 min. Absorbance of the
resulting solution was measured at 518 nm. Methanol (1
ml) added to 2.5 ml of each extract concentration was
used as blank, while 1 ml of 0.3 mM DPPH solution
added to 2.5 ml of methanol served as a negative control.
Gallic acid, prepared in the same concentrations as the
test extracts, were used as reference standards (positive
controls) for comparison.
Percentage DPPH scavenging activities of the extracts
and reference standards were determined using the formula.
% scavenging activity 100AAA100
 
where As = Absorbance of sample (extract or reference
standard), Ab = Absorbance of blank and Ac = Absorb-
ance of negative control [8].
2.4. Brine Shrimp Lethality Test (BST)
The brine shrimp lethality test (BST) was used to predict
the presence, in the extracts, of cytotoxic activity [9].
Both the crude extracts were tested for brine shrimp le-
thality. Solutions of the extract were made in DMSO and
incubated in duplicate vials with the brine shrimp larvae.
Ten brine shrimp larvae were placed in each of the du-
plicate vials. Control brine shrimp larvae were placed in
a third vial which contained sea water and DMSO only.
After 24 h the nauplii were examined against a lighted
background, and the average number of survived larvae
in each triplicate was determined. The mean percentage
mortality was plotted against the logarithm of concentra-
tions and the concentration killing fifty percent of the
larvae (LC50) was determined from the graph by taking
the antilogarithm of the concentration corresponding to
50% mortality rate of the test organisms. Etoposide was
used as a standard test drug.
3. Results
3.1. DPPH Radical Scavenging Assay
In this assay, the ability of the investigated extracts to act
as donors of hydrogen atoms or electrons in transforma-
tion of DPPH radical into its reduced form was investi-
gated. The results indicated that methanolic extract
(CBM) had IC50 396.205 and % radical scaven ging activ-
ity (%RSA) of 59.62% and while the dicholoromethane
extract (CBD) is inactive (Table 1). The scavenging ac-
tivities of the extracts were, however, lower than those
tested of gallic acid (93.13%).
Table 1. Antioxident activity of extracts.
Extract code Conc. mg/ml IC50 ± SEM.
µg/ml % RSA
CBM 0.5 396.205 ± 4.6 59.629
CBD 0.5 inactive 39.37
STD Gallic acid 0.094 4.3 ± 0.43 93.13
Copyright © 2013 SciRes. AJPS
Evaluation of Antioxidant and Cytotoxic Capacity of Croton bonplandianum. Baill 1711
3.2. Brine Shrimp Lethality Test
In the brine shrimp lethality test methanolic extracts was
toxic with an with a LD50 value of 115.76 (0.0048 - 13.76)
µg/ml. Dichloromethane extract was not toxic (Table 2).
4. Discussion
Plants have been mentioned as one of the most important
targets to search for natural antioxidants from the point
of view of safety [10,11]. The activities of antioxidants
have been attributed to various mechanisms including
prevention of chain initiation, decomposition of perox-
ides, radical scavenging and reducing capacity [12]. Con-
sequently, these activities vary with assay methods and a
single assay may be inadequate. It is for this reason that
antioxidant activities of the extracts in this study were
evaluated Phenols and flavonoids represent two phyto-
chemicals whose relative abundance in plant extracts has
been profusely linked to antioxidant activities. Phenols
and flavonoids in extracts may explain their high anti-
oxidant activities [12]. DPPH radical scavenging assay
provides an easy, rapid, and convenient method to evalu-
ate antioxidants and radical scavengers [13]. It is based
on the ability of 1,1-diphenyl-2-picryl-hydrazyl (DPPH),
a stable free radical, to decolorize in the presence of an-
tioxidants. The DPPH radical contains an odd electron,
which is responsible for the absorbance at 515 nm and
also for the visible deep purple colour. When DPPH ac-
cepts an electron donated by an antioxidant compound,
the DPPH is decolorized, and this effect can be quantita-
tively measured from the changes in absorbance [14].
The extracts tested in this study showed DPPH radical
scavenging activities.
Bioactive compounds are often toxic to shrimp larvae.
Hence, in vivo lethality to shrimp larvae can be used as a
rapid and simple preliminary monitor for bioactive com-
pounds during the isolation of natural products. The eggs
of the brine shrimp Artemia salina (Leach) are readily
available as fish food in pet shops. When placed in artifi-
cial sea water, the eggs hatch within 48 hours, providing
large numbers of larvae. These tiny shrimp larvae have
Table 2. Brine shrimp lethality test of extracts.
code Dose
µg/ml No. of
shrimp No. of
survivors LD 50
µg/ml STD.
Drug LD 50
1000 30 04
100 30 20
10 30 23
115.76 Etoposide 7.4625
1000 30 16
100 30 19 CBD
10 30 24
1327.85 Etoposide 7.4625
been extensively used as a tool to monitor the cytotoxic-
ity of samples under study. This is a rapid, inexpensive,
in-house, general bioassay which has been developed for
screening, fractionation and monitoring of physiologi-
cally active natural products[9].The methanolic extract
was toxic with LD50 value of 115.76 (0.0048 - 13.76)
µg/ml against Artemia salina when tested in vitro, indi-
cated a possibility that the extract may contain a toxic
5. Conclusion
The results of the present study indicate that the extracts
showed significantly different but appreciably potent
antioxidant and cytotoxic activity that cannot be neglec-
ted. These results support the traditional healers claim,
but it could also mean that methanolic extract is poten-
tially toxic, since brine shrimp lethality activity can also
be used as an indicator for toxicity. There is need to test
this compound on cancer cell lines and other tests in or-
der to establish its safety and the possibility of develop-
ing an anticancer agent. The study therefore not only
reveals these as accessible reservoirs of natural antioxi-
dants and cytotoxic compounds, but very importantly,
provides good scientific justification for the isolation of
pure bioactive compounds.
6. Acknowledgements
The authors are grateful to the Pharmacy Department,
Bahauddin Zakariya University Multan, Pakistan. Inter-
national Center of Chemical And Biological Sciences,
University of Karachi is also acknowledged for antioxi-
dant activity and brine shrimp lethality tests.
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