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European Journal of Applied Sciences – Vol. 10, No. 4
Publication Date: August 25, 2022
DOI:10.14738/aivp.104.12640. Ukwade, C. E., Ebuehi, O. A. T., Adisa, R. A., Odukoya, A., Agaba, O., Adesegun, S. A., Igietseme, J. U., & Omosun, Y. (2022).
Antioxidant and Cytotoxic Activities of Fractions of Ethanol Leaf Extract of Byrsocarpus Coccineus Schum. and Thonn.
(Connaraceae). European Journal of Applied Sciences, 10(4). 413-428.
Services for Science and Education – United Kingdom
Antioxidant and Cytotoxic Activities of Fractions of Ethanol Leaf
Extract of Byrsocarpus Coccineus Schum. and Thonn.
(Connaraceae)
Ukwade, Caroline E.
Department of Biochemistry, College of Medicine
University of Lagos, PMB 12003, Lagos, Nigeria
Ebuehi, Osaretin A. T.
Department of Biochemistry, College of Medicine
University of Lagos, PMB 12003, Lagos, Nigeria
Adisa, Rahmat A.
Department of Biochemistry, College of Medicine
University of Lagos, PMB 12003, Lagos, Nigeria
Odukoya, Abdulazeez
Department of Biochemistry, College of Medicine
University of Lagos, PMB 12003, Lagos, Nigeria
Agaba, Onyeka
Department of Biochemistry, College of Medicine
University of Lagos, PMB 12003, Lagos, Nigeria
Adesegun, Sunday A.
Department of Biochemistry, College of Medicine
University of Lagos, PMB 12003, Lagos, Nigeria
Igietseme, Joseph U.
Department of Biochemistry, Microbiology & Immunology
Morehouse School of Medicine, Atlanta, GA 30310, USA
Omosun, Yusuf
Department of Biochemistry, Microbiology & Immunology
Morehouse School of Medicine, Atlanta, GA 30310, USA
ABSTRACT
Medicinal plants have historically been used traditionally for their therapeutic
potentials, they are therefore investigated with the hope of developing novel drugs.
This study is to investigate the phytochemicals, antioxidant and cytotoxic activities
of Byrsocarpus coccineus fractions. The ethanol leaf extract of B. coccineus was
chromatographed on VLC over silica gel (Mesh 70-230) using n- hexane/EtOAc/MeOH eluent system; n-hexane/EtOAc (from100/0 to 0/100), then
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European Journal of Applied Sciences (EJAS) Vol. 10, Issue 4, August-2022
Services for Science and Education – United Kingdom
ethyl acetate (EtOAc)/MeOH (from 100/0 to 0/100) to yield seven (7) fractions.(F1-
F7). The phytochemical screening and antioxidant activities of the different
fractions were carried out. All the fractions were subjected to gas chromatography
and mass spectrometry (GC-MS) analysis and cytotoxic activities of the different
fractions were investigated using brine shrimp lethality assay. The phytochemical
result showed that fractions contain tannins, flavonoids, phenols, reducing sugar,
alkaloids, steroids, saponin and terpenoid. Antioxidant analysis show that F3 had
the highest DPPH (IC50=56.88μg/ml) and NO (IC50=53.02μg/ml) free radical
scavenging activities. F7 (IC50=56.25 μg/ml) and F3 (IC50 of 56.79 μg/ml), had the
highest lipid peroxidation free radical scavenging activity. The brine shrimp
toxicity assay result showed that F3 and F4 were cytotoxic to Artemia salina with a
LD50 value of 39.01μg/ml and 53.55 μg/ml respectively. The GC-MS analysis showed
the presence in the most active fraction with antioxidant (cis-vaccenic acid,
neophytadiene), antitumor and cancer preventive (squalene) and anticancer (9-
Octadecenoic acid, 9, 12-Octadecadienoic acid, methyl ester) potentials. This study
reveals that B. Coccineus contains pharmacologically bioactive compounds with
therapeutic potentials.
Keywords: Byrsocarpus coccineus, phytochemicals, antioxidant, brime shrimp lethality
assay, GC-MS
INTRODUCTION
Medicinal plants have been used as traditional remedies for ages, they are important source of
folk medicines. About 80% of the world population, especially those in the developing
countries, rely on medicinal plants for the treatment of various ailments (Vital and Rivera,
2009). Plants are reservoirs for bioactive secondary metabolites and provide a promising line
for therapeutics, as these are safer, ecofriendly, low-cost, readily available, biodegradable and
less toxic compared with conventional treatment methods (Iqbal et al., 2017).
Byrsocarpus coccineus Schum and Thonn (family Connaraceae) locally known in Western
Nigeria as “Amujewewe” (Burkill, 1985), is an ethno-medicinal plant used in traditionally in
several parts of West Africa (Dalziel, 1937). Various preparations of the plant; leaves, roots
(scraped bark and sap) and whole plant have been used traditionally to treat diverse ailments.
Previous studies reported that the leaf extract of B. coccineus had various pharmacological
activities such as analgesic activities (Akindele and Adeyemi, 2006a), antidiarrhea activities
(Akindele and Adeyemi, 2006b), antipyretic activities (Akindele and Adeyemi, 2007a), anti- inflammatory activities (Akindele and Adeyemi, 2007b), anxiolytic/sedative activities
(Akindele and Adeyemi, 2010) and antidiabetic activity (Dada et al., 2013). It was also shown
that ethanol root extract of B. coccineus had anti-inflammatory and in vitro antioxidant activities
(Dosseh et al., 2015). The aim of this present study is to investigate the phytochemical,
antioxidant, cytotoxic activities (against brine shrimp nauplii), and to possibly identify the
chemical components of active fractions of Byrsocarpus coccineus (B.C) ethanol leaf extract
using Gas Chromatography – Mass Spectrophotometry (GC-MS).
MATERIALS AND METHODS
Plant collection and Identification
Byrsocarpus coccineus (BC) is locally known as “Amujewewe” in Western Nigeria (Burkill,
1985), was collected from uncultivated land in Ibadan, Oyo state, Nigeria. The plant sample was
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Ukwade, C. E., Ebuehi, O. A. T., Adisa, R. A., Odukoya, A., Agaba, O., Adesegun, S. A., Igietseme, J. U., & Omosun, Y. (2022). Antioxidant and Cytotoxic
Activities of Fractions of Ethanol Leaf Extract of Byrsocarpus Coccineus Schum. and Thonn. (Connaraceae). European Journal of Applied Sciences,
10(4). 413-428.
URL: http://dx.doi.org/10.14738/aivp.104.12640
authenticated by Prof J.D. Olowokudejo, at the Department of Botany, University of Lagos,
Akoka, Lagos, Nigeria, with the voucher specimens number 7491.
Extraction and separation processes
Powdered BC leaf (1.5 kg) was macerated in 95% ethanol for 48 h in the dark under constant
agitation. The extract was filtered using Whatman filter paper (No.1) and the filtrate was
evaporated to dryness using a rotary evaporator at reduced pressure to yield the ethanol crude
extract (63.8 g).
Fractionation of BC ethanol crude extract
BC ethanol crude extract (60 g) was fractionated using vacuum liquid chromatography (VLC)
over silica gel (200–300 mesh) and eluting with different solvent ratios of increasing polarity:
100% N-Hexane (F1), 70:30 n-Hexane/Ethylacetate (F2), 30:70 n-Hexane/Ethylacetate (F3),
100% Ethylacetate (F4), 70:30 Ethylacetate/Methanol (F5), 30:70 Ethylacetate/Methanol (F6)
and 100% Methanol (F7).. All the fractions were evaporated to dryness using the rotary
evaporator.
Antioxidant Activities
DPPH Radical Scavenging Assay
DPPH scavenging effects of the extract and fractions were observed according to previous
method of Shimada (Shimada et al.,1992). Briefly 0.1 mM solution of DPPH in methanol was
prepared; 1ml of the solution was added to 1 ml of extract in methanol at different
concentrations (25-100 μg/ml). The mixture was shaken vigorously and allowed to stand at
room temperature for 30 min in the dark cupboard . The absorbance was measured at 517 nm
by using a UV-Visible Spectrophotometer. Lower absorbance of the reaction mixture indicated
higher free radical scavenging activity. The percent DPPH scavenging effect was calculated
using the following equation
DPPH Scavenging effect (%) = [(A0-A1)/A0] x 100
Where A0 was the absorbance of the control and A1 was the absorbance in the presence of the
standard sample or extract. The IC50 value represented the concentration of the compounds
that caused 50% inhibition of DPPH radical formation.
Lipid Peroxidation Assay
Lipid peroxidation assay was carried out by the method of Ohkawa et al. (1979). The reaction
mixture contained 1 mL of fowl egg yolk emulsified with phosphate buffer (pH 7.4) to obtain a
final concentration of 25 g/l, sample (different concentration of 20–100 μg/mL), and 100 μL of
1000 μM FeCl2. The mixture was incubated at 37°C for 1 h before being treated with 0.5 mL of
freshly prepared 15% trichloroacetic acid (TCA) and 1.0 mL of 1% thiobarbituric acid (TBA).
The reaction tubes were further incubated in boiling water bath for 10 min. Once cooled to
room temperature, the tubes were centrifuged at 3500 rpm for 10 min to remove precipitated
protein. The absorbance at 532 nm was determined and gallic acid was used as positive control.
The percentage inhibition was calculated from the following equation.
Inhibition (%) = [(Ab-As/Ab) X 100