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ABSTRACT
The in vitro antioxidant and antimicrobial activities of methanolic leaf extract of Dialium guineense were
studied in order to provide a pharmacological basis for their ethomedicinal applications. Antioxidant activity
was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and reducing
power assay. In addition, the total phenolic content was also analyzed. Antimicrobial activity was tested
against clinical isolates of Staphylococcus aureus, Streptococcus mutans, Escherichia coli, Bacillus cereus,
Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Salmonella typhi, Candida albicans,
Microsporium gypseum, Trichophyton mentagrophytes and Trichophyton rubrum using agar well diffusion
method. The results of the DPPH scavenging activity of the extract showed a concentration dependent
antioxidant activity with maximum scavenging activity (85.35%) observed at 250 g/ml concentration and
comparable to those of ascorbic (95.75%) and gallic acids (93.67%). The reducing potential of the extract
(0.069 0.003 nm) was also comparable to that of gallic acid (0.078 0.022 nm), while the total phenolic
content was 69.45 0.002 mg/g gallic acid equivalent. The antimicrobial inhibition zone and minimum
inhibitory concentration values of the extract ranged between 10.2 to 25.9 mm and 7.81 to 62.5 g/ml
respectively, with the Gram positive bacteria generally being most sensitive, followed by the fungi and the
Gram negative bacteria. This study indicates that D. guineense leaf extract has significant antioxidant and
antimicrobial properties, and thus substantiates its popular and wide traditional applications in diverse
ailments. The plant may therefore be exploited as a potential preservative in the pharmaceutical and food
industries.
Keywords: Dialium guineense, antioxidant properties, DPPH assay, total phenolics, gallic acid.
*Corresponding author. E-mail: gideonikechuku@yahoo.com. Tel: +2347037605625.
INTRODUCTION
Antioxidants have been found to play a major role in
protecting the human body against damage induced by
reactive free radicals (Halliwell and Gutteridge, 1990;
Mates et al., 1999; Omale and Omajali, 2010) by reacting
with free radicals, chelating and also by acting as oxygen
scavenger
(Shahidi
and
Wanasundara,
1992;
Buyukokuroglu et al., 2001). Some of the established
diseases of free radicals, resulting from products of
oxidative
stress,
included
rheumatoid
arthritis,
atherosclerosis, skin-aging, nephritis, reperfusion injury,
asthma,
diabetes
mellitus,
carcinogenesis,
neurodegenerative
diseases
(Alzheimers
and
Parkinsons diseases) and AIDS (Stadtman and Oliver,
1991; Cerutti, 1994; Feig et al., 1994; Florence, 1995;
Ogu et al.
Antifungal assay
The method formerly described by Ogu et al. (2011) was adopted.
Fungal spores were harvested after 7 days old SDA slant culture
was washed with 10 ml normal saline in 2% Tween 80 with the aid
of glass beads to help in dispersing the spores. The spore
suspensions were standardized to 105 spores/ml. Sabouraud
Dextrose Agar (SDA) (Lab M, India) was prepared according to
specifications, autoclaved (121C for 15 min), supplemented with
0.05% chloramphenicol and dispensed into 11 cm diameter Petri
dishes. 1 ml of each standardized spore suspension (105 spores/ml)
was evenly spread on the surface of the gelled SDA plates. Then,
sterile cork a borer (8 mm in diameter) was used to make well at the
center of each seeded plates. Thereafter, 100 l of the
reconstituted extracts (250, 125 and 62.5 g/ml) was applied into
each labeled well. 100 l each of 20% DLMSO and standard drug
griseofulvin (100 g/ml) (Clarion Medicals Ltd, Lagos, Nigeria),
served as negative and positive control, respectively. The plates
were incubated at ambient temperature for 1 to 7 days and
observed for growth. Anti-fungal activities of the extract as well as
the controls were measured and recorded as mean diameter of
zones of inhibition around the three wells.
Statistical analysis
The experiments were carried out in triplicates. The results were
given as mean standard deviation (SD). One way analysis of
variance (ANOVA) was carried out to test for significant differences
between the means of samples and standards at P < 0.05.
RESULTS
The determination of the free radical scavenging activity of each of
the crude extract was carried using the DPPH (2,2-diphenyl-1picrylhydrazyl) assay as described by Mensor et al. (2001) with
slight modifications. Various concentrations of 250, 125, 50, 25 and
10 g/ml of the extract in methanol were prepared. 1.0 ml of a 0.3
mM DPPH in methanol was added to 2.5 ml solution of the extract
or standard, and allowed to stand at room temperature in a dark
chamber for 30 min. The change in colour from deep violet to light
yellow was then measured at 518 nm on a spectrophotometer
(Jenway, 6025). The decrease in absorbance was then converted
to percentage Scavenging activity (%) using the formula:
100
Abs control
Blank = Methanol (1.0 ml) plus sample solution (2.0 ml), Negative
control = DPPH solution (1.0 ml, 0.25 mM) plus methanol (2.0 ml),
ascorbic acid and gallic acid were used as standards.
Figure 1. Inhibition of DPPH radical by methanolic leaf extract of Dialium guineese. *(P < 0.05) no significant
difference with control.
0.1
0.078
Absorbance
0.08
0.069
0.06
0.04
0.02
0
D. guineense
Gallic acid
Absorbance at 720 nm
2.5
y = 0.004x + 0.014
R = 0.996
2
1.5
1
0.5
0
0
200
400
Concentration (g/ml)
600
800
Ogu et al.
Table 1. Antimicrobial activity of methanolic leaf extract of D. guineense, Ciprofloxacin (Cip) and Griseofulvin (Gri).
D. guineense extract
S. aureus
S. mutans
E. coli
B. cereus
P. aeruginosa
K. pneumonia
P. mirabilis
S. typhi
C. albicans
M. gypseum
T. mentagrophytes
T. rubrum
62.5 g/ml
16.2 0.6
17.8 1.2
13.9 0.2*
16.9 2.2*
11.0 0.2
10.2 0.2
10.0 0.9*
12.9 1.2
15.0 0.2
13.2 0.9
14.9 0.2
11.0 1.7
ND = Not Determined, * Values significant (P < 0.05) compared with reference drugs.
DISCUSSION
There is a growing interest in the pharmacological
evaluation of various plants used in traditional system of
medicine globally. In the present study, the methanolic
leaf extract D. guineense exhibited comparable DPPH
free radical scavenging ability in a dose-dependent
manner. Similar findings were reported by Aliyu et al.
(2009) and Ibeh et al. (2013) in their studies on the
antioxidant activity of leaf extracts of Bauhinia rufescens
and Axonopus compressus, respectively. DPPH radical
scavenging activity assay assesses the capacity of the
extract to donate hydrogen or to scavenge free radicals.
DPPH radical is a stable free radical and on reacting with
an antioxidant compound which can donate hydrogen
becomes reduced to diphenylpicrylhydrazyl (DPPH). The
observed antioxidant of the extracts may be due to the
neutralization of free radicals (DPPH), either by transfer
of hydrogen atom or by transfer of an electron (Jao and
Ko, 2002; Naik et al., 2003). The scavenging ability of the
extracts may be a reflection of the total activities of
various components present in the plant (Omoregie and
Osagie, 2012). Earlier researchers have reported that the
antioxidant activity of most plants with therapeutic
Ogu et al.
Olukemi OA, Olukemi IO, Sofidiya MO, Aniunoh OA, Lawal BM, Tade
IO, 2005. Antioxidants activity of Nigerian dietary spices. Elect J
Environ Agric Food Chem, 496:1086-1093.
Omale J, Omajali JB, 2010. Evaluation of bio-safety and antioxidant
activity of the fruit and leaf of Saba florida (Benth.) from Ibaji forest.
Int J Med Med Sci, 2(3):100-105.
Omoregie ES, Osagie AU, 2012. Antioxidant properties of methanolic
extracts of some Nigerian plants on nutritionally-stressed rats. Nig J
Basic Appl Sci, 20(1):7-20.
Omotayo FO, 1999. Plant of South-Western Nigeria. University of
Ibadan, Nigeria, pp.139.
th
Pelczar MJ, Chan ECS, Kries NR, 1993. Microbiology, 5 Edition, MacGraw Hill Inc. pp. 221-224.
Rath CC, Dash SK, Mishra RK, 2002. Antimicrobial efficacy of six Indian
essential oils individually and in combinations. J Essential Oil Bearing
Plants, 5:99-107.
Rached W, Benamar H, Bennaceur M, Marouf A, 2010. Screening of
the antioxidant potential of some Algerian indigenous plants. J Biol
Sci, 10:316-324.
Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridh JB, 1995.
The relative
antioxidant activities of plant-derived polyphenolics
flavonoids. Free Radic Res, 22:375-383.
Stadtman ER, Oliver CN, 1991. Metal ion-catalyzed oxidation of
proteins, physiological consequences. J Biol Chem, 266:2005-2008.
Scalbert A, 1991. Antimicrobial properties of tannins. Phytochemistry,
30:3875-3883.
Shahidi F, Wanasundara PK, 1992. Phenolic antioxidants. Crit Rev
Food Sci Nutr, 32(1):67-103.
Stanly C, Bhatt A, Ali HMD, Keng CL, Lim BP, 2011. Evaluation of free
radical scavenging activity and total phenolic content in the petiolederived callus cultures of Zingiber zerumbet Smith. J Med Plant Res,
5(11):2210-2217.