Antioxidant and Antibacterial Properties of Lecaniodiscus cupanioides
Lecaniodiscus cupanioides Planks. ExBth (Sapindaceae) is widely used in Nigerian folk medicine for the treatment of inflammatory conditions, hepatomegaly and bacterial infections. This study investigated the antioxidant and antibacterial activity of the methanolic extract of the leaves to justify its use in traditional medicine. Extract exhibited strong DPPH and ABTS radical scavenging activity greater than BHT and comparable to ascorbic acid. 0.1 mg mL-1 extract inhibited DPPH and ABTS radicals up to 99.4 and 98.5%, respectively. Multiple antioxidant activity of extract was evident with moderate reducing power. TAE (37.678±1.66 mg g1 dry extract) was higher than that reported in many other plant extracts. Flavonoid and proanthocyanidin contents were 4.142±0.06 and 2.548±0.32 mg g-1, respectively. Strong correlation recorded; ABTS/TAE (R2 = 0.89), DPPH/TAE (R2 = 0.90). Antimicrobial activity was highest on gram +ve organisms B. cereus, S. aureus, M. kristine and S. pyrogens (MIC value < 1.0 mg mL-1). Gram-ve S. pooni and P. aeruginosa (MIC value = 2.0 mg mL-1). Results attributed the antioxidant potential of L. cupanioides leaf extract to its strong proton donating ability and justified its use for the treatment of bacterial infections in ethnomedicine.
to cite this article:
M.O. Sofidiya, F.O. Jimoh, A.A. Aliero, A.J. Afolayan, O.A. Odukoya and O.B. Familoni, 2008. Antioxidant and Antibacterial Properties of Lecaniodiscus cupanioides. Research Journal of Microbiology, 3: 91-98.
Oxygen centered free radicals and other reactive oxygen species are byproducts
of numerous physiological and biochemical processes in the human body. Overproduction
of such free radicals causes oxidative damage to biomolecules such as lipids,
proteins and DNA, eventually leading to many chronic diseases, such as atherosclerosis,
cancer, diabetes, aging and other degenerative diseases in humans (Halliwell,
1994). Oxygen free radicals contribute to tissue damage in the events following
skin injury and are known to impair healing process. It is consented that Reactive
Oxygen Species (ROS) are deleterious to wound healing process due to the harmful
effects on cells and tissues (Singh et al., 2006). Increase production
of reactive oxygen species, lipid peroxidation and ineffective scavenging play
a crucial role in various skin lesions (Niwa et al., 1986). Wound healing
is the process of repair that follows injury to the skin and other soft tissues.
It is fundamentally a connective tissue response. Initial stages of wound healing
involve an acute inflammatory phase followed by synthesis of collagen and other
extracellular matrix which are later remodeled to form scar. Molecular oxygen
plays a central role in the pathogenesis and therapy of chronic wounds. Overproduction
of Reactive Oxygen Species (ROS) results in oxidative stress thereby causing
cytotoxicity and delayed wound healing. Therefore, elimination of ROS could
be an important strategy in healing of chronic wounds (Dissemond et al.,
2002). Antioxidants are known to counter the excess proteases and ROS, which
are formed by neutrophil accumulation in the wound area and protect protease
inhibitors from oxidative damage. Fibroblasts and other cells may be killed
by excess ROS, thus making skin lipids less flexible; however, antioxidants
are known to reduce the possibility of occurrence of these adverse events (Houghton
et al., 2005). The search for natural remedies has drawn attention to
herbals. Proanthocyanidins and other tannins facilitate wound healing. A combination
of grape seed proanthocyanidin extract and resveratrol facilitates inducible
VEGF expression, a key element supporting wound angiogenesis. Strategies to
manipulate the redox environment in the wound are likely to be of outstanding
significance in wound healing (Sen et al., 2002).
In recent time, many studies have focused on medicinal plants in search for antioxidant principles. Many plants used in herbal preparation have been found to possess sufficient antioxidant compounds that can be used in the battle against cellular damage and disease.
Lecaniodiscus cupanioides Planch. ExBth (Sapindaceae) is a medicinal plant used in Western Nigeria for the treatment of wounds, boils, burns and bruises, tooth ache, fever and abdominal swelling caused by liver abscess (Gill, 1992; Iwu, 1993). Aqueous root extract of the plant has been reported to prevent strychnine-induced seizure, prolonged the latency and reduced severity of PTZ and picrotoxin induced seizure (Yemitan and Adeyemi, 2005). Pharmacological studies have also shown that the plant possessed analgesic and hepatotoxic properties (Yemitan and Adeyemi, 2004; Yemitan et al., 2005). Despite the various reports on the use of the extracts from this species for the treatment of various ailments, information on the antioxidant and antibacterial activities of its leaf extract is hitherto unavailable. Such type of information is vital in the understanding of its medicinal potential. The aim of this work was to evaluate the antioxidant and antibacterial activities of L. cupanioides leaf extract for its relevance since antioxidants hasten the process of wound healing by destroying the free radicals.
MATERIALS AND METHODS
The leaves of L. cupanioides were collected in January 2006 in Oyo
State, Nigeria. The plant was authenticated at the Forestry Research Institute
of Nigeria (FRIN), Ibadan courtesy Mr. Wale Ekundayo. Voucher specimens were
prepared and deposited as (FHI 107345) at the herbaria of both the Institute
and at the Pharmacognosy Department, University of Lagos.
Preparation of Crude Extracts
The plant material was air dried at room temperature for 21 days, pulverized
into powdered and a portion (100 g) of the material was extracted in methanol
at room temperature for 24 h. Extract was filtered through Whatman No.1 filter
paper and the filtrate evaporated into dryness at 40°C using rotary evaporator.
The chemicals used in this study included 2, 2-Diphenyl-2-picrylhydrazyl
(DPPH); potassium ferricyanide, catechin, butylated hydroxytoluene (BHT), Ascorbic
acid and FeCl3 and were purchased from Sigma Chemicals Co. (St Louis,
MO, USA). Vanillin was from BDH; Folin-Ciocalteuss phenol reagent and
sodium carbonate were from Merck Chemical Supplies (Darmstadt, Germany). All
other chemicals used were of analytical grade.
Determination of the Reducing Power of the Extract
The reducing capacity of the extract was determined by the method of Oyaizu
(1986). Varying concentrations of the extract in methanol (0.02-0.1 mg mL-1)
were mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide
(2.5 mL, 1%) and incubated at 50°C for 20 min. Aliquots (2.5 mL) of 10%
trichloroacetic acid were added to the reaction mixture, which was then centrifuged
at 1000 rpm for 10 min. The upper layer of the solution (2.5 mL) was mixed with
distilled water (2.5 mL) and ferric chloride (0.5 mL, 0.1%). The absorbance
was measured at 700 nm. The higher the absorbance value the stronger the reducing
Determination of DPPH Radical Scavenging Capacity
The effect of the extract on DPPH radical was estimated adopting the method
of Liyana-Pathirana and Shahidi (2005). A solution of 0.135 mM DPPH in methanol
was prepared and 1.0 mL of this solution was mixed with 1.0 mL of extract in
methanol containing 0.02-0.1 mg of extract. The reaction mixture was vortexed
thoroughly and left in the dark at room temperature for 30 min. The absorbance
of the mixture was measured spectrophotometrically at 517 nm. Ascorbic acid
and BHT were used as standards. The ability to scavenge DPPH radical was calculated
by the following Eq.:
DPPH radical scavenging activity (%) = [(Abscontrol -
Absorbance of DPPH radical + methanol
Absorbance of DPPH radical + sample extract/standard
ABTS Radical Cation Scavenging Activity
The free radical scavenging activity was determined by ABTS radical cation
decolorization assay (Re et al., 1999). It involved the generation of
ABTS+ chromophore by the oxidation of ABTS with potassium persulfate.
ABTS.+ radical cation was generated by reacting 7 mM ABTS and 2.45
mM potassium persulfate after incubation at room temperature in the dark for
16 h. The solution was then diluted by mixing 1 mL ABTS.+ solution
with 60 mL methanol to obtain an absorbance of 0.7074±0.001 at 734 nm
using the spectrophotometer. The reaction mixture (1 mL of extract and 1 mL
of ABTS.+) was allowed to stand at room temperature for 6 min and
the absorbance was immediately recorded at 734 nm. BHT standard solution was
prepared and assayed at the same conditions.
Determination of Total Phenolic Content
Total phenol contents in the extract were determined by the modified Folin-Ciocalteu
method of Wolfe et al. (2003). An aliquot of the extract was mixed with
5 mL Folin-Ciocalteu reagent (previously diluted with water 1:10 v/v) and 4
mL (75 g L -1) of sodium carbonate. The tubes were vortexed for 15
sec and allowed to stand for 30 min at 40°C for colour development. Absorbance
was measured at 765 nm using the Hewlett Packard UV-VS spectrophotometer. The
amount of total phenolics was calculated as tannic acid equivalents (TAE mg)
from the calibration curve. The experiment was replicated thrice.
Determination of Total Flavonoid
Total flavonoids were estimated using the method of Ordonez et al.
(2006). To 0.5 mL of sample, 0.5 mL of 2% AlCl3 ethanol solution
was added. After 1 h at room temperature, the absorbance was measured at 420
nm. Total flavonoid contents were calculated as quercetin equivalent from a
Determination of Proanthocyanidin
Proanthocyanidins were estimated by adopting the procedure of Sun et
al. (1998). 0.5 mL of 50 mg mL -1 of extract was mixed in 3 mL
of 4% vanillin-methanol solution and 1.5 mL hydrochloric acid and the mixture
was allowed to stand for 15 min. The absorbance was measured at 500 nm and the
result expressed as catechin equivalent.
Laboratory isolates of 10 bacteria species, which included five Gram positive
and five Gram negative strains were obtained from the Department of Microbiology,
Rhodes University. They were; Bacillus cereus, Staphylococcus epidermidus,
Staphylococcus aureus, Micrococcus kristinae, Streptococcus
pyrogens, Escherichia coli, Salmonella pooni, Serratia
marcescens, Pseudomonas aeruginosa and Klebsiella pneumonae. They
were maintained on nutrient agar slants and recovered by culturing in nutrient
broth (Biolab No. 2) for 24 h. Before use, each bacterial culture was diluted
1:100 with fresh sterile nutrient broth (Grierson and Afolayan, 1999). The bacteria
were streaked in radial patterns on the agar plates and incubated at 37°C
and examined after 24 and 48 h. The extract was tested at 2.0, 1.0, 0.5 and
0.1 mg mL -1 and complete suppression of growth by a specific concentration
of the extract was required to be declared active. The Minimum Inhibitory Concentration
(MIC) values were recorded, where no bacterial growth was observed. Blank plates
containing only nutrient agar and another set containing nutrient and 2% methanol
served as controls (Afolayan and Meyer, 1997).
Data were subjected to a one-way analysis of variance as applicable and
the significance of the difference between the means was determined by Duncans
Multiple Range Test (p<0.05).
RESULTS AND DISCUSSION
The antioxidant potential of the extract was assessed using standard in vitro antioxidant methods. The reducing power of L. cupanioides extract and the reference compounds increased with increasing concentration (Fig. 1). No significant difference in reducing activities was detected between the extract and BHT. However, reducing activity of catechin was significantly higher than that of the extract and BHT. This indicated that the antioxidant compounds are electron donors and could reduce the oxidized intermediates of lipid peroxidation processes, thus acting as primary and secondary antioxidants (Yen and Chen, 1995; Ordonez et al., 2006).
The proton-radical scavenging action has been known as an important mechanism
of antioxidation (Yun-Chin et al., 2005). The dose-response curves of
DPPH radical scavenging activity of methanol extract of L. cupanioides
compared with BHT and ascorbic acid are presented in Fig. 2.
The activity increased with increasing concentration of the extract up to 0.04
mg mL -1 and leveled off with further increase in concentration.
A similar trend was observed for ascorbic acid, though the activity of the extract
was higher than that of ascorbic acid. In all the concentrations tested, the
extract was found to be more active than BHT. A similar result on the antioxidant
potential of Ecklonia cava was reported (Senevirathne et al.,
2006). These results imply that, radical scavenging activity of the extract
may be attributed to its strong proton donating ability.
Reducing power of methanol extract from L. cupanioides.
||Free radical scavenging capacity of methanol extract from
L. cupanioides determined by DPPH method
|| ABTS.+ scavenging effects of methanol extract
from L. cupanioides
The ABTS scavenging activity of the extract was high (96%) at 0.02 mg mL -1, which was the lowest concentration, tested. The percentage inhibition of BHT at this concentration was 75.3%, which is comparatively lower than that of the extract. The activity increased with increasing concentration and was dose dependent (Fig. 3). High concentrations of the extract have been reported to be more effective in quenching free radical in the system (Ligangli et al., 2002). It is interesting to note that, the activity of L. cupanioides extract was higher than that of BHT.
Linear regression analysis of calibration plot gave a correlation coefficient
(R2) of 0.9277 for catechin, 0.9497 for tannic acid and 0.9812 for
quercetin used as standards in the determination of proanthocyanidin, total
phenol and flavonoid, respectively. The total phenolic content of the extract
was 37.678±1.66 mg g -1 TAE -1 of dry material,
which is higher than that reported in many other plant extracts, based on dry
extract (Muchuweti et al., 2006). The total phenolic contents of the
extract showed positive correlation with antioxidant activity. The correlation
coefficient between ABTS, DPPH assays and total phenol are 0.89 and 0.90, respectively.
This observation is similar to the reports of several authors who worked on
different plants (Sofidiya et al., 2006; Odukoya et al., 2005;
Velioglu et al., 1998). The total flavonoid and proanthocyanidin content
of the extract were 4.142±0.06 and 2.548±0.32, respectively and
they may contribute in a significant way to the radical scavenging activities
and reducing powers of the extract. Plant phenolics constitute one of the major
groups of compounds acting as primary antioxidants or free radical terminators
(Miliaus-Kas et al., 2004).
|| Antibacterial activity of methanol extract from the leaves
of L. cupanioides at 2 mg mL-1
|a: Minimum inhibitory concentration, b:
Not active at concentration = 2 mg mL-1
Synergism of polyphenolic compounds in an extract may contribute to the overall
antioxidant activity (Shahidi et al., 1994). The high activity of the
extract therefore, could be attributed to the presence of phenolic compounds
in the extract.
The antibacterial activity of methanol extract from the leaves of L. cupanoides showed growth inhibition against 6out of the 10 bacteria strains tested (Table 1). The strongest activity was found on Bacillus cereus, Staphylococcus aureus, Micrococcus kristinae and Streptococcus pyrogens with an MIC of 1.0 and 2.0 mg mL -1 for Salmonella pooni and Pseudomonas aeruginosa. The growth of Staphylococcus epidermidus, Escherichia coli, Serratia marcescens and Klebsiella pneumonae was not inhibited by the extract at the highest tested concentration. The activity of the extract was found to be higher on Gram-positive bacteria than the Gram-negative strains. The observed lower degree of activity against the Gram negative bacteria was not surprising as, in general, these bacteria are more resistant to plant extracts than the Gram positive ones (Rabe and Van Staden, 1997; Grierson and Afolayan, 1999). The antibiotic resistant patterns of Salmonella isolates have been reported (Gashe and Mpuchane, 2000; Urio et al., 2001). The susceptibility of Salmonella pooni to the methanol extract of this species is an indication of the potential of the extract as a drug that can be used against this organism. Although crude plant extracts have been reported to exhibit lower antimicrobial activity than pure substances (Navarro et al., 1996; Ebi and Ofoefule, 1997; Ibrahim et al., 1997), the activity exhibited by the extract in this study is noteworthy and is comparable to that of streptomycin and chloramphenicol standards with MIC value of 1.0 mg mL -1 on Staphylococcus aureus and Streptococcus pyrogens (Table 1). The bacterial strains used in this study, which showed susceptibility to the extract, have been implicated in some of the diseases against which this plant species is used in herbal remedy.
The significance of polyphenolic compounds of plant extracts has been recognized and is well known to exhibit a number of biological activities. They are reported to have multiple biological activities including vasodilatatory, anti-inflammatory, anticancerogenic, antiviral and antibacterial effects (Dorman et al., 2004). The presence of these compounds in L. cupanioides could be attributed to its wound healing and other pharmacological activities associated with free radicals. Although, there could be several mechanisms of action of the extract for its effectiveness in free radical implicated diseases, the antioxidant and free radical scavenging properties of this plant seem to be highly significant. The result of this study has revealed the antioxidant potential of L. cupanioides and has justified its use for the treatment of infectious diseases caused by bacteria.
The authors are grateful to the NRF, South Africa and the University of Lagos, Nigeria for support.
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