Abstract: Methanolic extract of Commiphora kerstingii Engl. stem bark was screened for antioxidant activity using DPPH (1,1-diphenyl-2-picryl hydrazyl) radical scavenging assay to obtain an IC50 value of 26.27±0.24 μg cm-3 compared to ascorbic acid with an IC50 value of 33.59±0.21 μg cm-3 used as control. Antibacterial activity against Bacillus subtilis and Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa and Pseudomonas fluorescens were tested. The result showed that Staphylococcus aureus was the most inhibited by the methanolic extract with diameter of inhibition zone of 30 mm while Pseudomonas fluorescens was the least inhibited with diameter of inhibition zone of 16 mm. Phytochemical investigation showed the presence of alkaloids, saponins, anthraquiniones, cardiac glycosides, tannins and flavonoids in the methanolic extract of Commiphora kerstingii stem bark.
INTRODUCTION
Oxidative stress is a problem in human beings, since it not only make our body cells age but cause diseases such as cancer that are difficult to treat. One of the ways to make the body healthier is to stop the ageing process. This can be done using certain chemicals and metals to prevent oxidation or mop up free radicals from the body known as antioxidant (Andlauer and Fürst, 1998). The human body produces endogenous antioxidants such as reduced glutathione (GSH), superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx), which are very important for counteracting or preventing oxidative stress (Sen, 1995), but the amount produced by the human body is insufficient. Therefore, supplementation had to be adopted in order to get enough antioxidants using natural exogenous antioxidants, such as vitamin C, vitamin E, flavone, beta-carotene, natural products in plants and so on (Diplok and Charleux, 1998; Rice-Evans et al., 1997). Exogenous antioxidants found in plants are safer compared to synthetic antioxidants (Grice, 1986; Sokmen et al., 2004) and they are considered to be useful agents for prevention of cardiovascular diseases (Duthie and Brown, 1994) and several kinds of cancer (Milner, 1994).
Due to the increasing interest in using plants as sources of safer antioxidants, the tropical plant Commiphora kerstingii was investigated. The plant is a tree about 9 m high distributed along the arid regions of Africa and it is often planted. Its stem bark is used traditionally in Northern Nigeria to treat fever, cancer, measles, asthma, rheumatism and venereal diseases (Mann et al., 2003). Earlier studies on Commiphora kerstingii showed that the plant contains classes of natural products like saponins, tannins and volatile oils. In addition, the plant was active against three bacteria namely Bacillus subtilis, Candida albican and Escherichia coli (Kubmarawa et al., 2007). The stem bark and leaves exudes resins (Mann et al., 2003).
The present study was carried out to investigate the antioxidant activity, phytochemical and antibacterial activity screening of Commiphora kerstingii in order to validate its claimed traditional medicine uses.
MATERIALS AND METHODS
General
Absorbance data were measured with a UV spectrometer Spectronic 20D+
instrument.
Extraction of Plant Materials
Stem bark of Commiphora kerstingii was collected at Ahmadu Bello
University, Zaria in July, 2007. The specimen was authenticated by Mallam
Gallah, Department of Biological Sciences, Faculty of Science, Ahmadu
Bello University, Zaria, Nigeria, through comparison with a voucher specimen
deposited at the herbarium unit of the department.
Twenty five grams of powdered air-dried stem bark of Commiphora kerstingii was extracted with 250 cm3 of methanol in a covered container left to stand for 4 days. The methanol extract was concentrated in vacuum using a rotatory evaporator to yield 2.20 g of residue. The residue was stored in a desiccator until when needed for analysis.
Antioxidant Activity Assay
Preparation of DPPH
0.004% 1, 1-diphenyl-2-picrylhydrazyl (DPPH) in methanol was prepared
and stored in the dark before use. The solution gives a deep purple color.
Preparation of Sample Solution
Various concentrations of the residue (extract) were prepared using
methanol as solvent. The concentrations of the samples are 10, 20, 40
and 80 μg cm-3, respectively.
DPPH Free Radical Activity
In the activity testing 3 cm3 of sample solution is poured
into 3 cm3 of DPPH solution and allowed to stand in the dark
for 30 min. After 30 min the absorbance is measured at 517 nm and recorded.
This experiment was carried out for each concentration of sample solution
that is 10, 20, 40 and 80 μg cm-3, respectively in triplicates
(Gupta et al., 2003).
The percentage inhibition of DPPH by methanolic extract was then determined by calculation as follows:
Where:
| ADPPH | = | Absorbance of DPPH only |
| ASample | = | Absorbance of DPPH and methanolic extract combined in the same vessel |
From the result obtained a plot of percentage inhibition of DPPH against concentration of methanolic extract is made and the IC50 determined. Control experiment was performed using ascorbic acid as sample and its IC50 also determined.
Antibacterial Activity Test
Preparation of Test Samples
In the study of the antibacterial activity, the methanol extract obtained
as described above in extraction of plant material was used. It was diluted
in dimethyl sulfoxide (DMSO): methanol (1:1, v/v) solvent. As a precaution
not to miss trace amount of antibacterials for preliminary screening,
a relatively high concentration of 200 mg cm-3 of the extract
was prepared for bioassay.
Test Microorganisms
The methanolic extract of the plant was assayed for antibacterial
activity against 6 clinical bacteria isolates, which were obtained from
the Department of Medical Microbiology, Ahmadu Bello University, Zaria-Nigeria.
The bacteria included Bacillus subtilis and Staphylococcus aureus,
Streptococcus pyogenes, Pseudomonas aeruginosa and Pseudomonas
fluorescens. The bacteria were rejuvenated on Mueller Hinton agar
medium (MHM, Merck, Germany) and subculture as needed.
Antibacterial Bioassay
Agar Well Diffusion Method
For bioassay suspension of approximately 1.5x108 cells
cm-3 in sterile normal saline were prepared as described by
Forbes et al. (1998) and about 1.5 cm3 of it was uniformly
seeded on MHM in 12x1.2 cm glass Petri dished, left for 15 min and excess
of suspension was then drained and discarded properly. Wells of 6 mm diameter
and about 2 cm apart were punctured in the culture media using sterile
borers. Respective concentrations of the extracts were administered to
fullness in each well. Culture plates were incubated at 37°C for 48
h. After 48 h bioactivity was determined by measuring Diameter of Inhibition
Zones (DIZ) in mm using a transparent ruler. Each experiment was done
in triplicates and the mean of the diameter of inhibition zones was calculated.
Controls included use of solvent without test compounds, although no antibacterial
activity was noted in the solvent used for the test.
Phytochemical Screening
A qualitative chemical analysis of methanol extract of the plant was
carried out to detect the presence of some classes of natural products
like alkaloids, tannins, flavonoids, saponins, anthraquiniones and cardiac
glycosides using earlier described methods by Harborne (1998) and Oyewale
et al. (2001).
RESULTS AND DISCUSSION
The methanol extract of Commiphora kerstingii was assayed for antioxidant activity using DPPH at various concentrations of the extracts in methanol as solvent. Table 1 shows the absorbance values obtained for DPPH only and that of methanolic stem bark extract of the plant at various concentrations. Table 1 also contains the percentage inhibition of DPPH by the methanol extract (sample) and that of ascorbic acid used as control at various concentrations. The concentration of the methanol extract that causes 50% inhibition of DPPH, which is known as IC50 was determined to be 26.27±0.24 μg cm-3. This value is less than the IC50 of control which is 33.59±0.21 μg cm-3. Despite this development, the statistical analysis result shows that the plant is as effective as the control when used as an antioxidant. A p-value of 0.600 (two-tail) was obtained using Microsoft Excel Program Data Analysis Toolpak as shown in Table 1. This p-value is greater than α = 0.05, which implies that there is no significant difference between the inhibition of sample (that is the methanolic extract of C. kerstingii) and inhibition of control.
| Table 1: | Antioxidant activity assay of Commiphora kerstingii stem bark methanolic extract |
| IC50 values presented are the SEM of three assays for each concentration of extract determined, **p-value shown in table was determined when means of percentage inhibition sample was compared to percentage inhibition control with Students` t-test. p≤0.05 implies significant difference, while p≥0.05 implies not significant difference | |
| Table 2: | Antibacterial activity of methanolic extracts of Commiphora kerstingii stem bark |
| DIZ >15 mm indicates very good antibacterial activity | |
| Table 3: | Phytochemical components of methanolic extract of Commiphora kerstingii stem bark |
| +++: Very high quantity, ++: Moderate quantity, +: Low quantity | |
Table 2 shows that the crude methanolic extract was active against all the bacteria tested with Staphylococcus aureus having the highest diameter of inhibition zone (30 mm), while the lowest is shown by Pseudomonas fluorescens with a diameter of inhibition zone (16 mm). This result indicates that the plant Commiphora kerstingii is highly effective in suppressing the growth of Staphylococcus aureus and the other bacteria tested. However, the result justifies the traditional medicine use of this plant for the treatment of fevers and asthma because of its antibacterial activity against all the 6 clinical bacteria isolates tested.
However, Table 3 shows that the plant has tannins as one of the classes of natural products detected during phytochemical screening. Tannins are mostly made up of phenols and phenolic compounds and they are classified into two classes, which are hydrolysable and condensed tannins (Cowan, 1999). Literature shows that there is high correlation between antioxidant activity and phenolic compounds (Odabasoglu et al., 2004). This implies that compounds that are tannins in nature are most likely to exhibit antioxidant activity. Although other phenolic compounds like flavonoids, which were found in the plant extract as shown in Table 3, also possess antioxidant activity and they are known to be in synergistic relationship with tannins in plants (Rice-Evans et al., 1997).
The positive result as shown in Table 3 shows that the methanolic plant extract contains phenolic compounds like tannins that are very good antimicrobial agents (Scalbert, 1991). In the above paragraph, it had been said that phenolic compounds possess significant antioxidant activity, which is likely due to the presence of tannins detected and the result of this experiment shows that the plant C. kersingii antibacterial agents in it that are highly active against six clinical bacteria isolates. Thus, it maybe summarized that the class of natural product present in the compound that exhibit both antioxidant and antibacterial activity are likely due to the presence of tannins, although other classes of natural products detected can also exhibit antioxidant and antibacterial activity.
CONCLUSION
The result reported in this study may be considered as the preliminary report on the in vitro antibacterial and antioxidant activities of Commiphora kersingii. Since the plant showed significant antibacterial and antioxidant activities, which are suspected to be phenolic compounds such as tannins from result obtained from the phytochemical screening, it become necessary to expand the work so as to carry out chromatographic separation to isolate the active compounds and characterize their structures using IR, UV, Mass and NMR spectroscopic methods. In addition toxicity assay is required to determine the safety level of the plant extract.
ACKNOWLEDGMENTS
The author wishes to thank Mallam Abdullahi Munkailu of Microbiology Unit, National Research Institute for Chemical Technology, Basawa, Zaria-Nigeria for helping to carry out the antibacterial activity test. This research is dedicated to the soul of my late mother Mrs. Aishetu Musa Enakere for introducing me to the local application of this plant and encouraging me to investigate it, God bless you.