0189-6016 © 2007 ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF PLANTS

Ten Nigerian plants suggested from their ethnomedical us activities were studied for their anti-microbial and anti-oxidant p against Escherichia coli NCTC 10418, Pseudomonas aeruginosa Candida albicans, Candida pseudotropicalis and Trichophyton ru leaf extract showed both antibacterial and antifungal activities and w bacteria tested. Boerhavia diffusa, Markhamia tomentosa and T. negative bacteria E.coli and P. aeruginosa strains whereas th Sphenoceutrum jollyamum root inhibited at least one of the fung hexane and chloroform fractions of T. heudelotti extract inhibited bacteria isolates from clinical infectins, respectively. At ≤ 5mg/ fractions of T. heudelotti and those of CHCl3 (34%) and EtOAC inhibition that was stronger than their corresponding methanol extra on M. acuminata, T. heudelotti, E. senegalensis and M. tomentosa w the TLC staining and 1,1-dipheyl-2-picry-hydrazyl (DPPH) free extracts of M. tomentosa and T. heudelotti, especially the latter, p activities and should be further investigated. These activities justifie


Introduction
Out of a total of 25 plants growing in Nigeria and selected based on their local uses, 15 of them have already been examined for their anti-microbial properties (Dalziel, 1956;Aladesanmi et al. 1986;Abbiw, 1989;Igbal et al., 1998;Kokuraro, 1993;Aderinokun et. al, 1999;Oluronke et al., 1999;Okafor et al., 2001;Wu et al., 2001) and hence were dropped.The remaining ten plants were investigated, some of which were used by the traditional healers for diseases such as diabetes, inflammation, loss of appetite, heart troubles, sores and skin infections (Oliver-Bever, 1986) as shown in Table 1.Eleven methanol extracts from these plants were studied for their antibacterial and antifungal properties against reference strains and multi-drug resistant clinical bacteria isolates.Their antioxidant activities were also assessed.This screening is of significant importance because of the urgent need for compounds that would be added to or replace the current antimicrobial agents to which microbes have become largely resistant (Chopra et. al., 1997).

Plant material and extracts preparation
The dried (500 g) powder of the eleven plant parts was extracted with MeOH in a Soxhlet apparatus.The MeOH extracts of the 2 most antimicrobial active plants, T. heudelotti and M. tomentosa, were partitioned successively with n-hexane, CHCl 3 , EtOAc and H 2 O to obtain their corresponding fractions.Each extract or fraction (1.0 g) was taken to make stock solutions for the antimicrobial and antioxidant tests.(Murray et al., 1995) The strains used were; E.coli NCTC 10418, P. aeruginosa ATCC 10145, S. aureus NCTC 6571 and B. subtilis NCIB 3610 for bacteria and C. albicans, C. pseudotropicalis NCYC 6 and T. rubrum for fungi.The standard strains were from stocks of culture collections maintained in our laboratory.Bacteria were maintained on nutrient agar slants, and fungi on Sabouraud Dextrose agar slants at 4 o C and subcultured monthly.Each extract (900 mg) was dissolved in 4 ml MeOH/H 2 O. Discs of Whatman No 1 filter paper (φ 6 mm) were soaked with 2 drops of the extract using a sterile Pasteur pipette and allowed to dry at room temperature.

Antimicrobial Activity Disc Diffusion Assay
Two colonies of a 24-hour plate culture of each organism were transferred aseptically into 10 ml sterile distilled water in a test tube and mixed thoroughly, using an electric shaker, for uniform distribution.A sterile cotton swab was then used to spread the resulting suspension uniformly on the surface of oven-dried Isosensitest agar (Oxoid) and Sabouraud dextrose agar plates (Sterillin) for bacteria and fungi, respectively.These were incubated for an hour at 37 o C and 25 o C for bacteria and fungi, respectively.Sterile forceps were used to place each of the discs on the agar plates aseptically and the plates were then refrigerated for 30 min at 4 o C following which, the inoculated plates were incubated at 37 o C for 24 hours for bacteria strains and at 25 o C for 72 hours for the fungal strains.Antimicrobial activity was evaluated by noting the zone of inhibition against the test organisms.Those extracts showing any inhibition at all were noted for further tests for the quantitative assessment of their activity.

Agar diffusion assay
Dilutions of 40, 20, 10 and 5 mg/ml were prepared from 225 mg/ml stock solutions of the four extracts that inhibited the microorganisms.Volumes (20 ml) of molten nutrient agar were seeded with 1 ml portions of overnight cultures of microorganisms and poured into sterile Petri dishes (85 mm) and allowed to set.Holes of diameter 9 mm were made in the agar plates using a sterile metal cup-borer.Two drops of each extract and control were put in each hole under aseptic condition, kept at room temperature for 1 hour to allow the agents to diffuse into the agar medium and incubated accordingly (Reeves et al., 1979).Chloramphenicol (100 µg/ml) and acriflavine (6.3 mg/ml) were used as positive controls for bacteria and fungi respectively, MeOH/H 2 O was the negative control.The plates were then accordingly incubated at 37 o C for 24 hours for the bacterial strains and at 25 o C for 72 hours for the fungal strains.The zones of inhibition were measured and extracts that gave significant activity against gram-negative isolates (E. coli and P. aeruginosa) were further tested against multidrug resistant bacteria including uropathogenic E. coli and P. aeruginosa wound isolates.

Determination of the antibiotic susceptibility of bacteria isolates
Forty-eight clinical isolates of bacteria from wound, stool and sputum of patients diagnosed with wound, gastro-intestinal tract and respiratory tract infections and apparently healthy individuals and two Bacillus species from the environment were used.The organisms were isolated on blood agar prepared with 5% human blood on Nutrient agar (Oxoid) and identified using their morphological characteristics and standard biochemical tests (Barrow and Feltham, 1993).
The Disc diffusion method (NCCLS, 2003) was used for the determination of microbial sensitivity.The antibiotic discs employed were: Nalidixic acid, Ofloxacin, Augmentin, Tetracycline, Nitrofurantoin, Chloramphenicol and Cephalothin all at 30 µg, Amoxycillin and Cotrimoxazole at 25 µg, Erythromycin at 15 µg, Gentamicin, Ciprofloxacin and Penicillin-V at 10 µg.The zones of inhibition were measured and interpretation was in accordance with manufacturer's instructions (AB, Biodisc; PDM Interpretative chat).

Determination of Minimum Inhibitory Concentrations (MICs) of the fractions of M. tomentosa and T. heudelotti on multi drug resistant clinical isolates
The MIC were determined by the agar dilution method on Nutrient Agar (Oxoid) using a stock solution (50 mg/ml) of each fraction and extract dissolved in 50% MeOH/H 2 O (Murray et al., 1995).The final plate concentrations were 5.0, 2.5, 1.25, 0.625 and 0.3125 mg/ml for the extract/fractions and 0.5, 0.25, 0.125, 0.0625, 0.03125mg/ml for chlorocresol (BDH), the +ve control.The hexane and CHCl 3 fractions were dissolved in Tween 80 (Sigma-Aldrich Co., USA) and the rest in 50% MeOH/H 2 O and these vehicles were the -ve controls.Bacteria were grown for 18 hours in Nutrient broth (Oxoid) and culture suspensions of 10 8 cfu/ml were applied to the surface of the nutrient agar plates containing dilutions of the fractions, chlorocresol, Tween 80 and methanol employing a multipoint inoculator.Plates were incubated at 37 o C for 24 hrs, after which all plates were observed for growth.The minimum dilution of fractions completely inhibiting the growth of each organism was taken as the MIC.

DPPH photometric assay
The free radical scavenging activities of each extract and ascorbic acid were analysed by the DPPH assay (Sanchez-Moreno, et al 1998).A 1.0 ml of the test extract, at gradient final concentrations of 0.5 -500 µg/ml, was mixed with 2 ml of 0.3 mM DPPH solution in MeOH in a cuvette.The absorbance was taken at 517 nm after 20 minutes of incubation in the dark at room temperature.The experiment was done in triplicates.The percentage antioxidant activity was calculated as follows: %Antioxidant Activity [AA] = 100 -[{(Abs sample -Abs blank ) X 100}/Abs control ].Where Abs sample was the absorbance of sample solution (2.0 ml) + DPPH solution (1.0 ml, 0.3 mM), Abs blank was the absorbance of Methanol (1.0 ml) + sample solution (2.0 ml), Abs control was the absorbance of DPPH solution (1.0 ml, 0.3 mM) + methanol (2.0 ml).

Results
As shown in Table 2, among the 10 plants studied, only M. tomentosa and T. heudelotti leaf MeOH extracts showed both antifungal and antibacterial activities.The leaf MeOH extract of B. diffusa gave only antibacterial activity whereas that of S. jollyanum root showed only antifungal properties (Table 2).In addition, T. heudelotti extract showed significant activity against the gram-negative bacteria, P. aeruginosa (MIC = 10mg/ml) and E. coli (MIC = 20mg/ml).The result of the susceptibility study showed a high level of antibiotic resistance by the clinical and environmental isolates (Table 3).There was a high level of antibiotic resistance to the commonly used antibiotics such as penicillin V (100%), cephalothin (98%), tetracycline (82%), and augmentin (77%).Some resistance was also demonstrated against fluoroquinolones e.g., ofloxacin (6.3%), ciprofloxacin (21%) and erythromycin (100%).Generally, 64% of the isolates were resistant to more than 50% of the antibiotics tested (Table 3).The activity of the crude MeOH extracts and partition fractions of M. tomentosa and T. heudelotti against the multi-drug resistant isolates are given in Table 3.At concentrations of ≤ 1.25 mg/ml, the CHCl 3 fractions of M. tomentosa and T. heudelotti showed the best activity, with the latter inhibiting the growth of most of the multi-drug resistant bacteria at a concentration of 312µg/ml.The intensity of the spots and reaction time of the DPPH radical scavenging capacities of the ten MeOH extracts using the TLC method showed that M. tomentosa, T. heudelotti and M. accuminata gave the highest antioxidant activities (Table 4).The EC 50 of the extracts using DPPH photometric assay were M. acuminata 4.00, AA 4.65, T. heudelotti 6.50, E. senegalensis 13.33, M. tomentosa 16.50, P. barteri 66.67 and S. jollyamum 133.34 µg/ml (Figure 1).

Discussion
All the plants investigated have been reported to be used in ethnomedicine (Table 1).. Studies have been carried out to test the hypoglycaemic, antifungal and antioxidant activities of B. diffusa extracts (Chude et. al., 2001, Agrawal et. al., 2004, Amarnath and Pari, 2004).Similarly, Abo and Ashidi (1999) examined the antimicrobial and antifungal effects of the plant.Our results confirm the insignificant antimicrobial activity.As shown in Table 2, B. diffusa leaf extract has little activity against the bacteria and no activity against the fungi tested and therefore validates the traditional uses of the plant in treating boils and abscess.The root and leaf       2).Thus, this plant could be a source of useful antifungal agent.The other six plants investigated (Table 2) did not show any significant antibacterial or antifungal activity; however, activity has been reported for the Ethiopian P. zeylanica root on some pneumonia causing pathogens (Lemma et al., 2002).The observation of weak and moderate antioxidant activities of B. diffusa and S. jollyanum extracts (Table 4) is in consistent with those reported (Mutho et al., 1998;Chude et. al., 2001, Oke andHamburger, 2002;Agrawal et. al., 2004, Amarnath andPari, 2004).The leaf extract of M. tomentosa showed good anti-staphylococcal activity as well as activities against P. aeruginosa and B. subtilis.It also showed encouraging activities against the fungus C. pseudotropicalis thereby demonstrating activity against a broad spectrum of organisms (Table 2).Extract of T. heudelotti leaves showed better antifungal and antibacterial activities than M. tomentosa (Table 2).T. heudelotti revealed an interesting activity against S. aureus (MIC = 5 mg/ml), P. aeruginosa (MIC = 10 mg/ml) and some activities against E. coli (MIC = 20 mg/ml), B. subtilis and T. rubrum (MIC = 40 mg/ml).Leaf extract of T. heudelotti also showed weak activities against the remaining 2 fungi tested (Table 2).The extracts of the two most active plants M. tomentosa and T. heudelotti were further fractionated to ascertain the properties of the active ingredients.These were tested against 50 multidrug resistant isolates from clinical and environmental sources including the organisms from the family Enterobacteriaceae which are commonly involved in clinical infections and are known to be highly versatile at acquiring resistance characteristics (Lamikanra et al., 1989).Two versatile pathogens, P. aeruginosa, a major nosocomial pathogen with low intrinsic susceptibility to antimicrobial agents and very high ability to acquire resistance, and S. aureus were also among those tested The organisms studied covered gram positive and gram-negative isolates (Table 3) and the demonstrated activities of the fractions showed that these plants have a wide spectrum of activity.
The bacteria used in Table 3 were not only resistant to the routinely used antibiotics in Nigeria such as Penicillin V and Erythromycin (100%); Cephalothin (98%), Tetracycline (82%), Augmentin (77%) and Cotrimoxazole but also showed resistance to the newer generation antibiotics like the fluoroquinolones e.g.ofloxacin (6.3%) and ciprofloxacin (21%).Sixty-four (64%) percent of the isolates was resistant to more than 50% of the antibiotics tested (Table 3).The resistance properties of these isolates showed the worsening situation of antibiotics resistance in the Nigerian environment and lead credence to the search for substances that could be added to or replace the antibiotics in current clinical use, which are becoming less useful with every hour (Chopra et al., 1997, Okeke andSosa, 2003).The better activities observed for the extracts and fractions of the two most active plants may provide an answer to this phenomenon.
The partition fractions, n-hexane, CHCl 3 , EtOAc, H 2 O together with the very active crude MeOH extracts of M. tomentosa and T. heudelotti showed MIC of < 5mg/ml against clinical and environmental isolates, especially against microbes that showed > 60% resistance against commonly used antibiotics (Table 3).Hence, extracts and active fractions of these plants may therefore be considered as alternative antimicrobial agent(s) against possible multiple antibiotic resistant strains that are widespread in the communities, especially in ethnomedicine (Chambers, 2003).At ≤ 1.25 mg/ml, the orders of activity presented for the fractions and extracts of M. tomentosa and T. heudelotti were MeOH < n-hexane = H 2 O < EtOAc < CHCl 3; and EtOAc = MeOH = H 2 O < n-hexane < CHCl 3 respectively (Table 3).The better activities of the fractions over the mother MeOH extracts showed that definite compounds present in these fractions were responsible for these activities.The M. tomentosa and T. heudelotti CHCl 3 fractions showed good anti-staphylococcal activity and MIC values of ≤ 5 mg/ml against multidrug resistant wound isolate of Ps. aeruginosa.The M. tomentosa CHCl 3 fractions also showed better activities against E. coli, B. subtilis, P. vulgaris and Salmonella enterica var.choleraesius, while that of T. heudelotti gave additional activities against A. haemolyticus and Staphylococcus epidermidis (Table 3).
Aladesanmi and Odediran (2000) had reported the isolation of antimicrobial phenolic acids from the EtOAc fraction of T. heudelotti.However, activities at < 5mg/ml given by the CHCl 3 fraction against 66% of the multidrug resistant organisms suggest the possibility of some other active constituent(s), whose activities are synergistic with the phenolic acids of the fractions.The frequent occurrence of variations in the MIC values within species and between related organisms suggests that resistances to M. tomentosa and T. heudelotti, when they occur, are due to intrinsic properties rather than acquired characters of the species.For these reasons, it would be useful if the extracts of the plants or active principles can be exploited for development into antimicrobial chemotherapeutic agents.Similarly there is the need to characterize the active principles in M. tomentosa.These plant extracts and fractions may serve as a good source of some cheap and highly effective antimicrobial agents for bacteria infections caused by multiresistant organisms.
The generation of the reactive oxygen species (ROS) beyond what the ability of the body can cope with leads to oxidative stress (Sies 1985, Gutteridge and Halliwell 1994, Maxwell 1995).Free radical oxidative stress has been implicated in the pathogenesis of a variety of human diseases like: artherosclerosis, diabetes mellitus, hypertension, inflammation, cancer and AIDS (Halliwell and Gutteridge 1989).The use of DPPH scavenging assays in assessing the cell membrane integrity/cell membrane stabilising capacities of plant constituents has given explanations as to the possible ways by which phytomedicines could help to reduce diseases caused by infections, inflammation and oxygen radicals generation affecting the cell membrane (Sadique et al 1989, Tsuda, 1998, 2000).The models of scavenging DPPH free radicals used are the rapid screening and the photometric assay methods commonly employed for evaluating antioxidants activities based on their abilities to donate hydrogen ion (Kumazawa, et al 2002).The DPPH is a free radical stable at the room temperature.The methanolic solution gives a purple colouration which when reduced by an antioxidant molecule gives rise to a yellow solution.On comparison with ascorbic acid (AA), a standard antioxidant drug, the antioxidant activities of the extracts in decreasing order were M. acuminata > AA > T. heudelotti > E. senegalensis > M. tomentosa > P. barteri > S. jollyamum.The EC 50 values of M. acuminata, AA, T. heudelotti and E. senegalensis antioxidant activities were higher than that of 14.16 + 0.20 µg/ml reported for rutin, a pure standard antioxidant compound (Mensor et al 2001).However, only M. tomentosa and T. heudelotti gave both strong radical scavenging abilities (Table 4) and antimicrobial activities (Tables 2, 3).It is therefore desirable to isolate and characterise the antioxidant agents from these two plants, T. heudelotti and M. tomentosa and determine whether or not the same constituents are responsible for both the antimicrobial and antioxidant activities.

Conclusion
The methanolic extracts of E. senegalensis, G. arboreum, M. acuminata, P. zeylanica, P. barteri and P. guajava have no antibacterial and antifungal activities.The leaf extracts of M. tomentosa and T. heudelotti showed relatively high antimicrobial activities complemented with impressive antioxidant activities.M. acuminata with the highest antioxidant activity showed no antimicrobial activity.The antimicrobial activities were found in the CHCl 3 partition fractions of T. heudelotti and M. tomentosa leaves.The former would be a better choice for antimicrobial activity, the later, M. acuminata leaf, would be good as an antioxidant agent.These results showed that the active plants M. tomentosa and T. heudelotts, especially the latter, could be further exploited for chemotherapeutic agents that could be used against infections caused by multiple antibiotic resistance strains very common in Nigeria.Alternatively, the extracts and active fractions of these plants may be used directly in ethnomedicine as antimicrobial agents.
The methanolic extracts of Ekebergia senegalansis leaf, Gossypium arboretum bark, Massularia acuminata leaf, Pleioceras barteri root and leaf, Plumbago zeylanica root and Psidium guajava bark were inactive to all the organisms tested.N.T.:Not tested.

Figure 1 :
Figure 1: Antioxidant activity of six medicinal plants

Table 1 :
Local Therapeutic uses of some Nigerian medicinal plants.

Table 2 .
In-vitro Antimicrobial activity of the methanolic extracts of some Nigerian medicinal Plants.

Table 3 :
Antibiotic resistant properties of organisms and the MIC values of the methanolic extracts and their partition fractions of M. tomentosa and T. heudelotti leaves.178