Antioxidant and antimicrobial activities of Canarium schweinfurthii Engl. Essential oil from Centrafrican Republic

Centre de Recherche en Sciences Biologiques, Alimentaires et Nutritionnelles, Laboratoire de Microbiologie et de Biotechnologie (CRSBAN), Université de Ouagadougou 03 BP7131 Ouagadougou Burkina Faso. Centre de Recherche en Pharmacopée et Médecine Traditionnelle (CERPHAMETRA), Université de Bangui, BP 1450 Bangui République Centrafricaine. Centre International de Recherches Médicales du Gabon, BP769 Franceville, Gabon Ecole Normale Supérieure, Laboratoire pluridisciplinaire de chimie, BP7131 Libreville Gabon.


INTRODUCTION
In the last years, scientists have focused on increasing human infections caused by pathogen bacteria, fungi and viruses. Microorganisms have unfavourable effects on the quality and safety of life. Synthetic chemicals are widely used against these microorganisms; unfortunately they develop resistance to many antibiotics due to the indiscriminate use of commercial antibiotics (Service, 1995;Mukherjee et al., 2002). In addition, these antibiotics sometimes cause allergic reaction and immunity suppression. Therefore the use of essential oils and plant extracts is less damaging to the human health and environment (Isman, 2000;Misra and Pavlovstathis, 1997). Canarium schweinfurthii Engl. (Burceracea) is a tree growing in the equatorial forest region from Cameroon, Centrafrican Republic, Gabon to Congo (Tchiégang, 2001;Tchouamo et al., 2000). The fruit pulp contains 30 to 50% of oil used for the manufacture of *Corresponding author. E-mail: jean_koudou@yahoo.fr. Fax: +226 50 36 85 73.
shampooing and bio fuel (Tchiégang, 2001;Ajiwe et al., 2000). The rhizhomes and leaves are used as stimulant and against fever, constipation, malaria, diarrhoea, sexual infections, post-partum pain and rheumatism (Koudou et al., 2005;Aké Assi and Guinko, 1991). Previous studies on the isolation of lipids and fatty acids from the fruit and the human food, the chemical composition and the significant analgesic effect of the resin essential oil of C. schweinfurthii have been reported (Koudou et al., 2005;Agbo et al., 1992). However there is so far no report about the antimicrobial activities. In other hand, the traditional use of the plant suggested an antioxidant activity.
The role of free radicals and active oxygen is becoming increasingly recognized in the pathogenesis of the many human diseases, including cancer, aging and atherosclerosis (Perry et al., 2000). Free radicals can also cause lipid peroxidation in foods that leads to their deterioration. Although there are some synthetic antioxidant compounds such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), these compounds are associated with some side effects (Ito et al., 1983).
There is no information in literature about the antioxidant activity of any Canarium species. So the works in the determination of natural sources of antioxidants and the antioxidant potential of plants is important.
As mentioned above, the antimicrobial and antioxidant activities of the C. schweinfurthii essential oil have not been studied to date. Therefore, the aim of the present study is to assess the antibacterial and antifungal activities of the resin essential oil and to determine its antioxidant activity.

Plant material
The resin of C. schweinfurthii was obtained from the tree growing in the equatorial rain forest near Boukoko village (Centrafrican Republic) in July 2006. Voucher specimens have been deposited in Cerphametra, University of Bangui (Centrafrican Republic).

Isolation of essential oil
Essential oil of C. schweinfurthii Engl was obtained by hydrodistillation of resin. The chemical composition of essential oil has been reported (Koudou et al., 2005).

Determination of antioxidant activity
The antioxidant activity was evaluated by two different methods: DPPH radical scavenging activity and -carotene-linoleic acid test.

2,2-Diphenylpicrylhydrazyl (DPPH) essay
The hydrogen atoms or electron-donating ability of the essential oil and BHT was determined from the bleaching of purple-colored methanol solution of DPPH. This spectrophotometric assay uses the stable radical DPPH as a reagent (Burits and Bucar, 2000). Experiments were carried out as described previously (Kordali et al., 2005). Briefly, 0.5 mM DPPH (Fluka) radical solution in methanol was prepared, and then 1 ml of this solution was mixed with 3 ml of the sample solution in ethanol. Various concentrations of extracts were obtained. BHT (Sigma) was used as a positive control at 100 g.ml -1 concentration. After incubation for 30 min in the dark, the absorbance was measured at 517 nm. Decreasing the absorbance of the DPPH solution indicates an increase in DPPH radical scavenging activity. This activity is given as percent DPPH radical scavenging, which is calculated with the equation: % DPPH radical scavenging = [(control absorbance-sample absorbance)/control absorbance] X 100 Control contained 1 ml of DPPH solution and 3 ml of ethanol. The measurements of DPPH radical scavenging activity were carried out for three sample replications, and values are an average of three replicates.

-carotene-Linoleic acid assay
In this assay antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxide formation from linoleic acid oxidation (Dapkevicius et al., 1998). A stock solution of -carotene-linoleic acid mixture was prepared as follows: 0.5 mg of -carotene was dissolved in 1 ml of chloroform (HPLC grade); 25 µl of linoleic acid and 200 mg of tween 40 were added as emulsifier becausecarotene is not water soluble. Chloroform was completely evaporated using a vacuum evaporator. Then, 100 ml of distilled water saturated with oxygen was added with vigorous shaking at a rate of 100 ml/min for 30 min; 2500 µl of this reaction mixture was dispersed to test tubes, and 350 µl portions of extracts, prepared in 2 g.l -1 concentrations, were added. The emulsion system was incubated for up to 48 h at room temperature. The same procedure was repeated with a positive control BHT and a blank. After this incubation time, the absorbance of the mixture was measured at 490 nm. Antioxidant capacities of extracts were compared with those at the BHT and the blank. Tests were carried out in triplicate. Inhibition of coloration of -carotene in percentage (I %) was calculated as: Where Ablank is the absorbance of the control reaction (containing all of the reagents except the test compound) and Asample is the absorbance of the test compound.

Bacterial and fungal strains
The micro organisms used were:

Disk diffusion essay
The tests were performed using Miller-Hinton medium for bacterial strains and saboureaud dextrose agar for fungal strains using disk diffusion method following the National Committee for Clinical Laboratory Standards methods (Kiehlbauch et al., 2000). The sterile Petri dishes (90 mm diameter) containing solid and sterile Mueller-Hinton agar medium (Becton Dickinson, USA) was used. The oil absorbed on sterile Whatman paper disks (5 l per disk of 6mm diameter), was placed on the surface of the media previously inoculated with 0.1 ml of microbial suspension (1 g per Petri dish). One filter paper disk was placed per Petri dish in order to avoid a possible additive activity exhibited via the vapour phase of the components from more than one disk. Every dish was sealed with laboratory film to avoid evaporation, and then incubated aerobically at 30 or 37°C according to strain for 24 h. Positive and negative growth controls were performed for every test. The bacterial and fungal sensitivities to the essential oil were assessed by measuring the diameter of inhibition zone. The inhibition zones were compared with that of tetracycline and ticarcilline (Bio-Rad Marnes-la coquette-France), fluconazole and griseofulvin (Bio-Rad-la coquette-France). All tests were performed in triplicate.

Antimicrobial activity essay
A broth microdilution method was used to determine the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) and the minimum fungicidal concentration (MFC),

Antioxidant activity
The antioxidant activity of C. schweinfurthii essential oil was investigated with two different methods: 2,2diphenylpicrylhydrazyl radical scavenging assay andcarotene bleaching test. The DPPH radical scavenging activity of essential oil was high but relatively lower than that of BHT (Figure 1). The ability of essential oil to inhibit the lipid peroxidation and evaluated by -carotene bleaching test (Figure 2) showed that the peroxidation of lipids was effectively inhibited by C. schweinfurthii essential oil. The essential oil showed higher values than the negative control and a strong activity, but this activity remained weak in comparison to the activity of a 100 µg.ml -1 concentration of BHT.

Antimicrobial activity
The results showed that almost all of bacterial strains were sensitive to the essential oil (Table 1). Only P. Obame et al. 2321 mirabilis CIP 104588 was not sensible (zone of inhibition 9 mm). The best sensitivity to essential oil was respectively obtained on S. enterica CIP 105150 (27 mm), S. pyogenes (25 mm) and S. aureus (24 mm). The other strains tested had sensitivities between 14 -22 mm. Following the results in Table 1, the different strains were more sensitive to essential oil than tetracycline, but were less sensitive to essential oil than tircacilline. The essential oil exhibited more activity on S. enterica CIP 105150 (27 mm) than tetracycline (S. enterica CIP 105150, 16 mm). The essential oil was tested against Candida albicans as pathogenic fungal species in human body and compared with fluconazole and griseofulvin. The result showed that the growth of fungal species was significantly inhibited by the essential oil (Table 2). Clinic origin C. albicans was more sensitive to the essential oil (23 mm) than reference C. albicans strain. It was also interesting to find that the inhibition effect of the oil against C. albicans (23 mm) were higher than that of fluconazole (C. albicans, 9 mm) and griseofulvin (C. albicans, 11 mm).
The MICs, MBCs and MFCs of the essential oil for all the strains tested are presented in Table 2. The essential oil failed to inhibit E. coli CIP NCTC11602 and P. aeruginosa obtained from hospital at the highest concentration (8%). L. innocua LMG 1135668, S. aureus ATCC9244, S. camorum LMG13567 BHI, S. aureus (clinic strain), C. albicans ATCC90028, C. albicans (clinic strain) were inhibited at the lowest MIC of 0.25%. The results of MBC and MFC demonstrated a bactericidal and fungicidal effect. The essential oil was bactericidal for E. faecalis, L. innocua, S. enterica, S. aureus, S. camorum (reference strains) and S. aureus (clinic strains). Furthermore the oil was fungicidal for C. albicans ATCC10231 and C. albicans (clinic strain). The MIC and MBC values showed that the essential oil was most effective against Gram-positive bacteria than Gram-negative bacteria. Previous reports show that the presence of oxygenated monoterpenes as 1, 8-cineole, linalool, -terpineol, nerolidol, spathulenol in high proportions exhibits antibac-terial and antifungal activities (Chalchat et al., 1997;Kordali et al., 2005;Setzer et al., 2004;Yoshihiro et al., 2004). C. schweinfurthii essential oil was composed of relatively lower proportions of these compounds and had antimicrobial activity. These reports are compatible with our results in the present study. Furthermore the essential oils consist of complex mixtures of numerous constituents. Possible synergistic effects of compounds in the essential oil should also be taken into consideration.
In conclusion, this study shows in vitro high antimicrobial activities and low antioxidant activity of the C. schweinfurthii essential oil. It was bactericidal and fungicidal for most of the reference strains and some clinic strains tested. Its effect is most effective against Grampositive bacteria than Gram-negative bacteria tested. The essential oil exhibits also antioxidant activity. These results indicate that the essential oil of C. schweinfurthii  could be used as a natural antimicrobial agent for human and infectious diseases and in food preservation. Furthermore, the development of natural antimicrobial agents will help to decrease negative effects (pollution of environment, resistance) of synthetic chemicals and drugs.