Vol 6 | Issue 1 | 2016 | 68-75.

ISSN 2249 - 7641 Print ISSN 2249 - 765X International Journal of Pharmacology Research

www.ijprjournal.org

OVICIDAL AND LARVICIDAL ACTIVITIES AGAINST ANOPHELES GAMBIAE, AND ANTIBACTERIAL PROPRIETIES OF AUCOUMEA KLAINEANA PIERRE, SCHWEINFURTHII ENGL AND EDULIS (G. DON) H. J. LAM ESSENTIAL OILS FROM

Louis-Clément Obame-Engonga1,2,4*, Joseph-Privat Ondo1,2, Guy-Stéphane Padzys2,3, Timoléon Andzi-Barhé1, Télérance Kounga1,2, Jean-Bernard Bongui1, Thierry Ndong Mba2,3, Cheikna Zongo4and Alfred S. Traoré4

1Laboratoires de Substances Naturelles et de Synthèses Organométalliques (LASNSOM), Université des Sciences et Techniques de Masuku B.P. 943, Franceville, Gabon. 2Laboratoire de Recherche en Biochimie (LAREBIO), Université des Sciences et Techniques de Masuku B.P. 769, Franceville, Gabon. 3Laboratoire de Physiologie Animale: Electrophysiologie-Pharmacologie-URAB, Université des Sciences et Techniques de Masuku, BP 913, Franceville Gabon. 4Centre de Recherche en sciences Biologiques, Alimentaires et Nutritionnelles (CRSBAN); UFR / SVT; Université de Ouagadougou ; 03 BP 7021 Ouagadougou 03, Burkina Faso.

ABSTRACT As part of ongoing research on the ovicidal and larvicidal activities against Anopheles gambiae, antioxidant and antimicrobial proprieties of a mixture of essential oils extracted from Aucoumea klaineana Pierre, Engl. and (G. Don) H. J. Lam with an aim regard in contributing to the search for beneficial uses of the previous mentioned medicinal of Gabon, antioxidant activities were evaluated with 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The essential oils were tested against 4 bacteria by using disc diffusion and micro-dilution methods. Based on the ovicidal efficacy and larvicidal activity tested by WHO in (2005), the standard protocol against 3rd and 4th instar larvae of field-collected mosquitoes vectors of human disease members of the Anopheles gambiae was revealed. The Antioxidant Activity Index (AAI) of essential oils ranged from 0.82 to 2.07 and could now be compared to AAI of vitamin C (11.32) and BHT (7.85) while those of combination ranged from 0.48 to 1.93. The essential oils exhibited a strong DPPH free radical scavenging action, IC50 values ranged from 48 to 78 µg/mL for essential oils and 20.37 to 82.61 µg/mL for the combined essential oils. Essential oils of A. klaineana, C. schweinfurthii and D. edulis, showed antimicrobial effects against all microorganisms tested. The FIC indices ranged from 0.11 to 7.13 for paired combinations of A. klaineana, C. schweinfurthii and D. edulis essential oils. All the paired combinations showed synergistic effects against Streptococcus pyogenes and Staphylococcus aureus. Combinations of A. klaineana with D. edulis or C. schweinfurthii had showed some antagonism effects on the inhibition of Bacillus cereus and E. coli. Only the combinations of A. klaineana with C. schweinfurthii had revealed some indifferent effects against Bacillus cereus. Somewhat, ovicidal and larvicidal activities were demonstrated. The A. klaineana, C. schweinfurthii and D. edulis essential oils exhibited also larvicidal activity. The median lethal concentration (LC50) of Anopheles gambiae larvae ranged between 9.94-22.98 µg/mL. The LC50 value of Anopheles gambiae eggs ranged between 22.08-67.64 µg/mL. Dacryodes edulis showed the highest activity against the Anopheles gambiae eggs and larvae. Over all the three plants, essential oils of Aucoumea klaineana had showed the lowest activity of Anopheles gambiae eggs and larvae. Larvae were more susceptible than eggs. The present study reveals the potential of ovicidal efficiency and larvicidal activity against Anopheles gambiae using the combination of Aucoumea klaineana, Canarium schweinfurthii and Dacryodesedulis essential oils to increase antioxidant and antimicrobial proprieties.

Keywords: Ovicidal efficacy, larvicidal activity, Anopheles gambiae, antioxidant and antimicrobial proprieties, combination of essential oils.

Corresponding Author:- Louis-Clément Obame-Engonga EMail Id: [email protected]

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INTRODUCTION Guinea [14]. Bark from the young is used in Malaria in Tropical area kills about 3 million each traditional medicine for rectal injections. It produces a year, including 1 child every 30 sec [1]. Mosquito vectored greenish/white , with an odour similar to that of pathogens infect more than 700 million people annually turpentine and the is ovoid, purplish in colour, at around the word through diseases such as malaria, maturity [4]. The oil is used in the manufacturing of filariosia, dengue, yellow fever, rift valley fever and shampoo, waxes or as a biocarburant [13, 15]. In Gabon, Japanese encephalitis [2]. An alternative approach for the resin is used to make torches or to make smoke to repel mosquito control is the use of natural product of mosquitoes [16]. The resin is used for the treatment of origin. The vast majority of essential oils are pleasant smell various diseases such as wounds and microbial infections. and its metabolic and evolutionary significance lies in the It is also valued for its emollient, stimulate and diuretic role they play as attractor of pollinating agents for its properties [17]. pleasant aroma, constitute elements of defense against the The present work reports results of ovicidal and attack of parasites, herbivorous animals and insects. larvicidal activities against Anopheles gambiae, antioxidant Aucoumea klaineana, Canarium schweinfurthii and and antimicrobial proprieties of Gabonese essential oils Dacryodes edulis, belong to the Family , which extracted from Aucoumea klaineana Pierre, Canarium has about 660 genera and 9000 species [3]. Aucoumea schweinfurthii Engl. and Dacryodes edulis (G. Don) H. J. klaineana or “Okoumé”, is largely distributed in equatorial Lam medicinal plants. Such a study may help in the forest from Gabon to . It is an important contribution of the ongoing search for beneficial uses of medicinal plant, widely used as a home remedy for several those plants to eradicate various infections and dry-skin diseases in Gabon. The roots and leaves are used to treat resistance of African diseases. fever, constipation, malaria, diarrhea and jaundice. The resin of the plant is used to purify water and as disinfectant MATERIALS AND METHODS [4]. Previous works have shown that A. klaineana contained Plant material triterpenoid compounds and its resin was used in the The of Aucoumea klaineana, Canarium cosmetic and pharmaceutical fields [5]. The essential oil schweinfurthii and Dacryodes edulis were collected in isolated from the resin of A. klaineana were contained August 2014 from Mebane Endama Minkong 7, Oyem, mainly monoterpenoids (96.06%) in which p-acetyl anisole Gabon. Plants were identified at the Herbarium is the single benzenic compound (0.18%). The predominant IPHAMETRA. Voucher specimens were deposited under constituent in the essential oil was δ-3-carene (72.31%) numbers 132, 133 and 135 related to Dacryodes edulis, [6].Aucoumea klaineana resin essential oil could be a Aucoumea klaineana and Canarium schweinfurthii at the natural antimicrobial and antifungal agent [7]. Laboratory of Natural Substances and Organometallic Dacryodes edulis or “Safou”, is a tropical synthesis (LASNSOM), the University of Sciences and producing a consumable fruit. The native area of Safou Technology of Masuku, Franceville, Gabon, respectively. extends from to the east of until the The resins were hydro-distilled during 3 h in a clevenger- south of border. The wounded bark exudes a limpid type apparatus. The essential oils were dried after resin that becomes opaque while solidifying. The burning decantation using anhydrous sodium sulfate and then resin releases a strong odour [4]. Traditional healers in filtered and stored in the dark at 4°C of the scaled vial prior and in Democratic Republic of Congo use the plant to the analysis. The essential oil yield was calculated on the to treat various infections as a remedy for parasitic skin basis of report/ratio (mass of dry oil extracted to the mass diseases, jigger, mouthwash, tonsillitis and drepanocytosis of the resin). [8]. Essential oil of untreated, boiled and roasted contains many constituents among which α-pinene, β- Microbial strains pinene, myrcene, limonene and sabinene were found to be The resin essential oils from Aucoumea klaineana, the main compounds [9]. The stem bark essential oil Canarium schweinfurthii and Dacryodes edulis were tested contains predominantly terpinen-4-ol, α-thujene and α- against a panel of resistant microorganisms, including pinene, whilst aphellandrene is the major component of the clinical strains of Bacillus cereus, Escherichia coli, root bark oil. β-caryophyllene is a dominant constituent of Staphylococcus aureus and Streptococcus pyogenes which the leaf oil [10]. The resin has been reported to yield a were isolated at the Laboratory of Medical Biology Saint peppery essential oil that is rich in sabinene, β-phellandrene Camille, Ouagadougou, Burkina Faso. and limonene. Information concerning in vitro antioxidant and antimicrobial activities of the Gabonese essential oil Mosquito eggs and larvae from the resin of D. edulis has been reported by Obame et Anopheles gambiae larvae and eggs were collected al., (2008) [11]. in various sites in Franceville Gabon, and they were reared The geographical distribution of Canarium to adults in the insectary that were used in the bioassays. schwe²infurthii Engl or “Aiélé”, is very widespread Members of the Anopheles gambiae complex were throughout [12; 13]. Aié lé is present in forests in identified from a previous collection in Goden.

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Antioxidant Activity Index determined using micro-well dilution assay method [19- The Antioxidant Activity Index (AAI) was 22]. A serial doubling two-fold dilution of either essential assessed according to the method described by Scherer and oil was prepared in a microtiter tray over the range 10– Godoy (2009) [18]. This method is based on the DPPH 0.075 mg/mL in 100 µL Mueller–Hinton broth. The broth radical test. Briefly, the working reagent was prepared by was supplemented with ethanol absolute at a concentration dissolving 10 mg of DPPH in 100 mL ethanol. Graded of 0.5% in order to enhance essential oils solubility. concentrations of essential oils ranging from 0.781 to 100 Overnight broth cultures of each strain were prepared from μg/mL obtained by two-fold dilutions were prepared and 18 h broth cultures and suspensions were adjusted to 0.5 100 μL of each dilution were mixed with 100 μL of the McFarland standard turbidity. An aliquot 100 µL of the working solution of DPPH in a 96-well plate. Absorbencies inoculums was added to diluted essential oil. The final were measured at 517 nm after 15 min incubation at room volume in each well was 200 µL. The plate was covered temperature in the dark. Ascorbic acid (Vitamin C) and with a sterile plate sealer. Positive and negative growth butylated hydroxyl toluene (BHT) were used as references. controls were included in every test. The tray was incubated The ability to scavenge DPPH radical was calculated using aerobically at either 30◦C or 37◦C according to bacteria for the following equation: 18–24 h. The MIC is defined as the lowest concentration of the essential oil at which the microorganism tested does not % RSA= 100 x [(Acontrol – Asample)]/ Acontrol demonstrate visible growth in the broth. Bacterial growth was indicated by turbidity. A= Absorbance at 517 nm. The IC50 (concentration providing 50% inhibition) of oils and standards was The checkerboard method determinate from regression curves in the linear range of The checkerboard method was performed using concentrations. The AAI was then calculated as follows: 96-well microtitre plates as described previously [19-23], to AAI= ([DPPH] (μg/ mL)) /IC50 (μg/mL), [DPPH] is the obtain the FIC (Fractional inhibitory concentration) index. final concentration of DPPH. We considered criteria of The microplate assay was arranged as follows: Essential oil Scherer and Godoy (2009), according to which plant A (EOA) was diluted two-fold along the x-axis, whilst EOB extracts show poor antioxidant activity when AAI < 0.5, B was diluted two-fold along the y-axis. The final volume moderate antioxidant activity when AAI between 0.5 and in each well was 100 µL comprising 50 µL of each EO 1.0, strong antioxidant activity when AAI between 1.0 and dilution. Subsequently, 100 µL of media containing 2 × 106 2.0, and very strong when AAI >2.0. CFU/mL of the indicator strain, were added to all wells. The plates were then incubated at 30 ◦C or 37 ◦C for 18 h. Antimicrobial assay The FIC indices were calculated as FICA+FICB, where Disk diffusion assay FICA and FICB 18 are the minimum concentrations that The agar disk diffusion method was employed for inhibited the bacterial growth for essential oils A and B, the screening of antimicrobial activities of the essential oils respectively. Thus, FICs were calculated as follows: FICA [19-21]. The test was performed in sterile Petri dishes (90 = (MICA combination/MICA alone) and FICB = (MICB mm diameter) containing solid and sterile Mueller–Hinton combination/MICB alone). The results were interpreted as agar medium (Becton, Dickinson, USA). The essential oils synergy (FIC < 0.5), addition (0.5 ≤ FIC ≤ 1), indifference absorbed on sterile paper discs (5 µL per Whatman disc of (1 < FIC ≤ 4) or antagonism (FIC > 4). All experiments 6 mm diameter), were placed on the surface of the media were done in triplicate. previously inoculated with 100 µL of overnight microbial suspension (108 CFU/mL). One filter paper disc was placed Bioassays per Petri dish in order to avoid a possible additive activity. Serial dilutions (V/V) of each essential oil were Every dish was sealed with laboratory film to avoid made in absolute alcohol. A series of five dilutions and an evaporation and then incubated aerobically at either 30°C alcoholic control were selected for testing. Test or 37°C according to bacteria for 18–24 h, followed by concentrations varied from 40 to 90 ppm for essential oil. measurement of the zone diameter of the inhibition expressed in mm. Antibiotic discs of Ciprofloxacin (Cip. = Larvicidal effect bioassays 5 µg/disc), Penicillin (Pen. = 10 µg/disc) and Gentamicin Essential oils of plant were tested according to the (Gen. = 10 µg/disc) were used as positive controls. standard WHO protocol (1970) [24], with slight modifications [1]. For the experimental treatment, 1 mL of Determination of minimal inhibitory concentration test solution was mixed with 224 mL of distilled water in a (MIC): plastic cup. Then, twenty 3rd and 4th instar larvae gathered The minimal inhibition concentration (MIC) in 25 mL of distilled water were transferred to the cup. values were studied for the bacterial strains which were Each replicate set included two Anopheles gambiae batched sensitive to the essential oil in disc diffusion assay. for each dose tested and one control which consisted of 1 Minimal inhibition concentration (MIC) values were mL of absolute ethanol in 249 mL of distilled water.

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After a period of 24 h, mortality counts were Also poor effects were observed between A. klaineana and performed. Dead larvae were identified, when they did not C. schweinfurthii. The strong DPPH radical scavenging arouse after probing with a needle on the siphon or the action of Aucoumea klaineana and Dacryode sedulis cervical region. Moribund larvae were those unable to rise essential oils alone as well as in combination, might be to the surface (within a reasonable period of time) or unable attributed to the presence of some components within the to show the characteristic, diving reaction when the water antioxidant activity as the 1,8-cineole, α-pinene and β- was disturbed. They also showed discoloration, unnatural pinene [7, 11, 17; 25]. positions, tremors, uncoordinated or rigor. After each replicate, moribund and dead larvae were combined and Antimicrobial activity expressed as percent mortality at each concentration. Each The antimicrobial proprieties of the combination test spanned 3 or 4 replicates. Replicates with ≥15% of Aucoumea klaineana, Canarium schweinfurthii and mortality in the control were discarded from the analysis. Dacryodes edulis essential oils were evaluated against a set of 4 microorganisms (Table 2). Their potency was assessed Ovicidal efficacy bioassays qualitatively and quantitatively by the presence or absence 1 mL of test solution was mixed with 29 mL of of inhibition zones diameters (ZDs). The inhibition zone of distilled water in six plastic cups (of 115 mm diameter and essential oils varies from 10 to 23 mm for the bacteria. The 80 mm depth). Then, 20 recently-laid eggs were held in the essential oil of Canarium schweinfurthii exhibited more insectary and egg mortality was scored 24 h post-treatment. activity on E. coli (17 mm) and Bacillus cereus (19 mm) Eggs that did not hatch after the incubation period (24h) than Aucoumea klaineana and Dacryodes edulis essential were considered as dead. A total of three replicate were oils. All the paired combinations showed antibacterial carried out. activity in the inhibition zone diameters of bacterial strains tested. Combinations of D. edulis with C. schweinfurthii Statistical analysis exhibited more activity to Bacillus cereus (19 mm), For comparison of MIC and FIC values, tests were Streptococcus pyogenes (17 mm) and Staphylococcus made in triplicate. Analysis of variance was performed. aureus (15 mm) than the mixture of the A. klaineana with Significant differences between means were determined by C. schweinfurthii or the D. edulis. It is interesting to note Fisher’s test at the threshold of (p < 0.05). that the mixture of D. edulis with C. schweinfurthii presents an antibacterial activity stronger than Ciprofloxacin and RESULTS AND DISCUSSION Gentamicin in the inhibition zones diameters of The essential oils Air-dried resins of Escherichia coli, Streptococcus pyogenes and Aucoumeaklaineana, Canarium schweinfurthii and Staphylococcus aureus. The best inhibition zone diameter Dacryodes edulis medicinal plants that subjected to hydro- in combination of A. klaineana with C. schweinfurthii was distillation using a Clevenger-type apparatus and the obtained for E. coli (19 mm). The mixture of C. specific coloured oils, yielded of 3.62% (w/w), 6.86% schweinfurthii with A. klaineana had an antibacterial (w/w) and 10.0% (w/w) for Aucoumea klaineana, activity stronger than Ciprofloxacin against Escherichia Canarium schweinfurthii and Dacryodes edulis, coliand Streptococcus pyogenes. The correlation between respectively. Comparing of the results of [7; 11; 17], this two different essential oils was generally related to larger was better as medicinal resin oils extraction remains a inhibition zone diameters. The Table 2 indicates that challenging to be isolated. essential oils displayed a variable degree of antimicrobial activity overall the different strains tested [7; 11; 17]. Antioxidant activity The antioxidant activities of Aucoumea klaineana, Minimum inhibitory concentration (MIC) and minimum Canarium schweinfurthii and Dacryodes edulis essential bactericidal concentration (MBC) oils are pointed out in Table 1. As it can be seen, the AAI The MICs and MBCs values are summarized in of essential oils ranged from 0.82 to 2.07 and can be Table 3 that are confirmed the antibacterial activity of the compared to AAI of Vitamin C and BHT (AAI values of plants. The essential oil of C. schweinfurthii exhibited the 11.32 and 7.85, respectively) while those of combination best antibacterial activity for Staphylococcus aureus ranged from 0.48 to 1.93. In addition to that, Table 1 shows (CMI=0.5 mg/mL), Bacillus cereus (CMI=3.09 mg/mL), the DPPH radical inhibition effect. The essential oils Streptococcus pyogenes (CMI=4 mg/mL) and E. coli exhibited a strong DPPH free radical scavenging action (CMI=5.88 mg/mL). The C. schweinfurthii essential oil (IC50 values ranged from 48 to 78 µg/mL for essential oils obtained bactericidal activity for Staphylococcus aureus and 20.37 to 82.61 µg/mL for combination). This showed a and Streptococcus pyogenes. It was found as a weak scavenging activity in comparison to the activity of bacteriostatic against Bacillus cereus and E. coli. The oil of vitamin C and BHT (IC50 values of 3.48 µg/mL and 6.30 D. edulis was found within bactericidal properties with µg/mL, respectively). However, combinations of D. edulis MIC and MBC equal to 1 and 8 of Staphylococcus aureus with A. klaineana or C. schweinfurthii had strong effects. and Streptococcus pyogenes, respectively.

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The D. edulis essential oil was found bacteriostatic oils. Also, the results showed that one or more synergistic against Bacillus cereus. The essential oil of Aucoumea components can produce the desired antibacterial effect. klaineana showed a strong antibacterial activity with MIC and MBC equal to 2, 6.08 and 16 on Staphylococcus Ovicidal and larvicidal activities against Anopheles aureus, Bacillus cereus and Streptococcus pyogenes. A. gambiae klaineana oil was found bacteriostatic against Escherichia A.klaineana, C. schweinfurthii and D .edulis coli. These results were similar to those of previous studies essential oils exhibited larvicidal activity as revealed in in Obame et al., [7; 11; 17], which have reported that WHO (2005) [1], where standard protocols against 3rd and essential oils extracted from resins of Aucoumea klaineana, 4th instar larvae of field-collected mosquitoes vectors of Canarium schweinfurthii and Dacryodes edulis exhibited human disease members of the Anopheles gambiae were both antibacterial activities against bacteria Gram negative described. The median lethal concentration (LC50) of and positive. Anopheles gambiae larvae ranged between 9.94-22.98 µg/mL (Table 4). Ovicidal efficacy against Anopheles Interaction studies gambiae was demonstrated. The LC50 value of Anopheles The fractional inhibitory concentration (FIC) gambiae ranged between 22.08-67.64 µg/mL. Dacryodes indices ranged from 0.11 to 7.13 for paired combinations of edulis showed the highest activity against the Anopheles Aucoumea klaineana, Canarium schweinfurthii and gambiae eggs (LC50=22.08 µg/mL) and larvae (LC50=9.94 Dacryodes edulis essential oils (Table 3). All the paired µg/mL). Among the three plants, resin essential oils from combinations showed synergistic effects against Aucoumea klaineana had the lowest activity to Anopheles Streptococcu spyogenes and Staphylococcus aureus except gambiae eggs (LC50=63.60 µg/mL) and larvae (LC50=22.98 for the combination of A. klaineana and C. schweinfurthii. µg/mL). Larvae were more susceptible than eggs. All the Combinations of A. klaineana with D. edulis or C. paired combinations showed lethal activity against the schweinfurthii antagonized effects on the inhibition of Anopheles gambiae eggs and larvae. The differences in the Bacillus cereus and E. coli. Only combinations of D. edulis observed LC50 values are likely to indicate that the with C. schweinfurthii encountered some antagonism Anopheles gambiae eggs and larvae experienced different effects against Bacillus cereus while A. klaineana levels of susceptibility to plant essential oils. The results combined to C. schweinfurthii got indifferent effects support of previous data by Bassolé et al. (2003) [28], against Bacillus cereus. The interaction synergy, indifferent reported that essential oils extracted from leaves of and antagonism as well as addition effects, between two Cymbopogon proximus, Lippia multiflora and Ocimum compounds were depended on the concentrations of the canum, exhibited both ovicidal and larvicidal activities single component and the overall susceptibility of the target against Aedes aegypti and Anopheles gambiae. microorganism. This may explain variation of interaction The larvicidal activity of selected plant essential observed between combinations and strains. The efficacy of oils against important vector mosquitoes: Dengue Vector, combinations appears to be related to chemical composition Aedes aegypti, malarial vector, Anopheles stephensi and of combined essential oils and to possible interactions filarial vector, Culex quinquefasciatus were tested against between their major components [19,26,27]. mature-stage larvae of Anopheles funestus [23;30]. Ahadji- The present study demonstrated the potential of Dabla et al. (2015) [31], showed that natural botanical the combination of Aucoumea klaineana, Canarium Biostop Mosquitoes (BM) and physiological changes had a schweinfurthii and Dacryodes edulis essential oils to high larvicidal activity against both strains of Anopheles increase antibacterial activity. The best synergistic effects gambiae and elicited a wide range of physiological among essential oils were obtained with combined essential changes.

Table 1. Antioxidant Activity Index (AAI) of essential oils by DPPH free radical scavenging method. 2 Essential oils Equations R IC50 (µg/mL) AAI Activity AK Y=2.4111X+4.0410 0.9887 48.00± 2.3 2.07 Very strong DE Y=1.4114X–2.3630 0.9203 78.00± 0.5 1.06 strong CS Y=1.0337X+0.2778 0.9910 68.00± 3 0.82 moderate AK-DE Y=2.1392X+2.0003 0.9948 22.44± 0.26 1.76 strong AK-CS Y=0.6177X-1.0292 0.9664 82.61± 0.3 0.48 poor DE-CS Y=2.4479X+0.1312 0.9999 20.37± 0.6 1.93 strong Vitamin C Y=14.559X-0.6130 0.9996 3.48± 11.32 Very strong BHT Y=5.659X+11.5130 0.9966 6.30± 7.85 Very strong AK = Aucoumea klaineana; DE = Dacryodes edulis; CS = Canarium schweinfurthii, BHT= butylated hydroxyl toluene: positive control.

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Table 2. Antimicrobial activities of essential oils: Screening by inhibition zone diameters (mm). Hospital strains Essential oils Escherichia coli Bacillus cereus Streptococcus pyogenes Staphylococcus aureus AK 10 ± 1.15 12 ± 2.08 13 ± 1.41 14 ± 1 DE 13 ± 2 12 ± 2.65 17 ± 0.71 10 ± 1 CS 17 ± 0.58 19 ± 2.31 11 ± 0.58 23 ± 2.52 AK-DE 15 ± 0.71 14 ± 2.08 13 ±0.71 13 ± 1.73 AK-CS 19 ± 2.12 14 ± 1.15 16 ± 1.53 13 ± 2.08 DE-CS 17 ± 1.15 19 ± 2.65 17 ± 1.41 15 ± 1 Cip 13 ± 2.04 35 ± 2.64 15 ± 5.93 ND Pen ND 12± 1.74 14± 2.06 19± 2.30 Gen ND ND ND 14 ± 1.90 AK = Aucoumea klaineana; DE = Dacryodes edulis; CS = Canarium schweinfurthii; Cip. = Ciprofloxacin (5 µg); Pen= Penicillin (10 µg), Gen=Gentamicin (10 µg); ND = Non determined.

Table 3. Antibacterial activities (MIC, MBC), Fractional Inhibitory Concentration (FIC) and interaction between essential oils. Hospitalstrains Essential oils Escherichia coli Bacillus cereus Streptococcus pyogenes Staphylococcus aureus MIC 22 6.08 16 2 AK MBC 41.60 6.08 16 2 MIC 1 40.30 8 1 DE MBC 1 76.50 8 1 MIC 5.88 3.09 4 0.50 CS MBC 11.20 5.88 4 0.50 FIC ND 4.58 0.22 0.17 AK-DE Interaction ND An S S FIC 4.01 1.94 ND 0.30 AK-CS Interaction An I ND S FIC ND 7.13 0.21 0.25 DE-CS Interaction ND An S S Cip. = Ciprofloxacin (5 µg) ; Gen. = Gentamicin (10 µg) ; AK = Aucoumea klaineana ; DE = Dacryodes edulis ; CS = Canarium schweinfurthii ; Ad= addition; An=antagonism; I= indifference; S= synergism; ND = Non determined.

Table 4. Ovicidal and larvicidal activities against Anopheles gambiae. Ovicidal activity Larvicidal activity Essential oils CL₅₀ CL₅₀ Equations R2 Equations R2 (µg/mL) (µg/mL) AK 63.60 Y=0.6512X+8.577 0.9612 22.98 Y=1.8362X+7.796 0.9548 CS 67.64 Y=1.4503X–48.101 0.9969 10.14 Y=3.7512X+11.945 0.9037 DE 22.08 Y=1.9058X+7.910 0.9529 9.94 Y=3.8801X+11.429 0.9098 AK = Aucoumea klaineana, CS = Canarium schweinfurthii, DE = Dacryodes edulis

CONCLUSION The use of natural products of plant origin increase ovicidal efficiency and larvicidal ability against constitutes an alternative approach for malaria control and Anopheles gambiae, antioxidant and antimicrobial better efficacy for combating various infections and dry- proprieties. This overlapped the multiplicity of the uses of skin resistance. The useful results assessed subsequently for plants in treating many infectious diseases in Tropical areas the combination of Aucoumea klaineana, Canarium including Africa and particularly in Gabon. schweinfurthii and Dacryodes edulis essential oils against pathogen bacteria and the Anopheles gambiae eggs as well ACKNOWLEDGEMENTS as larvae. The present study has demonstrated the potential The authors are very much thankful to the Shell of the combination of Aucoumea klaineana, Canarium Gabon for the financial support of materials in Laboratoire schweinfurthii and Dacryodes edulis essential oils to de Recherche en Biochimie (LAREBIO) USTM.

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CONFLICT OF INTEREST The authors declare that there are no competing interests. All the authors read and approved the final version.

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27. Goñi P, López P, Sánchez C, Gómez-Lus R, Becerril R, Nerín C. Antimicrobial activity in the vapour phase of a combination of cinnamon and clove essential oils. Food Chem, 116, 2009, 982–989. 28. Bassolé IH, Guelbeogo WM, Nebie R, Constantini C, Sagnon N, Kabore Z, Traore SA. Ovicidal and larvicidal activity against Ae aegypti and An gambiae complex mosquitoes of essential oils extracted from three spontaneus plants of Burkina Faso. Parassitologia, 45(1), 2003, 23-6. 29. Pugazhvendan SR and Elumali K. Larvicidal activity of selected plant essential oil against important vector mosquitoes: dengue vector, Aedes aegypti (L.), malarial vector, Anopheles stephensi (Liston) and filarial vector, Culex quinquefasciatus (Say) (Diptera: Culicidae). Middle-East J. of Scientific Research, 18 (1), 2013, 91-95. 30. Akono-Ntonga P, Baldovini N, Mouray E, Mambu L, Belong P and Grellier P. Activity of Ocimum basilicum, Ocimum canum, and Cymbopogon citratus essential oils against Plasmodium falciparum and mature-stage larvae of Anopheles funestus s.s. Parasite, 21, 2014, 33. 31. Ahadji-Dabla KM, Brunet JL, Ketoh GK, Apétogbo GY, Glitho IA and Belzunce LP. Larvicidal activity of a natural botanical biostopmoustiques and physiological changes induced in susceptible and resistant strains of Anopheles gambiae Giles (Diptera: Culicidae). The Open Entomol J, 9, 2015, 12-19.

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