Academia Journal of Medicinal Plants 6(10): 331-341, October 2018 DOI: 10.15413/ajmp.2018.0166 ISSN 2315-7720 ©2018 Academia Publishing

Research Paper

In vitro antisalmonellal and antioxidant activities of Canarium schweinfurthii stem bark extracts

Accepted 24th September, 2018

ABSTRACT

The use of antibiotics has been, and continues to be the main control strategy for avian salmonellosis, as well as typhoid and paratyphoid fevers in humans. Resistance of Salmonella to commonly used antimicrobials is increasing both in the veterinary and human health sectors and has emerged as a global problem. This resistance has been linked to the inappropriate use of antibiotics, especially in poultry farm. New control strategies of salmonellosis are needful. This study therefore aimed at evaluating in vitro antisalmonellal and antioxidant power of a medicinal plant, Canarium schweinfurthii Engl. (Burseraceae) commonly used as antityphoid plant in oder to implement it in poultry farm. For antisalmonellal assay, serial microdilution method was used to determine the MIC and MBC of plants extracts against four isolates and one strain of Salmonella; the antioxidant activities of the extracts, as well as the phytochemical screening, were performed using different standard methods. The result showed that most of the extracts exhibited minimum inhibitory concentrations (MICs) ranging from 64 to 1024 1 Jean Baptiste Sokoudjou , Guy Sedar µg/ml; ethanolic and hydro-ethanolic extract being the most effective as compared Singor Njateng1, Siméon Pierre Chegaing Fodouop2, Norbert Kodjio1, Serge Secco with aqueous extracts. As far as the antioxidant activities are concerned, all the Atsafack1, Alain Bertrand Fowa1, Merline tested concentrations of ethanolic and hydro-ethanolic extracts exhibited good Namekong Djimeli1 and Donatien DPPH scavenging activities as compared with the aqueous extracts. All extracts 1 Gatsing * exhibited a comparable NO radical scavenging activity at 100 µg/ml. The

1Laboratory of Microbiology and antioxidant activities were positively associated with the total phenolic contents. Antimicrobial Substances (LAMAS), The phytochemical results showed that stem bark extracts of C. schweinfurthii Faculty of Science, University of Dschang, contain various secondary metabolites such as alkaloids, anthocyanins, phenols, P. O. Box 67, Dschang, Cameroon. flavonoids, saponins, steroids, tannins and terpenoids. The obtained results 2Department of Biomedical Sciences, University of Ngaoundéré, P. O. Box 454, showed that, hyro-ethanolic stem back extract of C. schweinfurthii could be used to Ngaoundéré, Cameroon. control avian salmonellosis as well as typhoid and paratyphoid fevers.

*Corresponding author. E-mail: Key words: Salmonellosis, antisalmonellal activity, antioxidant activity, Canarium [email protected]. Tel: +237 677 51 67 40. schweinfurthii.

INTRODUCTION

Salmonellosis caused by Salmonella sp., a Gram negative Salmonella sp. have been detected in poultry. The most bacterium, is an important disease of chicken all over the important are Salmonella Typhimurium and Salmonella world. Avian salmonellosis is an important disease causing Enteritidis (Ali and Sultana, 2012) known to be infectious serious impediment to the development of poultry industry to human. Human salmonellosis caused by these serovars especially in developing countries of Asia and Africa generally appears as enteric illness (gastroenteritis), (Rajagopal and Mini, 2013). Poultry can become infected whereas other serovars are causative agent of typhoid and with many different types of Salmonella; about 10% of all paratyphoid fevers in humans. Salmonellosis is one of the Academia Journal of Medicinal Plants; Sokoudjou et al. 332

most common causes of food poisoning in humans. Moreover, extracts were obtained by macerating the same quantity of typhoid fever caused by Salmonella Typhi constitutes a major powder in different hydroethanolic (v/v) mixture of solvent public health problem in developing countries, where it (30/70, 50/50 and 70/30). After 48 h, these mixtures were remains an endemic disease (Tsafack et al., 2017a). In animals, filtered using Whatman №1 filter paper. The filtrates were gastrointestinal infections can be controlled using antibiotics, concentrated at 60C in a rotary evaporator under reduced but their use is strongly discouraged in many countries pressure. The residues which constitute the crude extracts because of the growing awareness of antimicrobial-resistance were recovered in flasks and then left in an oven at 40C problems. In many countries, the use of those antibiotics as until complete evaporation of the solvent. All extracts were growth promoters in farm animals is prohibited and therefore an interest in alternative products with antibacterial or anti- then kept at 4C until further use. inflammatory properties has increased (Varmuzova et al., 2015). Salmonella infection can leads to production of superoxide and nitric oxide radical which react together to Antisalmonellal assay form peroxynitrite, a strong biological oxidant (Rastaldo et al., 2007). Consequently, pathological conditions characterized by Chemicals for antisalmonellal assay oxidative stress can greatly result from Salmonella infection. Therefore, medicinal plants with both antimicrobial and Ciprofloxacin (Sigma-Aldrich, St Quentin Fallavier, France) antioxidant properties are important because they can manage and oxytetracyclin (pantex, Holland) were used as oxidative stress linked to Salmonella infection (Hanasaki et al., reference antibiotic. p-iodonitrotetrazolium chloride (INT) 1994; Cook and Samman, 1996). Today, there is a renewed was used as microbial growth indicator. interest in traditional medicine and an increasing demand for more drugs from plant sources. This revival of interest in plant-derived drugs is mainly due to the fact that these Test bacteria and culture media medicinal herbs are safe and more dependable than the costly synthetic drugs, many of which have adverse side effects The clinical bacterial isolates (Salmonella Typhi, Salmonella (Pamplona, 1999 ; Ayachi et al., 2009). In this order, several Paratyphi A, Salmonella Paratyphi B and Salmonella investigations are focused on medicinal plants as new Typhimurium) were collected from Pasteur Centre, strategies to control human (Tala et al., 2015 ; Kodjio et al., 2016 ; Atsafack et al., 2016) or avian salmonellosis (Ayachi et Yaoundé-Cameroon; the strains Salmonella Typhi al., 2009 ; Varmuzova et al., 2015 ; Abiala et al., 2016 ; Tchoua, ATCC6539 was obtained from American Type Culture 2016). Among these plants is C. schweinfurthii Engl. Collection. The culture media used, namely Salmonella- (Burseraceae), used by traditional healers and elders to treat Shigella Agar (SSA) and Mueller Hinton Broth (MHB), were typhoid fever in West region of Cameroun (Tsobou et al., manufactured by AccumixTM (Belgique). SSA was used for 2013). This study aimed at evaluating the in vitro effect of C. the activation and isolation of the Salmonella species, and schweinfurthii Engl. stem barks extracts on bacteria belonging MHB was used for antibacterial tests. to Salmonella genus known to be responsible for the economic losses in poultry production and for public health hazard in developing countries. In addition, in vitro antioxidant activities Determination of minimal inhibitory concentrations of extracts were assessed. (MICs) and minimal bactericidal concentrations (MBCs)

The MIC values of the C. schweinfurthii extracts and MATERIALS AND METHODS reference antibiotics (that is, oxytetracyclin and ciprofloxacin used as positive control) on the aforemention Plant collection and identification bacteria were determined using rapid INT colorimetric assay (Eloff, 1998; Mativandlela et al., 2006). Briefly, each C. schweinfurthii stem barks were harvested in Bamougoum, extracts was dissolved in 5% Dimethyl-sulfoxide situated in Mifi division, West region of Cameroon, in July (DMSO)/Mueller Hinton Broth (MHB). The solution 2016. This plant was identified at the National Herbarium at Yaoundé-Cameroon, where a voucher specimen was deposited obtained was then added to 100 µl of MHB, and two-fold under the reference Number 16929/SRF/Cam. serially diluted (in a 96-wells microplate). This was 6 followed by the addition of 100 µl of inoculum (1.5 × 10 CFU/ml) prepared in MHB. The plates were covered with a Preparation of different extracts of Canarium sterile plate sealer, agitated to mix the contents of the wells schweinfurthii using a shaker and incubated at 37C for 18 h. Wells containing MHB and 100 µl of inoculum served as negative The aqueous extracts (macerated, infused and decocted) control. The total volume in each well was 200 µl. The MICs were prepared using the method described by Duke (2000). of samples were detected after 18 h incubation at 37C, The ethanolic extract was obtained by macerating 50 g of following addition of 40 µl of INT (0.2 mg/ml) and powder in 500 ml of 95° ethanol, whereas hydroethanolic reincubation at 37C for 30 min. Viable bacteria reduced Academia Journal of Medicinal Plants; Sokoudjou et al. 333

Table 1: Specific reagents and reactions of phytochemical screening.

Phytochemical group Specific reagent or reaction Positive results Alkaloids Mayer’s reagent Turbidity of the resulting precipitate

Phenolic compounds Tannins Stiany reaction (FeCl3) Dark green solution Flavonoids Shinoda reaction Appearance of a reddish color

Phenols FeCl3 + potassium ferrocyanide Appearance of the greenish-blue color

Quinonic Coumarins Bornträger reaction-UV Intense inflorescence compounds anthraquinons 10% of HCl, CHCl3 and 10% NH3 Formation of rose-pink color Anthocyanins Ice-cold solution of HCl 1% Colors ranging from orange red to greenish blue Saponins Foam index Formation of strong and stable foam (>1 cm height) Steroids and terpenoids Lieberman-Buchard reaction (chloroform, Purplish red color, blue color glacial acetic anhydride, and H2S04)

the yellow dye to pink. MIC was defined as the lowest acid (Vitamin C) was used as control. Each assay was done concentration of the sample that prevented this change and in triplicate and the results, recorded as mean ± standard exhibited complete inhibition of microbial growth. The MBC deviation of three findings, were illustrated in tabular form. was determined by adding 50 µl aliquots of the The percentage of DPPH scavenging activity (%DPPHsa) preparations, which did not show any growth after was calculated according to the following equation: incubation during MIC assays, to 150 µl of adequate broth. These preparations were then incubated at 37C for 48 h. %DPPHsa = [(Absorbance of DPPH - Absorbance of The MBC was recorded as the lowest concentration of sample)/Absorbance of DPPH] × 100. extract which did not produce a color change after addition of INT as previously described. These tests were performed The radical scavenging percentages were plotted against in triplicates. The ratio MBC/MIC was calculated to logarithmic values of the concentration of test samples and determine the bactericidal (MBC/MIC ≤ 4) or bacteriostatic a linear regression curve (y = ax) was established in order (MBC/MIC > 4) activities of the extracts (Carbonelle et al., to calculate IC50, which is the amount of sample necessary 1987; Gatsing et al., 2006). to inhibit by 50% the free radical (DPPH).

Phytochemical screening Ferric reducing antioxidant power (FRAP) assay

After obtaining the crude extracts from plant material, The ferric reduction potential (conversion potential of Fe3+ qualitative phytochemical screening was performed as to Fe2+) of C. schweinfurthii extracts was determined described in Table 1 to obtain an idea regarding the type of according to the method described by Padmaja et al. phytochemicals existing in each extract. (2011). To do this, 1 ml of extract at different concentrations (200, 100, 50, 25 and 12.5 μg/ml) was mixed with 2.5 ml of a 0.2 M phosphate buffer solution (pH Antioxidant assay 6.6) and 2.5 ml of a solution of potassium ferricyanide K3Fe(CN)6 at 1%. The mixture was incubated in a water 2, 2-Diphenyl-picryl-hydrazyl (DPPH) radical scavenging bath at 50C for 20 min; then 2.5 ml of 10% trichloroacetic assay acid was added to stop the reaction, and tubes were centrifuged at 3000 rpm for 10 min. An aliquot (2.5 ml) of The free radical scavenging activities of the C. schweinfurthii supernatant was combined with 2.5 ml of distilled water stem barks extracts were evaluated using the DPPH assay and 0.5 ml of 0.1% methanolic FeCl3 solution. The method as described by Mensor et al. (2001) and Noghogne absorbance of the reaction medium was read at 700 nm et al. (2015). Briefly, the extracts (2000 µg/ml) was two- against a similarly prepared control, replacing the extract fold serially diluted with methanol. 100 µl of diluted extract with distilled water. The positive control was represented were mixed with 900 µl of 0.3 mM 2,2-diphenyl-1- by a solution of a standard antioxidant, ascorbic acid whose picrylhydrazyl (DPPH) methanol solution to give a final absorbance was measured under the same conditions as extract concentration of 12.5, 25, 50, 100 and 200 µg/ml. the samples. Increased absorbance of the reaction mixture After 30 min of incubation in the dark at room temperature, indicates higher reduction capacity of the tested extracts the optical densities were measured at 517 nm. Ascorbic (Mohammed et al., 2013). Academia Journal of Medicinal Plants; Sokoudjou et al. 334

Table 2: Yields and physical appearance of Canarium schweinfurthii stem barks extracts.

Physical characteristics Extracts Yields (%) Color Physical appearance Decoction 1.372 Dark brown Tender Infusion 1.228 Dark brown Bright powder Maceration 0.396 Dark Powder EE 95° 1.474 Dark Tender HEE 30/70 1.843 Dark Tender HEE 50/50 1.466 Dark brown Crystal HEE 70/30 1.760 Dark Crystal

EE: 95°ethanolic extract; HEE: Hydro-ethanolic Extract (v/v).

Nitric oxide (NO) radical scavenging activity gallic acid (0.2 g/l; 0-70 µl).

Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates NO (Green et al., 1982), which Evaluation of total flavonoids contents interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent (1% sulphanilamide, 0.1% The determination of total flavonoids content was done naphthylethylenediamine dichloride in water). Briefly, to using the colorimetric aluminum chloride method. In fact, 750 µl of sodium nitroprusside, 500 µl of extract or ascorbic 100 µl of extract (2 mg/ml) were mixed with 1.49 ml of acid (used as a positive control) was added at different distilled water and 30 µl of NaNO2 (5%). After 5 min concentrations (12.5, 25, 50, 100 and 200 µg/ml). The incubation at room temperature, 30 µl of AlCl3 (10%) was mixture was then incubated at room temperature for 120 added and the mixture was reincubated for 6 min; then 200 min. The blank was prepared by replacing extract with µl of sodium hydroxide (0.1 M) and 240 µl of distilled water methanol. At the end of the incubation time, 1.25 ml of were added. The solution was well mixed and the Griess reagent was added. The absorbance of the absorbance was read at 510 nm using a spectrophotometer. chromophores was formed during the diazotization of Each test was performed in triplicate and the results were nitrite with sulphanilamide, and subsequent coupling with expressed as milligrams of Equivalents Catechin (mgECat) naphthylethylenediamine dichloride (NED) was read at 540 per gram of extract using a standard calibration curve of nm using a spectrophotomer and referred to the Catechin (0.1 g/l; 0-70 µl). absorbance of Ascorbic Acid. Inhibition percentages of the nitrite oxide generated were measured by comparing the absorbance values of control and test samples. The Statistical analysis percentage of inhibition was calculated according to the following equation: The results were expressed as mean ± Standard deviation. Statistical analysis was carried out using Statistical Package Nitric oxide scavenged (%) = [(A1 – A2)/A1] × 100 for Social Sciences Software program (SPSS 16.0). Statistical analysis of data was performed by one way analysis of A1=Absorbance of negative control, A2=Absorbance of the variance (ANOVA), followed by Waller-Duncan test. P extract or standard (Ascorbic Acid) values < 0.05 were considered as significant.

Evaluation of total phenolic contents RESULTS

The Folin-Ciocateu method was used for the quantitative Yields and physical features of Canarium schweinfurthii determination of total phenolic compounds. The reaction extracts mixture consisted of 0.02 ml of extract (2000 µg/ml), 1.38 ml of distilled water, 0.02 ml of 2N Folin-ciocalteu reagent After the extraction of C. schweinfurthii Engl. stem bark, and 0.4 ml of a 20% sodium carbonate solution. The differences were observed at the level of their yields as well mixture was then incubated at 40C for 20 min and the as their physical features. Table 2 shows the yields, the absorbance was measured at 760 nm. The blank was color and physical appearance of the various extracts of C. prepared with distilled water instead of plant extracts. Each schweinfurthii: yields varied from 0.396 to 1.843, while the test was performed in triplicate and the results were color varied from brown to dark. As far as the physical expressed as milligrams of Equivalents Gallic Acid (mgEGA) appearance is concerned, the extracts were tender, powder per gram of extract using a standard calibration curve of or crystal. Academia Journal of Medicinal Plants; Sokoudjou et al. 335

Table 3: Inhibition parameters (MIC, MBC) of Canarium schweinfurthii stem barks extracts against different test microorganisms.

Strain/isolates Tested samples Studied parameter ST STs STM SPA SPB Decoction MIC (µg/ml) 256 256 512 128 256 MBC (µg/ml) >1024 1024 1024 1024 1024 MBC/MIC / 4 2 8 4

Infusion MIC (µg/ml) 1024 1024 512 1024 512 MBC (µg/ml) 1024 >1024 >1024 1024 >1024 MBC/MIC 1 / / 1 /

Maceration MIC (µg/ml) 256 512 128 128 1024 MBC (µg/ml) >1024 1024 >1024 1024 >1024 MBC/MIC / 2 / 8

EE 95° MIC (µg/ml) 128 128 64 128 256 MBC (µg/ml) 512 512 1024 1024 512 MBC/MIC 4 4 16 8 2

HEE 30/70 MIC (µg/ml) 512 128 128 256 128 MBC (µg/ml) >1024 512 256 1024 1024 MBC/MIC / 2 2 4 8

HEE 50/50 MIC (µg/ml) 256 128 64 64 64 MBC (µg/ml) 512 512 256 512 1024 MBC/MIC 2 4 4 8 16

HEE 70/30 MIC (µg/ml) 1024 512 1024 512 512 MBC (µg/ml) >1024 >1024 >1024 1024 >1024 MBC/MIC / / / 2 /

MIC (µg/ml) 8 8 4 4 8 Oxytetracyclin MBC (µg/ml) 32 64 32 64 64 MBC/MIC 4 8 4 16 8

MIC (µg/ml) 0,5 1 4 0,5 1 Ciprofloxacin MBC (µg/ml) 2 2 8 4 4 MBC/MIC 4 2 2 8 4

ST: Salmonella Typhi, STs: Salmonella Typhi ATCC6539, SPA: Salmonella Paratyphi A, SPB: Salmonella Paratyphi B, STM: Salmonella Typhimurium. EE: 95°ethanolic extract; HEE: Hydro-ethanolic Extract, MIC = Minimum inhibitory concentration; MBC = Minimum bactericidal concentration.

Antimicrobial assay activity (64≤MIC≤1024 µg/ml) on the tested microorganisms. The ethanolic (EE 95°) and hydro- Inhibition parameters (MIC, MBC, MBC/MIC ratio) of C. ethanolic extracts (HEE 50/50, v/v) have the lowest MIC schweinfurthii stem barks extracts against pathogenic values (64≤MIC≤256 µg/ml), indicating greater bacteria namely Salmonella Typhi (ST); Salmonella Typhi antisalmonellal activity. As compared with all the tested ATCC6539 (STs) ; Salmonella Paratyphi A (SPA); extracts, the hydro-ethanolic extract (HEE 50/50) have the Salmonella Paratyphi B (SPB) and Salmonella Typhimurium best actisalmonellal activity with MIC value of 64 µg/ml (STM) are shown in Table 3. The extracts have variable against Salmonella Typhimurium, Salmonella Paratyphi A Academia Journal of Medicinal Plants; Sokoudjou et al. 336

Table 4: Phytochemical composition of aqueoux, éthanolic and hydro-éthanolic extracts of Canarium schweinfurthii.

Phytochemical groups Decoction Infusion Maceration EE 95° HEE 30/70 HEE 50/50 HEE 70/30 Alcaloïds - + + + - - - Anthocyanins - - - + - - - Anthraquinons ------Phenols + + + + + + + Flavonoïds + + + + + + + Saponins - - + - - + + Steroïds - - + + + + - Tannins - - - + + - - Terpenoids + + + + - + +

EE: 95°ethanolic extract; HEE: Hydro-ethanolic Extract (v/v): +: Présence; - : Absence.

and Salmonella Paratyphi B; 128 and 256 µg/ml against activities of the aqueous extracts are lower than that of Salmonella Typhi ATCC6539 and Salmonella Typhi (ST), vitamin C, while those of the hydro-ethanolic and ethanolic respectively. MIC values of other hydro-ethanolic extracts extracts are greater than that of the vitamin C. range between 128 and 1024 µg/ml, while decoction, infusion and maceration are the less active extracts with MIC ranging from 128 to 1024 µg/ml (Table 3). Ferric reducing antioxidant power (FRAP)

The results of ferric reducing antioxidant power (FRAP) of Phytochemical composition of Canarium schweinfurthii extracts at different concentrations, determined through extracts the reduction of Fe3+ (that is, Fe3+↦Fe2+), are shown in Table 6. It appears from this table that at all concentrations, The qualitative phytochemical composition of each extract ascorbic acid (standard) exhibited the greatest ferric was evaluated and the results are shown in Table 4. The reducing activity. The 50/50 hydro-ethanolic extract phytochemical screening of the various extracts of C. exhibited the lowest activity at 12.5, 25 and 50 μg/ml as schweinfurthii showed the presence of different groups of compared with all other extracts. At a concentration of 12.5 secondary metabolites, including alkaloids, anthocyanins, μg/ml, the maceration and HEE 30/70 proved to be more phenols, flavonoids, saponins, steroids, tannins and active than all the other extracts. At a concentration of 200 terpenoids. Phenols and flavonoids were presents in all the μg/ml, except decoction and infusion, the other extracts extracts. Anthocyanins were presents only in EE 95°, while showed comparable activity but this activity was lower terpenoids were only absent in HEE 30/70. compared to that of vitamin C.

Antioxidant activities Nitric oxide (NO) radical scavenging activity

2, 2-Diphenyl-picryl-hydrazyl (DPPH) radical scavenging The radical scavenging activity of various extracts of C. assay schweinfurthii against NO radical is shown in Table 7. The nitric oxide scavenging activity of the extracts of C. The DPPH radical scavenging activity of the C. schweinfurthii ranged from 26.33 to 47.80%. At 200 µg/ml, schweinfurthii extracts is shown in Table 5. It appears that all the extracts exhibited a comparable NO radical at all the tested concentrations, the activities of ethanolic scavenging activity lower than that of vitamin C. The most and hydro-ethanolic extracts are higher than the activity of active extracts at 12.5µg/ml and 200 µg/ml were obtained, aqueous extracts. The HEE 30/70 (v/v) extract showed the respectively with infusion and HEE 30/70. best radical scavenging activity (ranging from 83.06 to 96.19%) as compared with all the other extracts and vitamin C. The antiradical activity of the ethanolic extract Total phenolic and flavonoid contents of Canarium and those of all hydroethanolic extracts were significantly schweinfurthii stem barks extracts (p <0.05) greater than that of vitamin C at all concentrations, except at the concentration of 12.5 μg/ml Table 8 shows the total phenolic and flavonoid contents of where the activities of ethanolic, hydro-ethanolic 70/30 C. schweinfurthii stem barks extracts. The total phenolic extract and vitamin C were not statistically different. When content ranged from 172.80 mgEGA/g of extract to 410.68 the concentration of the extracts is equivalent to IC50, the mgEGA/g of extract, while the flavonoids content ranged Academia Journal of Medicinal Plants; Sokoudjou et al. 337

Table 5: Reducing power activities of the extracts of Canarium schweinfurthii as well as vitamin C.

Concentration (µg/ml) and IC50 (µg/ml) of extracts Extracts 12.5 25 50 100 200 IC50 Decoction 42.95±1.06d 68.47±3.01c 84.56±2.60c 89.72±0.84d 90.76±0.95e 13.69 Infusion 45.54±3.68d 66.21±1.57c 87.53±0.73b 91.53±0.29c 91.92±0.55d 13.13 Maceration 62.57±1.08c 86.23±0.93b 87.21±0.39b 87.78±0.08e 87.99±0.39f 11.81 EE 95° 72.09±1.16b 91.92±0.29a 93.54±0.22a 95.09±0.11a 95.41±0.40a 09.73 HEE 30/70 83.06±1.16a 93.51±0.09a 93.83±0.09a 94.80±0.18ab 96.19±0.37a 09.29 HEE 50/50 83.11±1.82a 93.56±0.48a 93.72±0.55a 93.89±0.55b 94.37±0.16bc 09.45 HEE 70/30 75.56±1.58b 94.10±0.49a 94.80±0.24a 95.23±0.24a 95.33±0.16ab 09.47 Vitamin C 76.17±6.69b 86.18±0.62b 87.26±0.75b 90.15±1.03d 93.46±0.37c 10.61

EE: 95°ethanolic extract; HEE: Hydro-ethanolic Extract (v/v). In each column, values with the different letter are significantly different (p<0.05 ; Waller-Duncan test).

Table 6: Ferric reducing power of the extracts of Canarium schweinfurthii stem barks.

Concentration of extracts (µg/ml) and absorbance of plant extracts Extracts 12.5 25 50 100 200 Decoction 0.48±0.120a 0.59±0.040b 0.68 ±0.017ab 0.84 ±0.069a 0.90 ±0.075a Infusion 0.23±0.211a 0.96±0.038c 0.72 ±0.091ab 0.97 ±0.085ab 1.25 ±0.235ab Maceration 0.56±0.342b 0.74±0.303bc 0.91±0.277b 1.24±0.327bc 1.52 ±0.108b EE 95° 0.34 ±0.126b 0.70 ±0.350bc 0.81 ±0.472ab 1.55 ±0.106d 1.48 ±0.167b HEE 30/70 0.54±0.248b 0.61±0.103bc 0.71 ±0.211ab 1.39 ±0.046cd 1.53 ±0.123b HEE 50/50 0.12±0.006a 0.19±0.021a 0.39 ±0.114a 0.93 ±0.025a 1.54 ±0.082b HEE 70/30 0.21±0.093a 0.52±0.123ab 0.86 ±0.032b 1.34 ±0.119cd 1.63 ±0.015b Vit C 1.82±0.133c 2.01±0.0301d 2.01 ±0.010c 1.99 ±0.023e 2.36 ±0.572c

EE: 95°ethanolic extract; HEE: Hydro-ethanolic Extract (v/v); Vit C: vitamin C. In each column, values with the different letter are significantly different (p<0.05 ; Waller-Duncan test).

Table 7: Nitric oxide (NO) radical scavenging of the extracts of Canarium schweinfurthii stem barks.

Concentration (µg/ml) of extracts Extracts 12.5 25 50 100 200 Decoction 32.36±0.76b 39.06±1.79bc 41.53±1.15b 38.23±9.55a 44.46±1.12b Infusion 37.36±0.76c 40.90±1.01c 45.23±0.87c 38.13±5.94a 47.16±0.76cd Maceration 30.90±1.35b 32.36±0.76a 35.83±1.60a 40.46±0.46a 42.23±1.41a EE 95° 26.86±1.60a 32.46±2.25a 41.56±0.92b 44.96±0.25a 45.86±0.57bcd HEE 30/70 26.33±1.52a 37.56±3.63bc 43.00±0.50bc 45.50±0.50a 47.80±0.96d HEE 50/50 30.23±1.32b 38.16±1.60bc 41.46±1.16b 39.33±8.08a 46.23±1.27bcd HEE 70/30 30.80±1.93b 35.50±2.64ab 40.73±1.96b 42.00±1.73a 45.23±0.40ab Vitamin C 54.20±0.20d 54.83±0.25d 54.76±0.55d 56.10±0.36b 56.76±0.66e

EE: 95°ethanolic extract; HEE: Hydro-ethanolic Extract (v/v). In each column, values with the different letter are significantly different (p<0.05 ; Waller-Duncan test).

Table 8: Total phenolic and flavonoid contents of Canarium schweinfurthii stem barks extracts.

Total phenolic content (mgEGA/g Total flavonoids content Extracts of extract) (mgECat/g of extract) Decoction 172.80±4.48a 23.37±2.63a Infusion 286.13±4.89b 24.31±0.74c Maceration 251.72±5.75c 43.99±3.28a

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Table 8 Contd: Total phenolic and flavonoid contents of Canarium schweinfurthii stem barks extracts.

Total phenolic content (mgEGA/g Total flavonoids content Extracts of extract) (mgECat/g of extract) EE 95° 416.29±10.09e 37.97±0.90b HEE 30/70 410.68±23.56e 48.37±1.57d HEE 50/50 355.70±13.46d 38.57±1.93b HEE 70/30 372.91±8.42d 47.85±1.81cd

EE: 95°ethanolic extract; HEE: Hydro-ethanolic Extract (v/v). In each column, values with the different letter are significantly different (p < 0.05; Waller-Duncan test). mgEGA/g: milligrams of Gallic Acid Equivalents per gram of extract ; mgECat/g: milligrams of Catechin Equivalents per gram of extract.

from 23.37 mgECat/g of extract to 48.37 mgECat/g of and elders to treat typhoid fever. Furthermore, many plants extract. Briefly, the total phenolic contents of ethanolic and and their structurally diverse compounds are known for hydroethanolic extracts are significantly higher than those their antimicrobial activities that have been routinely of aqueous extracts. The highest quantity of phenolic assessed against different microbes (Iroha et al., 2010) and compound was recorded with EE 95°, whereas the highest particularly various extracts from different plants showed flavonoid content was recorded with HEE 30/70. The lower significant activities against a variety of Salmonella total phenolic and flavonoid contents were observed with (Noghogne et al., 2015; Kodjio et al., 2016; Atsafack et al., the decoction which eventually showed the lower 2016). The extracts of C. schweinfurthii showed variable antioxidant activities. These results also showed that antibacterial activities against the tested microorganisms. flavonoids are not the only phenolic compounds present in Antimicrobial substances are considered as bactericidal the plant extracts. agents when the ratio MBC/MIC ≤ 4 and bacteriostatic when the ratio MBC/MIC > 4. For the C. schweinfurthii extracts used, the ratio MBC/MIC was less than or equal to DISCUSSION 4 for certain extracts and more than 4 for others suggesting that some may be classified as bactericidal agents, whereas The stem bark extracts of C. schweinfurthii showed others are bacteriostatic against the bacteria interesting antibacterial and antioxidant activities. In fact, strain/isolates. Differences in observed antibacterial many extracts from different parts of C. schweinfurthii, that activities with respect to the different isolates and strain is, resin (Obame et al., 2007; Dongmo et al., 2010), leaves could be due either the constitutional or structural (Nvau et al., 2011), fruits (Dzotam et al., 2015) and stem variability of the treated bacteria or to the difference in the barks (Tsobou et al., 2015) had been presented to possess chemical composition of the genetic resistance elements antibacterial as well as antioxidant activities. In the same transferable between strains such as plasmids (Gatsing et line ,plants belonging to the family of Burseraceae showed Adoga, 2007). According to Kuete scale (2010), an extract various interesting activities (Obame et al., 2007; Murthy et have a significant activity when MIC is lower than 100 al., 2016) and many bioactive compounds have been μg/ml, moderate activity when 100 < CMI ≤ 625 µg/ml and isolated (Mogana and Wiart, 2011). weak activity when MIC is higher than 625 μg/ml. Hence the most active extract (HEE 50/50) has a significant activity against STM, SPA and SPB. The wide range of Antisalmonellal activities antisalmonellal properties can be explained by the qualitative and/or quantitative variation of various groups In this study, we assessed the antibacterial activity of C. of potentially active secondary metabolites. In reality, the schweinfurthii (used in traditional medicine) against four existence of these antimicrobial substances was confirmed isolates and one strain of Salmonella. In fact, the use of by the phytochemical screening which revealed the plants in therapy (phytotherapy) is very old and is presence of certain classes of compounds whose members currently experiencing a renewed interest in the public; it is have already been known to exhibit antimicrobial activities. possible to use the whole plants or the products of Tannins have been found to form irreversible complexes extraction that they provide. Waihenya et al. (2002) and with proline-rich proteins (Adejuwon et al., 2011) resulting Ayachi et al. (2009) showed in their respectively study that in the inhibition of cell protein synthesis. Tannins prevent Aloe secundiflora and Thymus vulgaris are traditionally used bacterial growth by precipitating their proteins (Gatsing for the treatment of human or avian salmonellosis. Since and Adoga, 2007). Some flavonoids have shown several plants are reservoirs of many bioactive compounds, their pharmacological activities including antibacterial and exploration could help in the development of new and more antifungal (Tittikpina et al., 2013). Alkaloids have been effective drugs. Tsobou et al. (2013) have already reported to exhibit antisalmonellal activities (Gatsing et mentioned in an ethnobotanical survey that C. Adoga, 2007). Many saponins are known to be schweinfurthii is used traditionally by traditional healers antimicrobial, to inhibit mould (George et al., 2002). The Academia Journal of Medicinal Plants; Sokoudjou et al. 339

antisalmonellal property of C. schweinfurthii could be could therefore serve as free radical inhibitors. Hence, the attributed to the presence of secondary metabolites, such observed radical scavenging activity could be linked to the as the flavonoids, tannins, triterpenes, or saponins, which presence of polyphenols and flavonoids in the extracts. may have a synergistic action. The difference in the activity Antioxidative properties of polyphenols arise from their of each extract may be due to the effect of each solvent on high reactivity as hydrogen or electron donors which can solubility, diffusion kinetics and mass transfer of bioactive stabilize and delocalize the unpaired electron (Bruneton, compounds (Cacace and Mazza, 2003). Absolutely, it is well 2009). Flavonoids protective effects in biological systems known that the affinity between solvent polarity and the are linked to their ability to transfer electrons to free compound of interest plays an important role in extraction radicals, chelate metals, activate antioxidant enzymes, processes (Złotek et al., 2016); the presence of reduce radicals of alpha-tocopherol or to inhibit oxidases anthocyanins only in EE 95° may be due to the high polarity (Ramde-Tiendrebeogo et al., 2012). of this class of compounds. According to Vega Arroy et al. (2017), pure methanol exhibited the greatest capability for extracting both total anthocyanins and phenolic Ferric reducing antioxidant power compounds. In the FRAP assay, we measured the capacity of an antioxidant substance to reduce the ferric ion to the ferrous Antioxidant assay form. Absolutely, in the physiology of living organism, the interaction between ferric ion and superoxide anion The role of free radicals and active oxygen is becoming induces the formation of hydroxyl radical which could increasingly recognized in the pathogenesis of many human initiate the oxidation of DNA, lipids peroxidation, oxidation diseases, including cancer, aging and artherosclerosis. The of proteins and the activation of kinases (Favier, 2013). In antioxidant potential of C. schweinfurthii extracts was this study, HEE 30/70 proved to be more active than all the tested by various methods because multiple mechanisms other extracts based on their ferric reducing capacity, are involved in the initiation of the oxidative stress. indicating that the phytochemical constituents with redox potential were more concentrated in the that extract. Others extract such as maceration and EE 95° also DPPH radical scavenging assay significantly reduced the Fe2+ ions. The obtain results showed that C. schweinfurthii extracts may prevent Antioxidants can be reductants, and inactivation of oxidants oxidative damage induced by ferric ion in biological system. by reductants can be described as oxido-reduction The ferric reducing power could be explain by the presence reactions, therefore an extract exhibits its free radical of phenolic compounds or flavonoids as revealed by the scavenging activity through neutralization of free radical by titration of total phenolic compounds and total flavonoid either a transfer of hydrogen or an electron (Amarowicz et content. al., 2004; Huang et al., 2005). The highest and lowest IC50 were respectively observed with decoction (13.69 µg/ml) and HEE 30/70 (09.29 Nitric oxide (NO) radical scavenging activity µg/ml). The extracts showing high IC50 values were less active (that is, less scavenging activity) than extracts having Nitric oxide (NO) is a signaling molecule that regulates low IC50 value (means that they are more potent scavengers many functions, such as vascular tone, blood pressure, of DPPH radical). According to Souri et al. (2008), scale the neurotransmission, immune response, synaptic plasticity antioxidant potential of a plant extract is divided into tree and oxidation-sensitive mechanisms (Bredtand and Snyder, ranges: significant when IC50 < 20 µg/ml, moderate when 1994 ; Alderton et al., 2001; Napoli et al., 2001; Bian et al., 20 µg/ml ≤ IC50 ≤ 75 µg/ml and weak when IC50 > 75 µg/ml; 2003). Besides the various function of NO, it has been this suggest that all the extracts from C. schweinfurthii used implicated in many pathophysiologic states. Indeed, several in this experiment had significant antiradical activity. The studies have indicated that low relative concentrations of effect of antioxidants on DPPH has been thought to be due NO seem to favor cell proliferation and anti-apoptotic to their hydrogen donating ability. Many study showed that responses, and higher levels of NO favor pathways inducing DPPH have been used as a substrate to evaluate anti- cell cycle arrest, mitochondria respiration, senescence, or oxidative or free radical scavenging activity of plant apoptosis (Napoli et al., 2013). Overproduction of NO can extracts (Noghogne et al., 2015; Kodjio et al., 2016; Tsafack mediate toxic effects, for example, DNA fragmentation, cell et al., 2017b ; Djimeli et al., 2017). In our experiment, the damage, and neuronal cell death (Moncada et al., 1991; significant DPPH radical scavenging activities of the various Dawson et al., 1992). So tracking of NO could prevent the extracts which were comparable to the standard negative effects of its overproduction. Lower antioxidant, vitamin C, suggested that the extracts have concentrations of NO have been suggested to exert a direct some compounds with high proton donating ability and effect on processes such as cell proliferation and survival, Academia Journal of Medicinal Plants; Sokoudjou et al. 340

whereas higher concentrations have an indirect effect molecule. Ann. Rev. Biochem. 63: 175–195. through both oxidative and nitrosative stresses. In this Bruneton J (2009). Pharmacognosie: phytochimie, plantes médicinales. 4eEd. Éditions médicales internationales (Tec & Doc). Paris, France, study, our plant extracts showed a relative NO scavenging 1288. activity, suggesting that this plant can be used to control or Cacace JE, Mazza G (2003). Mass transfer process during extraction of manage oxidative conditions during bacterial infection. phenolic compounds from milled berries. J. Food Eng. 59(4): 379-389. These results are in correlation which those observed by Carbonelle B, Denis F, Marmonier A, Pinon G, Vorgue R (1987). Techniques Usuelles de Bactériologie Médicale. Edition SIMEP: Paris, France. pp.330. Kodjio et al. (2016) who showed that NO scavenging Chimalizeni Y, Kawaza K, Molyneux E (2010). The epidemiology and activities of extracts and fractions of C. longa increased in a management of non typhoidal Salmonella infections. In Finn et al. (Eds.), dose dependant manner. The NO scavenging activity may Hot Topics in Infection and Immunity in Children VI. New-York: Springer. be due to the presence of bioactive principles in those pp. 33-46 Cook NC, Samman S (1996). Flavonoids - Chemistry, metabolism, extracts which allow them to act as reducing agents, cardioprotective effects, and dietary sources. J. Nutr. Biochem. 7(2): 66– hydrogen donors and singlet oxygen quenchers. 76. Dawson TM, Dawson VL, Snyder SH (1992). A novel neuronal messenger molecule in brain: the free radical, nitric oxide. Ann. Neurol. 32(3): 297–311. Conclusion Djimeli NM, Nkodo MJM, Njateng GSS, Fokunang C, Kodjio N, Atsafack SS, Lonfouo NAH, Sokoudjou J B, Fokeng GM, Gatsing D (2017). In vitro This study showed that C. schweinfurthii extracts can be Antioxidant and Anti-salmonellal activities of Stem Bark extracts of successfully used in the treatment of salmonellosis and Enantia chlorantha (Annonaceae). Res. J. Pharm. Biol. Chem. Sci. 8(6): 1- 12. management of the oxidative stress caused by this Dongmo P, François T, Bernadin N, Wilson A, Bertrand S, Paul HAZ, Chantal infection. Further study needs to be conducted to evaluate M. (2010). Chemical characterization, antiradical, antioxidant and anti- the in vivo therapeutic effect of the most active extract, its in inflammatory potential of the essential oils of Canarium schweinfurthii vivo antioxidant potential, as well as isolation and and Aucoumea klaineana (Burseraceae) growing in Cameroon. Agric. Biol. J. North Am. 1:606-611. identification of active principles. Duke JA (2000). Le pouvoir des plantes. Encyclopédie des Plantes Médicinales du Département de Phytothérapie de Bobigny, France. Dzotam KJ, Touani KF, Kuete V (2015). Antibacterial activities of the ACKNOWLEDGEMENTS methanol extracts of Canarium schweinfurthii and four other Cameroonian dietary plants against multi-drug resistant Gram-negative bacteria. Saudi J. Biol. Sci. 23(5): 565–570. The authors gratefully acknowledge the Cameroon National Eloff JN (1998). A sensitive and quick microplate method to determine the Herbarium (Yaounde) for the plant identification. We also minimal inhibitory concentration of plant extracts for bacteria. Planta wish to express our gratitude to the Laboratory of Medica. 64: 711-713. Gatsing D, Adoga GI (2007). Antisalmonellal activity and phytochemical Bacteriology of Centre Pasteur du Cameroun for providing screening of the various parts of Cassia petersiana Bolle Salmonella isolates. (Caesalpiniaceae). Res. J. Microbiol. 2(11): 876-880. Gatsing D, Mbah JA, Garba IH, Tane P, Djemgou P, Nji-Nkah B. F. (2006). An antisalmonellal agent from the leaves of Glossocalyx brevipes Benth (Monimiaceae). Pakistan J. Biol. Sci. 9(1): 84-87. REFERENCES George F, Zohar K, Harinder PS, Makkar, Klaus B (2002). The biological action of saponins in animal systems: a review. Br. J. Nutr. 88(6): 587- Abiala M, Olayiwola J, Babatunde O, Aiyelaagbe O, Akinyemi. 2016. 605. Evaluation of therapeutic potentials of plant extracts against poultry Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum bacteria threatening public health. BMC Complement Altern. Med. SR (1982). Analysis of nitrate, nitrite and 15N nitrate in biological 16(417): 1-8. fluids. Anal. Biochem. 126(1):131-138. Adejuwon AO, Agbaje EO, Idika N. 2011. Antifungal and antibacterial Hanasaki Y, Ogawa S, Fukui S (1994). The correlation between active activities of aqueous and methanolic root extracts of Carica papaya oxygens scavenging and antioxidative effects of flavonoids. Free Radic. Linn. (Caricaceae). Int. Res. J. Microbiol. 2(8):270-277. Biol. Med. 16: 845–850. Alderton WK, Cooper CE, Knowles RG (2001). Nitric oxide synthases: Huang D, Ou B, Prior RL (2005). The chemistry behind antioxidant structure, function and inhibition. Biochem. J. 357(Pt 3): 593–615. capacity assays. J. Agric. Food Chem. 53(6):1841-1856. Ali MZ, Sultana S (2012). Avian salmonellosis, newcastle disease and Iroha IR, ILang DC, Ayogu TE, Oji AE, Ugbo EC (2010). Screening for anti- aspergillosis. Technical Report. pp. 25. typhoid activity of some medicinal plants used in traditional medicine in Amarowicz R, Pegg RB, Rahimi-Moghaddam P, Barl B, Weil JA (2004). Ebonyi state, Nigeria. Afr. J. Pharm. Pharmacol. 4(12): 860-864. Free-radical scavenging capacity and antioxidant activity of selected Kodjio N, Atsafack SS, Fodouop SPC, Kuiate JR, Gatsing D (2016). In vitro plant species from the Canadian prairies. Food Chem. 84(4): 551–562. antisalmonellal and antioxidant activities of extracts and fractions of Atsafack SS, Kodjio N, Njateng GSS, Sokoudjou JB, Kuiate J-R, Gatsing D Curcuma longa L. Rhizomes (Zingiberaceae). Int. J. Biochem Res. 11(3): (2016). Anti-infectious and in vivo antioxidant activities of albizia 1-14. gummifera aqueous stem bark extract against salmonella typhi-induced Kodjio N, Atsafack SS, Njateng GSS, Sokoudjou JB, Kuiate J-R, Gatsing D typhoid fever in rats. Int. J. Pharm. 6(2): 20-30. (2016). Antioxidant Effect of Aqueous Extract of Curcuma longa Ayachi A, Alloui N, Bennoune O, Yakhlef G, Daas Amiour S, Bouzid W, Rhizomes (Zingiberaceae) in the Typhoid Fever Induced in Wistar Rats Djemai Zoughlache S, Boudjellal K, Abdessemed H (2009). Antibacterial Model. J. Adv. Med. Pharm. Sci. 7(3): 1-13. activity of some fruits; berries and medicinal herb extracts against Kuete V. (2010). Potential of Cameroonian plants and derived products poultry strains of Salmonella. Am. Eurasian J. Agric. Environ. Sci. against microbial infections: a review. Planta Medica. 76(14): 1-13. 6(1):12-15. Mativandlela SPN, Lall N, Meyer JJM (2006). Antibacterial, antifungal and Bian K, Murad F (2003). Nitric oxide (NO)-- dbiogeneration, regulation, antitubercular activity of Pelargonium reniforme (CURT) and andrelevance to human diseases. Front Biosci. 8:d264-78. Pelargonium sidoides (DC) (Geraniaceae) root extracts. South Afr. J. Bot. Bredtand DS, Snyder SH (1994). Nitricoxide: aphysiological messenger 72(2): 232-237. Academia Journal of Medicinal Plants; Sokoudjou et al. 341

Mensor LL, Menezes FS, Leitao GG, Reis ASO, Dos Santos TC, Coube Tsafack ND, Arfat YM, Njateng GSS, Fokunang C, Nyemb JN, Nighat F, CS, Leitao SG (2001). Screening of Brazilian plant extracts for Gatsing D (2017a). GC/MS analysis, antisalmonellal potential of antioxidant activity by the used DPPH free radical method. Phytother. methanol leaf extracts of Tristemma mauritianum and effects on Res. 15(2): 127-130. hematological parameters on Wistar rats infected with Salmonella Mogana R, Wiart C (2011). Canarium L.: A Phytochemical and Typhi. Int. J. Pharm. 7(2): 120-131. Pharmacological Review. J. Pharm. Res. 4(8): 2482-2489 Tsafack ND, Kodjio N, Njateng GSS, Fankam AG, Fokunang C, Tala DS, Mohammed AI, Neil AK, Shahidul IM (2013). In vitro anti-oxidative Gatsing D. (2017b). In vitro antisalmonella and antioxidant effects of activities and gc-ms analysis of various solvent extracts of cassia various extracts from leaves and stem of Tristemma mauritianum singueana parts. Drug Res. 70(4): 709-719. (Melastomataceae). Res. J. Pharm. Biol. Chem. Sci. 8(3): 1916- 1924. Moncada S, Palmer RMJ, Higgs EA (1991). Nitricoxide: physiology, Tsobou R, Mapongmetsem P M, Van Damme P (2013). Medicinal Plants pathophysiology, and pharmacology. Pharmacol. Rev. 43(2): 109–142. Used Against Typhoid Fever in Bamboutos Division, Western Cameroon. Murthy KSR, Reddy MC, Rani SS, Pullaiah T (2016). Bioactive principles Ethnobot. Res. Appl. 11:163–174. and biological properties of essential oils of Burseraceae: A review. J. Tsobou R, Mapongmetsem P-M, Voukeng KI, Van Damme P (2015). Pharmacogn. Phytochem. 5(2): 247-258. Phytochemical screening and antibacterial activity of medicinal plants Napoli C, Ignarro LJ (2001). Nitric oxide and atherosclerosis. Nitric Oxide. used to treat typhoid fever in Bamboutos division, West Cameroon. J. 5(2):88–97. Appl. Pharm. Sci. 5(6): 34-49. Napoli C, Paolisso G, Casamassimi A, Al-Omran M, Barbieri M, Sommese L, Varmuzova K, Matulova ME, Gerzova L, Cejkova D, Gardan-Salmon D, Infante T, Ignarro LJ (2013). Effects of nitric oxide on cell proliferation Panhéleux M, Robert F, Sisak F, Havlickova H, Rychlik I (2015). Curcuma novel insights. J. Am. Coll. Cardiol. 62(2): 89-95. and Scutellaria plant extracts protect chickens against inflammation and Nvau J, Gushit J, Orishadipe T, Kolo I (2011). Antimycobacterial activity of Salmonella Enteritidis infection. Poult. Sci. 94(9): 2049–2058 the leaves extract of Canarium schweinfurthii Engl. Cont. J. Pharm. Sci. 5: Vega Arroy JD, Ruiz-Espinosa H, Luna-Guevara JJ, Luna-Guevara ML, 20-24. Hernández-Carranza P, Ávila-Sosa R, Ochoa-Velasco CE (2017). Effect of Obame LC, Koudou J, Kumulungui BS, Bassolé IHN, Prosper E, Aboubakar solvents and extraction methods on total anthocyanins, phenolic SO, Alfred ST. (2007). Antioxidant and antimicrobial activities of compounds and antioxidant capacity of renealmia alpinia (Rottb.) Maas Canarium schweinfurthii Engl. Essential oil from Centrafrican Republic. peel. Czech J. Food Sci. 35: 456-465. Afr. J. Biotechnol. 6(20): 2319-2323. Waihenya RK, Mtambo MMA, Nkwengulila G, Minga UM (2002). Efficacy Padmaja M, Sravanthi M, Hemalatha KPJ (2011). Evaluation of of crude extract of Aloe secundiflora against Salmonella gallinarum in Antioxidant Activity of Two Indian Medicinal Plants. J. Phytol. 3(3): 86- experimentally infected free-range chickens in Tanzania. J. 91. Ethnopharmacol. 79(3): 317–323 Pamplona RG (1999). Guide des Plantes Médicinales, Tome 1. Encyclopédie Złotek U, Mikulska S, Nagajek M, Swieca M (2016). The effect of different vie et santé: Paris. pp. 398. solvents and number of extraction steps on the polyphenol content and Rajagopal R, Mini M (2013). Outbreaks of salmonellosis in three different antioxidant capacity of basil leaves (ocimum basilicumL.) extracts. Saudi poultry farms of Kerala, India. Asian Pac. J. Trop. Biomed. 3(6): 496-500. J. Biol. Sci. 23(5): 628-633. Ramde-Tiendrebeogo A, Tibiri A, Hilou A, Lompo M, Millogo-Kone H, Nacoulma OG, Guissou IP (2012). Antioxidative and antibacterial activities of phenolic compounds from Ficus sue Forssk. Int. J. Biol. Chem. Sci. 6(1):328-336. Rastaldo R, Pagliaro P, Cappello S, Mancardi D, Westerhof N, Losano G (2007). Nitric oxid and cardiac function. Life Sci. 81(10): 779-93 Souri E, Amin G, Farsam H, Barazandeh TM (2008). Screening of antioxidant activity and phenolic content of 24 medicinal. J. Pharm. Sci. 16(2): 83-87. Tala DS, Gatsing D, Fodouop SPC, Fokunang C, Kengni F, Djimeli MN (2015). In vivo anti-salmonella activity of aqueous extract of Euphorbia prostrata Aiton (Euphorbiaceae) and its toxicological evaluation. Asian Cite this article as: Pac. J. Trop. Biomed. 5(4): 310-318. Tchoua N (2016). Evaluation des activités antisalmonelles et Sokoudjou JB, Njateng GSS, Fodouop SPC, Kodjio N, Atsafack SS, antioxydantes des extraits de Erica mannii chez le poulet de chair. Thèse Fowa AB, Djimeli MN, Gatsing D (2018). In vitro antisalmonellal and de Mater, Université de Dshang. Faculté des sciences, école doctorale antioxidant activities of Canarium schweinfurthii stem bark extracts. des Sciences Fondamentales et Technologiques. pp.97 Acad. J. Med. Plants. 6(10): 331-341. Tittikpina NK, Agban A, Gbogbo KA, Hoekou YP, Pereki H, Batawila K, Akpagana K (2013). Évaluation des propriétés antimicrobiennes de Submit your manuscript at: Pterocarpus erinaceus Poir (Faboïdeae) et Daniellia oliveri (Rolfe) http://www.academiapublishing.org/ajmp Hutch. et Dalz (Caesalpinoïdeae), utilisées en médecine traditionnelle au Togo. Int. J. Biol. Chem. Sci. 7(4):1586-1594.