INTRODUCTION

The world is currently experiencing challenges of increased resistance development against available antimicrobials. The AIDS pandemic has also resulted in large numbers of immunocompromised patients susceptible to opportunistic bacterial and fungal infections. Toxicity of currently used antimicrobial drugs, is a limiting factor in their use. Additionally, the cost of effective antimicrobials plays a vital role in their availability, mainly in developing countries¹. Moreover, bacterial infections are the cause of a large burden of diseases and bacteria are listed in the first position among the common microorganisms responsible for opportunistic diseases occurring associated with AIDS. Therapy of bacterial infections is a frequent problem due to the emergence of bacterial strains resistant to numerous antibiotics^2,3. Even though pharmaceutical companies have produced a number of new antibacterials in the last years, resistance to these drugs has increased and has now became a global concern⁴. Therefore, assist to the global emergence of multi-drug resistant (MDR) bacteria is increasingly limiting the effectiveness of current drugs and significantly causing treatment failure⁵. Due to the increase of resistance to antibiotics, there is serious need to develop new antimicrobial agents that are very effective with minimal unwanted side effects and higher plants represent a potential source of novel antibiotic prototypes⁶. Medical plants have shown a promising alternative for the treatment of infectious diseases. In the antibacterial research, the vast majority, 78% of the new chemical entities are natural or natural products derived molecules⁷. In effect, at last 35,000 plant species are used for medicinal purposes throughout the world⁸. The most important industrial medicines nowadays are based on about 90 species of herbs and in developing countries, traditional remedies are usually based on mixtures of herbs collected from nature⁹. Among such plants, Boswelli dalzielii Hutch, is a plant broadly distributed in tropical and subtropical areas. In Burkina Faso, the aqueous extracts of Boswelli dalzielii Hutch are mostly used in popular folk medicine for the treatment of several diseases¹⁰. Ethnobotanical investigations in the central region of Burkina Faso and certain recent studies performed in laboratory have shown that Boswelli dalzielii Hutch is frequently and widely used in traditional medicine to treat various kinds of diseases such as infectious diseases in children, malaria, fever, pain, variola, antibacterial, anti-inflammatory, analgesic activities and hepatoprotective¹⁰. According the scientific literature, different fractions of stem bark have not been systemically studied. The aim was to justify the traditional antimicrobial use of this species and to produce scientific data for a future project for manufacturing phytomedicines, for use in combination with conventional antimicrobial drugs to better manage resistant bacteria infectious diseases. So, the present investigation was performed to evaluate antimicrobial and antipyretic activities of bioactive fraction (dichloromethane fraction) of stem bark from Boswelli dalzielii Hutch.

MATERIAL AND METHODS

Plants material: The vegetable materials (Fresh stem barks) of Boswelli dalzielii Hutch (Burseraceae) were collected in August, 2014 in Dedougou, 230 Km West of Ouagadougou, capital of Burkina Faso. This plant was botanically identified by Dr. Traoré Lassina from the plants Biology Department of the University of Koudougou.

Bacterial strains and antibiotic: Microorganisms used in this study were isolated from clinical samples at Laboratory of the General Hospital of Ouagadougou in Burkina Faso. Commercially available antibiotic diffusion discs (Ciprofloxacin: 10 μg/disc) were purchased from Alkom Laboratories LTD. Clinical isolates were MDR Gram’negative bacteria: Shigella dysenteriae, Shigella boydii, Shigella flexneri, Salmonella thyphi, Klebsiella pneumonia, Klebsiella arogenes, Escherichia coli, Pseudomonas stuartii, Pseudomonas aeruginosa and Proteus mirabilis. The following microorganisms were all identified by the use of their biochemical profiles as recommended by the manual¹¹.

Animals handling: Swiss NMRI mice (25-30 g) of both sexes were used for acute toxicity and Wister albino rats (180-240 g) of both sexes were used for antipyretic activity. All animals were housed in cages under controlled conditions of 12 h light and 12 h without light and 25°C. They received pellets of food enriched with 20% protein and water ad libitum. They were deprived of food for 15 h (but with access to drinking water) and weighed before the experiments. Experiments on the animals were performed according to the protocols already approved by the Institute of Health Sciences Research/University of Ouagadougou (Burkina Faso) and met the international standards for animal study¹².

Preparation of aqueous acetone extract for acute toxicity study: The field grown fresh samples (stem barks) were washed with tap water followed by distilled water to remove the adhering dust particles. After blotting, samples were air dried in shade. The dried plant materials (stem barks) were ground to fine powder and stored in clean air tight containers. A sample of 50 g of stem barks was placed in the soxhlet and run by using 80% aqueous acetone (500 mL) in 1/10 ratio (w/v) for 24 h under mechanic agitation at room temperature. After filtration all the extracts were dried in vacuum rotary evaporator at 40°C under reduced pressure. Extracts were weighed and stored at 4°C for further analysis. Fractionation of bioactive fraction (dichloromethane fraction) Fifty grams (50 g) of powdered plant material were extracted with 80% aqueous acetone (500 mL) in 1/10 ratio (w/v) for 24 h under mechanic agitation (SM 25 shaker, Edmund BÜHLER, Germany) at room temperature. After filtration, acetone was removed under reduced pressure in a rotary evaporator (BÜCHI, Rotavopor R-200, Switzerland) at approximately 40°C. The aqueous extracts were subjected to sequential liquid-liquid extraction with oil ether to remove chlorophyll and other low molecular weight compounds and dichloromethane. This fraction (dichloromethane fraction) was then collected and concentrated to dryness under reduced pressure to obtain phenol acids (dichloromethane fraction). The fraction was freeze-dried by Telstar Cryodos 50 freeze-dryer. The fraction residues were packed in water proof plastic flasks and stored at 4°C until use. For the tests, lyophilized sample was dissolved with 10% DMSO in water at the desired concentration¹⁰.

In vitro antimicrobial profile
Preparation of inocula: The susceptibility tests were performed by Mueller Hinton agar-well diffusion method¹³. The bacterial strains grown on nutrient agar at 37°C for 18 h were suspended in a saline solution (0.9%, w/v) NaCl and adjusted to a turbidity of 0.5 Mac Farland standard (10⁸ CFU mL kg^–1). To obtain the inocula, these suspensions were diluted 100 times in Muller Hinton broth to give 10⁶ colony forming units (CFU)/Ml¹⁴.

Preparation of discs: The stock solutions of dichloromethane fraction of stem bark from Boswelli dalzielii Hutch, was dissolved in 10% dimethyl sulfoxide (DMSO) in water¹⁵ at a final concentration of 100 μg mL^–1 after a serial two-fold dilution. Each stock solution of phenol acid-rich fractions was sterilized by filtration through 0.22 μm sterilizing Millipore express filter. The sterile discs (6 mm) were impregnated with 10 μL of the sterile phenol acid-rich fractions. Negative controls were prepared using discs impregnated with 10% DMSO in water and commercially available antibiotic diffusion discs (Ciprofloxacin) from Alkom Laboratories LTD) were used as positive reference standards (10 μg/disc) for all bacterial strains.

Disc-diffusion assay: Petri plates (9 cm) were prepared with 20 mL of a base layer of molten Mueller Hinton agar (DIFCO, Becton Dickinson, USA). Each Petri plate was inoculated with 15 μL of each bacterial suspension (10⁶ CFU mL^–1). After drying in a sterile hood, 6 mm diameter discs soaked with 10 μL of the different dichloromethane fraction dilutions were placed on the agar.

Discs containing Ciprofloxacin (10 μg/disc) were used as positive controls and 10% DMSO was used as a negative control. The plates were incubated for 24 h at 37°C and at 44°C for Escherichia coli because this bacterium is thermoresistant. The diameters of the inhibition zones were evaluated in millimeters. The dichloromethane fraction inducing inhibition zone >3 mm around disc were considered as antibacterial. All tests were performed in triplicate and the bacterial activity was expressed as the mean of inhibition diameters (mm) produced¹⁶.

Micro-well dilution assay: Minimum Inhibitory Concentration (MIC) was determined by the microdilution method in culture broth as recommended by NCCLS¹⁷. Eight serial two-fold dilutions of dichloromethane fraction were prepared as described before, to obtain final concentration range of 400-3.125 μg mL^–1. The 96 well micro-plates (NUNC, Danemark) containing 100 μL of Mueller Hinton (MH) broth were used. For each bacteria strain, three columns of eight wells to the micro-plate were used. Each well has getting: The culture medium+dichloromethane fraction+inoculum (10 μL of inocula) and INT (50 μL; 0.2 mg mL^–1). The plates were covered and incubated at 37 and at 44°C for Escherichia coli for 24 h. All tests were performed in triplicate and the bacterial activity was expressed as the mean of inhibitions produced. Inhibition of bacterial growth was judged by rose or yellow colour. The MIC was defined as the lowest concentration of extract or fraction of extract at which no colony was observed after incubation. So, the MIC was defined as the lowest concentration at which no visible growth was observed.

Minimal Bactericidal Concentration (MBC): Minimum Bactericidal Concentration (MBC) was recorded as a lowest dichloromethane fraction concentration killing 99.9% of the bacterial inocula after 24 h incubation at 37°C. Each experiment was repeated at least three times. MBC values were determined by removing 100 μL of bacterial suspension from subculture demonstrating no visible growth and inoculating nutrient agar plates. Plates were incubated at 37°C for a total period of 24 h. The MBC is determined with the wells whose the concentrations are >MIC^{16, 18}. The MBC were determined in Mueller Hinton (MH) agar (DIFCO, Becton Dickinson, USA) medium.

Evaluation of bactericidal and bacteriostatic capacity: The action of an antibacterial on the bacterial strains can be characterized with two parameters such as Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). According to the ratio MBC/MIC, we appreciated antibacterial activity. If the ratio MBC/MIC = 1 or 2, the effect was considered as bactericidal but if the ratio MBC/MIC = 4 or 16, the effect was defined as bacteriostatic¹⁹.

Time-kill assay: A bactericidal effect is defined as a 3 Log decrease in the CFU mL^–1 or a 99.9% kill over a specified time²⁰. The definition of kill for this study has been used as per²¹. Kill-time can be determined at 6 h²². A 90% kill at 6 h is equivalent to a 99.9% kill at 24 h²³. In this study the kill measurement was determined by the actual reduction in viable counts at 6 h for each isolate. Bacteria strains possessing the bactericidal effect were chosen to perform time-kill assay. Thus, 0.5 Mac Farland standards suspensions of the microorganisms were diluted to have 50 mL of approximately 10⁶ CFU mL^–1 in nutriment broth and the concentration corresponding to the best MIC, were respectively added to the corresponding culture. The cultures were incubated at 37°C. At 0, 1, 2, 3, 4, 5 and 6 h, an aliquot of 100 μL was removed and diluted with 10 ml sterile broth. The obtained suspension was used to inoculate 9 cm diameter Petri plates with a sterile non toxic cotton swab on a wooden applicator as indicated before in the agar-well diffusion assay. After 24 h incubation at 37°C, the viability of the microorganisms was evaluated by the presence of colonies on the plates. The experiment was carried out twice following²⁴ method with light modifications.

Acute toxicity study in mice of aqueous acetone extract: Healthy male and female Swiss mice (25-30 g) were randomly divided into 7 groups (1 control group and 6 treated assay groups) of 6 animals (3 male and 3 female). The control group received water containing 10% dimethyl sulfoxide (DMSO) administered intra-peritoneally. The water/acetone of extract of stem bark from Boswelli dalzielii Hutch, suspended in 10% DMSO was administered respectively intra-peritoneally at doses of 1, 2, 2.5, 3, 4, 5 and 6g kg^–1. The general behaviour of the mice was observed for 120 min after the treatment. The animals were observed for morbidity and mortality once a day for 14 days. The number of survivors after the 14 days period was noted. The toxicological effect was assessed on the basis of mortality for 14 days, which was expressed as the median lethal dose (LD50) (Lethal Dose 50) was estimated from the regression of log-probit mortality rate²⁵.

Antipyretic activity test: The method described by Alperman²⁶ was used for studying the antipyretic effect of dichloromethane fraction of stem bark from Boswelli dalzielii Hutch. Thirty rats of both sex weighing 180-240 g were divided into five groups of six rats in each. All rats were made hyperthermic by subcutaneous injection of brewer’s yeast in a physiological saline in a dose of 1.5 g kg^–1 b.wt. After 17 h, the initial body temperature of each rat was measured rectally using a medical thermometer. The first group was kept as control; the second group was given metamizole sodium 50 mg kg^–1 b.wt., as a standard antipyretic. The third and fourth groups were used to reveal the antipyretic effect of the tested dichloromethane fraction of stem bark from Boswelli dalzielii Hutch, when given orally in a dose of 200 and 300 mg kg^–1 b.wt., respectively. The body temperature of each rat was then recorded every hour for 3 successive hours.

Statistical analysis: Percentages reduction of hyperthermia were expressed as Mean±SEM. The significance of difference between the controls and treated groups were determined using ANOVA test. P (ANOVA with a statistical significance level set at p<0.05 and linear regression) with XLSTAT 7.1.