Medicinal plants have been used for many decades around the world to prevent or  treat  infectious  and  non-infectious diseases. As a result of the resistance of disease causing bacteria to conventional therapy, the use of medicinal plants has been on an increase (Notka et al., 2004).

Around 80% of the world population relies on alternative medicines from plants with approximately 70,000 plants being utilised (Thomas, 2000). A variety of plant parts such as flowers, roots, stems and fruits have been found useful in drug development because of their medicinal properties (Prior, 2003).

Medicinal plants have great pharmacological importance because they possess bioactive molecules. These molecules are produced by metabolic pathways and genetic structures that are unique to each plant species. It has been proved that the environment can influence the amount of phytochemicals that can be found in each plant species (Thomas, 2000). Phyto-chemicals are therefore being used in the production of novel medicines as an alternative to synthetically produced drugs.

Pastoral farming remains very active in Botswana not only for the sustenance of families but also as a viable source of income through beef exports. Veterinary diseases remain a great threat to livestock production in Botswana. The diseases can adversely impact livestock health, lead to loss of income, lack of food security and cause productivity losses. There are different ways through which veterinary diseases can be managed. These include among others quarantining of diseased animals, breeding control, regulation of entry into farm lots and the development of better antibiotics, diagnostics tools, vaccines and vector control techniques (Sharma, 2016).

As Sharma (2016) explains, diseases cause poor livestock performance and medicines are too expensive and inaccessible to most smallholder farmers. Because of the cultural and ecological diversity of Botswana, there is substantial knowledge on the proper management and utilization of the different medicinal plants in the country to alleviate diseases that affect livestock. Therefore, without modern medicine, traditional medicinal plant extracts will continue to be an important component of smallholder farming in Botswana for the foreseeable future (Sharma, 2016).

Because of the great potential of medicinal plants in the manufacturing of antimicrobial drugs, this study screened the plant species Albizia anthelmintica for the antibacterial and antifungal activity of its extracts. A. anthelmintica belongs to the kingdom Plantae (Order Fabales; family Fabaceae; subfamily Mimosoideae) (Grade et al., 2008). Albizia species have high content of phenolic compounds such as triterpenoids and saponins.

In Africa, Albizia plant species are commonly used in the treatment of conditions such as diarrhea, coughs and    rheumatism. Plants such as these have proved crucial  to the identification of novel agents of therapy (Grade et al., 2008). The study aimed to determine the antibacterial potential of A. anthelmintica as a medicinal plant on pathogenic veterinary isolates. Additionally, its potential antioxidant properties were investigated.

Sample collection and extract isolation

The sample site for this study was Sikwane village in Kgatleng district, Botswana. It is located at 55 km to the east of Gaborone. A. antihelmintica roots and bark samples were randomly collected and their identity confirmed at the University of Botswana Herbarium (Voucher No: 009). Four bacterial veterinary pathogenic strains identified as Escherichia coli, Clostridium perfringens, Salmonella enterica serovar Typhimurium (S. Typhimurium) and Proteus mirabilis were collected from the Department of Biological Sciences, University of Botswana, Gaborone. The bacterial strains were previously isolated from diseases cattle and goats in Botswana. Cultures of these bacteria were maintained in Mueller-Hinton nutrient agar slants at 28°C. Roots and bark samples were washed, dried and blender ground into powder. The powder was then soaked in 100% hexane, 100% chloroform, 100% ethanol and 70% ethanol. The solvents were then rota evaporated to retain crude extract. 

Determination and measurement of antioxidant activity of extracts

Free radical scavenging activity of plant extracts was determined using the semi quantitative TLC- DPPH method. Briefly, 1 mg/1000 µl methanol solutions for each extract were prepared. To determine the scavenging capacity of extracts for the free radical 2, 2-diphenyl-1-picryl hydrazyl (DPPH), thin layer chromatography was used as described by Yeboah and Majinda (2004). Methanol was used as a control. The following equation was used to plot a bar graph of inhibition concentration at 500 µg/ml.

*A_sample and A_control are absorbances of extract samples and the control, respectively.

Phytochemical screening and antimicrobial activity determination

Phytochemical screening was performed according to the methods described by Mazimba et al. (2006). Extracts were screened for the presence of the following: flavanoids, tannins, saponins, alkaloids,

terpenoids, quinones and fatty acids phenols. Antimicrobial activities of A. anthelmintica extracts (100% hexane, 100% chloroform 100% ethanol and 70% ethanol) were determined using a modified Kirby-Bauer disc diffusion method (Bauer et al., 1966). Antimicrobial assays were performed in duplicates and discs impregnated with 100 μl Trimethoprim and 100 μl of distilled water were used as positive and negative controls, respectively. Results were  interpreted  as  follows:   Sensitive   (S),   Intermediary  Resist  (IR) and Resistant (R) which is in accordance with the standard measurement of inhibitory zones in millimetre (mm).

Minimum inhibition concentrations (MIC) determination

MIC values were determined using the micro-titre broth-dilution method. Muller Hinton broth was used as the primary medium for the tube dilution to determine the MIC for each microorganism as described in Wikler (2008). MIC values of A. anthelmintica extracts were determined using two-fold broth micro-dilution to prepare extract concentrations of 80 and 40 mg/ml. Trimethoprim and Muller-Hinton broth containing bacterial isolates were used as positive and negative controls, respectively.

Statistical analysis

The data of various analyses were expressed as mean ± standard deviation. All tests were carried out in triplicate to improve accuracy. Data was analysed using one way analysis of variance (ANOVA) followed by Dunnet’s test. In the experiments, P<0.05 was taken to be significant.

Plant extract concentrations (15.6 to 1000 µg/ml) in different solvents were observed on the TLC sheet (Figures 1 and 2). These concentrations showed moderate activities, indicated by the faint yellow colouration over the purple DPPH background in comparison with the bold colouration of the gallic acid standard. In the bark extracts, the highest activities were observed in the 100% chloroform extracts, 70% ethanol extracts and 100% ethanol extracts while the 100% hexane extracts showed little activity (Figure 1). Similar trends were observed in roots extracts (Figure 2).

Percentage inhibitions of bark and roots extracts were determined at 500 µg/ml concentrations (Figure 3). From the highest inhibition to the lowest inhibition, the observed inhibition percentages for the different extracts were as follows: 70% ethanol roots (66.9%); 70% ethanol barks (58.9%); ethanol 100% roots (49.2%); chloroform barks (49%); hexane bark (25.5%); ethanol 100% barks (14.6%); chloroform roots (7.3%) and hexane roots (4.9%). The standard (gallic acid) displayed 86.7% DPPH inhibition at a dose of 500 µg/ml Phytochemical screening of all extracts was performed. Both 100% chloroform bark and roots extracts contained only one type of compound (terpenoids). Ethanol (100%) bark extracts contained all phenolic compounds except flavonoids (Table 1). Among all the compounds, terpenoids are the only ones present in all extracts. Chloroform barks showed the greatest antimicrobial activity of all the extracts. Barks of 100% ethanol extracts and 70% ethanol extracts showed the least antimicrobial activities (Table 2). All bacterial isolates were resistant to 100% ethanol roots extracts. Therefore, 100% ethanol roots displayed minimal antimicrobial activity.

MIC values determination showed a 0.625 mg/ml for chloroform  roots  against  S.  Typhimurium.  The  highest recorded MIC value was 5 mg/ml for both chloroform barks and hexane barks against P. mirabilis and C. perfringens, respectively (Table 3). 100% hexane barks recorded an MIC value of 2.5 mg/ml against E. coli.

In this study, 100% ethanol roots extract inhibited the growth of all bacterial isolates. C. perfringens is often involved in diseases in most domestic animals and some wildlife, including, poultry, sheep, goats, cattle, ostriches, dogs and cats (Niilo, 1993). Multidrug resistant S. Typhimurium and E. coli have been widely reported as causative agents of diarrhoea in cattle and small stock and still are a major cause of productivity and economic loss to cattle producers worldwide (Voetsch et al., 2004; Cho and Yoon, 2014).

P. mirabilis is one of the most common bacteria infecting the urinary tract in humans and dogs (Abe et al., 2017). S. Typhimurium showed intermediary inhibition and resistance to all ethanol extracts. MIC value of 0.625 mg/ml was observed on chloroform roots against S. Typhimurium. These MIC results were in agreement with the phytochemical screening results which indicated that chloroform roots contained only terpenoids.

In conclusion, findings of this study have shown that extracts of the A. anthelmintica plant species may be reliable sources of antioxidants and antimicrobials which can be used the development of novel drugs and the treatment of multi drug resistance veterinary pathogens.

Furthermore,  this  study  provides valuable information that can not only guide future studies on medicinal plants but can also be an educational reference for students and scholars of Botswana.

The authors have not declared any conflict of interests.

The authors are thankful to office of Research and Development, University of Botswana for funding this study.