Multidisciplinary Journal of TUM 1 (2) 2020 31-36 DOI: https://doi.org/10.48039/mjtum.v1i1.12 Original Article Vernoa hildebranditii, Acacia stuhlmannii and Moringa oleifera Leaf and Root-bark Extracts Exhibit Antimicrobial Effects on Escherichia coli and Staphylococcus aureus Bacteria Ronald Mwabonje1, Huxley Makonde2, Ali Juma Kanga1, Aggrey Adem3, Suleiman Saidi1, Victor Tunje Jeza1* 1Department of Medical Sciences, Technical University of Mombasa, P.O. Box 90420 - 80100 Mombasa, Kenya; 2Department of Pure and Applied Sciences, Technical University of Mombasa, P.O. Box 90420 – 80100 Mombasa, Kenya; 3Department of Maths and Physics, Technical University of Mombasa, P.O. Box 90420 – 80100 Mombasa, Kenya *Corresponding author: vjeza@tum.ac.ke Abstract I nfectious diseases are the main causes of morbidity and mortality worldwide. There is increasing concern of indiscriminate use of antibiotics and incidences of multiple antibiotic resistances in human pathogens. The potential of higher plants as source of new drug leads has been demonstrated but is still under explored. In Africa and most developing countries, traditional medicine still forms the backbone of rural medicinal practice. Although a number of American and Asian countries medicinal herbs have been evaluated scientifically and their medicinal properties demonstrated. In Africa, attempts to evaluate medicinal plants in relation to their biological activities and medicinal usefulness are limited. The emergence of antibiotic resistance has led to increased use of herbal medicine as an alternative to combat various ailments. This study aimed at determining the antimicrobial activity of Vernoa hildebrandtii, Acacia stuhlmannii and Moringa oleifera leafy and root bark extracts using disc diffusion technique. Crude extracts were obtained from dried powder by single solvent maceration with ethanol and water. Bioassays were used to evaluate the bioactivity of the extracts against Escherichia coli and Staphylococcus aureas. Antimicrobial activity was determined by measuring the zones of growth inhibition in mm. Moringa aleifera root water extract was the most potent fraction with bioactivity arrange of between 6-32 mm followed by Acacia stuhlmannii root bark water extracts with bioactivity range between 12-31 mm and Moringa oleifera root bark with bioactivity range of between 6-29 mm. Vernoa hildebrandtii leave alcohol and Acacia stuhlmannii Taub root bark- alcohol extract had bioactivity range of between 9-28 and 5-28, respectively. However, Acacia stuhlmannii leaf extracts did not show any antimicrobial activities. The antimicrobial effects of the plant extracts were dose dependent. These findings validate what have been known about Moringa oleifera. They also demonstrate potential biochemical agents in Vernoa hildebrandtii and Acacia stuhlmannii extracts in the management of gram positive and gram negative bacteria. Key Words: Antimicrobial Effects, Vernoa hildebrandtii, Acacia stuhlmannii, Moringa oleifera Introduction Antibiotic and antimicrobial agents have been used to treat infectious diseases for the last 70 years(Anwar et al., 2000). Since 1940s, antibiotics were reported to reduce the burden that infectious diseases pose to human health (Runyoro et al., 2006). However, the long term use of these drugs has given a chance to microorganisms to adapt and make the drugs less effective. The emergence of multidrug resistant pathogens such as Escherichia coli , Klebsiella pneumaniae and Candida albicans has been a major challenge in the management of these diseases caused by these pathogens (Diallo et al., 2002). Vernoa hildebrandtii is a tropical plant found in the coastal counties of Kenya; Tana-River, Kilifi and Kwale counties and belong to the family Compositae. This plant grows in the humid tropics or hot dry land with average height that ranges from 0.5m to 5m. It is a woody creeping plant with 31 Published: December 2020 Multidisciplinary Journal of TUM 1 (1) 2020 31-36 white and purple floral parts. It is locally known as Mrusapung in Swahili, (Chi)watsa in Digo, Mlazakoma in Giriama and Mlalapiri in Chonyi (Pakia et al., 2003). For a long time, the Mijikenda community have used this plant to treat diarrhea, stomach ache, vomiting, and other ailments (Greenway et al., 1969a). Acacia stuhlmannii is a member of the family Minosaceae. It is a multipurpose nitrogen fixing tree legume which occurs from sea level to over 2000 m (Adnan et al., 2018). It withstands extreme temperatures of above 50˚C and air dryness but sensitive to frost when young (Bargali et al., 2009). It is widely spread in subtropical and tropical Africa from Egypt to Mauritania southwards to South Africa and in Asia (Runyoro, et al., 2006; Jigam et al., 2010). In Kenya, the plant is found in Tsavo East and the counties of Kwale and Kilifi (Greenway et al.,1969). Moringa oleifera is one of the best known medicinal plants. It belongs to the family Moringaceae (Jigam et al., 2010). It is also known as horse-radish tree, drumstick. Another vernacular name is Mzungi as it is well known among the Mijikenda communities of Kilifi in coastal Kenya. It is a small, fast growing, evergreen, or deciduous tree that usually grows up to 10 or 12m in height. The species is native to Kilifi and also grows in tropical and subtropical regions. It is distributed among sub-Saharan Africa and India. It has its beneficial role in cancer, diabetes, liver treatment, ulcer and various other diseases. Similarly, it has applications as an antioxidant, immunosuppressant, cytoprotective, and gastro protective (Farooq et al., 2012) In Africa and most developing countries, traditional medicine still form the backbone of rural medicinal practice (Toda et al., 2016). Medicinal herbs are used extensively for various ailments in these countries. Although a number of American and Asian countries’ medicinal herbs have been evaluated scientifically and their medicinal properties known, up to now there are no serious attempts to evaluate African medicinal plants in a collection of their biological activities and medicinal usefulness (Runyoro et al., 2006). With the emergence of antibiotic resistance, herbal medicine has been the target alternative to combat various ailments. This study aimed at determining the antimicrobial activity of V. hildebrandtii, A. stuhlmannii and M. oleifera leaf and root-bark extracts. This research will ignite the advantage of Original Article the exciting medicinal plants in and their application to modern medicine and improvement of the usage of these herbs which are under-utilized in Kenya and other parts of sub-Saharan Africa. Materials and Methods Plant Materials Acacia stuhlmannii leaves, and root barks, and Moringa leaves were collected from Kwale County in Msambweni area located at 4.57˚S longitude and 39.28˚ E latitude and at an altitude of 2.2 m above the sea level, while V. hildebrandtii leaves and root barks were collected from Kilifi County in Chonyi area located at 3.78˚S longitude and 39.7˚ E latitude and at an altitude of 2.7 m above the sea level. Test Microorganisms Escherichia coli and Staphylococcus aureas were obtained from the American Type Culture Collection (ATCC) and propagated under appropriate conditions. The cultures were maintained on nutrient agar slants at 40C and subcultured before use. Preparation of Plant Extracts The plant samples were washed with sterile water, and kept under a shade till drying. Extraction from leaves and root barks were carried out by simple maceration process as described by Savithramma et al., (2011) with some adjustments. The specimens were grounded into coarse powder then suspended in 75% ethanol and water for 3 to 7 days at 37˚C in extraction bottles. Filtration of extract was done twice using Whatman-41 filter paper. Concentrations of crude extracts were done at a temperature range of 40˚C-50˚C using R209 rotary evaporator. The ethanol and water were completely evaporated by rotary evaporation to obtain the extracts. The extracts were stored at 4˚c till use. Preparation of Samples Each extract was dissolved serially in 1 ml of 10%v/v DMSO to make concentrations covering 15, 20, 30, 40 and 50 mg. Using DMSO, adjustments were made such that 15 µl of each was delivered on to sets of filter paper disks, each receiving approximately 15µl of solution. 32 Multidisciplinary Journal of TUM 1 (1) 2020 31-36 Preparation of Media for Bacteria Nutrient broth medium was prepared by dissolving 0.4 g/50 ml of distilled water for the growth of bacteria inoculums; pH was adjusted at 7.0 and autoclaved. Nutrient agar medium was prepared by dissolving 2.3g/100 ml of distilled water at pH 7.0 then autoclaved at 121˚C. Antimicrobial Susceptibility Test Antimicrobial susceptibility was determined by agar well diffusion and disc methods (Kabbashi et al., 2016). Muller Hinton Agar (MHA) medium was prepared and sterilized by autoclaving at 1210C for 15 min as described by Kabbashi, (2016). The media was poured into sterile Petri plates and allowed to solidify. Escherichia coli and Staphylococcus aureas were inoculated by spreading onto the surface of the MHA media by using sterile cotton swabs. Sterile discs impregnated with plant extract were seeded on the plates and incubated at 370C for 24 hrs. Antimicrobial susceptibility test was done in duplicate using Gentamicin, Ciprofloxacin, Ceftriazone and Nitrofuranton as positive controls and 10%v/v DMSO as negative control based on the CLSI susceptibility protocol. Zones of growth inhibition in mm were evaluated after 24 hrs of incubation. The lowest concentration of the crude extract that shows zones of growth inhibition is the minimum inhibitory concentration. Antimicrobial activity was evaluated by measuring the zones of growth inhibition against the tested microorganisms. Original Article were grinded and dissolved in water for 7 days at 37oC. The solvent was evaporated and extracts applied to E. coli and S. aureas. Results showed that the leaf extracts of V. hildebrandtii and M. oleifera inhibit the growth of E. coli and S. aureas bacteria in a dose dependent fashion (Fig. 1a & b). At the same time, A. stuhlmannii did not show any antimicrobial activity trend against E. coli and S. aureas (p = 0.034; Figs. 1a & b). Leaf alcohol extracts has antimicrobial properties against E. coli and S. aureas The potential of alcohol leaf extracts of V. hildebrandtii, A. stuhlmannii and M. oleifera to inhibit bacteria growth was explored. Leaves were grinded and dissolved in alcohol for 7 days at 370C. The solvents were evaporated and extracts applied. Screening antimicrobial activity revealed inhibitory activity by V. hildebrandtii and M. oleifera against E. coli and S. aureas (Fig. 2a & b). Acacia stuhlmannii leaf extracts did not show any antimicrobial activity trend against E. coli and S. aureas (p <0.01; Fig. 2a & b). Data Analysis Antimicrobial activity was an average of zones of growth inhibition in mm recorded from the duplicates. The mean diameter score was analyzed as a fraction of the extract concentration and solvent type. Statistical analysis was done using 1Way ANOVA in Minitab software. P values ≤ 0.05 were considered significant. Results Leaf Water Extracts of Vernoa hildebrandtii, Acacia stuhlmannii and Moringa oleifera inhibit Bacteria growth Here we explored the potential of water leaf extracts of V. hildebrandtii, A. stuhlmannii and M. oleifera to inhibit the growth of bacteria. Leaves Figure 1. Antimicrobial activity of water leaf extracts against a) E. coli and b) S. aureas by disc diffusion method 33 Multidisciplinary Journal of TUM 1 (1) 2020 31-36 Original Article Discussion Figure 2. Antimicrobial activity of leaf alcohol extracts against a) E. coli and b) S. aureas by disc diffusion method Root-bark Water Extracts of Moringa oleifera has the Highest Antimicrobial Activity To determine antimicrobial property of water root bark extracts of V. hildebrandtii, A. stuhlmannii and M. oleifera, root-bark were grinded and dissolved in water for seven days at 370C. The solvents were evaporated and extracts applied. Screening antimicrobial activity revealed inhibitory activity by V. hildebrandtii, A. stuhlmannii and M. oleifera against E. coli and S. aureas with M. oleifera recorded as the highest fraction with bioactivity range of between 6-32 mm (p <0.01; Fig. 3 a & b). Healing properties of medicinal plants that are used to manage infectious diseases has been proven by several studies conducted to evaluate antimicrobial activities of medicinal plants (Mahesh et al., 2008; Kabbashi et al., 2016). The first step towards development of new chemotherapeutic agents is in vitro antimicrobial activity assay (Mahesh et al., 2008). When tested by the disc diffusion method, M. oleifera root water extracts were the most potent fraction with bioactivity range of 6-32 mm (Fig. 3a) against E. coli followed by A. stuhlmannii root-bark water extracts which had bioactivity range of 12.5-31.5 mm against S. aureas (Fig. 3b). Moringa oleifera root-bark water extract had bioactivity range of 6.5-29.0 mm (Fig. 3a). Vernoa hildebrandtii leaf alcohol (Fig. 2a) and A. stuhlmannii root-bark alcohol extract (Fig. 4a) had bioactivity range of 9.5-28 and 5.8-28, respectively. However, A. stuhlmannii leaf extracts using both water and alcohol against E. coli and S. aureus did not show any antimicrobial activities (Figs. 1 & 2) contrary to Acacia aroma which has been shown to have antimicrobial activities against some gram-negative bacteria in other studies (Mattana et al., 2010). Vernoa hildebrandtii root-bark extracts did not show antimicrobial activities against E. coli and S. aureas at lower doses of 1015mg (p = 0.670; Figs 4a & b). Taken together these results clearly indicate that, antimicrobial activity varies with the species of the plants, extraction solvent used and specific parts of the plant material used. Further these results showed a dose dependent antimicrobial activity of V. hildebrandtii, A. stuhlmannii and M. oleifera against gram positive and gram negative bacteria. Vernoa hildebrandtii Root-bark Alcohol Extract does not have Antimicrobial Activities at Lower Concentration Figure 4 a & b shows a consistent increase in microbial activity as the dose increases at higher doses starting from 20 mg. There was no inhibition recorded between 10-15 mg concentration (p = 0.670). 34 Multidisciplinary Journal of TUM 1 (1) 2020 31-36 Original Article Conclusion Our results suggest that, V. hildebrandtii, A. stuhlmannii and M. oleifera possess compounds with antimicrobial properties which can be useful in designing new therapeutic agents. Further work is under way to elucidate the mechanisms of action. Acknowledgment This work was supported by Technical University of Mombasa internal grant number 40352-010M140. 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