|Emebet Zeleke*, Gashaw Enbiyale, Gezahegne Mamo, Mirutse Giday and Adonyas Luelseged|
|Corresponding Author: Emebet Zeleke, College of Veterinary Medicine and Agriculture, Addis Ababa University, Addis Ababa, Ethiopia|
|Received: June 01, 2019; Accepted: July 03, 2019;|
|Citation: Zeleke E, Enbiyale G, Mamo G, Giday M & Luelseged A. (2019) The In Vitro Antibacterial Sensitivity Test of Selected, Medicinal Plants. J Genomic Med Pharmacogenomics, 5(1): 402-412.|
|Copyrights: ©2019 Zeleke E, Enbiyale G, Mamo G, Giday M & Luelseged A. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.|
An experimental study was conducted from November 2015 to May 2016 at Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa and College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoftu on the in vitro antibacterial sensitivity test of selected medicinal plants. The study was carried out with the objective of determining and comparing the in vitro antibacterial effect of 80% ethanol and methanol extract of Calpurnia aurea leaf (“Digita”) and Cordia africana root (“Wanza”) at various concentrations against Staphylococcus aureus, E. coli and Salmonella species by using agar disc diffusion method on Muller-Hinton agar and broth serial micro dilution methods with the help of resazurin. The plant materials were collected from Bishoftu. The mean zone of inhibition results showed that all of the extracts were active against tested bacteria. A wider mean zone of inhibition was demonstrated by ethanol extract of Calpurnia aurea leaf against Staphylococcus aureus that showed 15.20 ± 1.3; mean ± S.D followed by methanol extract was showed, 14.20 ± 2.04 at a concentration of 200 mg/ml. Both ethanol and methanol extracts of this plant at concentration of 200 mg/ml have showed 13.60 ± 1.34 and 13.60 ± 1.14, respectively against E. coli. The results also indicated that the root of C. africana inhibits the tested organisms. Moreover, ethanol extract of 200 mg/ml against E. coli showed wider inhibition zone of 12.60 ± 2.14 than S. aureus 11.20 ± 2.168 and Salmonella species 11.0 ± 1.58. Furthermore, each plant extract was tested with broth serial micro dilutions and the obtained concentration was ranged from 25 mg/ml to 0.78 mg/ml against tested bacteria. Besides this, the mean of higher MIC of C. aurea showed 2.35 mg/ml ± 0.91 against Staphylococcus aureus and Cordia africana against Salmonella species had (9.38 mg/ml ± 3.61). In conclusion, most of the extracts have shown considerable activities against test bacteria. These may justify the traditional uses of these two plants have the potential for discovery of novel antimicrobial agents from medicinal plants. Therefore, further study is required to isolate and identify as well as to assess the bioactive ingredients responsible for this effect at in vitro and in vivo level.
Keywords: C. aurea, C. africana, E. coli, In vitro anti-bacterial effects, Medicinal plant, Salmonella species, S. aureus
The most important group of bacteria causing calf diarrhea are Escherichia coli (E. coli), Salmonella species and Clostridium perfringens. E. coli occurs as normal flora in the gastrointestinal tract of humans and animals. Pathogenic variants cause intestinal and extra-intestinal infections, including gastroenteritis, urinary tract infection, meningitis, peritonitis and septicemia .
Salmonellosis in dairy calves has many impacts on animal and human health that are considered as a major worldwide problem. Substantial economic losses were manifested through mortality and poor growth of infected animals as well as it has potential for zoonotic transmission .
Bovine mastitis has remained the most economically damaging disease that severely reduces milk production and often difficult to treat due to antimicrobial resistance . In addition to heavy losses in milk quality and quantity, it is also causes irreversible damage to the udder tissue and less occasional fatalities . The most important major pathogens that cause bovine mastitis are Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and Klebsiella species . From these organisms, Staphylococcus aureus (S. aureus) is a major problem associated with milk causing diseases of humans and animals .
Antimicrobial agents are essential for the maintenance of health and welfare in animals as well as humans. However, the use of antimicrobials can be linked to the emergence and increasing prevalence of antimicrobial resistant bacteria. In addition to this, the development of antimicrobial resistance in bacteria of animal origin reduces the efficacy of veterinary antimicrobial drugs . These bacteria are characterized by great resistance to antibacterial drugs, either by their natural resistance or by the development of different defense mechanisms against the host response . Hence, the development of antimicrobial-resistance imposes the need to search for new effective drugs to circumvent the problem. Thus, there has been a resurgence of interest recently in the development of drugs from plants, especially from those of the developing countries that have a rich heritage of botanical ethno-pharmacopoea .
Traditional medicinal plants are widely used in different part of the world for curing diseases. They have maintained their popularity in developing world. These medicinal plants are also rapidly spreading in the industrialized countries . In the developing countries, many people rely on traditional healing practices and medicinal plants for their daily healthcare needs. In some Asian and African countries, 80% of the population depends on traditional medicine for primary healthcare. This is because traditional medicine is a more affordable and accessible health care system when compared to modern medicine . The acceptance of traditional medicine as an alternative form of health care and the development of microbial resistance to the available conventional antibiotics have led researchers to investigate the antimicrobial activity of herbal extracts .
In Ethiopia, traditional remedies represent not only part of the struggle of the people to fulfill their essential drug needs but also they are integral components of cultural beliefs and attitudes . The Ethiopian flora is estimated to contain between 6500 and 7000 species of higher plants of which about 12% are endemic. More than 95% of traditional preparations in the country are of plant origin . Despite their vital role in catering for the health of human and livestock population, large part of the knowledge of ethno-medicinal plants is on the verge of irreversible loss and declining to deterioration due to oral passage of herbal heritage from generation to generation verbally rather than in writings . Consequently, the majority of those raising stock in rural areas are far from site of veterinary stations so that applying veterinary herbal medicine is the only option they have . Concerns have risen about drug-resistant pathogens originating from an animal related source (food borne infections, direct contact) as well as bacteria of human origin acquiring resistance genes through gene transfer from these organisms and causing infections that are difficult to treat. The rapid emergence of multiple drug resistance strains of pathogens to current antimicrobial agents has generated an urgent intensive for new antibiotics from medicinal plants. Many medicinal plants have screened extensively for their antimicrobial potential worldwide .
Medicinal herbs have been used in one form or another under indigenous systems of medicine . A range of medicinal plants with anti-microbial properties has been widely used by traditional healers. Among these plants, leaf of Calpurnia aurea (local name “Digita”) has been used In Ethiopia. Traditionally, the leaf of Calpurnia aurea are used for the treatment of syphilis, malaria, rabies, diabetes, hypertension, diarrhea, leishmaniasis, trachoma, elephantiasis, fungal diseases and different swellings, stomach-ache, bowel and bladder disorders [30,33]. Different parts of Cordia africana (local name “Wanza”) are used for skin disease, wound, diarrhea and ascaris infection in human and animals . Although, there are few studies that evaluated the antimicrobial activity of C. aurea and C. africana, more experiments are needed to discover new antimicrobial agents and thus, recent information is important to evaluate the antimicrobial activity of medicinal plants.
Therefore, the objectives of this study were:
· To determine the in vitro antimicrobial effects of leaf extracts of Calpurnia aurea (Digita) and root extracts of Cordia africana (Wanza);
· To determine the minimum inhibitory concentration of each plant extracts, and;
· To evaluate the efficacy of selected medicinal plants on bacterial isolates.
MATERIALS AND METHOD
A study of an in vitro antimicrobial effect of Phytopreparations was carried out from November 2015 to May 2016 in Bishoftu. The two plant materials were collected from Bishoftu (Debrezeit) and were identified at Aklilu Lemma Institute of Pathobiology (ALIPB), Addis Ababa University, (AAU). Bishoftu is located at 47 km South-east of Addis Ababa. The area has an altitude of 1,860 m above sea level with an average annual rain fall of 866 mm. It has a bimodal rainy seasons; a main rain season extends from the month of June to September and a short rainy season from March to May. The annual average minimum and maximum temperature is 11°C and 26°C, respectively. Day length is constant throughout the year (12-13 h) with about 6 h of sunshine during the rainy season and 8-10 h for the rest of the year. Humidity is about 50.9% .
The study design was an experimental design of anti-microbial efficacy with extract of medicinal plants. During the study period two different medicinal plants namely, Calpurnia aurea leaf (local name “Digita”) and Cordia africana root (Local name “Wanza”) were selected for their in vitro inhibitory effect against Salmonella species, E. coli and S. aureus and for those with the highest antibacterial activity on disc diffusion assay in which MIC was determined. Finally, the antibacterial activities of two solvent extracts with high efficacy were compared with standard antibiotics namely; streptomycin for E. coli and Salmonella species and tetracycline for S. aureus.
Description of study plants and their uses
Calpurnia aurea (“Digita” in Amharic): It belongs to the group of flowering plants within the family Fabaceae. It is a shrub or small tree growing in or along the margin of forests in many parts of Ethiopia . In Ethiopia, the leaf of C. aurea is traditionally used for the treatment of syphilis, malaria, rabies, diabetes, hypertension, diarrhoea, leishmaniasis, trachoma, elephantiasis, fungal diseases and different swellings, stomachache, bowel and bladder disorders .
Cordial africana (“Wanza” in Amharic): It is belongs to the family of Boraginaceae; which is a small to medium-sized evergreen tree, 4-15 m high, heavily branched with a spreading umbrella-shaped or rounded crown. The bark is grayish-brown to dark brown, smooth in young trees, but soon becoming rough and longitudinally fissured with age; young branchlets with sparse long hairs. Leaves are broad thinly dark green. It grows at altitude of 550-2,600 masl and mean annual rainfall 700-2,000 mm large leafed Cordia thrives in forest soil. Mature fruits have a sweet, mucilaginous, edible pulp .
Bacterial species used for the experiment
Bacterial species used in the study consisted of three pathogenic bacteria namely Salmonella species and E. coli isolates obtained from calves with diarrhea and S. aureus was from bovine mastitis. These organisms cause food borne diseases in human and diarrhea and mastitis in animals. Their effect on public health is high. Moreover, they have proven to be ideal for screening studies since it is easy to culture and generally present in large numbers in animal feces and milk. These bacteria streaked onto nutrient agar to obtain pure isolated colonies following a standard aseptic technique and the four-way streak plate inoculation .
Collection of plant materials and pre extraction preparation: The fresh leaves of Calpurnia aurea and roots of Cordia africana were collected from Bishoftu. The preparation of the plants and extractions were carried out following a protocol described by Gemechu et al. . After collection, the plants were washed with tap water to eliminate any foreign matter. Each plant material chopped into small pieces, air-dried under shade and grounded to fine powder using a wooden pestle and mortar. The material sieved and weighed before maceration.
Preparation of crude extracts: The grounded leaves and roots were weighed to be 100 g using an electronic balance and the powder was put into beaker. 400 ml (1:4 ratios) of 80% ethanol was added and macerated for 72 h with automatic shaker and the solution was filtered using a conical flask, a filter funnel and Whatman No. 1 filter paper (Camlab, UK). Filtered extract poured in a 500 ml round bottom flask. The solvents were evaporated from the filtered extracts in Rota vapor. Temperature of the water bath in the Rota vapor was set at 60°C to remove the remaining solvent. This temperature used because the evaporation under reduced pressure makes it possible to evaporate at much lower temperatures. Same procedure was repeated with 80% methanol for each plant.
The dried methanolic and ethanolic extracts obtained from each plant were taken out, put in petridishes and kept in a dry oven at 40°C to remove the remaining solvent until all solvents were evaporated. To determine the yielded quantities of extracts in two solvents, the weight of petridishes were subtracted from the weight of the petridishes and samples. The percentage weight yields were calculated as described by Emtinan et al. .
% yield = (weight of extract obtained)/ weight of plant powder
Preparation of antimicrobial discs from extracts: Preparation of paper disks: Paper disks with approximate diameter of 6 mm were punched out one by one from a sheet of paper Whatman Filter paper #1 using an ordinary office two-hole puncher. Precautions were taken to avoid overlapping of holes. Before the discs were impregnated they were kept on aluminium foil and autoclaved a 121°C for 15 min . A stock solution of 200 mg/ml (20%) in 2% Tween 80 was made for each extract. Concentration was recorded on a weight by volume (w/v) basis. Stock solution was stored in regular refrigerator set at +4°C. Working solutions made from the stock solution. To prepared 20% (20 g/100 ml), taken 1 g of crude extract and it was mixed with 5 ml of 2% Tween 80 by using vortex. Then, 2.5 ml from the first test tube (20%) was transferred to a second test tube by using micropipette to prepare 100 mg/ml (10%). The procedure continued by transferring 2.5 ml of solution taken from the 10% to prepare 50 mg/ml (5%) and added 2.5 ml 2% Tween 80 to the third test tube then mixed by using vortex. Reconstituted extracts were prepared for the evaluation of antimicrobial properties against the bacteria.
This was done by taking 20 µl of each extract of various concentrations and impregnated at 6 mm blank paper disc and leaving them in the biological hood (LABCONCO Purifier Class II bio-safety cabinet; Kansas, Missouri) to dry . After dried they were used for screening the antibacterial activity. Tween80 (2%) was used as negative control while standard antibiotic discs were used for Salmonella species and E. coli (streptomycin) and for S. aureus (tetracycline) as a positive control. The medicinal plants were chosen based on their frequent use for the treatment of those organisms.
Preparation of the test bacteria: The plant extracts were tested against E. coli and Salmonella species obtained from diarrhea of calves while S. aureus from bovine mastitis. These bacterial isolates obtained from the Department of Microbiology, College of Veterinary Medicine and Agriculture. The bacterial isolates were maintained on nutrient agar and sub cultured every three days. Each bacterial isolates was suspended in 5 ml of saline solution and adjusted to give a concentration of bacterial cells equivalent to 0.5% McFarland turbidity standard prior to the antibacterial testing .
Determination of in vitro antibacterial susceptibility
Principle: When a filter paper disc impregnated with a chemical placed on agar medium, the chemical will diffuse from the disc into the agar. Multiple factors can alter the size of the no-growth-zone around the disc. The solubility of the drug in question, its molecular size and its antimicrobial potency will be determined. If an organism is placed on the agar medium suitable for its growth, it will not grow in the area around the disc, if it is susceptible to that antibacterial agent. If there is no growth around the disc, it is known as ‘zone of inhibition’.
Procedure: Muller-Hinton agar (38 g) (Biotech, UK) medium was used for antimicrobial sensitivity test, and was mixed with 1litter of distilled water boiled to dissolve completely and autoclaved at 121°C for 15 min. Mueller-Hinton agar medium was prepared by pouring sterile agar plates and left to set. The agar plates incubated for 24 h at 37°C to confirm their sterility. When no growth occurred after 24 h, the plates considered sterile and used for antimicrobial sensitivity test . The colonies of the same morphology were scooped using a wire loop from the nutrient agar and mixed using sterile normal saline, and agitated with a vortex mixer. The turbidity of the bacterial suspension adjusted by comparing with 0.5 McFarland turbidity standards (1.5 × 108 CFU/ml) . The standard and the test suspension were placed in a 10 ml sized test tubes and compared against a white background with contrasting black lines until the turbidity of the test suspension equates to that of the turbidity of the standard. Adjustments of the turbidity made by adding saline or colonies depending on the degree of turbidity.
For each bacterium, a sterile applicator swab was dipped into its standardized cell suspension and squeezed gently by rotating the swab against the inside of the glass tube above the liquid to remove the excess fluid. The entire surface of each agar plate inoculated by streaking the swab in different directions to ensure a uniform growth. Additional plates for each organism were incubated by this fashion. A sterile disposable syringe needle used to pierce in the centre of a plain filter paper disc. The syringe was held upright with disc above. The required concentration of the plant extraction taken by a micropipette and discharged gently on the disc until the disc was thoroughly soaked. Then, the disc stamped on the surface of Mueller-Hinton agar medium. Changing the tips of micropipette, the processes were repeated . All the discs placed approximately the same distance from the edge of the plate and from each other.
The inoculated plates kept inverted in incubator at 37°C for 24 h. After 24 h, the plates viewed against a black background and illuminated with reflected light and zones of inhibition measured by ruler and recorded. The experiment repeated for five times and the result expressed as average value of zone of inhibition of each plant .
Determination of minimum inhibitory concentration (MIC)
Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. The micro dilution broth method was used to determine the MIC . Microorganisms were tested for their ability to produce visible growth on a series of agar plates at different concentrations of crude extract with incubation at 37°C for 18 h. The lowest concentration of crude extract that inhibited the visible growth of microorganisms designed as the MIC of that agent. The 80% ethanol and methanol leaf extract of C. aurea and root extract of C. africana inhibited growth of microorganisms were tested for MIC. The working solution of extracts (50 mg/ml) were diluted out across a 96-well in a serial dilution to give final testing concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.36, 0.18 and 0.09 mg/ml. The first column (C1) were filled by