Determinations of Antibacterial activity of Zambian Diospyros kirkii stem bark Miyanda petty M * Hatwiiko Haanzoma Department of Pharmaceutics, Department of pharmacy CT Institute of Pharmaceutical Sciences, University of Zambia lecturer Shahpur ,144020. Email: miyay35@gmail.com Email-Hatwiikohanzoma@yahoo.com Abstract The antibacterial activity of bark of Diospyros kirkii. (Ebenaceae) was evaluated against four strains of bacteria. Ethanol bark extract was prepared and studied for antibacterial activity using agar dilution method. Although the extract showed promising antibacterial activity against three of the tested strains with maximum activity against E.coli .It showed no activity against pseudomonas aeruginosa .Minimum inhibitory concentration(MIC) values for the extract ranged from 0.12mg/ml to 5 mg/ml. Keywords: Diospyos kirkii, Ethanol, Antibacterial activity, Agar dilution, MIC Introduction Plants have proven to be the most useful in curing diseases and provide an important source of medicine. The medicinal importance of plants lies in some of the chemical substances that they contain that may produce distinct physiological responses in humans (Okwu and hill, cited in Karmjit 2009)1. The major chemical constituents of importance in plants are steroids, terpenoids, tannins, carotenoids, flavonoids, alkaloids and glycosides. Saponins, glycosides, flavonoids and alkaloids have been reported to possess both antibiotic and antimicrobial activity (Karjimt, 2009). The chemical metabolites in plants act as chemotherapeutic, bactericidal and bacteriostatic agents (Purohit et al., 1998)2. The WHO has been a strong supporter for traditional medicines as safe remedies for ailments of both microbial and non-microbial origins (WHO, 2014).3 Scientists are shifting their attention to folk medicine in order to find new leads for better drugs against microbial infections. Plant materials are known as sources of new antimicrobial agents, as a result search has been to discover new antibacterial drugs of plant origin. various compounds like vincristine, quinine, salicyclic acid, eligitalis, morphine, and codeine have been isolated from plants which have enormous therapeutic benefit (Parekh and Chanda, 2007).4 The present study will focus on assessing the antibacterial potential of crude ethanol extracts of stem bark from Diospyros kirkii against six pathogenic bacteria, namely Staphylococcus aureus, Escherichiacoli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus pneumonia. Erythromycin will be used as a positive control while ethanol will be used as the negative control. Diospyros kirkii belongs to the family Ebenaceae it is indigenous to tropical Africa, and some parts of west Africa (Ruffo et al,. 2002).5 Diospyros species are valuable traditional medicines to Chinese herbal medicine, Tibetan medicine, and Ayurvedic (Maridass, (2008)6 .In Zambia, Diospyros kirkii is well known for its edible fruits gathered from the wild and enjoyed by both adults and children alike. The fruit is sold in local markets and also for timber and fuel (Soepadmo.E at al., 2002).7 Figure 1: Diospyros kirkii plant Diospros kirkii grows as a wild plant under natural conditions. Collected in a farm from kalomo, Zambia. Methods Plant Collection The Fresh sample of stem bark from Diospyros kirkii was collected from a farm in Kalomo, Southern Province Zambia were it grows as a wild plant and authenticated at the University of Zambia, department of biological sciences by the botanist and a herbarium kept. Stem bark of Diospyros kirkii was collected during the rainy season (March, 2016) by debarking part of the stem using an axe. The maceration process of crude drug extraction was adopted in this study. The identified plant part was washed with tap water and air-dried under a shade away from direct sunlight .The dried part was chopped into pieces, milled into fine powder by pounding manually with a clean and sterile pestle and mortar. The powdered samples were collected into sterile cellophane bag and labelled. The sample was kept in cool dry place till further use. The powdered sample was used for extraction purposes. (Shagal et al., 2012).8 Extraction procedure 320g of powder was measured out and placed in 1000ml of high grade ethanol (HI media, India) as outlined in samie et al (2005)9 and was left to stand at room temperature for 72 hours with frequent agitation.Whatman® Cellulose Filter Paper Number 1 was used to filter the mixture. The extraction process was repeated twice so as to ensure complete extraction of chemical metabolites. The filtrate that was obtained was then concentrated to dryness by use of rotary evaporator under reduced pressure and low temperature of 370C, the powder extract was put in an air tight, sterile glass container and placed in the refrigerator until further use. Reconstitution in dimethylsulphoxide (DMSO) solvent was done immediately before antibacterial assay. Bacteria Strain The plant extract was tested against four standard bacterial species. Two Gram positive bacteria; Staphylococcus aureus (ATCC 25923) and Streptococcus pneumonae with two Gram negative bacteria; (Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 49619). All bacteria that were used in this study were obtained from Food and Drugs control microbiology laboratory and a microbiologist assisted with the culture plates for the organisms. Agar diffusion Method and antibacterial assay Screening for antibacterial activity was carried out by agar diffusion method (samie et al., 2005). Prior to performing antibacterial assay; the bacterial strains were preserved on nutrient agar. Bacteria was sub cultured on Blood agar and 0.5% McFarland standard was prepared by suspending bacteria in 5ml of Mueller Hinton broth .Suspended bacteria was then incubated for 24hours at 370C. Adjustments were done to 24hour cultures to deliver a concentration of bacterial cells equivalent to McFarland No. 0.5 (Samie et al., 2005). Sterilised Muller-Hinton agar plates were inoculated with 0.1 ml of 24hour old bacterial pathogens whose concentration was equivalent to McFarland standard of 0.5% .Using a sterile inoculation loop, while holding the Petri dish at 450angle, the inoculation loop was run across the plate moving from outside to inside in a zigzag motion until the entire plate surface was covered in bacteria. 25mg of dried crude extract was dissolved in 1ml of DMSO to make a stock solution (25mg/ml). 0.01ml of this stock solution was added to each disc and was allowed to dry. After drying the disc containing the extract and the antibiotic disc they were individually and aseptically added onto the inoculated Muller-Hinton agar medium. Each disc contained 0.5mg of the crude extract. The plates were subsequently incubated at 370C for 24hours for the bacterial growth. The antibacterial activity was determined by the measurement of diameter of clear zone of inhibition using a measuring rule. The procedure was carried out three times so as to obtain consistent results. 10mg/ml of Gentamycin disc were used as positive controls and 0.01ml of ethanol coated disc was used as negative control. Determination of Minimum Inhibitory Concentration (MIC). The minimum inhibitory concentration (MIC) of the extracts that showed growth inhibition activity against the selected organism was determined by the agar dilution method as described by Lalitha (2009)10 with modifications. Briefly, MHA was prepared as per manufacturer’s instruction. After autoclaving, 19 mL of the media was dispensed into sterile test tubes with caps and maintained at 45 ºC. To each test tube 1 mL of the extracts already diluted to give the following concentrations 0.12mg/mL, 0.25 mg/mL, 0.5mg/mL, 1mg/mL, 2.5 mg/mL and 5 mg/mL was added. They were properly mixed for even distribution of the extracts within the agar, and the agar was poured into plates and allowed to set. The plates were then dried in a fume hood to remove steam. Each plate was divided into five sections. A loopful of the undiluted overnight broth culture was used to inoculate each section of the solidified agar/extract mixture. A media/DMSO plate with organisms and a plate with media/gentamycin containing organisms served as negative and positive control respectively. The plates were incubated at 37 ºC for 24 hours. They were then examined for the presence of colonies after the incubation period. The lowest concentration that gave no visible colonies was taken as the minimum inhibitory concentration of the crude extract. 3.4 Data analysis This was a quantitative experimental study; the data from the laboratory experiments was categorised, organised and presented using tables, bar charts constructed using Microsoft excel windows 2010 4.0 Results The antibacterial assay of the plant was carried out against four standard strains of bacteria namely Staphylococcus aureus (25923), Escherichia coli (27853), Streptococcus pnemoniae and Pseudomonas aeruginosa (49619). From the results it was revealed that the plant extract was active against the test bacteria at a concentration of 25mg/ml. The antibiotic used as positive control showed a varying range of activity against the respective bacteria strains that were tested against. The mean triplicate values of zones of inhibition of the bark extract of Diospyros kirkii and of the controls are shown in the proceeding table 1. Table 1: Showing diameter zones of inhibition of experiment 1   Diameter of zone of inhibition (mm) Test organisms Extract GN Ethanol S.aureus (ATCC25923) 8.5 20 0 P.aeruginosa (ATCC 49619) 0 20.5 0 E.coli (ATCC 27853) 9.8 19 0 Streptococcus pnemoniae 7.2 18 0 CN= Gentamycin, Table 2: Showing diameter zones of inhibition of experiment 2   Diameter of zone of inhibition (mm) Test organism Extract GN Ethanol S.aureus (ATCC25923) 8 18.5 0 P.aeruginosa (ATCC 49619) 0 22 0 E.coli (ATCC27853) 10.5 NT 0 Streptococcus pnemoniae 7.8 17 0 Table 3: Showing diameter zones of inhibition of experiment 3   Diameter of zone of inhibition (mm) Test organisms Extract GN Ethanol S.aureus (ATCC 25923) 8.5 18 0 P.aeruginosa (ATCC 49619) 0 NT 0 E.coli (ATCC27853) 10 20 0 Streptococcus pnemoniae 7.5 18 0 Table 4: Values representing mean of triplicate Antimicrobial activity of extracts stem-bark of Diospyros kirkii. Showing diameter of zone of inhibition (millimetre) on different isolates Diameter of zone of inhibition (mm) Test organisms Stem-bark extract gentamycin Positive control Ethanol Negative control Streptococcus pneumonae 8.3 18 _ Staphylococcus aureus(ATCC25923) 7.5 18.8 _ Pseudomonas aeruginosa (ATCC 27853) _ 21 _ Echerlicia coli (ATCC 49619) 10.1 19.5 _ Figures indicate Zone of Inhibition in mm. Inactive (-): weak activity (<7) moderately active (7-13): highly active (> 14) GN= Gentamycin, NT= Not tested NB: 6mm is the diameter of the disc Table 5: Minimum inhibitory concentration (MIC) of crude extracts activity against the tested microbes Test concentration Echerlicia coli(ATCC 49619) Streptococcus pneumonia Staphylococcus aureus(ATCC25923) 0.12mg/ml _ _ _ 0.25mg/ml _ _ 0.5mg/ml _ 1mg/ml 2mg/ml 5mg/ml 1ml DMSO _ _ _ 5.0 Discussion The antimicrobial activity of different crude extracts of D. kirkii bark was determined against four human pathogenic bacterial strains which are reported in Table 1. The extracts of D. kirkii bark in ethanol showed promising antibacterial activities against almost all bacterial strains. Both gram positive and gram negative bacteria were susceptible to the plant extract which is in contradiction to earlier reports by some workers (Rabe and Van Staden, 199711; Vlietinck et al., 1995)12 that plant extracts were most active against gram positive microorganisms than gram negative ones. In the present work, among gram positive bacteria Streptococcus pneumonae was the most susceptible with inhibition zones of 8.3 mm in ethanol extract,0mm ethanol and 18mm in gentamycin. whereas in case of gram negative bacteria, Escherichia coli was most susceptible with inhibition zones of 10.1mm, 0mm and 17mm for the extract, ethanol and gentamycin respectively. Minimal inhibitory concentration (MIC) of extracts with positive responses was tested on the bacterial strains which are given in Table 5. In the present study, MIC was determined by agar dilution method as described by Lalitha (2009) with modifications. where DMSO was used as a solvent. MIC values ranged from 0.12mg/ml to 5 mg/ml, The MICs for the plant extract that showed growth inhibition activity are shown in Table 5 indicated in red ticks. Echerlicia coli (ATCC 49619) was inhibited at the lowest concentrations of 0.25mg/ml of all the test organisms that showed activity followed by Streptococcus pneumonae at 0.5mg/ml and Staphylococcus aureus at 1mg/ml.There have been reports of antimicrobial activity of root, leaf and bark of D. anisandra (Borges-Argaez et al.,2007)13 and D. peregrina fruits (Dewanjee et al., 2007)14. Reports of antipyretic properties of D. mespiliformis (bark) and D. variegata (stem) (Adzu et al., 2002; Trongsakul et al., 2003)15-16 are also reported. The present paper reports antimicrobial activity of the bark of D.kirkii. In the present study both gram positive and gram negative bacteria were selected for screening of antimicrobial effect of ethanol of a single plant part to determine the antimicrobial spectrum and also to authenticate the ethnomedicinal uses. Diospyros species bark has been used against diarrhoea, dyspepsia by ethnomedicial practiotioners (Arunendra et al 2004).17 Thus, present findings of antimicrobial activity of D.kirkii have fairly good degree of correlation with ethnomedicinal uses of the plant. Preliminary results of this investigation appear to indicate that bark of D. kirkii have high potential antimicrobial activity. Novel bioactive compounds from the bark need to be isolated and screened for their pharmaceutical and biotechnological applications in order to cure chronic and infectious diseases. 5.1 Conclusion The demonstration of activity against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 49619 and Streptococcus pneuomoniae provides scientific evidence for the traditional usage of Diospyros kirkii in the treatment of ailments such as boils,wound infections and respiratory tract infections such as pneumonia. The fact that diospyros kirkii stem bark extract showed activity against gram negative and gram positive bacteria is an indication that it possesses broad spectrum of activity which is an important aspect because of the possibility of developing future therapeutic agents. The wide spectrum antibacterial activity and the low MICs exhibited indicate that D.kirkii can be used to treat diseases caused by the susceptible organisms. This study has also highlighted the interest in herbal plants as alternative therapies and hence act as an entry point for the development of plant based products into useable drugs, which would be of benefit to the country especially those in rural areas. NO conflict of interest from authors References Karmjit (2009). Phytochemical determination and antibacterial activity of Trichosanthesdioica Roxb (patal), Cucurbita Maxima (pumpkin and Abelmoschusesculentus Moench (Okra)plant seeds, thesis, Rourkela, National institute of technology. journal of phytochemistry. 12(4) , 614-624. Purohit p, Bohora A ( 1998). Effect of some plants extract on conidial germination of some important phyto pathogenic fungi. Geobios New Report. 17:183-184. World Health Organisation 601-607 (2014).The promotion and development of traditional medicines.Geneva:WHO; p.622. Parekh J, Chanda S (2007). In Vitro antibacterial activity of the crude methanol extract of Woodfordia fruticosa Kurz. Flower (Lythraceae).Braz. J. Micr. 38: 204-207. Ruffo, C.K.: Birnie, A. &Tengnas, B (2002). Edible Wild Plants of Tanzania. Regional Land Management Unit; Nairobi. ISBN-9966-896-60-0. M. Maridass, S. Ghanthikumar and G. Raju (1999). Preliminary Phytochemical Analysis of Diospyros Species. Animal Health Research Unit, St. Xavier’s College, Palayamkottai-627002, India. Soepadmo E.; Saw L.G.; Chung R.C.K ( 2002).Tree Flora of Sabah and Sarawak Forest Research Institute Malaysia; Kuala Lumpur.1– 6: 983-2181-27-5. Shagal.M.H, D. Kubmarawa, and H. Alim ( 2012). Preliminary phytochemical investigation and antimicrobial evaluation of roots, stem-bark and leaves extracts of Diospyros mespiliformis . International Research Journal of Biochemistry and Bioinformatics. 2(1). 011-015. Samie A., Obi C.L.,Bessong P.O, Namiita. L (2005).Activity profiles of fourteen selected medicinal plants from rural venda communities in south Africa against fifteen clinical bacterial species. African journal of Biotechnology 4 (12):1443-145. Lalitha MK.(2008).Manual on Antimicrobial susceptibility testing. 1st ed. Vellore, Tamil Nadu: Department of Microbiology, Christian Medical College: pp.10-13 Rabe T, Van Staden J (1997). Antibacterial activity of South African plants used for medicinal purposes. J. Ethnopharmacol. 56: 81-87. Vlietinck AJ, Van Hoof L, Totte J, Lasure A, Vanden Berghe D,Rwangobo PC, Mvukiyuniwami J (1995). Screening of hundred Rwandese medicinal plants for antimicrobial and antiviral properties.J. Ethnopharmacol. 46: 31-47. Borges-Argaez R, Canche-Chay IC, Pena-Rodriguez LM, SaidFernandez S, Molina-Salinas GM (2007.) Antimicrobial activity of Diospyros anisandra. Fitoterapia 78: 370-372. Dewanjee S, Kundu M, Maiti A, Majumdar R, Majumdar A, Mandal SC (2007). In vitro evaluation of antimicrobial activity of crude extract from plants Diospyros peregrina, Coccinia grandis and Swietenia macrophylla. Trop. J. Pharm. Res. 6(3): 773-778. Adzu B, Amos S, Dzarma S, Muazzam I, Gamaniel KS (2002). Pharmacological evidence favouring the folkloric use of Diospyros mespiliformis Hoehst in the relief of pain and fever. J.Ethnopharmacology. 82: 191-195. Trongsakul S, Panthong A, Kanjanapothi D, Taesotikud T (2003). The analgesic, antipyretic and anti-inflammatory activity of Diospyros variegata Kurz. J. Ethnopharmacol. 85: 221-225. Ganapaty S. Thomas, PS, Karagianis , G. Peter, G.W and Brun R (2006). Evaluation of five diospyros species for their antimicrobial activity. Phytochemistry :67(17), 1950.