Antibacterial activity of extracts from myrtus communis l. (ades) and dodoneae angustifolia l.f. (kitkita) using bioautography method – journals morphology of staphylococcus aureus

The increasing prevalence of antibacterial drug resistant organisms in our globe and high prevalence of infectious diseases in developing countries has led to new efforts in the search of bioactive compounds from complex chemical composition of plant extracts. A bioautographic agar overlay assay using Staphylococcus aureous as the indicator organism for the detection of antimicrobial compounds from ten extracts of

L was analyzed. Hexane, dichloromethane, acetone, methanol and water solvents are used as extractant and ethyl acetate: methanol: water, chloroform: ethyl acetate: acetic acid and benzene: ethanol: ammonia solvent systems were used to separate the components from all the extract of

L had bioactive constituents responsible for their antibacterial potentials. Water solvents extracted small number of antibacterial compounds from both plants, followed by hexane extractant; while dichloromethane, acetone and methanol extractant shared similarities in bioactive compounds on bioautograms, and extracted the highest number of antibacterial compounds with variety of polarities.

Chloroform: ethyl acetate: acetic acid solvent system separated the largest number of biologically active components in all extractants. As a high number of antibacterially active compounds were found in

infectious diseases. It was the major causes of mortality prior to the modern era of antimicrobial chemotherapy, and even today, infectious diseases worldwide account for over one-third of all deaths (NIC, 2000). In developing countries, more than one-half of all deaths annually were reported to be due to infectious diseases alone and Ethiopia is not exceptional (NIC, 2000; WWI, 2005). Even though antimicrobial agents were used to manage infectious diseases for many decades, in recent years, man is facing challenges with controlling and treating of these diseases due to the fact that resistance has developed to almost every group of antibiotics (Fish and Ohlinger, 2006). It is true that because of this antibiotic resistance many people are dying from previously curable infectious diseases (Fish and Ohlinger, 2006). The challenges due to high prevalence and antimicrobial resistance of infectious diseases necessitate many researchers to continuously search for novel antimicrobial drugs for the treatment of infectious diseases

., 2001). Traditional medicine is an important source of alternative medicine. Natural products especially, medicinal herbs are the source of inspiration for researchers in their search of novel compounds for the development of new antibacterials because they contain complex chemical constituents of therapeutic values (Cowan, 1999; Rios and Recio, 2005). Many of commercially used modern antimicrobial drugs were initially used in crude form in traditional practices. It is estimated that today, plant materials have supplied approximately more than one quarter of prescribed drugs for the treatment of human diseases (Cowan, 1999; Rios and Recio, 2005). However, this is only a few contribution for medicine as the traditionally used medicinal plants are thought to be store houses of complex undiscovered biologically active compounds having antimicrobial potentials (Leeds

., 2006). In Ethiopia, natural products, have been the basis of treatment of human diseases since time immemorial (Dawit Abebe and Ahadu Ayehu, 1993). As elsewhere in the world; the majority of Ethiopian population are dependent on medicinal plants for the treatment of infectious diseases (Dawit Abebe and Ahadu Ayehu, 1993). The country is rich in natural products having flora that is estimated to contain around 7000 species of higher plants and more than 800 are estimated to have therapeutic values (Dawit Abebe and Ahadu Ayehu, 1993; Sebsebe Demissew, 1993). However, the country is unable to get medical and economic advantage from these natural products and has been facing infectious diseases in the presence of therapeutic natural

L.f. were collected from North Shewa and Bale Zone, Ethiopia on September, 2008. Authentication of the plants identity was done in the Department of Drug Research, Ethiopian Health and Nutrition Research Institute, Ethiopia. The voucher specimens were deposited in the Herbarium of Department of Drug Research.

were dried in the shade and grounded to a fine powder. Samples from each plant were individually extracted by weighing five aliquots of 1g of finely ground plant material and extracting with 10ml of hexane, dichloromethane, acetone, methanol and water separately in centrifuge tubes. These were vigorously shaken and then centrifuged at 3500 rpm for 10 minutes; the supernatant was decanted into labeled containers. The procedure was repeated three times (Eloff, 1999). The solvent was removed under a cold-air stream and a different yield of dried extract was obtained. The crude dry extract was weighed and re-dissolved in

plant materials. Chemical tests for tannin, saponins, flavonoids, phenolic glycosides, chromophers, free anthra-quinones, anthraquinone glycosides, alkaloid and poly phenols were carried out on the powdered specimens, aqueous and alcoholic extract using standard procedures adopted in our phytochemicstry laboratory to identify plants secondary metabolite constituents (Asfaw Debella, 2002). Normal phase aluminum backed silica gel G60 F254 plates (10cm X 10cm) were used to develop chromatogram in order to separate the components of plant extracts. Each chromatogram was loaded with 5µL of 20mg/ml of solutions having the spot size of 4mm and developed in duplicate (with one plate intended for bioautography and another for reference chromatogram) simultaneously in a saturated, twin trough developing tank to minimize variation in chromatographic conditions. Ethylacetate: methanol: water (8:1:1) (polar/neutral); chloroform: ethylacetate: acetic acid (5:4:1) (intermediate polar/acidic); benzene: ethanol: ammonia (9:1:0.1) (non-polar basic) solvent system were used for the separation of chemical constituents. The UV active absorbing spots were detected at 366nm wave length on the reference chromatogram,

which was finally stained by vanillin-sulfuric acid (0.1g vanillin: 28ml methanol: 1ml sulfuric acid) in order to detect the separated chemical component of each extract. The sprayed TLC chromatogram was compared with the bioautogram for the identification of active bands or compounds.

C for 4 h. Plates were then examined for the presence of zone of inhibition of bacterial growth that could be seen around the active chromatogram spot representing separated compound. White areas that were produced due to the absence of reduction of methyl tetrazolium chloride to the colored formazan that happens in the presence of bacterium, indicated the presence of active antibacterial compounds that inhibited the growth of

extracts by different extractant and solvent systems (Fig. 1). Out of the five extractants used for the extraction, the smallest numbers of compounds from the plant materials were extracted by water solvents followed by hexane solvent while the other extractants shared many similarities in number of compounds extracted from both plants. As it can be clearly seen from this figure, the highest polar and the highest non polar solvents extracted the