Extraction and Phytochemicals Determination of Traditional Medicinal Plants for Anti-microbial Susceptibility Test

The study were conducted at Dilla University, College of Natural Sciences, Biological Sciences laboratories. Background: Ethiopia is a country with many ethnic groups, cultures and beliefs which in turn have contributed to the high diversity of traditional health care knowledge and practices of traditional medicine from local growth plants, animals and minerals for various physical and mental disorders of human and livestock population that passed from generation to generation for centuries. Medicinal plants contributors to pharmaceutical, agricultural and food industries in the world. The use of medicinal plants in the industrialized societies has been traced to extraction and development of several drugs used in order to heel some diseases having inhibiting effect against pathogenic microorganism. Objective: The main objective of this study was Extraction and Phytochemicals determination of traditional medicinal plants for anti microbial susceptibility test. Methodology: The extraction and identification of some phytochemicals crude compound which used for antimicrobial susceptibility test from plant sample such as Ocimum lamiifolium (OL), Croton maerosth (Cm) and Ruta chalepesis (RC) were conducted. Plant samples are collected, powdered using mortal and pistil and extracted using ethanol and some susceptibility tests were performed to identify some phytochemicals compound. Result: The main result of Antimicrobial activity test showed that the crude extract of OL has the highest zone of inhibition. The highest yield of crude extract (38.21%) was obtained from Croton maerosth (CM) which followed by Ruta chalepesis (RC) (32.43%). However, the lowest yield (28.37%) was obtained from Oscpmum lamifolium (OL). Conclusion: Traditional Medicine is used by many people to managing numerous conditions; it’s accessible and effective on antimicrobial activity. Therefore, it plays a significant role by reducing life-threatening ailments of people and other animals.


INTRODUCTION
Historically, learning via observation and experimentation indicates that plants have played as a major role for traditional medication and used to promote health of human beings. Traditional medicinal plants are used as remedies of safe for health, cost effective, almost free from serious side effect and also used as health practice, approaches, knowledge and beliefs. They are obtained from plant, animal and mineral based which used for spiritual therapies, being the plants are the most important source of medication. They used to apply in single or in combination to treat, diagnose and prevent illness and maintain well-being ( The people did not have the scientific insight to explain the use of traditional medicinal plants are found in many of the healing cultures of the world based on the assumption that the appearance of plant may give clues to their medicinal properties and interpreted as God's signature on the plant. For example, red juice and sap, is associated with blood and menstrual elements and also yellow flower and alkaloid containing latex of some plants associated with bile and jaundice is crude extract used successfully to treat jaundice (Gurib-Fakim, 2006). The traditional medicinal plant bioactive compounds are obtained mostly from plant leaves and used as an Firew AH et al. Medicinal Plants & Anti-microbial Susceptibility Test alternative medication for the treatment of tonic diuretic, antiphlogistic and blood purifier and also used as remedy against chronic ulcers, chronic eczema, chronic nervous disease, chronic rheumatism, cholera amenorrhea, madness, piles and fistula. The powder of the dried leaves is often given with milk in mental disability for the improvement of the memory. The fresh juice of leaves is grown as alterative in jaundice, fevers and gonorrhea. The same is also useful for children's in cutenaceous diseases and for the improvement of nervous system (Mazid, et al., 2012). Ailments have over the years been a scourge and a threat to mankind. People from different cultural backgrounds have used different herbal plants, plant extracts, animal products and mineral substances (Addae-Mensah, 1992) as the means to care, cure and treat illhealth, with disease prevention, and with health promotion (Curtis and Taket, 1996) since pre-historic times. Definition of traditional medicine by WHO as diverse health practices, approaches, knowledge and beliefs incorporating plant, animal, and/or mineral based medicines, spiritual therapies, manual techniques and exercises applied singularly or in combination to maintain well-being, as well as to treat, diagnose or prevent illness (WHO, 2002;2000).
There are a number of pathogenic microorganisms such as bacteria, fungi, viruses, etc are the main causative agents of infectious diseases that is a critical challenge to health and they are believed to be one of the main causes of increasing the rates of morbidity and mortality worldwide (Drusano, 2004

Description of Study Area
Dilla town is located at Gedeo zone, southern nation and nationality people region of Ethiopia. The town has latitude and longitude 38 0 18'36"E and 6 0 24 ' 30 " N with an elevation of 1570 meter above from sea level and 360km distance from the capital city of Ethiopia, Addis Ababa. The climate of Gedeo zone is characterized as worm humid temperature. The Gedeo zone is endowed with two rain season from March to May and July to December with interruption of 3 to 4 dry season. The climate is suitable for abundant forest cover. The population of Dilla town was around 59,150, the total number of male 31,068 and the women is 28,082 (PS, 2012). Gedeo zone contain one hospital established by government. The main economic activity and source of income in the district is farming. Antimicrobial Susceptibility Testing. Antibacterial activity test : the standard Kirby-Bauer disk diffusion method was used to determine the antimicrobial susceptibility testing (Bauer et al., 1996). Media such as nutrient agar (NA) and nutrient broth (NB) was used as cultural medium. Bacterial inoculums were prepared by suspending the freshly grown bacteria in 5 ml sterile NB and the turbidity was adjusted to that of a 0.5 Mcfar land standard. A concentration of 1-2x10 8 CFU/ml of bacterial inoculate was used. The media were poured onto 60mm diameter Petri plates (4mm agar thickness). A 0.1 ml of each bacterial suspension were obtained from nutrient broth (NB) and inoculated on to fresh media of nutrient agar (NA). The bacterial suspensions were uniformly distributed using swabs. Plates were allowed to stand for 15 min. Then, 6mm diameter disks were impregnated with the crude extract of medicinal plant. The impregnated disks were symmetrically placed onto the medium by using sterile tweezers. The plates were incubated aerobically at 28 o C for 24hrs. Based on the method described by Ayoola et al. (2008) the plant leaf samples phytochemical compositions were analyzed. Antimicrobial Susceptibility Testing. Antibacterial activity test: the standard Kirby-Bauer disk diffusion method was used to determine the antimicrobial susceptibility testing (Bauer et al., 1996). Media such as nutrient agar (NA) and nutrient broth (NB) was used as cultural medium. Bacterial inoculums were prepared by suspending the freshly grown bacteria in 5 ml sterile NB and the turbidity was adjusted to that of a 0.5 Mcfar land standard. A concentration of 1-2x10 8 CFU/ml of bacterial inoculate was used. The media were poured onto 60mm diameter Petri plates (4mm agar thickness). A 0.1 ml of each bacterial suspension were obtained from nutrient broth (NB) and inoculated on to fresh media of nutrient agar (NA). The bacterial suspensions were uniformly distributed using swabs. Plates were allowed to stand for 15 min. Then, 6mm diameter disks were impregnated with the crude extract of medicinal plant. The impregnated disks were symmetrically placed onto the medium by using sterile tweezers. The plates were incubated aerobically at 28 o C for 24hrs. Based       The weight of all samples are 37g, but after rotary evaporator Croton maerosth higher weight of crude extract but Oscpmum lamifolium lower weight of crude extracts and the medium was Ruta chalepesis.  In these study crude extract is high low inhibition zone.  The crude extract very low is high inhibition zone of medicinal plants. Some traditional medicinal plants contain some bioactive and phytochemical compounds. The present study has been shown that Oscpmum lamifolium (OL) Croton maerosth (Cm) and Ruta chalepesis (Rc) contain some phytochemical compound. The crude extract of all the studied plants leaves contain phytosterol, steroids and tannin phytochemical compounds which are bluish green, yellow with green fluorescence and yellow precipitate in color after screening have been conducted, respectively.

ACKNOWLEDGMENTS
First of all we would like to thank Almighty God (Allah) for letting as to be healthy and accomplish this study. Secondly, we would like to thank Dilla University, Zoology and microbiology laboratory staff for their supporting as during study time. Finally, we would like to thank our friends from chemistry staff for sharing and providing as some laboratory materials.

AUTHORS' CONTRIBUTIONS
The participation of each author corresponds to the criteria of authorship and contributorship emphasized in the Recommendations for the Conduct, Reporting, Editing, and Publication of Scholarly work in Medical Journals of the International Committee of Medical Journal Editors. Indeed, all the authors have actively participated in the redaction, the revision of the manuscript, and provided approval for this final revised version.