Determination of Antimicrobial Activity and Phytochemical Screening of Selected Medicinal Plants in Tigray region of Northern Ethiopia

Present paper evaluates the antimicrobial activities and preliminary phytochemical screening of the extracts of Beciumgrandiflorum, Meriandrabengalensis, Tamarindusindica, Balanitesaegyptiaca and Otostegiaintegrifolia. Comparative study was made for the selected traditional medicinal plants. The results of phytochemical test indicate that the bioactive chemical components show presence of alkaloids, flavonoids, phenols, carbohydrates, saponins and terpenoids. The antimicrobial activities of aqueous and ethanol extracts of leaf and stem were determined on the selected bacteria strains. The results indicate that the aqueous and ethanol leaf extracts of Meriandrabengalensis show better antimicrobial activity against Staphylococcus aureus and Staphylococcus pneumonia with inhibition zone diameters of 15.97 ± 0.09 and 19.03 ± 0.15 mm respectively, significant at (P<0.05) compared to other extracts. From the phytochemical components tested, carbohydrate was present in Becium grandiflorum extract. The lowest inhibition zone (4.5 ± 0.29 mm) was achieved for the extract of Balanitesaegyptiaca. The phytochemical component saponinis significantly present in Meriandrabengalensis in the aqueous leaf extract. In the comparative study made for the extraction solvents aqueous and ethanol, the aqueous extraction had strong inhibition zone. Therefore, a study need to be done on the isolation, identification, and quantitative determination of antimicrobial components present in the Meriandrabengalensis for its application in both animal and human pharmaceutical industries.

During the second half of the 20th century, taking traditional medicine as a substitute form of health care and the development of microbial resistance to the classical antibiotics led researchers to investigate the antimicrobial activities of medicinal plants.Antimicrobials of plant origin have enormous healing potential (Cowan, 1999).One of the vital activities possessed by these traditionally used medicinal plants is antimicrobial.The scarcity of infective diseases in plants is in itself signal of the successful defense mechanisms developed by them (Bolla et al., 2011).The substances that can either inhibit the growth of bacteria or kill them, with no toxicity or minimum toxicity to host cells are considered entrants for developing new antimicrobial drugs (Lee et al., 1998).Some of the bioactive compounds could deter the life processes of diseasecausing bacteria, either by itself or in combination with other therapeutic agents (Sivananthan, 2013).In recent years, antimicrobial properties of medicinal plants are being progressively reported from different parts of the world (Grosvenor et al., 1995;Ahmad and Beg, 2001;Abu-Shanab et al., 2008).It is expected that plant extracts showing target sites other than those used by antibiotics will be active against drug resistant microbial pathogens.However, very little information is available on such activity of medicinal plants (Lee et al., 1998).
In third world countries, medicinal plants serve as alternative resources for health care of human diseases and animal ailments (Alexiadesand Lacaze, 1996;Lozoya, 1996;Robineau and Soejarto, 1996).Most of the world population depends on the natural traditional medicinal Gebru, E.,Gereziher, G., Abraha, G., Tsehaye, A and Gidey, Y (MEJS) Volume 10( 2 plants.Different kinds of medicinal plants have active biochemical components that can inhibit growth of pathogenic organisms (Chitravadivu et al., 2009).The number of diseases that infect human not only increasing throughout the world (Gupta et al., 2004) but also the frequency of life threatening infection caused by pathogenic organisms is also increasing worldwide.In the last decade, research related to medicinal plants has been a burning issue for biological science disciplines (Pennaet al., 2003).Medicinal plants are known to be the main health care sources for large number of people throughout the world (Reddy and Jose, 2010).Traditional medicinal plants and their-derived medicines are widely used in old-style cultures in most of the world population, and are also becoming progressively common in modern societies as natural options to synthetic chemicals.About 70-80% of the rural population in many tropical countries depends on traditional medicines for their health care (Farnsworth et al., 1985).
Modern antibiotics are at times related with side effects whereas it is minimum or insignificantin using antimicrobial component of medicinal plants.Though, there can be smaller amount side effects, but have better tolerance and somewhat less expensive (Bari et al., 2010).Antibacterial components of medicinal plants and their use to cure the microbial illness as potential alternatives to artificial drugs to which many infectious microorganisms have become resistant seem to be very much promising (Singh et al., 2010).However, several studies have indicated that medicinal plants contain bioactive components e.g.peptides, unsaturated fatty acid, aldehydes, flavonoids, alkaloids, essential oils, phenols and water or ethanol soluble compounds.
These secondary metabolites produced by plants are organic chemicals of high structural density which play diverse functions including chemotherapeutic, bactericidal, bacteriostatic and antimicrobial functions (Samuelsson, 2004).
Plants have always formed an important component in providing of primary healthcare in many communities in Africa.The HIV and AIDs pandemic, increase of resistance by pathogens to modern drugs (Susana et al., 2007), coupled with the rise in the number of immunocompromised individuals suffering from opportunistic infections necessitates a search for alternative cure for these life threatening scourge.Therefore, the medicinal plants provide an alternative for potential cure for these challenges.In recent years, the studies on the plants having different values in their medicinal properties have been evaluated for possible antimicrobial activity and potential cures for a variety of ailments especially of microbial origin Gebru, E.,Gereziher, G., Abraha, G., Tsehaye, A and Gidey, Y (MEJS) Volume 10( 2 ii)to screen out phytochemical (the bioactive ingredients) of the plants extracts; and iii) to assess inhibitory activity of each of the traditional medicinal plant extracts.

Description of the Study Area
The study was conducted in Mekelle University, Mekelle, capital city of the Tigray regional state of northern Ethiopia.The areas around Mekelle city are selected for medicinal plants collection.
Mekelle is located about 780 kilometers north of Addis Ababa,Ethiopian capital, geographically lies at 13°29'N latitude, 39°28'E longitude, with an elevation of 2084 meters above mean sea level.The agro-ecological condition of the study areas around Mekelle is almostmiddle highland.Among themedicinal plants identified in different areas around Mekelle, the medicinal plant species that are found in almost all agro-ecological ecosystems were chosen for the study.
Details are given in tables 1 to 3.

Selection of the Traditional Medicinal Plants
The medicinal plants were selected from different areas around Mekelle based on informants' consensus factor (ICF).This was performed by gathering information about the traditional use of the medicinal plants from local informants.First, some traditional medicinal plants were selected based on the local healers.Then, seven informants asked on which medicinal plant cures for a given disease so that the more repeatedly chosen plant will be selected.Based on the informants' consensus factor method (Martin, 1995)  The information documented in table 2 is obtained from the local informants of the traditional medicinal plant species collected from different places around Mekelle (Table 3).The local traditional healers know the indigenous medicinal plants by transferring information from generation to generation orally.

Collection and Preparation of Plant Materials
The fresh plant parts samples were rinsed thoroughly to remove unnecessary materials two to three times using distilled water, cut into small pieces with sterile blade and dried in a cool dry area for about 7 to 10 days (25 -30 o C).While drying the plant parts were out of reach of sunlight in order to prevent loss of the bioactive components.The dried plant parts were crushed to fine powder and stored in air tight dark bottles at room temperature.Ten gram of each plant parts was extracted by mixing with distilled water (100 ml) influx for 48 hours at room temperature.
Extracts were filtered through Watman No.1 paper.All the extracts were evaporated in rotary evaporator (Laborota-4000-efficient made in Germany) material in Mekelle University Organic chemistry laboratory.The alcoholic extracts were concentrated in a soxhlet (made in UK) apparatus at 60 o C for 30 min and aqueous extracts were freeze dried.All extracts were stored at 4 o C until further analysis (Wijeratne et al., 2006;Hatamnia et al., 2013).

Microorganisms
The clinical isolates of Staphylococcus aureus and Staphylococcus pneumonia used in the study were obtained from College of Veterinary Medicine, Department of Microbiology, and MekelleUniversity.These isolates were identified on the basis of morphological, cultural and biochemical characteristics (Collee and Marr, 1996).

Qualitative Phytochemical Screening of the Plants
To test the qualitative screening of the phytochemicals of the medicinal plants the procedure of Adedeji et al. ( 2012) was followed.

Statistical Data Analysis
All results of the measurements were done in triplicates to test the reproducibility of the experimental data.SPSS 16 (One Way ANOVAs) was used for statistical analysis of the results.
All results are presented as mean ± Standard Error of mean.The values of P<0.05 were considered statistically significant.

Evaluation of Antimicrobial Activities of Plant Extracts
As    Gebru, E.,Gereziher, G., Abraha, G., Tsehaye, A and Gidey, Y (MEJS) Volume 10( 2 To observe the qualitative test of biologically active components of the plants extracts,leaves, barks, stems, roots and seeds for some was subjected to be dried in air without exposing to full sun light in order to prevent loosing of some volatile components from the parts of the plants.
The plant part materials were crushed using metal grinding equipment to increase surface area to volume ratio.The powder of the plant materials was subjected to the solvents of ethanol and water in flasks over 48 hours and filtered using Watman No.1 paper as indicated in figure 1.
Then, the filtrate was subjected for farther crude oil extraction in the laboratory using soxhlet evaporator.
In the antibacterial activities of aqueous leaf extracts of the medicinal plant Meriandrabengalensis inhibited growth of Staphylococcus pneumonia with 19.03 mm zone which is maximum inhibition zone when compared to the growth of Staphylococcus aureus which is 15.97mm (Table 4).The reason for this might be that the crude extracts of the plant species Meriandrabengalensis could be reactive with the organic solvent ethanol so that less amount of the crude extracts have low action on the organisms.When the leaf extracts of Meriandrabengalensis using water and ethanolare compared, the water extracts of the medicinal plant have higher inhibition zone than that of ethanol extracts against growth of the bacterial speciesStaphylococcus aureus (Tables 4 and 5).The 1:1 ratio of the mixtures of the extracts of the plant parts had higher zone of inhibition when compared with their Gebru, E.,Gereziher, G., Abraha, G., Tsehaye, A and Gidey, Y (MEJS) Volume 10( 2 single activities.This may be due to the activities of both mixed parts of the extracts might have supplementary activities to inhibit growth of the microbial organisms.

Phytochemical Screening of the Plants
Qualitative Phytochemical screening test was performed using standard procedures (Edeoga et al., 2005;and Adedeji et al., 2012).Of the bioactive components screened from the plants carbohydrate was observed highly in the species Beciumgrandiflorum (leaf) and the component saponins was highest in the plant extract of Meriandrabengalensis (leaf).Other components like Alkaloids, Flavonoids and Phenols are not clearly observed, may be due to the method/procedure followed is not accurately implemented or there may be some technical problems (table 6).

DISCUSSION
Ethanol used in the present study as an organic solvent to assess the antimicrobial activities of the traditionally used medicinal plants is commonly used solvent to test ethanol extracts of leaf of medicinal plants against Staphylococcus aureus bacterial species (Demetrio et al., 2015).Data on the traditional use of the medicinal plants is collected by the local people (Tables 1 and 2) during field work using consensus informant factor method.During the survey, different informants indicated the same plants and same usesespecially for some widely used medicinal plants.The informant consensus is helpful tosee the similarity of information given by the informants to confirm the authenticity ofinformation by comparing it with other information given by other informants on thesame medicinal plant.Accordingly, diseases of humans, numbers of citations and percentages arerecorded.The tested traditional medicinal plants were selected following a standard manual.The Informant Consensus Factor (ICF) is followed based on Martin (1995).Of the bioactive components screened from the plants, carbohydrate was observed highly in the species Beciumgrandiflorum (leaf) and the component saponin was highest in the plant extract of Meriandrabengalensis (leaf).The other components like alkaloids, flavonoids and phenols were not clearly observed.This may be due to the method/procedure that is followed might not be accurately implemented.There might be some technical problems (Table 5).
The results for the antimicrobial activities were observed after applying 10 µL/disc of the extracts oil which completely inhibited up to diameters of 4.5-19.03mm.Furthermore, the minimum inhibitory concentration (MIC) was estimated to be lower than 5 µL /disc.Some plants contain many biologically active compounds which are widely used to meet certain primary healthcare needs especially in most rural communities.They could serve as sources of lead compounds for the development of putative antimicrobial agents especially now that some synthetic drugs are barely efficacious against some pathogenic bacteria.
In the present case, the aqueous leaf, stem and bark extracts of Beciumgrandiflorum, Meriandrabengalensis, Tamarindusindica, Balanitesaegyptiaca and Otostegiaintegrifolia were investigated against some selected pathogenic bacteria.Meriandrabengalensis, the bacterial strain had better resistance to the extracts.This might be due to the properties of the solvent used (Table 5).
The maximum inhibition zone of aqueous extract of leaf of Meriandrabengalensis against the bacterial species of Staphylococcus pneumonia is 19.03 mm.The minimum inhibition zone of aqueous extract of Balanitesaegyptiaca stem is 4.5mm.The reason for this lower inhibition zone is considered to be due to the resistance of the bacterial species.
Moreover, in comparative study of ethanol leaf and stem extracts against Staphylococcus aureus all the extracts have lower inhibition zones.Carbohydrate, saponins and terpenoids were present in the aqueous extracts of all the tested traditional medicinal plants.The evidence for the sign ++ (double positive) was the results obtained when observed manually.Because the phytochemical screening was done following the standard procedure, the results indicated were observed manually and expressed aspositive, negative and double positive (Tables 6 to 9).
The qualitative test of biologically active components of the medicinal plants was done following a standard manual (Kensa et al., 2011).The organic solvent methanol was also used as a solvent to observe presence of the biological component/compounds (Demetrio et al., 2015).
The methanol extract of Meriandrabengalensis (leaf) was observed easily for the presence of terpenoids.The other medicinal plants were also observed for the presence of terpenoids whereas ):[255][256][257][258][259][260][261][262][263][264][265][266][267][268][269][270] 2018    © CNCS, Mekelle University 258 ISSN: 2220-184X(Kesavan,2007).There is no or limited research study conducted on the medicinal plants in Tigray region of northern Ethiopia.A study was therefore undertaken to assess the presence of the secondary biological components in the traditionally used selective medicinal plants present in Tigray region so that it will serve as starting point for researchers who need to conduct related work activities.The aim of the study was three fold, i)to measure the inhibitory activity of plant species,Beciumgrandiflorum, Meriandrabengalensis, Tamarindusindica, Balanitesaegyptiacaand Otostegiaintegrifolia extracts on Staphylococcus aureus and Staphylococcus pneumonia; shown in table 4, there is formation of clear zone that indicates the medicinal plants have inhibition zones of different sizes.The Maximum Inhibition Concentration (MIC) and Minimum Inhibition Concentration (MIC) of the extracts were 10 µl/disc and 5µl/disc respectively.All the experimental results obtained are the average of triplicates.

Figure 1 .
Figure 1.Preparation of the crude extracts in the laboratory.

Table 1 .
the traditionally used medicinal plants were collected.The medicinal plants reported by five and more informants (≥17%) around the areas where the plants are collected fromthe surveyed local areas of the study (Table1).A total of around 51 informants were participated during the assessment of the traditionally used medicinal The informants were consulted about the traditionally used medicinal plantsand the first selected by ten and more informants were examined for their antimicrobial activities and phytochemical screening test.Out of which, top five selected by 33% and more were considered for the laboratory extraction test activities.The five tested traditionally used medicinal plants are given in table 2. List of traditional medicinal plantspresent in areas around Mekelle.

Table 2 .
Traditional use of themedicinal plants by the local people.

Table 3 .
Collection sites of the medicinal plant materials around Mekelle.

Table 4 .
Antibacterial activity of aqueous leaf extracts against pathogenic organisms.

Plant Species Parts used Test Organisms Staphylococcus aureus Staphylococcus pneumonia
Data is expressed as Mean zone of inhibition in millimeter ± SE(Standard Error).

Table 5 .
Comparative study of ethanol leaf and stem extracts against Staphylococcus aureus.Data is expressed as Mean zone of inhibition in millimeter ±SE (Standard Error).

Table 7 .
Tested bioactive components of ethanol extracts of medicinal plants.

Table 8 .
Tested bioactive components of methanol extracts of medicinal plants.

Table 9 .
Tested bioactive components of acetone extracts of medicinal plants.
The results indicate that the aqueous and ethanol leaf extracts of Meriandrabengalensis show strong antimicrobial activity against Staphylococcus aureus and Staphylococcus pneumonia with inhibition zone diameters of 15.97 ± 0.09 and 19.03 ± 0.15 mm (Table 4) respectively, significant at P<0.05 compared to the other tested extracts.Only aqueous extracts of the plants were used on antimicrobial activities.In the comparative study indicated in table 4 only antimicrobial activity against the bacterial strain of Staphylococcus aureus was made.The reason for this might be theparticular bacterial strain has low inhibition zone when compared to the other bacterial strain, Staphylococcus pneumonia.In the comparative study of ethanol leaf and stem extracts against Staphylococcus aureus of