ANTIBACTERIAL AND PHYTOCHEMICAL SCREENING OF ROOT EXTRACTS OF EUCLEA RACEMOSA SUBSP. SCHIMPERIHTML Full Text
ANTIBACTERIAL AND PHYTOCHEMICAL SCREENING OF ROOT EXTRACTS OF EUCLEA RACEMOSA SUBSP. SCHIMPERI
Teklay Gebremariam 1, Teferra Abula 2 and Mebrahtom Gebrelibanos * 3
Department of Pharmacology 1, College of Health Sciences, Aksum University, Aksum, Ethiopia.
Department of Pharmacology 2, School of Medicine, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia.
Department of Pharmacy 3, Pharmacognosy Course and Research Unit, College of Health Sciences, Mekelle University, Mekelle, Ethiopia.
ABSTRACT: Emergence of multidrug-resistant strains of pathogens and adverse effects of antibiotics have rapid lead search for new antimicrobials. Medicinal plants have gained more importance as a source of alternative and effective drugs. Euclea racemosa sub sp. schimperi (DC.) Dandy (Ebenaceae) has many traditional uses against infections and related disorders. The objective of this study was to evaluate the antibacterial potential and identify major phytochemical groups in root extracts of E. racemosa. Root part of the plant was extracted by maceration using methanol, acetone, and chloroform. Extracts were subjected to antibacterial screening against seven bacteria strains: Staphylococcus aureus (ATCC215223), Streptococcus pneumonia (ATCC49619), Streptococcus pyogenes (ATCC19615), Escherichia coli (ATCC259292), Klebsiella pneumonia (ATCC70060), Pseudomonas aeruginosa (ATCC27853), and Salmonella typhi (ATCC1912/R). Well, the diffusion method was used to perform the antibacterial screening. Determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and phytochemical screening was also done on the extracts. Results indicated that the different extracts displayed significant (P<0.05) antibacterial activities and the methanol extract were more active. Flavonoids, glycosides, phenols, saponins, steroids, tannins, and triterpenes were detected in the root extracts of E. racemosa. Lowest MIC (300 mg/ml) was exhibited by methanol extract against S. pneumonia, and chloroform extract against S. typhi; and lowest MBC of 400 mg/ml was displayed by methanol extract against S. pneumonia and S. pyogenes, and chloroform extract against S. pyogenes and S. typhi. It was also shown that the tested extracts seem to demonstrate bactericidal activities. Thus, it was concluded that root extracts of E. racemosa demonstrated antibacterial activity against both grams positive and gram- negative bacteria strains; and this may partly justify the traditional use of the plant against infection and related disorders.
Euclea racemosa, Antibacterial activity, Phytochemical screening
INTRODUCTION: Adverse effects of popular antibiotics and multidrug-resistant strains of pathogens have to lead the rapid search for new antimicrobials.
Because of the long history of plants in the treatment of different human ailments, most of the herbal drugs are believed to be safer than the synthetic drugs with no side effects; therefore medicinal plants have gained more importance as the possible source of alternative and effective drugs. Plants and natural products remain as an untapped reservoir of potentially useful chemical compounds not only as drugs but also as unique templates that could serve as a starting point for synthetic analogs 1. Although, Ethiopia has rich plant biodiversity, its traditional medicine has not been investigated either from their chemical composition or in terms of their pharmacological activities 2, 3, 4. Euclea racemosa sub sp. schimperi (DC.) Dandy (Ebenaceae) is traditionally used in the treatment of the wound, teeth infections, eye disorders, head ache, pain, spasm, and also in smoking milk products. The wood of E. racemosa produces, when burned, thick, black smoke that was considered ideal for repelling insects and other pests 5. In Ethiopia, the root/stem part of E. racemosa is locally used as a toothbrush and to repel evil eye 6. Therefore, the objective of the present work was to screen the antibacterial activity and phytochemical properties of E. racemosa root extracts.
Collection of Plant Materials: Root part of E. racemosa was collected from Central Zone of Tigray, Northern Ethiopia. Mrs. Shoa authenticated the plant, and a specimen (voucher number, TG001/2006) was deposited in the National Herbarium at the Department of Biological Sciences, Addis Ababa University, Addis Ababa, Ethiopia.
Extraction: The collected roots of E. racemosa were washed with tap water until the sand and mud were removed from the parts, dried, size reduced using a hammer, and powdered using the grinder. Different extracts were prepared by maceration using methanol, chloroform, and acetone as solvents. Each time, the extracts were filtered, concentrated under reduced pressure using a rotary evaporator, and dried in an oven at a temperature of 35 °C. The dried extracts were then transferred into vials and stored at room temperature for further use.
Phyto-chemical Analysis: The preliminary phyto-chemical analyses of the methanol, chloroform, and acetone extracts were carried out using the methods described by Idris et al., (2009) 7 and Shakeri et al., (2012) 8.
Test Bacteria, Culturing and Antibacterial Activity Screening: The antibacterial activity screening of extracts was performed against seven bacteria species of both gram-positive and gram-negative strains: Escherichia coli (ATCC259292), Salmonella typhi (ATCC1912/R), Staphylococcus aureus (ATCC215223), Klebsiella pneumonia (ATCC70060), Streptococcus pyogenes (ATCC 19615), Pseudomonas aeruginosa (ATCC27853) and Streptococcus pneumonia (ATCC49619). The bacteria were obtained from the University of Gondar, Department of Microbiology and maintained on nutrient agar slope/slant at -20 °C, checked for purity by standard microbiological culture, biochemical tests and then used for their sensitivity to test samples. Stock culture was prepared by inoculating each culture from the slants to a flask in sterile broth (brain heart infusion - BHI) and then incubated for 24 h at 37 °C.
The stock culture was serially diluted by ten-fold with sterile BHI broth, and 0.1 ml of each dilution was spread over nutrient agar plates and incubated at 37 °C for 24 h. Antibacterial activity testing of the different extracts was done using well diffusion following the method described by Valgas et al., (2007) 9 with slight modification.
One loop full (loop diameter 3 mm) of each bacterial suspension was uniformly spread using a sterile cotton swab on a sterile petri dish Muller Hinton (MH) agar. 5 wells (each 6 mm diameter) were made on the MH agar of each Petri dish. Three concentrations (200, 400, and 800 mg/ml) from each sample extract were prepared using dimethylsulphoxide (DMSO). 100 µl of sample extracts and negative control (DMSO) were added to the formed wells. Standard Ciprofloxacin disc (5µg/ml) was used as a positive control. After 24 h of incubation at 37 ºC, the zone of inhibition (millimeter) of each test sample was measured using a digital calibrator. Tests were performed in triplicates.
MIC and MBC Determination: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations were done using different dilutions (100, 200, 300, 400, 500, 600, 700, and 800µg/ml) from each extract. Inoculums were added to test tubes containing the different dilutions and DMSO (control) and then incubated at 37 °C for 24 h. MIC was determined as the lowest concentration of an extract that inhibited visual growth in the liquid media. To determine the MBC, 20 μl samples from the tubes with higher than or equal to the MIC were sub-cultured on nutrient agar plates and incubated overnight at 37 °C. A reduction in colony counts by 99.9% from the original inoculum size was considered to represent the MBC.
Data Entry and Statistical Analysis: Data were analyzed using SPSS version 20. Inhibition zones were expressed as mean ± Standard deviation. One way ANOVA followed by Dunnett’s multiple comparisons was employed to compare results between extracts and between bacteria. Results were considered statistically significant at 95% confidence level and P-value <0.05.
RESULTS AND DISCUSSION:
Extraction: The percentage yield (w/w) of different extracts from roots of E. racemosa is summarized in Table 1. The methanol root extract showed higher percentage yield, and percentage yield decreased as solvent polarity decreased.
TABLE 1: PERCENTAGE YIELD OF DIFFERENT EXTRACTS OF E. RACEMOSA ROOTS
|Type of extract||Percentage yield (w/w)|
Phytochemical Screening: The phytochemical screening results Table 2 showed the possible presence of flavonoids, glycosides, phenols, saponins, steroids, tannins, and triterpenes in E. racemosa root as those were detected in at least one extract of the plant; whereas alkaloids and carbohydrates were not detected. Some of the phytochemical groups that have been detected in the extracts may contribute to the observed antibacterial activities of the extracts.
TABLE 2: PRELIMINARY PHYTOCHEMICAL SCREENING RESULTS OF DIFFERENT ROOT EXTRACTS OF E. RACEMOSA
(+) Denotes phytochemical group was detected and (-) not detected.
Antibacterial Activity Screening: Different extracts from the root of E. racemosa demonstrated various degrees of activities against standard bacteria of both gram-positive and gram-negative strains. The inhibition zones of the different extracts are summarized in Tables 3 and 4. As can be seen from Tables 3 and 4, all concentrations from all tested extracts showed antibacterial activity compared to the negative control (DMSO) which had inhibition zone of 6 mm (size of formed well).
The methanol extracts of E. racemosa exhibited a maximum zone of inhibition against gram-positive bacteria followed by chloroform and acetone extracts. Streptococcus pyogenes was most susceptible gram-positive bacteria followed by Streptococcus pneumonia and Staphylococcus aureus; and from the gram-negative bacteria, Salmonella typhi was the most susceptible followed by Pseudomonas aeruginosa, Klebsiella pneumonia, and Escherichia coli. The antibacterial activity of most extracts was statistically significant (P≤0.05) compared to the negative control (DMSO) and had more or less equivalent potency to that of ciprofloxacin, a standard drug used as positive control in this study.
Thus, the present study shows that the different extracts of E. racemosa possess significant antibacterial activity and could serve as a possible justification for the traditional use of the plant against different infectious disorders. Some of the phytochemical groups that were detected in the root extracts of the plant could be responsible for the displayed antibacterial activities.
MIC and MBC Determination: Determination of minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation, and minimum bactericidal concentrations (MBCs) are the lowest concentration of an antimicrobial that will prevent the growth of an organism after subculture on to antibiotic-free media 10. Accordingly, MICs and MBCs of different extracts from roots of E. racemosa against standard bacteria of both gram-positive and gram-negative strains have been done, and the results are shown in Tables 5 and 6.
As can be seen from Tables 5 and 6, the lowest observed MIC was 300 mg/ml against S. pneumonia (methanol extract) and S. typhi (chloroform extract); and the lowest MBC was 400 mg/ml against S. pneumonia, S. pyogenes (methanol extract), and S. typhi (chloroform extract). It has been indicated by Djeussi et al., (2013) 11 that a sample is bactericidal when the ratio MBC/MIC ≤ 4 and bacteriostatic when this ratio is >4. In the present study, MBC/MIC ≤4 values have been shown for all the dilutions in which MIC and MBC have been determined indicating that the tested extracts may be acting as bactericidal.
TABLE 3: MEAN ZONES OF INHIBITION OF DIFFERENT EXTRACTS OF E. RACEMOSA ROOT AT DIFFERENT CONCENTRATIONS AGAINST GRAM-POSITIVE BACTERIA
|Mean zone of inhibition ± S.D (mm)||P-
CIP = Ciprofloxacin (5µg/ml)
TABLE 4: MEAN ZONES OF INHIBITION OF DIFFERENT EXTRACTS OF E. RACEMOSA ROOT AT DIFFERENT CONCENTRATIONS AGAINST GRAM-NEGATIVE BACTERIA:
|Mean zone of inhibition + S.D (mm)||P-
CIP = Ciprofloxacin (5µg/ml), (-) = Statistically Not Significant
TABLE 5: MINIMUM INHIBITORY CONCENTRATIONS (MICS) AND MINIMUM BACTERICIDAL CONCENTRATIONS (MBCS) OF DIFFERENT EXTRACTS OF E. RACEMOSA ROOT EXTRACT AT DIFFERENT DILUTIONS AGAINST GRAM-POSITIVE BACTERIA
|Extracts||Concentrations (mg/ml)||MIC (mg/ml)||MBC (mg/ml)|
N = Determined
TABLE 6: MINIMUM INHIBITORY CONCENTRATIONS (MICS) AND MINIMUM BACTERICIDAL CONCENTRATIONS (MBCS) OF DIFFERENT EXTRACTS OF E. RACEMOSA ROOT EXTRACT AT DIFFERENT DILUTIONS AGAINST GRAM-NEGATIVE BACTERIA
|Extracts||Concentrations (mg/ml)||MIC (µg/ml)||MBC
N = Determined
CONCLUSION: Root extracts of E. racemosa demonstrated antibacterial activity against both gram positive and gram negative bacteria strains, possibly by bactericidal action. Flavonoids, glycosides, phenols, saponins, steroids, tannins, and triterpenes were detected in the extracts and may contribute to their antibacterial action. This anti-bacterial action may serve as partial justification to the traditional use of the plant against various infectious disorders.
ACKNOWLEDGEMENT: One of the authors (TG) is thankful to the University of Gondar for hosting his study and Aksume University for the study leave. He is also very grateful to Mr. Biruk Sintayehu for his critical and valuable comments, Mr. Kibrom Legesse for his help in statistical analysis, Mr. Teklay G/cherkos for his help during sensitivity testing, Mrs. Shoa for authentication of the study plants; at last but not least to Dr. Tewodros Haile, Dean College of Medicine and Health Sciences, Aksum University for his moral support and encouragements in the course of his study.
CONFLICT OF INTEREST: Nil
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How to cite this article:
Gebremariam T, Abula T and Gebrelibanos M: Antibacterial and phytochemical screening of root extracts of Euclea racemosa Subsp. Schimperi. Int J Pharmacognosy 2015; 2(2): 66-70. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.2(2).66-70.
This Journal licensed under a Creative Commons Attribution-Non-commercial-Share Alike 3.0 Unported License.
T. Gebremariam, T. Abula and M. Gebrelibanos*
Department of Pharmacy, Pharmacognosy Course and Research Unit, College of Health Sciences, Mekelle University, Mekelle, Ethiopia.
25 December 2014
18 January 2015
27 January 2015
01 February 2015