«In Vitro Antibacterial Activity of Allium roseum (Wild Allium or Rosy Garlic) against Some Clinical Isolates Katsa M., Gyar ...»
Journal of Microbiology Research and Reviews
Vol. 2(4): 30-33, July, 2014
In Vitro Antibacterial Activity of Allium roseum (Wild
Allium or Rosy Garlic) against Some Clinical Isolates
Katsa M., Gyar SD and Reuben CR*
Department of Science Laboratory Technology, Nasarawa State Polytechnic, Lafia, Nigeria
Email for Correspondence: firstname.lastname@example.org, email@example.com
Current research trends have placed medicinal plants as new resources for production of agents that could act as alternatives to antibiotics in the treatment of antibiotic-resistant infections. This study sought to evaluate the antibacterial activity of Allium roseum bulb extracts, using disk diffusion and micro dilution broth assays. It was demonstrated that A. roseum methanolic extracts were more effective against the tested isolates, with zones of inhibition ranging between 13 and 17mm while ethanolic extracts had zones of inhibition ranging between 10 and 14mm. Staphylococcus aureus (gram positive bacterium) was more susceptible to the test extracts than Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae (gram negative bacteria). The minimum inhibition concentration (MIC) of the extracts which inhibited bacterial growth varied from 1.85 mg/ml (S. aureus) to 6.95 mg/ml (E. coli). This concentration was not much different from the concentration that was safe for mammalian cells, suggesting that the extract of A. roseum may be a safe and strong antibacterial agent.
Key words: Allium roseum; Antibacterial activity; Microbial infection; Bulb extract
INTRODUCTIONIn recent years there has been an increased interest in the use of natural compounds, and issues concerning the safety of synthetic compounds have encouraged more detailed studies on plant resources. Sulfur compounds, the extracts and volatile products of plant secondary metabolism, have a wide application in folk medicine, food flavouring and preservation as well as in the fragrance industry (Radu et al., 2012). The indiscriminate use of antibiotics in agriculture and medicine has led to the emergence of highly resistant pathogenic microorganisms, which now present a very serious public health problem (Walsh, 2000; Cole et al., 2002). The problems of multidrug resistances exhibited by human pathogenic microorganisms and the side effects of antibiotics have led scientists to search for alternatives such as medicinal plants for the treatment of some bacterial related infections. The phenolic compounds present in most plants have a broad spectrum of biological activities whereby their antimicrobial actions stand out (Fernández et al., 1996).
The use of Allium genus members such as garlic and onion in the treatment of various ailments has been reported worldwide. Many members in this genus have been proved to possess antibacterial, antifungal, antiprotozoal, and anthelmintic activities (Harris et al., 2001; Ariga and Seki, 2006; Taran et al., 2006). In addition, Allium plants are believed to heal diabetes, arthritis, colds and flu, stress, fever, coughs, headache, hemorrhoids, asthma, arteriosclerosis, cancer, rheumatic, and inflammatory disorders (Hirsch et al., 2000; Eidi et al., 2006).
In Nigeria, local people in the rural area where A. roseum or rosy garlic occurs have extensively developed uses for this species both as a cooking ingredient and a sauce (Najjaa et al., 2011). A. roseum leaves are the main edible part, with a distinctive pungent odour and strong flavour. Besides its culinary use, rosy garlic is also used in folk medicine. Le Floc’h (1983) reported its use for the treatment of headaches and rheumatism. It is also used for the treatment of bronchitis, 31 colds as an inhalation, fever diminution and as an appetizer (Le Floc’h, 1983).
While several studies have provided information about A. roseum, detailed studies documenting compositional, nutritional and functional properties are very limited, if not lacking. The objective of the present study was to determine antimicrobial activities of A. roseum grown in central Nigeria.
MATERIALS AND METHODSCollection of Plants Samples The bulbs of A. roseum (rosy garlic) were collected from a botanical garden in Lafia, Nasarawa State, Nigeria between August to October 2013. The samples were cleaned from any strange plants, dust, or any other contaminants and finally washed with distilled water before been deposited in the Herbarium of Biology Laboratory of Nasarawa State Polytechnic, Lafia.
Microorganisms Clinical isolates of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa were used for this study. Strains were obtained from the Microbiology laboratory of Dalhatu Araf Specialist Hospital, Lafia.
Extraction Procedure The plant materials (A. roseum bulbs) were washed, air dried for 7-8 days, and ground into powder to increase the surface area of extraction. Thirty grams (30g) of the fine powder were weighed and dissolved in 60ml of each of the solvents (methanol and ethanol) in a closed conical flask for 24hours at 20OC after which the suspension were filtered for subsequent used (Prashant et al., 2011).
Antibacterial Susceptibility Test
The in vitro antimicrobial activity was screened using the disc diffusion method according to the protocol by Zaidan et al.
(2005) with minor modifications. Three hundred μl (300ml) of bacterial culture (suspended in tryptic soy broth) (Oxoid) adjusted to 0.5 McFarland standard was spread on Muller-Hinton agar plates evenly using a sterile swab and allowed to dry for 15 minutes. The different concentrations of extract (60 mg/mL) filtered by 0.45 μm millipore filters (Orange Scientific, Belgium) were impregnated on 6 mm sterile discs (Whatman paper number 1) with 20 μl per disc. The impregnated discs were placed on the surface of inoculated medium. The plates were incubated at 37°C for 24 hours.
The plates were examined and areas showing clear zones around the disc were measured and recorded. Gentamycin (Oxoid) was used as the positive control. The average of each zone of inhibition was calculated and recorded.
Minimum Inhibitory Concentration Determination
The procedure was implemented as described by Khan et al. (2009) with slight modifications. Different concentrations of extract in two-fold serial dilutions were prepared in tryptic soy broth. About 100 l of inoculum suspension with the optical density in the range of 0.08–0.10 (adjusted using spectrophotometer (Beckman Coulter Inc., Fullerton) at 0.5 Mcfarland standard (1.5 × 108 CFU/ml) was added into each well of a 96-well microtitre plate. Afterwards, 100 l of extract was added to the previous wells giving a final volume of 200 l. The 96- well microtiter plate was shaken for 20 seconds at O 300 rpm and incubated at 37 C for 24 hours. The inoculum suspension was used as the negative control. Gentamycin (10 g/ml) was used as the positive control, while tryptic soy broth alone was used as blank.
MIC is the lowest concentration which inhibits bacterial growth or it is the lowest concentration of the extract at which the microorganism does not demonstrate visible growth. Confirmatory test, MBC, (minimum bactericidal concentration) was performed by loading 5 l of each well onto nutrient agar (Merck, Germany). MBC is the concentration at which there was no bacterial growth and the result was recorded.
Aeruginosa K. pneumoniae, indicating their sensitivity to the extract as shown in Table 1. The inhibition zones from Methanolic extract ranged between 13 to 17 mm whereas the inhibition zones from Ethanolic extract ranged 10 to 14 mm respectively. The MIC and MBC values of the extract varied among the tested bacteria. The MIC values ranged between 1.85- 6.95 while the MBC values were between 3.59- 15.00 respectively (Table 2).
DISCUSSION In terms of the range of the zones of inhibition as well as the MIC and MBC values, it was generally observed that the extract was more effective against S. aureus which is a gram-positive bacterium as compared to E. coli, P. aeruginosa, and K. pneumoniae which are gram-negative bacteria. This might be due to the differences in their cell wall structures.
Specifically, gram-positive bacteria lack outer membrane while the outer membrane possessed by gram-negative bacteria might act as a barrier to many types of environmental substances which also include antibiotics (Joshi et al., 2009). This in agreement with the previous report that showed that the gram-positive bacteria were more sensitive to the different types of A. roseum extracts with the mean of growth inhibition zone ranged between 8 and 15 mm (Najjaa et al.,
2007. Chikwem et al. (2008) also reported that the A. sativum extract could inhibit the growth of gram-positive bacteria with a larger size of inhibition zone which was more than 20 mm in diameter by disk diffusion assay compared to gramnegative bacteria. This result was in contrast to other A. sativum extracts, which mainly exhibited their antibacterial activity on gram-negative bacteria (Iwalokun et al., 2004; Bakri and Douglas, 2005).
Although both methanolic and ethanolic extracts exhibited antimicrobial activity against the tested isolates, susceptibility was more in methanolic extract. In fact, it was demonstrated that A. roseum extracts, particularly when methanol or acetone were present (75 %) in the extraction solvent, exhibited more antimicrobial potential (Najjaa et al., 2011).
Antibacterial activity using microdilution assay showed that the MIC of the extract against S. aureus been a grampositive bacteria was 1.85 mg/ml which is almost similar to nontoxic concentration to normal mammalian cells. As for other pathogens used in this study, the MIC and MBC values were higher than the nontoxic concentration to normal cells. However, this controversy might be solved if further study on the mode of actions of individual compounds of the extract is carried out (Ali et al., 2011) or some appropriate chemical and structure modifications are applied on the extract (Suffredini, et al., 2006; Krishna and Jayakumaran, 2010). Moreover, in a study by Levison (Levison, 2004), it was observed that the effective MIC and MBC values used in vivo were lesser than obtained values in vitro. Therefore, further investigation is required to be carried out for the A. roseum extract to be used as a safe antibacterial agent.
antimicrobial activity of A. roseum extracts against various bacteria, this plant and its derivatives could be a potential source of new antibacterial agents. Further molecular testing is needed to typify or characterize the pharmacological properties A. roseum plant.
Ali MS., Saiful M., Masudur RM., Rabiul IM., Sayeed A and Rafikul IM (2011). Antibacterial and cytotoxic activity of ethanol extract of Mikania Cordata (Burm.F.) B.L. Robinson leaves. J Basic Clin Pharm., 2(2): 103–107.
Ariga T and ST (2006). Antithrombotic and anticancer effects of garlic-derived sulfur compounds: a review, BioFactors, 26(2): 93–103.
Bakri IM and Douglas CWI (2005). Inhibitory effect of garlic extract on oral bacteria, Archives of Oral Biology, 50(7):
Bradford MM (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Ann. Biochem. 72: 248–254.
Chikwem AJ., Chikwem JO and Swinton JD (2008). Aqueous extraction of dried and fresh garlic, and comparative antimicrobial susceptibility testing of garlic extracts on selected bacteria. BIOS, 79(2): 56–60.
Cole AM., Hong T and Boo LM (2002). Retrocyclin: A primate peptide that protects cells from infection by T- and Mtropic strains of HIV-1. Proc. Natl. Acad. Sci USA. 99:1813-1818.
Eidi A., Eidi M and Esmaeili E (2006). Antidiabetic effect of garlic (Allium sativum L.) in normal and streptozotocininduced diabetic rats. Phytomedicine, 13(9-10): 624–629.
Fernández MA., García MD and Sáenz MT (1996). “Antibacterial activity of the phenolic acids fractions of Scrophularia frutescens and Scrophularia sambucifolia,” J. Ethnopharmacol., 53(1): 11–14.
Harris JC., Cottrell SL., Plummer S and Lloyd D (2001). Antimicrobial properties of Allium sativum (garlic). Appl Microbiol Biotechnol., 57(3): 282–286.
Hirsch K., Danilenko M and Giat J (2000). Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation. Nutr Cancer., 38(2): 245–254.
Iwalokun BA., Ogunledun O., Ogbolu DO., Bamiro BS and Jimi-Omojola J (2004). In vitro antimicrobial properties of aqueous garlic extract against multidrug-resistant bacteria and Candida species from Nigeria. J Med Food., 7(3): 327– 333.
Joshi B., Lekhak S., and Sharma A (2009). Antibacterial property of different medicinal plants:ocimum sanctum, Cinnamomum zeylanicum, Xanthoxylum armatum and Origanum majorana. Kathmandu University Journal of Science, Engineering and Technology, 5(1): 143–150.
Khan R., Islam B and Akram M (2009). Antimicrobial activity of five herbal extracts against Multi Drug Resistant (MDR) strains of bacteria and fungus of clinical origin. Molecules, 14(2): 586–597.
Kojuri J., Vosoughi AR and Akrami M (2007). Effects of anethum graveolens and garlic on lipid profile in hyperlipidemic patients. Lipids in Health and Disease, 6(5): 34-39.
Koneman EW., Allen SD and Janda WM (1997). Color Atlas and Textbook of Diagnostic Microbiology Hiladelphia.
Philadelphia: Lippincott-Raven Publ., Pp. 785-856.
Krishna MS and Jayakumaran NA (2010). Antibacterial, cytotoxic and antioxidant potential of different extracts from leaf, bark and wood of Tectona grandis. International Journal of Pharmaceutical Sciences and Research, 2:155–158.
Le Floc’h E (1983). Contribution à une étude ethnobotanique de la flore tunisienne. Programme flore et végétation tunisienne. Tunis : Ministère de l’Enseignement Supérieur et de la Recherche Scientifique. Pp. 87-88.
Levison ME (2004). Pharmacodynamics of antimicrobial drugs. Infect Dis Clin North Am., 18(3): 451–465.
Najjaa H., Neffati M., Zouari S and Ammar E (2007). Essential oil composition and antibacterial activity of different extracts of Allium roseum L., a North African endemic species. Comptes Rendus Chimie, 10 (9): 820–826.