تاريخ النشر : 2016-09-24 21:36:01
عدد المشاهدات : 422
IN – VITRO EVALUATION OF ANTIBACTERIAL ACTIVITY OF SILYBUM MARIANUM
SEEDS OIL, ETHANOLIC EXTRACT AND ANTIBIOTICS AGAINST ISOLATED
BACTERIA
KHULOOD ABDUL KAREEM & WASFI DHAHIR ABID ALI
Department of Basic Medical Sciences, College of Nursing, University of Basrah, Iraq
ABSTRACT
The present study aimed to investigate the antibacterial activity of ethanolic extract of Silybum marianum seeds,
it
,
s oil, Chloromphincol, Rifampin and Methicillin against isolated bacteria, evaluation is done in-vitroby using hole plate
method for appearance zone of inhibition. Antimicrobial susceptibility test showed that their were no antibacterial activity
of siylmarin extract while the oil showed very mildeffect against Enterbacter ludwigii, Klebsiella pneumonia andPontoea
gaviniae. Chloromphincol showedeffects aginst most the isolated bacteria except Citrobacter freundii, Enterobacter
cloacae and Psudomonous aerogenosa, Rifampin affect only on Pontoea gaviniae while Methicillin didn’t show any
affects .Chloromphenicol showed intermediate effect on Enterbacter ludwigii, Enterobacter sakazakii, Klebsiella
pneumonia, Pantoea agglomeruns, Proteus mirabilis, Salmonella typhomurium,, while Staphylococcus homin ins were
Susceptible, most the isolated bacteria were resistance to Rifambin except Pontoea gaviniae gave intermediate inhibition
zone. in case of Rifampin no effect were recorded
.
KEYWORDS: Silymarin, Chloromphincol, Rifampin and Methicillin
INTRODUCTION
Silybum marianum is a wild growing annually herb that grow in many part of the world include the north part of
Iraq and some area north Baghdad city (Rajiha 2012). The seeds contain 22% oil ((hydrocarbons, sterols, and fatty acids)
Hammouda et al., 1994).in the Nile region (Delta) and Fayium region near water streams. The plant has been locally
cultivated successfully medical purposes and huge amounts of the oil-rich seeds, (Hassan et al 2003).Silymarin, obtained
from Silybum marianum is used for hepatoprotection (Parveen et al., 2012). Its antioxidant, anti-inflammatory and
anti-apoptotic effects these properties have implicated this compound as a potential renoprotective agent (Zeynab 2012).
Michelin et al., ( 2005), Zuanazzi, and Montanha (2004) pointed the synergistic drug-modifying effect when
silymarin and silibinin were combined with antibiotics and as adjuvant, against the different bacterial strains. Dayanne et
al.,(2015)Their results for the antifungal activity of silymarin and silibinin demonstrated a MIC of 1024 µg/mL
Chloramphenicol known as a broad-spectrum antibiotic produced by Streptomyces (Lin et al., 2005). Thus,
chloramphenicol is being reconsidered as an option for treatment of certain infections in critically ill patients
(Falagas and Kopterides 2007). Rifampin, is a antibiotic used to treat a number of bacterial infections includes
tuberculosis, leprosy. Often it is used along with other antibiotics. Rifampin used to prevent Haemophilus influenzaetype b
and meningococcal disease (The American Society of Health-System Pharmacists.2015). Meticillin was developed by
Beecham in 1959, It was previously used to treat infections caused by susceptible Gram-positive bacteria, Staphylococcus
BEST: International Journal of Humanities,
Arts, Medicine and Sciences (BEST: IJHAMS)
ISSN (P): 2348-0521, ISSN (E): 2454-4728
Vol. 3, Issue 12, Dec 2015, 43-48
© BEST Journals
44 Khulood Abdul Kareem & Wasfi Dhahir Abid Ali
Index Copernicus Value: 3.0 – Articles can be sent to editor.bestjournals@gmail.com
aureus (Graham 2009)
MATERIAL AND METHODS
Some Gram-positive bacteria and Gram-negative bacteria were used throughout this study which kindly supplied
by research laboratory from Department of Basic medical sciences- college of nursing - University of Basrah. Silybum
marianum seeds were brought from the north of Iraq (mossel),Seeds were grinded using grinder prior to extraction.50
grams each time were defatted in a soxhlet apparatus, using normal hexane (boiling point of 40C°) for 3 hr ,the oil was
separated by distillation, 150 ml of absolute ethanol was added to the remainder amount of defatted seed powder and
stirrered for 72 hr. by magnetic stirrer at room temperature. After filtration and concentration of the silymarin fraction
under vacuum, the yellow residue was dissolved in 20 ml of toluene and evaporated for one hour. 20 ml of diisopropyle
ether add to the crystals and refluxed for 1hr. and cooled for 1 hr. The mixture filtrated by vacuum and the remained
crystals were collect, dried, weighted and Stored in deep freezing.(Wallace et al ., 2003).
For Antibacterial activity the well agar diffusion method was used to determine the antimicrobial activity of the
prepared extracts oil and antibiotics .mixed 0.6 mlof the standardized bacterial stock suspension (108-109) colony forming
units per mL with 60mL of sterile nutrient agar thoroughly. Poured 20ml .inoculated nutrient agar intosterile petri dish
Left the agar to set and four well 10mm in diameterwas made in each of these plates using sterile cork borer NO 8 and
then removed agar disc. Filled the entire well with0.1 of extract using micropipette and allowed to diffuse at room
temperature for two hours. The plats were then incubated at 37°C for 24 hours. three replicates were then also performed
for each extract and antibiotics against each of the test bacteria (Lee et al ., 2003;Hanna et al ., 2008) .For other antibiotic
(Chloromphincol, Rifampin and Methicillin) use Kirby-Bauer disc diffusion (DD) methods (Woods et al.,1995).A
suspension of the colonies with 0.85 per cent normal saline was made, opacity adjusted to 0.5 McFarland and used for
performance of the procedure as described previously, after the inoculated plates had dried sufficiently the discs were
placed on the medium, gently pressed and plates incubated at 37°C for 24 hours, each zone size was interpreted with
reference standards as susceptible, intermediate and resistant.
RESULTS AND DISCUSSIONS
The ethanolic extract from Silybum marianum seed and it’s oil did not show any bacterial effects against any of
the isolated bacteria used the this study (Table 1).
Mukarram Shah et al.,( 2011)showed that silymarin both from blue and white capitulum’s seeds of S.marianum
has not shown zone of inhibition against fungus.andall the gram-negative bacteria also show resistance to silymarin while
Silymarin has been found very active against all gram positive bacteria.
Dayanne et al., ( 2015)indicated the possibility of the usage of silymarinand silibinin as adjuvants in the antibiotic
therapy against multidrug resistant bacteria (MDR), being a promising choice against the concerning problem of the
antibiotic resistance.
Enterbacter ludwigii, Enterobacter sakazakii, Klebs iella pneumonia, Pantoea agglomeruns, Proteus mirab ilis,
Salmonella typhomurium,, gave indeterminate(14-16)inhibition zone and Staphylococcus hominins were Susceptible(17)
to Chloramphenicol while the other bacteria were resistance on other hand most the isolated bacteria were resistance to
rifambin except Pontoea gaviniae gave intermediate inhibition zone. Methicilin showed negative effect on all isolated
bacteria(table 1).
Methicillin, a β-lactam antibiotic, acts by inhibiting penicillin-binding proteins (PBPs) that are involved in the
synthesis of peptidoglycan, an essential mesh-like polymer that surrounds the cell. Methicillin resistance in clinical isolates
has been reported to arise from expression of a methicillin-hydrolysing β-lactamase and through the expression of an
altered form of PBP2 that has a lower penicillin-binding affinity and higher rates of release of the bound drug compared to
the normal PBP ,methicillin resistance is affected by the inactivation of genes that affect the autolytic enzyme activities of
the cell. Inactivation of the llm gene, coding for a protein of unknown function, converts a homogeneous strain to a
heterogeneous phenotype and is associated with increased autolytic activity (Maki et al., 1994; Montanari et al., 1996;
Tschierske et al., 1997).
Moreover, Resistance to rifampicin (RIF) is a broadsubject covering not just the mechanism of clinical resistance,
nearly always due to a genetic change in the β subunit of bacterial RNA polymerase (RNAP), but also how studies of
resistant polymerases have helped us understand thestructure of the enzyme, the intricacies of the transcription process and
its role in complex physiological pathways. RIF-resistant (RIF
r
) clinical isolates of several different bacterial species, and a
single mutation predominates in mycobacteria (Goldstein, 2014).
Chloramphenicol binds to the 50S ribosomal subunit and inhibits the peptidyl transferase step in protein synthesis.
Resistance to chloramphenicol is generally due to inactivation of the antibiotic by a chloramphenicol acetyltransferase,
various enzymes have been described and are coded for by the cat genes found in gramnegative and gram-positive bacteria
and usually show little homology (Traced et al., 1993 ; Kehrenberg et al., 2001).
Table 1: Diameter of Zone Inhibition of S. marianum Extract. S, marianum oil Chloramphenicol,. Rifampin and
Methicillin against Gram Positive and Gram-NegativeBacteria
- -
C=Chloromphincol, RA= Rifampin, M= Methicillin, Resistance =R≤ 13, Susceptible =S≥ 17,
indeterminate= >14-16
CONCLUSIONS
The recent study concluded that silymarin extract, silymarin oil, Rifampin and mithicilin have no bacterial activity
against studied bacteria, Chloramphenicol showed mild effects on most of the bacteria Staphylococcus hominins detected
46 Khulood Abdul Kareem & Wasfi Dhahir Abid Ali
Index Copernicus Value: 3.0 – Articles can be sent to editor.bestjournals@gmail.com
the more Susceptible to Chloramphenicol.
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