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World J Microbiol Biotechnol (2009) 25:465–473 DOI 10.1007/s11274-008-9911-3 ORIGINAL PAPER Comparative activity against pathogenic bacteria of the root, stem, and leaf of Raphanus sativus grown in India Syed Sultan Beevi Æ Lakshmi Narasu Mangamoori Æ Naveen Anabrolu Received: July 2008/Accepted: November 2008/Published online: December 2008 Abstract Aqueous, methanol, ethyl acetate, and chloroform extracts of the root, stem, and leaf of Raphanus sativus were studied for antibacterial activity against foodborne and resistant pathogens All extracts except the aqueous extracts had significant broad-spectrum inhibitory activity The ethyl acetate extract of the root had the potent antibacterial activity, with a minimum inhibitory concentration (MIC) of 0.016–0.064 mg/ml and a minimum bactericidal concentration (MBC) of 0.016–0.512 mg/ml against health-damaging bacteria This was followed by the ethyl acetate extracts of the leaf and stem with MICs of 0.064–0.256 and 0.128–0.256 mg/ml, respectively and MBCs of 0.128–2.05 and 0.256–2.05 mg/ml, respectively The ethyl acetate extracts of the different parts of R sativus retained their antibacterial activity after heat treatment at 100°C for 30 min, and their antibacterial activity was enhanced when pH was maintained in the acidic range Hence this study, for the first time, demonstrated that the root, stem, and leaf of R sativus had significant bactericidal effects against human pathogenic bacteria, justifying their traditional use as anti-infective agents in herbal medicines Keywords Raphanus sativus Á Antibacterial activity Á Human pathogenic bacteria Introduction S S Beevi Á L N Mangamoori (&) Á N Anabrolu Centre for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500 085, Andhra Pradesh, India e-mail: mangamoori@jntuap.ac.in; mangamoori@rediffmail.com Despite the tremendous progress in human medicine, infectious diseases caused by microbes are still a major threat to public health, because of the emergence of widespread drug resistance (Okeke et al 2005) Concern over pathogenic and spoilage microorganisms in foods is increasing, because of the increase in outbreaks of foodborne disease (Tauxe 1997) Currently much attention has been focused on natural antimicrobial compounds, especially those extracted from plants, because they can serve as major sources of innovative therapeutic agents for infectious diseases and as potential natural agents for food preservation (Cowan 1999) Raphanus sativus L., which belongs to cruciferous family, is widely grown in India for its culinary and medicinal properties Although roots are the most valuable and edible part of R sativus, the stem and leaves have been used for food flavoring and as food preservative Medicinal uses of R sativus have been documented in India since the tenth century (Nadkarni 1976) Different parts of the plant are used in the indigenous system of medicine for treatment of various human ailments such as stomach disorders, liver dysfunction, infectious diseases, and bronchitis, and for burns, bruises, and smelly feet (Nadkarni 1976; Kapoor 1990) R sativus has received much attention in recent years because of its nutritional and health-protective value Previous investigations revealed the presence of cysteinerich peptides (De Samblanx et al 1996), isoperoxidases (Lee and Kim 1994), peroxidases (Kim and Kim 1996), pyrrolidine, isoquinoline, phenethylamine, pyrrolidine thionylcarboxylic acid, tetrahydrocarboline (Duke 1994; Villamar 1994), sinigrin, allyl isothiocyanate (AITC), methylthiobutenyl isothiocyanate (MTBITC), phenethyl isothiocyanate (PEITC), and benzyl isothiocyanate (BITC) 123 466 (Duke 1994; Villamar 1994; Nakamura et al 2001) in R sativus root, and methyl linolenate, phytol, sinapic acid ester, and kaempferol in R sativus sprouts (Takaya et al 2003) Previous studies have reported the antibacterial activity of various crucifers including cabbage, broccoli, wasabi, and watercress Inhibition of bacterial growth by these cruciferous plants is linked to biologically active degradation products of glucosinolates, the isothiocyanates (ITCs), whose antibacterial properties have been reported since 1937 Ward et al (1998 ) reported that AITC extracted from fresh horseradish root had species-specific activity against bacterial strains as a result of inhibiting the growth of pathogenic organisms without retarding the growth of bacteria present in the normal microflora Ono et al (1998 ) demonstrated the antibacterial activity of 6methylsulfinylhexyl ITC, a volatile fraction from wasabi stem, toward Escherichia coli and Staphylococcus aureus Haristoy et al (2005) reported that sulforaphane, an ITC from broccoli, had a bactericidal effect against intracellular Helicobacter pylori in a human epithelial cell line The health benefits of R sativus have been promoted for centuries, but few studies have been conducted to prove its medicinal and pharmaceutical value There have been very few studies of the antibacterial activity of R sativus Abdou et al (1972) described the antibacterial activity of an aqueous extract of R sativus root against Escherichia coli, Pseudomonas pyocyaneus, Salmonella typhimurium, and Bacillus subtilis Esaki and Onozaki (1982) identified the pungent principle of R sativus root as antimicrobial to Escherichia coli, Staphylococcus aureus, Saccharomyces cerevisiae, and Aspergillus oryzae In fact, in most of this literature there is an apparent lack of data about R sativus grown in India except that published by Khan et al (1985), who reported the antibacterial activity of the roots, flowers, and pods against bacteria such as Staphylococcus aureus and Bacillus subtilis Their study of the antibacterial activity of R sativus was performed on two Gram-positive bacteria only, however, and the number of bacterial species tested was limited Furthermore, there has been hardly any research on the antibacterial activity of the stem and leaf of R sativus Hence we have carried out a detailed and extensive study to evaluate and compare the antibacterial activity of extracts of R sativus root, stem, and leaf obtained with a variety of extraction solvents against diverse microorganisms (both Gram-positive and Gramnegative bacteria) including resistant strains and foodborne pathogens Because the main antibacterial activity has been attributed to the ITC content, we quantified the total ITC content of different parts of R sativus and studied a possible relationship between them Further, the effect of pH and heat treatment on the antibacterial activity of R sativus was also studied in an attempt to understand the 123 World J Microbiol Biotechnol (2009) 25:465–473 chemical nature of components that could contribute to its inhibitory effects Materials and methods Chemicals and reagents Benzyl isothiocyanate (BITC), 1,2-benzenedithiol, penicillin, and streptomycin were procured from Sigma– Aldrich (USA) Mueller–Hinton broth (MHB) and Mueller–Hinton agar (MHA) were bought from HiMedia (India) All other chemicals and reagents used in this study were of analytical or HPLC-grade and obtained from Merck (India) Plant materials and preparation of extracts Raphanus sativus L was purchased fresh from the local supermarket in Hyderabad city It was separated into root, stem, and leaf, washed thoroughly with distilled water, and freeze dried Freeze dried root, stem, and leaf of R sativus (5.0 g of each) were extracted three times with 100 ml each of selected solvents—methanol, ethyl acetate, and chloroform—and concentrated at 40°C under vacuum on a rotary evaporator (Heidolph-Rotacool, Germany) An aqueous extract of R sativus root, stem, and leaf was prepared by soaking 5.0 g dried powder in distilled water (3 100 ml) and mixing with a magnetic stirrer at low rpm for 24 h The extract was then filtered through Whatman No paper and was subsequently lyophilized in a lyophilizer at lm Hg pressure at -50°C (ScanVac-Coolsafe, Denmark) Extracts were sterilized by filtration using a 0.22 lm membrane and were stored at -80°C until use Determination of total isothiocyanates in R sativus The total isothiocyanate content of different parts of R sativus was evaluated by use of the method of Zhang et al (1992) Each extract (5.0 ll) was added rapidly to a tube containing 2.0 ml methanol, 1.8 ml 50 mM sodium borate buffer (pH 8.5) and 0.2 ml mM 1,2-benzenedithiol The mixture was heated at 65°C for h, cooled to 25°C, and absorbance was measured at 365 nm using a Shimadzu (Japan) UV 2450 spectrophotometer The isothiocyanate content was calculated from a linear standard equation derived from benzyl isothiocyanate Test organisms and culture conditions A collection of ten organisms including four Gram-positive and six Gram-negative organisms were used for this study Bacillus subtilis (MTCC 2391), Escherichia coli (MTCC World J Microbiol Biotechnol (2009) 25:465–473 1563), and Pseudomonas aeruginosa (MTCC 6642) were obtained from the Microbial Type Culture Collection, IMTECH, Chandigarh, India Clinical isolates such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes, and Enterobacter cloacae were obtained from the Microbiology Laboratory of Global Hospital, Hyderabad, India All the strains were tested for purity by standard microbiological methods Bacterial stock cultures were maintained on MHA (HiMedia, India) slants and were stored at 4°C Determination of antibacterial activity An agar-well diffusion method was employed for evaluation of antibacterial activity (Perez et al 1990) The bacterial strains were reactivated from stock cultures by transferring into MHB (HiMedia, India) and incubating at 37°C for 18 h A final inoculum containing 10 colonyforming units (1 10 CFU/ml) was added aseptically to MHA medium (HiMedia, India) and poured into sterile Petri dishes Different test extracts at a concentration of mg/ml were added to wells (8 mm diameter) punched into the agar surface Plates were incubated overnight at 37°C and the diameter of the inhibition zone (DIZ) around each well was measured in mm All experiments were performed in triplicate Antibiotics such as penicillin (100 lg/well) and streptomycin (100 lg/well) were used as positive reference standards to determine the sensitivity of the microorganisms tested Negative controls were prepared using the solvents methanol, chloroform, and ethyl 467 Effect of pH on inhibitory zone against pathogenic bacteria Ethyl acetate extracts of root, stem, and leaf at a concentration of 100 mg/10 ml were adjusted with sterile 0.1 M HCl and NaOH to pH ranging from 3.0 to 9.0 The pHadjusted extracts were then filtered through 0.22-lm membranes and used within 60 Sterile distilled water adjusted to different pH as above was used as acid or alkali control solution to ascertain whether observed changes in bacterial growth were because of acidic or alkaline pH or because of the extracts pH-unadjusted extracts were used as controls Experiments were performed in triplicate Effect of temperature on inhibitory zone against pathogenic bacteria Ethyl acetate extracts of root, stem, and leaf at a concentration of 100 mg/10 ml were incubated in a water bath for 30 at 25, 50, 75, and 100°C The incubated extracts were then cooled and stored at -80°C until use Untreated extracts were used as controls Experiments were performed in triplicate Statistical analysis Results calculated from triplicate data were expressed as means ± standard deviations The data were compared by least significant difference test using Statistical Analysis System (SAS, ver 9.1) acetate Results Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of R sativus extracts Antibacterial activity of R sativus extracts MIC and MBC for all extracts and antibiotics were determined by tube broth dilution assay (Muroi and Kubo 1996) Serial twofold dilutions ranging from 4.06 to 0.008 mg/ml of the extracts and antibiotics (positive control) were prepared and 500 ll of each dilution was incubated with 2.5 ml MHB (HiMedia, India) containing 50 ll inoculum (1 10 CFU/ml) at 37°C for 24 h The MIC was determined as the lowest concentration that resulted in no visible growth as assessed by macroscopic evaluation After determination of MIC, tubes showing no turbidity were diluted 100-fold with drug-free MHB and incubated at 37°C for 48 h The lowest concentration of the tube that resulted in no visible growth in the drug-free cultivation was regarded as the MBC Both MIC and MBC assays were performed in triplicate Ten bacterial strains were used as taxonomical representatives to evaluate the effect of candidate antimicrobial components against specific target microbes: Gram-positive spore-forming rods (Bacillus subtilis), Gram-positive cocci (Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis), Gram-negative enterobacteria (Escherichia coli, Salmonella typhimurium, Enterobacter cloacae, Enterobacter aerogenes, and Klebsiella pneumoniae), and Gram-negative non-enterobacteria (Pseudomonas aeruginosa) As shown in Table 1, extracts from root, stem, and leaf of R sativus had different growth-inhibitory activity Except for the aqueous extracts, all the extracts had significant antibacterial activity in the agar well diffusion assay against all the bacteria tested In particular, the ethyl acetate extract of R sativus root, stem, and leaf resulted in exceptionally large diameters of the inhibition zones, comparable with those obtained by use of standard 123 468 World J Microbiol Biotechnol (2009) 25:465–473 Table Antibacterial activity of R sativus root, stem, and leaf extracts against pathogenic bacteria, by the agar well diffusion method Pathogenic organism Part tested Inhibition zone (mm)a Antibiotic (100 lg/ml) Aqueous Methanol Ethyl acetate Chloroform Penicillin Streptomycin Root –b 17.97 ± 0.42 26.80 ± 0.36 17.93 ± 0.25 29.83 ± 0.29 24.33 ± 0.58 Stem – 16.47 ± 0.50 18.30 ± 0.26 15.90 ± 0.79 Leaf Root – – 15.17 ± 0.15 19.83 ± 0.55 16.90 ± 0.36 23.83 ± 0.21 13.10 ± 0.10 14.13 ± 0.49 35.03 ± 0.06 32.97 ± 0.06 Stem – 15.80 ± 0.20 20.50 ± 0.50 14.83 ± 0.72 Leaf Root – – 19.90 ± 0.36 21.27 ± 0.40 18.73 ± 0.25 25.87 ± 0.41 16.20 ± 0.20 24.73 ± 0.47 31.67 ± 0.58 19.67 ± 0.29 Stem – 16.97 ± 0.15 18.30 ± 0.52 15.63 ± 0.41 E faecalis Leaf Root – – 20.10 ± 0.22 19.90 ± 0.36 20.77 ± 0.25 26.13 ± 0.15 12.17 ± 0.21 16.57 ± 0.89 S typhimurium Stem Leaf Root – – – 12.03 ± 0.15 12.97 ± 0.23 24.37 ± 0.49 18.17 ± 0.15 17.17 ± 0.2 26.37 ± 0.31 11.03 ± 0.15 12.10 ± 0.10 18.63 ± 0.59 26.27 ± 0.46 20.23 ± 0.40 K pneumoniae Stem Leaf Root – – – 15.70 ± 0.36 15.57 ± 0.51 17.43 ± 0.67 18.50 ± 0.56 18.67 ± 0.29 24.53 ± 1.01 11.93 ± 0.40 15.97 ± 0.25 16.83 ± 0.49 23.33 ± 0.58 27.97 ± 0.05 Stem – 15.67 ± 0.58 18.53 ± 0.51 14.87 ± 0.15 Leaf Root – – 18.13 ± 0.32 18.07 ± 0.21 18.70 ± 0.30 19.67 ± 0.60 14.17 ± 0.15 15.57 ± 0.83 32.67 ± 0.57 19.97 ± 0.06 Stem – 14.77 ± 0.49 17.13 ± 0.32 12.10 ± 0.26 Leaf Root – – 13.73 ± 0.25 18.03 ± 0.55 18.30 ± 0.20 23.40 ± 1.04 18.40 ± 0.36 18.43 ± 1.21 26.30 ± 0.36 14.17 ± 0.29 Stem – 16.67 ± 0.58 20.73 ± 0.25 14.80 ± 0.20 E cloacae Leaf Root – – 13.73 ± 0.38 27.97 ± 0.57 18.33 ± 0.31 34.20 ± 0.66 12.63 ± 0.32 23.40 ± 0.26 22.83 ± 0.72 24.97 ± 0.15 P aeruginosa Stem Leaf Root – – – 19.90 ± 0.56 20.40 ± 0.40 20.77 ± 0.38 21.67 ± 0.59 25.30 ± 0.10 24.90 ± 0.60 18.97 ± 0.25 18.53 ± 0.21 19.43 ± 0.70 Stem – 14.37 ± 0.47 19.53 ± 0.55 16.53 ± 0.42 Leaf – 15.17 ± 0.38 18.33 ± 0.31 15.23 ± 0.25 B subtilis S aureus S epidermidis E coli E aerogenes – – – 21.33 ± 0.57 Each value is the mean ± standard deviation from three replicates The concentration of all the extracts used was 1.0 mg/ml a b Inhibitory zones in mm, including diameter of the well (8.0 mm); mean ± standard deviation of three replicates No inhibition or inhibition zone was less than mm antibiotics, thus demonstrating strong inhibitory activity toward all the pathogenic bacteria tested Methanol and chloroform extracts had moderate to high antibacterial activity E faecalis (resistant to penicillin and streptomycin) and P aeruginosa (resistant to penicillin) were significantly inhibited by ethyl acetate extracts of R sativus (DIZ = 17.17–26.13 mm) The other extracts had different inhibitory activity towards these resistant strains which was significantly lower than that of the ethyl acetate extracts E cloacae was found to be highly sensitive organism with DIZ in the range 18.53–34.20 mm Selected food-borne pathogens used in this study were susceptible to all 123 extracts, but were highly sensitive to the ethyl acetate extracts Further, both Gram-positive and Gram-negative bacteria were equally susceptible, demonstrating the broadspectrum inhibitory effect of R sativus Of the different parts of R sativus used in this study, root extracts tended to be more active than the stem and leaf extracts in inhibiting bacterial growth In contrast, the inhibition zones of three solvent controls, methanol, ethyl acetate, and chloroform were below 9.0 mm, indicating they were inactive against all the microorganisms tested The antibiotics penicillin (100 lg/ml) and streptomycin (100 lg/ml) were effective against most organisms, except penicillin had no activity World J Microbiol Biotechnol (2009) 25:465–473 469 against E faecalis and P aeruginosa, and streptomycin had no effect against E faecalis MIC and MBC of R sativus The results obtained for the MIC and MBC of R sativus root, stem, and leaf extracts are presented in Table Of the three solvents, extracts obtained with ethyl acetate had the lowest MIC and MBC, followed by the methanol and chloroform extracts The ethyl acetate extract of root had a notable inhibitory effect compared with stem and leaf extracts Over half of the MICs and MBCs for the ethyl acetate extract of root were close to or equal to those of positive controls (penicillin and streptomycin) and were in the ranges 0.016–0.064 and 0.016–0.512 mg/ml, respectively MICs and MBCs for ethyl acetate extracts of stem and leaf ranged from 0.064–0.256 to 0.128–2.05 mg/ml, respectively The methanol and chloroform extracts of root, stem, and leaf also had substantial antibacterial activity, with MICs in the range 0.064–1.02 mg/ml and MBCs in the range 0.256–4.10 mg/ml These were, however, significantly higher than the MICs and MBCs of the ethyl acetate extracts of R sativus Total ITC content of R sativus The total ITC content of root, stem, and leaf extracts are listed in Table ITCs were detected in substantial amounts in the root, stem, and leaf of R sativus Root extracts contained the highest levels of ITC, followed by Table MIC and MBC of R sativus root, stem, and leaf extracts against health-damaging bacteria Pathogenic organism Part tested MIC (MBC) mg/ml Methanol Ethyl acetate Chloroform Penicillin Streptomycin Root 0.256 (0.512) 0.032 (0.032) 0.256 (0.512) 0.016 (0.032) 0.032 (0.032) Stem 0.512 (2.05) 0.256 (0.512) 0.512 (2.05) Leaf Root 0.512 (2.05) 0.064 (0.256) 0.256 (1.02) 0.032 (0.128) 1.02 (4.10) 0.512 (2.05) 0.008 (0.016) 0.008 (0.008) Stem 0.512 (2.05) 0.128 (0.256) 0.512 (2.05) Leaf Root 0.064 (0.256) 0.128 (0.256) 0.256 (1.02) 0.032 (0.032) 0.512 (2.05) 0.128 (0.512) 0.016 (0.016) 0.064 (0.064) Stem 0.256 (1.02) 0.256 (1.02) 0.512 (2.05) E faecalis Leaf Root 0.128 (0.512) 0.256 (2.05) 0.128 (0.512) 0.064 (0.128) 1.02 ([4.10) 0.512 (4.10) NTa NT S typhimurium Stem Leaf Root 1.02 ([4.10) 1.02 (4.10) 0.064 (0.256) 0.256 (2.05) 0.256 (2.05) 0.032 (0.128) 1.02 ([4.10) 1.02 ([4.10) 0.256 (1.02) 0.032 (0.128) 0.064 (0.128) K pneumoniae Stem Leaf Root 0.256 (1.02) 0.256 (1.02) 0.256 (1.02) 0.256 (1.02) 0.256 (1.02) 0.064 (0.256) 1.02 ([4.10) 0.512 (2.05) 0.256 (1.02) 0.064 (0.128) 0.032 (0.032) Stem 0.512 (2.05) 0.128 (0.512) 0.512 (2.05) E coli Leaf Root 0.256 (1.02) 0.256 (1.02) 0.128 (0.512) 0.064 (0.512) 0.512 (2.05) 0.512 (4.10) 0.008 (0.008) 0.064 (0.128) E aerogenes Stem Leaf Root 0.512 (2.05) 0.512 (4.10) 0.256 (1.02) 0.256 (2.05) 0.256 (1.02) 0.064 (0.512) 1.02 ([4.10) 0.256 (1.02) 0.256 (2.05) 0.032 (0.064) 0.128 (0.512) Stem 0.256 (1.02) 0.128 (0.512) 0.512 (2.05) E cloacae Leaf Root 0.512 (4.10) 0.064 (0.256) 0.256 (1.02) 0.016 (0.016) 1.02 (4.10) 0.256 (0.512) 0.064 (0.064) 0.032 (0.032) P aeruginosa Stem Leaf Root 0.128 (0.512) 0.128 (0.256) 0.128 (1.02) 0.128 (0.512) 0.064 (0.128) 0.064 (0.512) 0.256 (0.512) 0.256 (0.512) 0.256 (4.10) NT 0.064 (0.256) Stem 0.512 (2.05) 0.128 (1.02) 0.256 (2.05) Leaf 0.512 (2.05) 0.256 (2.05) 0.512 ([4.10) B subtilis S aureus S epidermidis The results shown are means from three measurements obtained on separate occasions a Not tested 123 470 World J Microbiol Biotechnol (2009) 25:465–473 Table Total isothiocyanate content of the root, stem, and leaf of R sativus Part used Total ITC content (mg/g dry extract) Water Methanol Ethyl acetate Chloroform Root 0.42 ± 0.04 0.49 ± 0.01 0.76 ± 0.02 Stem Leaf 0.08 ± 0.002 0.09 ± 0.00 0.12 ± 0.007 0.14 ± 0.006 0.12 ± 0.008 0.13 ± 0.006 0.16 ± 0.009 0.21 ± 0.004 1.18 ± 0.07 Each value is the mean ± standard deviation from three replicates leaf and stem extracts A significantly greater total amount of ITC was recovered from the ethyl acetate and chloroform extracts than from the aqueous and methanol extracts, irrespective of whether the extracts were from the root, stem, or leaf The ITC content of the chloroform extract was more than that of ethyl acetate extract; the water and methanol extracts seemed to contain more or less similar amounts of ITCs Effect of pH and heat treatment on the antibacterial activity of R sativus The ethyl acetate extracts of root, stem, and leaf of R sativus, which had potent inhibitory activity and more effective bactericidal activity than methanol and chloroform extracts, were further studied to determine the effects of pH and temperature on their antibacterial activity The effects of pH and heat treatment on the antibacterial activity of ethyl acetate extracts of R sativus are shown in Table At pH 3.0 the inhibitory activity of the ethyl acetate extracts was slightly higher than that of the control (pH 4.2) At pH 6.0 antibacterial activity seemed to be slightly lower than that of the control extract At pH 9.0 the inhibitory effect was much lower than that of the control Thus the fractions studied had excellent antibacterial activity when the pH was maintained around 3.0–6.0 and tended to lose their activity when the pH was increased towards alkaline The acid and alkali control solutions were not inhibitory to any of the bacteria tested (data not shown) The inhibitory effect of heat-treated extracts was not significantly different from that of untreated extracts when the extracts were incubated at or below 75°C for 30 However, boiling the extracts at 100°C for 30 significantly reduced, but did not abolish, their antibacterial activity Discussion Ever increasing demands from consumers for use of natural agents as additives and food preservatives, and the increased incidence of new and re-emerging infections, has 123 led to a search for new and more effective antimicrobial compounds that have diverse chemical structure and novel mechanism of action Plants are an invaluable source of pharmaceutical products, because they have an almost infinite ability to synthesize compounds with different antimicrobial activity against various pathogenic and opportunistic microorganisms (Cowan 1999) R sativus root, stem, and leaf extracts had excellent bactericidal activity against both Gram-positive and Gramnegative bacteria Successful extraction of bioactive compounds from plant material depends on the solvent used in the extraction procedure In this study it was observed that extraction of the plant with the organic solvents methanol, ethyl acetate, and chloroform resulted in much greater antibacterial activity against all the health-damaging bacteria than extraction with water In particular, the ethyl acetate extracts of R sativus root, stem, and leaf were very active These observations can be explained by different active compounds being extracted with each solvent These findings are in contrast with the results of Abdou et al (1972), who described the antibacterial activity of an aqueous extract of R sativus tubercle against E coli, P pyocyaneus, S typhimurium, and B subtilis Because no appreciable inhibitory activity was found for an aqueous extract of R sativus at a concentration of mg/ml, it is supposed the aqueous extract used by Abdou et al (1972) was of higher concentration than those used in this study This study included E faecalis resistant to penicillin and streptomycin and P aeruginosa resistant to penicillin, because these opportunistic bacteria can cause life-threatening infections in humans, especially in a nosocomial environment (Toye et al 1997; Hancock 1998) Interestingly, this study recorded a notable susceptibility of these resistance strains, especially to root extract, suggesting that the components contained in that particular extract may provide an alternate strategy for combating these organisms and thus could improve the treatment of infections caused by these organisms Further, different parts of R sativus appeared to have potent inhibitory activity toward the food-borne pathogens used in this study Many previous studies reported the inability of natural antimicrobial agents to inhibit the growth of Gram-negative bacteria (Alzoreky and Nakahara 2003; Hansen et al 2001; Weseler et al 2002), perhaps because of the presence of the complex cell wall structure that usually reduces penetration of bacterial cells by extracts Thus the remarkable finding of this study was that R sativus was effective against both Gram-positive and Gram-negative bacteria Isothiocyanates (ITCs) are regarded as the main constituents responsible for the antibacterial activity of cruciferous plants Glucosinolates, precursors of ITCs, are found in different proportions in different parts of plants in response to different forms of synthesis pattern and World J Microbiol Biotechnol (2009) 25:465–473 471 123 472 environment stress (Ciska et al 2000) This study detected the presence of different amounts of ITCs in the root, stem, and leaf of R sativus Root extracts seemed to contain larger amounts of ITCs, followed by leaf and stem Further, it was noted that the ITC content was strongly dependent on the solvent used, because chloroform and ethyl acetate extracted more ITCs than methanol and water Despite similar ranges of total ITC content of methanol and aqueous extracts, all water extracts were less effective at inhibiting the growth of bacteria Similarly, the inhibitory activity of the ethyl acetate extract was higher than that of the chloroform extract, even though the amount of ITCs was less than that in the chloroform extract, thus excluding the possibility that the presence of ITCs in this plant was solely responsible for the antibacterial activity observed Shin et al (2004) recently demonstrated that phenolic compounds, in addition to isothiocyanates, could be responsible for the antibacterial activity of wasabi The acid tolerance and thermal stability of plant extracts are critical aspects of their use in food-processing applications as natural preservatives to control bacterial growth In this study it was observed that R sativus had excellent antibacterial activity at acidic pH, and that increasing the pH of the extracts toward alkaline led to a significant drop in their inhibitory action It has been reported that antibacterial compounds seemed to be stabilized in cationic forms that may interact with and disrupt the negatively charged bacterial cells (Rhodes et al 2006 ) Hence the dependence of the antibacterial activity of R sativus on low pH suggests that molecular structure or charge of the antibacterial species may be vital for its inhibitory effect Heat treatment of the R sativus extracts at 100°C for 30 reduced their antibacterial activity, but these extracts still retained some of their inhibitory effect These results suggest that the extracts have significant thermal stability, which is regarded as an important property for compounds to be used in food preservation Conclusions The results obtained in this study lead to the conclusion that the root, stem, and leaf of R sativus have substantial antibacterial activity against both Gram-positive and Gram-negative bacteria, thus justifying its traditional use in herbal medicines ITCs are present in different amounts in the different parts of R sativus, with significant amounts being present in the root The antibacterial activity of R sativus did not, however, seem to be directly dependent on total ITC content R sativus extracts also had substantial acid tolerance and thermal stability R sativus may thus be an economical source of natural antibacterial substances 123 World J Microbiol Biotechnol (2009) 25:465–473 that could be of significant importance in food-processing applications and for use against pathogens Acknowledgment This study was supported by funding under the Technology Education Quality Improvement Program (TEQIP) of the World Bank to the Center for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological University, Hyderabad, India Syed Sultan Beevi is in receipt of a Research Fellowship from the J.N.T University References Abdou IA, Abou-Zeid AA, El-Sherbeeny MR, Abou-El-Gheat ZH (1972) Antimicrobial activities of Allium sativum, Allium cepa, Raphanus sativus, Capsicum frutescens, Eruca sativa, Allium kurrat on bacteria Qual Plant Material Veg 22(1):29–35 Alzoreky NS, Nakahara K (2003) Antibacterial activity of extracts from some edible plants commonly consumed in Asia Int J Food Microbiol 80:223–230 doi:10.1016/S0168-1605(02)00169-1 Ciska E, Przybyszewska 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