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: 3 July 2008 / Accepted: 8 November 2008 / Published online: 9 December 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Aqueous, methanol, ethyl acetate, and chloro-
form extracts of the root, stem, and leaf of Raphanus
sativus were studied for antibacterial activity against food-
borne 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 concen-
tration (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 bacteri-
cidal effects against human pathogenic bacteria, justifying
their traditional use as anti-infective agents in herbal
medicines.
Keywords Raphanus sativus
Antibacterial activity

Human pathogenic bacteria
S. S. Beevi
L. N. Mangamoori (&)
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
Introduction
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 food-
borne disease (Tauxe 1997). Currently much attention has
been focused on natural antimicrobial compounds, espe-
cially 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 cysteine-
rich 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)
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(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 degra-
dation 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 6-
methylsulfinylhexyl 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. Ab-
dou 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 Gram-
negative bacteria) including resistant strains and food-
borne 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
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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 chloro-
form—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 9 100 ml)
and mixing with a magnetic stirrer at low rpm for 24 h. The
extract was then filtered through Whatman No. 1 paper and
was subsequently lyophilized in a lyophilizer at 5 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 8 mM 1,2-benzenedith-
iol. The mixture was heated at 65°C for 1 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 (Hi-
Media, India) slants and were stored at 4°C.
Determination of antibacterial activity
An agar-well diffusion method was employed for evalua-
tion 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 106 colony-
forming units (1 9 106 CFU/ml) was added aseptically to
MHA medium (HiMedia, India) and poured into sterile
Petri dishes. Different test extracts at a concentration of
1 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 pre-
pared using the solvents methanol, chloroform, and ethyl
acetate.
Minimum inhibitory concentration (MIC)
and minimum bactericidal concentration (MBC)
of R. sativus extracts
MIC and MBC for all extracts and antibiotics were deter-
mined 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 pre-
pared and 500 ll of each dilution was incubated with
2.5 ml MHB (HiMedia, India) containing 50 ll inoculum
(1 9 106 CFU/ml) at 37°C for 24 h. The MIC was deter-
mined 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.
467
Effect of pH on inhibitory zone against pathogenic
bacteria
Ethyl acetate extracts of root, stem, and leaf at a concen-
tration 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 pH-
adjusted extracts were then filtered through 0.22-lm
membranes and used within 60 min. 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 concen-
tration of 100 mg/10 ml were incubated in a water bath for
30 min 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 per-
formed 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).
Results
Antibacterial activity of R. sativus extracts
Ten bacterial strains were used as taxonomical represen-
tatives to evaluate the effect of candidate antimicrobial
components against specific target microbes: Gram-posi-
tive 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 pneumo-
niae), 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 dif-
fusion 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
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Table 1 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

B. subtilis 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 – 15.17 ± 0.15 16.90 ± 0.36 13.10 ± 0.10
S. aureus Root – 19.83 ± 0.55 23.83 ± 0.21 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 – 19.90 ± 0.36 18.73 ± 0.25 16.20 ± 0.20
S. epidermidis Root – 21.27 ± 0.40 25.87 ± 0.41 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
Leaf – 20.10 ± 0.22 20.77 ± 0.25 12.17 ± 0.21
E. faecalis Root – 19.90 ± 0.36 26.13 ± 0.15 16.57 ± 0.89 – –
Stem – 12.03 ± 0.15 18.17 ± 0.15 11.03 ± 0.15
Leaf – 12.97 ± 0.23 17.17 ± 0.2 12.10 ± 0.10
S. typhimurium Root – 24.37 ± 0.49 26.37 ± 0.31 18.63 ± 0.59 26.27 ± 0.46 20.23 ± 0.40
Stem – 15.70 ± 0.36 18.50 ± 0.56 11.93 ± 0.40
Leaf – 15.57 ± 0.51 18.67 ± 0.29 15.97 ± 0.25
K. pneumoniae Root – 17.43 ± 0.67 24.53 ± 1.01 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 – 18.13 ± 0.32 18.70 ± 0.30 14.17 ± 0.15
E. coli Root – 18.07 ± 0.21 19.67 ± 0.60 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 – 13.73 ± 0.25 18.30 ± 0.20 18.40 ± 0.36
E. aerogenes Root – 18.03 ± 0.55 23.40 ± 1.04 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
Leaf – 13.73 ± 0.38 18.33 ± 0.31 12.63 ± 0.32
E. cloacae Root – 27.97 ± 0.57 34.20 ± 0.66 23.40 ± 0.26 22.83 ± 0.72 24.97 ± 0.15
Stem – 19.90 ± 0.56 21.67 ± 0.59 18.97 ± 0.25
Leaf – 20.40 ± 0.40 25.30 ± 0.10 18.53 ± 0.21
P. aeruginosa Root – 20.77 ± 0.38 24.90 ± 0.60 19.43 ± 0.70 – 21.33 ± 0.57
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
Each value is the mean ± standard deviation from three replicates
The concentration of all the extracts used was 1.0 mg/ml
a
Inhibitory zones in mm, including diameter of the well (8.0 mm); mean ± standard deviation of three replicates
b
No inhibition or inhibition zone was less than 9 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 streptomy-
cin) 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
extracts, but were highly sensitive to the ethyl acetate
extracts. Further, both Gram-positive and Gram-negative
bacteria were equally susceptible, demonstrating the broad-
spectrum 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

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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 2. 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, respec-
tively. 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, signifi-
cantly 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 3. 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 2 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

B. subtilis 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 0.512 (2.05) 0.256 (1.02) 1.02 (4.10)
S. aureus Root 0.064 (0.256) 0.032 (0.128) 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 0.064 (0.256) 0.256 (1.02) 0.512 (2.05)
S. epidermidis Root 0.128 (0.256) 0.032 (0.032) 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)
Leaf 0.128 (0.512) 0.128 (0.512) 1.02 ([4.10)
E. faecalis Root 0.256 (2.05) 0.064 (0.128) 0.512 (4.10) NTa NT
Stem 1.02 ([4.10) 0.256 (2.05) 1.02 ([4.10)
Leaf 1.02 (4.10) 0.256 (2.05) 1.02 ([4.10)
S. typhimurium Root 0.064 (0.256) 0.032 (0.128) 0.256 (1.02) 0.032 (0.128) 0.064 (0.128)
Stem 0.256 (1.02) 0.256 (1.02) 1.02 ([4.10)
Leaf 0.256 (1.02) 0.256 (1.02) 0.512 (2.05)
K. pneumoniae Root 0.256 (1.02) 0.064 (0.256) 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)
Leaf 0.256 (1.02) 0.128 (0.512) 0.512 (2.05)
E. coli Root 0.256 (1.02) 0.064 (0.512) 0.512 (4.10) 0.008 (0.008) 0.064 (0.128)
Stem 0.512 (2.05) 0.256 (2.05) 1.02 ([4.10)
Leaf 0.512 (4.10) 0.256 (1.02) 0.256 (1.02)
E. aerogenes Root 0.256 (1.02) 0.064 (0.512) 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)
Leaf 0.512 (4.10) 0.256 (1.02) 1.02 (4.10)
E. cloacae Root 0.064 (0.256) 0.016 (0.016) 0.256 (0.512) 0.064 (0.064) 0.032 (0.032)
Stem 0.128 (0.512) 0.128 (0.512) 0.256 (0.512)
Leaf 0.128 (0.256) 0.064 (0.128) 0.256 (0.512)
P. aeruginosa Root 0.128 (1.02) 0.064 (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)
The results shown are means from three measurements obtained on separate occasions
a
Not tested

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Table 3 Total isothiocyanate content of the root, stem, and leaf of
R. sativus
Part Total ITC content (mg/g dry extract)
used
Water Methanol Ethyl acetate Chloroform
Root 0.42 ± 0.04 0.49 ± 0.01 0.76 ± 0.02 1.18 ± 0.07
Stem 0.08 ± 0.002 0.09 ± 0.00 0.12 ± 0.007 0.14 ± 0.006
Leaf 0.12 ± 0.008 0.13 ± 0.006 0.16 ± 0.009 0.21 ± 0.004
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 chloro-
form 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 chloro-
form 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 4. 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 min. However, boiling the extracts at 100°C for 30 min
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
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World J Microbiol Biotechnol (2009) 25:465–473
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 Gram-
negative bacteria. Successful extraction of bioactive com-
pounds 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 bac-
teria 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 1 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-threat-
ening infections in humans, especially in a nosocomial
environment (Toye et al. 1997; Hancock 1998). Interest-
ingly, 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 organ-
isms 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 anti-
microbial 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 con-
stituents 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
 Table 4 Effect of pH and heat treatment of ethyl acetate extracts on inhibitory zone (mm) against resistant and food-borne pathogens
Organism Part tested Control pH Temperature (°C)
3.0 6.0 9.0 25 50 75 100

B. subtilis Root 26.80 ± 1.36a 26.92 ± 1.54 25.15 ± 2.24 16.73 ± 0.64 26.30 ± 0.26 26.75 ± 1.30 26.15 ± 0.24 13.45 ± 0.62
Stem 18.30 ± 0.26 19.66 ± 0.92 17.19 ± 0.31 10.72 ± 0.42 17.57 ± 0.40 18.54 ± 0.56 18.63 ± 0.32 10.26 ± 0.10
Leaf 16.90 ± 0.36 17.30 ± 1.50 14.83 ± 0.31 9.57 ± 0.60 16.70 ± 0.26 17.00 ± 0.10 16.54 ± 0.55 10.10 ± 0.21
S. aureus Root 23.83 ± 1.21 23.87 ± 2.64 22.31 ± 1.14 12.10 ± 0.08 23.37 ± 0.35 23.80 ± 1.20 23.40 ± 0.40 12.65 ± 0.40
Stem 20.50 ± 0.50 21.82 ± 0.74 19.10 ± 0.06 12.30 ± 0.15 19.80 ± 0.26 19.50 ± 0.84 20.10 ± 0.30 12.15 ± 0.33
Leaf 18.73 ± 0.25 19.10 ± 0.81 17.03 ± 0.25 11.07 ± 0.60 18.23 ± 0.25 18.55 ± 2.15 17.93 ± 0.12 10.50 ± 0.50
S. epidermidis Root 25.87 ± 0.41 25.70 ± 1.58 23.96 ± 1.53 14.12 ± 0.35 24.93 ± 0.25 24.75 ± 1.97 24.60 ± 0.40 13.27 ± 0.62
Stem 18.30 ± 0.52 18.50 ± 0.60 16.93 ± 0.57 11.58 ± 0.63 17.43 ± 0.51 18.49 ± 0.82 18.30 ± 0.20 11.20 ± 0.40
Leaf 20.77 ± 1.25 20.87 ± 1.35 19.63 ± 1.60 12.13 ± 0.32 20.07 ± 0.38 20.40 ± 2.35 20.00 ± 0.10 11.35 ± 0.94
World J Microbiol Biotechnol (2009) 25:465–473

E. faecalis Root 26.13 ± 0.15 28.50 ± 0.50 24.78 ± 0.42 15.21 ± 0.31 26.36 ± 0.54 26.10 ± 0.00 26.12 ± 0.15 NDb
Stem 18.17 ± 0.15 17.25 ± 1.08 16.75 ± 0.46 10.05 ± 0.16 18.40 ± 0.20 18.24 ± 1.34 17.57 ± 0.68 ND
Leaf 17.17 ± 0.21 18.00 ± 0.00 16.27 ± 0.31 10.40 ± 0.85 17.13 ± 0.26 17.20 ± 0.60 17.02 ± 0.15 ND
S. typhimurium Root 26.37 ± 2.31 27.54 ± 2.42 24.98 ± 0.63 14.13 ± 0.21 25.60 ± 0.24 25.83 ± 2.61 25.87 ± 0.63 12.47 ± 0.33
Stem 18.50 ± 0.56 18.98 ± 1.87 16.81 ± 0.50 12.17 ± 0.36 18.05 ± 0.4 18.50 ± 1.50 17.94 ± 0.55 10.89 ± 0.43
Leaf 18.67 ± 0.29 18.50 ± 0.82 16.13 ± 0.32 12.07 ± 0.35 18.60 ± 0.35 17.45 ± 0.67 17.68 ± 0.37 11.20 ± 0.30
K. pneumoniae Root 24.53 ± 1.01 24.80 ± 2.50 23.55 ± 0.40 13.18 ± 0.29 24.58 ± 0.23 25.00 ± 0.50 24.00 ± 0.00 10.55 ± 0.45
Stem 18.53 ± 0.51 19.00 ± 0.50 17.06 ± 0.08 12.69 ± 0.62 17.90 ± 0.36 18.43 ± 1.73 17.94 ± 0.72 10.20 ± 0.58
Leaf 18.70 ± 0.30 18.92 ± 0.68 16.63 ± 0.60 11.03 ± 0.25 17.85 ± 0.46 17.50 ± 1.20 18.20 ± 0.40 10.20 ± 0.74
E. coli Root 19.67 ± 0.60 20.51 ± 1.42 18.16 ± 0.20 11.64 ± 0.32 18.69 ± 0.34 19.25 ± 2.50 19.24 ± 0.53 12.27 ± 1.67
Stem 17.13 ± 0.32 17.50 ± 1.20 16.10 ± 0.29 11.30 ± 0.47 16.90 ± 0.20 17.24 ± 0.87 16.81 ± 0.68 10.35 ± 0.82
Leaf 18.30 ± 0.20 18.90 ± 0.71 16.80 ± 0.61 11.90 ± 0.56 18.67 ± 0.19 18.44 ± 0.68 18.50 ± 0.00 10.40 ± 0.20
E. aerogenes Root 23.40 ± 1.04 24.89 ± 1.62 21.79 ± 2.43 10.33 ± 0.21 23.50 ± 0.75 23.15 ± 2.82 22.80 ± 0.42 10.82 ± 0.98
Stem 20.73 ± 0.25 22.10 ± 0.24 18.75 ± 0.25 13.00 ± 0.99 19.80 ± 0.42 20.70 ± 0.69 20.15 ± 0.83 10.10 ± 0.22
Leaf 18.33 ± 0.31 18.53 ± 0.15 16.23 ± 1.25 9.17 ± 0.31 18.34 ± 0.76 18.30 ± 1.45 18.28 ± 0.17 9.80 ± 0.60
E. cloacae Root 34.20 ± 2.66 36.80 ± 1.48 32.58 ± 0.73 16.91 ± 0.30 32.27 ± 0.35 32.73 ± 1.68 33.40 ± 0.20 12.50 ± 0.2
Stem 21.67 ± 0.59 22.43 ± 0.56 19.53 ± 0.41 16.10 ± 0.32 20.94 ± 0.17 20.97 ± 0.48 20.05 ± 0.41 10.22 ± 0.42
Leaf 25.30 ± 0.10 26.41 ± 1.38 23.77 ± 0.93 14.33 ± 0.45 24.60 ± 0.25 25.44 ± 1.24 24.83 ± 0.72 10.64 ± 0.63
P. aeruginosa Root 24.90 ± 0.60 25.66 ± 0.84 21.74 ± 0.23 13.55 ± 0.53 24.43 ± 0.58 25.30 ± 0.60 24.05 ± 0.50 12.00 ± 0.00
Stem 19.53 ± 0.55 20.43 ± 0.92 17.47 ± 0.56 10.58 ± 0.33 19.30 ± 0.20 19.50 ± 1.46 18.97 ± 0.63 ND
Leaf 18.33 ± 0.31 18.70 ± 1.20 16.10 ± 0.66 9.53 ± 0.35 17.52 ± 0.62 18.27 ± 1.61 18.22 ± 0.14 ND
The concentration of all extracts used was 1.0 mg/ml
a
Inhibitory zones in mm, including diameter of the well (8.0 mm), are mean ± standard deviation from three replicates
b
Not detected
471

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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 aque-
ous 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 appli-
cations 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 anti-
bacterial 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 min 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.
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