Chemical composition and antimicrobial activity of turnip

Full text

Chemical composition and antimicrobial activity of essential oils
and extracts of two varieties of Turnip (Brassica rapa)
root and leaves in Fars-Iran
INTRODUCTION
Foodborne infections have been one of the major public
health concerns worldwide and account for considerably
[1]
high cases of illnesses . Also the increasing antibiotic resistance
of some pathogens associated with diseases has increased the
interest in development of new types of effective and nontoxic
[2]
antimicrobial compounds . Addition of chemical preservatives
has long been an effective method to control microbial
contamination and development of oxidative reactions, although
in recent years, popular demand has shown a negative vision to
such synthetic chemical preservatives. This has resulted in a
growing demand for natural products, principally, plant extracts,
which are, in the consumers mind, safer, functional and provide
nutritional and health benefits. This demand has increased the
importance of searching for alternative sources of natural
[3]
preservatives . Use of natural antimicrobials such as organic
acids, essential oils, plant extracts and bacteriocins could be a
[4]
good alternative to ensure food safety .
Plant secondary metabolites such as essential oils and plant
extracts, are studied for their antimicrobial activities and most
essential oils derived from plants are known to possess
antibacterial, insecticidal, antifungal, acaricidal and cytotoxic
[5,6]
activities . Essential oils (EOs) are complex mixtures of
biologically active substances used for a long time as flavoring
[7, 8]
agents and constituents of a number of commercial products .
The antimicrobial activity of EOs has created the opportunity to
[9, 10]
use them in pharmacological field and food preservation .
B. rapa belongs to the Cruciferae (Brassicaceae) family, also
known as the mustard family. The name Crucifer comes from the
shape of flowers, with four diagonally opposite petals in the form
[11]
of a cross . This vegetable is usually grown in regions that
[12]
experience temperate climates . Turnip edible parts are
commonly consumed as a boiled vegetable, being used in the
[13]
preparation of soups and stews, too . The antimicrobial activity
[14]
of turnip was previously tested . The highest antimicrobial
Submitted : 02.01.2017 Accepted : 04.04.2017 Published : 30.04.2017
The aim of this study was to evaluate the chemical composition of essential oil (EO) and antimicrobial activity of essential oils and extracts
of two varieties of Turnip (Brassica rapa) root and leaves in Fars-Iran. The EO oil was obtained by hydro-distillation and analyzed by gas
chromatography-mass spectrometry (GC-MS) which revealed 16 compounds in which methyl chavicol, transanethole, linalool and α-pinene were the
main components. The antimicrobial activity was measured by disk-diffusion and micro-dilution method for determination of MIC and MBC. The
highest inhibition zone was recorded against Staphylococcus aureus (33±2 mm) for EO of leaves from shirazi Turnip. The highest MIC (S.aureus and
Bacillus cereus = 0.312 mg/ml) and MBC (S. aureus=0.625 mg/ml) values were detected for Shirazi Turnip leaves. Results presented suggest that the
EOs and extract of turnip root and leaves possess antibacterial properties and potential application in food industry.
Abstract
Asian Journal of Biological and Life Sciences Original Research
399
Key words : GC-MS, Turnip, Methyl chavicol, Anethole, Linalool, α-Pinene.
1 2
Khalil Behnam , Ali Mohamadi Sani*
1 Department of Food Science and Technology, Quchan Banch, Islamic Azad University, Quchan, Iran.
2 Young Researchers and Elite Club, Quchan Branch, Islamic Azad University, Quchan, Iran.
E-mail : mohamadisani@yahoo.com
activity was observed by methanolic extracts on Micrococcus
species while mold was resistant to this extract. Other alcoholic
extracts also showed such higher activity. Another research tried
to examine the antibacterial effect of crude extracts of turnip
species grown in northern Iran showed a significant antimicrobial
activity of B napus L. against pseudomonas aeruginosa
[15].Therefore, the aims of the present study were to examine the
chemical composition of the EOs and to evaluate the antibacterial
activities of EOs and extracts (aqueous and ethanolic) from leaves
and roots of two varieties of B. rapa native to Fars province in Iran
against food-borne pathogenic bacteria.
Ethanol and dimethyl sulfoxide (DMSO) (Merck, Germany)
MHA (Muller Hinton Agar) and MHB (Muller Hinton Broth)
(Merck, Germany) were purchased. Aerial parts (leaves) and
roots of Turnip were collected in 2014 from Shiraz (GPS location:
Latitude: 29° 36' 37.12" N, Longitude: 52° 31' 52.07" E) and
Jahrom (GPS location: Latitude: 28° 56' 59.99" N, Longitude:
53° 56' 59.99" E) cities from Fars province in Iran. The plant was
identified by voucher specimens (No.2383) in herbarium of
department of biology, Shiraz university-Iran. The roots were cut
into small pieces and dried in the shade also the leaves of plant
[16]
were dried at shade .
The air-dried leaves and the roots of the plant (100 g) were
powdered, and the EOs were isolated by hydro-distillation for 3 h
using a Clevenger-type apparatus. The EOs were dried over
anhydrous Na SO and preserved in a sealed vial at 4ºC until
2 4
[5, 17]
further analysis .
Aqueous and ethanolic extracts of leaves and roots from
Turnip were prepared by putting 50 g of each leave or root powder
MATERIALS AND METHODS
Chemicals and plant materials
EO preparation
Preparation of extracts

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400
[18]
in 250 ml of pure water and ethanol. The mixture was agitated
by orbital shaker for 24 h at 240 rpm and then was filtered over a
Buchner funnel. The filtrates were put in oven at 40°Cafterward
[19, 20]
powder of extracts collected in sterile glass tubes and the
yield of extraction was determined. All the dried extracts were
preserved in refrigerator at 4ºC until further use. Plant extracts
were dissolved in the solvent before use in the antimicrobial assay
[21]. Dry matter contents of the extracts were in the range of 1.58-
1.73% which varied between varieties.
EOs were analyzed using an Agilent gas chromatograph
Model 6890, coupled to an Agilent MS model 5973, equipped
with a HP-5MS column (30 mm × 0.25 mm, coating thickness
0.25 μm) (Restek, Bellefonte, PA). Oven temperature program
was from 50ºC (30 min) to 240ºC at 3/min. Helium (99.99 %) was
the carrier gas at a flow rate of 1 ml/min. Diluted samples (1/100
[10]
in hexane, v/v) were injected manually . Injector and detector
temperatures were 220ºC. The electron impact mode, electron
multiplier and ion source temperature of MS worked respectively
[10, 22]
at 70 eV, 1500 V and 240ºC .
The identification of the compounds was performed by
matching their recorded mass spectra of the GC-MS data system.
Quantitative data were obtained from electronic integration peak
areas and comparing their retention time and mass spectra library
with those found in the literature and supplemented by the Wiley
th
(Wiley7 Mass Spectral Library) & NIST MS Search 2.0
(National Institute of Standards and Technology) GC-MS
[23]
libraries .
Four bacterial strains including Staphylococcus aureus
(PTCC 1431), Bacillus cereus (PTCC 1212), Salmonella
typhimurium (PTCC 1074) and Escherichia coli (PTCC 1330)
were obtained from Persian Type Culture Collection, Iranian
Research Organization for Science and Technology (PTCC, Iran).
The bacterial strains were first grown on MH medium at 37°C for
[24]
24 h prior to seeding on to the nutrient agar . Finally,
suspensions were adjusted to 0.5 Mc-Farland standard turbidity.
Bacterial suspensions were standardized to concentrations of
8 [25]
1.5×10 CFU/ml .
[26, 27]
The antibacterial activity was studied using disk diffusion
[28]
and micro-dilution methods . All tests were performed in
Triplicate.
[29]
Sterile 6 mm filter paper disks were placed on lawn culture
prepared on Muller-Hinton (MH) agar (Merck) using sterile
[30] [20]
cotton swab impregnated with 15 μl of each bacterial
suspension. The plates were put at room temperature for about 1 h
to allow the extract and EO to diffuse from the discs into the
medium, and then incubated at 37ºC for 24 h and after that the
diameter of the zone from bacterial growth inhibition around each
[30]
dick were measured and recorded in millimeter .
96-wellsmicro-plates were prepared by dispensing into each
well with 100 μL of extracts and EOs. Then, 100 μL of MH broth
[10] [31]
and 20μl of each bacterial suspension were added to the wells
Gas chromatography-mass spectrometry analysis
Organisms and inoculation conditions
Antimicrobial assay
Disk diffusion method
Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC) Test
6
(standardized at 1.5×10 CFU/ml by adjusting the optical density
to 0.1 at 600 nm by Shimadzu UV-120-01 spectrophotometer).
The final volume in each well was 220μl. The plates were covered
with sterile plate sealer and followed by shaking for 30s and then
incubated at 37°C for 24 h. After incubation, wells were examined
for microbial growth. MIC was defined as the lowest
concentration of the extract or essential oil in the medium in
[32]
which there was no visible growth after incubation .Using the
results of the MIC assay, the concentrations showing complete
absence of visual growth of bacteria were identified and 50 µL of
each culture broth was transferred on to the agar plates and
incubated at 37ºC for 24 h. the complete absence of growth on the
agar surface in the lowest concentration of sample was defined as
[33, 34]
MBC .
The EOs were extracted by the hydro-distillation of the dried
aerial parts (leaves) and roots of Shirazi and Jahromi B. rapa and
the constituents were analyzed by GC-MS. The essential oil of B.
rapa was analyzed to determine their constituents (Table 1).
GC/MS analyses showed 16 compounds in which the main
components were Methyl chavicol (35.41%), trans-anethole
(23.87%), linalool (18.32%), α-pinyin (9.28%) and β-pinyin and
(5.11 %).
Data are summarized in Table 2.All samples were virtually
effective on pathogens especially essential oil of leaves which
showed the largest inhibition zone on S. aureus (33±2 mm) and
then on B. cereus (31±1 mm). The results indicated that essential
oils at all tested concentrations significantly inhibited the growth
of S. aureus, B. cereus, S. typhimurium and E. coli.
The Inhibition zone for ethanolic/aqueous extracts of Jahromi
Turnip leaves and root are shown in table 2. All of the extracts had
antimicrobial activity against tested bacteria. The inhibition zone
of gram-positive bacteria were larger than gram-negative
bacteria. These results coincided with the results obtained by
[35] [15]
Tenore et al. (2012) and Beltagy et al. (2014) . B. cereus was
sensitive to ethanolic extract of leaves (16±0.6 mm) while
aqueous extracts of leaves and root did not show strong
antibacterial activity on E. coli (7±1 mm). Numerous surveys
have highlighted the potential importance of extracts from
Brassica species as sources of polyphenolics (flavonoids,
phenolic acids and related analogues) able to exert antimicrobial
[36, 37]
effects . Actually, it is well known that phenolic acids are
present in ionized form at the buffer H value (7.0) and are too
polar to penetrate the semi permeable bacterial membrane and
[38]
react with the cytoplasm or cellular proteins . This is the same
reason why the lipidic wall of gram-negative pathogens
represents a great barrier for most polyphenols and hence only
[15]
slight inhibition is achieved .
Findings are presented in table3which shows the MIC values
in the ranges of 3.125 (for S. aureus and B. cereus) and50 mg/ml
(for E. coli). Essential oils had higher inhibitory effects against
all of the microorganisms. The MIC value was in range of 0.312-
2.5 mg/ml and the essential oil of Shirazi Turnip leaves had the
strongest bacteriostatic effect on the bacterial strains.
Table 4, reports the MBC values of the extracts and essential
RESULTS
Chemical composition of Turnip EOs
Disk diffusion results
MIC and MBC data

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Table 1: Chemical composition of turnip essential oils (%).
Table 2: Inhibition zone in diameter (mm) for ethanolic and aqueous extracts and EOs of Shirazi
and Jahromi turnip leaves and root
Values are the mean of triplicate.
oils against bacterial strains which were prevented at different
rates. The most sensitivity was observed for Bacillus cereus and S.
aureus and the least for S. typhimurium and E. coli. The MBC
value was between 0.625-50 mg/ml.
The antimicrobial activity of extracts obtained from spices,
DISCUSSION
herbs and other aromatic plants have been recognized for many
[39]
years . Various publications have documented the antibacterial
[31, 40, 41]
activity of essential oil constituents and plant extracts . In
this study, the essential oil and extracts exhibited remarkable
activity against some of the representative food borne and
spoilage pathogenic bacteria such as S. aureus, B. cereus, S.
typhimurium, E. coli. The Gram-positive bacteria were found to

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Table 3: MIC for ethanolic and aqueous extracts and EOs of Shirazi and Jahromi turnip
leaves and root (mg/ml).
Values are the mean of triplicate.
Table 4: MBC for ethanolic and aqueous extracts and EOs of Shirazi and Jahromi turnip
leaves and root (mg/ml)
be more susceptible to the essential oil and various solvent
extracts than Gram-negative bacteria. This is probably due to the
cell membrane of Gram-positive bacteria, which can interact
directly with hydrophobic compounds of essential oils, whereas
the external cell wall around the cell membrane of Gram-negative
bacteria is hydrophilic and blocks the penetration of hydrophobic
oil and avoids the accumulation of essential oils in target cell
[31, 42]
membrane .
Regarding the antibacterial activity of plant extracts/essential
oils, the researchers have described several mechanisms of
action, including cell membrane damage resulting in increased
Values are the mean of two replicate.
permeability, changes in intracellular pH and membrane
potential, dissipation of cellular components, decrease in the
cytoplasmic ATP concentration, which together induce bacterial
death. Secondary effects that may be involved seem to be the
inhibition of enzymes, loss of turgor pressure, alterations in
[43]
macromolecules synthesis, and other cellular processes .
The results presented in this study indicated that essential oils
and extracts obtained from leaves and roots of turnip (B. rapa)
possess antibacterial properties. On the basis of the experimental
results, it can be postulated that the extracts of turnip (B. rapa)
CONCLUSION

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have the potent antibacterial properties against some
representative food-borne pathogens. Specifically, turnip leaves
essential oils and extracts obtained by alcoholic solvent which
presence of more phenolic compounds than others is reason.
Therefore, they could be used as possible food antimicrobial
preservative in food industry, but the in vivo studies should be
done to evaluate the probable adverse effect on food sensory
properties.
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