J Med Bacteriol. Vol. 5, No. 1, 2 (2016): pp.29-35 jmb.tums.ac.

ir

The Study of Antimicrobial Activities of Partially Purified Cyclotide

Content and Crude Extracts from Viola tricolor

Zahra Khoshkam 1, Mahboobeh Zarrabi 1*, Zargham Sepehrizade 2,

Maryam Keshavarzi 3

1
Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.

2
Department of Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

3
Department of Plant Sciences, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.

ARTICLE INFO ABSTRACT

Article type: Background: Searching for the potent natural antibiotics will improve the treatment of most infectious
Original Article diseases. Cyclotides are potent active peptides derived from some plant families like Violaceae. Viola
tricolor (the pansy) has been found to have numerous cyclotides. Nowadays cyclotides attract more
Article history: attention for their antibacterial activities. The current research studied the antimicrobial properties of
Received: 12 Dec 2015 semi-purified cyclotides from V. tricolor.
Revised: 22 Jan 2016
Methods: Extraction and purification of cyclotides from Iranian Viola tricolor were performed by
Accepted: 14 May 2016 fractionation and solid phase extraction methods, and their antimicrobial effects were studied against
Published: 15 Aug 2016 several bacterial strains using diffusion assays. Also, attendance of cyclotide in the extract was verified
Keywords: by Tricine-SDS page and spectroscopic methods.
Viola tricolor, Results: Antimicrobial effects of semi-purified cyclotides and crude extracts resulted in the
Cyclotide , Extraction, antibacterial activity potential of V. tricolor totally extracted samples against gram negative bacteria,
Antibacterial effect E.coli and P. aeruginosa. However, there is need for optimizing the assay method and the culture media.
Conclusion: Viola tricolor as a remedy represents the antibacterial potential, which may not be
unrelated to its cyclotide content although the effectiveness of cyclotides may also differ because of
their synergism, natural structure and bioactivities, the amount of purified content, and the way they
were assayed.

 Please cite this paper as: Khoshkam Z, Zarrabi M, Sepehrizade Z, Keshavarzi M. The Study of Antimicrobial Activities of Partially
Purified Cyclotide Content and Crude Extracts from Viola tricolor. J Med Bacterial. 2016; 5 (1, 2): pp.29-35.

*Corresponding Author: Mahboobeh Zarrabi, Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.
Tel: +98-021-85692722. E-mail: [email protected]
The Study of Antimicrobial Activities … Khoshkam Z, et al.

Introduction This study aimed the extraction and semi-

purification of cyclotides from Iranian Viola
Treatment of most infectious diseases using tricolor, and then examination of their
different synthetic and natural antibiotics should be antimicrobial effects against several bacterial
better than the past centuries. However in spite of strains. The presence and quality of the partially
these advances, newer problems such as purified cyclotides were evaluated by Tricine-
nosocomial infections have come and this is the SDS-PAGE and Bradford methods.
main reason for more interest of the researchers for
finding more and better drugs (1). Cyclotides are Material and method
fascinating circular proteins ranging from 28 to 37
amino acid residues that are naturally expressed in Plant material
plants (2). They are believed to have an important
role in the plants, defense system by their The herbs of Viola tricolor were collected from
insecticidal effects (3); they have also been shown Absard region located in east of Tehran Province
to be cytotoxic (4), anti-HIV (5), antimicrobial (6), by our botanist. Total extraction and semi-
and hemolytic agents (7). All of these mini- purification for the aerial parts of V. tricolor
peptides share a unique head-to-tail circular (leaves & flowers separately) were performed
knotted topology of three disulfide bridges, called using fractionation protocol (FP), solvent-solvent
“cystine knot” topology resulting in their partitioning (SSP) and solid phase extraction (SPE)
significant thermal, chemical and enzymatic methods.
resistance (2, 8). Cyclotides are present in many
plants from the Violaceae, Rubiaceae, Fabaceae Plant total extraction
and Cucurbitaceae families (9). Viola tricolor (the
pansy) is widely cultivated as an ornamental plant. About 15 g of powdered plant powdered material
Previous investigations have determined several leaves and flowers were separately extracted 5
special cyclotides like tricyclon A & B, vitri A-F, times by 200 ml dichloromethane. The solution
Cycloviolacin O12 & O2, kalata S, varv (A, D, E, was discarded, and the plant residues were dried at
F, H, Hm & He) from V.tricolor till now (10-12). room temperature overnight. The dried plant
Many plants of the Violaceae family have been remnant was extracted 3 times with 300 ml of 50%
used in traditional remedies, and V. tricolor have ethanol; then the solution was concentrated to 400
been used in folk medicine externally and ml and acidified by 2% acetic acid. By using a
internally as an adjuvant in treating various skin polyamide column, tannins were removed. The
conditions such as eczema, impetigo, acne and tannin free extract was concentrated and
pruritus also internally as an auxiliary agent to lyophilized. Solvent-solvent partitioning for
promote metabolism (13). Most of medicinal completion of extraction was performed for about
properties of V. tricolor have been previously 1 g of previously achieved extract powder solved
reported in association with the contribution of in 100 ml water, then partitioned 3 times with 100
nonpeptidic compounds (14, 15). Antimicrobial ml butanol. The resulting water and butanolic
activity of this herb for several crude extracts with phases were collected and concentrated to half the
different polarity has been evaluated before, which initial volume and then lyophilized (16).
suggests a synergistic interaction between the Cyclotides mostly share hydrophobic properties;
compounds of V. tricolor herb, showing its low to hence, they must enter butanolic phase. However,
significant activity against most of the tested water phase may also include some important
microorganisms (15). cyclotides.

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The Study of Antimicrobial Activities … Khoshkam Z, et al.

Solid phase extraction Pränting group (6) for some purified cyclotides. In
the current study, firstly, we examined the RDA
Semi-purification of cyclotides with method used by the Pränting group, for this
hydrophobic properties separated by butanolic purpose, bacteria were cultured on TSB (Tryptic
phase was achieved by solid phase extraction Soy Broth); after reaching to optimum density they
(SPE) using C18 flash cartridges. 25 mg of the were plated and washed by cold sodium phosphate
butanolic extract powder, which obtained from buffer. Nearly 4 × 106 cfu were added to the bottom
previous step was dissolved in ammonium acetate layer of the media containing 0.03% w/v TSB, 1%
buffer (50 mmol.l-1, pH 8) and loaded onto C18 w/v LE agarose and 0.02% v/v tween 20. The
SPE (MACHERY-NAGEL, Germany) cartridge. mixture was added to Petri dish (85 mm). Different
The cartridge was first activated with methanol and amounts of crude samples and semi-purified
equilibrated with the same buffer (16, 17). For cyclotides were added to 3 mm gel punch wells.
releasing of hydrophobic cyclotides, the column The mixture was incubated at 37˚C for 3 hours in
was washed with 4 ml of 20%, 50% and 80% order to diffuse the peptides. Then the upper layer
aqueous Ethanol. of the media (6% w/v TSB, 1% w/v agarose in 10
mM SPB) was added to the plates. The plates were
Bradford assay & Tricine-SDS-PAGE incubated at 37 °C overnight. Due to the some
contradictory results of the mentioned
The presence and concentration of the peptides in antimicrobial assays and better comparison of
total ethanol extract, its water and butanolic phase, appropriate media for cyclotides and their crude
and semi-purified fractions were determined by extracts, we used MHA and modified MHB based
using Bradford assay (18). In order to determine culture media ( including MHB, agarose LE 1%,
the purity and molecular weight of the partially phosphate buffer 0.01 M and 0.02% Tween 20)
purified cyclotides, Tricine-SDS-PAGE was within the well diffusion assay against the standard
performed using the protocol described by bacteria strains. Total protein quantities obtained
Schagger and Jagwa (19, 20). from Bradford assay and the antibacterial activity
results are reported in Table 1.
Bacterial strains, extracts and media
Results
Examination of the antimicrobial effects of the
crude extracts and their both separated water and Attendance of partially purified cyclotides in
butanolic phase of leaves (named as EL, WL and different extracts was determined using the above
BL), and flowers (named as EF, WF, BF) as well mentioned methods. Tricine-SDS-PAGE resulted
as their partially purified cyclotides using C18 SPE in the presence of peptides with a molecular
column was performed by Radial diffusion assay weight of about 3500 to 4600 Dalton (Figure 1),
(6) and also its modified method using MHB based which is compatible with the small size of
culture media against human pathogenic bacteria, Cyclotides. The concentration and purity of
Escherichia coli (ATCC 25922), Pseudomonas peptides in 50% elution of SPE column were
aeruginosa (ATCC 27853), Staphylococcus more than in 80% elution. However, all crude and
aureus (ATCC 25923). fractionated extracts were investigated for their
protein quantity and antibacterial activities. The
Radial diffusion assay (RDA) and well diffusion quantities (TPQ) for most, and no protein
assay

The modified version of Radial diffusion assay

(RDA) was previously used by Lehrer (21) and
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The Study of Antimicrobial Activities … Khoshkam Z, et al.

Table 1. Antibacterial activities and total protein

quantities of crude extracts and fractionated ones by
RDA and WDA methods.

Figure 1. Tricine- SDS- PAGE. lane1: molecular

weight of protein marker. Lane2: Solution obtained
with 50% aq EtOH. Arrow: Cyclotide.

results showed different amounts of total protein

attendance for some fractions washed with C18
column especially 20% elution. Table 1 presents
the results of TPQ for some crude extracts and
fractions with antimicrobial activity. For special
substances like cyclotides mostly sharing
hydrophobic charged residues with different
polarity, it is better to use appropriate culture
media and test method. However, not all
cyclotides even in almost pure samples may have
antibacterial effects or show their effective
potential within the crude extracts better than
partially purified samples because of their
synergism. Here, we used two culture media for
better comparison for crude extracts, especially
the crude butanolic ones. For semi-purified
samples, only RDA method was used, and via the
mentioned method, some of the 50% and 80%
semi-purified cyclotide elutions, flowers and
leaves had no effective antibacterial potential;
however, their including crude butanolic extract
presented antibacterial properties using only
MHA culture medium. The results could be
explained by low amount of cyclotides in the
samples or their natural non -effectiveness against
the tested bacteria, or even the use of
inappropriate assay methods or culture media.

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Strain sensitivity to Cyperus rotundus tuber odorata against E.coli, P. aeruginosa and S.
extract aureus. The results showed that the most
susceptible bacterium was S. aureus (24).
In the blank disc method, zone of growth Similarly, Roshan et al. reported the same results
inhibition appeared at a minimum inhibitory (25). However, there are some contradictory
concentration of 25 mg/ml for all bacterial strains results for the antibacterial activities of cyclotides
(Table 2). have been studied till now. In the current study,
In broth microdillution methods, the MIC value using RDA method, the most susceptible
for all bacterial strains was at a concentration of bacterium to the semi-purified peptides extracted
0.1 mg/ml (Table 3). from V. tricolor’s flowers was E.coli, with the
inhibitory zone diameter of 13 mm. The crude
Discussion ethanol and butanolic extracts also showed better
activities against the gram negative E. coli and P.
Cyclotides have multiple biological activities, aeruginosa than S. aureus. It is possible that the
and their anti-microbial activity has been susceptibility between the studied strains as well
examined in many studies. In the current study, as the natural activity of all cyclotides could be
we focused on the extraction and semi- different. Other explanation could be the
purification of cyclotides from Iranian Viola synergism effect of the total cyclotides used in
tricolor, and then examination of their some studies.
antimicrobial effects against several human Cyperus rotundus tuber extract has been shown
pathogenic bacteria. Antimicrobial activity of this to possess antimicrobial activity (28). While its
herb for several crude extracts with different inhibitory effect against Streptococcus pyogenes
polarity has been evaluated before, suggesting a growth was demonstrated by Mehta et al. (29), the
synergistic interaction between the compounds of whole plant extract has been shown to be
V. tricolor herb and showing its low to significant ineffective against strains of E. coli,
activity against most tested of the microorganisms Pseudomonas aeruginosa and Salmonella typhi
(15). The antibacterial properties of some plant (30). A MIC value of 12.5 mg/ml has indeed been
derived cyclotides have been reported in several determined for certain bacterial strains treated
previous studies. Pränting et al. studied the with the rhizomes oil (17), whereas using the disc
antibacterial activity of cycloviolacin O2 and diffusion method, zone of growth inhibition was
kalata B1 against some gram positive and gram revealed in Pseudomonas aeruginosa and
negative bacteria, which resulted in the Staphylococcus aureus treated with the ethanolic
effectiveness of cyclotide against the gram extract of the plant (31). In a separate study, using
negative E. coli and no activity against the gram the agar disk diffusion method, the ethanolic
positive S. aureus (6). Gran et al. also were extract of Cyperus rotundus revealed a zone of
achieved to the same result by Pränting group; growth inhibition for E. coli and C. albicans (32).
they reported that kalata B1 has no effect against
S.aureus while it is active against the gram Acknowledgements
negative bacteria (22). In another study, Tam
group examined the antibacterial activities of four We appreciate Iran National Science
synthetic cyclotides against different bacteria Foundation (INSF) to the financial support of this
strains, which were active against the gram project (project 88000118).
positive bacteria such as S. aureus (23); however,
they were almost not active against gram negative
bacteria. Zarrabi et al. examined the anti-bacterial
activity of semi-purified cyclotides from V.
J Med Bacteriol. Vol. 5, No. 1, 2 (2016): pp.29-35 jmb.tums.ac.ir
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The Study of Antimicrobial Activities … Khoshkam Z, et al.

Conflict of interests 7. Craik DJ, Daly NL, Mulvenna J, Plan MR,

Trabi M. Discovery, structure and biological
None of the authors has any conflicts of interest activities of the cyclotides. Curr Protein
to disclose and all authors support submission to Pept Sci 2004; 5: 297-315.
this journal. 8. Colgrave ML, Craik DJ. Thermal, chemical,
and enzymatic stability of the cyclotide
Financial disclosure
kalata B1: the importance of the cyclic
cystine knot. Biochemistry 2004; 43: 5965-
Financial support of this was project provided by
Iran National Science Foundation (INSF). 5975.
9. Koehbach J, Attah AF, Berger A, Hellinger
R, Kutchan TM, et al. Cyclotide discovery
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