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In vitro anti-proliferative, anti-bacterial potential and induction of DNA strand break of partially purified Cuscuta reflexa Roxb.

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Cuscuta reflexa is an important medicinal plant, mentioned in Ayurveda, an ancient Indian system of medicine. The plant is selected to evaluate the possibility for novel pharmaceuticals for anticancer and antibiotics drugs. Since most of these drugs had developed resisitance against currently used chemotherapeutics. This study describes the in vitro anti-proliferative, anti-bacterial and single stand DNA break of the holoprasitic plant Cuscuta reflexa. Bioassay-guided fractionation and partial purification of the plant were done and evaluated for antiproliferative activity against human cancer cell lines by SRB assay and single strand DNA break by comet assay. Further antibacterial activity was also performed by agar well diffusion assay. The alcoholic extract, chloroform fraction and partially purified ethylacetate-methanol (1:1) sub-fraction of C. reflexa showed anti-proliferative potential against IMR-32 and 502713 human cancer cell lines. Alcoholic extract exhibited anti-proliferative activity of 74% and 72%, chloroform fraction demonstrated 91% and 95% against neuroblastoma (IMR-32) and colon (502713) cancer cell lines at 100 μg/ml. Single strand DNA break of the chloroform fraction was also demonstrated using comet assay, indicating that possible mode of cell death may be apoptosis. Anti-microbial properties were evaluated against eight species of pathogenic and non-pathogenic microorganisms and maximum zone of inhibition for anti-bacterial activity was found against Staphylococcus aureus (22 mm) by alcoholic extract, 21 mm by chloroform fraction and 12 mm by ethylacetate-methanol (1:1) sub-fraction. Minimum inhibitory concentration (MIC) of the chloroform fraction was 1500 μg/ml for S. aureus. The plant was found to be equally effective against gram-positive and negative bacteria. Studies are well underway to isolate and identify active compounds from chloroform fraction and ethyl acetate:methanol (1:1) sub-fraction, which can be used as effective drug for various diseases.
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International Journal of Green Pharmacy
| October-December 2011 |
307
ORiginAl ARticle
In vitro anti-proliferative, anti-bacterial potential
and induction of DNA strand break of partially
purified Cuscuta reflexa Roxb.
Madhulika Bhagat, Ajit K. Saxena
1
School of Biotechnology, Jammu University,
1
Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Jammu Tawi, India
Cuscuta reflexa is an important medicinal plant, mentioned in Ayurveda, an ancient Indian system of medicine. e plant is selected
to evaluate the possibility for novel pharmaceuticals for anticancer and antibiotics drugs. Since most of these drugs had developed
resisitance against currently used chemotherapeutics. is study describes the in vitro anti-proliferative, anti-bacterial and single stand
DNA break of the holoprasitic plant Cuscuta reflexa. Bioassay-guided fractionation and partial purification of the plant were done
and evaluated for antiproliferative activity against human cancer cell lines by SRB assay and single strand DNA break by comet assay.
Further antibacterial activity was also performed by agar well diffusion assay. e alcoholic extract, chloroform fraction and partially
purified ethylacetate–methanol (1:1) sub-fraction of C. reflexa showed anti-proliferative potential against IMR-32 and 502713 human
cancer cell lines. Alcoholic extract exhibited anti-proliferative activity of 74% and 72%, chloroform fraction demonstrated 91% and
95% against neuroblastoma (IMR-32) and colon (502713) cancer cell lines at 100 µg/ml. Single strand DNA break of the chloroform
fraction was also demonstrated using comet assay, indicating that possible mode of cell death may be apoptosis. Anti-microbial
properties were evaluated against eight species of pathogenic and non-pathogenic microorganisms and maximum zone of inhibition
for anti-bacterial activity was found against Staphylococcus aureus (22 mm) by alcoholic extract, 21 mm by chloroform fraction and
12 mm by ethylacetate–methanol (1:1) sub-fraction. Minimum inhibitory concentration (MIC) of the chloroform fraction was 1500
µg/ml for S. aureus. e plant was found to be equally effective against gram-positive and negative bacteria. Studies are well underway
to isolate and identify active compounds from chloroform fraction and ethyl acetate:methanol (1:1) sub-fraction, which can be used
as effective drug for various diseases.
Key words: Anti-bacterial, anti-proliferation, bioassay-guided fractions, comet assay, Cuscuta reflexa, minimum inhibitory
concentration
Address for correspondence: Dr. Madhulika Bhagat, School of Biotechnology, University of Jammu, Jammu - 180 006, India.
E-mail: madhulikabhagat@rediffmail.com
Received: 12-10-2011; Accepted: 02-01-2012
INTRODUCTION
Natural products and related drugs are used to treat
87% of all categorised human diseases including
cancer, bacterial infection and immunological
disorders.
[1]
Approximately 15 million people die
each year due to infectious diseases nearly all live
in developing countries.
[2]
Chemotherapy is one of
the most widely used approaches for the treatment of
many cancers, but the long-term use of chemotherapy
can lead to drug resistance via several different
mechanisms, such as gene mutation, DNA methylation
and histone modification. Overuse of antibiotics
has become the major factor in the emergence and
dissemination of multi-drug resistant strains of several
groups of microorganisms. Patients are gradually
developing resistance to widely used and standard
chemotherapeutic agents, such as 5-uorouracil, taxol,
doxorubicin, cisplatin, campothecin, paclitaxel and
topotecan. Due to this resistance to cancer drugs, it is
important to nd new anti-cancer and antibiotic agents
in order that they can be developed into novel drugs that
can circumvent the existing resistance mechanisms.
[3]
Since plants possess bioactive substances, which are
safer to use without any side eects, there is need to
screen out medicinal plants for potent anti-cancer and
anti-microbial activities.
[4]
Cuscuta reflexa Roxb. (Family: Convolvulaceae,
Amervel in Hindi). It is a common parasite found in
several part of India and also well known as medicinal
plant reported in Indian system of medicine and used
for various ailments.
[5,6]
The chemical compounds
isolated from the plants are mainly avonoids.
[7,8]
Various studies conducted on the plant reported
for anti-bacterial activity,
[9]
anti-fertility activity
[10]
and anti-oxidant activity.
[11]
Whereas petroleum
ether extract and methanolic extract from stem
part had psychopharmacological effect
[12]
and
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anti-steroidogenic eect on mice.
[13]
The present work
was undertaken to evaluate the anti-proliferative and
anti-bacterial properties of C. reexa and to identify the
cause of cell death.
MATERIALS AND METHODS
Plant Collection
Whole plant of C. reexa Roxb., Family: Convolvulaceae,
was collected from Nagrotra region of Jammu (India) in
the month of December and was authenticated at source
by taxonomist of the institute. A voucher specimen has
been deposited at the herbarium of the Institute vide IIIM
collection No.17148, Acc. No.17719. The authenticated
and freshly collected whole plant was chopped and dried
under shade.
Extraction
Three extracts of the plant material were made with 95%
alcohol, alcohol–water (1:1) and water by using repeated
solvent extraction procedure. Dried powdered plant
material (1 kg) was percolated in 95% ethanol (5 L) at
ambient temperature for 16 h. The solvent was decanted
and the process was repeated four times. The pooled
solvent was evaporated under reduced pressure to yield
alcoholic extract 160 g. Similarly, hydro-alcoholic extract
was prepared. The dried plant material (200 g) was soaked
in alcohol–water (1:1, 1 L) and the extract obtained was
72 g. The dried powdered plant material (200 g) was
heated with distilled water (1.5 L) on steam bath for 2 h
for the preparation of aqueous extract, the supernatant
was decanted and filtered through celite powder and
the process was repeated four times, pooled extract was
concentrated on rotavapour and dried in a lyophilizer, 40
g extract was obtained.
Partial Purication of the Crude Alcoholic Extract of C.
Reexa by Solvent Partitioning with Dierent Polarity
Solvents
The alcoholic extract was fractionated sequentially with
n-hexane, chloroform, n-butanol and water. The dried
alcoholic extract (20 g) was macerated with n-hexane
(4×500 ml). The combined solvent portion was evaporated
under reduced pressure to yield hexane fraction (1.5 g).
The residue was further macerated with chloroform (4×500
ml). The combined organic layer was evaporated under
reduced pressure to yield chloroform fraction (2.25 g). The
residue obtained was dissolved in distilled water (1 L) and
partitioned between n-butanol and water. The process was
repeated four times (4×500 ml); the organic layer was dried
over anhydrous sodium sulphate and concentrated under
reduced pressure to yield n-butanol fraction (8.55 g). The
aqueous part was concentrated under reduced pressure to
give aqueous fraction (6.4 g).
Chromatography
Flash chromatography
The chloroform fraction was sub-fractionated on flash
chromatography using silica gel 230–400 (mesh size).
Elution was carried out using petroleum ether with
increasing concentration of ethyl alcohol and methanol.
Eight fractions were prepared CR-1 (petroleum ether), CR-2
(petroleum ether: ethyl acetate 3:1), CR-3 (petroleum ether:
ethyl acetate 1:1), CR-4 (petroleum ether: ethyl acetate 1:3),
CR-5 (ethyl acetate 100%), CR-6 (ethyl acetate: methanol
3:1), CR-7 (ethyl acetate: methanol 1:1) and CR-8 (methanol
100%) were collected.
Thin layer chromatography
One-dimensional thin layer chromatography (TLC) was
used in order to group the obtained components and to
determine the purity of fractions. A silica gel plate (10 cm in
height) was used as the stationary phase, and the respective
extracts or fractions were spoed at the starting line at
0.5–1 cm intervals. The mobile phase solvents used (one
per TLC plate) were 1:0, 3:1 and 1:1 (v/v) ratio of CH
2
Cl
2
:
hexane. When the mobile phase had almost reached the top
of the plate, the samples were visualised under UV light
(254 nm). Alternatively, the gel plate was sprayed by a 5%
(v/v) H
2
SO
4
/0.03% (w/v) a-naphtal methanolic solution,
dried in an oven or hot plate and visualised under UV
light (350 nm).
Bioassay-guided isolation
Each of the three crude extracts (alcoholic, 50% alcoholic
and aqueous) of C. reexa, obtained as detailed above,
were evaluated for their in vitro anti-proliferative activity
against two selected cancer cell lines, i.e. neuroblastoma
(IMR-32) and colon (502713) using the SRB assay as detailed
below. The extract which provided the best selective
anti-proliferative activity, which is the highest activity
on the cancer cell lines, was selected for further partial
purication by ash chromatography. In the same way,
each fraction obtained from the ash chromatography was
likewise assayed for selective anti-proliferative activity on
the cell lines.
Cell lines and culture
The human cancer cell lines were obtained either from
National Center for Cell Science, Pune, India or National
Cancer Institute, Fredrick, USA. The colon (502713) cancer
cell line was grown and maintained in RPMI-1640 medium,
pH 7.4, whereas DMEM was used for neuroblastoma
(IMR-32) cell line. The media were supplemented with FCS
(10%), penicillin (100 units/ml), streptomycin (100 µg/ml)
and glutamine (2 mM).
Anti-proliferative activity against human tumour lines
The anti-proliferative activity of extracts, fractions and
Bhagat and Saxena: Anti-proliferative, mechanistic and anti-bacterial properties of Cuscuta reexa
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partially purified fractions were determined using
sulforhodamine-B (SRB) as described previously.
[14]
The cells
were grown in tissue culture asks in growth medium at
37°C in an atmosphere of 5% CO
2
and 95% relative humidity
in a CO
2
incubator. The cells at subconuent stage were
harvested from the ask by treatment with trypsin (0.05%
trypsin in PBS containing 0.02% EDTA) and suspended in
the growth medium. Cells with more than 97% viability
(trypan blue exclusion) were used for determination of
cytotoxicity. 100 µl of cell suspension (10
5
to 2×10
5
cells/ml)
was seeded in 96-well tissue culture plate and incubated for
24 h; further test materials (100 µl) were added to the wells
and again incubated for another 48 h. The cell growth was
stopped by adding 50 µl of 50% trichloroacetic acid and
again incubated at 4°C for an hour. The plates were washed
with distilled water and air-dried. Sulforhadamine B (100 µl,
0.4% in 1% acetic acid) was added to each well and plates
were incubated at room temperature for 30 min and washed
with 1% acetic acid. Plates were air dried, tris-HCL buer
(100 µl, 0.01 M, pH 10.4) was added to all the wells and plates
were gently stirred for 5 min on a mechanical stirrer. The
optical density was recorded on ELISA reader at 540 nm.
Suitable blanks and positive controls were also included.
Each test was done in triplicate. The value reported here in
are mean of two experiments.
COMET assay (Single cell gel electrophoresis)
Drug-induced DNA damage was analyzed using the comet
assay with modications.
[15]
Cell pellets (treated with 0,
10, 30 and 100 µg/ml of chloroform fraction for 72 h) were
collected by centrifugation and re-suspended with 200 µl
PBS and 800 µl of 1% low melting point (LMP) agarose. The
mixture was then pipeed onto a frosted glass microscope
slide pre-coated with a layer of 1.0% normal melting
point agarose, prepared in PBS, covered with cover slips
and incubated at 4°C for 10 min. Aer the LMP agarose
solidied, the cover slips were gently removed, then 0.8%
LMP agarose pre-coated cover slips were added and the
slides were allowed to solidify at 4°C for 10 min. Aer
10 min, the cover slips were removed and the cells were
lysed in high salt solution (2.5 M NaCl, 10 mM tris–HCl,
100 mM EDTA, pH 10, with 1% Triton and 10% dimethyl
sulfoxide added fresh) for 1 h. The slides were then placed
in a horizontal electrophoresis unit containing fresh buer
(1 mM EDTA, 300 mM NaOH, pH 13) and incubated
for 20 min to allow unwinding of DNA. Electrophoresis
was then conducted in freshly prepared electrophoresis
buer (pH 13) for 20 min at 25 V and 300 mA (0.8 V/cm)
at 4°C. Subsequently, the slides were gently washed with
neutralization solution (0.4 M tris–HCl, pH 7.5) for 20
min and stained with 20 µl ethidium bromide (15 µg/ml).
Stained nucleoids were scored visually using a uorescence
microscope equipped with a digital camera. 100 comets on
two slides were acquired using the IM50 soware image
analysis system. Tail length was calculated and expressed
in mean±S.E.M.
Microbial Cultures
Eight pathogenic bacteria, Staphylococcus aureus (MTCC
1144), Escherichia coli (MTCC 1089), Bacillus subtilis (MTCC
7164), Bacillus licheniformis (MTCC 7425), Staphylococcus
epidermidis (MTCC 3615), Bacillus brevis (MTCC 7404), Vibrio
cholerae (MTCC 3904) and Pseudomonas aeruginosa (MTCC
1034) used in the study, were obtained from Institute of
Microbial Technology (IMTECH), Chandigarh. Organisms
were maintained on nutrient agar (Hi-Media, India) slopes
at 4°C and sub-cultured before use. Active cultures for
experiments were prepared by transferring a loopful of
cells from stock cultures to test tubes of Muller-Hinton
broth (MHB) that were incubated without agitation for 24
h at 37°C.
Anti-bacterial Assay
In vitro anti-bacterial activities of all extracts, fractions and
sub-fractions of C. reexa were determined by standard agar
well diusion assay.
[16]
Petri dishes (100 mm) containing
25 ml of Mueller–Hinton Agar (Merck) seeded with 100 µl
inoculum of bacterial strain (inoculum size was adjusted so
as to deliver a nal inoculum of approximately 10
6
CFU/ml).
Media were allowed to solidify and then individual petri
dishes were marked for the bacteria inoculated. Wells of
6 mm diameter were cut into solidied agar media with
the help of sterilised cup-borer. 100 µl of each extract was
poured in the respective wells and the plates were incubated
at 37°C for overnight. DMSO and sterilised distilled water
were used as negative control, while kanamycin antibiotic
(1 U strength) was used as positive control. The experiment
was performed in triplicate under strict aseptic conditions
and the anti-bacterial activity of each extract was expressed
in terms of the mean of diameter of zone of inhibition
(in mm) produced by the respective extract at the end of
incubation period.
MIC for the Bacteria
The anti-bacterial activity of the chloroform fraction
was examined by determining the minimum inhibitory
concentration (MIC) in accordance with Clinical and
Laboratory Standard Institute (CLSI) methodology.
[17]
All
tests were performed in Mueller–Hinton broth supplemented
with DMSO at a nal concentration of 10% (v/v) to enhance
their solubility. The chloroform fraction was dissolved in
MHB. Test strains were suspended in MHB to give a nal
density of 5×10
5
CFU/ml and these were conrmed by
viable counts. Dilutions ranging from 100 to 2000 mg/ml of
the fraction was prepared in tubes, including one growth
control, MHB+DMSO 10% (v/v) and one sterility control
MHB+DMSO 10% (v/v+test extracts). The MIC values
initially recorded were from visual examinations as being
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the lowest concentration of the chloroform fraction with
no bacterial growth. Plates were incubated under normal
atmospheric conditions at 37°C for 24 h for bacteria.
RESULTS
In this research, the therapeutic potential of C. reexa was
evaluated against cancer and also for various infective
diseases caused by bacteria. Anti-proliferative activity of
the plant was demonstrated on cell lines derived from
neurobalstoma (IMR-32) and colon (502713) cancer,
whereas anti-bacterial potential was confirmed against
eight pathogenic and non-pathogenic strains of the bacteria.
Additional studies using the comet assay permitted
determination of the role in which apoptosis may play the
role in the anti-cancer activity. Bioassay-guided fractionation
of C. reexa was done and active fraction was further partially
puried using ash choromatography. The anti-proliferative
potential at 10, 30 and 100 µg/ml showed growth inhibition
in a dose-dependent manner against both the cell lines by
all the three extracts [Figure 1]. Among them the aqueous
extract was found to be least eective against both the cell
lines, whereas alcoholic extract showed more pronounced
cell growth inhibition against neuroblastoma cancer cell line
(24, 53 and 74 at 10, 30 and 100 µg/ml, respectively) and also
at colon cancer cell line (18, 64 and 72 at 10, 30 and 100 µg/ml,
respectively), whereas hydro-alcoholic and aqueous extract
had showed activity only at 100 µg/ml.
In case of fractions, it was observed that all the four fractions
of the alcoholic extract had demonstrated strong growth
inhibition in dose-dependent manner in all the cell lines at
10, 30 and 100 µg/ml [Figures 2 and 3] and among all the
four fractions the chloroform fractions was most active than
rest of the fractions (11, 52 and 91 at 10, 30 and 100 µg/ml,
respectively) and aqueous fraction was least effective
for neuroblastoma cancer cell line [Figure 3]. Similarly,
chloroform fraction (14, 92 and 95 at 10, 30 and 100 µg/ml,
respectively) was also most active for colon cancer cell line,
followed by n-butanol and n-hexane fractions, whereas
aqueous extract was least active [Figure 2].
Further partially puried chloroform fraction was also
evaluated for anti-proliferative potential, it was observed
that ethylacetate and methanol (1:1) sub-fraction (CR -7)
was found to be more potential for both the cancer cell
lines [Figure 4]. Petroleum ether: ethyl acetate (1:3) (CR-4)
and ethyl acetate 100% (CR-5) sub-fractions had shown 50
and 54% growth inhibition at 100 µg/ml for neuroblastoma
cell lines and rest of the sub-fractions had shown less than
50% growth inhibition. For colon cancer cell lines rest of the
sub-fractions had shown more than 52% growth inhibition
except ethyl acetate: methanol (3:1) (CR-6), which has shown
least growth inhibition.
Figure 1: In vitro cytotoxicity of the extracts of Cuscuta reflexa against
neuroblastoma and colon human cancer cell lines
Figure 2: In vitro cytotoxicity of the fractions of Cuscuta reexa against colon
(502713) human cancer cell lines
Figure 3: In vitro cytotoxicity of the fractions of Cuscuta reflexa against
neuroblastoma (IMR-32) human cancer cell lines
Figure 4: In vitro cytotoxicity of the sub-fractions of Cuscuta reexa against
neuroblastoma (IMR-32) and colon (502713) human cancer cell lines
Bhagat and Saxena: Anti-proliferative, mechanistic and anti-bacterial properties of Cuscuta reexa
0
20
40
60
80
100
120
100
µg/ml
30
µg/ml
10
µg/ml
100
µg/ml
30
µg/ml
10
µg/ml
1x10
-6
M
2x10
-5
M
Alcoholic extract
Hydro-aqueous extract
Aqueous extract
Adriamycin
5-FU
IMR-32
502713
0
20
40
60
80
100
120
n-hexane cholroform n-butanolaqueous
5-FU
10 µg/ml 30 µg/ml 100 µg/ml (2x10
-5
M)
% Growth inhibition
0
20
40
60
80
100
120
n-hexane cholroform n-butanolaqueous Adriamycin
10 µg/ml 30 µg/ml 100 µg/ml (1x10
-6
M)
% Growth inhibition
0
20
40
60
80
100
120
CR-2
CR-3
CR-4
CR-5
CR-6
CR-7
CR-8
IMR-32 (100 µg/ml) 502713 (100 µg/ml)
Adriamycin (1x10
-6
M) 5-FU (2x10
-5
M)
% Growth inhibition
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In this work, assessment of DNA damage of the cancer cells
was also performed by using comet assay in neuroblastoma
and colon cancer cells. The comet assay is a sensitive
method used to monitor single strand (ss) DNA breaks
at the single-cell level. Any DNA damage is represented
as a tail length (tail migration) of the DNA strand. When
cancer cells were treated with 0, 10, 30 and 100 µg/ml
of the chloroform fraction for 72 h, ssDNA damage was
signicant as indicated by the increased tail length when
compared with the controls [Figure 5]. Moreover, the
DNA damage induced by the chloroform extract was also
dose-dependent.
Anti-infective potential of the plant was demonstrated by
the plant against eight pathogenic and non-pathogenic
strains. Anti-bacterial property of the alcoholic extract
was better than those of hydro-alcoholic and aqueous
extract. Aqueous extract was found almost ineffective
against bacterial strains producing zone of inhibition ≤9
mm. Hydro-alcoholic extract showed zone of inhibition
ranging from 13 to 10 mm, whereas alcoholic extract
showed maximum zone of inhibition of 22–18 mm. It was
found that alcoholic extract was equally inhibitory for
gram-positive bacteria as well as for gram-negative bacteria.
Among all the bacterial strains tested, S. aureus was found
most susceptible with maximum inhibition by alcoholic
extract producing zone of inhibition 22 mm followed by
hydro-alcoholic extract and least activity was observed by
aqueous extract [Table 1]. Further, chloroform fraction of
the active alcoholic extract was comparatively more active
producing zone of inhibition of 21–18 mm, followed by
n-hexane fraction demonstrating zone of inhibition of 19–15
mm, whereas n-butanol and aqueous fraction showed
least anti-bacterial activity following in range of 11–9
mm, respectively [Table 2]. Kanamycin (a positive control)
showed inhibition diameters ranging from ~30 to 32 mm
against all test microorganisms. Control experiments using
sterile distilled water and DMSO (negative control) showed
no inhibition of any bacteria.
Again the anti-bacterial potential of partially puried
sub-fractions of chloroform fraction was evaluated and
it was observed that ethyl acetate and methanol (1:1)
sub-fraction (CR-7) followed by ethyl acetate: methanol
(3:1) sub-fraction was comparatively more active by
producing zone of inhibition of ≤12 mm and ≤10 mm,
whereas rest of the sub-fractions (CR-6) showed very
low activity or were not detectable [Table 3]. Fractions
and sub-fractions obtained aer purication have low
anti-microbial activity and the zone of inhibition was
found to have decreased by extract purication. It was
noted during purication that some compounds are lost,
which may explain the lesser anti-microbial properties
observed with puried sub-fractions than with crude
Table 1: Results of anti-microbial screening of Cuscuta
reexa extracts determined by agar diffusion method
Bacterium name Alcoholic
extract
Hydro-alcoholic
extract
Aqueous
extract
Staphylococcus aureus
22±0.24 13±0.16 8±0.16
Escherichia coli
19±0.22 10±0.16 8±0.14
Bacillus subtilis
18±0.21 11±0.14 9±0.13
Bacillus licheniformis
21±0.24 11 ±0.11 9±0.16
Staphylococcus epidermidis
18±0.20 12±0.13 8±0.16
Bacillus brevis
19±0.22 10±0.13 9±0.16
Vibrio cholera
19±0.22 10±0.10 9±0.14
Pseudomonas aeruginosa
20±0.23 11±0.11 8±0.13
Kanamycin 31±1.5 32±2.2 30±2.1
DMSO NA NA NA
Zone of inhibition (in mm diameter) including the diameter of well (6 mm) in agar well
diffusion assay. In each well, the sample size was 100 µl. Kanamycin (positive control)
1 U strength. DMSO (negative control) 100 µl was used NA (no activity). Data are
represented in the form of mean of three tests±SEM of the standard group
Figure 5: Evaluation of single strand DNA break of the chloroform fraction against
IMR-32 and 502713 human cancer cells
Table 2: Results of anti-microbial screening of Cuscuta
reexa fractions determined by agar diffusion method
Bacterium
name
Alcoholic extract
n-Hexane
fraction
Chloroform
fraction
n-Butanol
fraction
Aqueous
fraction
Staphylococcus
aureus
19±0.20 21±0.15 11±0.16 10±0.15
Escherichia coli
17±0.19 20±0.14 10±0.14 10±0.14
Bacillus subtilis
18±0.20 18±0.11 10±0.13 11±0.13
Bacillus
licheniformis
15±0.21 20±0.12 11±0.16 10±0.16
Staphylococcus
epidermidis
17±0.20 20±0.13 10±0.16 ND
Bacillus brevis
18±0.23 19±0.13 10±0.16 9±0.16
Vibrio cholera
17±0.20 18±0.14 9±0.14 9±0.14
Pseudomonas
aeruginosa
18±0.21 21±0.13 10±0.13 10±0.13
Kanamycin 31±1.3 30±2.2 31±2.2 30±2.0
DMSO NA NA NA NA
Zone of inhibition (in mm diameter) including the diameter of well (6 mm) in agar well
diffusion assay. In each well, the sample size was 100 µl. Kanamycin (positive control) 1
U strength. DMSO (negative control) 100 µl was used. Data are represented in the form
of mean of three tests±SEM of the standard group. NA - No activity; ND - Not detected
Bhagat and Saxena: Anti-proliferative, mechanistic and anti-bacterial properties of Cuscuta reexa
0
10
20
30
40
50
60
70
80
01030100
IMR-32
502713
Concentration of chloroform extract (µg/ml)
Tail length ( µm)
extract, or may suggest the presence of more than one
active compound. MIC against susceptible bacterial species
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by chloroform extract was evaluated [Table 4]. MIC of
chloroform fraction was 1,500 µg/ml against S. aureus,
1000 µg/ml against E. coil, 2000 µg/ml against B. subtilis
and 1200 µg/ml against B. licheniformis, while it was 800
µg/ml against B. brevis and S. epidermidis and 700 µg/ml
against V. cholera and P. aeruginosa. All anti-microbial
activity occurred in a concentration-dependent manner as
suggested by MIC determination. However, the ecacy
of fraction was less than that of the standard antibiotic,
Kanamycin.
DISCUSSION
Above-mentioned results have provided knowledge that a
holoparasite C. reexa might be potential source for producing
anti-cancer and anti-infective drug. These ndings may be
aributed to the nature of biological active compounds
and their strong solubility with appropriate solvent. It is
well documented that alcohols (ethanol, methanol) are
used as a solvent for preparation of plant extract for their
strongly extraction power. Many researchers had already
used methanol or ethanol as a solvent for cytotoxicity,
phytotoxicity, anti-bacterial and anti-tumour activities in
several plant species.
[18-22]
As there might be compounding
eects due to the potential presence of catatonic agents at
high bioactive concentrations mixed in with the desired
bioactivity compounds, we further enriched the extracts.
Solvent partitioning based upon dierent solvent polarities
was used for further purication and the anti-proliferative
[Figures 2 and 3] and anti-bacterial activities [Table 2] of
active compounds and they were likely to be non-polar or
low-polar chemicals in nature. Since high cytotoxicity was
principally observed in the chloroform partitioned fraction,
it was then further fractionated by ash chromatography
with elution based upon solvents of increasing polarity to
obtain seven dierent sub-fractions among them the ethyl
acetate and methanol (1:1) sub-fraction (CR -7) Figure 4
and Table 4 demonstrated signicant activity. Furthermore,
considering the TLC separation paern, more purication
steps that were performed, beer observed separation and
migration of compounds was observed (data not shown).
Anti-cancer activity of the plant primarily was elucidated
by ss DNA break with help of comet assay. Here, the extent
of DNA damage increased proportionately with chloroform
fractions of alcoholic extract of C. reflexa. Overall, the
bioassay-guided isolation appeared suitable in order to
obtain the puried active compounds, as reported before
[23]
and use of non-polar solvents for the preparation of extracts
provides more consistent results as compared to polar
solvents. We know that plants synthesise various bioactive
substances by secondary metabolism to defend themselves
when aacked by bacteria, fungi, parasites, viruses or other
agents.
[24]
These substances have also been shown to be
eective in preventing malignant transformation of cells
in culture and experimentally induced tumourigenesis in
various animal models.
[25]
As reported by various workers,
the presence of phytochemical like cuscutin, cuscutalin and
a large quantity of avonoids in the methanol extract of C.
reexa.
[7,26]
However, considering the results presented here,
it is possible that the anti-proliferation activity and DNA
damage demonstrated by the plant may be derived from
a combination of compounds. Since various avanoids
have been reported to possess cytotoxic and anti-bacterial
eect,
[27,28]
it may be considered the active fractions reported
here may be due to the presence of such compounds. Further
studies are in progress for the purication to homogeneity
and analysis of the chemical structures of each bioactive
component should be performed in order to investigate
which exact compounds in the holoprasitic plant C. reexa
are responsible for the anti-proliferation, anti-bacterial
activity and mechanistic involve in the cell death due to
these compounds so that it can be further design as template
for future drug design.
ACKNOWLEDGMENT
Authors are grateful to National Centre for Cell Science, Pune
Table 3: Results of anti-microbial screening of Cuscuta
reexa sub-fractions determined by agar diffusion
method
Bacterium name CR-7 Sub-fraction CR-6 Sub-fraction
Staphylococcus aureus
12±0.10 10±0.15
Escherichia coli
10±0.11 9±0.10
Bacillus subtilis
11±0.10 8±0.11
Bacillus licheniformis
9±0.9 8±0.9
Staphylococcus epidermidis
9±0.11 8±0.9
Bacillus brevis
8±0.10 ND
Vibrio cholera
8±0.11 8±0.14
Pseudomonas aeruginosa
9±0.10 ND
Kanamycin 31±1.3 30±2.0
DMSO NA NA
Zone of inhibition (in mm diameter) including the diameter of well (6 mm) in agar well
diffusion assay. In each well, the sample size was 100 µl. Kanamycin (positive control)
1 U strength. DMSO (negative control) 100 µl was used. Data are represented in the
form of mean of three tests±SEM of the standard group. NA no activity, ND not detected
Table 4: Results of minimum inhibitory concentration of
chloroform fraction of Cuscuta reexa
Bacterium name Chloroform
fraction
Positive
control
(Broth+TO)
Negative
control
(Broth+CF)
Staphylococcus aureus
1500 G NG
Escherichia coli
1000 G NG
Bacillus subtilis
2000 G NG
Bacillus licheniformis
1200 G NG
Bacillus brevis
800 G NG
Vibrio cholera
700 G NG
Pseudomonas aeruginosa
700 G NG
Staphylococcus epidermidis
800 G NG
TO - Test organisms; CF - Chloroform fraction; G - Growth; NG - No growth
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(India), and National Cancer Institute, Frederick, MD, USA, for
providing human cancer cell line. We are thankful to Dr. B.K.
Kapahi for their help and support.
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Bhagat and Saxena: Anti-proliferative, mechanistic and anti-bacterial properties of Cuscuta reexa
How to cite this article: Bhagat M, Saxena AK. In vitro anti-proliferative,
anti-bacterial potential and induction of DNA strand break of partially puried
Cuscuta reexa Roxb.. Int J Green Pharm 2011;5:307-13.
Source of Support: Nil, Conict of Interest: None declared.
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