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Original
Research Paper
I
ntroduction
Herbal plants are being used as medicine from
ancient age and usefulness of them are recorded
in human history. Herbal plants are reported to be
excellent source of several nutrients
[1]
. The use of
herbal drugs in treatment of diseases is found
among all sections of people in India.
The plant Pistia stratiotes, commonly known as
water cabbage or water lettuce, belongs to the
family Araceae, is an edible, aquatic, floating
ornamental plant with widely distributed across
tropical and sub-tropical areas around the world.
The plant leaves are light green, obovate with
prominent longitudinal veins at its base
[2]
. P.
stratiotes is widely distributed and is being loathed
in Asia and Africa. Many organic compounds,
metabolites and pigments like vicenin and lucenin
have been isolated from P. stratiotes
[3]
. This plant
and its extracts are potentially believed to have
medicinal effects. P. stratiotes leaves are
traditionally used against ringworm infection of
scalp, boils and syphilitic eruptions. Traditionally, oil
extracts of this plant are used for treatment of
tuberculosis, asthma and dysentery
[4]
.
Water lettuce is proved to be superior to most
other plants in nutrient removal efficiency in the
environmental cleanup, owing to its rapid growth
and high biomass yield potential
[5]
. Water lettuce
appears to be one of the most effective aquatic
plants for the bioremediation of mercury and
cadmium
[6]
. This plant is proven to be antiseptic,
antitubercular and antidysentric. In various parts
of the world it is also used as anodyne for
eyewash. The leaves are used in eczema, leprosy,
ulcers and piles
[4]
. The plant is bitter, pungent
flavor, having cooling, laxative property. It is used
in ‘Tridosha’ fever and diseases of blood. The root
is laxative, emollient and has diuretic effect. Leaf
Copyright © 2014 By IYPF
All rights reserved
Open Access Contents
Int. J. Drug Dev. & Res.
| October - December 2014 | Vol. 6 | Issue 4 | ISSN 0975-9344 |
Cytotoxicity and Antimicrobial effects of Pistia stratiotes Leaves
www.ijddr.in
Jayanthi Abraham*
Pritha Chakraborty
Anish Mathew Chacko
Kaustubh Khare
Microbial Biotechnology Lab,
School of Biosciences and
Technology, VIT University,
Vellore 632014, Tamil Nadu,
India.
Corresponding Authors:
Email:
jayanthi.abraham@gmail.com
A
bstract:
Pistia stratiotes belongs to family Araceae. Pistia stratiotes
leaves are traditionally
used against ringworm infection of scalp, boils and syphilitic eruptions.
Traditionally, oil extracts of Pistia stratiotes a
re used for treatment of tuberculosis,
asthma and dysentery. As the effect of Pistia stratiotes
extracts on the area of
cytotoxicity is unexplored, the present work was aimed at evaluating the effect of
extracts on cancer cell line and also on clinical pat
hogens. Phytochemical study
was done with methanol extract of Pistia stratiotes leaves. Analytical study (GC-
MS
and TLC) was done with methanol, chloroform and hexane extracts of
Pistia
stratiotes
leaves. Antimicrobial activity by well diffusion method and
antioxidant
activity by DPPH assay were also done. Antimicrobial study was done against
eight clinical pathogens namely, Proteus mirabilis, Enterococcus sp.
, Salmonella
sp., Shigella sp., Staphylococcus aureus, Serretia sp., Klebsiella sp.
, Pseudomonas
aeuroginosa
. As bone cancer is an aggressive form of cancer MG63 cell line of
osteocarcoma was choosen for the study.
K
eywords:
Araceae, antimicrobial activity, antioxidant activity, osteocarcoma,
MG 63 cell line, anticancer activity, pathogens, TLC, Gas Ch
romatogrphy, Mass
Spectroscopy.
Page 208
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© 2014 Jayanthi Abraham et al, publ
isher and licensee IYPF. This is an Open Access article which permits unrestricted
noncommercial use, provided the original work is properly cited.
infusions have been mentioned in the folklore to
be used for dropsy, bladder complaints, kidney
afflictions, hematuria, dysentery and anemia
[7]
.
The studies over antioxidant effects on the cancer
cell line cultures are not much established. This
study is undertaken to evaluate the antimicrobial,
anticancer and antioxidant scavenging effects of
Pistia stratiotes leaf extracts by using experimental
approach.
M
aterials and methods
Chemicals
All chemicals and solvents used in this study were
of high purity and analytical grade.
Sample extraction
The Pistia stratiotes leaves were collected dried
and powdered. Powdered sample were
extracted with methanol, hexane, chloroform
using soxhlet apparatus. The extracted solvent is
later collected, dried and stored for further
studies.
Phytochemical studies
Phytochemical studies were done with methanol
extract of plant leaves to primarily detect the
presence of various compounds.
Detection of alkaloids
Solvent free extract, 5 mg was stirred with few ml
of diluted hydrochloric acid and filtered. The
filtrate was tested carefully with various alkaloidal
reagents
[8]
.
A. Hager’s Test: Filtrates were treated with Hager’s
reagent (saturated picric acid solution). Presence
of alkaloids was confirmed by the formation of
yellow coloured precipitate.
B. Wagner’s Test: To few ml of filtrate, few drops of
Wagner’s reagent were added along the side of
test tube. A reddish brown precipitates indicated
positive test.
[9]
.
Wagner’s reagent: iodine (1.27g) and potassium
iodide (0.92 g) was dissolved in 5 ml of water and
made up to 100 ml with distilled water.
Detection of carbohydrates and glycosides
5 mg of extract was dissolved in 5 ml of water and
filtered. The filtrate was subjected to the following
tests.
[10]
A. Fehling’s Test: 1 ml of filtrate was boiled on
water bath with 1 ml of each of Fehling’s solutions
A and B. Appearance of red precipitate
confirmed the presence of sugar.
Fehling’s solution A: Copper sulphate (34.66 g)
was dissolved in distilled water and made up to
500 ml using distilled water.
Fehling’s solution B: Potassium sodium tartarate
(173 g) and sodium hydroxide (50g) was dissolved
in water and made up to 500 ml.
B. Molish Test: to 2 ml of filtrate, two drops of
alcoholic solution of α napthol were added, the
mixture was shaken well and 1 ml of conc.
sulphuric acid was added slowly along the sides of
test tube and allowed to stand. Formation of
violet ring indicated the presence of
carbohydrates.
Detection of phytosterols
Libermann Burchard’s Test: The extract (5 mg) was
dissolved in 2 ml acetic anhydride. To this, one or
two drops of conc. sulphuric acid were added
slowly along the sides of the test tube. An array of
colour changes indicated the presence of
phytosterols
[11]
.
Detection of phenolic compounds
Ferric chloride test: The extract (2 mg) was
dissolved in 5 ml of distilled water. To this, few
drops of neutral 5% ferric chloride solution was
added. Appearance of green colour indicates
the presence of phenolic compounds
[12]
.
Jayanthi Abraham et al; Cytotoxicity and Antimicrobial effects of Pistia stratiotes Leaves
Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., October-December 2014, 6 (4): 208-217
© 2014 Jayanthi Abraham et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted
noncommercial use, provided the original work is properly cited.
Page 209
Detection of flavonoids
Alkaline Reagent Test: Extracts were treated with
few drops of sodium hydroxide solution. Formation
of intense yellow colour, which becomes
colourless on addition of dilute acid, which
indicated the presence of flavonoids.
Lead acetate Test: To 1ml of the plant extract
was added in a test tube. To this 1ml of 5% lead
acetate and the mixture was allowed to stand for
few minutes. The formation of precipitates in
samples confirmed the presence of flavonoids.
Detection of Tannins
About 0.5 g of extract was stirred with about 10 ml
of distilled water and then filtered. Few drops of
1% ferric chloride solution were added to 2 ml of
the filtrate. Formation of a bluish black, bluish
green or green precipitate confirmed the
presence of tannins.
Detection of terpenoids (Salkowski test)
2 ml of chloroform of chloroform was added to
the extract. Concentrated H
2
SO
4
(3 ml) was
carefully added to form a layer. Appearance of
reddish brown colouration on the interface
indicates the presence of terpenoids.
A
nalytical methods
Fractionation of the crude extract using TLC
Using pre-coated TLC F254 plates, the crude
extract was fractionated using different
combinations of hexane/chloroform/methanol
solvents (2:1:1) and methanol/hexane (3:2) as the
mobile phase. Separated components were
viewed in visible light, under UV at 360 nm, by
fluorescence quenching less than 254 nm.
Separation done with mobile phase of hexane:
chloroform: methanol was fractionated and
recorded.
Gas Chromatography Mass Spectrometry
Methanol and hexane extract of Pistia stratiotes
leaves were analyzed by GC-MS. Perkin Elmer
Clarus 680 gas chromatographic instrument
equipped with a mass spectrometer detector
(Clarus 600 model) and an Elite-5MS (30.0 m, 0.25
mmID, 250 µm df) column was used. The carrier
gas used was helium at a flow rate of 1 ml min
-1
.
The following temperature program was used:
initially the oven temperature was held at 60°C for
2 min and then ramped from 10°C/min to 300°C
with hold time for 4 min, total run time 30 min. The
temperature of the injector was maintained at
300°C. The ion trap was operated at 70 eV with a
scan range of m/z from 50 to 600. A sample of 1 µl
was injected in split mode (10:1). The intermediate
and end product was identified based on the
Wiley registry of mass spectral data.
Antimicrobial study
The antibacterial activity of three different
extracts (methanol, chloroform and hexane) of
Pistia stratiotes plant leaves against eight bacterial
pathogens was evaluated by using agar well
diffusion method
[13,14]
. Muller Hinton Agar (MHA)
plates were inoculated with selected bacterium.
Wells of 8 mm size were made with sterile borer on
agar plates. Four different volumes (25 µl, 50µl, 75
µl, 100 µl) of the plant extract were poured into
each well of inoculated plates. Respective solvent
for particular solvent extracts was used as a
negative control. Then they were left at room
temperature for ten minutes allowing the diffusion
of the plant extract into the agar
[15]
. After
incubation for 24 hrs at 37
o
C, the plates were
observed for clear zone. Antibacterial activity of
the extract was identified by an inhibition zone
surrounding the well containing the plant extract.
The zone of inhibition was measured and
expressed in millimeters (mm). Clear zone of the
Page 210
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Original
Research Paper
Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., October-December 2014, 6 (4): 208-217
© 2014 Jayanthi Abraham
et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted
noncommercial use, provided the original work is properly cited.
plant extract and comparison with negative
control was recorded
[16]
.
Antioxidant Study
The antioxidant activity of the methanol,
chloroforn and hexane extracts was evaluated by
DPPH radical scavenging assay which was
originally described by Blois
[17]
. DPPH (2, 2-
diphenyl-1-picrylhydrazyl) is a synthetic free
radical with deep violet colour when is in form of
solution which has a λ
max
at 517nm. It can accept
an electron or hydrogen radical to become
stable diamagnetic molecule and appear as light
purple in colour which indicates the scavenging
of DPPH and the substance has antioxidant
activity.
Methanol solutions were prepared with all the
three extracts. Methanol solution of DPPH was
used as negative control. 500µl of each sample
and 500 µl of DPPH solution was allowed to react
and incubated at room temperature for 30mins
under dark conditions. Absorbance was taken at
λ
max
i.e. 517nm against a blank which was 500µl of
methanol. Percentage inhibition was calculated
by the following equation to conclude the
presence of antioxidant activity of the extracts.
. Percentage of inhibition = (OD control – OD
sample / OD control) x 100
Anticancer study
Cell line:
The human osteosarcoma cell line (MG 63) was
obtained from National Centre for Cell Science
(NCCS), Pune and grown in Eagles Minimum
Essential Medium containing 10% fetal bovine
serum (FBS). The cells were maintained at 37
°
C, 5%
CO
2
, 95% air and 100% relative humidity.
Maintenance cultures were passaged weekly,
and the culture medium was changed twice a
week.
Cell treatment procedure:
The monolayer cells were detached with trypsin-
ethylenediaminetetraacetic acid (EDTA) to make
single cell suspensions and viable cells were
counted using a hemocytometer and diluted with
medium containing 5% FBS to give final density of
1x10
5
cells/ml. One hundred microlitres per well of
cell suspension were seeded into 96-well plates at
plating density of 10,000 cells/well and incubated
to allow for cell attachment at 37
°
C, 5% CO
2
, 95%
air and 100% relative humidity. After 24 h the cells
were treated with serial concentrations of the test
samples. They were initially dissolved in neat
dimethylsulfoxide (DMSO) and an aliquot of the
sample solution was diluted to twice the desired
final maximum test concentration with serum free
medium. Additional four serial dilutions were
made to provide a total of five sample
concentrations. Aliquots of 100 µl of these different
sample dilutions were added to the appropriate
wells already containing 100 µl of medium,
resulting in the required final sample
concentrations. Following sample addition, the
plates were incubated for an additional 48 h at
37
°
C, 5% CO
2
, 95% air and 100% relative humidity.
The medium containing without samples were
served as control and triplicate was maintained
for all concentrations.
MTT assay:
After 48 h of incubation, 15µl of MTT (5mg/ml) in
phosphate buffered saline (PBS) was added to
each well and incubated at 37
°
C for 4h. The
medium with MTT was then flicked off and the
formed formazan crystals were solubilized in 100µl
of DMSO and then measured the absorbance at
570 nm using micro plate reader.
The percentage of cell viability was then
calculated with respect to control as follows
Percentage of cell viability = [A] Test / [A]control x
100
Jayanthi Abraham et al; Cytotoxicity and Antimicrobial effects of Pistia stratiotes Leaves
Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., October-December 2014, 6 (4): 208-217
© 2014 Jayanthi Abraham et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted
noncommercial use, provided the original work is properly cited.
Page 211
The percentage of cell inhibition was determined
using the following formula.
Percentage of cell inhibition = 100- Abs
(sample)/Abs (control) x100.
Nonlinear regression graph was plotted between
percentage of cell inhibition and Log
concentration and IC50 was determined using
GraphPad Prism software.
R
esults
Phytochemical study
In recent years, secondary plant metabolites
extensively investigated as a source of medicinal
agents. It has been accepted that natural
compounds play an important role in health care.
Water cabbage leaves are source of various
bioactive compounds. Result of phytochemical
studies of plant extract with methanol is presented
in a table form in table 1. According to the result,
alkaloids, phytosterols, phenols, flavonids and
tannins are present in Pistia stratiotes leaves.
However Fehling’s and Molish test for
carbohydrate and Salkowski test for terpenoids
showed negative response. According to Singh et
al.
[18]
phenolic compounds possess biological
properties such as antiapoptosis, antiaging,
anticarcinogen, antiinflammation, as well as
inhibition of angiogenesis and cell proliferation
activities. Phytosterol acts as growth hormones in
plants. The plant has medicinal property due to
presence of these phytochemicals
[19]
.
Analytical studies
TLC
TLC was done to fractionate each components of
the extract by its characteristic Rf values.
Separated spots were observed under UV light
(fig.1). Two separated spots have been observed
under UV light for methanol and chloroform
extract of Pistia stratiotes leaves. But no separation
was observed for hexane extract. Separated spots
on TLC plates indicate presence of different
compounds which were further analyzed by GC-
MS.
Fig 1: Separated spots on TLC plates visualized
under UV light; A: methanol extract, B: chloroform
extract, C: hexane extract.
GC-MS
In order to determine the compounds present in
the extract of Pistia stratiotes leaves, GC-MS
analysis was done. This analysis revealed that
methanol, chloroform and hexane extract of P.
stratiotes leaves contain different compounds.
Some of them are known for their biological
activity whereas activity of a few compounds
remains unknown. The GC-MS chromatogram of
methanol, chloroform and hexane extracts are
presented in fig 2, fig 3, and fig 4 respectively.
From GC-MS analysis, it has been seen that methyl
17-methyl-octadecanoate is present in chloroform
extract of P. stratiotes leaves, which is reported to
possess antimicrobial, antioxidant and antitumor
activity
[20]
. N-hexadecanoic acid (palmitic acid),
found in methanol and hexane extract of Pistia
Page 212
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noncommercial use, provided the original work is properly cited.
stratiotes leaves which posses antibacterial and
cholesterolaemic effects, selective toxicity to
human leukemic cells. It also has shown in
vivo antitumor activity in mice by making a target
to DNA topoisomerase I
[21,22]
. Whereas activity of
Z,Z-6,28-hepatatriactontadien-2-one present in
both methanol and hexane extract remained
unknown. Activities of these compounds are
believed to be responsible for the activities shown
by P. stratiotes leaves extracts.
Antimicrobial Test
Antimicrobial activity of P. stratiotes leaves was
checked against eight clinical pathogens.
Antimicrobial activity was determined by
measuring zone of inhibition formed after
incubation period. No inhibition zone was
observed for chloroform and hexane extracts
against eight test pathogens. The methanol
extract showed characteristic zone of inhibition
against five pathogens including Pseudomonas
aeruginosa, Shigella sp., Serretia sp., Salmonella
sp. and Klebsiella sp. among eight test pathogens.
The zone of inhibition was very pronounced.
Highest zone of inhibition was observed at
100mg/ml concentration against Klebsiella sp.
Methanol extract of P. stratiotes leaves showed
antimicrobial activity against both Gram positive
and Gram negative
[23]
. Table 2 indicates the
result of the antimicrobial activities of methanol
extract of P. stratiotes leaves.
Antioxidant Test
The antioxidant activity of the extracts was
evaluated by DPPH radical scavenging assay
which was originally described by Blois
[17]
. DPPH (2,
2-diphenyl-1-picrylhydrazyl) is a synthetic free
radical with deep violet colour when is in form of
solution which has a λ
max
at 517nm. Among
methanol, hexane and chloroform extract of Pistia
stratiotes leaves, methanol extract has shown
highest antioxidant activity where hexane and
chloroform extract has shown moderate activity
(table 3). The reducing power of methanol extract
indicates presence of some compounds in Pistia
stratiotes extracts which can donate electron and
could react with free radicals to convert them into
more stable products and to terminate radical
chain reactions. Increased absorbance of
reaction mixture indicates increased reducing
power of the extract.
[24]
Anticancer study
Anticancer study of the Pistia stratiotes leaves was
done against human osteosarcoma cell line (MG
63). Anticancer activity of methanol and
chloroform extract was checked. The activity of
methanol and chloroform extract was shown in fig
5 and fig 6 respectively. In methanol extract cell
viability is decreased with increased
concentration of extract, which indicates the
moderate activity of the extract. On the other
hand,1.0 µg/ml concentration of extract showed
less than 20% of cell inhibition while at 2.5 µg/ml
concentration of extract, percentage of cell
inhibition reaches 60%, which indicates that
percentage of cell inhibition is increased with
increasing concentration of chloroform extract
[25]
.
It indicates the presence of anticancer activity of
both methanol and hexane extract of P. stratiotes
leaves.
D
iscussion
From the above study, the plant Pistia stratiotes, is
proven to have good antimicrobial, antioxidant
and anticancer activities. As the above result are
very promising and encouraging to continue
research on Pistia stratiotes leaves and there is a
need to purify the compounds to enhance their
Jayanthi Abraham et al; Cytotoxicity and Antimicrobial effects of Pistia stratiotes Leaves
Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., October-December 2014, 6 (4): 208-217
© 2014 Jayanthi Abraham et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted
noncommercial use, provided the original work is properly cited.
Page 213
activity and make them suitable for further clinical
approach in near future.
Table 1: Phytochemical properties of methanol
extract of Pistia stratiotes leaves.
Sl. No.
Phytochemical test Result
1 Alkaloids
Hager’s test +
Wagner’s test +
2 Carbohydrates
Fehling’s test -
Molish Test -
3 Phytosterols
Libermann Burchard’s Test +
4 Phenols
Ferric chloride test +
5 Flavonoids
Alkaline Reagent Test -
Lead acetate Test: +
6 Tannin +
Terpenoids
7 Salkowski test -
Table 2: Zone of inhibition of methanol extract of
Pistia stratiotes leaves against eight pathogens.
Sl
no
Organisms Zone of inhibition (cm)
25mg/ml
50mg/ml
75mg/ml
100mg/ml
1 Pseudomonas
aeruginosa 0.9 1.0 1.2 1.3
2 Shigella sp. - 0.7 1.0 1.3
3 Serretia sp. - 1.0 1.3 1.5
4 Salmonella sp. - 1.0 1.3
5 Klebsiella sp. 1.0 1.2 1.3 1.5
6 Enterobacter
sp. - - - -
7 Proteus mirabilis
- - - -
8 Staphylococcus
sp. - - - -
Table 3: Percentage of inhibition of different
extracts of Pistia stratiotes leaves.
Sample OD Sample At
517nm
Percentage Of
Inhibition
Standard 0.823
Methanol
extract 0.445 45%
Chloroform
extract 0.483 34%
Hexane extract
0.740 10%
Fig. 2: GC-MS chromatogram of Pistia stratiotes leaves methanol extract.
Page 214
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Research Paper
Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., October-December 2014, 6 (4): 208-217
© 2014 Jayanthi Abraham
et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted
noncommercial use, provided the original work is properly cited.
Fig 3: GC-MS chromatogram of Pistia stratiotes leaves chloroform extract.
Fig 4: GC-MS chromatogram of Pistia stratiotes leaves hexane extract.
Fig 5: Anticancer activity of methanol extract of Pistia stratiotes leaves
Jayanthi Abraham et al; Cytotoxicity and Antimicrobial effects of Pistia stratiotes Leaves
Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., October-December 2014, 6 (4): 208-217
© 2014 Jayanthi Abraham et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted
noncommercial use, provided the original work is properly cited.
Page 215
Fig 6: Anticancer activity of chloroform extract of Pistia stratiotes leaves
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Article History: ------------------------
Date of Submission: 16-12-2014
Date of Acceptance: 29-12-2014
Conflict of Interest: NIL
Source of Support: NONE
Jayanthi Abraham et al; Cytotoxicity and Antimicrobial effects of Pistia stratiotes Leaves
Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., October-December 2014, 6 (4): 208-217
© 2014 Jayanthi Abraham et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted
noncommercial use, provided the original work is properly cited.
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