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340 Indian Journal of Pharmacology | June 2011 | Vol 43 | Issue 3
Studies on the activity of
Cyperus rotundus
Linn. tubers against
infectious diarrhea
Poonam G. Daswani, Brijesh S., Pundarikakshudu Tetali1, Tannaz J. Birdi
Short Communication
The Foundation for Medical
Research, Worli,
Mumbai, 1Naoroji Godrej Centre
for Plant Research, Lawkin Ltd.
Campus, Shirwal, Satara, India
Received:
Received: 07-10-2010
Revised:
Revised: 24-12-2010
Accepted:
Accepted: 23-02-2011
Correspondence to:
Correspondence to:
Dr. Tannaz J. Birdi,
E-mail: fmr@fmrindia.org
ABSTRACT
To study the antidiarrheal activity of the decoction of
Cyperus rotundus
Linn. tubers using
representative assays of diarrheal pathogenesis and understand its mechanism of action.
Antibacterial, antigiardial and antirotaviral activities were studied. Effect on adherence of
enteropathogenic
Escherichia coli
(EPEC) and invasion of enteroinvasive
E. coli
(EIEC) and
Shigella flexneri
to HEp-2 cells was evaluated as a measure of effect on colonization. Effect
on enterotoxins such as enterotoxigenic
E. coli
(ETEC) heat labile toxin (LT), heat stable toxin
(ST) and cholera toxin (CT) was also assessed. The decoction showed antigiardial activity,
reduced bacterial adherence to and invasion of HEp-2 cells and affected production of CT
and action of LT. The decoction of
C. rotundus
does not have marked antimicrobial activity
and exerts its antidiarrheal action by mechanisms other than direct killing of the pathogen.
KEY WORDS:
KEY WORDS: Bacterial adherence,
Cyperus rotundus
, HEp-2 cells, infectious diarrhea
Introduction
Diarrheal diseases, one of the most common infectious
worldwide, are predicted to remain a leading health problem.[1]
Oral rehydration therapy has been the key strategy for effective
case management. However, it often fails in high stool output
state. With contraindications of antimotility agents in infectious
diarrhea and an increasing threat of drug resistance, various
attempts for developing vaccines against diarrheal pathogens
have been made.[2,3] However, the response to vaccines in
developing countries has not been encouraging.[3] In the recent
past, attempts have been made to treat infectious diarrhea
with supportive therapy such as probiotics; but these are still
under development.[4] Therefore, medicinal plants may provide
a cost-effective alternative for treatment of diarrhea.
Most of the studies on antidiarrheal medicinal plants have
focused on intestinal motility and/or antibacterial activity.[5]
Hence, there is limited information on their mechanism(s)
of action against pathogenicity of infectious diarrhea. In this
study, we have evaluated the effect of crude decoction of
tubers of
Cyperus rotundus
Linn. (family Cyperaceae) on
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DOI:
DOI: 10.4103/0253-7613.81502
various parameters, viz., bacterial adherence to and invasion
of epithelial cells and production and action of enterotoxins, in
addition to their antimicrobial activity.
Materials and Methods
Plant Material
Tubers of
C. rotundus
were collected from the Parinche
valley near Pune, Maharashtra, India, and authenticated by Dr.
P. Tetali. A voucher specimen has been deposited at Botanical
Survey of India (Western Circle), Pune, India, under herbarium
number 124666. Tubers were shade dried and stored at 4°C
until further use. All experiments were performed with the
same dried material.
Preparation of the Crude Aqueous Extract (Decoction)
The decoction was prepared by boiling 1 g of the powdered
dried plant material in 16 mL double-distilled water till the
volume was reduced to 4 mL.[6] To replicate field conditions,
the decoction was freshly prepared every time. To minimize
variability, similar boiling conditions were maintained for each
preparation and the dry weight was recorded. The decoction
was centrifuged and filtered through a membrane of 0.22-μm
pore size before use. The decoction was diluted 1:100, 1:20 and
1:10 in appropriate media for each experiment (referred to as
1%, 5% and 10%, respectively, throughout the text).
Phytochemistry
The qualitative phytochemical analysis of the decoction was
carried out using standard methods.
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341Indian Journal of Pharmacology | June 2011 | Vol 43 | Issue 3
Microorganisms Used
Six bacteria, viz., enteropathogenic
Escherichia coli
(EPEC) strain B170, serotype 0111:NH; enterotoxigenic
E. coli
(ETEC) strain B831-2, serotype unknown (heat labile toxin,
LT, producer) and strain TX1, serotype 078:H12 (heat stable
toxin, ST, producer); enteroinvasive
E. coli
(EIEC) strain E134,
serotype 0136:H-;
Vibrio cholerae
C6709 El Tor Inaba, serotype
01 (cholera toxin, CT, producer) and
Shigella flexneri
M9OT,
serotype 5 were used.
Giardia lamblia
P1 trophozoites and
simian rotavirus SA-11 were also studied.[7,8]
Biological Assays
The assays briefly described below were undertaken using
methods and positive controls described previously.[7,8] Each
experiment was done in duplicate/triplicate and repeated at
least three times.
Antimicrobial Action
The decoction was assessed for its antibacterial, antigiardial
and antirotaviral activities using agar dilution, trypan blue
and neutral red assay, respectively. Ofloxacin (1 μg/mL) and
metronidazole (10 μg/mL) were used as controls for the former
two assays, respectively.
Effect on Bacterial Colonization
Adherence of EPEC and invasion of EIEC and
S. flexneri
to HEp-2 cells was assessed. HEp-2 cells were incubated in
the absence (control) and presence of various dilutions of the
decoction, either 18–20 hours prior to infection (pre-incubation)
or simultaneously with infection (competitive inhibition).
Results were compared with that of lactulose, a prebiotic
oligosaccharide.
Effect on Bacterial Enterotoxins
The production of LT/CT and their binding to ganglioside
monosialic acid receptor were assessed by ganglioside
monosialic acid enzyme linked immunosorbent assay GM1-
ELISA. Results were compared with those of 2-mercaptoethanol
and gallic acid, respectively. The production and action of ST
was assessed by suckling mouse assay.
Approval from the Institutional Ethics Committee and
the Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA, registration No. 424/01/a/
CPCSEA, June 20, 2001) was obtained for the study.
Statistical Analysis
Data were expressed as mean standard error of the
percentage values from three independent experiments. The
percentage in each experiment was calculated using the formula
(C or T)/C 100, where C is the mean value of the duplicate/
triplicate readings of the control group and T is mean value of
the duplicate/triplicate readings of the test (dilutions of the
decoction) groups. Hence, the values of the test groups were
represented as percentages relative to control (100%). Data
were analyzed by analysis of variance (ANOVA) and Dunnett’s
post-test using the software Prism 4.0 (GraphPad, Inc.,
San Diego, CA, USA).
P
≤ 0.05 was considered statistically
significant.
Results
Dry Weight/Yield
The variability between different preparations of the
decoction was within acceptable limits as indicated by the
standard error of the dry weights. The average dry weight was
52 ± 2.8 mg/mL (
n
= 25), the yield being 21 ± 1.12% (w/w)
with respect to the starting material. Thus, the concentrations
of the different dilutions used in the biological assays were
0.52 ± 0.028 mg/mL (1%), 2.6 ± 0.14 mg/mL (5%) and 5.2 ±
0.28 mg/mL (10%).
Phytochemistry
The decoction contained carbohydrates, reducing sugars,
proteins, amino acids, flavonoids, tannins and saponins,
whereas glycosides, alkaloids, and phytosterols were absent.
Antimicrobial Activity
C. rotundus
did not exhibit antibacterial and antirotaviral
activity (data not shown). The multiplication of
G. lamblia
was
restricted at all the concentrations tested in a dose-dependent
manner with maximum inhibition (43.81 ± 2.54%) at 10%
concentration [Figure 1]. However, the decrease was less than
that caused by metronidazole.
Effect on Bacterial Colonization
The adherence of
E. coli
B170 and invasion of
E. coli
E134
and
S. flexneri
to HEp-2 cells was significantly affected when the
HEp-2 cells were incubated with the decoction either prior to or
simultaneously with the infection [Figure 2a–c]. In comparison
to lactulose, the decoction showed greater decrease in bacterial
colonization.
Effect on Bacterial Enterotoxins
The decoction showed an overall increase (statistically
insignificant) in the production of LT by
E. coli
B831-2 at all the
concentrations tested. The binding of LT to GM1 was marginally
decreased at 10% concentration. The decrease, however, was
lesser than that observed with gallic acid [Figure 3a].
Th e decoction significantly decreased CT production by
V.
cholerae
at 5 and 10% concentrations which was comparable
to that of 2-mercaptoethanol. How ever, there was no effect on
the binding of CT to GM1 at any of the concentrations tested
[Figure 3b].
Figure 1: Antigiardial activity of the decoction of C. rotundus [C:
Control, trophozoites in medium alone; M: trophozoites incubated
in medium with metronidazole (10 μg/mL)]. Values represent mean
standard error (n = 3) of percentage viable trophozoites relative to
control (100%); *P < 0.05
Daswani, et al.: Antidiarrhoeal activity of C. rotundus
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342 Indian Journal of Pharmacology | June 2011 | Vol 43 | Issue 3
The production and action of ST was not affected at any
concentration (data not shown).
Discussion
C. rotundus
Linn., commonly known as nut grass and locally
Daswani, et al.: Antidiarrhoeal activity of C. rotundus
Figure 2: Effect of the decoction of C. rotundus on bacterial colonization to HEp-2 cells: (a) Adherence of EPEC strain B170; (b) Invasion of EIEC
strain E134; (c) Invasion of S. fl exneri (C: Control, bacterial adherence/invasion to HEp-2 cells in medium alone; L1: adherence/invasion to HEp-2
cells in medium with 2.5 mg/mL lactulose; L2: adherence to HEp-2 cells in medium with 15 mg/mL lactulose in the competitive protocol). Values
represent mean ± standard error (n = 3) of percentage adherence/invasion relative to respective control (100%); *P < 0.05
as Musta, is said to possess antidiarrheal, anti-inflammatory
and antipyretic activities.[9,10] The tubers are used in Ayurvedic
medicine and have been mentioned in ancient texts for various
ailments.
Our previous study conducted on
C. rotundus
tubers
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343Indian Journal of Pharmacology | June 2011 | Vol 43 | Issue 3
Figure 3: Effect of the decoction of C. rotundus on bacterial enterotoxins: (a) Production of heat labile toxin (LT)
by E. coli B831-2 and its binding to GM1; (b) Production of cholera toxin (CT) by V. cholerae and its binding to
GM1 (C: Control, toxin in medium alone; M1: LT in medium with 5 mM 2-mercaptoethanol; M2: CT in medium with
1 mM 2-mercaptoethanol; G: LT/CT in medium with 50 mM gallic acid). Values represent mean standard error
(n = 3) of percentage production/binding relative to respective control (100%); *P < 0.05
collected from Madhya Pradesh, India, had reported selective
action against ETECs with no direct killing of bacteria.[11] We
expanded the study with this plant to include multiple pathogens
and parameters. In the present study, tubers from Parinche,
Maharashtra, were used. Though the antibacterial profile of
the two batches was similar, differential effects were seen on
adherence of EPEC, LT and ST. Despite the mode of extract
preparation being similar, the difference in the results of the
two studies can be attributed to different ecotypes and the
time of collection.
Some other studies have also reported antidiarrheal activity
of
C. rotundus
. Antidiarrheal action in castor oil-induced
diarrhea and in irritable bowel syndrome in animal models
has been demonstrated.[12,13] However, there is a dearth of
information regarding its mechanism(s) of action in controlling
infectious diarrhea. We, therefore, undertook the present study.
The present study was intentionally restricted to crude
extract as it is our belief that the different biological activities
may not be due to a single constituent. This has also been
highlighted in recent studies on
Psidium guajava
and
Alchornea
cordifolia
.[8,14] Previous studies with the essential oil of
C.
rotundus
showed it to be more bactericidal against Gram-
positive bacteria.[15,16] In this study, however,
C. rotundus
showed
no antibacterial activity which could be due to a difference in
the extract used and/or a difference in the test strains, all the
strains being Gram negative. The major constituents present in
C. rotundus
are essential oil, triterpenes, polyphenol, alkaloids
and flavonoids. However, none of these have been attributed
with antidiarrheal activity.[10] The decoction used herein showed
the presence of carbohydrates, reducing sugars, proteins,
amino acids, tannins, flavonoids and saponins. Tannins and
flavonoids, in general, have been reported to have antidiarrheal
activity.[17-19] Thus, these compounds may be responsible for the
observed activity. However, it may be noted that since tannins
and flavonoids have not been studied for their activities
vis-à-
vis
colonization of enteric pathogens to the gut epithelium and/
or production and action of enterotoxins, further investigations
with isolated constituents are necessary.
The results show that
C. rotundus
has limited antimicrobial
action. Since bacterial colonization was reduced when the
Daswani, et al.: Antidiarrhoeal activity of C. rotundus
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344 Indian Journal of Pharmacology | June 2011 | Vol 43 | Issue 3
HEp-2 cells were incubated with the decoction prior to and
simultaneously with infection, it is likely that
C. rotundus
affects
metabolism of HEp-2 cells and/or modifies its receptors to
prevent bacterial adherence/entry. Since the decoction did not
kill
V. cholerae,
the suppression of CT production could be due to
its effect on bacterial metabolism. LT production was, however,
not affected. Since LT and CT have antigenic similarities, the
differential effect on binding of these toxins (inhibiting the
binding of only LT) suggests that the decoction may not be
affecting the common antigenic moiety of these toxins.[20]
To conclude, the results suggest that
C. rotundus
has limited
activity against different forms of infectious diarrhea due to its
selective activity against diarrheal pathogens. In the absence
of a marked antimicrobial activity, this plant seems to exhibit
the antidiarrheal action because of its action on some features
of bacterial virulence viz., bacterial colonization, production of
CT and action of LT.
Acknowledgements
We are thankful to Aviansh Gurav, Santosh Jangam of Foundation
for Research in Community Health, Pune for collection of plant material;
Dr. N. F. Mistry, Foundation for Medical Research for her critical
suggestions in the study design; staff and students, Pharmacognosy
Department, Principal KM Kundanani College of Pharmacy, Mumbai
for assistance in qualitative phytochemical studies.
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Cite this article as: Daswani PG, Brijesh S, Tetali P, Birdi TJ. Studies on the
activity of Cyperus rotundus Linn. tubers against infectious diarrhea. Indian J
Pharmacol 2011;43:340-4.
Source of Support: Grant No. 91283, Department of Science and
Technology, Ministry of Science and Technology, Government of
India. Confl ict of Interest: None declared.
Daswani, et al.: Antidiarrhoeal activity of C. rotundus
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