Study on effect of anti-diarrheal medicinal plants on enteropathogenic Escherichia coli induced interleukin-8 secretion by intestinal epithelial cells

Abstract

Diarrhea is a major health concern in developing countries with enteropathogenic Escherichia coli (EPEC) being a leading cause of infantile diarrhea. Much of the pathology of EPEC infection is due to the inflammatory responses of infected intestinal epithelium through secretion of pro-inflammatory cytoki - nes such as interleukin (IL)-8. With medicinal plants gaining popularity as prospective antidiarrheal agents, we aimed to evaluate the effect of anti-diarrheal medicinal plants on secretion of IL-8 by epithelial cells in response to EPEC infection. The effect of the decoctions of four anti-diarrheal medicinal plants viz. Aegle marmelos , Cyperus rotundus , Psidium guajava and Zingiber officinale was studied on secretion of IL-8 by a human colon adenocarcinoma cell line, HT-29 infected with E. coli E2348/69. Two protocols were used viz. pre-incubation and post-incubation. The data obtained demonstrated that out of the four plants used, only P. guajava decreased secretion of IL-8 in the post-incubation protocol although in the pre-incubation protocol an increase was observed. A similar increase was seen with C. rotundus in the preincubation protocol. No effect on IL-8 secretion was observed with A. marmelos and Z. officinale in both protocols and with C. rotundus in the post-incubation protocol. The post-incubation protocol, in terms of clinical relevance, indicates the effect of the plant decoctions when used as treatment. Hence P. guajava may be effective in controlling the acute inflammatory response of the intestinal epithelial cells in response to EPEC infection.
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[page 64] [Alternative Medicine Studies 2011; 1:e16]
Study of effect of
anti-diarrheal medicinal plants
on enteropathogenic
Escherichia coli
induced
interleukin-8 secretion by
intestinal epithelial cells
Brijesh S.,1Pundarikakshudu Tetali,2
Tannaz J. Birdi1
1The Foundation for Medical Research,
84A, RG Thadani Marg, Worli, Mumbai,
2Naoroji Godrej Centre for Plant
Research, Lawkin Ltd. Campus,
Shindewadi, Shirwal, Satara, India
Abstract
Diarrhea is a major health concern in devel-
oping countries with enteropathogenic
Escherichia coli (EPEC) being a leading cause
of infantile diarrhea. Much of the pathology of
EPEC infection is due to the inflammatory
responses of infected intestinal epithelium
through secretion of pro-inflammatory cytoki -
nes such as interleukin (IL)-8. With medicinal
plants gaining popularity as prospective
antidiarrheal agents, we aimed to evaluate the
effect of anti-diarrheal medicinal plants on
secretion of IL-8 by epithelial cells in response
to EPEC infection. The effect of the decoctions
of four anti-diarrheal medicinal plants viz. Aegle
marmelos, Cyperus rotundus, Psidium guajava
and Zingiber officinale was studied on secretion
of IL-8 by a human colon adenocarcinoma cell
line, HT-29 infected with E. coli E2348/69. Two
protocols were used viz. pre-incubation and
post-incubation. The data obtained demonstrat-
ed that out of the four plants used, only P. guaja-
va decreased secretion of IL-8 in the post-incu-
bation protocol although in the pre-incubation
protocol an increase was observed. A similar
increase was seen with C. rotundus in the pre-
incubation protocol. No effect on IL-8 secretion
was observed with A. marmelos and Z. officinale
in both protocols and with C. rotundus in the
post-incubation protocol. The post-incubation
protocol, in terms of clinical relevance, indicates
the effect of the plant decoctions when used as
treatment. Hence P. guajava may be effective in
controlling the acute inflammatory response of
the intestinal epithelial cells in response to
EPEC infection.
Introduction
Diarrheal diseases are a major health con-
cern in developing countries with an estimated
1.8 million deaths per annum.1Despite improve-
ments in public health and economic well being,
it remains an important clinical problem in
developed countries as well.2It is estimated that
infectious diarrhea will remain a cause of global
health concern in the next 2-3 decades.3
Diarrhea is an etiologically diverse condi-
tion caused by a variety of enteric pathogens.4
E. coli is recognized to be a common cause of
gastroenteritis and accounts for nearly 30% of
total diarrheal pathogens in some regions.5
There are seven different categories of diar-
rheagenic E. coli strains based on epidemiolo-
gy, clinical syndromes, and virulence proper-
ties.6The enteropathogenic E. coli (EPEC) is
an important category that is a leading cause
of infantile diarrhea in developing countries.7
EPEC colonize the intestinal epithelial sur-
face and cause histopathological mucosal
changes, the physiological end results of which
are disruption of the intestinal epithelial barri-
er, alterations in intestinal transport, and
inflammation.8Much of the pathology of EPEC
infection has been linked to inflammatory
responses by the infected epithelium which
occurs through activation of the nuclear tran-
scription factor NF-κB. Since EPEC is a non-
invasive pathogen, NF-κB is activated by solu-
ble factors secreted or shed by the pathogen or
translocated type three secretory system-
dependent effectors.9Activation of NF-κB in
turn promotes the expression of proinflamma-
tory cytokines such as interleukin (IL)-810,11
which result in substantial recruitment of neu-
trophils and other PMNs to the site of in vivo
infection.12 Although the inflammatory
response is not the mechanism that initiates
EPEC mediated diarrhea, it may contribute to
the duration and severity of the diarrheal
response.8The PMNs and other inflammatory
cells recruited to the infected intestine also
cause considerable tissue damage through the
release of toxic inflammatory mediators.13
In recent years, medicinal plants have
gained popularity as prospective antidiarrheal
agents, with a large numbers of studies being
published in the past decade.14 Whilst a few
studies have reported antimicrobial activity, a
majority have focused on physiological diar-
rhea and thereby reporting their effect on
intestinal motility in experimental models.
Hence, we studied the activity of selected
antidiarrheal medicinal plants against patho-
genicity of infectious diarrhea, including
parameters such as colonization of epithelial
cells and production and action of enterotox-
ins.15-18 In the present study we aim to screen
medicinal plants for their effect on inflamma-
tory responses to infections by enteric
pathogens. These plants, if active, may prove to
be an effective therapy towards control of the
intestinal inflammation associated with infec-
tious forms of diarrhea.
Different approaches have been used to
analyze the generally anti-inflammatory poten-
tial of plants and plant-derived compounds
which include acute and chronic inflammatory
models. The acute models, which include both
in vitro and in vivo studies, are designed to
assess the effect of the plants on secretion of
pro-inflammatory cytokines such as IL-8,
changes in vascular permeability, leukocyte
migration and chemotaxis, macrophage activi-
ty, and measurement of rat paw edema.19
Therefore, in the present study we have evalu-
ated the in vitro activity of hot aqueous
extracts (decoction) of four selected antidiar-
rheal medicinal plants i.e. Aegle marmelos (L.)
Correa (Family Rutaceae), Cyperus rotundus
Linn., (Family Cyperaceae), Psidium guajava
L. (Family Myrtaceae) and Zingiber officinale
(Roscoe) (Family Zingiberaceae) against
secretion of the pro-inflammatory cytokine IL-
8 by HT-29 cells in response to EPEC infection.
These plants were chosen on the basis of an
ethonobotanical survey in the Parinche valley,
near Pune, Maharashtra, India.20 All four
plants are widely used in indigenous systems
of medicine across the world for their various
medicinal properties. They are widely used for
treatment of diarrhea and have been reported
to have immuno-modulatory properties includ-
ing anti-inflammatory activity.21-24
Alternative Medicine Studies 2011; volume 1:e16
Correspondence: Dr. Tannaz J. Birdi, Deputy
Director, The Foundation for Medical Research,
84A, RG Thadani Marg, Worli, Mumbai - 400018,
India.
Tel. +91.22.249.349.89 - Fax: +91.22.24932876
E-mail: fmr@fmrindia.org
Key words: Aegle marmelos, Cyperus rotundus,
Psidium guajava, Zingiber officinale, IL-8,
enteropathogenic Escherichia coli.
Contributions: BS carried out the laboratory stud-
ies, analyzed the data and prepared the manu-
script; PT collected the plant materials, authenti-
cated them and obtained voucher specimen num-
bers; TJB has overall responsibility for the study.
Conflict of interest: the authors report no con-
flicts of interest.
Received for publication: 17 May 2011.
Revision received: 28 September 2011.
Accepted for publication: 21 October 2011.
This work is licensed under a Creative Commons
Attribution NonCommercial 3.0 License (CC BY-
NC 3.0).
©Copyright Brijesh S. et al., 2011
Licensee PAGEPress, Italy
Alternative Medicine Studies 2011; 1:e16
doi:10.4081/ams.2011.e16
Non-commercial use only

[Alternative Medicine Studies 2011; 1:e16] [page 65]
Materials and Methods
Media, reagents, plastic ware and
instrumentation
The bacteriological media were purchased
from HiMedia laboratory, Mumbai, India.
Dulbecco’s modified Eagle medium (DMEM)
and fetal calf serum (FCS) were procured from
GibcoBRL, UK. The Quantikine human
CXCL8/IL-8 immunoassay kit was purchased
from R&D Systems, USA. All chemicals were
from SD Fine Chemicals, Mumbai. The 24-well
flat bottomed tissue culture plates were pur-
chased from Nunclon, Denmark, and the 55
mm diameter tissue culture plates were
obtained from Tarsons, Kolkata, India.
Bacterial strain
For the present study, EPEC strain E2348/69,
serotype 0127:H6 (kindly provided by Dr. S.
Knutton, Institute of Child Health, University
of Birmingham, Birmingham, UK) was used.
The bacterial strain was stored at -80°C in
brain heart infusion (BHI) containing 20%
glycerol. For each assay a frozen stock of the
bacterial strain was revived in BHI.
Cell culture
The human colon adenocarcinoma cell line,
HT-29, was obtained from the National Centre
for Cell Sciences, Pune, India. The cell line was
maintained by passage every 4-5 days in
DMEM supplemented with 10% FCS, at 37°C in
5% CO2atmosphere.
Plant material
The unripe fruits of A. marmelos, rhizomes of
C. rotundus and Z. officinale and the leaves of P.
guajava were collected from the Parinche valley,
about 53 km south east of the city of Pune in the
state of Maharashtra, India. The plant materials
were authenticated by Dr. P. Tetali, Naoroji
Godrej Centre for Plant Research (NGCPR).
Voucher specimens of A. marmelos, C. rotundus
and P. guajava were deposited at the Botanical
Survey of India (BSI), Western Circle, Pune,
Maharashtra, India, and that of Z. officinale at
the herbarium at the NGCPR (Table 1). The
plant materials were shade dried and powdered,
and stored at 4°C until used.
Preparation of extract
Crude aqueous extracts (decoctions) were
used for the study since this represents the
nearest form to traditional preparations. The
decoctions were prepared as described in the
Ayurvedic text:25 1 g of the powdered plant
materials were boiled in 16 mL double distilled
water till the volume reduced to 4 mL. The
decoctions were centrifuged and filtered
through a 0.22 μm membrane before use. To
replicate field conditions, each assay was per-
formed with freshly prepared decoctions. The
decoctions were diluted 1:1000, 1:100, 1:20 and
1:10 in DMEM for each experiment and have
been referred to as 0.1%, 1%, 5%, and 10%,
respectively throughout the text.
The dry weight of the decoctions was record-
ed to determine the concentration of the
extracts in each dilution. The qualitative phy-
tochemical analysis was performed using stan-
dard methods.26
IL-8 secretion by intestinal
epithelial cells in response to EPEC
infection
The effect of the decoctions on the inflamma-
tory response of intestinal epithelial cells to
infection by EPEC was measured by estimation
of IL-8 secreted by HT-29 cells in response to E.
coli E2348/69 supernatant by IL-8 enzyme
linked immunosorbent assay (ELISA).9Briefly,
E. coli E2348/69 was grown overnight in BHI at
37°C. The culture supernatant obtained by cen-
trifugation at 2,000 rpm was stored at -20°C till
used for the assay. For each assay, 0.2 mL of the
thawed culture supernatant of E. coli E2348/69
was added directly to HT-29 cells (5¥105/well)
grown overnight in 24-well tissue culture plates
in a total assay volume of 0.5 mL containing 0.3
mL DMEM with 10% FCS. Following incubation
for 18-20 h, the culture supernatant was collect-
ed and centrifuged at 14,000 rpm. The super-
natant were either immediately estimated for
IL-8 using a Quantikine IL-8 immunoassay kit
as per the manufacturer’s instructions or stored
at -20ºC for estimation of IL-8 at a later date.
The optical density was measured at 450 nm
(reference 540 nm) on an ELISA plate reader
(Labsystems, Finland).
The IL-8 assay was performed using two dif-
ferent protocols: pre-incubation protocol and
post-incubation protocol. In the pre-incubation
protocol the HT-29 cells were incubated with
different concentrations of the plant decoc-
tions for 3 h prior to addition of the EPEC
supernatant whereas in the post-incubation
protocol the plant decoctions were added into
the assay system 3 h after the addition of the
EPEC supernatant to the HT-29 cells.
Statistical analysis
The results have been expressed as the
mean ± standard error of the percentage val-
ues of the test groups relative to control
(100%) from three independent experiments.
Data were analyzed by analysis of variance
(ANOVA) and Dunnett’s post test. A value of
P≤0.05 was considered to be statistically sig-
nificant. The EC50 values, wherever applicable,
were calculated by non-linear regression
analysis using the equation for a sigmoid con-
centration-response curve. All statistical analy-
ses were performed using the software Prism
4.0 (GraphPad Software, Inc., USA).
Results
Phytochemical analysis
The percentage yield (mg/mL) for each
decoction with respect to the respective start-
ing material is shown in Table 1. The qualita-
tive phytochemical analysis revealed the pres-
ence of constituents such as carbohydrates,
flavonoids and tannins in the decoctions of all
four plants. While saponins were detected in
the decoctions of A. marmelos, C. rotundus and
P. guajava, phytosterols and anthraquinone
glycosides were detected in the decoction of A.
marmelos only (Table 2).
Effect on IL-8 secretion in response
to EPEC infection
The effect of the decoctions on EPEC
induced secretion of IL-8 by HT-29 cells is
shown in Figures 1-4. The baseline secretion
of IL-8 by HT-29 cells in the culture medium
was estimated to be 56.55±5.12 pg/mL where-
as the secretion of IL-8 in presence of EPEC
supernatant increased to 477.53±8.84 pg/mL
(represented as 100%). The decoctions of A.
marmelos and Z. officinale had no effect on
EPEC induced secretion of IL-8 by the HT-29
cells in either of the two protocols used
(Figures 1 and 4, respectively) whereas the
decoction of C. rotundus had no effect in the
post-incubation protocol (Figure 2). In com-
parison the decoction of P. guajava resulted in
a significant decrease (EC50 value
Article
Table 1. Details of the plant material.
Herbarium Botanical name Common name Family name Part Used % yield of
N. decoction
(w/w)
BSI-124675 A. marmelos Wood Apple, Rutaceae Unripe fruit pulp 20.4±2.11
Bengal Quince
BSI-124666 C. rotundus Nutgrass Cyperaceae Rhizomes 20.9±1.12
BSI-124672 P. guajava Guava Myrtaceae Leaves 10.8±0.5
NGCPR-642 Z. officinale Ginger Zingiberaceae Rhizomes 8.84±0.01
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[page 66] [Alternative Medicine Studies 2011; 1:e16]
2.09±0.78%) in EPEC induced IL-8 secretion in
the post-incubation protocol with a maximum
decrease at 10% concentration (Figure 3). On
the contrary, in the pre-incubation protocol the
decoctions of both C. rotundus and P. guajava
resulted in a significant increase in EPEC
induced IL-8 secretion (Figures 2 and 3,
respectively).
Discussion
The anti-inflammatory activity of four anti-
diarrheal medicinal plants used in traditional
medicine, i.e. A. marmelos, C. rotundus, P. gua-
java and Z. officinale were assessed against
EPEC induced secretion of IL-8 by intestinal
epithelial cells. Analysis of the results of the
study shows that the decoctions of A. marmelos
and Z. officinale had no effect on EPEC
induced IL-8 secretion by HT-29 cells in both
pre- and post-incubation protocols. On the
other hand, the IL-8 secretion by HT-29 cells
was increased in the pre-incubation protocol
by decoctions of C. rotundus and P. guajava and
inhibited in the post-incubation protocol by the
decoction of P. guajava.
Extracts from several plants have been
reported to inhibit IL-8 secretion from epithe-
lial cells.27-29 Our observations in the post-
incubation protocol on the inhibitory activity of
aqueous extract of P. guajava leaves on IL-8
secretion from epithelial cells confirm the
results reported by Peng et al.30 who used a
similar protocol. However, in contrast to the
observation made in the present study in
which the decoction of Z. officinale showed no
inhibition of IL-8 secretion, several studies
have reported on the inhibitory effect of the
ethanolic,31 50% ethanolic32 and methanolic33
extracts of Z. officinale on either IL-8 or IL-8
and NF-κB modulation in vivo and in vitro.
These differences in observations could be due
to the use of aqueous decoction in the present
study compared to the use of organic extracts
by other workers. Major bioactive constituents
from Z. officinale such as [6]-shogaol, [6]-
gingerol, [8]-gingerol, and [10]-gingerol,34
and constituents such as ar-curcumene and α-
pinene isolated from volatile oil,35 have also
been reported to have an inhibitory effect on
IL-8. However, these belong to the class of con-
stituents that are not soluble in water. In addi-
tion, the differences in the ecotype and/or vari-
ety of the Z. officinale used could also be
responsible for the observed difference in the
activity.
Inhibition of IL-8 secretion by epithelial
cells has been attributed to polyphenolic com-
pounds present in the extracts.36 Different
modes of action have been proposed for their
inhibitory activity on IL-8 secretion which
include: i) inhibition of inhibitory factor IKK
Article
Table 2. Results for the qualitative phytochemical analysis of the decoctions of the plants
used for the study.
Phytochemical Phytochemical Inference
Constituent Test A. marmelos C. rotundus P. guajava Z. officinale
Carbohydrates Molisch’s test ++++
Reducing sugars Fehling’s test ++++
Benedict’s test ++++
Starch Iodine test ++-+
Phytosterols Salkowski’s test +---
Cardiac glycosides Legal’s test ----
Anthraquinone Modified
glycosides Borntrager’s test +---
Saponins Foam test +++-
Flavonoids Shinoda’s test ++++
Tannins Ferric chloride test ++++
Lead acetate test ++++
Alkaloids Wagner’s test +-+-
Figure 1. Effect of A. marmelos on IL-8 secretion by HT-29 cells in response to EPEC
infection in the pre-incubation and the post-incubation protocols. Values represent mean
± standard error (n=3) of percentage secretion of IL-8 by HT-29 cells in presence of dif-
ferent concentrations of the decoction relative to control (100%).
Figure 2. Effect of C. rotundus on IL-8 secretion by HT-29 cells in response to EPEC
infection in the pre-incubation and the post-incubation protocols. Values represent mean
± standard error (n=3) of percentage secretion of IL-8 by HT-29 cells in presence of dif-
ferent concentrations of the decoction relative to control (100%). * P<0.05.
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[Alternative Medicine Studies 2011; 1:e16] [page 67]
activity, e.g. curcumin;37 and ii) inhibition of
MAPK activation, e.g. Punica granatum fruit
extract.38 It is known that different polypheno-
lic compounds influence different molecular
targets and pathways to exhibit their anti-
inflammatory activities.39 Though polyphenolic
compounds such as tannins and flavonoids
were detected in the crude decoctions of all
four plants, only the decoction of P. guajava
leaves inhibited IL-8 secretion by HT-29 cells.
Since EPEC is a non-invasive pathogen, the
activation of NF-κB has been linked to soluble
factors secreted or shed by the pathogen or
translocated type three secretory system-
dependent effectors.9Much of the pathology of
EPEC infection which is responsible for trig-
gering IL-8 release from epithelial cells has
been linked to flagellin, the flagellar structural
protein. Flagellin interaction with Toll-like
receptors (TLR)-5 results in activation of the
NF-κB which in turn promotes the expression
of IL-8.9The increase in IL-8 secretion by HT-
29 cells in the presence of the decoctions of C.
rotundus and P. guajava in the pre-incubation
protocol may be due to components that are
similar to flagellins structurally and/or func-
tionally which may be interacting with these
receptors and inducing expression of IL-8
mRNA. The effect could be similar to that
observed with green tea extract (GTE) which
was reported to induce de novo synthesis of IL-
8 in Caco-2 cells.40 Interestingly, Netsch et al.
reported that GTE, while inducing expression
of IL-8 mRNA, specifically inhibited its extra-
celluar secretion.40 Similarly, the decoction of
P. guajava, in addition to components that
induce expression of IL-8 mRNA, may also con-
tain constituents that may be specifically
inhibiting the extracellular secretion of IL-8.
This could probably be the reason for the inhi-
bition of IL-8 secretion observed in the post-
incubation protocol even when IL-8 expression
is induced. However, no decrease in extracellu-
lar secretion of IL-8 in the pre-incubation pro-
tocol following induction of IL-8 mRNA expres-
sion could be due to the removal of the decoc-
tion from the assay system prior to addition of
EPEC supernatant resulting in washing off the
components responsible for inhibiting the
extracellular secretion of IL-8.
It is interesting to note that the P. guajava
decoction had a biphasic effect on expression
of IL-8 mRNA in the pre-incubation protocol.
There was a dose dependent increase in secre-
tion of IL-8 with maximum secretion at a 5%
concentration. However, at a 10% concentra-
tion of decoction the IL-8 secretion was lesser
than that at 5%, though it was still much high-
er than the control. The observed biphasic
activity may probably be due to possible inter-
action between constituents of the crude
extract. Such biphasic activities of plant con-
stituents have been reported previously.41-44
However, as explained before, since the secre-
tion of IL-8 was specifically inhibited by cer-
tain plant constituents in the post-incubation
protocol in a dose dependent manner, the
biphasic effect on expression of IL-8 mRNA
was not apparent.
It is hypothesized that in terms of clinical
relevance the results of the pre-incubation pro-
tocol indicate a preventive effect of the plant
decoctions on the host whereas the post-incu-
bation protocol indicates the effect of the plant
decoctions when used as treatment. The
results, therefore, indicate that amongst the
plants tested, while none of them may be effec-
tive as preventive measures, P. guajava can be
used for the treatment of inflammatory diar-
rheal episodes. Our previous studies with
these four plants, however, have demonstrated
their efficacy against other parameters of
infectious forms of diarrhea, such as coloniza-
tion of epithelial cells, and production and
action of enterotoxins15-18 which still render
them effective for treatment of diarrhea.
In conclusion, it may be stated that of the
four antidiarrheal medicinal plants used in the
present study, only P. guajava is effective in
controlling the acute inflammatory response of
the intestinal epithelial cells in response to
EPEC infection.
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Figure 3. Effect of P. guajava on IL-8 secretion by HT-29 cells in response to EPEC infec-
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standard error (n=3) of percentage secretion of IL-8 by HT-29 cells in presence of differ-
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