Newer insights into the mechanism of action of Psidium guajava L. leaves in infectious diarrhoea

Abstract

Background
Psidium guajava L., Myrtaceae, is used widely in traditional medicine for the treatment of diarrhoea, dysentery, gastroenteritis, stomachaches, and indigestion. However, the effect of the leaf extract of P. guajava on the pathogenesis of infectious diarrhoea has not been studied. The present study evaluates the effect of a hot aqueous extract (decoction) of dried leaves of P. guajava on parameters associated with pathogenicity of infectious diarrhoea. The aim was to understand its possible mechanism(s) of action in controlling infectious diarrhoea and compare it with quercetin, one of the most reported active constituents of P. guajava with antidiarrhoeal activity.
Methods
The crude decoction and quercetin were studied for their antibacterial activity and effect on virulence features of common diarrhoeal pathogens viz. colonization of epithelial cells and production and action of enterotoxins. Colonization as measured by adherence of enteropathogenic Escherichia coli (EPEC) and invasion of enteroinvasive E. coli (EIEC) and Shigella flexneri was assessed using HEp-2 cell line. The production of E. coli heat labile toxin (LT) and cholera toxin (CT) and their binding to ganglioside monosialic acid (GM1) were studied by GM1-ELISA whereas the production and action of E. coli heat stable toxin (ST) was assessed by suckling mouse assay.
Results
The decoction of P. guajava showed antibacterial activity towards S. flexneri and Vibrio cholerae. It decreased production of both LT and CT and their binding to GM1. However, it had no effect on production and action of ST. The decoction also inhibited the adherence of EPEC and invasion by both EIEC and S. flexneri to HEp-2 cells. Quercetin, on the other hand, had no antibacterial activity at the concentrations used nor did it affect any of the enterotoxins. Although it did not affect adherence of EPEC, it inhibited the invasion of both EIEC and S. flexneri to HEp-2 cells.
Conclusion
Collectively, the results indicate that the decoction of P. guajava leaves is an effective antidiarrhoeal agent and that the entire spectrum of its antidiarrhoeal activity is not due to quercetin alone.

Full text

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Open Access
RESEARCH ARTICLE
Newer insights into the mechanism of action of
Psidium guajava L. leaves in infectious diarrhoea
© 2010 Birdi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons At-
tribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Research article
Tannaz Birdi*1, Poonam Daswani1, S Brijesh1, Pundarikakshudu Tetali2, Arvind Natu3 and Noshir Antia1,4
Abstract
Background: Psidium guajava L., Myrtaceae, is used widely in traditional medicine for the treatment of diarrhoea,
dysentery, gastroenteritis, stomachaches, and indigestion. However, the effect of the leaf extract of P. guajava on the
pathogenesis of infectious diarrhoea has not been studied. The present study evaluates the effect of a hot aqueous
extract (decoction) of dried leaves of P. guajava on parameters associated with pathogenicity of infectious diarrhoea.
The aim was to understand its possible mechanism(s) of action in controlling infectious diarrhoea and compare it with
quercetin, one of the most reported active constituents of P. guajava with antidiarrhoeal activity.
Methods: The crude decoction and quercetin were studied for their antibacterial activity and effect on virulence
features of common diarrhoeal pathogens viz. colonization of epithelial cells and production and action of
enterotoxins. Colonization as measured by adherence of enteropathogenic Escherichia coli (EPEC) and invasion of
enteroinvasive E. coli (EIEC) and Shigella flexneri was assessed using HEp-2 cell line. The production of E. coli heat labile
toxin (LT) and cholera toxin (CT) and their binding to ganglioside monosialic acid (GM1) were studied by GM1-ELISA
whereas the production and action of E. coli heat stable toxin (ST) was assessed by suckling mouse assay.
Results: The decoction of P. guajava showed antibacterial activity towards S. flexneri and Vibrio cholerae. It decreased
production of both LT and CT and their binding to GM1. However, it had no effect on production and action of ST. The
decoction also inhibited the adherence of EPEC and invasion by both EIEC and S. flexneri to HEp-2 cells. Quercetin, on
the other hand, had no antibacterial activity at the concentrations used nor did it affect any of the enterotoxins.
Although it did not affect adherence of EPEC, it inhibited the invasion of both EIEC and S. flexneri to HEp-2 cells.
Conclusion: Collectively, the results indicate that the decoction of P. guajava leaves is an effective antidiarrhoeal agent
and that the entire spectrum of its antidiarrhoeal activity is not due to quercetin alone.
Background
Infectious diarrhoea accounts for nearly 3.2% of all deaths
globally [1] and is the second largest cause of years of pro-
ductive life lost due to premature mortality and disability
[2]. It is a major health concern in developing countries
and remains an important clinical problem even in devel-
oped countries despite improvements in public health
and economic wealth [3]. It is estimated that during the
next 20-30 years, diarrhoea along with other infectious
diseases will remain a cause of global health concern [4].
Diarrhoea is an etiologically diverse condition unlike
some other infectious diseases such as tuberculosis, HIV/
AIDS and malaria since it is caused by a variety of enteric
pathogens including bacteria, viruses and protozoa [5-7].
Oral rehydration therapy has been the key strategy for
effective case management. However, it often fails in high
stool output state. Moreover, symptomatic therapy with
antimotility agents is contraindicated in infectious diar-
rhoea and there is an increasing threat of drug resistance
to antibiotics [8]. Various attempts for developing vac-
cines against diarrhoea causing organisms have been
made [6,9-11]. However, the responses to vaccines in
developing countries have not been encouraging [12-15].
In the recent past there have been advances towards the
treatment of infectious diarrhoea with supportive therapy
such as the use of probiotics; but these are still under
development [3]. Hence, medicinal plants may aid in
developing cost effective alternative approaches for treat-
ment of diarrhoea.
* Correspondence: fmr@fmrindia.org
1 The Foundation for Medical Research, 84A, RG Thadani Marg, Worli, Mumbai
400018, Maharashtra, India
^ Deceased
Full list of author information is available at the end of the article

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Medicinal plants have recently gained popularity as
prospective antidiarrhoeal agents as can be judged by the
number of studies that have been undertaken. An online
search on PubMed shows that more than 200 studies on
the antidiarrhoeal activity of medicinal plants have been
published in the last decade. Gutierrez et al. [16] have
reviewed more than 50 such studies conducted during
the period 2000 to 2007. Whilst a few studies have
reported antimicrobial activity, a majority of the studies
have focused on the effect of the plants on intestinal
motility in experimental models. Hence, though data is
available on the effect of medicinal plants on physiologi-
cal diarrhoea as studied in animal models there is a pau-
city of information on their mode of action on infectious
diarrhoea.
Psidium guajava L., Myrtaceae, is used widely in tradi-
tional medicine throughout Latin America and the Carib-
bean for the treatment of diarrhoea, dysentery,
gastroenteritis, stomachaches, and indigestion [17]. It is
also used for diarrhoea and dysentery in countries such as
China, Philippines, Senegal and USA, as an antiamoebic
in Congo, antispasmodic in India and Ghana, antiseptic
in China and as an antibiotic in USA. However, this plant
is not very popular in India as an antidiarrhoeal agent. An
ethnobotanical survey carried out by us in Parinche val-
ley located 55 km south-east of Pune, Maharashtra, India
revealed that P. guajava had very limited usage by the
local community as a treatment for diarrhoea [18]. Only 2
out of 24 traditional healers interviewed had knowledge
of P. guajava as an antidiarrhoeal agent. The antidiar-
rhoeal activity of P. guajava though widely reported has
been mainly investigated with respect to its antimicrobial
action and/or its effect on physiological diarrhoea in ani-
mal models [17]. Thus its mechanism(s) of action in
infectious diarrhoea is largely unknown. To the best of
our knowledge there have been no studies till date on the
effect of the leaf extract of P. guajava on the pathogenesis
of infectious diarrhoea.
Hence, in the present study the crude aqueous extract
of the leaves of P. guajava was studied for its effect on
various virulence parameters of infectious diarrhoea viz.,
colonization on intestinal epithelial cells and production
and action of enterotoxins. The activity of the crude
extract was compared with that of quercetin to ascertain
its role in the observed biological activity. Quercetin is a
major flavonoid present in P. guajava leaves and has been
previously reported to have antidiarrhoeal activity [17].
Quercetin has also been used as a marker for standard-
ization of crude aqueous leaf extract [19].
Methods
Plant material, preparation of decoction, quercetin
The leaves of P. guajava, variety Sardar which is amongst
the 5 common varieties found in India, were collected
from Parinche valley, Maharashtra, India. A voucher
specimen of the plant material has been deposited at the
Botanical Survey of India, Western circle, Pune, India
with herbarium number 124672. The leaves were shade
dried, powdered, and stored at 4°C. All experiments were
performed with the same plant material.
As described in the Ayurvedic texts [20], the decoction
was prepared by boiling 1 g of the plant material in 16 ml
double distilled water till the volume reduced to 4 ml. To
replicate field conditions a fresh decoction was prepared
every time. The decoction was centrifuged and filtered
through a membrane of 0.22 μm pore size before use. For
each experiment, 0.1% (1:1000 dilution), 1% (1:100 dilu-
tion), 5% (1:20 dilution), and 10% (1:10 dilution) (v/v)
concentrations of the decoction and corresponding dilu-
tions of quercetin in appropriate media were used. The
amount of quercetin present in the decoction was esti-
mated by a method as described under the section of
phytochemical analysis.
Media, reagents, plastic ware and instrumentation
The bacterial media was purchased from HiMedia labo-
ratory, Mumbai, India. Dulbecco's modified Eagle
medium (DMEM) and fetal calf serum (FCS) were pro-
cured from GibcoBRL, UK. Quercetin, anti-cholera toxin
and bovine serum albumin were purchased from Sigma,
USA. Peroxidase labeled swine anti-rabbit immunoglobu-
lin was from Dako, Denmark. All chemicals were from
SD Fine Chemicals, Mumbai. Gallic acid was kindly pro-
vided by Dr. K. S. Laddha, Institute of Chemical Technol-
ogy, Mumbai, India. Lactulose was a product of Intas
Pharmaceuticals, Ahmedabad, India. The 24- and 96-well
tissue culture plates and the 96-well ELISA plates were
purchased from Nunclon, Denmark, the 55 mm diameter
tissue culture plates were obtained from Tarsons, Kolk-
ata, India and the ELISA plate reader was purchased from
Labsystems, Finland. The pre-coated Silica gel plates for
high performance thin layer chromatography, G60 F254
TLC, were obtained from Merck, Germany.
Cell culture
The human laryngeal epithelial cell line, HEp-2 was
obtained from the National Centre for Cell Sciences,
Pune. The cell line was maintained in DMEM supple-
mented with 10% FCS, at 37°C in a 5% CO2 atmosphere.
The cells were maintained in logarithmic growth by pas-
sage every 3-4 days.
Microorganisms used
Six bacteria viz., enteropathogenic Escherichia coli
(EPEC) strain B170, serotype 0111:NH, enterotoxigenic
E. coli (ETEC) strains B831-2, serotype unknown (heat
labile toxin producer) and TX1, serotype 078:H12 (heat
stable toxin producer) (all strains obtained from Centre

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for Disease Control, Atlanta, USA), enteroinvasive E. coli
(EIEC) strain E134, serotype 0136:H- (kindly provided by
Dr. J. Nataro, Veterans Affairs Medical Centre, Maryland,
USA); Vibrio cholerae C6709 El Tor Inaba, serotype 01
(kindly provided by Dr. S. Calderwood, Massachusetts
General Hospital, Boston, USA) and Shigella flexneri
M9OT, serotype 5 (kindly provided by Dr. P. Sansonetti,
Institut Pasteur, France) were used for the present study.
Phytochemical analysis
The decoction was qualitatively assayed for presence of
carbohydrates, glycosides,
saponins, flavonoids, alkaloids and tannins [21].
High performance thin layer chromatography (HPTLC)
fingerprinting of the methanol soluble fraction of the
decoction was carried out on pre-coated Silica gel G60
F254 TLC plates with quercetin as a standard. The plates
were developed in solvent system consisting of Dichlo-
romethane:Ethyl acetate:Methanol:Acetic acid (8:4:2:0.1).
The chromatogram was derivatized with ferric chloride
which is specific for quercetin and scanned using TLC
Scanner 3 (CAMAG) in visible light. The amount of
quercetin in P. guajava decoction was estimated by pre-
paring a standard graph by running different concentra-
tions of standard quercetin and measuring the intensity
of the spots by using the software Multigauge version 3.0
(Fugifilm). The intensity of the corresponding spot
obtained with the decoction (hydrolysed using 1N
Hydrochloric acid) was measured and extrapolated onto
the standard graph to estimate the amount of total quer-
cetin in the decoction. The acid hydrolysed decoction
was used so as to obtain the conjugated quercetin in free
form.
The concentration of quercetin in the decoction was
also confirmed by high performance liquid chromatogra-
phy (HPLC) using HPLC system from Dionex Corp.,
USA. The column used was 250 mm × 4.6 mm 15 μC18
120 Å Acclaim. 20 μl of sample diluted 1:1 with Water:
Acetonitrile:Phospohoric Acid (50:50:0.1) was run at a
flow rate of 1 ml/min with Water: Acetonitrile:Phospo-
horic Acid as the mobile phase at the ratios of 90:10:0.1
and 10:90:0.1 in Phase A and Phase B respectively. The
column oven temperature was adjusted to 30°C and the
readings were taken at a wavelength of 210 nm.
Antibacterial activity
The antibacterial activity was determined by a microtitre
plate based assay [22]. The bacterial strains were incu-
bated with the decoction/quercetin in nutrient broth and
the optical density was measured after 24 h as a measure
of growth. Ofloxacin (1 μg/ml) was used as the antibiotic
control. Three independent experiments were carried
out. In each experiment, triplicate wells were set up for
proteins, phytosterols,
control as well as for each dilution of the decoction/quer-
cetin.
Effect on bacterial colonization
Effect on adherence
The effect on the adherence of E. coli B170 to epithelial
cells was assayed by a method described by Cravioto et al.
[23]. Briefly, a 48 h culture of HEp-2 cells on glass cover-
slips was infected with a log phase culture (5 × 107/ml) of
the bacteria in DMEM and incubated for 3 h. Non-adher-
ent bacteria were washed off, the coverslips fixed in 10%
formaldehyde and stained with toluidine blue stain (0.1%
w/v). HEp-2 cells having typical EPEC microcolonies [24]
were counted under light microscope. Three independent
experiments were carried out. In each experiment, dupli-
cate coverslips were set up for control as well as for each
dilution of the decoction/quercetin.
The effect of the decoction/quercetin on adherence of
E. coli B170 to HEp-2 cells was compared with that of
lactulose, a prebiotic oligosaccharide, known to inhibit
adherence of EPEC to tissue culture cells [25].
Effect on invasion
The effect on invasion of E. coli E134 and S. flexneri to
epithelial cells was studied by a method described by
Vesikari et al. [26]. Briefly, a 48 h culture of HEp-2 cells
grown in a 24-well tissue culture plate was infected with
log phase culture (108/ml) of the bacteria in DMEM and
incubated for 2 h. The extracellular bacteria were then
washed off and the culture was further incubated with
DMEM containing gentamycin (100 μg/ml) for 3 h. The
medium containing gentamycin was washed off and the
epithelial cells were then lysed using chilled distilled
water and the released bacteria were enumerated by plat-
ing on nutrient agar. Three independent experiments
were carried out. In each experiment, duplicate wells
were set up for control as well as for each dilution of the
decoction/quercetin.
The effect of the decoction/quercetin on invasion of S.
flexneri was also compared with that of lactulose, as it has
been used for the treatment of shigellosis and inflamma-
tory bowel disease [27]. Since the mechanism of invasion
of both EIEC and S. flexneri is almost identical [28], the
effect of the decoction on invasion of E. coli E134 was
also compared with that of lactulose.
Two different protocols were performed for both the
adherence and the invasion assays to understand whether
the bacterial adherence and invasion respectively were
affected by the effect of the decoction/quercetin on the
epithelial cells or through competitive inhibition. The
HEp-2 cells were incubated in absence (control) and pres-
ence of different dilutions of the decoction/quercetin
either for 18-20 h prior to infection (pre-incubation) or

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simultaneously with infection (competitive inhibition)
respectively.
Effect on bacterial enterotoxins
Effect on E. coli heat labile toxin (LT) and cholera toxin (CT)
LT, which is localized in the cell membrane of E. coli
B831-2 was obtained by lysing the bacterial cells with pol-
ymyxin B sulphate (1 mg/ml) whereas CT, which is
released extracellularly, was obtained as a culture super-
natant of V. cholerae. LT and CT were assayed by the gan-
glioside monosialic acid enzyme linked immunosorbent
assay (GM1-ELISA) [29]. Briefly, the toxins were added to
ELISA plates pre-coated with 1.5 μmol/ml of GM1. Anti-
cholera toxin (1:300) and peroxidase labeled swine anti-
rabbit immunoglobulin (1:200) were used as primary and
secondary antibodies respectively. Orthophenylene
diamine was used as the substrate. The intensity of the
color thus developed was read at 492 nm in an ELISA
plate reader.
The effect of the decoction/quercetin on production of
these toxins was compared with that of 2-mercaptoetha-
nol since thiols such as 2-mercaptoethanol, L-cysteine
monohydrochloride and sodium thioglycolate have been
reported to inhibit production of LT, CT and ST [30,31].
Similarly, their effect on binding of these toxins to GM1
was compared with that of gallic acid, a polyphenol,
which has been reported to block the binding of LT to
GM1 [32]. As LT and CT are antigenically closely related
[33], the effect of the decoction on the binding of CT was
also compared to that of gallic acid.
To study the effect on production of the toxins, the
respective bacteria were grown in Casein hydrolysate
yeast extract (CAYE) in absence (control) and presence of
different dilutions of the decoction/quercetin and the
toxins produced were assayed by GM1-ELISA. To study
the effect on the binding of the toxins to GM1, the toxins
obtained by growing the respective bacteria in CAYE
were added to the assay system in absence (control) and
presence of the decoction/quercetin. Three independent
experiments were carried out. In each experiment, tripli-
cate wells were set up for control as well as for each dilu-
tion of the decoction/quercetin.
Effect on stable toxin (ST)
ST, which is released extracellularly was obtained as the
culture supernatant of E. coli TX1 and assayed by the
method originally described by Gianella [34]. Briefly, the
toxin was inoculated intragastrically in 2-3 day old Swiss
White suckling mice. Following an incubation of 3 h at
room temperature, the animals were sacrificed and the
ratio of gut weight to that of the remaining carcass weight
was calculated. Ratio of ≥ 0.083 was considered as posi-
tive for fluid accumulation.
To study the effect on production of ST, the bacterium
was grown in CAYE in absence (control) and presence of
different dilutions of the decoction/quercetin and the
toxin produced was assayed. To study the effect on the
action of ST, the toxin obtained by growing the bacterium
in CAYE was intragastrically injected in absence (control)
and presence of different dilutions of the decoction/quer-
cetin. CT was used as a negative control. Three indepen-
dent experiments were carried out. In each experiment,
three animals were inoculated for control as well as each
dilution of the decoction/quercetin.
The Institutional Ethics Committee and the Committee
for the Purpose of Control and Supervision of Experi-
ments on Animals (CPCSEA) cleared the use of animals
in the study. The Foundation for Medical Research (FMR)
is registered with CPCSEA (registration No. 424/01/a/
CPCSEA, June 20th, 2001).
Statistical analysis and presentation of data
The results for each assay have been expressed as the
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/tripli-
cate readings of the control group and T is mean value of
the duplicate/triplicate readings of the test (dilutions of
the decoction/quercetin) groups. Hence, the value of con-
trol is 100% and the values of the test groups have been
represented as percentages relative to control.
Data were analyzed by analysis of variance (ANOVA)
and Dunnett's post test. P ≤ 0.05 was considered to be
statistically significant. The EC50 values, wherever appli-
cable, were calculated by nonlinear regression analysis
using the equation for a sigmoid concentration-response
curve. All statistical analyses were performed using the
software Prism 4.0 (GraphPad, Inc.).
Results
Phytochemistry
The percent yield of the decoction with respect to the
starting dried plant material was 10.8% ± 0.5% (w/w). The
dry weight of the decoction was 27.0 mg/ml ± 1.25 mg/
ml. Thus the concentrations of the different dilutions
used in the biological assays were 0.027 mg/ml ± 0.001
mg/ml (0.1%), 0.270 mg/ml ± 0.013 mg/ml (1%), 1.850
mg/ml ± 0.063 mg/ml (5%) and 2.700 mg/ml ± 0.125 mg/
ml (10%).
The amount of total quercetin was estimated to be 2 mg
per gram of the dried leaves of P. guajava. Quercetin was
used at concentrations of 2 μg/ml, 20 μg/ml, 100 μg/ml
and 200 μg/ml which corresponded to the amount of
quercetin present in the different concentrations of the
decoction used for the study.
The classes of compounds present in the decoction
were carbohydrate, reducing sugars, proteins, saponins,

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flavonoids, tannins, and traces of alkaloids. Phytosterols
and glycosides were absent.
The HPTLC profile of the methanol soluble fraction of
the decoction is presented in Figure 1. The fingerprint
shows presence of quercetin as one of the constituents of
the decoction (Rf value 0.58). The HPLC profile has been
provided in additional file 1.
Antibacterial activity
The decoction inhibited the growth of two of the six bac-
terial strains tested viz., S. flexneri (EC50 value 0.98% ±
0.2%) and V. cholerae (EC50 value 2.88% ± 0.36%) (Figure
2) whereas there was no effect of quercetin at any of the
concentrations tested on the growth of all six strains
tested (data not shown). Ofloxacin at a concentration of 1
μg/ml completely inhibited the growth of all the bacterial
strains.
Effect on bacterial colonization
Adherence of E. coli B170
In the competitive protocol, 15 mg/ml of lactulose was
used as a positive control whereas in the pre-incubation
protocol 2.5 mg/ml of lactulose was used since it was
toxic to HEp-2 cells even at 5 mg/ml when incubated
overnight with the cells. Below this concentration lactu-
lose had no effect on the adherence.
As compared to lactulose the decoction showed a
greater decrease in the adherence of E. coli B170 to HEp-
2 cells in both the pre-incubation (EC50 value 0.37% ±
0.05%) and the competitive protocols (EC50 value 1.27% ±
0.18%) (Figure 3a). However, quercetin had no effect on
bacterial adherence (Figure 3b). This suggests that the
crude decoction contains active components(s), other
than quercetin, which may be responsible for the
decrease in adherence. Though the decoction reduced
the adherence of E. coli B170 in both the protocols, the
extent of decrease was greater in the pre-incubation pro-
tocol. The decrease in adherence in the competitive pro-
tocol cannot be attributed to antibacterial activity since
Figure 1 HPTLC profile of the decoction of P. guajava. (A) Metha-
nol fraction of the decoction. (B) Quercetin.
Figure 2 Antibacterial activity of decoction of P. guajava against
S. flexneri and V. cholerae. C: Control, O: Ofloxacin (1 μg/ml). Values
represent mean ± standard error (n = 3) of percentage bacterial
growth relative to control (100%); * P < 0.05.
CO0.11510CO0.115 10
0
20
40
60
80
100
120
Shigella flexneri Vibrio cholerae
*
*
*
*
*
*
*
Decoction (%)Decoction (%)
% Growth

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the decoction was not cidal to E. coli B170. Thus it is
likely that the components of the decoction act either on
HEp-2 cell metabolism or block the receptors on the cell
surface specific for bacterial adhesins thereby preventing
the attachment of the bacteria.
Invasion of E. coli E134 and S. flexneri to HEp-2 cells
As compared to lactulose (2.5 mg/ml) the crude decoc-
tion as well as quercetin showed greater inhibition of the
invasion of E. coli E134 and S. flexneri in both the pre-
incubation and the competitive protocols. The EC50 val-
ues for inhibition of invasion of E. coli E134 with the P.
guajava decoction in the pre-incubation (0.23% ± 0.02%)
and competitive (0.25% ± 0.01%) protocols were signifi-
cantly lower as compared to that of the EC50 values with
quercetin (39.36 μg/ml ± 3.27 μg/ml and 55.87 μg/ml ±
13.14 μg/ml respectively which correspond to 1.97% ±
0.16% and 2.79% ± 0.66% of the crude decoction). Simi-
larly, the EC50 values for inhibition of invasion of S. flex-
neri in the competitive (0.19% ± 0.06%) protocol with P.
guajava decoction was significantly lower as compared to
that of the EC50 value with quercetin (41.6 μg/ml ± 9.11
μg/ml corresponding to 2.08% ± 0.46% of the crude
decoction ) whereas in the pre-incubation protocol the
EC50 value with the decoction (0.19% ± 0.06%) was com-
parable with that of the EC50 value with quercetin (0.81
μg/ml ± 1.03 μg/ml corresponding to 0.04% ± 0.05% of
the crude decoction).
Since the extent of decrease in invasion was similar in
both the protocols it suggests that both the decoction and
quercetin probably affect HEp-2 cell metabolism. As dis-
cussed earlier, since the decoction and quercetin had no
effect on viability of E. coli E134, the reduction in inva-
sion of this bacterium in the competitive protocol cannot
be attributed to antibacterial activity. Likewise though
the decoction was cidal to S. flexneri, the decrease in
invasion of this strain in the competitive protocol even at
1% concentration (at which it had no bactericidal activity)
indicates that the effect of the decoction on invasion of
this bacterium was not only due to direct killing of the
bacteria.
It may be noted that the extent of decrease with querce-
tin was less than the decoction (Figure 4 and Figure 5). In
fact 2 μg and 20 μg of quercetin corresponding to 0.1%
and 1% respectively of the decoction showed no signifi-
cant reduction in invasion of E. coli E134 (Figure 4b) in
the two protocols used and surprisingly increased the
invasion of S. flexneri in the competitive protocol (Figure
5b).
Figure 3 Effect on adherence of E. coli B170 to the HEp-2 cells in
the pre-incubation and the competitive protocols with a) the de-
coction of P. guajava; b) quercetin. C: Control, bacterial adherence
to HEp-2 cells in medium alone; L1: Bacterial adherence to HEp-2 cells
when pre-incubated in medium with 2.5 mg/ml lactulose; L2: Bacterial
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 relative to control (100%); * P < 0.05.

a)
C0.11510C0.11510
0
20
40
60
80
100
120
Pre-incubation of HEp-2Competitive
*
*
* *
*
*
*
*
Decoction (%) Decoction (%)
L1
L2
*
% Adherence of E. coli B170


b)
C220100 200C220100200
0
20
40
60
80
100
120
Pre-incubation of HEp-2Competitive
Quercetin (μ μ μ μg/ml) Quercetin (μ μ μ μg/ml)
L1
L2
*
% Adherence of E. coli B170
Figure 4 Effect on invasion of E. coli E134 to the HEp-2 cells in the
pre-incubation and the competitive protocols with a) the decoc-
tion of P. guajava; b) quercetin. C: Control, bacterial invasion to HEp-
2 cells in medium alone; L1: Bacterial invasion to HEp-2 cells in medium
with 2.5 mg/ml lactulose. Values represent mean ± standard error (n =
3) of percentage invasion relative to control (100%); * P < 0.05.
a)
C0.11510C0.115 10
0
50
100
150
200
****
Pre-incubation of HEp-2Competitive
*
*
*
*
Decoction (%)Decoction (%)
L1
L1
*
% Invasion of E. coli E134


b)
C220100200C220100200
0
50
100
150
200
*
*
*
*
Pre-incubation of HEp-2 Competitive
Quercetin (μ μ μ μg/ml)
Quercetin (μ μ μ μg/ml)
L1
L1
*
% Invasion of E. coli E134

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Effect on bacterial enterotoxins
LT
The decrease in the production of LT by E. coli B831-2
(EC50 value 1.03% ± 0.32%) and its binding to GM1 (EC50
value 0.06% ± 0.02%) by the decoction was comparable to
that of 2-mercaptoethanol (5 mM) and gallic acid (50
mM) respectively (Figure 6a). Quercetin, on the other
hand, increased the production of LT at concentration of
100 μg/ml (Figure 6b) but did not affect the binding to
GM1. These results suggest that the decoction, in addi-
tion to quercetin, contains compounds which nullify the
enhancing effect of quercetin resulting in an overall
decrease in the production of LT. The effect of the decoc-
tion and quercetin on LT production by E. coli B831-2 is
indicative of their effect on bacterial metabolism.
CT
The decoction showed an overall greater decrease in pro-
duction of CT (EC50 value 2.69% ± 1.18%) and its binding
to GM1 (EC50 value 2.51% ± 1.07%) (Figure 7a) as com-
pared to 2-mercaptoethanol (1 mM) and gallic acid (50
mM) respectively. Quercetin did not inhibit the produc-
tion of CT but increased the binding of CT to GM1 (Fig-
ure 7b). As LT and CT are largely antigenically related
[33], the difference in the pattern of binding of LT and CT
to GM1 in presence of quercetin is suggestive of the addi-
tional affinity of quercetin to determinants specific to CT.
Figure 6 Effect on production of LT and its binding to GM1 with a)
the decoction of P. guajava; b) quercetin. C: Control, LT in medium
alone; M1: LT in medium with 5 mM 2-mercaptoethanol; G: Toxin in
medium with 50 mM gallic acid. Values represent mean ± standard er-
ror (n = 3) of percentage production/binding relative to control
(100%); * P < 0.05.
a)
C 0.11510C0.115 10
0
50
100
150
200
250
0
50
100
150
200
250
Production of LTBinding of LT to GM1
*
*
*
* *
*
*
Decoction (%)
Decoction (%)
*
*
G
M1
% Production of LT
% Binding of LT to GM1



b)
C220100200C220100200
0
50
100
150
200
250
0
50
100
150
200
250
Production of LTBinding of LT to GM1
Quercetin (μ μ μ μg/ml)
Quercetin (μ μ μ μg/ml)
*
*
*
G
M1
% Production of LT
% Binding of LT to GM1

Figure 7 Effect on production of CT and its binding to GM1 with
a) the decoction of P. guajava; b) quercetin. C: Control, toxin in me-
dium alone; M2: CT in medium with 1 mM 2-mercaptoethanol; G: Toxin
in medium with 50 mM gallic acid. Values represent mean ± standard
error (n = 3) of percentage production/binding relative to control
(100%); * P < 0.05.
a)

C0.11510C0.11510
0
50
100
150
200
250
0
50
100
150
200
250
Production of CTBinding of CT to GM1
*
*
*
*
Decoction (%)Decoction (%)
*
*
GM2
% Production of CT
% Binding of CT to GM1


b)

C220100200C220100200
0
50
100
150
200
250
0
50
100
150
200
250
Production of CTBinding of CT to GM1
Quercetin (μ μ μ μg/ml) Quercetin (μ μ μ μg/ml)
*
*
*
*
GM2
*
*
% Production of CT
% Binding of CT to GM1

Figure 5 Effect on invasion of S. flexneri to the HEp-2 cells in the
pre-incubation and the competitive protocols with a) the decoc-
tion of P. guajava; b) quercetin. C: Control, bacterial invasion to HEp-
2 cells in medium alone; L1: Bacterial invasion to HEp-2 cells in medium
with 2.5 mg/ml lactulose. Values represent mean ± standard error (n =
3) of percentage invasion relative to control (100%); * P < 0.05.
a)
C0.11510C0.11510
0
50
100
150
200
*
*
*
*
Pre-incubation of HEp-2Competitive
*
*
*
*
Decoction (%)Decoction (%)
L1
L1
*
% Invasion of S. flexneri



b)
C220100200C220100200
0
50
100
150
200
*
*
*
Pre-incubation of HEp-2 Competitive
*
*
*
*
Quercetin (μ μ μ μg/ml)Quercetin (μ μ μ μg/ml)
*
L1
L1
*
% Invasion of S. flexneri

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It may be noted that the decrease in production of CT
(unlike LT) by the decoction may be due to the cidal
action of the decoction against V. cholerae at 5% and 10%
concentrations. Thus analogous to the observations made
in the colonization assays, these results are also sugges-
tive of the presence of other 'active components' in the
decoction in addition to quercetin.
ST
The production and action of ST was unaffected by the
decoction as well as quercetin (data not shown). This sug-
gests the selective action of the decoction and quercetin
against LT and CT.
Discussion
The pathogenesis of infectious diarrhoea has been widely
studied. Enteric pathogens have evolved a remarkable
array of virulence traits that enable them to colonize the
intestinal tract. These organisms colonize and disrupt
intestinal function to cause malabsorption or diarrhoea
by mechanisms that involve microbial adherence and
localized effacement of the epithelium, production of
enterotoxin(s) and direct epithelial cell invasion [35].
Adherence is a means of colonizing the appropriate eco-
logical niche, which enables the organism to resist being
swept away by mucosal secretions, with subsequent pro-
liferation and colonization in the gut and may be followed
by enterotoxin production or invasion [36]. Thus, these
are important parameters in the pathogenesis of diar-
rhoea and can be used for understanding the varied
mechanism(s) of action of antidiarrhoeal medicinal
plants. These parameters have been employed by us [37-
42] to assess the antidiarrhoeal activity of selected medic-
inal plants. In the present study the antidiarrhoeal action
of P. guajava leaves has been assessed using a similar
approach. It may be noted that our studies deviate from a
number of other studies on antidiarrhoeal activity of
medicinal plants which are mostly restricted to intestinal
motility and antimicrobial activity [43-53] and thus over-
look the pathogenesis of infectious diarrhoea.
P. guajava is not only popular as a tropical plant which
is widely consumed and processed, but also as an impor-
tant food crop with several commercial applications and
medicinal properties [17]. It is considered as a native to
Mexico and extends throughout the South America,
Europe, Africa and Asia. It grows in all the tropical and
subtropical areas of the world and adapts to different cli-
matic conditions. Recent ethnopharmacological studies
have shown various medicinal uses for P. guajava apart
from its traditional use in gastrointestinal disorders, in
dermatologic conditions, as an anti-inflammatory, for
diabetes, hypertension, caries, wounds, pain relief, and
reducing fever [17]. Although all parts of this plant are
known to possess several medicinal properties, the leaves
and fruits, in particular, have been widely studied and
attributed with various pharmacological properties.
The restriction of crude extract for the study was inten-
tional and based on the belief that it would represent the
nearest possible form to traditional preparations that can
be used by the locals. This has also been emphasized
recently by Rosales-Reyes et al. [54]. Studies reporting the
antidiarrhoeal activity of the aqueous extracts of P. gua-
java leaves both in animal models as well as in clinical tri-
als [17] further strengthen the use of decoction in the
present study. Additionally, the safety of the crude aque-
ous extract of P. guajava leaves has been well demon-
strated. A LD50 value of 1534 ± 69 mg/kg (IP) in mice was
obtained in a recent study carried out by Ojewole et al.
[55]. Oral administration of 100-500 mg/Kg body weight
of the aqueous extract of P. guajava leaves has been
shown to have no significant harmful effect in Winstar
rats after 72 h [56]. The water extract of P. guajava leaves
was found to be effective in inactivating the mutagenic
effects of agents such as 4-nitro-o-phenylenediamine,
sodium azide and 2-aminofluorene on S. typhimurium in
Ames assay [57]. In vivo and in vitro cytotoxicity and
mutagenicity tests of aqueous extract of the leaves in
Winstar rat bone marrow cells and human peripheral
blood lymphocytes respectively did not show any statisti-
cally significant alterations in either the cell cycle or the
number of chromosome alterations [58].
Quercetin one of the most abundant flavonoids and
biologically active constituent present in P. guajava leaves
[17] has also been reported to have many beneficial
effects on human health, including cardiovascular pro-
tection, anti-cancer activity, anti-ulcer effects, anti-
allergy activity, cataract prevention, antiviral activity, and
anti-inflammatory effects [59]. Quercetin has been
reported to be antispasmodic [19,60,61] and also to
inhibit gastrointestinal release of acetylcholine [62].
Hence, in this study quercetin was also screened along
with the crude decoction of P. guajava leaves to elucidate
its contribution towards the efficacy of the decoction in
the bioassays employed herein.
Through the results of the present study, the antidiar-
rhoeal activity of the crude aqueous decoction of P. gua-
java is evident even in the absence of marked
antibacterial action. The decrease in bacterial coloniza-
tion of HEp-2 cells was seen in both the protocols (pre-
incubation and competitive) used. This indicates that P.
guajava affects HEp-2 cell metabolism. Similarly, the
decrease in production of LT without arresting the
growth of E. coli B831-2 is suggestive of P. guajava also
being able to affect bacterial metabolism.
On the other hand, quercetin had no effect on adher-
ence of E. coli B170 to HEp-2 cells in both the protocols
but affected the invasion of both E. coli E134 as well as S.

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flexneri in the two protocols used at 100 μg/ml and 200
μg/ml. Thus like the crude decoction, quercetin seems to
affect HEp-2 cell metabolism. However as quercetin did
not decrease bacterial adherence it is probable that unlike
the crude decoction it does not block the cell surface
receptors of HEp-2 cells. It is also possible that the modu-
lation of HEp-2 cell metabolism by quercetin may be at a
post adherence stage. Interestingly, the decrease in inva-
sion of S. flexneri as compared to E. coli E134 in the pre-
incubation protocol was noted at the lower concentra-
tions (2 μg/ml and 20 μg/ml) of quercetin (Figure 5b).
Since the mechanism of invasion of both EIEC and S.
flexneri is almost identical [28], at present it is difficult to
explain this observation. Though quercetin increased the
production of LT, like the crude decoction it seems to
affect bacterial metabolism as well.
Thus the comparative results of the crude decoction of
P. guajava leaves and quercetin illustrate that quercetin
alone is not responsible for all the observed biological
activities of the crude leaf decoction. It has been reported
previously that constituents such as tannins, flavonoids
and saponins in general have antidiarrhoeal activity
[43,47-50,53,63-66] which have been attributed to their
antimotility and antisecretory effects [45,50,51,64] and
antimicrobial action [47,48,52]. As tannins, flavonoids
and saponins were found to be present in the crude
decoction of P. guajava leaves, these constituents may be
responsible for the observed activities in the present
study. Therefore, it is speculated that the efficacy of crude
extract may be due to the interplay between the different
active constituents that may be present in the extract
leading to better activity and/or decrease in potential tox-
icity of some individual constituents. Alternatively, the
individual action of different constituents present in the
extract may collectively contribute to the efficacy of the
extract. This is illustrated by observations obtained from
the present study wherein the extent of decrease of bacte-
rial invasion with crude extract was greater than that with
quercetin alone and the ability of the crude decoction
(and not quercetin) to inhibit adherence and LT/CT. The
effect of the interplay of different constituents is also evi-
dent from the observation that the enhancement of inva-
sion of S. flexneri to HEp-2 cells in the presence of 2 μg/
ml and 20 μg/ml of quercetin (Figure 5b) was abrogated
in the crude decoction (Figure 5a). A similar observation
has been made by Mavar-Manga et al. [67] who demon-
strated that different constituents of crude extracts act on
different mechanisms.
Conclusion
The present study demonstrates the usefulness of P. gua-
java leaves in different forms of infectious diarrhoea. In
the absence of marked antibacterial activity, the inhibi-
tion of the production and the action of E. coli heat labile
toxin and cholera toxin and reduction of bacterial coloni-
zation (both adherence and invasion) to epithelial cells
are the probable modes of action of P. guajava leaves. The
study also showed that the crude decoction of P. guajava
leaves contains components other than quercetin which
contribute to its antidiarrhoeal action.
The mechanisms of antidiarrhoeal activity of P. guajava
leaves as proposed above along with other reported
mechanism(s) of action viz., antimicrobial activity, anti-
spasmodic activity, inhibition of increased watery secre-
tion and inhibition of acetylcholine release [17],
strengthen the ethnomedical usage of P. guajava leaf in
different forms of diarrhoea. Hence this study besides
providing newer insights into the varied possible antidi-
arrhoeal mechanisms of P. guajava leaves also adds cre-
dence to the existing traditional knowledge about this
plant and justifies its continued use globally.
Additional material
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TB and NA were responsible for the study. PD and BS carried out the laboratory
studies, analyzed the data and were involved in the preparation of the manu-
script. PT collected the plant material, authenticated it and obtained the
voucher specimen number. AN guided the phytochemical studies. All the
authors except Late Dr. Noshir Antia have read and approved the final version
of the manuscript.
Acknowledgements
This work has been supported by Department of Science and Technology,
Ministry of Science and Technology, Government of India (Grant No. 91283)
and Indian Council of Medical Research (Grant No. 59/10/2005/BMS/TRM). We
are thankful to Avinash Gurav and Santosh Jangam of Foundation for Research
in Community Health, Pune, for collection of plant material; Dr. Nerges Mistry,
Foundation for Medical Research, Mumbai, for her critical suggestions in the
study design; staff and students of Pharmacognosy Department, Principal K. M.
Kundanani College of Pharmacy, Mumbai; Dr. D. Charegaonkar of Anchrom
Enterprises (I) Pvt. Ltd., Mumbai; Mr. B. N. Vyas and Mr. R. Iyer of Godrej Agrovet
Ltd., Mumbai; and Dr. V. R. Bhate, Analytical Solutions, Navi Mumbai; for assis-
tance in phytochemical studies. We also thank Prof. S. V. George of St. Xavier's
College, Mumbai for assistance in statistical analysis.
Author Details
1The Foundation for Medical Research, 84A, RG Thadani Marg, Worli, Mumbai
400018, Maharashtra, India, 2Naoroji Godrej Centre for Plant Research, Lawkin
Ltd. Campus, Shindewadi, Shirwal, Satara 412801, Maharashtra, India, 3Indian
Institute of Science Education and Research, Central Tower, Sai Trinity, Garware
Circle, Sutarwadi, Pashan, Pune 411021, Maharashtra, India and 4The
Foundation for Research in Community Health, 3-4, Trimiti-B Apartments, 85,
Anand Park, Pune 411 007, Maharashtra, India
Additional file 1 HPLC profile of the decoction of P. guajava. HPLC pro-
file of the acid hydrolysed decoction of P. guajava shows the presence of
the standard reference compound quercetin.
Received: 22 April 2010 Accepted: 28 June 2010
Published: 28 June 2010
This article is available from: http://www.biomedcentral.com/1472-6882/10/33© 2010 Birdi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
BMC Complementary and Alternative Medicine 2010, 10:33

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Pre-publication history
The pre-publication history for this paper can be accessed here:
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Cite this article as: Birdi et al., Newer insights into the mechanism of action
of Psidium guajava L. leaves in infectious diarrhoea BMC Complementary and
Alternative Medicine 2010, 10:33