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BioMed Central
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BMC Complementary and
Alternative Medicine
Open Access
Research article
Studies on the antidiarrhoeal activity of Aegle marmelos unripe fruit:
Validating its traditional usage
S Brijesh†1, Poonam Daswani†1, Pundarikakshudu Tetali2, Noshir Antia^1,3
and Tannaz Birdi*1
Address: 1The Foundation for Medical Research, 84A, R. G. Thadani Marg, Worli, Mumbai 400018, Maharashtra, India, 2Naoroji Godrej Centre
for Plant Research, Lawkin Ltd. Campus, Shindewadi, Shirwal, Satara 412801, Maharashtra, India and 3The Foundation for Research in
Community Health, 3-4, Trimiti-B Apartments, 85, Anand Park, Pune 411 007, Maharashtra, India
Email: S Brijesh - brijeshsuku@rediffmail.com; Poonam Daswani - poonam0214@yahoo.co.in; Pundarikakshudu Tetali - ptetali@godrej.com;
Noshir Antia - frchpune@bsnl.in; Tannaz Birdi* - fmr@fmrindia.org
* Corresponding author †Equal contributors ^Deceased
Abstract
Background: Aegle marmelos (L.) Correa has been widely used in indigenous systems of Indian
medicine due to its various medicinal properties. However, despite its traditional usage as an anti-
diarrhoeal there is limited information regarding its mode of action in infectious forms of diarrhoea.
Hence, we evaluated the hot aqueous extract (decoction) of dried unripe fruit pulp of A. marmelos
for its antimicrobial activity and effect on various aspects of pathogenicity of infectious diarrhoea.
Methods: The decoction was assessed for its antibacterial, antigiardial and antirotaviral activities.
The effect of the decoction on adherence of enteropathogenic Escherichia coli and invasion of
enteroinvasive E. coli and Shigella flexneri to HEp-2 cells were assessed as a measure of its effect on
colonization. The effect of the decoction on production of E. coli heat labile toxin (LT) and cholera
toxin (CT) and their binding to ganglioside monosialic acid receptor (GM1) were assessed by GM1-
enzyme linked immuno sorbent assay whereas its effect on production and action of E. coli heat
stable toxin (ST) was assessed by suckling mouse assay.
Results: The decoction showed cidal activity against Giardia and rotavirus whereas viability of none
of the six bacterial strains tested was affected. It significantly reduced bacterial adherence to and
invasion of HEp-2 cells. The extract also affected production of CT and binding of both LT and CT
to GM1. However, it had no effect on ST.
Conclusion: The decoction of the unripe fruit pulp of A. marmelos, despite having limited
antimicrobial activity, affected the bacterial colonization to gut epithelium and production and
action of certain enterotoxins. These observations suggest the varied possible modes of action of
A. marmelos in infectious forms of diarrhoea thereby validating its mention in the ancient Indian
texts and continued use by local communities for the treatment of diarrhoeal diseases.
Published: 23 November 2009
BMC Complementary and Alternative Medicine 2009, 9:47 doi:10.1186/1472-6882-9-47
Received: 14 July 2009
Accepted: 23 November 2009
This article is available from: http://www.biomedcentral.com/1472-6882/9/47
© 2009 Brijesh 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 2009, 9:47 http://www.biomedcentral.com/1472-6882/9/47
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Background
India has a rich heritage of traditional knowledge and is
home to several important time-honored systems of
health care like Ayurveda, Siddha and Unani. It has been
estimated that the proportion of medicinal plants in India
(7,500 of the 17,000 higher plant species are medicinal
plants) is higher than any country of the world with
respect to the existing flora of that respective country
[1,2].
Aegle marmelos (L.) Correa commonly known as Bael/Bilva
belonging to the family Rutaceae has been widely used in
indigenous systems of Indian medicine due to its various
medicinal properties. Although this plant is native to
northern India it is also widely found throughout the
Indian peninsula and in Ceylon, Burma, Thailand and
Indo-China. A. marmelos tree is held sacred by Hindus and
offered in prayers to deities Lord Shiva and Parvati and
thus the tree is also known by the name Shivaduma (the
tree of Shiva) [3]. Hindus also believe that goddess Lak-
shmi resides in Bael leaves. It is therefore widely cultivated
and commonly found in the vicinity of temples.
All parts of this tree, viz. root, leaf, trunk, fruit and seed are
useful in several ailments. The root is an important ingre-
dient of the 'Dasmula' (ten roots) recipe [4]. The decoction
of the root and root bark is useful in intermittent fever,
hypo-chondriasis, melancholia, and palpitation of the
heart [5]. The leaves and bark have been used in medi-
cated enema. The leaves are also used in diabetes mellitus.
The greatest medicinal value, however, has been attrib-
uted to its fruit [4] and the unripe fruit is said to be an
excellent remedy for diarrhoea and is especially useful in
chronic diarrhoeas [4-6]. The effectiveness of A. marmelos
fruit in diarrhoea and dysentery has resulted in its entry
into the British Pharmacopoeia [4]. Moreover, Chopra [4]
has appropriately stated that "No drug has been longer
and better known nor more appreciated by the inhabit-
ants of India than the Bael fruit." Charaka has described
this plant as a Rasayana [7].
Despite the traditional use of A. marmelos unripe fruit as
an antidiarrhoeal, few studies have reported its antidiar-
rhoeal activity. According to Chopra [4], A. marmelos is
effective in chronic cases of diarrhoea due to the presence
of large quantities of mucilage, which act as a demulcent.
Additionally, A. marmelos has been shown to be effective
in experimental models of irritable bowel syndrome and
physiological diarrhoea [8-10]. However, to the best of
our knowledge, besides antiprotozoal studies [11], effect
of A. marmelos unripe fruit in infectious diarrhoea has not
been reported.
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 mal-absorption or diarrhoea
by mechanisms that involve microbial adherence and
localized effacement of the epithelium, production of
toxin(s) and direct epithelial cell invasion [12]. Adher-
ence which is a means of colonizing the appropriate eco-
logical niche enables the organism to resist being swept
away by mucosal secretions. Adherence also aids in subse-
quent proliferation and colonization of the gut and may
be followed by toxin production or invasion [13]. The
importance of using colonization and production and
action of enterotoxins as specific parameters reflecting the
pathogenesis has been earlier used by us as an approach
towards understanding the varied mechanism(s) of action
of antidiarrhoeal medicinal plants against infectious diar-
rhoea [14-17]. The studies highlighted the necessity of
including multiple parameters for assessing effectiveness
of medicinal plants against infectious forms of diarrhoea,
especially in the absence of antimicrobial activity. Thus
our studies deviate from a number of other studies that
are restricted to intestinal motility and antibacterial activ-
ity as markers for antidiarrhoeal activity [18-29]. In this
study, we evaluated the decoction of dried unripe fruit
pulp of A. marmelos for its effect on various parameters of
diarrhoeal pathogenicity, viz., adherence to and invasion
of intestinal epithelium and production and action of
enterotoxins to elucidate its mechanism(s) of action in
infectious diarrhoea.
Methods
Plant material and preparation of decoction
A February collection of the unripe fruits of A. marmelos
collected from Pangare village in the Parinche valley,
about 53 km south east of Pune city in the state of Mahar-
ashtra, India, was used for the present study. February was
chosen as the month of collection since the fruits achieve
full size at this time but are still unripe. The plant material
was authenticated by Dr. P. Tetali, Head, Research and
Development, Naoroji Godrej Centre for Plant Research,
Shirwal, Maharashtra, India. A voucher specimen has
been deposited at the Botanical Survey of India (Western
Circle), Pune, India, under herbarium number 124675.
The fruits were cut into small pieces, shade dried and
stored at 4°C.
A crude aqueous extract (decoction) was used for the
study since it represents the nearest form to traditional
preparations. The decoction was prepared as described in
Ayurvedic text [30]. 1 g of the powdered dried fruit pulp
was boiled in 16 ml double distilled water till the volume
reduced to 4 ml. It was centrifuged and filtered through a
0.22 μm membrane before use. To replicate field condi-
tions, each assay was performed with freshly prepared
decoction.
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The dry weight of the decoction thus obtained was 51.1
mg/ml ± 0.5 mg/ml and 20.4% ± 2.11% (w/w) with
respect to the starting dried plant material. The decoction
was diluted 1:100, 1:20 and 1:10 in appropriate media for
each experiment and has been referred to as 1%, 5%, and
10% respectively throughout the text. The dry weight con-
tents of these dilutions of the decoction were 0.51 mg/ml
± 0.005 mg/ml, 2.55 mg/ml ± 0.025 mg/ml and 5.11 mg/
ml ± 0.05 mg/ml respectively.
Media, reagents, plastic ware and instrumentation
The bacterial media and the Minimal Essential Medium
(MEM) were purchased from HiMedia laboratory, Mum-
bai, India. Dulbecco's Modified Eagle's Medium (DMEM)
and fetal calf serum (FCS) were procured from GibcoBRL,
UK. The constituents of the Diamond's TYI-SS medium
were procured from local Indian manufacturers, as were
the antibiotics. Trypan blue, neutral red, ganglioside mon-
osialic acid (GM1), anti-cholera toxin, ortho-phenylene
diamine, bovine serum and bovine serum albumin were
purchased from Sigma, USA. Peroxidase labeled swine
anti-rabbit immunoglobulin was purchased from Dako,
Denmark. All chemicals were from SD Fine Chemicals,
Mumbai. Standard marmelosin was purchased from Nat-
ural Remedies, Bangalore, India. Gallic acid was kindly
provided by Dr. KS Laddha, University Institute of Chem-
ical Technology, 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,
Kolkata, India, and the ELISA plate reader was purchased
from Labsystems, Finland.
Cell culture
The human laryngeal epithelial cell line, HEp-2, and the
embryonic monkey kidney derived cell line, MA-104,
were obtained from National Centre for Cell Sciences,
Pune, India. The cell lines were maintained in DMEM and
MEM respectively, supplemented with 10% FCS, at 37°C
in a 5% CO2 atmosphere. The cells were maintained in
logarithmic growth by passage 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 strain TX1, serotype 078:H12 (heat
stable toxin producer) (all strains obtained from Centre
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.
In addition, Giardia lamblia P1 trophozoites (kindly pro-
vided by Dr. P. Das, National Institute for Cholera and
Enteric Diseases, Kolkatta, India) and simian rotavirus SA-
11 (kindly provided by Dr. S. Kelkar, National Institute of
Virology, Pune, India) were also included.
Phytochemical analysis
Qualitative phytochemical analysis of the decoction was
carried out for assaying presence of carbohydrates, glyco-
sides, proteins, amino acids, phytosterols, saponins, fla-
vonoids, alkaloids and tannins [31]. High performance
thin layer chromatography (HPTLC) fingerprinting of the
methanol soluble fraction of the decoction was carried
out with the solvent system n-Hexane:Ethyl acetate:Acetic
acid (40:60:0.5). Marmelosin was used as a phytochemi-
cal reference standard for fingerprinting.
Antimicrobial activity
Antibacterial activity
The antibacterial activity was determined by a microtitre
plate-based assay [32]. The bacterial strains were incu-
bated in the absence (control) and presence of different
dilutions of the decoction in nutrient broth and the opti-
cal density was measured after 24 h as a measure of
growth. Three independent experiments were carried out.
In each experiment, triplicate wells were set up for control
as well as each dilution of the decoction.
Antigiardial activity
A 24 h culture of G. lamblia P1 trophozoites was incubated
in the absence (control) and presence of different dilu-
tions of the decoction in Diamond's TYI-SS medium. The
number of viable trophozoites after 24 h was counted in
a haemocytometer using trypan blue [33]. Three inde-
pendent experiments were carried out. In each experi-
ment, duplicate tubes were set up for control as well as
each dilution of the decoction.
Antirotaviral activity
The entry and subsequent survival of rotavirus SA-11 in
MA-104 cells was assayed by the neutral red uptake assay
[34]. Briefly, MA-104 cells were grown in 96-well tissue
culture plates for 72 h after which they were infected with
rotavirus for 90 min in absence (control) and presence of
different dilutions of the decoction. Subsequently, the
extracellular virus and the decoction were washed off and
the culture was further incubated for 72 h. Thereafter, the
cells were incubated with 0.03% neutral red dye for 30
min. The intracellular dye was released with 1:1 solution
of 100 mM acetic acid and ethanol and the intensity meas-
ured at 540 nm (reference 630 nm) in an ELISA plate
reader. Three independent experiments were carried out.
In each experiment, triplicate wells were set up for control
as well as each dilution of the decoction.
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Effect on bacterial colonization
Effect on adherence
The effect on the adherence of E. coli strain B170 to epithe-
lial cells was assayed by the method described by Cravioto
et al. [35]. Briefly, a 48 h culture of HEp-2 cells on glass
coverslips was infected with a log phase culture of the bac-
terium (5 × 107/ml) and incubated for 3 h. Non-adherent
bacteria were washed off, the coverslips were fixed in 10%
formaldehyde and stained with toluidine blue stain (0.1%
w/v). HEp-2 cells having typical EPEC micro-colonies [36]
were counted under light microscope. Three independent
experiments were carried out. In each experiment, dupli-
cate cover-slips were set up for control as well as each dilu-
tion of the decoction.
Effect on invasion
The effect on invasion of E. coli E134 and S. flexneri into
epithelial cells was studied by the method described by
Vesikari et al. [37]. Briefly, a 48 h culture of HEp-2 cells
grown in a 24-well tissue culture plate was infected with
log phase culture of the bacteria (108/ml) and incubated
for 2 h. The culture was further incubated with gentamy-
cin (100 μg/ml) for 3 h. The epithelial cells were then
lysed by cold shock with chilled distilled water and the
released bacteria were counted by plating on nutrient
agar. Three independent experiments were carried out. In
each experiment, duplicate wells were set up for control as
well as each dilution of the decoction.
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 on the epithelial
cells or through competitive inhibition. The HEp-2 cells
were incubated in absence (control) and presence of dif-
ferent dilutions of the decoction either for 18-20 h prior
to infection (pre-incubation) or 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 bacterial cell membrane, was
obtained by lysing E. coli B831-2 with polymyxin B sul-
phate (1 mg/ml) whereas CT, which is released extracellu-
larly, was obtained as a culture supernatant of V. cholerae.
LT and CT were assayed by the GM1-enzyme linked
immunosorbent assay (GM1-ELISA) [38]. Briefly, the tox-
ins were added to ELISA plates pre-coated with 1.5 μmol/
ml of GM1. Anti-cholera toxin and peroxidase labeled
swine anti-rabbit immunoglobulin used at dilutions of
1:300 and 1:200 were used as primary and secondary anti-
bodies respectively. Ortho-phenylene diamine (6 mg)
with hydrogen peroxide (4 μl) in 10 ml citrate buffer (pH
5.5) was used as the substrate. The intensity of the color
thus developed was read at 492 nm in an ELISA plate
reader.
To study the effect on production of the toxins, the respec-
tive bacteria were grown in Casein Hydrolysate Yeast
Extract (CAYE) in absence (control) and presence of dif-
ferent dilutions of the decoction 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. Three independent experiments were carried
out. In each experiment, triplicate wells were set up for
control as well as each dilution of the decoction.
Effect on E. coli heat stable toxin (ST)
ST was assayed by the method originally described by
Gianella [39]. Briefly, ST, which is released extracellularly,
was obtained as the culture supernatant of E. coli TX1. The
toxin was inoculated intra-gastrically in 2-3 days old Swiss
White suckling mice. Following an incubation of 3 h at
room temperature, the pups were sacrificed and the ratio
of gut weight to that of the remaining carcass weight was
calculated. Ratio of ≥ 0.083 was considered as positive.
To study the effect on production of ST, the bacterium was
grown in CAYE in absence (control) and presence of dif-
ferent dilutions of the decoction 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
intra-gastrically injected in absence (control) and pres-
ence of different dilutions of the decoction. CT was used
as a negative control. Three independent experiments
were carried out. In each experiment, three animals were
inoculated for control as well as each dilution of the
decoction.
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/CPC-
SEA, 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 inde-
pendent 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 read-
ings of the control group and T is mean value of the dupli-
cate/triplicate readings of the test (dilutions of the
decoction) groups. Hence, the value of control is 100%
and the values of the test groups have been represented as
percentages relative to control.
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Data were analyzed by analysis of variance (ANOVA) and
Dunnett's post-test. Statistical analyses were performed
using the software Prism 4.0 (GraphPad, Inc.). P ≤ 0.05
was considered to be statistically significant.
Results
Phytochemistry
The decoction contained carbohydrates, glycosides,
amino acids, proteins, tannins, flavanoids, and phytoster-
ols. The results were similar to earlier reports [40,41]. The
chromatogram of the HPTLC fingerprinting analysis of
the methanol soluble fraction of the decoction scanned at
254 nm has been presented in Fig 1.
Antimicrobial activity
In comparison to ofloxacin (1 μg/ml), which completely
inhibited all the six bacterial strains tested, the decoction
did not inhibit the growth of any of the bacteria (data not
shown). The decoction, however, affected growth of G.
lamblia. The number of viable trophozoites was signifi-
cantly lower (approximately 50%) at 10% dilution of the
decoction (Fig 2), as observed by trypan blue staining. The
surviving trophozoites failed to multiply when provided
with fresh medium indicating that A. marmelos was cidal
for giardia. However, the decrease though statistically sig-
nificant was less than that observed with metronidazole
(10 μg/ml), which resulted in almost 90% killing of the
trophozoites.
Rotavirus, following entry, lyses MA-104 cells and hence
the number of viable cells remaining at the end of the
assay is an indirect measure of antirotaviral activity of the
decoction. It was observed that compared to the control,
cell death was decreased following infection with the virus
in the presence of 10% dilution of the decoction (Fig 3)
indicating that the decoction was inhibitory to the virus at
this dilution.
Effect on bacterial colonization
Fig 4A shows characteristic micro-colony formation typi-
cal of EPEC whereas Fig 4B shows a representative image
of the effect on the micro-colony formation on HEp-2
cells in presence of 10% dilution of the decoction. As can
be seen in Fig 4B, the decoction was not cytotoxic to HEp-
2 cells. The adherence of E. coli B170 to HEp-2 cells was
significantly reduced by the decoction in both the proto-
cols (Fig 4C).
The effect of the decoction on adherence of E. coli B170 to
HEp-2 cells was compared with that of lactulose, a prebi-
otic oligosaccharide, known to inhibit adherence of EPEC
to tissue culture cells [42]. As compared to the control the
adherence of E. coli B170 to the HEp-2 cells in the pres-
ence of 10% dilution of the decoction in the competitive
HPTLC of the decoction of A. marmelosFigure 1
HPTLC of the decoction of A. marmelos. (A) Methanol
fraction of the decoction. (B) Marmelosin.
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protocol was 43.42% ± 2.51% whereas it was 45.44% ±
5.44% in the presence of lactulose (15 mg/ml). In the pre-
incubation protocol, lactulose was toxic to HEp-2 cells
even at 5 mg/ml when incubated overnight with the cells.
Below this concentration lactulose had no effect on the
adherence. The decoction, on the other hand, showed
47.49% ± 1.82% adherence of E. coli B170 to HEp-2 cells
at 10% dilution in the pre-incubation protocol.
The decoction also significantly reduced the invasion of
both E. coli E134 and S. flexneri in both protocols (Fig 5A
and Fig 5B respectively). The effect of the decoction on
invasion of S. flexneri was compared with that of lactulose,
as it has been used for the treatment of shigellosis and
inflammatory bowel disease [43]. Since the mechanism of
invasion of both EIEC and S. flexneri is almost identical
[44], the effect of the decoction on invasion of E. coli E134
was also compared with that of lactulose. The decoction
showed maximum decrease in invasion at 10% dilution
with 28.87% ± 7.37% invasion of E. coli E134 and 14.78%
± 6.84% invasion of S. flexneri in the competitive proto-
col. In comparison, lactulose (2.5 mg/ml) showed
60.38% ± 5.94% and 31.68% ± 8.29% invasion for E. coli
E134 and S. flexneri respectively. In the pre-incubation
protocol, as seen in the adherence assay, lactulose was
found to be toxic to HEp-2 cells even at 5 mg/ml when
incubated overnight with the cells. Below this concentra-
tion lactulose had no effect on the invasion of either strain
to the HEp-2 cells. The decoction, on the other hand,
showed 14.13% ± 4.65% and 12.93% ± 7.68% invasion
of E. coli E134 and S. flexneri respectively to HEp-2 cells at
10% dilution in the pre-incubation protocol.
Effect on bacterial enterotoxins
On incubation of V. cholerae with the decoction, the pro-
duction of CT was inhibited (Fig 6B). The decoction
showed maximum inhibition of production of CT at 10%
dilution with the percent production being 55.56% ±
9.72% compared to control. However, production of LT
by E. coli B831-2 was not affected (Fig 6A). The effect of
the decoction on production of these toxins was com-
pared with that of 2-mercaptoethanol since thiols such as
2-mercaptoethanol, L-cysteine monohydrochloride and
sodium thioglycolate have been reported to inhibit pro-
duction of LT, CT and ST [45,46]. The production of LT
and CT in presence of 2-mercaptoethanol (5 mM and 1
mM respectively) was 53.27% ± 5.30% and 51.63% ±
3.40% respectively. The bacterial growth was not affected
at these concentrations of 2-mercaptoethanol (data not
shown).
The binding of both LT (Fig 6A) and CT (Fig 6B) to GM1,
on the other hand, was affected by the decoction. The
decoction showed maximum inhibition at 10% dilution
with the binding of these toxins to GM1 being 75.42% ±
7.34 and 56.58% ± 5.99% respectively compared to con-
trol. The effect of the decoction on binding of these toxins
to GM1 was compared with that of gallic acid, a polyphe-
nol, as it is reported to block the binding of LT to GM1
[47]. As LT and CT are antigenically closely related [48],
the effect of the decoction on the binding of CT was also
compared to that of gallic acid. The binding of LT and CT
to GM1 in presence of gallic acid (50 mM) was 48.36% ±
6.35% and 50.04% ± 6.56% respectively.
The production and action of ST was not affected by the
decoction at any of the dilutions tested (data not shown).
Discussion
Diarrhoeal diseases are amongst the most common infec-
tious diseases worldwide resulting in 3.2% of all deaths
killing about 1.8 million people globally each year [49].
Annually, diarrhoeal diseases kill over 1.5 million chil-
dren globally [50]. Even though economic development
and progress in health care delivery are expected to cata-
lyze substantial improvements in infectious disease
related morbidity and mortality by the year 2020, it is pre-
dicted that diarrhoea will remain a leading health prob-
lem [51]. It affects mostly children in developing
countries and can lead to dehydration and death and in
survivors to impaired growth and malnutrition [52]. In
adults, while the impact is less severe, it nevertheless can
lead to nutritional deficiencies especially in the case of
persistent diarrhoea [53].
A. marmelos has been used for centuries in India not only
for its dietary purposes but also for its various medicinal
properties [4-6]. The fruit is widely consumed as 'serbet'
Antigiardial activity of the decoction of A. marmelosFigure 2
Antigiardial activity of the decoction of A. marmelos.
C: Control, trophozoites in medium alone; M: trophozoites
incubated in medium with metronidazole (10 μg/ml); D: tro-
phozoites incubated in medium with decoction. Values rep-
resent mean ± standard error (n = 3) of percentage viable
trophozoites relative to control (100%); * P < 0.05.
C M 1% D 5% D 10% D
0
50
100
150
*
*
% Viable Trophozoites
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(liquid fruit concentrate) and 'murbha' (jam) and the
unripe fruit is highly recommended for diarrhoea and is
especially for chronic diarrhoea [3-6,54]. Hence, it is gen-
erally considered safe and few studies have been carried
out with respect to its toxicity. Nevertheless, aqueous
extract of A. marmelos fruit has been reported to be non
mutagenic to Salmonella typhimurium strain TA 100 in the
Ames assay [55]. In addition, acute toxicity studies have
reported that a hydroalcoholic extract of A. marmelos fruit
is non-toxic up to a dose of 6 g/kg body weight in mice
[56]. Pharmacological studies on animal models involv-
ing repeated doses of A. marmelos fruit extract over a
period of up to 30 days have not reported any adverse
effect up to a maximum dose of 250 mg/kg body weight
[56-59]. The decoction of A. marmelos showed no cyto-
toxic activity on HEp-2 cells in the present study even at
the highest concentration tested (Fig. 4B).
Though a few studies have been carried out on the antidi-
arrhoeal activity of A. marmelos [8-10], no reports are
available pertaining to its activity in infectious diarrhoea.
The present work with the crude aqueous extract of dried
unripe fruit pulp of A. marmelos provides an insight into
its possible mechanism of action in infectious diarrhoea
and validates its traditional use as an antidiarrhoeal. The
study has intentionally been undertaken using a crude
aqueous extract as it is our belief that the different biolog-
ical activities assayed herein may not be due to a single
constituent. This has also been highlighted by Mavar-
Manga et al. [60] who have stated that crude extracts con-
tain several compounds acting on different mechanisms.
In addition, interplay of the constituents in the crude
extract may result in better activity due to synergism or
lead to decrease in toxicity and it is possible that pure
compound(s) may not necessarily behave in the same
manner as the natural extract [61,62].
The decoction of A. marmelos exhibited antigiardial and
antirotaviral activity whereas it did not show any antibac-
terial activity. The results show that despite not being bac-
tericidal, the antidiarrhoeal effect of this plant is possibly
due to its ability to affect other bacterial virulence param-
eters.
A. marmelos prevented the colonization by E. coli B170, E.
coli E134 and S. flexneri. The reduction in colonization is
probably due to its effect on the metabolism of HEp-2
cells and/or modification of cell receptors to prevent
adherence or bacterial entry as seen on the pre-incubation
of HEp-2 with the decoction. The decoction exhibited
greater inhibition of invasion of E. coli E134 and S. flexneri
as compared to adherence of E. coli B170 in both proto-
cols. This indicates that the decrease in invasion may not
merely be due to the inhibition of initial attachment of
the bacteria to the epithelial cells by the plant decoction
but also could be due to its effect on the engulfment proc-
ess of the bacteria at a post adherence stage. Thus the
results of both adherence and invasive assays, representa-
tive of the colonization of the pathogens to the intestinal
epithelium, indicate that A. marmelos does not permit the
pathogens to establish themselves. It may be noted that
since the adherence of the pathogen to the gut epithelium
is the foremost stage of the disease process, inhibition of
adherence could be a very important aspect in the antidi-
arrhoeal activity of the plant.
The decoction also reduced the binding of both LT and CT
to the GM1 thereby inhibiting their action. LT and CT are
known to be antigenically similar [48]. Hence, the effect
of the decoction on their binding suggest that it may con-
tain some compound(s), which either bind to the com-
mon antigenic moiety of these toxins or may directly
block the GM1 on the cell membrane thereby inhibiting
their binding to the receptor. In addition, though the
decoction had no effect on production of LT it inhibited
the production of CT. Since the decoction had no cidal
activity against V. cholerae, suppression of CT production
suggests that the decoction affected the bacterial metabo-
lism.
Literature shows presence of mucilage, pectin, coumarins
such as marmelosin and marmelide, and tannins in A.
marmelos fruits [11,54,63,64]. In the current study, the
qualitative phytochemical analysis of the decoction
showed presence of carbohydrates, glycosides, amino
acids, proteins, tannins, flavanoids, phytosterols and the
HPTLC analysis showed the presence of marmelosin. Tan-
nins and flavonoids in general have been reported to have
Antirotaviral activity of the decoction of A. marmelosFigure 3
Antirotaviral activity of the decoction of A. marmelos.
C: Control, rotavirus infected MA-104 cell s in medium alone;
D: Rotavirus infected MA-104 cells in medium with decoc-
tion. Values represent mean ± standard error (n = 3) of per-
centage viable MA-104 cells relative to control (100%); * P <
0.05.
C1% D 5% D 10% D
0
20
40
60
80
100
*
% Death of MA-104 cells
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Page 8 of 12
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antidiarrhoeal activity through inhibition of intestinal
motility, antimicrobial action and antisecretory effects
[20,23,24,26-28,65]. However, none of the isolated
chemical constituents from the plant have been specifi-
cally studied for their antidiarrhoeal activity including
effect on colonization and production and action of enter-
otoxins.
Conclusion
The present study validates the use of unripe fruit of A.
marmelos as an anti-diarrhoeal agent in traditional medi-
cine. The results obtained in the study suggest that the
decoction of A. marmelos can control several forms of
infectious diarrhoeal diseases caused by EPEC, EIEC, LT
producing ETEC, V. cholerae, S. flexneri and to some extent
it can also control giardiasis and rotaviral infections.
However, it may not be effective against diarrhoea caused
by ST producing ETEC.
The study emphasizes that the bioassays used in the
present study which represent intestinal pathology can be
employed as possible novel targets for studying antidiar-
Effect of the decoction of A. marmelos on bacterial adherence to HEp-2 cellsFigure 4
Effect of the decoction of A. marmelos on bacterial adherence to HEp-2 cells. (A) E. coli B170 microcolonies (arrows)
on HEp-2 cells in medium alone. (B) E. coli B170 microcolonies (arrow heads) on HEp-2 cells when incubated in medium with
10% dilution of the decoction. (C) Adherence of E. coli B170 to the HEp-2 cells in the pre-incubation (HEp-2 cells incubated
with the decoction prior to infection) and the competitive (HEp-2 cells incubated with the decoction simultaneously with the
infection) protocols. C: Control, adherence to HEp-2 cells in medium alone; L1: Adherence to HEp-2 cells when pre-incubated
in medium with 2.5 mg/ml lactulose; L2: Adherence to HEp-2 cells in medium with 15 mg/ml lactulose in the competitive proto-
col; D: Adherence to HEp-2 cells in medium with decoction. Values represent mean ± standard error (n = 3) of percentage
adherence relative to control (100%); * P < 0.05.
C1% D 5% D 10% D C1% D 5% D 10% D
0
20
40
60
80
100
120
140
*
*
*
*
Pre-incubation Competitive
*
L
1
L
2
C)
% Adherence of E. coli B170
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Page 9 of 12
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rhoeal activity of medicinal plants, especially in absence
of antimicrobial activity. It, therefore, provides a new
basis for the development of potent antidiarrhoeal ther-
apy from medicinal plants. In addition, the study also
highlights the importance of using relevant and where
necessary multiple bioassays covering the entire spectrum
of activities that can provide a more reliable evaluation of
the biological efficacy of medicinal plants.
Effect of the decoction of A. marmelos on bacterial invasion to HEp-2 cellsFigure 5
Effect of the decoction of A. marmelos on bacterial invasion to HEp-2 cells. (A) Invasion of E. coli E134 to HEp-2 cells
in the pre-incubation (HEp-2 cells incubated with the decoction prior to infection) and the competitive (HEp-2 cells incubated
with the decoction simultaneously with the infection) protocols. (B) Invasion of S. flexneri to HEp-2 cells in the pre-incubation
(HEp-2 cells incubated with the decoction prior to infection) and the competitive (HEp-2 cells incubated with the decoction
simultaneously with the infection) protocols. C: Control, invasion to HEp-2 cells in medium alone; L1: Invasion to HEp-2 cells in
medium with 2.5 mg/ml lactulose; D: Invasion to HEp-2 cells in medium with decoction. Values represent mean ± standard
error (n = 3) of percentage invasion relative to respective control (100%); * P < 0.05.
C1% D 5% D 10% D C1% D 5% D 10% D
0
20
40
60
80
100
120
140
*
*
*
Pre-incubation Competitive
*
L
1
L
1
A)
% Invasion by E. coli E134
C1% D 5% D 10% D C1% D 5% D 10% D
0
20
40
60
80
100
120
140
*
*
*
*
Pre-incubation Competitive
*
L1L1
B)
% Invasion by S. flexneri
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Page 10 of 12
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Competing interests
The authors declare that they have no competing interests.
Authors' contributions
BS and PD carried out the laboratory studies, helped in
analysis of data and preparation of manuscript. PT col-
lected the plant material, authenticated it and obtained a
voucher specimen number. NA and TB were responsible
for the study. All the authors except Late Dr. Noshir Antia
have read and approved the final version of the manu-
script.
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 Foun-
Effect of the decoction of A. marmelos on bacterial enterotoxinsFigure 6
Effect of the decoction of A. marmelos on bacterial enterotoxins. (A) Production of E. coli heat labile toxin (LT) and its
binding to GM1. (B) Production of cholera toxin (CT) 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: Toxin in medium with 50 mM
gallic acid; D: Toxin in presence of decoction. Values represent mean ± standard error (n = 3) of percentage production/bind-
ing relative to respective control (100%); * P < 0.05.
C1% D 5% D 10% D C1% D 5% D 10% D
0
20
40
60
80
100
120
140
0
20
40
60
80
100
120
140
**
Effect on Production Effect on Binding to GM1
G
M
1
*
*
A)
% Production of LT
% Binding of LT to GM1
C1% D 5% D 10% D C1% D 5% D 10% D
0
20
40
60
80
100
120
140
0
20
40
60
80
100
120
140
**
*
*
Effect on Production Effect on Binding to GM1
GM
2
*
*
B)
% Production of CT
% Binding of CT to GM1
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Page 11 of 12
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dation for Research in Community Health, Pune for collection of plant
material; Dr. Nerges Mistry, Foundation for Medical Research, for her crit-
ical suggestions in the study design; staff and students, Pharmacognosy
Department, Principal K. M. Kundanani College of Pharmacy, Mumbai, for
assistance in qualitative phytochemical studies and Anchrom Enterprises,
Mumbai, for help in the HPTLC analysis.
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