Vol. 9(33), pp. 884-891, 3 September, 2015
Article Number: CEFF46755111
Copyright © 2015
Author(s) retain the copyright of this article
Full Length Research Paper
Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand, Private Bag
Received 5 March, 2015; Accepted 19 August, 2015
Antibacterial, antidiarrheal, antimotility, phytochemicals.
Diarrheal infections are major causes of morbidity and
among infants and children. There are approximately 1.5
billion episodes of diarrheal infections per year. More
than one in ten deaths of children under the age of 5
years are due to diarrhoeal infections (WHO and
UNICEF, 2009). Diarrhoea is gastrointestinal disorder
that is characterized by a decrease in the stool
consistency and an increase in frequency, fluidity, or
volume of the faeces during defecation for a period of
*Corresponding author. E-mail: email@example.com.
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days or weeks (Mazzolin et al., 2013). The most common
symptoms of diarrhoeal infections range from mild and
self-limiting symptoms (Mims et al., 2004).
However, severe diarrhoea may lead to the disordered
gastrointestinal tract (GIT) motility, dehydration,
electrolyte imbalance, acidosis and malnutrition (Dyer
and Gould, 2011). Diarrhoea often occurs due to the
damage of the intestinal mucosal cells by exotoxins and
endotoxins of microbial origin in contaminated food and
water and metabolic disorder in gastrointestinal tract
(Kumar et al., 2001).
Several studies have reported the beneficial effects of
fruit pulps and seed extracts in the treatment of
diarrhoeal infections (Ashorobi and Umukoro, 2005;
Maha et al., 2013). The antibacterial and antidiarrheal
activitiv of fruit pulp and seed extracts depend on the
presence and concentrations of phytochemicals (Arup et
al., 2012). According to Neethiriajan et al. (2012),
phytochemicals have strong antibacterial and
antidiarrheal properties. Although fruits and seeds are
excellent sources of therapeutic phytochemicals, pulps
and seeds have been rarely used as medicine (van Wyk
et al., 2009; Kossah et al., 2011; Srividhya et al., 2013).
However, the prohibitive costs and the negative side
effects of allopathic medicine used against diarrheal
infections have recently elevated fruits and seeds as
sources for novel antidiarrheal agents.
(RSA). They are widely distributed in the Eastern Cape,
KwaZulu-Natal, across northern part of the RSA and in
areas with high rainfall (Orwa et al., 2009). The bark and
trees have been proven to
possess antidiarrhoeal properties (Sibandze
et al., 2010;
Amabeoku and Deliwe, 2013).
The fruits of
have only been used for
consumption and not for pharmacological purposes such
as treatment of diarrheal infections. The fruits are purple,
ovoid and fleshy up to 2 cm fruits with 2.8 cm thick seeds
(Downs and Wilson, 2012). The fruiting season is usually
from October to June in Republic of South Africa (RSA)
(Drummond and Moll, 2002).
The study was undertaken to evaluate the antibacterial
and antidiarrheal activities of
pulp and seed
extracts as to find novel sources that can be developed
for treatment of diarrhoeal infections.
were randomly harvested in summer
(February, 2014) from the trees at the main campus of the
University of Zululand (UZ), KwaZulu-Natal, RSA. The fruits were
washed with distilled water, seeds and pulps were manually
separated. The fruit pulps and seeds were air-dried at room
temperature. The dried
fruit-pulps and seeds were
separately ground to a coarse powder form using an electric grinder
and filtered with a filter of mesh size 1.0 mm to increase the surface
area for solvents during the extraction process. The grounded
samples were stored at 4°C until required for use.
Sidney et al. 885
was done according to Bii
some modifications. The ground
fruit-pulp sample (100
g) was subjected to Soxhlet extraction using 400 ml of methanol
(Univ.AR). The sample was put on a mechanical shaker at a speed
of 200 rpm at 37°C for 12 h. The extract obtained was filtered
through Whatman filter paper and concentrated using a Bȕchi
rotary evaporator at 45°C. The yield of the extract was weighed and
re-dissolved in 100 ml of 10% dimethyl sulfoxide (DMSO) to the
volume concentration of 100 mg/ml. The extracts were stored at
4°C until they were to be used. The percentage yield from
fruit-pulp extract was calculated using the formula below
that was used by Shahid (2012).
Weight of the extract (g)
Weight of powdered sample (g)
The extracted crude
fruit pulp and seed extracts were
screened for phenolics, alkaloids, flavonoids, tannins, phenols,
terpenoids, cardiac glycoside, saponins and betulinic acid. The
phytochemical screening was done in all the extracts (except for
betulinic acid) using the methods of Harborne (1973).
An original line of 2 cm from the edge, across the plate was drawn.
standard indicator followed by loading of methanol extract of
fruit-pulp. The thin-layer chromatography plate was
placed in a chromatography tank containing mixture of hexane and
ethyl acetate in the ratio of 7:3, respectively, covering about 1 cm of
the plate. The chromatography was allowed to proceed until the
hexane-ethyl acetate reaches the top of the plate. At that point, the
chromatogram was removed from the tank and dried using hot air
dryer. The plate was viewed under ultra violet light at 354 nm. It
was then sprayed with 5% sulphuric acid-methanol solution. The
appearance of a pink colour indicated the presence of betulinic acid
The total phenolic contents were determined by the Folin-
Ciocalteau method according to Makkar et al. (1993). An aliquot
(0.2 ml) of 500 μg/ml methanolic fruit-pulp and seed extracts were
made up to 1.0 ml with distilled water, respectively. 0.5 ml of Folin-
Ciocalteau reagent (1N) was added, followed by 2.5 ml of sodium
carbonate solution (20%). The mixtures were mixed properly, and
then incubated at room temperature for 40 min. The absorbance of
the blue-colored complex formed was measured at 725 nm against
the appropriate blank. The total phenollic content was determined
from the standard curve of tannic acid and expressed as
equivalents of tannic acid (μg/mg).
The bacterial strains known to cause GIT infections used in this
Revival of the selected bacterial strains
The selected bacteria were inoculated into nutrient broth and
incubated at 37°C for overnight. Afterwards, 1 ml from each of the
bacteria species was pipetted into 9 ml of fresh prepared nutrient
broth in separate test tubes labelled with corresponding
microorganism. The test tubes were then incubated at 37°C for
overnight .After overnight incubation, absorbance of the selected
bacterial strains was read in the spectrophotometer (600 nm) for
determination of their turbidity. The turbidity of the resulting
suspensions was diluted with nutrient broth to obtain an
absorbance of 0.132. This absorbance was taken as comparable to
0.5 McFarland turbidity standard. The turbidity was estimated to be
equivalent to 1.5 x colony forming unit (CFU)/ml (Qaralleh et al.,
A serial microdilution method was adapted as described by Eloff
(1998) and Qaralleh et al. (2012) to measure the MIC of the fruit-
pulp extract. The MIC is the lowest concentration of the extract
required to inhibit microbial growth. 96-well microplate was used to
quantitatively determine the MIC of the extract. The sterile nutrient
broth (50 μl) was added to all the wells of the 96-well microplate
and 50 μl of the extract (50 mg/ml, in 10% DMSO) was poured in
the wells in the first rows and mixed well. The extract mixture (50 μl)
were removed from all the wells in the row A to perform a 3-fold
serial dilution down the columns. The last 50 μl, in the last column
was discarded so that the total volume solution of each well was 50
μl. The selected bacterial strains (50 μl) were transferred into the
corresponding wells. 10% DMSO was used as negative control
while ciprofloxacin (20 μg/ml) was used as a positive control. The
plate was covered and incubated at 37°C for overnight. 0.2 mg/ml
of P-iodonitrotetrazodium violet (INT) solution was used after the
incubation period. 40 μl of 0.2 mg/ml INT solution was added to
each well and incubated at 37°C for 30 min. A reddish colour which
was the result of INT being reduced by the metabolic activity of
microorganism to formazan indicated microbial activity. The clear
colour was to be the indication of the absence of bacterial activity
since the INT was not broken-down to form formazan. The test was
replicated three times and the mean value was reported.
Minimum bactericidal concentration (MBC)
For the determination of MBC, the agar dilution method was used.
The MBC of the extract was determined by removing a loop full of
each culture medium from the wells that had no bacterial growths.
They were streaked on different sterile nutrient agar plates. The
agar plates were incubated at 37°C for 12 h. The lowest
concentration of the
fruit-pulp extract that exhibited the
complete killing of test microorganisms was considered as the MBC
(Qaralleh et al., 2012).
Antidiarrheal and antimotility activities
Ethical clearance for the use of animals was collected from the
rats (150 to 260 g) were collected
from the animal house in the Department of Biochemistry and
Microbiology at the same institution. Prior to the determination of
standard food and given free access to water for one week to adapt
to the laboratory conditions (temperature 23±2°C and 12 h light
dark cycle). The rats were then fasted for 18 h before the start of
the experiment to empty the GIT and to increase their
responsiveness to the extracts and drugs used, but allowed free
access to water (Orhan et al., 2013).
The method used in for determination of antidiarrheal activity was
were divided into four groups of four rats each namely: Group A,
Group B, Group C and Group D. Group A served as a negative
control. It received vehicle distilled water (2 ml /kg) orally. Group D
served as a positive control. It received atropine at the dose of 5
mg/kg orally by gavage. Group B and Group C received the seed
and fruit-pulp extracts (400 mg/kg), respectively. Each rat was put
in its own cage. Diarrhoea was introduced to each rat by orally
administering 0.2 ml of castor oil. After 30 min of administration of
castor-oil, observation of the defecation was done for 5 h. The
onset time of faeces and number of normal and wet faeces were
the determined parameters. A score based on stool consistency
was assigned as follows; normal-stool = A and wet-stools = B. The
presence of normal stools was recorded as a positive result,
indicating protection offered by the controls and the extracts from
diarrhea while the presence of watery stools was recorded as
The method used in the antimotility test was adopted from Teke et
four groups of four rats each namely: Group A, Group B, Group C
and Group D. Diarrhoea was introduced to each rat in all groups by
orally administering 0.2 ml of castor oil. After 30 min of
administration of castor-oil, all rats received different treatments.
Group A served as a negative control and received distilled water (2
ml /kg) orally. Group D served as a positive control. It received
atropine at the dose of 5 mg/kg orally by gavage. Group B and C
received the seed extract and fruit-pulp extract of 400 mg/kg,
respectively. Thereafter, each rat was put in its own cage after the
administration of 2 ml of charcoal meal (3 % deactivated charcoal in
distilled water) orally. The rats were sacrificed 30 min thereafter for
determination of gastrointestinal motility. The intestinal distance
moved by the charcoal meal from pylorus to caecum was measured
and expressed as a percentage of distance travelled from pylorus
to caecum. The mean movement of charcoal meal in ratio to the
intestinal length and percentage of inhibition were arithmetically
measured. The following formulas were used:
% travelled = × 100
Total length of small intestine
% inhibition = × 100
Mean length of duodenum
The use of methanol as an extracting solvent resulted in
Preliminary phytochemical screening of
Betulinic acid (BA) TLC
concentration, +++ denotes high concentration, TLC denotes Thin layer
chromatography and PE denote fruit-pulp extract.
a good percentage yield of 10 for fruit-pulp extract and 6
for seed extract. Phytochemicals are non-nutritional
bioactive chemicals from plants that help plants to survive
biotic and abiotic environmental changes and have
therapeutic properties in humans. The total phenolic
content of 16.4±1.8 and 21.4±1.4 µg/mg were obtained in
pulp and seed extracts, respectively. The qualitative and
quantitative analysis of phytochemicals from
fruit-pulp and seed samples and extracts are presented in
The antibacterial results are as presented in Table 3.
Pulp extract showed the lowest MIC value of 3.13 mg/ml
isolates while the seed extract had the lowest MIC value
(6.25 mg/ml) on
all gram-positive bacteria.
fruit-pulp and seed extracts exhibited different percentage
of inhibition against the diarrheal activity in castor oil
fruit-pulp and seed extracts
both reduced the number of wet stools, total stools and
onset time generally in comparison to the negative
control (distilled water).
fruit-pulp and seed
extracts, in a dose-related manner (400 mg/kg of rat),
exerted the antidiarrhoeal properties by reducing
intestinal motility. The results are tabled in Tables 4 and 5
The use of methanol as an extracting solvent resulted in
for seed extract. The good percentage yields implied that
methanol is an important solvent to be used when
determining the biological activities of the extracts. The
ability of methanol solvent to extract good yields is owed
to its polarity.
Phytochemicals have been reported to possess strong
antibacterial, antidiarrheal and gastroprotective properties
(Neethirajan et al., 2012). The phytochemicals detected
Total phenolic content in 500 µg/ml of crude
pulp and seed extract.
results Concentration (µg/mg) original sample) ± SER
TAE: Pulp-extract, seed extract
Values are the average of duplicates experiments and represented as mean ± standard error (SER) and were expressed
MIC and MBC (mg/ml) of the
pulp and seed extracts on the selected bacterial strains known to cause
3.13 6.25 6.25
12.5 3.13 6.25
6.25 6.25 12.5
12.5 3.13 6.25
6.25 12.5 25 50
3.13 6.25 12.5
12.5 3.13 3.13
3.13 3.13 6.25
12.5 1.56 3.13
12.5 3.13 12.5
Effects of the crude methanolic
PE and SE extracts on castor oil-induced rats.
Onset times (min)
Distilled water plus Co
SE plus Co
PE plus Co
Atropine plus Co
Key: Values are represented as mean ± standard error. PE denotes fruit-pulp extract, SE denotes seed extract and Co denotes
in both extracts were phenolic compounds, alkaloids,
cardiac glycosides, phytosterols, flavonoids, saponins,
terpenoids and betulinic acid (Table 1). The quantitative
analysis showed the significant amount of the total
phenolic compounds (16.4±1 μg/mg) in pulp extract and
(21.4±1.4 µg/mg) in seeds extract (Table 2). The
detected phytoconstituent implied that
substances with therapeutic and preventive applications
against bacteria that may cause diarrheal infections.
Ciprofloxacin is a broad-spectrum antibiotic which is
effective against gram-negative and gram-positive
bacteria (Volans and Wiseman, 2010). Ciprofloxacin has
bactericidal effect against
E. coli, Salmonella spp
.strains (Paw and
Shulman, 2010). Ciprofloxacin is widely used to treat
urinary and respiratory infections as well as
gastroenterities. Ciprofloxacin (20 μg/ml) was used as a
positive control on the tested bacteria in this study.
Ciprofloxacin had the inhibitory effects on all the selected
bacteria with the lowest MIC values of (1.56 mg/ml) on
(AL 019) and S
(ATCC 700030). The highest MIC value
(3.13 mg/ml) of ciprofloxacin was recoded on all other
selected bacterial strains.
Many naturally occurring compounds found in pulp
extract have been reported to possess antibacteriall
pulp extract showed broad-spectrum
antibacterial action with the lowest MIC value of 3.13
(ATCC 8043) and
pulp extract was more pronounced on all gram-
positive bacterial strains, the extract also show
remarkable antibacterial activity against gram-negative
(ATCC 7700) as well with the
same MIC value of 3.13 mg/ml. Gram-negative bacteria,
in addition to a thin peptidoglycan layer (2 to 7 nm),
possess about 7 to 8 nm of the outer membrane. This
outer membrane composes of additional protective
lipopolyssachride layer that exhibits toxicity and
Antimotility activity of crude methanolic
PE and SE extracts on castor oil-induced rats.
Doses (ml/kg or
Mean total length
of small intestines
Mean distance travelled
PE plus Co
Key: Values are represented as mean ± standard error. PE denotes fruit-pulp extract, SE denotes seed extract and Co denotes castor
antigenicity against antimicrobials or chemotherapeutic
agents (Martinko and Madigan, 2006). It was concluded
that the high resistance shown by some gram-negative
bacteria as compared to gram-positive bacteria to both
pulp extract was due to the mechanism of
action of this layer. Gram-positive bacteria do not
possess this layer and therefore, they were generally
sensitive to the action of the antibacterial action of the
detected phytochemicals. Gram-positive bacteria allow
the direct contact of the extract constituents with the
phospholipid bilayer of the cell membrane, enabling the
antibacterial compounds to inhibit bacterial growth easily.
The low MIC values displayed by the fruit-pulp extract
indicated its higher efficacy against bacteria causing GIT
infections than the seed extract. According to Jayashree
et al. (2014), the good and promising potency of
methanolic fruit extract has the MIC value ranging
between 3.125 to 12.5 mg/ml. This implied that
pulp extract has a potential to be used as
sources of novel antibacterial agent. Antimicrobial
substances are considered as bactericidal agents when
the ratio is MBC/MIC ≤ 4 and bacteriostatic agents when
the ratio is MBC/MIC > 4 (Erhabor et al., 2013).
fruit-pulp extract exhibited bactericidal effect on
all selected bacterial species. However, the standard
drug-ciprofloxacin showed bactericidal effect on all
selected bacterial species with the exception on
(AL 042) where it showed the bacteriostatic
Castor oil is an effective emollient laxative agent.
Castor oil causes a decrease in fluid and nutrient
absorption, increase in the electrolyte secretion and
water and produces alterations in intestinal motility (Priff
and Harold, 2005). The diarrheal activity of castor oil is
attributed to its active cathartic glyceride known as
ricinoleic acid (Chambers et al., 2015). Thus, castor oil-
induced diarrhoea is as a result of the action of ricinoleic
acid formed from the hydrolysis of its triglyceride in the
duodenum by pancreatic lipase. The ricinoleic acid
stimulates intestinal hypersecretion, hypermotility and
decreases gastrointestinal transit time (Schellack, 2004).
In this study, castor oil was used to induce diarrhoea in
the test rats. Atropine was used in this study as a positive
antidiarrheal activity. Atropine is a
tertiary amine belladonna alkaloid (Chambers et al.,
2015). Atropine is a racemic hyoscyamine tropic acid
ester of the base tropine. Atropine has high affinity for
muscarinic receptors (Hollinger, 2008; Champe and
Harvey, 2009). Atropine exerts its pharmacodynamic
effect by binding competitively at the muscarinic
receptors to prevent acetylcholine to bind, and thus
reversing excessive secretions of fluids and electrolytes.
Atropine actions reduce the interstinal hypertonicity and
hypermotility of the GIT and thus act as an antidiarrheal
agent (Lehne, 2004)
Group C was fed
pulp extract at a dose of
400 mg/kg of a rat weight. Group C had stool onset time
of 68 minutes, total normal stools of 8.25±0.17 and the
total wet stools of 2.75±0.3 while Group B (seed extract)
at the same dose as Group C and had the stool onset
time of 98 minutes, total normal stool of 5.25±0.20 and
the total wet stools of 1.5±0.41 in comparison to the
Group A (distilled water) which had stool onset time of 51
min, total normal stools of 13±0.15 and the total wet
stools of 10.3±0.2. Group D had the longest onset time
(127 min) and the least total number of normal stools
(1.25±0.19) with wet stools not being observed.
fruit-pulp and seed extracts exhibited the
antidiarrheal activity by reducing the number of wet
stools, total stools and onset time generally.
Phytochemicals mediate antidiarrheal activity through
antisecretory and antimotility mechanisms (Cowan,
2015). It was therefore esteemed that the antidiarrheal
activity observed in Group B and C was due to the
presence of these phytochemicals in
antidiarrheal activity was esteemed to mimic that of an
Group D was given an antimuscarinic drug (atropine)
and had the highest inhibitory percentage of 64 followed
by Group C with 49 of inhibition. Group B (seed extract)
had the inhibitory percentage of 41. There was no
inhibition observed in Group A (distilled water). The
reduction in the distance travelled by charcoal in the
fruit-pulp and seed extracts treated groups
pulp and seed extracts
exerted antidiarrheal activity by decreasing the GIT
motility. The reduction of GIT motility of extracts in
comparison to the negative control (distilled water) was
attributed to the presence of the detected phytochemicals
(saponins, alkaloids, triterpenoids, flavonoids, tannins
and betulinic acid). Phytochemicals exert similar mode of
action as antimotility agents (Ahmad et al., 2006; Saleem
et al., 2010; Chollet and Gleason, 2012).
pulp and seed extracts might have
exhibited the antimotility action through the same
mechanism of action exerted by the drug-atropine. The
results scientifically support
pulp and seed
extracts as potential sources for effective, novel
antibacterial and antidiarrheal agents.
pulp and seed extracts demonstrated the
therapeutic and biological efficacy (antibacterial,
antidiarrheal and antimotility activities). Due to the
pharmacodynamic effects revealed by
pulps and seeds can be
viewed as satisfactorily beneficial sources of therapeutic
compounds against diarrheal infections. Further studies
will focus on the purification and identification of some of
the bioactive compounds that are responsible for the
antibacterial and antidiarrheal activities.
Conflict of Interest
The authors have not declared any conflict of interest.
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