Antibacterial and Cytotoxicity Studies of
Isaac John Umaru1*, Fasihuddin A Badruddin1, Zaini B Assima2, Hauwa A Umaru2 and Dluya Thagriki3
1Faculty of Resource Science and Technology Sarawak, Federal University, Malaysia
2Department of Biochemistry, Federal University, Malaysia
3Department of Biochemistry Moddibo Adama University Science and Technology, Malaysia
Submission: July 12, 2018; Published: August 08, 2018
*Corresponding author: Isaac John Umaru, Faculty of Resource Science and Technology Sarawak, Kuching, Federal University, Malaysia,
How to cite this article: Isaac John Umaru, Fasihuddin A Badruddin, Zaini B Assima, Hauwa A Umaru, Dluya Thagriki. Antibacterial and Cytotoxicity
Studies of Barringtonia Asiatica. Anatomy Physiol Biochem Int J: 2018; 5(3): 555662. DOI: 10.19080/APBIJ.2018.05.555662.
Methods:Barringtonia asiatica extract was evaluated for its antibacterial activity. Antibacterial activity assessment was performed by Disc diffusion assay the leaves of the plant were extracted with n-hexane, dichloromethane, ethyl acetate, chloroform and methanol and then vaporized to give respective extracts. Antimicrobial activity against Escherichia coli, salmonella typhi, Staphylococcus aureus and Klebsielia pneumonia, was determined. The optical density of the broth using UV mini spectrophotometer and zone of inhibition by the crude extract were determined.
Results:The results showed that of n-hexane extracts of varying concentration the 500ppm and 1000ppm displayed more activity with 4.00 ± 0.10, 4.30 ± 0.10b, 3.70 ± 0.10, 4.07 ± 0.12mm and 4.67 ± 0.12a, 4.35 ± 0.07a, 4.05 ± 0.07a, 4.55 ± 0.07mm respectively on all the pathogen subjected to the studies displayed where aSignificantly (p<0.05) higher compared to different extract at the same concentration b Significantly (p<0.05) lower compared to the control, than others at 25-1000 ppm per disc of the extracts concentration tested. However, the result of the cytotoxicity showed that Barringtonia asiatica Leaf extract were toxic on brine shrimp larvae with LC50 value of 208.091 when compared with the control 7.455 thus having toxicity when referred to the fact that LC50 value of less than 1000μg/mL is toxic while LC50 value of greater than 1000μg/mL is non-toxic.
Conclusion: The present results showed the potential of the medicinal plant used by traditional herbal medical practitioners as natural antimicrobial agents, thus can be further used to determine the bioactive products that may provide as leads in the development of new drugs.
Plants are important sources of medicinal products, they are recognized for their ability to produce a rich source of secondary metabolites and humans have long before now used many species to treat various kind of disease and ailment . Barringtonia asiatica is a species of Barringtonia native to mangrove habitats on the tropical, it is a common plant in the Malaysian Mangroves and wetlands such as the Kuching wetlands Sarawak and Bako National Park,
It is also found in tropical Africa, Nigeria and Madagascar. Its large pinkish-white, pompon flowers give off a sickly-sweet smell to attract bats and moths which pollinate the flowers at night. It is grown along streets for decorative and shade purposes
in some parts of Sarawakian houses and it’s also known as Box Fruit due to the distinct box-shaped of the fruit, it is a medium-sized tree growing to 7-25 m tall. [2,3].
The leaves are narrow obovate, 20–40 cm in length and 10–20 cm in width matured foliage colour is green, smooth glossy shiny leathery thick simple and evergreen. It is used as sausage food among the native of sarawakian in the kampong as well as a medicinal plant, inhabitants of several West African countries, Nigeria and the Polynesian Islands use liquid from the crushed bark of Barringtonia asiatica to treat chest pains and heart troubles. The same plant is used in Papua New Guinea to treat stomach-aches, the top leaves from this tree are squeezed into water and the liquid taken orally . The plant when mature the bark texture is smooth and woody with the root type fibrous and
In cytotoxicity study, the brine shrimp cytotoxicity assay is
considered as a convenient method for preliminary assessment
of toxicity, testing. However, limited studies have reported
bioactivities of Barringtonia asiatica and the antimicrobial
Thus, this in-vivo lethality assay is the simplest zoological
organism (brine shrimp) which can be used as a convenient
monitor for screening and fractionation in the discovery and
monitoring of bioactive natural product, it is a general assay
and capable of detecting various bioactivity present in crude
extracts of medicinal plants and has been used as an indicator
for general toxicity and as a guide for the detection of antitumor
and pesticidal compounds. Since its introduction by Meyer et al.
All chemicals used in this investigation were of analytical
grade and were obtained from SIGMA. Standard antibacterial
agent (30μg) tetracycline, antimicrobial susceptibility test discs
and Nutrient agar (CM0003) were obtained from Oxoid Ltd,
Wade Road, Basingstoke, Hants, RG2 8PW, UK.
The bacterial was sub-cultured in a 10 mL of broth, each in
universal glass bottle for 16 hours inside an incubator equipped
with shaker at 37°C . After 16 hours incubation, turbidity
(optical density/OD) of the bacterial broth was measured by
using UV mini spectrophotometer (model 1240 of Shimadzu
brand), comparable to that of nutrient broth standard tube for
further use . Measurement was performed at wavelength
575 nm and the bacterial broth was ready to be used when its
turbidity was between OD 0.6 to 0.9. Nutrient broth was used to
adjust the turbidity until the desired value was obtained.
Inoculation of the bacteria was carried out in a biohazard
cabinet and the procedure was based on method described by
Pundir & Jain . Approximately 1 mL of the ready bacterial
broth were transferred into mini centrifuge tubes. A sterile
cotton swap was dipped into the mini centrifuge tube containing
bacteria broth and streaked over entire of the agar plate surface,
performed in 4 different directions. The agar plate was then left
for 5-10 minutes before applying the test samples.
The disc used was 6 mm diameter. A volume of 10 μL of the
test samples of concentration 25, 50, 100, 250, 500, 1000 ppm
were each pupated onto the discs and placed onto the agar plate
by using sterile forceps and gently pressed to ensure contact.
Next to be placed on the agar plate was the disc pupated with
methanol as negative control, followed by 30 μg of tetracycline as
standard antibacterial agent (positive control). The plates were
left at room temperature for 10 minutes to allow the diffusion
of the test samples and the standards into the agar. Each crude
extract was tested in triplicate for each bacterium used. The
plate samples were then incubated at 37°C for 24 hours before
the inhibition zone around every sample disc being examined.
The inhibition zone was measured in diameter to indicate the
presence of antibacterial activity for each sample, as compared
to the positive control.
Toxicity test against brine shrimp (Artemia salina) developed
by  was used in this study. The brine shrimp hatch, 1.5 g of
Artemia salina cysts (Sanders Great Salt Lake, Brine Shrimp
Company U. S. A.) was aerated in 1 L capacity glass container
containing filtered seawater (collected from Damai beach in
Air pump was fitted to the water to ensure complete aeration
of the cysts after 48 hrs. of incubation at room temperature
between 27-29°C under continuous illumination of fluorescence
lamp, newly hatched free-swimming nauplii were harvested
from the bottom of the glass container. The freshly hatched
nauplii were used for the bioassay.
Exactly 5mg of sample was dissolved in 5 mL methanol, and
the mixture was sonicated to ensure homogeneity of the extract.
six different volumes of 500, 100, 50, 25, 10 and 1μL each from
the stock solution were transferred into NUNC multidisc in
triplicate. Solvent could evaporate under a running fume hood
for overnight and followed by the addition of 0.2 mL DMSO and
4.8 mL seawater to give final concentration of 500, 100, 50, 25,
10 and 1 μg/mL, respectively.
Ten brine shrimp nauplii were transferred into each
concentration in NUNC multidisc, and was observed every 6 hours for 24 hours. The amount of dead nauplii were calculated.
Thymol was used as positive control, whereas 0.2 mL DMSO and
4.8 mL seawater was used as negative control. The data was
analyzed to determine the concentration of the samples that kill
50% of brine shrimp at 24 hours or known as LC50.
The results were expressed as means ± Standard deviation
(SD) of three parallel measurements with one-way ANOVA. The
LC50 values for toxicity assay was calculated and determined
by performing Profit analysis in IBM SPSS Statistic software of
In the antibacterial and cytotoxicity studies, the hexane
extract of Barringtonia asiatica exhibited the presence of
antibacterial bioactive component (Table 1). The antibacterial
activity of the extract was in concentration dependent manner.
Activity was gradually increased with the concentration, from
low concentration level to higher concentration level. The hexane
extract exhibited dose dependent inhibition of bactericidal in
comparison to the control.
Determination Values are Mean ± SD for five
aSignificantly (p< 0.05) higher compared to different concentration on same organism in each row bSignificantly (p< 0.05) higher compared to at the
same organism at different concentration in each column.
The extract showed 4.00 ± 0.10mm, 4.30 ± 0.10mm, 3.70 ±
0.10mm, 4.07 ± 0.12 mm and 4.67 ± 0.12mm, 4.35 ± 0.07mm,
4.05 ± 0.07mm, 4.55 ± 0.07mm inhibition of activity at the
doses of 500 and 1000 ppm, respectively while tetracycline
showed 5.50±0.91mm, 5.68±0.59mm, 5.83±0.29mm, 6.73±0.77
inhibition of bacteria. While, at various concentration of the
extract (1, 10, 25, 50, 100 and 500ppm) the average death of
Artemia salina of hexane crude extract of the Leaf caused the
death rate to increase with increase in concentration, given rise
to LC50 208.091 μg/mL when compared to the test control thymol
with LC50 7.455μg/mL. The result is mean+SD. N = 30 (Table 2).
The hexane extract of Barringtonia asiatica various
concentration gave an impressive inhibition against the pathogen
(Salmonella typhi, E. coli, Staphylococcus aureus, Klebsielia
Pneumonia) with a diameter of inhibition within the range
of 2.35 ± 0.07mm and 4.67 ± 0.12mm for 25pmm-1000ppm.
However, the crude extract showed a greater antibacterial
activity against Salmonella typhi with inhibition zone of 4.67
± 0.12mm at concentrations of 1000ppm, when compared to
positive standard of tetracycline as well as other concentration
with the inhibition zone.
The inhibition of Escherichia coli (E. coli) by the crude extract at
various concentration is within the diameter range of 2.60 ± 0.10
to 4.35 ± 0.07mm for 25-1000ppm. Most of the crude extract
inhibition gave an increase in the inhibition with increase in
concentration, this was followed by Staphylococcus aureus and
Klebsielia Pneumonia as shown in the (Table 1). Barringtonia
asiatica at 1000ppm is significant and active on all the pathogen,
with aSignificantly (p< 0.05) higher compared to different
concentration in each rows and bSignificantly (p< 0.05) higher
compared to different extract at the same concentration in each
However, the result of the cytotoxicity showed that
Barringtonia asiatica Leaf extract were toxic on brine shrimp
larvae with LC50 value of 208.091 when compared with the
control thymol at LC50 7.455 thus having toxicity when referred
to the fact that LC50 value of less than 1000μg/mL is toxic while
LC50 value of greater than 1000μg/mL is non-toxic.
The hexane extract of the leaves of Barringtonia asiatica
indicated varied levels of antibacterial activities. The
concentration of the plant extracts at various concentration
level exhibited a high cytotoxicity activity on Brine shrimps.
Thus, the plant is said to have a reasonable potential as
antimicrobial compounds against microorganisms especially
in the case of Salmonella typhi, E. coli, Klebsielia Pneumonia and
lastly Staphylococcus aureus with increase in concentration.
However, the plant extracts can be used as a novel drug against
the development of resistance strains and in the treatment of
infectious diseases caused by resistance bacteria.