Antibacterial Activity of Aqueous Root, Seed, Flower and Stem Bark Extracts of Acronychia pedunculata Grown in Sri Lanka

Abstract

In vitro antibacterial activity of six(06) different aqueous water extracts of Acronychia pedunculata found in Sri Lanka were investigated against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 35218). Disk diffusion antimicrobial susceptibility testing method was used to determine antibacterial activity of each aqueous extract. The results showed that there was a marked antibacterial activity against S.aureus (ATCC 25923) strain. The highest activity observed for the combined aqueous sample which had all ingredients(roots, stem barks, leaves, flowers and seeds) in the same aqueous solution and the antibacterial activity was observed in a concentration dependent (r 2 = 0.97) manner. Lowest activity was recorded for seeds (r 2 = 0.87) while root (r 2 = 0.99) and stem bark (r 2 = 0.87) aqueous solution showed second most and third most antibacterial activity against S.aureus (ATCC 25923) on a concentration dependent manner. Gentamicin (10 µg/disk) and distilled water were used as positive and negative controls. When compared with Gentamicin inhibition zone (14.03 ± 0.03mm), the combined aqueous sample at a concentration of 1000µg/disk, showed some potent, significant antibacterial activity with an inhibition zone of 11.26 ± 0.03 mm ,where aqueous root sample has an inhibition zone of 10.90 ± 0.06 mm. These two aqueous samples (combined sample and root sample) could be used further experiments as lead compounds for the development of potent, novel antibacterial agents from Acronychia pedunculata. However there was no antibacterial activity observed for all aqueous extracts against E.coli (ATCC 35218) and this observed phenomena needs to be further investigated with other Gram negative bacterial strains.

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International Journal of Pharmaceutical Research & Allied Sciences, 2016, 5(2): 21-25
Research Article
ISSN : 2277-3657
CODEN(USA) : IJPRPM
21
Antibacterial Activity of Aqueous Root, Seed, Flower and Stem Bark Extracts
of Acronychia pedunculata Grown in Sri Lanka
Chathuranga Bharathee Ranaweera
1
, Nimantha Karunathilaka
2
, Athige Rajith Niloshan
Silva
3
, Sagara Karunarathna
4
, Ranjith Pathirana
3
and W. D. Ratnasooriya
3*
1
General Sir John Kotelawala Defence University, Faculty of Allied Health Sciences, Department of Medical
Laboratory Sciences, Ratmalana, Sri Lanka
2
General Sir John Kotelawala Defence University, Faculty of Allied Health Sciences, Department of Nursing and
Midwifery, Ratmalana, Sri Lanka
3
General Sir John Kotelawala Defence University, Faculty of Allied Health Sciences, Department of Basic Sciences,
Ratmalana, Sri Lanka
4
Technical Officer,
General Sir John Kotelawala Defence University, Faculty of Medicine, Ratmalana, Sri Lanka
* Email: cbr2704@kdu.ac.lk
_____________________________________________________________________________________________
ABSTRACT
In vitro antibacterial activity of six(06) different aqueous water extracts of Acronychia pedunculata found in Sri
Lanka were investigated against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 35218). Disk
diffusion antimicrobial susceptibility testing method was used to determine antibacterial activity of each aqueous
extract. The results showed that there was a marked antibacterial activity against S.aureus (ATCC 25923) strain.
The highest activity observed for the combined aqueous sample which had all ingredients( roots, stem barks, leaves,
flowers and seeds) in the same aqueous solution and the antibacterial activity was observed in a concentration
dependent (r
2
= 0.97) manner. Lowest activity was recorded for seeds (r
2
= 0.87) while root (r
2
= 0.99) and stem
bark (r
2
= 0.87) aqueous solution showed second most and third most antibacterial activity against S.aureus (ATCC
25923) on a concentration dependent manner. Gentamicin (10 µg/disk) and distilled water were used as positive
and negative controls. When compared with Gentamicin inhibition zone (14.03 ± 0.03mm), the combined aqueous
sample at a concentration of 1000µg/disk, showed some potent, significant antibacterial activity with an inhibition
zone of 11.26 ± 0.03 mm ,where aqueous root sample has an inhibition zone of 10.90 ± 0.06 mm. These two aqueous
samples (combined sample and root sample) could be used further experiments as lead compounds for the
development of potent, novel antibacterial agents from Acronychia pedunculata. However there was no
antibacterial activity observed for all aqueous extracts against E.coli (ATCC 35218) and this observed phenomena
needs to be further investigated with other Gram negative bacterial strains.
Key words: Acronychia pedunculata, Aqueous extract, Antibacterial activity, Disk diffusion method, folk
medicine, Sri Lanka.
_____________________________________________________________________________________________
INTRODUCTION
Acronychia pedunculata (Family: Rutacea) is a small tree with pale smooth bark and glabrous branches. This plant
is also known as , Claw-flowered Laurel & Laka wood in English, Ankenda in Sinhala language and Kattukanni,
Muttainari in tamil language.
The plant is distributed in parts of India, Malaysia, Sri Lanka and Philippine Islands.
This plant is common in Sri Lanka up to 5000 feet elevation.
1

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22
The leaves are arranged in simple, opposite or some alternate pattern where normal size of leaves vary between 7.5
cm -12.5 cm in length. Naturally plant flowers from February to April and flowers are in yellowish green in 1.8cm
in length on long pedicels where flowers are arranged loosely in pyramidal, divaricately on long straight axilarry
peduncles. The fruit is globular shaped in 1.2- 1.8 cm in length. The fruit has four chambers and has a seed per
chamber.
1
In traditional and folk medicine in Sri Lanka, the bark is used externally on swelling, fractures, sores and tonic for
scabies and ulcers. The bark has also been used internally as purgative.
1,2
Extracts of A.pedunculata exhibit
antibacterial activity against Enterococcus faecalis and Pseudomonas aeruginosa.
3
According to studies carried out
by Pathmasiri and colleagues , methanolic extracts of stem and root bark exhibit significant cytotoxicity in human
K3 tissue culture assay.
4
It is also worth stating here that dichloromethane extracts of stem bark of this plant showed
inhibition of cyclooxygenase-2 (CoX-2)enzyme.
4
Therefore this study was undertaken to investigate in vitro antibacterial activity of aqueous extracts of roots, seeds,
flower, and leaves, stem barks of A.pedunculata separately and identify the most active aqueous extract/s with idea
of using these extract/s for development of novel pharmacophores from plant sources against infectious bacterial
pathogens.
MATERIALS AND METHODS
Collection & Authentication:
Whole matured plants with flowers and fruits were collected in Mallawapitiya area in Kurungegala district in Sri
Lanka (GPS 7°28'21.7"N 80°23'23.8"E), in December 2015. The whole plant had been identified and authenticated
by a botanist at National Herbarium at Peradeniya Sri Lanka.
A voucher specimen of whole plant of A.pedunculata (AP/ 01/2015), stem bark of A.pedunculata (AP/ 02/2015),
seeds of A.pedunculata (AP/ 03/2015), roots of A.pedunculata (AP/ 04/2015), flowers of A.pedunculata
(AP/05/2015), leaves (AP/06 / 2015) were deposited at the Department of Medical Laboratory Sciences, General Sir
John Kotelawala Defence Sri Lanka.
Preparation of aqueous extracts of A.pedunculata:
The roots, seeds, flowers, leaves, stem bark were removed from the plants and were air dried in shade for 2-4 days
and were cut in to small pieces separately. In order to prepare aqueous extracts for each sample separately, twenty
three grams (23 g) from each sample was boiled slowly in 92 ml of distilled water for approximately for 3 hours
until the volume is reached to 18 ml. The prepared aqueous sample was left for cooling down and stored securely for
later use.
There was another separate aqueous sample prepared by adding 23 g of roots, 05 g of seeds, 05 g of flower, 23g of
leaf and 23 g of stem bark in to 316 ml of distilled water. This sample was boiled for about 5 hours until the final
volume of the solution reached 62 ml. This sample was labelled as combined sample.
Antibacterial Activity Screening:
Prepared aqueous samples (labelled as roots, seeds, flowers, leaves, stem bark and all) were evaluated for
antibacterial activity using disc diffusion methods as described by clinical and laboratory institute .
5
Distilled water
was used as the negative control while Gentamicin (10 µg/disk) was used as positive control. The growth medium
use for this experiment was Muller Hinton Agar and the antibacterial activities were determined against
Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 35218) at following concentrations of 500 µg of
plant extract /disk,750 µg of plant extract /disk and 1000 µg of plant extract /disk. The experiment was carried out in
triplicates for each sample and the diameter of the inhibition zone (in mm) for each extract against above mentioned
strains were measured and recorded.
Statistical Analysis:
The data was statistically analysed by using Non Parametric-Kruskal-Wallis Test in SPSS (17
th
Version) software
package. Data expressed as mean inhibition zone diameter ± Standard Error of the Mean (SEM) and coefficient of
determination (r
2
value). Significance level was set at p < 0.05.
RESULTS
The results are summarized in table 1. As shown, Gentamicin was used as the positive control at a concentration of
10 µg/disk for each extract and distilled water was used as the negative control. There was no antibacterial activity

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observed against E.coli strains (ATCC 35218) for any of the prepared extracts at any concentration used for this
investigation.
At 500 µg/disk concentration, there was no inhibitory zone observed for seed extract where mild inhibitory zones
were observed for leaves (mean 5.03 mm), flowers (mean 6.06 mm) and stem bark (mean 6.10 mm) extracts. Root
(mean 7.96 mm) and combined sample (mean 9.06 mm) showed moderate anti bacterial activity against S.aureus
Strains (ATCC 25923). Same pattern of antibacterial activity was observed for all extracts at 750 µg/disk and 1000
µg/disk concentrations. However when compared with positive Gentamicin control at concentration of 10 µg/disk
(mean inhibition of 13.98 mm ) ,there was a strong antibacterial activity ( mean inhibition 11.26 mm) was observed
for the combined sample against S.aureus Strains, at the concentration of 1000 µg/disk. Further, the observed
antibacterial activity for each extract was concentration dependent (r
2
values from 0.87 to 0.99, p < 0.05).
Table 1: Antibacterial Screening Results of Plant Extracts
Plant Extract
Antibacterial activity (inhibition Zone diameter – mm )
S.aureus Strains (ATCC 25923) E.coli Strains (ATCC 35218)
500 µg/disk 750 µg/disk 1000 µg/disk r
2
Value 500 µg/disk 750 µg/disk 1000 µg/disk
Roots 7.96 ± 0.08 9.53 ± 0.15 10.90 ± 0.06 0.99 - - -
Seeds 0.00 ±0.00 2.1 ± 0.05 2.53 ± 0.03 0.87 - - -
Flowers 6.06 ± 0.03 7.00 ± 0.05 7.53 ± 0.03 0.97 - - -
Leaves 5.03 ± 0.03 7.10 ± 0.05 8.06 ± 0.03 0.95 - - -
Stem Barks 6.10 ± 0.05 8.10 ± 0.05 8.53 ± 0.03 0.87 - - -
Combined Sample 9.06 ± 0.03 9.86 ± 0.03 11.26 ± 0.03 0.97 - - -
Gentamicin 14.00 ± 0.02 14.03 ± 0.03 13.98 ± 0.04 N/A 15.00 ± 0.02 15.02 ± 0.03 15.04 ± 0.02
Distilled water 0.00 0.00 0.00 N/A 0.00 0.00 0.00
Data presented as mean Inhibition zone diameter ± SEM (n=3).
Sign (-) indicates no antibacterial activity. Sign (N/A) indicates not applicable.
Gentamicin was used as Positive control. Distilled Water was used as Negative control.
DISCUSSION
This study examined in vitro antibacterial activity of aqueous extracts of A.pedunculata. This experiment was
carried out with a view of scientific justification of its use in traditional and folk medicine in Sri Lanka. At the same
time, there is a huge demand for novel, cheap, antibiotics due to the rapid emergence of novel antibiotic resistant
microorganisms.
6,7,8
On the other hand, there are only limited number of peer reviewed published reports on antibacterial activity of Sri
Lankan medicinal plants.
9
In our previous studies we have shown that there are many Sri Lankan medicinal plants
which possess significant antibacterial activity with a huge potential of development of novel antibacterial, anti-
arthritic and anti-oxidant drugs from medicinal plants in Sri Lanka.
9,10,11,12,13
In general there are other extracts , such
as hexane, methanol , chloroform are commonly being used for antibacterial screening tests.
14
we have decided to
use water extracts for this experiment since aqueous extracts (hot or cold) are the most common form of extract
being used in traditional and folk medicine.
21, 22
There was no significant antibacterial activity observed against E.coli (ATCC 35218) strains for all aqueous extracts
prepared. This was an interesting observation since there was no antibacterial activity observed even for the sample
where roots, leaves, flowers, seeds and stem barks have been mixed together. Therefore this observed phenomena
needs to be further investigated to see the effect of the extract on other Gram negative bacterial strains. These further
investigations will provide a general idea about the its activity against other gram negative bacterial strains and
based on those future results, it would be possible to classify its activity generally against Gram negative bacterial
strains.
All aqueous extracts of A.pedunculata showed marked antibacterial activity against Gram positive S.aureus (ATCC
25923) strains. Aqueous seed extract showed the lowest antibacterial activity while ‘all sample’ showed the highest
antibacterial activity. Aqueous extracts of roots and stem barks showed second most and third most activities
respectively. The general order of potency of aqueous extracts, based on the results of this experiment, could be
stated as combined sample > root > stem bark > leaves > flowers > seeds. The observed highest antibacterial activity
for combined sample could be due to the synergistic activity of each compound/s from each plant material used in
this experiment. Therefore it is worth to investigate each extract individually using various chromatographic
techniques and identify active compound/s separately. Those identified compound/s could be further investigate for
their in vitro synergistic activities later.

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Gentamicin was selected as the positive drug as it inhibits the growth of wide variety of Gram negative and Gram
positive microorganisms by inhibiting bacterial protein synthesis.
6
Gentamicin is also recognized as one of the most
important medications needed in basic health system by World health Organization .
15
It is worth mentioning here
that when compared to a pure compound as Gentamicin at 10 µg/disk concentration (inhibition zone of 13.98 mm) ,
the combined sample of 1000 µg/disk showed some potent, note worthy antibacterial activity (inhibition zone of
11.26 mm) against S.aureus (ATCC 25923) strains. Therefore it is fair to assume that a further chemically purified,
combined aqueous sample would produce relatively higher antibacterial activity when compared to its crude
aqueous sample and even to Gentamicin at 10 µg/disk concentration. This crude, combined aqueous sample could be
further investigated to identify potential novel antibacterial compounds from natural resources.
The stem bark and roots contain polyphenol (Acrovestone), triterpene alcohols and acetophenones.
16, 17
These
compounds might have played a vital role in observed high antibacterial activity in stem bark and root extracts and
the results of this and previous studies collaborate with the use of these plants in traditional medicine.
18, 20
It is also
worth stating here that oils from aerial parts of A.pedunculata possess a broad spectrum of antimicrobial activity
against both Gram positive and Gram negative strains, particularly against Salmonella enterica and Staphylococcus
epidermidis.
19
Thirty four (34) compounds have been identified based on Gas Chromatography Mass Spectrometry (GC-MS) and
Carbon-13 Nuclear Magnetic Resonance (
13
C –NMR) studies .The major constituents were α-pinene and β-
caryophyllene and these oils have shown to possess a broad spectrum of antimicrobial activity against both Gram
positive and Gram negative microorganisms.
19
CONCLUSION
In conclusion, the results of this study show for the first time, that aqueous extracts (mainly combined sample ,roots
and stem bark) of A.pedunculata found in Sri Lanka, posses marked antibacterial properties against S.aureus
(ATCC 25923) strain. The observed relatively higher activity (when compared to Gentamicin) for the combined
sample must be further investigated as this could be used as a potential lead compound for the development of
potent, novel antibacterial agents from Acronychia pedunculata. However, the activity of aqueous extracts against
Gram negative microorganisms needs to be further investigated. At the same time this study rationalizes the use of
this plant in traditional and folk medicine treatments in Sri Lanka. At the same time this plant could also be used for
screening processes in identifying novel, potent antibacterial compounds from natural resources.
Acknowledgements
Thanks are due to Mr. Weranga Rajapaksha for collection of plants and Dr A.K. Chandana for assistance in
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