Bioactivity of Syzygium jambos methanolic extracts:
Antibacterial activity and toxicity
Author
Mohanty, S, Cock, IE
Published
2010
Journal Title
Pharmacognosy Research
DOI
https://doi.org/10.4103/0974-8490.60577
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Address for correspondence:
I. E. Cock, Biomolecular and Physical Sciences, Nathan
Campus, Grifth University, 170 Kessels Rd., Nathan, Brisbane,
Queensland 4111, Australia.
E-mail: i.cock@grifth.edu.au
DOI:10.4103/0974-8490.60577
Bioactivity of Syzygium jambos methanolic extracts:
Antibacterial activity and toxicity
S. Mohanty
1
, I. E. Cock
1,2
1
Department of Biomolecular and Physical Sciences, Nathan Campus, Grifth University, 170 Kessels Rd., Nathan, Brisbane, Queensland 4111,
2
Genomics Research Centre, Gold Coast Campus, Grifth University, Parklands Drive, Southport, Queensland 4222, Australia
Submitted: 23-11-2009 Revised: 08-12-2009 Published: 13-03-2010
O R I G I N A L A R T I C L E
INTRODUCTION
Plants have long been used as medicines for treating a variety
of different diseases and complaints. Phytotherapy in Asia is
particularly widespread. Plant preparations and medications
continue to be used in the treatment of numerous disorders,
including eczema, malaria, respiratory disorders and
infectious diseases.
[1]
For some of these plant treatments, the
antimicrobial activity has been proven; however, for many
plant-based antiseptics, the evidence is anecdotal or, at best,
epidemiological. Many traditionally used antiseptic agents
have yet to be subjected to thorough scientic investigation.
Syzygium jambos Alston (syn. Eugenia jambos L.; Jambosa
jambos Millsp.; Jambosa vulgaris DC.; Caryophyllus jambos
Stokes) is an evergreen tree of the family Myrtaceae. It is
native to Southeast Asia but has been naturalized in India,
especially in the state of Kerala, where it is grown both
for its fruit and for its medicinal properties. S. jambos has a
long history of use in Indian traditional medicine for the
treatment of numerous ailments. The fruit has been used
as a tonic for the brain and liver and as a diuretic.
[2]
The
owers are believed to reduce fever, and the seeds were
used to treat diarrhea, dysentery and catarrh.
[2]
In South-
American cultures, the seeds have additionally been used
as an anesthetic,
[2]
and recent studies have shown S. jambos
extracts to have a similar analgesic efcacy to morphine in
rats.
[3]
S. jambos leaf decoctions were also used traditionally
in the treatment of diabetes,
[2]
although some studies have
shown leaf extracts to be ineffective as antihyperglycemic
agents.
[4]
In Indian traditional medicinal systems, S. jambos
leaves were also used as a diuretic, an expectorant in
the treatment of rheumatism; to treat sore eyes; and as
a febrifuge.
[2]
Bark of the S. jambos tree is used to treat
asthma, bronchitis and hoarseness.
[2]
Cuban healers have
also used the root to treat epilepsy.
[2]
S. jambos leaf extracts
have also been shown to possess antiviral activity towards
herpes simplex type 1 and type 2 and towards vesicular
somatitis virus.
[5,6]
The antiseptic properties of some members of the genus
Syzygium have been extensively studied. In the commercially
most important species Syzygium aromaticum (clove), the
antiseptic properties are well known. Numerous studies have
reported on the antibacterial
[7]
and antifungal
[8]
activities of oils
and extracts from this plant. Other Syzygium species from India
(Syzygium lineare, Syzygium cumini and Syzygium travancoricum)
[9,10]
have also been shown to have antimicrobial activity. Two
Australian species have also recently been shown to possess
Methanol extracts from S. jambos leaves were tested for antimicrobial activity and toxicity. S. jambos leaf extract inhibited
the growth of 4 of the 14 bacteria tested (29%). Both gram-positive and gram-negative bacterial growths were inhibited
by S. jambos leaf extract, although gram-positive bacteria appeared more susceptible. Two of the 10 gram-negative
bacteria (20%) and 2 of the 4 gram-positive bacteria (50%) tested had their growths inhibited by the extract. The leaf
extract also proved to be toxic in the Artemia franciscana bioassay, with a 48-h LC
50
of 387.9 6 38.8 mg/mL, making it
slightly more toxic than Mevinphos (505.3 6 37.7 mg/mL) and approximately 5-fold less toxic than potassium dichromate
(80.4 6 4.3 mg/ mL). Whilst potassium dichromate’s LC
50
remained constant across the 72-hour test period (24-h LC
50
,
86.3 6 5.1; 72-h LC
50
, 77.9 6 4.9), the extract and Mevinphos LC
50
values decreased by 72 hours (87.0 6 11.3 mg/mL
and 103.9 6 12.8 mg/mL, respectively), indicating their similar levels of toxicity in the assay.
Key words: Antibacterial, medicinal plants, methanolic extracts, phytotoxicity, rose apple, Syzygium jambos
A B S T R A C T
P H C O G R E S .
Pharmacognosy Research | January 2010 | Vol 2 | Issue 1 5
Mohanty and Cock: Bioactivity of Syzygium jambos methanolic extracts
antibacterial activity.
[11]
However, despite its numerous
traditional medicinal uses, the antiseptic properties of S. jambos
remain largely unexamined. Some studies have demonstrated
the antibacterial
[12]
and antifungal
[13]
activities of S. jambos
bark extracts. To the best of our knowledge, no studies have
examined the antibacterial nature of S. jambos leaf extracts.
Therefore, the current study reports on the antibacterial
properties of S. jambos leaf extracts, as well as examining
their toxicity, to determine their potential as antibiotic agents.
MATERIALS AND METHODS
Plant material
Collection of plant samples
Syzygium jambos leaves were a gift from Mervyn Cooper of
the Queensland Tropical Fruit Association. Leaves were
obtained from a single tree, washed in deionized water and
processed within 4 hours of collection.
Preparation of crude extracts
Syzygium jambos leaves were dried in a Sunbeam food
dehydrator, and the dried material was ground to a coarse
powder. One gram of each of the dried plant materials
was extracted extensively in 50 mL methanol (Ajax, AR
grade) for 24 hours at 4°C with gentle shaking. The extract
was ltered through lter paper (Whatman no. 54) under
vacuum, followed by drying by rotary evaporation in an
Eppendorf concentrator 5301. The resultant pellet was
dissolved in 10 mL 20% methanol. The extract was passed
through 0.22-mm lter (Sarstedt) and stored at 4°C.
Antibacterial screening
Test microorganisms
All microbial strains were obtained from Michelle Mendell
and Tarita Morais, Grifth University, Australia. Stock
cultures of Aeromonas hydrophilia, Alcaligenes faecalis, Bacillus
cereus, Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae,
Proteus mirabilis, Pseudomonas uorescens, Salmonella newport,
Serratia marcescens, Shigella sonnei, Staphylococcus aureus,
Staphylococcus epidermidis and Streptococcus pyogenes were
subcultured and maintained in nutrient broth at 4°C.
Evaluation of antimicrobial activity
Antimicrobial activity of all plant extracts was determined
using a modied Kirby-Bauer
[14]
disc diffusion method.
Briey, 100 mL of the test bacteria was grown in 10 mL
of fresh media until they reached a count of approximately
10
8
cells/mL. One hundred microliters of microbial
suspension was spread onto nutrient agar plates.
The extract components were tested using 5-mm sterilized
lter paper discs. Discs were impregnated with 10 mL of the
test sample, allowed to dry and placed onto inoculated plates.
The plates were allowed to stand at 4°C for 2 hours before
incubation with the test microbial agents. Plates inoculated
with Alcaligenes faecalis, Aeromonas hydrophilia, Bacillus cereus,
Citrobacter freundii, Klebsiella pneumoniae, Proteus mirabilis,
Pseudomonas uorescens, Serratia marcescens were incubated at
30°C for 24 hours, and then the diameters of the inhibition
zones were measured in millimeters. Plates inoculated with
Escherichia coli, Salmonella newport, Shigella sonnei, Staphylococcus
aureus, Staphylococcus epidermidis and Streptococcus pyogenes were
incubated at 37°C for 24 hours, and then the diameters of
the inhibition zones were measured. All measurements were
to the closest whole millimeter. Each antimicrobial assay was
performed in at least triplicate. Mean values are reported in this
study. Standard discs of ampicillin (2 mg) and chloramphenicol
(10 mg) were obtained from Oxoid Ltd. and served as positive
controls for antimicrobial activity. Filter discs impregnated
with 10 mL of distilled water were used as a negative control.
Minimum inhibitory concentration determination
The minimum inhibitory concentration (MIC) of the
S. jambos extract was determined by the disc-diffusion
method across a range of doses. The plant extracts were
diluted in deionized water across a concentration range
of 5 mg/mL to 0.1 mg/mL. Discs were impregnated with
10 mL of the test dilutions, allowed to dry and placed
onto inoculated plates. The assay was performed as
outlined above, and graphs of the zone of inhibition
versus concentration were plotted for each extract. Linear
regression was used to calculate the MIC values.
Toxicity screening
Reference toxins for toxicity screening
Potassium dichromate (K
2
Cr
2
O
7
) (AR grade, Chem-Supply,
Australia) was prepared as a 1.6 mg/mL solution in distilled
water and was serially diluted in articial seawater for
use in the Artemia franciscana nauplii bioassay. Mevinphos
(2-methoxycarbonyl-1-methylvinyl dimethyl phosphate)
was obtained from Sigma-Aldrich as a mixture of cis
(76.6%) and trans (23.0%) isomers and prepared as a
4 mg/ mL stock in distilled water. The stock was serially
diluted in articial seawater for use in the bioassay.
Artemia franciscana nauplii toxicity screening
Toxicity was tested using the Artemia franciscana nauplii lethality
assay developed by Meyer et al.
[15]
for the screening of active
plant constituents with the following modications. Artemia
franciscana Kellogg cysts were obtained from North American
Brine Shrimp, LLC, USA (harvested from the Great Salt
Lake, Utah). Synthetic seawater was prepared using Reef
Salt, AZOO Co., USA. Seawater solutions at 34 g/L distilled
water were prepared prior to use. Two grams of A. franciscana
cysts were incubated in 1 L synthetic seawater under articial
light at 25°C, 2000 lux with continuous aeration. Hatching
commenced within 16-18 hours of incubation. Newly
hatched A. franciscana (nauplii) were used within 10 hours
6 Pharmacognosy Research | January 2010 | Vol 2 | Issue 1
Mohanty and Cock: Bioactivity of Syzygium jambos methanolic extracts
of hatching. Nauplii were separated from the shells and
remaining cysts and were concentrated to a suitable density
by placing an articial light at one end of their incubation
vessel, and the nauplii-rich water closest to the light was
removed for biological assays. Four hundred microliters of
seawater containing approximately 40 (mean, 39.8; n 5 175;
SD, 19.0) nauplii was added to wells of a 48-well plate and
immediately used for bioassay. The plant extracts were
diluted to 2 mg/mL in seawater for toxicity testing, resulting
in a 1 mg/mL concentration in the bioassay. Four hundred
microliters of diluted plant extracts and the reference toxins
were transferred to the wells and incubated at 25 6 1°C under
articial light (1000 lux). A negative control (400 mL seawater)
was run in at least triplicate for each plate. All treatments
were performed in at least triplicate. The wells were checked
at regular intervals and the number of dead was counted.
The nauplii were considered dead if no movement of the
appendages was observed within 10 seconds. After 72 hours,
all nauplii were sacriced and counted to determine the total
number per well. The LC
50
with 95% condence limits for
each treatment was calculated using probit analysis.
[16]
RESULTS
Antibacterial activity
One gram of powdered dried S. jambos leaves was extensively
extracted with methanol and dried under vacuum, resulting
in 656 mg of dried extracted material. Resuspension of
the dried fraction in 10 mL of 20% methanol resulted in
the crude test extract concentration of 65.6 mg/mL. The
extract was diluted to a concentration of 15 mg/mL for
testing for antimicrobial activity. Ten microliters of extract
(150 mg) was tested in the disc-diffusion assay against
14 bacteria [Table 1]. The S. jambos leaf extract inhibited
the growth of 4 of the 14 bacteria tested (28.6%).
Numbers indicate the mean diameters of inhibition (mm)
of triplicate experiments 6 standard deviation. ‘-’ indicates
no growth-inhibition. Chloramphenicol (10 mg) and
ampicillin (2 mg) were used as positive controls.
Both gram-positive and gram-negative bacteria were
affected by S. jambos leaf extract, although the gram-
positive bacteria appeared more susceptible. Of the
10 gram- negative bacteria tested, only A. faecalis and
A. hydrophilia (20%) were inhibited by S. jambos leaf extract.
The leaf extract also inhibited the growth of 2 (B. cereus
and S. aureus) of the gram-positive bacteria tested (50%).
The relative level of antibacterial activity was evaluated by
determining the MIC values for each extract against the
bacteria which were shown to be susceptible by disc- diffusion
assays. MICs were evaluated in the current studies by disc
diffusion across a range of concentrations. This has
previously been determined to be a valid method of MIC
determination, as MIC values determined by disc diffusion
correlate well with those determined by broth-dilution
assays.
[17]
The antibacterial activity was strongest against
gram-positive bacteria, especially B. cereus (as determined by
the minimum inhibitory concentration) [Table 2].
Numbers indicate the mean MIC values of at least triplicate
determinations.
Quantication of toxicity
The S. jambos leaf extract was diluted to 2000 mg/mL in
articial seawater for toxicity testing, resulting in a 1000-
mg/ mL concentration in the Artemia franciscana lethality
bioassay [Figure 1a]. For comparison, the reference
Table 1: Antibacterial activity of Syzygium jambos leaf extract
Zone of inhibition (mm)
Syzygium jambos leaf extract Ampicillin Chloramphenicol
Gram-negative rods
A. faecalis
12.6 6 0.5 15.2 6 1.2 6.3 6 0.6
A. hydrophilia
9.7 6 0.8 12.0 6 1.0 28.7 6 1.6
C. freundii -
8.3 6 0.6 15.7 6 1.2
E. coli -
14.7 6 0.6 17.3 6 0.6
K. pneumoniae -
10.3 6 0.6 21.3 6 1.5
P. mirabilis -
17.3 6 0.6 8.7 6 0.6
P. uorescens -
18.2 6 0.5 21.2 6 1.2
S. newport -
18.7 6 0.6 20.3 6 0.6
S. marcescens - -
14.7 6 0.6
S. sonnei -
14.0 6 0 14.3 6 0.6
Gram-positive rods
B. cereus
10.2 6 0.5 26.7 6 0.6 13.3 6 1.2
Gram-positive cocci
S. aureus
9.0 6 0 11.7 6 2.1 16.0 6 1.0
S. epidermidis -
26.3 6 1.5 12.3 6 0.6
S. pyogenes -
17.0 6 1.0 24.0 6 1.0
