FULL LENGTH RESEARCH PAPER - Ahmadu Bello University

Int. Jor. P. App. Scs., 2(4):1-5, 2008__________________________________www.ijpas.com
INTERNATIONAL JOURNAL OF PURE AND APPLIED SCIENCES
FULL LENGTH RESEARCH PAPER
Phytochemical and antibacterial properties of Ludwigia suffruticosa
(Willd.) Oliv. ex. O. Ktze (Onagraceae)
A.B. Aliyu, 1* A. M. Musa, 2 M. S. Abdullahi 3 and A. O. Oyewale 1
1 Department of Chemistry, Ahmadu Bello University, Zaria-Nigeria.
2 Department of Pharmaceutical and Medicinal Chemistry, Ahmadu Bello University, Zaria-Nigeria.
3 Department of Leather & Biotechnology, National Research Institute for Chemical Technology- Basawa, Zaria-
Nigeria.
*Corresponding author’s e-mail: babandoaliyu@yahoo.com Phone: +2348057371917
________________________________________________________________________
ABSTRACT
The antibacterial activities of the ethanol extract, ethyl acetate and n-butanol fractions of
Ludwigia suffruticosa were evaluated using the disc diffusion technique against three
gram-positive bacteria (Staphylococcus aureus, Bacillus. subtilis, Streptococcus
pyogenes) and three gram-negative bacteria (Escherichia coli, Salmonella typhi,
Klebsiella pneumoniae) using ampiclox and streptomycin as reference standards. The
extract and fractions have demonstrated significant antibacterial activity. The ethanol
extract was most potent against all the test organisms with zone of inhibition ranges from
19.0-31.0mm (MIC, 30-50 mgmL -1 ), the n-butanol fraction; 19.0-21.7mm (MIC 40
mgmL -1 ) was also active except on S. pyogenes and E.coli that displayed some resistance.
Similarly, ethyl acetate fraction exhibited activity with zone of inhibition ranges 14.0-
19.0mm (MIC 50 mgmL -1 ) on the organisms except on B. subtilis and K. pneumoniae.
Phytochemical screening revealed the presence of flavonoids, tannins, steroids and
saponins. The spectra of activities displayed by the extract and fractions can be
attributed to the presence of these phytochemicals which signifies the potential of L.
suffruticosa as a source of therapeutic agents and may provide leads in the on going
search for antimicrobial agent from plants.
Keywords: Ludwigia suffruticosa, antibacterial, phytochemical properties.
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INTRODUCTION
Medicinal plants have been used for centuries as
remedies for human diseases because they contain
components of therapeutic values (Nostro et al.,
2000). About 80% of the world population relies on
the use of traditional medicine which is
predominantly based on plant material (WHO, 1993).
The scientific studies available on a good number of
medicinal plants indicates that promising
phytochemicals can be developed for many human
health problems (Gupta, 1994; Dahiru et al., 2005),
including diabetes, cancer and infections diseases.
The continued investigation into the secondary plant
metabolites for anti-infective agents has gained
importance because of the alarming increase in the
rate of resistance of pathogenic microorganism to
existing antibiotics. Therefore the need to develop
efficient, safe and inexpensive drugs from plant
sources is of great importance.
Ludwigia suffruticosa (Onagraceae) or water primrose
is an aquatic plant with a cosmopolitan but mainly
tropical distribution. It is a robust, variable herb with
leaves linear 1-2 mm broad and the stems are up to 2
m high by 1 cm diameter (Burkill, 1997). In Northern
Nigeria the plant is commonly known as Shashatau
(Hausa). The plant is used as vermifuge, laxative, and
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Int. Jor. P. App. Scs., 2(4):1-5, 2008__________________________________www.ijpas.com
for dysentery. It is believed to have analgesic
properties alongside other drug-plants in the treatment
of rheumatic pain (Hutchinson and Dalziel, 1953).
Previous studies on Ludwigia hyssopifolia
(Onagraceae) have indicated that an alkaloid 1-[5-(1,
3-benzodioxol-5-yl)-1-oxo-2, 4-pentadienyl]
piperidine, was isolated and found to exhibit
antibacterial activity (Das et al., 2007). Thirteen
chemical compounds: β-sitosterol, oleanolic acid, α-
hydroxy ursolic acid, tormentic acid, daucosterol,
maltol, luteolin, quercetin, apigenin, methyl
brevifolincarboxylate, gallic acid, ellagic acid and 3,
4, 8,9, 10-pentahydroxydibenzo [b, d] pyran-6-one
(Yan and Yang, 2005) were also isolated from
Ludwigia octovalvis. Although no pharmacological
study has been reported on Ludwigia suffruticosa; this
study was designed to evaluate the phytochemical and
antibacterial properties of Ludwigia suffruticosa, in
order to establish the molecular basis for some of its
therapeutic properties in folkloric use.
MATERIALS AND METHODS
Plant material
Whole plant of Ludwigia suffruticosa was collected in
the month of February, 2006 at Samaru-Zaria,
Nigeria. The plant was taxonomically authenticated at
the herbarium of the Biological Sciences Department,
Ahmadu Bello University, Zaria. A Voucher
specimen (No. 917) was deposited there for future
reference.
Extraction and preliminary fractionation
A 250 g weight of the powdered sample (aerial part of
plant) was extracted exhaustively with 50% ethanol
(cold extraction) for two weeks. The extract was
filtered using Whatman filter paper no. 2, and
concentrated at reduced pressure to afford 12.5 g of
aqueous ethanol extract. 10 g of the extract was
suspended in distilled water and partitioned with ethyl
acetate (3 x 250ml) and n-butanol (3 x 250ml) to give
a yield of 1.22 g and 1.35g respectively.
Phytochemical screening
Phytochemical screening of the extract and fractions
were carried out to identify the constituents, using
standard phytochemical methods as described by
Harborne (1973), Trease and Evans (1989) and
Sofowora (1993).
Test organisms
Six clinical bacterial strains: Escherichia coli,
Klebsiella pneumoniae, Bacillus subtilis,
Streptococcus pyogenes, Staphylococcus aureus, and
Salmonella typhi were obtained from the Department
of Medical Microbiology Ahmadu Bello University
Teaching Hospital (ABUTH) Zaria-Nigeria. The
bacterial strains were maintained on nutrient agar and
sub-cultured every three days. An inoculum of each
bacterial strain was suspended in 5 ml of Mueller
Hinton broth (MHB) and incubated overnight at 37
ºC. The over night cultures were diluted with Mueller
Hinton broth and adjusted to give a concentration of
bacterial cells equivalent to a McFarland 0.5 standard
prior to the bacterial testing (Samie et al., 2005).
Determination of antibacterial activity
The disc diffusion method was used (Nostro et al.,
2000). Stock solution (100 mg mL -1 ) of each extract
and fractions were prepared using the extractants.
Disc (6 mm diameter) were prepared using Whatman
filter paper and sterilized by autoclaving. The blank
sterile discs were placed on the inoculated Mueller
Hinton Agar (MHA) surface and impregnated with
15µL of stock solutions (1500µg dics -1 ). Antibiotic
discs of ampiclox (75µg dics -1 ) and streptomycin
(30µg dics -1 ) were used as control. The plates were
incubated at 37ºC for 24h. All tests were performed in
duplicate and the antibacterial activity was expressed
as the mean diameter of inhibition zones (mm)
produced by the plant extracts.
Minimum inhibitory concentration [MIC]
Minimum inhibitory concentration was carried out
using broth dilution as previously reported by
Lennette et al (1974). Dilution (10-90 mgmL -1 ) of
concentrations of extract and fractions that exhibited
sensitivity against the test organisms were prepared
using test tubes containing 9 ml of double strength
broth. The test tubes were inoculated with the
suspension of the standardized inocula and incubated
at 37ºC for 24h. MICs were recorded as the lowest
concentration of extract showing no visible growth of
the broth.
RESULTS
The results of the phytochemical screening revealed
the presence of flavonoids, tannins, steroids and
carbohydrates in the ethanolic extract, ethyl acetate
and n-butanol fractions. Saponin was present in only
ethanolic extract and n-butanol fraction (Table 1). The
ethanolic extract exhibit activity on all the test
organisms producing zones of inhibition ranging from
19.0-31.0 mm (Table 2). The activity was most on S.
aureus (31.0 mm) and least on E. coli (19.0 mm). The
ethyl acetate fraction also exhibited activity with zone
of inhibition range 14.0-19.0mm except on B. subtilis
and K. pneumoniae. Similarly, the n-butanol fraction
was found to be very active with zone of inhibition
range 19.0-21.7mm except on S. pyogenes and E. coli
(Table 2).The minimum inhibitory concentration
(MIC) showed that ethanolic extract exhibit the
lowest MIC value 30 mgmL -1 against S. aureus, the n-
butanol fraction exhibits 40 mgmL -1 against S. aureus,
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Int. Jor. P. App. Scs., 2(4):1-5, 2008__________________________________www.ijpas.com
B. subtilis, S. typhi and K. pneumoniae. The ethyl
acetate fraction however, showed MIC value of 50
mgmL -1 against S. aureus, S. pyogenes, S. typhi and
E.coli (Table 3).
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Table 1: Results of phytochemical screening
Plant constituents Ethanol Ethyl acetate n-butanol
Extract fraction fraction
Alkaloids - - -
Flavonoids + + +
Saponins + - +
Tannins + + +
Steroids + + +
Carbohydrates + + +
+=positive
- = negative
Table 2: Results of Antibacterial activity of extract and fractions.
Zone of growth inhibition (mm)
Gram
Test organisms
+/-
EE EAF NBF Ampiclox Streptomycin
(75µgdics -1 ) (30µg dics -1 )
S. aureus + 31.0 19.0 20.0 20 27
S. pyogenes + 21.0 14.0 0 0 22
B. subtilis + 27.2 0 21.7 30 30
S. typhi - 20.0 17.0 19.0 0 0
E. coli - 19.0 18.0 0 14 0
K. pneumoniae - 22.2 0 21.0 0 20
EE= Ethanol Extract, EAF = Ethyl acetate fraction, NBF = n-butanol fraction
Table 3: Results of Minimum inhibitory concentration (MIC)
MIC concentrations in mgmL -1
Test organisms EE EAF NBF
S. aureus 30 50 40
S. pyogenes 40 50 NT
B. subtilis 40 NT 40
S. typhi 40 50 40
E. coli 50 50 NT
K. pneumoniae 40 NT 40
EE= Ethanol Extract, EAF = Ethyl acetate Fraction, NBF = n-butanol Fraction, NT = Not tested
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Int. Jor. P. App. Scs., 2(4):1-5, 2008__________________________________www.ijpas.com
DISCUSSION
The crude extract and solvent fractions have
demonstrated significant antibacterial activity. The
ethanol extract was most potent against all the test
organisms with MIC range 30-50 mgmL -1 . The lowest
MIC (highest potency) exhibited was 30 mgmL -1
against S. aureus (Table3). The antibacterial activities
of ethyl acetate and n-butanol fractions showed good
response on both gram-positive and gram-negative
bacteria. Although gram-negative bacteria tend to
have higher intrinsic resistance to most antimicrobial
agents (Ndukwe et al., 2005); In spite of this,
impressive activity against gram-negative bacteria
was observed with ethyl acetate fraction (E.coli
18.0mm, S. typhi 17.0mm) and n-butanol fraction (K.
pneumoniae 21.0mm, S. typhi 19.0mm) (Table 2).
The MIC exhibited by the ethyl acetate (50 mgmL -1 )
and n-butanol (40 mgmL -1 ) fractions against the test
organisms showed that the result was also remarkable.
The potency of the extract and fractions based on the
zones of inhibitions, compared with standard
antibiotics such as ampiclox at 75µgdics -1 and
streptomycin at 30µgdics -1 as shown in Table 2. It was
however observed that the antibacterial activity of
crude ethanolic extract is higher than those of the
solvent fractions; which indicate that the activity may
be due to combined effects of the fractions or
synergism.
Flavonoids, saponins, tannins and steroids were
present in the extract and fractions. Antibacterial
properties of several plant extracts have been
attributed to secondary metabolites such as some of
these (Cowan, 1999; Okoli and Iroegbu, 2005). Thus,
the spectra of activities displayed by these extracts
can be explained by the presence of flavonoids,
tannins, saponins and steroids. It signifies the
potential of Ludwigia suffruticosa as a source of
therapeutic agents which may provide leads in the on
going search for antimicrobial agent from plants.
In conclusion, the significant activity exhibited by the
extracts against clinical bacterial strains that are
associated with various infectious diseases, may
provide scientific justification for the ethnomedicinal
uses of the plant.
ACKNOWLEDGEMENTS
To Mallam Umar S. Gallah for identifying the plant,
and Mrs. Asiya Abba Mohammad of Federal College
of Education, Zaria; for typesetting the manuscript.
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