World Applied Sciences Journal 16 (1): 22-29, 2012

ISSN 1818-4952
© IDOSI Publications, 2012

Antioxidant and Antibacterial Activity of Ludwigia octovalvis on

Escherichia coli O157:H7 and Some Pathogenic Bacteria

Haidar Kadum Yakob, Shaida F. Sulaiman and Abd M. Uyub

School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia

Abstract: Ludwigia octovalvis (Jacq.) P. H. Raven (Family: Onagraceae) is traditionally used to treat skin
diseases, diarrhea and flatulence. This study assayed twelve extracts of L. octovalvis for their total phenolic
content (TPC), antioxidant and antibacterial activity. The highest TPC at 264.76 ± 0.23 GAE mg/g dry weight
and antioxidant activity (evaluated by 2, 2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power
assays) at 1080.84 ± 6.07 µM TE/mg dry weight and 1256.88 ± 5.38 µM TE/mg dry weight, respectively, were
detected in 80% methanol extract of the leaf. A strong correlation between TPC and antioxidant activities of
both assays was observed (r > 0.98). Eighty percent methanol extract of the leaf gave the lowest minimum
inhibitory concentration (MIC, 62.5 µg/mL) and minimum bactericidal concentration (MBC, 125.0 µg/mL) against
Escherichia coli O157:H7, Escherichia coli ATCC 25922 and Bacillus spizizenii (ATCC 6633), while 80%
methanol extract of the root gave similar MIC and MBC values against Pseudomonas aeruginosa
(ATCC 27853). Our findings suggest that leaf of L. octovalvis as a possible new source of natural mixture of
antioxidant and anti-E. coli O157:H7.

Key words: E. coli O157:H7 Ludwigia octovalvis Total phenolic DPPH FRAP

INTRODUCTION flatulence [12, 13]. A review performed on major published

series such as PUB MED (http:www.pub.med, accessed
E. coli O157:H7 is a foodborne pathogen that causes on 3rd October 2011) revealed that reported antibacterial
diarrhea, hemorrhagic colitis and, in a subset of patients, activities of this plant are limited only to against
hemolytic uremic syndrome, which is a life-threatening Streptococcus mutans [14], Helicobacter pylori [15] and
complication [1]. It was first isolated in the USA in 1975 dermatological bacteria [16].
from sporadic cases of hemorrhagic colitis, but is now Similarly, antioxidant activity of L. octovalvis is only
implicated with disease outbreaks around the globe [2]. limited to one report [17] and there was no mentioned of
Initial outbreak was associated with undercooked chemical compounds associated with the activity.
hamburgers and unpasteurized milk, but currently a Subsequent studies [18-20] reported on the isolation of
variety of foods, cow-manure contaminated water and new compounds from L. octovalvis with anticancer
vegetables, swimming in contaminated waters and activity and some of these compounds like luteolin,
indirect or direct animal contact have been implicated as quercetin, apigenin and gallic acid are known to have
sources of the pathogen [3-6]. Plant extracts have been antibacterial and antioxidant activity. Emerging evidence
developed and used in foods as natural antioxidants is pointing to the beneficial dual role of phenolic
and antimicrobials [7]. There have been studies on phytochemicals having antioxidant activity as well as anti-
antibacterial activity against E. coli O157:H7 and other E. coli O157:H7 agent [21]. The synergistic contribution
pathogens by plant extracts or metabolites for their of phenolics and antioxidant activity may have
potential use in controlling foodborne pathogens [8-10]. implications for extending the shelf life of foods as well as
Ludwigia octovalvis (Jacq.) P. H. Raven (Family: reduction of foodborne diseases due to E. coli O157:H7.
Onagraceae) is a medicinal plant that is widely distributed In the present study we reported on the antioxidant
in Malaysia [11]. A poultice of an entire plant is externally and antibacterial activity of methanol extract of L.
applied to heal dermatitis, boil, ulcer, impetigo and pimple. octovalvis against E. coli O157:H7 and other bacteria.
Its decoction is drunk as a health drink and to treat Antioxidant activity correlates strongly with total
gastrointestinal complaints such as diarrhea and phenolic content.

Corresponding Author: Abd M. Uyub, School of Biological Sciences, Universiti Sains Malaysia, 11800,
USM, Pulau Pinang, Malaysia. Tel: +604-6534002, Fax: +604-6565125, E-mail: uyub@usm.my.
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 World Appl. Sci. J., 16 (1): 22-29, 2012

MATERIALS AND METHODS spectrophotometer (Hitachi Model U-1900) with 10 %

(v/v) DMSO as blank. The procedure was repeated using
Plant Material: The whole plant of L. octovalvis was different concentrations of standard gallic acid solutions
harvested at its mature stage from the wet area of the (0.05–0.2 mg/ml).
Universiti Sains Malaysia main campus, Pulau Pinang. A Experiments were carried out in triplicate and the
voucher specimen number (11090) was deposited at the mean value was recorded. The TPC was calculated as
herbarium of the School of Biological Sciences, Universiti gallic acid equivalent (GAE) using the following equation:
Sains Malaysia.
Y (absorbance) = 8.982 X (µg gallic acid) – 0.01412, r2 =
Chemicals and Reagents: The following chemicals and 0.9925 that was obtained from the standard gallic acid
reagents were used in this study: Fisher Scientific, graph. The absorbance value was inserted in the
Springfield, USA: n-Hexane, chloroform, ethyl acetate and abovementioned equation and the total amount of
methanol. Sigma-Aldrich Chemical, St. Louis, USA: Folin- phenolic compound was calculated.
Ciocalteau’s (FC) reagent, 2, 2-diphenyl-1-picrylhydrazyl
(DPPH), gallic acid (98% purity),2,4,6-tris (2-pyridyl)-1,3,5- Determination of Antioxidant Activity
triazine (TPTZ), ferric chloride hexahydrate, sodium 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH) Assay: The free
acetate, acetic acid glacial, Trolox (6-hydroxy-2,5,7,8- radical scavenging activity of all the extracts was
tetramethylchroman-2-carboxylic acid) and p- measured using DPPH scavenging activity [24, 25], with
iodonitrotetrazolium chloride (INT). QRec, Germany: slight modifications. Briefly, 150 µl of 300 µM ethanolic
Dimethylsulphoxide (DMSO). Oxoid, England:nutrient DPPH solution was added to 50 µl of 1 mg/ml extracts
broth and nutrient agar. Bendosen Laboratory Chemicals, (diluted in DMSO) in 96-microwell plates. DMSO was
England: Anhydrous sodium carbonate. used as negative control. The reaction mixture was
incubated in the dark at 37°C for 30 min. The decrease in
Plant Extraction: The leaves, stems and roots of the plant absorbance value was then measured at 515 nm using a
were separated and washed. The samples were then microplate reader (Thermo, Multiskan Ex, Finland). All
dried at 40°C for 24 h and ground into powder. Each measurements were carried out in triplicate.
powdered plant part (30 g) was sequentially extracted
(cold extraction) in a flask with 200 ml of n-hexane by Ferric Reducing Antioxidant Power (FRAP) Assay:
continuous shaking at 40°C for 8 h. The extract was Reducing power was determined using a FRAP assay [26],
filtered using 150 mm diameter filter paper (Whatman, with slight modifications. The FRAP reagent was prepared
U.K.). The residue was then dried, similarly and by mixing 300 mM acetate buffer (pH 3.6), 10 µM TPTZ
successively extracted using chloroform, ethyl acetate solution in 40 mM HCl and 20 mM ferric chloride
and finally 80% (v/v) methanol all at 40°C. Each extract hexahydrate at a proportion of 10:1:1 (v:v). The FRAP
was concentrated using a rotary evaporator and stored at reagent was freshly prepared before analysis and warmed
-4°C until further use. Overall, a total of 12 extracts was to 37°C prior to use. The extracts were dissolved in DMSO
obtained. at a concentration of 1 mg/ml. Extracts (20 µl) were allowed
to react with 180 µl of FRAP solution for 5 min in the dark.
Determination of Total Phenolic Content: The total The absorbance of the reaction mixture was then
phenolic content (TPC) in all the extracts was estimated measured at 593 nm using a microplate reader (Thermo,
by a colorimetric assay [22, 23], with slight modifications. Multiskan Ex, Finland). All measurements were carried out
The TPC was calculated from the calibration curve using in triplicate.
gallic acid as a standard. The results are expressed as
milligram of gallic acid equivalents per gram dry weight of Determination of FRAP and DPPH Inhibition Values:
extract (mg GAE/g d.w.). DMSO (10%v/v) was used to Trolox was used as reference in both assays. Two
dilute the extract to obtain an initial concentration of 1 different standard curves were obtained using Trolox
mg/ml. Briefly, 0.5 ml of each extract was pipetted in a test standard solution in 10 % (v/v) DMSO at various
tube followed by 1.0 ml of 10% dilution of FC reagent. concentrations. The absorbance of the reaction sample
The contents of the test tube were thoroughly mixed. was compared to that of the Trolox standard. The results
After 3 min, 3 ml of sodium carbonate (1% w/v) was are expressed in terms of microMolar of Trolox
added. The mixture was kept in the dark for 2 h at 25°C. equivalents per milligram dry weight of extract (µM TE/mg
The absorbance was measured at 760 nm using a d.w.).

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 World Appl. Sci. J., 16 (1): 22-29, 2012

Antibacterial Activity against E. coli O157:H7 in a total volume of 200 µl [28].

Bacterial Strains and Growth Media: Antibacterial Cells (5×105) were cultured in nutrient broth containing
activity was evaluated on following strains of bacteria, final concentration of the extract ranging from 1000 µg/ml
Gram positive bacteria: Bacillus cereus (ATCC 10876), to 7.8 µg/ml. In two positive controls, cells (5×105) were
Bacillus licheniformis (ATCC 12759), Bacillus spizizenii cultured in nutrient broth containing final concentration
(ATCC 6633), Staphylococcus aureus (ATCC 12600), of chloramphenicol or doxycycline ranging from 250 µg/ml
Staphylococcus epidermidis (ATCC 12228) and to 2.0 µg/ml. A negative control with cells (5×105) cultured
Streptococcus mutans (ATCC 25175), Gram-negative in nutrient broth only and another control containing
bacteria: Escherichia coli (ATCC 25922), Escherichia coli extract and nutrient broth but without cells were also
0157:H7, Klebsiella pneumoniae (ATCC 13883), included in the 96-well plate. All tests were in triplicates.
Pseudomonas aeruginosa (ATCC 27853), Pseudomonas The plates were covered and incubated aerobically at
stutzeri (ATCC 17588) and Shigella boydii (ATCC 9207). 37°C. After 18h, 40 µl of 0.2 mg/ml INT was added to each
E. coli O157:H7 was obtained from National Laboratory well and the plates were further incubated for 30 min.
for Food Safety and Quality of Malaysia. All bacteria Bacterial growth in the wells was indicated by
strains were cultured on nutrient agar at 37°C for 18 h. and development of red-pink color, while growth inhibition
stored on nutrient agar slant at 4°C until use. was indicated by no change in the colour of cell
suspensions. The MIC of each extract is defined as the
Disc Diffusion Method: A disc diffusion method [27] was lowest concentration inhibiting growth. To determine the
used to determine the antibacterial activities of all the MBC, samples from all wells showing no growth as well as
extracts. Stock solutions of each extracts were prepared sample from the lowest concentration showing growth in
by dissolving 20 mg of the extract in 1 ml 10% DMSO and the MIC assay, were subcultured on freshly prepared
filtered (0.2 µm, Whatmann, UK). Stock solution of nutrient agar. Plates were incubated for 24h at 37°C to
antibiotics (chloramphenicol or doxycycline) used as check for any macrocolonies developing. The MBC is the
positive controls were prepared at 1mg/ml. lowest concentration of extract that killed viable cells,
For a particular bacterial species tested, cells from a hence no macrocolonies were formed on the recovery
18h culture at 37°C on nutrient agar, were suspended in medium.
saline solution (0.85% w/v) and turbidity was adjusted to
a scale of 0.5 on the MacFarland standard (Biomerieux, Statistical Analysis: All data are reported as the mean ±
France). Briefly, 100 µl of the bacterial suspension was S.D. of triplicate determinations. The statistical analysis of
inoculated on nutrient agar and swabbed three times, the data was carried out using one-way ANOVA, followed
rotating the plates 120 degrees between swabbing to by post hoc least-significant difference (LSD) test using
distribute the cells uniformly. Seven discs were gently SPSS 17.0 for Windows (SPSS Inc., Chicago, IL) at a
pressed on the agar with sterile forceps viz. two as confidence level higher than 95% (p < 0.05). Pearson’s
negative controls (each impregnated with 20 µl water or correlation test was conducted to determine the
10%v/v DMSO) and one impregnated with 20 µl of correlation between antioxidant and TPC. The test was
chloramphenicol (20 ug/disc) and the remaining 4, each carried out using the Prism 3.02 statistical software of
impregnated with 20 µl of n-hexane, chloroform, ethyl GraphPadPrism (San Diego, CA).
acetate and 80% methanol extract of leaf (400µg
extract/disc). This was done in triplicate and repeated but RESULTS
using doxycycline as a positive in place of
chloramphenicol. The whole test was then repeated but Total Phenolic Content (TPC): Eighty percent of
using extracts of stem and roots. These plates were methanol extracts of the leaf and stem contained
incubated aerobically overnight at 37°C and the diameters significantly (p < 0.05) highest amount of TPC (264.76 ±
of the inhibition zones were measured. 0.23 GAE mg/g d.w. and 239.05 ± 0.29 GAE mg/g dry
weight, respectively. Generally TPC of extract declined
Broth Micro-Dilution Method: Stock solution of 80% with decreasing polarity of the solvent used, i.e 80%
methanol extract of leaf was prepared in 10% DMSO and methanol, ethyl acetate, chloroform and n-hexane. But for
filtered (0.2 µm, Whatmann UK). A broth micro-dilution the root, TPC was significantly higher (p<0.05) in ethyl
bioassay in 96-well plates was used to determine MIC and acetate extract compared to that of 80% methanol extract
MBC of the extract or antibiotic at two-fold dilution (Table 1).

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 World Appl. Sci. J., 16 (1): 22-29, 2012

Table 1: TPC and antioxidant activity of Ludwigia octovalvis extracts

Antioxidant activity (µM TE/mg d.w.)
------------------------------------------------------------
Plant part Extract TPC (mg GAE/g d.w.) DPPH FRAP
Leaf Methanol 264.76±0.23a 1080.84±6.07a 1256.88±5.38 a
Ethyl acetate 68.45±0.17e 301.48±8.92d 253.45±5.97 d
Chloroform 47.85±0.28i 113.04±1.58j 136.83±3.48g
n-Hexane 34.64±0.11j 91.00±0.31k 142.04±3.53g
Stem Methanol 239.05±0.29b 905.00±7.37b 912.17±5.06b
Ethyl acetate 70.23±0.23d 206.68±2.71f 166.02±2.3f
Chloroform 68.45±0.17e 182.30±5.02g 188.30±6.73e
n-Hexane 57.65±0.17g 146.15±5.87i 113.40±6.65h
Root Methanol 62.32±0.17f 225.32±1.84e 203.67±7.05e
Ethyl acetate 98.88±0.19c 369.28±8.76c 303.36±4.76 c
Chloroform 70.08±0.28d 174.52±5.71gh 159.90±2.07f
n-Hexane 50.00±0.22h 162.06±4.7hi 195.73±6.32de
Values are mean ± standard deviation of three replicates. Values in the same columns with different superscript letters are significantly different (p < 0.05) based
on one-way ANOVA and post hoc least-significant difference (LSD) test

Antioxidant Activity (AOA): The results of antioxidant

activity from both assays are expressed as µM Trolox
equivalent (µM TE)/mg dry weight which is a more
meaningful and descriptive expression, compared with
antioxidant activity expressed as percentage of activity at
a specific concentration (Table 1). Results may provide a
direct comparison of the antioxidant activity with that of
Trolox equivalent [29].
The 80% methanol extract of leaf exhibited the
highest antioxidant activity with DPPH and FRAP values
at 1080.84 ± 6.07 µM TE/mg dry weight and 1256.88 ± 5.38 Fig. 1: Correlation between TPC and antioxidant
µM TE/mg dry weight, respectively (Table 1). These activity using DPPH assay of 12 extracts of
values were followed by the 80% methanol extract of the Ludwigia octovalvis
stem with DPPH and FRAP values at 905.00 ± 7.37 µM
TE/mg d.w. and 912.17 ± 5.06 µM TE/mgd.w., respectively.
Like the TPC, generally antioxidant activity of extract also
decreased with decreasing polarity of the solvents used
except for the root when antioxidant activity was
significantly higher (p < 0.05) in ethyl acetate extract than
in 80% methanol extract.

Correlation Between TPC and AOA: The correlation

between TPC and antioxidant activities of the extracts was Fig. 2: Correlation between TPC and antioxidant
tested using Pearson’s correlation test. The TPC values activity using FRAP assay of 12 extracts of
(mg GAE/g dry weight) of all the extracts were plotted Ludwigia octovalvis.
separately against those of DPPH and FRAP. Figures 1
and 2 show high correlation coefficients were observed possessed antibacterial activity, but they were unable to
between TPC and DPPH values (r = 0.9926, p <0.0001) and inhibit all the bacteria tested, unlike the control
between TPC and FRAP values (r = 0.9814, p < 0.0001). antibiotics. Generally methanol extracts were active
against both gram-positive and gram-negative bacteria
Sensitivity of Bacteria Against Plant Extract: By disc and methanol extract of leaf inhibited both the E. coli
diffusion assay, both positive control antibiotics inhibited strains used. With additional factor that methanol extracts
all the bacteria tested (Table 2). For the extract, only contained high TPC, they were preferred for determination
methanol extract of stem and roots and all leaf extracts of MIC and MBC against the tested bacteria.

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 World Appl. Sci. J., 16 (1): 22-29, 2012

Table 2: Antibacterial activities of extracts of Ludwigia octovalvis based on disc diffusion assay
Bacteria Diameter of inhibition zone (mm)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------
Leaf Stem Root Antibiotics
----------------------------------------------------------------------------- ----------- ----------- ------------------------------------------
Gram-positive Methanol Ethyl- acetate Chloroform n-Hexane Methanol Methanol Chloramphenicol Doxycycline
B. cereus 10.8±0.8b 7.2±0.4a x x
7.6±0.8a 10.0±0.6b 17.5±0.3fg 16.5±0.5f
B. licheniformis 12.2±1.2c 8.4±0.5a x x
9.7±0.6b x
15.2±0.8e 22.2±0.3i
B. spizizenii 14.0±0.8de 7.0±0.5a x x
10.2±0.3b 12.2±1.3c 24.0±0.4i 27.1±0.3j
Staph. aureus 7.8±1.7a 9.2±1.2b 7.3±0.4a 7.0±0.0a 7.0±0.6a 10.5±0.7b 17.4±0.6f 19.4±0.6h
Staph.epidermidis 17.8±1.2fg 9.5±0.5b 8.3±0.5a 7.0±0.0a 11.3±1.0c 10.0±0.5b 20.0±0.5h 13.5±0.5cd
Strep. mutans 15.3±1.6e 9.1±1.2b 8.0±0.4a 7.4±0.3a 11.4±0.8c 10.2±0.8b 18.0±0.0fg 22.0±0.4i
Gram negative
E. coli O157H7 12.0±0.4c 10.6±0.5b 9.5±1.5b 7.0±0.0a x x
15.8±1.2e 14.7±0.8e
E. coli ATCC 14.8±0.8e 11.0±1.8c 9.0±0.5b 8.2±1.3a 10.3±0.6b 10.6±0.7b 20.6±2.2h 16.6±0.5f
Kl. pneumoniae x x x x x
8.8±0.5a 18.4±1.7g 15.4±0.8f
Ps.aeruginosa 9.8±1.4b 8.0±1.0a 7.6±0.1a 7.0±0.0a 8.2±0.2a 14.2±1.4e 16.4±1.5f 18.4±0.5g
Ps. Stutzeri 15.7±1.1e 13.2±0.8cd 10.7±1.1b 9.3±0.8b 11.3±0.8c 7.7±1.0a 12.9±1.5cd 11.9±0.4c
Sh. Boydii 8.0±0.8a 9.4±0.3b 9.0±0.0b 8.7±1.0a 7.0±0.0a 10.2±0.8b 17.0±0.2f 18.0±0.2g
Values are means of three replicates ± standard deviation. Values with different superscript letters are significantly different (p < 0.05) based on one-way ANOVA
and post hoc least-significant difference (LSD) test. xno inhibition

Table 3: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 80% methanol extracts of Ludwigia octovalvis
Bacteria MIC and MBC values (µg/mL)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------
Leaf Stem Root Chloramphenicol Doxycycline
-------------------- ----------------------- ----------------------- --------------------- -------------------------
Gram-positive MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
B. cereus 125.0 250.0 500.0 1000.0 125.0 250.0 15.60 31.25 15.60 31.25
B. licheniformis 500.0 1000.0 250.0 500.0 >1000 >1000 15.60 31.25 15.60 31.25
B. spizizenii 62.5 125.0 500.0 1000.0 125.0 500.0 7.80 15.60 7.80 15.60
S. aureus 500.0 1000.0 1000.0 >1000 250.0 1000.0 15.60 31.25 15.60 31.25
S. epidermidis 250.0 500.0 125.0 250.0 250.0 500.0 7.80 15.60 31.25 62.50
S. mutans 125.0 500.0 125.0 250.0 125.0 250.0 15.60 31.25 7.80 15.60
Gram- negative
E. coli O157H7 62.5 125.0 >1000 >1000 >1000 >1000 15.60 31.25 15.60 31.25
E. coli ATCC 62.5 125.0 125.0 250.0 125.0 250.0 15.60 31.25 7.80 15.60
K. pneumoniae 125.0 250.0 >1000 >1000 125.0 500.0 15.60 31.25 15.60 31.25
P. aeruginosa 125.0 1000.0 500.0 1000.0 62.5 125.0 15.60 31.25 7.80 15.60
P. stutzeri 125.0 250.0 125.0 250.0 125.0 250.0 31.25 62.50 31.25 62.50
S. boydii 125.0 500.0 500.0 1000.0 125.0 250.0 15.60 31.25 15.60 31.25

In view of the limitation of disc diffusion assay to DISCUSSION

objectively indicate actual activity of the plant extracts,
broth microdilution technique was applied to determine In this study, three different parts of L. octovalvis
the MIC and MBC of methanol extracts. The lowest MIC were separately and successively extracted by increasing
and MBC were 62.50 µg/mL and 125.00 µg/mL, the polarity of solvents and each extract was quantified
respectively and this was demonstrated by leaf methanol for its TPC and evaluated for its antioxidant and
extract against both E. coli strains and B. spizizenii and antibacterial activities. Our finding that 80% methanol
root methanol extract against P. aeruginosa. Other extract of stem and leaf contained high amount of TPC
extracts gave higher MIC and MBC values and were explains its broad spectrum antibacterial activity as shown
considered ineffective (Table 3). by disc diffusion assay. It is known that plant phenolics
None of the extracts demonstrated comparable exert their bactericidal activity by accumulating at the cell
MIC and MBC values with the positive controls. The membrane [30] and disrupting the membrane fluidity [31]
antibacterial activity of leaf methanol extract against leading to efflux of K + ions, a major cytoplasmic action
E. coli O157:H7 is of great importance considering that it and involved in several key functions of growing bacterial
is an emerging human pathogen. cells.

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 World Appl. Sci. J., 16 (1): 22-29, 2012

Result from the study also showed some bacteria role of phenolic phytochemicals having an antioxidant
were less sensitive towards 80% methanol extract. This is activity as well as antibacterial activity especially E. coli
not unexpected as earlier report [32] showed similar result, O157:H7. Emerging epidemiological evidence is
albeit using a different Ludwigia species that is L. increasingly pointing to the beneficial effects of phenolic
adscendens. Our result that polar extract were active phytochemicals having antioxidant activity in managing
against wide range of bacteria also concur with earlier infectious diseases [38]. Synergistic phenolics and
report on polar solvent extract of L. octovalvis which antioxidant activity inhibits bacterial growth and this has
exhibited significantly higher antibacterial activity against implications for diet-based management of infectious
dermatological bacteria than did the non-polar extracts disease which is an inexpensive blocker against infectious
such as petroleum ether, hexane and chloroform [16]. diseases [39]. A mixture of phenolics phytochemicals in
For the root, TPC and AOA of 80%methanol extract whole foods is effective in protectively supporting human
was lower than that of ethyl acetate, which may be due to health compared to isolated individual phenolic
the differences in morphological and anatomical phytochemicals. L. octovalvis is potentially useful as
characteristics of the different parts of a plant. This sources of phenolics and therefore future studies should
necessitates a different extraction solvent system to identify the profile of phenolic phytochemicals which
ensure optimum recovery of TPC [33]. TPC in ethyl contributes to the functionality of the plant from
acetate extract is three times lower than in leaves and synergistic interaction of constituent phenolic
stem, so its contribution to the observed antibacterial phytochemicals.
activity was possibly minor.
To the best of our knowledge, activity of L. CONCLUSIONS
octovalvis against E. coli O157:H7, a newly emerging
pathogen appears lacking. By microdilution assay, only Extracting solvents and plant parts influenced the
80% methanol extract of leaf inhibited both E. coli strains TPC, antioxidant and antibacterial activities of the
at the lowest MIC (62.5µg/ml) and MBC (125 µg/ml) extracts. The most polar solvent used (80% methanol)
values. This finding suggests the potential of leaf extract extracted the most total phenolics from leaf and stem,
as a natural source of both, phenolics which are which correlates strongly with their high antioxidant and
antibacterials and antioxidants. Only one publication explains why the extract has antibacterial activity. Eighty
reported the MIC of L. octovalvis and that was against percent methanol extract of leaf inhibited both E. coli
S.mutans [14]. Our study showed that MIC of all 80% 0157:H7 and E. coli ATCC 25922 and Bacillus spizizenii
methanol extracts against S. mutans was 125.0 µg/ml, (ATCC 6633), while that of the root inhibited
much lower than that of the aqueous extract of the entire Pseudomonas aeruginosa (ATCC 27853) at MIC and
plant at 2000 µg/ml. This showed 80% methanol as the MBC values of 62.5 ug/ml and 125 ug/ml, respectively.
preferred solvent for extracting antibacterial phenolic Other extracts were considered ineffective as the MIC and
compounds from L. octovalvis. Overall, antioxidant MBC were higher than these values. We conclude that
and antibacterial activities of the extracts vary with the 80% methanol extract of the leaf of L. octovalvis could
parts of L. octovalvis and types of solvent used [34]. possibly be a new source of natural mixture of antioxidant
Bioactive components of different species and parts of and anti E. coli O157:H7.
plants have different solubility levels in different
solvents [35]. ACKNOWLEDGMENTS
The extracts were also examined for their antioxidant
activities using DPPH and FRAP assays. Despite This study was supported by an incentive grant
representing different mechanism of actions, these assays (Grant number: 1001/PBiologi/822151) and postgraduate
generated a similar conclusion that TPC correlates grant scheme (1001/PBiologi/843096) from the Universiti
strongly (r > 0.98) with AOA. These results supported Sains Malaysia.
earlier studies [36, 37], who found a significant correlation
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