Journal of Apicultural Research

ISSN: 0021-8839 (Print) 2078-6913 (Online) Journal homepage: http://www.tandfonline.com/loi/tjar20

Antibacterial activity of polyphenol-rich extract of

selected wild honey collected in Sabah, Malaysia

Philip Yap, Mohd Fadzelly Abu Bakar, Herbert Lim & David Carrier

To cite this article: Philip Yap, Mohd Fadzelly Abu Bakar, Herbert Lim & David Carrier (2016):
Antibacterial activity of polyphenol-rich extract of selected wild honey collected in Sabah,
Malaysia, Journal of Apicultural Research, DOI: 10.1080/00218839.2016.1151633

To link to this article: http://dx.doi.org/10.1080/00218839.2016.1151633

Published online: 11 Mar 2016.

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 Journal of Apicultural Research, 2016
http://dx.doi.org/10.1080/00218839.2016.1151633

ORIGINAL RESEARCH ARTICLE

Antibacterial activity of polyphenol-rich extract of selected wild honey collected in
Sabah, Malaysia
Philip Yapa, Mohd Fadzelly Abu Bakara,b*, Herbert Limc and David Carrierd
a
Institute for Tropical Biology and Conservation, University Malaysia Sabah, Kota Kinabalu, Malaysia; bFaculty of Science, Technology and
Human Development (FSTPi), Universiti Tun Hussein Onn Malaysia (UTHM), Batu Pahat, Malaysia; cLagud Sebrang Agriculture Research
Station, Tenom, Malaysia; dCentre for Plant Sciences, School of Molecular and Cellular Biology, University of Leeds, Leeds, UK
(Received 22 April 2013; accepted 18 February 2014)

Four types of Sabah wild honey produced by bee species native to Sabah; namely Apis cerana, Apis andreniformis, Apis
nuluensis and Apis koschevnikovi were collected from January to April 2012. This study was conducted to determine the
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antibacterial activity, minimum inhibitory concentration (MIC) activity, total phenolic and flavonoid contents of this wild
honey. All samples were extracted using 80% and absolute methanol. For antibacterial activity, wild A. cerana honey
showed the strongest effect against all five different bacteria strains tested with its greatest activity against Bacillus cer-
eus with the value of 16.00 mm (100% concentration) in 80% methanol extract. As for the MIC activity, wild A. cerana
and wild A. nuluensis honey have the lowest MIC value against the tested bacteria with the lowest against Bacillus cereus
and Bacillus subtilis. Total phenolic and total flavonoid contents were highest in the wild A. cerana honey with the values
of 10.43 ± .02 and 8.76 ± .00 mg/g in 80% methanol extract. These findings suggested that the wild honey from Sabah
have a wide range of phytochemical content and antibacterial activities against different strains of bacteria which might
contribute to the development of pharmaceuticals and nutraceuticals products.

Actividad antibacteriana del extracto rico en polifenoles de miel seleccionada

silvestre recogida en Sabah, Malasia

Se recogieron cuatro tipos de miel silvestre producida por especies de abejas nativas de Sabah; a saber, Apis cerana,
Apis andreniformis, Apis nuluensis y Apis koschevnikovi entre enero y abril de 2012. Este estudio se realizó para determinar
la actividad antibacteriana, la actividad de la concentración mı́nima inhibitoria (CMI), y el contenido de fenoles totales y
de flavonoides de esta miel silvestre. Todas las muestras fueron extraı́das con metanol al 80% y absoluto. Para la activi-
dad antibacteriana, la miel silvestre de A. cerana mostró el efecto más fuerte contra todas las cinco cepas de diferentes
bacterias probadas con su mayor actividad contra Bacillus cereus con el valor de 16,00 mm (concentración 100%) en
extracto de metanol 80%. En cuanto a la actividad de CMI, la miel silvestre de A. cerana y A. nuluensis tienen el valor
más bajo de CMI contra las bacterias probadas con el nivel más bajo contra Bacillus cereus y Bacillus subtilis. Los con-
tenidos totales de fenólico y de flavonoides fueron más altos en la miel silvestre de A. cerana con los valores de 10,43
± 0,02 mg/g y 8,76 ± 0,00 mg/g en extracto de metanol al 80%. Estos hallazgos sugieren que la miel silvestre de Sabah
tiene una amplia gama de contenido fitoquı́mico y actividad antibacteriana contra diferentes cepas de bacterias que
podrı́an contribuir al desarrollo de productos farmacéuticos y nutracéuticos.
Keywords: Sabah honey; antibacterial activity; MIC activity; total phenolic and flavonoid contents

Introduction and actions of active biological compounds, honey can

Honey is a sweet, viscous liquid produced by honey
bees. It is made by the bees from nectar gathered from
flowers, and consequently contains various phytochemi-
cals (Al-Waili, 2004). Sabah has four types of honey
bees from the genus Apis (Koeniger, Koeniger, & Tingek,
2010). New species of Apis have recently been discov-
ered, namely A. nuluensis that can be found in the region
near mount Kinabalu (Koeniger et al., 2010).
Physicochemical and phytochemical properties of
wild honey can be due to natural variations composi-
tions as they originate from different plant varieties and
collected from different geographical locations (Yannio-
tis, Skaltsi, & Karaburnioti, 2006). Due to its content
be used as a natural therapeutic substance (Gullett
et al., 2010). The application and used of honey in medi-
cine as a natural remedy has been performed for thou-
sands of years with recorded use by many different
ancient societies (Zumla & Lulat, 1989). Thus, the
potential applications for honey in primary health care
are very promising especially in the pharmaceuticals and
nutraceuticals fields (Burkill, 1935). Modern scientific
research strategies into the medicinal properties of
honey will undoubtedly result in a beneficial contribu-
tion to human health worldwide.
Honey has been reported to have at least 181 sub-
stances, which include phenolic acids, flavonoids, certain

*Corresponding author. Email: fadzelly@uthm.edu.my

© 2016 International Bee Research Association

 2 P. Yap et al.

Table 1. Antimicrobial activity of wild Sabah honey against S. aureus in 80% and absolute methanol extracts.

Inhibition zone (mm) Inhibition zone (mm) of Inhibition zone (mm) of

of wild A. cerana wild A. andreniformis Inhibition zone (mm) of wild A. koschevnikovi
honey honey wild A. nuluensis honey honey
Concentration of 80%
methanol extracts (v/v)
(%)
25 4.00 ± .06d 1.00 ± .02d 2.00 ± .00d 1.00 ± .05d
50 5.00 ± .24c 2.00 ± .12c 4.00 ± .02c 2.00 ± .00c
75 6.00 ± .00b 3.00 ± .06b 5.00 ± .02b 4.00 ± 2.00b
100 8.00 ± .03a 5.00 ± .21a 6.00 ± .03a 5.00 ± 1.00a
Concentration of
absolute methanol
extracts (v/v)
25 1.00 ± .01b 1.00 ± .00d 1.00 ± .00c 1.00 ± .02b
50 1.00 ± .04b 2.00 ± .01c 1.00 ± .03bc 2.00 ± .00b
75 2.00 ± .00b 3.00 ± .03b 2.00 ± .05b 3.00 ± .02b
100 3.00 ± .04a 4.00 ± .01a 3.00 ± .00a 4.00 ± .06a
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Note: Values presented as mean ± standard deviation (n = 3), results in column with different alphabets are significantly different (p < .05).

Table 2. Antimicrobial activity of wild Sabah honey against B. cereus in 80% and absolute methanol extracts.

Inhibition zone (mm) Inhibition zone (mm) of Inhibition zone (mm) of

of wild A. cerana wild A. andreniformis Inhibition zone (mm) of wild A. koschevnikovi
honey honey wild A. nuluensis honey honey
Concentration of 80%
methanol extracts (v/v)
(%)
25 2.00 ± .01d 2.00 ± .01d 3.00 ± .01d 1.00 ± .05d
50 3.00 ± .24c 3.00 ± .01c 4.00 ± .04c 2.00 ± .02c
75 6.00 ± 2.00b 4.00 ± .26b 5.00 ± .02b 3.00 ± .04b
100 16.00 ± .33a 5.00 ± .05a 7.00 ± .02a 5.00 ± .02a
Concentration of
absolute methanol
extracts (v/v)
25 1.00 ± .01d 1.00 ± .22c 1.00 ± .00d 1.00 ± .02b
50 2.00 ± .05c 2.00 ± .01b 2.00 ± .02c 1.00 ± .03b
75 4.00 ± .07b 2.00 ± .01b 3.00 ± .22b 2.00 ± .01b
100 8.00 ± .02a 3.00 ± .06a 4.00 ± .01a 3.00 ± .02a
Note: Values presented as mean ± standard deviation (n = 3), results in column with different alphabets are significantly different (p < .05).

Table 3. Antimicrobial activity of wild Sabah honey against B. subtilis in 80% and absolute methanol extracts.

Inhibition zone (mm) Inhibition zone (mm) of Inhibition zone (mm) of

of wild A. cerana wild A. andreniformis Inhibition zone (mm) of wild A. koschevnikovi
honey honey wild A. nuluensis honey honey
Concentration of 80%
methanol extracts (v/v)
(%)
25 2.00 ± .00d 1.00 ± .06d 2.00 ± .03d 1.00 ± .02d
50 3.00 ± .03c 2.00 ± .02c 4.00 ± .02c 2.00 ± .01c
75 4.00 ± .02b 3.00 ± .03b 5.00 ± .01b 3.00 ± .02b
100 5.00 ± .01a 4.00 ± .01a 6.00 ± .03a 4.00 ± .08a
Concentration of
absolute methanol
extracts (v/v)
25 2.00 ± .01c 1.00 ± .03c 2.00 ± .01d 1.00 ± .03a
50 2.00 ± .02c 1.00 ± .02bc 3.00 ± .02c 1.00 ± .03a
75 3.00 ± .11b 2.00 ± .03b 4.00 ± .05b 2.00 ± .00a
100 4.00 ± .00a 3.00 ± .01a 5.00 ± .02a 2.00 ± .00a
Note: Values presented as mean ± standard deviation (n = 3), results in column with different alphabets are significantly different (p < .05).
 Antibacterial of Sabah honey 3

Table 4. Antimicrobial activity of wild Sabah honey against E. coli in 80% and absolute methanol extracts.

Inhibition zone (mm) Inhibition zone (mm) of Inhibition zone (mm) of

of wild A. cerana wild A. andreniformis Inhibition zone (mm) of wild A. koschevnikovi
honey honey wild A. nuluensis honey honey
Concentration of 80%
methanol extracts (v/v)
(%)
25 2.00 ± .00d 1.00 ± .01d 1.00 ± .05c ND
50 3.00 ± .02c 2.00 ± .00c 2.00 ± .01bc ND
75 4.00 ± .01b 3.00 ± .02b 2.00 ± .00b ND
100 5.00 ± .02a 4.00 ± .00a 3.00 ± .02a ND
Concentration of
absolute methanol
extracts (v/v)
25 1.00 ± .03d 1.00 ± .00b 1.00 ± .00a ND
50 2.00 ± .00c 1.00 ± .02b 1.00 ± .00a ND
75 3.00 ± .01b 2.00 ± .01b 2.00 ± .00a ND
100 4.00 ± .02a 3.00 ± .03a 2.00 ± .04a ND
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Note: Values presented as mean ± standard deviation (n = 3), results in column with different alphabets are significantly different (p < .05), ND = not
detected.

Table 5. Antimicrobial activity of wild Sabah honey against S. enteritidis in 80% and absolute methanol extracts.

Inhibition zone (mm) Inhibition zone (mm) of Inhibition zone (mm) of

of wild A. cerana wild A. andreniformis Inhibition zone (mm) of wild A. koschevnikovi
honey honey wild A. nuluensis honey honey
Concentration of 80%
methanol extracts (v/v)
(%)
25 2.00d ± .04c ND 2.00 ± .08d ND
50 3.00c ± .02b ND 3.00 ± .01c ND
75 3.00 ± .22ab ND 4.00 ± .01b ND
100 4.00 ± .01a ND 5.00 ± .00a ND
Concentration of
absolute methanol
extracts (v/v)
25 1.00 ± .03d ND 2.00 ± .00c ND
50 2.00 ± .00c ND 3.00 ± .02b ND
75 3.00 ± .01b ND 4.00 ± .04a ND
100 4.00 ± .00a ND 4.00 ± .05a ND
Note: Values presented as mean ± standard deviation (n = 3), results in column with different alphabets are significantly different (p < .05), ND = not
detected.

enzymes, ascorbic acids, organic acids and protein com-
pounds that possess a health-promoting potential
through their antibacterial activities (Us National Honey
Board, 2003). The phytochemical compounds in honey
have become the aim of the past studies due to the cor-
relation exists between the phenolic and flavonoid con-
tents with their biological properties (Martos et al.,
2000; Tomás-Barberán, Martos, Ferreres, Radovic, &
Anklam, 2001; Yao et al., 2003). Analysis of phenolic
compounds has been regarded as the promising way in
determination of floral and geographical origins of honey
due to their stable presence in the flower nectar
(Yao et al., 2003; Alvarez-Suarez, Tulipani, Romandini,
Vidal, & Battino, 2009). The natural antioxidants com-
pound such as flavonoids, exhibits a wide range of bio-
logical effects including antibacterial, anti-inflammatory,
anti-allergic, antithrombotic and vasodilator actions. It
also possess other antibacterial properties due to its
osmotic effect, acidity, hydrogen peroxide activity as
well as phytochemicals factor mentioned (Molan, 1992).
The presence of hydrogen peroxide in honey is formed
by the glucose oxidation process catalysed by glucose
oxidase present in the honey and makes honey more
likely to become antibacterial agent (Snowdon & Cliver,
1996). In fact, the major antimicrobial influence of honey
is due to the presence of hydrogen peroxide; however,
when used medicinally, the non-peroxide compounds
such as phenolic and flavonoids are important since the
potency of the antibacterial activity to be reduced by
the action of catalase in human body by breaking down
the hydrogen peroxide (Molan, 1992). Differences in the
antibacterial activity of honey extracts due to the
 4 P. Yap et al.
differences in content of their active compounds which,
in turn, can be influenced by the methods of extractions
(Snowdon & Cliver, 1996) with crude extractions show-
ing the strongest activity as compared to esters extrac-
tions (Okeke, Iroegbu, Eze, Okoli, & Esimone, 2001).
These properties of honey can inhibit the growths of
both pathogenic and non-pathogenic bacteria (Molan,
1992). To date, honey has been reported to have an
inhibitory effect on up to 60 species of bacterial includ-
ing both aerobic and anaerobic, and Gram-positive and
Gram-negative strains (Molan, 1992).
Honey has been proven to be effective in gastroin-
testinal disorder, in the treatment of some serious
burns and wound cases, as an antimicrobial agent as well
as providing gastric protection against acute and chronic
gastric lesions (Abd-El Aal, El-Hadidy, El-Mashad, and
El-Sebaie. 2007). Along with the antimicrobial properties
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mentioned, the recovery of the cell and the hearing pro-
cess is accelerated (Molan, 1992) due to the osmotic
characteristics in honey. Besides the internal factors, the
diversity of flora, origin and substrate condition makes
honey as antibacterial agent (Molan, 1992). These fac-
tors are related with the analysis of chemical com-
pounds especially phenolic and flavonoids which become
the floral markers and as antimicrobial agent (Tan, Wilk-
ins, Holland, & McGhie, 1989). By having these proper-
ties, it will destroy the growth of some pathogenic
bacteria (Chick, Shin, & Ustunol, 2001). This study was
conducted to determine the antimicrobial activity and
phytochemicals (phenolic and flavonoid) contents of
selected honey of Sabah and investigate the correlation
between the parameters.
Materials and methods
Wild honey samples
From January to April 2012, wild honey samples from
four different bee species (Apis cerana, Apis andreniformis,
Apis nuluensis, Apis koschevnikovi) were collected around
Sabah. The locations were Agricultural Research Station
(ARS), Tenom, Sabah, Malaysia; Mesilau, Sabah, Malaysia
and Bundu Tuhan, Ranau, Sabah, Malaysia. Manuka honey
and sugar analogue (consists of 40% fructose, 30% glu-
cose, 10% maltose and 20% water) were used as posi-
tive and negative control for all assays. The bee
specimens were deposited into the insect collection
room, BORNEENSIS, University Malaysia Sabah (UMS).
Sample extraction
Wild honey samples (.7 g) were extracted for 1 h with
70 ml of solvents (80% methanol or absolute methanol)
at room temperature. The solvents were evaporated
using rotary evaporator (El-Gendy, 2010). The extracts
were decanted into vials and tested for their antibacte-
rial and phytochemical activities.
Antimicrobial Study
Preparation of test materials
Test materials were prepared by diluting the wild honey
extracts at different concentrations (v/v), 25, 50, 75 and
100 l/ml, in distilled water.
Disc diffusion method (Burt, 2004)
Antimicrobial activity was determined using disc
diffusion method as described by Burt (2004). Sterile
agar plates were inoculated with five different bacte-
rial strain, Staphylococcus aureus ATCC 1721 (S. aur-
eus), Bacillus cereus ATCC 10702 (B. cereus), Bacillus
subtilis ATCC 6633 (B. subtilis), Escherichia coli ATCC
25922 (E. coli) and Salmonella enteritidis ATCC 4931
(S. enteritidis), A six-millimeter diameter of Whatmann
No.5 filter paper soaked with each of wild honey at
different concentration and placed into the agar.
Different honey concentration (w/v) were used along
with standards antibiotics, Ampicillin (AMRESCO,
0339) 100 l/ml and Canamycin (AMRESCO, 0408)
100 l/ml and disc soaked in 80% methanol as stan-
dards controls, respectively. The agar plates were
incubated at 37 ˚C for 24 h. The diameter of the
clear zone surrounding the paper disc of inhibited
bacterial growth was measured (mm). All tests were
performed in triplicate.
Minimum inhibitory concentration (MIC) (Broth dilution
method)
The MICs of the wild honey extracts were determined
by broth dilution method as described by Amsterdam,
1996;. MIC values of the four wild honey samples were
determined against the same five bacterial strains as the
disc diffusion method; S.aureus, E.coli, S.enteritidis, B.cereus
and B.subtilis. In this method, the honey extracts that
have strong antimicrobial effect (inhibitory
zone > 1.00 mm) regardless concentration, were further
analysed using broth system (Heunvelink et al., 1998).
Graded doses (v/v) sample of honey were used along
with the initial concentration of 250 l/ml, followed by
125, 62.5, 31.25 and 15.625 l/ml. Overnight broth cul-
tures (diluted to the concentration of working inocu-
lums) of each bacterial strain were prepared in nutrient
broth in which .1 ml of wild honey extracts was added
to each tube. The turbidity was visually compared
against McFarland value of .5 standards (Heunvelink
et al., 1998). McFarland standards are used as visual tur-
bidity standards for preparation of suspensions of
micro-organisms and bacteria inoculated for antibacterial
activities. The lowest concentration of honey in the ser-
ies that inhibited the growth rate of bacteria was con-
sidered to be the MIC, expressed in l/ml. All tests were
performed in triplicate.
 Antibacterial of Sabah honey 5

Determination of total phenolic content Statistical analysis

Total phenolic content of the honey extracts were
determined as described according to Beretta, Granata,
Ferrero, Orioli, and Facino (2005), using Folin-Ciocal-
teu’s reagent. .7 g of honey sample with 70 ml of 80%
methanol was subjected to vacuum rotary evaporator
for 1 h. The extract was transferred into the vials and
used for total phenolic assay. About 100 l of extract
was mixed with .75 ml of Folin-Ciocalteu’s reagent (pre-
viously diluted 10-fold with distilled water); vortex for
2 min, .75 ml of sodium bicarbonate added to the mix-
ture and allowed to stand for 90 min at 22 ˚C. The mix-
ture was the transferred to a cuvette and the
absorbance measured at 725 nm using uv-spectropho-
tometer. The total phenolic content of the samples
were expressed as gallic acid equivalents (mg of GAE/g
of honey).
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All experiments were carried out in triplicates and the
values were presented as mean ± standard deviation
(SD). One-way analysis variance (ANOVA) followed by
Pearson’s coefficient correlation (r) were evaluated
using Prism 5 statistical software. The confidence level
of statistical significance was at p < .05.
Results
Antimicrobial activity
The antimicrobial activity of four wild Sabah honey
against five different bacteria strains; i.e. S. aureus, B. sub-
tilis, B. cereus, E. coli and S. enteritidis, were determined
by their ability to inhibit the growth of the bacteria is
shown in Tables 1–5, for both 80% and absolute metha-

nol extracts. The antimicrobial activities were greatest

Determination of total flavonoid content
Total flavonoid content of honey was measured as
described by Zhishen, Mengcheng, and Jianming (1999),
with slight adjustments. About 100 l of honey extract
was added to 4 mL of distilled water and .3 ml of 5%
NaNi3 immediately added. At 5 min, .6 mL 10% AlCl3
was added and at 6 min, 2 ml of 1 M NaOH and 2.1 ml
of distilled water added before thorough mixing. The
mixture was transferred to a cuvette and the absor-
bance measured against a blank at 510 nm using UV-
spectrophotometer. Total flavonoid contents of the
samples were expressed as rutin equivalents (mg of
RUE/g of honey).
in 80% methanol extracts as compared to absolute
methanol extracts. The largest clearer zone of inhibition
(mm) of the bacteria regardless the concentration, con-
sidered to be the greatest inhibition was with wild A.cer-
ana honey extracts against Gram-positive bacteria B.
cereus, where it showed 16.00 mm inhibition zone at
100% concentration (80% methanol extract). The
absence of zones of inhibition indicated that the antimi-
crobial activity could not be detected (ND) for this wild
honey as the bacteria was resistant against the concen-
tration of the wild honey extracts. Standard antibiotics
(Canamycin and Ampicillin) and positive control, Manuka
honey showed greater inhibition zone against all the
tested bacteria while sugar analogue did not show inhi-

Table 6. Antibacterial activity of positive control against tested bacteria.

Canamycin (g/ml) inhibition zones (mm) Ampicillin (g/ml) inhibition zones (mm)
Bacteria 25% 50% 75% 100% 25% 50% 75% 100%
S. aureus 12.00 ± .01d 14.00 ± .01c 16.00 ± .01b 20.00 ± .01a 8.00 ± .01d 9.00 ± .01c 10.00 ± .01b 12.00 ± .01a
B. cereus 12.00 ± .01d 14.00 ± .01c 16.00 ± .01b 18.00 ± .01a 10.00 ± .01c 12.00 ± .01b 13.00 ± .01ab 14.00 ± .01a
B. subtilis 10.00 ± .01d 12.00 ± .01c 14.00 ± .01b 16.00 ± .01a 8.00 ± .01d 10.00 ± .01c 11.00 ± .01b 14.00 ± .01a
E. coli 10.00 ± .01c 12.00 ± .01b 13.00 ± .01ab 14.00 ± .01a 6.00 ± .01d 8.00 ± .01c 10.00 ± .01b 12.00 ± .01a
S. enteritidis 12.00 ± .01c 14.00 ± .01b 15.00 ± .01b 18.00 ± .01a 10.00 ± .01d 12.00 ± .01c 14.00 ± .01b 16.00 ± .01a
Note: Values presented as mean ± standard deviation (n = 3), results in column with different alphabets are significantly different (p < .05).

Table 7. Antibacterial activity of positive control against tested bacteria.

Manuka honey inhibition zones (mm) (absolute

Manuka honey inhibition zones (mm) (80% methanol) methanol)
Bacteria 25% 50% 75% 100% 25% 50% 75% 100%
a a a a c bc ab
S. aureus 4.00 ± .01 5.00 ± .01 6.00 ± .01 8.00 ± .01 2.00 ± .01 4.00 ± .01 5.00 ± .01 6.00 ± .01a
B. cereus 4.00 ± .01d 6.00 ± .01c 8.00 ± .01b 10.00 ± .01a 3.00 ± .01c 5.00 ± .01b 6.00 ± .01b 7.00 ± .01a
B. subtilis 2.00 ± .01d 4.00 ± .01c 6.00 ± .01b 8.00 ± .01a 2.00 ± .01d 3.00 ± .01c 4.00 ± .01b 5.00 ± .01a
E. coli 2.00 ± .01d 3.00 ± .01c 4.00 ± .01b 6.00 ± .01a 1.00 ± .01d 2.00 ± .01c 4.00 ± .01b 5.00 ± .01a
S. enteritidis 2.00d ± .01 3.00c ± .01 4.00b ± .01 5.00a ± .01 1.00 ± .01c 2.00 ± .01bc 3.00 ± .01b 4.00 ± .01a
Note: Values presented as mean ± standard deviation (n = 3), results in column with different alphabets are significantly different (p < .05).
 6 P. Yap et al.

Table 8. Minimum Inhibitory Concentration (MIC) activity of wild Sabah honey extracts against different bacteria strains in 80%
methanol extracts.

Bacteria S. aureus B. cereus B. subtilis E. coli S. enteritidis

Honey
Wild A. cerana 62.50 ± .00a 31.25 ± .00a 62.50 ± .00a 125.00 ± .00a 62.50 ± .00a
Wild A. andreniformis 125.00 ± .00a 62.50 ± .00a 62.50 ± .00a 125.00 ± .00a
Wild A. nuluensis 62.50 ± .00a 62.50 ± .00a 31.25 ± .00a 125.00 ± .00a 125.00 ± .00a
Wild A. koschevnikovi 250.00 ± .00a 125.00 ± .00a 125.00 ± .00a
Manuka 31.25 ± .00a 62.50 ± .00a 62.50 ± .00a 62.50 ± .00a 62.50 ± .00a
Note: Values presented as mean ± standard deviation (n = 3), results in column with different alphabets are significantly different (p < .05).

Table 9. Minimum Inhibitory Concentration (MIC) activity of wild Sabah honey extracts against different bacteria strains in absolute
methanol extracts.

Bacteria S. aureus B. cereus B. subtilis E. coli S. enteritidis

Honey
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Wild A. cerana 125.00 ± .00a 62.50 ± .00a 125.00 ± .00a 250.00 ± .00a 125.00 ± .00a
Wild A. andreniformis 250.00 ± .00a 125.00 ± .00a 125.00 ± .00a 250.00 ± .00a
Wild A. nuluensis 125.00 ± .00a 125.00 ± .00a 62.50 ± .00a 250.00 ± .00a 250.00 ± .00a
Wild A. koschevnikovi 250.00 ± .00a 250.00 ± .00a 250.00 ± .00a
Manuka 62.50 ± .00a 62.50 ± .00a 62.50 ± .00a 125.00 ± 00a 125.00 ± .00a
Note: Values presented as mean ± standard deviation (n = 3), results in column with different alphabets are significantly different (p < .05).

in 80% and absolute methanol extracts. The MICs were

Table 10. Total phenolic content and total flavonoid contents lowered in 80% methanol extract compared to absolute
of four wild Sabah honey.
methanol extract. The screening results showed that
Samples Total phenolic1 Total flavonoid2 the overall inhibitory concentrations of the four wild
Sabah honey extracts against S. aureus ranged from
80% methanol extracts
Wild A. cerana honey 10.43 ± .02b 8.76 ± .00b
62.50 to 250 l/ml, whereas for B. cereus and B. subtilis
Wild A. andreniformis honey 9.90 ± .01c 8.09 ± .00c ranged from 31.25 to 250 l/ml, for both 80% and
Wild A. nuluensis honey 8.78 ± .01d 6.67 ± .00d absolute methanol extract, respectively. As for Gram-
Wild A. koschevnikovi honey 7.93 ± .02e 5.48 ± .01e negative bacteria, E. coli ranged from 125.00 to 250 l/ml,
Manuka honey 10.61 ± .01a 9.84 ± .00a whereas S. enteritidis ranged from 62.5 to 250 l/ml for
Absolute methanol extracts
Wild A. cerana honey 9.72 ± .03a 7.77 ± .00b
both 80% and absolute methanol extraction,
Wild A. andreniformis honey 9.32 ± .00b 6.14 ± .00c respectively. Positive control, Manuka honey screening
Wild A. nuluensis honey 8.40 ± .01d 5.27 ± .00d results showed the overall concentrations against five
Wild A. koschevnikovi honey 7.80 ± .01e 4.67 ± .00e tested bacteria ranged from 31.25 to 250 l/ml, in both
Manuka honey 8.81 ± .01c 8.07 ± .00a 80% and absolute methanol extraction, respectively
Note: Values are presented as mean ± standard deviation (n = 3) (Tables 8 and 9).
which with different alphabets are significantly different at p < .05.
1
Total phenolic content was expressed as mg gallic acid equivalents in
1 g of honey (mg GAE/g honey). Total phenolic content
2
Total flavonoid content was expressed as mg rutin equivalents in 1 g
of honey (mg RE/g honey). The result of this study showed that the total phenolic
content levels were highest in the wild A. cerana honey
closely followed by A. andreniformis honey, then A. nulu-
bitory effect against all of the tested bacteria (data not ensis and A. koschevnikovi honey for 80% and absolute
shown) (Tables 6 and 7). methanol extracts, respectively. Manuka honey phenolic
content level was highest in 80% methanol extract as
compared to absolute methanol extract while sugar ana-
Minimum inhibition concentration (MIC) activities
logue did not have phenolic content (data not shown)
The MICs of the four wild Sabah honeys were deter- (Table 10).
mined against five different bacterial strains via visual
observation against McFarland standard (Heunvelink
et al., 1998). The MICs were determined according to Total flavonoid content
the turbidity clearness via visual observation. The MICs The result of this study showed that total flavonoid con-
of four wild Sabah honey extracts showed marked varia- tent was highest in the wild A. cerana honey followed by
tion between tested micro-organisms (Tables 8 and 9) A. andreniformis, A. nuluensis and A. koschevnikovi honey

for both 80% and absolute methanol extracts, respec-
tively. Manuka honey flavonoid content was highest in
80% methanol extract as compared to absolute metha-
nol extract while sugar analogue did not have flavonoid
content (data not shown) (Table 10).
Discussion
Honey has been shown to display potent antimicrobial
activity in the treatment of wound infections due to its
high osmotic properties and the presence of phenolic
and flavonoids compounds (Kirnpal-Kaur, Tan, Boukraa,
& Gan, 2011). In this study, the antibacterial activity,
MIC, total phenolic and total flavonoid contents of
selected wild honey from Sabah were tested for the
first time and revealed their potential to become medic-
inally beneficial antimicrobial agent.
Downloaded by [Australian National University] at 02:41 14 March 2016
In the preliminary antimicrobial studies, the four wild
Sabah honey extracts showed promising effect against
the different bacteria strains tested, especially against
Gram-positive bacteria where B. cereus was the most
susceptible bacteria since the inhibitory zones were the
largest, followed by S. aureus, B. subtilis, S. enteritidis and
E. coli in both 80% and absolute methanol extract
(Tables 1–5). The variation in the bacterial susceptibility
might be due to the physiological structure of the cell
membrane. The presence of a thick lipopolysaccharides
layer as the outer membrane in Gram-negative bacteria
such as E. coli and S. enteritidis might explain why it were
not susceptible to Gram-positive bacteria (Alzoreky &
Nakahara, 2003). The thick layer could act as the
mechanical layer that prevents the invasion of other
compounds thus reducing the injury or lethality in the
bacterial cells (Nostro, Germano, Marino, & Canatelli,
2000). According to Abd-El Aal, El-Hadidy, El-Mashad,
and El-Sebaie (2007) and El-Sukhon, Abu-Harfeil, and
Sallal (1994), honey has a greater inhibitory effect on
isolated Gram-positive bacteria as compared to Gram-
negative bacteria due to the differences in the polarity,
solubility and method of extractions. This is in agree-
ment with Willix, Molan, and Harfoot (1992) and Bilal,
Molan, and Harfoot (1998), who found that wild honey,
inhibited the growth of S. aureus. Molan (2001) stated
that S. aureus is one of the bacterial species that most
likely susceptible to the antimicrobial activity of honey.
This is due to the susceptibility of S. aureus against the
osmotic effect, pH effect and occurrence of hydrogen
peroxides and non-peroxides compounds which inhibit
the bacteria growth (Postmes, Van Den Bogaard, &
Hazen, 1993).
The zone of inhibition of the 80% methanol extract
was greater compared to absolute methanol extract
against all five bacteria (Tables 1–5) with wild A. cerana
honey extracts showed the greater antimicrobial activity
(16.00 mm against B. cereus), whereas wild A. koschevni-
kovi honey extracts showed the weakest antimicrobial
activity (no inhibition against Gram-negative bacteria).
According to Chinakwe (2006), the antimicrobial
Antibacterial of Sabah honey 7
properties of honey properties increased when diluted
with organic solvent or water due to the combination
of hydrogen peroxide (Taormina, Niemira, & Beuchat,
2001). Hydrogen peroxide has the ability to penetrate
and specified protein and polysaccharide molecules
(Sapers & Simmon, 1998). It could generate a short-lived
singlet oxygen species that biocidal against Gram-posi-
tive and Gram-negative bacteria (Taormina et al., 2001).
Ukuku (2004), reported that the Salmonella population
on cantaloupe was significantly reduced after washing it
with 2.5 and 5% hydrogen peroxide solutions. According
to Adil, Cetin, Yener, and Bayindirli (2007), the addition
of small amount of organic solvents creates more polar
medium which facilitates phenolic and flavonoids extrac-
tion. In addition, the mixture of alcohol and water sol-
vent were said to be more efficient in phytochemicals
extractions compared to mono-component solvent. The
difference in the antimicrobial activity might be due to
the differences in the source of plants phenolic and fla-
vonoid (Taormina et al., 2001). Besides that, the crude
phenolic phytochemicals extracts should have strong
antimicrobial activity due to the combination of active
and non-active components in extracts (Okeke et al.,
2001). The presence of non-peroxides compounds like
flavonoids degrading the cytoplasm membrane of the
bacteria and lead to cell autolysis due to loss of potas-
sium ions (Hamouda & Marzouk, 2011). This was in
agreement with Mirzoeva, Grishanin, and Calder (1997)
who reported that flavonoid contents in honey
increased membrane permeability and bacterial lose the
ability to synthesis energy, membrane transport and lead
to motility. In addition, Mahopatra, Thakur, and Brar
(2010) described that methanol extract was the most
effective as an antimicrobial agent showing maximum
inhibitory activity when compared to ethanol and ethyl
acetate. All four of the wild Sabah honey extracts
showed significance different in 80% methanol extract
against Gram-positive bacteria for antimicrobial activity
(p < .05).
The MICs studies showed that the most susceptible
bacteria to honey were Pseudomonas spp. followed by
E. coli, B. cereus, B. subtilis and S. aureus (Cooper &
Molan, 1999). This was in agreement with the prelimi-
nary MIC’s result where all four wild Sabah honey
extracts sensitive against all five different bacteria strains
in 80% and absolute methanol extracts. B. cereus was
the most susceptible micro-organism, followed by S. aur-
eus, B. subtilis, E. coli and S. enteritidis (Tables 8 and 9).
The differences in the concentration ranges showed that
higher concentrations extract were required to inhibit
Gram-negative bacteria. All the four wild Sabah honey
extracts exhibited weak inhibitory effect against E. coli
and S. enteritidis as higher concentration of extracts
(125–250 l/ml) were required to exert the inhibitory
effects on this bacteria. These differences in MIC’s activ-
ity might due to the differences in the plant source from
which the raw material of the honey was collected
.Taormina et al. (2001) also reported that different
 8 P. Yap et al.
plants contain different phenolic content that can exert
different antibacterial activities.
The presence of glucose oxidase increased the
antibacterial activity due to the presence of glucose oxi-
dase at the surface of honey in which reduces the atmo-
spheric oxygen into hydrogen peroxide which acts as
antimicrobial barrier and giving the low values of MIC
(Stinson, Subers, Petty, & White, 1960). Normally, low
antibacterial activity will have a high MIC value but some
honey extracts that produced large inhibition zones also
have high MIC value. Thus, broth dilution method was
the best method to determine a more accurate MIC
value in their antibacterial activity against the test
micro-organisms due to the effectiveness indirectly giv-
ing results precisely (Kim, Davidson, & Chung, 2001).
However, all four of the wild Sabah honey extracts
showed no significance different in both extract for
Downloaded by [Australian National University] at 02:41 14 March 2016
MICs activity (p < .05).
Honey comprises phenolic acids, esters and flavo-
noids were the mostly abundant in phytochemicals com-
pounds (Yao et al., 2003). The colorimetric assay based
on the reaction of Folin-Ciocalteu reagent widely used
as determination of phenolic contents in honey (Aljadi &
Kamaruddin, 2004; Alvarez-Suarez et al., 2009; Bal-
trušaitytė, Venskutonis, & Čeksterytė, 2007; Beretta
et al., 2005; Bertoncelj, Dobersek, Jamnik, & Golob,
2007; Henriques, Jackson, Cooper, & Burton, 2006;
Kim, Jeong, & Lee, 2003; Meda, Lamien, Romito, Millogo,
& Nacoulma, 2005; Prior, Wu, & Schaich, 2005; Single-
ton, Orthofer, & Lamuela-Raventós, 1999) .The results
showed that the total phenolic content was higher in
wild A. cerana honey while lower in wild A. koschevnikovi
honey in both 80% and absolute methanol extract
(Table 8). According to Chye and Ng (2008), wild
Malaysian honey was significantly higher in phenolic acids
compared to cultured Malaysian honey for both types
and geographical locations factors. Highlands were
mostly surrounded by primary forests with abundant
botanical resources compared to lowlands. Thus, the
yield of the extracts obtained differs from each other
since the plant sources visited by the bees were diverse
(Taormina et al., 2001). There was a significant different
of total phenolic content among the four wild Sabah
honey (p < .05).
Flavonoids were subclasses to the phenolic com-
pounds (Ferreres et al., 1994). The aluminium chloride
method was used to determine the flavonoid contents
in honey (Aralas, Mohamed, & Abu Bakar, 2009;
Arvouet-Grand, Vennat, Pourrat, & Legret, 1994; Mark-
ham, 1982; Meda et al., 2005; Nagai, Sakai, Inoue, Inoue,
& Suzuki, 2001; Nor Qhairul Izreen & Mohd Fadzelly,
2013; Özkök, D’arcy, & Sorkun, 2010; Tomas-Barberan,
Ferreres, Garcia-Viguera, & Tomas-Lorente, 1993;
Zhishen et al., 1999). The results showed that the total
flavonoid content was higher in wild A.cerana honey
while lower in wild A. koschevnikovi honey in both 80%
and absolute methanol extract (Table 8). According to
Chye and Ng (2008), wild Malaysian honey was signifi-
cantly higher in flavonoids content as compared to cul-
tured Malaysian honey for both types and geographical
locations factors. The differences in percentage of yield
of phenolic and flavonoids between these samples were
due to botanical resources of the geographical origins.
Plus, different honey bee species have different capacity
of ability to cover the access of nectar in much wider
areas, such as wild A. dorsata or giant honey bee which
can cover up to 2 km compared to the dwarf honey
bee or A. andreniformis (Koeniger & Koeniger, 2000).
There was a significant different of total phenolic con-
tent among the four wild Sabah honey (p < .05).
The phenolic and flavonoids might be phytochemicals
that suspected to contribute to the antibacterial activity
of wild honey of Sabah. Hence, the correlation analysis
was performed to investigate the relationship between
the phytochemicals and antibacterial activity in wild honey
of Sabah. The analysis showed that antimicrobial activity
was strongly positive correlated with the phenolic con-
tent for both 80% and absolute methanol extract
(r = .904, p < .05). Total flavonoid content was moder-
ately positive correlated with antibacterial activity in both
80% and absolute methanol extract (r = .622, p < .05).
The results were acceptable since both were subclasses
of phenolic compounds (Alcaraz, Blanco, Puig, Tomas, &
Ferretti, 2000). In addition, the exceptionally high correla-
tion between phenolic and flavonoid extracts were an
indication that most compounds in the total phenolic con-
tent comprised of flavonoids (Ferreres et al., 1994).
In conclusion, this study showed that all four wild
honey samples from Sabah possessed an active antibac-
terial and polyphenol-rich extract that based on the his-
torical background of the health properties of honey
and the health benefits of phenolic and flavonoids that
honey contents, has the potential to be used in medici-
nal field. However, further studies are needed to maxi-
mize the potential and discover the new unreported
benefits of honey.
Acknowledgements
We are indebted to Agriculture Research Station (ARS),
Tenom, Sabah, for the for the accommodation and technical
assistance, to Institute for Tropical Biology and Conservation
(ITBC) and School of Science and Technology (SST) University
Malaysia Sabah, Malaysia for the use of the laboratory facilities
and technical assistance.
Disclosure statement
No potential conflict of interest was reported by the
authors.
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