Advances in Environmental Biology

Advances in Environmental Biology, 8(18) Special 2014, Pages: 1-7
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Antioxidant Properties, Enzyme Activities and Inhibitory Effects of Melaleuca
Honey against Cariogenic Bacteria Growth and Biofilm Formation
1,2Wen-Jie
1
Ng, 1Tze-Jian Chin, 1Betty, Hui-Yee Khoo
Faculty of Science, 2Centre for Biodiversity Research, Universiti Tunku Abdul Rahman, 31900, Perak, Malaysia
ARTICLE INFO
Article history:
Received 30 September 2014
Received in revised form
17 November 2014
Accepted 25 November 2014
Available online 6 December 2014
Keywords:
Honey, Melaleuca, Gelam
Antioxidant, Antibacterial
ABSTRACT
Background: Several studies on the potential effects of honey have been conducted;
however such properties are highly dependent on the geographical origins and botanical
sources of honey. Current knowledge of the antioxidant, enzyme activities and
antibacterial effects of Malaysian local honey, specifically Melaleuca honey, is still
confined. Objectives: In this study, other than the assessment of antioxidant capacities
and enzyme activities on three honey types, antibacterial effects of honey against
cariogenic bacteria, Streptococcus mutans together with the ability in preventing
biofilm formation were investigated. Results: As compared to other honey samples,
Melaleuca honey showed the highest amount of total phenolic compounds (561.67 ±
10.41 mg GAE/kg) and flavonoid content (67.50 ± 2.78 mg CEQ/kg), with the greatest
DPPH scavenging activity (76.99 ± 0.89%) and FRAP value (668.33 ± 2.89 µM Fe
[II]/100 g). Besides that, Melaleuca honey with 1 g/ml concentration was able to inhibit
bacterial growth completely (100%) while biofilm biomass was the least (71.05%) at
0.5 g/ml concentration of honey. Conclusion: Melaleuca honey was found to exhibit
high antioxidant capacity, other than inhibiting the growth of bacteria, the honey was
also able to prevent the formation of biofilm.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Wen-Jie Ng, Tze-Jian Chin, Betty, Hui-Yee Khoo., Antioxidant Properties, Enzyme Activities and Inhibitory Effects
of Melaleuca Honey against Cariogenic Bacteria Growth and Biofilm Formation. Adv. Environ. Biol., 8(18), 1-7, 2014
INTRODUCTION
Honey is a collection of nectar that processed by honey bees which is mainly composed of a complex
mixture of carbohydrate with some minor but important constituents including proteins, organic acids, enzymes,
amino acids, lipids, volatile chemicals, phenolic acids, minerals, flavonoids, carotenoid-like substances and
vitamins [23]. The variation of the chemical composition makes each type of honey has its unique flavor, color,
aroma and texture.
Honey is one of the well-known natural products with high nutritional and prophylactic medicinal values,
which include antimicrobial, in healing of wounds, burns and gastrointestinal disorders as well [30,19,4,26].
Partly of the therapeutic values of honey has been credited to its antioxidant properties [19,3].
Honey contains a number of different enzymes either introduced by the bees or originated from the floral
nectar. Forager honeybees secrete many enzymes from their glands after filling up their honey crop with floral
nectar [5]. The foragers mix the enzymes with nectar thoroughly and then pass to housebees through
trophallaxis [5]. Before the storage of honey in hive, more enzymes are added into the nectar for the ripening of
nectar into honey [28]. The common enzymes that can be found in honey are diastase, invertase, catalase,
glucose oxidase and others. They are the important components which responsible for the conversion of nectar
and honeydew to honey [32]. However, the final amount of the enzymes in honey is highly associated with
many factors such as physiological stage, age and diet of the bees, temperature, abundance of nectar flow, pollen
consumption and etc.
Diastase which is added into the honey by bees catalyzes the transformation of starch into dextrin, oligo-,
di- and monosacharides like maltose. While invertase is responsible for converting sucrose in nectar into
glucose and fructose. Both diastase and invertase are very sensitive to heat and storage damage. Together with
diastase, it is also used as a parameter for the freshness of honey in few European countries [32].
The hot wet climate of Malaysia supports its tropical rainforests which cover 59% to 70% of its total land
area. Around 14500 species of flowering plants and trees can be found in Malaysia [2]. Thus, many types of
Corresponding Author: Wen-Jie Ng, Faculty of Science, Centre for Biodiversity Research, Universiti Tunku Abdul
Rahman, 31900, Perak, Malaysia.
E-mail: [email protected]
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Malaysian honeys can be easily found including Coconut honey, Durian honey, Melaleuca honey, Nenas honey,
Tualang honey and etc. However, different aspects of study on Malaysian honeys are still very limited.
On the other hand, as a non-shedding surface, teeth permit the plaque to form a “structure” that contains
numerous layers of bacteria. This “structure” is also known as biofilm which is described as a collective of
bacteria that can hold on to each other to a solid surface. The gel-like matrix of extracellular polysaccharides
produced by the oral bacteria are the one that make up around 90% of the biofilm and hold it together which
make it progressively more arduous to be eradicated. Furthermore, biofilm can cause a cell to experience
changes in its gene expression and become more resistant to the antibiotics and other antimicrobial agents than
its planktonic counterparts (Higham, n.d.). Nevertheless, Streptococci (most notably Streptococcus mutans) are
one of the common cariogenic bacteria that are associated with biofilm formation on teeth and dental caries.
Since the study of antioxidant capacity of Malaysian local honey and its potential effect against these cariogenic
bacteria is still limited; hence, the goals of this study were: (1) to screen the antioxidant capacities of Malaysian
local honey samples; and (2) to evaluate the inhibitory effects of the honey on bacterial growth and biofilm
formation of S. mutans.
1. Methodology:
Honey Samples and Bacterial Strain:
Three types of honey samples were screened in this study, Melaleuca honey is a wild honey produced by
Apis spp., from the plant Melaleuca cajuputi Powell or locally known as Gelam tree. While Durian and Nenas
honey from the plants of Durio zibethinus Murr. and Ananas comosus (L.) Merr., respectively. All of the honey
samples were collected or harvested in Malaysia by authorized bee farmers under the supervision of the
Ministry of Agriculture and Agro Agrobased Industry. All of these honey samples were screened for antioxidant
and enzyme properties, then honey sample with the strongest antioxidant capacity was furthered with
antibacterial assays. A strain of Gram-positive bacteria, Streptococcus mutans (ATCC 25175) was obtained
from Faculty of Science, Universiti Tunku Abdul Rahman. Bacteria were cultured and maintained on nutrient
agar and mitis salivarius agar.
Analysis of Antioxidant Properties:
Determination of Total Phenolic Compounds:
The concentration of total phenolic compounds in honey was estimated using modified Folin-Ciocalteu
method [16]. Briefly, 0.5 ml of the diluted honey sample (0.2 g/ml) was mixed with 0.5 ml of Folin and
Ciocalteu’s phenol reagent. After 3 minutes, 0.5 ml of 10% sodium carbonate solution was added to the mixture
and adjusted to 5 ml with distilled water. The reaction mixture was kept in dark for 90 minutes, after which the
absorbance was read at 725 nm using spectrophotometer against the blank. A set of gallic acid standard
solutions (20, 40, 60, 80 and 100 μg/ml) was prepared to construct a standard curve. The concentration of
phenolic compounds was measured in triplicate. The results were reported as the mean ± standard deviation and
expressed as mg of gallic acid equivalents (GAEs) per kg of honey.
Determination of Total Flavonoid Content:
Total flavonoid content of honey sample was determined by a spectrophotometric assay with some
modifications by Khalil et al. [16]. One millilitre of diluted honey sample (0.2 g/ml) was mixed with 4 ml of
distilled water and 0.3 ml of sodium carbonate (5% w/v). After 5 minutes, 0.3 ml of aluminum chloride (10%
w/v) was added, followed by 2 ml of sodium hydroxide (1 M) 6 minutes later. Then, the total volume was then
topped up to 10 ml with the addition of 2.4 ml distilled water. The mixture was vigorously shaken to ensure
adequate mixing, and the absorbance was read at 510 nm against a blank. A set of catechin standard solutions
(20, 40, 60, 80 and 100 μg/ml) was prepared to construct a standard curve. The content of flavonoid was
measured in triplicate. The results were reported as the mean ± standard deviation and the values were expressed
as mg catechin equivalents (CEQ) per kg of honey.
Free Radical-Scavenging Activity Assay:
The antioxidant capacity of the honey sample was also studied through the evaluation of free radicalscavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) as described by Aljadi and Kamaruddin, [3];
Mohamed et al., [20]. Diluted honey (0.1 g/ml), 0.75 ml was mixed with 1.5 ml of DPPH methanol solution
(0.09 mg/ml). The mixture was left to stand for 30 minutes in the dark at room temperature and the absorbance
of the mixture was measured at 517 nm. The radical-scavenging activity (RAS) was calculated as the percentage
of DPPH discoloration using the equation: % RSA = (AC – AS / AC) x 100, where Ac is the absorbance of the
control and As is the absorbance of the sample.
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Ferric Reducing-Antioxidant Power (FRAP) Assay:
The FRAP assay was performed according to a modified method by Benzie and Strain [6]. FRAP reagent
was freshly prepared by mixing 10 ml of 300 mM/L acetate buffer (pH 3.6) with 1 ml of 10 mM 2,4,6-tris (1pyridyl)-5-triazine (TPTZ) solution and 1 ml of 20 mM ferric chloride solution. The resulting mixture was then
pre-warmed at 37°C. Firstly, 200 μl of diluted honey (0.1 g/ml) and each ferrous sulfate standard solution were
mixed with 1.5 ml of FRAP reagent. Next, the reaction mixtures were incubated at 37 oC for 4 minutes and the
absorbance was read at 593 nm against the blank. A standard curve was plotted, using a set of ferrous sulfate
aqueous solutions (100, 200, 400, 600 and 1000 μM/L). The FRAP value was expressed as micromoles of
ferrous equivalent (μM Fe [II]) per 100 g of honey.
Analysis of Enzyme Activities:
Diastase Assay:
The diastase assay was performed by using Amylazyme tablet with adopting the procedures from
Megazyme. In this assay, 1 ml of the honey sample (0.05 g/ml) that diluted in 100 mM sodium maleate buffer
was pre-incubated for 5 minutes at 40oC. An Amylazyme tablet was then added into the sample and incubated
for another 10 minutes. Ten millilitre of Trizma base solution was added into the mixture and left at room
temperature for 5 minutes. The mixture was filtered and the absorbance of sample solution was measured at 590
nm against a reaction blank. The diastase activity of honey is expressed as diastase number (DN) which
indicates the amount of diastase that hydrolyze 1 ml of 1% of starch solution using 1 g of honey per hour at
40oC.
Invertase Assay:
The invertase activity of honey sample was determined with Abnova invertase assay kit according to the
principle stated by Huang, Picha and Johnson [11]. Honey sample (40 µl) was added into a flat-bottom 96-well
plate. Next, 5 µL of diluted sucrose was added into the sample and incubated for 20 minutes at 30°C. Then, 90
µl of working reagent was added into each well and incubated again for another 20 minutes in the dark. Lastly,
the absorbance value was read at 570 nm. A glucose standard (30, 60, 100 µM) curve was plotted and the
enzyme activity was calculated and expressed in U/L which one unit of invertase catalyzes the formation of 1
µmole glucose per minutes at pH 4.5 under the assay conditions.
Assessment of Antibacterial Effects:
Bacterial Growth Assay:
One hundred microliter of prepared 0.5 McFarland bacterial suspension and 100 µl of honey sample (0.5
g/ml, 0.25 g/ml, 0.125 g/ml and 0.0625 g/ml) were added into 96-well flat-bottom microtiter plate. Instantly, 20
µl of the mixture from the wells was diluted into 1:100 and plated evenly on agar medium. The agar plates and
the microtiter plate were then incubated at 37°C overnight. On the next day, the numbers of colony forming unit
(CFU) for each concentration of honey in 0 minute and 24 hours were determined using a colony counter.
Biofilm Assay:
Fifty microliter of prepared bacterial suspension and 50 µl of honey sample (0.5 g/ml, 0.25 g/ml, 0.125 g/ml
and 0.0625 g/ml) were added into 96-well flat-bottom microtiter plate. The plate was incubated at 37°C for 24
hours without shaking. The planktonic cells were then discarded and the wells were gently washed with
phosphate buffered saline (PBS). The biofilm was fixed by adding 100 µl of 10% formaldehyde solution and left
overnight at room temperature. On the next day, the formaldehyde was removed and the wells were washed with
PBS gently. Next, 100 µl of 0.1% crystal violet was added for an hour. The wells were then gently washed twice
with PBS and left dried overnight. Then, 200 µl of 30% acetic acid was added to solubilise the dye for 15
minutes. Lastly, the content of each well was briefly mixed and 150 µl of the solution from each well was
transferred to another plate. The absorbance of solution in each well was read at 570 nm [8,25].
2. Results:
The antioxidant properties and enzyme activities of three Malaysian honey samples are tabulated in Table 1.
The strongest antioxidant capacity of Melaleuca honey can be observed with the greatest amount of phenolic
compounds and flavonoids; highest radical scavenging and ferric reducing activities. Melaleuca honey which
showed the strongest antioxidant capacity was furthered with antibacterial assays.
As shown in Figure 1, all of the concentrations of Melaleuca honey had successfully inhibited the growth of
S. mutans where 1 g/ml concentration showed a whopping 100% inhibition rate while the concentrations of 0.5
g/ml, 0.25 g/ml and 0.125 g/ml came with 99% of inhibition as well. Nevertheless, the lowest inhibition rate
(93.5%) was observed at 0.0625 g/ml honey concentration.
The effect of Melaleuca honey on biofilm formation is summarized in Figure 2. As shown in the figure,
reduction of biofilm mass formation can be seen with the concentration of 0.5 g/ml achieved the greatest effect
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followed by 0.25 g/ml, 1 g/ml and then 0.125 g/ml respectively. However, in the concentration of 0.0625 g/ml,
instead of reduction, an increment of 10.4% in the biofilm mass was observed.
Table 1: Antioxidant properties and enzyme activities of Malaysian honeys.
Antioxidant properties
Melaleuca honey
Total phenolic compound
561.67 ± 10.41
(mg GAE/kg honey)
Total flavonoid content
67.50 ± 2.78
(mg CEQ/kg honey)
DPPH inhibition (%)
76.99 ± 0.89
FRAP value
668.33 ± 2.89
(µM Fe [II]/100 g honey)
Enzyme activities
Diastase activity
7.04 ± 0.17
(Diastase Number, DN)
Invertase activity (U/L)
0.38 ± 0.01
*1 U/L corresponds to 7.345 Invertase
Number, IN
Data are mean ± standard deviation of triplicate measurements
Durian honey
343.33 ± 20.82
Nenas honey
316.67 ± 16.07
53.83 ± 7.97
54.67 ± 2.75
30.00 ± 0.87
205.00 ± 5.00
20.95 ± 1.62
170.00 ± 5.00
11.20 ± 0.33
2.64 ± 0.08
0.55 ± 0.13
0.18 ± 0.01
Data are mean ± standard deviation of triplicate measurements
*Error bar denotes standard deviation
Fig. 1: Inhibitory effect of Melaleuca honey on bacterial growth.
Data are mean ± standard deviation of triplicate measurements
*Error bar denotes standard deviation
Fig. 2: Effect of Melaleuca honey on biofilm formation.
3. Discussion:
Both diastase and invertase activities of honey can be influenced by storage condition and high temperature;
thus they are commonly used as indicators for quality and freshness of honey [28,32,5]. But, diastase and
invertase activities of the tested honey sample in this study did not fit in the quality criteria standard. The
general diastase activity of honey should be more than 8 DN and 10 IN and for honey with natural low enzyme
content should be higher than 3 DN and 4 IN [7,31]. Low levels of enzymes activities found in the tested honey
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sample could be accounted for an inadequate processing or storage conditions. Prolonged storage period and
exposure to high temperature during processing could degrade the enzyme [35]. However, the variation of
enzymes levels in honey is also depending on its geographic origin with the differences of botanical sources,
climate and also the honey bees. Different floral sources and the climatic atmospheric condition can affect the
amount of nectar released by plants. This subsequently influences the amount of enzyme as the nectar with thick
consistency needs less manipulation by the bees and vice versa [29]. Different bee species may also affect the
production of enzymes due to the differences in genetic make-up that lead to different protein expressions in
hypopharyngeal gland [13,1]. Presently, there is no much information about the enzyme activities of Malaysian
honey.
Nevertheless, Melaleuca honey showed to contain the highest level of phenolic compounds. The phenolic
compounds that can be found in honey including caffeic acid chlorogenic acid, chrysin, ellagic acid, ferulic acid,
gallic acid, herperetin, kaempferol, luteolin, myricetin, p-coumaric acid and quercetin [14,12]. However, the
amount and types of phenolic compounds in honey vary depending on its botanical source since the phenolic
compounds generally are derived from plants. In year 2005, Yao, et al. found out the phenolic compounds in
honey are largely depending on its botanical origin and they were able to demonstrate that phenolic compounds
profile can be used in floral authentication for certain floral honeys.
Same as phenolic compounds, flavonoids also have a significant relation to the antioxidant capacity of
honey [23]. From the result, generally, the total flavonoid content of Melaleuca honey was not high which
around 12% of the total phenolic compounds. Flavonoids are the components in phenolic compounds but its
composition varies depending on the botanical sources which explain the reason of phenolic compounds content
was higher in Melaleuca honey but with lower flavonoid proportion [12,16,23].
Besides that, the antioxidant activities of all tested honey samples were also demonstrated in DPPH and
FRAP analyses. The outcomes of both assays were well-correlated with the result of total phenolic compounds
as Melaleuca honey with the strongest radical inhibition and ferric reducing activities was proven to contain the
highest amount of total phenolic compounds among the tested honey samples.
As shown in both Figure 1 and 2, Melaleuca honey with the strongest antioxidant capacity was able to
inhibit the growth of both planktonic bacterial cells and biofilm formation of S. mutans where greater inhibition
was observed in higher concentration of honey. Molan [22] and Ansari et al. [4] claimed that the
hyperosmolarity in honey owing to high sugar content is one of the factors that prevent the growth of microbes.
As bacteria require water to survive, so the bacteria will not be able to sustain the osmotic effect and the
constant loss of water molecules thereby causing a reduction in the biofilm mass [21,4,27].
On top of that, the special component of natural honey that plays a vital role in limiting bacterial growth is
hydrogen peroxide which generated by glucose oxidase in the honey. However, it can be observed that, the
percentage of biofilm mass reduction in the highest concentration of honey was lower than that found in other
concentrations. Such outcome could be explained with the inactivation of glucose oxidase in higher
concentration of honey as this enzyme requires sufficient amount of water to be activated. Hence, probably less
hydrogen peroxide was generated to reduce the biofilm mass [24,27].
Acidity is another factor that can contribute to the antibacterial property of Melaleuca honey. The reaction
of glucose with oxygen and water produces gluconic acid and this is the one that lead to acidity in the honey. In
normal condition, the pH of honey is acidic as it has a pH of 3.2 to 4.5 that can inhibit many bacteria [21].
However, the more diluted the honey, the lesser the acidity. It means that the diluted honey would have greater
pH value and it could reduce the antibacterial effect of honey [24]. This explains well the results in Figure 1 and
2 which clearly show that as the concentration of the honey decreases, the percentage of inhibition decreases as
well.
Ansari et al. [4] further elaborated that besides demonstrating the antioxidant activity, flavonoids, a natural
antioxidant that can be found in the honey can affect bacterial growth factors at their receptor binding sites and
indirectly reduce the opportunity of biofilm establishment [4,27]. Moreover, Melaleuca honey also contains
some unique phenolic compounds like gallic acid and ferulic acid. Gallic acid is a very potent free radical
scavenger which is capable to decrease divalent ions. It can act as a catalyst towards lipid peroxidation process
too [17]. For ferulic acid, it plays its role as peroxynitrite scavenger by nitration. These two special compounds
are thought to be able to reduce the biofilm mass as well [14].
In addition, the inhibition of bacterial quorum sensing could be one of the factors that prevent formation of
biofilm [17,34]. Lee et al. [17] claimed that honey was able to reduce the quorum sensing genes like AI-2
import genes which are vital in influencing the biofilm formation in E. coli. Moreover, they also reported that
natural honey was capable of suppressing quorum sensing signaling as previous studies had shown that chestnut
honey was able to restrain quorum sensing N-acyl-L-homoserine lactones (AHLs) and biofilm formation in
Yersinia enterocoliticia, Aeromonas hydrophilia and Erwinia carotovora. Thus, it is possible that Melaleuca
honey used the same principle in preventing the formation of S. mutans biofilm.
However, the reason behind the increment of biofilm mass in the lowest concentration of honey could be
due to the utilization of sugars by S. mutans as fuel and energy source to promote growth and thereby forming
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more biofilm. This can show that the antibacterial effect of honey is not solely depending on the
hyperosmolarity property. Besides that, researchers had observed that the biofilms are more resistant to
antibiotics than planktonic cells which the magnitude of resistance can increase by 10-1000 times [18]. This
could explain the reason why low concentrations of honey with lower quantity of active compounds did not act
well to prevent the formation of biofilm.
4. Conclusions:
Conclusively, the enzymes levels of all tested honeys were lower than the recommended standard range
which could be due to processing or prolonged storage. However, this study was able to reveal the presence of
high antioxidant potential in Malaysian honeys, specifically Melaleuca honey. Other than possessing the
strongest antioxidant capacity, Melaleuca honey was able to inhibit the growth and prevent biofilm formation of
S. mutans which is one of the major cariogenic agents. This shows that natural honey could be potentially used
to prevent dental caries.
Acknowledgment
The authors wish to thank Universiti Tunku Abdul Rahman (UTAR) for the facilities and financial support.
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