Vol.4, No.5B, 46-51 (2013) Agricultural Sciences
Physicochemical and radical scavenging activities of
honey samples from Malaysia
Norul Liza A-Rahaman, Lee Suan Chua*, Mohamad Roji Sarmidi, Ramlan Aziz
Metabolites Profiling Laboratory, Institute of Bioproduct Development, Universiti Teknologi Malaysia, Johor Bahru, Johor, Malaysia;
*Corresponding Author: chualeesuan@utm.my
Received 2013
The physical properties, total phenols, total fla-
vonoid content and free radical scavenging ac-
tivities of honey samples from Malaysia were
investigated. The physical properties of Tualang,
Gelam and Acacia honey samples, in terms of pH,
color, moisture, electrical conductivity and total
soluble solid were significantly different (p =
0.000). Gelam honey was reported to have the
highest total phenols (606.17 mg GAE/kg honey)
and flavonoid content (183.43 mg RE/kg honey).
Tualang honey was reported to have the highest
free radical scavenging activity with the IC50,
72.75 g/L compared to Gelam (77.41 g/L) and
Acacia (90.83 g/L). There is no significant dif-
ference has been revealed among honey samples
for radical scavenging activity (p = 0.827). Nev-
ertheless, strong correlation was obtained be-
tween pH, color, electrical conductivity and total
soluble solid with the scavenging activity of all
honey samples with the correlative coefficient, r
= 0.979, 0.902, 0.917 and 0.957, respectively. The
establishment of the statistical correlation could
be useful for honey related industry.
Keywords: Total Phenolic Content; Total Flavonoid
Content; Radical Scavenging Activity
Honey is a natural food source that widely used as a
traditional medicine. To the best of our knowledge, more
than 200 substances including complex sugar and small
amount of other substances have been found in honey.
The common types of honey found in lowland rain forest
of Peninsular Malaysia are Tualang and Gelam honey,
originates from Tualang (Koompassia excels) and Gelam
(Melaleuca cajuputi) trees. Another common source of
honey in Malaysia is Acacia honey and this honey is
easily available all over the world with different physical
and chemical characteristics.
Honey is widely used as antioxidant to reduce the risk
of heart disorder, cancer and to increase the immune
system [1]. The antioxidant activity of honey is believed
mainly contributed by the presence of phenolic acids,
flavonoids, catalases, peroxides, carotenoids and non-
peroxidal components. According to Gheldof and Enge-
seth [2] and Turkmen et al. [3], the composition of honey
is highly dependent on the botanical and geographical
origin, as well as the handling process during harvesting
and storage. There are also studies reported that the bo-
tanical origin is the critical factor influencing the anti-
oxidant activity of honey [4,5].
There are several methods commonly used for the
measurement of antioxidant activity of honey based on
different principles and experimental condition. Accord-
ing to Bertoncelj et al. [6], the methods include FRAP
assay (ferric reducing antioxidant power), TEAC (Trolox
equivalent antioxidant activity), DPPH (1, 1-diphenyl-
2-picrylhydrazyl) and ORAC (oxygen radical absorbance
capacity). DPPH method is likely to be the most reliable
technique to determine the antioxidant activity of honey,
since DPPH produces stable free radicals for testing [7].
Since honey varies according to their origin, the physical
properties of honey samples including pH, color, mois-
ture, electrical conductivity and total soluble solid were
studied and correlated statistically with their total phe-
nols, total flavanoids and antioxidant activity. The estab-
lishment of the physicochemical correlation enhances the
understanding of honey characteristics from tropical
country which is beneficial to honey related industry.
The findings from this study could be compared to the
data reported previously which found the physical prop-
erties were highly correlated with the antioxidant activity
of honey [6,8,9]. Therefore, this study was investigated
the physical properties, phenolic and flavonoid contents,
as well as the antioxidant activity of honey samples from
2.1. Honey Samples
All honey samples were collected from the West Coast
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N. L. A-Raham an et al. / Agricultura l Sciences 4 (2013) 46-51 47
of Peninsular Malaysia, which consist of Tualang, Gelam
and Acacia honey. Tualang and Gelam honey was ob-
tained from the Federal Agricultural Marketing Author-
ity (FAMA), Kedah, whereas MB An-Nur Apiary, Johor,
supplied the Acacia honey. Honey samples were stored
in glass containers at room temperature (25 - 30) in
the dark place without any processing or treatment prior
to analysis. The analyses were carried out within six
months after harvesting.
2.2. Reagents and Standard
All reagents are of analytical grade otherwise stated.
HPLC-grade of methanol from Merck (Darmstadt, Ger-
many). Hydrochloric acid, sodium hydroxide and alu-
minium trichloride were purchased from Fisher Scien-
tific (Pittsburg, USA). Deionized water was produced by
Barnstead NANOpure Diamond water purification sys-
tem at 18.2 M-cm resistivity (State of Illinois, USA).
The standard reagents such as Folin-Ciocalture reagent,
2-2-diphenyl-1-picryhydrazyl and gallic acid were pur-
chased from Sigma-Aldrich (Missouri, USA). Ascobic
acid (98 %) and rutin (97 %) were obtained from Across
Organics (Pittsburg, USA).
2.3. Physical Analysis
Determination of pH: The pH value of honey was
determined based on the method described by Saxena et
al. [9] using a pH meter (Mettler Toledo Delta 320) for the
measurement. A 10% w/v of honey solution was prepared
in aqueous medium.
Determination of color: The color of honey was meas-
ured by using Lovibond Honey Photometer based on the
AOAC Official Method 985.25 (2000) with minor modi-
fication. Honey samples were placed in a 10 mm path-
length plastic cuvette. The color values was determined
in triplicate and expressed in millimeter Pfund scale.
Determination of moisture content: The moisture con-
tent was measured based on the AOAC Method 969.38
(2000). A handheld Refractometer (Atago, Japan) was
used to determine the water content in honey based on
the refractive index. The readings were corrected for a
standard temperature of 20 by adding the correction
factor of 0.00023/℃. The moisture content was calcu-
lated by using Wedmore’s table referring to the refractive
index. All measurements were carried out in triplicate
and the moisture content is expressed in percentage (%).
Determination of total soluble solid: The total soluble
solid was measured by using a refractometer (Atago
Pocket Refractometer) at ambient temperature. The re-
sults were expressed in % Brix.
Determination of electrical conductivity: The electrical
conductivity was determined based on the electrical re-
sistance by using a conductivity meter (EcoScan COND
6+ Conductivity Meter from Eutech Instrument) [9]. All
honey samples were analyzed in triplicate and the results
are expressed in mS/cm.
2.4. Analysis of Chemical and Scavenging
Total phenolic content in honey samples was determined
by using the Folin-Ciocalteu method described by Sin-
gleton et al. [10] with minor modification. A 0.5 mL of
honey solution (0.1 g/mL) was mixed with 2.5 mL of 0.2
N Folin-Ciocalteu reagent and incubated for five minutes.
Then, 2 mL of sodium carbonate (75 g/L) was added and
incubated for two hours. After incubation, the absorbance
was measured against methanol as blank at 760 nm using
a UV-Vis spectrophotometer (Perkin Elmer, Lamda 25).
The calibration curve was constructed using gallic acid
(0 - 100 mg/L). The measurement was performed in trip-
licate and the total phenolic content was expressed in mg
of gallic acid equivalents (GAE) per kg of honey.
Total flavonoid content was determined by using the
method described by Singleton et al. [10] with minor
modification. A 5 mL of honey solution (0.1 g/mL) was
mixed with 5 mL of 2% aluminium trichloride (AlCl3).
The complex formation (flavonoid-aluminium) was meas-
ured at 415 nm by using a UV-Vis spectrophotometer
(Perkin Elmer, Lambda 25) after ten minutes incubation.
The standard curve for total flavonoids content was plot-
ted using rutin (0 - 100 mg/L) as a standard chemical.
The measurement was performed in triplicate and the
total flavonoids content was expressed in mg rutin equi-
valents (RE) per kg of honey.
The radical scavenging activity of honey was meas-
ured according to the 2, 2-diphenyl-1-picryhydrazyl (DPPH)
method as described by Velazquez et al. [11] with minor
modification. This method measures the reduction of
DPPH purple color to yellow color. A 0.75 mL of honey
solution (0.02 - 0.04 g/mL methanol) was mixed with 1.5
mL of 0.02 mg/mL DPPH solution. The complex mixture
against DPPH was measured by using a UV-Vis spectro-
photometer (Perkin Elmer, Lambda 25) at 517 nm after
15 minutes incubation at 25. The sample blank con-
sisted of 0.75 mL methanol and 1.5 mL of methanolic
DPPH. The ascobic acid standard curve (0 - 10 mg/L)
was plotted for calibration. The free radical scavenging
activity was expressed as IC50.
2.5. Statistical Analysis
The data were analyzed by the analysis of variance
(ANOVA) using SPSS software (SPSS 16.0). Correla-
tions were carried out by using the Pearson’s correlation
coefficient (r) in bivariate linear correlations.
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N. L. A-Raham an et al. / Agricultura l Sciences 4 (2013) 46-51
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3.1. Physical Properties of Honey
From Table 1, the pH values of honey samples; Tualang,
Gelam and Acacia, were ranging from 3.14 to 3.52, where
Tualang honey showed the most acidic value in pH. The
pH values of all honey samples were significantly dif-
ferent (p = 0.000) due to the difference in botanical and
geographical origin of honey. The parameter of pH is
crucial as it is strongly related to the stability, life
expectancy of honey product and fermentation process
due to storage [8,12]. The pH values for Malaysian honey
reported by Khalil et al. [13]] was slightly higher, which
ranged from 3.44 to 3.89 compared to the pH values
reported by Alqarni et al. [14]. However, honey samples
from Serbian, Brazil and Uruguayan showed the com-
parable findings with this study, the pH were ranging from
3.1 - 4.14, 2.9 - 3.7 and 3.00 - 4.30, respectively[8,15,16].
Gelam honey was reported to have darker color
compared to Acacia and Tualang honey with Pfund scale,
139.00, 97.00 and 74.00 mm, respectively. All honey
samples were statistically different (p = 0.000) in color
depending on the type of floral fed by bees. Based on the
National Honey Board [1], the color of Tualang, Gelam
and Acacia honey was categorized as light amber to dark
amber. The color of honey might be contributed by
pigments such as chlorophylls, carotenoids, flavonoids
and derivatives of tannis and polyphenols [17]. The total
soluble solid are primarily consisted of sugars; fructose,
glucose, and sucrose. Moreover, organic compounds
such as acids and minerals also contributed to the total
soluble solid in honey. Acacia honey showed the highest
soluble solid (74.80%), followed by Gelam (74.07%) and
Tualang (72.93%). The difference of soluble solid might
due to the difference in chemical composition of honey.
Acacia honey was reported to have the lowest mois-
ture content compared to Tualang and Gelam honey;
19.53, 24.07, 25.20%, respectively. Tualang and Gelam
honey contained high moisture content, which exceeding
20%, while the moisture content of Acacia honey was
within the permitted ranged [18]. The moisture content
of honey is a crucial parameter to determine the quality
and shelf-life of honey product. The factor of storage
combined with the high moisture content in honey could
increase the fermentation rate, and leading to the forma-
tion of acidified honey because of acetic acid formation.
Somehow, the moisture content of honey is highly de-
pending on the climatic change, maturity of honey and
harvesting season [19]. Based on Kayacier and Karaman
[20], the moisture content might indicate the origin of
honey. The tropical country of Malaysia with rainy sea-
son all over the year leads to high moisture content in
honey. Therefore, Malaysian honey is always treated by
evaporation to reduce the water content, simultaneously
increase the quality of honey.
The electrical conductivity was the highest in Acacia
honey, followed by Gelam and Tualang honey. All honey
samples were significantly different in electrical conductive-
ity (p = 0.045). The electrical conductivity measures the
ability of honey solution to conduct an electric current.
This electric current was carried by ions and the chemi-
cal changes in the solution. The factors that contributed
to the high value of electrical conductivity might be storage,
time, temperature, water content and concentration of
ions and minerals [14,21,22]. The electrical conductivity
affects the pH of honey, which indicated in the acidity
value, depending on the dissolved chemical compound
and biochemical process [23].
3.2. Total Phenols, flavonoids and
Antioxidant Properties
The amount of total phenolic contents was predicted
by using the Folin-Ciocalteu reagent from methanolic
honey solution. The total phenolic contents in Malaysian
honey were expressed in milligram gallic acid equivalent
to kilogram of honey. The result obtained (Table 1) showed
that the total phenolic contents for honey samples were
ranged from 383.79 to 606.17 mg GAE/kg. Floral honey,
Gelam was reported having the highest phenolic contents,
followed by Acacia (honeydew honey) and Tualang (floral
honey) with 606.17, 550.00 and 383.79 mg GAE/kg,
Table 1.
Honey (Mean ± SD)
Parameters (Unit) Tualang Gelam Acacia
pH 3.14 ± 0.06c 3.52 ± 0.07a 3.33 ± 0.08b
Color (mm Pfund) 74.00 ± 0.00c 139.00 ± 0.00a 97.00 ± 0.00b
Moisture (%) 25.20 ± 0.17a 24.07 ± 0.12b 19.53 ± 0.12c
Electrical Conductivity, (mS/cm) 0.91 ± 0.0012c 1.02 ± 0.0089b 1.09 ± 0.0030a
Total soluble solid, (%) 72.93 ± 0.06c 74.07 ± 0.15b 74.80 ± 0.10a
Total phenolics content, (mggallic acid/kg) 383.79 ± 13.57a 606.17 ± 20.36a 550.00 ± 21.93a
Total flavonoids content, (mgrutin/kg) 49.04 ± 0.53c 183.43 ± 1.70a,b 115.96 ± 4.69a,b
DPPH Radical Scavenging Activity (IC50, g/L) 72.75 ± 4.52a 77.41 ± 62.29a 90.83 ± 12.31a
a,b,cSuperscript in the same row was compared based on LSD’s test (p < 0.05). Same superscript means no significant difference.
N. L. A-Raham an et al. / Agricultura l Sciences 4 (2013) 46-51 49
respectively. However, there is no significant difference
(p = 0.471) between floral and honeydew honey for total
phenolic contents. The similar finding was reported by
Beretta et al. [24] and Bertoncelj et al. [6] using floral
and honeydew honey for their studies.
The total flavonoid contents of all honey samples were
expressed in mg rutin equivalent/kg by using rutin stan-
dard curve (r2 = 0.995). Once again, Gelam honey was
reported to have the highest flavonoid contents (183.43
mg RE/kg), followed by Acacia (115.96 mg RE/kg) and
Tualang (49.04 mg RE/kg). There is no significant dif-
ference between Gelam and Acacia honey (p = 0.650) for
total flavonoid content. The amount of flavonoid con-
tents was lower than the phenolic contents in all honey
samples. The values of phenolic and flavonoid contents
in honey samples were in close agreement with the re-
sults reported by Meda et al. [25] and Yao et al. [26]
The free radical scavenging activity was determined
based on the DPPH method using the stable radical 2,2-
diphenyl-1-picryhydrazyl. The radicals are scavenged by
delocalization of the spare electron over the molecule.
This delocalization would give the purple color in metha-
nol solution and turned to pale yellow after reacting with
the substance by donating hydrogen atom. The free radi-
cal scavenging activity typically expressed as IC50, to
represent the quantity of antioxidant required to reduce
the initial concentration of DPPH by 50 %. By using IC50,
the antioxidant activity increase with the de- creasing of
IC50 value [27]. From the Ta b l e 1 , the IC50 values were
ranged from 72.75 to 90.83 g/L, where Acacia honey
reported to have the highest value (90.83 g/L), followed
by Gelam (77.41 g/L) and Tualang (72.75 g/L). Acacia
honey was the least reactive honey to trap the free radical,
compared to Gelam and Tualang honey. However, there
is no statistically different between all honey samples (p
= 0.827) for the radical scavenging activity. Somehow, it
is important to note that, the presence of peptide, organic
acids, enzymes and the product of Maillard reaction
would contribute to the antioxidant activity in honey [6].
3.3. Correlation between Physical,
Chemical and Antioxidant
The physical properties in honey closely related to the
presence of antioxidant properties. Indeed, the physical
properties of honey depend on the geographical and
botanical origin of the nectar. This present study reported
that the antioxidant properties have strong correlation
with pH, color, electrical conductivity and total soluble solid.
Nevertheless, the moisture and free radical scavenging
activity of honey samples showed a negative correlation.
From Ta bl e 2, the total phenolic contents showed the
strong correlation between pH, color and total soluble
solid with correlation coefficient, r = 0.979, 0.902 and
0.957, correspondingly. Besides, the total flavonoid con-
tent also exhibited similar correlation with the pH (r =
0.997), color (r = 0.986) and total soluble solid (r =
0.839). The presence of phenolic acids and flavonoids in
honey samples might act as a proton donator leading to the
for- mation of acidified honey with low pH value. The pig-
ments of polyphenolics and flavonoids that exist in honey
might contribute to the variable color of honey [17]. The
dark honey contributed by the higher content of phenolic
compounds [28]. These organic compounds and acids
also explained to the strong correlation with the total
soluble solid. The free radical scavenging activity exhib-
ited strong correlation with electrical conductivity and
total soluble solid with correlation coefficient, r = 0.917
and 0.921, respectively. This electric current was carried
by ions and controlled by dissolved chemical compound
and biochemical process [23]. Strong negative correla-
tion was reported between free radical scavenging active-
ity and moisture content (r = -0.998). The high moisture
content could lead to the fermentation process that could
decrease the antioxidant capacity.
Table 2.
pH Color Moisture EC TSS TPC TFC DPPH-IC50
pH 1
Color 0.971 1
Moisture -0.188 -0.016
EC 0.710 0.474 -0.881
TSS 0.878 0.738 -0.675 0.944 1
TPC 0.979 0.902 -0.432 0.807 0.957 1
TFC 0.997 0.986 -0.165 0.612 0.839 0.961 1
DPPH-IC50 0.246 0.083 -0.998 0.917 0.921 0.624 0.246 1
Correlations were carried out by using Pearson’s correlation coefficient (r); EC: Electrical conductivity, TSS: Total soluble solid, TPC: Total phenolic content,
TFC: Total flavonoid content.
Copyright © 2013 SciRes. Openly accessible at http://www.scirp.org/journal/as/
N. L. A-Raham an et al. / Agricultura l Sciences 4 (2013) 46-51
The physical properties of honey are closely related
with the chemical composition of honey. The composi-
tion is dependent on the botanical and geographical ori-
gins, in addition to the shelf life of honey. Gelam was
reported to have high phenolic and flavonoid contents.
The high scavenging activity was also reported in Tu-
alang honey. The physical properties could be used to
predict the antioxidant activity in honey based on the
strong correlation for pH, color, electrical conductivity
and total soluble solid. The establishment of the correla-
tion could be the easiest way to determine the quality of
The authors would like to thank Ms. Nur Ardawati Adnan and Ms.
Norfahana Abd-talib for their support and guidance. This research work
was granted by GUP (Tier 1) Fund under the Research Alliance of
Biotechnology (7125.00H05), UTM.
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