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Analysis of Antioxidant Properties and Volatile Compounds of Honey from Different Botanical and Geographical Origins

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  • Universiti Malaya

Abstract and Figures

Honey has been consumed since ancient time due to its nutritional and therapeutic values. Studies showed that honey possesses antioxidant properties which can inhibit oxidation and cell damage in the body. However, the chemical contents and antioxidant properties of honeys are varied, depending on botanical and geographical origins of honey. In this study, we analysed the total phenolic content (TPC), total flavonoid content (TFC), antioxidant properties (DPPH, ABTS, FRAP and TAOC) and volatile profiles of several commercial honeys originated from Malaysia, Turkey, and Yemen. The results showed that sample H4 (Pine honey) from Turkey was the highest in TPC (0.84 µg GAE/mg honey), ABTS (63.15% inhibition) and FRAP (0.45 µg FeSO 4 equivalent/mg honey) values, while H2 (Acacia honey) from Malaysia showed the highest values in TFC (0.11 µg quercetin equivalent/mg honey) and DPPH (45.13 mg/mL IC 50). Meanwhile, H5 (Marai honey) from Yemen recorded the highest TAOC value (24.14 µg ascorbic acid equivalent/mg honey). Twenty-four volatile compounds were identified using gas chromatography-mass spectrometry (GC-MS), among others are 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, linoleic acid ethyl ester, 2,5-dimethyl-4-hydroxy-3(2H)-furanone, and 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one which contribute to chemical characteristics of certain honeys. In regards to the TPC, TFC, and antioxidant assays, the honey samples were ranked based on the chemical properties level as follows: H4 (Pine honey) > H2 (Acacia honey) > H7 (Kelulut 2) > H3 (Kelulut 1) > H6 (Sumar honey) > H1 (Tualang honey) > H5 (Marai honey). This finding increases the knowledge of the chemical compositions, volatile compounds, and antioxidant activities of several commercial honeys derived from different botanical and geographical origins.
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Sains Malaysiana 51(4)(2022): 1111-1121
http://doi.org/10.17576/jsm-2022-5104-13
Analysis of Antioxidant Properties and Volatile Compounds of Honeys from
Dierent Botanical and Geographical Origins
(Analisis Antioksidan dan Sebatian Meruap Madu daripada Asal Usul Botani dan Geogra yang Berbeza)
KASFUL ASRA SAKIKA1, MOHD ZUWAIRI SAIMAN2,3*, NOR HISAM ZAMAKSHSHARI3, IDRIS ADEWALE AHMED3,
MUHAMMAD NAZIL AFIQ NASHARUDDIN3 & NAJIHAH MOHD HASHIM3,4*
1Institute for Advanced Studies, Universiti Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia
2Institute of Biological Sciences, Faculty of Science, Universiti Malaya, 50603 Kuala Lumpur, Federal Territory,
Malaysia
3Centre for Natural Products Research and Drug Discovery (CENAR), Universiti Malaya, 50603 Kuala Lumpur,
Federal Territory, Malaysia
4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universiti Malaya, 50603 Kuala Lumpur, Federal
Territory, Malaysia
Received: 5 May 2021/Accepted: 20 August 2021
ABSTRACT
Honey has been consumed since ancient time due to its nutritional and therapeutic values. Studies showed that honey
possesses antioxidant properties which can inhibit oxidation and cell damage in the body. However, the chemical contents
and antioxidant properties of honeys are varied, depending on botanical and geographical origins of honey. In this
study, we analysed the total phenolic content (TPC), total avonoid content (TFC), antioxidant properties (DPPH, ABTS,
FRAP and TAOC) and volatile proles of several commercial honeys originated from Malaysia, Turkey, and Yemen.
The results showed that sample H4 (Pine honey) from Turkey was the highest in TPC (0.84 µg GAE/mg honey), ABTS
(63.15% inhibition) and FRAP (0.45 µg FeSO4 equivalent/mg honey) values, while H2 (Acacia honey) from Malaysia
showed the highest values in TFC (0.11 µg quercetin equivalent/mg honey) and DPPH (45.13 mg/mL IC50). Meanwhile,
H5 (Marai honey) from Yemen recorded the highest TAOC value (24.14 µg ascorbic acid equivalent/mg honey).
Twenty-four volatile compounds were identied using gas chromatography-mass spectrometry (GC-MS), among others are
4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, linoleic acid ethyl ester, 2,5-dimethyl-4-hydroxy-3(2H)-furanone,
and 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one which contribute to chemical characteristics of certain honeys. In
regards to the TPC, TFC, and antioxidant assays, the honey samples were ranked based on the chemical properties level
as follows: H4 (Pine honey) > H2 (Acacia honey) > H7 (Kelulut 2) > H3 (Kelulut 1) > H6 (Sumar honey) > H1 (Tualang
honey) > H5 (Marai honey). This nding increases the knowledge of the chemical compositions, volatile compounds,
and antioxidant activities of several commercial honeys derived from dierent botanical and geographical origins.
Keywords: Antioxidant properties; avonoids; honeybee; phenolics; stingless bee; volatile compounds
ABSTRAK
Madu telah digunakan sejak zaman dahulu disebabkan nilai nutrisi dan terapeutiknya. Kajian menunjukkan bahawa
madu mempunyai kandungan antioksidan yang boleh menghalang pengoksidaan dan kerosakan sel dalam badan. Walau
bagaimanapun, kandungan kimia dan antioksidan madu adalah berbeza-beza, bergantung kepada punca botani dan
geogra madu tersebut. Dalam kajian ini, kami telah menganalisis jumlah kandungan fenol (TPC), jumlah kandungan
avonoid (TFC), sifat antioksidan (DPPH, ABTS, FRAP dan TAOC) dan prol sebatian meruap daripada beberapa madu
komersial yang berasal dari Malaysia, Turki dan Yaman. Keputusan kajian menunjukkan bahawa H4 (madu Pain) dari
Turki adalah paling tinggi bagi TPC (0.84 µg GAE/mg madu), ABTS (63.15% penghambatan) dan FRAP (0.45 µg FeSO4/
mg madu), manakala H2 (madu Akasia) dari Malaysia menunjukkan nilai tertinggi dalam TFC (0.11 µg kuersetin/mg
madu) dan DPPH (45.13 mg/mL IC50). Sementara itu, H5 (madu Marai) dari Yaman mencatatkan nilai TAOC tertinggi
(24.14 µg asid askorbik/mg madu). Dua puluh empat sebatian meruap telah dikenal pasti menggunakan kromatogra
gas-spektrometri jisim (GC-MS), antara lain adalah 4H-piran-4-one, 2,3-dihidro-3,5-dihidroksi-6-metil, asid linolik etil
1112
ester, 2,5-dimetil-4-hidroksi-3(2H)-furanon, dan 2,4-dihidroksi-2,5-dimetil-3(2H)-furan-3-one yang menyumbang
kepada ciri-ciri kimia bagi madu-madu tertentu. Berdasarkan kepada TPC, TFC dan ujian antioksidan, sampel madu
disusun mengikut tahap sifat kimia seperti berikut: H4 (madu Pain) > H2 (madu Akasia) > H7 (Kelulut 2) > H3 (Kelulut
1) > H6 (madu Sumar) > H1 (madu Tualang) > H5 (madu Marai). Hasil kajian ini dapat menambah pengetahuan tentang
komposisi kimia, sebatian meruap dan aktiviti antioksida bagi beberapa madu komersial yang berasal daripada punca
botani dan geogra yang berbeza.
Kata kunci: Fenol; avonoid; kelulut; lebah madu; sebatian meruap; sifat antioksidan
INTRODUCTION
Honey is a natural sweet substance produced by both
honeybees and stingless bees. There are 10 species of
honeybees which belongs to the genus Apis (Gupta et al.
2014), and over 500 species of stingless bee have been
identied worldwide (Michener 2013). Honey produced
by stingless bees is dierent from that honeybee’s honey
in terms of its colour, taste, and viscosity. Bees collect
nectar from plants, then transform and combine it with
their own substances and store it in honey comb till
ripen and mature (Biluca et al. 2017). Based on the oral
source of nectar, honeys can be classied into monooral
or polyoral origin. Monooral honey is predominantly
derived from one plant species but minor nectar can also
be derived from other plants. Besides, polyoral honey
involves several sources of plant species and none of which
is predominant (Gašić et al. 2014).
The use of honey by mankind can be traced back from
8,000 years ago as pictured by a cave painting in Valencia,
Spain (Purbafrani et al. 2014). The ancient civilizations
have been reported to utilize honey in various applications.
For instance, the ancient Vedic civilization used honey for
treating insomnia, cough, skin disorder such as wound and
burns, eye ailment to prevent cataract, and for keeping
healthy teeth and gum (Eteraf-Oskouei & Naja 2013).
In ancient Egypt, honey was used to heal burn together
with Aloe vera and tannic acid as prescribed in the Smith
papyrus, an Egyptian text dated back between 2600
and 2200 B.C. (Pećanac et al. 2013). Meanwhile, the
ancient Greeks believed that consumption of honey could
help one to live longer (Arawwawala & Hewageegana
2017). In the Islamic medicine, honey is considered as
a healthy food since the holy Quran mentioned about its
potential therapeutic values. The great Muslim scientist and
physician, Avicenna had recommended honey as one of the
remedies to treat tuberculosis (Asadi-Pooya et al. 2003).
The therapeutic values of honey have been
contributed by its phenolic compounds (Cianciosi et
al. 2018). These compounds are responsible for the
antioxidant property of honey with their ability to scavenge
free radicals. Excessive free radical in cellular system
can cause oxidative damage to the cells and break down
the essential macromolecules such as nucleic and amino
acids, protein, and lipid. This may lead to the development
of diseases such as cancer, metabolic disorders, and
cardiovascular dysfunction (Rahal et al. 2014). The
composition of phenolic compounds in honey depends on
the source of the oral origin. Phenolic compounds and
other bioactive substances are transferred from plant’s
nectar to honey as a nal product, making its composition
are highly determined by the oral origin (Kaškoniene
& Venskutonis 2010). Moreover, honey composition is
inuenced by seasonal and environmental factors in the
geographical area, as well as the processing method of
honey (Manyi-Loh et al. 2011).
The source of oral origin mainly determines the
diversity of compounds found in honey. Therefore, the
presence of certain volatile compounds could be used as
oral markers to dierentiate certain monooral honeys.
Pattamayutanon et al. (2017) have listed 2,2-dimethyl
butanal, hexadecane, tetradecane, and pentadecane
as floral markers for longan honey (Apis mellifera)
from Thailand. While Seisonen et al. (2015) suggested
2H-pyran, tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-
(9CI) as a compound marker for Lindin honey from
Estonia. Volatile compounds could also contribute to the
bioactivities of honey, especially the antioxidative
effect due to their natural radical scavenging potential
(Manyi-Loh et al. 2011). In the present study, the total
phenolic contents (TPC), total avonoid content (TFC),
antioxidant properties (DPPH, ABTS, FRAP and TAOC) and
volatiles prole of several commercial honeys derived
from dierent botanical and geographical origins were
analysed.
MATERIALS AND METHODS
SAMPLE COLLECTION
A total of seven honey samples were used in this study
(Table 1). Samples H1-H3 were obtained from Federal
1113
Agricultural Marketing Authority (FAMA) Kedah,
Malaysia. Samples H4 were from Turkey and samples
H5-H6 were from Yemen, which all samples were
acquired from the market in Malaysia. Sample H7 was
obtained from the Malaysian Agricultural Research and
Development Institute (MARDI), Selangor, Malaysia. All
samples were obtained or purchased between September
and November 2018 and were kept at 4 °C until further
analysis. The information on the bee species and oral type
of samples H1-H3, and H7 were provided by FAMA and
MARDI, respectively. While the information for samples
H4-H6 were stated on the product label.
TABLE 1. Description of honey samples analysed in this study
Samples Name Species Floral type Geographical origin Source
H1 Tualang honey Apis dorsata Monooral (Koompassia excels)Malaysia (Kedah) FAMA
H2 Acacia honey Apis melliferaMonooral (Acacia mangium)Malaysia (Kedah) FAMA
H3 Kelulut 1 Heterotrigona
itama Polyoral Malaysia (Kedah) FAMA
H4 Pine honey Apis mellifera Monooral (Pinus spp) Turkey
(not mentioned) Market
H5 Marai honey Apis mellifera Polyoral Yemen
(not mentioned) Market
H6 Sumar honey Apis mellifera Monooral (Vachellia tortilis)Yemen
(not mentioned) Market
H7 Kelulut 2 Heterotrigona
itama Polyoral Malaysia (Terengganu) MARDI
FAMA: Federal Agricultural Marketing Authority, MARDI: Malaysian Agricultural Research and Development Institute
REAGENTS AND CHEMICALS
ABTS (2,2´-Azino-bis (3-ethylbenzothiazoline-6-
sulfonic acid), potassium persulfate, sodium phosphate,
ammonium molybdate, gallic acid, Folin-Ciocalteu
reagent, sodium carbonate, quercetin hydrate, sodium
acetic acid, ascorbic acid and 2,4,6-tris (2-pyridyl)-s-
triazine (TPTZ) were purchased from Sigma-Aldrich
(Steinheim, Germany), 2,2’-diphenyl-1-picrylhydrazil
(DPPH) from Merck KGaA (Darmstadt, Germany), ferric
chloride from R & M Chemical (Essex, England) and
dimethyl sulfoxide (DMSO) from Fisher Scientific
(Loughborough, England). All reagents and chemicals
were of analytical grade.
ANALYSIS OF TOTAL PHENOLIC CONTENT (TPC)
Total phenolic content (TPC) was measured according to
Biluca et al. (2016) with slight modication. Briey, 20
µL (10 mg/mL) of the samples was inserted into a 96-well
plate and added with 50 µL Folin-Ciocalteu (F-C) reagent.
The F-C reagent was diluted with distilled water with a
ratio of 1:10. The mixture was left for 3 min before 100 µL
(60 g/L) of Na2CO3 was added. Finally, the plate was left
in the dark for 60 min before the absorbance was read at
750 nm using VersaMaxTM microplate reader. Gallic acid
was used to calculate standard curve (3.90, 7.81, 15.62,
31.25, 62.5, and 250 µg/mL; r2 = 0.9827), and the TPC
values were expressed as µg of gallic acid equivalents
(GAEs) per mg of honey.
ANALYSIS OF TOTAL FLAVONOID CONTENT (TFC)
The total flavonoid content (TFC) was determined
according to the method by Ranneh et al. (2018) with
slight modication. Briey, 2% of AlCl3 solution in
methanol was prepared in the beaker before transferred
to the 50 mL centrifuge tube. Then, 100 µL (10 mg/mL)
of samples were inserted into 96-well microplates and
added with 100 µL 2% v/v AlCl3 solution. The plate was
incubated in the dark for 1 hour and the absorbance was
measured at 430 nm using VersaMaxTM microplate
reader. Quercetin was used to calculate standard curve
(3.90, 7.81, 15.62, 31.25, 62.5, and 250 µg/mL; r2 =
0.9998). The TFC values were expresses as µg of
quercetin equivalent per mg of honey.
1114
ANALYSIS OF 2,2’-DIPHENYL -1- PICRYLHYDRAZIL (DPPH)
The DPPH assay was carried out according to Salonen
et al. (2017). One gram of honey was dissolved in 1 mL
methanol. This stock solution was then further diluted to
eight dierent concentrations (1.95, 3.90, 7.81, 15.62,
31.25, 62.5, 125, and 250 mg/mL) with methanol. The
samples were then centrifuged at 10000 rpm for three
min at 4 °C (Eppendorf 5415R centrifuge, Hamburg,
Germany). Then, 60 µL of each concentration of samples
was mixed with 60 µL of DPPH (0.1 mg/mL methanol) in
the 96-well plates and incubated at room temperature for
30 min. The absorbance was measured at 517 nm using
VersaMaxTM microplate reader. Methanol was used as a
blank and the percentage of the radical-scavenging assay
(RSA) was calculated as:
%RSA = [Absorbance blankAbsorbance sample] / [Absorbance blank] × 100
The graph of these percentages against various
concentrations (7.81, 15.62, 31.25, 62.5, 125, and 250
mg/mL) was then plotted and the results are expressed as
IC50 mg/mL (required concentration of honey sample for
50% inhibition of free radicals). Butylated hydroxytoluene
(BHT) was used as a reference.
ANALYSIS OF FERRIC REDUCING ANTIOXIDANT POWER
(FRAP)
Ferric reducing antioxidant power (FRAP) was measured
according to Habib et al. (2014). FRAP reagent was
prepared with the ratio of 10:1:1 of solution 1 (300 mM
acetate buer), solution 2 (10 mM TPTZ) and solution 3 (20
mM FeCl3.6H2O), respectively. Then, 10 µL of the sample
(10 mg/mL) were loaded to each well of the 96-well plate
and added with 300 µL of FRAP reagent. The plate was
incubated in the dark at 35 ºC and the absorbance was
read at wavelength 470 nm using VersaMaxTM microplate
reader after 4 min. Iron sulfate (FeSO4) was used to
calculate standard curve (25, 50, 100, 200, 600 and 800
µg/mL; r2 = 0.9950). FRAP values were expressed as µg
of FeSO4 equivalent per mg of honeys.
ANALYSIS OF TOTAL ANTIOXIDANT CAPACITY (TAOC)
Phosphomolybdate assay procedure as described by
Jan et al. (2013). The reagent solution was prepared
by mixing 28 mM sodium phosphate, 4 mM ammonium
molybdate and 0.6 M sulphuric acid. One millilitre of the
reagent and 0.1 mL of the sample (10 mg/mL) were loaded
into a 1.5 mL microcentrifuge tube and were incubated
at 95 ºC for 90 min. After the tube was cooled to room
temperature, 300 µL of each mixture was transferred to
the 96-well microplate and the absorbance was analysed
at 695 nm using VersaMaxTM microplate reader. Ascorbic
acid was used to calculate standard curve (3.90, 7.81,
15.62, 31.25, 62.5 and 250 µg/mL; r2 = 0.9971), and the
values were expressed as µg of ascorbic acid equivalent
per mg of honey.
ANALYSIS OF 2,2’-AZINO-BIS
(3-ETHYLBENZOTHIAZOLINE-6-SULPHURIC ACID) (ABTS)
Free radical scavenging activity of the honey sample
was determined using ABTS assay according to Silva
et al. (2013) with slight modication. ABTS+● solution
(7mM) was first prepared by dissolving 38 mg of
2,2-azino-bis (3-ethylbenzothiazoline-6-sulphuric acid)
diammonium salt (ABTS) in 10 mL distilled water and
added by 10 mL of 20 mM potassium persulfate. The
solution was incubated in the dark for 12 h prior to use
at room temperature and underwent dilution (by adding
distilled water) to reach absorbance of 0.68 (± 0.008485)
at 743 nm. Then, 10 µL (10 mg/mL) of samples were
transferred into a 96-well plate and added by 300 µL of
ABTS+● solution. The plate was incubated for 10 min at
room temperature and the absorbance was read at 743 nm
wavelength using VersaMaxTM microplate reader. DMSO
was used as a blank. The values were calculated as:
Percentage (%) of inhibition = [(Absorbance blank- Absorbance sample)/
Absorbance blank] × 100
GC-MS ANALYSIS
GC-MS analysis was performed according to Mohamad
Shah et al. (2013) with minor modication. Initially,
honey was weighed (0.5 g) into a microcentrifuge tube and
mixed with 2.5 mL methanol. The mixture was vortexed for
3 min before centrifuged at 2500 rpm for 5 min. Finally,
the mixture was ltered using a syringe lter (0.22 µm)
into a GC vial for analysis.
GC-MS analysis was performed using a Shimadzu
QP2010 ULTRA coupled with mass spectrometer. The
column was an Rtx-5MS fused-silica capillary column
(30 m × 0.25 mm i.d.; 0.25 µm lm thickness) with helium
as a mobile phase at a ow rate of 50 mL/min. The injection
volume was 5 µL volume using a split mode at an injector
temperature of 300 °C. The oven temperature was held
(5 min) at 40 °C and ramped to 160 and 280 °C (15 min
hold) with rate 4 and 5°C/min, respectively. The total run
time for each sample was 74 min. All peaks were identied
based on mass matching (≥ 90%) from the NIST libraries.
Only compounds with 90% or greater spectral matching
accuracy were reported.
1115
STATISTICAL ANALYSIS
Assays were performed in triplicate (n = 3) and the
results were expressed as the mean values with standard
deviation (SD). The significant difference (p ˂ 0.05)
represented by dierent letters were obtained by one-
way analysis of variance (ANOVA) followed by Duncan
Multiple Range Test (DMRT). Pearson product-moment
coecient test was used to determine the relationship of
variables (TPC, TFC, DPPH, ABTS, FRAP and TAOC). All
statistical analysis was conducted using SPSS software
version 15.
RESULTS AND DISCUSSION
In this study, seven samples of honey from dierent
types and sources were analysed for their total phenolic
content (TPC) and total flavonoid content (TFC),
antioxidant properties and volatile compounds. Two
honey samples i.e. H3 and H7 (Kelulut) were produced by
Heterotrigona itama, the highly propagated stingless
bee (Meliponini) species in Malaysia. The other honey
samples were produced by the honeybee (Apini) subfamily
derived from Malaysia, Turkey and Yemen (Table 1). The
TPC and TFC values are presented in Figure 1.
FIGURE 1. (A) total phenolic content (TPC); (B) total avonoid content (TFC) of
several honey samples (H1: Tualang; H2: Acacia; H3: Kelulut 1; H4: Pine; H5: Marai;
H6: Sumar; H7: Kelulut 2). Values presented are mean ± SD (n = 3). Bars having
dierent letters are signicantly dierent (p ˂ 0.05)
a
b
c
b
aa
b
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
H1 H2 H3 H4 H5 H6 H7
Total phenolic (µg GAE/mg extract)
Honey samples
(A)
a
b
c
b
a
d
c
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
H1 H2 H3 H4 H5 H6 H7
Total flavonoid (µg Quercetin/mg extract)
Honey samples
(B)
Generally, the phenolic compounds are classied
into two main families: phenolic acids with their related
derivatives; and flavonoids. Flavonoids itself can
be further classied in a number of subfamilies such
as flavones, flavonols, isoflavones, flavanones, and
avanonols (Ciulu et al. 2016). The TPC values found in
this study range between 0.54 ± 0.16 and 0.84 ± 0.07 µg
GAE/mg honey (gallic acid equivalent per milligram of
honey) with the highest value shown by H4 (Pine honey)
followed by H2 (Acacia honey) and H7 (Kelulut 2) which
were signicantly dierent (p ˂ 0.05) from other samples.
However, a lower range was found for TFC value (0.03
1116
± 0.01 to 0.11 ± 0.24 µg quercetin equivalent/mg honey)
with the signicant highest value (p ˂ 0.05) was recorded
in H2 (Acacia honey) followed by H4 (Pine honey).
Moniruzzaman et al. (2013) reported that the TPC
values of their Tualang and Acacia honey samples
collected from the tropical rain forests of Kedah and
Johor (Malaysia) are 0.35 ± 0.81 and 0.19 ± 0.84 µg
gallic acid equivalent/mg honey, respectively. While the
TFC values are 0.07 ± 0.74 and 0.02 ± 1.73 µg catechin
equivalent/mg honey for Tualang and Acacia honey,
respectively. Both TPC and TFC values reported by
Moniruzzaman et al. (2013) are lower compared to our
present study, except for the TFC value of Tualang honey.
In another study of samples collected from the forests of
Kedah and Johor (Malaysia), the Kelulut honey showed
a signicantly stronger antioxidant capacity compared
to Tualang honey, which the result was correlated to the
higher TFC and TPC level in Kelulut honey (Ranneh et
al. 2018). The variations of chemical composition and
level inferred that oral origins (Cianciosi et al. 2018)
and environmental/seasonal factors (Cheung et al. 2019)
contribute to the phenolic and avonoid content in honey.
Antioxidant properties of honey samples were
investigated by DPPH, ABTS, FRAP, and TAOC assays.
DPPH and ABTS assays are based on the free radical
scavenging properties of honey and the decolourization
of DPPH and ABTS radicals occurs during the presence
of antioxidant compounds. Meanwhile, FRAP and TAOC
assays measure the potential of honey as a reducing
agent. In the presence of a reducing agent (antioxidant),
Fe3+TPTZ complex and Molybdenum VI are reduced to
Fe2+TPTZ complex and Molybdenum V. The results of the
antioxidant properties of honeys are presented in Figure 2.
FIGURE 2. Antioxidant properties of (A) DPPH; (B) ABTS; (C) FRAP; and (D)
TAOC of several honey samples (H1: Tualang; H2: Acacia; H3: Kelulut 1; H4: Pine;
H5: Marai; H6: Sumar; H7: Kelulut 2). Values presented are mean ± SD (n = 3). Bars
having dierent letters are signicantly dierent (p ˂ 0.05)
a
a
a
a
a
a
b
010 20 30
H1
H2
H3
H4
H5
H6
H7
TAOC (µg ascorbic acid/mg extract)
Honey samples
(D)
a
b
c
d
e
e
c
020 40 60 80
H1
H2
H3
H4
H5
H6
H7
ABTS (% inhibition)
Honey samples
(B)
a
b
b
c
a
a
b
00.1 0.2 0.3 0.4 0.5
H1
H2
H3
H4
H5
H6
H7
FRAP (µg FeSO4/mg extract)
Honey samples
(C)
a
b
c
b
a
d
b
c
050 100 150 200 250
H1
H2
H3
H4
H5
H6
H7
ST
DPPH/IC50 (mg/ml)
Honey samples
(A)
1117
The DPPH IC50 values (Figure 2(A)) indicate the
concentration of honey that has caused 50% scavenging
of free radical, which means the lower the IC50 value,
the higher the antioxidant properties of the honey.
Our result shows that the DPPH IC50 values range from
45.12 (H2) to 182.56 mg/mL (H1). Sample H3 (Kelulut
1) showed an antioxidant activity (IC50 = 83.38 mg/mL)
comparable to the reference butylated hydroxytoluence
(BHT) (IC50 = 80.98 mg/mL). H2 (Acacia honey) showed
the highest activity (IC50 = 45.12 mg/mL), followed by H4
(Pine honey) (IC50 = 60.50 mg/mL), H7 (Kelulut 2) (IC50 =
73.80 mg/mL), H6 (Sumar honey) (IC50 = 109.76 mg/mL),
H5 (Marai honey) (IC50 = 159.74 mg/mL) and H1 (Tualang
honey) (IC50 = 182.56 mg/mL). Samples that possess
IC50 values ranging from 10 to 50 mg/mL is considered
to have strong antioxidant activity, 50 to 100 mg/mL is
intermediate, and > 100 mg/mL is considered as weak
(Phongpaichit et al. 2007). Therefore, in this study, H2
(Acacia honey) possesses a strong antioxidant activity.
While H4 (Pine honey), H7 (Kelulut 2), and H3 (Kelulut
1) shows intermediate antioxidant activity. The lowest
antioxidant activity corresponds to H1 (Tualang honey)
and H5 (Marai honey). The free radical scavenging
activity of the honey samples was also evaluated
through ABTS assay and reported as % inhibition. In
accordance with DPPH results, H2 and H4 exhibited the
best ABTS inhibition activity (46.06 and 63.15%) among
the samples (p ˂ 0.05). The ndings from both assays are
also consistent with the TPC and TFC results, indicating
that the samples with higher TPC and TFC values would
also have higher antioxidant free radical scavenging
properties (Gašić et al. 2014; Ranneh et al. 2018).
FRAP and TAOC assays measure the antioxidant
properties based on the reducing power of the samples.
In the present study, the FRAP value is between 0.29 ±
0.06 (H1) and 0.45 ± 0.02 (H4) FeSO4 equivalence in
milligram of honey. In contrast to DPPH and ABTS results,
the second-highest value was H7 (Kelulut 2) and not H2
(Acacia honey). The TAOC which was determined using
phosphomolybdate assay, however, shows dierent
results from the other antioxidant assays. The imported
honey from Yemen which are Marai (H5) and Sumar
(H6) recorded the highest TAOC values with 24.14 ±
0.43 and 24.11 ± 0.04 ascorbic acid equivalence in
milligram of honey, respectively. However, this result was
not statistically signicant with other samples except for
H7 (Kelulut 2) which showed signicantly lower value
(p ˂ 0.05).
Pearson product-moment coefficient test was
conducted to identify the correlation between variables
of phenolic compounds and antioxidant activities. As
shown in Table 2, TPC and TFC had a strong positive
correlation with ABTS and FRAP and a strong negative
correlation with DPPH. A similar correlation was also
found by Khalil et al. (2012) and Moniruzzaman et al.
(2014) suggesting that phenolic compounds are important
antioxidant factors. However, this does not imply that
other compounds are not involved in the prevention of
oxidation processes. As evidenced by a weak and very
weak negative correlation between TAOC, TPC, and TFC,
a non-phenolic antioxidant may also responsible for the
antioxidant activity of honey (Bertoncelj et al. 2007;
Bogdanov 2012).
TABLE 2. Correlation coecient results with Pearson product-moment coecient test
TPC TFC FRAP ABTS TAOC DPPH
TPC 1.916** .907** .899** -.498 -.833*
TFC .916** 1.778* .881** -.132 -.812*
FRAP .907** .778* 1.947** -.544 -.873*
ABTS .899** .881** .947** 1-.323 -.865*
TAOC -.498 -.132 -.544 -.323 1.388
DPPH -.833* -.812* -.873* -.865* .388 1
* Correlation is signicant at p ˂ 0.05
** Correlation is signicant at p ˂ 0.01
1118
Proling and identication of the volatile compounds
was conducted using a gas chromatography-mass
spectrometry (GC-MS). Based on a comparison of the
mass spectra with the NIST library, 24 peaks of the volatile
compounds with 90% or greater spectral matching
accuracy have been identied from the honey samples
(Table 3). These volatiles belong to dierent chemical
classes such as oxygenated compound (aldehyde, ketones,
TABLE 3. Major volatile compound in honey samples
Peaks RT (min) Molecular
formula [M-W](Frag. MS m/z)Compounds Samples
13.152 – 3.291 CH6N246 (14,15,22,28,31,42,46) Hydrazine, methyl H3,H5
23.363 – 3.434 CH2O246 (12,19,29,31,46) Formic acid H1,H2
33.366 C2H6N2O290 (30,33,44,45,49,60,61) Methanamine, N-methoxy-N-nitroso H3
43.514 – 3.674 C2H4O260 (2,15,18,26,43,55,60) Acetic acid H1,H3
54.381 – 4.412 C4H6O286 (15,27,29,39,50,55,57,71,85) Butane, 1,2:3,4-diepoxy-, (.+/-.) H3,H4
64.514 C4H6O286 (14,26,29,31,38,50,55,59,68,85) 2,2’-Bioxirane H3
7 5.093 – 5.413 C4H10O3106 (15,26,31,33,41,45,59,75,77,105) Glycolaldehyde dimethyl acetal All except H4
8 5.686 – 6.260 C5H4O296 (14,24,29,34,39,40,49,67,96) Furfural All
97.203 – 7.394 C5H6O2
98 (14,24,27,39,41,48,53,55,61,69,79,8
1,85,98) 2-Furanmethanol All
10 8.586 C6H12O3
132 (29,31,41,45,53,59,69,72,85,101,1
02,131) Furan, tetrahydro-2,5-dimethoxy H4
11 9.248 – 9.300 C6H6O2110 (27,38,39,44,51,63,77,93,95,110) Ethanone, 1-(2-furanyl) H3,H4,H6
12 9.527 C4H4O284 (27,29,33,39,41,49,55,56,67,83,84) 2(5H)-Furanone H5
13 10.069 – 10.195 C5H6O298 (26,35,41,51,55,57,66,69,72,79,96,98) 1,2-Cyclopentanedione H1,H2,H4,H7
14 10.832 – 11.997 C6H8O4144 (43,45,55,59,73,84,97,101,144) 2,4-Dihydroxy-2,5-dimethyl-3(2H)-
furan-3-one H3,H7
15 11.398 – 11.472 C6H6O2110 (15,27,29,39,51,53,61,69,81,95,110) 2-Furancarboxaldehyde, 5-methyl All
16 12.596 C8H8O 120 (29,39,51,65,66,77,89,91,118,120) Benzene acetaldehyde H7
17 17.014 C6H8O3
128 (15,29,31,43,45,57,60,72,85,87,10
0,128)
2,5-Dimethyl-4-hydroxy-3(2H)-
furanone H1
18 20.403 C6H8O4
144 (39,43,46,55,58,69,73,87,97,101,11
5,126,144)
4H-Pyran-4-one, 2,3-dihydro-3,5-
dihydroxy-6-methyl H2
19 21.769 C5H12O3120 (15,29,41,47,48,59,60,75,89,90,119) Ethane, 1,1,2-trimethoxy H5
20 23.993 – 25.389 C6H6O3126 (29,38,41,50,68,69,80,97,109,126) 5-Hydroxymethylfurfural All
21 46.973 C18H34O2
282 (43,55,69,83,97,111,123,137,165,17
9,222,264) 9-Octadecenoic acid, (E) H4
22 47.335 C20H36O2
308 (41,55,67,81,95,109,123,136,150,22
0,263,308) Linoleic acid ethyl ester H1
23 56.230 C44H90
618 (29,57,71,113,211,267,351,407,491
,533,618) Tetratetracontane H7
24 60.546 C20H38O2
310 (43,55,69,82,96,109,
137,166,194,235,267,310) Z-14-Octadecen-1-ol acetate H7
RT: Retention time; [M-W]: Molecular weight; H1: Tualang; H2: Acacia honey; H3: Kelulut 1; H4: Pine honey; H5: Marai honey; H6: Sumar honey; H7: Kelulut 2
1119
esters, alcohol, and carboxylic), hydrocarbon (aliphatic,
aromatic, and cyclic) and heterocyclic compounds
(pyran and furan derivatives). Furan derivatives which
are produced as a result of sugar degradation (Aljohar et
al. 2018) are found as the most encountered compound
with furfural, 2-furanmethanol, 2-furancarboxaldehyde,
5-methyl and 5-hydroxymethylfurfural (HMF) detected
in all samples. HMF can be found in sugar-containing
food and is associated with sugar degradation due to heat
or long storage. This compound is commonly low and
minimal concentration in honey (<40 or <80 mg/kg for
honeys from tropical region) but when HMF is detected
at higher level than the concentration limit, this indicates
a poor storage condition, or exposure to high temperature
and excessive heat treatment (Shapla et al. 2018). However,
in this study, quantitative study was not conducted to
compare HMF level among the samples.
Of the 24 volatile compounds that have been
identified, only 4H-Pyran-4-one, 2,3-dihydro-3,5-
dihydroxy-6-methyl (DDMP) and 2,4-dihydroxy-2,5-
dimethyl-3(2H)-furan-3-one have been reported to have
antioxidant activities (Kanzler et al. 2016; Wong & Kern
2011). In the present study, DDMP was only detected
in sample H2 (Acacia honey). This compound has
been reported in several foodstus including prunes
(Čechovská et al. 2011), heated pear (Hwang et al. 2013),
whole wheat bread (Bin & Peterson 2016), and honey
(Awasum et al. 2015). Besides antioxidant activity, DDMP
has also been associated with autonomic nerve activity
of rats (Beppu et al. 2012).
The antioxidant compound 2,4-dihydroxy-2,5-
dimethyl-3(2H)-furan-3-one was only detected in the
sample H3 (Kelulut 1) and H7 (Kelulut 2). Chukwu et al.
(2007) reported that 2,4-dihydroxy-2,5-dimethyl-3(2H)-
furan-3-one was found in the Sysepalum dulcicum fruit
extract. Therefore, this compound could be derived from
a certain plant species which its nectar consumed by the
stingless bees. Nevertheless, though both honey samples
were produced by the same species of stingless bee
Heterotrigona itama, their volatiles prole are dierent.
This implies that the foraging activities of the same bee
species is dierent and depends on the geographical and
botanical origins.
CONCLUSION
In this study, Pine honey from Turkey (H4) showed the
highest phenolic contents and antioxidant properties
followed by Acacia honey (H2), Kelulut 2 (H7), Kelulut 1
(H3), Sumar honey (H6), Tualang honey (H1) and Marai
honey (H5). A strong correlation was found between
the phenolic content and their antioxidant properties:
DPPH, ABTS and FRAP except for TAOC (a negative
weak correlation). In terms of volatile compounds, only
DDMP and 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-
one have been reported possess antioxidant activities.
DDMP was only identied in Acacia honey (H2), while
2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one was
detected only in Kelulut samples. Moreover, linoleic acid
and furaneol which were identied in Tualang honey (H1)
could be considered as possible oral markers. However,
further studies are required particularly related with their
physicochemical properties and bioactive components for
a better understanding. Overall, the present study provides
valuable information on the analysis and proling of
honeys from dierent botanical and geographical origins.
ACKNOWLEDGEMENTS
The authors would like to thank the Ministry of Higher
Education, Malaysia for the financial support under
Fundamental Research Grant Scheme (Project No. FP035-
2021 [FRGS/1/2021/STG01/UM/02/9]).
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*Corresponding authors; email: zuwairi@um.edu.my
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