Physicochemical, antioxidant capacity and color analysis of six honeys from different origin

Abstract

Six honey samples from different origin were examined and compared for their physicochemical proprieties (pH, moisture content, TSS, water activity, amino acid and refractive index) and antioxidant activity antioxidant content, (total phenolic and total flavonoids). In addition, colore intensityABS635, Hunter color values, lycopene, β – carotene and HMF were also studed. Clover honey, Egypt) was the most acidic (pH 3.26). (Sider honey, Yemen) and (Sider, Saudi) showed the lowest moisture content (16.81 and 16.91) respectively. Among all the samples, acacia honey showed the lowest HMF content (3.6mg/kg) while sider honey-Saudi recorded the highest HMF content (22.47 mg/kg). All the tested samples showed high content of phenolic and flavonoid compounds. Out of the six tested honey, Sider-Saudi honey recorded the highest phenolic and flavonoid content and color intensity flowed by pine honey, Germany). The results of the antioxidant proprieties of the different honey samples as assed by DPPH method showed strong positive correlation between the antioxidant content and antioxidant activity and the polyphenolic content and the color of the honey. The darker honeys (sider honey, Saudi and pine honey, Germany) recorded higher antioxidant activety.

Full text

Original article
Physicochemical, antioxidant capacity and color analysis of six honeys
from different origin
Iryna Smetanska
a
, Salman S. Alharthi
b
, Khaled A. Selim
c,
⇑
a
Department of Plant Food Processing, Agric. Faculty, Univ. of Applied Science, Weihenstephan-Triesdorf, Steingruberstraße 2, 91746 Weidenbach, Germany
b
Department of Chemistry, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
c
Department of Food Science and Technology, Faculty of Agricultuer., Fayoum University, P.O. Box 6351, Fayoum, Egypt
article info
Article history:
Received 27 September 2020
Revised 31 March 2021
Accepted 13 April 2021
Available online 22 April 2021
Keywords:
Honey
Phenolic content
Antioxidant activity
Color intensity
abstract
Six honey samples from different origin were examined and compared for their physicochemical
proprieties (pH, moisture content, TSS, water activity, amino acid and refractive index), antioxidant activ-
ity (total phenolic and total flavonoids) and antioxidant content. In addition, colore intensity ABS
635
,
Hunter color values, lycopene, b– carotene and HMF were also studed. (Clover honey, Egypt) was the
most acidic (pH 3.26). (Sider honey, Yemen) and (Sider, Saudi) showed the lowest moisture content
(16.81 and 16.91) respectively. Among all the samples, acacia honey showed the lowest HMF content
(3.6 mg/kg) while sider honey-Saudi recorded the highest HMF content (22.47 mg/kg). All the tested sam-
ples showed high content of phenolic and flavonoid compounds. Out of the six tested honey, Sider-Saudi
honey recorded the highest phenolic and flavonoid content and color intensity flowed by pine honey,
Germany). The results of the antioxidant proprieties of the different honey samples as assed by DPPH
method showed strong positive correlation between the antioxidant activity, the polyphenolic content
and the color of the honey. The darker honeys (sider honey, Saudi and pine honey, Germany) recorded
higher antioxidant activity.
Ó2021 The Authors. Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Honey is a natural supersaturated sugar solution produced by
honeybees. the major constituents in honey are carbohydrates
(glucose, fructose and sucrose). In addition, honey also contains
different minor compounds such as pigments (carotenoids and
anthocyanins), polyphenolic compounds (phenolic acids, flavo-
noids), amino acids, vitamins (C and E), enzymes, and organic acids
and minerals. These components give honey the functional proper-
ties in the human body minor constituents gives the honey (Saxena
et al., 2010; Djebli et al., 2020).
Man has used honey since ancient times in traditional medicine
(skin ulcers, asthma and infected wounds). as for food and in pre-
serving food from spoilage delaying deterioration and preserving
the color demolishing of foods by light (National honey board,
2003; Kücük et al., 2011; Meda et al., 2005). Honey could be used
as a functional food because of its biological properties (Kolayli
et al., 2020, Gomes et al., 2010).
There has been an interest in adding natural antioxidants to
food for the role it played in resisting oxidative damage of fat
and preventing free radicals forming inside the body which pro-
tecting the body from the risk of numerous disease (The National
Honey Board, 2003).
It was shown in several studies that honey contains a wide
range of constituents which had antioxidant activity, such as caro-
tenoids, flavonoids, phenolic acids, vitamins and enzymes which
have encouraging effect in the curing of some chronic illness. The
presence and quantity of these ingredients in honey depends on
many factors, which include the geographical location, floral
source, climate, entomological source, season and processing of
honey (Pauliuc et al., 2020; Baltrusaityte et al., 2007). A positive
relationship between the honey color, which is mainly refer to
the presence of different pigments and its antioxidant potential.
It was reported that the darker colored honey had higher total phe-
nolic content than the light one and consequently provide higher
antioxidant capacities (Bertoncelj et al., 2007; Alves et al., 2014).
https://doi.org/10.1016/j.jksus.2021.101447
1018-3647/Ó2021 The Authors. Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
⇑
Corresponding author.
E-mail address: kas00@fayoum.edu.eg (K.A. Selim).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
Journal of King Saud University – Science 33 (2021) 101447
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Interest in investigating the antioxidant potentials of honeys
and analysing their phenolic and flavonoids compounds was
increased (Gül and pehlivan, 2018; Can et al., 2015). The honeys
produced from mountains with dry and hot climate (sider honey)
usually darker and have lower moisture content than the other
honeys. The purpose of the current study is to investigate the phys-
ical and chemical properties and antioxidant activities six different
honey types collected from different botanical origin and also to
study their possible interdependence.
2. Experiments
2.1. Honey samples and classification
Six different types of floral honey samples (about 300 g for
each) were selected for the present investigation. The honey sam-
ples were collected and ported to Troisdorf University, Germany.
The information concerning local name, location of collection,
botanical data were presented in Table 1. Botanical classification
was achieved when the pollen spectrum contained > 455 of the
corresponding dominant pollen according to Louveaux et al.
(1978). The pollen was studied a Motic microscope (Motic, Xiamen,
China).
2.2. Chemicals and reagents
Ascorbic acid, catechin, 2,2- diphenyl-1-picrylhydrazyl (DPPH),
Folin–Ciocalteu’s reagent, standard phenoilc acids and amino acids
were purchased from Sigma-Aldrich (St. Louis, Mo., U.S.A.). Metha-
nol, ethanol, hexane and acetone were purchased from Merck
(Darmstadt, Germany). Aluminum chloride, Sodium carbonate,
sodium hydroxide and sodium nitrite (NaNO
2
) were purchased
from Merck (Darmstadt, Germany). All chemicals used were of
analytical grade.
2.3. Water activity
The water activity of the samples was determined using a
w
meter (Novasina Lab Touch-aw meter, Novasina AG, Neuheim-
strasse 12, CH 8853 Lachen, Switzerland) according to (Chirife,
et al., 2006).
2.4. Moisture, sugar and Hydroxymethyl furfural content (HMF)
The moisture content, Sugar content (fructose, glucose and
sucrose and refractive indices of honey samples were measured
according to (AOAC, 1990).
Hydroxymethyl furfural (HMF) was determined using UV spec-
trometric method described by White (1984) and calculated using
the following equation:
HMF = [ (A284-A336) / (sample wight)] 74.87
The pH value was measured using a pH meter (Toledo AL20,
Mettler Toledo Group, 8603 Schwezenboch, Switzerland) for a
10% (w/v) solution of honey prepared in milli Q water.
2.5. Total phenolic and flavonoids content
Total phenolic content of the investigated samples was mea-
sured by Folin–Ciocalteu method as described by (Singleton,
et al., 1999). The total phenolic content was calculated using gallic
acid standard curve (0–200 mg/l; Y = 0, 0014X + 0.0338;
R
2
= 0.9935). Flavonoids content was determined using AlCl3
method (Barros et al., 2007) The concentration was calculated as
mg of (+)-catechin equivalents (CEs) /kg honey using (+)-Catechin
was used to calculate the standard curve (20–100 mg/ml).
2.6. Carotenoids determination
Total Carotenoids and Lycopene was determined according to
Boussaid et al. (2018) using Beta-carotene for the calibration curve.
2.6.1. Color intensity: ABS
635nm
Five grams of each honey sample was diluted with distilled
water to 50% (w/v) and homogenized. The solution was centrifuged
at 3200 rpm for five minutes and the absorbance was measured at
635 nm. The color intensity was determined using the Pfund scale
(Pfund HI, Hanna Instruments, USA) after conversion of the absor-
bance values: mm Pfund = 38.70 + 371.39 Abs (White, 1984;
Ferreira et al., 2009). The pfund scale is ranged from < 9 for water
white color to > 114 for dark Amber color. The color value of the
honeys (CIE L*a*b*) were determined using a hunter spectrometer
(CR-400, Konica Minolta, Tokyo, Japan).
2.7. Determination and identification of free amino acids
The determination and identification of FAA in the investigated
honey samples was done by High Performance Liquid Chromatog-
raphy according to the procedure of Pawlowska and Armstrong
(1994).
2.8. Recommended procedures for analysis of of phenolic compounds
Extraction was carried out as described previously (Yao et al.,
2004). Honey samples (25–50 g) were dissolved in 250 ml of dis-
tilled water and the pH of the solution was adjusted to pH 2.0. sug-
ars and other polar compounds of honey solution were removed by
filtration through the column with Amberlite XAD- 2 resin
(Supelco, Bellefonte, PA, USA). The phenolic compounds were
eluted from the sorbent with methanol and the methanol extracts
were dried under reduced pressure at 35 °C in a Speed Vac Concen-
trator, SPD111V 230 (Thermo scientific, USA) The residue was re-
dissolved in methanol to a known final concentration and stored
at-80 °C until further analysis.
2.8.1. HPLC analysis of phenolic compounds
HPLC analysis of the phenolic extracts was carried out as
described by (Baltrusaityte et al., 2007): L C 10 AD HPLC eluent
pump (Shimadzo, Kyoto, Japan), DAD SPDM10 AVP, UV–Vis SPD
10AVP detectors (Shimadzo, Kyoto, Japan) and LCMS 2010 EV Mass
spectrometer. The time of HPLC run was over 45 min. UV–Vis
detector was operating at 254 nm /280 nm wavelength. Mass spec-
tra of phenolic compounds were scanned with ion trap MS after
electrospray ionization (ESI) in negative and positive mode (Yao
et al., 2004).
Table 1
Characterization of honey samples.
Samples Local name Dominant pollen Production
aria
Sample 1 Acacia, Germany Robinia pseudoacacia L. Nurnberg,
Germany
Sample 2 El-sider, Libya Ziziphus Lotus L. Cyrene, Libya
Sample 3 Clover, Egypt Trifolium alexandrinum L. Fayoum, Egypt
Sample 4 Sider, Yemen Ziziphus Spina-christi L.Hadramout,
Yemen
Sample 5 Pine Germany Pinus halepensis L. Pinus
brutia
Munchin,
Germany
Sample 6 Sider Saudi Arabia
(KSA)
Ziziphus Spina-christi L.South, Saudi
Arabia
I. Smetanska, S.S. Alharthi and K.A. Selim Journal of King Saud University – Science 33 (2021) 101447
2

2.8.2. Determination of antioxidant content
The antioxidant content calculated as ascorbic acid equivalent
antioxidant content (AEAC) was determined using the method of
Meda et al. (2005) using DPPH solution and the absorbance was
measured at 517 nm using a spectrophotometer. the mean value
of absorbance (triplicate) was expressed as mg of (AEAC) /100 g
of honey.
2.8.3. Determination DPPH radical scavenging of the tested honey
samples
The free radical scavenging activity (RSA) was evaluated accord-
ing to the method of Brand-Williams et al. (1995) using Jenway
Genova UV/vis Spectrophotometer (HS service GmbH, Switzer-
land). The absorbency of the remaining DPPH was determined at
515 nm. The extract concentration providing 50% of radicals scav-
enging activity (SC50) was calculated by interpolation from the
graph of RSA percentage against extract concentration.
3. Results and discussion
3.1. pH value of honey samples
The results in Table 2 summarized the physiochemical analysis
of the investigated honey samples. All of the honey samples ana-
lyzed were found to be acidic in character. Their pH values ranged
from 3.26 to 4.01. The pH of the honeys is correlated with its stor-
age stability and microorganism’s growth (Alves et al., 2014). The
highest pH values were observed for sider honey from KSA
(4.91). The high acidity of honey could be attributed to the fermen-
tation of sugar into organic acids, in addition to the presence of
inorganic ions such as phosphate and chloride ions which are
responsible for the stability of honey’s against microbial spoilage
(Ahmida et al., 2013). Our results were within the limit that indi-
cated the freshness of the honey samples and comparable with
those previously reported by other researchers (Feás et al., 2010;
Alves et al., 2014). The low pH value of the honey could reflect
its ability to inhibit the microorganism’s growths. The pH values
of Algerian, Brazilian, Spanish, Malaysian, Indian and Turkish hon-
eys have been found to vary between, 3.10 to 5.01, respectively
(Saxena et al., 2010; Moniruzzaman et al., 2013). The variation in
the pH among the honey samples could be returned to the plant
floral types.
3.2. Water activity
The water activity is an important factor, which governs the
food stability by preventing or limiting microbial growth. The
osmotolerant yeasts are able to grow at a minimal water activity
of 0.6 (Chirife et al., 2006). The water activity of the investigated
honey samples varied from 0.504 to 0.565 (Table 2). El-sider Haney
from Libya recorded the lowest a
w
value while, pine honey found
to have the highest aw value among the investigated samples.
Our results are in agreement with those found for Greek honey
(a
w
0.53 to 0.67) and lower than the Indian and Brazilian honeys
for which the a
w
values ranged from 0.57 to 0.70 and 0.67 to
0.77 respectively, (Saxena et al., 2010; Silva et al., 2013).
3.3. Moisture content
The moisture content of honey is very important factor, control-
ling its storage stability and granulation during storage. The high
moisture content may lead to fermentation of honey during stor-
age which gives the honey sour taste (Kucuk et al., 2007 and
Ahmida et al., 2013). Moisture content (%) in the investigated sam-
ples ranged from 16.81 to 19.3. The highest moisture contents
were recorded for Pine honey (Pinus halepensis L.) from Germany
and Clover honey (Trifolium alexandrinum L) from Egypt which
recorded moisture contents (%) of 19.10 and 19.30 respectively.
Moisture content (%) of Sider honey from Yemen was
16.81 ± 0.75 which was the lowest among all the investigated sam-
ples which gives it longer shelf life during storage. The results also
showed that all tested honeys recorded moisture contents <20%,
which is the maximum limit for the moisture content according
to the Codex standard for honey quality (Codex Alimentarius,
2001). The results showed varietal differences in moisture contents
among the analysed samples. These differences could be due to
various factors such as degree of maturity in hive, harvesting sea-
son, environmental condition and manipulation by beekeepers
during harvest period (Alvarez-Suarez et al., 2010). The moisture
levels of the analyzed samples were similar to those reported by
other workers for different honey types including Malaysian honey
12.79% to 22.32% (Moniruzzaman et al., 2013), Indian honeys 18.7%
to 21.8% and Turkish honeys (Kucuk et al., 2007) and Romanian
honeys 14.3 to 20.2% (Marghitas et al., 2010).
3.4. HMF content of honey samples
HMF is important indicator in evaluating the freshness and the
purity of the honey (Khalil et al., 2012). It is usually found in a very
small amounts in honeys and increases during the storage or by
heating process (Can et al., 2015)). Results showed significant dif-
ference between the HMF contents of the tested honeys. HMF
levels of the investigated honeys varied from 3.69 to 22.47
(Table 2). Acacia honey recorded the lowest HMF content (3,6mg/
kg while, sider honeys from KSA and Yemen had the highest HMF
contents of 22.47 and 12.82 mg/kg honey respectively. However,
all values were within the recommended level according to the
codex Alimentarius (<80 mg/Kg). The higher HFM recorded for
KSA and Yemen honeys could be attributed to the tropical climate
of these countries which led to increase the HMF in hives. HMF
found to be affected by many factors such as sucrose content, type
of sugars, temperature, pH, age of the honey, floral source and fruc-
tose /glycose ratio (Islam et al., 2012; Can et al., 2015). Our results
are similar to those previously reported for honeys originating
from other tropical countries such as Malaysian honeys (6.07 to
67.94 mg/kg), Morocco honeys (0.09 to 53.38 mg/kg) ant that for
Table 2
Physical properties of the different honey samples.
CharacteristicsSamples Water activity Moisture Content pH R.I.* Total reducing sugars (%) Sucrose
(g/100 g)
HMF (mg/kg) TSS
**
Acacia, Germany 0.561 ± 0.01
a
18.83 ± 1.02
a
4.10 ± 0.02
b
1.4863 67.57 ± 1.98
c
3.82 ± 0.61
b
3.69 ± 0.21
d
81.06 ± 1.52
a
EL-Sider Libya 0.504 ± 0.01
b
17.52 ± 0.85
b
4.87 ± 0.04
a
1.4935 69.29 ± 2.45
b
2.07 ± 0.08
c
11.28 ± 0.53
b
80.27 ± 2.05
ab
Clover Egypt 0.559 ± 0.02
a
19.1 ± 0.94
a
3.26 ± 0.02
c
1.4908 66.81 ± 2.11
c
6.38 ± 0.57
a
10.94 ± 0.94
bc
78.57 ± 1.61
b
Sider Yemen, 0.554 ± 0.00
a
16.81 ± 0.75
b
4.66 ± 0.05
ab
1.4995 71.245 ± 2.81
a
2.15 ± 0.44
c
12.82 ± 1.03
b
81.64 ± 1.92
a
Pine, Germany 0.565 ± 0.01
a
19.30 ± 0.87
a
4.05 ± 0.03
b
1.4930 66.57 ± 2.03
c
2.17 ± 0.42
c
9.37 ± 0.58
c
78.71 ± 2.17
b
Sider, Saudi Arabia 0.543 ± 0.02
a
16.91 ± 0.72
b
4.91 ± 0.06
a
1.4990 70.95 ± 2.47
a
2.43 ± 0.37
c
22.47 ± 1.78
a
81.41 ± 2.35
a
* Rrefractive index, ** Total soluble solids
I. Smetanska, S.S. Alharthi and K.A. Selim Journal of King Saud University – Science 33 (2021) 101447
3

Bangladeshi honeys (3.06 to 43.81). (Moniruzzaman et al., 2013;
Islam et al., 2012).
3.5. Sugar content
The results showed that the total reducing sugar in the honey
samples ranged from 71.245 to 66.57% and represented the largest
portion of the honey composition. Data indicated that sucrose con-
tent in the investigated honeys ranged from 6.38 to 2.15%. All sam-
ples had sucrose levels below the maximum allowable limit of 5%
presented by codex standard except for clover honey sample
(6.38). The higher sucrose content of the clover honey could be
attributed to over feeding of honey bee with sucrose syrup or the
early harvest of honey (Azeredo et al., 2003; Saxena et al., 2010).
On the other hand, the higher total sugar content in Sider Yemen
and Sider Saudi Arabia could be because of the low levels of their
moisture content (Can et al., 2015). Our results are inconstant with
those reported by Khalil et al. (2012) for Algerian honeys (68.80 to
70.0%) and higher than that founded for Malaysian honeys (63.33
to 68.40) (Moniruzzaman et al., 2013).
3.6. Amino acid content of analyzed honey
The amino acid profile of the studied honey samples is summa-
rized in Fig. 1 A-B. RP- HPLC ensured the separation and evaluation
of 18 amino acid in the analyzed hones and the concentration of
each amino acid is shown in Table 3. The obtained data showed
that the most common essential amino acids found in these honeys
were lysine, histidine, leucine and threonine in all the investigated
honeys. Lysine found to be the highest essential amino acid in all
samples and ranged from 2.29 to 17.12 mg/100 g. The results
showed significant difference between clover from Egypt and sider
honey from KSA and the other honeys in the lysine content. El-
sider honeys from Libya and pine and acacia honey from Germany
recorded similar lysine content with no significant difference. Lib-
yan El-Sider honey and KSA honey recorded threonine content
higher than the other honey types. The results also revealed no sig-
nificant differences between all samples in the histidine content.
This is agreement with the results reported by Sun et al. (2017)
for Chinese unifloral honey and Kowalski et al. (2017) for polish
and Slovak honeys. Phenylalanine found in pine honey with quan-
tity higher than that in all the other honey types. Isoleusine, valine
and arginine are also found in small and similar quantities in all the
analyzed honeys. Our results are similar to that reported by
Kowalski et al. (2017) for Polish honeys.
The dominant non-essential amino acids in the investigated
honeys were proline ranged from (48.32 to 31.15 mg/100 g) glu-
tamic acid from (1.69 to 1.21 mg/100 g) and aspartic acid from
(1.29 to 1.13 mg/100 g), glycine from 1.08 to 0.52 and serine from
0.97 to 0.87 mg/100 g) honey. Lower but also important quantities
of tyrosine, glutamine and alanine were present in all the honey
types. Proline which produced from bee salivary secretion is an
important amino acid used as assign for honey ripeness and the
adulteration of honey with sugar (Bogdanov et al., 2004 and
Kowalski et al., 2017). The results showed that proline was the
highest amino acid in all the honey types and ranged from 48.32
to 27.78 mg/100 honey. Clover honey from Egypt recorded the
highest proline content while Yemeni sider honey recorded the
lowest content. Likewise, pine honey showed higher glutamic acid
content higher than the other samples. The proline contents
recorded in our study are similar to those founded for rosemary,
lavender and thyme honeys from Span and that reported by
Moniruzzaman et al. (2013) for Gelam and Sourwood honeys from
Malaysia. Kowalski et al (2017) reported proline content of Slo-
vakian acacia honey of 331.42 mg /kg which is very close to our
finding. The results showed that the proline contents recorded in
our samples were higher than those reported for Indonesian, some
Indian and Algerian honeys (Noor et al., 2019;Khalil et al., 2012).
Methionine and cysteine which are sulfur containing amino acids
were not detected in all the analyzed honey samples. Similar find-
ing was reported by Sun et al. (2017).
3.7. B- carotene, lycopene and color characteristics
The honey color analysis and color characteristic are presented
in (Table 4). Β- carotene and lycopene content of the investigated
samples were noticeably different and varied from 9.10 to 26.604
and from 4.75 to 11.89 mg/kg respectivly. The higest Β- carotene
concentration was recorded for the clover honey from Egypt fol-
lowed by acacia honey from Germany while sider honey KSA and
pine honey from Germany had the lowest B- carotene among all
the tested samples. Pine honey from Germany had the highest
lycopene content comparing with the other samples. Our results
are similar to that reported by Ferreira et al. (2009) for Portuguese
honeys and higher than those reported by Alvarez-Suarez et al.
(2010) for Cuban honeys (5.57 to 1.17 mg B-carotene E/kg honey).
The color of the honey is usually affected by many factors such as
its pigments content (carotenoids and flavonoid), total phenolic
content, age, botanical origins, contacts with metals and the way
of handling (Moniruzzaman et al., 2013).
According to USDA approved color stander, the honey had
Pfund value ranged from 8 to 16 is classified as extra white honey.
With increasing the Pfund value the honey gets more dark color
and classified as amber honey when its Pfund value was between
86 and 114. In the present work, clover and acacia honeys were
the brightest with the lowest Pfund values of (20.281 and
26.508 mm) and classified as white honey meanwhile, pine honey
and Sider KSA honey recorded the highest Pfund values 94.843 and
92.588 respectively and classified as amber honeys.
Parameters L*, a* and b* recorded for the different types of
honey are presented in Fig. 2. The results summarized that clover
and acacia honey had the highest L* values of 64.43 and 61.83
respectively, which exhibits lightness. There were significant dif-
ferences between these two honeys and the other tested honeys.
Sider honey KSA pine honey from Germany recorded the lowest
L* average values of (22.03 and 20.67) with no significant differ-
ence between them and classified as amber honey. The results
showed negative correlation between the L* values and the found
values of the tested honeys samples which mean darker honeys
had lower L* values because of their high contents of total flavo-
noids and polyphenolic compounds. The results also showed that
parameter a* and b* were varied among the different honey sam-
ples and ranged from 0.69 to 11.32 and from 3.08 to 10.13 respec-
tively. The L values of the samples ranged from 20.57 to 63.91 with
lower L value indicating a darker honey color. Our findings are sim-
ilar to those reported by Can et al. (2015) for acacia and clover
Turkish honeys and by (Bertoncelj et al., 2007) for Lithuanian hon-
eys. The dark-colored honeys could be because of the high levels of
pigment, pollen, phenolic compounds, minerals and Maillard reac-
tion products (Bertoncelj et al., 2007; Pauliuc et al., 2020).
3.8. Extraction yield, total flavonoids and total phenolic content of
honey samples
The extraction yields of different honey samples with metha-
nol: (80:20 v/v) were ranged from 115 ± 4.28 to 211 ± 4.33 m
g/100 g honey. Pine honey recorded the highest yield followed
by acacia and clover hones. On the other hand, the lowest
extraction yield was observed for the Yemeni sider honey of
115 mg/100 g honey.
I. Smetanska, S.S. Alharthi and K.A. Selim Journal of King Saud University – Science 33 (2021) 101447
4

010 20 30 40 50 min
0
25
50
75
100
125
150
175
200
225
250
275
300
mV
Asp
Glu
(U1)
Asn
Ser
U2
Gln
His
Gly
Thr
Arg
U3
(U4) U5
Ala
U6
Tyr
Lys-1
U7
Val
Met
Norv al
Ile Phe Leu
Lys-2
U8
U9
010 20 30 40 50 min
-25
0
25
50
75
100
125
150
175
200
225
250
275
300
325
350
375
400
425
450
475
500
525
550
mV
Asp
Glu
U1
Asn
Ser
U2
Gln
His
Gly
Thr
Arg
U3
(U4) U5
Ala
U6
Tyr
Lys-1
U7
Val
Met
Norv al
Ile Phe Leu
Lys-2
U8
U9
Fig. 1. HPLC chromatogram for amino acid analysis (A) acacia honey from Germany and (B) sider honey from KSA.
I. Smetanska, S.S. Alharthi and K.A. Selim Journal of King Saud University – Science 33 (2021) 101447
5

3.8.1. Total phenolic content (TP)
The polyphenol compounds are considered as very important
components in honey because of their influence in the honey color
and its functional properties. The main total phenolic content (mg
gallic acid equivalent GAE/kg) determined by using Folin-
Ciocalteau method for the studied mono floral honey samples are
specified in table (5). It was varied from 18.574 mg GAE/100 g in
clover honey to 53.314 mg/100 g in sider honey from KSA. Among
the tested honey samples, sider honey from KSA exhibited the
highest TP content (53.314 mg/100 g) followed by pine honey
which recorded TP content of (44.828 mg GAE /100 g). On the con-
trary, the clover honey found to have the lowest TP content
(18.574 mg GAE/ kg of honey). The results showed significant dif-
ferences between all the investigated samples. This difference
could be due to the difference in the geographical origin and the
floral sources. our results are compatible with that reported by
Kowalski et al. (2017); Can et al., 2015 for Turkish honey which
ranged from 24.20 to 124.05 mg/ 100 g . Likewise, Bertoncelj
et al. (2007) obtained TP content ranged from 44.8 to 233.9 mg kg
1
for Slovenian honey. Silici et al. (2010) indicated that rhododen-
dron honeys from turkey had phenolic content ranged from 0.24
to 141.83 mg/100 g honey. The obtained results elucidate positive
correlation between the honey color and its phenolic content in
which the darker honey contain more phenolic than the lighter
one. Our results are agreed with that presented by Ferreira et al.,
2009).
3.8.2. Flavonoid content
The main total flavonoids of the investigated honeys varied
from 41. 704 to 120.857 mg catechin/kg honey Table (5). The total
flavonoids content of the tested honey exhibited the order: KSA
sider honey > pine honey > sider, Yemen > El-sider,
Libya > acacia, Germany > clover, Egypt. As with phenolic content,
sider honey from KSA recorded the highest flavonoid content
among all the samples. The results showed no significant differ-
ences between acacia honey and clover honey and between El-
sider honey and pine honey. Our results are considerably higher
than that reported for Malaysian honey, Indian honey, and Cuban
honey (Moniruzzaman et al., 2013;Alvarez-Suarez et al., 2010).
However similar results were observed for sunflower honey, Geren
honey, and manuka honey (Das et al., 2013). In study by Meda et al.
(2005) for Burkina Fasan honey, the flavonoid content found to be
varied from 17 to 83.5 mg of quercetin / kg
1
of honey.
3.8.3. Analysis of phenolic compounds in the honey extracts
The HPLC analysis of the studied honey samples presented in
Table 6 and Fig. 3 indicated that chlorogenic acid, caffeic acid, p-
coumaric acid, the main plenolic acids in most of honey extracts
while kaempferol, Chrysin were the main flavonoids in all the
tested samples. chlorogenic acid found to be the predominate phe-
nolic acid in all the studied samples except that for Libyan El-sider
Table 3
Amino acids content of analyzed honey samples (mg/100 g).
Name Acacia, G. El-sider, L. Clover, EG. Sider, Y. Pine, G. Sider, KSA
Average Average Average Average Average Average
Asp 1.17 ± 0.03 1.13 ± 0.01 1.21 ± 0.03 1.17 ± 0.01 1.25 ± 0.01 1.29 ± 0.04
Glu 1.21 ± 0.00 1.38 ± 0.06 1.32 ± 0.06 1.36 ± 0.02 1.69 ± 0.03 1.40 ± 0.08
Asn 0.49 ± 0.00 0.54 ± 0.02 0.47 ± 0.01 0.47 ± 0.00 0.65 ± 0.01 0.46 ± 0.01
Ser 0.88 ± 0.01 0.90 ± 0.02 0.89 ± 0.00 0.85 ± 0.01 0.91 ± 0.01 0.97 ± 0.03
Gln 0.62 ± 0.00 0.68 ± 0.07 0.62 ± 0.07± 0.17 ± 0.24 1.01 ± 0.03 1.06 ± 0.27
His 2.70 ± 0.04 2.51 ± 0.08 2.70 ± 0.03 2.53 ± 0.00 2.64 ± 0.18 2.92 ± 0.01
Gly 0.59 ± 0.03 0.83 ± 0.08 0.74 ± 0.01 0.52 ± 0.01 0.77 ± 0.01 1.08 ± 0.18
Thr 0.75 ± 0.01 1.03 ± 0.06 0.92 ± 0.05 0.69 ± 0.01 0.82 ± 0.01 1.11 ± 0.14
Arg 0.29 ± 0.00 0.33 ± 0.02 0.39 ± 0.03 0.23 ± 0.01 0.48 ± 0.01 0.52 ± 0.07
Ala 0.30 ± 0.01 0.50 ± 0.03 0.40 ± 0.03 0.36 ± 0.01 0.48 ± 0.01 0.53 ± 0.08
Tyr 0.32 ± 0.01 0.35 ± 0.01 0.32 ± 0.03 0.41 ± 0.02 0.60 ± 0.01 0.40 ± 0.05
Lys 7.51 ± 0.15 6.27 ± 0.19 12.03 ± 0.40 12.29 ± 0.01 6.44 ± 0.03 17.12 ± 0.54
Val 0.47 ± 0.00 0.55 ± 0.03 0.54 ± 0.01 0.46 ± 0.00 0.48 ± 0.00 0.70 ± 0.05
Met 0.00 ± 0.00 0.00 ± 0.04 0.00 ± 0.00 0.00 0.00 0.00 ± 0.00
Ile 0.39 ± 0.00 0.43 ± 0.02 0.41 ± 0.01 0.36 ± 0.00 0.41 ± 0.00 0.50 ± 0.04
Phe 0.31 ± 0.01 0.47 ± 0.04 0.49 ± 0.12 0.39 ± 0.02 1.78 ± 0.06 1.24 ± 0.39
Leu 0.61 ± 0.03 0.52 ± 0.08 0.74 ± 0.00 0.51 ± 0.05 0.65 ± 0.03 2.28 ± 0.63
Pro 31.96 ± 1.97 31.15 ± 4.80 48.32 ± 2.39 27.78 ± 1.87 32.77 ± 2.46 34.59 ± 6.99
Table 4
b-carotene, Lycopene and Color (mm Pfund) of the different honey samples.
Samples b-carotene (mg/kg) Lycopene (mg/kg) Color (mm Pfund) color
Acacia, Germany 18.015 10.227 26.508 White
El-Sider Libya 13,070 7.394 49.905 Extra light Amber
Clover Egypt 26.604 9.702 20.281 White
Sider, Yemen 12.199 6.216 51.019 Light Amber
Pine, Germany 9.104 11.899 94.843 Amber
Sider, Saudi Arabia 11.255 4.752 92.588 Amber
Fig. 2. L*, a* and b* parameters for investigated honey samples G. = Germany,
EG = Egypt, Y = Yamen, L = Libya, KSA = Saudi Arabia.
I. Smetanska, S.S. Alharthi and K.A. Selim Journal of King Saud University – Science 33 (2021) 101447
6

honey. The results showed no significant differences between
clover, pine and KSA sider honey in the content of chlorogenic acid.
Among all the honey samples, acacia honey recorded the lowest
content of chlorogenic acid. Chlorogenic acid was detected in many
honey types such as Serbian honey (keckes et al., 2013), Lithuanian
honey (Baltrusaityte et al., 2007). and northeast Portugal honeys
(Feás et al., 2010). Similar results were also reported by
Trautvetter et al. (2009) for clover honey. The chromatogram of
the phenolic compounds in our samples indicated that cinnamic
acid and ferulic acid were not detected in acacia, clove and pine
honeys while p-coumaric acid was absent only in Libyan El-sider
honey. These results are agreed with that reported by Yao et al.
(2004) for the Australian honey. These phenolic acids were
detected in different types of honeys by some authors (Kassim et
al., 2010).
P-coumaric acid represented about 34.93 and 20.42% of the
total phenolic compounds in clover and pine honey respectively.
According to the results in Table 6, kaempferol and chrysin was
detected in all tested honey samples. Acacia honey was superior
to all of other investigated samples in its kaempferol and chrysin
content while EL-sider honey recorded the lowest kaempferol
and chrysin content. The differences between the honey types in
their phenolic compounds profile could be related to the floral
sources and the geographical origin.
0.0 5. 0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min
-5
0
5
10
15
20
25
30
35
40
45 mAU
5.329
7.241
10.392
10.789
13.951
17.274
18.856
20.374
21.495
27.395
32.629
G.=Germany, EG = Egypt, Y = Yamen, L = Libya, KSA = Saudi Arabia
Fig. 3. Results of HPLC chromatogram of phenolic acids and flavonoids detected in acacia honey (290 nm): (1) chlorogenic acid = 5.32 min, (2) caffeic acid = 10.39 min, (3) p-
coumaric acid = 13.95 min, (4). Kaempferol = 18.85 min, (5). Chrysin = 27.39, and binocambrin = 32.62 min.
Table 5
Yield of methanolic extract (mg/100 g), Total phenolic (mg/100 g), Total Flavonoids (mg/kg) and EC
50
of the different honey samples.
Parameters
samples
Yield of methanolic extract
(mg/100 g)
AEAC
(mg/100 g)
Total Flavonoids
(mg Catechin /kg)
Total phenolic
(mg GAE/100 g)
SC
50 mg/ml
Acacia, Germany 203 ± 5.02
b
12.368 ± 1.30
d
45.714 ± 2.03
d
21.457 ± 5.10 35.35
El-sider Libya 167 ± 3.58
c
16.375 ± 2.08
c
97.429 ± 2.85
c
29.252 ± 5.68
d
18.22
Clover Egypt 201 ± 8.71
b
11.001 ± 1.07
d
41.704 ± 1.58
d
18.574 ± 4.37
f
55.31
Sider, Yemen 115 ± 2.61
c
21.013 ± 1.15
b
103.143 ± 4.61
b
33.885 ± 5.25
c
10.61
Pine, Germany 211 ± 4.33
a
20.192 ± 1.24
b
106.002 ± 4.59
b
44.828 ± 6.89
b
9.94
Sider, Saudi Arabia 175 ± 3.62
c
27.983 ± 1.82
a
120.857 ± 5.25
a
53.314 ± 6.90
a
4.17
Table 6
Concentration of identified phenolic acid and flavonoids in tested honeys (a. u. 10
3
) of the total beaks area.
Honey Compounds Acacia, G. El-sider L. Clover, EG Sider, Y. Pine, G. Sider, KSA
Chlorogenic acid 11.328 6.625 32.817 28.123 34.069 33.995
p-coumaric acid 4.772 nd 34.939 8.936 20.422 14.448
Caffeic acid 7.878 20.929 11.994 25.021 10.956 21.431
Ferulic acid nd 24.158 nd 7.438 nd 9.272
Cinnamic acid nd 18.298 nd 6.074 nd nd
Chrysin 4.165 1.431 1.579 3.114 1.956 3.189
Kaempferol 17.986 2.205 3.057 11.312 6.329 5.284
Sinapic acid nd nd nd nd nd 6.868
Binocembrin 6.849 nd nd nd nd nd
Beta-phenyl acetic nd nd nd 4.269 nd nd
a.u. = arbitrary units, G = Germany, EG = Egypt, L = Libya, Y = Yemen, KSA = Saudia Arabia, nd = not identified.
I. Smetanska, S.S. Alharthi and K.A. Selim Journal of King Saud University – Science 33 (2021) 101447
7

3.9. Antioxidant activity of honeys methanolic extracts
The antioxidant activity of the phenolic extracts of the different
honey samples measured using DPPH assy. From the results in Fig.
4, it could conclude that all the tested samples exhibited good anti-
oxidant activity however, different honey revealed varying degrees
of antioxidant capacity.
Six different concentrations of each honey extract were used in
this assay from 2.83 to 18.87 mg/ml solution and the results
showed that the scavenging activity was increased with increasing
the phenolic content in the solution. From the results in Table 5
and Fig. 4, the amount of antioxidant required to decrease the con-
centration of DPPH by 50% (EC
50
) values varied from 4.17 to
55.31 mg/ml.
KSA Sider honey showed the highest antioxidant activity and
lowest EC
50
(4.17 mg/ml) followed by pine honey with EC
50
of
(9.94 mg/ml) while clover honey exhibited the lowest antioxidant
activity with the highest EC
50
of (55.31 mg/ml). The high radical
scavenging activity of KSA sider honey and pine honey could be
due to their high phenolic and flavonoid contends.
All of our honey samples showed EC
50
lower that those reported
by other authors for different honey types including Indian honey,
Malaysian honey and Portuguese honey (Das et al., 2013;
Moniruzzaman et al., 2013; Ferreira et al., 2009).
Our results for acacia honey agreed wit that reported by
Bertoncelj et al. (2007) for the Slovenian acacia honey and lime
honey. The results indicated that there was a significant positive
correlation between the color of the honey and its antioxidant
capacity in which the darker honey had the higher antioxidant
activity. It could be summarized that the antioxidant activity of
the honeys is not only consequent to the phenolic and flavonoids
content but also their content of carotenoids and vitamin E and C.
4. Conclusion
Our results proved that there is a positive correlation between
the color of honey and its phenolic, flavonoids, carotenoids and
HMF content. All the investigated samples showed high antioxi-
dant activity. Saudi honey was superior to all other honey in its
content of total polyphenols and flavonoids. This difference could
be due to the difference in the geographical origin and the floral
sources.
Acknowledgment
This work was financial supported by Cultural Affairs and Mis-
sions Sector, Egypt and Taif University Researchers Supporting Pro-
ject number (TURSP-2020/90), Taif University, Taif, Saudi Arabia.
Our thank also for the beekeepers for providing the honey samples
used in this study.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
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