ArticlePDF Available

Abstract and Figures

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.
Content may be subject to copyright.
Original article
Physicochemical, antioxidant capacity and color analysis of six honeys
from different origin
Iryna Smetanska
, Salman S. Alharthi
, Khaled A. Selim
Department of Plant Food Processing, Agric. Faculty, Univ. of Applied Science, Weihenstephan-Triesdorf, Steingruberstraße 2, 91746 Weidenbach, Germany
Department of Chemistry, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
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
Phenolic content
Antioxidant activity
Color intensity
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
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 (
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).
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 (
Corresponding author.
E-mail address: (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
Contents lists available at ScienceDirect
Journal of King Saud University Science
journal homepage:
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,
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
) 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
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;
= 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
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
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
Sample 1 Acacia, Germany Robinia pseudoacacia L. Nurnberg,
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,
Sample 5 Pine Germany Pinus halepensis L. Pinus
Sample 6 Sider Saudi Arabia
Ziziphus Spina-christi L.South, Saudi
I. Smetanska, S.S. Alharthi and K.A. Selim Journal of King Saud University Science 33 (2021) 101447
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
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
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
0.53 to 0.67) and lower than the Indian and Brazilian honeys
for which the a
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
18.83 ± 1.02
4.10 ± 0.02
1.4863 67.57 ± 1.98
3.82 ± 0.61
3.69 ± 0.21
81.06 ± 1.52
EL-Sider Libya 0.504 ± 0.01
17.52 ± 0.85
4.87 ± 0.04
1.4935 69.29 ± 2.45
2.07 ± 0.08
11.28 ± 0.53
80.27 ± 2.05
Clover Egypt 0.559 ± 0.02
19.1 ± 0.94
3.26 ± 0.02
1.4908 66.81 ± 2.11
6.38 ± 0.57
10.94 ± 0.94
78.57 ± 1.61
Sider Yemen, 0.554 ± 0.00
16.81 ± 0.75
4.66 ± 0.05
1.4995 71.245 ± 2.81
2.15 ± 0.44
12.82 ± 1.03
81.64 ± 1.92
Pine, Germany 0.565 ± 0.01
19.30 ± 0.87
4.05 ± 0.03
1.4930 66.57 ± 2.03
2.17 ± 0.42
9.37 ± 0.58
78.71 ± 2.17
Sider, Saudi Arabia 0.543 ± 0.02
16.91 ± 0.72
4.91 ± 0.06
1.4990 70.95 ± 2.47
2.43 ± 0.37
22.47 ± 1.78
81.41 ± 2.35
* Rrefractive index, ** Total soluble solids
I. Smetanska, S.S. Alharthi and K.A. Selim Journal of King Saud University Science 33 (2021) 101447
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
010 20 30 40 50 min
(U4) U5
Norv al
Ile Phe Leu
010 20 30 40 50 min
(U4) U5
Norv al
Ile Phe Leu
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
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
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.,
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
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
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
45 mAU
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
of the different honey samples.
Yield of methanolic extract
(mg/100 g)
(mg/100 g)
Total Flavonoids
(mg Catechin /kg)
Total phenolic
(mg GAE/100 g)
50 mg/ml
Acacia, Germany 203 ± 5.02
12.368 ± 1.30
45.714 ± 2.03
21.457 ± 5.10 35.35
El-sider Libya 167 ± 3.58
16.375 ± 2.08
97.429 ± 2.85
29.252 ± 5.68
Clover Egypt 201 ± 8.71
11.001 ± 1.07
41.704 ± 1.58
18.574 ± 4.37
Sider, Yemen 115 ± 2.61
21.013 ± 1.15
103.143 ± 4.61
33.885 ± 5.25
Pine, Germany 211 ± 4.33
20.192 ± 1.24
106.002 ± 4.59
44.828 ± 6.89
Sider, Saudi Arabia 175 ± 3.62
27.983 ± 1.82
120.857 ± 5.25
53.314 ± 6.90
Table 6
Concentration of identified phenolic acid and flavonoids in tested honeys (a. u. 10
) 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
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
) values varied from 4.17 to
55.31 mg/ml.
KSA Sider honey showed the highest antioxidant activity and
lowest EC
(4.17 mg/ml) followed by pine honey with EC
(9.94 mg/ml) while clover honey exhibited the lowest antioxidant
activity with the highest EC
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
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
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.
Ahmida, M.S., Elwerfali, S., Agha, A., Elagori, M., Ahmida, N.S., 2013.
Physicochemical, Heavy Metals and Phenolic Compounds Analysis of Libyan
Honey Samples Collected from Benghazi during 2009–2010. Food Nutr. Sci. 4,
Alvarez-Suarez, J.M., GonzaLez-Parma, A.M., Santos-Buelga, C., Battino, M., 2010.
Antioxidant Characterization of Native Monofloral Cuban Honeys. J Agric Food
Chem 58, 9817–9824.
Alves, J.P.; Alves da, LAMC; Reis da, SSJ., Adriana, F., 2014. Color, phenolic and
flavonoid content, and antioxidant activity of honey from Roraima, Brazil. Food
Sci. Technol, vol.34, n.1, pp.69-73..
AOAC, 1990. Official methods of analysis. Association of official Analytical Chemists
Inc., Arlington, VA, USA.
Azeredo, L.D.C., Azeredo, M.A.A., De Souza, S.R., Dutra, V.M.L., 2003. Protein content
and physicochemical properties in honey samples of Apis Mellifera of different
floral origins. Food Chem. 80, 249–254.
Baltrusaityte, V., Venskutonis, P.R., Ceksteryt, V., 2007. Radical scavenging activity
of different floral origin honey and beebread phenolic extracts. Food Chem. 101,
Barros, L. et al., 2007. Effect of Lactarius piperatus fruiting body maturity stage on
antioxidant activity measured by several biochemical assays. Food and
Chemical Toxicology 45, 1731–1737.
Bertoncelj, J., Dobersek, U., Jamnik, M., Golob, T., 2007. Evaluation of the phenolic
content, antioxidant activity and colour of Slovenian honey. Food Chem 105,
Bogdanov, S. et al., 2004. Propolis: composition, health, medicine: a review. Bee
Product Sci., 1–40.
Boussaid, A., Chouaibi, M., Rezig, L., Hellal, R., Donsı, F., Ferrari, G., Hamdi, S., 2018.
Physicochemical and bioactive properties of six honey samples from various
floral origins from Tunisia. Arabian J. Chem. 11, 265–274.
Brand-Williams, W., Culivier, M.E., Berset, C., 1995. Use of a free radical method to
evaluate antioxidant activity. LWT –. Food Science and Technology 28 (1), 25–30.
Can, Z., Yildiz, O., Sahin, H., Turumtay, E.A., Silici, S., Kolayli, S., 2015. An
investigation of Turkish honeys: their physico-chemical properties,
antioxidant capacities and phenolic profiles. Food Chem. 180, 133–141.
Chirife, J., Zamora, M. C., Motto, A., 2006. The correlation between water activity and
% moisture in honey: Fundamental aspects and application to Argentine honeys.
Journal of Food Engineering, 72, 287–292.
Codex Alimentarius Commission Standards, 2001. CODEX STAN 12-1981, Rev.1
(1987), Rev.2.
Das, A., Mukherjee, A., Dhar, P., 2013. Characterization of Antioxidants and
Antioxidative Properties of Various Unifloral Honeys Procured From West
Bengal, India. J. Environ. Sci., Toxicol. Food Technol. 7 (3), 56–63.
Djebli, N., Mustafa, M.R., Keskin, M., Kolayli, S., 2020. Anti Ulcerogenic and
Cytoprotective Effects of Saharian (Sidr) Honey from Algeria. Comb. Chem.
High Throughput Screening.
Feás, X., Pires, J., Iglesias, A., Estevinho, M.L., 2010. Characterization of artisanal
honey produced on the Northwest of Portugal by melissopalynological and
physico-chemical data. Food Chem. Toxicol. 48, 3462–3470.
Ferreira, C.F.I., Aires, E., Barreira, J., Estevinho, M.L., 2009. Antioxidant activity of
Portuguese honey samples: Different contributions of the entire honey and
phenolic extract. Food Chem. 114 (4), 1438–1443.
Gomes, S., Dias, L.G., Moreira, L.L., Rodrigues, P., Estevinho, L., 2010.
Physicochemical, microbiological and antimicrobial properties of commercial
honeys from Portugal. Food Chem. Toxicol. 48, 544–548.
Gül, A., Pehlivan, T., 2018. Antioxidant activities of some monofloral honey types
produced across Turkey. Saudi J. Biol. Sci. 25, 1056–1065.
Islam, A.1, Khalil I., Islam N., Moniruzzaman M., Mottalib, A., Sulaiman, S. A., and
Gan, S.H., 2012. Physicochemical and antioxidant properties of Bangladeshi
honeys stored for more than one year. BMC Complementary and Alternative
Medicine 12:177..
Kassim, M., Achoui, M., Mustafa, M.R., Mohd, M.A., Yusoff, K.M., 2010. Ellagic acid,
phenolic acids, and flavonoids in Malaysian honey extracts demonstrate in vitro
antiinflammatory ctivity. Nutr. Res. 30, 650–659.
keckes, S. et al., 2013. The determination of phenolic profiles of Serbian
unifloralhoneys using ultra-high-performance liquid chromatography/high
resolutionaccurate mass spectrometry. Food Chem. 138, 32–40.
Khalil, M.I., Moniruzzaman, M., Boukraâ, L., Benhanifia, M., Islam, M.A., Islam, M.N.,
Sulaiman, S.A., Gan, S.H., 2012. Physicochemical and Antioxidant Properties of
Algerian Honey. Molecules 17 (9), 11199–11215.
Kolayli, S.E., Palabiyik, I., Atik, D.S., Keskin, M., Bozdeveci, A., Karaoglu, S.A., 2020.
Comparison of Antibacterial and Antifungal Effects of Different Varieties of
Honey and Propolis Samples. Acta Alimentaria 49 (4), 515–523.
Kowalski, S., Kopuncova, M., Ciesarova, Z., Kukurova, K., 2017. Free amino acids
profile of Polish and Slovak honeys based on LC–MS/MS method without the
prior derivatization. J. Food Sci Technol. 54 (11), 3716–3723.
Fig. 4. Antioxidant activity of different concentration of phenolic extracts from the
honey samples.
I. Smetanska, S.S. Alharthi and K.A. Selim Journal of King Saud University Science 33 (2021) 101447
Küçük, M. et al., 2007. Biological activities and chemical composition of three
honeys of different types from Anatolia. Food Chemistry 100, 526–534.
Kücük, M., Kolayli, S., Karaoglu, S., Ulusoy, E., Baltaci, C., Candan, F., 2011. Biological
activities and chemical composition of three honeys of different types from
Anatolia. Food Chem. 100, 526–534.
Louveaux, J., Maurizio, A., Vorwohl, G., 1978. Methods of Melissopalynology. Bee
World 59, 139–157.
Marghitas, L.A., Dezmirean, D.S., Pocol, C.B., Ilea, M., Bobis, O., Gergen, I., 2010. The
development of a biochemical profile of acacia honey by identifying
biochemical determinants of its quality. Not Bot Hort Agrobot Cluj 38 (2), 84–
Meda, A., Lamien, C.E., Romito, M., Millogo, J., Nacoulma, O.G., 2005. Determination
of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as
well as their radical scavenging activity. Food Chem 91, 571–577.
Moniruzzaman, M., Sulaiman, A.S., Khalil, I.M., Gan, H.S., 2013. Evaluation of
physicochemical and antioxidant properties of sourwood and other Malaysian
honeys: a comparison with manuka honey. Chem. Cent. J. 7, 138.
Noor, A., Mahmud, M., Ahmad, A., Arfah, R.A., 2019. Amino acids
characterization of forest honeys from some area of South Sulawesi. The
3rd International Conference on Science, Journal of Physics: Conference
Series, 1341, 1-8..
Pauliuc, D.; Dranca, F., and Oroian, M., 2020. Antioxidant Activity, Total Phenolic
Content, Individual Phenolics and Physicochemical Parameters Suitability for
Romanian Honey Authentication Foods 2020, 9, 306;.
National Honey Board: Honey and Bees; 2003.
Pawlowska, M., Armstrong, D.W., 1994. Evaluation of enantiomeric purity of
selected amino acids in honey. Chirality 6, 270–276.
Saxena, S., Gautam, S., Sharma, A., 2010. Physical, biochemical and antioxidant
properties of some Indian honeys. Food Chem. 118 (2), 391–397.
Silici, S., Sagdic, O., Ekici, L., 2010. Total phenolic content, antiradical, antioxidant and
antimicrobial activities of Rhododendron honeys. Food Chem. 121, 238–243.
Silva, T.M.S., Santos, F.A.R., Evangelista-Rodrigues, A., Silva, E.M.S., Silva, G.S., Novais,
J.S., Santos, F.A.R., Camara, C.A., 2013. Phenolic compounds,
melissopalynological, physicochemical analysis and antioxidant activity of
jandaıra (Melipona subnitida) honey. J. Food Compos. Anal. 29, 10–18.
Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M., 1999. Analysis of total phenols
and other oxidation substrates and antioxidants by means of Folin–Ciocalteu
reagent. Methods in Enzymology 299, 152–178.
Sun, Z., Zhao, L., Chenga, N., Xuec, X., Wuc, L., Zhenga, J., Cao, W., 2017. Identification
of botanical origin of Chinese unifloral honeys by free amino acid profiles and
hemometric methods. J. Pharm. Anal. 7, 317–323.
Trautvetter, S., Koelling-Speer, I., Speer, K., 2009. Confirmation of phenolic acids and
flavonoids in honeys by UPLC-MS. Apidologie 40, 140–150.
White, J.W., 1984. Instrumental color classification of honey: collaborative study. J.
Assoc. Off. Anal. Chem. 67, 1129–1131.
Yao, L., Jiang, Y., Singanusong, R., D’Arcy, B., Datta, N., Caffin, N., 2004. Flavonoid in
Australian Malaleuca, Guioa, Lophostemon, Banksia and Helianthus honeys and
their potential for floral authentication. Food Res. Int. 37 (2), 166–174.
I. Smetanska, S.S. Alharthi and K.A. Selim Journal of King Saud University Science 33 (2021) 101447
... The main compounds of honey are sugars, and therefore one of its properties is to be hygroscopic. A high moisture content of honey can cause fermentation and spoilage, and these processes negatively change the physicochemical properties and undoubtedly lower the quality and price, shortening the shelf life of the product [9,10,17,21,49,52]. The moisture content of honey samples can be determined by measuring the refractive index. ...
... In its composition, there are natural compounds that yield strong antioxidant properties, such as the polyphenolic compounds (phenolic acids, catechins, flavonoids, etc.) [27,71]. These compounds qualitatively and quantitatively varied, and are directly linked with the rich flora, the environment, and the area around the beehive, due to their plant-honey-nectar traceability [13,19,43,44,49,56,72,73]. The chestnut honey sample had the highest average concentration of phenols of 73.2 mg GAE/100 g (Table 3). ...
... Rapeseed honey (RP3) of dark color (light amber) also had a high content of ash and antioxidants. The high content of polyphenolic compounds, pollen, pigments (carotenoids and flavonoid), and minerals present in honey can contribute to the appearance of a dark color of the honey [8,49,73]. In this study, comparable values of phenols concentrations in the acacia honey sample (A3) and rapeseed honey sample (RP3) were observed, but the flavonoid content was 3.3 times higher in the RP3 sample compared to sample A3, confirming the conclusions of several studies of a strong positive correlation between color and flavonoid content. ...
Full-text available
The objective of this research was to evaluate some quality-defining physicochemical parameters (moisture, specific gravity, pH, free acidity, ash, electrical conductivity, total phenols, and total flavonoids content, K, Ca, Mg, Na, and P) of seven Romanian monofloral honeys (linden, acacia, rapeseed, sunflower, mint, raspberry, and chestnut) collected in 2017. The investigated quality parameters are mainly within the recommended limits set by standards for honey. Sample analyses indicate the presence of antioxidants, such as TPC (17.9–73.2 mg GAE/100 g) and TFC (0.84–4.81 mg QE/100 g), and high amounts of K (101–1462 mg kg−1), Ca (58.3–167.5 mg kg−1), Mg (24.8–330.6 mg kg−1), Na (94.5–233.3 mg kg−1), and P (34.1–137.2 mg kg−1). The Pearson’s correlations between some parameters (such as color/TFC, color/Mg, color/P, EC/Ash, mm Pfund/TFC, TPC/TFC, K/Ash, P/Mg), together with PCA, HCA, and ANOVA statistics, highlight three main factors that explain the variability in the dataset and could be attributed to stability, mineral, and color/antioxidant contributions. FTIR spectra confirm the authenticity of all the monofloral honeys. The results and data processing confirm the influence of environmental elements (soil, water, air) on the honey composition and highlight the quality of honey, as a complete food and a therapeutic product.
... In addition, Smetanska et al. analyzed the pine honey from Germany in a study evaluating authentic honeys from various countries and found the TPC value of 44.828±6.89 mg GAE/100 g. [32] This value is lower than the average data in the current study. All of these results indicate that Turkish pine honey has a significantly high TPC capacity. ...
... [33] In a different study, the TFC values of the studied pine honey samples were different than those of German pine honey (106.002±4.59 mg catechin/kg), [32] which could be explained by differences in the use unit and expression standards. Also, that situation should be evaluated honey. ...
... Karabagias et al. [11] and Smetanska et al. [32] in terms of the number of phenolic compounds analyzed by HPLC. Although protocatechuic acid wasn't analyzed in those studies, it gave some information about the general phenolic characteristics of pine honey including phenolic acid and flavonoid types. ...
Full-text available
The purpose of this research was to identify the biological activity of Turkish pine honeys and the most abundant phenolic compound(s) to guide future studies to be done about this type of honey. Moreover, this will provide the chance to introduce Turkish pine honeys by elucidating a distinct parameter compared to other forms of honey reported in the literature. To this end, microscopic examination based on the number of honeydew elements (NHE) and the number of total pollen (NTP) grains was performed on 17 samples of pine honeys from the Aegean region of Turkey. Then 18 commonly‐occurring phenolic standards were used in high‐performance liquid chromatography (HPLC) to examine the samples. Each sample was tested for individual phenolic compounds, as well as total phenolic content (TPC), total flavonoid content (TFC), and ferric reducing antioxidant power (FRAP). As shown by the numerical data, the values for NHE and NTP were 6600–144000 and 3000–75000, respectively. In addition, TPC, TFC, and FRAP values were 53.000 mg GAE/100 g fresh sample, 1.944 mg QE/100 g fresh sample, and 3.455 µmol FeSO4/g fresh sample, respectively. Protocatechuic acid predominated with a range of 197.841–780.227 µg phenolic /100 g sample in all samples. According to the general mean for values detected in the samples, p‐hydroxybenzoic acid and benzoic acid were the second and third markers for Turkish pine honey samples. It is believed that these results will help efforts to set standards for pine honey on a national and global scale.
... The moisture content of honey samples studied in other research publications [2,29,[37][38][39] ranged between 14.45% and 22.8%; there were some honey samples reported with moisture contents exceeding the value of 20% established by the legislation (Table 4). A low content of moisture provides quality assurance for a long time period; high moisture content is a favorable condition for the occurrence of the fermentation phenomenon, thus leading to changes in both sensory and physicochemical properties, and thus, to the decrease in the quality of the bee product [12,34]. [39] Milek 3 ) were also found in rapeseed/acacia samples. ...
... Honey pH and acidity are correlated with the internal amount of microorganisms, enzymatic activity and the presence of organic acids. A low pH indicates a good environment that inhibits microorganism growth, while the free acidity level is a biochemical marker for honey samples freshness [34,51]. Similar values of free acidity were found among Serbian acacia honey samples [47]; these were the lowest values when compared to other types of honey ( Table 4). ...
... Honey can be considered a healing food due to the antioxidant role conferred mainly by phenolic compounds, such as phenolic acids and flavonoids. The amount of antioxidant compounds depends mainly on the floral source and on the geographical origin [34,35]. The highest amplitude of variation for the phenolic content was found in the linden honey (8.99 mg GAE/100 g of phenolic content between the analytical limits). ...
Full-text available
Since ancient times, honey has been appreciated not only for its sensorial traits, but also for the observed effects in rejuvenation and treatment against several bad health conditions, when used externally or internally, along with other beehive products, such as pollen, propolis and royal jelly. Today, it is known that such effects are generated by compounds bearing antimicrobial, anti-inflammatory, and antioxidative features (enzymes, polyphenolic molecules). The purpose of this study was to assess the total phenolic and flavonoid contents of 28 samples of Romanian raw monofloral honey (acacia; linden; rapeseed, sunflower and mint), and to establish their correlations with several qualitative parameters. Pearson’s test revealed a strong positive correlation between total phenolic content and total flavonoids (r = 0.76) and color intensity (r = 0.72). For total flavonoid content, correlations were strongly positive with color intensity (r = 0.81), ash content (r = 0.76) and electrical conductivity (r = 0.73). The relevant levels of polyphenols and flavonoids identified in the analyzed honey types demonstrate its antioxidant potential, with essential nutritional and sanogenic features in human nutrition.
... Without focusing on specific individual compounds, a study conducted by Smetanska et al. [82] revealed that the total phenolic content in Egyptian clover honey (T. alexandrinum L.), determined using the Folin Ciocalteau method, varied from 18.574 mg to 53.314 mg gallic acid equivalent/g honey. ...
... Similarly, the authors also investigated the total flavonoid content in Egyptian clover honey by a complexation reaction with AlCl 3 and found it to be 26.604 mg catechin/kg honey [82]. By extension, these unspecific quantifications can be seen as a broad assessment of the antioxidant activity of Egyptian clover honey. ...
... More specific bioactivity studies to date have mainly investigated the antioxidant and antimicrobial activities of honeys. In the case of red clover honeys, they have been found in two studies to exhibit antioxidant activity to different degrees [38,82]. In one, red clover honey showed moderate radical scavenging activity in the DPPH assay, where DPPH (2,2-diphenyl-1-picrylhydrazyl) is used as a stable free radical to capture the antiradical activity of honey [38]. ...
Full-text available
This review covers a comprehensive overview of the phytoconstituents and bioactivities reported to date for clover honeys produced from various Trifolium spp. against the backdrop of a more general discussion of the chemistry and bioactivity of these important agricultural species. While research into the phytochemical composition of various honeys and their associated bioactivities is growing, this review demonstrates that the literature to date has seen only a limited number of studies on clover honeys. Surprisingly, there appear to be no comparative data on the concentration of flavonoids in general or isoflavonoids specifically in different clover honeys, although the latter have been identified as a main group of bioactive compounds in red clover plants. Based on the findings of this review, the presence of phytoestrogenic isoflavonoids (e.g., formononetin, biochanin A, genistein, daidzein, glycitein) in clover plants and, by extension, in clover honeys should be further investigated, specifically of clover species outside the three popular perennial clovers (red, white and alsike clovers) to exploit new opportunities of potential benefit to both the pharmaceutical and apiculture industries.
... All samples had an HMF value below the recommended limit of 40 mg/kg. In the same line, Smetanska and coworkers investigated the physicochemical quality of organic honeys from different middle eastern areas and showed values below 40 mg/kg [48]. Accordingly, these samples had HMF contents of 10.94 mg/kg for Egyptian honey with a predominance of Trifoliumalexandrinum L. pollen, 12.82 mg/kg for Yamani (ZiziphusSpina-christi L.), and 22.47 mg/kg for Saudi Arabia (Ziziphus spina-christi L.). ...
Full-text available
This work aimed to characterize and compare the physicochemical, ascorbic acid, phe-nolic, and flavonoid compounds, as well as the antioxidant properties, pollen spectra, and sugar profiles of twenty-three organic honeys produced in the Middle Atlas of Morocco. As results, the pollen analysis showed 22 taxa and revealed the dominance of Ziziphus lotus pollens for all mon-ofloral honeys. The moisture content ranged from 15.9 to 19.0%, pH values werebetween 3.9 and 4.8, electrical conductivity varied from 100 to 581 µs/cm, ash content varied from 0.1 to 2.4%, and the invertase activity ranged from 3.5 to 36 U/kg. Moreover, hydroxymethylfurfural(HMF) varied from 1.2 to 13.5 mg/kg, which confirmed the freshness of our honey samples. For the sugar profiles, there were no significant differences between the examined groups of honeys (p > 0.05) for both fructose and glucose. Additionally, our study showed good antioxidant properties (total antioxi-dant activity ranged from 34.18 to 131.20 mg AAE/g; DPPH IC50 values ranged from 8.14 to 45.20 mg/mL; ABTS IC50 values ranged from 8.19 to 32.76 mg/mL) and high amounts of phenolic compounds ranging between 20.92 ± 0.03 and 155.89 ± 0.03 mg GAE/100 g, respectively; flavonoid compounds ranged from 5.52 to 20.69 mg QE/100 g, and ascorbic acid ranged from 8.01 to 23.26 mg/100 g. Overall, the proximate composition and the general characterization of organic monofloral and polyfloral honeys as sustainable and health-promising functional products may increase their commercial values, promote their marketability, and might have a significant impact on the basic circu-lar/sustainable economy as a solid lever for solidarity economic development, especially in the ru-ral/poor Moroccan communities. The investigated features may allow and support the incorporation of Moroccan organic honeys and their biovaluable ingredients in the nutraceutical and food industries for multiple purposes.
... 16,[28][29][30][31] and serine; but proline is the most abundant. 26,52 A study reported that after 24 h, 10 mM of L-phenylalanine reduced food intake and plasma ghrelin in control and obese rats (p < 0.01). 53 A total of 100 mM L-phenylalanine stimulated glucagon-like peptide-1 (GLP1) and gastrointestinal (GI) hormone peptide YY (PYY) (p < 0.01) in ileal organoid cell culture from C57BL/6 mice via the activation of a calciumsensing receptor (CaSR), a promiscuous amino-acid-sensing receptor expressed in the GI tract. ...
Full-text available
Honey has a long history of therapeutic properties for multiple diseases, including inflammation and oxidative stress. This review aimed to provide a better understanding and renewed interest in the potential role of honey in obesity control, obesity-related diseases treatment and weight management, with specific reference to its components and the effect of honey overall. There is compelling evidence that honey possesses the desired properties for this purpose, as seen in the in vitro, in silico, in vivo and clinical analyses discussed in this review. This review also highlights the components potentially responsible for the health benefits of honey. Honey and its components reduce blood sugar levels, improve insulin sensitivity and lipid metabolism by reducing triglycerides, and reduce total cholesterol and LDL levels while increasing HDL levels that prevent excessive weight gain and reduce the risk of obesity and its complications. Further controlled studies are necessary to validate the role of honey in the management of obesity, both as a preventive and as a therapeutic agent.
... Proline is the essential amino acid among other amino acids found in honey. It is used for the characterization of honey and the location of botanical origin and a criterion for determining honey quality [41]. ...
Full-text available
This research was aimed to investigate and evaluate the phytochemical, physicochemical, sensory, microbiological and pharmacological properties such as antioxidant, anticancer, anti elastase, anti-melanogenic and anti-tyrosinase activity in connection with health, safety, food and nutritional benefits of raw (RHs) and regular (ReHs) Nigerian honey. Eighteen raw honey (RHs) samples and twelve regular honey (ReHs) samples were collected from the Republic of Nigeria. Tannin content, total phenolic content, total flavonoid content, moisture, ash, pH, EC, free acidity, proline, diastase, HMF, invertase, glucose, fructose, and sucrose were the tested phytochemical and physicochemical parameters. Fiehe, Lund and Lugol qualitative test were performed to test the purity of honey samples. The results obtained from the research showed that the quality of honey samples studied were within the recommended standard (Codex). The existence of the research findings showed that RHs proved exceptional pharmacological (anticancer, antibacterial, anti-melanogenic and antioxidant) activities against the cell line (MCF-7). The highest microbial count was observed in RHs (5.5 × 0.47 × 102 cfu/100 g). There were no observable coliform growths in the samples. The findings in this current study showed that honey samples contain better consumable quality. In conclusion, honey can be potentially used as alternative treatments for controlling infectious diseases.
Honey crystallization is a common occurrence, while the underlying mechanisms are not clear. Here we evaluated natural crystallization and induced crystallization of honey. By using the method of microscopy, colorimetry, rheology, texture, and calorimetry to investigate the main physical and structural properties and compare with honey seed crystals-induced crystallization in six unifloral honey. The results showed honey transformed into a pseudoplastic fluid during natural crystallization, with the increase of L-value, firmness and cohesiveness and the fastest crystallization rate occurring at 14°C and water content of 16%. The effect of the addition of seed crystals in the honey crystallization can be summarized as the acceleration of crystallization rate (3 times) and the improvement of crystallization characteristics including uniform particles and reducing stratification. This study provides insight into honey crystallization theory and a resource for the further exploration and development of the new performance of honey products.
Full-text available
he present study was carried out to make new healthy synbiotic flavored fermented skim milk drinks (SFFSD) supplemented with either Doum (Hyphaene thebaica L.) or Carob (Ceratonia siliqua) fruits powder for nutritional, antioxidant and antimicrobial activities. The HPLC analysis showed higher phenolic and flavonoid content in Doum extract than that of Carob. The antimicrobial activity of the methanolic extracts of both Doum and Carob against foodborne pathogens showed that the most sensitive indicators were Bacillus cereus, C. albicans and S. aureus, followed by T. mentagrophyte and E. coli wherein, the diameter of clear zones was, 29, 27, 24, 23 and 13 mm, respectively. While no effect was noticed against Aspergillus flavus. On the other hand, the methanolic extract of Carob fruit exhibited only a weak antibacterial effect against B. cereus (20 mm). Six treatments of SFFSD were made using both Doum or Carob fruit powder as prebiotic and Lactobacillus paracasei as probiotic bacteria. The added levels from Doum were 2%, 4% and 6%, while that added from Carob were 5%, 10% and 15%. Adding Doum and Carob powder increased the containing of minerals; Ca, K, Mg and iron compared with control. Both viscosity and viability of L. paracasei for the SFFSD were increased with increasing the added levels from either Doum or Carob, compared with the control. Samples of SFFSD containing Carob powder, show superior sensory for all parameters and total score points during storage period, especially at a level of 10%. It seems evident that samples supplemented with Doum powder were also acceptable.
Full-text available
Our aim in this study was to outline phenolic and color characteristic that characterize the labeled unifloral resin spurge (Euphorbia resinifera) honey. With respect to phenolic composition, 17 phenolic compounds have been analyzed in the 29 honey samples. The proposed markers (syringic acid, ethyl gallate, m-coumaric acid and naringenin) might help to the enhancement of this honey type and thus, guarantee its commercial value. The color characterization by diffuse reflectance spectrophotometry revealed typical values of light amber honey (lightness ranged from 36 to 70 units, and chroma from 18 to 30 units). On the other hand, many correlations between the color attributes and phenolic acids, total phenolic compounds, caffeic acid, p- and m-coumaric acids and hesperidin have been demonstrated, also, correlation between phenols, color parameters and percentage of pollen of E. resinifera has been found. This study is one of the rare researches which have correlated the CIELAB color parameters with the individual phenolic acids and flavonoid compounds within the same unifloral honey.
Full-text available
Honey is the most important bee product. There are many secondary metabolites, carbohydrates, enzymes, and vitamins in honey, thus, honey has antimicrobial activity. In this study, in vitro antimicrobial activity of forty-two honey and eight propolis ethanolic extracts (PEE) were investigated against 16 microorganisms. Total phenolic content ranged between 20.00–124.10 mg GAE/100 g and 103–232 mg GAE/g for honey and raw propolis samples, respectively. Pine and oak honeydew honeys had higher antimicrobial activity than four different grades of Manuka Honeys up to 18 mm minimum inhibition zone diameters. The ethanolic propolis extracts showed much higher antimicrobial activity than the honey samples. Fungi species were inhibited by the propolis samples. Helicobacter pylorii ( H. pylorii ) was the most sensitive, whereas Streptococcus agalactiae was the most resistant bacteria among the studied microorganisms. Brazilian and Zonguldak propolis had the closest antimicrobial activity to ampicillin, streptomycin, and fluconazole. It can be concluded that both honey and propolis could be used in preservative and complementary medicine.
Full-text available
The present study aimed to evaluate the physicochemical characteristics of honey (raspberry, mint, rape, sunflower, thyme and polyfloral) produced in Romania. The honey samples were from the 2017 to 2018 harvest and were subjected to melissopalynological analysis, alongside the determination of the following physicochemical parameters: moisture content, pH, free acidity, electrical conductivity (EC), hydroxymethylfurfural (HMF) content, color, total polyphenols content (TPC), flavonoids content (FC), DPPH radical scavenging activity, phenolic acids, flavonols, sugars and organic acids in order to evaluate the usefulness of this parameters for the classification of honey according to botanical origin. The results of the melissopalynological analysis revealed that five types of honey samples had a percentage of pollen grains above the minimum of 45%, which was required in order to classify the samples as monofloral honey. The total polyphenols content reached the maximum value in the case of dark honey such as mint honey, followed by raspberry, thyme and polifloral honey. Fructose, glucose, maltose, sucrose, turanose, trehalose, melesitose, and raffinose were identified and quantified in all samples. Gluconic acid was the main organic acid in the composition of all honey samples. Principal component analysis (PCA) confirmed the possibility of the botanical authentication of honey based on these physicochemical parameters.
Full-text available
This study was conducted with the aim of determining the chemical, biochemical properties, and antimicrobial capabilities of some of the monofloral honeys produced in Turkey. In this study, 23 different monofloral honey samples were obtained from diverse geographical regions of Turkey. Floral origin of the honey samples was determined by melissopalinological analyses. Additionally, antioxidant properties were determined. To determine the antioxidant properties of honey samples, four test methods of total phenolic content, DPPH, iron reduction power and β-carotene linoleic acid emulsion method were used. As a result of the antioxidant activity analysis among the honey samples, rhododendron and parsley honey showed most prominent results in terms of the amount of phenolic compounds and antioxidant activity. On the other hand, acacia and citrus honey samples showed least antioxidant activity. A positive correlation was determined between four methods. Differences between antioxidant activities of honey samples were significantly found (P < 0.01).
Full-text available
The amino acid contents of five floral sources Chinese honeys (jujube, rape, chaste, acacia, lungan) were measured using reversed phase high performance liquid chromatography (RP-HPLC). The results showed that proline was the main amino acid in most of the analyzed samples. Phenylalanine was present at highest content in chaste honey samples, and the total amino acid contents of chaste honeys were also significantly higher than those of other honey samples. Based on the amino acid contents, honey samples were classified using chemometric methods (cluster analysis (CA), principal component analysis (PCA), and discriminant analysis (DA)). According to the CA results chaste honeys could be separated from other honeys, while the remaining samples were correctly grouped together when the chaste honey data were excluded. By using DA, the overall correct classification rates reached 100%. All of these results revealed that amino acid contents could potentially be used as indicators to identify the botanical origin of unifloral honeys.
Aim and Objective: This study aimed at investigating the gastro-protective effects of Algerian Sahara (Sidr) honey from Apis mellifera intermissa against HCl/Ethanol-induced gastric ulcers in rats. Material and Methods: Total phenolic content, antioxidant activity and phenolic compounds were determined. Than, Three groups of rats (control, HCl/ Ethanol induced ulcer, and orally administered honey) were used for the determination of gastro protective effect of Sidr honey. Results: Total phenolic content, total flavonoid content, and DPPH activity of honey sample was determined as 47.35±3.35 mg GAE/ 100 g, 2.13±0.17 mg QE/ 100 g, and 229.24±0.02 mg/mL, respectively. Oral pretreatment of rats with honey (1.2 g/Kg body weight orally at interval of 2 days) protected gastric mucosa against HCl/Ethanol induced damage by decreasing ulcer score, the volume and acidity of gastric juice and increasing pH. Conclusion: These results were confirmed by the histological assessment which demonstrated a significant gastro protective activity of Saharian (Sidr) honey against HCl/Ethanol-induced stomach ulcer. Plasma tumor necrosis factor-!, IL-6 and PGE2were also measured. Sahara honeys significantly decreased the plasma TNF-!, PGE2, and IL-6 concentrations. Keywords: Gastric ulcer, Sahara honey, HCl/Ethanol, ulcer score, TNF, IL-6, PGE2
A collaborative study was carried out to test the use of the Lovibond 2000 honey color comparator. Fourteen collaborators classified 6 honeys in the test. Results were generally favorable and the method has been adopted official first action.
LC–MS/MS method was applied for determination of free amino acids in honey without derivatisation steps. Twenty free amino acids including aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, glycine, leucine, histidine, hydroxyproline, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine and ornithine were analyzed in thirty honey samples from Poland and Slovakia. The analysis covered: acacia, lime, rape, multifloral and forest types of honey. Applied method was characterized to had good sensitivity with limit of detection ranged from 3.0 ng/cm³ for valine to 13.0 ng/cm³ for hydroxyproline. Average content of proline (main amino acid component in honey) ranged from 151.46 μg/g (rape honey from Slovakia) to 389.66 μg/g for forest honey (honeydew honey) from Poland. In analyzed honeys large quantities of glutamine, glutamic acid, lysine, phenylalanine, asparagine, alanine, and valine were also found.