Saudi Journal of Biological Sciences 27 (2020) 2902–2911

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Saudi Journal of Biological Sciences

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Original article

Phytochemicals, mineral contents, antioxidants, and antimicrobial

activities of propolis produced by Brunei stingless bees Geniotrigona
thoracica, Heterotrigona itama, and Tetrigona binghami
Nurul Aliah Abdullah a, Nadzirah Zullkiflee a, Siti Nurul Zahirah Zaini a, Hussein Taha b, Fatimah Hashim c,
Anwar Usman a,⇑
a
Department of Chemistry, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong BE1410, Brunei
b
Environmental and Life Sciences Programme, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong BE1410, Brunei
c
Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: The present study focused on the evaluation of phytochemical properties, essential mineral elements, and
Received 9 March 2020 heavy metals contained in raw propolis produced by stingless bees Geniotrigona thoracica, Heterotrigona
Revised 20 July 2020 itama, and Tetrigona binghami found in the same ecological conditions and environment in Brunei
Accepted 6 September 2020
Darussalam. The results indicated that propolis of the three stingless bee species mainly consisted of
Available online 12 September 2020
lipids (45.60–47.86%) and very low carbohydrate (0.17–0.48%) and protein contents (0.18–1.18%). The
propolis was rich in mineral elements, thus good sources of minerals, while they contained low concen-
Keywords:
trations of all heavy metals. Propolis of the different bee species could be distinguished based on their
Stingless bee propolis
Phytochemicals
mineral compositions. The vibrational and absorption spectra suggested that propolis contains p-
Mineral elements conjugated aliphatic and aromatic compounds as well as aromatic acids having amine, ester, carbonyl,
Antioxidant alkyl, and hydroxyl functional groups which might be attributed to the presence of phenolic and flavo-
Antibacterial noid compounds. The antioxidant capacity of the propolis, based on radical scavenging activity of their
ethanol extract, was in line with their total phenolic content. The ethanol extract of the propolis also
showed antimicrobial activities against four bacterial strains (Bacillus subtilis, Staphylococcus aureus,
Escherichia coli, and Pseudomonas aeruginosa). The propolis showed slightly higher antibacterial activity
against Gram-positive (B. subtilis and S. aureus) bacteria, indicating that the antimicrobial active com-
pounds could be associated with flavonoids, which were quantified to be approximately comparable in
all the propolis.
Ó 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction been the focus of sociality, phylogeny, and colony evolutionary

study (Rasmussen and Camargo, 2008; Lichtenberg et al., 2010;
Stingless bees (Hymenoptera; Apidae) with more than 500 spe- Rasmussen et al., 2017). The stingless bees are also of interest,
cies within the tribe Meliponini are found in tropical dry and owing to their honey, wax, and propolis (Ajibola et al., 2012).
humid forests and some subtropical regions throughout the world Stingless bee honey is consumed and utilized in health care
(Michener, 2013). The stingless bees can be classified into 2 genera, products since ancient time to the modern era (Meo et al., 2017),
namely, the Melipona and the Trigona. They are ecologically active due to its important biologically active compounds, such as
and play an important role in the forest ecosystem, and they have polyphenols, carotenoids, minerals, proteins, free amino acids,
and vitamins (Alvarez-Suarez et al., 2013). Stingless beeswax
⇑ Corresponding author. which is mainly comprised by fats is utilized in cosmetics, includ-
E-mail address: [email protected] (A. Usman). ing body lotions, facial creams, and lip balms, as well as in pharma-
Peer review under responsibility of King Saud University. ceutical and health products and coating materials for tablets and
capsules. Besides, beeswax is also applied in the manufacture of
textiles, polishes, and candles. Stingless bee propolis is a resinous
adhesive, composed mainly by plant resins, pollen, and bud
Production and hosting by Elsevier excretions, which are collected from various trees and utilized by

https://doi.org/10.1016/j.sjbs.2020.09.014
1319-562X/Ó 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
N.A. Abdullah et al. Saudi Journal of Biological Sciences 27 (2020) 2902–2911

stingless bees to coat, seal, and protect their hives from predators Thailand (Sanpa et al., 2015), Turkey (Keskin et al., 2001), and Viet-
as well as to prevent them from bacteria, viruses, or parasites nam (Georgieva et al., 2019). Based on the literature, the different
(Castaldo and Capasso, 2002; Massaro et al., 2014; Bankova et al., species, botanical origins, and surrounding environment are gener-
2014). Propolis has been found to have antiseptic effects ally pointed out to be the reasons for the different phytochemical
(Marcucci, 1995; Righi et al., 2011; Kakino et al., 2012), and it properties and biological activities of propolis. To the best of our
has long been traditionally used as medicines and remedies knowledge, reports on a comparative study of phytochemical prop-
(Carvalho et al., 2015). Several studies have revealed that propolis erties, chemical compositions, and biological activities of propolis
has antimicrobial (Velikova et al., 2000; Barrientos et al., 2013), from different species of stingless bees are limited (Silici and
antioxidant (Sawaya, 2009; Guimarães et al., 2012; Campos et al., Kutluca, 2005; Przybyłek and Karpiński, 2019). Driven by the
2014), anti-inflammatory (Barbarić et al., 2011; Cavendish et al., importance of obtaining as much information and comparative
2015), and antifungal properties (Viuda-Martos et al., 2008). The properties of the stingless bee propolis as possible, in this study,
biological activities of propolis have been attributed to its flavo- the phytochemical properties and mineral contents in propolis of
noid, phenolic, diterpenic acid, and aromatic acid contents three stingless bees, G. thoracica, H itama, and T. binghami, found
(Coneac et al., 2008), and the chemical composition of the stingless in the same ecological conditions and environment in Brunei
bee honey, wax, and propolis varied depending on the bee species, Darussalam were investigated. The antioxidant and antibacterial
botanical origin, environmental, and storage conditions (Gheldof activities of the ethanol extract of the propolis were also evaluated.
et al., 2002; Alvarez-Suarez et al., 2013; Biluca et al., 2016; Lim
et al., 2019). 2. Materials and methods
Different countries and regions may have their own specific and
unique stingless bee species, which adapted to the local ecosystem. 2.1. Chemicals, reagents, and standard solutions
In Borneo, including Brunei, Kalimantan, Sabah, and Sarawak, for
instance, there are at least 50 species of stingless bees, and their All chemicals and reagents were of analytical grade and were
genetic and behavioral characteristics have been well documented used without further purification. 2,2-Diphenyl-1-picrylhydrazyl
(Rasmussen and Cameron, 2007; Silvestre et al., 2008). Among (DPPH) radical, ascorbic acid, boric acid, gallic acid, D-glucose, phe-
them, the stingless bee species Geniotrigona thoracica, Heterotrig- nol, hydrochloric acid (HCl), nitric acid (HNO3), sodium carbonate
ona itama, and Tetrigona binghami (Fig. 1) are commonly domesti- (Na2SO4), aluminum chloride (AlCl3), sulfuric acid (H2SO4), and
cated, because their log hives can be found and collected from sodium hydroxide (NaOH) were purchased from Sigma-Aldrich
natural forests, and they can be easily cultivated and maintained (USA), while ethanol (96%) and multielement standard solution
in a suburban area. A number of studies focusing on the physico- (ICP TraceCERT) were purchased from Merck (Darmstadt-
chemical property, biological activity against Gram-positive Germany). Ultrapure water was used throughout the sample
(Mycobacterium smegmatis, Bacillus subtilis, and Staphylococcus aur- preparations and measurements. Standard solutions of each gallic
eus) and Gram-negative (Escherichia coli and Pseudomonas aerugi- acid, ascorbic acid, and quercetin were prepared in ethanol.
nosa) bacteria, and toxicity of stingless bee propolis from Borneo
have been scarcely reported, compared with those from Brazil
2.2. Propolis samples
(Sforcin et al., 2000), Bulgaria (Bankova et al., 2014), India
(Kasote et al., 2019), Indonesia (Trusheva et al., 2011), Malaysia
The propolis of three stingless bee species, G. thoracica, H. itama,
(Ibrahim et al., 2016), Mexico (Guzmán-Gutiérrez et al., 2018),
and T. binghami, were collected from Tasbee Meliponiculture Farm

Fig. 1. The images of stingless bees (A) Geniotrigona thoracica, (B) Heterotrigona itama, and (C) Tetrigona binghami along with their respective propolis.

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in Tutong District, Brunei Darussalam (Fig. 2). It is noteworthy to raw propolis in distilled water, followed by sonication. Once
mention that this stingless bee farm is located in a suburban area cooled, the absorbance of the solution was measured at 490 nm
far from high industrial or agricultural activities. The propolis using a UV-Vis spectrophotometer (Shimadzu UV-1900, Japan).
was scrapped from their beehives during the nectar flow season The analysis was performed in duplicates. A linear regression plot
in July-September 2019. The propolis collection did not disrupt of absorbance as a function of the concentration of D-glucose was
any endangered or protected species. constructed and it was used as a calibration curve to calculate the
Ethanolic extract of the propolis was obtained by macerating total carbohydrates contained in the raw propolis, where the per-
the dried raw propolis in 96% ethanol (Abdullah et al., 2019). The centage of total carbohydrates was calculated as the ratio between
suspension was agitated at 150 rpm at 37 °C for 18 h. The suspen- the concentration of D-glucose and mass of raw propolis.
sion was then passed through vacuum filtration to remove partic- The acid hydrolysis and semi-continuous solvent extraction
ulates, and the supernatant was rotary evaporated until were carried out according to the procedure described by Nielsen
approximately half solvent volume reduction, followed by oven (2010). Here, 0.1 g of the dried raw propolis was weighed in a glass
drying at 40 °C. capsule fitted with filter paper. The capsule was then placed in the
boiling stand before subjected to acid hydrolysis. The hydrolysis
beaker (SoxCapTM, Sweden) was filled up with 800 mL of 3 M
2.3. Analytical characterizations HCl. The boiling stand along with the capsule was lowered into
the beaker, and the solution was allowed to boil gently for 1 h.
The phytochemical properties, including total carbohydrates, The capsule was then transferred to a drying stand and left to
lipids, and crude proteins of propolis of stingless bees, G. thoracica, dry overnight in a convection oven at 60 °C. The dried sample
H. itama, and T. binghami, were determined using the phenol–sul- was then transferred into a paper thimble and it was subjected
furic acid, the acid hydrolysis and semi-continuous solvent extrac- to extraction using Soxhlet (SoxtecTM, Sweden) with petroleum
tion, and Kjeldahl method, respectively, while the mineral contents ether as extraction solvent. The extracted sample was allowed to
in the propolis were analyzed using inductively coupled plasma- dry in an oven at 100 °C for 30 min, and it was put in a desiccator
optical emission spectrometry (ICP-OES) analyzer (iCAP 7200, before weighing. The percentage of lipids was then calculated
Thermo Fisher Scientific) with the detection limit being based on the ratio between the extracted lipids and mass of raw
0.1 lg kg1. propolis.
The phenol-sulfuric acid method was performed according to The protein analysis is divided into three stages; e.g. digestion,
the procedures reported by Albalasmeh et al. (2013) with some distillation, and titration, according to the procedures reported by
modifications. In this method, D-glucose was used as a standard, Bradstreet (1965) and Owusu-Apenten (2002) with some modifica-
where 1 mL of six different concentrations of D-glucose (0, 20, tions. Here, 0.1 g of the raw propolis was first subjected to diges-
40, 60, 80, and 100 mg L–1) was mixed with 50 mL of 80% phenol tion with H2SO4 and Kjeldahl tablet (Gerhard KjeldathermÒ,
in water, followed by addition 5 mL of 97% H2SO4. The solution Germany) at 200 °C for 1 h. The solutions were then allowed to cool
mixture was left to cool at room temperature. The same procedure and filtered, followed by neutralization using NaOH and then dis-
was repeated for homogenous suspensions of raw propolis (ap- tillation using 1% boric acid solution. The protein content was cal-
proximately 100 mg L–1), which were prepared by soaking the culated based on the spent borate ions, which was determined by
titration.
The mineral elements, including aluminium (Al), calcium (Ca),
cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe),
potassium (K), magnesium (Mg), manganese (Mn), sodium (Na),
nickel (Ni), lead (Pb), zinc (Zn), and arsenic (As), were determined
according to the procedure described by González-Martín et al.
(2015) with some modifications. Here, 1 g of the dried raw propolis
was ground and then digested using concentrated HNO3 followed
by 2.5 mL of 37% HCl. The sample was cooled to room temperature
and was diluted with ultrapure water. The calibration was per-
formed with ICP-multielement standard solutions in the range of
10–200 lg kg1.

2.4. Scanning electron microscopy, UV-vis absorption and Fourier-

transform infrared spectroscopy (FTIR) spectroscopy

The morphological characteristics of the propolis were investi-

gated using a scanning electron microscope (SEM; JEOL JSM-
6490LA, Japan) operating at 50 kV. The SEM images with a magni-
fication of 700 were recorded. The absorption spectra of ethano-
lic extract of propolis of the different stingless bees were measured
using UV-Vis spectrophotometer (Shimadzu UV-1900, Japan). The
spectra were recorded in the range of 200–500 nm. The vibrational
spectra of the propolis were analyzed using FTIR spectrophotome-
ter (Shimadzu Prestige-21, Japan). Approximately 2 mg of the raw
propolis was added into 200 mg pre-heated KBr powder. The mix-
ture was pressurized using the SPECAC hydraulic pellet press, and
Fig. 2. Borneo island, indicating Brunei map and the location of Tasbee Melipon-
the pellet was then subjected to the measurement. The vibrational
iculture Farm (locality; latitude 4°490 02.000 N, longitude 114°450 48.200 E) where the spectrum was recorded in the range of 400–4000 cm1 with a res-
propolis of stingless bees G. thoracica, H. itama, and T. binghami was collected. olution of 2 cm1.
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2.5. Antioxidant assays (Escherichia coli ATCC-11775 and Pseudomonas aeruginosa ATCC-
27853), which were cultured separately for 24 h at 37 °C in Nutri-
The antioxidant capacity of ethanolic extract of propolis of the ent Broth (Merck) and then diluted 10 fold, equivalent to 0.5
different stingless bees was determined using the 2,2-diphenyl- McFarland standard. Similarly, discs containing streptomycin or
1-picrylhydrazyl (DPPH) free radical scavenging assay according rifampicin at 2 g L–1 were used as positive controls (reference stan-
to reported procedures (Moreira et al., 2008). In this measurement, dards) and discs containing water were used as a negative control.
solutions with various concentrations of the ethanolic extract were The Petri dishes were then placed in an incubator at 37 °C for 24 h
prepared, and 0.5 mL of each solution was then mixed with 3.5 mL before the diameter of inhibition zone as defined by the bacterial
of DPPH solution (50 mg L–1) in ethanol. The mixture was vigor- growth inhibition was measured. The results were expressed as
ously vortexed and then kept at room temperature for 30 min in average diameter ± standard deviation of 3 to 4 replicates for the
the dark. The decrease of DPPH radical was monitored by measur- propolis samples and of 8 to 9 replicates for the controls.
ing the absorbance of the mixture at 517 nm using an ELISA micro-
plate reader (Biobase EL-10A, China) with ethanol acting as a blank. 2.8. Data analysis
The IC50 was defined as the concentration of the ethanolic extract
of propolis to scavenge 50% initial DPPH radical, as reflected by a All measurements have been performed at least in triplicates,
50% reduction of absorbance. Based on the IC50 values of the propo- and all of the data were carefully analyzed. Certain data were
lis and ascorbic acid as the standard, the total antioxidant capacity checked for normal distribution using Shapiro-Wilk test and statis-
(TAC) of the propolis was calculated. The TAC value was then tical analysis was carried out using an unpaired t-test to compare
expressed as milligrams (mg) ascorbic acid equivalent (AAE) per the significant difference between two means at a significance
gram (g) of propolis. level of p < 0.05. The data were presented as the mean values along
with the standard deviation.
2.6. Total phenolic and flavonoid contents

The presences of phenols and flavonoids in ethanolic extract of 3. Results and discussion
propolis were analyzed using colorimetric methods. The total phe-
nolic contents (TPC) were determined using the Folin-Ciocalteau 3.1. Surface morphology of propolis
analysis as adapted by Singleton et al. (1999) and Kumazawa
et al. (2002) with some modifications. Three different concentra- Surface morphology of the raw propolis of stingless bee species
tions of propolis in ethanol (10, 50, and 100 mg L–1) were prepared. G. thoracica, H. itama, and T. binghami was assessed using SEM
A 100 mL of the solutions were mixed with equivolume of 7.5% Na2- imaging. As shown in Fig. 3(A)–(C), SEM images indicated that
CO3 in a 96 well plate. A 50 mL of Folin-Ciocalteau reagent was the propolis has different surface morphologies. The surface of H.
added into the mixture, which was then incubated in the dark itama propolis tends to be smooth, and that of G. thoracica propolis
for 30 min at room temperature. The absorbance of the mixture contains some spherical grain size textures embedded on the sur-
was measured at 760 nm. A similar procedure was carried out face. In contrast, the surface of T. binghami propolis is rough, sug-
for ten different concentrations of gallic acid (0–100 mg L–1). A lin- gesting that the propolis contained the agglomeration of
ear regression plot of absorbance as a function of the concentration irregularly shaped particles. This finding may imply different nat-
of gallic acid was used as a calibration curve to calculate the TPC ure of raw materials, compositions, surface properties, and struc-
contained in the raw propolis. The results were expressed as mg tures of propolis of the different stingless bee species.
gallic acid equivalent (GAE) per gram (g) of propolis.
The total flavonoid content (TFC) of propolis was investigated 3.2. Phytochemical properties
using the spectrophotometric method based on flavonoid-AlCl3
complexation as described by Franchin et al. (2012) with modifica- The phytochemical properties, particularly different nutritional
tions. A 100 lL each solution of propolis in ethanol (10, 50, and parameters, including lipid, protein, and carbohydrate content of
100 mg L–1) was mixed with an equivolume of 2% AlCl3 solution the raw propolis of stingless bee species (G. thoracica, H itama,
in a 96 well plate and was left to incubate for 30 min at room tem- and T. binghami) are summarized in Table 1. The total lipids, pro-
perature. The absorbance of the mixture was then measured at teins, and carbohydrates of the propolis varied in the range of
420 nm. The absorbance of quercetin solutions (0–100 mg L–1) 45.60–47.86%, 0.18–1.18%, and 0.17–0.48%, respectively. This high-
was recorded, plotted as a function of the concentration, and used lights that lipids were the major component of the propolis irre-
as a calibration curve. The TFC of the raw propolis was expressed as spective of their different stingless bee species, and they can be
mg quercetin equivalent (QE) per gram (g) of propolis. considered to be originated from plant waxes and resin (Bonvehí
et al., 1994; Kalogeropoulos et al., 2009; Nedji and Loucif-Ayad,
2.7. Antibacterial analysis 2014). Within the uncertainty, the lipid content of the propolis of
three stingless bee species in this study is comparable with each
The antibacterial activity of ethanolic extract of propolis of the other. It is, however, noteworthy that the lipid content of the stin-
three different stingless bee species, G. thoracica, H itama, and T. gless bee propolis was 3–5 fold higher compared with those of the
binghami, were evaluated using the disc diffusion method (Bauer Apis melifera honeybees (8.19–15.61%) (Devequi-Nunes et al.,
et al., 1966). The ethanolic extract of propolis was suspended in 2018). The higher lipid content makes the propolis of stingless bees
water, thereby the concentration of the suspension was 2 g L–1. to be more water-resistant compared with honeybee hives. The
Sterile filter paper discs (Whatman No. 1; 6 mm in diameter) were significant difference in the lipid content may be due to the differ-
fully soaked into the suspension, and they were then allowed to ent floral sources used by the stingless bees and honeybees to build
dry at ambient condition. The dried discs containing the propolis their propolis. In particular, the propolis of stingless bees is made
particles were then placed in Petri dishes containing Mueller- of a mixture of beeswax and resins collected from a variety of plant
Hinton agar (Bio-Rad) which were inoculated with 100 mL of the parts with the addition of the mandibular secretion (dos Santos
diluted bacterial culture. The bacteria were two Gram-positive et al., 2009; Simone-Finstrom and Spivak, 2010). This water-
bacterial strains (Staphylococcus aureus ATCC-29213 and Bacillus resistant cerumen is used as a storage pot for honey which has a
subtilis ATCC-11774) and two Gram-negative bacterial strains water content of 14.74 g/100 g (Shamsudin et al., 2019) and as a
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Fig. 3. SEM images of the raw propolis of stingless bee (a) G. thoracica, (b) H. itama, and (c) T. binghami. The scale bar represents 5 lm size.

Table 1 Table 2
Phytochemical parameters of G. thoracica, H. itama, and T. binghami propolis. Mineral element and heavy metal mean values (lg g1) of G. thoracica, H. itama, and T.
binghami propolis.
Parameters Stingless bee
propolis Elements Stingless bee propolis
G. thoracica H. itama T. binghami G. thoracica H. itama T. binghami
Total Lipids (%) 47.86 ± 1.61 45.60 ± 1.45 46.82 ± 1.85 Al 7.92 33.03 48.59
Crude Proteins (%) 1.18 ± 0.04 0.18 ± 0.02 0.22 ± 0.01 Ca 150.06 18.11 550.05
Total Carbohydrates (%) 0.17 ± 0.01 0.43 ± 0.03 0.24 ± 0.01 Cd — 0.599 0.664
Co 0.149 0.438 0.774
Cr 0.884 2.74 2.08
Cu 24.25 14.58 10.85
Fe 28.37 19.84 69.64
depository for mummified intruders to keep the sterile environ- K 402.14 974.24 728.50
ment in the hive. On the other hand, honeybee hive is made of a Mg 92.16 357.99 378.23
mixture of beeswax and resins without the mandibular secretion, Mn 6.98 34.49 106.03
Na 7.64 273.26 48.06
and it is used as an internal layer and a sealer surrounding the
Ni 0.102 2.20 2.10
hexagon-shaped nest combs. Pb 0.195 0.75 1.00
The crude protein of G. thoracica propolis was found to be Zn 2.97 0.24 –
approximately 5 times higher than those of H. itama and T. bing- As 0.716 4.45 4.45
hami propolis. On the other hand, the total carbohydrate of H.
itama was twice higher than those of G. thoracica and T. binghami minerals in propolis are originated from pollens collected by stin-
propolis. These slight variations of nutritional compositions might gless bees from various flowering plants in the surrounding area of
depend on the flowering season and the availability of plants sur- their hives (Campos et al., 2008; Dagaroglu, 2004; De-Melo et al.,
rounding the stingless bee hives. It is important to highlight that, 2015). This notion is also supported by the fact that K, Mg, and
all the raw propolis of stingless bees, regardless of the species, Ca were the most metal elements found in bee pollen collected
shows the same trend, i.e. low contents of total crude fiber, carbo- from Turkey (Altunatmaz et al., 2017), Brazil (Campos et al.,
hydrate, and crude protein being <1% of the dry weight of propolis 2008; Morgano et al., 2012), Serbia (Kostic et al., 2015), and Chile
(Abdullah et al., 2019). It can be postulated that the low fiber, car- (González-Martín et al., 2015), although bee pollens were also very
bohydrate, and protein contents are the key important factor to rich with respect to P, Fe, and Zn. In contrast, the mineral elements
prevent the propolis from fermentation process (Alibardi and present in stingless bee honey are K, Zn, P, Ca, Na, Mg, S, Cu, Fe, and
Cossu, 2016), which shortens the shelf life of the propolis and influ- Mn (Rao et al., 2016). This highlights the different minerals con-
ences the phytochemical properties of its honey (Temaru et al., tained in the stingless bee propolis, pollen, and honey
2007; Massaro et al., 2011). (Dagaroglu, 2004; De-Melo et al., 2015).
It is noteworthy that Ca content in T. binghami propolis
(550 lg g1) is much higher than those in G. thoracica (150.1 lg g1)
3.3. Mineral contents
and H. itama (18.1 lg g1). Since the three stingless bee species are
found at the same location, implying equivalent geological forma-
Mineral elements detected in the raw propolis of stingless bee
tions, the high Ca content suggested that T. binghami propolis could
in this study were Al, Ca, Cu, Fe, K, Mg, Mn, Na, and Zn. As summa-
belong to geopropolis and the stingless bee acquired the minerals
rized in Table 2, for all propolis of the three different stingless bee
from Ca-rich soil. An extremely high Ca content has also been
species, the greatest composition of mineral elements per gram
reported in propolis of stingless bees classified as geopropolis
propolis was found to be K. This finding is generally in agreement
found in South Spain (Bonvehí and Orantes-Bermejo, 2013), and
with those reported by González-Martín et al. (2015) on propolis
the ethnobiological and ecological perspectives of the stingless
collected from different geographical origins in Chile and Spain,
bees have been reported (Reyes-González et al., 2014). Thus, fur-
and the high content of this macro element has also been found
ther detailed studies to interrelate propolis, biology, classification,
in stingless bee propolis obtained from China and USA (Gong
and behavior of the stingless bee species in this study are pursued
et al., 2012).
in the future.
In general, the main mineral elements in propolis are K, Mg, Ca,
Mn, Na, and Al, suggesting that the propolis are very rich with
respect to these mineral elements. However, the sequence going 3.4. Heavy metal contents
from the greatest to the smallest amount of these mineral ele-
ments depends on the stingless bee species as well as the geo- As summarized in Table 2, the ICP analysis also showed that the
graphical origin (Popov et al., 2017), demonstrating that the stingless bees G. thoracica, H. itama, and T. binghami propolis also
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contains traceable heavy metals, including cadmium (Cd; 0.599–0.

664 lg g1), cobalt (Co; 0.149–0.774 lg g1), chrome (Cr; 0.884–2.
74 lg g1), nickel (Ni; 0.1–2.20 lg g1), lead (Pb; 0.195–1.00 lg g
1
), and arsenic (As; 0.716–4.45 lg g1). All the heavy metal ele-
ments were detected at low concentrations, two to three orders
lower than the mineral contents. Irrespective of the stingless bee
species in this study, Cd content in their propolis was much higher
as compared to those observed in propolis found in Macedonia (0.
012–0.038 lg g1) (Popov et al., 2017) and in Poland (0.012–0.05
5 lg g1) (Formicki et al., 2013). In addition, Pb content in the pro-
polis was also much higher compared to those in propolis found in
Macedonia (0.033–0.041 lg g1) (Popov et al., 2017), but they
tended to be lower than those in propolis found in Poland (0.89–
2.94 lg g1) (Formicki et al., 2013). Considering that stingless bees
may collect waxes, resin, nectar, and minerals from the plants and
soil over a distance of sub-kilometer from their hives
(Przybyłowski and Wilczyńska, 2001), the low contents of heavy
metals in the propolis reflect low contaminations of heavy metals
in plants, soil, and atmosphere in the surrounding area where the
propolis was collected. This finding also supported the notion that
the heavy metal contents can be used to monitor environmental
contamination (Gilbert and Lisk, 1978; Uren et al., 1998), where
high contents of heavy metals in bee products have been attributed
to the beehives being located in densely populated areas with high
industrial or agricultural activities. Nevertheless, the major path-
ways of the presence of heavy metals in propolis might be figured
out by surveying the plants and soil surrounding the stingless bee-
hives from which the bees collect resins, buds, pollen, nectar, and
minerals.

3.5. Vibrational and absorption spectroscopies of propolis

FTIR spectroscopy has been applied to identify functional

groups corresponding to the chemical bonds of constituents pre-
sent in the propolis which inevitably correlate to the chemical
components potent in the propolis of stingless bees G. thoracica,
H. itama, and T. binghami. As shown in Fig. 4(A), propolis of the
three stingless bee species show different vibrational spectra to
some extent, which can be attributed to their distinct compositions
Fig. 4. (A) The FTIR of raw propolis and (B) UV-Vis absorption spectra of ethanolic
of compounds. Table 3 summarizes the vibrational bands and their
extract of propolis of (a) G. thoracica, (b) H. itama, and (c) T. binghami.
tentative vibrational assignments. Despite the different FTIR spec-
tral pattern, in general, the spectral bands of all the propolis can be
assigned to the stretching, bending, wagging, and out-of-plane niques in the future. Nevertheless, it might be interesting to recall
vibrations of OAH, NAH, CAH, CAO, CAN, CAC, C@C, C@O, CAH that, as it has been documented in the literature, in general, the
of alcohols, phenols, carboxylic acids, alkenes, and aromatic rings, chemical compounds in various stingless bee propolis are mainly
suggesting that all the raw propolis contains alkyl and aromatic the phenolic and flavonoid compounds, including tocopherol,
compounds having amine, ester, carbonyl, alkyl, and hydroxyl quercetin, vanilic, caffeic acid, ferulic acid, coumaric acid, benzoic
functional groups. This may further indicate the presences of aro- acid, cinnamic acid, pinobanksin 5-methyl ether, apigenin, kaemp-
matic acids, terpenes, flavonoids and phenolic acids in the raw ferol, pinobanksin, cinnamylideneacetic acid, chrysin, pinocem-
propolis. In particular, the total phenolics and flavonoids in propo- brin, galangin, pinobanksin 3-acetate, phenethyl caffeate,
lis were quantified further using chemical assay, as described in cinnamyl caffeate, and tectochrysin (Marcucci and Bankova,
the following section. 1999; Medić-Šarić et al., 2004; Coneac et al., 2008; Bonamigo
The absorption spectra of ethanolic extract of stingless bees G. et al., 2017).
thoracica, H. itama, and T. binghami propolis are shown in Fig. 4
(B). The absorption spectra exhibited elastic Rayleigh scattering,
suggesting that the ethanolic extract of propolis forms colloidal 3.6. Antioxidant capacity
suspensions. The G. thoracica propolis clearly showed an absorp-
tion maximum at 293 nm and a shoulder at 350 nm, H. itama pro- The antioxidant capacity of the raw propolis was represented by
polis exhibited a peak at 272 nm and a shoulder at 310 nm DPPH radical scavenging activity (RSA) of their ethanol extract. The
respectively, and T. binghami propolis showed a low-intensity peak IC50 value along with total phenolic content (TPC) and total flavo-
at 270 and a slight shoulder at 308 nm. The absorption peaks can noid content (TFC) are summarized in Table 4. The IC50 values were
be assigned to the presences of p-conjugated aliphatic and aro- 570.2, 76.5, and 1975.0 mg L–1 for G. thoracica, H. itama, and T. bing-
matic compounds as well as aromatic acids in the propolis. How- hami propolis particles, respectively, revealing their relative
ever, the chemical structures and compositions in the propolis of amounts of antioxidants. By comparing the IC50 value of ethanolic
stingless bees G. thoracica, H itama, and T. binghami in this study extract of the propolis with that of ascorbic acid (24.3 ± 0.1 mg L–1)
will be precisely quantified in detail using chromatographic tech- as the standard, the total antioxidant capacity (TAC) of the raw
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N.A. Abdullah et al. Saudi Journal of Biological Sciences 27 (2020) 2902–2911

Table 3 among the phenolics, flavonoids, terpenes, and aromatic acids con-
Vibrational peaks and assignments of FTIR spectra of G. thoracica, H. itama, and T. tained in propolis (Coneac et al., 2008; Bonamigo et al., 2017),
binghami propolis.
regardless the synergetic effects, the phenolic compounds play an
Stingless bee propolis Vibrational modes important role in the antioxidant activity (Sousa et al., 2008;
H. itama G. T. Lima et al., 2009).
thoracica binghami
3312(b) 3466(b) 3425(b) mO-H of alcohols, phenols and 3.7. Antimicrobial activity
carboxylic acids
2926(s) 2918(s) 2920 mN-H of amines In order to evaluate the potential of propolis of the stingless
2853(s) 2851(s) 2851 mCH3, mCH2 and mCH of alkenes
1693(b) 1721(l) 1710 mC = O of carbonyl bees G. thoracica, H. itama, and T. binghami as an antimicrobial
1603(b) 1646(s) 1644 mC = C of conjugated aromatic rings agent, they were screened against two Gram-positive (B. subtilis
1515(w) 1538(s) 1545(b) dN-H of amines; mC-C aromatic ring and S. aureus) and two Gram-negative bacteria (E. coli and P. aerug-
1443(s) 1430(b) 1459(s) dCH of aromatic rings inosa). The inhibitory activities of ethanol extract of the propolis
1379(s) 1383(s) 1382 mC-O of carboxylic acids
were assessed by disc diffusion assay which is considered as a
— 1315(s) 1315(w) mC-O alcohols; xCH2 of alkenes
1283(w) 1243(w) 1244 mC-O phenols qualitative screening method. However, comparison of the inhibi-
1221(s) — 1213 mC-O of phenols tion zones would likely suggest that larger inhibition zone has a
1160(w) 1157(s) 1160 mC-N of amines better antibacterial activity compared to smaller inhibition zone.
1073(w) 1082(s) 1069 mC-C aromatic rings Ultrapure water which has no inhibition was used as a negative
1033(b) 1029(s) 1032 mC-O of esters
813(w) 817 — cCH2 of alkenes control, and two different antibiotics (rifampicin and strepto-
771(s) — 757 cC-H of alkenes mycin) were used as positive controls. The inhibition zone for
717 714 — cC-H of alkenes the different propolis and the two standard antibiotics was deter-
687 — 667 cO-H of alcohols and phenols mined (Table 5), under the same experimental conditions.
The present result indicated that all of the ethanol extracts of pro-
polis showed distinct antibacterial strengths against the four bacte-
Table 4 rial strains, suggesting that the propolis have strong antibacterial
The IC50, the total antioxidant capacity (TAC), total phenolic content (TPC), and total activities to some extent against B. subtilis, S. aureus, and P. aerugi-
flavonoid content (TFC) of ethanolic extract of G. thoracica, H. itama, and T. binghami
propolis.
nosa, as reported by Kujumgiev et al. (1999), Moreno et al. (1999),
and Sforcin et al. (2000). This consideration was supported by the
Parameters Stingless bee propolis inhibition zone of the ethanol extract of propolis which was not sig-
G. thoracica H. itama T. binghami nificantly different compared to the standard antibiotic, rifampicin.
IC50 (mg L-1) 570.2 ± 6.1 76.5 ± 1.3 1975 ± 22.5 However, when compared to another antibiotic, streptomycin, the
TAC (mg AAE g1) 42.5 ± 0.5 317.6 ± 5.4 12.3 ± 0.3 propolis, in general, has significantly lower inhibitory activities
TPC (mg GAE/g propolis) 2192.7 ± 12.3 2391.0 ± 16.1 2151.9 ± 12.1 against S. aureus and P. aeruginosa but the activities were mostly
TFC (mg QE/g propolis) 299.4 ± 2.6 275.2 ± 3.5 275.9 ± 2.1
found to be comparable against B. subtilis and E. coli. This bacterial
Note: IC50 of ascorbic acid 24.3 mg L-1
strain-dependent antibacterial activity reflects the variations of
bioactive compounds contained in propolis of the different stingless
bee species. Although the propolis in a few of the tests against Gram-
propolis of G. thoracica, H. itama, and T. binghami was estimated to positive and Gram-negative bacteria showed less activities than any
be 42.5, 317.6, and 12.3 mg AAE g1, respectively. Among the pro- of the two standard antibiotics, nevertheless, as natural products,
polis in this study, H. itama shows the highest TAC, equivalent to 7- they are potentially applicable for various biomedical applications
and 25-fold higher than that of G. thoracica and T. binghami, respec- (Tosi et al., 1996; Sforcin and Bankova, 2011).
tively. A similar trend of TAC of propolis of the three different stin- The understanding of the antimicrobial activity of the propolis
gless bee species G. thoracica, H. itama, and T. binghami found in particles is important. In the literature, the antimicrobial activity
Malaysia has also been reported by Awang et al. (2018). of propolis is related to the phenolic and flavonoid compounds
The variations in the TAC values might be attributed to the dif- with various polarities and their synergetic effect. Although the
ferent types and contents of phenolic and flavonoid compounds in exact mechanism of the antibacterial activities is still unknown
the raw propolis, as each phenolic and flavonoid compound has (Santos et al., 2002), it may be attributed to polar and lipophilic
different RSAs (Aljadi and Kamaruddin, 2004; Kucuk et al., 2007). phenolic and flavonoid compounds. In particular, those compounds
This revealed that the considerable amount of phenolic compounds having electronegative carbonyl, amine, imine, sulfide, thiol, meth-
contained in propolis of stingless bees can be attributed to its high oxyl, and hydroxyl groups are highly polar and lipophilic and could
antioxidant capacity, as it has been pointed out by Araujo et al. be responsible for the contact with bacterial cells and induce struc-
(2016). In other words, the RSA of the propolis is strongly deter- tural damage to the cell wall and membrane, leading to the leakage
mined by the TPC, rather than the TFC. It further suggested that of cellular contents and cell death (Cushnie et al., 2003; Cushnie

Table 5
Antibacterial activities of ethanolic extract of G. thoracica, H. itama, and T. binghami propolis along with the two standard antibiotics, rifampicin and streptomycin, for comparison.
Negative control (water) did not show any inhibition zone as expected. R and S denote significant difference (p < 0.05) between the inhibition zones of the propolis and that of
rifampicin (RIF) and streptomycin (STR), respectively.

Bacterial strain Inhibition zone (mm)

G. thoracica propolis H. itama propolis T. binghami propolis Antibiotics
RIF STR
B. subtilis 10.8 ± 1.0 13.0 ± 4.6 11.0 ± 2.7 14.0 ± 4.4 14.0 ± 4.1
S. aureus 7.8 ± 0.5S 9.7 ± 4.6S 7.0 ± 0.8RS 12.9 ± 4.6 16.8 ± 4.2
E. coli 11.3 ± 1.0 10.8 ± 1.0 10.0 ± 4.4 13.8 ± 4.6 12.4 ± 4.0
P. aeruginosa 9.0 ± 1.2RS 9.8 ± 1.3S 8.8 ± 1.3S 14.0 ± 5.1 14.8 ± 2.6

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N.A. Abdullah et al. Saudi Journal of Biological Sciences 27 (2020) 2902–2911

and Lamb, 2005; Kim and Chung, 2011; Sanpa et al., 2015; data; AU wrote the paper. All authors read and approved the final
Echeverría et al., 2017), followed by aggregation (Pohjala and manuscript.
Tammela, 2012).
The aforementioned slight higher sensitivity of the Gram- Declaration of Competing Interest
positive bacteria compared with Gram-negative bacteria indicates
the important role of flavonoids, rather than phenolic compounds, The authors declare that they have no known competing finan-
in the antibacterial activities of the propolis, since flavonoids such cial interests or personal relationships that could have appeared
as galangin and quercetin derivatives have minimum inhibitory to influence the work reported in this paper.
concentrations as low as sub-ppm to ppm levels against the
Gram-positive bacteria. This notion is supported by the similar References
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