Bee propolis or bee glue is a very sticky valuable resinous mixture produced
by honeybees from trees bubs and various plants sources around the hive, it
is masticated by the bees, salivary enzymes and beeswax added, then used as
a construction material in bee hives for filling cracks and repairing combs
thereby insulating and reinforcing the hives, also protecting the hive and its
nutritious contents from attack by micro-organisms (Burdock,
1998; Johnson et al., 1994). Due to biological
and pharmacological activities, propolis has been extensively used in folk medicine
since ancient times (Galvao et al., 2007) and
is now known to be a natural medicine with antibacterial, antifungal, antitumoral,
antioxidative, imunomodulatory and other beneficial activities (Bankova
et al., 2002; El-Kott and Owayss, 2008).
Now is presently used in health food and various pharmaceutical and cosmetic
products such as mouthwash preparations, face creams, lotions and tablets (Burdock,
1998). Propolis contains a diversity of compounds of plant origin basically
is composed of 55% vegetable resins and balsam, 30% bee wax, 10% essential oil
and 5% pollen (Cizmarik and Matel, 1970; Pepeljnjak
et al., 1985; Serkedjieva et al., 1992).
The chemical composition and antioxidant capacities of propolis of many countries
have been widely studied by a lot of scientific research groups (Zadeh
et al., 2007; Moreira et al., 2008;
Gulcin et al., 2010) but only a few reports can
be found in literature on Algerian propolis. This motivated us to explore the
antioxidant capacity of Algerian propolis and its total polyphenol and flavonoid
The aim of this study is to measure the in vitro antioxidant activity
of the methanolic extract of south Algerian propolis. We used the following
three assay systems: (1) 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
activity assay, (2) reducing antioxidant power assay (RP) and (3) Cyclic Voltammetry
assay (CV). Total polyphenol and Flavonoid contents of the methanolic extract
of propolis were determined by standard colourimetric methods. (Chang
et al., 2002; McDonald et al., 2001;
Mello et al., 2010).
MATERIALS AND METHODS
Chemical: Methanol (99%), Folin Ciocalteu reagent, trichloroacetic acid
(99%), potassium chloride (99.8%) were all purchased from Biochem Chemopharma
Co. (Canada). 1,1-Diphenyl-2-picryl hydrazyl (DPPH) (99%),potassium ferricyanide
(99%), ascorbic acid (99.7%), gallic acid (99%) ferric chloride (99%), sodium
carbonate (99%), AlCl3 (99%), rutin (99%) were all purchased from
Merck Co.Orthophosphoric acid (85%) was purchased from Riedel-de Haen Co., all
other reagents used were of analytical grade.
Propolis: Crude propolis sample was brought from hives of honeybees located in El-Oued (south of Algeria) in May-April, 2010. Samples, once received were stored at 4°C in airtight/dark plastic containers until analysis.
Instrument: UV-Visible spectrophotometer (PRIM Advanced SCHOTT Instruments Gmbh), centrifuge Machine (SLW centryge, Ultra-8TL), PGP301 potentiostat with voltamaster 4 version 7.08 software (radiometer analytical SAS), rotary evaporator (IKA Evaporator RV 06-ML).
Extraction of propolis compounds: Extraction of propolis contents was achieved using methanol as a solvent. The propolis, is cut into small portions; ground into a coarse powder; dived in methanol (1 g/30 mL) for 24 h, the mixture was then centrifuged for 30 min at 3500 rpm. The insoluble residue (mostly beeswax) was removed by filtering through Whatman No. 4 paper and evaporated to 40°C.
Determination of total polyphenolics: Total polyphenolic content was
determined using Folin-Ciocalteu reagents according to the method of Scalbert
et al. (1989), briefly described as 0.5 mL of Folin and Ciocalteus
phenol reagent was mixed with 100 μL extract solution. After 3 min, 2 mL
of 20% aqueous sodium carbonate solution was added to the mixture and adjusted
to 10 mL with distilled water. The reaction was kept in the dark for 30 min,
after which the absorbance was read at λ = 760 nm.
Gallic acid was used to calculate the standard curve (0.03-0.3 mg mL-1;
y = 3.3974x; R2 = 0.994) and the results were expressed as mg of
Gallic Acid Equivalents (GAEs) g-1 of extract. The amount of total
phenols found for the methanolic propolis extracts was equal to 10.99 mg g-1
of GAEs. This value is very low compared to that of Portuguese propolis samples
from Bornes and Fundão regions which were of 329 and 151 mg g-1
of GAE, respectively (Moreira et al., 2008).
Present values still also lower than the Korean propolis from Yeosu, Yangpyeong,
Boryung and Cheorwon regions which was, respectively equal to 212.7, 160.6,
172.3 and 180.3 mg g-1 of GAEs (Choi et al.,
Determination of total flavonoids: For flavonoid contents determination,
the methanol extracts of propolis was retaken in 1 mL of methanol and treated
with AlCl3 methanol solution (2%, 1 mL). After 30 min the solution
was mixed well and the intensity of pink color was measured at λ = 430
nm. Rutin was used to calculate the standard curve, the calibration graph obtained
for rutin presents linearity between 0.1 and 0.02 g L-1 (y = 13.686
x where y represents the value of absorbance and x, the value of rutin concentration
expressed as g L-1; R2 = 0.999) and the results were expressed
as mg of Rutin Equivalents (REs) g-1 of extract. All the samples
and the standards were analyzed in triplicate. The amount of total flavonoids
found for the methanolic propolis extracts was equal to 2.12 mg g-1
of REs. Present results still lower compared with Brazilian propolis samples
which contained 6.95 mg g-1 of flavonoids using quercetin as standard
(Mello et al., 2010). Also lower than propolis
from Greece and Cyprus which contained flavonoids at levels of and 8.8 mg g-1
using caffeic acid as standard (Kalogeropoulos et al.,
Evaluation of antioxidant capacity by spectrophotometrical techniques
Using the free radical scavenging determination: The free radical scavenging
capacity of propolis was measured in terms of hydrogen donating or free radical
scavenging ability by using the stable 1, 1-diphenyl-2-picryl hydrazyl radical
(DPPH) (Molyneux, 2004), propolis extract and standard
ascorbic acid solution (0.1 mL) of different concentrations viz., 0.1, 0.2,
0.4, 0.6, 0.8 and 1 mg L-1 was added to 1 mL of a 0.004% methanol
solution of DPPH. An equal amount of methanol and DPPH served as control. After
30 min incubation in the dark, absorbance was recorded at 517 nm and the percentage
inhibition capacity was calculated from the following relation:
where, A0 is the absorbance of the control and A1 is
the absorbance of the extract/standard.
The antioxidant capacity of the extract was expressed as IC50. The
IC50 value was defined as the concentration (in μg mL-1)
of extracts that inhibits the formation of DPPH radicals by 50%. All the tests
were performed in triplicate and the graph was plotted with the average of three
observations. The equation obtained from the linear calibration graph in the
studied concentration range for ascorbic acid is y = 271.04 x (where y represents
the value of absorbance and x, the value of ascorbic acid concentration, expressed
as g L-1) with a correlation coefficient of R2 = 0.9942.
Under the same conditions the Eq of the calibration graph for propolis is y
= 1954 x with a correlation coefficient of R2 = 0.9982. According
to IC50 values, the propolis extract has a higher antioxidant capacity;
the results are summarized in Table 1.
Using Reducing Power (RP) determination: Different concentrations of
propolis extract and standard ascorbic acid solution viz., 10, 20, 40, 60, 80
and 100 mg L-1 in 1 mL of methanol were mixed with phosphate buffer
(2.5 mL, 0.2 M pH 6.6) and potassium ferricyanide K3Fe (CN)6
(2.5 mL, 1%). The mixture was incubated at 50°C for 20 min. A volume of
2.5 mL of aqueous tricholoroacetic acid solution (10%) was added to the mixture.
A volume of 2.5 mL of the resulting mixture was mixed with 2.5 mL distilled
water and (0.5 mL, 0.1%) of ferric chloride. The absorbance was then recorded
at 700 nm. All the tests were performed in triplicate and the graph was plotted
with the average of three observations (Chevion et al.,
Both calibration graphs for ascorbic acid and the propolis are linear in the range of the studied concentrations, with an standard curve equation for the ascorbic acid of y = 673 x with a correlation coefficient of R2 = 0.9937 (where y represents the value of absorbance and x, the value of acid ascorbic or propolis concentration, expressed as mM). The standard curve equation for the propolis is, y = 0.9314 x with a correlation coefficient of R2 = 0.9994, (where y represents the value of absorbance and x, the value of the inverse of the dilution coefficient of propolis concentration).
The result of Reducing Power (RP) of the propolis extract, in terms of ascorbic Acid Equivalent Antioxidant Capacity (AEAC) calculated from the calibration graph using linear regression analysis is found to be equal to 1.384 mM.
Evaluation of antioxidant capacity by electrochemical techniques: The
measurement of the antioxidant capacity of the studied samples of propolis was
performed using an electrochemical method based on cyclicvoltammetry techniques
(Campanella et al., 2001; Cosio
et al., 2006). Cyclicvoltammetry measurements were performed in an
electrochemical cell with a volumetric capacity of 50 mL containing a Glassy
Carbon Electrode (GCE) working electrode (radiometer analytical SAS), a Pt wire
counter electrode and an Hg/Hg2Cl2 reference electrode
(saturated with KCl). The potential was swept in inverse scanning mode starting
from-200 to +800 mV with a scanning rate of 100 mV sec-1. To avoid
reducing the sensitivity of the working electrode, the latter was polished after
each cycle by rubbing its surface using alumina oxide (particle size 0.3 μm)
before every electrochemical assay. After polishing it was rinsed thoroughly
with bidistilled water for 30 sec.
The antioxidant capacity of the studied samples of propolis was obtained using
the area below the anodic curve of the voltammogram. The calibration graph is
obtained by plotting the area below the anodic curve of the voltammogram of
each sample of the standard versus its concentration. Ascorbic and gallic acids
were used as standards in the calculation of antioxidant capacity of the studied
sample of propolis because of their wide spreading in nature and also because
their anodic area displays excellent linearity toward ascorbic or Gallic acids
concentrations (Laskar et al., 2010; Oyaizu,
RESULTS AND DISCUSSION
The cyclic voltammetry voltammograms obtained for 1 mM of ascorbic and gallic acids in pH 7, 0.1 M phosphate buffer solution (pH = 2, Britton-Robinson buffer solution for gallic acid) and 0.1 M KCl as a supporting electrolyte using a 3 mm-diameter glassy carbon electrode present typical irreversible oxidation processes with the existence of an irreversible one oxidation peak at 0.26 V for ascorbic acid (Fig. 1A) and two oxidation peaks at 0.58 and 0.85 V for gallic acid (Fig. 1B).
The same irreversible electrochemical behavior was observed for propolis sample extract (Fig. 2) although, with oxidation potential value of propolis extract is more positive than ascorbic acid, around 0.44 V and less positive than gallic acid, however these results do not indicate that, under the electrochemical conditions used, the propolis extract has an antioxidant capacity less than gallic acid and more than ascorbic acid but it indicates that the propolis extract do not contain any of the constituents of the standards ascorbic nor gallic acids.
Although the oxidation potential value of propolis extract is less positive
than gallic acid, the antioxidant capacity of propolis is higher than gallic
acid, this stands in sharp contrast with the results of Kilmartin
and Hsu (2003) (extracts with lower oxidation potential values have higher
antioxidant capacity). This may be due to the fact that the obtained voltammograms
do not have the same allure.
||Cyclic voltammograms obtained in 1 mM of ascorbic acid (A)
and gallic acid (B) in pH 7 and pH 2 buffer solutions containing 0.1 mol
L-1KCl at scan rate 100 mV sec-1
||Cyclic voltammogram of propolis extract in pH 7, 0.1 M phosphate
buffer solution containing 0.1 mol L-1 KCl at scan rate 100 mV
The antioxidant activity of propolis methanolic, ethanolic and aqueous extracts
has been investigated by many scientific research groups in different countries
and has been reported in different spectrophotometrical methods including Ferric
Reducing Antioxidant Potential (FRAP) (Kalogeropoulos
et al., 2009), b-carotene-linoleic acid system (Ahn
et al., 2007) and the free radical-scavenging capacity with reduction
of radical diphenylpicrylhydrazyl (DPPH) (Russo et al.,
2004), but only a few reports can be found in literature using electrochemical
assays as means of evaluation of the antioxidant capacity of propolis or any
other natural materials.
||Cyclic voltammograms referring to different ascorbic (A) and
gallic acids (B) concentrations
The cyclic voltammograms, at different concentrations of ascorbic and gallic
acids, are shown in Fig. 3a, b. As can be seen there is an
increase in peak current with the increase in ascorbic or gallic acids concentrations
which leads to a linear relation between these two parameters.
In order to express the antioxidant capacity of the propolis extract in equivalent
terms of Ascorbic Acid Equivalent Antioxidant Capacity (AEAC) and Gallic Acid
Equivalent Antioxidant Capacity (GEAC), different concentrations of the standards
ascorbic and gallic acids were plotted verses the Area of the Anodic Wave (AAW).
The anodic area displays excellent linearity toward both ascorbic and Gallic
acids concentrations Fig. 4A, B. The values are presented
in Table 2.
The equation obtained from the linear calibration graph in the studied concentration
range for ascorbic and gallic acids is, respectively, y = 5.0628 x + 1.7674
and y = 8.418 x + 1.169 (where y represents the value of the area of the anodic
wave and x, the value of standards concentration, expressed as g L-1),
with a correlation coefficient of R2 = 0.999 for both equation.
||Area of the anodic wave obtained from cyclic voltammetry of
ascorbic and gallic acids
||The antioxidant capacity of propolis calculated using (RP),
(DPPH) values and (AEAC), (GEAC) equivalent
|*calculated using the current density of the anodic peak
||Calibration curve obtained by cyclic voltammetry method expressed
as ascorbic (A) and gallic (B) acids equivalents/L
The Ascorbic Acid Equivalent Antioxidant Capacity (AEAC) and Gallic Acid Equivalent
Antioxidant Capacity (GEAC) of the propolis extract calculated from the calibration
graphs is equal to 5.125 and 8.8205 mg g-1. Table 3
summarizes the values of antioxidant capacity of propolis extract estimated
by spectrophotometrical and electrochemical assays.
Laskar et al. (2010) have studied the antioxidant
capacity of Indian propolis by cyclic voltammetry assay and found that the propolis
ethanolic extracts reducing power values of Indian samples, expressed as mg
ascorbic acid equivalents, ranged between 15.32 and 23.95 mg g-1
ascorbic acid equivalents, these values are relatively higher values than Algerian
propolis samples. This may be due to its higher polyphenol content. Hence aqueous
extract may well be a substitute of organic solvent extracts of propolis.
The antioxidant capacity of Italian propolis was also quantified by Buratti
et al. (2007) using galangin as reference. Quantification was based
on peak height using amperometric flow injection analysis. The antioxidant capacity
of propolis samples varies from 2 to 169 mg g-1 galangin equivalents,
these values still relatively higher than those observed in Algerian propolis
samples. The differences in values of the antioxidant capacity may be attributed
to the standards used.
Both the spectrophotometrical (DPPH and RP) and electrochemical (AEAC and GEAC) assays suggest that the methalonic extract of propolis shows in vitro antioxidant activities by inhibiting DPPH and reducing power ability which may be due to presence of flavonoids and phenolic compounds found in the preliminary phytochemical screening. The results show that the antioxidant capacity, expressed in terms of ascorbic (AEAC) and gallic acids (GEAC) equivalent antioxidant capacity obtained from electrochemical experiments is higher than that obtained from spectrophotometrical experiment using (RP) and (DPPH). This outcome can be attributed to the overestimation of the total polyphenolic content due to the interferences of other non-phenolic species like reduction sugars.