Abstract (English):
Introduction. This study aimed to evaluate the antioxidant and anti-diabetic activity of aqueous and hydroalcoholic extracts of pomegranate (Punica granatum L.) leaves in vitro, as well as to determine the content of polyphenols, flavonoids, and flavonols. Study objects and methods. The antioxidant activity was determined by the DPPH test using the free radical 1,1-diphenyl-2-picrylhydrazyle and the FRAP method, as well as by measuring total antioxidant capacity and the hydrogen peroxide scavenging activity. Results and discussion. The content of total polyphenols varied between 4.43 ± 0.3 and 12.66 ± 1.6 mg EAG/g. The highest content of flavonoids was observed in the hydroalcoholic extract of P. granatum leaves (P < 0.05). The flavonol contents in the hydroalcoholic and aqueous extracts were 7.68 ± 0.6 and 9.20 ± 2.8 mg EQ/g, respectively. The IC50 of the antioxidant potential of the hydroalcoholic and aqueous extracts was 32.4 ± 1.109 and 35.12 ± 4.107 mg/mL, respectively. According to the DPPH test, the aqueous extract was the least active (IC50 = 14.15 ± 1.513 mg/mL). The highest percentage of hydrogen peroxide trapping was found in the aqueous extract (45.97 ± 6.608 %). The inhibition of α-amylase showed an IC50 of between 9.804 ± 0.67 and 19.011 ± 9.82 mg/mL in the aqueous and hydroalcoholic extracts, respectively. The inhibition of glucose uptake by yeast recorded a high inhibitory capacity at 50 mg/mL of glucose. Conclusion. We found that the antioxidant and anti-diabetic activity of P. granatum leaves extracts was due to the presence of bioactive compounds such as flavonoids, which is why they are effective in preventing diabetes and its complications.

Keywords:
Punica granatum L., plant extracts, antioxidant activity, anti-diabetic activity, flavonoids

INTRODUCTION
The pomegranate (Punica granatum L.) is a shrub
that belongs to the Lythraceae family. It is between 5
and 10 m tall and is characterized by deciduous fruiting
leaves. The pomegranate is used to prevent cancer,
cardiovascular disease, diabetes, dental conditions,
and erectile dysfunction, as well as against ultra violet
radiation. Pomegranate leaf extracts contain high total
phenols, tannins, and triterpenoids [1].
Numerous studies have demonstrated the in
vitro antioxidant activity and polyphenol content of
pomegranate. According to Amjad et al., the antioxidant
activity of pomegranate leaves is directly related to
the presence of phenolic compounds and antioxidant
components which act as hydrogen donors, contributing
to the concentration of total phenols [2]. These authors
demonstrated that pomegranate n-butanol, ethyl acetate,
hydroethanol, and aqueous leaf extracts contained
ellagic acid, an efficient free radical scavenger [2]. Vinodhini et al. reported that the aqueous extract
of pomegranate leaves had the greatest antioxidant
activity and contained significant levels of total phenols
and flavonoids [3]. The leaf extracts showed antioxidant
activity in vivo by protecting yeast cells against
oxidative stressing agent H2O2. The authors found
pomegranate a good source of natural compounds with
health benefits, which makes it possible to use it in diets
to reduce oxidative stress.
In the study by Bekir et al., the methanolic extract
of pomegranate leaves displayed high antioxidant, antiinflammatory,
anti-cholinesterase, and antiproliferative
activities [4]. These results showed that pomegranate
leaves could be a potential source of active molecules
intended for applications in pharmaceutical industry.
The aim of the study was to evaluate the antioxidant
and anti-diabetic activity of aqueous and hydroalcoholic
extracts of pomegranate leaves in vitro.
STUDY OBJECTS AND METHODS
The pomegranate (Punica granatum L.) leaves
were collected in September 2017 in Chlef, Algeria.
The collected samples were dried at room temperature
away from sunlight and then powdered using an electric
mortar.
Preparation of aqueous extract. The aqueous
extract of pomegranate leaves was prepared according
to the method described by Diallo et al., with some
modifications [5]. 15 g of powdered leaves in 150 mL of
boiling water was heated for 15 min and filtered through
filter paper. The filtrate was placed in an oven at 40°C
until obtaining a dry extract and stored at 4°C.
Preparation of hydroalcoholic extract. The
hydroalcoholic extract of pomegranate leaves was
prepared by maceration of 15 g of powdered leaves in
100 mL of a hydroalcoholic solution (70%) at room
temperature away from light, with maximum agitation
for 72 h [6]. Then the mixture was filtered through filter
paper. The filtrates were placed in an oven at 40°C. The
dry extract was stored in a refrigerator at 4°C.
Total polyphenols were determined spectrophotometrically
following the Folin-Cioclateu method [7].
For this, 0.2 mL of each leaf extract was mixed in a test
tube with 1.0 mL of Folin-Cioclateu reagent and 0.8 mL
of a 7.5% sodium carbonate solution (Na2CO3). After
incubation in the shade and at room temperature for
30 min, absorbance was measured at 760 nm. The
results were expressed in milligram equivalent of
gallic acid per gram of extract (mg EAG/g extract)
from a calibration curve prepared using gallic acid as a
standard.
Flavonoid levels were measured using the method
described by Mbaebie et al. [8]. For this, 1.0 mL of each
extract was added to 1.0 mL of a 2% ethanol solution
of aluminum chloride (AlCl3) and then incubated for an
hour at room temperature. Absorbance was measured
by a UV-visible spectrophotometer at 420 nm. The
concentrations of flavonoids in the extracts were
calculated from the calibration curve and expressed in
milligram equivalent of quercetin per gram of extract
(mg EQ/g extracted).
Flavonols were determined according to the method
described by Kosalec et al. [9]. For this, 0.3 mL of the
extract was mixed with 0.3 mL of aluminum chloride
(AlCl3) and 0.45 mL of sodium acetate. The mixture
was vigorously stirred and then incubated for 40 min.
Absorbance was measured at 440 nm. The quantification
of flavonols was based on a calibration curve made by
quercetin. The content of flavonols was expressed in
milligram equivalent of quercetin per gram of extract
(mg EQ/g).
Total antioxidant capacity. Determination of
total antioxidant capacity is a technique based on the
reduction of molybdate Mo (VI) to molybdenum Mo (V)
in the presence of an antioxidant with the formation of
a green complex (phosphate/Mo (V)) at acidic pH [10].
The phosphomolybdate reagent was prepared from a
mixture of 0.6 M sulfuric acid (H2SO4), 28 mM sodium
phosphate (Na3PO4), and 4 mM ammonium molybdate
((NH4) 6Mo7O24 • 4H2O). 1.0 mL of this reagent was
added to 100 μL of each extract with concentrations of
10, 25, 50 and 100 mg/mL. The tubes were incubated
at 95°C for 90 min. After cooling, absorbance was
measured at 695 nm. Total antioxidant capacity was
expressed in milligrams of ascorbic acid equivalent per
gram of extract (mg Eq AA/g extract) from a calibration
curve of ascorbic acid.
Ferric Reducing Antioxidant Power (FRAP).
The FRAP method involves measuring the ability
of a sample to reduce the tripyridyltriazine ferric
complex to tripyridyltriazine at a low pH. This ferrous
tripyridyltriazine complex has an intense blue color
measured by a spectrophotometer at 593 nm [11].
The FRAP reagent was prepared by mixing a
300 mM sodium acetate buffer (pH 3.6), a solution of
10 mM TPTZ in 40 mM HCl and 20 mM FeCl3 in a ratio
of 10:1:1 (v/v/v). 200 μL of each extract (10, 25, 50 and
100 mg/mL) was added to 3 mL of the FRAP
reagent. After incubation in the dark at 37°C for
30 min, absorbance was measured at 593 nm against
the blank [11].
Hydrogen peroxide scavenging activity. The
scavenging capacity of hydrogen peroxide is based on
the reduction of the H2O2 concentration by scavenger
compounds, the absorbance value of the latter at
230 nm also reduces [12]. A 40 mM hydrogen peroxide
solution was prepared in a 50 mM phosphate buffer
(pH 7.4). 4.0 mL of each extract with a concentration of
10 mg/mL was mixed with 0.6 mL of the H2O2 solution.
After 10 min incubation, absorbance was measured
at 230 nm. Ascorbic acid was used as a positive
control [13]. The percent inhibition was calculated using
the following equation:
Percent inhibition (%) = [(A control – A sample) /
A control] × 100 (2)
where A is absorbance of the control and experimental
samples.
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DPPH test. To p repare a 0.004% solution of DPPH,
250 μL of extracts at concentrations of 10, 25, 50, and
100 mg/mL or standard (ascorbic acid) was added to
1 mL of the DPPH solution. After incubation in the
dark at room temperature for 30 min, absorbance
was measured at 517 nm against a blank sample that
contained pure methanol [14]. The antioxidant activity
evaluated with the DPPH method was expressed in
percentage according to the following formula:
% antioxidant activity = [(A control – A sample) /
A control] × 100 (2)
where A is absorbance of the control and experimental
samples.
Inhibition test of α-amylase enzymatic activity.
The inhibition test of α-amylase enzymatic activity
followed the method of Daksha et al. [15]. For t his, t wo
solutions had been prepared, namely a 1% starch stock
solution and a 1% amylase solution in a 0.1 M phosphate
buffer at pH 7.2, both solutions preserved at 4°C. The
reaction mixture contained 2.0 mL of a phosphate
buffer, 1.0 mL of each extracts (aqueous and hydroalcoholic)
at concentrations of 10, 25, 50, and 100 mg/mL,
1 mL of amylase, and 1 mL of starch. The mixture
was incubated for an hour. The enzymatic reaction
was stopped by the addition of 0.1 mL of the iodide
indicator. All experiments were performed in triplicate.
Absorbance was measured at 565 nm.
The inhibitory activity of each extract was calculated
according to the following formula:
% inhibition activity = (A sample – A control /
A sample) × 100 (3)
where A is absorbance of the control and experimental
samples.
To determine effects of the extracts on glucose
uptake by yeast, we prepared yeast cells according
to the method described in [16]. 1 g of commercial
baker’s yeast was washed by centrifugation (4200 rpm,
5 min) in 5 mL of distilled water until the supernatant
liquid was clear. Then a 10% suspension (v/v) was
prepared in distilled water. Different concentrations
of plant extracts (10 to 100 mg/mL) were added to
1 mL of glucose solution (10, 25 and 50 mg/mL) and
incubated together for 15 min at 37°C. Then, 100 μL of
the yeast suspension was added, followed by a vortex
and a new incubation at 37°C for 60 min. After one
hour, the tubes were centrifuged (2500 rpm, 5 min)
and glucose was estimated in the supernatant by the
iodine reagent [17]. Metformin was taken as a standard
antidiabetic drug. Absorbance was measured at 540 nm
and all experiments were performed in triplicate. The
percentage increase in glucose uptake by yeast cells was
calculated using the following formula [18]:
% inhibition of glucose uptake = (A sample – A control /
A sample) × 100 (5)
where A is absorbance of the control and experimental
samples.
The data presented in our study were analyzed using
XL Stat Pro 7.5 statistical software. The experiments
were performed in triplicate. The results were presented
as mean values and a standard deviation. ANOVA
test was conducted to determine any significance
differences. P < 0.05 was considered as statistically
significant.
RESULTS AND DISCUSSION
Table 1 demonstrates total phenolic, flavonoid
and flavonol contents of the pomegranate (Punica
granatum L.) extracts. The hydroalcoholic extract
showed a significantly (P < 0.05) higher content of total
phenolic compounds compared to the aqueous extract,
with values of 12.66 ± 0.10 and 4.43 ± 0.01 mg EAG/g
extract, respectively (Table 1). These results were not
consistent with those found by Sinha et al., namely
9.85 ± 0.82 and 14.78 ± 2.10 mg EAG/g extract for the
pomegranate aqueous and hydroalcoholic extracts,
respectively [19].
The hydroalcoholic extract showed a significantly
(P < 0.05) higher content of flavonoids than the
aqueous extract (24.78 ± 1.59 and 8.76 ± 0.90 mg EQ/g,
respectively). These results were closer to those reported
by [19], namely 12.7 ± 0.23 and 26.08 ± 1.24 mg EQ/g
for the aqueous and methanolic extracts, respectively.
According to quantitative analyses, pomegranate leaves
contained a higher amount of flavonoids compared to
phenolic compounds. These results were confirmed
by [19], where pomegranate leaf extracts showed a lower
content of total polyphenols and a higher content of
flavonoids compared to pomegranate bark, flower, and
seed extracts.
Our results indicated that the aqueous extract was
richer in flavonols compared to the hydro-alcoholic
extract; with contents of 9.20 ± 2.80 and 7.68 ± 0.60 mg
EQ/g of extract, respectively (Table 1). The statistical
analyses did not show any significant difference between
the two extracts (P > 0.05).
Table 2 shows the antioxidant capacity of the
pomegranate extracts. The aqueous extract of
pomegranate leaves had a significantly higher (P < 0.05)
total antioxidant capacity with an IC50 value of 12.404 ±
0.136 mg/mL, while the hydroalcoholic extract showed a
significantly lower (P > 0.05) antioxidant capacity with
an IC50 of 18.719 ± 1.001 mg/mL.
Table 1 Total phenolic, flavonoid and flavonol contents
of pomegranate L. extracts
Extract TPC,
mg GAE/g
TFC,
mg QE/g
TFLC,
mg QE/g
Aqueous extract 4.43 ± 0.01b 8.76 ± 0.90b 9.20 ± 2.80a
Hydroalcoholic
extract
12.66 ± 0.10a 24.78 ± 1.59a 7.68 ± 0.60a
Values with different lowercase letters mean they are significantly
different (P < 0.05) (a > b > c)
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In a study of three local varieties of Piper betle
leaves by Dasgupta et al., the Kauri variety showed
the highest total antioxidant capacity expressed in
milligrams of ascorbic acid equivalent per milligram of
extract [20].
According to the FRAP test results, the antioxidant
potential of iron was almost the same for both
hydroalcoholic and aqueous extracts, with IC50 of 32.4 ±
1.109 and 35.12 ± 4.107 mg/mL, respectively (Table 2).
While there were no significant differences
(P > 0.05) between the hydroalcoholic and aqueous
extracts, there was a significant difference (P < 0.05)
between the extracts and ascorbic acid, which showed a
reducing power with an IC50 of 55.531 ± 1.133 mg/mL.
These results were not consistent with those recorded
by [19], namely IC50 of 348.68 ± 24.69 and 293.63 ±
15.29 mg/mL for the aqueous and methanolic extracts of
pomegranate leaves, respectively.
The percentage of hydrogen peroxide scavenging
activity of the hydroalcoholic and aqueous extracts
was 43.57 ± 10.145% and 45.97 ± 6.608%, respectively.
There was no significant difference between the extracts
(P > 0.05) (Table 2).
Compared to the extracts, ascorbic acid showed
a significantly higher (P < 0.05) percentage, namely
85.663 ± 5.024%.
According to the DPPH test results, the
hydroalcoholic extract was significantly the most potent
extract (P < 0.05) w ith a n IC50 of 9.40 ± 1.586 mg/mL,
followed by the aqueous extract with an IC50 of
14.15 ± 1.513 mg/mL (Table 2).
Compared to the extracts, the standard antioxidant
(ascorbic acid) showed a significantly higher (P < 0.05)
antioxidant activity, with an IC50 of 2.27 ± 0.012 mg/mL
(Table 2).
These results were in agreement with the data
reported by Bekiretal, where the methanolic extract
of pomegranate leaves showed a greater antioxidant
activity than the ethanolic extract, with an IC50 of
5.62 ± 0.23 mg/L and 9.25 ± 0.72 mg/L, respectively [4].
The study also revealed comparable antioxidant activity
between the methanolic extract and quercetin (2.86 ±
0.09 mg/L). The dichloromethane extract showed lower
antioxidant activity (IC50 = 71.57 ± 3.65mg/L). However,
the extract obtained with hexane had the lowest DPPH
activity with an IC50 value of 263.44 ± 12.72 mg/L.
According to Fig. 1, the aqueous extract showed
an α- amylase inhibitory concentration of 9.804 ±
0.67 mg/mL. This value was significantly lower
(P < 0.05) than that for acarbose and hydroalcoholic
extracts, with IC50 values of 17.179 ± 4.26 and 19.011 ±
9.82 mg/mL, respectively. On the other hand, there was
no significant difference between the IC50 of acarbose
and the IC50 of the hydroalcoholic extract (P > 0.05).
These inhibition results were not in agreement with
those found by Kam et al., who recorded IC50 inhibitory
concentrations of 0.19 and 0.65 mg/mL for aqueous and
alcoholic extracts of pomegranate, respectively [21].
This inhibitory power can be explained by the fact
that the hydroalcoholic and aqueous extracts have
compounds that bear functional groups close to those of
the substrate (starch), which occupies the active site of
the enzyme.
Figure 2 demonstrates the inhibition of glucose
uptake by yeast. At a concentration of 10 mg/mL of
glucose, metformin showed a significant difference
from the extracts (P < 0 .05), w ith a n I C50 of 5.442 ±
0.047 mg/mL. However, we found no significant
Table 2 Antioxidant activity of pomegranate extracts
Extract Total antioxidant
capacity, mg/mL
FRAP,
mg/mL
Hydrogen peroxide
scavenging, %
DPPH,
mg/mL
Aqueous 12.404 ± 0.136a 35.12 ± 4.107a 45.97 ± 6.608b 14.15 ± 1.513c
Hydroalcoholic 18.719 ± 1.001b 32.4 ± 1.109a 43.57 ± 10.145b 9.40 ± 1.586b
Ascorbic acid / 55.531 ± 1.133b 85.663 ± 5.024a 2.27 ± 0.012a
Values with different lowercase letters mean they are significantly different (P < 0.05) (a < b < c)
Figure 1 Inhibition of α-amylase, IC50, mg/mL Figure 2 Inhibition of glucose uptake by yeast, IC50, mg/mL
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difference between the hydroalcoholic and aqueous
extracts (P > 0.05), with IC50 values of 7.267 ± 0.644 and
6.975 ± 0.394 mg/mL, respectively.
At a concentration of 25 mg/mL of glucose, there
was no significant difference (P > 0.05) between the
aqueous extract, metformin, and hydro-alcoholic extract,
with IC50 values of 7.297 ± 0.76, 5.353 ± 0.11, and 8.509 ±
2.94 mg/mL, respectively (Fig. 2).
At a concentration of 50 mg/mL of glucose, there
was a significant difference between metformin and
the extracts (P < 0.05) and no significant difference
(P > 0 .05) b etween t he e xtracts. T he I C50 values of
metformin, aqueous and hydroalcoholic extracts were
5.499 ± 0.073, 8.379 ± 2.4, and 8.937 ± 2.892 mg/mL,
respectively (Fig. 2).
Based on these results, metformin showed a higher
inhibition capacity than the aqueous and hydroalcoholic
extracts.
According to the results, the antioxidant property
varied according to the extraction solvent. The
antioxidant properties of plant extracts can be explained
by various factors: the presence of natural ascorbic
acid (vitamin C), α-tocopherol (vitamin E), β-carotene
(a precursor of vitamin A), flavonoids, and other
phenolic compounds [22, 23].
These phenolic compounds are capable of acting as
antioxidants that can neutralize free radicals by donating
an electron or a hydrogen atom [24, 25].
The antioxidant capacity of phenolic compounds
is also attributed to their ability to chelate ionic metals
involved in the production of free radicals. For example,
when attaching a ligand (phenolic compound) to Fe+3 in
the FRAP test, polyphenols can reduce iron to Fe+2 [26].
Antioxidants act as “sensors” of free radicals,
fighting against radical oxidation. Antioxidants of
phenolic type react according to a mechanism proposed
by Sherwin in 1976: an antioxidant formally yields a
hydrogen radical, which may be an electron transfer
followed, more or less rapidly, by a proton transfer [27].
Polyphenolic compounds are increasingly being
used in therapeutics [28]. Many studies suggest
that polyphenols participate in the prevention of
cardiovascular diseases. They inhibit the oxidation
of low density lipoproteins and platelet aggregation
involved in the phenomenon of thrombosis that can
lead to occlusion of the arteries [29]. These compounds
show antioxidant activities: they have anti-inflammatory,
antiatherogenic, antithrombotic, analgesic, antibacterial,
and antiviral effects and can act as anticarcinogens, antiallergens,
or vasodilators [30, 31].
Flavonoids also perform many biological functions
that are attributed in part to their antioxidant properties.
These compounds not only inhibit free radicals, but
also neutralize oxidative enzymes and chelate metal
ions responsible for the production of reactive oxygen
species [32].
As for tannins, they are defined as sources of plant
origin because they can precipitate proteins, inhibit
digestive enzymes, and decrease the use of vitamins and
minerals. On the other hand, tannins are also considered
as “health promoting” components in plant-derived
foods and beverages. For example, tannins have been
reported to have anti-carcinogenic and antimutagenic
potential, as well as antimicrobial properties.
The antioxidant activity of pomegranate leaves is
due to their richness in phenolic compounds (tannins,
flavones, glucosides). In fact, the work by Kang et al.
suggested that polar polyphenolic molecules present
in the plant’s extract contributed to the increase in
antiradical activity [33].
As for anti-diabetic activity, Patel et al. reported that
pomegranate extract regulates post-ponderal glucose by
its inhibitory effect on α-amylase [34].
Flavonoids have a high nutritional value because they
are part of our usual diet, which could be explained by
their rapid metabolism, elimination, and relatively low
bioavailability [35].
The reaction mechanisms of α-amylase enzyme
inhibition remain unclear. However, flavonoids in foods
can interact with starch and react with nitrous acid
derived from the oral cavity in the stomach before being
transported to the intestine [36]. This review mainly
deals with: (a) the inhibition of α-amylase activity by
flavonoids, suggesting the mechanisms of inhibition,
and (b) the suppression of starch digestion by flavonoids
by forming starch-flavonoid complexes through
hydrophobic interactions.
The inhibition potential for flavonoids and tannins is
correlated with the number of hydroxyl groups in their B
cycles. These compounds inhibit α-amylase by forming
hydrogen bonds between its hydroxyl groups and the
residues of the active site of this enzyme. Flavonoids or
flavonoid-rich foods can reduce the risk of diabetes by
modulating glucose uptake and insulin secretion [37].
The transport of glucose through the yeast cell
membrane occurs by facilitated diffusion, a passive
mechanism without energy input. Glucose transport is
continued if intracellular glucose is effectively reduced
or used [38].
Scientific evidence shows that apical or luminal
GLUT 2, facilitating the intestinal transport of
glucose, is the major route of glucose uptake and thus
an attractive target for some plant-based inhibitory
agents [39].
Calystegine, a compound found in the pomegranate,
exerts an antidiabetic effect by acting on the absorption
of glucose by a competitive mechanism because of their
structural analogy with glucose [40].
CONCLUSION
Our study demonstrated that pomegranate (Punica
granatum L.) leaf extracts are rich in phenolic
compounds which play a very important role in the
scavenging of free radicals, it makes a significant
contribution to the justification of the antioxidant and
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anti-diabetic activity. It gives the extracts a power to
protect the body against stress and manifestations linked
to diabetes. The hydroalcoholic leaves extract was
effective in preventing diabetes due to its high flavonoid.
Therefore, there is a need for further in vivo studies to
better understand the mechanism of their action.
CONTRIBUTION
M. Cheurfa and A. Azouzi performed the extraction
and chemical characterization. A. Mariod, A. Azouzi,
and M. Cheurfa performed the biological experiments
and wrote the manuscript. M. Cheurfa and M. Achouche
analyzed the data. All the authors revised the manuscript
for publication.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interests.

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