INTRODUCTIONThe pomegranate (Punica granatum L.) is a shrubthat belongs to the Lythraceae family. It is between 5and 10 m tall and is characterized by deciduous fruitingleaves. The pomegranate is used to prevent cancer,cardiovascular disease, diabetes, dental conditions,and erectile dysfunction, as well as against ultra violetradiation. Pomegranate leaf extracts contain high totalphenols, tannins, and triterpenoids [1].Numerous studies have demonstrated the invitro antioxidant activity and polyphenol content ofpomegranate. According to Amjad et al., the antioxidantactivity of pomegranate leaves is directly related tothe presence of phenolic compounds and antioxidantcomponents which act as hydrogen donors, contributingto the concentration of total phenols [2]. These authorsdemonstrated that pomegranate n-butanol, ethyl acetate,hydroethanol, and aqueous leaf extracts containedellagic acid, an efficient free radical scavenger [2]. Vinodhini et al. reported that the aqueous extractof pomegranate leaves had the greatest antioxidantactivity and contained significant levels of total phenolsand flavonoids [3]. The leaf extracts showed antioxidantactivity in vivo by protecting yeast cells againstoxidative stressing agent H2O2. The authors foundpomegranate a good source of natural compounds withhealth benefits, which makes it possible to use it in dietsto reduce oxidative stress.In the study by Bekir et al., the methanolic extractof pomegranate leaves displayed high antioxidant, antiinflammatory,anti-cholinesterase, and antiproliferativeactivities [4]. These results showed that pomegranateleaves could be a potential source of active moleculesintended for applications in pharmaceutical industry.The aim of the study was to evaluate the antioxidantand anti-diabetic activity of aqueous and hydroalcoholicextracts of pomegranate leaves in vitro.STUDY OBJECTS AND METHODSThe pomegranate (Punica granatum L.) leaveswere collected in September 2017 in Chlef, Algeria.The collected samples were dried at room temperatureaway from sunlight and then powdered using an electricmortar.Preparation of aqueous extract. The aqueousextract of pomegranate leaves was prepared accordingto the method described by Diallo et al., with somemodifications [5]. 15 g of powdered leaves in 150 mL ofboiling water was heated for 15 min and filtered throughfilter paper. The filtrate was placed in an oven at 40°Cuntil obtaining a dry extract and stored at 4°C.Preparation of hydroalcoholic extract. Thehydroalcoholic extract of pomegranate leaves wasprepared by maceration of 15 g of powdered leaves in100 mL of a hydroalcoholic solution (70%) at roomtemperature away from light, with maximum agitationfor 72 h [6]. Then the mixture was filtered through filterpaper. The filtrates were placed in an oven at 40°C. Thedry extract was stored in a refrigerator at 4°C.Total polyphenols were determined spectrophotometricallyfollowing the Folin-Cioclateu method [7].For this, 0.2 mL of each leaf extract was mixed in a testtube with 1.0 mL of Folin-Cioclateu reagent and 0.8 mLof a 7.5% sodium carbonate solution (Na2CO3). Afterincubation in the shade and at room temperature for30 min, absorbance was measured at 760 nm. Theresults were expressed in milligram equivalent ofgallic acid per gram of extract (mg EAG/g extract)from a calibration curve prepared using gallic acid as astandard.Flavonoid levels were measured using the methoddescribed by Mbaebie et al. [8]. For this, 1.0 mL of eachextract was added to 1.0 mL of a 2% ethanol solutionof aluminum chloride (AlCl3) and then incubated for anhour at room temperature. Absorbance was measuredby a UV-visible spectrophotometer at 420 nm. Theconcentrations of flavonoids in the extracts werecalculated from the calibration curve and expressed inmilligram equivalent of quercetin per gram of extract(mg EQ/g extracted).Flavonols were determined according to the methoddescribed by Kosalec et al. [9]. For this, 0.3 mL of theextract was mixed with 0.3 mL of aluminum chloride(AlCl3) and 0.45 mL of sodium acetate. The mixturewas vigorously stirred and then incubated for 40 min.Absorbance was measured at 440 nm. The quantificationof flavonols was based on a calibration curve made byquercetin. The content of flavonols was expressed inmilligram equivalent of quercetin per gram of extract(mg EQ/g).Total antioxidant capacity. Determination oftotal antioxidant capacity is a technique based on thereduction of molybdate Mo (VI) to molybdenum Mo (V)in the presence of an antioxidant with the formation ofa green complex (phosphate/Mo (V)) at acidic pH [10].The phosphomolybdate reagent was prepared from amixture of 0.6 M sulfuric acid (H2SO4), 28 mM sodiumphosphate (Na3PO4), and 4 mM ammonium molybdate((NH4) 6Mo7O24 • 4H2O). 1.0 mL of this reagent wasadded to 100 μL of each extract with concentrations of10, 25, 50 and 100 mg/mL. The tubes were incubatedat 95°C for 90 min. After cooling, absorbance wasmeasured at 695 nm. Total antioxidant capacity wasexpressed in milligrams of ascorbic acid equivalent pergram of extract (mg Eq AA/g extract) from a calibrationcurve of ascorbic acid.Ferric Reducing Antioxidant Power (FRAP).The FRAP method involves measuring the abilityof a sample to reduce the tripyridyltriazine ferriccomplex to tripyridyltriazine at a low pH. This ferroustripyridyltriazine complex has an intense blue colormeasured by a spectrophotometer at 593 nm [11].The FRAP reagent was prepared by mixing a300 mM sodium acetate buffer (pH 3.6), a solution of10 mM TPTZ in 40 mM HCl and 20 mM FeCl3 in a ratioof 10:1:1 (v/v/v). 200 μL of each extract (10, 25, 50 and100 mg/mL) was added to 3 mL of the FRAPreagent. After incubation in the dark at 37°C for30 min, absorbance was measured at 593 nm againstthe blank [11].Hydrogen peroxide scavenging activity. Thescavenging capacity of hydrogen peroxide is based onthe reduction of the H2O2 concentration by scavengercompounds, the absorbance value of the latter at230 nm also reduces [12]. A 40 mM hydrogen peroxidesolution was prepared in a 50 mM phosphate buffer(pH 7.4). 4.0 mL of each extract with a concentration of10 mg/mL was mixed with 0.6 mL of the H2O2 solution.After 10 min incubation, absorbance was measuredat 230 nm. Ascorbic acid was used as a positivecontrol [13]. The percent inhibition was calculated usingthe following equation:Percent inhibition (%) = [(A control – A sample) /A control] × 100 (2)where A is absorbance of the control and experimentalsamples.331Cheurfa M. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 329–336DPPH test. To p repare a 0.004% solution of DPPH,250 μL of extracts at concentrations of 10, 25, 50, and100 mg/mL or standard (ascorbic acid) was added to1 mL of the DPPH solution. After incubation in thedark at room temperature for 30 min, absorbancewas measured at 517 nm against a blank sample thatcontained pure methanol [14]. The antioxidant activityevaluated with the DPPH method was expressed inpercentage according to the following formula:% antioxidant activity = [(A control – A sample) /A control] × 100 (2)where A is absorbance of the control and experimentalsamples.Inhibition test of α-amylase enzymatic activity.The inhibition test of α-amylase enzymatic activityfollowed the method of Daksha et al. [15]. For t his, t wosolutions had been prepared, namely a 1% starch stocksolution and a 1% amylase solution in a 0.1 M phosphatebuffer at pH 7.2, both solutions preserved at 4°C. Thereaction mixture contained 2.0 mL of a phosphatebuffer, 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 mixturewas incubated for an hour. The enzymatic reactionwas stopped by the addition of 0.1 mL of the iodideindicator. All experiments were performed in triplicate.Absorbance was measured at 565 nm.The inhibitory activity of each extract was calculatedaccording to the following formula:% inhibition activity = (A sample – A control /A sample) × 100 (3)where A is absorbance of the control and experimentalsamples.To determine effects of the extracts on glucoseuptake by yeast, we prepared yeast cells accordingto the method described in [16]. 1 g of commercialbaker’s yeast was washed by centrifugation (4200 rpm,5 min) in 5 mL of distilled water until the supernatantliquid was clear. Then a 10% suspension (v/v) wasprepared in distilled water. Different concentrationsof plant extracts (10 to 100 mg/mL) were added to1 mL of glucose solution (10, 25 and 50 mg/mL) andincubated together for 15 min at 37°C. Then, 100 μL ofthe yeast suspension was added, followed by a vortexand a new incubation at 37°C for 60 min. After onehour, the tubes were centrifuged (2500 rpm, 5 min)and glucose was estimated in the supernatant by theiodine reagent [17]. Metformin was taken as a standardantidiabetic drug. Absorbance was measured at 540 nmand all experiments were performed in triplicate. Thepercentage increase in glucose uptake by yeast cells wascalculated 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 experimentalsamples.The data presented in our study were analyzed usingXL Stat Pro 7.5 statistical software. The experimentswere performed in triplicate. The results were presentedas mean values and a standard deviation. ANOVAtest was conducted to determine any significancedifferences. P < 0.05 was considered as statisticallysignificant.RESULTS AND DISCUSSIONTable 1 demonstrates total phenolic, flavonoidand flavonol contents of the pomegranate (Punicagranatum L.) extracts. The hydroalcoholic extractshowed a significantly (P < 0.05) higher content of totalphenolic compounds compared to the aqueous extract,with values of 12.66 ± 0.10 and 4.43 ± 0.01 mg EAG/gextract, respectively (Table 1). These results were notconsistent with those found by Sinha et al., namely9.85 ± 0.82 and 14.78 ± 2.10 mg EAG/g extract for thepomegranate aqueous and hydroalcoholic extracts,respectively [19].The hydroalcoholic extract showed a significantly(P < 0.05) higher content of flavonoids than theaqueous extract (24.78 ± 1.59 and 8.76 ± 0.90 mg EQ/g,respectively). These results were closer to those reportedby [19], namely 12.7 ± 0.23 and 26.08 ± 1.24 mg EQ/gfor the aqueous and methanolic extracts, respectively.According to quantitative analyses, pomegranate leavescontained a higher amount of flavonoids compared tophenolic compounds. These results were confirmedby [19], where pomegranate leaf extracts showed a lowercontent of total polyphenols and a higher content offlavonoids compared to pomegranate bark, flower, andseed extracts.Our results indicated that the aqueous extract wasricher in flavonols compared to the hydro-alcoholicextract; with contents of 9.20 ± 2.80 and 7.68 ± 0.60 mgEQ/g of extract, respectively (Table 1). The statisticalanalyses did not show any significant difference betweenthe two extracts (P > 0.05).Table 2 shows the antioxidant capacity of thepomegranate extracts. The aqueous extract ofpomegranate 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 asignificantly lower (P > 0.05) antioxidant capacity withan IC50 of 18.719 ± 1.001 mg/mL.Table 1 Total phenolic, flavonoid and flavonol contentsof pomegranate L. extractsExtract TPC,mg GAE/gTFC,mg QE/gTFLC,mg QE/gAqueous extract 4.43 ± 0.01b 8.76 ± 0.90b 9.20 ± 2.80aHydroalcoholicextract12.66 ± 0.10a 24.78 ± 1.59a 7.68 ± 0.60aValues with different lowercase letters mean they are significantlydifferent (P < 0.05) (a > b > c)332Cheurfa M. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 329–336In a study of three local varieties of Piper betleleaves by Dasgupta et al., the Kauri variety showedthe highest total antioxidant capacity expressed inmilligrams of ascorbic acid equivalent per milligram ofextract [20].According to the FRAP test results, the antioxidantpotential of iron was almost the same for bothhydroalcoholic 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 aqueousextracts, there was a significant difference (P < 0.05)between the extracts and ascorbic acid, which showed areducing power with an IC50 of 55.531 ± 1.133 mg/mL.These results were not consistent with those recordedby [19], namely IC50 of 348.68 ± 24.69 and 293.63 ±15.29 mg/mL for the aqueous and methanolic extracts ofpomegranate leaves, respectively.The percentage of hydrogen peroxide scavengingactivity of the hydroalcoholic and aqueous extractswas 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 showeda significantly higher (P < 0.05) percentage, namely85.663 ± 5.024%.According to the DPPH test results, thehydroalcoholic extract was significantly the most potentextract (P < 0.05) w ith a n IC50 of 9.40 ± 1.586 mg/mL,followed by the aqueous extract with an IC50 of14.15 ± 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 datareported by Bekiretal, where the methanolic extractof pomegranate leaves showed a greater antioxidantactivity than the ethanolic extract, with an IC50 of5.62 ± 0.23 mg/L and 9.25 ± 0.72 mg/L, respectively [4].The study also revealed comparable antioxidant activitybetween the methanolic extract and quercetin (2.86 ±0.09 mg/L). The dichloromethane extract showed lowerantioxidant activity (IC50 = 71.57 ± 3.65mg/L). However,the extract obtained with hexane had the lowest DPPHactivity with an IC50 value of 263.44 ± 12.72 mg/L.According to Fig. 1, the aqueous extract showedan α- amylase inhibitory concentration of 9.804 ±0.67 mg/mL. This value was significantly lower(P < 0.05) than that for acarbose and hydroalcoholicextracts, with IC50 values of 17.179 ± 4.26 and 19.011 ±9.82 mg/mL, respectively. On the other hand, there wasno significant difference between the IC50 of acarboseand the IC50 of the hydroalcoholic extract (P > 0.05).These inhibition results were not in agreement withthose found by Kam et al., who recorded IC50 inhibitoryconcentrations of 0.19 and 0.65 mg/mL for aqueous andalcoholic extracts of pomegranate, respectively [21].This inhibitory power can be explained by the factthat the hydroalcoholic and aqueous extracts havecompounds that bear functional groups close to those ofthe substrate (starch), which occupies the active site ofthe enzyme.Figure 2 demonstrates the inhibition of glucoseuptake by yeast. At a concentration of 10 mg/mL ofglucose, metformin showed a significant differencefrom the extracts (P < 0 .05), w ith a n I C50 of 5.442 ±0.047 mg/mL. However, we found no significantTable 2 Antioxidant activity of pomegranate extractsExtract Total antioxidantcapacity, mg/mLFRAP,mg/mLHydrogen peroxidescavenging, %DPPH,mg/mLAqueous 12.404 ± 0.136a 35.12 ± 4.107a 45.97 ± 6.608b 14.15 ± 1.513cHydroalcoholic 18.719 ± 1.001b 32.4 ± 1.109a 43.57 ± 10.145b 9.40 ± 1.586bAscorbic acid / 55.531 ± 1.133b 85.663 ± 5.024a 2.27 ± 0.012aValues 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/mL333Cheurfa M. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 329–336difference between the hydroalcoholic and aqueousextracts (P > 0.05), with IC50 values of 7.267 ± 0.644 and6.975 ± 0.394 mg/mL, respectively.At a concentration of 25 mg/mL of glucose, therewas no significant difference (P > 0.05) between theaqueous 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, therewas a significant difference between metformin andthe extracts (P < 0.05) and no significant difference(P > 0 .05) b etween t he e xtracts. T he I C50 values ofmetformin, aqueous and hydroalcoholic extracts were5.499 ± 0.073, 8.379 ± 2.4, and 8.937 ± 2.892 mg/mL,respectively (Fig. 2).Based on these results, metformin showed a higherinhibition capacity than the aqueous and hydroalcoholicextracts.According to the results, the antioxidant propertyvaried according to the extraction solvent. Theantioxidant properties of plant extracts can be explainedby various factors: the presence of natural ascorbicacid (vitamin C), α-tocopherol (vitamin E), β-carotene(a precursor of vitamin A), flavonoids, and otherphenolic compounds [22, 23].These phenolic compounds are capable of acting asantioxidants that can neutralize free radicals by donatingan electron or a hydrogen atom [24, 25].The antioxidant capacity of phenolic compoundsis also attributed to their ability to chelate ionic metalsinvolved in the production of free radicals. For example,when attaching a ligand (phenolic compound) to Fe+3 inthe FRAP test, polyphenols can reduce iron to Fe+2 [26].Antioxidants act as “sensors” of free radicals,fighting against radical oxidation. Antioxidants ofphenolic type react according to a mechanism proposedby Sherwin in 1976: an antioxidant formally yields ahydrogen radical, which may be an electron transferfollowed, more or less rapidly, by a proton transfer [27].Polyphenolic compounds are increasingly beingused in therapeutics [28]. Many studies suggestthat polyphenols participate in the prevention ofcardiovascular diseases. They inhibit the oxidationof low density lipoproteins and platelet aggregationinvolved in the phenomenon of thrombosis that canlead to occlusion of the arteries [29]. These compoundsshow 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 functionsthat are attributed in part to their antioxidant properties.These compounds not only inhibit free radicals, butalso neutralize oxidative enzymes and chelate metalions responsible for the production of reactive oxygenspecies [32].As for tannins, they are defined as sources of plantorigin because they can precipitate proteins, inhibitdigestive enzymes, and decrease the use of vitamins andminerals. On the other hand, tannins are also consideredas “health promoting” components in plant-derivedfoods and beverages. For example, tannins have beenreported to have anti-carcinogenic and antimutagenicpotential, as well as antimicrobial properties.The antioxidant activity of pomegranate leaves isdue to their richness in phenolic compounds (tannins,flavones, glucosides). In fact, the work by Kang et al.suggested that polar polyphenolic molecules presentin the plant’s extract contributed to the increase inantiradical activity [33].As for anti-diabetic activity, Patel et al. reported thatpomegranate extract regulates post-ponderal glucose byits inhibitory effect on α-amylase [34].Flavonoids have a high nutritional value because theyare part of our usual diet, which could be explained bytheir rapid metabolism, elimination, and relatively lowbioavailability [35].The reaction mechanisms of α-amylase enzymeinhibition remain unclear. However, flavonoids in foodscan interact with starch and react with nitrous acidderived from the oral cavity in the stomach before beingtransported to the intestine [36]. This review mainlydeals with: (a) the inhibition of α-amylase activity byflavonoids, suggesting the mechanisms of inhibition,and (b) the suppression of starch digestion by flavonoidsby forming starch-flavonoid complexes throughhydrophobic interactions.The inhibition potential for flavonoids and tannins iscorrelated with the number of hydroxyl groups in their Bcycles. These compounds inhibit α-amylase by forminghydrogen bonds between its hydroxyl groups and theresidues of the active site of this enzyme. Flavonoids orflavonoid-rich foods can reduce the risk of diabetes bymodulating glucose uptake and insulin secretion [37].The transport of glucose through the yeast cellmembrane occurs by facilitated diffusion, a passivemechanism without energy input. Glucose transport iscontinued if intracellular glucose is effectively reducedor used [38].Scientific evidence shows that apical or luminalGLUT 2, facilitating the intestinal transport ofglucose, is the major route of glucose uptake and thusan attractive target for some plant-based inhibitoryagents [39].Calystegine, a compound found in the pomegranate,exerts an antidiabetic effect by acting on the absorptionof glucose by a competitive mechanism because of theirstructural analogy with glucose [40].CONCLUSIONOur study demonstrated that pomegranate (Punicagranatum L.) leaf extracts are rich in phenoliccompounds which play a very important role in thescavenging of free radicals, it makes a significantcontribution to the justification of the antioxidant and334Cheurfa M. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 329–336anti-diabetic activity. It gives the extracts a power toprotect the body against stress and manifestations linkedto diabetes. The hydroalcoholic leaves extract waseffective in preventing diabetes due to its high flavonoid.Therefore, there is a need for further in vivo studies tobetter understand the mechanism of their action.CONTRIBUTIONM. Cheurfa and A. Azouzi performed the extractionand chemical characterization. A. Mariod, A. Azouzi,and M. Cheurfa performed the biological experimentsand wrote the manuscript. M. Cheurfa and M. Achoucheanalyzed the data. All the authors revised the manuscriptfor publication.CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterests.