potential antioxidant and antibacterial properties of a popular jujube fruit: apple kul (zizyphus...

POTENTIAL ANTIOXIDANT AND ANTIBACTERIALPROPERTIES OF A POPULAR JUJUBE FRUIT: APPLE KUL(ZIZYPHUS MAURITIANA)RIZWANA AFROZ1, E. M. TANVIR1, MD. ASIFUL ISLAM2, FAHMIDA ALAM2, SIEW HUA GAN2 andMD. IBRAHIM KHALIL1,3,4

1Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh2Human Genome Centre and 3Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan,

Malaysia

4Corresponding author.TEL: 880-171-103-4983;FAX: 8802-770-8069;EMAIL: [email protected]

Received for Publication June 02, 2014Accepted for Publication October 07, 2014

doi:10.1111/jfbc.12100

Conflicts of InterestThe authors declare that they have nocompeting interests.

ABSTRACTThe purpose of the present study was to investigate the antioxidant and antimi-crobial activities of a methanolic extract of Apple kul (Zizyphus mauritiana) asit has not been studied extensively. Apple kul was found to be a rich sourceof polyphenols (52.19 ± 2.38 mg gallic acid equivalents/100 g), flavonoids(13.19 ± 1.31 mg catechin equivalents/100 g), ascorbic acid (48.17 ± 2.04 mgascorbate equivalent/100 g) and tannins (50.20 ± 3.61 mg tannic acid equivalents/100 g). The estimated protein and reducing sugar contents in Apple kul were1.21 ± 0.04 g/100 g and 1.96 ± 0.15 g/100 g, respectively. The high ferric-reducingantioxidant power value (6336.71 ± 554.88 μmol Fe [II]/g) also indicated a highantioxidant potency for Apple kul. Apple kul showed highest activity towardsPseudomonas aeruginosa and Staphylococcus aureus.

PRACTICAL APPLICATIONSApple kul is full of vital potential antioxidants and can act as an antimicrobialagent, which is beneficial to fight against oxidative stress associated diseases as wellas against harmful bacteria to maintain a healthy human life.

INTRODUCTION

There are some reactive oxygen species (ROS) ( [hydroxylradicals, peroxide anions, peroxy radicals, superoxide anionradicals, singlet oxygen, hydrogen peroxide [H2O2], ozone[O3] and hypochlorous acid), which exert harmful effectson living cells. Mitochondria are the major source of theseROS, which are formed as by-products of oxygen metabo-lism following mitochondrial oxidative phosphorylation.If ROS accumulate in the body, they can damage macrobiomolecules such as carbohydrates, lipids, proteins andeven nucleic acids (Shodehinde and Oboh 2013). Theinduced oxidative damage by ROS can further initiate thedevelopment of aging as well as play role in many diseasesincluding cancer, liver injury and cardiovascular diseases(Liao and Yin 2000).

Antioxidants are either endogenous or exogenous sub-stances that prevent the deterioration or damage to cellscaused by oxidation. The normal human body has a good

defense system including enzymatic antioxidants such assuperoxide dismutase, catalase and glutathione peroxidase,as well as nonenzymatic antioxidants including glutathione,ascorbic acid, vitamin E and alpha-tocopherol. These anti-oxidants play an important role by scavenging oxidants andthus protecting against oxidative damage. When the defensesystem is disrupted, usually in the presence of differentpathologic conditions, the body depends on exogenousantioxidants to scavenge free radicals (Singh and Guizani2012). For example, synthetic chemicals such asbutylhydroxyanisole and butylhydroxytoluene added asantioxidants were reported to induce stomach and livertumors, respectively, when administered to animals (Imaidaet al. 1984; Maeura and Williams 1984). In contrast, naturalantioxidants such as vitamin E (tocopherol) haveanticarcinogenic effects (Kahl and Kappus 1993). Therefore,today, many researchers and health critics are questioningthe safety of synthetic compounds used as antioxidants.Hence, scientists are interested in investigating the quality,

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Journal of Food Biochemistry ISSN 1745-4514

1Journal of Food Biochemistry •• (2014) ••–•• © 2014 Wiley Periodicals, Inc.

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quantity and safety issues of using natural antioxidants aspreservatives (retinoids [vitamin A], bioflavonoids [citrin],polyphenols [hydroxytyrosol], tocopherols [vitamin E] andascorbic acid [vitamin C] ), which are abundantly present inmany natural products including tea, vegetables, fruits andhoney. The antioxidant and antimicrobial properties ofplants and plant products are mainly attributable to thepresence of a wide range of phytochemicals including poly-phenols, ascorbic acid, alpha-tocopherol and tannins.

“Bers” or “Jujubes,” locally known as “Boroi” or “Kul,” orsometimes collectively called “Kul boroi,” are one of themost common and popular fruits in Bangladesh. Apple kul(Zizyphus mauritiana) is an improved version of thisvariety, which belongs to the Rhamnaceae family. It is actu-ally a hybrid of the delicious plum or ‘Kul boroi.” The Applekul fruit is oval-shaped and is reddish-green in color(Fig. 1). It is fleshy and juicy and has a smooth skin and asweet taste. Its whole weight is approximately 9.6 g and itcontains 85.94% pulp, 14.06% stone and 40.00% juice of itswhole weight (Ibrahim et al. 2009). Apple kul has a highyielding capacity and is suitable for cultivation. It is usuallymultiplied by vegetative propagation. According to a previ-ous study, the fruits from the Rhamnaceae family have highnutritional values and anti-infectious properties (Al-Rezaet al. 2010).

Generally, Zizyphus species are widely used as medicinalplants in Asian countries, particularly in Taiwan and China,for the treatment of various liver diseases, urinary troubles,allergies, constipation, depression, chronic bronchitis andinsomnia (Li et al. 2005). Despite this fact, the medicinalvalue of Apple kul remains scientifically unproven.Although it is believed that Apple kul may be a rich sourceof polyphenols, flavonoids, vitamins, carbohydrates and

antimicrobial agents, this fact remains to be confirmed. Toour knowledge, there is no available data on the antioxidantand antimicrobial properties of Apple kul to date.

MATERIALS AND METHODS

Chemicals and Reagents

Gallic acid, catechin, 1, 1-diphenyl-2-picrylhydrazyl radical(DPPH) and 2, 4, 6-tris (2-pyridyl)-1, 3, 5-triazine (TPTZ)standards were purchased from Sigma-Aldrich (St. Louis,MO). Tannic acid, L-ascorbic acid, trichloroacetic acid,ammonium molybdate, sodium carbonate (Na2CO3), alu-minum chloride (AlCl3), sodium nitrite (NaNO2), ferroussulfate heptahydrate (FeSO4.7H2O), sodium hydroxide(NaOH) and methanol were purchased from Merck Co.(Darrmstadt, Germany). Folin-Ciocalteu’s phenol reagentwas purchased from LOBA Chemie (Mumbai, India), whileMueller Hinton Agar was purchased from HIMEDIA(Mumbai, India). All of the chemicals and reagents used inthis study were of analytic grade.

Sample Collection

Mature Apple kul fruits were purchased from the localmarkets in Savar, Dhaka, Bangladesh during the late winterof February 2012 and were authenticated by ProfessorNuhu Alam from the Botany Department, JahangirnagarUniversity.

Preparation of the Extract

The fresh matured Apple kul fruits were thoroughly rinsedwith cold water and then cut into small pieces using a sterilestainless steel knife. Then, small pieces of the pulp weremashed using a household blender. To prepare themethanolic extract, the mashed fruit (200 g) was extractedwith methanol by a soxhlet extractor for 6 h. The crudeextract was concentrated in a rotary evaporator (Buchi,Tokyo, Japan) under reduced pressure (100 psi) and at acontrolled temperature (40C). Following this procedure,22.50 g of the extract was collected and finally preserved at−20C for the subsequent studies.

Phytochemical Analysis

Estimation of the Total Polyphenol Contents. Thetotal polyphenol content in the Apple kul extract was deter-mined using a modified Folin-Ciocalteu’s method (Aminet al. 2006). Briefly, the solution (0.4 mL) containing 1 mgof the extract was mixed with 1.6 mL of 7.5% of Na2CO3

solution. After mixing, 2 mL of the 10-fold dilutedFIG. 1. APPLE KUL (ZIZYPHUS MAURITIANA)

ANTIOXIDANT AND ANTIBACTERIAL PROPERTIES OF APPLE KUL R. AFROZ ET AL.

2 Journal of Food Biochemistry •• (2014) ••–•• © 2014 Wiley Periodicals, Inc.

Folin-Ciocalteu’s reagent was added. The final reactionmixture was incubated in the dark for 1 h. In an alkalinesolution, any phosphotungustomolybdic acid present inFolin-Ciocalteu’s reagent is reduced by the polyphenolspresent in the extract to produce a mixture with a verystrong blue color. The intensity of the blue-colored complexwas measured at 765 nm using a PD-303S Spectrophotom-eter (APEL, Angyouryou Negishi, Kawaguchi Saitama,Japan). The concentration of the total polyphenol contentwas determined as gallic acid equivalents (GAEs) at severalconcentrations (5, 10, 20, 40, 80 μg/mL) and is expressed asmg of GAEs per 100 g of Apple kul.

Estimation of the Total Flavonoid Content. In analkaline solution, any flavonoid molecules present in theextract will react with sodium nitrite and aluminum chlo-ride to form a colored flavonoid–aluminum complex. Theflavonoid content in the Apple kul extract was estimatedaccording to the aluminum chloride colorimetric assaymethod (Shiv 2011). Briefly, 1 mL of the extract solutioncontaining 1 mg of Apple kul extract was mixed with 4 mLof distilled water. Then, 0.3 mL of 5% NaNO2 was added tothe reaction mixture and after approximately 5 min, 0.3 mLof 10% AlCl3 was added. Six minutes later, 2 mL of 1 MNaOH was added, followed by the immediate addition of2.4 mL of distilled water to make a total volume of 10 mL.Then, the reaction mixture was properly mixed and theintensity of the colored flavonoid–aluminum complex wasmeasured at 510 nm. The concentration of the total flavo-noid was determined as catechin equivalents (CEQs; 5, 10,20, 40, 80 0μg/mL) and the results are expressed as mg ofCEQs per 100 g of Apple kul.

Estimation of the Ascorbic Acid Content. The ascor-bic acid content in the Apple kul extract was estimated by themethod described by Omaye et al. (1979) with some slightmodifications. The ascorbic acid present in the sample extractwas converted to dehydroascorbic acid before coupling to 2,4-dinitrophenyl hydrazine. This conversion was followed bythe formation of bis-2, 4-dinitrophenyl hydrazones, which isvisible as a stable brownish-red solution based on the follow-ing chemical reactions:

L-ascorbic acid Cu Dehydro-L-ascorbic acid Cu+ ↔ ++ +2 22

Dehydro-L-ascorbic acid Cu

Diketo-L-gulonic acid Cu

+↔ +

+

+2

2

2

Where, Cu2+ = Copper ions.The intensity of the colored compound was measured at

520 nm. The concentration of the ascorbic acid was deter-mined as ascorbate equivalents (AEs; 1.25, 2.50, 5.00, 10.00,

20.00 μg/mL) and the ascorbic acid content is expressed asmg of AEs per 100 g of Apple kul.

Estimation of the Total Tannin Content. The totaltannin content in the Apple kul extract was estimatedaccording to Folin-Ciocalteu’s method (Folin and Ciocalteu1927). Briefly, 0.1 mL of solution containing 1 mg of theextract was mixed with 7.5 mL of distilled water and then0.5 mL of Folin-Ciocalteu’s reagent was added to the solu-tion. Then, 1.0 mL of 35% Na2CO3 and 0.9 mL of distilledwater were added to the solution. The solution was properlymixed and incubated for 30 min. In an alkaline solution, thephosphotungustomolybdic acid present in the Folin-Ciocalteu’s reagent is reduced by the tannin moleculespresent in the extract to produce a very strong blue color.The intensity of this developed blue-colored complex wasmeasured at 725 nm. The concentration of the total tanninswas estimated as tannic acid equivalents (TEs; 12.5, 25.0,50.0, 100.0, 200.0 μg/mL) and the results are expressed asmg of TEs per 100 g of Apple kul.

Estimation of the Total Protein Content. The totalprotein content in the Apple kul extract was estimated usingLowry’s method (Lowry et al. 1951). This method is basedon the formation of a copper–protein complex because ofthe reduction of phosphomolybdate and phosphotungstate(present in Folin-Ciocalteu’s reagent) to hetero-polymolybdenum blue and tungsten blue, respectively.Bovine serum albumin (BSA) (0.05–1.00 mg/mL) was usedas a standard to prepare a calibration curve and the finalresults are expressed as g of BSA equivalents per 100 g.

Estimation of Reducing Sugar Content. The contentof reducing sugars in the Apple kul extract was estimatedaccording to the Nelson-Somgi method. Briefly, 2 mL of theextract (0.25 mg/mL) and standards were transferred intodifferent test tubes followed by the addition of 2 mL ofcopper reagent to each tube. The tubes were heated for15 min in a water bath at 100C before a cooling step. Finally,an arsenomolybdate color reagent (1 mL) was added andthe solution was mixed. The absorbance was read at520 nm. Dextrose was used as a standard for the prepara-tion of the calibration curve (6.25, 12.50, 25.00, 50.00,100.00 μg/mL) and the reducing sugar content is expressedas g of D-glucose per 100 g of Apple kul.

Antioxidant Activity Analysis

DPPH Free-Radical Scavenging Activity. The antioxi-dant potential of the Apple kul extract was investigated byestimating its free radical scavenging effects on DPPH

R. AFROZ ET AL. ANTIOXIDANT AND ANTIBACTERIAL PROPERTIES OF APPLE KUL


radical. The DPPH free radical activity of the Apple kulextract was estimated according to the modified method ofBraca et al. (2002). DPPH is a stable free radical thatstrongly absorbs at 517 nm because of the presence of itsodd electron. In the presence of a free radical scavengingantioxidant (an electron donor), the odd electron of DPPHwill be paired up, thus decreasing the intensity of theabsorption at 517 nm. The extract solution (1 mL) wasmixed with 1.2 mL of 0.003% DPPH in a methanolic solu-tion at different concentrations (31.25, 62.50, 125.00,250.00, 500.00 μg/mL) and the percentage of DPPH inhibi-tion was calculated using the following equation:

% of DPPH inhibition A A ADPPH S DPPH= −( )[ ]×100

where ADPPH = absorbance of DPPH in the absence of theextract; and AS = absorbance of DPPH in the presence ofeither the extract or the standard.

The DPPH scavenging activity is expressed as the concen-tration of the extract required to decrease the DPPH absor-bance by 50% (IC50) and was graphically determined byplotting the absorbance (% of inhibition of DPPH radical)against the log concentration of DPPH using the slope ofthe nonlinear regression.

H2O2 Scavenging Activity. The H2O2 scavenging activ-ity of the Apple kul extract was estimated by the “replace-ment titration method” as described by Zhang with slightmodifications (Zhang 2000). Briefly, 1.0 mL of the extract atvarious concentrations (62.50–1,000.00 μg/mL) was mixedwith 5.0 mL of sulfuric acid (2.0 M) and 3.5 mL of potas-sium iodide (1.8 M) followed by the addition of 50 μL ofH2O2 (8.8 M) and 200.0 μL of 3.0% ammonium molybdate.The solution was mixed well and then titrated with5.09 mM sodium thiosulfate (Na2S2O3) until the disappear-ance of the yellow color was observed. The percentage ofscavenging activity of H2O2 was calculated as follows:

% inhibition V V V= −( ) ×[ ]0 1 0 100

Where, V0 = volume of Na2S2O3 solution used to titrate thecontrol (containing no extract) in the presence of H2O2; andV1 = volume of Na2S2O3 solution used in the presence ofplant extract.

The H2O2 scavenging activity is expressed as the percent-age of inhibition in a concentration-dependent manner.

Ferric-Reducing Antioxidant Power Assay. A ferric-reducing antioxidant power (FRAP) assay was performedbased on the modified method of Benzie and Strain (1999).At low pH, the ferric tripyridyl triazine complex is reducedto the ferrous form, producing an intense blue color that

can be monitored by measuring the change in the absor-bance at 593 nm. Briefly, 200 μL of the solution at differentconcentrations (62.50 125.00, 250.00, 500.00 and1,000.00 μg/mL) was mixed with 1.5 mL of the FRAPreagent. Then, the reaction mixture was incubated at 37Cfor 4 min. The change in the absorbance was monitored at593 nm against a blank that was prepared by using distilledwater. The FRAP reagent was prepared by mixing 10volumes of 300 mM acetate buffer (pH 3.6) with 1 volumeof 10 mM TPTZ solution in 40 mM HCl and 1 volume of20 mM FeCl3.6H2O. The FRAP reagent was pre-warmed at37C and was always freshly prepared. A standard curve wasplotted using an aqueous solution of ferrous sulfateFeSO4.7H2O (100, 200, 400, 600 and 1,000 μmol), whereFRAP was expressed as moles of ferrous equivalent (μmolFe [II] ) per gram of Apple kul.

Antibacterial Activities

Sterility of the Extracts. The Apple kul extracts were fil-tered using Millipore (Millipore, Billerica, Massachusetts,USA) nylon membranes (0.45 μm) and were tested for ste-rility by introducing 2 mL of the extract into 10 mL ofsterile nutrient broth. This step was followed by incubationof the mixture at 37C for 24 h. The sterile extract was indi-cated by the absence of turbidity (i.e., broth clarity) after theincubation period (Atlas 1995).

Bacterial Strains. Five pathogenic bacterial strains(Salmonella paratyphi, Escherichia coli, Chromobacteriumviolaceum, Staphylococcus aureus and Pseudomonasaeruginosa) were used in the antibacterial activity tests. Thestrains were obtained from the Bangladesh Institute forResearch and Rehabilitation in Diabetes, Endocrine and Meta-bolic Disorders. All of the microorganisms were maintainedat 4C on nutrient agar slants. As an additional confirmatorystep, the bacterial strains were reidentified on the basis oftheir morphologic, cultural and biochemical characteristicsaccording to the method established by Cheesbrough (2006).

Determination of Antibacterial Activity. The anti-bacterial activities of the Apple kul extract were determinedusing the agar well diffusion method based on the previ-ously established method by Perez et al. (1990) with someslight modifications. Briefly, a fresh 24-h culture of bacteriawas suspended in sterile distilled water to obtain a turbidityof 0.5 McFarland units. The final inoculum size wasadjusted to 5 × 105 CFU/mL. The test microorganisms wereinoculated on the Mueller Hinton Agar media by spreadingthe bacterial inoculums on its surface. Wells of 8 mm indiameter were punched in the agar plate and were filled



with 100 μL of the Apple kul extracts (500 mg/mL inwater). The negative control wells contained only distilledwater. Phenol (14%) and a standard antibiotic solution ofstreptomycin (100 μg/mL) were run in parallel on the sameplate as positive controls for comparison. The plates werethen incubated at 37C for 24 h. The antibacterial activitieswere determined by measuring the diameters of the zonesof inhibition for the respective positive controls andthe extract. The relative antibacterial potency of the givenextract was calculated by comparing its diameter of zone ofinhibition with those of the positive controls.

Determination of the Minimum InhibitoryConcentration of the Extract. The Apple kul extract(500 mg/mL) was serially diluted to 450, 400, 350, 300, 250,200, 150, 100 and 50 mg/mL using a sterile nutrient broth toobtain a total volume of 3 mL. After obtaining the differentconcentrations of the extract, each concentration wasinoculated with 0.05 mL of a standardized bacterial cell sus-pension (approximately 106 CFU/mL) followed by an incu-bation at 37C for 24 h. The lowest concentration of theextract that inhibited the growth of the test organism wasdefined as the minimum inhibitory concentration (MIC).The experiments were conducted first using (1) nutrientbroth only and then repeated with (2) nutrient broth andsterile Apple kul extract; and (3) nutrient broth and a testorganism.

Statistic Analysis

All analyses were carried out in triplicate and the data areexpressed as mean ± standard deviation (SD). The data wereanalyzed using SPSS (Statistical Packages for Social Science,version 20.0, IBM Corporation, Armonk, New York) andMicrosoft Excel 2007 (Redmond, Washington).

RESULTS AND DISCUSSION

Phytochemical Analysis

Total Polyphenol Contents. Polyphenols serve as pow-erful antioxidants because of the hydrogen-donating abilityof their hydroxyl groups as well as their ability to donateelectrons to arrest the production of free radicals as a resultof oxidative stress (John and Shahidi 2010).

The estimated total polyphenol content of Apple kul was52.19 ± 2.38 mg GAE/100 g (Table 1) (r2 = 0.99) which ishigher than three other edible fruits from Western Ghats ofIndia, namely, Zizyphus rugosa (41.80 ± 0.20 mg GAE/100 g), Flueggea leucopyrus (37.70 ± 4.92 mg GAE/100 g)and Grewia tiliaefolia (44.10 ± 1.81 mg GAE/100 g), which

are consumed in both fresh and dried forms by the localsthroughout the year (Karuppusamy et al. 2011).

Zizyphus species display a robust genetic diversitybecause of natural cross-pollination and self-incompatibility. The difference in total polyphenol contentspresent in Apple kul compared with other variants ofZizyphus species may be due to variations in the geneticmakeup of the Zizyphus species. Apple kul was confirmed tocontain a considerable amount of phenolic compounds andmay thus provide beneficial effects against ROS-induceddamage.

Total Flavonoid Content. Flavonoids have beenreported to provide many beneficial health effects, includingantimicrobial activities, anti-inflammatory effects, inhibi-tion of platelet aggregation and inhibition of histaminemast cells (Nandave et al. 2005).

The estimated flavonoid content of Apple kul(13.19 ± 1.31 mg CEQ/100 g) is higher than those of manyIndian Z. mauritiana varieties, including Chuhara(8.36 ± 1.47 mg CEQ/100 g), Urman (10.76 ± 0.85 mg CEQ/100 g), Sonaur-5 (11.47 ± 1.83 mg CEQ/100 g), Mundia(12.70 ± 1.10 mg CEQ/100 g), Thornless (12.38 ± 0.45 mgCEQ/100 g), but is similar to an Indian Zizyphus genotype,Kaithali (13.09 ± 3.93 mg CEQ/100 g) (Koley et al. 2011)(Fig. 2).

Ascorbic Acid Content. Apple kul also showed a highascorbic acid content (48.17 ± 2.04 mg AE/100 g). The valueis much higher than in the Indian Zizyphus genotypesUrman (19.54 ± 1.85 mg AE/100 g), Seb (21.95 ± 0.50 mgAE/100 g), Sonaur-5 (36.22 ± 0.51 mg AE/100 g) andRashmi (39.29 ± 3.57 mg AE/100 g) (Koley et al. 2011) andis also higher than Z. rugosa (35.00 ± 3.21 mg AE/100 g)(Karuppusamy et al. 2011) and Masau (Z. mauritiana)(15.00 ± 0.00–43.8 ± 0.02 mg/100 g) from the ZambeziValley in Zimbabwe (Nyanga et al. 2012). Ascorbic acid actsas both a reducing and chelating agent that scavenges free

TABLE 1. AMOUNTS OF POLYPHENOLS, FLAVONOIDS, ASCORBICACID, TANNIN, TOTAL PROTEIN AND REDUCING SUGARS PRESENT IN100 g OF APPLE KUL

Phytochemicals Amounts present in Apple kul

Polyphenols (mg/100 g GAEs) 52.19 ± 2.38Flavonoids (mg/100 g CEs) 13.19 ± 1.31Ascorbic acid (mg/100 g AEs) 48.17 ± 2.04Tannin (mg/100 g TEs) 50.20 ± 3.61Total protein (g/100 g) 1.21 ± 0.04Reducing sugars (g/100 g) 1.96 ± 0.15

Data are presented as mean ± standard deviation.AE, ascorbate equivalent; CE, catechin equivalents; GAE, gallic acidequivalents; TE, tannic acid equivalent.



radicals. It directly interacts with ROS and terminates thechain reaction because of ROS-mediated electron transferand is also involved in the regeneration of vitamin E oralpha-tocopherol (Chan 1993).

Total Tannin Content. Tannins are water-soluble sec-ondary metabolites present in plants and display greatstructural diversity. These complex polyphenolic com-pounds are responsible for both the antioxidant and antimi-crobial activities of many plants.

The total tannin content of Apple kul was50.20 ± 3.61 mg TEs/100 g (Table 1) (r2 = 0.998). The highcontent of tannins may contribute to the antibacterial activ-ity of Apple kul. Tannins present in the fruit extract ofZ. mauritiana species have been shown to have significantantibacterial activities against two bacterial pathogenicspecies, E. coli and S. aureus (Das 2012).

Total Protein Content. Apple kul contained1.21 ± 0.04 g protein/100 g of the fruit (Table 1) (r2 =0.996). This protein content is much higher than that of avariety of Indian jujube (Z. mauritiana) (0.80 g/100.0 g)(Sunil 2013) and is similar to that of a jujube variety fromPakistan called “Yazman local” (Z. mauritiana) (1.23 g/100 g) (Malik et al. 2012). The high protein content inApple kul suggests that it has high nutritional value, whichmay be attributed to the presence of many enzymes andother protein-derived components, such as amino acids.

Reducing Sugar Content. Fruits are among the richestsources of different types of carbohydrates. The estimatedamount of reducing sugars present in Apple kul was

1.96 ± 0.15 g/100 g (Table 1) (r2 = 0.962), which is signifi-cantly higher than that in Zizyphus spina-christi from Africa(0.15 ± 0.01 g/100 g) (Amoo and Atasie 2012), an Indianjujube (Z. mauritiana) (1.4–6.2 g/100 g) (Sunil 2013), but issimilar to a jujube variety from Pakistan, namely, Dil-Bahar(Z. mauritiana) (1.99 g/100 g) (Malik et al. 2012). The highlevels of reducing sugars in Apple kul indicate the presenceof glucose, sucrose, fructose, maltose, galactose or othertypes of reducing sugars, but this hypothesis needs to beconfirmed in further investigations. Thus, Apple kul may bea good source of daily dietary carbohydrates for consumers.

Antioxidant Activity Analysis

DPPH Free-Radical Scavenging Activity. The DPPHfree-radical scavenging activity assay is one of the mostcommon methods for investigating the free-radical scaveng-ing activities of plant products.

Different concentrations of Apple kul extract were testedfor DPPH radical scavenging activities and the scavengingactivity was found to increase with increasing concentra-tions of Apple kul. The inhibitory concentration at 50%(IC50) value of Apple kul was 0.73 mg/mL, which is signifi-cantly higher than the values reported for catechin (3.30 μg/mL), ascorbic acid (2.80 μg/mL) and gallic acid (3.00 μg/mL), which were used as standards in the present study.However, this value is much lower than the IC50 of an Indianjujube variety (Z. mauritiana) (6.15 mg/mL) and is similarto the IC50 value of Z. nummularia extract (0.74 mg/mL)(Gupta et al. 2011).

H2O2 Scavenging Activities. H2O2 is a weak oxidizingagent, but may give rise to hydroxyl radicals within cells andthus becomes detrimental to animal tissues. It is postulatedto react with Fe2+ and Cu2+ ions to form hydroxyl radicals(Narayanasamy and Ragavan 2012). H2O2 can oxidize essen-tial thiol (−SH) groups of many important enzymes,thereby inactivating them. Therefore, the neutralization ofhydroxyl radicals or removal of H2O2 is considered vital forthe body’s protection against oxidative stresses. The extractof Apple kul was also able to scavenge H2O2 in aconcentration-dependent manner (Fig. 3).

FRAP Assay. The FRAP assay is a simple and directmethod for the detection of the antioxidant potentials ofdifferent types of samples. Antioxidants present in differenttypes of test samples have the ability to reduce ferric toferrous ions.

The calculated FRAP value of Apple kul was6336.71 ± 554.88 μmol Fe (II)/g of Apple kul, which ismuch higher than that reported in some cultivars of

8.36

10.76 11.47

12.38 12.70 13.09 13.19

0

2

4

6

8

10

12

14

16

18 To

tal f

lavo

noid

con

tent

(mg

CE

/100

g)

Zizyphus species

FIG. 2. THE FLAVONOID CONTENT IN APPLE KUL (GREEN BAR) WASTHE HIGHEST (13.19 ± 1.31 MG CEQ/100 G) COMPARED WITHOTHER INDIAN VARIETIES OF ZIZYPHUS SPECIES



Chinese jujube (Zizyphus jujuba), namely, “Yazao,”“Jianzao,” “Jinsixiaozao,” “Junzao” and “Sanbianhong” withFRAP values that ranged from 342.0 ± 45.8 to1173.0 ± 112.0 μmol Fe (II)/g (Li et al. 2007). The FRAPvalue of Apple kul is also comparatively much higher than15 other promising jujube genotypes (Z. jujube Mill.)from the Mediterranean regions in Turkey with FRAPvalues that ranged from 779 to 1237 μmol Fe (II)/g (Önderet al. 2009), while the value reported for an Indian jujubegenotype (Z. mauritiana Lamk.) was from 7.41 to13.93 trolox/g (Koley et al. 2011). Basically, there are anumber of possibilities for differences in FRAP values asseen. One of them could be the day/night temperature com-binations of different regions as previously suggested byWang and Zheng (2001). Another possibility may be differ-ences in soil temperature, climate and weather, which canaffect fruit quality as suggested by Barrett et al. (Barrettet al. 2007).

Antibacterial Activity. Plant products show significantantimicrobial activities due to the presence of differentphytochemicals. The Apple kul extract showed potent anti-bacterial activity against the five investigated pathogenicbacteria (Table 2).

The largest zone of inhibition was illustrated againstP. aeruginosa (30.50 ± 0.50 mm) and the smallest zoneof inhibition against S. paratyphi (16.67 ± 0.58 mm).P. aeruginosa, an aerobic gram-negative bacterium, isthought to be the main causative agent for lung infectionsor pneumonia (Tortora et al. 2007). Apple kul displayedhigher activity against P. aeruginosa compared withZizyphus abyssinica fruit (11.3 ± 0.3 mm zone of inhibition)(Nyaberi et al. 2012) and should be further investigated as apotential antibacterial agent.

A much larger zone of inhibition is found againstC. violaceum (19.67 ± 0.76 mm) (Table 2) when themethanolic extract of Apple kul is tested for its antibacterialpotential. C. violaceum is a gram-negative facultativeanaerobic protobacterium that rarely infects humans;however, occasionally, this organism may cause severe sys-temic infections by entering the bloodstream via an openwound. Interestingly, the activity of Apple kul againstC. violaceum indicates that the fruit could also be used totreat rarely found severe infections caused by C. violaceum.Conversely, S. aureus is a group of gram-positive bacteriacommonly found on the skin and mucus membranes(Tortora et al. 2007). When methanolic extract of Apple kulwas used against S. aureus, 16.83 ± 0.29 mm zone of inhibi-tion was observed. The antimicrobial activity of Apple kulagainst this commonly found infectious bacteria is com-paratively higher than two other medicinal plants, namely,Tinospora cordifolia and Asparagus racemosus. No measur-able zone of inhibition was observed against S. aureus whenmethanolic extracts of the T. cordifolia stem and theA. racemosus root were investigated (Maharjan et al. 2013).Therefore, it would be particularly useful to investigate theApple kul methanolic extract against P. aeruginosa andS. aureus infections in clinical studies because of the poten-tial activities shown.

Apple kul also had potential activities against E. coli andS. paratyphi. E. coli has been reported as a causative agent ofa number of infections including wound, urinary tract,lung, meningeal and septicemic infections (Olaniran et al.2011). Apple kul extract displayed robust effectivenessagainst E. coli (17.5 ± 1.32 mm zone of inhibition) com-pared with the Z. oenoplia root extract, which showed nomeasureable zone of inhibition against E. coli. The activity

38.89

22.22

16.67

12.22

8.89

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

0 100 200 300 400 500 600 700 800 900 1000

% o

f inh

ibiti

on

Concentration (µg/mL)

FIG. 3. A CONCENTRATION-DEPENDENT H2O2 SCAVENGE APPLE KULEXTRACT WAS SEEN, WHERE, IT CLEARLY ILLUSTRATES THAT, THEHIGHER THE CONCENTRATION OF APPLE KUL EXTRACT, THE HIGHERTHE PERCENTAGE OF H2O2 INHIBITION

TABLE 2. THE ZONES OF INHIBITION FOR THE APPLE KUL EXTRACT AGAINST FIVE PATHOGENIC BACTERIAL SPECIES

Test organismsSparatyphiparatyphi Escherichia coli

Chromobacteriumviolaceum

Staphylococcusaureus

Pseudomonasaeruginosa

Zones ofinhibition (mm)

Apple kul 16.67 ± 0.58a 17.50 ± 1.32a 19.67 ± 0.76b 16.83 ± 0.29a 30.50 ± 0.50b

Streptomycin 16.33 ± 0.58a 18.16 ± 1.04a 16.67 ± 0.58a 15.83 ± 0.76a 16.00 ± 0.50a

Phenol 23.83 ± 0.29b 28.83 ± 0.76b 26.83 ± 0.76c 30.00 ± 1.00b 31.33 ± 1.52b

Data are expressed as mean ± standard deviation. Significantly different values are represented by different letters (superscripts) within the samecolumn (a, b, c) (P < 0.01).



of Apple kul against E. coli is not only better than theZ. oenoplia root extract, but is also better than the Acoruscalamus rhizome, T. cordifolia stem and A. racemosus rootextracts. The absence of a measurable zone of inhibition hasbeen reported in the case of all three previously statedmedicinal plants (Maharjan et al. 2013). Paratyphoid feversare a group of enteric illnesses that are very similar totyphoid fevers caused by serotypic strains of the bacterialgenus Salmonella, namely, S. paratyphi. S. paratyphi hasbeen reported to be resistant to methanolic extracts fromdifferent parts of many other medicinal plants, such as theAegle marmelos fruit, Woodfordia fruticosa flower, A. calamusrhizome, T. cordifolia stem and A. racemosus root extracts(Maharjan et al. 2013). Although the methanolic extract ofApple kul inhibited S. paratyphi (16.67 ± 0.58 mm zone ofinhibition) to a similar extent compared with streptomycin,it showed inferior activity against E. coli compared withstreptomycin. All the previously stated information stronglysupports the medicinal value of Apple kul, which displayedpotential antimicrobial activity against different types ofbacteria.

MIC

MIC is important in laboratories either to confirm the resis-tance of a microorganism to a drug or to monitor the activ-ity of a new antimicrobial agent. The actual mechanism forantimicrobial activity may involve a number of cellular pro-cesses that may lead to an increase in plasma membranepermeability and finally to ion leakage from the cell (Walshet al. 2003).

The lowest MIC for Apple kul is found againstP. aeruginosa and S. aureus (250 mg/mL), while Apple kulexhibited higher MICs (300 mg/mL) against C. violaceum,E. coli and S. paratyphi (Fig. 4), which is consistent with thezones of inhibition previously found. These MIC valueswere similar with many other medicinal plants, e.g., MICvalue of essential oil extracted from leaves of Hyptispectinata (used as a traditional medicine and commonlydistributed throughout America, West Africa and WesternIndia) against P. aeruginosa was 200 mg/mL (Santos et al.2008). Zehneria scabra (traditionally used as medicinalplant) leaf extract MIC value against E. coli was 250 mg/mL(Abew et al. 2014) and MIC value of the methanolic extractof garlic root (commonly used as spice and to treat manydiseases) against S. aureus was 100 mg/mL (Garba et al.2014). Overall, our findings confirmed that the methanolicextract of Apple kul has the potential to be used againstP. aeruginosa and S. aureus infections. Future studies toidentify the chemical constituents present as well as thesafety of the extract in a clinical setting are needed toincrease the number of effective compounds to combat

bacterial infections despite the increase in resistance againstcommonly used antibiotics.

CONCLUSION

Apple kul contained high levels of polyphenols, flavonoids,FRAP and ascorbic acid contents, β-carotene, tannins, totalproteins, reducing sugars and DPPH free radicals, and dis-played strong H2O2 scavenging activities, indicating itshigh antioxidant potential. The free-radical scavengingactivity was observed in a concentration-dependentmanner. The methanolic extract of Apple kul also showedpotent activities against P. aeruginosa and S. aureus as wellas C. violaceum, E. coli and S. paratyphi. The presence ofhigh levels of several bioactive phytochemicals and theresults of antioxidant activity assays indicate that Apple kulis a robust and promising source of natural antioxidantsand antimicrobials.

ACKNOWLEDGMENT

This study was financially supported by a JahangirnagarUniversity research grant, 2012–2013.

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a 300

a 300

a 300

b 250

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S. paratyphi E. coli C. violaceum S. aureus P. aeruginosa

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FIG. 4. THE MICS OF APPLE KUL AGAINST FIVE INVESTIGATEDORGANISMS REVEALS THAT APPLE KUL EXHIBITED HIGHER MICS(300 G/ML) AGAINST CHROMOBACTERIUM VIOLACEUM,ESCHERICHIA COLI AND SPARATYPHI PARATYPHI, WHEREAS, LOWER(250 MG/ML) AGAINST PSEUDOMONAS AERUGINOSA ANDSTAPHYLOCOCCUS AUREUS. SIGNIFICANTLY DIFFERENT VALUES AREREPRESENTED BY DIFFERENT LETTERS (A, B) (P < 0.01)



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potential antioxidant and antibacterial properties of a popular jujube fruit: apple kul (zizyphus...

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