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Journal of Food Biochemistry ISSN 1745-4514

POTENTIAL ANTIOXIDANT AND ANTIBACTERIAL PROPERTIES OF A POPULAR JUJUBE FRUIT: APPLE KUL (ZIZYPHUS MAURITIANA) RIZWANA AFROZ1, E. M. TANVIR1, MD. ASIFUL ISLAM2, FAHMIDA ALAM2, SIEW HUA GAN2 and MD. IBRAHIM KHALIL1,3,4 1 2

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

Malaysia

4

Corresponding author. TEL: 880-171-103-4983; FAX: 8802-770-8069; EMAIL: [email protected] Received for Publication June 02, 2014 Accepted for Publication October 07, 2014 doi:10.1111/jfbc.12100 Conflicts of Interest The authors declare that they have no competing interests.

ABSTRACT The purpose of the present study was to investigate the antioxidant and antimicrobial activities of a methanolic extract of Apple kul (Zizyphus mauritiana) as it has not been studied extensively. Apple kul was found to be a rich source of 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 mg ascorbate 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 were 1.21 ± 0.04 g/100 g and 1.96 ± 0.15 g/100 g, respectively. The high ferric-reducing antioxidant power value (6336.71 ± 554.88 μmol Fe [II]/g) also indicated a high antioxidant potency for Apple kul. Apple kul showed highest activity towards Pseudomonas aeruginosa and Staphylococcus aureus.

PRACTICAL APPLICATIONS Apple kul is full of vital potential antioxidants and can act as an antimicrobial agent, which is beneficial to fight against oxidative stress associated diseases as well as against harmful bacteria to maintain a healthy human life.

INTRODUCTION There are some reactive oxygen species (ROS) ( [hydroxyl radicals, peroxide anions, peroxy radicals, superoxide anion radicals, singlet oxygen, hydrogen peroxide [H2O2], ozone [O3] and hypochlorous acid), which exert harmful effects on living cells. Mitochondria are the major source of these ROS, which are formed as by-products of oxygen metabolism following mitochondrial oxidative phosphorylation. If ROS accumulate in the body, they can damage macro biomolecules such as carbohydrates, lipids, proteins and even nucleic acids (Shodehinde and Oboh 2013). The induced oxidative damage by ROS can further initiate the development of aging as well as play role in many diseases including cancer, liver injury and cardiovascular diseases (Liao and Yin 2000). Antioxidants are either endogenous or exogenous substances that prevent the deterioration or damage to cells caused by oxidation. The normal human body has a good 592

defense system including enzymatic antioxidants such as superoxide dismutase, catalase and glutathione peroxidase, as well as nonenzymatic antioxidants including glutathione, ascorbic acid, vitamin E and alpha-tocopherol. These antioxidants play an important role by scavenging oxidants and thus protecting against oxidative damage. When the defense system is disrupted, usually in the presence of different pathologic conditions, the body depends on exogenous antioxidants to scavenge free radicals (Singh and Guizani 2012). For example, synthetic chemicals such as butylhydroxyanisole and butylhydroxytoluene added as antioxidants were reported to induce stomach and liver tumors, respectively, when administered to animals (Imaida et al. 1984; Maeura and Williams 1984). In contrast, natural antioxidants such as vitamin E (tocopherol) have anticarcinogenic effects (Kahl and Kappus 1993). Therefore, today, many researchers and health critics are questioning the safety of synthetic compounds used as antioxidants. Hence, scientists are interested in investigating the quality, Journal of Food Biochemistry 38 (2014) 592–601 © 2014 Wiley Periodicals, Inc.

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quantity and safety issues of using natural antioxidants as preservatives (retinoids [vitamin A], bioflavonoids [citrin], polyphenols [hydroxytyrosol], tocopherols [vitamin E] and ascorbic acid [vitamin C] ), which are abundantly present in many natural products including tea, vegetables, fruits and honey. The antioxidant and antimicrobial properties of plants and plant products are mainly attributable to the presence of a wide range of phytochemicals including polyphenols, ascorbic acid, alpha-tocopherol and tannins. “Bers” or “Jujubes,” locally known as “Boroi” or “Kul,” or sometimes collectively called “Kul boroi,” are one of the most common and popular fruits in Bangladesh. Apple kul (Zizyphus mauritiana) is an improved version of this variety, which belongs to the Rhamnaceae family. It is actually a hybrid of the delicious plum or ‘Kul boroi.” The Apple kul fruit is oval-shaped and is reddish-green in color (Fig. 1). It is fleshy and juicy and has a smooth skin and a sweet taste. Its whole weight is approximately 9.6 g and it contains 85.94% pulp, 14.06% stone and 40.00% juice of its whole weight (Ibrahim et al. 2009). Apple kul has a high yielding capacity and is suitable for cultivation. It is usually multiplied by vegetative propagation. According to a previous study, the fruits from the Rhamnaceae family have high nutritional values and anti-infectious properties (Al-Reza et al. 2010). Generally, Zizyphus species are widely used as medicinal plants in Asian countries, particularly in Taiwan and China, for the treatment of various liver diseases, urinary troubles, allergies, constipation, depression, chronic bronchitis and insomnia (Li et al. 2005). Despite this fact, the medicinal value of Apple kul remains scientifically unproven. Although it is believed that Apple kul may be a rich source of polyphenols, flavonoids, vitamins, carbohydrates and

ANTIOXIDANT AND ANTIBACTERIAL PROPERTIES OF APPLE KUL

antimicrobial agents, this fact remains to be confirmed. To our knowledge, there is no available data on the antioxidant and 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), aluminum chloride (AlCl3), sodium nitrite (NaNO2), ferrous sulfate heptahydrate (FeSO4.7H2O), sodium hydroxide (NaOH) and methanol were purchased from Merck Co. (Darrmstadt, Germany). Folin-Ciocalteu’s phenol reagent was purchased from LOBA Chemie (Mumbai, India), while Mueller Hinton Agar was purchased from HIMEDIA (Mumbai, India). All of the chemicals and reagents used in this study were of analytic grade.

Sample Collection Mature Apple kul fruits were purchased from the local markets in Savar, Dhaka, Bangladesh during the late winter of February 2012 and were authenticated by Professor Nuhu Alam from the Botany Department, Jahangirnagar University.

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

Phytochemical Analysis

FIG. 1. APPLE KUL (ZIZYPHUS MAURITIANA)

Journal of Food Biochemistry 38 (2014) 592–601 © 2014 Wiley Periodicals, Inc.

Estimation of the Total Polyphenol Contents. The total polyphenol content in the Apple kul extract was determined using a modified Folin-Ciocalteu’s method (Amin et al. 2006). Briefly, the solution (0.4 mL) containing 1 mg of the extract was mixed with 1.6 mL of 7.5% of Na2CO3 solution. After mixing, 2 mL of the 10-fold diluted 593


Folin-Ciocalteu’s reagent was added. The final reaction mixture was incubated in the dark for 1 h. In an alkaline solution, any phosphotungustomolybdic acid present in Folin-Ciocalteu’s reagent is reduced by the polyphenols present in the extract to produce a mixture with a very strong blue color. The intensity of the blue-colored complex was measured at 765 nm using a PD-303S Spectrophotometer (APEL, Angyouryou Negishi, Kawaguchi Saitama, Japan). The concentration of the total polyphenol content was determined as gallic acid equivalents (GAEs) at several concentrations (5, 10, 20, 40, 80 μg/mL) and is expressed as mg of GAEs per 100 g of Apple kul. Estimation of the Total Flavonoid Content. In an alkaline solution, any flavonoid molecules present in the extract will react with sodium nitrite and aluminum chloride to form a colored flavonoid–aluminum complex. The flavonoid content in the Apple kul extract was estimated according to the aluminum chloride colorimetric assay method (Shiv 2011). Briefly, 1 mL of the extract solution containing 1 mg of Apple kul extract was mixed with 4 mL of distilled water. Then, 0.3 mL of 5% NaNO2 was added to the reaction mixture and after approximately 5 min, 0.3 mL of 10% AlCl3 was added. Six minutes later, 2 mL of 1 M NaOH was added, followed by the immediate addition of 2.4 mL of distilled water to make a total volume of 10 mL. Then, the reaction mixture was properly mixed and the intensity of the colored flavonoid–aluminum complex was measured at 510 nm. The concentration of the total flavonoid was determined as catechin equivalents (CEQs; 5, 10, 20, 40, 80 0μg/mL) and the results are expressed as mg of CEQs per 100 g of Apple kul. Estimation of the Ascorbic Acid Content. The ascorbic acid content in the Apple kul extract was estimated by the method described by Omaye et al. (1979) with some slight modifications. The ascorbic acid present in the sample extract was converted to dehydroascorbic acid before coupling to 2, 4-dinitrophenyl hydrazine. This conversion was followed by the formation of bis-2, 4-dinitrophenyl hydrazones, which is visible as a stable brownish-red solution based on the following chemical reactions:

L-ascorbic acid + 2 Cu 2+ ↔ Dehydro-L-ascorbic acid + 2 Cu + Dehydro-L-ascorbic acid + 2 Cu 2+ ↔ Diketo-L-gulonic acid + 2 Cu +

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20.00 μg/mL) and the ascorbic acid content is expressed as mg of AEs per 100 g of Apple kul. Estimation of the Total Tannin Content. The total tannin content in the Apple kul extract was estimated according to Folin-Ciocalteu’s method (Folin and Ciocalteu 1927). Briefly, 0.1 mL of solution containing 1 mg of the extract was mixed with 7.5 mL of distilled water and then 0.5 mL of Folin-Ciocalteu’s reagent was added to the solution. Then, 1.0 mL of 35% Na2CO3 and 0.9 mL of distilled water were added to the solution. The solution was properly mixed and incubated for 30 min. In an alkaline solution, the phosphotungustomolybdic acid present in the FolinCiocalteu’s reagent is reduced by the tannin molecules present in the extract to produce a very strong blue color. The intensity of this developed blue-colored complex was measured at 725 nm. The concentration of the total tannins was estimated as tannic acid equivalents (TEs; 12.5, 25.0, 50.0, 100.0, 200.0 μg/mL) and the results are expressed as mg of TEs per 100 g of Apple kul. Estimation of the Total Protein Content. The total protein content in the Apple kul extract was estimated using Lowry’s method (Lowry et al. 1951). This method is based on the formation of a copper–protein complex because of the reduction of phosphomolybdate and phosphotungstate (present in Folin-Ciocalteu’s reagent) to heteropolymolybdenum blue and tungsten blue, respectively. Bovine serum albumin (BSA) (0.05–1.00 mg/mL) was used as a standard to prepare a calibration curve and the final results are expressed as g of BSA equivalents per 100 g. Estimation of Reducing Sugar Content. The content of reducing sugars in the Apple kul extract was estimated according to the Nelson-Somgi method. Briefly, 2 mL of the extract (0.25 mg/mL) and standards were transferred into different test tubes followed by the addition of 2 mL of copper reagent to each tube. The tubes were heated for 15 min in a water bath at 100C before a cooling step. Finally, an arsenomolybdate color reagent (1 mL) was added and the solution was mixed. The absorbance was read at 520 nm. Dextrose was used as a standard for the preparation of the calibration curve (6.25, 12.50, 25.00, 50.00, 100.00 μg/mL) and the reducing sugar content is expressed as g of D-glucose per 100 g of Apple kul.

Antioxidant Activity Analysis Where, Cu2+ = Copper ions. The intensity of the colored compound was measured at 520 nm. The concentration of the ascorbic acid was determined as ascorbate equivalents (AEs; 1.25, 2.50, 5.00, 10.00, 594

DPPH Free-Radical Scavenging Activity. The antioxidant potential of the Apple kul extract was investigated by estimating its free radical scavenging effects on DPPH Journal of Food Biochemistry 38 (2014) 592–601 © 2014 Wiley Periodicals, Inc.

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radical. The DPPH free radical activity of the Apple kul extract was estimated according to the modified method of Braca et al. (2002). DPPH is a stable free radical that strongly absorbs at 517 nm because of the presence of its odd electron. In the presence of a free radical scavenging antioxidant (an electron donor), the odd electron of DPPH will be paired up, thus decreasing the intensity of the absorption at 517 nm. The extract solution (1 mL) was mixed with 1.2 mL of 0.003% DPPH in a methanolic solution at different concentrations (31.25, 62.50, 125.00, 250.00, 500.00 μg/mL) and the percentage of DPPH inhibition was calculated using the following equation:

% of DPPH inhibition = [( A DPPH − A S ) A DPPH ] ×100 where ADPPH = absorbance of DPPH in the absence of the extract; and AS = absorbance of DPPH in the presence of either the extract or the standard. The DPPH scavenging activity is expressed as the concentration of the extract required to decrease the DPPH absorbance by 50% (IC50) and was graphically determined by plotting the absorbance (% of inhibition of DPPH radical) against the log concentration of DPPH using the slope of the nonlinear regression. H2O2 Scavenging Activity. The H2O2 scavenging activity of the Apple kul extract was estimated by the “replacement titration method” as described by Zhang with slight modifications (Zhang 2000). Briefly, 1.0 mL of the extract at various concentrations (62.50–1,000.00 μg/mL) was mixed with 5.0 mL of sulfuric acid (2.0 M) and 3.5 mL of potassium iodide (1.8 M) followed by the addition of 50 μL of H2O2 (8.8 M) and 200.0 μL of 3.0% ammonium molybdate. The solution was mixed well and then titrated with 5.09 mM sodium thiosulfate (Na2S2O3) until the disappearance of the yellow color was observed. The percentage of scavenging activity of H2O2 was calculated as follows:

% inhibition = [( V 0 − V1) V 0 × 100] Where, V0 = volume of Na2S2O3 solution used to titrate the control (containing no extract) in the presence of H2O2; and V1 = volume of Na2S2O3 solution used in the presence of plant extract. The H2O2 scavenging activity is expressed as the percentage of inhibition in a concentration-dependent manner. Ferric-Reducing Antioxidant Power Assay. A ferricreducing antioxidant power (FRAP) assay was performed based on the modified method of Benzie and Strain (1999). At low pH, the ferric tripyridyl triazine complex is reduced to the ferrous form, producing an intense blue color that Journal of Food Biochemistry 38 (2014) 592–601 © 2014 Wiley Periodicals, Inc.


can be monitored by measuring the change in the absorbance at 593 nm. Briefly, 200 μL of the solution at different concentrations (62.50 125.00, 250.00, 500.00 and 1,000.00 μg/mL) was mixed with 1.5 mL of the FRAP reagent. Then, the reaction mixture was incubated at 37C for 4 min. The change in the absorbance was monitored at 593 nm against a blank that was prepared by using distilled water. The FRAP reagent was prepared by mixing 10 volumes of 300 mM acetate buffer (pH 3.6) with 1 volume of 10 mM TPTZ solution in 40 mM HCl and 1 volume of 20 mM FeCl3.6H2O. The FRAP reagent was pre-warmed at 37C and was always freshly prepared. A standard curve was plotted using an aqueous solution of ferrous sulfate FeSO4.7H2O (100, 200, 400, 600 and 1,000 μmol), where FRAP was expressed as moles of ferrous equivalent (μmol Fe [II] ) per gram of Apple kul.

Antibacterial Activities Sterility of the Extracts. The Apple kul extracts were filtered using Millipore (Millipore, Billerica, Massachusetts, USA) nylon membranes (0.45 μm) and were tested for sterility by introducing 2 mL of the extract into 10 mL of sterile nutrient broth. This step was followed by incubation of the mixture at 37C for 24 h. The sterile extract was indicated by the absence of turbidity (i.e., broth clarity) after the incubation period (Atlas 1995). Bacterial Strains. Five pathogenic bacterial strains (Salmonella paratyphi, Escherichia coli, Chromobacterium violaceum, Staphylococcus aureus and Pseudomonas aeruginosa) were used in the antibacterial activity tests. The strains were obtained from the Bangladesh Institute for Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders. All of the microorganisms were maintained at 4C on nutrient agar slants. As an additional confirmatory step, the bacterial strains were reidentified on the basis of their morphologic, cultural and biochemical characteristics according to the method established by Cheesbrough (2006). Determination of Antibacterial Activity. The antibacterial activities of the Apple kul extract were determined using the agar well diffusion method based on the previously established method by Perez et al. (1990) with some slight modifications. Briefly, a fresh 24-h culture of bacteria was suspended in sterile distilled water to obtain a turbidity of 0.5 McFarland units. The final inoculum size was adjusted to 5 × 105 CFU/mL. The test microorganisms were inoculated on the Mueller Hinton Agar media by spreading the bacterial inoculums on its surface. Wells of 8 mm in diameter were punched in the agar plate and were filled 595


with 100 μL of the Apple kul extracts (500 mg/mL in water). The negative control wells contained only distilled water. Phenol (14%) and a standard antibiotic solution of streptomycin (100 μg/mL) were run in parallel on the same plate as positive controls for comparison. The plates were then incubated at 37C for 24 h. The antibacterial activities were determined by measuring the diameters of the zones of inhibition for the respective positive controls and the extract. The relative antibacterial potency of the given extract was calculated by comparing its diameter of zone of inhibition with those of the positive controls.

Determination of the Minimum Inhibitory Concentration 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 to obtain a total volume of 3 mL. After obtaining the different concentrations of the extract, each concentration was inoculated with 0.05 mL of a standardized bacterial cell suspension (approximately 106 CFU/mL) followed by an incubation at 37C for 24 h. The lowest concentration of the extract that inhibited the growth of the test organism was defined as the minimum inhibitory concentration (MIC). The experiments were conducted first using (1) nutrient broth only and then repeated with (2) nutrient broth and sterile Apple kul extract; and (3) nutrient broth and a test organism.

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

RESULTS AND DISCUSSION Phytochemical Analysis Total Polyphenol Contents. Polyphenols serve as powerful antioxidants because of the hydrogen-donating ability of their hydroxyl groups as well as their ability to donate electrons to arrest the production of free radicals as a result of oxidative stress (John and Shahidi 2010). The estimated total polyphenol content of Apple kul was 52.19 ± 2.38 mg GAE/100 g (Table 1) (r2 = 0.99) which is higher than three other edible fruits from Western Ghats of India, 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 596

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TABLE 1. AMOUNTS OF POLYPHENOLS, FLAVONOIDS, ASCORBIC ACID, TANNIN, TOTAL PROTEIN AND REDUCING SUGARS PRESENT IN 100 g OF APPLE KUL Phytochemicals

Amounts present in Apple kul

Polyphenols (mg/100 g GAEs) Flavonoids (mg/100 g CEs) Ascorbic acid (mg/100 g AEs) Tannin (mg/100 g TEs) Total protein (g/100 g) Reducing sugars (g/100 g)

52.19 ± 2.38 13.19 ± 1.31 48.17 ± 2.04 50.20 ± 3.61 1.21 ± 0.04 1.96 ± 0.15

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

are consumed in both fresh and dried forms by the locals throughout the year (Karuppusamy et al. 2011). Zizyphus species display a robust genetic diversity because of natural cross-pollination and selfincompatibility. The difference in total polyphenol contents present in Apple kul compared with other variants of Zizyphus species may be due to variations in the genetic makeup of the Zizyphus species. Apple kul was confirmed to contain a considerable amount of phenolic compounds and may thus provide beneficial effects against ROS-induced damage. Total Flavonoid Content. Flavonoids have been reported to provide many beneficial health effects, including antimicrobial activities, anti-inflammatory effects, inhibition of platelet aggregation and inhibition of histamine mast 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 many Indian 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 mg CEQ/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 high ascorbic acid content (48.17 ± 2.04 mg AE/100 g). The value is much higher than in the Indian Zizyphus genotypes Urman (19.54 ± 1.85 mg AE/100 g), Seb (21.95 ± 0.50 mg AE/100 g), Sonaur-5 (36.22 ± 0.51 mg AE/100 g) and Rashmi (39.29 ± 3.57 mg AE/100 g) (Koley et al. 2011) and is 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 Zambezi Valley in Zimbabwe (Nyanga et al. 2012). Ascorbic acid acts as both a reducing and chelating agent that scavenges free Journal of Food Biochemistry 38 (2014) 592–601 © 2014 Wiley Periodicals, Inc.

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Total flavonoid content (mg CE/100 g)

18 16 14 12 10

10.76

11.47

12.38

12.70

13.09

13.19

8.36

8 6 4 2

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

0

Antioxidant Activity Analysis Zizyphus species

FIG. 2. THE FLAVONOID CONTENT IN APPLE KUL (RED BAR) WAS THE HIGHEST (13.19 ± 1.31 MG CEQ/100 G) COMPARED WITH OTHER INDIAN VARIETIES OF ZIZYPHUS SPECIES

radicals. It directly interacts with ROS and terminates the chain reaction because of ROS-mediated electron transfer and is also involved in the regeneration of vitamin E or alpha-tocopherol (Chan 1993). Total Tannin Content. Tannins are water-soluble secondary metabolites present in plants and display great structural diversity. These complex polyphenolic compounds are responsible for both the antioxidant and antimicrobial activities of many plants. The total tannin content of Apple kul was 50.20 ± 3.61 mg TEs/100 g (Table 1) (r2 = 0.998). The high content of tannins may contribute to the antibacterial activity of Apple kul. Tannins present in the fruit extract of Z. mauritiana species have been shown to have significant antibacterial activities against two bacterial pathogenic species, E. coli and S. aureus (Das 2012). Total Protein Content. Apple kul contained 1.21 ± 0.04 g protein/100 g of the fruit (Table 1) (r2 = 0.996). This protein content is much higher than that of a variety of Indian jujube (Z. mauritiana) (0.80 g/100.0 g) (Sunil 2013) and is similar to that of a jujube variety from Pakistan called “Yazman local” (Z. mauritiana) (1.23 g/ 100 g) (Malik et al. 2012). The high protein content in Apple kul suggests that it has high nutritional value, which may be attributed to the presence of many enzymes and other protein-derived components, such as amino acids. Reducing Sugar Content. Fruits are among the richest sources of different types of carbohydrates. The estimated amount of reducing sugars present in Apple kul was Journal of Food Biochemistry 38 (2014) 592–601 © 2014 Wiley Periodicals, Inc.

DPPH Free-Radical Scavenging Activity. The DPPH free-radical scavenging activity assay is one of the most common methods for investigating the free-radical scavenging activities of plant products. Different concentrations of Apple kul extract were tested for DPPH radical scavenging activities and the scavenging activity was found to increase with increasing concentrations of Apple kul. The inhibitory concentration at 50% (IC50) value of Apple kul was 0.73 mg/mL, which is significantly 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 Indian jujube variety (Z. mauritiana) (6.15 mg/mL) and is similar to the IC50 value of Z. nummularia extract (0.74 mg/mL) (Gupta et al. 2011). H2O2 Scavenging Activities. H2O2 is a weak oxidizing agent, but may give rise to hydroxyl radicals within cells and thus becomes detrimental to animal tissues. It is postulated to react with Fe2+ and Cu2+ ions to form hydroxyl radicals (Narayanasamy and Ragavan 2012). H2O2 can oxidize essential thiol (−SH) groups of many important enzymes, thereby inactivating them. Therefore, the neutralization of hydroxyl radicals or removal of H2O2 is considered vital for the body’s protection against oxidative stresses. The extract of Apple kul was also able to scavenge H2O2 in a concentration-dependent manner (Fig. 3). FRAP Assay. The FRAP assay is a simple and direct method for the detection of the antioxidant potentials of different types of samples. Antioxidants present in different types of test samples have the ability to reduce ferric to ferrous ions. The calculated FRAP value of Apple kul was 6336.71 ± 554.88 μmol Fe (II)/g of Apple kul, which is much higher than that reported in some cultivars of 597


40.00

38.89

35.00

% of inhibition

30.00 25.00 22.22

20.00 16.67

15.00 12.22

10.00

8.89

5.00 0.00 0

100

200

300

400

500

600

700

800

900

1000

Concentration (µg/mL)

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

Chinese jujube (Zizyphus jujuba), namely, “Yazao,” “Jianzao,” “Jinsixiaozao,” “Junzao” and “Sanbianhong” with FRAP values that ranged from 342.0 ± 45.8 to 1173.0 ± 112.0 μmol Fe (II)/g (Li et al. 2007). The FRAP value of Apple kul is also comparatively much higher than 15 other promising jujube genotypes (Z. jujube Mill.) from the Mediterranean regions in Turkey with FRAP values that ranged from 779 to 1237 μmol Fe (II)/g (Önder et al. 2009), while the value reported for an Indian jujube genotype (Z. mauritiana Lamk.) was from 7.41 to 13.93 trolox/g (Koley et al. 2011). Basically, there are a number of possibilities for differences in FRAP values as seen. One of them could be the day/night temperature combinations of different regions as previously suggested by Wang and Zheng (2001). Another possibility may be differences in soil temperature, climate and weather, which can affect fruit quality as suggested by Barrett et al. (Barrett et al. 2007). Antibacterial Activity. Plant products show significant antimicrobial activities due to the presence of different phytochemicals. The Apple kul extract showed potent antibacterial activity against the five investigated pathogenic bacteria (Table 2).

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The largest zone of inhibition was illustrated against P. aeruginosa (30.50 ± 0.50 mm) and the smallest zone of inhibition against S. paratyphi (16.67 ± 0.58 mm). P. aeruginosa, an aerobic gram-negative bacterium, is thought to be the main causative agent for lung infections or pneumonia (Tortora et al. 2007). Apple kul displayed higher activity against P. aeruginosa compared with Zizyphus abyssinica fruit (11.3 ± 0.3 mm zone of inhibition) (Nyaberi et al. 2012) and should be further investigated as a potential antibacterial agent. A much larger zone of inhibition is found against C. violaceum (19.67 ± 0.76 mm) (Table 2) when the methanolic extract of Apple kul is tested for its antibacterial potential. C. violaceum is a gram-negative facultative anaerobic protobacterium that rarely infects humans; however, occasionally, this organism may cause severe systemic infections by entering the bloodstream via an open wound. Interestingly, the activity of Apple kul against C. violaceum indicates that the fruit could also be used to treat rarely found severe infections caused by C. violaceum. Conversely, S. aureus is a group of gram-positive bacteria commonly found on the skin and mucus membranes (Tortora et al. 2007). When methanolic extract of Apple kul was used against S. aureus, 16.83 ± 0.29 mm zone of inhibition was observed. The antimicrobial activity of Apple kul against this commonly found infectious bacteria is comparatively higher than two other medicinal plants, namely, Tinospora cordifolia and Asparagus racemosus. No measurable zone of inhibition was observed against S. aureus when methanolic extracts of the T. cordifolia stem and the A. racemosus root were investigated (Maharjan et al. 2013). Therefore, it would be particularly useful to investigate the Apple kul methanolic extract against P. aeruginosa and S. aureus infections in clinical studies because of the potential activities shown. Apple kul also had potential activities against E. coli and S. paratyphi. E. coli has been reported as a causative agent of a number of infections including wound, urinary tract, lung, meningeal and septicemic infections (Olaniran et al. 2011). Apple kul extract displayed robust effectiveness against E. coli (17.5 ± 1.32 mm zone of inhibition) compared with the Z. oenoplia root extract, which showed no measureable zone of inhibition against E. coli. The activity

TABLE 2. THE ZONES OF INHIBITION FOR THE APPLE KUL EXTRACT AGAINST FIVE PATHOGENIC BACTERIAL SPECIES Test organisms Zones of inhibition (mm)

Apple kul Streptomycin Phenol

Sparatyphi paratyphi

Escherichia coli

Chromobacterium violaceum

Staphylococcus aureus

Pseudomonas aeruginosa

16.67 ± 0.58a 16.33 ± 0.58a 23.83 ± 0.29b

17.50 ± 1.32a 18.16 ± 1.04a 28.83 ± 0.76b

19.67 ± 0.76b 16.67 ± 0.58a 26.83 ± 0.76c

16.83 ± 0.29a 15.83 ± 0.76a 30.00 ± 1.00b

30.50 ± 0.50b 16.00 ± 0.50a 31.33 ± 1.52b

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

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of Apple kul against E. coli is not only better than the Z. oenoplia root extract, but is also better than the Acorus calamus rhizome, T. cordifolia stem and A. racemosus root extracts. The absence of a measurable zone of inhibition has been reported in the case of all three previously stated medicinal plants (Maharjan et al. 2013). Paratyphoid fevers are a group of enteric illnesses that are very similar to typhoid fevers caused by serotypic strains of the bacterial genus Salmonella, namely, S. paratyphi. S. paratyphi has been reported to be resistant to methanolic extracts from different parts of many other medicinal plants, such as the Aegle marmelos fruit, Woodfordia fruticosa flower, A. calamus rhizome, T. cordifolia stem and A. racemosus root extracts (Maharjan et al. 2013). Although the methanolic extract of Apple kul inhibited S. paratyphi (16.67 ± 0.58 mm zone of inhibition) to a similar extent compared with streptomycin, it showed inferior activity against E. coli compared with streptomycin. All the previously stated information strongly supports the medicinal value of Apple kul, which displayed potential antimicrobial activity against different types of bacteria.

MIC MIC is important in laboratories either to confirm the resistance of a microorganism to a drug or to monitor the activity of a new antimicrobial agent. The actual mechanism for antimicrobial activity may involve a number of cellular processes that may lead to an increase in plasma membrane permeability and finally to ion leakage from the cell (Walsh et al. 2003). The lowest MIC for Apple kul is found against P. aeruginosa and S. aureus (250 mg/mL), while Apple kul exhibited higher MICs (300 mg/mL) against C. violaceum, E. coli and S. paratyphi (Fig. 4), which is consistent with the zones of inhibition previously found. These MIC values were similar with many other medicinal plants, e.g., MIC value of essential oil extracted from leaves of Hyptis pectinata (used as a traditional medicine and commonly distributed throughout America, West Africa and Western India) against P. aeruginosa was 200 mg/mL (Santos et al. 2008). Zehneria scabra (traditionally used as medicinal plant) leaf extract MIC value against E. coli was 250 mg/mL (Abew et al. 2014) and MIC value of the methanolic extract of garlic root (commonly used as spice and to treat many diseases) against S. aureus was 100 mg/mL (Garba et al. 2014). Overall, our findings confirmed that the methanolic extract of Apple kul has the potential to be used against P. aeruginosa and S. aureus infections. Future studies to identify the chemical constituents present as well as the safety of the extract in a clinical setting are needed to increase the number of effective compounds to combat Journal of Food Biochemistry 38 (2014) 592–601 © 2014 Wiley Periodicals, Inc.


310 300

a 300

a 300

a 300

290 MIC (mg/mL)

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280 270 260 250

b 250

b 250

S. aureus

P. aeruginosa

240 230 220 S. paratyphi

E. coli

C. violaceum Microorganisms

FIG. 4. THE MICS OF APPLE KUL AGAINST FIVE INVESTIGATED ORGANISMS 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 AND STAPHYLOCOCCUS AUREUS. SIGNIFICANTLY DIFFERENT VALUES ARE REPRESENTED BY DIFFERENT LETTERS (A, B) (P < 0.01)

bacterial infections despite the increase in resistance against commonly used antibiotics.

CONCLUSION Apple kul contained high levels of polyphenols, flavonoids, FRAP and ascorbic acid contents, β-carotene, tannins, total proteins, reducing sugars and DPPH free radicals, and displayed strong H2O2 scavenging activities, indicating its high antioxidant potential. The free-radical scavenging activity was observed in a concentration-dependent manner. The methanolic extract of Apple kul also showed potent activities against P. aeruginosa and S. aureus as well as C. violaceum, E. coli and S. paratyphi. The presence of high levels of several bioactive phytochemicals and the results of antioxidant activity assays indicate that Apple kul is a robust and promising source of natural antioxidants and antimicrobials.

ACKNOWLEDGMENT This study was financially supported by a Jahangirnagar University research grant, 2012–2013.

REFERENCES ABEW, B., SAHILE, S. and MOGES, F. 2014. In vitro antibacterial activity of leaf extracts of Zehneria scabra and

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Ricinus communis against Escherichia coli and methicillin resistance Staphylococcus aureus. Asian Pac. J. Trop. Biomed. 4, 805–809. AL-REZA, S.M., YOON, J.I., KIM, H.J., KIM, J.S. and KANG, S.C. 2010. Anti-inflammatory activity of seed essential oil from Zizyphus jujube. Food Chem. Toxicol. 48, 639–643. AMIN, I., NORAZAIDAH, Y. and HAINIDA, K.I.E. 2006. Antioxidant activity and phenolic content of raw and blanched Amaranthus species. Food Chem. 94, 47–52. AMOO, I.A. and ATASIE, V.N. 2012. Nutritional and functional properties of Tamarindus indica pulp and Zizyphus spina-christi fruit and seed. J. Food Agric. Environ. 10, 16–19. ATLAS, R.M. 1995. Micro-Organisms in Our World, p. 765, Mosby-Year Book, St. Louis, MO. BARRETT, D.M., WEAKLEY, C., DIAZ, J.V. and WATNIK, M. 2007. Qualitative and nutritional differences in processing tomatoes grown under commercial organic and conventional production systems. J. Food Sci. 72, C441–C451. BENZIE, I.F. and STRAIN, J.J. 1999. Ferric reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299, 15–27. BRACA, A., SORTINO, C., POLITI, M., MORELLI, I. and MENDEZ, J. 2002. Antioxidant activity of flavonoids from Licania licaniaeflora. J. Ethnopharmacol. 79, 379–381. CHAN, A.C. 1993. Partners in defense, vitamin E and vitamin C. Can. J. Physiol. Pharmacol. 71, 725–731. CHEESBROUGH, M. 2006. District Laboratory Practice In Tropical Countries, Vol 2, pp. 256–267, Cambridge University Press, Cambridge, NY and Melbourne, Australia. DAS, S. 2012. Antimicrobial and antioxidant activities of green and ripe fruits of Averrhoa carambola Linn. and Zizyphus mauritiana Lam. Asian J. Pharm. Clin. Res. 5, 102–105. FOLIN, O. and CIOCALTEU, V. 1927. On tyrosine and tryptophane determinations in proteins. J. Biol. Chem. 73, 627–650. GARBA, I., UMAR, A., ABDULRAHMAN, A., TIJJANI, M., ALIYU, M., ZANGO, U. and MUHAMMAD, A. 2014. Phytochemical and antibacterial properties of garlic extracts. BAJOPAS. 6, 45–48. GUPTA, D., MANN, S., JAIN, I. and GUPTA, R.K. 2011. Phytochemical, nutritional and antioxidant activity evaluation of fruits of Ziziphus nummularia Burm. F. Int. J. Pharm. Biol. Sci. 2, 629–638. IBRAHIM, M., SHAFIQUE, M., HELALI, M., RAHMAN, M., BISWAS, S. and ISLAM, M. 2009. Studies on the physiological and biochemical composition of different ber (Zizyphus mauritiana Lamk.) cultivars at Rajshahi. Bangladesh J. Sci. Ind. Res. 44, 229–232. IMAIDA, K., FUKUSHIMA, S., SHIRAI, T., MASUI, T., OGISO, T. and ITO, N. 1984. Promoting activities of butylated hydroxyanisole, butylated hydroxytoluene and sodium

600

R. AFROZ ET AL.

L-ascorbate on forestomach and urinary bladder carcinogenesis initiated with methylnitrosourea in F344 male rats. Gan 75, 769–775. JOHN, J.A. and SHAHIDI, F. 2010. Phenolic compounds and antioxidant activity of Brazil nut (Bertholletia excelsa). J. Funct. Foods 2, 196–209. KAHL, R. and KAPPUS, H. 1993. [Toxicology of the synthetic antioxidants BHA and BHT in comparison with the natural antioxidant vitamin E]. Z. Lebensm. Unters. Forsch. 196, 329–338. KARUPPUSAMY, S., MUTHURAJA, G. and RAJASEKARAN, K.M. 2011. Antioxidant activity of selected lesser known edible fruits from Wester Ghats of India. Indian J. Nat. Prod. Resour. 22, 174–178. KOLEY, T.K., KAUR, C., NAGAL, S., WALIA, S., JAGGI, S. and SARIKA, J. 2011. Antioxidant activity and phenolic content in genotypes of Indian jujube (Zizyphus mauritiana Lamk.). Arabian J. Chem. DOI:10.1016/j.arabjc.2011.11.005. LI, J., DING, S. and DING, X. 2005. Comparison of antioxidant capacities of extracts from five cultivars of Chinese jujube. Process Biochem. 40, 3607–3613. LI, J.W., FAN, L.P., DING, S.D. and DING, X.L. 2007. Nutritional composition of five cultivars of Chinese jujube. Food Chem. 103, 454–460. LIAO, K.L. and YIN, M.C. 2000. Individual and combined antioxidant effects of seven phenolic agents in human erythrocyte membrane ghosts and phosphatidylcholine liposome systems: Importance of the partition coefficient. J. Agric. Food Chem. 48, 2266–2270. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. and RANDALL, R.J. 1951. Protein measurement with the Folin Phenol reagent. J. Biol. Chem. 193, 265–275. MAEURA, Y. and WILLIAMS, G. 1984. Enhancing effect of butylated hydroxytoluene on the development of liver altered foci and neoplasms induced byN-2-fluorenylacetamide in rats. Food Chem. Toxicol. 22, 191–198. MAHARJAN, N., SINGH, A., MANANDHAR, M.D., BASNYAI, S., LEKHAK, B. and KALAUNI, S.K. 2013. Evaluation of antibacterial activities of medicinal plants. Nepal J. Sci. Technol. 13, 209–214. MALIK, M.A., NASEEM, S. and TANWEER, A.M. 2012. Quality evaluation of promising ber (Zizyphus mauritiana) varieties under climatic conditions of faisalabad. Pakis. J. Agr. Res. 50, 401–408. NANDAVE, M., OJHA, S.K. and ARYA, D.S. 2005. Protective role of flavonoids in cardiovascular diseases. Indian J. Nat. Prod. Resour. 4, 166–176. NARAYANASAMY, K. and RAGAVAN, B. 2012. In-vitro antioxidant activity of Zanthoxylum tetraspermum W.A. stem bark. Int. J. Eng. Sci. Technol. 4, 155–162. NYABERI, M.O., ONYANGO, C.A., MATHOOKO, F.M., MAINA, J.M., MAKOBE, M. and MWAURA, F. 2012. Evaluation of phytochemical, antioxidant and antibacterial activity of edible fruit extracts of Ziziphus abyssinica. J. Anim. Plant Sci. 6, 623–629.


R. AFROZ ET AL.

NYANGA, L.K., GADAGA, T.H., NOUT, M.J., SMID, E.J., BOEKHOUT, T. and ZWIETERING, M.H. 2012. Nutritive value of masau (Ziziphus mauritiana) fruits from Zambezi Valley in Zimbabwe. Food Chem. 138, 168–172. OLANIRAN, A.O., NAICKER, K. and PILLAY, B. 2011. Toxigenic Escherichia coli and Vibrio cholerae: Classification, pathogenesis and virulence determinants. Biotechnol. Mol. Biol. Rev. 6, 94–100. OMAYE, S.T., TURNBULL, J.D. and SAUBERLICH, H.E. 1979. [1] Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids. Methods Enzymol. 62, 3–11. ÖNDER, K., SEZAI, E., MEMNUNE, E., CELIL, T. and SEDAT, S. 2009. Total phenolics and antioxidant activity of jujube (Zizyphus jujube Mill.) genotypes selected from Turkey. Afr. J. Biotechnol. 8, 303–307. PEREZ, C., PAULI, M. and BAZERQUE, P. 1990. An antibiotic assay by agar well diffusion method. Acta Biologica Medica Exp. 15, 113–115. SANTOS, P.O., COSTA, M.D.J., ALVES, J.A., NASCIMENTO, P.F., DE MELO, D.L., BARBOSA, A.M., JR., TRINDADE, R.D.C., BLANK, A.F., ARRIGONI-BLANK, M.F. and ALVES, P.B. 2008. Chemical composition and antimicrobial activity of the essential oil of Hyptis pectinata (l.) Poit. Quim. Nova 31, 1648–1652.



SHIV, K. 2011. Free radicals and antioxidants: Human and food system. Adv. Appl. Sci. Res. 2, 129–135. SHODEHINDE, S. and OBOH, G. 2013. Distribution and antioxidant activity of polyphenols in boiled unripe plantain (Musa paradisiaca) pulps. J. Food Biochem. 38, 293–299. SINGH, V. and GUIZANI, N. 2012. Comparative analysis of total phenolics, flavonoid content and antioxidant profile of different date varieties (Phoenix dactylifera L.) from Sultanate of Oman. Int. Food Res. J. 19, 1063–1070. SUNIL, P. 2013. Nutritional composition of jujube fruit. Emir. J. Food Agric. 25, 463–470. TORTORA, G.J., FUNKE, B.R. and CASE, C.L. 2007. Microbiology: An Introduction, 9th Ed., Pearson Education, San Francisco, CA. WALSH, S.E., MAILLARD, J.Y., RUSSEL, A.D., CATRENICH, C.E., CHARBONNEAU, A.L. and BARTOLO, R.G. 2003. Activity and mechanism of action of selected biocidal agents on gram-positive and -negative bacteria. J. Appl. Microbiol. 94, 240–247. WANG, S.Y. and ZHENG, W. 2001. Effect of plant growth temperature on antioxidant capacity in strawberry. J. Agric. Food Chem. 49, 4977–4982. ZHANG, X.Y. 2000. Principles of Chemical Analysis, pp. 275–276, China Science Press, Beijing, China.

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