evaluation of antioxidant and antimicrobial capacity of

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Journal of Food Biochemistry ISSN 1745-4514

EVALUATION OF ANTIOXIDANT AND ANTIMICROBIAL CAPACITY OF SYZYGIUM CUMINI L. LEAVES EXTRACTED SEQUENTIALLY IN DIFFERENT SOLVENTS jfbc_614

168..176

1

MITAL KANERIA and SUMITRA CHANDA

Phytochemical, Pharmacological and Microbiological Laboratory, Department of Biosciences, Saurashtra University, Rajkot-360 005 Gujarat, India

1

Corresponding author. TEL: +919426247892; EMAIL: [email protected] Accepted for Publication June 20, 2011 doi:10.1111/j.1745-4514.2011.00614.x

ABSTRACT Syzygium cumini is commonly used in India as a traditional medicine. In this study, the antioxidant and antimicrobial potentials of different solvent extracts of S. cumini leaves were investigated. Extraction was done sequentially in Soxhlet apparatus, using various solvents (petroleum ether, toluene, ethyl acetate, acetone and water). Antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay, hydroxyl radical scavenging assay, superoxide anion radical scavenging assay and reducing capacity assessment. Total phenol and flavonoid content was also measured. The antimicrobial activity was done by agar well diffusion method against some of the tested foodborne, pathogenic and skin disease causing microorganisms. The acetone extract had more total phenol content and more antioxidant activities, conforming to the hypothesis that phenol content and antioxidant activity has a direct correlation. The acetone extract had notable antimicrobial effect on microorganisms. The results obtained appeared to confirm the antioxidant and antimicrobial potentials of the S. cumini.

PRACTICAL APPLICATIONS Food safety is a fundamental concern of both consumers and the food industry, especially as the number of reported cases of food-associated infections continues to increase. It has been estimated that as many as 30% of people in industrialized countries suffer from foodborne diseases each year. Microorganisms play a major role in contamination of stored foods deteriorating them quantitatively as well as qualitatively. Fungi are significant destroyer of foodstuffs during storage, rendering them unfit for human consumption by retarding their nutritive value and sometimes by producing mycotoxins. In the present study, antioxidant and antimicrobial activities of different solvent extracts of Syzygium cumini leaves was evaluated. Acetone extract had good antioxidant activity comparable with standard and remarkable antimicrobial activity. The results indicate that S. cumini may become important natural source of compounds with health protective potential and antimicrobial agent, which can be used in pharmaceutical, nutraceutical and food preparation.

INTRODUCTION The oxidative stress, defined as “the imbalance between oxidants and antioxidants in favor of the oxidants potentially leading to damage”, has been suggested to be the cause of aging and various diseases in human. In fact oxidative stress 168

results from an imbalance between the generation of reactive oxygen species (ROS) and endogenous antioxidant systems. Excessive amount of ROS is harmful because they initiate bimolecular oxidation, which leads to cell death and cause inadvertent enzyme activation and oxidative damage to cellular system (Wiseman and Halliwell 1996). Antioxidants can Journal of Food Biochemistry 37 (2013) 168–176 © 2011 Wiley Periodicals, Inc.

M. KANERIA and S. CHANDA

protect the human body from ROS effects. There are many synthetic antioxidants in use but gradually their use is causing a concern because of many side effects and carcinogenicity (Namiki 1990). Therefore, the use of natural antioxidant is increasing and people are searching for natural antioxidant because of their presumed safety, nutritional and therapeutic value (Ajila et al. 2007). Natural antioxidants have been reported in different plant materials (Alesiani et al. 2010; Chanda and Baravalia 2010; Vaghasiya and Chanda 2010). Infectious diseases are leading cause of death world wide due to multidrug-resistant strains of bacteria, reduced susceptibility to antimicrobics and increase in untreatable bacterial infections. The frequency of life-threatening infections caused by pathogenic microorganisms has increased worldwide and is becoming an important cause of morbidity and mortality in immunocompromised patients in developing countries (Ai-Bari et al. 2006). The increasing prevalence of multidrug-resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raised the specter of “untreatable” bacterial infections and adds urgency to the search for new infection-fighting strategies (Rojas et al. 2006). Despite the existence of potent antibiotic and antifungal agents, resistant or multiresistant strains are continuously appearing, imposing the need for a permanent search and development of new drugs (Fritsche et al. 2005; Nathwani 2005). It is therefore very necessary that the search for newer antibiotic sources be a continuous process. Plants are the cheapest and safer alternative sources of antimicrobials. Syzygium cumini is reported to possess many pharmacological activities like antidiabetic (Kumar et al. 2008a; Patel et al. 2009), anti-inflammatory (Lima et al. 2007; Kumar et al. 2008b), antimicrobial (Duraipandiyan et al. 2006; Oliveira et al. 2007), antibacterial (Shafi et al. 2002), antifungal (Braga et al. 2007), antioxidant (Ruan et al. 2008; Kaneria et al. 2009), antigenotoxicity (Bartolome et al. 2006), antileishmanial (Braga et al. 2007), brine shrimp lethality (Krishnaraju et al. 2006), antihyperlipidemic (Patel et al. 2009), central nervous system (Kumar et al. 2007), antiallergic (Brito et al. 2007), antifertility (Rajasekaran et al. 1988), gastroprotective (Ramirez and Rao 2003) and radioprotective (Jagetia and Baliga 2003). Few researchers have already worked on antioxidant and antimicrobial property of S. cumini leaves but it was preliminary work; for example, it was part of a screening program, the solvents used were one or two, extraction techniques were different and the microorganisms used were few and also different. Our earlier work (Kaneria et al. 2009) suggested S. cumini as a potent antioxidant agent; hence, it was thought of interest to investigate the antioxidant and antimicrobial potential of S. cumini leaves thoroughly. The objectives of this study were to quantify the total phenol content and to investigate antioxidant and antimicroJournal of Food Biochemistry 37 (2013) 168–176 © 2011 Wiley Periodicals, Inc.

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF SYZYGIUM CUMINI

bial activities of various solvent extracts from S. cumini leaves by using sequential Soxhlet extraction method.

MATERIALS AND METHODS Chemicals 2,2-Diphenyl-1picrylhydrazyl (DPPH), nitroblue tetrazolium (NBT), phenazine methosulphate (PMS), nicotinamide adenine dinucleotide reduced (NADH), gallic acid, ascorbic acid, quercetin, Folin–Ciocalteu’s reagent, aluminium chloride, potassium acetate, 2-deoxy-D-ribose, thiobarbituric acid, trichloroacetic acid, hydrogen peroxide, ethylenediaminetetraacetic acid (EDTA), ferric chloride, potassium ferricyanide, Tris-HCl, dimethyl sulfoxide (DMSO), nutrient broth, Sabouraud dextrose broth, Muller Hinton no. 2, Sabouraud dextrose agar, agar powder were obtained from Hi-Media, Mumbai, India; petroleum ether, toluene, acetone, ethyl acetate, methanol, etc., were obtained from Merck, Mumbai, India.

Plant Material Fresh leaves of S. cumini L. belonging to family Myrtaceae were collected in the month of August 2008, from JamJodhpur, Jamnagar, Gujarat, India. The plant was compared with voucher specimen (voucher specimen No. PSN295) deposited at Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India. The leaves were separated, washed thoroughly with tap water, shade dried, homogenized to fine powder and stored in airtight bottles.

Sequential Extraction Method The dried powder of the leaves was extracted sequentially (Wiart et al. 2004) by Soxhlet apparatus (Lin et al. 1999), using different solvents depending upon their polarities like petroleum ether, toluene, ethyl acetate, acetone and water (Fig. 1). The extracts were concentrated and freed of solvent under reduced pressure, using a rotary evaporator. The dried crude concentrated extracts were weighed to calculate the extractive yield and stored in a refrigerator (4C) in airtight bottles.

Quantitative Phytochemical Analysis Determination of Total Phenol Content. The amount of total phenol content, in different solvent extracts was determined by Folin–Ciocalteu’s reagent method (Mc Donald et al. 2001). A total of 0.5 mL of extract and 0.1 mL (0.5 N) Folin–Ciocalteu’s reagent was mixed and the mixture was incubated at room temperature for 15 min. Then 2.5 mL 169


Plant powder subjected to sequential extraction in Soxhlet apparatus

Extraction with petroleum ether

Residue

Petroleum ether extract

Extraction with toluene

Residue

Toluene extract


measured by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) by the modified method of Mc Cune and Johns (2002). The reaction mixture 3.0 mL, which consisted of 1.0 mL of DPPH (0.3 mM), 1.0 mL of extract (different concentrations) and 1.0 mL of methanol, was incubated for 10 min, in dark, after which the absorbance was measured at 517 nm. Ascorbic acid was used as positive control. The assay was carried out in triplicate and the mean values with ⫾SEM are presented. The percentage inhibition was determined by comparing the results of the test and the control. Percentage of inhibition was calculated using the formula

Extraction with ethyl acetate

% Inhibition = ([ B − A] B ) × 100 Residue

Ethyl acetate extract

Extraction with acetone

Residue

Acetone extract

Extraction with water

Residue

Aqueous extract

FIG. 1. SYSTEMATIC REPRESENTATION OF DIFFERENT SOLVENT EXTRACTS OF SYZYGIUM CUMINI LEAVES BY SEQUENTIAL EXTRACTION METHOD

saturated sodium carbonate solution was added and further incubated for 30 min at room temperature, and the absorbance was measured at 760 nm. Gallic acid was used as a positive control. Total phenol values are expressed in terms of gallic acid equivalent (mg/g of extracted compounds).The assay was carried out in triplicate and the mean values with ⫾standard error of the mean (SEM) is presented. Determination of Total Flavonoid Content. The amount of flavonoid content in different solvent extracts was determined by aluminium chloride colorimetric method (Chang et al. 2002). The reaction mixture 3.0 mL consisted of 1.0 mL of sample (1 mg/mL), 1.0 mL methanol, 0.5 mL of (1.2%) aluminium chloride and 0.5 mL (120 mM) potassium acetate and was incubated at room temperature for 30 min. The absorbance of all samples was measured at 415 nm. Quercetin was used as positive control. The flavonoid content is expressed in terms of quercetin equivalent (mg/g of extracted compound). The assay was carried out in triplicate and the mean values with ⫾SEM are presented.

Antioxidant Assays DPPH Free Radical Scavenging Activity. The free radical scavenging activity of different solvent extracts was 170

where B is the absorbance of blank (DPPH, plus methanol); A is the the absorbance of sample (DPPH, methanol plus sample).

Hydroxyl Radical Scavenging Activity. The hydroxyl radical scavenging activity of different solvent extracts was measured by studying the competition between deoxyribose and test compound for hydroxyl radicals generated by Fe+3ascorbic acid-EDTA-H2O2 system (Fenton reaction) according to the method of Kunchandy and Rao (1990). The reaction mixture 1.0 mL, which consisted of 100 mL of 2-deoxy-D-ribose (28 mM in 20 mM KH2PO4-KOH buffer, pH 7.4), 500 mL of the various solvent extracts, 200 mL EDTA (1.04 mM) and 200 mM FeCl3 (1:1 v/v), 100 mL 1.0 mM H2O2 and 100 mL ascorbic acid (1.0 mM), was incubated at 37C for 1 h. One milliliter of thiobarbituric acid (1%) and 1.0 mL of trichloroacetic acid (2.8%) was added and incubated at 100C for 20 min. After cooling, the absorbance of pink color was measured at 532 nm against a blank sample. Gallic acid was used as a positive control. The assay was carried out in triplicate and the mean values ⫾ SEM are presented. The percentage inhibition was determined by comparing the results of the test and the control.

Superoxide Anion Radical Scavenging Activity. The superoxide anion radical scavenging activity of different solvent extracts was measured by the method as described by Robak and Gryglewski (1988). Superoxide radicals are generated by oxidation of NADH and assayed by the reduction of NBT. The reaction mixture 3.0 mL consisted of 0.5 mL of NBT (0.3 mM), 0.5 mL of Tris-HCl buffer (16 mM, pH 8), 0.5 mL NADH (0.936 mM), 0.5 mL PMS (0.12 mM) and 1.0 mL of different concentrations of different solvent extracts. The superoxide radical generating reaction was started by the addition of 0.5 mL of PMS solution to the mixture. The reaction mixture was incubated at 25C for 5 min and then the absorbance was measured at 560 nm against a blank sample. Gallic acid was used as a positive Journal of Food Biochemistry 37 (2013) 168–176 © 2011 Wiley Periodicals, Inc.



control. The assay was carried out in triplicate and the mean values with ⫾ SEM are presented. The percentage inhibition was determined by comparing the results of the test and the control. Reducing Capacity Assessment. The reducing capacity assessment of different solvent extracts was determined using method as described by Athukorala et al. (2006). 1.0 mL of different concentrations of solvent extracts was mixed with 2.5 mL of potassium phosphate buffer (200 mM, pH 6.6) and potassium ferricyanide (2.5 mL, 30 mM). The mixture was then incubated at 50C for 20 min. There after 2.5 mL of trichloroacetic acid (600 mM) was added to the reaction mixture and then centrifuged for 10 min at 3,000 rpm. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and 0.5 mL of FeCl3 (6 mM) and the absorbance was measured at 700 nm. Ascorbic acid was used as positive control. The assay was carried out in triplicate and the mean values with ⫾SEM are presented.

Antimicrobial Assay Microorganisms Tested. The bacterial and fungal strains used to assess the antibacterial properties of different solvent extracts of S. cumini included five gram-positive bacteria (Bacillus megaterium ATCC9885, Bacillus subtilis ATCC6633, Corynebacterium rubrum ATCC14898, Staphylococcus aureus ATCC25923, Staphylococcus epidermidis ATCC12228), five gram-negative bacteria (Citrobacter freundii ATCC10787, Enterobacter aerogenes ATCC13048, Klebsiella pneumoniae NCIM2719, Proteus mirabilis NCIM2241, Salmonella typhimurium ATCC23564) and five fungi (Candida albicans ATCC2091, Candida glabrata NCIM3448, Cryptococcus leuteolus ATCC32044, Candida neoformans NCIM3542, Candida tropicalis ATCC4563). The investigated bacterial and fungal strains were obtained from National Chemical Laboratory, Pune, India. The microorganisms were maintained on nutrient agar and Sabouraud dextrose agar (Hi-Media, Mumbai, India) for bacteria and fungi respectively at 4C and subcultured before use. The microorganisms studied are the clini-

TABLE 1. EXTRACTIVE YIELD, TOTAL PHENOL AND FLAVONOID CONTENT OF DIFFERENT SOLVENT EXTRACTS OF LEAVES OF SYZYGIUM CUMINI

cally important ones causing several infections, foodborne diseases, spoilages, skin infection, and it is essential to overcome them through some active therapeutic agents. Determination of Antimicrobial Assay. In vitro antimicrobial activity of the different solvent extracts of S. cumini was studied against 15 microbial strains by the agar-well diffusion method (Perez et al. 1990; Parekh and Chanda 2007). Muller Hinton no. 2 and Sabouraud dextrose agar (Himedia) was used for the antibacterial and antifungal susceptibility test, respectively. The extracts were diluted in 100% DMSO at the concentration of 20 mg/mL. The microbial activity was evaluated at the concentration 2.0 mg/well. The Muller Hinton agar/Sabouraud dextrose agar was melted and cooled to 48–50C, and a standardized inoculum (1.5 ¥ 108 cfu/mL, 0.5 McFarland) was then added aseptically to the molten agar and poured into sterile Petri dishes to give a solid plate. Wells were prepared in the seeded agar plates. The test compound (100 mL) was introduced in the well (8.5 mm). The plates were incubated overnight at 37C for 24 h and 28C for 48 h for bacteria and fungi, respectively. The antimicrobial spectrum of the extract was determined for the bacterial and fungal species in terms of zone sizes around each well. DMSO was used as negative control. The control zones were subtracted from the test zones and the resulting zone diameter is shown in the Table 5. The experiment was performed three times to minimize the error and the mean values ⫾ SEM are presented.

RESULTS AND DISCUSSION Extractive Yield The extractive yield of different solvent extracts of S. cumini leaves is given in Table 1. The extractive yield varied among different solvents used. Aqueous extract showed highest extractive yield than the other extracts. The percentage extractive yield can be ranked from high to low in the following order: aqueous extract (11.35) > acetone extract (3.59) > petroleum ether extract (1.61) > ethyl acetate extract (1.05) > toluene extract (0.63). There are many reports in the

Different solvent extracts

% Yield (w/w)*

Total phenol content (mg/g of extracted compound)*

Flavonoid content (mg/g of extracted compound)*

Petroleum ether Toluene Ethyl acetate Acetone Aqueous

1.61 ⫾ 0.03 0.63 ⫾ 0.04 1.05 ⫾ 0.04 3.59 ⫾ 0.27 11.35 ⫾ 0.74

ND 9.43 ⫾ 0.15 75.17 ⫾ 1.13 217.73 ⫾ 5.18 104.42 ⫾ 1.94

ND 17.11 ⫾ 0.50 95.69 ⫾ 0.88 104.78 ⫾ 0.30 44.46 ⫾ 0.17

* Values are expressed in mean ⫾ standard error of the mean (n = 3). ND, not done.

Journal of Food Biochemistry 37 (2013) 168–176 © 2011 Wiley Periodicals, Inc.

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IC50 values (mg/mL) Antioxidant assays DPPH OH SO

Standard

Extracts

Gallic acid

Ascorbic acid

Toluene

Ethyl acetate

Acetone

Aqueous

ND 140 185

11.4 ND ND

– – –

44 290 950

12.3 410 75

38 200 330

TABLE 2. HALF MAXIMAL INHIBITORY CONCENTRATION (IC50) VALUES OF DPPH, HYDROXYL (OH) AND SUPEROXIDE ANION (SO) RADICAL SCAVENGING ACTIVITIES OF DIFFERENT SOLVENT EXTRACTS OF LEAVES OF SYZYGIUM CUMINI

–, >1,000 mg/mL. DPPH, 2,2-diphenyl-1-picrylhydrazyl free radical; ND, not done.

literature where extractive yield varied with different solvents (Vaghasiya and Chanda 2007; Yang et al. 2007). The extractive yield in some solvents was more than that reported earlier by Kaneria et al. (2009). This may be because of difference in the extraction technique used. In the present work, Soxhlet extraction technique is used – which may facilitate the extraction of phytoconstituents completely – which may not be so in the cold percolation technique.

Total Phenol and Flavonoid Contents In the present work, total phenolic content was more than the flavonoid content in polar solvent extracts, while in nonpolar solvent extracts, flavonoid content was more than phenols (Table 1). Highest total phenolic and flavonoid content was present in acetone extract (Table 1). As stated earlier, the technique used in the present work was better for extracting total phenol and flavonoid content as compared with the cold percolation method, reported by Kaneria et al. (2009).

Antioxidant Activities DPPH Free Radical Scavenging Activity. Out of four extracts investigated, only toluene extract showed half maximal inhibitory concentration (IC50) value more than 1,000 mg/mL (Table 2); the remaining three extracts showed varied levels of DPPH free radical scavenging activity (Table 3). IC50 values ranged from 12.3 to 44 mg/mL (Table 2). The IC50 value of acetone extract was 12.3 mg/mL, which was almost similar to that of standard ascorbic acid 11.4 mg/mL (Table 2). The IC50 value of aqueous extract was 38 mg/mL while that of ethyl acetate extract was 44 mg/mL (Table 2). Low IC50 value indicates high antioxidant activity. Therefore, it can be stated that acetone extract possess strong antioxidant activity. Ruan et al. (2008) and Kaneria et al. (2009) also reported a good DPPH activity in S. cumini leaves. Silva et al. (2000) reported the significant scavenging effects of phenolic compounds against the DPPH free radical. It is generally

TABLE 3. 2,2-DIPHENYL-1-PICRYLHYDRAZYL FREE RADICA (DPPH), HYDROXYL (OH) AND SUPEROXIDE ANION (SO) RADICAL SCAVENGING ACTIVITIES OF DIFFERENT SOLVENT EXTRACTS OF LEAVES OF SYZYGIUM CUMINI Assay

DPPH

OH

SO

Standard (ascorbic acid/gallic acid)

Ethyl acetate extract

Acetone extract

Aqueous extract

Conc. (mg/mL)

% Inhibition*

Conc. (mg/mL)

% Inhibition*

Conc. (mg/mL)

% Inhibition*

Conc. (mg/mL)

% Inhibition*

2 4 8 12 14 16 20 60 100 140 160 50 100 150 200 225

8.60 16.60 31.33 48.65 59.70 69.79 8.67 ⫾ 1.469 23.31 ⫾ 0.872 29.54 ⫾ 0.419 49.96 ⫾ 1.328 75.38 ⫾ 0.395 11.88 ⫾ 1.459 24.94 ⫾ 0.355 41.37 ⫾ 0.546 52.17 ⫾ 0.055 66.66 ⫾ 0.000

10 20 30 40 50 60 200 400 600 800 1,000 200 400 600 800 1,000

10.99 ⫾ 0.008 22.90 ⫾ 0.002 35.51 ⫾ 0.005 45.71 ⫾ 0.010 55.60 ⫾ 0.003 64.37 ⫾ 0.004 34.14 ⫾ 0.001 45.56 ⫾ 0.001 53.48 ⫾ 0.001 55.39 ⫾ 0.001 59.09 ⫾ 0.001 5.86 ⫾ 0.003 15.24 ⫾ 0.031 30.91 ⫾ 0.048 37.63 ⫾ 0.024 54.90 ⫾ 0.003

3 6 9 12 15 18 200 400 600 800 1,000 20 40 60 80 100

12.70 ⫾ 0.006 25.58 ⫾ 0.007 35.86 ⫾ 0.006 45.85 ⫾ 0.005 59.39 ⫾ 0.000 68.51 ⫾ 0.003 37.58 ⫾ 0.001 48.92 ⫾ 0.000 60.70 ⫾ 0.001 64.02 ⫾ 0.001 66.27 ⫾ 0.000 20.57 ⫾ 0.015 27.11 ⫾ 0.011 39.80 ⫾ 0.010 51.92 ⫾ 0.006 68.84 ⫾ 0.015

10 20 30 40 50 60 200 400 600 800 1,000 100 200 300 400 500

16.11 ⫾ 0.007 28.72 ⫾ 0.001 40.63 ⫾ 0.005 51.57 ⫾ 0.001 59.89 ⫾ 0.003 72.06 ⫾ 0.003 50.42 ⫾ 0.000 56.43 ⫾ 0.002 62.65 ⫾ 0.001 67.61 ⫾ 0.001 69.09 ⫾ 0.000 11.58 ⫾ 0.006 30.95 ⫾ 0.011 47.51 ⫾ 0.006 60.17 ⫾ 0.002 75.64 ⫾ 0.008

* Values are expressed in mean ⫾ standard error of the mean (n = 3).

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20 40 60 80 100 120 140 160 180 0.330 ⫾ 0.012 0.669 ⫾ 0.000 0.942 ⫾ 0.002 1.208 ⫾ 0.002 1.430 ⫾ 0.000 1.695 ⫾ 0.001 1.856 ⫾ 0.009 1.897 ⫾ 0.001 1.927 ⫾ 0.002 20 40 60 80 100 120 140 160 180 0.065 ⫾ 0.002 0.142 ⫾ 0.010 0.193 ⫾ 0.001 0.239 ⫾ 0.000 0.282 ⫾ 0.001 0.325 ⫾ 0.000 0.368 ⫾ 0.000 0.414 ⫾ 0.003 0.463 ⫾ 0.000 20 40 60 80 100 120 140 160 180 * Values are expressed in mean ⫾ SEM (n = 3).

20 40 60 80 100 120 140 160 180 0.186 ⫾ 0.000 0.365 ⫾ 0.001 0.542 ⫾ 0.022 0.670 ⫾ 0.005 0.762 ⫾ 0.020 0.897 ⫾ 0.007 1.083 ⫾ 0.031 1.298 ⫾ 0.001 1.429 ⫾ 0.006 20 40 60 80 100 120 140 160 180

0.000 ⫾ 0.000 0.012 ⫾ 0.001 0.017 ⫾ 0.001 0.016 ⫾ 0.000 0.150 ⫾ 0.045 0.016 ⫾ 0.000 0.018 ⫾ 0.001 0.022 ⫾ 0.001 0.021 ⫾ 0.002

Conc. (mg/mL) Conc. (mg/mL) Conc. (mg/mL)

Reducing power (Absorbance at 700 nm)*

Conc. (mg/mL)


Conc. (mg/mL)


Aqueous extract Acetone extract Ethyl acetate extract Toluene extract

Reducing Capacity Assessment. Out of four studied extracts, toluene extract showed poor reducing capacity. In S. cumini leaves, there was a concentration-dependent increase in the absorbance of reaction mixture for all the three extracts and the standard ascorbic acid (Table 4). The acetone extract showed maximum absorbance followed by aqueous extract, and hence maximum reducing capacity assessment, even better than the standard ascorbic acid (Table 4). The reducing capacity assessments of extracts were in this order: acetone extract > aqueous extract > ascorbic acid > ethyl acetate extract > toluene extract. According to these results, there

Standard Ascorbic acid

Superoxide Anion Radical Scavenging Activity. Out of the four extracts investigated, only toluene extract showed IC50 value more than 1,000 mg/mL (Table 2); the remaining three extracts showed varied levels of superoxide anion radical scavenging activity (Table 3). IC50 values ranged from 75 mg/mL to 950 mg/mL (Table 2). The IC50 value of aqueous extract was 330 mg/mL while that of ethyl acetate extract was 950 mg/mL (Table 2). The IC50 value of acetone extract was 75 mg/mL, which was better than that of the standard gallic acid 185 mg/mL (Table 2). These data showed that the acetone extract of S. cumini is a strong superoxide anion scavenger. This result indicates that a constituent within S. cumini extract, especially in acetone extract, is capable of scavenging superoxide anion.

TABLE 4. REDUCING CAPACITY ASSESSMENT OF THE DIFFERENT SOLVENT EXTRACTS OF LEAVES OF SYZYGIUM CUMINI

Hydroxyl Radical Scavenging Activity. Out of four extracts investigated, toluene extract showed IC50 value more than 1,000 mg/mL (Table 2), and the remaining three extracts showed varied levels of hydroxyl radical scavenging activity (Table 3). The best hydroxyl radical scavenging activity was shown by aqueous extract, and its IC50 value was 200 mg/mL (Table 2). The IC50 value of ethyl acetate extract was 290 mg/mL while that of aqueous extract was 410 mg/mL (Table 2). Gallic acid was used as a standard with IC50 value 140 mg/mL (Table 2). This antioxidant assay showed weak correlation with phenolic content. The acetone extract had high amount of phenol content, but did not show the best hydroxyl radical scavenging activity, while aqueous and ethyl acetate extracts showed low amounts of phenol content but showed better hydroxyl radical scavenging activity than the acetone extract. This suggests that nonphenolic compounds may also be responsible for the observed antiradical activity as also suggested by Yam et al. (2008).


believed that plants that have more phenolic content show good antioxidant activity, that is there is a direct correlation between total phenol content and antioxidant activity (Baravalia et al. 2009; Gholivand et al. 2010).

0.123 ⫾ 0.000 0.387 ⫾ 0.000 0.526 ⫾ 0.002 0.758 ⫾ 0.001 0.932 ⫾ 0.003 1.088 ⫾ 0.000 1.227 ⫾ 0.000 1.352 ⫾ 0.005 1.485 0.002




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is good relationship between total phenols and antioxidant activity. Overall, the antioxidant activities of the extracts were highly correlated with their total phenolic contents, and these results are similar to those of previous findings (Ebrahimabadi et al. 2010; Park and Jhon 2010).

Antimicrobial Activity The antimicrobial activity has been studied against five grampositive, five gram-negative and five fungi strains in five different solvent extracts. The results of antimicrobial activity are shown in Table 5. The acetone and aqueous extracts showed better activity amongst all the extracts studied. Highest inhibition zone was shown by ethyl acetate extract against B. subtilis. The acetone extract showed activity against all the gram-positive and gram-negative bacteria except E. aerogenes and S. typhimurium. In the present work, the plant extracts showed more antibacterial activity than antifungal activity, and that gram-positive bacteria were more susceptible than gram-negative bacteria. This is similar to the general belief that gram-positive bacteria are more susceptible to herbal drugs than gram-negative bacteria (Parekh et al. 2005; Oliveira et al. 2008). The methanolic extract of S. cumini showed antifungal activity against C. neoformans (Braga et al. 2007); the hydroalcoholic extract of S. cumini leaves showed good antimicrobial activity (Oliveira et al.


2008) and Shafi et al. (2002) reported antibacterial activity of essential oils from S. cumini leaves. The results of the present study and other reported results suggest the antibacterial potential of S. cumini leaves. In the present investigation, antioxidant potential of the extract was evaluated by four different assays and compared with that of total phenol content. Due to the complex nature of phytoconstituents, the antioxidant activities of crude extract cannot be measured by a single method, and it is suggested that at least two assays should be used (Chanda and Dave 2009). Chanda and Nagani (2010) suggested that it is imperative to evaluate more than one antioxidant methods and in more than one solvent in a single plant. Therefore, in the present work, four commonly accepted assays were used to evaluate the antioxidant effects of S. cumini in five different solvents. The acetone extract of S. cumini showed remarkable antioxidant activity comparable with that of the standards; it also showed significant antimicrobial activity. The results of the present work and the results reported by others strongly support the antioxidant nature of S. cumini leaves. Perhaps it is for the first time that the antioxidant and antibacterial potentials of S. cumini leaves are reported.

CONCLUSION The result obtained from present study showed that the acetone extract of S. cumini has remarkable antioxidant

Zone of inhibition (mm)* Microorganisms

Petroleum ether Toluene

Gram positive

– – – – – – – 10.5 ⫾ 0.00 – – – – – 9.5 ⫾ 0.29 –

BM BS CR SA SE Gram negative CF EA KP PM ST Fungi CA CG CL CN CT

– – – – – – – 10.5 ⫾ 0.00 11 ⫾ 0.00 – – – – – –

Ethyl acetate Acetone 11 ⫾ 0.00 14 ⫾ 0.00 10 ⫾ 0.00 – 10.5 ⫾ 0.29 – – 10 ⫾ 0.29 – – – – – – –

Aqueous

11 ⫾ 0.00 9.5 ⫾ 0.29 11.5 ⫾ 0.29 11.5 ⫾ 0.29 10.5 ⫾ 0.29 9.5 ⫾ 0.29 – 11.5 ⫾ 0.00 11.5 ⫾ 0.29 – – – – – –

11.5 ⫾ 0.29 – 11 ⫾ 0.00 10 ⫾ 0.00 11 ⫾ 0.00 – – 11 ⫾ 0.29 10.5 ⫾ 0.29 – – 9 ⫾ 0.00 – – –

TABLE 5. ANTIMICROBIAL ACTIVITY OF DIFFERENT SOLVENT EXTRACTS OF LEAVES OF SYZYGIUM CUMINI AGAINST SOME PATHOGENIC MICROORGANISMS

–, no inhibition. * Values are presented in mean ⫾ SEM (n = 3). BM, Bacillus megaterium ATCC9885; BS, Bacillus subtilis ATCC6633; CA, Candida albicans ATCC2091; CF, Citrobacter freundii ATCC10787; CG, Candida glabrata NCIM3448; CL, Cryptococcus leuteolus ATCC32044; CN, Candida neoformans NCIM3542; CR, Corynebacterium rubrum ATCC14898; CT, Candida tropicalis ATCC4563; EA, Enterobacter aerogenes ATCC13048; KP, Klebsiella pneumoniae NCIM2719; PM, Proteus mirabilis NCIM2241; SA, Staphylococcus aureus ATCC25923; SE, Staphylococcus epidermidis ATCC12228; ST, Salmonella typhimurium ATCC23564.

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activity as well as potential antimicrobial activity. The extract had high phenolic content and significant antioxidant activity, which was either comparable or better than the standard. More importantly, the results indicated that the acetone extract of S. cumini rich in phenolics also had antibacterial and antifungal activities. However, the components responsible for the activities of acetone extract of S. cumini are currently not known. Therefore, it is suggested that further work be performed on the isolation and identification of the bioactive compounds, which may be useful for therapeutic purpose.

ACKNOWLEDGMENTS The authors thank Prof. S.P. Singh, Head, Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India for providing excellent research facilities. One of the authors, Mital Kaneria, is thankful to University Grants Commission, New Delhi, India for providing financial support as Junior Research Fellowship.

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