IAJPS 2016, 3 (2), 92-97
Sonia Zaman
CODEN (USA): IAJPBB
ISSN 2349-7750
ISSN: 2349-7750
INDO AMERICAN JOURNAL OF
PHARMACEUTICAL SCIENCES Available online at: http://www.iajps.com
Research Article
EXPLORING THE ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF ELAEOCARPUS FLORIBUNDUS LEAVES Sonia Zaman* Department of Pharmacy, Southeast University, Dhaka-1213, Bangladesh Abstract: Background: Medicinal plants are store house of phytochemicals for treatment of various diseases from the ancient time to present day, which lead to the discovery of pharmakon. Aim: The aim of the present study is to verify the antibacterial and antioxidant activities of the ethanolic, chloroform and petroleum ether extracts of the Elaeocarpus floribundus leaves. Method: Antibacterial activity was evaluated by disc diffusion method using kanamycin as standard against five Gram positive (Staphylococcus aureus, Bacillus cereus, Bacillus megaterium, Bacillus subtilis and Sarcina luteae) and five Gram negative (Escherichia coli, Vibrio parahemolyticus, Shigella dysenteriae, Salmonella paratyphi and Pseudomonus aeruginosa) bacteria. Antioxidant activity of the extracts was determined by using DPPH as free radical and ascorbic acid as standard. Results: The ethanol extract showed over all good activity against all microorganisms compared to chloroform and pet ether extract. The ethanol extract showed significant activity against S. aureus, B. subtilis, S. luteae, S. dysenteriae, P. aeruginosa and E. coli with zone of inhibition of 22mm, 20mm, 20mm, 18mm, 18mm, and 16mm respectively. The chloroform extract also showed good activity against S. aureus and B. cereus with 10mm zone of inhibition. The pet ether extract showed good activity only against S. aureus with zone of inhibition 14mm. Both chloroform and petroleum ether extract showed moderate activity against other bacteria. Among the three extracts, ethanol and petroleum ether extracts showed significant antioxidant activity with IC50 value of 45.23 and 60.18 Âľg/ml compared to ascorbic acid 41.78Âľg/ml. Conclusion: This study suggested that Elaeocarpus floribundus leaves have promising antibacterial and antioxidant activity. Keywords: Pharmakon, Elaeocarpus floribundus, antibacterial activity, antioxidant, DPPH.
*Corresponding author: Sonia Zaman, Department of Pharmacy Southeast University E-mail: sonia_6995@yahoo.com Mobile: +8801722448229
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Please cite this article in press as Sonia Zaman, Exploring the Antibacterial and Antioxidant Activities of Elaeocarpus Floribundus Leaves, Indo Am. J. Pharm. Sci, 2016; 3(2).
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IAJPS 2016, 3 (2), 92-97 INTRODUCTION: Nature is the paramount source of about 87% of drugs that are used to treat all types of human diseases. Plants are the basis of Almost 25% of prescribed drugs [1]. Plants, more specifically medicinal plants are deliberated as rich resources of ingredients which can be used in drug development and synthesis. Plants are always thought to be a principal basis of drugs in complementary and alternative system of medicine in various forms such as crude form, juice, decoction, and crude extracts. According to WHO (World Health organization) in the world about 80% of people, predominantly in the rural areas of developing countries, continue using complementary and alternative medicine for their primary health care[2]. Some plants are contemplated as essential source of nutrition and on account of that these plants suggested for their therapeutic values. Active ingredients are commonly extracted from medicinal plants, which are being used in the synthesis of different types of therapeutic agents including laxatives, blood thinners, anticancer, antioxidants, antidiabetics, antibiotics and antimalaria medications etc possess ingredients from plants [3]. In health care services bacterial infections are accountable for 90% of infections. The major reason of treatment failure is the emergence of multi drug resistance (MDR) bacterial strains [4]. Medicinal plants have been evidenced to be useful in the treatment of infectious diseases with having little or no side effects as practiced with synthetic drugs [5]. Infectious diseases that are caused by bacteria, fungi, and viruses are still in rise and they are now the principal threat to public health. Efforts are always ongoing to investigate for novel organically active compounds from natural bases as new antimicrobial agent in order to invention new chemical structures which could defeated the multiple resistances developed by the pathogenic microbes in the direction of existing antimicrobial agents. Without oxygen cannot live any of aerobic organisms; however, oxidative stress may induce damage to cellular biomolecules and is implicated in many diseases [6]. Free radical and reactive oxygen species (ROS) are essentially the major cause of various disorders in humans that are produced by an imbalance between oxidants and antioxidants in human body system resulting in oxidative stress. Several chronic diseases, including cancer, diabetes, aging, and other degenerative diseases in humans [6, 7] along with lipid peroxidation are mainly caused by oxidative damage to lipids, proteins, and DNA. To defend the adverse effects of free radical, human cells produce enzymes such as superoxide dismutase (SOD) and catalase or compounds like ascorbic acid,
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Sonia Zaman
ISSN 2349-7750
tocopherol, and glutathione [8]. Plants that are rich in antioxidants; so much consideration has been focused towards the development of botanical medicines as they comprise tannins, vitamins, phenols, alkaloids, terpenoids, and flavonoids many more phytochemicals accountable for different pharmacological activities [9]. Elaeocarpus floribundus is a sour tasted fruit which is available all regions in Bangladesh. Locally this plant is commonly known as Jolpai and belonging to the family Elaeocarpaceae. It is native to South and East Asian region. It is an evergreen medium sized plant. Leafs of this plant are oblong, elliptical, ovate, etc shaped, having pointed tip and toothed margin. Jolpai flowers are always available as cluster. Color of the flower is white, creamy white, etc. Young olive leafs are tasted sour too. Jolpai fruits are 2-5cm in length and having a smooth surface. Taste of the fruit is acidic and is used make pickle [10]. Leaves are used in rheumatism. Fruits are generally prescribed in dysentery and diarrhoea. Infusion of the bark and leaves are widely used as a mouth-wash for inflammed gums [11]. Leaves comprise vitamin C, myricetin, myricitrin, mearnsetin and ellagic acid. Fruits comprise tannin and large amount of plant acids including citric acid [12, 13]. Acidic portions of water-soluble dietary fibre of the fruits comprise rhamnose (11%), arabinose (26%), galactose (35%) and uronic acid (27%). The polymeric portion is composed of Îą-n-galactopyranosyl uronic acid chains with side chains of rhamnose, galactose and arabinose residues. A skin care product containing extracts from E. floribundus exhibited excellent active-oxygen scavenging actions, and is believed to have excellent anti-aging and skin-whitening activities [14]. The present study was designed to assess the antibacterial and antioxidants activities of ethanolic, chloroform and petroleum ether extracts of Elaeocarpus floribundus leaves. MATERIALS AND METHODS: Drugs and Chemicals DPPH (1,1-diphenyl-2-picryl hydrazyl) was obtained from Sigma Aldrich USA. Ascorbic acid was obtained from SD Fine Chem. Ltd, Biosar, India. DMSO (dimethylsulfoxide) was purchased from Merck, Germany. Kanamycin was collected from Square Pharmaceuticals Ltd., Bangladesh. Collection and Identification of the Plant The fresh leaves of E. floribundus were collected in the month of April, 2013 from Mymensing, Bangladesh and were taxonomically identified by Bangladesh National Herbarium.
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IAJPS 2016, 3 (2), 92-97
Drying and Grinding of Plant Material The fresh leaves of the plants were first washed with water to remove adhering dirt. Then leaves were sun dried for 7 days and finally dried in an oven at temperature not more than 50°C for better grinding. The dried leaves were ground into course powder by a grinder machine and were stored in an airtight container for further use. Extraction of the Plant Material Powdered sample having a weight of 400 g of E. floribundus was extracted by cold extraction process using ethanol (1000 ml) with daily shaking and stirring for 15 days at room temperature. After 7 days the extract was filtered through cotton followed by filter paper (Double filter paper 102, 11.0 cm). Then the concentrated liquid extract was dried at 37°C to obtain a greenish mass. The weight of the crude extract obtained from leaves was 30 grams. The whole process was repeated with chloroform and pet ether instead of ethanol using as solvent. Antimicrobial Activity Test Microorganism Antimicrobial activity was carried out against five Gram positive (S. aureus, B. cereus, B. megaterium, B. subtilis and S. luteae) and five Gram negative (E. coli, V. parahemolyticus, S. dysenteriae, S. paratyphi and P. aeruginosa) bacteria. These bacteria were chosen to be studied as they are important pathogens and also due to rapidly developed antibiotic resistance. The microorganisms were collected as pure cultures from the Institute of Nutrition and Food Science (INFS), University of Dhaka, Bangladesh. For bacteria, the culture media was prepared by nutrient agar, reconstituting with distilled water according to specification (2.8% w/v). Preparation of Inoculum Preparation of inoculum of the test organisms was done by using the colony suspension method [15]. The bacterial stock cultures were incubated for 24 hours at 37°C on nutrient agar and potato dextrose agar (PDA) medium at 4°C. The bacterial strains were grown in Mueller-Hinton agar (MHA) plates at 37°C. The stock cultures were maintained at 4°C. The bacterial strains were adjusted to a turbidity of 0.5 McFarland standards approximately 108 CFU/ml for bacteria with the addition of sterile saline (0.9 % NaCl) based on the optical density (OD) measurement at 530 nm. Antibacterial Activity by Disc Diffusion Method Antibacterial activity of E. floribundus was carried out by the standard disc diffusion method [16].
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Sonia Zaman
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Solution of known concentration (500 μg /disc) of the test sample was made by dissolving measured amount of the sample (50 mg) in 1 ml of methanol. Then sterile filter paper disc (5 mm diameters) was impregnated with known test substance and dried. The dried disc was placed on plates (Petri dishes, 120 mm diameter) containing a suitable medium (nutrient agar) seeded with the test organisms. Standard disc of kanamycin (30μg/disc) and blank discs (impregnated with solvents followed by evaporation) were used as positive and negative control. These plates were kept at low temperature (4ºC) for 24 hours to allow maximum diffusion. The plates were then kept in an incubator (37ºC) for 24 hours to allow the growth of microorganisms. Antibacterial activity of the test sample was observed by growth inhibition of organisms forming clear, distinct zone surrounding the discs. The antibacterial activity was expressed in terms of millimeter by measuring the diameter of the zone of inhibition. The greater zone of inhibition indicates the greater activity of the test material against the test organism. DPPH Radical Scavenging Activity The free radical scavenging activity (antioxidant capacity) of the three leaves extracts on the stable radical 1,1-diphenyl-2- picrylhydrazyl (DPPH) was determined by the method of Brand-Williams [1719]. The stock solution (24 mg DPPH/100 mL methanol) was diluted with methanol to get an absorbance of 1.1 at 515 nm using UV spectrophotometer (Model NO. 1501PC Shimadzu, Japan). 0.6 mL of the sample extracts at different concentrations, blank and ascorbic acid as standard were permitted to react with 3 mL of the DPPH working solution for 20 min under dark conditions. Then, the absorbance was taken at 515 nm. DPPH free radical scavenging capacity was calculated from the absorbance of sample, blank, and ascorbic acid as standard. Percent scavenging of the DPPH free radical was measured using the following equation% DPPH radical scavenging = [1 – (As/Ac)] × 100 Here, Ac = absorbance of control, As = absorbance of sample/standard solution. Statistical Analysis Results were expressed as mean ± standard deviation (SD) from three separate observations. Student’s t test was used to find the significance of standard and sample for IC50 values. Microsoft Excel 2010 (Roselle, IL, USA) was used for the statistical and graphical evaluations. A probability of p < 0.05 was considered as significant.
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Zone of inhibition (mm)
RESULTS AND DISCUSSION: Antibacterial Activity The ethanol, chloroform and pet ether extract of E. floribundus was subjected against gram positive and Gram negative bacteria. Most bacteria were sensitive to the extracts as shown in Figure1 and Figure 2. The ethanol extract showed significant antimicrobial activities against both gram positive and gram negative bacteria. The chloroform and pet ether showed moderate antimicrobial activity compared to standard kanamycin. The ethanol extract showed significant activity against S. aureus, B. subtilis, S. luteae, S. dysenteriae, P. aeruginosa and E. coli with
35 30 25 20 15 10 5 0
ISSN 2349-7750
22mm, 20mm, 20mm, 18mm, 18mm and 16mm zone of inhibition respectively. The chloroform extract also showed good activity against S. aureus and B. cereus with 10mm zone of inhibition. The pet ether extract showed good activity only against S. aureus with zone of inhibition 14mm. Chloroform and pet ether showed mild antibacterial activity against the remaining bacteria.
Ethanol Chloroform Extract Petroleum Ether Extract Kanamycin
Gram positive bacteria
Zone of inhibition (mm)
Fig 1: Antibacterial activity of E. floribundus leaves extracts against gram positive bacteria. Values are expressed as mean Âą SD (n = 3).
30 25 20 15 10 5 0
Ethanol Chloroform Extract Petroleum Ether Extract Kanamycin
Gram negative bacteria
Fig 2: Antibacterial activity of E. floribundus leaves extracts against gram negative bacteria. Values are expressed as mean Âą SD (n = 3).
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IAJPS 2016, 3 (2), 92-97 Antioxidant Activity The antioxidant activity of E. floribundus was determined spectrophotometrically using DPPH free radical and ascorbic acid as standard. The percentages of DPPH free radical scavenging are given in Figure 3 that shows that the scavenging effects were in the following order: Ethanol extract > Petroleum ether extract > Chloroform extract. The percentage of scavenged DPPH radical is directly proportional to the dose of the extracts that is, as the
Sonia Zaman
ISSN 2349-7750
concentration of the extracts increase, the percentage of scavenged DPPH radical increases. Then % of inhibitions was plotted against respective concentrations used and from the graph IC50 was calculated. The ethanolic, chloroform and petroleum ether extracts showed significant antioxidant activity with IC50 of 45.23µg/ml, 79.14 µg/ml and 60.18 µg/ml respectively as compared with 41.78µg/ml, IC50 of ascorbic acid (Figure 4).
Fig 3: DPPH radical scavenging activity of standard and E. floribundus leaves extracts. Values were expressed as mean ± SD (n = 3).
Fig 4: IC50 values of standard and E. floribundus leaves extracts. Values were expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01 significance difference from standard
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IAJPS 2016, 3 (2), 92-97 In this study, among all the extracts tested, the maximum capacity to scavenge DPPH radical was found in the ethanolic extract with IC50 value 45.23 ± 2.14 μg/ml compared to chloroform and petroleum ether extracts respectively. CONCLUSION: The present antibacterial and antioxidant studies on this plant have proved the traditional acceptance of this plant, although all the extracts were not same important. Among the extracts ethanol extract showed significant antibacterial and antioxidant activities. The evaluation of antibacterial and antioxidant activities of E. floribundus would play a significant role in the findings of more chemical entities and their bioactivities. ACKNOWLEDGEMENT: The author wish to acknowledge the phytochemical research laboratory of the Department of Pharmacy, Southeast University, Dhaka-1213, Bangladesh. COMPETING INTERESTS: The author proclaim that there is no competing interests exist about the content of this article. AUTHORS’ CONTRIBUTIONS: SZ designed the study, wrote the protocol, managed the analyses of the study, carried out the tests, managed the literature searches and prepared the draft of the manuscript. REFERENCES: 1. K Amina, Rahman M, Haque T, Rahman MM, Akter M, Akter S, Jhumur A. Cytotoxicity potentials of eleven bangladeshi medicinal plants. The Scien Wor J, 2014; 2014:1-7. 2. Poonamand K, Singh GS. Ethnobotanical study ofmedicinal plants used by the Taungya community in Terai Arc Landscape, India. J of Ethnopharma, 2009; 123(1):167-176. 3. Rasool Hassan BA. Medicinal Plants (Importance and Uses) Pharmaceut Anal Acta, 2012; 3:e139. 4. Koyama J. Anti-infective quinone derivatives of recent patents,” Recent Patents on Anti-infective Drug Discovery, 2006; 1(1):113-125,. 5. Iwu MW, Duncan AR, Okunji OO. 1999. New antimicrobials of plant origin,” in perspective on new crops & new uses. Alexandria, Va, USA: ASHS Press.
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6. Harman D. Aging: phenomena and theories. Ann of the New York Acad of Sci, 1998; 854:1-7. 7. Maxwell S.R.J. Prospects for the use of antioxidant therapies. Drugs, 1995; 49(3):345-361. 8. Niki E, Shimaski H, Mino M. Antioxidantism-Free Radical and Biological Defense, Gakkai Syuppn Center, Tokyo, Japan, 1994. 9. Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham J.B. The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radi Res, 1995; 22(4):375-383. 10. Chowdhury KA, Ghosh, SS. 1958. Indian woods, their identification, properties and uses. Delhi, India: Manager of Publications. 11. Yusuf M, Begum J, Hoque MN, Chowdhury JU.2009. Medicinal Plants of Bangladesh. BCSIR, Chittagong. 12. Ghani A. 2003. Medicinal Plants of Bangladesh. Asiatic Society of Bangladesh, Dhaka, Bangladesh. 13. Rastogi RP, Mehrotra BN. 1993. Compendium of Indian Medicinal Plants. New Delhi, India: Publication and Information Directorate. 14. Aggarwal S. 2001. Elaeocarpus L. In: van Valkenburg, J.L.C.H. & Bunyapraphatsara, N. (Editors). Plant Resources of South-East Asia No 12(2): Medicinal and poisonous plants. Backhuys Publishers, Leiden, Netherlands. 241-246. 15. European Committee for Antimicrobial Susceptibility Testing (EUCAST). Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by broth dilution. Clin J Microbiol Infect 2003; 9:1-7. 16. Bauer AW, Kirby WMM. Sherries, Tuck M. Antibiotic susceptibility testing by a standardized disc diffusion method. Ame J of Cli Patho, 1966; 45:493-49. 17. Brand-Williams W, Cuvelier ME, Berset C. Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie. 1995; 28:25-30. 18. Aher AN, Pal SC, Yadav SK, Patil UK, Bhattacharya S. Antioxidant activity of isolated phytoconstituents from Casuarina equisetifolia Frost (Casuarinaceae). J. Plant Sci, 2009: 4:15-20. 19. Ham YM, Kim KN, Lee WJ, Lee NH, Hyun CG. Anti-inflammatory effects of apo-9′-fucoxanthinone from the brown alga, Sargassum muticum. Int. J. Pharmacol, 2010; 6:147-151.
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