The in vitro antibacterial and antioxidant properties of hydromethanolic extract of peel from Citrus sinensis (Sweet orange) was investigated. In this study the antibacterial activity of Citrus sinensis peel extract against different gram positive and gram negative bacteria by disc diffusion method and antioxidant activity by Fenton reaction was undertaken. Citrus sinensis peels had shown mild activity against gram positive and gram negative bacteria and the MIC value was measured. In another experiment antioxidant activity was observed and IC50 was shown at 600 Ã‚Âµg/ml

Agrawal1, Rahul Verma1 and Rahi Jain1, Dayanand Dubey1*, K. Balamurugan2, R.C.

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Recent Research in Science and Technology 2011, 3(11): 22-25 ISSN: 2076-5061 www.scholarjournals.org   www.recent-science.com 22  RRST-Pharmacology Evalution of Antibacterial and Antioxidant Activity of Methanolic and Hydromethanolic Extract of Sweet Orange Peels  Dayanand Dubey1*, K. Balamurugan2, R.C. Agrawal1, Rahul Verma1 and Rahi Jain1  1Jawaharlal Nehru Cancer Hospital & Research Centre, Idgah Hills, Bhopal, India. 2Annamalai University, Dept. of Pharmacy, Chidambaram, Tamilnadu, India  Article Info  Abstract Article History  The in vitro antibacterial and antioxidant properties of hydromethanolic extract of peel from Citrus sinensis (Sweet orange) was investigated. In this study the antibacterial activity of Citrus sinensis peel extract against different gram positive and gram negative bacteria by disc diffusion method and antioxidant activity by Fenton reaction was undertaken. Citrus sinensis peels had shown mild activity against gram positive and gram negative bacteria and the MIC value was measured. In another experiment antioxidant activity was observed and IC50 was shown at 600 µg/ml.   Received : 20-04-2011  Revised : 27-04-2011  Accepted : 03-05-2011  *Corresponding Author  Tel : +91-8085605968       Email:  dayananddubey04@gmail.com   ©ScholarJournals, SSR  Key Words: Orange peel, Antioxidant activity, Antibacterial activity    Introduction Citrus is one of the most important commercial fruit crops grown in all countries of the world [1]. Antioxidants are the substances, compounds or nutrients in our foods which can prevent or slow oxidative damage to our bodies. These agents are able to remove the deleterious effects of free radicals within our body. Nowadays, considerable interest is focused on the development and evaluation of natural antioxidants and radical scavengers from plant materials which are rich in polyphenolic compounds. It is well known that plant derived polyphenols have remarkable antioxidant and free radical scavenging activity resulting in multiple beneficial nutritional physiological effects in the humans. The essential oil obtained from citrus fruits is having excellent antimicrobial properties and is used in cosmetic industry [2]. An antimicrobial is a substance that kills or inhibits the growth of microorganism such as bacteria, fungi or protozoans. Antimicrobial drugs either kill microbes (microbicidal) or prevent the growth of microbes (microbistatic). Disinfectants are antimicrobial substances used on nonliving objects. Citrus fruit products are known to potent antimicrobial agents against bacteria and fungus. These citrus fruits are rich source of flavonones and many polymethoxylated flavones which are very rare in other plants [3]. Antimicrobial and antioxidant properties of peel and pulps of some citrus fruits were investigated [4]. These compounds, not only play an important and physiological role, but are also of commercial interest because of their multitude of applications in the food and pharmaceutical industries activities of the world.     Materials and Methods Plant material The fresh sweet orange fruits were collected from local shops of Bhopal (M.P), India. Preparation of Extract The whole peels of sweet orange were fresh dried and were mechanically crushed into powder and extracted with distilled water at room temperature up to 48 h. The extract was filtered and concentrated in water bath under reduced pressure to obtain semisolid material which was then powdered. The powdered material was soaked separately in methanol solvent and hydro-methanol (50 % v/v) separately to obtain the extracts [5]. Test organism The following gram negative and gram positive bacteria viz. Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Shigella flexineri, Bacillus subtilis and Escherichia coli were used for antibacterial activities which were from stock culture of our laboratory. Antibacterial activity Antibacterial activities of 50 % methanolic, extract from fruit of Orange peels were investigated using Disk diffusion method [6]. 20ml of sterile Nutient agar was added in the test tube after that petri plates were prepared and cultures were swabbed on the top of the solidified media and allowed to dry. The test was conducted at four different concentrations of the crude extract 25 %, 50%, 75% and 100% (100µl per disk), the disk which was soaked with extract were placed on the surface of the medium the placed on the surface of the medium the Dayanand Dubey et al. /Rec Res Sci Tech 3 (2011) 22-25     23plate and were incubated for 12-18 h at 37ºC and the zone of inhibition was recorded. Antioxidant activity The hydroxyl radical attached deoxyribose and initiated a series of reaction that eventually resulted in the formation of Thiobarbituric acid reaction substance (TBARs). The measurement of TBARs gives an index of free radical scavenging activity. the reaction mixture consisted of a deoxiribose (3mM, 100µl), ferric chloride (Fe3+, 0.2 mM 50µl), EDTA (0.1 mM 50 µl), ascorbic acid (0.1 mM 100 µl), stock solution of orange peel extract 20 mg/ml were prepared from which 200-2000 µl were added in reaction mixture, the final volume was made up to 1ml by adding adequate quantity of phosphate buffer saline (pH 7.4) and incubated for 1 h at 37°C. The reaction was stopped by adding 0.5 ml of 5 % TCA and 0.5 ml of 1 % TBA the mixture was than incubated for 20 min in a boiling water bath. The absorbance was measured at 532 nm. Ascorbic acid was used as positive control. The results are expressed as the percentage inhibition of TBARS as reported [7]. Results and Discussion  Antibacterial activity  Hydromethanolic extract of orange peel at the different concentration i.e. 25%, 50%, 75%, 100% exhibits antibacterial against Staphylococcus aureus (6-14mm), Bacillus subtilis (6-9mm), Staphylococcus epidermis (5-10mm), E. coli (7-12 mm), Shigella flexineri (9-12mm), Pseudomonas aeruginosa (6-9 mm). The results are recorded in Table 1 and Figure 1-6. Antioxidant activity  The extract of orange peel showed good antioxidant property in Fenton reaction model, the test drug was compared with a low concentration of ascorbic acid. Table 2 showed the percent TBARS inhibition of Hydromethanolic extract of orange peel and Ascorbic acid. It is clear from the above data that the Orange peels which are considered as waste material of the fruit consist of good Antibacterial and Antioxidant activity.  Table 1:  Antibacterial activity of orange peel extract against different bacterial strains S.No Microorganism Concentration of Hydromethanolic extract  Zone of inhibition Concentration of Methanolic extract Zone of inhibition   25% 50% 75% 100% 25% 50% 75% 100% 1. Staphylococcus aureus 8mm 12mm 14mm 14mm 9mm 13mm 16mm 16mm 2 Bacillus subtilis NA 8mm 9mm 9mm 6mm 8mm 9mm 10mm 3. Staphylococcus epidermis NA 7mm 8mm 10mm NA 10mm 10mm 13mm 4. E. coli 7mm 9mm 10mm 12mm 8mm 12mm 14mm 16mm 5. Shigella flexineri 6mm 9mm 10mm 12mm 8mm 9mm 10mm 14mm 6. Pseudomonas  aeruginosa NA 6mm 8mm 9mm 7mm 8mm 9mm 13mm  Table 2:  In vitro Antioxidant activity of Hydromethanolic Extract of orange peel S. No. Concentration (20mg in1ml ) % of  TBARS Concentration (20mg In 1ml ) Of Orange Peels Extract  Ascorbic acid  Hydromethanolic % of TBARs Methanolic % of TBARs 1. 200 50.3 200 45.30 200 45.85 2. 400 51.6 400 45.85 400 46.96 3. 600 81.8 600 49.72 600 63.53 4. 800 85.2 800 55.24 800 113.25 5. 1000 95.3 1000 59.11 1000 74.58 6. 1200 69.7 1200 66.85 1200 82.87 7. 1400 122 1400 63.54 1400 85.63 8. 1600 110.7 1600 67.95 1600 109.39 9. 1800 139.5 1800 72.38 1800 110.01 10. 2000 142.2 2000 74.59 2000 151.9   Methanolic extract of Orange peels consist of good antioxidant and antibacterial activity as compared to Hydromethanolic extract. Among many other bacterial species, S. aureus showed good antibacterial activity at 100 % methanolic extract concentration. Both (Hydromethanolic and Methanolic extracts) showed good antioxidant activity when done by Fenton reaction. Orange peel showed IC50 at 600µg/ml and standard Ascorbic Acid showed IC50 at 200 µg/ml. The results of antimicrobial and antioxidant activity are found to be correlated with some previous work [4].  Dayanand Dubey et al. /Rec Res Sci Tech 3 (2011) 22-25     24               Figure 1: Inhibition of Staphylococcus aureus in a different concentration of Hydromethanolic and Methanolic extract of Orange peel Extract               Figure 2: Inhibition of Bacillus subtilis in a different concentration of Hydromethanolic and   Methanolic extract of Orange peel Extract               Figure 3: Inhibition of Staphylococcus epidermis in a different concentration of Hydromethanolic and Methanolic extract of Orange peel Extract               Figure 4: Inhibition of E coli in a different concentration of Hydromethanolic and Methanolic extract of Orange peel Extract.                 Figure 5: Inhibition of Shigella flexineri in a different concentration of Hydromethanolic or    Methanolic extract of Orange peel Extract              Figure 6: Inhibition of Pseudomonas aeruginosa in a different concentration of Hydromethanolic and Methanolic extract of Orange peel Extract  Conclusion From the present investigation it may be concluded that hydromethanolic extract of sweet orange peel has potent antibacterial activity against gram positive and gram negative bacteria. Hydromethanolic extract of Citrus sinensis showed good antioxidant activity and IC50 was found at 600 µg/ml concentration. Further studies are however needed to isolate the active molecule responsible for both antimicrobial and antioxidant activity. The studies thus may lead to the formulation of a potent antimicrobial and antioxidant agent.  References  [1] Tao K. 2007. Chemical composition of essential oil from the peel of Satsuma mandarin. African   Journal of   Biotechnology. 7(a):1261-1264. [2] Caccioni DRL. 1998. Relationship between volatile components of citrus essential oils and antimicrobial action on Penicillium digitatum and Penicillium italicum. Int. J. Food Microbiology. 43:73-79. [3] Ahmed A. 2006. Genetic variability to essential oil composition in four Citrus fruit species. Pak. J. Bot. 38(2): 319-324 [4] Mathur A., S.K. Verma, R. Purohit, GBKS. Prasad, D. Mathur, V. Gupta, V.K. Dua and S. Singh. 2011. Evaluation of in vitro antimicrobial and antioxidant properties of some citrus fruits. IJPI’s Journal of Biotechnology & Biotherapeutics. 1(2):1-17. Dayanand Dubey et al. /Rec Res Sci Tech 3 (2011) 22-25     25[5] Alade P.I. and O.N. Irobi. 1993. Antimicrobial activity of crude leaf extract of Acalypha wilkesiana. J. Ethanopharmacol.39: 170-174. [6] Nehru S.S., Z. Zuraini, S. Sasidharan and S. Suryani. 2008. Antimicrobial activity of Cassia surattensis and Cassia fistula. J. Mol. Bio. & Biotechnology. 1:1-4. [7] Kamran G., G. Yousuf and M.A. Ebrahimzadehb. 2009. Antioxidant activity, phenol and flavonoid contents of 13 citrus species peels and tissues. Dept. of horticulture, Faculty of Agriculture. 22 (3): 277-281.  

Evalution of Antibacterial and Antioxidant Activity of Methanolic and Hydromethanolic Extract of Sweet Orange Peels

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Evalution of Antibacterial and Antioxidant Activity of Methanolic and Hydromethanolic Extract of Sweet Orange Peels

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