Presented by: A Srikanth sai Nitesh Banerjee Rakesh Ranjan Pradeep kr Raut Avinash kr singh

Guided by: K. R. Ethiraj

Content
Introduction Aim and Objective Structure of chalcones Literature review Materials and Methods Results and Discussion Conclusion References

Introduction
Several oxidative agents are formed in body during normal metabolic processes that are believed to be etiological factor for many diseases e.g. Cancer, Diabetes, Renal disease & Neurodegenerative diseases (Fauci et al 2006, Kumar et al. 2008, Mark Percival 1998).
Thus bringing in the need to develop and access the compounds having antioxidant property in order to develop an effective and efficient therapeutics to deal with oxidative stress. Chalcones are known to possess antioxidant character at various extents which is related to a number of different mechanism such as free radical scavenging, hydrogen donation, singlet oxygen quenching, metal ion chelation and acting as a substrate for free radicals such as superoxide and hydroxide (Chetana B. Patil et al 2009) .

Aim and objective

Screening of flouro and chloro substituted synthetic chalcones for anti oxidative property by following in vitro biochemical assay: 1. DPPH radical scavenging activity method 2. Ferrous ion chelating activity 3. Hydrogen peroxide radical scavenging activity

Structure of chalcones
Chalcones
Chalcones are 1,3 diphenyl1-2-propene-1-one, in which two aromatic rings are linked by a three carbon ,- unsaturated carbonyl system.

Synthetic chalcone

Literature review
Kankate R.S. et al reported their study on the Evaluation of Antioxidant Activity of Chalcones and Flavonoids that 2-hydroxychalcones, 2- Bromochalcone and 2Chlorochalcones showed significant activity and 3-hydoroxyflavones, 4- methoxy and 4 chloroflavone were highly potent.
Anastasia Detsi et al synthesized and evaluated the antioxidant and soybean lipoxygenase inhibitory activity of Natural and synthetic 2-hydroxy-chalcones and aurones. 2hydroxy-chalcones and their cyclization products were tested for their antioxidant and soybean lipoxygenase inhibitory activity. An extensive structure activity relationship studies were carried out in order to understand the appealing pharmacological profile combining high antioxidant and lipid peroxidation activity possessed by chalcones. Ruby John Anto et al reported cytotoxic, anti neoplastic and antioxidant activities of various synthetic chalcones and structurally related compounds. Methyl and hydroxyl substituted chalcones were found to be cytotoxic in- vitro condition whereas hydroxyl substituted were found to have anti neoplastic activity in laboratory animals. Although most of the compounds were found to possess antioxidant property but those with hydroxyl and methyl substitution were highly potent as an antioxidant.

Materials & Methods

Chemicals
Ascorbic acid 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) Ferrous sulphate Hydrogen peroxide Ferrozine Methanol Phosphate buffer was prepared according to Indian pharmacopeia

Contd
Methods
DPPH radical scavenging activity method (S.D. Sanja et al. 2009) Different dilution of stock solution (10, 25, 50, 75 and 100 g/ml) was prepared. 3 ml of the test sample was taken from each and 1 ml of 0.05mM DPPH solution was added. The mixture was incubated at room temperature for 15 minutes and absorbance was taken in UV- visible spectrophotometer (UV 1601, SHIMADZU). Methanol was used as blank for base line correction. Ferrous ion chelating activity (Bektas Tepe et al 2011) different concentration of 10, 25, 50, 75 and 100 g/ml of test sample and standard was prepared. 0.05 ml of 2mM solution of FeCl2 was added to the test sample. 5mM ferrozine was added to this solution to start the reaction; total volume was adjusted to 5 ml with methanol. The mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance was taken at 562 nm in a UV- visible spectrophotometer (UV 1601, SHIMADZU). Hydrogen peroxide radical scavenging activity (Vilasrao et al. 2010) The test sample was diluted to give different concentration of 10, 25, 50, 75 and 100 g/ml. 3.4 ml of each concentration was taken and 0.6 ml of hydrogen peroxide solution in phosphate buffer (pH- 7.4) was added to it. This mixture was incubated at 37C for 10 minutes and absorbance was taken at 230 nm in a UV- visible spectrophotometer. % activity = [(control absorbance sample absorbance)/control absorbance]*100

Result and Discussion

Percentage DPPH Radical scavenging activity
Concentration (g/ml) Percentage DPPH Radical scavenging activity of Ascorbic acid. Percentage DPPH Radical scavenging activity of choloro substituted chalcone. Percentage DPPH Radical scavenging activity of floro substituted chalcone.

10 25
50 75 100

4.173 10.483
20.509 30.789 40.967

3.014 7.447
14.539 21.764 29.034

4.223 10.581
21.063 31.148 41.927

DPPH Radical scavenging activity

45 40 35 30 25

percentage activity

%Ascorbic acid
20 15 10 %Activity chalcon1 %Activity chalcon2

5
0 0 20 40 60 concentration 80 100 120

Contd
Percentage hydrogen peroxide Radical scavenging activity
Concentration (g/ml) Percentage hydrogen peroxide Radical scavenging activity of Ascorbic acid. Percentage hydrogen peroxide Radical scavenging activity of choloro substituted chalcone. Percentage hydrogen peroxide Radical scavenging activity of floro substituted chalcone.

0 10 25

0 1.108 2.467

0 0.889 2.159

0 2.160 4.218

50 75
100

4.230 6.042
8.056

3.683 6.2586
8.190

7.922 11.523
15.432

H2O2 Radical scavenging activity

18 16 14 percentage activity 12 10 8 6 4 2 0 0 20 40 60 concentration 80 100 120

%Activity ascorbic acid

%Activitychalcon1 %Activitychalcon2

Contd
Percentage ferrous ion chelating activity
Concentration (g/ml) Percentage ferrous ion chelating activity of Ascorbic acid . Percentage ferrous ion chelation of choloro substituted chalcone. Percentage ferrous ion chelating activity of floro substituted chalcone.

0 10 25

0 11.745 27.223

0 10.509 24.096

0 13.990 23.840

50 75
100

45.621 65.648
82.960

49.096 71.218
88.889

45.967 66.096
85.939

. Ferrous ion chelating activity

100 90

80
70 percentage activity 60 50 40 30 20 10 0 0 20 40 60 concentration 80 100 120

%Activity ascorbic acid

%Activitychalcon1 %Activitychalcon2

Discussion
DPPH method centered stable free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecule. It changes its color from violet to yellow upon reduction. Substance that can donate electron or hydrogen to reduce the DPPH can be considered as antioxidant and thus accepted as free radical scavengers. DPPH radical reduction capability was determined at 516 nm which shows decrease in absorbance, maximum being for stable DPPH in ethanol (control) at 516. Thus it was found that the antioxidant activity of the test compounds increased with increase in concentration. Hydroxyl radicals are produced by hydrogen peroxide. These radicals are scavenged by the test sample which is indication of their antioxidant property and the basic principle behind the assay. Decrease in the absorbance at 230 nm with increasing concentration of test sample is due to the reduction of these radical. The scavenging activity of the chalcones and ascorbic acid is directly proportional to the concentration. Ferrozine quantitatively forms complex with Fe2+. Chelating agent disrupts the complex formation between ferrozine and Fe2+ thus the intensity of red color decreases. Degree of decrease in red color intensity is proportional to the concentration of chelating agent and thus helpful in measurement of chelating activity of test sample.

conclusion
The percentage DPPH radical scavenging activity, the hydrogen peroxide radical scavenging activity, the percentage ferrous ion chelating activity of flouro substituted chalcone was found to be greater than the chloro substituted chalcone and ascorbic acid at all the concentration.
At 100 g/ml concentration percentage DPPH radical scavenging activity of flouro substituted chalcone, chloro substituted chalcone and ascorbic acid was respectively 40%, 30% and 39%. At 100 g/ml concentration the hydrogen peroxide radical scavenging activity was found to be 15.8% that seemed to be poor. The hydrogen peroxide radical percentage scavenging activity of ascorbic acid was found to be 8% at 100 g/ml. The maximum ferrous ion chelating activity was found to be 89.9% for flouro, 90% for chloro and 89.5 for ascorbic acid at 100 g/ml.

Reference
Dr. Mark Percival (1998) Antioxidant; Nut031 1/96 Rev. 10/98, Clinical Nutrition Insights
Anastasia Detsi, Maya Majdalani, Christos A. Kontogiorgis, Dimitra Hadjipavlou- Litina, Panagiotis Kefalas, (2009), Natural and synthetic 2-hydroxy-chalconees and aurones: synthesis, characterization and evaluation of the antioxidant and soybean lipoxygenase inhibitory activity; Bioorganic & Medicinal Chemistry, vol. 17, 8073-8085 Belsare D.P., Pal S.C., Kazi A.A., kankate R.S., Vanjari S.S, (April-June 2010), Evaluation of Antioxidant Activity of Chalconees and Flavonoids; Inernational Journal of ChemTech Research, Vol.2, No.2, pp 1080-1089, Bektas Tepe, H. Askin Akpulat, and Munevver Sokmen, (2011) Evaluation of the chemical composition and antioxidant activity of the essential oils of Peucedanum longifolium and P. palimbioides; records of natural products, 5:2, 108-116. Chetana B. Patil, S.K. Mahajan, Suvarna A. Katti; Chalconee (2009): A Versatile Molecule; journal of Pharmaceutical Sciences and Research vol.1(3), 11-22. Fauci, Braunwald, Kasper, Hauser, Longo, Jameson, Loscalzo,(2006) Harrisons principle of internal medicine; 17th edition, pg.70; McGraw-Hills Access Medicine. H. M.M. Hassan and Nahla M.M. Hassan(2010) in vitro Antioxidant and Free Radical scavenging activities of Red Grape seeds extracts; Global Journal of Biotechnology and Biochemistry (5): 106-115.

Contd.

Hua-Yew Cheng, Ta-Chen Lin, Kuo-Hua YU, Chien-Min Yang, and Chun- ching Lin; Antioxidant and Free Radical Scavenging Activities of Terminalia chebula; Biol. Pharm. Bull. 26(9) 1331-1335. Kumar, Abbas, Fausto, Mitchell, (2008) Robbins Basic Pathology 8th edition, pg 7& 280-281, ELSEVIER. O. Gursoy Kol and H. Yuksek, (2010) synthesis and in vitro Antioxidant evaluation of some novel 4,5Dihydro-1H,2,4-triazol-5-one Derivatives; E-Journal Chemistry,7(1), 123-136. P. Arulpriya, P. Lalitha and S. Hemalatha,( 2010) cyclic voltametric assessment of the antioxidant activity of petroleum ether extract of Samanea saman; Advances in applied Science Research, 1 (3): 24-35. Ron Kohen and Abraham Nyska, (2002) Oxidation of Biological systems: Oxidative stress phenomena, Antioxidants, Redox Reaction and Methods for their Quantification; Toxicol Pathol/ 30:620. Ruby John Anto, K. Sukumaran, Girija Kuttan, M.N.A. Rao, V. Subbaraju, Ramadasan Kuttan, (1995) Anticancer and Antioxidant activity of synthetic Chalconees and related compounds; Cancer Letter 33-37. S.D. Sanja, N.R. Sheth, N.K. Patel, Dhaval Patel, Biraju Patel, ( 2009) Characterization and evaluation of antioxidant activity of Portulaca oleracea; International Journal of Pharmacy and pharmaceutical sciences, vol. 1, issue 1. Vilsrao j. kadam, Yadunath M. Joshi, Harshand P. Sawant, Tej A. Jadhav, (2010) Free radical scavenging activity of aqueous solution of black salt; International Journal of Pharmacy and Pharmaceutical Sciences, vol. 2 suppl. 2.