“Antioxidant activity of Nigella sativa and protection for Biological Damages Due to Radiation Effects” 1 Presented by Mr.Halavath Ramesh M.A,M.sc,B.ED,PGDCAQM,PGDCA,M.Phil,(P.HD)(UoH) University of Madras Dept. of Chemistry Loyola College,Chennai.

Introduction Phytochemicals screening Estimation of phenol and Flavanoids Antioxidant activity Genomic DNA extraction DNA protection assay Expected out come of the proposed study Equipment and budget Conclusion References Acknowledgement 2 CONTENT

1. Radiation effect of biological damage in living organisms is one of the major problems in the world. 2.The interaction of ionizing radiation with living organisms can lead to harmful health consequences such as tissue damage, cancer, and cataracts in space and on earth. 3.High radiation doses tend to kill cells while low doses tend to damage or alter the genetic code(DNA) of irradiated cells. 4.Challenges in the field phytochemistry including isolating specific compounds and determining their structure and identifying what specific Phytochemicals is primarily responsible for biological activity. 5. The secondary metabolites contribute significantly towards the biological activities of medicinal plants such as antioxidant, antidiabetic, antimicrobial and antileprosy, anti- inflammatory. 6. Antioxidants are molecules capable of reducing the causes or effects of oxidative stress. 7. The development of radioactive substance has attracted an increasing interest because of their potential use in radiotherapy counteracting occupational radiation hazards, space exploration, and in research. 8. The Nigella sativa plant extracts show a wide range of biological activities. 3 INTRODUCTION

Radiation Effects on Human Body Effects of Radiation on Human Body Hair Brain Thyroid Reproductive Track Gastrointestinal Track Anticancer Heart Blood System

5 MEDICO ETHNO BOTANICAL SURVEY OF Nigella Sativa KingdomPlantae (unranked)Angiosperms (unranked)Eudicots (unranked)Asterids Order Gentianales FamilyRanunculales GenusNigella SpeciesN.Sativa

Sample preparation and extraction Black cumin seeds(N.sativa) + Grind + Powder by using mortar pestle Powder dissolve in three solvent(Petroleum ether,EA,Methanol) Filter by whatsman no.4 filter paper (vacuum filtration) Evaporate three solvents by using rota vapor at different temperature After dried and weight the above three crude extract Sample is use for further studies

Phytochemicals screening Tannin Glycoside Xanthoprotein Terpenoids Flavanoids Phenol Alkaloids Saponin

1. Phytochemical Screening: Sl.NoPhytochemicalsPetroleum ether extractEthyl acetate extractMethanol extract 1Alkaloid +/- 2Amino acid +/ - 3Anthraquinones +/ - 4Catechin +/ - 5Fixed oil +/ - 6Flavonoid +/ - 7Glycoside +/- 8Phenols +/- 9Quinone +/- 10Reducing sugars +/- 11Saponin +/- 12Steroids +/ - 13Tannin +/- 14Terpenoids +/- 15Xanthoprotein +/ - 9 Note:( +) presence(-) Absence Ref: Abdurohaman, A. J. of Life Sciences 2015; 3(5):

Estimation phenol Method Aliquot of sample were pipetted out in series of test tubes and volume was made up to 3 ml with distilled water. Folin-Ciocalteau reagent (0.5ml) was added to each tube and incubated for 3 min. at room temperature Sodium carbonate (20%; 2ml) solution was added, mixed thoroughly and the tubes were incubated for 1 min. in boling water bath. Absorbance was measured at 650nm against a reagent blank. Standard curve using different concentrations of standard phenolic -catechol was prepared. From the standard curve, concentration of phenols in the test samples was determined and expressed as µg of catechol equivalent Results Table: Concentration of Phenolics in given sample Test Sample μg/mg of catechol equivalent phenolics MD 170 Reference Malick, C.P. and Singh, M.B. (1980). In: Plant Enzymology and Histoenzymology. Kalyani Publishers. New Delhi. P 286)

Estimation of Flavanoids Method Known volume of sample was pipetted out in series of test tubes and volume was made up to 0.5 ml with distilled water. Sodium nitrite (5%; 0.03ml) was added to each tube and incubated for 5 min. at room temperature Aluminium chloride solution (10%; 0.06ml) solution was added and incubated for 5 min. at room temperature. Sodium Hydroxide solution (1 M; 0.2ml) solution was added and total volume was made up to 1 ml with distilled water. Absorbance was measured at 510nm against a reagent blank. Standard curve using different concentrations of rutin was prepared. From the standard curve, concentration of flavonoids in the test samples was determined and expressed as mg of rutin equivalent. Results Table:Concentration of flavonoids in given sample Test Sample μg/mg of rutin equivalent flavonoid MD 10 Reference Helmja K., Vaher M., Gorbatšova J. and Kaljurand M. (2007). Characterization of bioactive compounds contained in vegetables of the Solanaceae family by capillary electrophoresis. Proc. Estonian Acad. Sci. Chem., 56; (4) 172–186.

Antioxidant activity Free radical scavenging activity Method Stock extract sample was prepared by dissolving 10mg/ml in Dimethyl Sulphoxide. Different concentrations (50 μg, 100μg, 250μg and 500 μg) of extract and Butylated hydroxy anisole (BHA: reference standard) was taken in series of test tubes. The volume was adjusted to 500 μl by adding Methanol. To these tubes, 5 ml of 0.1 mM methanolic solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH: Sigma-Aldrich, Bangalore) was added and shaken vigorously. A control without the test compound, but with an equal amount of methanol was maintained. The tubes were allowed to stand at room temperature for 20 min. The absorbance of the samples was measured at 517 nm. Radical scavenging activity was calculated using the formula: Percent radical scavenging activity = (Control Absorbance - sample Absorbance) ×100. Control Absorbance

Hydroxyl radical scavenging acitivity Various concentrations (10 μg, 500 μg, 100μg and 500 μg) of extract was taken in different test tubes and made up to 250μl with 0.1M phosphate buffer. One milliliter of iron-EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 ml of EDTA (0.018%), and 1 ml of Dimethyl sulphoxide (0.85% v/v in 0.1 M phosphate buffer, pH 7.4) were added to these tubes, and the reaction was initiated by adding 0.5 ml of 0.22% ascorbic acid. These reaction mixtures were incubated at room temperature for 15 min. The reaction was terminated by the addition of 1 ml of ice-cold TCA (17.5% w/v). Three milliliters of Nash reagent (150 g of ammonium acetate, 3 ml of glacial acetic acid, and 2 ml of acetyl acetone were mixed and raised to 1 L with distilled water) was added to all of the tubes and left at room temperature for 15 min for color development. The intensity of the yellow color formed was measured spectrophotometrically at 412 nm against reagent blank. The percentage hydroxyl radical scavenging activity was calculated by the following formula: % hydroxyl radical scavenging activity = 1 - Difference in absorbance of sample × 100. Difference in absorbance of blank)

Ferric reducing antioxidant power Various concentrations (10 μg, 500 μg, 100μg and 500 μg) of extract was taken in different test tubes and made up to 250μl with 0.1M phosphate buffer. One milliliter of iron-EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 ml of EDTA (0.018%), and 1 ml of Dimethyl sulphoxide (0.85% v/v in 0.1 M phosphate buffer, pH 7.4) were added to these tubes, and the reaction was initiated by adding 0.5 ml of 0.22% ascorbic acid. These reaction mixtures were incubated at room temperature for 15 min. The reaction was terminated by the addition of 1 ml of ice-cold TCA (17.5% w/v). Three milliliters of Nash reagent (150 g of ammonium acetate, 3 ml of glacial acetic acid, and 2 ml of acetyl acetone were mixed and raised to 1 L with distilled water) was added to all of the tubes and left at room temperature for 15 min for color development. The intensity of the yellow color formed was measured spectrophotometrically at 412 nm against reagent blank. The percentage hydroxyl radical scavenging activity was calculated by the following formula: % hydroxyl radical scavenging activity = 1 - Difference in absorbance of sample × 100. Difference in absorbance of blank)

Nitric oxide radical scavenging Various concentrations (10 μg, 50μg, 100μg and 500 μg) of extract was taken in different test tubes and made up to 3ml with 0.1M phosphate buffer (pH 7.2). Sodium Nitroprusside (5mM) prepared in buffered saline (pH7.2) was added (1 ml) to each tube. The reaction mixture was incubated for 30 min at RT. A control without the test compound, but with an equivalent amount of methanol was maintained. After 30 min, 1.5 ml of above solution was mixed with 1.5 ml of Griess reagent (1% Sulphanilamide, 2% phosphoric acid and 0.1% N-1- Naphthylethylenediamine dihydrochloride). The absorbance of the samples was measured at 546 nm. Nitric oxide radical scavenging activity was calculated using the following formula: % radic al scavenging activity = (control absorbance - sample absorbance) ×100. Control absorbance

Genomic DNA extraction Preheat the extraction buffer + Tris-Hcl(PH8)+ 20mM EDTA (CTAB method) Transfer ground material+ Centrifuge + Incubate in water bath at 60 0 C hr Supernatant add equal vol. of Chloroform+ Isoamyl alcohol + Mix inversion Supernatant add twice the volume of chilled isopropanol to ppt DNA Wash the pellet with ethanol and Dry pellet in room temperature TE buffer + Dissolve DNA ( Store at C for future use.

DNA damage protection assay Extracted DNA + Phosphate buffer + Feso 4 DNA is analyzed with one percentage of agarose gel electrophoresis Plant sample Add 4µl of 0.1mM H 2 O 2 Incubate 37 0 c for 30 min. 5µl of garlic acid added DNA sample read

DNA Quality DNA should be intact and free of contaminants that inhibit amplification. – Contaminants can be purified from the original DNA source. Heme from blood, humic acid from soil and melanin from hair – Contaminants can be introduced during the purification process. Phenol, ethanol, sodium dodecyl sulfate (SDS) and other detergents, and salts.

DNA Quantity DNA quantity More template is not necessarily better. – Too much template can cause nonspecific amplification. – Too little template will result in little or no PCR product. The optimal amount of template will depend on the size of the DNA molecule.

A schematic picture of an agarose gel Plant AMarkerPlant B - + Plant C Monomorphic bandsorphic bands Polymorphic bands Presence of a band ” 1” f a band, ”1” Absence of a band ”0’’nd, ”0”

Examples

Expected out come of the proposal study Objective: To extract the Phytochemicals present in Nigella Sativa plant using N- Hexane, Chloroform and ethyl acetate solvent. To perform the Phytochemicals screening of all three extracts of Nigella Sativa plant. Isolation of phytochemicls by SFE method best among the other methods. Thymoquinone Phytochemicals may be acts as good Antioxidant Agent. Hopefully we will get good result such Ferric Ion Reducing Antioxidant Power Assay(FRAP).Modified FRAP Assay, Modified Cupric Ion Reducing Antioxidant Capacity Assay,DPPH Radical Scavenging methods. To anlayse the Immunomodulatory effects and boost the antioxidant defense mechanisms in the body. It is directly associated with utilization of the non-polluting,Eco-friendly Sources of the universe.

Equipments and Budgets

Conclusion Preliminary phytochemical analysis is that the Petroleum ether extract of nigella sativa will contain alkaloids,carbohydrates,tannins,phenolic compounds,flavonoids,terpenoids, and glycosides. Estimation of phenol and Flavanoids also performed. In addition,the NSE extract will show significant free radicals scavenging and antioxidant activities and it would be shown protection activity against Fenton's reagent and DNA damage potential is conformation by the agarose gel electrophoresis. Further isolation and testing of phytoconstituents for their individual biological activity will undoubtedly bring promising results in the discovery and development of novel drugs.

Any suggestions

ACKNOWLEDGEMENTS My sincere thanks to my guide Prof. AbaniK Bhuyan for taking special interest in giving me valuable suggestions, encouragement and generous assistance throughout this project Ideas. I extend my thanks to Senior Labments &PDF Madam, My Labments and M.sc Projects Students for helping me lot.

“Sometimes a good idea comes to you when you are not looking for it. Through an improbable combination of coincidences, inexperience and lucky mistakes, such a revelation came to me one Friday night in April, 1983, as I gripped the steering wheel of my car and snaked along a moonlit mountain road into northern California's redwood country. That was how I stumbled across a process that could make unlimited numbers of copies of genes, a process now known as the polymerase chain reaction (PCR).” (Kary B. Mullis, “The Unusual Origin of the Polymerase Chain Reaction,” Scientific American,