1. PHYTOCHEMICAL INVESTIGATION AND THE STUDY OF DRUG POTENTIAL OF PHYTO COMPOUND IN Nicotiana tobaccum Presented by Mr.Halavath Ramesh M.A,M.sc,B.ED,PGDCAQM,PGDCA,M.Phil,(P.HD)(UoH) E-mail: halavathramesh39@gmail.com University of Madras Dept. of Chemistry Loyola College,Chennai. 1

2. Introduction Experimental section Results and discussion Summary and conclusions References Acknowledgements 2 CONTENT

3. 1. Phytochemicals are chemical compounds produced by plants. 2.Challenges in the field phytochemistry including isolating specific compounds and determining their structure and identifying what specific Phytochemical is primarily response in biological activity. 3. In India Herbal medicines play an important role in the treatment of various diseases by trational methods practiced such as Ayurveda, Unani and Siddha. 4. Over 2,48,000 species of the higher plants have been identified 12,000 plants are known to have medicinal properties. 5. The knowledge of pharmacology in essential for understanding action of drugs on animals and human system. 6. The specific natural products of the primary and secondary metabolites are very important for chemists. 7. Alkaloids are a group of naturally occuring chemical compound. 8. Nicotine is the most abundant of the volatile alkaloids in the tobacco leaf. Molecular Formula : C 10 H 14 N 2 3 INTRODUCTION

4. 4 MEDICO ETHNO BOTANICAL SURVEY OF Nicotiana tobaccum KingdomPlantae (unranked)Angiosperms (unranked)Eudicots (unranked)Asterids OrderSolanales FamilySolanaceae GenusNicotiana SpeciesN. tabacum

5. Medicinal uses of tobacco Toothaches Insect Repellent Rattle Snake and Insect Bites COLD Tooth Paste Anticancer Agent Skin rashes Resist Infection

6. To extract the phytochemicals present in Nicotina tobaccum plant using N-Hexane, Chloroform and ethyl acetate solvent. To perform the Phytochemicals screening of all three extracts of Nicotina tobaccum plant. To determine the physiochemical parameters such as Ash values, Fluorescence Analysis and the Qualitative determination of Phytochemical such as Alkaloids, Ascorbic Acid. To study the Antibacterial and Antioxidant Properties of three solvent extract of Nicotina Tobaccum. To anlayse the Phytochemical constituents using analytical techniques:GC-MS and HPTLC fingerprinting. Characterization of Plant extract using IR spectroscopy, HRMS spectroscopy they use in determination of functional groups, and mass and Ionization state. 6 AIMS AND OBJECTIVES

7. EXPERIMENTAL SECTION 7

8. drywash Add solvent STEP-1 HRMS FT-IR Spectral Studies Fluorescence, Quantitative determination, TLC, GC-MS. Phytochemical screening test Analysis STEP-2 Biological Activity Antioxidant Antibacterial STEP-3

9. RESULTS AND DICUSSION 1. Phytochemical screening tests. 2. Fluorescence Analysis 3. Estimation of Alkaloids, Ascorbic acid, Phenolics 4. TLC Analysis 5. Antibacterial Activity 6. Antioxidant Activity 7. HPTLC Analysis 8. GC-MS Analysis 9. FT-IR Analysis 10. High Resolution–Mass Spectroscopy 9

10. 1. Phytochemical Screening: Sl.NoPhytochemicalsN-Hexane ExtractEthyl Acetate ExtractChloroform Extract 1Alkaloid + + + 2Amino acid - - - 3Anthraquinones - - - 4Catechin - - - 5Fixed oil - - - 6Flavonoid - - - 7Glycoside - - - 8Phenols + + + 9Quinone + + + 10Reducing sugars - - - 11Saponin + + + 12Steroids - + + 13Tannin + + + 14Terpenoids - - - 15Xanthoprotein - - - 10

11. Sl.NoParticulars of treatment Colour Appearance Visible lightUV-Light.1Powder as suchBrown 2Powder +1N NaOHBrownGreen 3Powder+1N NaOH(aq)BrownGreen 4Powder + HCL (1:1)BrownGreen 5Powder + HNO 3 (1:1)BrownGreen 6Powder + H 2 SO 4 (1:1)BrownDark 2. Fluorescent Analysis of the powder of Nicotiana tobaccum.. Figure. 1 Flourescence analysis of samples under UV light (365 nm) (NaOH, HCL, H2SO 4, HNO 3, CHCl 3, H 2 O) 11

12. 3.Quantitative Parameters Moisture content - 3.9% Total Ash - 39.6% Acid soluble Ash - 44% Water-soluble Ash - 69% Qualitative Determination of Mineral Constituents: 12 6. Thin layer chromatography analysis

13. 1.Estimation of Alkaloids by Singh and Archana Sahu Method. 13 2.Estimation of Phenol 3.Estimation of Ascorbic Acid(VitaminC) by 2.4-DNPH Method.

14. HPTLC Analysis The HPTLC analysis of N-Hexane, Ethyl Acetate, Chloroform extract shows the presence of seven different phytochemicals in extract. Figure. 2. HPTLC analysis of N-hexane, ethyl acetate and chloroform extract. 14

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16. GC-MS Analysis: The results from GC-MS analysis leads to the identification of number of pharmacologically important compounds from the GC fractions of the N-Hexane, Ethylacetate and Chloroform extract of Nicotina Tobaccum. The constituents of the plant extract were identified by comparing of the mass fragmentation Figure. 5.8. GC-MS analysis 16

17. Infra Red Spectroscopy Analysis 17

18. High Resolution Mass Spectrometers (HRMS) Analysis Fig.4 HRMS spectrum of N-Hexane Solvent extract of Nicotina Tobaccum and the proposed fragmentation paths. 18

19. Absorbance Antioxidant activity: ferric reducing power method Ferric Reducing Antioxidant Power Results Absorbance at 700 nm Series 1.indicate-plant extract, series 2.indicate – standard. 19 Modified ferric iron reducing antioxidant power assay (Modified FRAP assay)

20. Antibacterial activity of N-Hexane, Ethylacetate, Chloroform extracts: Antibacterial activity of aqueous leaf extract of Nicotiana tobaccum against five human pathogens as tested by paper disc method. Bacterial strainsZone of Inhibition in mm HexaneEthyl acetateChloroformstandard 1. B.Sabtilis5 mm6 mm 10 mm 2. S.epidermis6 mm 10 mm 3. E.Aerogens7 mm5 mm6 mm10 mm 4. Seudomonas aeruginosa6 mm5 mm 10 mm 5. Klebsiella aerogenus.6 mm5 mm6 mm10 mm 20

21. SUMMARY AND CONCLUSION The work carried out on Nicotina Tobaccum, showed the presence of various Phytochemical constituents and bioactive compounds in all the extracts in varying range and quantity. The quality and quantity of phytochemicals are based on the selection of solvent system. The biological activities are attributed to Phytochemical constituents of Nicotina Tobaccum and the fluorescent analysis of powdered drug can play a remarkable role in the determination of quality and purity of the raw drug. These types of studies have vital role because of the commercial and research interest. The various functional groups, detected from FT-IR analysis of N-Hexane, Chloroform and EA extracts, are responsible for the medicinal properties of plant. The compounds identified by GC-MS in N-Hexane extract of Nicotina Tobaccum are medicinally valuable and possess a wide variety of pharmacological applications. Further isolation and testing of phytoconstituents for their individual biological activity will undoubtedly bring promising results in the discovery and development of novel drugs. 21

22. References 1..Duangsri P, Juntarapun K, and Satirapipathkul C: The tobacco leaf extract and antibacterial activity in textile. International Conference: Textiles & Fashion 2012; 3-4. 2.Fransworth NR, Akerele O, Bingel AS, Soejarto DD and Guo: Medicinal plants in therapy. Bulletin of the World Health Organization 1985; 63:965–981. 3.Sharma Y, Dua D and Srivastva SN: Comparative study of different parts of Azadirachta indica (neem) plant on the basis of anti-bacterial activity, phytochemical screening and its effect on rat PC–12 (Pheochromocytoma) cell line. International Journal of Biotechnology and allied fields 2014; 2 (7):144 – 154. 4.Bakht J, Tayyab M, Ali H, Islam A and Shafi M: Effect of different solvent extracted samples of Allium sativum on bacteria and fungi. African Journal of Biotechnology 2011; 10:5910–5915. 5.Nascimento GG, Locatelli J, Freitas PC and Silva GL: Antibacterial activity of plant extracts and phytochemicals on antibiotic resistant bacteria. Brazilian Journal of Microbiology 2000; 31:247-256. 22

23. ACKNOWLEDGEMENTS. My sincere thanks to my guide Dr. M.F.Valan for taking special interest in giving me valuable suggestions, encouragement and generous assistance throughout this project. I am grateful to Rev.Father Dr.JohanPragasm,SJ,LIFE,Director Loyola College for providing all the necessary facilities. I extend my thanks to HOD, Dr.M.GeorgeJohnson & Department of chemistry all faculty members. 23

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