Dheeraj Antioxidant Seminar


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About categorising antioxidant and in vitro antioxidant assay

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Dheeraj Antioxidant Seminar

  1. 1. <ul><li>ANTIOXIDANT AND SCREENING MODELS </li></ul>
  3. 4. <ul><li>Free radicals having single electron in their outer shell and becomes more reactive. </li></ul><ul><li>They need to satisfy their pair with surplus electron and therefore they heat the electron reach centers like protein, lipid etc. </li></ul><ul><li>Free radical attack on lipid, protein leads to the alteration of its functions. </li></ul><ul><li>Oxidative stress leads to the structural changes of the cell wall and hamper the process of maturation of cellular structures. Its also lead to the change in the function and death eventually. </li></ul>
  4. 5. FREE RADICALES <ul><li>A molecule with one or more unpaired electron in its outer shell </li></ul><ul><li>They are easily available to react with various targets like lipid, protein, DNA. </li></ul><ul><li>ENDOGENOUS SOURCE </li></ul><ul><ul><ul><li>Body's normal use of oxygen such as respiration and some cell mediated immune functions </li></ul></ul></ul><ul><ul><ul><li>Immune cell activation </li></ul></ul></ul><ul><ul><ul><li>Inflammation </li></ul></ul></ul><ul><ul><ul><li>Mental stress </li></ul></ul></ul><ul><li>EXOGENOUS SOURCE </li></ul><ul><ul><ul><li>Environmental pollutants </li></ul></ul></ul><ul><ul><ul><li>Cigarette smoke </li></ul></ul></ul><ul><ul><ul><li>Automobiles fumes </li></ul></ul></ul><ul><ul><ul><li>Radiations </li></ul></ul></ul>
  5. 6. TYPES OF FREE RADICALS <ul><li>Superoxide radical </li></ul><ul><li>Hydroxide radical </li></ul><ul><li>Nitric oxide radical </li></ul><ul><li>Peroxide radicals </li></ul><ul><li>H2O2(non radical)..etc </li></ul>
  6. 7. SUPEROXIDE RADICAL <ul><li>It is generated via several cellular oxidase system like… </li></ul><ul><ul><ul><li>NADPH oxidase </li></ul></ul></ul><ul><ul><ul><li>Xanthin oxidase </li></ul></ul></ul><ul><ul><ul><li>Peroxidase </li></ul></ul></ul><ul><li>It starts the process of generation of other free radicals. </li></ul>
  7. 8. HYDROXYL RADICAL <ul><li>It is most dangerous radical </li></ul><ul><li>It is most reactive radical in biological system </li></ul><ul><li>It is formed by the reaction of O2 with H2O2 in the presence of Fe 2+ </li></ul>
  8. 9. <ul><li>HYDROGEN PEROXIDE </li></ul><ul><ul><ul><li>It is produced by the action of several oxidase enzyme, including amino acid oxidase and xanthin oxidase </li></ul></ul></ul><ul><li>NITRIC OXIDE </li></ul><ul><ul><li>It is formed in biological tissue from the oxidation of L-argenine to citrulline by nitric oxide synthtase </li></ul></ul>
  9. 11. BENEFICIAL ACTIVITIES <ul><li>At low and moderate level they helps in maturation process of cellular structures. </li></ul><ul><li>Act as a weapon for host defense system. </li></ul><ul><li>Phagocytes realizes free radicals to destroy invading pathogenic microbes as a part of body defense system. </li></ul><ul><li>They play physiological role in functions of number of cellular signaling system </li></ul>
  10. 12. DELETERIOUS   ACTIVITY <ul><li>Responsible oxidative stress </li></ul><ul><li>Damaging DNA proteins </li></ul><ul><li>Lipid peroxidation </li></ul><ul><li>Structural damage to cell and tissue. </li></ul><ul><li>Mutation deformities </li></ul><ul><li>Responsible for so many life threatening disorders and diseases </li></ul><ul><li>Can participates in enhancing to disease conditions like…. </li></ul><ul><ul><li>Cancer </li></ul></ul><ul><ul><li>Cardiovascular disease </li></ul></ul><ul><ul><li>Parkinsonism </li></ul></ul><ul><ul><li>Arthritis </li></ul></ul><ul><ul><li>Diabetes </li></ul></ul>
  11. 13. Cancer and oxidative stress…….. <ul><li>Cancer constitute major health problem in all industrialized countries where it is the main cause of premature mortality </li></ul><ul><li>The development of cancer in complex process including cellular and molecular changes mediated by endogenous and exogenous stimuli </li></ul><ul><li>Voluminous literature suggest that the free radicals are directly or indirectly proved harmful and can cause the carcinogenesis </li></ul><ul><li>The oxidative DNA damage is responsible for cancer </li></ul><ul><li>Cancer initiation and promotion are associated with chromosomal defect and oncogene activation </li></ul><ul><li>Free radical formation interferes with normal cell growth by causing genetic mutation and altering normal gene transcription </li></ul>
  12. 14. Arthritis and oxidative stress …. <ul><li>It is a relapsing immuno-inflammatory multisystem disease with synovial proliferation and destruction of articular cartilage </li></ul><ul><li>1-2% population get affected </li></ul><ul><li>Incidence increases with age </li></ul><ul><li>Etiology of RA remains unknown </li></ul>
  13. 15. <ul><li>The pathogenesis of RA is multifactorial and recent research has implicated oxygen free radicals as mediators of tissue damage </li></ul><ul><li>Several other pathways can lead to increased the formation of free radicals at inflamed joint. </li></ul>
  14. 16. <ul><li>Free radicals=------Lipid peroxidation----cellular component alteration -----tissue damage and synovial fluid collapse---RA </li></ul><ul><li>Free radicals ----decreases glutathione---damage defense system </li></ul><ul><li>Activated phagocyte in the inflamed joint have been implicated along with mediators of inflammation and pathogenesis of tissue destruction. </li></ul>
  15. 17. Atherosclerosis and Oxidative stress…. <ul><li>Activation of macrophages </li></ul><ul><li>It contains some low density receptor </li></ul><ul><li>Cholesterol rapidly accumulate on macrophages and convert it to foam cell. </li></ul><ul><li>Endothelial cell damage. </li></ul><ul><li>Lipid peroxidation by product might be act as chemo tactic factor for blood monocytes and recruit them at atherosclerotic lesion. </li></ul>
  16. 18. Diabetes and Oxidative Stress… <ul><li>Insulin break down </li></ul><ul><li>Inactivation of pancreatic cell </li></ul><ul><li>Structural and functional damage of pancreatic cell </li></ul><ul><li>COMPLICATIONS </li></ul><ul><li>Etiology of complication involves oxidative stress perhaps as hyperglycemia. </li></ul><ul><li>Elevated glucose itself increased protein glycosylation are the important source of free radicals. </li></ul>
  17. 19. Neurodegenerative disorders <ul><li>Schizophrenia </li></ul><ul><li>Parkinsonism </li></ul>
  18. 20. Erectile dysfunction and oxidative stress……… <ul><li>Penile erection depends upon the vascular smooth muscles relaxation in erectile tissue. </li></ul><ul><li>The principle mediator of relaxation is NO . </li></ul><ul><li>Increased the inactivation of NO . by super oxide results in ED. </li></ul><ul><li>Propagation of endothelial dysfunction by ROS may results in chronic impairment of penile vascular function </li></ul><ul><li>The literature suggest that antioxidant therapy may prove beneficial in the management of ED </li></ul>
  19. 21. What are Antioxidants
  20. 22. Antioxidants may be classified as………. <ul><li>ENZYMATIC </li></ul><ul><ul><ul><li>SOD </li></ul></ul></ul><ul><ul><ul><li>Catalase </li></ul></ul></ul><ul><ul><ul><li>Glutathione peroxidase </li></ul></ul></ul><ul><ul><ul><li>Glutathione reductase </li></ul></ul></ul><ul><li>NON-ENZYMATIC </li></ul><ul><ul><li>Metabolic antioxidants </li></ul></ul><ul><ul><ul><li>Lipoid acid </li></ul></ul></ul><ul><ul><ul><li>Uric acid </li></ul></ul></ul><ul><ul><ul><li>Melatonin </li></ul></ul></ul><ul><ul><li>Nutrient antioxidants </li></ul></ul><ul><ul><ul><li>Carotenoids </li></ul></ul></ul><ul><ul><ul><li>Tannins </li></ul></ul></ul><ul><ul><ul><li>Flavonoids etc. </li></ul></ul></ul>
  21. 23. Mechanism of Antioxidants <ul><li>Reducing agents </li></ul><ul><li>Chain breakers </li></ul><ul><li>Free radical scavenger </li></ul><ul><li>Metal ions cheletor </li></ul>
  22. 26. Need of screening for antioxidants
  23. 28. CRITERIA FOR SELECTION OF SCREENING MODEL <ul><li>The screening models should fit into the following criteria </li></ul><ul><ul><ul><li>1. Utilization of biological relevant molecule </li></ul></ul></ul><ul><ul><ul><li>2. Technically simple </li></ul></ul></ul><ul><ul><ul><li>3. Crystal clear end point and mechanism </li></ul></ul></ul><ul><ul><ul><li>4. Readily available instrumentation </li></ul></ul></ul><ul><ul><ul><li>5. Good reproducibility </li></ul></ul></ul><ul><ul><ul><li>6. Adaptable for both lipophilic and hydrophilic antioxidants. </li></ul></ul></ul><ul><ul><ul><li>Two main categories </li></ul></ul></ul><ul><ul><ul><li>Scavenging capacity assay against specific ROS/NOS </li></ul></ul></ul><ul><ul><ul><li>Scavenging capacity assay against stable and non-biological radical. </li></ul></ul></ul>
  24. 29. Target Oxidized Target Oxidized antioxidant Antioxidant 1. Competitive Reactive species Reduced reactive species
  25. 30. Oxidized antioxidant Antioxidant 2. Non-competitive Reactive species Reduced reactive species
  26. 31. <ul><li>SREENING MODELS </li></ul>
  27. 32. Hydroxyl radical scavenging activity [OH - ] <ul><li>OH is highly reactive species and most dangerous to biological system. </li></ul><ul><li>Involved in atherosclerosis , oncogenesis, DNA mutation etc. </li></ul><ul><li>Reagents :- </li></ul><ul><ul><ul><li>Fecl3 </li></ul></ul></ul><ul><ul><ul><li>EDTA </li></ul></ul></ul><ul><ul><ul><li>Ascorbic acid </li></ul></ul></ul><ul><ul><ul><li>H2O2 </li></ul></ul></ul><ul><ul><ul><li>Thiobarbituric acid </li></ul></ul></ul><ul><ul><ul><li>Buffer (7.4pH) </li></ul></ul></ul>
  28. 33. Before addition of antioxidant FeCl3 H2O2 EDTA OH Deoxyribose Chromogen TBA
  29. 34. After addition of antioxidant FeCl3 H2O2 EDTA OH Deoxyribose Chromogen (532 nm) TBA
  30. 35. Superoxide radical anion (O2 .- ) scavenging assay <ul><li>This radical arise from several metabolic processes </li></ul><ul><li>It is also generated in biological system by enzyme hypoxanthine and xanthin oxidase pH7.4 </li></ul><ul><li>Reagents- </li></ul><ul><ul><ul><li>1. Phenazine methosulphate </li></ul></ul></ul><ul><ul><ul><li>2. Nicotinamide adenine dinucleotide </li></ul></ul></ul><ul><ul><ul><li>3. Nitro blue terazolium </li></ul></ul></ul><ul><ul><ul><li>4. Buffer (7.4) </li></ul></ul></ul>
  31. 36. Before addition of antioxidants agent O2 - Nitro blue terazolium reduction Formazan (560 nm)
  32. 37. After addition of antioxidants agent Nitro blue terazolium Reduction Formazan
  33. 38. <ul><li>We can use the ferricytochrome –c as a target in place of NBT </li></ul><ul><li>The scavenging capacity against O2 .- radical can be measured by using electron spin resonance spectrometry. </li></ul><ul><li>Here O2 .- is trapped by 5,5-dimithyl-1-pyrroline-n-oxide (DMPO) </li></ul><ul><li>DMPO-OOH adduct is detected by ESR. </li></ul>
  34. 39. Nitric oxide scavenging activity [NO . ]………. <ul><li>Sodium nitroprusside ------aqueous solution---- </li></ul><ul><li>physiological ph------NO . radicals---nitrite---Griess </li></ul><ul><li>reagent----chromophore---546nm </li></ul>
  35. 40. DPPH . Scavenging assay <ul><li>It is a purple colored stable free radical </li></ul><ul><li>Antioxidant compound convert it to colorless moiety. </li></ul><ul><li>The amount of DPPH reduced could be quantified by measuring a decrease in absorbance and may be attributed to its hydrogen donating ability </li></ul><ul><li>The assay should be performed at 517 nm </li></ul>
  36. 41. Peroxyl radicals {ROO . } scavenging activity <ul><li>This is an hydrogen atom transfer reaction. </li></ul><ul><li>Thermo labile azo-compound --- carbon centered radicals ----with fast reaction of O2-------ROO . ----target---florescence </li></ul><ul><li>Both water soluble and lipid soluble antioxidants can be screened </li></ul><ul><li>AAPH ( 2,2 – azobis-2-amidinopropane, dihydrochloride ) </li></ul><ul><li>AMVN ( 2,2 – azobis-2,4 dimethylvaleronitrile ) </li></ul>
  37. 42. ORAC <ul><li>The principle is based upon the deceasing florescence along with the Pyroxyl radicals. </li></ul><ul><li>The radicals are formed due to thermal decomposition of AAPH in aqueous buffer. </li></ul><ul><li>This assay is generally used to evaluate the chain breaking activity </li></ul><ul><li>B-phycoerithrin was used as a florescent target </li></ul><ul><li>Now a days flurescein is used. </li></ul>
  38. 43. Ferric reducing antioxidant power (FRAP) assay <ul><li>This assay helps to measure the ability of antioxidant compound to reduce ferric ions into ferrous complex </li></ul><ul><li>It required the acid medium and absorbance is calculated at 593 nm </li></ul><ul><li>This assay can give response to compound having reducing power and metal chelating capacity </li></ul>
  39. 44. Folin-ciocalteu capacity <ul><li>It contains phosphomolybdic /phosphotungstic acid complex. </li></ul><ul><li>Transfer of electron in alkaline medium from phenolic compound to molybdenum forming blue complex that can be detected spectrometrycally at 760 nm </li></ul><ul><li>This assay is non-specific to phenolic compound as it can be reduced by many non phenolic compound like aromatic amines, ascorbic acid etc. </li></ul>
  40. 45. Other Antioxidant Models <ul><li>Hypoclorous acid (HOCL) scavenging activity </li></ul><ul><li>Peroxynitrite (ONOO) scavenging activity </li></ul><ul><li>Singlet oxygen scavenging capacity assay </li></ul><ul><li>Total radical-trapping antioxidant parameter (TRAP) </li></ul><ul><li>Total oxyradical scavenging capacity </li></ul><ul><li>H2O2 scavenging assay using Horseradish peroxidase . </li></ul>
  41. 46. List of Antioxidant plants from “RASAYANA” Acorus calamus Aloe vera Asparagus racemosus Azardirachta indiaca Bacopa monnieri Desmodium gangeticum Phyllanthus emblica Terminalia chebula Tinospora cordifolia Withania somnifera
  42. 47. Other Ayurvedic plants Piper betel Santalum album Piper nigrum Swertia chirata Plumbago zeylanica Andrographis paniculata Curculigo Orchioides Glycyrrhiza glabra Hygrophila auriculata Hemidesmus indicus Cassia fistula Punica granatum Mangifera indica Shorea robusta Curcuma longa Cassia sophera Emblica officinalis Calicarpa macrophyla Momordica charantia Allium sativum
  43. 48. Ayurvedic plants-----unfolded antioxidant potential Pluchea lanceolata Prunus cerasoides Salix caprea Hyoscymus niger Litea glutinosa Semecarpus anacardium Salmalia malabarica
  44. 49. These plants can be acts as best antioxidants. Steppe plants Dessert plants Alpine plants Mountain plants
  45. 50. <ul><li>Very high carbon assimilation </li></ul><ul><li>Short growing period </li></ul><ul><li>Extreme climate conditions </li></ul><ul><ul><li>Low or high temperature </li></ul></ul><ul><ul><li>Rapid temperature changes </li></ul></ul><ul><ul><li>Unfavorable conditions for photosynthesis </li></ul></ul><ul><ul><li>Light intercity etc.. </li></ul></ul>
  47. 54. Conclusion <ul><li>Free radicals are the most harmful entities and can be act as main culprit in almost each and every disorders and diseases. </li></ul>
  48. 55. <ul><li>We have to go step by step to screen the actual mechanism and total antioxidant activity of compound against specific ROS. </li></ul><ul><ul><li>Reducing power </li></ul></ul><ul><ul><li>DPPH assay </li></ul></ul><ul><ul><li>OH - radical scavenging assay </li></ul></ul><ul><ul><li>O2 .- radical scavenging assay </li></ul></ul><ul><ul><li>H 2 O 2 radical scavenging assay </li></ul></ul><ul><ul><li>NO . radical scavenging assay </li></ul></ul><ul><ul><li>Peroxyl radical scavenging assay </li></ul></ul><ul><ul><li>Total phenolic content capacity </li></ul></ul>
  49. 56. <ul><li>All in-vitro models can give an idea about the role and potency of compound as antioxidant. However, It is very difficult to correlate with biological system. </li></ul>
  50. 57. <ul><li>Dietary and Natural source will be the best source of antioxidant. </li></ul>