1、ANTIOXIDANT AND SCREENING MODELS,INTRODUCTION,Free radicals having single electron in their outer shell and becomes more reactive.They need to satisfy their pair with surplus electron and therefore they heat the electron reach centers like protein, lipid etc.Free radical attack on lipid, protein lea
2、ds to the alteration of its functions. 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.,FREE RADICALES,A molecule with one or more unpaired electron in its
3、outer shell They are easily available to react with various targets like lipid, protein, DNA. ENDOGENOUS SOURCE Bodys normal use of oxygen such as respiration and some cell mediated immune functions Immune cell activation Inflammation Mental stress EXOGENOUS SOURCE Environmental pollutants Cigarette
4、 smoke Automobiles fumes Radiations,TYPES OF FREE RADICALS,Superoxide radicalHydroxide radicalNitric oxide radicalPeroxide radicalsH2O2(non radical)etc,SUPEROXIDE RADICAL,It is generated via several cellular oxidase system like NADPH oxidase Xanthin oxidase PeroxidaseIt starts the process of generat
5、ion of other free radicals.,HYDROXYL RADICAL,It is most dangerous radical It is most reactive radical in biological systemIt is formed by the reaction of O2 with H2O2 in the presence of Fe2+,HYDROGEN PEROXIDE It is produced by the action of several oxidase enzyme, including amino acid oxidase and xa
6、nthin oxidase NITRIC OXIDE It is formed in biological tissue from the oxidation of L-argenine to citrulline by nitric oxide synthtase,BENEFICIAL ACTIVITIES,At low and moderate level they helps in maturation process of cellular structures. Act as a weapon for host defense system.Phagocytes realizes f
7、ree radicals to destroy invading pathogenic microbes as a part of body defense system.They play physiological role in functions of number of cellular signaling system,DELETERIOUS ACTIVITY,Responsible oxidative stress Damaging DNA proteins Lipid peroxidation Structural damage to cell and tissue. Muta
8、tion deformities Responsible for so many life threatening disorders and diseases Can participates in enhancing to disease conditions like. Cancer Cardiovascular disease Parkinsonism Arthritis Diabetes,Cancer and oxidative stress,Cancer constitute major health problem in all industrialized countries
9、where it is the main cause of premature mortalityThe development of cancer in complex process including cellular and molecular changes mediated by endogenous and exogenous stimuliVoluminous literature suggest that the free radicals are directly or indirectly proved harmful and can cause the carcinog
10、enesisThe oxidative DNA damage is responsible for cancer Cancer initiation and promotion are associated with chromosomal defect and oncogene activation Free radical formation interferes with normal cell growth by causing genetic mutation and altering normal gene transcription,Arthritis and oxidative
11、 stress .,It is a relapsing immuno-inflammatory multisystem disease with synovial proliferation and destruction of articular cartilage1-2% population get affected Incidence increases with ageEtiology of RA remains unknown,The pathogenesis of RA is multifactorial and recent research has implicated ox
12、ygen free radicals as mediators of tissue damageSeveral other pathways can lead to increased the formation of free radicals at inflamed joint.,Free radicals=-Lipid peroxidation-cellular component alteration -tissue damage and synovial fluid collapse-RAFree radicals -decreases glutathione-damage defe
13、nse systemActivated phagocyte in the inflamed joint have been implicated along with mediators of inflammation and pathogenesis of tissue destruction.,Atherosclerosis and Oxidative stress.,Activation of macrophages It contains some low density receptorCholesterol rapidly accumulate on macrophages and
14、 convert it to foam cell.Endothelial cell damage.Lipid peroxidation by product might be act as chemo tactic factor for blood monocytes and recruit them at atherosclerotic lesion.,Diabetes and Oxidative Stress,Insulin break down Inactivation of pancreatic cell Structural and functional damage of panc
15、reatic cell COMPLICATIONS Etiology of complication involves oxidative stress perhaps as hyperglycemia. Elevated glucose itself increased protein glycosylation are the important source of free radicals.,Neurodegenerative disorders,Schizophrenia Parkinsonism,Erectile dysfunction and oxidative stress,P
16、enile erection depends upon the vascular smooth muscles relaxation in erectile tissue. The principle mediator of relaxation is NO. Increased the inactivation of NO. by super oxide results in ED. Propagation of endothelial dysfunction by ROS may results in chronic impairment of penile vascular functi
17、on The literature suggest that antioxidant therapy may prove beneficial in the management of ED,What are Antioxidants,Antioxidants may be classified as.,ENZYMATIC SOD Catalase Glutathione peroxidase Glutathione reductase NON-ENZYMATIC Metabolic antioxidants Lipoid acid Uric acid Melatonin Nutrient a
18、ntioxidants Carotenoids Tannins Flavonoids etc.,Mechanism of Antioxidants,Reducing agents Chain breakersFree radical scavengerMetal ions cheletor,Need of screening for antioxidants,CRITERIA FOR SELECTION OF SCREENING MODEL,The screening models should fit into the following criteria 1. Utilization of
19、 biological relevant molecule 2. Technically simple 3. Crystal clear end point and mechanism 4. Readily available instrumentation 5. Good reproducibility 6. Adaptable for both lipophilic and hydrophilic antioxidants. Two main categories Scavenging capacity assay against specific ROS/NOS Scavenging c
20、apacity assay against stable and non-biological radical.,Target,Oxidized Target,Oxidized antioxidant,Antioxidant,Reactive species,Reduced reactive species,1. Competitive,Oxidized antioxidant,Antioxidant,Reactive species,Reduced reactive species,2. Non-competitive,SREENING MODELS,Hydroxyl radical sca
21、venging activity OH-,OH is highly reactive species and most dangerous to biological system. Involved in atherosclerosis , oncogenesis, DNA mutation etc. Reagents :- Fecl3 EDTA Ascorbic acid H2O2 Thiobarbituric acid Buffer (7.4pH),Before addition of antioxidant,FeCl3,H2O2,EDTA,OH,Deoxyribose,Chromoge
22、n,TBA,FeCl3,H2O2,EDTA,OH,Deoxyribose,Chromogen (532 nm),TBA,After addition of antioxidant,Superoxide radical anion (O2.-) scavenging assay,This radical arise from several metabolic processes It is also generated in biological system by enzyme hypoxanthine and xanthin oxidase pH7.4 Reagents- 1. Phena
23、zine methosulphate 2. Nicotinamide adenine dinucleotide 3. Nitro blue terazolium 4. Buffer (7.4),Before addition of antioxidants agent,O2-,Nitro blue terazolium,reduction,Formazan (560 nm),After addition of antioxidants agent,Nitro blue terazolium,Reduction,Formazan,We can use the ferricytochrome c
24、as a target in place of NBTThe scavenging capacity against O2.- radical can be measured by using electron spin resonance spectrometry. Here O2.- is trapped by 5,5-dimithyl-1-pyrroline-n-oxide (DMPO)DMPO-OOH adduct is detected by ESR.,Nitric oxide scavenging activity NO.,Sodium nitroprusside -aqueous
25、 solution-physiological ph-NO. radicals-nitrite-Griess reagent-chromophore-546nm,DPPH. Scavenging assay,It is a purple colored stable free radicalAntioxidant compound convert it to colorless moiety.The amount of DPPH reduced could be quantified by measuring a decrease in absorbance and may be attrib
26、uted to its hydrogen donating ability The assay should be performed at 517 nm,Peroxyl radicals ROO. scavenging activity,This is an hydrogen atom transfer reaction. Thermo labile azo-compound - carbon centered radicals -with fast reaction of O2-ROO. -target-florescence Both water soluble and lipid so
27、luble antioxidants can be screened AAPH (2,2 azobis-2-amidinopropane, dihydrochloride) AMVN (2,2 azobis-2,4 dimethylvaleronitrile),ORAC,The principle is based upon the deceasing florescence along with the Pyroxyl radicals. The radicals are formed due to thermal decomposition of AAPH in aqueous buffe
28、r. This assay is generally used to evaluate the chain breaking activity B-phycoerithrin was used as a florescent target Now a days flurescein is used.,Ferric reducing antioxidant power (FRAP) assay,This assay helps to measure the ability of antioxidant compound to reduce ferric ions into ferrous com
29、plexIt required the acid medium and absorbance is calculated at 593 nmThis assay can give response to compound having reducing power and metal chelating capacity,Folin-ciocalteu capacity,It contains phosphomolybdic /phosphotungstic acid complex. Transfer of electron in alkaline medium from phenolic
30、compound to molybdenum forming blue complex that can be detected spectrometrycally at 760 nm This assay is non-specific to phenolic compound as it can be reduced by many non phenolic compound like aromatic amines, ascorbic acid etc.,Other Antioxidant Models,Hypoclorous acid (HOCL) scavenging activit
31、yPeroxynitrite (ONOO) scavenging activitySinglet oxygen scavenging capacity assayTotal radical-trapping antioxidant parameter (TRAP)Total oxyradical scavenging capacityH2O2 scavenging assay using Horseradish peroxidase.,List of Antioxidant plants from “RASAYANA”,Other Ayurvedic plants,Ayurvedic plan
32、ts-unfolded antioxidant potential,These plants can be acts as best antioxidants.,Very high carbon assimilation Short growing periodExtreme climate conditionsLow or high temperature Rapid temperature changes Unfavorable conditions for photosynthesis Light intercity etc,DIETARY ANTIOXIDANT,Conclusion,
33、Free radicals are the most harmful entities and can be act as main culprit in almost each and every disorders and diseases.,We have to go step by step to screen the actual mechanism and total antioxidant activity of compound against specific ROS.Reducing power DPPH assay OH- radical scavenging assay
34、 O2.- radical scavenging assay H2O2 radical scavenging assay NO. radical scavenging assay Peroxyl radical scavenging assay Total phenolic content capacity,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.,Dietary and Natural source will be the best source of antioxidant.,
