EVALUATION SEMINAR“EVALUATION OF ANTIOXIDANT” PRESENTED BY Mr. Rahul R. Lavhale (M.Pharm-II), Department of Pharmacognosy, KLEU College of Pharmacy, Vidyanagar, Hubli-31. CONTENTS INTRODUCTION REACTIVE OXYGEN SPECIES (ROS) SOURCES OF FREE RADICALS MECHANISM OF CELL DAMAGE ANTIOXIDANT NATURAL ANTIOXIDANTS ANTIOXIDANT PROTECTION SYSTEM OXIDATIVE STRESS AND HUMAN DISEASE SCREENING MODEL OF ANTIOXIDANT ACTIVITY IN VITRO METHODS IN VIVO METHODS LIST OF ANTIOXIDANT PLANTS REFERENCES INTRODUCTION Oxygen is highly reactive atom that is capable of becoming part of potentially damaging molecule commonly called “free radical.” Free radicals are capable of attacking cells of the body, causing them to lose their structure and function. Free radial formation is controlled naturally by various compounds known as antioxidants. Free radicals are electrically charged molecules. i.e. they have an unpaired electron, which cause them to seek out and capture electron form other substance in order to neutralize themselves. Although the initial attack cause the free radical to become neutralized, another free radical is formed in this process, causing a chain reaction is occur. And until subsequent free radicals are deactivated, thousand of free radical reaction can occur within second of the initial reaction. Antioxidants are capable of stabilizing or deactivating, free radical before they attack cells. Antioxidants are absolutely critical for maintaining optimal cellular and systemic health and well-being. nucleic acids.e. i. ROS are generated by a number of pathways. All are capable of reacting with membrane lipids. oxygen-containing molecules. enzymes and other small molecules. detoxification of toxic substances. proteins. . including free radicals.. Most of the oxidants produced by cells occur as: Normal aerobic metabolism: Approximately 90% of the oxygen utilized by the cell Oxidative burst from phagocytes (white blood cells) Xenobiotic metabolism.REACTIVE OXYGEN SPECIES (ROS) Reactive oxygen species (ROS) is define as all highly reactive. resulting in cellular damage. Glutathione. Flavonoids. Glutathione peroxidase . Vitamin E. Glutathione. Beta carotene. Flavonoids. Lipoic acid. Vitamin E. Lipoic acid Superoxide radical Vitamin C. Glutathione. Lipid peroxides Beta carotene.VARIOUS ROS AND CORRESPONDING NEUTRALIZING ANTIOXIDANTS REACTIVE OXYGEN SPECIES NEUTRALIZING ANTIOXIDANTS Hydroxyl radical Vitamin C. Flavonoids. SOD Hydrogen peroxide Vitamin C. Flavonoids. EXTERNAL SOURCES: Cigarette smoke Radiation U.V.SOURCES OF FREE RADICALS INTERNAL SOURCE: Mitochondria Phagocytes Xanthine oxidase Exercise Inflammation Ischemia and reperfusion. light Certain drugs Reagents and industrial solvents pollution . MECHANISM OF CELL DAMAGE . Oxidation is a process that causes damage in our tissues through the work of free radicals. Antioxidants are chemical substances that donate an electron to the free radical and convert it to a harmless molecule. Antioxidants are substances used by the body to protect itself from damage caused by oxidation.ANTIOXIDANT Antioxidant is substance that prevents or slows the breakdown of another substance by oxygen. An antioxidant is a chemical that prevents the oxidation of other chemicals. . Amla. Minerals:. their mechanism and site of action. Manganese.Albumin. Apple. Zinc Vitamins:-VitaminA. Superoxide dismutase(SOD). Grapes and wines.Ascorbic acid. b) Water soluble antioxidants:. C.NATURAL ANTIOXIDANTS Naturally occurring antioxidants differ in their composition. Tomato. their physical and chemical properties.Reduced glutathione (GSH). Copper.Selenium.Tocopherol. . Plants antioxidants:. Transferin. Ashwagandha. Catalase(CAT). Liquorice. Uric acid and Some Polyphenols. Enzymes:. Quinine and Some Polyphenols.Soyabean. Sesame seed. Ceruplasmin. Orange. Carotenoids. Citrus peel. and E. Glutathione peroxidase(GPx) High molecular weight compounds(proteins):. Carrot. Haptoglobin Low molecular weight compounds: a) Lipid soluble antioxidants:. flavonoids. and proanthocyanidins Metal Binding Proteins Albumin (copper). Vitamin E. e. N-acetyl cysteine. glutathione. e.g. Ceruloplasmin (copper). Beta carotene and other carotenoids and oxycarotenoids. Myoglobin (iron). Metallothionein (copper). Thiols. Transferrin (iron) ..ANTIOXIDANT PROTECTION SYSTEM Endogenous Antioxidants Bilirubin. e. flavones. lipoic acid. Lycopene and lutein Polyphenols.. Uric acid Enzymes: – Copper/Zinc and manganese-dependent super oxide dismutase (SOD) – Iron-dependent catalase – Selenium-dependent glutathione peroxidase Dietary Antioxidants Vitamin C. flavonols.g. NADPH and NADH.g. Ferritin (iron). OXIDATIVE STRESS AND HUMAN DISEASE . .MAJOR GROUPS OF ANTIOXIDANT 20 METABOLITES Important groups of antioxidant activity are Phenol Phenolic acids Anthocyanins Flavones Flavonoids Flavonols Tannins Isoflavanoids These group of compound show antioxidant activity & plant defense mechanisms against pathogenic microoranism. SCREENING MODEL OF ANTIOXIDANT ACTIVITY . ELECTRON TRANSFER REACTION : Trolox equivalent antioxidant capacity (TEAC) Ferric reducing antioxidant power (FRAP) α.IN VITRO METHODS In vitro methods divided into two major groups. α. HYDROGEN ATOM TRANSFER REACTION : Oxygen radical absorbance capacity (ORAC) Total radical trapping antioxidant potential (TRAP) β-carotene bleaching.-diphenyl -β-picryl-hydrazyl redical scavenging assay (DPPH) Superoxide anion radical scavenging assay Hydrogen radical Scavenging assay Nitric oxide radical scavenging assay Total phenol assay . This method is based on the reduction of DPPH in methanol solution in the presence of a hydrogen-donating antioxidant due to the formation of the non radical from DPPH-H. This transformation results in color change from purple to yellow. The decrease in the absorbance is directly proportional to the radical scavenging activity .FREE RADICAL SCAVENGING ASSAYS (1-1-DIPHENYL-2-PICRYL-HYDRAZYL) DPPH DPPH is a molecule containing a stable free radical. DPPH is reduced by donating hydrogen to the antioxidant molecule and resulting in the absorbance decrease. which is measured spectrophotometrically. . 1-diphenyl-2-picryl hydroxyl (DPPH). The degree of discoloration indicates the scavenging potential of the antioxidant extract.MECHANISM Free radical scavenging potentials of the extracts were tested against a methanolic solution of 1. Antioxidants react with DPPH and convert it to 1-1-diphenyl -2picryl hydrazine . The change in the absorbance produced at 517nm has been used as a measure of antioxidant activity. BHT.0 ml) consist of 1.Absorbance of control A1:. The percentage of inhibition can be calculated using the formula: Inhibition (%) = (A0 – A1 / A0) × 100 Where : A0 :.PROCEDURE: The reaction mixture (3. Catechin. . Gallic acid.0 ml of the extract and 1. It is incubated for 10 min in dark. Positive controls: Ascorbic acid.Absorbance of test. then the absorbance is measured at 517 nm.3 mM).0 ml of methanol.0ml of DPPH in methanol (0. 1. α-tocopherol. Rutin. Quercetin. BHA. anthocyanins. flavonols. a diverse group of phenolic compounds (flavanols. etc.) possess an ideal structural chemistry for free radical scavenging activity.TOTAL PHENOLIC CONTENT (TPC) Plant polyphenols. Antioxidative properties of polyphenols arise from their high reactivity as hydrogen or electron donors from the ability of the polyphenol derived radical to stabilize and delocalize the unpaired electron (chain-breaking function) and from their potential to chelate metal ions (termination of the Fenton reaction) The amount of total phenol content can be determined by Folin-Ciocateu reagent method . phenolic acids. 5 N) are mixed and incubated at room temperature for 15 min. Tannic acid.Gallic acid . Pyrocatechol. Chlorogenic acid.1 ml of Folin-Ciocalteu reagent (0. Then 2.5 ml of extract and 0. Positive control:. Quercetin. .5 ml saturated sodium carbonate(2%) is added and further incubated for 30 min at room temperature and absorbance measured at 760 nm.PROCEDURE 0. The total phenolic content is expressed in terms of standard equivalent (mgg -1 of extracted compound). SUPEROXIDE ANION RADICAL SCAVENGING (SO) ASSAY Superoxide anion is a weak oxidant. Reduction of NBT is the most popular method. The capacity of extracts to inhibit the colour to 50% is measured in terms of EC50 . it gives rise to generation of powerful and dangerous hydroxyl radicals In-vitro super oxide radical scavenging activity is measured by riboflavin/NBT (Nitro blue tetrazolium) reduction. The super oxide radical reduces NBT to a blue colored formazan that can be measured at 560nm. The method is based on generation of super oxide radical by auto oxidation of riboflavin in presence of light. BHA.5 ml Tris-HCl buffer (0.5 ml phenazine methosulfatenicotinamide adenine dinucleotide (PMS-NADH) solution (0. containing 0. The reactionis started by adding 0.0 ml extract and 0. α-tocopherol.3). Quercetin or Trolox.3). pH 8. 1.Procedure The superoxide anion radicals are generated in 3.0 ml of TrisHCl buffer (0.02 M. incubated at 25°C for 5 min and then the absorbance is measured at 560 nm against a blank sample.5 ml of NBT (0.3mM). Ascorbic acid. 0. Positive control: Gallic acid. pH 8.12 mM) to the mixture.5 ml NADH (0. Curcumin. .02 M.936 mM) solution. . water. human body. Human beings exposed to H2O2 indirectly via the environment are estimated as 0. microorganisms. plants.28 mg/kg/day with intake from leaf crops contributing most to this exposure. food and beverages. In the body. H2O2 is rapidly decomposed into oxygen and water and this may produce hydroxyl radicals (OH˙) that can initiate lipid peroxidation and cause DNA damage. Hydrogen peroxide enters the human body through inhalation of vapour through eye or skin contact.HYDROGEN PEROXIDE RADICAL SCAVENGING (H2O2) ASSAY Hydrogen peroxide occurs naturally at low concentration levels in the air. . Positive control: Ascorbic acid. pH 7. The percentage of hydrogen peroxide scavenging is calculated as follows: % Scavenged (H2O2) = (A0 – A1 / A0) X 100 Where: A0 is the absorbance of control and A1 is the absorbance of test. α–tocopherol.60 µg/ml) in distilled water is added to hydrogen peroxide and absorbance at 230 nm is determined after10 min against a blank solution containing phosphate buffer without hydrogen peroxide.4). The concentration of hydrogen peroxide is determined by absorption at 230 nm using a spectrophotometer. Quercetin. Rutin. Extract(20 . BHA.Procedure: A solution of hydrogen peroxide (40 mM) is prepared in phosphate buffer (50mM. In vitro inhibition of nitric oxide radical is also a measure of antioxidant activity. because of its unpaired electron. In presence of scavengers. is classified as a free radical and displays important reactivity’s with certain types of proteins and other free radicals. the absorbance of the chromophore is evaluated at 546nm. NITRIC OXIDE RADICAL SCAVENGING (NO) ASSAY Nitric oxide. The activity is expressed as % reduction of nitric oxide. . This method is based on the inhibition of nitric oxide radical generated from sodium nitroprusside in buffer saline and measured by Griess reagent. 5.0 ml of the incubated sample. POSITIVE CONTROL: Curcumin. Sodium nitrite.0 ml of extract and reference compound in different concentrations (20 – 100 µg/ml). Ascorbic acid.5) is added to 2. 0.1% naphthyethylene diamine dihydrochloride (NED) in 2% H3PO3) is added and pink chromophore generate during diazotization of nitrite ions with sulphanilamide and subsequent coupling with NED was measured spectrophotometrically at 540nm. BHA. Percent inhibition of the nitrite oxide generated is measured by comparing the absorbance values of control and test preparations. Rutin. To 5. The resulting solutions are then incubated at 25°C for 60 min A similar procedure is repeated with methanol as blank.0ml of Griess reagent (1% sulphanilamide.0 ml of 10mM sodium nitroprusside in phosphate buffer (pH-7. which serves as control. Caffeic acid. BHT or α-tocopherol .PROCEDURE 3. . Antioxidant activity can be measured using both serum sample as well as tissue homogenate.IN VIVO SCREENING METHODS OF ANTIOXIDANT ACTIVITY Various in-vivo methods are used to evaluate the ability of antioxidant to reduce the radical. The different in vivo antioxidant screening methods Lipid peroxidation (LPO) Reduced glutathione (GSH) Glutathione peroxidase (GPx) Glutathione reductase (GR) Glutathione-s-transferase (GST) Superoxide dismutase (SOD) Catalase (CAT) Total protein Lactate dehydrogenase (LDH) Ornithine decarboxylase Glucose 6 phosphatase (G6P) Creatinine phosphokinase(CPK) etc. Which is accepted as an indicator of lipid peroxidation. which is a common cause of cell death. . This process may cause peroxidative tissue damage in inflammation. MDA is one of the end products in the lipid per-oxidation process .LIPID PEROXIDATION (LPO) LPO is an autocatalytic process. Malondialdehyde (MDA) is formed during oxidative degeneration as a product of free oxygen radicals. cancer and toxicity of xenobiotics and aging. 0 ml of the mixture of n-butanol and pyridine (15: 1.1% sodium dodecyl sulfate (SDS). After centrifugation at 4000 rpm for 10 min. 1.1 ml of sample.2 ml of 8. 0. v/v) were added. the absorbance of the organic layer (upper layer) was measured at 532 nm against blank without the sample. and in the pH range of 1. The mixture was finally made up to 4. After cooling with tap water. 20% acetic acid containing 0.0. The pH of 20% acetic acid solution was adjusted with NaOH above pH 3. 1.27 M HCl was adjusted to the specified pH with NaOH.PROCEDURE: The reaction mixture contained 0. .0 ml of distilled water and 5.5 ml of 20% acetic acid solution of various pHs.0 ml with distilled water.8% aqueous solution of thiobarbituric acid (TBA). The level of lipid peroxidase were expressed as thiobarbituric acid reactive substance (TBARS)/mg protin.5 ml of 0. and 1.0.0-3. and the mixture was shaken vigorously. and heated at 95°C for 60 min. GSH is an intra-cellular reductant and plays major role in catalysis. REDUCE GLUTATHIOL(GSH) If deficiency of GSH within living organisms can lead to tissue disorder and injury. It protects cells against free radicals. peroxides and other toxic compounds. Glutathione also plays an important role in the kidney and takes part in a transport system involved in the reabsorption of amino acids. Glutathione was also implicated in diseases like cataract and aminoaciduria. Thus GSH monoesters are also useful in the treatment of aminoacidouria. . metabolism and transport. Deficiency of GSH leads to cataract formation in mice and rats. Absorbance values were compared with a standard curve generated from standard curve from known GSH.8ml of the 5.4) was taken. solutions were measured at 412 nm against blank. .PROCEDURE To measure the GSH level. The mixture was centrifugation for 10 min at 200 rpm allowed to stand for 5 min.1 M phosphate buffer pH 7. The supernatant (200 μl) was then transferred to a new set of test tubes and added 1.1mM) was prepared in 0. the tissue homogenate (in 0. 5'-dithiobis-2-nitrobenzoic acid (0. After completion of the total reaction. The homogenate was added with equal volume of 20% trichloroacetic acid (TCA) containing 1 mM EDTA to precipitate the tissue proteins.3 M phosphate buffer with 1% of sodium citrate solution). Then all the test tubes make upto the volume of 2ml with double distilled water. 0. After incubation at 30°C for 90 s.1ml of sample. 1. then addition of 0.0ml of n-butanol.1 ml phenazine methosulphate (186 μM).SUPEROXIDE DISMUTASE (SOD) SOD is a metalloprotein and is the first enzyme involved in the antioxidant defence by lowering the steady-state level of O2. 0. Reaction was started by addition of NADH. centrifuged and butanol layer was separated.2 ml NADH (750 μM).3 ml of 300 μM nitroblue tetrazolium.3.052 M). 0. . Mixture was allowed to stand for 10min. Colour intensity of the chromogen in the butanol layer was measured at 560 nm spectrophotometrically and concentration of SOD was expressed as units/mg. 0.1ml glacial acetic acid to stop reaction. Procedure : Assay mixture contained 0.2ml of sodium pyrophosphate buffer (pH 8. Reaction mixture was stirred vigorously with 4. CATALASE (CAT) CAT is a hemeprotein. . localized in the peroxisomes or the microperoxisomes. CAT is a key component of the antioxidant defense system. Inhibition of these protective mechanisms results in enhanced sensitivity to free radical-induced cellular damage. This enzyme catalyses the decomposition of H2O2 to water and oxygen and thus protecting the cell from oxidative damage by H2O2 and OH. 9 ml of 50 mM phosphate buffer (pH 7.1 ml of supernatant was added to cuvette containing 1.0 ml of freshly prepared 30 mM H2O2 The rate of decomposition of H2O2 was measured spectrophotometrically from changes in absorbance at 240 nm.0) Reaction was started by the addition of 1. Absorbance is calculated was expressed as units/mg protin.PROCEDURE: 0. . 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 . 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 . AYURVEDIC PLANTS UNFOLDED ANTIOXIDANT POTENTIAL Pluchea lanceolata Prunus cerasoides Salix caprea Hyoscymus niger Litea glutinosa Semecarpus anacardium Salmalia malabarica . . DPPH 4’-O-methyl epigallocatechin Cinnamomum zeylanica PART : Leaves EXTRACT: 50% Ethanolic. . Superoxide Leucasin radical. TPC. ABTS Garcinol. Lipid Peroxidation. polyisoprenylated benzophenone Garcinia indica PART: Fruits EXTRACT: Methanolic. Kempferol Leucas aspera PART: Leaves EXTRACT: Methanolic.7-diphenyl-5hydroxy-6-hepten-3one. DPPH. DPPH. Flavokawin B. Ethyl Acetate. Reducing Power Quercetin.PLANT NAME PART & EXTRACT MODEL USED CHEMICAL CONST. Quercetrin. Ethyl Acetate. Cassine transvaalesis PART: Root EXTRACT: Ethanolic. Methyl cinnamate ORAC. TPC Alpinia rafflesiana PART: Leaves EXTRACT: Methanolic. Ethyl Acetate.6 dehydrokawain. 5. FRAP. DPPH 1. kernels) ME. Water TPC. Water DPPH. ME. Water DPPH Rosmarius officinalis Leaves ME TEAC. DPPH . EA. Flower. seeds. NO. Raw fruit. TBARS Cassia tora L.PLANT NAME PART Solvent Model Used Aegle marmelos Correa(Rutaceae) Fruit ME DPPH. RP Polygonum paleaceum Rhizomes BT. (Caesalpiniaceae) Seeds ME RP.SO Azadirachta indica Leaves. EA. Fruits (pulp. Water DPPH. Total antioxidant activity. peels. DPPH. ME. Ripe fruit. DPPH. TPC. RP. Stem bark HE. TPC. (Ebenaceae) Leaves PE. TF Mangifera indica L. A. Metal Chelating Diospyros ebenum Roxb. 21: 213-238. Text book of Pathology. Food Chem. van Doornen. Rice-Evans C. Ortuño A. L. B. Paganga G. . J. Chang C. 2002 10: 178-182.1996. 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