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 leads 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 RADICALESA molecule with one or more unpaired electron in its outer shellThey are easily available to react with various targets like lipid,

protein, DNA. ENDOGENOUS SOURCE

Body's normal use of oxygen such as respiration and some cell mediated immune functions

Immune cell activation Inflammation Mental stress

EXOGENOUS SOURCE Environmental pollutants Cigarette smoke Automobiles fumes Radiations

TYPES OF FREE RADICALS Superoxide radical

Hydroxide radical

Nitric oxide radical

Peroxide radicals

H2O2(non radical)..etc

SUPEROXIDE RADICALIt is generated via several cellular oxidase system

like… NADPH oxidaseXanthin oxidasePeroxidase

It starts the process of generation of other free radicals.

HYDROXYL RADICAL

It is most dangerous radical

It is most reactive radical in biological system

It 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 xanthin oxidase

NITRIC OXIDEIt is formed in biological tissue from the

oxidation of L-argenine to citrulline by nitric oxide synthtase

BENEFICIAL ACTIVITIESAt low and moderate level they helps in

maturation process of cellular structures.

Act as a weapon for host defense system.

Phagocytes realizes free 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 stressDamaging DNA proteins Lipid peroxidationStructural damage to cell and tissue.Mutation deformities Responsible for so many life threatening disorders and

diseasesCan 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 where it is the main cause of premature mortality

The development of cancer in complex process including cellular and molecular changes mediated by endogenous and exogenous stimuli

Voluminous literature suggest that the free radicals are directly or indirectly proved harmful and can cause the carcinogenesis

The oxidative DNA damage is responsible for cancerCancer 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 stress ….

It is a relapsing immuno-inflammatory multisystem disease with synovial proliferation and destruction of articular cartilage

1-2% population get affected

Incidence increases with age

Etiology of RA remains unknown

The pathogenesis of RA is multifactorial and recent research has implicated oxygen free radicals as mediators of tissue damage

Several 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---RA

Free radicals ----decreases glutathione---damage defense system

Activated 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 receptor

Cholesterol rapidly accumulate on macrophages and 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 downInactivation of pancreatic cellStructural and functional damage of

pancreatic cellCOMPLICATIONS 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………Penile 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 function

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-ENZYMATICMetabolic antioxidants

Lipoid acid Uric acid Melatonin

Nutrient antioxidants Carotenoids Tannins Flavonoids etc.

Mechanism of AntioxidantsReducing agents

Chain breakers

Free radical scavenger

Metal 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 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 1. Scavenging capacity assay against specific ROS/NOS2. Scavenging capacity assay against stable and non-

biological radical.

TargetOxidized Target

Oxidized antioxida

nt

Antioxidant

Reactive species

Reduced reactive species

1. Competitive

Oxidized antioxida

ntAntioxidant

Reactive species

Reduced reactive species

2. Non-competitive

SREENING MODELS

Hydroxyl radical scavenging 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 EDTAOH Deoxyribose

Chromogen

TBA

FeCl3 H2O2 EDTAOH Deoxyribose

Chromogen(532 nm)

TBA

After addition of antioxidant

Superoxide radical anion (O2.-) scavenging assayThis radical arise from several metabolic

processes It is also generated in biological system by

enzyme hypoxanthine and xanthin oxidase pH7.4

Reagents- 1. Phenazine 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 as a target in place of NBT

The 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 solution----

physiological ph------NO. radicals---

nitrite---Griess

reagent----chromophore---546nm

DPPH. Scavenging assay

It is a purple colored stable free radical

Antioxidant compound convert it to colorless moiety.

The amount of DPPH reduced could be quantified by measuring a decrease in absorbance and may be attributed to its hydrogen donating ability

The assay should be performed at 517 nm

Peroxyl radicals {ROO.} scavenging activityThis 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 soluble

antioxidants can be screened AAPH (2,2 – azobis-2-amidinopropane,

dihydrochloride)AMVN (2,2 – azobis-2,4 dimethylvaleronitrile)

ORACThe principle is based upon the deceasing

florescence along with the Pyroxyl radicals.The radicals are formed due to thermal

decomposition of AAPH in aqueous buffer. This assay is generally used to evaluate the

chain breaking activity B-phycoerithrin was used as a florescent

targetNow a days flurescein is used.

Ferric reducing antioxidant power (FRAP) assayThis assay helps to measure the ability of

antioxidant compound to reduce ferric ions into ferrous complex

It required the acid medium and absorbance is calculated at 593 nm

This 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 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 ModelsHypoclorous acid (HOCL) scavenging activity

Peroxynitrite (ONOO) scavenging activity

Singlet oxygen scavenging capacity assay

Total radical-trapping antioxidant parameter (TRAP)

Total oxyradical scavenging capacity

H2O2 scavenging assay using Horseradish peroxidase.

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 plantsPiper 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

These plants can be acts as best antioxidants.Steppe plants

Dessert plants

Alpine plants

Mountain plants

Very high carbon assimilation

Short growing period

Extreme climate conditions

Low or high temperature Rapid temperature changesUnfavorable conditions for photosynthesis Light intercity etc..

DIETARY ANTIOXIDANT

ConclusionFree 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 powerDPPH assay

OH- radical scavenging assayO2.- radical scavenging assayH2O2 radical scavenging assay

NO. radical scavenging assayPeroxyl radical scavenging assayTotal 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.