ANTIOXIDANT TEST In biological systems potentially harmful reactive oxygen species (ROS) are produced during the normal

aerobic metabolism. Antioxidants are deployed to prevent generation of ROS or to scavenge those formed. Deficiency of antioxidative defenses may lead to oxidative stress, which might be associated with a variety of disorders such as coronary heart diseases, neural disorders, diabetes, arthritis and cancers. Although organisms are bestowed with antioxidant and repair systems that have evolved to protect them against oxidative damage, these systems are insufficient to prevent the damage totally. Hence antioxidants in diet are of great importance as possible protective agents to help human body to reduce oxidative damage. Recently a large number of natural antioxidants have been isolated from different plants. Human diet containing m e d i c i n a l herb possessing antioxidative properties would be

potentially useful to help human body to reduce oxidative damage. Much research has concentrated on different plant extracts abilities to induce antioxidant effects. Natural antioxidative compounds from plants have aroused great attention due to concerns about the safety of synthetic antioxidants. Increasing efforts have been made to search for plant-derived antioxidants. Many herbals contain antioxidant compounds which protect cells against the damaging effects of reactive oxygen species, such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite. Overproduction of such free radicals can cause oxidative damage to biomolecules (e.g. lipids, proteins, DNA), eventually leading to many chronic diseases, such as atherosclerosis, cancer, diabetes, aging, and other degenerative diseases in humans. Plants (fruits, vegetables, medicinal herbs, etc.) may contain a wide variety of free radical scavenging molecules, such as phenolic compounds (e.g. phenolic acids, flavonoids, quinones, coumarins, lignans, stilbenes, tannins), nitrogen compounds (alkaloids, amines, betalains), vitamins, terpenoids (including carotenoids), and some other endogenous metabolites, which are rich in antioxidant. Free radical formation by oxygen Oxygen has double-edged properties, being essential for life; it can also aggravate the damage within the cell by oxidative events. Free radicals and its adverse effects were discovered in the last decade. These are dangerous substances produced in the body along with toxins and wastes which are formed during the normal metabolic process of the body. Oxidative stress and impaired antioxidant system have been implicated in the pathophysiology of diverse disease states.

The body obtained energy by the oxidation of carbohydrates, fats and proteins through both aerobic and anaerobic process leads the generation of free radicals. Over production of the free radicals can responsible for tissue injury. Cell membranes are made of unsaturated lipids and these unsaturated lipid molecules of cell membranes are particularly susceptible to free radicals. Oxidative damage can direct to a breakdown or even hardening of lipids, which composition of all cell walls. Breakdown or hardening is due to lipid peroxidation leads to death of cell or it becomes unfeasible for the cell to properly get its nutrients or get signals to achieve another. In addition, other biological molecules including RNA, DNA and protein enzymes are also susceptible to oxidative damage. Reactive oxygen-free radicals (ROS) have been implicated in many diseases and in aging process. These free radicals, which cause tissue damage via oxidative stress, are generated by aerobic respiration, inflammation, and lipid peroxidation. Antioxidant systems minimize or prevent deleterious effects of the ROS. High concentration of hydrogen peroxide is deleterious to cells, and its accumulation causes oxidation of cellular targets such as DNA, proteins, and lipids, leading to mutagenesis and cell death. Environmental agents also initiate free radical generation leads different complication in body. The toxicity of lead, pesticides, cadmium, ionizing radiation, alcohol, cigarette smoke, UV light and pollution may all be due to their free radical initiating capability. A free radical may defined as a molecule or molecular fragments containing one or more unpaired electrons in its outermost atomic or molecular orbital and are capable of independent existence.

Problem of free radical Free radical stress leads to a wide number of health problems which include tissue injury and progression of disease conditions such as arthritis, hemorrhagic shock, atherosclerosis, diabetes, hepatic injury, aging, and ischemia, reperfusion injury of many tissues, gastritis, tumor promotion, neuron degenerative diseases and carcinogenesis. Safer antioxidants suitable for long term use are needed to prevent or stop the progression of free radical mediated disorders. Free radical damage and oxidative stress are the major reasons for liver tissue damage. Lipid peroxidation is regarded as one of the basic mechanisms of tissue damage caused by free radicals .The antioxidants are the first line defense against such damage and provide protection against the deteriorating outcome. In addition to the antioxidant defense system of our body, antioxidants that are mainly supplied as dietary consumptions can also impede carcinogenesis by scavenging oxygen radicals or interfering with the binding of carcinogens to DNA, include vitamin C, vitamin E (-tocopherol, -tocopherol), -carotenoids (-carotene, -carotene, - cryptoxanthin, lutein, zeaxanthin, lycopene) and several polyphenolic compounds including flavonoids (catechins, flavonols, flavones, isoflavonoids). Several potential plant-derived antioxidants such as quercetin, carnosol, thymol, carnosic acid, hydroxytyrosol, gallic acid derivatives, tannins, catechins, rutin, morin, ellagic acid, eugenol, and rosemarinic acids have come to attention because of their extensive use in dietary supplementation, food preservation, and treating various free radical mediated diseases.

Methods of evaluating antioxidant activity Antioxidant property of the various fraction of the plant was determined by following methods Determination of DPPH radical scavenging assay (Quantitative analysis) Determination of total phenolic content Determination of total antioxidant capacity by phosphomolybdenum method Determination of total flavonoids content

DPPH radical scavenging assay

DPPH free radical scavenging activity of the plant fractions was determined following the method described by Braca et al. (2001). Principle DPPH (1,1-diphenyl-2-picrylhydrazyl) radical has been widely used to evaluate the free radical scavenging capacity of antioxidants. DPPH free radical is reduced to the corresponding

hydrazine when it reacts with hydrogen donors. DPPH can generate stable free radicals in aqueous or methanol solution. With this method it was possible to determine the antiradical power of an antioxidant by measuring the decrease in the absorbance of DPPH at 517 nm. Resulting from a color change from purple to yellow the absorbance decreased when the DPPH was scavenged by an antioxidant, through donation of hydrogen to form a stable DPPH molecule. In the radical form, this molecule has an absorbance at 517 nm which disappears after acceptance of an electron or hydrogen radical from an antioxidant compound to become a stable diamagnetic molecule. When the odd electron of DPPH radical becomes paired with hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H, then the color turns from purple to yellow as the molar absorptive of the DPPH radical reduces from 9660 to 1640 at 517 nm. Scavenging of DPPH free radicals by antioxidants decreases the absorbance. The DPPH assay was used to measure the level of antioxidants in a substance. DPPH is a well known radical and a trap ("scavenger") for other radicals. Therefore, rate reduction of a chemical reaction upon addition of DPPH is used as an indicator of the radical nature of that reaction.

Materials & Reagents 1,1-diphenyl-2-picrylhydrazyl UV- visible spectrophotometer L-Ascorbic acid Beaker (100 & 200ml) Distilled water Test tube Methanol Aluminum foil Pipette (5ml) Spatula Analytical balance 6.1.3. Methods 2.0 ml of a methanol solution of the extract at different concentration (400, 200,100, 50,25, 12.5,6.25,3.125,1.5625,0.78125}g/ml were made. After this all the testtubes were filled up by to 5 ml by mixing 3 ml DPPH solution A blank solution was made which contain only DPPH solution of 5 ml(20 g/ml). After 30 min reaction period at room temperature in dark place the absorbance was measured against at 517 nm against methanol as blank by using a UV- visible spetrophotometer. Inhibition free radical DPPH in percent (I%) was calculated as follows: (I%) = (1 Asample/Ablank) X 100 Where Ablank is the absorbance of the control reaction (containing all reagents except the

test material). Extract concentration providing 50% inhibition (IC50) was calculated from the graph plotted inhibition percentage against extract concentration. L-Ascorbic acid was used as positive control.

Estimation of Total Phenolic Content The total phenolic concentration of the extract of Spondias pinnata skin was determined by the modified Folin-Ciocalteu method. The process of measuring total phenolic content of the crude extract of Spondias pinnata fruit involves the use of Folin-Ciocalteu reagent. The Folin-Ciocalteu reagent is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric assay of phenolic and polyphenolic antioxidants. The FCR actually measures a samples reducing capacity. The exact chemical nature of the FC reagent is not known, but it is believed to contain heteropolyphosphotunstates - molybdates. Sequences of reversible one- or two-electron reduction reactions lead to blue species, possibly (PMoW11O40)4- . It is believed that the molybdenum is easier to be reduced in the complex and electron- transfer reaction occurs between reductants and Mo (VI): Mo (VI) + e Mo (V) It measures the amount of substance being tested needed to inhibit the oxidation of the Folin-Ciocalteu reagent. The reagent does not contain phenol. Rather, the reagent will react with phenols and nonphenolic reducing substances to form chromogens that can be detected spectrophotometrically. The generated chromogens give a strong absorption maximum at 760 nm. 6.2.2.2. Materials & Reagents Test tube Analytical balance Pipette UV- visible spectrophotometer Spatula Vortex Mixer Folin-Ciocalteu reagent Distilledwater

Sodium carbonate (Na2CO3) Methanol Gallic acid Aluminum foil Composition of Folin-Ciocalteu Reagent SL. No. Component 1 2 3 4 5 6 Water Lithium Sulfate Sodium Tungstate Dihydrate Hydrochloric Acid>=25% Phosphoric Acid 85% solution in water Molybdic Acid Sodium Dihydrate Quantity (%) 57.5 15.0 10.0 10.0 5.0 2.5

6.2.2.3. Methods 0.5ml of a methanol solution of the crude extract of concentration of 1mg/ml was mixed with 5ml Folin ciocalteu reagent (1:10 v/v distilled water) and 4 ml (75g/L) of Sodium carbonate. The mixture was vortexed for and allowed to stand for 30min in dark place for color development and the absorbance was measured at 760 nm against methanol as blank by using a UV- visible spetrophotometer. The total phenolics was expressed extract as gm of the acid GAE (gallic acid

equivalent)/100gm of the dried obtained from a

using Gallic

following

equation curve

standard

calibration

Y=0.0162x+0.0215;R=0.9972.

Total antioxidant capacity Principle The phosphomolybdenum method usually detects antioxidants such as ascorbic acid, some phenolics, -tocopherol, and carotenoids. The phosphomolybdenum method is based on the reduction of Mo(VI) to Mo(V) by the antioxidant compound and subsequent formation of a green phosphate/Mo(V) complex at acid pH. In essence, it is believed that the molybdenum is easier to reduce in the complex and electron-transfer reaction occurs between

reductants and Mo(VI) and the formation of a green phosphate/Mo(V) complex with a maximal absorption at 695 nm. Mo (VI) + e Mo (V) Sometimes a correlation analysis is performed between the total phenolic content and total antioxidant capacity to reveal the correlation. It is obvious that the plant phenolic compounds contribute to the major antioxidant activity. Based on the absorbance values of the extract solution, reacted with reagent solution (0.6 M Sulfuric acid,28mM Sodium Phosphate and 4mM Ammonium molybdate) and compared with the standard solutions of L-ascorbic acid equivalents, result of the colorimetric analysis of the total antioxidant capacity is given in table 6.9. Total antioxidant capacity of the sample is expressed as mg of L-ascorbic acid per gm of dried extract. Method 1. 300l of each fraction (200 g/ml) was taken in test tubes. 2. 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) was added into the test tubes. 3. The test tubes were incubated at 950 C for 90 minutes to complete reaction. 4. Then the absorbance of the solution was measured at 695 nm using a spectrophotometer against blank after cooling to room temperature.

Assay for total flavonoids concentration Aluminium chloride colorimetric method was used for determination of total flavonoids concentration in the samples of S. pinnata fruit skin. Each extract and fraction(0.5 ml, 1:10 gml-1) ) in methanol were separately mixed with 1.5 ml of methanol, 0.1 ml of 10%aluminum chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water It was allowed to stand for 30 min at room temperature and the absorbance of the reaction mixture was measured at 415 nm. Total flavonoids content was determined as mg of Quercetin equivalent per gram using the equation obtained from a standard Quercetin calibration curve Materials and Equipments Test tubes UV-2450 spectrophotometer Test tubeholder Beaker Electronic balance 6.5.2. Reagents Methanol Aluminumchloride Potassium acetate Quercetin Distilled water Method 1. 1.0 ml of each fraction (200 g/ml) and standard (quercetin) in different concentrations were taken in test tubes. 2. 3 ml of methanol was added into the test tubes.

3. 200 l of 10% aluminium chloride solution was added. 4. 200 l of 1 M potassium acetate solution was added to the mixtures in the test tubes. 5. Then 5.6 ml of distilled water was added into the test tubes. 6. The test tubes were incubated for 30 minutes at room temperature to complete reaction. 7. Then the absorbance of the solution was measured at 415 nm using a spectrophotometer (Shimadzu UV PC-1600) against blank. 8. Methanol was used as the blank. 9. Total flavanoid concentration o f the fractions was expressed as quercetin equivalents (QE) after calculation using the following equation: C = (c x V)/m Where:

C = total flavonoid contents, mg/g plant extract in QE, c = concentration of quercetin obtained from calibration curve (mg/ml), V = the volume of the sample solution (ml) m=weight of the sample(g)