A study on antioxidant nature of petai (parkia speciosa)

A STUDY ON ANTIOXIDANT NATURE OF PETAI (PARKIA SPECIOSA) BHEEMARAJU AMARNATH (M.Sc., Nagarjuna University) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2004 Dedicated to Sri Sreedhar & Srimati Jayalakshmi ii Acknowledgements I thank my supervisor Dr. Leong Lai Peng for her help, supervision and guidance. I convey my deepest thanks and regards to my parents for their constant support and encouragement. I also express my gratefulness to the god who showered his blessings upon me. I would like to express my gratitude to my lab friends Janaka, Abul, Vel, Guanghou and Caroline for their motivation. I thank my room mate Srinivas and house mates Sumod, Ravi, Guru and Mukthar for helping me in many ways. I express my appreciation for all the help of laboratory officers of FST Lab Lee Chooi Lan, Analytical Lab Francis, LCMS Lab Madam Wong, deputy lab officer Lew Huey Lee and supporting staff Rahman. I express my gratefulness to Assoc. Prof. Dr.Conrad O.Perera for his inspiration and help. I would like to express my thankfulness to Assoc. Professors Dr. P.J.Barlow and Dr. Zhou. My brother Vikram, sister-in-law Lalitha, sister Madhavi and brother-in-law Muralis’ cooperation is appreciated. Thanks to my uncles (Gopi, Mahendra, Narasimha, Ravindranath, Nagabhushana Verma and Vijayasimha), my cousins (Bharadwaja, Ravi, Meena, Yathi, Vishnu, Vasudha, Bhargavi, Aparna, Ajay, Sumanth, Deepthi, Neelima, Aruna, Anitha, Sunil and Vijaya), my nephews Kashyap and Rithvik, my grandparents and all other members of my family for their encouragement and love. Last but not least I would like to express my best wishes to my present room mate Rishi and house mates Ravi, Bedobrata and Anil. iii Table of Contents Abbreviations v Summary vi List of Tables viii List of Figures ix CHAPTER General Introduction 1.1 Radicals and their biological effects. 1 1.1.1 Free radicals and reactive oxygen species 1.1.2 Types of Reactive Oxygen Species and their generation 1.1.3 Biological effects of radicals 1.2 Importance of antioxidants 1.3 Antioxidants classification based on their sources 1.3.1 Natural antioxidants 1.3.2 Synthetic antioxidants 12 1.4 Different types of antioxidants 14 1.4.1 Primary antioxidants or chain breaking antioxidants 14 1.4.1.1 Important reactions of primary antioxidants 14 1.4.1.2 Important primary antioxidant compounds 15 1.4.2 Secondary antioxidants or preventive antioxidants 19 1.4.3 Synergistic antioxidants 20 1.5 Some important cellular antioxidants and low molecular weight 22 antioxidants 1.6 Methods of measuring the total antioxidant capacity 24 1.6.1 ABTS radical cation scavenging assay 24 1.6.2. DPPH radical scavenging assay 28 1.6.3. Ferric Reducing / Antioxidant Power 30 1.6.4 Oxygen Radical Absorption Capacity 32 iv 1.6.5 Total Radical Trapping Antioxidant Parameter (TRAP) 34 method 1.7 Pitfalls in the determination of total antioxidant capacity by 36 various methods 1.8 HPLC for the identification of antioxidant compounds 38 1.9 Overview of petai 40 1.9.1 Introduction to petai 40 1.9.2 Composition of petai 41 1.9.3 Medicinal properties of petai 42 1.10 Objectives CHAPTER Materials and Methods 2.1 Materials 45 48 48 2.1.1 Petai 48 2.1.2 Chemicals 48 2.1.3 Equipment 49 2.2 Methods 2.2.1 Pre-treatment of petai seeds and pods for further analysis 49 49 2.2.1.1 Extraction procedure for TAC analysis 50 2.2.1.2 Extraction procedure for Vitamin C analysis in 50 petai seeds and pods 2.2.1.3 Extraction procedure for antioxidant compounds 50 and for collection of fractions 2.2.1.4 Extraction procedure for analysis of compounds in 51 petai seeds and pods using LC-MS 2.2.2 Optimization of extraction parameters 51 2.2.3 Methods for determination of TAC 52 2.2.3.1 ABTS Assay 52 2.2.3.2 DPPH Assay 53 v 2.2.3.3 FRAP Assay 54 2.2.4 Folin-ciocalteu assay for total phenolic content 54 2.2.5 Total thiol content of Petai using Ellman’s assay 55 2.2.6 Identification of antioxidant compounds of Petai seeds and 55 pods using HPLC 2.2.7 Determination of vitamin C of petai using HPLC 56 2.2.8 Analysis of compounds in petai seeds and pods using 57 LC-MS 2.2.9 Analysis of important antioxidant fractions from seed 57 extract CHAPTER Results and Discussion 3.1 Determination of optimal conditions for extraction 3.1.1 Determination of optimal solvent combination for 58 58 60 extraction 3.1.2 Determination of optimal shaking parameters for 63 extraction 3.1.3 Effect of microwaves on the extraction 64 3.1.4 Determination of optimal temperature for extraction 66 3.2 Determination of total antioxidant capacity 3.2.1 Radical scavenging assays for determination of TAC 3.2.1.1 ABTS and DPPH assays 3.2.2 Reducing power 3.2.2.1 FRAP assay 3.2.3 Total phenolic content 3.2.3.1 Folin assay 68 69 70 73 74 75 75 3.2.4 Ellman’s assay for determination of total thiol content 79 3.3 Correlation between TAC and TPC of different batches of petai 81 seeds vi 3.4 Optimization of separation using HPLC 85 3.5 Identification of antioxidant compounds in petai based on the 87 reaction with ABTS+ and DPPH radical solutions using HPLC 3.6 Determination of Vitamin C by HPLC 90 3.7 Antioxidant capacity of petai pods 95 3.7.1 Optimization of extraction 95 3.7.1.1 Solvent for optimal extraction 95 3.7.1.2 Optimal heating parameters 96 3.7.2 TAC of petai pods 96 3.7.3 Comparison of TAC of petai seeds and pods 98 3.7.4 Correlations between TAC and TPC 101 3.7.5 Identification of antioxidant compounds in pods using 104 HPLC 3.7.6 HPLC analysis for Vitamin C in petai pods 105 3.8 Possible phenolic compounds from petai seed and pod extracts 106 3.9 Analysis of antioxidant nature of different fractions of petai 112 seeds using preparative HPLC CHAPTER Conclusions and Future research work 116 References 120 Appendix 150 Presentations / Conferences 160 vii Abbreviations AAPH 2,2’-azobis(2-amidino-propane) dihydrochloride ABAP 2,2’-azobis-(2-amidino propane) dihydrochloride ABTS 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonate AEAC Ascorbic acid equivalent antioxidant capacity DNA Deoxy ribo nucleic acid DPPH 2,2-diphenyl-1-picrylhydrazyl DTNB 5,5’-diphenyl picryl hydrazyl FRAP Ferric reducing / antioxidant power GAE gallic acid equivalents HAT Hydrogen atom transfer ORAC Oxygen radical absorption capacity. ROS Reactive oxygen species R-PE R-phycoerythrin SET Single electron transfer TAA Total antioxidant activity. TAC Total antioxidant capacity. TEAC Trolox equivalent antioxidant capacity TRAP Total radical absorption power TRAP Total radical-trapping antioxidant parameter. TROLOX 6-hydroxy-2, 5,7,8-tetramethyl-2-carboxylic acid viii Summary In this research the antioxidant nature of petai seeds and pods was studied. The effectiveness of petai as a natural source of antioxidants was evaluated using several methods. Antioxidant capacities of seeds and pods were compared, the active antioxidants were identified using HPLC and the nature of antioxidant compounds was studied. Furthermore, possible antioxidants present in pods and seeds were analyzed using LC-MS. Aqueous ethanolic extract of petai seeds showed high radical scavenging activity with ABTS+ (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonate) and DPPH (2,2-diphenyl-1picrylhydrazyl) radicals. It also showed good reducing ability with FRAP (Ferric reducing / antioxidant power) assay. Thus this seeds are significant in the diet, as they can effectively scavenge harmful radicals / reduce metal ions that induce Fenton reactions and protect the cells from damage. Petai seeds were found to show high phenolic content. They also showed some activity with Ellman’s reagent indicating the presence of thiol compounds. Vitamin C content was found to be high in petai seeds. This is one of the major compounds that contribute to the total antioxidant capacity (TAC) of the seeds. LC-MS analysis of the seed extract showed that there are some important flavonoids and polyphenolic compounds present in the seeds which contribute to the TAC. The correlation between the TAC and total phenolic content (TPC) was found to be high. This showed that a major portion of TAC was contributed by phenolic compounds. Further it was found that antioxidant activity increased on increasing the temperature. This increase ix was not due to the increase in extraction of vitamin C into the solution but might be due to the presence of Maillard reaction products formed on heating the solution. Antioxidant capacity of petai pods found by ABTS•+, DPPH• and FRAP methods was very high compared with the seeds. There was a 6-fold difference in the antioxidant capacities of petai seeds and pods found by radical scavenging assays while there was 19 -fold difference in the antioxidant capacities by FRAP assay. There was no vitamin C in pods which is in contrast to the seeds. Similar to seeds the TAC of pods also was found to correlate well with the TPC. This shows the contribution of phenolic compounds to the TAC. HPLC analysis shows many active antioxidant compounds. Several possible antioxidant compounds were identified in pods by LC-MS. x Suvachittanont W. and Pensook J. 2000. Mitogenic effect of parkia speciosa seeds Lectin on Human Lymphocytes. Planta Med., 66, 699-704. 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Sci., 307, 284-292. 149 APPENDIX 15 45 AEACABTS ( mg / 100 g ) 900 30 60 700 500 300 100 20 40 60 80 100 T (°C) Figure Effect of temperature on extraction of antioxidants from petai pods at different temperatures 1.6 1.4 1.2 A 0.8 0.6 0.4 0.2 0 500 1000 1500 2000 t (s) Figure Decay of ABTS+ radicals on addition of petai pod extract 150 1.4 1.2 A517 0.8 0.6 0.4 0.2 0 20 40 60 t (min) Figure Decay of DPPH radicals on addition of petai pod extract 1.8 1.6 1.4 A 1.2 0.8 0.6 0.4 0.2 0 50 100 150 t (min) Figure Increase in absorbance on addition of petai pod extract to FRAP reagent 151 0.35 0.3 0.25 A 0.2 0.15 0.1 0.05 0 20 40 60 t (min) 80 Figure Increase in absorbance at 765 nm on addition of petai pod extract to folin reagent AMAR APR8 SEEDS #127 RT: 5.49 AV: NL: 4.27E6 F: + c ESI Full ms [ 150.00-2000.00] 288.07 4200000 4000000 3800000 3600000 184.17 3400000 3200000 3000000 2800000 2600000 262.12 Intensity 2400000 2200000 2000000 1800000 508.73 1600000 304.20 1400000 629.84 1200000 1000000 1227.78 630.97 800000 476.10 359.36 425.63 600000 688.82 1132.37 792.09 931.90 1040.65 1229.35 1423.09 1518.31 1557.32 830.53 1730.91 1814.81 400000 1957.31 200000 200 400 600 800 1000 1200 wavelength (nm) 1400 1600 1800 Figure ESI-MS spectra of pseudo molecular ionic compound 152 2000 AMAR APR8 SEEDS #451 RT: 19.85 AV: NL: 1.55E6 F: + c ESI Full ms [ 150.00-2000.00] 519.11 1500000 1400000 1300000 312.98 1200000 1100000 1000000 224.25 Intensity 900000 800000 700000 600000 541.71 328.95 500000 482.98 557.01 300000 823.62 675.63 400000 906.98 1004.44 710.41 561.11 1123.43 1126.21 1272.03 1308.79 1506.84 1421.70 200000 1556.60 1617.61 1766.31 1826.48 100000 1954.16 1837.57 200 400 600 800 1000 1200 wavelength (nm) 1400 1600 1800 2000 Figure ESI-MS spectra of pseudo molecular ionic compound AMAR AP R S E E D S # 1 R T: .5 F: + c E S I Fu ll m s [ .0 -2 0 .0 ] AV: N L : .6 E 6 1 .0 5500000 5000000 4500000 4000000 3500000 Intensity 3000000 2500000 2000000 1500000 3 .2 1000000 500000 .2 .7 2 .3 4 .9 .9 .6 .6 .9 1 .7 .4 .3 1 .8 .5 .1 2 .6 .2 .7 .3 .7 8 .2 9 .0 200 400 600 800 1000 1200 w a ve le n g th (n m ) 1400 1600 1800 Figure ESI-MS spectra of pseudo molecular ionic compound 153 2000 AMAR APR8 SEEDS #1335 RT: 59.08 AV: NL: 2.18E6 F: + c ESI Full m s [ 150.00-2000.00] 351.10 2100000 2000000 1900000 1800000 1700000 1600000 1500000 1400000 472.95 1300000 1092.74 Intensity 1200000 1100000 468.00 1000000 900000 800000 412.83 312.92 249.04 918.31 1411.17 1123.21 880.57 599.60 192.25 1062.00 1099.47 1034.95 753.34 516.87 700000 600000 930.65 1197.42 602.09 500000 1434.39 1259.39 1287.18 1635.74 1477.29 1603.75 400000 1745.20 1678.71 1915.04 1990.02 1813.56 300000 200000 100000 200 400 600 800 1000 1200 wavelength (nm ) 1400 1600 1800 2000 Figure ESI-MS spectra of pseudo molecular ionic compound AMAR AP R S EE D S # R T: .7 AV: N L : .7 E F: + c ES I Fu ll m s [ .0 -2 0 .0 ] .0 1700000 1600000 1500000 1400000 1300000 1200000 .2 1100000 Intensity 1000000 900000 800000 700000 .8 6 .4 .2 .5 .9 600000 5 .0 .9 500000 .2 .2 400000 8 .9 1 .3 8 .7 9 .3 .6 .9 .2 .7 .8 300000 .4 200000 .4 9 .3 .3 .2 .0 .7 100000 200 400 600 800 1000 1200 w a ve le n g th (n m ) 1400 1600 1800 Figure 10 ESI-MS spectrum of pseudo molecular ionic compound 154 2000 AMAR APR SEED S #1 49 R T: .46 AV: N L: 7.88 E6 F: + c ESI Full m s [ 50 .00 -2 00 0.0 0] 32 9.95 75 00 00 70 00 00 65 00 00 60 00 00 55 00 00 50 00 00 65 .75 Intensity 45 00 00 40 00 00 62 .2 35 00 00 30 00 00 33 3.9 25 00 00 34 8.15 20 00 00 68 0.82 19 1.94 15 00 00 51 .9 1.0 46 2.80 10 00 00 74 6.4 16 .5 72 .02 52 0.10 50 00 00 11 13 .3 92 .85 67 .81 26 .37 79 .73 42 7.45 79 0.92 15 95 .66 93 5.5 18 41 .5 2 00 00 60 00 10 00 12 0 w a velen gth (n m ) 40 16 00 80 20 00 Figure 11 ESI-MS spectrum of pseudo molecular ionic compound AMAR APR SEED S #2 51 R T: 0.9 AV: N L : 2.3 2E7 F: + c ESI Full m s [ 50 .00 -2 00 0.0 0] 79 .9 30 00 00 20 00 00 10 00 00 00 00 00 90 00 00 80 00 00 70 00 00 60 00 00 50 00 00 40 00 00 Intensity 30 00 00 20 00 00 10 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 58 .68 82 .02 00 00 00 28 4.08 78 2.12 02 .04 67 .1 25 5.07 66 2.8 63 3.8 45 .2 79 6.69 2.9 11 81 .7 12 66 .27 11 02 .9 13 45 .25 15 43 .1 65 8.1 17 87 .9 18 98 .6 20 40 00 80 10 0 20 w a ve le ng th (nm ) 14 00 16 00 80 Figure 12 ESI-MS spectrum of pseudo molecular ionic compound 155 20 00 AMAR AP R S EE D S # R T: .8 AV: N L : .2 E F: + c ES I Fu ll m s [ .0 -2 0 .0 ] 3 .8 1200000 3 .0 1150000 1100000 1050000 1000000 950000 900000 850000 800000 .9 750000 Intensity 700000 650000 .7 .9 600000 550000 500000 .5 450000 .8 .4 .9 400000 8 .5 9 .7 350000 300000 1 0 .9 .3 .7 2 .5 .3 4 .8 .1 9 .3 1 .2 .9 .1 6 .5 250000 .3 200000 .1 150000 .2 100000 .4 50000 200 400 600 800 1000 1200 w a ve le n g th (n m ) 1400 1600 1800 2000 Figure 13 ESI-MS spectra of pseudo molecular ionic compound AMAR APR8 SEEDS #963 RT: 42.53 AV: NL: 4.37E6 F: + c ESI Full m s [ 150.00-2000.00] 565.03 4200000 4000000 3800000 3600000 3400000 3200000 3000000 2800000 2600000 Intensity 2400000 2200000 2000000 1800000 1600000 1400000 208.07 587.16 1200000 1000000 295.95 800000 314.98 600000 683.06 390.80 526.93 825.97 804.01 400000 879.25 1224.10 1054.86 938.82 1111.98 1339.09 1503.96 1590.93 1233.68 1427.87 1636.35 1743.51 1841.96 200000 1922.51 200 400 600 800 1000 1200 wavelength (nm ) 1400 1600 1800 Figure 14 ESI-MS spectrum of pseudo molecular ionic compound 156 2000 AMAR AP R S E E D S # R T: .0 F: + c E S I Fu ll m s [ .0 -2 0 .0 ] AV: N L : .3 E .9 7000000 6500000 6000000 5500000 5000000 4500000 Intensity 4000000 3500000 3000000 2500000 2000000 1500000 3 .1 .1 1000000 500000 6 .5 2 .1 .0 .8 6 .1 6 .4 7 .1 0 .1 5 .7 .6 1 .5 .4 .4 .2 .2 .6 .6 8 .3 8 .8 .3 200 400 600 800 1000 1200 w a ve le n g th (n m ) 1400 1600 1800 2000 Figure 15 ESI-MS spectrum of pseudo molecular ionic compound AMAR APR4 POD_040405102830 #113 RT: 4.97 AV: NL: 1.05E6 F: + c ESI Full m s [ 150.00-2000.00] 304.18 1000000 950000 900000 850000 800000 750000 700000 650000 Intensity 600000 550000 500000 450000 400000 350000 300000 250000 200000 150000 286.40 251.99 204.22 351.04 445.14 373.75 484.00 593.66 100000 50000 748.07 816.89 966.90 1025.87 1270.27 1328.05 1451.15 1526.48 1651.25 1371.88 1782.81 1913.13 1999.28 200 400 600 800 1000 1200 wavelength (nm ) 1400 1600 1800 Figure 16 ESI-MS spectrum of pseudo molecular ionic compound 157 2000 AMAR APR4 POD_040405102830 #707 RT: 31.16 AV: NL: 4.15E6 F: + c ESI Full m s [ 150.00-2000.00] 763.00 4000000 3800000 3600000 3400000 3200000 3000000 2800000 2600000 Intensity 2400000 2200000 2000000 764.02 1800000 1600000 785.00 1400000 1200000 1524.79 1000000 800000 600000 400000 200000 786.41 1547.94 425.10 236.86 304.92 409.29 442.91 459.05 1066.89 636.95 746.17 876.01 954.88 1174.88 1279.87 1419.97 1548.98 1851.79 1914.87 1969.11 200 400 600 800 1000 1200 1400 1600 1800 2000 m /z Figure 17 ESI-MS spectrum of pseudo molecular ionic compound AMAR APR4 POD_040405102830 #1626 RT: 71.29 AV: NL: 1.68E6 F: - c ESI Full m s [ 150.00-2000.00] 535.49 1600000 256.47 1500000 1400000 1300000 1200000 513.47 1100000 Intensity 1000000 900000 800000 700000 600000 500000 227.54 257.37 400000 300000 200000 349.45 484.45 387.15 100000 814.45 512.47 297.35 617.10 658.18 855.87 805.18 1093.15 907.59 1060.50 1149.10 1265.26 1335.34 1436.35 1679.96 200 400 600 800 1000 1200 1400 1600 1831.77 1800 m /z Figure 18 ESI-MS spectrum of pseudo molecular ionic compound 158 1959.43 2000 AMAR AP R P O D _ 4 # R T: .0 F: + c E S I Fu ll m s [ .0 -2 0 .0 ] 3 .0 AV: N L : .0 E 400000 380000 360000 340000 320000 300000 280000 3 .9 260000 240000 Intensity 220000 200000 180000 160000 .9 140000 120000 100000 3 .5 7 .0 .1 3 .4 .0 80000 .3 60000 .4 5 .4 7 .1 .5 .7 40000 1 .8 7 .5 .8 1 .7 .1 .0 8 .8 .8 .8 7 .6 5 .7 .3 20000 200 400 600 800 1000 1200 1400 1600 1800 m /z Figure 19 ESI-MS spectrum of pseudo molecular ionic compound 159 2000 Presentations / Conferences 1. Effect of temperature on the antioxidant activity of petai. Poster presentation, 2nd Asia pacific conference on anti-ageing medicine, Singapore. 2. Identification of antioxidants in petai, Poster presentation, RCYConference, Malaysia. 3. Analysis of active antioxidant compounds in petai. Poster presentation, Singapore International Chemical Conference - 3, Singapore. 4. Phenolic profile and antioxidant activity of seeds and pods of petai. Poster presentation, 3rd Asia pacific conference on anti-aging medicine, Singapore. 5. Antioxidant potential of the extract of petai pods. APRU Doctoral Conference, Sydney, Australia.(Abstract Submitted) 6. Identification of antioxidants in petai. Proceedings, RCYC 2004. 7. Studies on antioxidant potential of petai (Parkia Speciosa). Worldnutra 2004, San Francisco, USA. 8. Evaluation of antioxidant properties of petai. Publication. In preparation. 160 161 [...]... 1.25 A typical graph used in the calculation of TRAP value by 35 monitoring peroxidation reaction of a sample (plasma) and trolox Figure 1.26 Photograph of fresh petai Figure 3.1 Decay of ABTS+ radicals on addition of fresh petai seed 40 59 extract Figure 3.2 Rate of absorbance drop on addition of petai extract to 59 + ABTS radicals Figure 3.3 Calibration curve of L-ascorbic acid with ABTS assay 60... extract Figure 3.9 Total antioxidant capacity of petai extract in AEAC and 71 GAE values by ABTS and DPPH assays Figure 3.10 Increase in the absorbance at 593 nm on addition of petai 74 extract to FRAP solution xiii Figure 3.11 Increase in absorbance at 765 nm on addition of petai 76 extract to folin reagent Figure 3.12 AEAC / GAE values of petai extract with FRAP and 77 Folin assay Figure 3.13 Increase... synthetic antioxidants Natural antioxidants are extracted from plant and animal sources Synthetic antioxidants are prepared synthetically in the laboratory 1.3.1 Natural antioxidants Natural antioxidants such as tocopherols and vitamin C can act as primary antioxidants and are efficient radical scavengers, other naturally occurring antioxidants such as thiols, sulfides, free amino groups of proteins, carotenoids... tertiary butyl hydroquinone Figure 1.6 Structures of some artificial antioxidant compounds 13 1.4 Different types of antioxidants Based on the mechanism of reactions, antioxidants are classified into primary antioxidants, secondary antioxidants, and synergistic antioxidants 1.4.1 Primary antioxidants or chain breaking antioxidants Chain breaking antioxidants scavenge radicals, inhibit chain initiation,... their reaction with radicals is the same as that of phenolic antioxidant compounds, i.e they act as chain breaking antioxidants and involve transfer of a hydrogen atom or an electron to radicals Antioxidants of this category such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) stabilize by delocalization of electrons after the donation of a hydrogen atom (Figure 1.4) They form stable... absorbance at 412 nm on addition of petai 79 extract to Ellman’s reagent Figure 3.14 Correlation between GAE values by ABTS assay and 82 Folin assay Figure 3.15 Correlation between GAE values by DPPH and Folin 82 assay Figure 3.16 Correlation between the GAE values by FRAP and Folin 83 assay Figure 3.17 Correlation between the GAE values by ABTS assay and 84 concentration of thiols by Ellman’s assay... This class of antioxidants can react with ROS either by single electron transfer or hydrogen atom transfer (Ou et al., 2002) 1.4.1.1 Important reactions of primary antioxidants Primary antioxidants (AH) can react with free radicals [e.g lipid radicals (L•)] and form stable antioxidant radicals (A ) (Equation 1.2) This delays the initiation of free radicals AH + L• → A + LH (Equation 1.2) 14 They react... important information regarding its antioxidants nature In this chapter a detailed discussion about the biological effects of radicals, their generation, different types of antioxidants, methods used for measuring antioxidant activities, and importance of petai will be discussed in detail 1.3 Antioxidants classification based on their sources Antioxidants can be classified into two classes as natural or synthetic... peroxy or alkoxy radicals (Equation 1.3 and Equation 1.4) and prevent their reaction with lipids (Rajalakshmi and Narasimhan, 1996) AH + LOO• → A + LOOH (Equation 1.3) AH + LO• → A + LOH (Equation 1.4) 1.4.1.2 Important primary antioxidant compounds There are several naturally available primary antioxidant compounds The reaction mechanism of these compounds with radicals and their oxidation products... Figure 3.23 Overlaid spectra of the pure vitamin C and that identified 91 in extract Figure 3.24 Chromatogram of the petai extract without the addition 92 of DPPH radical solution Figure 3.25 Chromatogram of the petai extract after the addition of 93 DPPH radical solution Figure 3.26 AEAC and GAE values of petai pod extract by different 97 xiv assays Figure 3.27 The AEAC values of petai seeds and pods using . electron transfer TAA Total antioxidant activity. TAC Total antioxidant capacity. TEAC Trolox equivalent antioxidant capacity TRAP Total radical absorption power TRAP Total radical-trapping antioxidant. cooperation is appreciated. Thanks to my uncles (Gopi, Mahendra, Narasimha, Ravindranath, Nagabhushana Verma and Vijayasimha), my cousins (Bharadwaja, Ravi, Meena, Yathi, Vishnu, Vasudha, Bhargavi,. temperature on the extraction 66 Figure 3.8 Decay of DPPH radicals at 517 nm on addition of petai extract 70 Figure 3.9 Total antioxidant capacity of petai extract in AEAC and GAE values