UNIVERSITI PUTRA MALAYSIA ANTIOXIDANT AND ANTI ...psasir.upm.edu.my/4894/1/FBSB_2007_12.pdfAntioxidant and Anti-inflammatory Activities Of Leaves, Calli And Cell Suspension Of Putat

UNIVERSITI PUTRA MALAYSIA

ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES OF LEAVES

CALLI AND CELL SUSPENSION OF PUTAT (BARRINGTONIA RACEMOSA)

MANDANA BEHBAHANI

FBSB 2007 12



By

MANDANA BEHBAHANI

Thesis Submitted to the School of Graduate Studies Universiti Putra Malaysia in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

September 2007

Dedicated

To my dearly beloved family for all their love supports understanding and patient

ii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in

fulfilment of the requirements for the Degree of Doctor of Philosophy

ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES OF LEAVES CALLI AND CELL SUSPENSION OF PUTAT(BARRINGTONIA RACEMOSA)

By

MANDANA BEHBAHANI

September 2007

MANDANA BEHBAHANI

Chairman Abdul Manaf Ali PhD

Faculty Biotechnology

The medicinal plant of Barringtonia racemosa (Lecythidaceae family) has been

used widely in traditional medicine for anti-inflammation and anticancer in

Malaysia The present investigation was carried out to study anti-oxidant and

anti-inflammatory effects of leaves callus cell suspension and in vitro regenerated

shoots and roots of B racemosa

The results showed that different crude extracts of fully expanded leaf extracts of

B racemosa have a very strong nitric oxide (NO) inhibitory and antioxidant

activities In the Griess assay non polar extracts such as chloroform and hexane

extracts were found to be strong inhibitors of NO at different concentrations

(25 50 100 and 200 microgml) in comparison with polar extract (ethanol extract)

iii

Calli were aseptically obtained by placing surface sterilized leaf explants on

Woody Plant Medium (WPM) supplemented with different concentrations of

24-dichlorophenoxyacetic acid (24-D) On the shoot induction medium the callus

induced on the WPM medium containing 2 mgL (wv) KIN+02 mgl (wv) IBA

and 2 mgL (wv) of KIN + 04 mgL(wv) of NAA was the most effective

providing high shoot regeneration frequency of 856 and 765 respectively

In addition the highest number of shoots produced was 82 and 63 shoots per

explant respectively in the medium containing the mentioned plant growth

regulators The rooting percentage and number of roots per shoot which achieved

on WPM medium supplemented with 3gL (wv) of activated charcoal and 08

mgL (wv) of IBA were 62 and 56 respectively 96 of the in vitro rooted

plantlets with well developed shoots and roots were survived when transferred

to soil

Results obtained from this study revealed that Bracemosa is one of the important

sources of lycopene Lycopene has long been recognized as important antioxidants

both in vivo and in vitro Lycopene level was detected at a range of 002 to 414

mgg dry weight in in vitro regenerated shoots and roots respectively Lycopene

level was also successfully detected in the callus (034 to 212 mgg dry weight)

and cell suspension cultures (018 to 068 mgg dry weight) under dark and light

conditions and the amount was lower than that produced in the intact plant tissues

However manipulating the physical conditions feeding of precursor and elicitation

managed to increase the lycopene content in cultured tissues Studies on the effects

of the medium composition show that fully strength of the basal Woody Plant

Medium and B5 containing 3 (wv) of sucrose increased the lycopene content in

both callus and cell suspension cultures The precursor-feeding studies revealed

iv

that concentrations of 3 mgL (wv) of isopentenyl pyrophosphate and 2 to 4 mgL

(wv) of Mevalonate were preferred for lycopene production The elicitor studies

exhibited that the different elicitors showed distinctive effects on lycopene

production Nevertheless casein hydrolysate at 10 and 15 mgl (wv) was found to

be the best in increasing the lycopene production in callus and cell suspension

cultures The study further concluded that there was correlation between anti-

oxidant and anti-inflammatory activities and lycopene content in callus cell

suspension and in vitro regenerated organs of Bracemosa

v

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia bagai memenuhi keperluan untuk ijazah Doktor Falsafah

ANTI-OXIDADANT DAN ANTI-PEMBEKAKAN AKTIVITI DARI DAUN KALUS DAN SEL AMPAIAN DARI PUTAT (BARRINGTONIA RACEMOSA)

Oleh

MANDANA BEHBAHANI

September 2007

Pengerusi Abdul Manaf Ali PhD

Fakulti Bioteknologi

Barringtonia racemosa (Lecythidaceae family) adalah tumbuhan perubatan yang

biasa digunakan dalam perubatan traditional untuk anti-inflamasi dan anti-kanser di

Malaysia Kajian ini dijalankan untuk mengesan aktiviti anti-oksidan dan anti-

inflamasi dari daun kalus sel ampaian dan in-vitro regenerasi organ dari akar

Bracemosa

Keputusan telah menunjukkan pelbagai ekstrak kasar dari daun B racemosa

mempunyai kesan anti-oxidan and perencatan rdquonitric oxiderdquo (NO) Dalam kajian

esai rdquoGriessrdquo ekstrak yang tidak berpolar seperti ekstract dari kloroform dan

heksan telah menunjukkan kesan perencatan terhadap NO atas kepekatan yang

berlainan (25 50 100 200 microgml) jika dibandingkan dengan ekstrak dari bahagian

berpolar (ekstrak dari etanol)

vi

Kalus diperolehi setelah daun eksplan yang sterile diletak di atas rdquoWoody Plant

Mediumrdquo (WPM) yang mengandungi pelbagai kepekatan asid

dichlorophrnoxyacetic (24-D) Dalam medium perangsangan pucuk kalus didapati

tumbuh pada medium WPM yang mengandungi 2 mgl (wv) KIN + 02 mgl (wv)

IBA dan 2 mgl (wv) KIN + 04 mgl (wv) NAA menunjukkan kesan paling baik

dalam regenerasi pucuk iaitu dengan frekuensi 856 dan 765 Tambahan pula

jumlah bilangan pucuk yang dihasilkan di dalam media yang mengandungi fito-

hormon adalah 82 dan 63 per eksplan Peratusan pembentukkan akar dan nombor

bagi bilangan akar terhadap pucuk yang diperolehi melalui medium WPM yang

mengandungi 3gL (wv) arang aktif dan 08 mgL (wv) IBA adalah 62 and 56

96 plantlet yang membentuk akar dengan perkembangan pucuk telah berjaya

hidup apabila dipindahkan ke tanah

Keputusan yang diperolehi daripada kajian menunjukkan bahawa likopin adalah

bahan komponen aktif di dalam Bracemosa Lipokin telah lama dikenai sebagai

bahan anti-oxidan secara in-vitro dan in-vivo Tahap likopin dikesan pada kadar

002-414 mgg berat kering bagi regenerasi organ secara in vitro dari pucuk dan

akar Tahap likopin juga berjaya dikesan di dalam kalus (043-212 mgg berat

kering) kultur ampaian sel (018-068 mgg berat kering) dan jumlah ini adalah

rendah berbanding jumlah yang dihasilkan di dalam ldquointactrdquo tisu tumbuhan Walau

bagaimanapun manipulasi keadaan fizikal prekursor penyuapan dan elisitasi

berjaya meningkatkan kandungan likopin di dalam tisu kultur Kajian terhadap

kesan komposisi media menunjukkan bahawa media asas iaitu basal

WPM dan B5 yang mengandungi 3 (wv) sukrosa menunjuakkan kesan yang

vii

ketara dan dapat meningkatkan kandungan likopin di dalam kedua-dua kalus dan

sel ampaian kultur Kajian penyuapan prekursor menunjukkan bahawa kepekatan 3

mgl (wv) bagi isopentenyl pyrophosphate dan 2-4 mgl (wv) mevalonate adalah

sesuai untuk penghasilan likopin Kajian elisitor dengan pelbagai elisitor

menunjukkan bahawa kesan nyata keatas penghasilan likopin Walau

bagaimanapun casein hydrolysate pada 10-15 mgL (wv) didapati adalah terbaik

untuk meningkatkan penghasilan likopin di dalam kalus atau sel ampaian kultur

Kesimpulannya anti-oxidan and anti-inflamasi aktiviti dalam B racemosa adalah

berkaitan dengan kandungan likopen di dalam kalus ampaian sel and in-vitro

regenerasi organ dari B racemosa

viii

AKNOWLEDGEMENTS

My full praise to our God for enabling me to complete my study My sincere

appreciation to my supervisor and chair person of the supervisory committee Prof

Abdul Manaf Ali who was a great source of inspiration and encouragement throughout

the period of my study

I would like to express my deep thanks to my supervisory committee members Dr

Radzali Muse Dr Noorjahan Banu Mohd Aliteen and DrIntan Safinar for their

valuable contribution and suggestions

My deepest appreciation and gratitude to my dear family members for their spiritual

financial and moral support

I cannot leave this page without expressing my appreciation to Dr Radzali Muse for

reading the draft and offered numerous suggestions leading to the improvement of this

thesis

ix

I certify that an Examination Committee met on 18th Oct 2007 to conduct the final examination of Mandana Behbahani on her Doctor of Philosophy thesis entitled Antioxidant and Anti-inflammatory Activities Of Leaves Calli And Cell Suspension Of Putat (Barringtonia Racemosa) in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981 The Committee recommends that the candidate be awarded the relevant degree Members of the Examination Committee are as follows Janna Ong Abdullah PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Parameswari ap Namasivayam PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Azizah Abd Hamid PhD Associate Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Internal Examiner) Chong Ving Ching PhD Professor Institute of Biological Science University of Malaya (External Examiner)

HAMSHER OHAMAD RAMA DILI PhD GULAM RUSUL RAHMAT ALI PhD Professor Deputy Dean School of Graduate Studies

Universiti Putra Malaysia Date

x

This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the Degree of Doctor of Philosophy The members of the Supervisory Committee are as follows Abdul Manaf Ali PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Intan Safinar PhD Lecturer Faculty of Sciences Universiti Putra Malaysia (Member) Noorjahan Banu Mohd PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Radzali Muse PhD Lecturer Faculty of Chemistry and Biomolecular Sciences Universiti Putra Malaysia (Member)

AINERIS P h AINI IDERIS PhD Professor Dean School of Graduate Studies Universiti Putra Malaysia

Date

xi

DECLARATION

I hereby declare that this thesis is based on my original work except for quotations and citations which have been duly acknowledged I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions MANDANA BEHBAHANI

Date

xii

TABLE OF CONTENTS

DEDICATION i ABSTRACT Iii ABSTRAK v ACKNOWLEDGEMENTS APPROVAL DECLARATION TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS CHAPTER 1 INTRODUCTION

2 LITERATURE REVIEW 21 Lecythidaceae Family 211 Overview on Barringtonia spp 212 Bioactive Compounds from Lecythidaceae family 22 Plant Cell Cultures As A Source Of Secondary Metabolites 221 Callus and Cell Suspension Cultures 2211 Callus Characteristics 2212 Callus Growth 222 Organ Cultures 223 Physical Factors which Affect Secondary Metabolites 224 Selection of Tissue for in vitro Culture 23 Organogenesis 231 Direct Adventitious Shoot Formation 232 Callus-Mediated Production of Adventitious Shoots 24 Factors Affecting in vitro Shoot Production and Proliferation 241 Biotic Factors 2411 Explant 2412 Genotype 242 Abiotic Factors 25 Rooting 26 Yield improvement strategies

ii iii vi ix x xi xii xvii xviii xxi 1 5 5 5 7 8 8 9 10 11 11 12 13 13 14 15 16 17 18 18 19 20

xiii

261 Medium 262 Carbon Sources 2621 Sucrose 263 Sources and Concentration of Nitrogen 264 Gelling Agent 265 Vitamins 266 Organic Supplements 267 Precursor Feeding 268 Elicitor Treatment 269 Plant Growth Regulators 2691 Auxin 2692 Cytokinins 27 Free Radicals 271 Damage to Lipids Proteins and DNA Caused by Free Radicals 272 Lipid Oxidation 28 Antioxidants 281 Natural Antioxidants

2811 Tocopherols and Tocotrienols 2812 Ascorbic acid

282 Synthetic Antioxidants 283 Prevention and Repair of Oxidative Damage 29 Inflammation 291 Pro-inflammatory Cytokines 292 Reactive Oxygen Species (ROS) in Inflammatory Process 293 NFkB In Inflammatory Process 294 Mechanism of Anti-inflammatory of Carotenoids 210 Carotenoids 211 Lycopene 2111 Lycopene Biosynthetic Pathway 2112 Genes and Enzymes of Carotenoid Biosynthesis

21121 IPP isomerase 21122 GGPP Synthase 21123 Phytoene Synthase 21124 Phytoene Desaturase and Z-Carotene Desaturase 21125 Lycopene B-Cyclase

3 GENERAL METHODOLOGY

31 Extraction of Phytochemicals Using Different Organic Solvents 32 Total Antioxidant Activity

321 Ferric Thiocyanate Method (FTC) 322 Thiobarbituric Acid (TBA) Method 323 DPPH Free Radical Scavenging Activity 324 Statistical Analysis

33 Anti-inflammatory Activity

xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61

331 Culture of RAW 2647 Cells 332 Sample Dilution

333 Griess Assay for Nitric Oxide Inhibitory Activity 334 Measurement of Nitrite

34 Plant Tissue Culture 341 Medium preparation

3411 Stock solution of medium 342 Preparation of Stock Solutions of Plant Growth Regulators 3421 24-Dichlorophenoxyacetic acid(24-D) (01mgml) 3422 Naphthalene acetic acid (NAA)(01 mgml) 3423 6-furfurylaminopurine (Kinetin) (01 mgml) 3424 Benzyl amino purine (BAP) (01 mgml) 35 Plant Material 36 Aseptic Manipulation 37 Callus Induction of Barringtonia raceomosa 38 Experimental Design and Statistical Analysis 39 Determination of Callus Growth Curve 310 Determination of Cell Viability 311 Establishment of Cell Suspension Culture 312 Culture Conditions Studied 313 Three Established Basal Media 314 Different Media Strength 315 Incubation of Cultures under Light and Dark Conditions 316 Carbon Source Supplied and Their Concentrations 317 Plant Growth Regulators (PGRs) 318 Preparation of Bio-Elicitors 319 Preparation of Lycopene Precursors 320 In Vitro Shoot Regeneration from Callus Cultures of B raceomosa 321 In Vitro Root Regeneration from Callus Cultures of B racemosa 322 Preparation of Standard Lycopene and B-carotene 323 Spectrophotometric assay 324 Analysis of Lycopene by High performance Liquid Chromatography 325 Analysis of Lycopene by Thin Layer Chromatography 326 Total Antioxidant and Anti-inflammatory Activity of Callus and in vitro Regenerated organs 4 RESULTS AND DISCUSSION 41 The Effect of Different Leaf Extracts on Nitric Oxid Production in RAW 2647 Cells 42 The Effect of Chloroform Extract on NO Production in RAW 2647 Cells 43 Total Antioxidant Activity of B racemosa 431 Ferric Thiocyanate Method (FTC) 432 Thiobarbituric Acid Method (TBA) 433 DPPH Radical Scavenging Activity 44 Conclusion

xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97

45 Callus Induction and Cell Suspension of B racemosa Derived From Young Fully Expanded Leaves 451 Introduction 452 Callus Induction of Braceomosa 453 Cell viability of Callus Cultures 454 Determination of Callus Growth Curve 455 Production of Lycopene From Bracemosa Callus Cultures Under Light and Dark Conditions 456 Effects of Different Basal Media On The Biomass and

Lyco Lycopene Production in Callus Cultures of Bracemosa 457 Effects of Media Strength on The biomass and Lycopene Production in Callus Cultures of Bracemosa 458 Effects of Different Basal Media on The Biomass and Lycopene Production in Cell Suspension Cultures of Bracemosa 459 Production of Lycopene from Bracemosa Cell Suspension Cultures under Light and Dark Conditions 4510 Conclusion 46 Yield improvement strategies 461 Effects of Different Carbon Sources on The Biomass and Lycopene Production in vitro Callus Cultures of Bracemosa 462 Effects of Sucrose Concentrations on The Biomass and Lycopene Production in vitro Callus Cultures of Bracemosa 463 Effects of Different Carbon Sources on The Biomass and Lycopene Production in vitro Cell Suspension Cultures of Bracemosa 464 Effects of Sucrose Concentrations on The Biomass and Lycopene Production in vitro Cell Suspension Cultures of Bracemosa 465 Effects of Casein Hydrolysate on the Biomass and lycopene Production in vitro Callus Cultures of Bracemosa 466 Effects of Casein Hydrolysate On the Biomass and Lycopene Production in vitro Cell Suspension Cultures of Bracemosa 467 Effects of Yeast Extract Supplied on The Biomass and Lycopene Production in vitro Callus Cultures of Bracemosa 468 Effects of Yeast Extract Supplied on The Biomass and Lycopene Production in vitro Cell Suspension Cultures of Bracemosa 469 Effects of Isopentenyl Pyrophosphate (IPP) Supplied on Callus Biomass and Lycopene Production of Bracemosa 4610 Effects of Isopentenyl Pyrophosphate (IPP) Supplied on Cell Suspension Cultures and Lycopene Production of Bracemosa 4611 Effects of Mevalonate Supplied on Callus Biomass and Lycopene Production of Bracemosa 4612 Effects of Mevalonate Supplied on Cell Suspension Cultures and Lycopene Production of Bracemosa 4613 Conclusion 47 In Vitro shoot Regeneration of Bracemosa Callus culture 471 Effects of Three Established Basal Media 48 In Vitro Root Regeneration of Bracemosa Callus culture 49 Experimental Design

xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183

410 Determination of Lycopene Content 4101 TLC Analysis 4102 HPLC Analysis 4103 Lycopene Content of Callus Cell suspension and various parts of Bracemosa Using Spectrophotometric Assay 411 The Effect of Different Extracts on NO Production in RAW 2647 Cells 412 Ferric Thiocyanate Method (FTC) 4 SUMMARY AND CONCLUSIONS REFRENCES

APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203

LIST OF TABLES TABLE 21 Free Radicals and Their Effects 22 Antioxidant Components in Food 41 Anti-inflammatory activities of leaves of Bracemosa 42 Aantioxidant activity of leaves of B racemosa 43 Percentage of callus induction (CI) of callus formed from Bracemosa leaf

explants on WPM medium supplemented with different levels of 24-D and Kin under dark condition

44 Percentage of callus induction (CI) of callus formed from Bracemosa leaf



explants on WPM medium supplemented with different levels of 24-D and BAP under dark condition


explants in WPM medium supplemented with different levels of 24-D and BAP under light condition

47 Effects of different level of auxins (24-D NAA and IBA) supplied on callus

induction in leaf explants of Bracemosa after under dark condition 48 Effects of phytohormone combination (BAP and IBA) supplied on percentage

of shoot regeneration number of Bracemosa callus cultures 49 Effects of phytohormone combination (BAP and NAA) supplied on percentage

of shoot regeneration of Bracemosa callus cultures 410 Effects of three basal media on percentage of shoot regeneration of Bracemosa

callus cultures 411 Effect of Plant Growth Regulators on root regeneration of Bracemosa 412 Lycopene contents of extracts of different organs of Bracemosa 413 Anti-inflammatory activities of different organs of Bracemosa 414 Antioxidant activity of different samples of Bracemosa

xviii

LIST OF FIGURES FIGURE 21 Steps in Lipid Auxidation 12 Structures of Ascorbic acid And Tocopherol 23 Biosynthetis of Nitric Oxide 24 Synthesis of Isopentenyl pyrophosphate and dimethylallyl pyrophosphate via

mevalonate or via deoxy xyluse-5-phosphate 25 Biosynthetic formation of lycopene from geranylgeranyl diphosphate (GGPP) 31 B racemosa seedling grown in the mixed soil placed in a plastic pot (M=2X) 41 The chloroform extract inhibited the production of NO released into the media

of LPS-activated Raw cell 42 Absorbance value of leaf extracts of B racemosa at 200 ppm concentration

using FTC method 43 The total antioxidant activity of chloroform and Ethanol extract of Bracemosa

by using FTC and TBA method 44 Scavenging activity of chloroform hexane and ethanol extracts of leaves of

B racemosa 45 The induction of callus appeared at cut edge of leaf explants of Bracemosa on

the WPM basal medium containing 2mgL 24-D 46 Bracemosa callus growth on the basal WPM medium containing 2mgL 24-D

after five weeks of culture under dark and light conditions (M=2X) 47 Growth of Bracemosa callus on basal WPM medium supplemented with

1-3 mgL (wv) of 24-D incubated at 25plusmn2ordmC under dark and light conditions 48 Growth of Bracemosa callus on basal WPM medium supplemented with 1-3

mgL (wv) of 24-D and constant concentration of 1 mgL KIN incubated at 25plusmn2ordmC under dark and light conditions

49 Growth of Bracemosa callus on basal WPM medium supplemented with

1-3 mgL (wv) of 24-D and constant concentration of 2 mgml KIN 410 Growth of Bracemosa callus on basal WPM medium supplemented with

1-3 mgL (wv) of 24-D and constant concentration of 3 mgml Kin

29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123

411 Profile of Lycopene production in leaf derived callus of B racemosa cultured in

basal WPM medium supplemented with 2mgL (wv) of 24-D

xix

126 125 130 131 134 136 142 145 149

412 Effects of different basal media WPM MS and B5 on the biomass production in callus cultures of Bracemosa under dark and light conditions

413 LLycopene production of callus of Bracemosa cultured in different basal

medium (B5 MS and WPM) supplemented with 2mgL 24-D under dark and light conditions

414 Effects of different strength of WPM B5 and MS basal media on the biomass

production in cultured callus of Bracemosa 415 Effects of media strength of WPM B5 and MS basal media on lycopene

production in cultured callus of Bracemosa 416 Effects of different basal media WPM MS and B5 on the biomass production

in cultured cell suspension of Bracemosa and lycopene production under dark and light conditions

417 Profile of Lycopene production in cell suspension of B racemosa cultured in

basal B5 media supplemented with 2mgL (wv) of 24-D under dark and light conditions

418 Effects of different carbon sources on the biomass production and lycopene

content in callus cultures of Bracemosa in the basal WPM medium supplemented with 2 mgL of 24-D under dark and light conditions

419 Effects of different concentration of sucrose ( wv) supplied on the biomass

production and lycopene content in cultured callus of Bracemosa in the WPM medium supplemented with 2 mgL of 24-D under dark and light condition


content in cell suspension cultures of Bracemosa cultured in the basal WPM medium supplemented with 2 mgL of 24-D under dark and light conditions

421 Effects of sucrose concentrations ( wv) supplied on the biomass production

and lycopene content in cell suspension cultures of B racemosa in the WPM medium supplemented with 2 mgL of 24-D under dark and light condition

150

422 Effects of casein hydrolysate on the biomass and lycopene production in calluses of Bracemosa in the WPM medium supplemented with 3 sucrose under dark and light conditions

154

423 Effects of casein hydrolysate on the biomass and lycopene production in cell suspension of Bracemosa after three weeks in the WPM medium supplemented with 3 sucrose under dark and light conditions

155

424 Effects of yeast extract on the biomass and lycopene production in calluses of Bracemosa in the WPM medium supplemented with 3 sucrose 2 mgL of 24-D and incubated at 25plusmn2ordmC under dark and light conditions

158

xx

425 Effects of yeast extract on the biomass and lycopene production in cultured cell suspension of Bracemosa after five weeks in the WPM medium supplemented with 2 mgL of 24-D under dark and light conditions

426 Effects of IPP on the biomass and lycopene production in cultured callus of

Bracemosa after five weeks in the WPM medium supplemented 2 mgL of 24-D and incubated at 25plusmn2ordmC under dark and light conditions

427 Effect of IPP on the biomass production and lycopene production in cultured

cell suspension of Bracemosa after five weeks in the WPM medium supplemented with 2 mgL of 24-D under dark and light condition

428 Effects of Mevalonate supplied on the biomass and lycopene production in

cultured calluses of Bracemosa in the WPM medium supplemented with 2 mgL of 24-D under dark and light condition


cultured cell suspension of Bracemosa in the WPM medium supplemented with 2 mgL of 24-D under dark and light conditions

430 B racemosa leaf explants derived calluses regenerated shoots when cultured on

WPM with different concentrations of 24-D and IBA after 6 weeks (M=2X) 431 Rooted shoot of Bracemosa on the medium containing 08 mgL IBA after

2 months

161 163 165 168 170 175 184 187 190 191 192 193 194

432 TLC profiles of standard lycopene fully expanded leaf extracts callus produced

under dark and light conditions in vitro regenerated shoots and in vitro regenerated roots of Bracemosa

433 HPLC chromatogram of standard lycopene spectra 434 HPLC chromatogram of standard B-carotene spectra 435 HPLC chromatogram of fully expanded leaf extract of B racemosa 436 HPLC chromatogram of callus of Bracemosa 437 HPLC chromatogram of mixture of standard lycopene and callus of

Bracemosa

xxi

xxii

LIST OF ABBREVIATIONS

mg Milligram g Gram mL Millilitre L Litre microM Micromolar Percentage ordmC Degree Celsius vv Volume per volume wv Weight per volume FW Fresh weight DW Dry weight PGR(s) Plant Growth regulator(s) NAA Naphthalene acetic acid 24-D 24-Dichlorophenoxy acetic acid IBA Indol-3-butyric acid IAA Indol-3-butyric acid BAP 6-benzyl aminopurine IPP Isopentenyl pyrophosphate MVA Mevalonate acid CH Casein hydrolysate vv Volume per volume wv Weight per volume

CHAPTER 1

INTRODUCTION

Antioxidant is a chemical substance extremely useful to humans It helps us ward off

many kinds of disease related to lungs kidneys heart cardiovascular system muscle and

brain and it helps to retard the aging process Antioxidant has the ability to prevent or

delay the formation of free radicals and lipid peroxidation in the human bodies two main

causes of human disease and aging (Galati and Brien 2004)

Antioxidant can be obtained from synthesis and natural sources Antioxidant from natural

sources include a wide variety of compounds from a wide range of classes including

plant-based antioxidants amino acids peptides and protein hydrolyzates phytates

phospholipids vitamins and enzymes (Halliwell et al 1995) In the recent year there has

been an increase in the use of plants as sources of natural antioxidants for scavenging of

free radicals (Galati and Brien 2004)

Among the plants that have been known to provide antioxidant compounds are those

belonging to Lecythidaceae Lecythidaceae is a large mainly nut-bearing family of trees

which includes well known nuts such as Barringtonia (Berkov and Tavakilian 1998)

Barringtonia comprises some 39 species of trees and shrubs These are scattered over a

wide area extending from east Africa and India over the islands of the East Indian

Archipelago and of Oceania even to Australia (Edwin 1977)

The medicinal plant of B racemosa has been used widely in traditional medicine for

anti-inflammation and anticancer in Malaysia Previous studies on some Barringtonia

species including B asiatica Bacutangul and Blanceolata have showed that most of the

species possess medicinal properties (Grosvenor et al 1995 Khan and Omoloso 2002)

The literature survey revealed that there are no scientific studies carried out regarding

antioxidant and anti-inflammatory activity of the leaves of B racemosa In this study we

attempted to study the production of antioxidant and anti-inflammatory compound in

Bracemosa using plant cell culture In relation to the study and production of plant

secondary metabolites application of plant tissue culture technique offers many

advantages compared to the intact plant (Ushiyama 1991) such as relatively easy to

grow and can be maintained under strictly controlled nutrional and environmental

conditions simple and more convenient in experimental system than intact plant

very effective way of incorporating precursor materials which are often difficult to

administer to the entire plant the uncertainties of climate and soils can be avoided

Several products were found to be accumulated in cultured cells at a higher level than

those in intact plants through optimization of cultural conditions For example

ginsenosides by Panax ginseng (Palazoacuten et al 2003) rosmarinic acid by Lavandula vera

(Pavlov and Ilieva 2006) shikonin by Lithospermum erythrorhizon (Chung et al 2006)

diosgenin by Dioscorea spp (Katarina et al 1998) ubiquinone-10 by Nicotiana tabacum

(Hartmann and Bach 2001) were accumulated in much higher levels in cultured cells

than in the intact plants

2



By

MANDANA BEHBAHANI

Thesis Submitted to the School of Graduate Studies Universiti Putra Malaysia in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

September 2007

Dedicated


ii




By

MANDANA BEHBAHANI

September 2007

MANDANA BEHBAHANI













iii













to soil













iv









v



Oleh

MANDANA BEHBAHANI

September 2007







Bracemosa







vi
























vii











viii

AKNOWLEDGEMENTS












thesis

ix




x



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2

Dedicated


ii




By

MANDANA BEHBAHANI

September 2007

MANDANA BEHBAHANI













iii













to soil













iv









v



Oleh

MANDANA BEHBAHANI

September 2007







Bracemosa







vi
























vii











viii

AKNOWLEDGEMENTS












thesis

ix




x



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2




By

MANDANA BEHBAHANI

September 2007

MANDANA BEHBAHANI













iii













to soil













iv









v



Oleh

MANDANA BEHBAHANI

September 2007







Bracemosa







vi
























vii











viii

AKNOWLEDGEMENTS












thesis

ix




x



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2













to soil













iv









v



Oleh

MANDANA BEHBAHANI

September 2007







Bracemosa







vi
























vii











viii

AKNOWLEDGEMENTS












thesis

ix




x



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2









v



Oleh

MANDANA BEHBAHANI

September 2007







Bracemosa







vi
























vii











viii

AKNOWLEDGEMENTS












thesis

ix




x



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2



Oleh

MANDANA BEHBAHANI

September 2007







Bracemosa







vi
























vii











viii

AKNOWLEDGEMENTS












thesis

ix




x



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2
























vii











viii

AKNOWLEDGEMENTS












thesis

ix




x



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2











viii

AKNOWLEDGEMENTS












thesis

ix




x



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2

AKNOWLEDGEMENTS












thesis

ix




x



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2




x



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2



Date

xi

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2

DECLARATION


Date

xii

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2

TABLE OF CONTENTS




xiii









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2









xiv

21 21 22 22 23 23 24 24 25 26 27 28 29 30 32 35 35 38 39 40 41 41 43 44 45 46 49 50 52 56 56 56 56 57 57 58 58 59 60 58 61 61





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2





xv

61 62 63 64 66 66 66 66 67 67 67 67 68 70 71 72 73 73 74 75 75 76 76 76 77 77 77 78 79 80 80 79 81 83 83 84 86 89 89 92 94 97



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2



xvi

99 99 101 113 114 121 124 128 132 135 137 138 139 143 146 147 149 153 156 159 162 164 166 169 171 172 178 178 183


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2


APPENDIX

xvii

185 185 188 195 199 202 204 207 229

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2

31 37 87 91 102 103 104 105 110 176 177 181 178 195 201 203














xviii














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2














29 35 43 49 50 63 89 93

117 118 122

34 40 48 54 55 69 88 91 93 96 111 112 117 118 119 120 123



xix

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2

126 125 130 131 134 136 142 145 149


















150


154


155


158

xx












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2












2 months

161 163 165 168 170 175 184 187 190 191 192 193 194




Bracemosa

xxi

xxii



CHAPTER 1

INTRODUCTION







































2

xxii



CHAPTER 1

INTRODUCTION







































2

CHAPTER 1

INTRODUCTION







































2






















2

Documents

UNIVERSITI PUTRA MALAYSIA ANTIOXIDANT AND ANTI ...psasir.upm.edu.my/4894/1/FBSB_2007_12.pdfAntioxidant and Anti-inflammatory Activities Of Leaves, Calli And Cell Suspension Of Putat