UNIVERSITI PUTRA MALAYSIA

BIOLOGICAL ACTIVITIES OF BARRINGTONIA RACEMOSA (LECYTHIDACEAE) AND THE QUANTITATIVE ANALYSIS OF

PHENOLIC ACIDS AND FLAVONOIDS PRESENT IN THE VARIOUS BIOACTIVE EXTRACTS OF THE SPECIES

NURUL MARIAM HUSSIN

FBSB 2009 24

BIOLOGICAL ACTIVITIES OF BARRINGTONIA RACEMOSA (LECYTHIDACEAE) AND THE QUANTITATIVE ANALYSIS OF PHENOLIC

ACIDS AND FLAVONOIDS PRESENT IN THE VARIOUS BIOACTIVE EXTRACTS OF THE SPECIES

By

NURUL MARIAM HUSSIN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science

October 2009

DEDICATION

With love to all my family including my mother Ku Nadzrah Ku Aziz, my father Hussin

Mohd. Hanif, my sisters (Nurul Huda, Nurul Ashikin, Nurul ‘Ain Sakina, Nurul Suraya,

and Nurul Aiman), for enduring, with patience and understanding. Also not forgetting

my lovely friends (Nurazah Zain, Dayana Wazir, Rudi Hendra and to my entire lab

mates in lab 230) for their patience and guidance.

ii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

BIOLOGICAL ACTIVITIES OF BARRINGTONIA RACEMOSA (LECYTHIDACEAE) AND THE QUANTITATIVE ANALYSIS OF PHENOLIC

ACIDS AND FLAVONOIDS PRESENT IN THE VARIOUS BIOACTIVE EXTRACTS OF THE SPECIES

By

NURUL MARIAM HUSSIN

October 2009

Chairman : Radzali Muse, PhD

Faculty : Biotechnology and Biomolecular Sciences

Barringtonia racemosa (Family Lecythidaceae) posses several bioactivities and is used

in traditional medicine of Malaysia. However, its antioxidant, antimicrobial, anti-

inflammatory and allelopathic activities investigation are still lacking. The aims of this

study were therefore to determine those biological activities of methanolic, ethanolic and

hot water leaf, stem and bark extracts of B. racemosa and quantitative analysis of

phenolic acids and flavonoids present in the various bioactive extracts of the species.

The methanolic and ethanolic extracts of bark showed strong free radical scavenging

activity (89.23 ± 0.40 and 91.35 ± 0.89 %) and this was even higher than butylated

hydroxytoluene (BHT), ascorbic acid and α-tocopherol (antioxidant standards). The

results also showed that all methanolic extracts (leaf, stem, and bark; 84.47 ± 0.05, 84.41

± 0.28 and 84.03 ± 0.03 %) and ethanolic extracts of leaf and bark (82.58 ± 0.06 and

88.21 ± 0.06 %), respectively had similarly strong reducing power when compared to

the antioxidant standards, tested in this study. The leaf of methanolic and ethanolic

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extracts showed strong antioxidant activity (80.03 ± 5.56 and 88.93 ± 8.32 %) in the β-

carotene bleaching assay which were higher than the antioxidant standards. Although

most of the hot water extracts showed weak to moderately antioxidant activities, the hot

water extract of the leaf surprisingly showed strong reducing power potential (83.45 ±

0.07 %). Overall, the methanolic and ethanolic extracts of B. racemosa had effective

antioxidant activities compared to hot water extracts. In Griess assay, all the B.

racemosa extracts showed no significant activity on nitric oxide inhibition in RAW264.7

cell line. However, the extracts do not seem to be cytotoxic. In antibacterial assay, B.

racemosa aerial parts extracts showed weak to moderate inhibitory activities against

both Gram-positive and Gram-negative bacteria tested accept for boiling water extracts

which was inactive. For antifungal assay, methanolic extracts of B. racemosa leaf, stem

and bark exhibited the higher inhibitory activity among the four of seven fungi tested.

Among different fungi tested, all extracts were found to be more sensitive to Fusarium

sp. compared to the others. The seed germination assay results showed that B. racemosa

extracts were able to effect the germination of the Brassica nigra L. (allelopathic

activity). In the present study, quantitative analysis of phenolic compounds from B.

racemosa leaf, stem and bark were performed using reversed-phase high performance

liquid chromatography (RP-HPLC) technique. The results showed that gallic acid and

naringin were the major phenolic acids and flavonoids present in all different aerial parts

of B. racemosa. Ferulic acid, rutin, kaempferol and luteolin were also detected in several

aerial parts of B. racemosa. Overall results obtained from the biological assays

suggested that B. racemosa is a source of bioactive compounds endowed with interesting

biological activities, such as strongly antioxidants agents and can be rated as good

dietary sources of natural phenolic antioxidants.

iv

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

AKTIVITI-AKTIVITI BIOLOGI BARRINGTONIA RACEMOSA (LECYTHIDACEAE) DAN ANALISA KUANTITATIF ASID FENOL DAN

FLAVONOID YANG HADIR DI DALAM PELBAGAI EKSTRAK BIOAKTIF SPESIES

Oleh

NURUL MARIAM HUSSIN

Oktober 2009

Pengerusi : Radzali Muse, PhD Fakulti : Bioteknologi dan Sains Biomolekul

Barringtonia racemosa (keluarga Lecythidaceae) memiliki pelagai bioaktiviti dan

digunakan di dalam perubatan tradisional di Malaysia, walaubagaimanapun kajian

terhadap aktiviti antioksida, antimikrob, anti-radang dan allelopatik masih lagi kurang

dan tidak mendalam. Oleh itu, objektif utama kajian ini adalah untuk menentukan

aktiviti-aktiviti biologi tersebut dalam ekstrak metanol, etanol dan air panas daun,

ranting dan kulit kayu B. racemosa dan menjalankan ujian analisa kuantitatif asid fenol

dan flavonoid yang hadir di dalam pelbagai ekstrak bioaktif spesies tersebut. Ekstrak

metanol dan etanol pada bahagian kulit kayu menunjukkan kekuatan yang tinggi dalam

akitiviti pemerangkapan radikal bebas (89.23 ± 0.40 dan 91.35 ± 0.89 %), malah

keputusan tersebut adalah lebih tinggi dari hidroksitoluena butilat (BHT), asid askorbik

dan α-tokoferol. Keputusan juga menunjukkan kesemua ekstrak metanol (daun, ranting

dan kulit kayu; masing-masing 84.47 ± 0.05, 84.41 ± 0.28 and 84.03 ± 0.03 %) dan

v

ekstrak etanol pada daun dan kulit kayu (masing-masing 82.58 ± 0.06 and 88.21 ± 0.06

%) mempunyai kekuatan aktiviti kuasa penurunan yang hampir sama dengan kesemua

antioksidan piawai yang diuji. Dalam uji kaji aktiviti pelunturan β-kerotina, ekstrak

metanol dan etanol pada daun menunjukkan aktiviti yang sangat kuat (80.03 ± 5.56 and

88.93 ± 8.32 %) di mana lebih tinggi dari antioksidan piawai. Walaupun kebanyakan

ekstrak air panas menunjukkan aktiviti yang sederhana dalam kesemua uji kaji

antioksidan, salah satu daripada ekstrak air panas iaitu pada daun telah menunjukkan

kekuatan kuasa penurunan yang amat tinggi (83.45 ± 0.07 %). Secara keseluruhannya,

ekstrak metanol dan etanol B. racemosa mempunyai kesan yang amat efektif terhadap

aktiviti antioksidan berbanding ekstrak air panas. Dalam ujian Griess, kesemua ekstrak

B. racemosa tidak menunjukkan aktiviti yang siknifikan terhadap perencatan nitrik

oksida dalam sel RAW 264.7. Walaubagaimanapun, ekstrak tersebut tidak memberi

kesan sitotoksik pada sel. Kesemua ekstrak daun, ranting dan kulit kayu B. racemosa

menunjukkan aktiviti rencatan yang lemah hingga sederhana terhadap bacteria Gram-

positif dan Gram-negatif kecuali ekstrak air panas yang tidak menunjukkan sebarang

aktiviti rencatan terhadap kedua-dua bakteria tersebut. Dalam ujian antikulat, ekstrak

metanol pada daun, ranting dan kulit kayu B. racemosa mempamerkan activiti rencatan

yang tinggi terhadap empat jenis kulat daripada tujuh jenis kulat yang diuji kaji.

Kesemua ekstrak adalah sangat sensitif terhadap Fusarium sp. berbanding dengan lain-

lain kulat. Keputusan dari ujian germinasi biji benih juga menunjukkan bahawa ekstrak

metanol, etanol dan air panas B. racemosa berpontensi memberi kesan germinasi biji

benih Brassica nigra L. (biji benih sawi). Dalam kajian ini, analisa kuantitatif sebatian

fenol di dalam daun, ranting dan kulit kayu B. racemosa dijalankan dengan

menggunakan teknik kromatografi fasa-pembalikan HPLC. Keputusan menunjukkan

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asid galik dan naringin merupakan sebatian asid fenol dan flavonoid utama di dalam

semua bahagian arial B. racemosa (daun, ranting dan kulit kayu). Asid ferulik, rutin,

kemferol dan lutiolin juga dapat dikesan di dalam beberapa bahagian arial B. racemosa.

Secara keseluruhannya, dari keputusan yang diperolehi daripada ujian aktiviti biologi,

dapat disimpulkan bahawa B. racemosa adalah menjadi sumber sebatian bioaktif yang

mengandungi pelbagai aktiviti biologi yang istimewa, misalnya sebagai agen

antioksidan yang kuat dan mampu menjadi sumber diet berasaskan antioksidan fenolik

semulajadi.

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ACKNOWLEDGEMENTS

First and foremost, thank and praised God, for making this research a memorable one,

and for the time to finish it. I would like to express my deepest gratitude and boundless

acknowledgement to each and every one who has contributes in finalizing this thesis.

Special thanks go to Associate Professor Dr. Radzali Muse, my main supervisor, for his

patience, guidance, advice and his continuous support during this research. I also wish to

thank the other members of my supervisory committees, Dr. Syahida Ahmad and

Professor Dr. Maziah Mahmood for their valuable review and assistance to this research.

I am sincerely thankful and greatly appreciative for all of the help, encouragement,

guidance and friendship that I have gained during working with this project from the

staff of the Department of Biochemistry, Faculty of Biotechnology and Biomolecular

Sciences and research assistants of animal tissue culture laboratory, Biosciences

Institute, Universiti Putra Malaysia. I hereby to knowledge, with gratitude, Mr. Mohd

Fadhil Abd. Rahman for his help on the high performance liquid chromatography

(HPLC) techniques, Miss Mahfuzatunajla Hashim and Miss Chow Yuh Lit for their help

on the animal tissue culture techniques. Not forgetting the Government of Malaysia and

Universiti Putra Malaysia for the two years of financial support under the Pasca

Siswazah Fellowship from which I was able to complete my research work successfully.

Last but not least, I want to thank my family and friends for their love, support and

encouragement throughout my study.

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I certify that an Examination Committee has met on 13th October 2009 to conduct the final examination of Nurul Mariam Hussin on her Master of Science thesis entitled “Biological Activities of Barringtonia racemosa (Lecythidaceae) and The Quantitative Analysis of Phenolic Acids and Flavonoids Present In The Various Bioactive Extracts of The Species” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The committee recommends that the student be awarded the relevant degree. Members of the Examination Committee were as follows: Tan Soon Guan, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Khozirah Shaari, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner) Norihan Mohd Salleh, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Shaida Fariza Sulaiman, PhD Associate Professor School of Biological Sciences Universiti Sains Malaysia (External Examiner) ________________________ BUJANG KIM HUAT, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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This thesis was submitted to Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Radzali Muse, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Maziah Mahmood, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Syahida Ahmad, PhD Senior Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) ________________________________

HASANAH MOHD GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 14 January 2010

x

DECLARATION I declare that the thesis is my original work except for the quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution. ________________________ NURUL MARIAM HUSSIN Date:

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TABLE OF CONTENTS Page DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS vii APPROVAL ix DECLARATION xi LIST OF TABLES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS xviii CHAPTER

1 INTRODUCTION 1 2 LITERATURE REVIEW 2.1 Medicinal Plants 4 2.2 Malaysian Medicinal Plants 4 2.2.1 Barringtonia racemosa of ‘Putat Kampung’ 7 2.3 Phenolic Compounds

2.3.1 Phenolic Acids 2.3.2 Flavonoids 2.4 Antioxidant Activity 2.5 History of Phenolics Use In Antimicrobial Treatment 2.5.1 Antimicrobial Activity 2.6 Inflammation 2.6.1 Reports of Plant Phenolics Possessing Anti- inflammatory Activity 2.7 Allelopathic Activity

12 13 16 20 24 25

31

33 35

3 MATERIALS AND METHODS 3.1 Materials 38 3.2 Plant Materials 39 3.3 Extraction of Barringtonia racemosa aerial parts 39 3.4 Determination of Total Phenolic and Flavonoid Contents

3.4.1 Total Phenolics Assay 3.4.1 Total Flavonoids Assay

40 40 41

3.5 Determination of Antioxidant Activity of B. racemosa Extracts 3.5.1 DPPH Radical Scavenging Assay

41 41

xii

3.5.2 Reducing Power Assay 3.5.3 β-carotene Linoleic Acid Model System (β- CLAMS) Assay

42

43 3.6 Anti-inflammatory Activity

3.6.1 Sample Dilution 3.6.2 Cells Seedling and Stimulation 3.6.3 Measurement of Nitrite Formation (Griess Assay) 3.6.4 Cell Viability (MTT) Assay

44 44 44 45 46

3.7 Antimicrobial Assays 3.7.1 Preparation of Media 3.7.2 Antibacterial Activity Assay 3.7.3 Antifungal Activity Assay

47 47 47 48

3.8 Seed Germination Assay 49 3.9 Reversed-Phase High Performance Liquid

Chromatography (RP-HPLC) Method 3.9.1 Extraction of B. racemosa 3.9.2 Determination of Phenolic Compounds

50 50 51

3.10 Statistical Analysis 52

4 RESULTS AND DISCUSSION 4.1 Yield, Total Phenolic and Flavonoid Contents of B.

racemosa Extracts

54 4.2 Antioxidant Activity of B. racemosa Extracts

4.2.1 DPPH Radical Scavenging Activity 4.2.2 Reducing Power Activity 4.2.3 β-carotene Bleaching Activity

57 58 59 61

4.3 Anti-inflammatory Activity of B. racemosa Extracts 64 4.4 Antimicrobial Activity of B. racemosa Extracts 70 4.5 Allelopathic Activity of B. racemosa Extracts

4.5.1 Germination Percentage 4.5.2 Radicle Length and Seedling Weight

79 79 83

4.6 Analysis of Phenolic Acid and Flavonoid Compounds in B. racemosa by Using Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) Method

88 5 CONCLUSIONS 94

REFERENCES 97 APPENDICES 110 BIODATA OF THE STUDENT 129

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LIST OF TABLES

Table Page 1 Malaysian medicinal plants with their major traditional uses. 6 2 Phenolic compounds isolated from plant extracts and their

antioxidant activities.

22 3 Characteristics of flavonoid structure for most effective radical-

scavenging activity.

23 4 Antimicrobial activity of plant extracts. 29 5 Antimicrobial mechanism and the major sub classes of antimicrobial

compounds derived from plants.

30 6 HPLC solvent gradient elution programme. 52 7 Yield, total phenolic and flavonoid contents of hot water, methanolic,

and ethanolic extracts of Barringtonia racemosa leaf, stem and bark.

56 8 Effect of the B. racemosa extracts on NO synthesis. 69 9 In vitro antibacterial activitiesa of B. racemosa extracts. 71

10 Inhibition of mycelial growth of fungal species by the crude extracts of Barringtonia racemosa.

75

11 Effect of dilute extracts of B. racemosa leaves, stems, barks and four

phenolic standards on percentage of germination of mustard seed (Brassica nigra L.) after a week exposure time at each 4 mg/ml of concentration.

83

12 Effect of B. racemosa extracts and four phenolic standards on the radicle length, seedling weight of mustard seed (Brassica nigra L.) after one week exposure.

83

13 Contents (µg/g freeze-dried weight) of phenolic compounds in leaf, stem and bark extracts of B. racemosa.

89

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LIST OF FIGURES

Figure Page 1 The buds, flowers, fruits and leaves of B. racemosa. 8 2 Oleane-type isomeric triterpenoids from B. racemosa. 11 3 The common structure of phenolic acid. 15 4 Simplified schematic of the flavonoid pathway. 17 5 The skeleton structure of the flavones (a class of flavonoids), with

ring named and positions numbered.

18 6 The skeleton structures of main classes of flavonoids. 19 7 Free radical scavenging activity of hot water (HW), methanolic

(MeOH) and ethanolic (EtOH) extracts of leaf, stem and bark of B. racemosa, BHT, ascorbic acid and α-tocopherol by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals at concentration of 80 µg/ml.

59 8 Reducing power activity of hot water (HW), methanolic (MeOH) and

ethanolic (EtOH) extracts of leaf, stem and bark of B. racemosa, BHT, ascorbic acid and α-tocopherol using spectrophotometric detection of the Fe3+ - Fe2+ transformations at concentration of 80 µg/ml.

60 9 Inhibition of beta-carotene bleaching activity (%) of hot water (HW),

methanolic (MeOH) and ethanolic (EtOH) extracts of leaf, stem and bark of B. racemosa, BHT, ascorbic acid and α-tocopherol using β-carotene linoleate model system at the concentration of 80 µg/ml.

62

10 Nitric oxide (NO) production level (a-c) in the presence of B. racemosa methanolic extracts; basal level of nitrite concentration without IFN-γ/LPS treatment.

65

11 Nitric oxide (NO) production level (a-c) in the presence of B. racemosa ethanolic extracts; basal level of nitrite concentration without IFN-γ/LPS treatment.

66

12 Nitric oxide (NO) production level (a-c) in the presence of B. racemosa hot water extracts; basal level of nitrite concentration without IFN-γ/LPS treatment.

67

xv

13 Effect of methanolic (MeOH), ethanolic (EtOH) and hot water (HW) extracts of B. racemosa leaf at different concentration on growth of the B. nigra L..

80

14 Effect of methanolic (MeOH), ethanolic (EtOH) and hot water (HW) extracts of B. racemosa stem at different concentration on growth of the B. nigra L.

81

15 Effect of methanolic (MeOH), ethanolic (EtOH) and hot water (HW) extracts of B. racemosa bark at different concentration on growth of the B. nigra L.

82

16 B. racemosa plant aged two years old, that was taken from Pengkalan Hulu, Perak, Malaysia and then was growth at the herbs planting area, Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.

110

17 The different aerial parts of B. racemosa that were used in the present

study.

111

18 A standard curve for estimation of NaNO3 (µg NaNO3/ml) in B. racemosa extracts.

114

19 A standard curve for estimation of total phenolic concentration (µg

GAE/ml) in B. racemosa extracts.

115

20 A standard curve for estimation of total flavonoid concentration (µg rutin eq./ml) in B. racemosa extracts.

115

21 A standard curve for estimation of naringin concentration (mg

naringin/ml) in B. racemosa extracts.

116

22 A standard curve for estimation of rutin concentration (mg rutin/ml) in B. racemosa extracts.

116

23 A standard curve for estimation of kaempferol concentration (mg

kaempferol/ml) in B. racemosa extracts.

117

24 A standard curve for estimation of luteolin concentration (mg luteolin/ml) in B. racemosa extracts.

117

25 A standard curve for estimation of gallic acid concentration (mg galic

acid/ml) in B. racemosa extracts.

118

26 A standard curve for estimation of ferulic acid concentration (mg ferulic acid/ml) in B. racemosa extracts.

118

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27 HPLC profiles of phenolic acids from B. racemosa leaf extract

detected at 280 nm.

119

28 HPLC profiles of phenolic acids from B. racemosa stem extract detected at 280 nm.

120

29 HPLC profiles of phenolic acids from B. racemosa bark extract

detected at 280 nm.

121

30 HPLC profiles of flavonoids from B. racemosa leaf extract detected at 365 nm.

122

31

32

HPLC profiles of flavonoids from B. racemosa stem extract detected at 365 nm. HPLC profiles of flavonoids from B. racemosa bak extract detected at 365 nm.

123

124

33 Cidal effect (clear inhibition zone) of B. racemosa methanolic extracts (10, 20, 30 μl/disc) on Staphylococcus aureus.

125

34 Cidal effect (clear inhibition zone) of B. racemosa ethanolic extracts

(10, 20, 30 μl/disc) on Staphylococcus aureus.

125

35 Cidal effect (clear inhibition zone) of B. racemosa ethanolic extracts (10, 20, 30 μl/disc) on Proteus micrabilis.

125

36 Inhibition of mycelial growth of Ganoderma lucidum fungal species. 126

37 Inhibition of mycelial growth of Aspergillus sp. fungal species. 127

38 Inhibition of mycelial growth of Fusarium sp. fungal species. 128

xvii

LIST OF ABBREVIATIONS

A Absorbance ABTS 2,2’-azino-bis(3-ethylbenzythiazoline-6-sulphonic

acid) AlCl3 Aluminium chloride AP-1 Activator protein-1 BHA Butylate hydroxyanisole BHT Butylated hydroxytoluene HW Hot water CoA Coenzyme A CD50 Cluster of differentiation 50 Cu Cuprum Da Dalton DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPPH 1,1-diphenyl-2-picryl-hydrazyl EtOH Ethanol FCS Foetal calf serum FDW Freezed-dried weight Fe Ferrum/ion FTC Ferric thiocyanate FRAP Ferric reductant antioxidant power HCl Hydrochloric acid H202 Hydrogen peroxide HOCl Hypochlorous HOSC Hydroxyl radical scavenging capacity HPLC High performace liquid chromatography IC50 Half maximal inhibitory concentration (IFN)-γ Interferon-gamma -1β Interleukin -1β iNOS Nitric oxide synthase K3Fe(CN)6 Potassium ferricyanide L-NAME L-nitro-arginine methyl ester LPS Lipopolysaccharide MAPK Mitogen activated protein kinase MAP Mitogen activated protein MDA Malaondialdehyde MeOH Methanol mRNA Messenger ribonucleic acid MTT 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-

tetrazolium bromide NA Nutrient agar NaCO3 Sodium carbonate

xviii

xix

NaNO2 Sodium nitrate NaNO3 Sodium nitrite NaOH Sodium hydroxide NB Nutrient broth NF-κB Nuclear factor kappa-B nm Nanometer NMR Nuclear magnetic resonance NO Nitric oxide NO2- Nitrogen oxide OONO- Peroxynitrite O2

•- Superoxide •OH Hydroxyl 1O2 Singlet oxygen ONOO- Oxidant peroxynitrite ORAC Oxygen radical absorbance capacity PDA Potato dextrose agar PG Propyl gallate PGE2 Prostaglandin E2 RAW 264.7 Murine monocytic macrophage cell line RNS Reactive nitrogen species RSNO S-nitrosothiols SOD Superoxide dismutase TCA Trichloroacetic acid TFA Trifluoroacetic acid TNF-α Tumor necrosis factor-alpha TBA Thiobarbituric acid UV-B Ultraviolet-B

CHAPTER 1

INTRODUCTION

Natural phytochemicals derived from plants have been reported to possess a wide range

of biological effects, including antioxidant, anti-inflammatory and antimicrobial actions

(Ao et al. 2008; Brunet et al. 2009). Of which, phenolics were one of the most notable

groups. Among them, phenolic acids and flavonoids have been the object of the great

number of studies of their biological activities in traditional medicinal plants. The

antioxidative activity may be due to the both phenolic compounds which act as free

radical scavengers and/or metal chelators (Ao et al. 2008; Brunet et al. 2009). A positive

correlation was observed between the high phenolic content and the strong antioxidant

activity (Ao et al. 2008; Barros et al. 2007).

The search for natural antioxidants of plant origin is also being explored as an

alternative to the synthetic antioxidants, such as butylated hydroxyanisole (BHA),

propyl gallate (PG) and butylated hydroxytoluene (BHT), used in food and

pharmaceutical industries (Brunet et al. 2009; Gulcin et al. 2005) as the synthetic

antioxidants may posses some side effects and toxic properties such as carcinogenic to

human health (Manian et al. 2008). Natural antioxidants can protect the human body

from free radicals and retard the progress of many chronic diseases as well as lipid

oxidative rancidity in foods (Gulcin et al. 2004). Further more, the area of natural

antioxidants developed enormously in the past decade mainly because of the increasing

limitations on the use of synthetic antioxidants and enhanced public consumers because

they are considered safe (Madhavi et al. 1996).

Medicinal plants have been used for a large range of purposes, including medicine,

nutrition, flavourings, beverages, dyeing, repellents, fragrances, cosmetics, charms,

smoking and industrial uses. Nowadays, medicinal plant extracts are very attractive not

only in the modern phytotherapy but also for food industry (Brunet et al. 2009).

Barringtonia racemosa (Lecythidaceae) is a small tree capable of reaching 20 m with

leaves tufted at the ends of stout twigs. In Malaysia, it is commonly grow in the moist

low places, especially near the shores of back waters, lakes, rivers and the banks of

paddy field. The ethnopharmacological uses of B. racemosa indicate it to be rich source

of phytomedicine. B. racemosa is also widely used in the form of a decoction in

traditional medicine in Sri Lanka (Deraniyagala et al. 2003). The bark and leaves are

used for rat-snake bites, rat poisoning and on boils. The seeds along with other

ingredients are employed in the preparations for the treatment of itch, piles and thypoid

fever. The bark is claimed to be specific for gastric ulcers (Deraniyagala et al. 2003;

Khan et al. 2001). This species have been shown to contain polyhydroxylated

triterpenoids, saponins (Hasan et al. 2000) and phenolic compounds (Sun et al. 2006).

In view of the lack of investigation of antioxidant properties in B. racemosa, the present

study have investigated the activities of this plant extracts in inhibiting scavenging

DPPH radical, bleaching of β-carotene and potential in breaking the free radical chain by

donating a hydrogen atom and other biological activities. Antioxidants isolated from

2

3

plants also showed antibacterial, anticarcinogenic, anti-inflammatory, antiviral,

antiallergic, allelopathic, estrogenic and immune-stimulating effects (Larson, 1988).

Even though recent studies conducted internationally have confirmed the efficacy of B.

racemosa diterpenoids and triterpenoids (Hasan et al. 2000; Khan et al. 2001; Khan and

Omoloso, 2002), information on the content of phenolic acids and flavonoids content in

the plant is still lacking. These compounds have attracted considerable interest due to

their many potential health benefits and the B. racemosa possess many biological

activities that can be a new source of natural product in the future.

Objectives of The Study

1. To evaluate the biological activities of hot water, methanolic, and ethanolic

extracts of B. racemosa leaf, stem and bark using antioxidant, anti-inflammatory,

antimicrobial and seed germination assays.

2. To determine and to quantify the major phenolic compounds which are phenolic

acids and flavonoids, present in three different aerial parts of B. racemosa (leaf,

stem and bark) using reversed-phase high performance liquid chromatography

(RP-HPLC) technique.

CHAPTER 2

LITERATURE REVIEW

2.1 Medicinal Plants

The world is rich with natural and unique medicinal plants. Medicinal plants are now

getting more attention than ever because they have offer of many benefits to society or

indeed to all mankind, especially in terms of medicinal and pharmacological uses. The

medicinal value of these plants lies in the bioactive phytochemical constituents that

produce definite physiological action on the human body (Akinmoladun et al. 2007).

Medicinal plants and their chemical constituents have been used worldwide in folk

medicine as an alternative treatment of various diseases (Castelucci et al. 2007). Some

of the most important classes of bioactive phytochemical constituents are alkaloids,

essential oils, flavonoids, tannins, terpenoids, saponins, phenolic acids and many more

(Edeoga et al. 2005). These natural compounds formed the foundations of modern

prescription drugs as we know today (Krishnaiah et al. 2009).

2.2 Malaysian Medicinal Plants

Malaysian rainforest store a large number of plant species which are important as a

source of traditional medicine. From about 10 000 species of higher plants and 2000

species of lower plants available in Peninsular Malaysia, approximately 16 % are

claimed to be used for medicinal purposes (Lattif et al. 1984; Murakami et al. 2000;

Saha et al. 2004; Soepadmo, 1999). It is estimated that about 20% (3,000 species) of

angiosperma and gymnosperma plant species found in Malaysia have medicinal

properties and have been used in traditional medicine preparation for a considerable

time. Those species are from the families of Annonaceae, Apocynaceae, Araceae,

Compositae, Dioscoreceae, Ebenaceae, Euphorbiaceae, Flacourtiaceae, Lauraceae,

Leguminosae, Menispermaceae, Myrsinaceae, Myrtaceae, Rubiaceae, Rutaceae,

Simaroubaceae, Thymelaeaceae, Zingiberaceae and saveral others (Soepadmo, 1999).

Malay traditional vegetables in Malaysia (locally called ‘ulam’) comprise more than 120

species representing various families. The leaves, shoots or rhizomes of the vegetables

are eaten fresh as salad or cooked (Norhanom et al. 1999). They are consumed because

of their taste, which adds variety and flavour to the diet, as well as for their health

benefits. Nutritional studies have indicated that many of these vegetables are rich in

carbohydrates, proteins, minerals and vitamin (Abas et al. 2006). Some of the vegetables

are also claimed to have medicinal properties, such as blood cleansing, induction of

uterine contractions and prevention or cure of ailments such as diabetes, high blood

pressure, cardiovascular disease, arthritis, fever and coughs. In addition, it is also

believed that these vegetables play a vital role in lowering the incidence of cancer, as

well as the control of ageing and age-related diseases (Abas et al. 2006). Table 1

summarized some common Malaysian medicinal plants and their related traditional uses.

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