i

PHYSICOCHEMICAL PROFILING AND DETECTION OF PHENOLIC CONSTITUENTS WITH ANTIOXIDANT AND ANTIBACTERIAL

ACTIVITIES OF Myristica fragrans HOUTT.

by

MARDIATY IRYANI BINTI ABDULLAH

Thesis submitted in fulfillment of the requirements for the degree of Master of Science

July 2009

ii

To my late grandmother, Hajah Siti Rafeah bt Haji Ahmad

ا 234567

iii

Nutmeg in literatureNutmeg in literatureNutmeg in literatureNutmeg in literature

I had a little nut tree,

Nothing would it bear,

But a silver nutmeg,

And a golden pear.

The King of Spain’s daughter,

Came and visit me,

And all for the sake,

Of my little nut tree.

I skipped over ocean,

I danced over sea,

And all the birds in the air,

Couldn’t catch me.

iv

Bismillahirrahmanirrahim

(In the name of Allah, The Most Gracious, Most Merciful)

I am indebted to all of generous individuals for their efforts, encouragement and

kindness. I acknowledge with gratitude the assistance received from the following:

Associate Professor Dr Shaida Fariza Binti Sulaiman, my supervisor for continuous

support in the research project and for her interest and comments throughout the

project. Without her encouragement and valuable guidance, I could not have

finished this dissertation. Her passionate is my inspiration.

I am grateful to Associate Professor Dr Noryati Binti Ismail, my co - supervisor,

lecturer of School of Industrial Technology, for her generous help and assist me

whenever I needed help.

I would like to thank both Deans and staff of the School of Biological Sciences and

Institutes of Graduate Studies for giving me the opportunity to be part of the family.

I am so grateful for their kindness during the preparation of this dissertation.

Not forgotten, Mr. Shahabudin, from the Soil Science and Ecology Laboratory, who

create a safety place of work during the mineral analysis. I am also grateful to Mr.

Mutalib and Mrs. Nurul from the Microbiology Laboratory. Thanks also go to Mr.

Joseph Hemandry, who was keen to help and assist me during my working section in

the Undergraduate Laboratory, School of Industrial Technology. I would like to

v

extend my appreciation to Kak Khuzma from School of Industrial Technology for

assisting with the handling of the ultraviolet spectrometer and nutritional analysis.

I should like to express my special appreciation to Kak Loh, Kak Bing, Kak Marissa,

Kak Rozi and June, my generous senior of Phytochemical Laboratory. Also thanks

for their kindhearted that made the atmosphere in the workplace more joyful and for

the meaningful guidance and support. To my colleague, Eng Meng, Adlin, Ernawita

and Suhail you really deserve a warm appreciation from me. Allow me to say ‘thank

you’ for your courage, commenting and offering help whenever I needed help. Your

unfailing support had kept me on track to continue my works. I am so glad to be

with and working with you guys.

This thesis would not have been possible without the valuable courage given by my

parent, Tuan Haji Abdullah Bin Haji Hashim and Puan Hajah Zainab Binti Haji

Yusuf. I want to say ‘thank you’ for your continuous prayers and unconditional

supports and faith on me. Both of them who are always supporting me to come this

far and make me strong to face the future. Without them, it might not be possible for

me to come up to this level. To my sister and brother, you rock!!!.

I also acknowledge with gratitude the financial assistance, National Science

Fellowship (NSF) received from Ministry of Science, Technology and Innovation,

Malaysia.

vi

Finally, and by no means least, I also dedicate my thesis to all those who formally

and informally gave me benefit of their interest, knowledge, views and experiences.

Without all of them, I won’t be able to finish up my thesis.

Mardiaty Iryani Binti Abdullah

vii

TABLE OF CONTENT

Page

TABLE OF CONTENTS vii

LIST OF TABLES xiii

LIST OF FIGURES xv

LIST OF PLATES xviii

ABSTRAK xix

ABSTRACT xxi

CHAPTER 1 INTRODUCTION

1.1 Myristica fragrans Houtt. 1

1.1.1 Morphological description 1

1.1.2 Therapeutic values 2

1.1.3 Scientific biological and pharmacological studies 3

1.1.4 Phytochemical content 5

1.2 Nutritional aspect 6

1.3 Antioxidant activity 9

1.3.1 Oxidative damage and diseases 9

1.3.2 Phenolic compounds as natural antioxidant 10

1.4 Antibacterial activity 13

1.4.1 Diseases and antibacterial agents

1.4.2 Roles of phenolic compounds in treating bacterial diseases

13

14

1.5 Problem statement 17

1.6 Objectives of study 18

viii

CHAPTER 2 LITERATURE REVIEW

2.1 Physicochemical profiling of M. fragrans 19

2.2 Phenolic compounds of M. fragrans 20

2.3 Antioxidant properties of M. fragrans 22

2.4 Antibacterial properties of M. fragrans 24

CHAPTER 3 MATERIALS AND METHODS

3.1 Plant materials 26

3.2 Physicochemical study 26

3.2.1 Physical analysis 26

3.2.1.1 Color analysis 26

3.2.2 Chemical analysis 27

3.2.2.1 Proximate analysis 27

3.2.2.1.1 Moisture 27

3.2.2.1.2 Fat 27

3.2.2.1.3 Total ash 28

3.2.2.1.4 Crude fiber 29

3.2.2.1.5 Protein 30

3.2.2.1.6 Available carbohydrate 31

3.3.2.2 Mineral analysis 31

3.3 Extraction procedure 33

3.3.1 Acid hydrolysis 34

3.4 Antioxidant study 34

3.4.1 Folin - Ciocalteu assay 34

3.4.2 1, 1 - diphenyl - 2 - picryl hydrazyl (DPPH) radical

scavenging assay

35

3.4.3 Correlation between total phenolic content and antioxidant

activity

37

3.4.4 Identification of isolated compounds 38

3.4.4.1 Fractionation of crude extracts 38

3.4.4.1.1 Thin Layer Chromatography 38

ix

3.4.4.1.2 Paper Chromatography 39

3.4.4.1.3 Column Chromatography 40

3.4.4.1.4 Purification of fraction 40

3.4.4.2 Identification of isolated compounds 41

3.4.4.2.1 Compounds isolated from leaf extract 41

3.4.4.2.2 Compound isolated from pericarp extract 41

3.4.4.2.3 Spectrum analysis 42

3.4.4.2.4 Sugar analysis 44

3.4.4.2.4.1 Sample preparation 44

3.4.4.2.4.2 Paper Chromatography 44

3.5 Antibacterial study 45

3.5.1 Bacteria strains and culture condition 45

3.5.2 Screening test for antibacterial activity 46

3.5.2.1 Preparation of agar 46

3.5.2.2 Preparation of inoculum 47

3.5.2.3 Preparation of extracts 47

3.5.2.4 Diffusion sensitivity test 47

3.5.3 Minimum inhibitory concentration (MIC) assay 48

3.5.4 Minimum bactericidal concentration (MBC) assay 51

3.5.5 Identification of isolated compound 51

3.5.5.1 Partition procedure of 70% acetone extract of the seed 51

3.5.5.2 Fractionations of partition of seed extract 52

3.5.5.2.1 Chromatographic analysis 52

3.5.5.2.2 Column chromatography 52

3.5.5.2.3 Antibacterial activity of fractions on TLC plate 53

3.6 Comparative antioxidant and antibacterial evaluation of the active

phenolic fractions

54

3.7 Statistical analysis 54

x

CHAPTER 4 RESULTS

4.1 Physicochemical properties of M. fragrans 56

4.1.1 Physical analysis 56

4.1.1.1 Color parameter 56

4.1.2 Chemical composition 60

4.1.2.1 Proximate analysis 60

4.1.2.2 Mineral analysis 65

4.2 Antioxidant study 71

4.2.1 Screening test of various extracts of M. fragrans 71

4.2.1.1 Total phenolic content of various M. fragrans

extracts

4.2.1.2 Free radical scavenging activity of various M.

fragrans extracts

4.2.1.3 Correlation between total phenolic content and

antioxidant activity

71

75

81

4.2.2 Bioassay - guided fractionation 82

4.2.2.1 70% acetone extract of M. fragrans leaf 82

4.2.2.1.1 Fractionation of crude extract

4.2.2.1.2 Total phenolic content of fractions

4.2.2.1.3 Free radical scavenging activity of fractions

4.2.2.1.4 Refractionation of af4

4.2.2.1.5 Total phenolic content of sub - fractions

4.2.2.1.6 Free radical scavenging activity of sub -

fractions

82

84

86

91

92

93

4.2.2.2 Aglycone extract of M. fragrans pericarp 96

4.2.2.2.1 Fractionation of aglycone crude extract

4.2.2.2.2 Total phenolic content of fractions

4.2.2.2.3 Free radical scavenging activity of fractions

96

98

100

4.2.3 Identification of pure compounds 105

4.2.3.1 Compound af4i 105

4.2.3.2 Compound af4iii 107

4.2.3.3 Compound cf2 109

xi

4.3 Antibacterial study 115

4.3.1 Screening test of various extracts of M. fragrans 115

4.3.1.1 Diffusion sensitivity test of M. fragrans

extracts

4.3.1.2 Minimum inhibitory concentration (MIC) value of

M. fragrans extracts

4.3.1.3 Minimum bactericidal concentration (MBC) value of

M. fragrans extracts

115

120

121

4.3.2 Bioassay - guided fractionation

4.3.2.1 Determination of MIC and MBC values of partitions

of 70% acetone seed extract against Staphylococcus

aureus ATCC 12600

4.3.2.2 TLC chromatogram of ethyl acetate partition

4.3.2.3 Fractionation of ethyl acetate partition

4.3.2.4 Diffusion sensitivity test of fractions

4.3.2.5 Determination of MIC and MBC values of fractions

122

122

123

125

127

128

4.3.3 Bioautography screening 129

4.3.3.1 Antibacterial activity of fraction MFa10 on silica TLC

plate

4.3.3.2 Antibacterial activity of fraction MFa10 on cellulose

TLC plates

129

130

4.4 Determination of antioxidant and antibacterial activities of the active

phenolic fractions

131

4.4.1 Antioxidant activity

4.4.2 Antibacterial activity

131

134

CHAPTER 5 DISSCUSION

5.1 Physical evaluation of various parts of M. fragrans 135

5.1.1 Color analysis 135

5.2 Chemical analysis of various parts of M. fragrans 136

5.2.1 Proximate analysis 136

5.2.2 Mineral evaluation 140

xii

5.3 Total phenolic content of various M. fragrans extracts 146

5.4 Antioxidant activity 151

5.4.1 Antioxidant activity of various M. fragrans extracts 152

5.5 Correlation between total phenolic content and antioxidant activity 157

5.6 Antibacterial activity of various extracts of M. fragrans 159

5.7 Bioassay - guided fractionation 163

5.7.1 Antioxidant activity 163

5.7.2 Antibacterial activity 166

5.8 Structure elucidation 168

5.8.1 Compounds isolated from leaf of M. fragrans 169

5.8.2 Compound isolated from pericarp of M. fragrans 173

5.8.3 Compound isolated from seed of M. fragrans 175

CONCLUSION AND RECOMMENDATION FOR FUTURE RESEARCH

REFERENCES

xiii

LIST OF TABLES

Page

1.1 Comparison of constituents in M. fragrans 6

1.2 Phenolic antibacterial compounds and their mechanism of action 16

2.1 Analysis on M. fragrans 19

2.2 Total phenolic content of M. fragrans 21

2.3 Antioxidant evaluation of M. fragrans 23

3.1 Summary of serial dilution for MIC determination 49

3.2 Media and extract preparation for MIC micro - well dilution test 50

4.1 Color parameter of M. fragrans 59

4.2 Proximate composition of M. fragrans 61

4.3 Total phenolic content of various M. fragrans extracts 73

4.4 Antioxidant activity of various M. fragrans extracts and positive

controls

76

4.5 Chromatographic properties of fractions 83

4.6 Chromatographic properties of sub - fractions 91

4.7 Chromatographic properties of fractions 97

4.8 Rf values and colors of compound af4i 105

4.9 Rf values and colors of compound af4iii 107

4.10 Rf values and colors of sugar moiety of compound af4iii 108

4.11 Rf values and colors of compound cf2 109

4.12 UV - Visible spectra shifts for compound cf2 with different shift

reagents

112

4.13 The antibacterial activity of twenty four extracts of M. fragrans

against selected bacteria

116

4.14 MIC (µg/mL) value of five extracts of M. fragrans against five

bacteria

121

4.15 MBC (µg/mL) value of five extracts of M. fragrans against five

bacteria

122

4.16 MIC and MBC values (µg/mL) of partitions of 70% acetone seed 123

xiv

extract against Staphylococcus aureus ATCC 12600

4.17 Lignans identification of ethyl acetate partition of seed of M.

fragrans with n - hexane : ethyl acetate (7 : 3) as a mobile phase

124

4.18 Chromatographic properties of fractions 126

4.19 Antibacterial activity of fractions against Staphylococcus aureus

ATCC 12600

127

4.20 MIC and MBC values (µg/mL) of fractions against Staphylococcus

aureus ATCC 12600

128

4.21 Antibacterial activity of fraction MFa10 on silica TLC plate

developed using n - hexane : ethyl acetate (3 : 7)

129

4.22 Antibacterial activity of fraction MFa10 on cellulose TLC plates

developed using 50% acetic acid and Forestal

130

4.23 Antioxidant activity and total phenolic content of fraction MFa10 131

4.24 MIC and MBC values of isolated compounds against

Staphylococcus aureus ATCC 12600

134

xv

LIST OF FIGURES

Page

3.1 Overall flow chart of analyses which have been carried out in the

present study

55

4.1 The CIELAB color system 57

4.2 Comparison of major element concentrations (mg/100g dry weight)

in M. fragrans

66

4.3 Comparison of minor elements concentrations (mg/100g dry

weight) in M. fragrans

68

4.4 Gallic acid calibration curve for determination of total phenols

using Folin - Ciocalteau colorimetric assay

71

4.5 Antioxidant activities of M. fragrans extracts with the inhibition

percentage above 70% and positive controls at different

concentration defined as inhibition percentage of DPPH● in DPPH

assay

79

4.6 EC50 values of M. fragrans crude and aglycone extracts and

positive controls in DPPH free radical scavenging assay

80

4.7 Linear correlation between the DPPH free radical scavenging

activity and total phenolic content of various extracts of M.

fragrans

82

4.8 Total phenolic content of paper chromatographic fractions of M.

fragrans leaf and its crude extract, expressed as GAE (gallic acid

equivalent)

85

4.9 Antioxidant activities of 70% acetone extract of M. fragrans leaf,

its fractions and positive controls at different concentration defined

as inhibition percentage of DPPH● in DPPH assay

87

4.10 EC50 values of 70% acetone extract of M. fragrans leaf, its fractions

and positive controls in DPPH free radical scavenging assay

89

4.11 Linear correlation between the DPPH free radical scavenging

activity and total phenolic content of fractions and crude extract of

90

xvi

M. fragrans leaf

4.12 Total phenolic content of sub - fractions, fraction and crude extract

of M. fragrans leaf, expressed as GAE (gallic acid equivalent)

92

4.13 Antioxidant activities of sub - fractions, fraction and crude extract

of M. fragrans leaf and positive controls at different concentration

defined as inhibition percentage of DPPH● in DPPH assay

93

4.14 EC50 of sub - fractions, fraction and crude extract and positive

controls in DPPH assay

94

4.15 Linear correlation between the DPPH free radical scavenging

activity and total phenolic content of sub - fractions, fraction and

crude extract

95

4.16 Total phenolic content of aglycone of 70% acetone extract of M.

fragrans pericarp and its fractions, expressed as GAE (gallic acid

equivalent)

99

4.17 Antioxidant activities of aglycone of 70% acetone extract of M.

fragrans pericarp, its fractions and positive controls at different

concentration defined as inhibition percentage of DPPH● in DPPH

assay

101

4.18 EC50 values of aglycone of 70% acetone extract of M. fragrans

pericarp, its fractions and positive controls in DPPH free radical

scavenging assay

103

4.19 Linear correlation between the DPPH free radical scavenging

activity and total phenolic content of fractions and crude extract

104

4.20 Suggested structure of compound af4i, quercetin 106

4.21 Suggested structure of compound af4iii, quercetin - 3 - O -

glucoside

108

4.22 UV absorption spectrum of compound cf2 in 80% methanol and

shift reagents

111

4.23 The structure of pomiferin 113

4.24 Suggested structure of compound cf2, scandinone 114

4.25 Antibacterial activity of five extracts with the inhibition above

12mm against five bacteria

119

4.26 EC50 values and total phenolic contents of isolated compounds 132

xvii

5.1 Structural configuration of quercetin and its radical scavenging

activity

171

xviii

LIST OF PLATES

Page

1.1 Myristica fragrans Houtt. 2

4.1 Lignans identification of ethyl acetate partition of seed of M.

fragrans with n - hexane : ethyl acetate (7 : 3) as a mobile phase

124

4.2 Bioautography of fraction MFa10 against Staphylococcus aureus

ATCC 12600 developed using n - hexane : ethyl acetate (3 : 7)

129

4.3 Bioautography of fraction MFa10 against Staphylococcus aureus

ATCC 12600 developed using two solvent systems

130

xix

PEMPROFILAN FIZIKOKIMIA DAN PENGESANAN JUZUK FENOLIK

DENGAN AKTIVITI ANTIOKSIDAN DAN ANTIBAKTERIA BAGI Myristica

fragrans HOUTT.

ABSTRAK

Penyelidikan ini dijalankan untuk menilai ciri fizikal (warna) dan kimia (analisis

proksimat dan mineral) serta aktiviti antioksidan dan antibakteria ekstrak daun,

perikarpa, aril, biji, isi tempurung dan tempurung Myristica fragrans Houtt. Analisis

warna menunjukkan bahagian aril memiliki nilai a* dan C yang tinggi (28.14 ± 0.49

dan 29.92 ± 0.58, masing - masing) dengan sudut hue (ho) paling rendah (19.87 ±

0.35o), kulit perikarpa pula mencatatkan nilai b* paling tinggi (28.59 ± 0.78) dan isi

tempurung mencatatkan parameter L* paling tinggi (72.85 ± 0.16). Kandungan

lembapan adalah paling tinggi untuk semua sampel kecuali tempurung. Sementara

itu, kesemua sampel memiliki kandungan abu yang rendah kecuali bahagian daun

dan isi tempurung, di mana kandungan proteinnya lebih rendah berbanding abu.

Analisis unsur utama (Ca, Na, K, Mg) dan unsur surih (Cu, Mn, Fe, Zn)

menunjukkan kepekatan kalium (K) dan kalsium (Ca) yang tertinggi dalam perikarpa

manakala mangan (Mn) merupakan unsur surih paling tinggi dikesan. Ekstrak

mempamerkan julat kandungan fenolik total yang luas daripada 649.00 ± 2.16mg

GAE/g ekstrak kering hingga 8.66 ± 0.71mg GAE/g ekstrak kering. Aktiviti

antioksidan berbeza daripada 84.53 ± 0.89% hingga 12.57 ± 0.98%. Korelasi yang

signifikan dan positif telah direkodkan bagi kandungan fenolik total dan aktiviti

antioksidan (r2 = 0.7039, p < 0.0001). Ini menunjukkan bahawa fenolik merupakan

bahan antioksidan utama di dalam ekstrak. Sejumlah lapan fraksi diperolehi dari

xx

ekstrak krud aseton 70% bahagian daun dan af4 mempamerkan nilai terbaik bagi asai

antioksidan (nilai EC50 = 24.91 ± 0.29µg/mL) dan paling tinggi kandungan fenolik

total (526.68 ± 0.82mg GAE/g ekstrak kering). Sub - fraksi af4i pula menunjukkan

nilai terbaik untuk aktiviti antioksidan (nilai EC50 = 23.08 ± 0.61µg/mL) dan sub -

fraksi af4iii mengandungi kandungan fenolik total yang paling tinggi (579.05 ±

0.46mg GAE/g ekstrak kering). Daripada lapan fraksi dari kromatografi turus bagi

ekstrak aglikon aseton 70% bahagian perikarpa, cf2 mempamerkan nilai terbaik

untuk kedua - dua kandungan fenolik total (358.85 ± 1.32mg GAE/g ekstrak kering)

dan asai antioksidan (nilai EC50 = 38.91 ± 1.81µg/mL). Aktiviti antibakteria oleh

kesemua ekstrak adalah lebih menonjol ke atas bakteria Gram - positif berbanding

bakteria Gram - negatif. Diameter zon perencatan bagi ekstrak berada dalam julat

16.00 ± 0.00mm hingga 9.00 ± 0.00mm. MFa10 yang disisihkan daripada ekstrak

aseton 70% biji adalah juzuk yang paling poten dengan nilai MIC 37.50µg/mL dan

nilai MBC 150.00µg/mL terhadap Staphylococcus aureus ATCC 12600. Empat

komponen fenolik iaitu kuersetin, kuersetin - 3 - O - glukosida, skandinon dan

sebatian lignan yang tidak dikenalpasti telah disisihkan dan dicirikan sebagai juzuk

utama dengan aktiviti menyingkirkan radikal bebas dan antibakteria.

xxi

PHYSICOCHEMICAL PROFILING AND DETECTION OF PHENOLIC

CONSTITUENTS WITH ANTIOXIDANT AND ANTIBACTERIAL

ACTIVITIES OF Myristica fragrans HOUTT.

ABSTRACT

This study was conducted to evaluate physical (color) and chemical (proximate and

mineral analysis) characteristics as well as the antioxidant and antibacterial activity

of extracts from leaves, pericarps, maces, seeds, seed kernels and shells of Myristica

fragrans Houtt. The color analysis revealed that the mace has the highest a* and C

value (28.14 ± 0.49 and 29.92 ± 0.58, respectively) and the least hue angle (ho)

(19.87 ± 0.35o), skin of pericarp was detected to have the highest b* value (28.59 ±

0.78) and seed kernel exhibited the highest L* parameter (72.85 ± 0.16). Moisture

content was at the highest for all samples except for shell. Meanwhile, all samples

were low in ash content except for leaf and seed kernel, whereby their protein

contents were lower than ash. Analysis for the major elements (Ca, Na, K and Mg)

and for the minor and trace elements (Cu, Mn, Fe and Zn) showed the highest

concentration of potassium (K) and calcium (Ca) in the pericarp while manganese

(Mn) is the predominant microelement detected. These extracts exhibited a wide

range of total phenolic content varying from 649.00 ± 2.16mg GAE/g dry extract to

8.66 ± 0.71mg GAE/g dry extract. The antioxidant activity varied from 84.53 ±

0.89% to 12.57 ± 0.98%. Significant and positive linear correlation were recorded

for total phenolic content and antioxidant activity (r2 = 0.7039, p < 0.0001),

indicating that phenolics were the major antioxidant constituents in the extracts. A

total of eight fractions were collected from 70% acetone crude extract of the leaf and

xxii

af4 exhibited the greatest value in antioxidant assay (EC50 value = 24.91 ±

0.29µg/mL) and the highest in total phenolic content (526.68 ± 0.82mg GAE/g dry

extract). Sub - fraction af4i showed the greatest value in antioxidant activity (EC50

value = 23.08 ± 0.61µg/mL) and sub - fraction af4iii was the highest of total phenolic

content (579.05 ± 0.46mg GAE/g dry extract). From eight column chromatographic

fractions of the aglycone of 70% acetone extract of the pericarp, cf2 showed the

greatest values in both total phenolic content (358.85 ± 1.32mg GAE/g dry extract)

and antioxidant assay (EC50 value = 38.91 ± 1.81µg/mL). The antibacterial activity

of all the extracts was more pronounced against Gram - positive bacteria than Gram -

negative bacteria. The inhibition zone diameters of extracts were ranging from

16.00 ± 0.00mm to 9.00 ± 0.00mm. MFa10 that was purified from 70% acetone

extract of the seed was the most potent component with MIC value of 37.50µg/mL

and MBC value of 150.00µg/mL against Staphylococcus aureus ATCC 12600. Four

phenolic substances, which were quercetin, quercetin - 3 - O - glucoside, scandinone

and an unidentified lignan compound were purified and characterized as the main

constituents with free radical scavenging and antibacterial activities.

1

CHAPTER 1 INTRODUCTION

1.1 Myristica fragrans Houtt.

1.1.1 Morphological description

Myristica fragrans Houtt., locally known named pala in Malay is an evergreen tree

growing to a height of about 18m (Ong, 2004). It belongs to the Myristicaceae

family. The leaves are oval in shape, pinnately and alternately arranged with

fragrance odor when crush (Weiss, 2002). The aromatic flowers are pale yellow in

color and are clustered in sima umbellate inflorescence (Weiss, 2002; Ong, 2004).

M. fragrans produces drupe type fruits, pyriform in shape, 6cm to 9cm long,

yellowish skin with perpendicular groove around the fruit and whitish flesh (Weiss,

2002). The flesh is about 1.3cm thick and contributes 75% to 85% of total weight. It

splits when ripe revealing its red mace encasing its brown glossy seed (Felter &

Lloyd, 1898). Parts of M. fragrans fruit are shown in Plate 1.1.

1.1.2 The

The leaves

1999). The

The mace w

(Zaidi et a

rheumatism

beneficial e

is also usef

disease, liv

2002), flatu

the bark is a

Leaf

Skin

Mace

Flesh Seed

Plate 1.1 Myristica fragrans Houtt.

2

rapeutic values

are drunk as tea to relief flatulence and intestinal spasm (Flach & Willink,

shoots are also used medicinally to treat hypertension (Mustapa, 2008).

as also used as stomach tonic and for healing headache and migraine

l., 2009). In Indonesian folk medicine the mace is used for curing

. The seed kernel (nutmeg) is widely used as spice with possible health

ffects such as aphrodisiac, anthelmintic, anticonvulsant and antiseptic. It

ul in treating inflammation, vomiting, diarrhea, dysentery, asthma, heart

er and spleen disorder, insomnia, colic, menorrhagia (Sharma et al.,

lence, nausea and dyspepsia (Zaidi et al., 2009). A resin obtained from

pplied externally to treat polyarthritis and gout (Adams et al., 2009).

3

In addition to its medicinal uses, the sour ripe fruits are used for preparing pickles,

jams, sweets and jellies while the seed and mace are used as flavoring for fish, meat,

biscuits, cakes, sauces and soups (Ong, 2004).

1.1.3 Scientific biological and pharmacological studies

Various biological and pharmacological evaluations have been conducted to verify

the therapeutic values of M. fragrans. The ethanolic extract of the pericarp exhibited

hypolipidaemic effect by lowering the total cholesterol, low density lipoprotein

(LDL) cholesterol and triglycerides levels in the treated albino rabbits after 60 days.

In addition, the extract also showed platelet anti - aggregatory effect with no side

effects on various hematological and biochemical parameters (Ram et al., 1996).

Meanwhile, Ozaki et al. (1989) have reported the anti - inflammatory effect of

methanol extract of the mace and myristicin was detected as the active constituent.

Jannu et al. (1991) have tested the effectiveness of mace as chemotheraphy agent on

7, 12 - dimethylbenzanthracene (DMBA) - induced papillomagenesis in the skin of

male albino mice. The extract reduced 50% of papillomagenesis (as compared with

that of control (100%)). Hussain and Rao (1991) reported that at the dose of

10mg/mouse/day orally for seven days before and ninety following days, mace

exhibited good chemopreventive activity by reducing the cervical carcinoma

incidence from 73.90% (control) to 21.40%. Moreover, Kumari and Rao (1989)

reported the hepatoprotective effect of the mace based on a significant increase in

gluthathione - S - transferase (GST) and acid soluble sulfhydryl (SH) levels.

4

Another studied conducted by Sharma and colleagues (1995) demonstrated the

efficacy of seed extract as preventive agent for hypercholesterolemia and

atherosclerosis in rabbits. The extract assisted in reducing serum cholesterol, LDL

cholesterol and cholesterol/phosholipid ratio by 69.10%, 76.30% and 31.20%

respectively and also significantly elevated the decrease of high density lipoprotein

(HDL). Moreover, the extract also prevented the accumulation of cholesterol,

phospholipids and triglycerides in liver, heart and aorta, dissolved the atheromatous

plaques of aorta from 70.90% to 77.65% and increased the fecal excretion of

cholesterol and phospholipid. Furthermore, Olajide et al. (1999) found that the

chloroform extract of the seed showed anti - inflammatory activity by inhibiting the

rat paw oedema. The extract also has analgesic property by reducing writhings.

Sonavane et al. (2001) reported that the n - hexane extract of the seed has

anxiogenic, sedative and analgesic activities. The results obtained from Parle et al.

(2004) revealed that the n - hexane extract of the seed at lowest dose of 5mg/kg

administered for three days, improved learning and memory of young and aged mice.

This extract also reversed scopolamine - and diazepam - induced impairment in

learning process and memory. Goncalves et al. (2005) found that the aqueous seed

extract was able to inhibit only human rotavirus cell (HCR3) with 90.00% inhibition

at the maximum non - toxic concentration (MNTC) of 160.00µg/mL.

The crude suspension and petroleum ether extract of seed kernel possessed a good

antidiarrheal effect and sedative property, with weak analgesic effect (Grover et al.,

2002). An experimental study by Tajuddin et al. (2003) and Tajuddin et al. (2005)

were undertaken to evaluate the improving effect of 50% ethanolic extract of the

seed kernel on sexual function. The result indicated that the extract increased both

5

libido and potency, which might be attributed to its nervous stimulating property.

Apart from possessing aphrodisiac effect, the extract was observed to be devoided of

any adverse effects and acute toxicity. Meanwhile, Janssens et al. (1990) suggested

that eugenol and isoeugenol (the major components of seed kernel oil) play the major

role in the inhibiting platelet aggregation. Moreover, based on the plasma

aminotransferase activities, the seed kernel oil and myristicin showed a prominent

hepatoprotective activity (Morita et al., 2003). According to antidepressant study

conducted by Tan (2006) via tail suspension test (TST) and forced swimming test

(FST), the hexane, chloroform and 80% methanol extracts of pericarp, mace, kernel

and seed generally showed poor antidepressant effect.

Apart to its health beneficial effects on human, M. fragrans do play a role as insect

controlling agent. The results obtained from Huang et al. (1997) suggested that the

essential oil of the seed extract exhibited good grain protectant property as the oil

reduced the production of progeny of Tribolium castaneum and Sitophilus zeamais,

decreased in the percentage of eggs hatched and viable larvae after hatching at

various concentrations.

1.1.4 Phytochemical content

The identified constituents based on spectroscopic methods reported by numerous

authors were compiled in Table 1.1. As indicated in the Table, pericarp, mace and

seed kernel share constituents, such as α - pinene, β - pinene, limonene and sabinene.

Choo et al. (1999) asserted that the constituents present are similar in pericarp, mace

and seed kernel though the compositions are substantially different. As far as

6

literature survey could as certain, no report on chemical composition of essential oil

and extract of shell M. fragrans was obtained.

Plant part Constituents Leaf myristicin, quercetin and kaempferol (Suhaj, 2006). Pericarp α - pinene, β - pinene, γ - terpinene, α - terpinene, α - terpineol,

myristicin, limonene, sabinene, α - terpinolene, α - myrcene, terpinen - 4 - ol, isoeugenol, myristicin (Choo et al., 1999), ferulic acid (Wojdylo et al., 2007), caffeic acid, catechin (Shan et al., 2005), quercetin, and kaempferol (Suhaj, 2006).

Mace α - pinene, β - pinene, limonene, safrole, sabinene, lignans, neolignans (Hada et al., 1988), linoleic acid, palmitic acid, elemicin, isocroweacin, methoxyeugenol, isoeugenol (Singh et al., 2005), cyanidin, quercetin and kaempferol (Suhaj, 2006).

Seed isoeugenol, methyl-eugenol, eugenol, dihydroguaiaretic acid, γ - terpinene, terpinen - 4 - ol, myristic acid, oleanolic acid, palmitic acid, camphene, lauric acid, myrcene, quercetin and kaempferol (Suhaj, 2006)

Seed kernel α - pinene, β - pinene, sabinene, safrole, terpinen - 4 - ol, elemicin, myristicin, α - terpineol, myristicin, limonene, α - terpinene (Spricigo et al., 1999; Tomaino et al., 2005; Jukić et al., 2006), eugenol, isoeugenol (Janssens et al., 1990), neolignans (myrisfragransin) (Li & Yang, 2008), lignans (diarylbutane, 7 - methyl ether diarylbutane and aryltetralin) (Kwon et al., 2008), neolignan (myrislignan), macelignan (Chung et al., 2006).

1.2 Nutritional aspect

There are seven major classes of nutrients which are carbohydrates, fats, fiber,

minerals, proteins, vitamins, and water (Watkin, 1979). These nutrients can be

generally grouped into macronutrients and micronutrients (Whitney & Rolfes, 1996).

The macronutrients are carbohydrates, fats, fiber, proteins and water. On the other

hand, minerals and vitamins are called micronutrients. Minerals can be divided into

two groups. First group is macroelement (calcium, phosphorus, potassium, sodium

and sulfur) which are required in large quantities above 100mg/kg diet. Second

Table 1.1 Comparison of constituents in M. fragrans

7

group is microelements (ferum, cuprum, manganese, zinc, cobalt, molybdenum,

chromium, selenium, flour, iodine and nickel) which are required in small amount

below 100mg/kg diet.

Primarily, carbohydrates, proteins, and fats are metabolized to give energy. Protein

serves as the major structural component of all cells in the body, and functions as

enzymes, in membranes, as transport carriers, and as some hormones. Minerals are

essential chemical elements in human body which are involved in the formation of

skeletal structure, blood protein, enzymes and hormones, maintenance of colloidal

system and regulation of acid base equilibrium. They also act as component which

involved in enzyme activation, hemoglobin composition and lipid, amino acid and

carbohydrate metabolism (Mason, 2001).

Plants contribute notably to human nutrition and health, because they contain almost

all essential mineral and organic nutrients. Nutrient composition varies among

different plants’ parts and species (Sanchez - Castillo et al., 1998) and not all plants

contain essential nutrients needed for individual health. For instance, leafy

vegetables are good sources of most minerals and vitamins with less concentration of

protein and carbohydrates. Seeds are good sources of carbohydrates, proteins, lipids,

and lipid - soluble vitamins, but tend to have low concentrations of iron and calcium

(Grusak & DellaPenna, 1999). To ensure an adequate dietary intake of all essential

nutrients and to increase the consumption of various health - promoting plants, it is

an urgent need for researchers to quantify and compile all the nutritional information

of all food plants (Arzani et al., 2007).

8

The human body requires a number of minerals in order to maintain good health.

Malnutrition is major nutrition concern for tropical countries. Malnutrition is a

concept of nutrition disorder. The disorder may be due to excessive nutrition

(overnutrition) or deficiency nutrition (undernutrition) (McLaren, 1979). In the

developing world, many low - income families survive on a simple diet comprised

primarily of staple foods such as rice, wheat and maize that are poor in some

macronutrients and many micronutrients. As a result, 30% of the world’s population

is at risk for iron deficiency anemia (infants, children, and women at reproductive

age are particularly vulnerable) (Arzani et al., 2007). Hardisson et al. (2001)

reported that the risk of deficiencies depends on a number of factors such as the daily

dietary intake, the food content, the technical treatment of the products, the presence

of substances that limit or increase the bioavailability of minerals and the

physiological state of the food and overall health status of consumer.

The importance of optimal intakes of essential nutrient to maintain peak health is

widely recognized (Avioli, 1988). Optimal intakes of elements such as sodium,

potassium, magnesium, calcium, manganese, copper, zinc, and iodine could reduce

individual risk factors, including those related to cardiovascular disease (Mertz,

1982). It also has been recognized that some elements such as selenium could play a

protective role in decreasing the risk of some types of cancer (World Cancer Report,

1997). Thus, balance diet rich in minerals, fiber and vitamins are more than perfect

for the human health.

9

1.3 Antioxidant activity

1.3.1 Oxidative damage and diseases

Free radicals and other reactive species are waste products present in the body and

can be generated endogenously and exogenously (Gaté et al., 1999). Unhealthy

human diet containing mutagenic and carcinogenic substances and pathologic cell

metabolism also contribute to the formation of free radicals.

Free radicals are atom, molecule or mixture containing one or more unpaired electron

(Forrester et al., 1968). The species are capable to extract electron from other

molecules to stabilize the electron number, thus led to the formation of new free

radicals known as reactive oxygen species (ROS) (Stengler, 2001). Various ROS

such as singlet oxygen (1O2), superoxide radical (O-2●), hydroxyl radical (OH●) and

hydrogen peroxide (H2O2) are generated as by - products during aerobic metabolism

in cells (Gutteridge, 1994), which have the potential for bringing about extensive

damages to living cells (Darley - Usmar et al., 1995).

Within the cells, ROS will enter intercellular space and subsequently attack

oxidizable substrates such as DNA, lipids, proteins and carbohydrates (Halliwell,

1995). These will cause DNA lesions, lipid peroxidation, protein fragmentation

within the cells of biological macromolecules (Gutteridge, 1994) and subsequently

destroy membrane integrity and resulting cell lyses (Wei & Shiow, 2001). Vast

scientific reports acknowledged that the oxidative stress is an important contributor

to the pathophysiology of a variety of pathological conditions including

cardiovascular dysfunctions, atherosclerosis, carcinogenesis, inflammation,

10

neurodegenerative diseases such as such as Alzheimer’s disease, Parkinson’s disease

and Downs syndrome (Manach et al., 2004) and in natural aging processes

(Govindarajan et al., 2005).

1.3.2 Phenolic compounds as natural antioxidant

Phenolic substances are secondary metabolites compounds synthesized by plant and

derived from phenylalanine and tyrosine pathways (Shahidi & Naczk, 2004). Plant

phenolics include simple phenols, phenolic acids (both benzoic and cinnamic acid

derivatives), coumarins, flavonoid, stilbenes, hydrolysable and condensed tannins,

lignins and lignans. Structurally, phenolic compounds comprise an aromatic ring,

bearing one or more hydroxyl substituents and range from simple phenolic molecules

to highly polymerized compounds (Sakihama et al., 2002). These substances may

act as phytoalexins, pigments, antioxidants, attractant for pollinators and protective

agents against UV light (Harborne, 1967; McClure, 1975; Timberlake & Bridle,

1975; Heim et al., 2002). In food, phenolic substances may contribute to the

bitterness, astringency, flavor, odor and color (Shahidi & Naczk, 1995). Phenolic

compounds are not uniformly distributed in plant at the tissue, cellular and

subcellular levels (Maisuthisakul et al., 2008). The content in plants are differed due

to genetic and environmental factors as well as post - harvest and storage conditions

(Franke et al., 2004).

Halliwell (1995) defined antioxidant as a substance that, when present at low

concentrations compared to substrate, significantly inhibit or delay the oxidation of

substrates by inhibiting the initial or propagation of oxidation chain reactions.

11

Antioxidants are actively involved in preventing free radical damage (Seifried et al.,

2007). Antioxidants are divided into two main types according to their action.

Primary antioxidants can inhibit or delay oxidation by scavenging reactive oxygen

species. Secondary antioxidants function by binding metal ions, converting

hydroperoxides to non - radical species, absorbing UV radiations or deactivating

singlet oxygen (Gordon, 2001). Antioxidants are also believed to contribute to the

beneficial effects through stimulating the antioxidative defense enzyme activities

(Saha et al., 2004).

Among the various kinds of natural antioxidants, polyphenols have received much

attention (Luo et al., 2002). The ability of natural phenolic substances including

flavonoids and phenolic acids as antioxidant agents has been extensively investigated

(Rice - Evans et al., 1996; Shui & Lai, 2004; Kim et al., 2005; Duarte - Almeida et

al., 2007). Phenolic antioxidants in plants tend to be water soluble and most of them

appear as glycosides and they are located in the cell vacuoles (Harborne, 1998).

Dietary consumption of fruits and vegetables contain abundant of natural ROS

scavenging molecules including phenolic compounds (Shahidi & Naczk, 1995;

Aruoma, 2003). These exogenous antioxidants are required to maintain adequate

level of antioxidants in human body for disease prevention and health promotion.

These natural diets tend to be safer than synthetic antioxidant such as butylated

hydroxyl anisole (BHA) and butylated hydroxyl toluene (BHT), which are

extensively used in food processing industry (Mathew & Abraham, 2006).

Among the phenolic compounds, flavonoids are the most well - known antioxidant.

Phenolic structure provided a primer factor of antioxidant property (Rice - Evans &

Miller, 1998). The basic flavonoids structure is the flavan nucleus, which consists of

15 carbon atoms arranged in three rings (C6 - C3 - C6), labeled A, B and C. Vary in

plant antioxidant properties mostly due to hydroxylation, glycosylation, aromatic

substitution and conjugation with phenolic compounds or organic acid (Heim et al.,

2002). In addition to antioxidant properties of natural flavonoids, extensive

investigation has been done to reveal the pharmacological aspects such as

antiallergic, antiatherogenic, antiinflammatory, antimicrobial, antithrombotic,

cardioprotective and vasodilatory effects (Balasundram et al., 2006).

Natural phenolic antioxidants (NPH) hinder the oxidation process of substrates by

rapid donation of a hydrogen atom to radicals (RO●).

A stable intermediate substance, phenoxy radical (NP●) act as terminators of the

propagation route by reacting with other free radicals (Ferguson, 2001).

RO● + NPH → NP● + ROH

12

RO● + NP● → RONP

13

1.4 Antibacterial activity

1.4.1 Diseases and antibacterial agents

Throughout history, there has been a continual battle between humans and the

multitude of microorganisms that cause various types of infections and diseases. A

variety of microorganisms may also lead to food spoilage that will threaten for both

consumers and the food industries.

Cheesbrough (1984) defined antibacterial agents as any chemical substances that,

when present at certain concentration are capable to kill or inhibit the growth of

bacteria. Bacteriostatic agents prevent the growth of bacteria while bactericidal

agents are capable to kill the bacteria (Nester et al., 2007). Action of antibacterial

agents falls into four general categories; through inhibition of cell wall, protein and

nucleic acid synthesis or by disturbing the cell membrane function (Talaro & Talaro,

2002).

Multiple drug resistance has become a global concern (Westh et al., 2004) due to

indiscriminate use of commercial antimicrobial drugs in the treatment of infectious

diseases (Service, 1995). In a recent study done in New York City, up to 50% of

Streptococcus pneumonia isolates obtained from two institutions were resistant to

erythromycin (Lin et al., 2004). Resistance among bacteria genera are due to, first,

by prevention of interaction of drugs with target, secondly by effluxing of the

antibacterial agent from the cell and lastly, by modification of bacteria’s structure

(Mendonco - Filho, 2006).

14

Numerous studies have identified compounds within plants that are effective

antibacterial (Basile et al., 1997; Cowan, 1999; Basile et al., 2000; Mackeen et al.,

2000). Herbal remedies utilized in traditional healing systems around the world are

important resources for the discovery of new antibacterial compounds (Okpekon et

al., 2004).

1.4.2 Roles of phenolic compounds in treating bacterial diseases

Natural products have served as an important source of drugs since ancient times and

about half of the useful drugs today are derived from natural sources. However, the

development of bacterial resistance to the available known antibiotics, the emergence

of uncommon infections (Liu et al., 2008), the undesirable side effects of certain

antibiotics such as hypersensitivity, immunosuppression and allergic (Ahmad et al.,

1998; Sudha et al., 2001) and the acceptance of traditional medicine as an alternative

form for health care have led researchers to investigate the antibacterial activity of

medicinal plants (Mahasneh & El - Oqlah, 1999; Sahin et al., 2003; Baydar et al.,

2004; Venkat Reddy et al, 2004; Loziene et al., 2007; Adedapo et al., 2008).

According to Atlas (1984), the commercialization of antibacterial agents as

chemotherapeutic drugs is influenced by several factors such as solubility, stability,

excretion rate, allergic reaction and toxicity to cell.

The use of complementary and alternative medicine has increased dramatically. This

situation forced the scientists to validate the therapeutic values of various sources

medicinal plants thus ascertain new antimicrobial substances. For instance,

antibacterial activity of common herbal remedies of Texas: Achillea millefolium,

15

Berberis vulgaris, Commiphora molmol, Galium aparine, Glycyrrhiza glabra,

Matricaria chamomilla, Pimenta dioica, Salvia greggii, Uncaria tomentosa and Zea

mays were reported by Romero et al. (2005). Mothana and Lindequist (2005) have

screened the antimicrobial activity of extracts of 25 selected plants belonging to 19

families from the island Soqotra against several bacteria including Staphylococcus

strains. The results revealed the potential value of Punica protopunica, Boswellia

species, Commiphora parvifolia, Buxus hildebrandtii, Jatropha unicostata,

Kalanchoe farinacea and Withania species as antibacterial drugs against Gram -

positive bacteria.

Among the various kinds of secondary metabolites in plants polyphenols have

received much attention as antibacterial agents (Karamanoli, 2002). Extracts of

various medicinal plants containing flavonoids have been reported to possess

antimicrobial activity (El - Abyad et al., 1990; Singh & Nath, 1999; Cakir et al.,

2003; Sato et al., 2004). For centuries, preparations containing phenolic compounds

as the active constituents have been utilized by physicians and lay healers in attempt

to treat infectiuos diseases (Havsteen, 1983). Huang chin (Scutellaria baicalensis) is

yet a good example. This herb is believed for many thousands of years in China for

the treatment of periodontal abscesses and infected oral wounds, by applying

systemically and topically. A flavone, baicalein was reported as the antibacterial

compound of this plant (Tsao et al., 1982). A list of plants with antibacterial

phenolic compounds and its mechanism of action are shown in Table 1.2.

of a

ctio

n

s

ynth

esis

se IV

- de

pend

ent D

NA

cle

avag

e IV

- de

pend

ent d

ecat

enat

ion

activ

ity

d th

e re

lativ

ely

hom

olog

ous

gyra

se e

nzym

e ar

e in

volv

ed

ne fl

uidi

ty in

hyd

roph

ilic

and

hydr

opho

bic

regi

ons

thus

redu

ced

the

nn

er la

yers

of m

embr

anes

er

s by

dir

ectly

pen

etra

ting

them

and

dis

rupt

ing

the

barr

ier f

unct

ion

roce

ss th

at re

sults

in le

akag

e of

intr

amem

bran

ous

mat

eria

ls a

nd a

ggre

gatio

n

acte

rial

mem

bran

e

lity

of th

e ce

llula

r mem

bran

e an

d da

mag

ing

mem

bran

e fu

nctio

n m

embr

ane

dam

age

and

pota

ssiu

m le

akag

e ity

of t

he in

ner b

acte

rial

mem

bran

e an

d a

diss

ipat

ion

of th

e m

embr

ane

cter

ial m

otili

ty

y m

etab

olis

m,

mpt

ion

and

NA

DH

- cy

toch

rom

e c

redu

ctas

e m

etab

olis

m

ir m

echa

nism

of a

ctio

n (C

ushn

ie &

Lam

b, 2

005)

16 M

echa

nism

1) I

nhib

itio

n of

nuc

leic

aci

d sy

nthe

sis

Phe

nolic

com

poun

ds

Rob

inet

in, m

yric

etin

and

(-)

- epi

gallo

cate

chin

In

hibi

t DN

A a

nd R

NA

Que

rcet

in, a

pige

nin

and

3, 6

, 7, 3

', 4'

-

Inhi

bit D

NA

gyr

ase

Pen

tahy

drox

yfla

vone

Rut

in

Prom

ote

topo

isom

era

In

hibi

t top

oiso

mer

ase

Gal

angi

n T

opoi

som

eras

e IV

an

2) I

nhib

itio

n of

cyt

opla

smic

mem

bran

e fu

ncti

on

Phe

nolic

com

poun

ds

S

opho

rafl

avan

one

G a

nd n

arin

geni

n A

ltera

tion

of m

embr

a

flui

dity

of o

uter

and

i (

-) -

epig

allo

cate

chin

gal

late

Pe

rtur

b th

e lip

id b

ilay

m

embr

ane

fusi

on, a

p (

-) -

epic

atec

hin

galla

te a

nd 3

- O

- oc

tano

yl -

Act

on

and

dam

age

b (

+) –

cat

echi

n

2, 4

, 2' -

trih

ydro

xy -

5' -

met

hylc

halc

one

Cha

nge

the

perm

eabi

Gal

angi

n In

duce

s cy

topl

asm

ic

Nar

inge

nin

and

quer

cetin

In

crea

se in

per

mea

bil

po

tent

ial i

nhib

ited

ba

3)

Inh

ibit

ion

of e

nerg

y m

etab

olis

m

Phe

nolic

com

poun

ds

R

etro

chal

cone

s (l

icoc

halc

one

A a

nd C

) In

terf

erin

g w

ith e

nerg

In

hibi

t oxy

gen

cons

u L

onch

ocar

pol A

In

terf

eres

with

ene

rgy

Tab

le 1

.2 P

heno

lic a

ntib

acte

rial

com

poun

ds a

nd th

e

17

The antimicrobial mode of action is related with the phenolic compounds (Cakir et

al., 2004). It is also worth determining the antibacterial mechanism of action of

various phenolic compounds. Phenolics attack and disturb the structure of lipid

bilayers membrane by penetrating into them and disturbing the barrier function. This

may cause membrane fusion, a process that results in leakage of intramembranous

materials and aggregation (Ikigai et al., 1993), capable to change the permeability of

the cellular membrane and damaging membrane potential (Sato et al., 1997). They

also interfere with membrane function via electron transport, nutrient uptake, protein

and nucleic acid synthesis and enzyme activity (Denyer & Hugo, 1991). It is also

believed that chelation of transition reactive metals ions, such as iron and copper, by

phenolic compounds reduces bioavailability for bacterial growth (Jay, 1996).

1.5 Problem statement

None of the previous studies were ever highlighted on the physicochemical

characteristic including color parameter, proximate and mineral analyses of different

parts of M. fragrans. Even though many scientific studies have been conducted on

this plant, more emphasis was given to mace, seed and seed kernel extracts. There

have been no attempts to verify the therapeutic values of leaf, pericarp and shell

extracts. Comparative evaluation between extracts of different plant parts is also

lacking.

Due to the limited data, the aim of this study is to provide new information on the

physicochemical profiling, phenolic content, antioxidant activity and antibacterial

activity of the plant.

18

1.6 Objectives of study

The aims of the present study are as follows:

1. To quantify and compare the physicochemical properties of six parts of M.

fragrans.

2. To determine and compare the total phenolic content, free radical scavenging

activity and the antibacterial property of the extracts.

3. To correlate between total phenolic content and free radical scavenging

activity of the extracts.

4. To detect the active antioxidant and antibacterial compounds.

CHAPTER 2 LITERATURE REVIEW

2.1 Physicochemical profiling of M. fragrans

Limited data is available on physicochemical properties of M. fragrans. The only

data is on the general composition of seed kernel and mace that was obtained from

Gopalakrishnan (1992) and is presented in Table 2.1.

Bas

mac

fruc

kern

Table 2.1 Analysis on M. fragrans (Gopalakrishnan, 1992)

19

Composition Plant part Seed kernel (%) Mace (%) Moisture 40.00 40.00 Volatile oil 11.00 15.30 Non - volatile oil ether extract 33.60 21.98 Starch 30.20 44.05 Sugar Glucose 0.10 0.17 Fructose 0.07 0.10 Total reducing sugars 0.17 0.27 Sucrose 0.72 0.39 Total sugars 0.89 0.65 Protein 7.16 9.91 Crude fiber 11.70 3.93 Total ash 2.57 1.56 Ash insoluble in HCl 0.20 0.15 Polyphenols Total tannins 2.50 - True tannins 1.00 -

ed on the data in Table 2.1, the moisture content level of the seed kernel and

e were equal (40.00%). In mace, the amounts of volatile oil, starch, glucose,

tose, total reducing sugars, sucrose and protein were higher than that of the seed

el. The seed kernel has higher amount of non - volatile oil ether extract, sucrose,

20

total sugars, crude fiber, total ash and ash insoluble in acid hydrochloric (HCl).

Polyphenol was only detected in seed kernel (2.50% of total tannin and 1.00% of true

tannins).

2.2 Phenolic compounds of M. fragrans

Phenolic substances are ubiquitously distributed throughout the plant kingdom

especially in fruits and vegetables. Several studies have quantified the total phenolic

content in pericarp, mace and seed kernel of M. fragrans. The results are

summarized in Table 2.2. However, the result cannot be compared among one

another due to the different in extraction procedures and phenolic content estimation

protocols.

Various phenolic compounds have been isolated from M. fragrans. According to a

review by Suhaj (2006), quercetin and kaempferol were widely distributed in leaf,

pericarp, mace and seed. On the other hand, Shan et al. (2005) have detected the

presence of caffeic acid and catechin in the pericarp. Ferulic acid was also found in

the pericarp (Wojdylo et al., 2007). Hada et al. (1988) have isolated eight

neolignans and five lignans from the mace part. Kwon et al. (2008) reported the

presence of six diarylbutane lignans and one aryltetralin lignan in the 95% methanol

extracts of the seeds and 7 - methyl ether diarylbutane lignan was a novel compound.

Li and Yang (2008) found that myrislignan is a major acyclic neolignan in seeds.

21

Pla

nt p

art

Ext

ract

T

otal

phe

nolic

con

tent

R

efer

ence

s

Pe

rica

rp

80%

met

hano

l 1.

61 ±

0.0

0g G

AE

/100

g of

dry

wei

ght

Shan

et a

l. (2

005)

80%

met

hano

l 8.

95 ±

0.4

5mg

GA

E/1

00g

of d

ry w

eigh

t W

ojdy

lo et a

l. (2

007)

M

ace

A

ceto

ne

40m

g C

E/1

00g

of fr

esh

wei

ght

Cha

tterj

ee et a

l., (2

007)

80%

met

hano

l 1.

98g

GA

E/1

00g

dry

wei

ght

Surv

esw

aran

et a

l. (2

007)

Se

ed k

erne

l 80

% m

etha

nol

1.30

g G

AE

/100

g dr

y w

eigh

t Su

rves

war

an et a

l. (2

007)

80%

met

hano

l and

50%

ace

tone

2.

68 ±

0.1

20m

g G

AE

/g e

xtra

ct fo

r 50%

ace

tone

ext

ract

, Su

et a

l. (2

007)

2.

62 ±

0.0

1mg

GA

E/g

ext

ract

for 8

0% m

etha

nol e

xtra

ct

Met

hano

l 15

3.00

± 1

.00m

g G

AE

/g d

ry w

eigh

t H

o et al.

(200

8)

Tab

le 2

.2 T

otal

phe

nolic

con

tent

of M

. fra

gran

s

Dat

a ex

pres

sed

as g

allic

aci

d eq

uiva

lent

s (G

AE

) or c

atec

hin

equi

vale

nt (C

E).

22

2.3 Antioxidant properties of M. fragrans

Various researches were done to determine the antioxidant properties of the M.

fragrans. The results were compiled in Table 2.3. Several assays were used such as

2, 2’ - azino - bis - 3 - ethyl benzthiazoline - 6 - sulfonic acid (ABTS●+) and 1, 1 -

diphenyl - 2 - picryl hydrazyl (DPPH●) radical scavenging activities, ferric reducing/

antioxidant power (FRAP), ferric thiocyanate (FTC), thiobarbituric acid (TBA), in

vitro thiobarbituric acid reactive substances (TBARS) and oxygen radical absorbance

capacity (ORAC). The 2, 2’ - bipyridyl competition assay was also conducted to

measure the Fe2+ - chelating activity and hydroxyl radical (HO●) - scavenging

capacity was examined by the electron spin resonance (ESR) spectroscopy method.

The result obtained from FRAP and ABTS●+ assay were categorized into five main

groups. The sample with trolox equivalent antioxidant capacity (TEAC) over

500µM/100g is classified as containing extremely high activity, from 100 to

500µM/100g, 50 to 100µM/100g and 10 to 50µM/100g and less than 10µM/100g

were classified as containing high, good, low and very low antioxidant capacities,

respectively. Thus, the outcomes in Table 2.3 were discussed based on these

categories.

23

P

lant

par

t E

xtra

ct

Ant

ioxi

dant

test

O

utco

me

Ref

eren

ces

Pe

rica

rp

80%

met

hano

l A

BT

S●+

Ext

ract

has

low

ant

ioxi

dant

act

ivity

Sh

an et a

l. (2

005)

80%

met

hano

l FR

AP,

AB

TS●

+ E

xtra

ct h

as a

mod

erat

e an

tioxi

dant

val

ues

in A

BT

S●+ a

nd

DPP

H●

+ ass

ay a

nd h

igh

antio

xida

nt c

apac

ity in

FR

AP

test

W

ojdy

lo et a

l. (2

007)

an

d D

PPH

●+

Mac

e A

ceto

ne

FTC

, T

BA

an

d D

PPH

●+

Ext

ract

and

ess

entia

l oi

l sho

wed

str

onge

r ac

tivity

tha

n B

HA

an

d B

HT

Si

ngh et al.

(200

5)

Ace

tone

D

PPH

●+

and

β -

caro

tene

- lin

olei

c

acid

Ext

ract

sho

wed

bet

ter

radi

cal

scav

engi

ng a

ctiv

ity t

han

its

thre

e lig

nan

frac

tions

and

the

se f

ract

ions

wer

e ca

pabl

e to

in

hibi

t per

oxid

atio

n

Cha

tterj

ee et a

l.

(200

7)

80%

met

hano

l A

BT

S●+ , D

PPH

●+

and

FRA

P E

xtra

ct w

as c

onsi

dere

d ha

ving

goo

d an

tioxi

dant

cap

acity

Su

rves

war

an et a

l.

(2

007)

Seed

95

% m

etha

nol

TB

RA

S E

xtra

ct

has

the

abili

ty

to

prot

ect

hum

an

LD

L

agai

nst

Cu2+

indu

ced

pero

xida

tion

Kw

on et a

l. (2

008)

Seed

ker

nel

80%

met

hano

l A

BT

S●+ , D

PPH

●+

50%

ace

tone

ext

ract

has

low

er E

C50

val

ue,

high

er c

hela

ting

activ

ity a

gain

st F

e2+ a

nd s

cave

ngin

g ac

tivity

on

HO

● t

han

80%

met

hano

l ex

trac

t. 80

% m

etha

nol

extr

act

has

grea

ter

AB

TS●

+ and

OR

AC

val

ue th

an 5

0% a

ceto

ne e

xtra

ct

Su et a

l. (2

007)

and

50%

ace

tone

2,

2’ -

bip

yrid

yl

com

petit

ion

assa

y,

ESR

and

OR

AC

80

% m

etha

nol

AB

TS●

+ , DPP

H●

+ E

xtra

ct w

as c

onsi

dere

d ha

ving

goo

d an

tioxi

dant

cap

acity

Su

rves

war

an et a

l.

and

FRA

P

(200

7)

M

etha

nol

AB

TS●

+ , DPP

H●

+ E

xtra

ct w

as c

onsi

dere

d ha

ving

goo

d an

tioxi

dant

act

ivity

H

o et al.

(200

8)

and

OR

AC

Tab

le 2

.3 A

ntio

xida

nt e

valu

atio

n of

M. fra

gran

s

24

As indicated in Table 2.3, Surveswaran et al. (2007) have compared the antioxidant

activities of the 80% methanol extract of the mace and seed kernel through ABTS●+,

DPPH● and FRAP assays. It was found that the TEAC value of ABTS●+ assay of

the mace was higher than the seed kernel extracts with 26.03mmol trolox per 100g

dry weight (mmol trolox/100g) and 17.92mmol trolox/100g, respectively. Moreover,

the seed kernel (13.31mmol trolox/100g) was highly capable to scavenge DPPH free

radical as compared to mace extract (9.70mmol trolox/100g). The authors also found

that the 80% methanol extract of both parts exhibited more or less similar FRAP

capacity.

2.4 Antibacterial properties of M. fragrans

Based on anti - Helicobacter pylori comparative evaluation of various Thai

medicinal plants, M. fragrans mace extract gave the lowest minimum inhibitory

concentration (MIC) that was 12.50µg/mL (Bhamarapravati et al., 2003). The leaf

extract also has low MIC of 50.00µg/mL. Zaidi et al. (2009) found that 70% ethanol

extract of mace (minimum bactericidal concentration (MBC) value ranged from

62.50µg/mL to 31.20µg/mL) showed stronger bactericidal activity than 70% ethanol

extract of seed (MBC value ranged from 125.00µg/mL to 62.50µg/mL).

Screening by Rani and Khullar (2004) on some traditional Ayurvedic medicine

against resistant Salmonella thypi, resulted a strong antibacterial activity of the

methanol extract of the seed. Consequently, Mahady and colleagues (2005) found

that the MIC value of the methanol extract of the seed was 12.50µg/mL against

Helicobacter pylori. Chung et al. (2006) have investigated the antibacterial activities