UNIVERSITI PUTRA MALAYSIA

ANTIOXIDATIVE ACTIVITIES AND ISOLATION OF PHENOLIC COMPOUNDS FROM THE ROOT, FRUIT AND LEAF OF

MENGKUDU (Morinda citrifolia L.)

ZAMZAHAILA BT MOHD ZIN

FSMB 2003 25

ANTIOXIDATIVE ACTIVITIES AND ISOLATION OF PHENOLIC COMPOUNDS FROM THE ROOT, FRUIT AND LEAF OF

MENGKUDU (Morinda c;tri!o/;a L.)

By

ZAMZAIIAILA BT MOHD ZIN

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

October 2003

£Specially dedicated to ........ .

Ayah and ma Along and Abang 'Wan

Kak (9he, mujahid, Kakak, Abg mat, Kak (9hik and Adik Faiz <Dina, Suhaila and Irian

mohamad K'.hairi mohd Zainol and last but not least my princess

nur F atin Aliya

11

thank you for your love, guidance, encourageltleKt and doa 's given throughout my life ..... .

thank you for having faith in me .....

III

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

ANTIOXIDATIVE ACTIVITIES AND ISOLATION OF PHENOLIC COMPOUNDS FROM THE ROOT, FRUIT AND LEAF OF MENGKUDU

(Morinda citrifolill L)

Chairman

Faculty

.

.

By

ZAMZAHAILA BT MOHD ZIN

October 2003

Associate Professor Azizah Abdul Hamid, Ph.D

Food Science and Biotechnology

This study was conducted with the objective of evaluating the antioxidative activities of

extracts from different parts of Mengkudu (Morinda citrifolia L.) including leaf, fruit

and root Methanol, petroleum ether, water and ethyl acetate were used as extraction

solvent. The anti oxidative activities assays were done using ferric thiocyanate method

(FTC) and thiobarbituric acid test (TBA). Furthermore, the activities of these extracts

were then compared to that of a-tocopherol and butylated hydroxyl tolune (BHT). Effect

of solvent types and concentration of extract used on antioxidative activities of M.

citrifolia were also carried out. Meanwhile, isolation of phenolic compounds was carried

out using Sephadex LH-20 column chromatography. Identification and quantification of

the common flavonoids in the separated fractions were carried out using a reversed

phase high-performance liquid chromatography (RP-HPLC) procedures on a Symmetry

CIS colwnn with water-methanol gradient elution system. Results showed that methanol

root extract exhibited higher activity compared to that of fruit and leaf extract of the

IV

same with no significant (p<O.05) difference as compared to that of a-tocopherol and

BHT. The result also showed that methanol gave highest yield of extract compared to

that of water, petroleum ether and ethyl acetate extraction. Meanwhile, both polar and

non-polar extract of M citrifo/ia L. exhibited appreciable activity. Increasing

concentration of methanol root extracts from 1000 to 5000 ppm do not significantly

increased the anti oxidative activity. On the other hand, there was significant (p<0.05)

difference in antioxidative activities of methanol leaf and fruit extracts when

concentrations were increased. Similarly, increasing concentrations of water extracts

from 1000 to 5000 ppm do not significantly increased the antioxidative activity of root

and leaf of M cirrifolia L. However, there was significant (P<0.05) difference in

anti oxidative activities of fruit extract at 1000 ppm compared to 2000 ppm. At 3000 ppm

onwards there were no significant (p<0.05) difference in activities. Result also showed

that at 3000 ppm, water extract of root, fruit and leaf of M citrifolia L. exhibited no

significant (p<0.05) difference in activities dum that of either a -tocopherol or BHT.

Meanwhile, increasing concentration of petroleum ether extract significantly increased

the anti oxidative activity of all parts of M citrifolia. The result also revealed that the

antioxidative activities exhibited by petroleum ether root extract was significantly

(p<0.05) higher than that of either a -tocopherol or BHT at concentration more than

4000 ppm. Similar observation was found in fruit and leaf extract of same solvent at

5000 ppm, where fruit and leaf extract exhibited significantly (p<0.05) higher activity

than that of either a -tocopherol or BHT. Increasing concentration of ethyl acetate leaf

extract from 1000 ppm to 5000 ppm do not significantly increased the antioxidative

activities. On the other hand, at 3000 ppm onwards there was significant (p<0.05)

v

difference in anti oxidative activities of root and fruit extracts when concentrations were

increased. Result also showed that at 3000 ppm and 4000 ppm, there was no significant

(P<0.05) difference in the activities exhibited by root and leaf extract of same solvent

than that of either (l -tocopherol or BHT. Meanwhile ethyl acetate fruit extract exhibited

significantly higher activity than that of either (l -tocopherol or BHT at the same

concentration tested. From this study it can be seen that different parts of M citrifolia

exhibited varying degrees of antioxidative activities when different solvents were used

in extraction procedures. Of the four solvents used, ethyl acetate is the best solvent for

extracting antioxidative compounds from different parts (root, fruit and leaf) of Morinda

citrifolia L. compared to other solvent. Isolation of phenolic compounds mainly focused

on the separation and identification of antioxidants from methanol extract because of the

polar properties of phenolic compounds especially flavonoids, which are known to be

potent antioxidant. All fractions were se�n to contain different amount of total phenolic

compound and exhibited considerably high antioxidative activity. Identification of

flavonoid successfully determines the individual active compound present in M

citrifolia. Major component determine including catechin and epicatechin, which are

known to be potent antioxidant.

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

AKTIVITI ANTIOKSIDA DAN ISOLASI BAHAN FENOLIK DARIPADA AKAR, BUAH DAN DAUN MENGKUDU (Morlnda citrlfolia L.)

Oleh

ZAMZAHAILA BT MOHD ZIN

Oktober 2003

Pengerusi Profesor Madya Dr. Azizah Abdul Hamid, Ph.D.

Fakulti Sains Makanan dan Bioteknologi

VI

Kajian ini dijalankan bertujuan untuk mengenalpasti kesan aktiviti antioksida oleh

ekstrak daripada bahagian Mengkudu (Morinda citrifolia L.) yang berbeza termasuklah

daun, buah dan akar. Metanol, petroleum eter, air dan etil acetate telah digunakan

sebagai pelarut pengekstrakan. Ujian antioksida yang digunakan adalah ujian ferik

thiosianat (FTC) dan ujian asid tiobarbiturik (TBA). Seterusnya, basil daripada kajian ini

dibandingkan dengan a-tokoferol and butilated hidroksil tolune (BHT). Selain itu, kesan

jenis pelarut yang berlainan dan kesan kepekatan ekstrak yang digunakan juga dikaji.

Disamping itu juga, Sephadex LH-20 telah digunakan dalam proses isolasi bahan

fenolik dengan kaedah kromatografi turns. Seterusnya, pengenalpastian dan pengiraan

beberapa jenis flavonoid yang biasa terdapat dalam tumbuhan dikenalpasti didalam

fraksi-fraksi yang terhasil daripada proses kromatografi tUTUS. Ini dilakukan dengan

menggunakan kaedah kromatografi cecair bertekanan tinggi berfasa terbalik (RP-HPLC)

berfasa pegun tUTUS Symmetry CIS dengan air dan methanol sebagai fasa gerak. Kajian

ini mendapati, ekstrak akar dalam metanol menunjukkan kesan antioksida yang terbaik

VII

manakala buah dan daun menujukkan aktiviti yang lebih rendah yang juga tidak

menunjukkan kesan yang ketara berbanding a-tocopherol dan BHT. Kajian ini juga

mendapati, metanol merupakan pelarut yang paling baik dalam menghasilkan hasil

ekstraksi yang terbanyak berbanding air, petroleum eter dan eti! acetat. Selain itu, kedua­

dua ekstrak polar dan tak polar Mengkudu menunjukkan kesan antioksida yang agak

tinggi. Seterusnya, kajian ini juga mendapati peningkatan kepekatan ekstrak metanol

bagi akar dari ] 000 ppm ke 5000 ppm tidak meningkatkan aktiviti dengan ketara. Selain

daripada itu, terdapat perbezaan yang ketara dalam aktiviti antioksida ekstrak metanol

bagi daun dan buah apabila kepekatan bertambah. Kesan yang sarna berlaJru dalam

ekstrak air bagi akar dan daun, dimana peningkatan kepekatan ekstrak tidak

meningkatkan aktiviti antioksida dengan ketara. Tetapi, terdapat perbezaan yang ketara

dalam aktiviti antioksida ekstrak buah pada kepekatan 1000 ppm berbanding 2000 ppm.

Pada 3000 ppm keatas, tiada perbezaan yang ketara dalam aktiviti antioksida. Keputusan

juga menunjukkan pada 3000 ppm, ekstrak air bagi akar, buah dan daun Mengkudu

menunjukkan tiada perbezaan yang ketara dalam aktiviti dibandingkan dengan a­

tocopherol dan BHT. Manakala, peningkatan kepekatan ekstrak petroleum eter, secara

ketara meningkatkan aktiviti antioksida dalam semua bahagian Mengkudu. Keputusan

juga mendapati aktiviti antioksida yang ditunjukkan oleh ekstrak petroleum eter bagi

akar adalah lebih tinggi berbanding kedua-dua a-tocopherol dan BHT pada kepekatan

4000 ppm dan keatas. Pemerhatian yang sarna didapati bagi ekstrak buah dan daun pada

5000 ppm, dimana kedua-duanya telah menunjukkan aktiviti yang lebih tinggi

berbanding a-tocopherol dan BHT. Penigkatan kepekatan ekstrak etil acetate daun

daripada 1000 ppm - 5000 ppm tidak meningkatkan aktiviti antioksida secara ketara.

VIII

Seterusnya, pada kepekatan 3000 ppm dan keatas, terdapat perbezaan yang ketam dalam

aktiviti antioksida bagi akar dan buah bila kepekatan meningkat. Keputusan juga

menunjukkan pada 3000 ppm dan 4000 ppm, tiada perbezaan yang ketam dalarn aktiviti

yang ditujukkan oleh akar dan daun berbanding dengan kedua-dua a-tocopherol dan

BHT. Disarnping itu juga ekstrak etil acetate bagi buah rnenunjukkan aktiviti yang lebih

tinggi berbanding kedua-dua a-tocopherol dan BHT pada kepekatan yang sarna.

Daripada kajian ini, didapati bahagian-bahagian yang berbeza pada Mengkudu

menunjukkan darjah aktiviti antioksida yang berbeza bila pelarut yang berbeza

digunakan dalam pengekstrakan. Daripada empat pelarut yang digunakan, etil acetate

didapati merupakan pelarut yang paling baik untuk mengekstrak antioksida daripada

pelbagai bahagian Mengkudu berbanding dengan pelarut lain. Pengisolasi bahan fenolik

adalah ditumpukan kepada pemisahan dan pengenalpastian antioksida daripada ekstrak

metanol disebabkan ciri polar yang �rdapat pada komponen fenolik terutamanya

flavonoid yang terkenal sebagai komponen aktif antioksida .. Keseluruhan bahagian

ekstrak yang dipisahkan didapati mempunyai jumlah komponen fenolik yang berbeza

dan menunjukkan aktiviti antioksida yang tinggi. Pengenalpasti komponen-komponen

aktif yang hadir dalam M citrifolia mendapati komponen major yang hadir dalam M

citrifolia adalah catechin dan epicatechin, yang mana komponen ini telah dikenali

sebagai komponen yang aktif antioksida.

IX

ACKNOWLEDGEMENTS

Syukur Alhamdullillah to the Almighty Allah S.W.t for giving me strength, patience and

capability to complete this project and thesis write up and salawat and salam to his

righteous messenger, prophet Muhammad s.a. w.

First of all, I would like to express my deepest thanks and appreciation to the Chairman

of Supervisory Committee, Associate Professor Dr. Azizah Binti Abdul Hamid of the

Department of Food Science, Faculty of Food Science and Biotechnology, UPM for her

guidance, patience, advice, constructive criticisms and encouragement throughout this

interesting research and most importantly her kindness to give me a chance to gained

knowledge and experience through the seminar that I have attended. All the valuable

experiences that I gained as one of her graduate students and her continuous

commitment and willingness to help towards the success of my study will always be

remembered deep in my heart.

My deep sense of gratitude and respect is quoted to my co-supervisors, Associate

Professor Dr. Azizah Binti Osman and Associate Professor Dr. Nawnid Bin Saari, for

their sincere advice, support and encouraging interest in my study. My sincere thank also

extended to all the highly dedicated, friendly and helpful staffs of Faculty of Food

Science and Biotechnology, UPM and to all my fellow graduate students especially in

the Nutraceutical and EnzymoloW Lab. Sincere gratitude is also dedicated to Malaysian

Government for the financial support provided through IRP A fund and to Kolej

x

Universiti Sains Dan Teknologi Malaysia (KUSTEM) for providing me with the

scholarship to pursue the debJfee.

Paramount gratitude is also owed to my beloved husband Mohamad Khairi Bin Mohd

Zainol for his patience and encouragement to give me strength to complete this research

and to my cute daughter Nur Fatin Aliya for spices mama's life. Finally, my deepest

gratitude and appreciation to both my parents, Mohd Zin Bin Omar and Latifah Binti

Dollah for their continuous support, and all my brothers and sisters, thank you so much

for their love and support who have been inspiring my life.

XI

I certify that an Examination Committee met on 5th September 2003to conduct the final examination of Zamzahaila Mohd Zin on her Master of Science thesis entitled "Antioxidative Activities and Isolation of Phenolic Compounds from the Root, Fruit and Leaf of Mengkudu (Morinda citr(folia L.)" 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:

ABDUL MANAF BIN ALI, Ph.D. Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Chairman)

AZIZAH BTE ABDUL HAMID, Ph.D.

Associate Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

AZIZAH BTE OSMAN, Ph.D.

Associate Professor Faculty of Food Science and Biotechnology Vniversiti Putra Malaysia (Member)

NAZAMID BIN SAARI, Ph.D.

Associate Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

SUL RAHMAT ALI, Ph.D. ProfessorlDe uty Dean School of Graduate Studies Universiti Putra Malaysia.

Date: '2 '1' NOV 2003

XII

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirements for the degree of Master of Science. The members of the Supervisory Committee are as follows:

AZIZAH BTE ABDUL HAMID, Ph.D.

Associate Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Chairman)

AZIZAH BTE OSMAN, Ph.D.

Associate Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

NAZAMID BIN SAARI, Ph.D. Associate Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

AINI IDERIS, Ph.D.

ProfessorlDean School of Graduate Studies Universiti Putra Malaysia.

XJII

DECLARATION

J hereby declare that the 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 degree at UPM or other institutions.

Date: 3 1 OCT 2003

XIV

TABLE OF CONTENTS Page

DEDICATION ... ... '" . . . . . . ... ...... ... . . . ... ... ...... . .. .. . . . . . ,. ... ... ... . . . ... .. . . . . .... ii ABSTR.ACT . . . . . . . . , '" . . . . . . . . . .. . . . . . " . . . . . , ... ... . .. . . . . . . . . . .. . . . . . .. . . . .. , . . . . . . . . . . . . iii ABSTRAK ... ... ... ... ... ... . .. .. , ... ... . . . ... . . . ... .. . ... ... ... . . . ... ... ... . .. ... ... ... ... . .. vi ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX APPROVAL SHEETS ... .. . . . . '" ... ... ... ...... .. , ...... ... ... ... ... . , . ..... , ... ... .... XI DECLARATION FORM . .. '" .,. '" .. , ..... , ... ... ... ... ...... ... ... .. , ... ... ... ... ..... Xlll LIST OF TABLES ... . . . . . . . . . . . . . . . ... . . . . . . ... . . . . , . ... .. , ... .. . ... ... ... ... ... ... ... ... XVII LIST OF FIGURES . .. . . . . . . . . . . . . .. . . . . ... . . . . . . . . . . . . ... . , . ... .. , ... ... ... ... ... ... ... ... XVIll LIST OF PLATES ... ... .. , ... .. , ... ... ... ... ... ... ... ... ... ... .. . ... ... ... ... ... ... ... ... xx LIST OF ABBREVIATIONS . . . .. . . . . . . . . . . . . . . .. . .. .. . . . , ... .. , ... ... ... ... ... ... ...... XXI

CHAPTER

I GENERAL INTRODUCTION .... . . . .. ... . . . ... . .. . . . . . . .. , ... ... ... '" ..... ... 1

n LITERATURE REVIEW ...... ... ... . , . ... .. , ... ... . . . ... ... ... ... ... . .. ... 5 2.1 Botany, Distribution and Ethnobotany of Morinda Species... ... ... 5

2.1.1 Family Rubiaceae ... ... ... ... . . . ... .. . . . . ... ... ... ... . .. ... .. . 5 2.1.2 Genus Morinda . . . .. . .. . . . . . . . . . . . , . .. . . .. ..... , ... ... ... '" 5 2.1.3 Morinda citrifolia L. . . . ... . . . .. . . .. . . . ... . . . .. . .. . .. . ... . .. .... 8

2.1.3.1 Other Name ... ... ... ... ... .. . ... . . . ... 11 2.1.3.2 Health Benefits ...... ...... ... ... . . . .... 12 2.1.3.3 Health Related Compounds .. , ..... . . .. 14

2.1.4 A Review of Previous Work on Genus Morinda . ... . . ... 24 2. 1.4.1 Morinda e/liptica .. . . . . . . . . . . .. . . . . . .. . . . . . . .. 24 2.1.4.2 Morinda umbellate .. . . . . ... ... ... .. . .. . ... 25 2.1.4.3 Morinda pervifolia ... . . ' ..... . '" .. . ... ... 26 2.1.4.4 Morinda lucida . . . . ,. ... .. . . .. . .. .. . . . . 27 2.1.4.5 Morinda tomentosa .. . . . . . . . . . . . . . .. . '" ... 27

2.2 Autoxidation ... . .. ... ... .. , ... .. . ... ... ... ... .. . ... ... ... ... ... ... 28 2.2.1 Antioxidants ... . .. ..... . ... ... '" ... '" ... .. . ... ... ... . .. ... 32

2.2.1.1 Synthetic Antioxidants ... ... ... ... . .. ... 35 2.2.1.2 Natural Antioxidants ... ... ... ... ... ... 36

2.2.2 Pro-oxidant . . . ... . . . ... .. . ... . , . ... . , . . .. .. , . .. ... ... ... ... ... 38 2.2.3 Biological Activity of Antioxidant ... .. , ... ... '" ... ... '" 38 2.2.4 Methods for Determining Antioxidative Activities ... ... 40

2.2.4.1 Ferric Thiocyanate Method (FTC) ... ... ... 42 2.2.4.2 Thiobarbituric Acid Test (TBA)... ... ... ... 43 2.2.4.3 Linoleic Acid Model System ... ... ... ... 45 2.2.4.4 Electron Spin Resonance (ESR)

Spectroscopy... ... ... . .. ... ... ... ... ... ... ... 46 2.2.4.5 2,2-diphenyl-l-picrylhydrazyl (DPPH)

Radical Scavenging Method... ... ... ... ... 47

III ANTIOXIDA TIVE ACTIVITIES OF EXTRACTS FROM MENGKUDIJ (Morinda eitrifolia L.) ROOT, FRllIT AND LEAF... 49 3.] Introduction . .. . ,. ... ... .. . .. . .. . . .. .. . ... . . . ... . .. . ... .. . . . .. . ... . 49 3. 2 Materials and Methods . . . . .... . ... . . . .. . . . , .. ...... , . . . . . . . ,. ... . 51

3. 2. ] Extraction of Antioxidative Compounds .. . . .. . . , . . . . . . 5] 3.2. 2 Determination of Antioxidative Activities .. . .. . . . .. , . . . . . . 52

3. 2. 2. ] Ferric Thiocyanate Method (FTC) . .. . . . . . . . 52 3. 2.2. 2 Thiobarbituric Acid Test (TBA) . . .. .. . . . . .. 52

3. 2. 3 Statistical Analysis . . .. . . '" . . . . . . . . . . . . . . . . .. . . . . . . . ,. . . . 53 3.3 Results and Discussion . . . '" . , . .. . . . , . . . . . . . . . . . . . . , . . . . . . . ,. . . . 53 3.4 Conclusion ..... . . . . . .. . . , ... '" ......... '" ...... ..... , ... ... ... ... 64

IV EFFECT OF SOLVENT TYPES AND CONCENTRATION OF EXTRACT USED ON ANTIOXIDATIVE ACTIVITIES OF MENGKUDU (Morindtl eM/olia L) ROOT, FRUIT AND LEAF .. .. . , 66 4. 1 Introduction . ... . , . . . . . . . . . . . . . . , . . . . . . . . . . . . . . , . . . . . . . . . . . . . . , . . . . . . 66 4.2 Materials and Methods . . , '" . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . .. . . . . '" 68

4.2. 1 Extraction of Antioxidative Compounds .. .. .. . ,. . . . . . . 68 4. 2. 2 Determination of Antioxidative Activities ... . . . . . . . . . . . 71

4.2.2. 1 Thiobarbituric Acid (TBA) Direct Method . . 71 4.2. 3 Characterization Study .. ... . . . . . .. ... .. .. .. '" . . . . . . . . . '" 71 4. 2. 4 . Statistical Analysis .. . '" . .. . . . . . . . . . . . . .. . . .. . . . .. . . . . .. . .. . 72

4. 3 Results and Discussion '" . . . . . , . . . .. . ... .. .. . , . . . ... .. . . .. . . , . . . 72 4. 3. 1 Yield of Extracts . . . . . . . . . . . . . . . . .. . . . . . ,. . . . . . . . . . . .. . . . . . . .. . 72 4. 3. 2 Effect of Concentration of Extract on Antioxidative

Property of Different Parts of M citrifolia L.... ... . .. . . . 74 4.3. 3 Effect of Different Solvent Used for Extracting

Antioxidative Compounds of M citrifolia L... . .. . . . . . . . . . 82 4. 4 Conclusion . .. ' " . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , '" 86

V ISOLATION OF PHENOLIC COMPOUNDS FROM ROOT, FRUIT AND LEAF OF MENGKUDU (Morindtl eM/oM L) . . . . . , . . . . . . . 88 5. 1 Introduction .. .. , . .. . . . . . . . . . . . , . . . . .. . . . . . . . . ,. ... . . . . . . . . . . .. . . . . 88 5.2 Materials and Methods . . ... . '" ... ... ... ... ... ... ... ... ... ... ... 91

5.2. 1 Preparation of Morinda citrifolia L. Root, Fruit and Leaf Extract . . . ... .. . , '" . . . . . . . . . . . ,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

5.2. 2 Sephadex LH-20 Column Chromatography............... 91 5.2.3 Determination of Total Phenolic Compounds (TPCs).... ... 92 5.2. 4 Determination of Antioxidative Activities . . . '" . . . . . . . . . . . . 92

5.2. 4. 1 Ferric Thiocyanate method (FTC)....... . .. 92 5. 2. 4. 2 Thiobarbituric Acid Test (TBA)........ . . . 93

5.2. 5 Analytical HPLC .. . . .... .. . . .... . ... , . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

xv

5.2.5.1 Chemicals . .. .. . '" ... . , .... ... . ,. ... ... ... ... ... 93 5.2.5.2 Preparation of Standard Flavonoids... ... ... 94 5.2.5.3 Sample Preparation and HPLC System . . . . . , 94

5.2.6 Statistical Analysis ... . . . . . . ... . .. .. . . . .. .. . .. .. . ...... . . . '" ... 95 5 .3 Results and Discussion ... . .. . .. .. , ... ... .. , '" ... . .. ... ... ... ... ... 96

5.3.1 Isolation of Antioxidative Compound of the Fruit, Root

XV)

and Leaf Fractions of M citrifolia L... . . . . .. . . . . . .. ... 1 00 5.3.2 Total Phenolic Compounds of Fruit, Root and Leaf

Fractions of M citr!folia L. ..... ... ...... ...... ... ... .. , ... ... .. 102 5 .3 .3 Antioxidative Activity of the Fruit, Root and Leaf

Fractions of M citrifolia L. .. . .. . . . . . . . .. . .. . .. '" ... ... ... ... . 1 06 5.3.4 Relationship Between Phenolic Compound and

Antioxidative Activities of Fruit, Root and Leaf Fractions of M citrifolia . . . '" ... .. . . . . ... ... .. , ... .. . ... . . . . . . .. 1 19

5.3.5 Identification of the Antioxidative Compounds of Fruit, Root and Leaf Fractions by Reversed Phase High Performance Liquid Chromatography (RP-HPLC)... ... . .. . 122

5.4 Conclusion . . . . . . . . , ., . . . . . , . .. .... . " ... . .. ... ... ... . . . . .. . . . ... . . . 13 1

VI SUMMARY AND RECOMMENDATIONS... ... ... ... ... ... ... . .. ... ... . .. 132

BmLI()(;RAPHY ... .. , ... ... . " . . . ... . , . ... . .. .. , ... . .. .. . ... .. . ...... .. , .. . ... .. , . .. . .. ... 1 36 APPENDICES ... ... ... . .. ... ... ... ... .. , ... ... ... ... ... . ,. ... ... ... ... ... ... ... ... ... ... 1 61 BIODATA OF AUTHOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , ... ... ... . .. ... 169

XVll

LIST OF TABLES

Table Page

2.1 Morinda species native to Peninsular Malaysia 7

2.2 Factors affecting peroxidation 32

2.3 Measurements of lipid peroxidation 40

4.1 Extraction yield from different parts of M cirri/olia 73

5.1 Mobile phase composition the separation of flavonoid 95

5.2 Total phenolic content of fruit extract 103

5.3 Total phenolic content of root extract 104

5.4 Total phenolic content of leaf extract 105

XVJ1)

LIST OF FIGURES Figure Page

2.1 Major anthraquinone aglycones found in intact plant of M citrifolia 16 L.

2.2 Anthraquinones in the M citrifolia L. 17

2.3 New antileukemic anthraquinone from M parvifolia 26

3.1 Antioxidative activity of methanol extracts of different parts of 56 M citrifolia as measured by FTC method.

3.2 Antioxidative activity of methanol extracts of different parts of 60 M citrifolia as measured by TBA method.

4.1 Extraction of antioxidative compound 70

4.2 TBA value of methanol extract of (A)- root, (B)- fruit and (C)- leaf 75 extracts at different concentrations.

4.3 TBA value of water extract of (A)- root, (B)- fruit and (C)- leaf 77 extracts at different concentrations.

4.4 TBA value of petroleum ether extract of (A)- root, (B)- fruit and 79 (C)-leaf extracts at different concentrations.

4.5 TBA value of ethyl acetate extract of (A)-root, (B)-fruit and (C)- 81 leaf extracts at different concentrations.

4.6 Antioxidative activities of different parts of M citrifolia as affected 83 by different extracting solvent.

5.1 Chromatogram of a solution of 9 flavonoids standards monitored at 165 370nm.

5.2 Eluates following Sephadex LH-20 column chromatography of (a) 101 fruit, (b) root and ( c ) leaf of M cirrifolia.

5.3 Antioxidative activity of Sephadex LH 20 column chromatographic 107 fractions obtained from fruit extracts of M citrifolia as measured by FTC method.

5.4 Antioxidative activity of Sephadex LH 20 column chromatographic 108 fractions obtained from fruit extracts of M citrifolia as measured by TBA method.

XIX

5.5 Total phenolic compounds of Sephadex LH 20 column 113 chromatographic fractions obtained from root extracts of M citrifolia.

5.6 Antioxidative activity of Sephadex LH 20 column chromatographic 115 fractions obtained from root extracts of M citrifolia as measured by TBAmethod.

5.7 Antioxidative activity of Sephadex LH 20 column chromatographic 117 fractions obtained from leaf extracts of M citrifolia as measured by FTC method.

5.8 Antioxidative activity of Sephadex LH 20 column chromatographic 118 fractions obtained from fruit extracts of M citrifolia as measured by TBAmethod.

5.9 Relationship between phenolic compound and antioxidative 120 activities of fruit, root and leaf fraction of M citrifolia.

5.10 Chromatogram of fraction I, n, ill, IV, V, VI obtained from fruit 166 extracts of M citrifola monitored at 370 nm.

5.11 Flavonoid content of Sephadex LH 20 column chromatographic 124 fractions obtained from fruit extracts of M citrifolia.

5.12 Chromatogram of fraction I, n, ill, IV, V, VI obtained from root 167 extracts of Mcitrifolia monitored at 370 'nm.

5.13 Flavonoid content of Sephadex LH 20 column chromatographic 126 fractions obtained from root extracts of M citrifolia.

5.14 Chromatogram of fraction 1, II, ill, IV, V, VI obtained from leaf 168 extracts of MCitrifolia monitored at 370 nm.

5.15 Flavonoid content of Sephadex LH 20 column chromatographic 129 fractions obtained from .leaf extracts of M citrifolia.

xx

LIST OF PLATES

Plate Page

2. 1 Morinda citrifolia L. tree in UPM, Serdang. 16 1

2.2 M citrifolia L. leaves and mature fruit 162

2.3 Arrangement of M citrifolia L. leaves 162

2.4 M citrifolia L. fruit: young green fruit with flowers 163

2.5 M citrifolia L. seed 163

2.6 M citrifolia L. fruits: mature fruits 164

2.7 M citrifolia L. roots 164

BHA BHT DPPH FTC h HCI HPLC L LDL mg ml

run mm

J..I.g

J..Lm mM MAD MeOH Min N NaDEDTC PG RP-HPLC ppm rpm SAS TFA TBA UV UVNis v/v w/v

LIST OF ABBREVIATIONS

Butylated hydroxyanisole Butylated hydroxytoluene 2.2-diphenyl-l-picrylhydrazyJ Ferric thiocyanate Hour Hydrochloric acid Reverse phase high pressure liquid chromatography Liter L ow density lipoprotein Milligram Milliliter Nanometer MilIimeter Microgram Micrometer MilliMolar Malonaldehyde Methanol Minutes Nonnality Sodium diethyldithiocarbamic Propyl gallate Reverse phase high pressure liquid chromatography Part per million Revolution per minute Statistical Analysis System Trifluoroacetic acid Thiobarbituric acid Ultraviolet Ultraviolet / visible Volume per volume. Weight per volume

XXI

CHAPTER I

GENERAL INTRODUCTION

The tale of the Hawaiian noni (Mormda cltr(fhlia) plant likely began 1500 years ago

with the arrival of the early Polynesian settlers to Hawaii (Abbott and Shimazu.

1985). Archaeological evidence suggests that the first settlers were from the

Marquesas Islands (Feher. 1 %9; Mitchell. 1992). and in fact the Marquesans and

Hawaiian use the same name for the noni. compared to Samoans and Tongans (nonu)

or Rarotongans (nono) (Ube, 1974). The ancient Polynesians likely brought along the

noni for its use as a food. dye and medicine (Abbott and Shimazu. 1985). They

consumed the fruits during times of famine (Degener, 1973), coloured their kapa

(Hawaiian cloth) yellow and red with roots (Mitchell, 1992) and treated numerous

ailments with various portions of the plant (Chun, 1994).

Morinda citrifolia must have easily taken root in the new land because of the warm

tropical climate. Even to this day, it can be found growing near abandoned ancient

dwellings (Degener, 1973), scattered along rocky lava coastlines and cultivated in

lowland gardens (Merlin, 1977). Although the historical accounts are few, the most

intriguing aspect of the M. citrifolia is its professed healing power. It was highly

valued for its medicina1 properties (Kepler, 1983), in which virtually the whole plant

(fiuit, leaf, bark, root. flower and seed) had been used.

In the Tropics it seems to have much valued medicinally, the genus is cultivated for

its roots, also at times for leaves and fruits. Most part of the tree has been widely

used medicinally since ancient times. The plant portions treated numerous

2

conditions. including some that were infectious in origin. Combination of the bark,

fruit and other plant products was antidotal against tuberculosis. The flower or fruit

with additional plant material treated thrush, a fungal infection (Chun. 1994). The

fruit also acted as a poultice to "draw out the pus and core from an infected sore or

boil" (Krauss, 1981) and alleviated "itching of the anus due to pinworms" (Chun.

1994). Other sundry remedies included purifying the blood and stimulating the

appetite with a root concoction (Chun. 1994). healing broken bones, "especially if

there was a compound fracture" with the unripe fruit (Krauss, 1981), treating

rheumatic joints (Gutmanis, ] 994) or "sprains of knuckles or wrists and of the knees"

(Chun, 1994) with the leaf, helping kidney and bladder problems with the flower and

fruit (Gutmanis. 1994) and medicating heart troubles with the juice of the ripe fruit

(McBride, 1975).

The traditional healing applications of M cirrifolia in Hawaii are comparable with

other regions of the Pacific; This might be expected since the early Polynesian

immigrants would have brought with them their customs and knowledge of plants

from their former homeland. In general, the Polynesians mainly used the fruits or

leaves of the plants to treat infectious and inflammatory disorders, such as boils, oral

infections and rheumatism Sometimes the natives from one Pacific Island utilised a

different portion of plant to treat an analogous ailment. For example, in Hawaii the

noni fruit with other agents treated 'gripping pain of the stomach or intestines"

(Chun, 1994), whereas in Tonga it was the bark or leaves that medicated stomach­

aches (Whistler, 1992). Female gynecological problems such as pain after childbirth

were treated with the flowers in Ponape (Glassman, 1952), while the leaves were

used to alleviate pregnancy pain in Fiji (Cambie and Ash, ] 994). The application of

3

the various plant sections to treat similar conditions may indicate the existence of a

common active component throughout the plant. Also, it is interesting to note that the

plant occasionally treated opposing ailment. The fruit was useful for constipation in

Hawaii (Chun, 1994), and for diarrhoea in Gilbert Island (Cambie and Ash, 1994).

The bark and roots were used as homeostatic for menstruation in Penope (Glassman,

1952) whiJe the bark or leaves treated amenorrhea in Tonga (Singh et ai. , 1984).

Other regions of the world also recorded folkloric uses of the M. citrlfolia plant. The

M. citrifolia plants can be found in parts of tropical Africa, America, Asia and

Australia, although it may be originally native only to the island of Indonesia

(Whistler, 1992). In parts of Central America, it was commonly referred to as

"painkiller" because of the leaves, which were useful in relieving pain (Little and

Wadsworth, 1964). In Barbados, the fiuit was believe to be poisonous and was called

"Forbidden Fruit" (Gooding et ai., 19,65). This is in contrast to Bwma, where the

. fiuits were considered edible and were consumed raw with salt or cooked in curries

(Morton., 1992). In other neighbouring areas such as Vietnam, Laos and Cambodia,

the fruits, leaves and roots were used as medicine (Perry and Metzger, 1980). For the

most part, the M citrlfolia had similar medicinal application around the world. The

plants was commonly used for fevers, sore throats, cough, stomach-aches, ulcers,

diarrhea, dysentery, pain, boils, wounds, arthritis and worms. Less common was its

used for asthma, tuberculosis, hypertension, diabetes, malaria, liver ailments and

splenomegaly. The universal recognition of M. citrifolia as a traditional healing

remedy establishes a sense of credibility for the medicinal lore of this plant.