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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.