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UNIVERSITI PUTRA MALAYSIA
NOR’AISHAH ABU SHAH
FBSB 2013 8
CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF ESSENTIAL OIL FROM CHEMPAKA (Michelia alba De Candolle)
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CHEMICAL CONSTITUENTS
AND BIOLOGICAL ACTIVITIES OF ESSENTIAL OIL FROM CHEMPAKA
(Michelia alba De Candolle)
By
NOR'AISHAH BINTI ABU SHAH
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia
in Fulfilment of the Requirements for the Degree of Doctor of Philosophy
January 2013
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Dedication
My late beloved mother who had always prayed for my wellbeing.
This writing is specially dedicated to her.
To all my loves ones and those who have sacrificed and supported me
throughout my studies.
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ABSTRACT
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment
of the requirement for the Degree of Doctor of Philosophy
CHEMICAL CONSTITUENTS
AND BIOLOGICAL ACTIVITIES OF ESSENTIAL OIL FROM CHEMPAKA
(Michelia alba De Candolle)
By
NOR’AISHAH ABU SHAH
January 2013
Chairman: Associate Professor Janna Ong Abdullah, PhD
Faculty: Biotechnology and Biomolecular Sciences
Malaysia is one of the twelfth mega-diversity countries in the world with a lot of
colourful and aromatic flowers flowering throughout the year. Unfortunately many
plant species are not fully exploited towards perfume production. Michelia alba, an
aromatic plant with fascinating flower odour, has long been used as fragrance in
perfume and cosmetics products. However, there is still shortage of information on
the chemical profile their pharmacognosy values. The flower only budding mostly
during rainy season, concurrently with shoots formation. Furthermore, this local
plant is hardly produce fruit or seeds due to pollen shortage and exhibit low
efficiency in conventional propagation. This study was carried out to profile the
chemical and biological properties of the essential oils in fresh M. alba flowers and
leaves and establish essential oil production in its callus cultures. The chemical
compositions of the essential oils of M. alba flowers (from white bud to full bloom
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flower) and the leaves were determined using gas chromatography-mass
spectrometry (GC-MS) techniques. Approximately 100 compounds were detected
and 30 were chromatographically identified. Linalool was found to be dominant in
the leaf essential oils at 76.6% while it constituted 67.1% in the stage 8 flower. Other
major chemical constituents detected in the leaf oil were farnesol (5.5%), β-elemen
(3.7%) and nerolidol (2.2%). Other abundant chemical components in most of the
flower stages were 2-methyl-methyl-ester-butanoic acid, phenol, caryophyllene,
phenyl ethyl alcohol, ocimene and germacrene D. The compositions of the essential
oils in different parts of the flower revealed that the highest percentage of linalool
were from gynoecium, stamen and tepal at 74.5%, 74.4% and 38.8%, respectively.
Other major compounds found were 2-methyl-butanoic acid and 2-methyl-methyl-
ester-butanoic acid.
The essential oils from the fresh flowers and leaves of M. alba were specifically
extracted to further test their effects towards growth inhibition of different
pathogenic bacteria and fungi using disc diffusion assay. Two kind solvents of flower
and leaf extracts were used to examine the effect of extraction solvents with different
polarities on biological activities. Most of the extracts from different solvents used
inhibit the growth of all the bacteria tested. The extracts showed better antibacterial
effects on most of the tested bacteria with lower MIC values ranging from 1.25 to
10.00 μl/ml. The least inhibited bacteria was Pseudomonas aeroginosa, effective
only with leaf essential oil extracted in dichloromethane (MIC value 10.00 μl/ml).
All Gram-negative bacteria tested were more resistant compared to the Gram-
positive bacteria. Candida albicans was found to be more susceptible to the leaf
extract, while Fusarium oxysporium was more susceptible to the dichloromethane
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flower extract. Overall, the results showed that the leaf gave better inhibitory effect
from dichloromethane extract (DL) and the flower from n-pentane extract (PF),
consistent with the higher percentage of linalool in the leaf (DL: 76.6%) and the
flower (PF: 63.2%).
The antioxidant activity of the fresh flowers and leaves extracted with
dichloromethane and n-pentane were also investigated with three different
established methods: the conjugate diene method, the 2,2’-diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging method and -carotene bleaching method.
Approximately 20 mg/ml (v/v) of the essential oils of M. alba was found to exhibit
moderate antioxidant property (25% - 50%). Even though the essential oils was slow
in its scavenging effects either with dichloromethane or pentane, the scavenging
activity was independent of the solvent polarity. The oxidation of linoleic acid in -
carotene bleaching test was best inhibited by the leaf extracted in dichloromethane
(93.9%), compared to standard butylated hydroxytoluene (BHT) (83.3%). The
antioxidant activity tested using the conjugated diene method showed higher
inhibition of lipid peroxidation for both flower (PF: 90.7%) and leaf (PL: 77.1%)
extracts in n-pentane, which was equivalent to the standard antioxidant, BHT
(91.5%) and α-tocopherol (vitamin E) (77.2%). Overall, the essential oils from the
flower showed slightly higher antioxidant activity than the leaf oils, both extracted in
n-pentane.
In order to ensure sustainability of the essential oils due to seasonal flowering
problem, M. alba explants were induced for calli production for the corresponding
secondary metabolites. Tepal, gynoecium, stamen and leaf explants were separately
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cultured on Murashige and Skoog (MS) and Woody Plant (WP) basal media each
supplemented with various concentrations of NAA and kinetin in order to select the
best medium for callus proliferation and organogenesis. Both media supplemented
with phytohormones produced pale (colour) friable calli. However, the WPM
medium produced remarkable callus proliferation compared to the MS medium with
around 50% to 100% of the explants producing friable to compact nodular calli.
Chemical compositions of the essential oils extracted with dichloromethane using
simultaneous distillation extraction (SDE) from the callus cultures of gynoecium and
stamen of M. alba flowers and analyzed by GC-MS revealed there were 60 peaks
detected and approximately 15 compounds were identified in both oils. The
compound with the highest composition in both essential oils was linalool with
50.0% in gynoecium oil and 28.8% in stamen oil for 0.1g of dry weight callus. The
other abundant chemical components of gynoecium oil were β-elemen (10.0%),
caryophyllene oxide (8.4%), caryophyllene (6.4%), germacrene D (4.2%), δ-
cadinene (5.6%) and eugenol methyl ether (2.3%). The chemical constituents of
stamen oil were α-bergamotene (12.5%), β-elemen (4.9%), germacrene-D (4.0%), δ-
cadinene (4.8%), caryophyllene (3.4%) and caryophyllene oxide (2.9%). The major
compounds of the essential oils from the callus cultures of gynoecium and stamen
were not significantly different (p>0.05) from the essential oils of fresh gynoecium
and stamen of M. alba flower.
This study indicates that in vitro production of the oils via callus culture from
gynoecium and stamen explants has potential in mass fragrance production from M.
alba replacing the seasonal fresh flowers. In addition, the M. alba essential oils have
the potentials as antimicrobial and antioxidant agents useful for the cosmetic
industry.
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ABSTRAK
Abstrak tesis yang dikemukakan kepada Senat Univesiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Doktor Falsafah
KANDUNGAN KIMIA DAN AKTIVITI BIOLOGI MINYAK PATI
DARIPADA CHEMPAKA (Michelia alba De Candolle)
Oleh
NOR'AISHAH BINTI ABU SHAH
Januari 2013
Pengerusi: Profesor Madya Janna Ong Abdullah, PhD
Fakulti: Bioteknologi and Sains Biomolekul
Malaysia adalah satu daripada dua belas buah negara di dunia dengan kepelbagaian-
mega, mempunyai tumbuhan yang berbunga sepanjang tahun dengan pelbagai warna
dan aroma bunga. Malangnya tumbuhan ini tidak dimanafaatkan sepenuhnya dalam
penghasilan wangian. Michelia alba sejenis tumbuhan beraroma dengan bau yang
menarik telah lama digunakan sebagai pewangi dalam pembuatan minyak wangi dan
kosmetik. Namun profil kimianya masih lagi terhad dan nilai ubatannya belum ramai
yang ketahui. Bunga hanya berputik kebanyakannya ketika musim hujan, serentak
dengan pembentukan pucuk. Tambahan pula tumbuhan tempatan ini sukar
menghasilkan buah atau biji kerana debunganya yang terhad dan propagasi secara
konvensional kurang berkesan. Kajian ini dijalankan untuk menentukan profil kimia
dan aktiviti biologi minyak pati bunga dan daun segar M. alba dan penghasilan
minyak pati dari kultur kalusnya. Komposisi kimia minyak pati M. alba dari daun,
putik bunga putih hingga bunga mekar telah ditentukan dengan menggunakan
kromatografi gas (GC-MS). Lebih kurang 100 sebatian dikesan dan 30 sebatian telah
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dikenal pasti melalui proses kromatografi. Linalool merupakan sebatian yang
dominan dengan 76.6% dalam ekstrak daun dan 67.1% dalam ekstrak bunga
peringkat 6. Komponen kimia utama yang dikesan dalam daun ialah caryophyllene,
β-elemen dan 'caryophyllene' oksida. Komponen kimia utama yang dikesan di
kebanyakan peringkat bunga ialah 2-metil-metil-ester-butanoik asid, fenol,
'caryophyllene', alkohol fenil etil, 'ocimene' dan 'germacrene' D. Bahagian bunga
yang berlainan mengandungi berlainan peratusan komposisi minyak pati dan didapati
bahawa peratusan tertinggi komposisi minyak pati dalam ginoesium, stamen dan
tepal adalah linalool, masing-masing pada 74.5%, 74.4% dan 38.8%. Sebatian utama
lain ialah asid 2-metil-butanoik dan asid 2-metil-metil-ester-butanoik.
Minyak pati daripada bunga dan daun segar M. alba telah diekstrak secara khusus
untuk menguji kesan mereka terhadap perencatan pertumbuhan beberapa jenis kulat
dan bakteria patogenik dengan menggunakan kaedah resapan dari cakera. Dua jenis
ekstrak bunga dan daun telah digunakan untuk menguji kesan pengekstrakan pelarut
dengan polariti berlainan ke atas aktiviti-aktiviti biologi. Kebanyakan ekstrak dengan
pelarut yang berbeza yang digunakan, menyekat pertumbuhan semua bakteria yang
diuji. Pengekstrakan tersebut menunjukkan kesan antibakteria yang lebih baik
terhadap kebanyakan bakteria yang diuji pada nilai MIC sekitar 1.25 hingga 10.00
μl/ml. Pseudomonas aeroginosa merupakan bakteria paling sukar disekat
pertumbuhannya kecuali dengan minyak pati dari daun yang diekstrak dengan
dikloromethana sahaja (nilai MIC 10.00 μl/ml). Semua bakteria gram-negatif yang
diuji adalah lebih rentan berbanding dengan bakteria gram-positif. Candida albicans
didapati boleh direncatkan oleh ekstrak daun dengan diklorometana, manakala
Fusarium oxysporium adalah lebih mudah direncatkan oleh ekstrak bunga
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dari pelarut yang sama. Secara keseluruhannya, keputusan menunjukkan bahawa
ekstrak daun dengan diklorometana (DL) memberi kesan perencatan yang lebih baik
berbanding dengan pati bunga dari ekstrak n-pentana (PF), selaras dengan peratusan
linalool yang lebih tinggi di dalam daun (PL:76.6%) berbanding dengan bunga (PF:
63.2%).
Kajian antioksidan ke atas ekstrak bunga segar dan daun dengan diklorometana dan
n-pentana juga telah dijalankan melalui tiga kaedah yang berbeza iaitu kaedah diena
terkonjugat, kaedah pemusnahan radikal 2,2-'difenil-1-picrylhydrazyl (DPPH) dan
kaedah pelunturan β-karotena. Lebih kurang 20 mg / ml (v / v) minyak pati M. alba
telah didapati mempamerkan kesan antioksidan di antara sederhana kepada baik.
Walaupun minyak pati menunjukkan kesan pemusnah radikal yang perlahan dalam
pelarut diklorometana dan n-pentana, aktiviti pemusnah radikal adalah tidak
dipengaruhi oleh polariti pelarut. Pengoksidaan terbaik asid linoleik dalam ujian
pelunturan -karotena telah direncat oleh pengekstrakan daun dengan diklomethana
(93.9%), berbanding dengan piawai butylated hydroxytoluene (BHT) (83.3%).
Aktiviti antioksidan diuji dengan menggunakan kaedah diena terkonjugat telah
menunjukkan perencatan pengoksidaan lipid yang lebih tinggi di dalam kedua-dua
ekstrak bunga (PF: 90.7%) dan daun (PL: 77.1%), yang hampir sama dengan aktiviti
antioksidan piawai, BHT (91.5%) dan α-tokoferol (vitamin E) (77.2%). Secara
keseluruhan, minyak pati dari bunga menunjukkan aktiviti antioksidan yang tinggi
sedikit daripada minyak pati daun yang diekstrak dengan n-pentana.
Bagi memastikan kelestarian penghasilan minyak pati dan mengatasi masalah
pembungaan yang dipengaruhi musim, penghasilan kalus dengan pengeluaran
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metabolit sekunder yang sepadan telah dijalankan. Tepal, ginoesium, stamen dan
daun telah dikultur secara berasingan di atas media Murashige and Skoog (MS) dan
Woody Plant (WP) yang dibekalkan dengan pelbagai kepekatan NAA dan kinetin
untuk memilih media yang terbaik untuk percambahan kalus dan organogenesis.
Kedua-dua media yang dibekalkan dengan fitohormon telah menghasilkan kalus
rapuh dan berwarna pucat. Walau bagaimanapun, media WP menghasilkan
percambahan kalus yang lebih baik berbanding dengan media MS dengan
pertumbuhan sekitar 50% hingga 100% eksplan dengan penghasilan kalus dari yang
rapuh hingga kepada kalus padat nodular. Komposisi kimia minyak pati daripada
kultur kalus ginoesium dan stamen M. alba telah diekstrak dengan diklorometana
menggunakan pengekstrakan penulenan serentak (SDE) dan dianalisa dengan GC-
MS telah menunjukkan terdapat 60 sebatian yang dikesan dalam kedua-dua minyak
tersebut dan kira-kira 15 sebatian telah dikenalpasti. Komposisi sebatian yang
tertinggi dalam kedua-dua minyak pati tersebut adalah linalool dengan 49.98% dalam
ekstrak ginoesium dan 28.80% dalam ekstrak stamen untuk 0.1g kalus berat kering.
Sebatian kimia utama lain yang terdapat dalam minyak pati ginoesium adalah β-
elemen (10.0%), 'caryophyllene' oksida (8.4%), 'caryophyllene' (6.4%), 'germacrene'
D (4.2%), δ-cadinena (5.6%) dan eugenol metil eter (2.3 %). Manakala sebatian
kimia utama lain minyak pati stamen ialah 'α-bergamotene' (12.5%), β-elemen
(4.9%), 'germacrene-D' (4.0%), 'δ-cadinene' (4.8%), 'caryophyllene' (3.4%) dan
'caryophyllene' oksida (2.9 %). Sebatian utama minyak pati daripada kultur kalus
ginoesium dan stamen tidak mempunyai perbezaan yang ketara dari ginoesium dan
stamen bunga segar M. alba.
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Kajian ini menunjukkan bahawa pengeluaran in vitro minyak pati dari M. alba
melalui kultur kalus eksplan gynoecia dan stamen mempunyai potensi dalam
pengeluaran minyak wangi secara besar-besaran menggantikan pengeluaran bunga
segar yang bermusim. Di samping itu, minyak pati M. alba mempunyai potensi
sebagai agen antimikrob dan antioksidan yang berguna dalam industri kosmetik.
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ACKNOWLEDGEMENTS
Alhamdulillah.
Thank you Allah for showing me the path of your endless knowledge. And to Allah
belongs whatever is in heaven and whatever is on earth.
The earth He laid out for the creatures. Therein is fruit and palm trees having sheaths
of dates. And grains having husks and scented plants. Ar Rahman.
Great appreciation goes to Associate Professor Dr Janna for having me as her
student. Her willingness, guidance, motivation, suggestion and assistance has helped
me towards realizing the completion of this study. Appreciation also goes to
Professor Dr Mohd Aspollah Hj Sukari and Associate Professor Dr Maheran Abdul
Aziz, who have served as supervisory committee members from the beginning.
Appreciation also goes to my former supervisor Associate Professor Dr Radzali
Muse for his guidance, kindness advise, and assistance during my experimental
work. I pray sincerely for his speedy recovery and good health.
My gratitude is also extended to the Malaysian government and Universiti Teknologi
MARA for supporting my studies and to Universiti Putra Malaysia and MARDI for
supporting my research project.
My sincere thanks also goes to all lecturers, officers, technicians and laboratory
assistants in the departments of Biochemistry and Microbiology, Faculty of
Biotechnology and Biomolecular Sciences, and the Department of Chemistry,
Faculty of Science, in helping me directly or indirectly.
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Lastly, thanks to all the postgraduate students graduated from the Natural Product
Laboratory and my office mates in UiTM Negeri Sembilan for their cooperation,
patience and support. I cannot leave this space without expressing my appreciation to
my family and to my two beloved 'PhD born' sons who inculcate me challenges and
inspiration and I am grateful for all the intention and encouragement.
May Allah bless all of you.
.
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I certify that an Examination Committee has met on 8 January 2013 to conduct the
final examination of Nor'aishah binti Abu Shah on her PhD thesis entitiled
"Chemical constituents and biological activities of essential oil from Chempaka
(Michelia alba De Candolle)" in accordance with Universities and University
Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 10]
15 March 1998. The Committee recommends that the student be awarded the Doctor
of Philosophy.
Members of the Thesis Examination Committee were as follows:
Name of Chairperson, PhD
Prof. Dr. Norhani bt Abdullah
Laboratory of Animal Production
Institute of Tropical Agriculture
Universiti Putra Malaysia
Name of Examiner 1, PhD
Dr. Syahida binti Ahmad
Department of Biochemistry
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
Name of Examiner 2, PhD
Assoc. Prof. Dr. Muhajir bin Hamid
Department of Microbiology
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
Name of External Examiner, PhD.
Prof. Dr. Deepthi Chandrika Bandara
Department of Agriculture Biology
Faculty of Agriculture
University of Paradeniya
Peradeniya Peradeniya
Ceylon
_________________________
SEOW HENG FONG, PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Doctor of Philosophy.
The members of the Supervisory Committee were as follows:
Janna Ong Abdullah, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Mohd Aspollah Hj Sukari, PhD
Professor
Faculty of Science
Universiti Putra Malaysia
(Member)
Maheran Abdul Aziz, PhD
Associate Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Member)
____________________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis is my original work except for quotations and citations which
have been duly acknowledged. I also declare that it has not been previously and is
not concurrently, submitted for any other degree at Universiti Putra Malaysia or at
any other institution.
______________________________
NOR'AISHAH BINTI ABU SHAH
8 January 2013
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TABLE OF CONTENTS
Page
DEDICATION ii
ABSTRACT iii
ABSTRAK vii
ACKNOWLEDGEMENT xii
APPROVAL xiv
DECLARATION xvi
LIST OF TABLES xx
LIST OF FIGURES xxii
LIST OF APPENDICES
LIST OF ABBREVIATIONS
xxiv
xxv
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 5
2.1 Botanical aspects of Michelia alba DC
2.2 Plants volatiles
2.2.1 Volatile compounds
2.2.1.1 Terpenoids
2.2.1.2 Importance of terpenoids
5
11
12
13
14
2.2.2 Change in floral scent compositions
2.2.3 Site of biosynthesis and secretion
17
18
2.3 Volatile metabolism in plants
2.4 Monoterpenoids
2.4.1 Chemical structures of monoterpenoids
2.4.2 Monoterpenoids distribution
20
24
25
26
2.5 Extraction of volatile compounds
2.5.1 Traditional extraction of volatile oils
2.5.2 Conventional extraction technique
2.5.3 New extraction techniques
2.5.3.1 Supercritical and subcritical fluid extraction
2.5.3.2 Direct sampling of volatiles
26
27
28
30
31
33
2.6 Exploitable properties of essential oil
2.6.1 Antioxidant properties
2.6.2 Antimicrobial activities
2.6.3 Insecticidal activities
2.6.4 Termiticidal activities
2.6.5 Allelopathic properties
2.6.6 Adverse effect
34
34
37
39
41
42
42
2.7 In vitro production of secondary metabolites
43
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3 CHEMICAL COMPOSITIONS OF ESSENTIAL OILS FROM
LEAF AND FLOWER OF Michelia alba 3.1 Introduction
52
52
3.2 Methodology
3.2.1 Determination of leaf and flower growth and
development
3.2.2 Collection of plant samples
3.2.3 Isolation of the oils
3.2.4 Chemical analysis by Gas Chromatography -Mass
Spectrometry
54
54
54
55
55
3.3 Results and discussion
3.3.1 Growth development of leaf and flower of M. alba
3.3.2 Chemical composition of leaf and flower M. alba
3.3.3 Chemical profiles within each flower developmental
stages
3.3.4 Chemical profiles in specific flower parts
56
56
62
71
82
3.4 Conclusion
90
4 BIOLOGICAL ACTIVITIES OF ESSENTIAL OILS OF
Michelia alba 4.1 Introduction
92
92
4.2 Methodology
4.2.1 Essential oil preparation
4.2.2 Antimicrobial assay
4.2.3 Evaluation of antioxidant activity
94
94
94
96
4.3 Results and discussion
4.3.1 Antibacterial activity
4.3.2 Antifungal activity
4.3.3 Antioxidant activity
98
98
109
114
4.4 Conclusion
126
5 FRAGRANCE PRODUCTION FROM CALLUS CULTURES
OF Michelia alba
5.1 Introduction
129
129
5.2 Methodology
5.2.1 Explant preparation
5.2.2 Media preparation
5.2.3 Establishment of callus cultures
5.2.4 Establishment of suspension cultures
5.2.5 Callus proliferation for phytochemical induction
5.2.6 Extraction of essential oil from callus cultures
5.2.7 Analysis of volatile compounds from callus cultures
132
132
134
134
135
136
136
137
5.3 Results and discussion
5.3.1 Effect of explants position
5.3.2 Effect of illumination
5.3.3 Effect of basal media
5.3.4 Effect of plant growth regulators in different explants
5.3.5 Induction of organogenesis
5.3.6 In vitro regeneration of M. alba
5.3.7 Phytochemical evaluation of organogenic calli
137
138
140
142
145
148
153
157
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5.4 Conclusion
162
6 SUMMARY, GENERAL CONCLUSIONS AND
RECOMMENDATION FOR FUTURE RESEARCH
6.1 Summary
6.2 General conclusion
6.3 Recommendations for future research
165
165
174
175
REFERENCES 177
APPENDICES 214
BIODATA OF STUDENT 223
