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UNIVERSITI PUTRA MALAYSIA
SITI HUMEIRAH BINTI ABDUL GHANI
FBSB 2012 28
CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITIES OF ESSENTIAL OILS FROM Goniothalamus AND Xylopia
(ANNONACEAE)SPECIES
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CHEMICAL COMPOSITIONS AND BIOLOGICAL
ACTIVITIES OF ESSENTIAL OILS FROM
Goniothalamus AND Xylopia (ANNONACEAE)
SPECIES
SITI HUMEIRAH BINTI ABDUL GHANI
MASTER OF SCIENCE
UNIVERSITI PUTRA MALAYSIA
2012
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CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITIES OF
ESSENTIAL OILS FROM Goniothalamus AND Xylopia (ANNONACEAE)
SPECIES
By
SITI HUMEIRAH BINTI ABDUL GHANI
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in
fulfillment of the requirements for the Degree of Master of Science
March 2012
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In the name of ALLAH, the Beneficent, the Merciful
This thesis is dedicated to my beloved:
Dad (Ab Ghani bin Sudin)
Mom (Salmiza binti Ujang@ Baharin)
Brothers (Mohd Qayyum & Zhafri)
Supervisors (DR Puad, DR Muhajir, DR Nor Azah)
Datuk DR Latiff, DR Rasadah, Pn. Wan Rahmah, Pn
Liza Ismail, Pn Ruziah Ripin, Che Rohani and also all
Staff of Natural Products Division, FRIM
Friends
Zulkifli Anuar (Ajoi)
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of
the requirement for the degree of Master of Science
CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITIES OF
ESSENTIAL OILS FROM Goniothalamus AND Xylopia (ANNONACEAE)
SPECIES
By
SITI HUMEIRAH BINTI ABDUL GHANI
March 2012
Chairman : Associate Professor Mohd Puad Bin Abdullah, PhD
Faculty : Biotechnology and Biomolecular Sciences
The use of plant essential oil in daily care products, cosmetics and fragrances has
increasingly gained attention in recent years. However, detailed studies on the oil
especially in the aspects of chemical compositions, biological activity, safety and
efficacy as a raw material for daily care products are limited thus limiting its commercial
potential.
The essential oils of five plant species from the family Annonaceae, namely
Goniothalanus macrophyllus, G. tortilipetalus, Xylopia malayana, X. fusca and X.
elliptica were studied and their chemical compositions were analyzed by Gas
Chromatography (GC) and Gas Chromatography/Mass Spectrometry (GC/MS). The leaf
oil from X. malayana (1.39%) which was obtained from a water distillation produced the
highest yield of essential oil. The chemical compositions of the essential oils from both
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species were analyzed by comparing their Retention Indices (RI) with the literature
values, by comparing their mass spectral data with the mass spectral database from the
published or by co-chromatography with authentic samples. This is the first report on the
chemical compositions, antimicrobial activities and cytotoxicity effects of the essential
oils from G. tortilipetalus, X. fusca and X. elliptica. The chemical constituents of the oils
differed from species to species but were mainly of monoterpenoids and
sesquiterpenoids. A total of 239 components were identified, representing 42.5% to
91.6% of the total oils. The main monoterpenoids were α-pinene, linalool, terpinen-4-ol,
1,8-cineole and α-terpineol while geranyl acetate, geraniol, bicyclogermacrene and
spathulenol are the major sesquiterpenoids identified from the oils.
The essential oils together with eight standards: α-pinene, linalool, geraniol, geranyl
acetate, terpinen-4-ol, limonene, 1,8-cineole and β-pinene were evaluated for their
antimicrobial properties against nine microorganisms including Gram positive and Gram
negative bacteria, and yeast by using the broth microdilution method. The essential oils
exhibited antimicrobial activities (MIC values between 156-2500 µg/ml) against all of
the microorganisms tested, except for Pseudomonas aeruginosa and Escherichia coli.
The standards tested exhibited weak antimicrobial activities (MIC values >5000 µg/ml)
against all of the microorganisms tested except for α-pinene.
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The cytotoxicity of the oils was evaluated against the human normal Chang liver and
human normal skin fibroblast cell lines. The essential oils from the twig of X. elliptica
and the leaf of G. tortilipetalus were found to be non-cytotoxic towards the Chang liver
cell line even at higher concentrations (IC50 >1000 μg/ml). The other oils, however,
exhibited moderate to weakly cytotoxic activity against both cell lines with the IC50
values ranged from 97.7 μg/ml to 877.0 μg/ml. The study revealed the potential use of
the essential oils from ariel parts of Xylopia and Goniothalamus species as a new
ingredient for various practical applications.
Key Word: Annonaceae; Essential oil; Antimicrobial activity; Cytotoxicity activity
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk Ijazah Master Sains
KOMPONEN KIMIA DAN AKTIVITI BIOLOGI TERHADAP MINYAK PATI
DARI SPESIS Xylopia DAN Goniothalamus (ANNONACEAE)
Oleh
SITI HUMEIRAH BINTI ABDUL GHANI
Mac 2012
Pengerusi : Profesor Madya Mohd Puad Bin Abdullah, PhD
Fakulti :Bioteknologi dan Sains Biomolekul
Penggunaan minyak pati daripada tumbuhan beraroma dalam produk penjagaan harian,
barangan kosmetik dan haruman semakin mendapat perhatian masyarakat kebelakangan
ini. Namun demikian, kajian terperinci mengenai minyak pati khususya dari segi
komposisi kimia, aktiviti biologi, keselamatan serta keberkesanannya sebagai bahan
mentah dalam produk penjagaan harian adalah sangat sedikit sekaligus menghadkan
penggunaannya secara komersial.
Minyak pati daripada lima spesis pokok keluarga Annonaceae iaitu Goniothalamus
macrophyllus, G. tortilipetalus, Xylopia malayana, X. fusca dan X. elliptica telah dikaji
dan komponen kimianya telah dianalisis menggunakan teknik Kromatografi Gas (KG)
dan Kromatografi Gas/Spektrometer Jisim (KG/SJ). Proses penyulingan air terhadap
sampel daun dari X. malayana telah menghasilkan peratusan minyak pati yang tertinggi
iaitu sebanyak 1.39%. Komponen kimia minyak pati telah dikenal pasti melalui
pengiraan dan perbandingan nilai Indeks Penahanan (RI) dengan nilai daripada literatur,
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ko-kromatografi dengan sampel tulen serta perbandingan spekrum jisim yang diperolehi
terhadap spekrum jisim rujukan. Hasil kandungan kimia, aktiviti antimikrob serta kesan
sitoksiksiti daripada minyak pati sampel G. tortilipetalus, X. elliptica dan X. fusca adalah
pertama kali dilaporkan dalam kajian ini. Analisis kimia menunjukkan bahawa
komponen minyak pati kedua-dua spesis adalah berbeza dan terdiri daripada sebatian
monoterpenoid dan seskuiterpenoid. Sebanyak 239 komponen telah dikenalpasti iaitu
merangkumi sebanyak 42.5% hingga 91.6% daripada jumlah keseluruhan minyak pati
yang dikenalpasti. Kumpulan monoterpena utama adalah α-pinena, linalol, terpinen-4-ol,
1,8-sineol dan α-terpineol manakala geranil asetat, geraniol, bisiklogermakrena dan
spatulenol merupakan kumpulan sesquiterpena yang tertinggi dalam minyak yang dikaji.
Ujian antimikrob terhadap sembilan jenis mikroorganisma yang terdiri daripada bakteria
Gram positif, Gram negatif dan yis telah dijalankan ke atas minyak pati serta lapan
rujukan piawai iaitu α-pinena, linalol, geraniol, geranil asetat, terpinen-4-ol, limonena,
1,8-sineol, β-pinena melaui kaedah pencairan mikro berkaldu. Keputusan ujian
antimikrob menunjukkan bahawa minyak pati mempunyai aktiviti perencatan (nilai MIC
adalah diantara 156-2500 µg/ml) terhadap mikroorganisma yang diuji kecuali bakteria
Pseudomonas aeruginosa dan Escherichia coli. Semua piawaian tunggal kecuali α-
pinena menunjukkan aktiviti perencatan yang lemah (nilai MIC adalah >5000 µg/ml)
terhadap semua mikroorganisma yang diuji.
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Ujian sitotoksik telah dijalankan ke atas sel hati Chang dan sel kulit fibroblast. Minyak
pati daripada ranting X. elliptica dan daun G. tortilipetalus didapati tidak menunjukkan
kesan sitotoksik terhadap sel hati Chang walaupun pada tahap kepekatan yang tertinggi
(IC50 >1000 μg/ml). Minyak pati daripada spesis yang lain menunjukkan kesan
sitotoksik yang sederhana dan rendah terhadap kedua-dua sel yang diuji di mana nilai
IC50 adalah secara meningkat iaitu dari 97.7 μg/ml hingga 877.0 μg/ml. Kajian ini
menunjukkan bahawa minyak pati daripada spesis Xylopia dan Goniothalamus
mempunyai potensi untuk diaplikasikan dalam menghasilkan barangan untuk kegunaan
harian.
Kata Kunci: Annonaceae; Minyak Pati; Aktiviti Antimikrob; Aktiviti Sitotoksik
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ACKNOWLEDGEMENTS
In the name of Allah, only by His permission, grace and mercy this thesis can be
completed. First of all, I would like to extend my appreciation and most sincere
acknowledgement to my supervisory committee: Assoc. Prof. Dr. Mohd Puad bin
Abdullah (chairman), Assoc. Prof. Dr. Muhajir Hamid and Dr Nor Azah Mohd Ali (the
one responsible for the original idea of this research) for their professional guidance,
encouragement and constructive criticisms from the beginning of this research till the
final review of the thesis.
I would like also to express my sincere thanks to Dr. Mastura Mokhtar, Mr. Saiful Azmi
Johari, Mrs. Rohana Rashdan, Mr. Mohd Jemain from Bioactivity Programme, Natural
Products Division, FRIM for their technical support and discussion on the antimicrobial
and cytotoxicity aspects of the study. Much appreciation also goes to Dr. Zainah Adam
and Mr Beh Jo Ee from Animal Cell Culture laboratory, FBSB, UPM for lending me
their precious time to help me in the cytotoxicity analysis and discussion. I wish to
express my sincere thanks to FRIM for the financial support provided through the
Research fellowship scheme (RA-Scheme) and special thanks also due to the
Government of Malaysia under the MOSTI grant (Project number: 02-03-10-SF0047)
for supporting this study.
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Last but not least, I am very indebted to my beloved families: Ab Ghani bin Sudin
(papa), Salmiza binti Baharin@ Ujang (mama), Zhafri (dik Rung) and Mohd Qayyum
for their understanding and constant support throughout my study. I also would like to
extent my thanks to my beloved friends; Zulkifli Anuar (Ajoi) for their constant love and
support and also for Mrs Mailina, Husni, Majid, Aiman, Nik Yasmin, Faridz, Irman,
Qayyum, Ina, Nad Iah, Nomy, Adiana, Rashid Li and names which may not mentioned
for making my time an enjoyable one.
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I certify that a thesis Examination Committee has met on 5 Mac 2012 to conduct the
final examination of Siti Humeirah Binti Abd Ghani on her Master Degree thesis entitled
“Chemical compositions and biological activities of essential oils from Goniothalamus
and Xylopia (Annonaceae) species” in accordance with the Universities and University
Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106]
15 March 1998. The Committee recommends that the student be awarded the Master of
Science.
Members of the Examination Committee were as follows:
Janna Ong Binti Abdullah, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Shuhaimi Bin Mustafa, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Syahida Binti Ahmad, PhD
Lacturer
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Nor Hadiani Ismail, PhD
Professor
Faculty of Applied Science
Universiti Teknologi Mara
Malaysia
(External Examiner)
____________________________
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 fulfillment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows:
Mohd Puad Abdullah, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Muhajir Hamid, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Nor Azah Mohamad Ali, PhD
Senior Research Officer
Natural Product Division
Forest Research Institute of 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 University Putra Malaysia or other
institutions.
_____________________________________
SITI HUMEIRAH BINTI ABDUL GHANI
Date: 5 March 2012
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TABLE OF CONTENTS
Page
DEDICATION i
ABSTRACT ii
ABSTRAK v
ACKNOWLEDGEMENTS viii
APPROVAL x
DECLARATIONS xii
LIST OF TABLES xvi
LIST OF FIGURE xviii
LIST OF ABREVIATIONS xxi
CHAPTER
1 INTRODUCTION 1
1.1 Research Justifications 4
1.2 Objective of the studies 5
2 LITERATURE RIVIEW 6
2.1 Definition of Essential Oils 6
2.1.1 Uses of essential oil 7
2.1.2 Extraction and analysis of essential oil 8
2.2 The family Annonaceae 10
2.3 The Genus Goniothalamus 11
2.3.1 Traditional Medicinal Values of 12
Goniothalamus plants
2.3.2 Previous Works on Genus Goniothalamus 13
2.4 The Genus Xylopia 18
2.4.1 Traditional Medicinal Values of 19
Xylopia plants
2.4.2 Previous Work on Genus Xylopia 20
2.5 Botanical aspects of the plants study 29
2.5.1 The species Xylopia malayana 29
Hk. F.et. Thoms.
2.5.2 The species X. elliptica 30
Maing. ex Hk. & Thoms.
2.5.3 The species X. fusca 31
Maingay ex Hk. f. et Thomas.
2.5.4 The species Goniothalamus macrophyllus 32
(Blume) Hook.f. & Thomson
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2.5.5 The species G. tortilipetalus Henderson. 33
3 MATERIAL AND METHODS 34
3.1 Material 34
3.1.1 Plant materials 35
3.1.2 General instrument 35
3.1.2.1 Clavenger Types and 35
Dean-stark apparatus
3.1.2.2 Gas Chromatography (GC) 35
3.1.2.3 Gas Chromatography-Mass 35
Spectrometry (GC-MS)
3.1.2.4 ELISA Reader 35
3.1.3 Chemical and Reagents 36
3.1.4 Bacterial and Yeast Strains 36
for Antimicrobial Assay
3.1.5 Cell lines for cytotoxicity assay 37
3.2 Methods 39
3.2.1 Sampling Methods 39
3.2.2 Extraction of Essential Oils 39
3.2.3 Azeotropic process 40
3.2.4 Determination of Chemical 41
Composition of the Essential Oils
3.2.5 Antimicrobial Assay 44
3.2.5.1 Maintenance of bacteria 44
and yeast strains
3.2.5.2 Preparation of bacteria 44
and yeast inoculate
3.2.5.3 Preparation of sample 45
stock solution
3.2.5.4 Minimum Inhibitory 45
Concentration (MIC)
3.2.6 Cytotoxicity Assay 46
3.2.6.1 Maintenance of Cell Culture 47
3.2.6.2 Preparation of stock solution 48
3.2.6.3 Cell Viability Assay 48
3.2.7 Data Analysis 49
4 RESULTS AND DISCUSSION 50
4.1 Yield and physical appearance 50
of the essential oils
Analysis and identification of 53
essential oils study
4.2 Chemical composition of essential 54
oils from Goniothalamus sp.
4.2.1 The leaf oils of G. macrophyllus 54
4.2.2 The twig oils of G. macrophyllus 56
4.2.3 The root oils of G. macrophyllus 58
4.2.4 The leaves oils of G. tortilipetalus 60
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4.2.5 Chemical variation in the 62
essential oils of Goniothalamus species
4.2.5.1 Chemical variation in the leaves oils 62
of Goniothalamus species
4.2.5.2 Chemical variation in the twig and 64
root oil of G. macrophyllus 4.3 Essential oils from Xylopia sp. 66
4.3.1 The leaves oils of X. elliptica 66 4.3.2 The twig oils of X. elliptica 68 4.3.3 The leaves oils of X. fusca 70
4.3.4 The twig oils of X. fusca 72
4.3.5 The leaves oils of X. malayana 73
4.3.6 The twig oils of X. malayana 76
4.4 Chemical variation in the essential 79
oils of Xylopia species
4.4.1 Chemical variation between the essential oils 84
from Goniothalamus and Xylopia species with
another genera
4.4.2 Antimicrobial activities 85
4.5 Cytotoxicity activities 91
5 CONCLUSION 95
REFERENCES 98
APPENDICES 108
BIODATA OF STUDENT 119
LIST OF PUBLICATIONS 120