universiti putra malaysia chemical composition …psasir.upm.edu.my/33088/1/fbsb 2012...

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


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


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


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


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



(Internal Examiner)

Syahida Binti Ahmad, PhD

Lacturer



(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


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



(Chairman)

Muhajir Hamid, PhD

Associate Professor



(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


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.

_____________________________________


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

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