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ESSENTIAL OILS FROM MICHELIA ALBA, MICHELIA CHAMPAKA • AND JASMINUM SAMBAC
HABSAH MOHAMAD
This dissertation is submitted in partial fulfillment of the requirement for
the degree of Bachelor ofEducation with Honours
in Chemistry
Program of Resource Chemistry
Faculty of Resource Science and Technology
UNIVERSITY MALAYSIA SARA WAK
April 2005
ACKNOWLEDGEMENTS
... Bismillahirrahmanirrahim...
Firstly, I would like to express profound gratitude and sincere appreciation to my
supervisor, Assoc. Prof. Dr. Zaini B. Assim for his guidance and invaluable advice
during the preparation and execution of the research and also for my co-supervisor,
Assoc. Prof. Dr. Fasihuddin B. Ahmad and Mr. Chieng Tion Chin, for their substantial
contribution towards the completion of this research project.
I also would like to express special thanks and profound gratitude to the other lecturers
from Programme of Resource Chemistry and all laboratory assistants, especially to Mr.
Rajuna B. Tahir and Mr. Jahina, for their help throughout this project. I would like
aknowledgde Madam Durie Augustine and Mr. Mohd. Norizam from Dewan Bandaraya
Kuching Utara, Kuching for the permission given to us to collect a fresh plant samples
for this project. Special recognition is given to Mr. Ng. Siaw Chiung and Mr. Lee Choon
Lip from Pepper Marketing Board, Kuching for their invaluable help with the Gas
Chromatography/Mass Spectrometry analysis.
Not forgetting my dear friends, Haslina Marzoki, Madiana Bakar, Juhsep Langi and
Dayang Monira, as they are wonderful team to work with. A very special thanks to my
mom, Mdm Salus Bt. Amit, brothers; Mr. Daud and Mr. Abd. Rahman, sisters; Ms.
Hamidah and Ms. Amanah. Last but not least, my dearest husband, Mr. Mohd. Zulfikar
B. Dahlan for his financial support, encouragement, valuable advices and support, care
and love throughout my three years in UNIMAS.
Wassallam.
'1."
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,.. . ,
ABSTRACT
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Chemical components for essential oils from flowers, leaves, root and fiuits of M. alba. M. champaka and J. sambac were extracted using hydrodistillation method. The essential oils were then analysed using gas chromatography-mass spectrometry (GCfMS) and gas chromatography-flame ionization detector (GCIFID). Percentages of essential oils in all parts within species were in the ranged between 0.2% (v/w) - 3.9% (v/w) and the flowers ofM. alba gave the highest percentage ofalL The major component identified in essential oils for flowers and leaves of M alba was artemisia alcohol with 52.21 % and 56.94%, respectively. Cadina-l,4-dien-3-01 was a major component identified in root oil of M. alba. The major components identified in essential oils for flowers, leaves, root and fiuits of M. champaka was lauric aldehyde (33.15%), coumarin (28.07%), decyl alcohol (52.13%) and methylene bis (17.42%), respectively. The toxicity test toward Artemia salina showed that essential oils from leaves ofM. champaka gave the highest toxicity with LCso at 28.2 llg/mL.
Keywords: Essential oils, M alba, M. champaka, hydrodistillation, gas chromatographymass spectrometry, gas chromatography-flame ionization detector
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ABSTRAK
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Komponen kimia bagi minyak pati dart bahagian bunga. daun. akar dan buah dartpada spesies M alba, M. champaka dan J. sambac telah diekstrak dengan menggunakan kaedah penyulingan hidro. Hasil minyak pall telah dianalisis dengan menggunakan kromatograji gas-spektrometer jisim (KG/SJ) dan kromatogra/i gas- nyalaan pengesan ion (KG/NPI). Peratus minyak paa dalam kesemua spesies yang dikaji berada dalam julat 0.2% (v/i1;J - 3.9% (v/i1J dan bahagian bunga daripada spesies M. alba memberikan peratusan minyak pati yang terlinggi. Komponen kimia utama minyak pali pada bahagian bunga dan daun bagi spesies M. alba adalah alkohol artemisia dengan 52.21% dan 56.94%. masing-masingnya. Manakala. pada bahagian akar spesies M. alba pula. komponen kimia utamanya adalah kadina-1,4-dten-3-01 (31.79%). Komponen kimia utama minyak pall pada bahagian bunga, daun, akar dan buah bagi spesies M champaka adalah laurikaldehida (33.15%). koumarin (28.07%), alkohol desil (52.13%) dan metylena bis (17.42%), berturut-turut. Ujian ketoksikan terhadap Artemia salina memmjukkan bahawa minyak patl daripada bahagian daun bagi spesies M champaka memberikan kesan ketoksikan paling tinggi dengan nilai LC50 pada kepekatan 28.2 JiglmL.
Kala kunci: Minyak pali, M alba, M. champaka, penyulingan htdro, kromatograji gas spektrometer jisim, kromalograji gas- nyalaan pengesan ion
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TABLE OF CONTENTS
t
Declaration
Acknowledgement
Abstract
Abstrak
Table ofContents
List ofTables
List of Figures
CHAPTER ONE
1.1
1.2
CHAPTER TWO
2.1
2.2
2.3
2.4
2.5
2.6
11
III
iv
v
Vlll
IX
INTRODUCTION
General Introduction 1
Objectives of the Project 3
LITERATURE REVIEW 4
Introduction 4
Commercial Importance ofEssential Oils 4
Michelia spp. 5
Jasmine sp. 6
Extraction 7
2.5.1 Hydrodistillation 7
Fractionation 8
v
2.7
2.8
CHAPTER THREE
3.1
3.2
3.3
3.4
3.5
3.6
2.6.1 Gas Chromatography 9
2.6.2 Identification 9
Chemometric Analysis 10
Bioassay 11
MATERIALS AND METHOD 13
Sampling 13
Extraction ofEssential Oils 13
Gas Chromatography Analysis 14
3.3.1 Gas Chromatography - Flame Ionization
Detector (GCIFID) 15
3.3.2 Gas Chromatography - Mass Spectroscopy
(GC/MS) 15
Qualitative and Quantitative Analysis 16
3.4.1 Percentage ofEssential Oils 16
3.4.2 Qualitative Analysis 17
3.4.3 Semi quantitative Analysis 18
Chemometric Analysis 18
3.5.1 Cluster Analysis 19
Biological Activity ofEssential Oils 19
3.6.1 Toxicity Activities to Artemia salina 19
VI
CHAPTER FOUR RESULT AND DISCUSSION 21
4.1 Abundance ofEssential Oils in Michelia spp.
and J. sambac 21
4.2 Identification ofChemical Components in
Essential Oils 23
4.3 Chemical Components ofthe Essential Oils from
M. alba 26
4.4 Chemical Components ofthe Essential Oils from
M champaka 35
4.5 Similarity ofChemical Contents ofEssential Oils
between M. alba and M. champaka 45
4.6 Chemical Components of the Essential Oils in J. sambac 47
4.7 Cluster Analysis 54
4.8 Bioassay Against Brine Shrimp 55
CHAPTER FIVE CONCLUSION 58
REFERENCES 59
APPENDICES
Vll
LIST OF TABLES
Table 1 Percentage yield and physical properties ofessential oils 22
Table 2 Retention time for alkane based on GC/FID data, calculated
and predicted using graph 25
Table 3 Chemical components ofessential oils from M alba 28
Table 4 Chemical components ofessential oils from M. champaka 36
Table 5 The chemical components identified in essential oils for both
M. alba and M champaka 42
Table 6 Chemical components ofessential oils from 1. sambac 46
Vlll
LIST OF FIGURES
Figure 1 Gas chromatogram ofn-alkane standard analyzed using GC/MS 24
Figure 2 Gas chromatogram ofn-alkane standard analyzed using GC/FID 24
Figure 3 Gas chromatogram ofthe flowers oils from M. alba analyzed
using GC/FID 27
Figure 4 Gas chromatogram ofthe flowers oils from M alba analyzed
using GC/MS 27
Figure 5 Gas chromatogram ofthe leaves oils from M alba analyzed
using GC/FID 28
Figure 6 Gas chromatogram ofthe leaves oils from AI. alba analyzed
using GC/MS 28
Figure 7 Gas chromatogram ofthe root oils from M. alba analyzed
using GC/FID 29
Figure 8 Gas chromatogram ofthe leaves oils from M. alba analyzed
using GC/MS 29
Figure 9 Some of the structure of the chemical components identified
in essential oils ofM. alba 33
Figure 10 Gas chromatogram ofthe flowers oils from M. champaka
analyzed using GC/FID 36
Figure 11 Gas chromatogram of the flowers oils from M champaka
analyzed using GC/MS 36
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•
Figure 12 Gas chromatogram of the leaves oils from M champaka
analyzed using GCIFID 37
Figure 13 Gas chromatogram ofthe leaves oils fromM. champaka
analyzed using GCIMS 37
Figure 14 Gas chromatogram ofthe root oils from M. champaka
analyzed using GCIFID 38
Figure 15 Gas chromatogram ofthe root oils from M. champaka
analyzed using GCIMS 38
Figure 16 Gas chromatogram of the fruits oils from M champaka
analyzed using GCIFID 39
Figure 17 Gas chromatogram ofthe fruits oils from M champaka
analyzed using GCIMS 39
Figure 18 Some ofthe structure of the chemical components identified
in essential oils ofM. champaka 43
Figure 19 Some ofthe structure ofthe chemical components identified
in essential oils ofboth species 45
Figure 20 Gas chromatogram ofthe flowers oils from J. sambac
analyzed using GCIFID 49
Figure 21 Gas chromatogram of the flowers oils from J. sambac
analyzed using GC/MS 49
Figure 22 Gas chromatogram ofthe leaves oils from J. sambac
analyzed using GCIFID 50
x
Figure 23 Gas chromatogram ofthe leaves oils from J. sambac
analyzed using GelMS 50
Figure 24 Some ofthe structure ofthe chemical components identified
in essential oils ofJ. sambac 53
Figure 25 Dendogram ofcluster analysis ofM. alba parts extracted
using quantitative analysis as data 54
Figure 26 Dendogram ofcluster analysis ofM champaka parts extracted
Using quantitative analysis as data 54
Figure 27 Toxicity test of M. alba against Artemia Salina 56
Figure 28 Toxicity test of M. champaka against Artemia Salina 57
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CHAPTER ONE
INTRODUCTION
1.1 General Introduction
Essential oils are known as aromatic substances produced by specific plant species.
Most of these oils have been used as raw materials for fragrance and flavoring agents
since ancient times. The substance was called essential oil because it was thought that
each oil represented the essence ofthe original plant (Nakatsu et at., 2000).
Essential oils can be extracted from several parts of plant including flowers, leaves,
fruits, fruit peel, seeds twigs, stems and roots (Nakatsu et al., 2000). There are several
methods can be used to extract essential oils from plant. The techniques used
commonly to isolate essential oils from leaf material are steam or hydrodistillation and
solvent extraction (Milner et al., 1997).
Essential oils are the most studied chemical compounds in plants as regards their
composition and physical and chemical properties. Advances in organic chemistry
have allowed for the establishment of techniques to define the component profiles of
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many aromas and fragrances that permit the establishment of composition standards
for trade regulations and for the synthesis of aromas using less costly starting raw
materials (Hernandez, 2000). Moreover, aromatic plants are of interest because many
of these essentials oils obtained from them have a valuable properties and can have
several usage (Arrebola, 1997).
Several aromatic ornamental flowers around Kuching were investigated. However, as
a preliminary study two species in genus on Magnolia, M. alba and M. champaka
were studied in details. The study involved characterization of essential oils and the
toxicity activities against A. salina. The scent of M alba and M. champaka can be
considered strong and its indicate that it may contain a considerable quality of
essential oils. There is also limited information and studies on essential oils from this
two species. Therefore this two species were selected for characterization ofessential
oils composition and also to explore the potential ofthese essential oils for these two
speCIes.
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1.2 Objectives of the Project
The main aim of this project were to extract essential oils from the flowers, leaves,
roots and fruits of the M alba and M. champaka and subsequently characterize the
chemical composition ofessential oils extracted. The other objective was to carry out
statistical analysis on essential oils in order to find out whether it has a significance
value for chemotaxonomy purpose. Information on toxicity activities against A. salina
ofessential oils from M alba and M. champaka were documented.
The other objectives were to extract essential oils from Jasminum Sambac flowers and
its leaves as a comparison between the results obtained from the M alba and M
champaka.
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CHAPTER TWO
LITERATURE REVIEW
2.1 Introduction
Essential oils are natural volatile substances found in a variety of plants. When
isolated from plants, essential oils are not usually extracted as chemically pure
substances, but consist of mixtures of many compounds. It is well known that plant
derived natural products are extensively used as biologically active compounds.
Among them, essential oils were the first preservatives used by man, originally in their
natural state within plants tissue and then as oils obtained by hydrodistillation.
Essential oils composed of isoprenoid compounds, mainly monoterpenes and
sesquiterpenes where are responsible in generating odor for the aromatic plants
(Franzios et al., 1997).
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2.2 Commercial Importance of Essential Oils
Commercially, essential oils are used in four primary ways; as phannaceuticals, as
flavor enhancers in food products, as odorants in fragrances and as insecticides. For
example, the leaf and bark essential oils of C. japonica are have a potential as
larvicides against Aedes aegypti larvae (Cheng et al., 2003). Other uses of essential
oils are to add flavors to foodstuffs and beverages and to scent perfumes, lotions,
soaps, detergents and household cleaners (Hernandez, 2000). Essential oils compose
of many types or classes of molecules including terpenoids, phenolics, aromatics,
cyclic and acyclic compounds, acetonides, and sulfur and nitrogen containing
compounds, depending on the plant and the extraction method (Nakatsu et aI., 2000).
For instance, essential oils from the flowers ofMagnolia sieboldii has a positive effect
on lipopolysaccharide (LPS) - induced production of nitric oxide (NO) and
prostaglandin (POE2) by rat peritoneal macrophages (Soon et al., 2001).
2.3 Michelia spp.
The Magnoliaceae is a small family of 10 genera with approximately 100 species.
Over 70 species are included in the genus Magnolia. All of these species are woody
trees or shrubs with simple alternate and large bisexual flowers (Novak, 1967). There
are only two species in the genus ofMagnolia are found in Sarawak. They are White
5
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Cempaka (Michelia alba) and Orange Cempaka (Michelia champaka). These two
species are believed to origenate from .lava and India. It is a medium-size tree up to 20
m but usually the size is smaller ifplanted. The Cempaka is well known because ofits
very fragrant flowers (Chai, 1984).
M. alba or Cempaka Putih has white and very fragrant flowers, and the fruitlets in one
bunch are fused into one aggregate fruit. On the other hand, M. champaka or Cempaka
Merah has an orange or yellow fragrant flower and is sometimes knowns as Cempaka
Kuning. The fruits are hanging in grape like bunches. However, this species is less
common in Malaysia. The Cempaka is well known because of its very fragrant. The
flowers produce in great quantities and may be seen in villages and some house
garden. It is easily raised from seeds and requires some shade to grow well. Many
applications have recorded for these plants. The essential oils extracted from the
flowers ofCempakaMerah were used as a cosmetic. A decoction ofthe bark was used
as a febrifuge, for fever, and in childbirth; while the seeds were prescribed for
rheumatism (Chai, 1984).
2.4 Jasmine sp.
Jasmine is called the King ofFIOlvers and indeed this is probably the most masculine . of all the floral aromas. It is belongs to Oleaceae family and consists of200 species. It
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is believe that most of the Jasmine flowers in Malaysia come from India. J. sambac is
one of the well-known Jasmine due to its fragrant flowers. In China, the flowers ofJ.
Sambac are dried ofand mixed with tea (Noraini, 2000).
2.5 Extraction
The methods of extraction of essential oils from plants significantly affect the
chemical constituents and composition of the essential oil (Nakatsu et al., 2000).
Extraction using steam distillation, solvent extraction and supercritical CO2 produce
different result with regards to yield and composition profiles of the extracted
materials. Steam distillation usually gives the lowest yield of recovered aroma but
produces a concentrate that is a true essential oil. Solvent extraction with hexane or
alcohols produces the highest yield but there is always the possibility that unwanted
involatile materials might end up in the final product. Solvent extraction also
conducted high temperatures and there is possibilities lose of essential oiL
Supercritical C02 extraction produces a lower yield than conventional solvent
extraction but higher than steam distillation and it also has the advantage ofusing little
or no heat and no steam or moisture in the process. However, this method has the
disadvantage of being expensive since it involves high pressure and sophisticated
equipment and controls (Hernandez, 2000).
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2.5.1 HydrodistiUation
Hydrodistillation technique also known as direct-heating distillation. Suitably ground
plant materials are placed in a boiler with water completely covering them. As heat is
slowly applied, steam alone will be initially fOlmed and the distillate will be clear.
With continued heating the essential oil starts to distil over with the steam and the
distillate becomes milky white. Distillation is continued until the distillate becomes
clear, with no more oil distilling over the material. Even though the essential oils have
relatively high boiling points, codistillation like this brings about a satisfactory
recovery of the oil because, in accordance with Dalton's law, a mixture boils when the
sum of the vapour pressures of the individual components equals the atmospheric
pressure. However, this method is slow and requires close manual attention and
separation of oil and aqueous phases ofthe condensate (Macrae et al., 1993).
2.6 Fractionation
The component of a mixture, such as an extract from a living organism, can be
separated into groups ofcompounds sharing similar physico-chemical characteristics.
This process is called fractionation and can be carried out in various ways.
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Chromatographic procedures are the most diverse and the most widely used
techniques in the fractionation ofextracts (Houghton and Raman, 1998).
2.6.1 Gas Chromatography
Gas chromatography (GC) is one ofthe fastest and most useful techniques available in
the laboratory. Gas chromatographic analysis is basically limited to organic
compounds that are volatile and not thermally labile (Shugar and Ballinger, 1990). In
GC, samples are volatilized and transported by mobile phase (the carrier gas) to the
column, where separation takes place. The components of the mixtures will reach the
end of the column more or less separated in time; there they are detected and, in
appropriate, recovered (Macrae et ai., 1993). Furthermore, samples subjected to GC
analysis must be volatile at the analysis temperature to ensure that they remain in the
vapour phase, yet they must be sufficiently stable so that they do not undergo
alteration during the chromatography process. In some cases samples must be
derivatized in order to enhance their volatility and heat stability (Macrae et aI., 1993).
2.6.2 Identification
Since the main part of the oil consists of volatile components, gas chromatography
equipped with Flame Ionization Detector (Fill) or Mass Spectrometer (MS) was the
most used technique to identifY the essential oil components (Bicchi, 2000).
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Eventhough, since essential oils are generally very complex mixtures, reliable GC
location and identification oftheir components can only be through retention indices,
calculated by the Kovats method or with the van den Dool algorithm. These make
retention values independent ofGC conditions. Identification through retention indices
in general only considered significant when two successful matches are obtained from
different - polarity stationary phases (Bicchi, 2000).
2.7 Chemometrics Analysis
Chemometrics is defined as "the use of computational and mathematical methods to
extract information from analytical data" (Hibbert, 1997). Its also can be defined as
"the chemical discipline that uses mathematical and statistical methods to design or
select optimal measurement procedures and experiments and to provide maximum
chemical infOlmation by analyzing chemical data" (Otto, 1999).
Principle Component Analysis (PCA) is one of the methods for analyzing data
included in multivariate analysis (Arrebola, 1997). It is successful in analyzing
multivariate data, since it can investigate relationships between large numbers of
variables, and it is useful for reducing the numbers of variables in data set by finding
linear combinations of those variables that explain most of the variability. These
characteristics make PCA successful in comparing and discriminating groups of
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essential oils, for instance versus a series ofreference samples, in particular for routine
purposes (Bicchi, 2000).
Cluster analysis tries to group objects by their proximity in this variable space. A
typical aggregation method would have the following step: find the two objects closest
together and place them by a cluster with coordinates midway between them. This step
is repeated until the entire set is clustered. The groupings that build up may be
displayed as a graph, called a dendogram, showing the groups as a function of
similarity. In the essential oil literature where hierarchical clustering is one ofthe most
commonly used pattern recognition methods (Hibbert, 1997).
2.8 Bioassay
According to Palmer (2004), bioassay is a toxicity test used to determine the dose or
concentration ofa toxicant. In dealing with toxins, a frequent relative danger indicator
is the LDso. For example, the LDso of sugar in rats is 30 grams; that is, out of 100
laboratory rats, 50 would be expected to die at levels of30 grams of sugar/kg ofbody
weight. Nicotine has an LDso in rats of 0.05g, which is more toxic than sugar.
Palmer (2004) also stated that a similar measure to LDso that is often used in labs is
the LCso, where LC stands for Lethal Concentration. The brine shrimp lethality assay
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is considered a useful tool for preliminary assessment of toxicity. It has also been
suggested for screening phannacological activities in plants extracts (Carballo et al.,
2002). For instance, the brine shrimp (Arternia salina) bioassay was used to test the
toxicity of5 volatile oils (Deans et ai., 1992).
The brine shrimp is a crustacean found in saline water worldwide. The availability of
eggs, the ease ofhatching them into larvae, the rapid growth ofnauplii and the relative
ease ofmaintaining a population under laboratory conditions, make the brine shrimp a
simple and effective test animal in bioassays and toxicology studies (Doganca et aI.,
1997).
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p
CHAPTER THREE
MATERIALS AND METHOD
3.1 Sampling
The samples collection for two species of Michelia was carried out on August and
October 2004 from along Demak Road, Kuching. The flowers and leaves ofJasmine
sp. were collected from the garden of Sarawak State Library on January 2005. The
plants samples were kept in the plastic bags and stored in cool room until further
analysis. Herbarium samples were prepared for taxonomical identification and
deposited at Unimas Herbarium.
3.2 Extraction of Essential Oils
Prior to extraction, the plant samples will cut into small pieces. The extractions of
essential oils were based on method as described by Datta (1987). Essential oils were
extracted and isolated using hydrodistillation method in a Clevenger-type apparatus.
The flowers and leaves ofthe plant samples were cut into small pieces. The root ofthe
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