essential oils and biological activities of three selected wild alpinia
TRANSCRIPT
ESSENTIAL OILS AND BIOLOGICAL ACTIVITIES OF THREE
SELECTED WILD ALPINIA SPECIES
DEVI ROSMY BINTI SYAMSIR
INSTITUTE OF BIOLOGICAL SCIENCES
FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR
2009
ii
ESSENTIAL OILS AND BIOLOGICAL ACTIVITIES OF THREE
SELECTED WILD ALPINIA SPECIES
DEVI ROSMY BINTI SYAMSIR
THESIS SUBMITTED IN FULFILMENT
OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
INSTITUTE OF BIOLOGICAL SCIENCES
FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR
2009
iii
ACKNOWLEDGEMENT
Praise to Allah the Most Merciful and Compassionate for giving me the strength in
completing this research and thesis.
First and foremost, I would like to express my appreciation to my supervisor, Prof. Dr.
Halijah Ibrahim from Institute of Biological Sciences (IBS) and Prof. Dr. Khalijah Awang
from Department of Chemistry, Faculty of Science, University of Malaya for their
supervision, advice, guidance and patience throughout my research. Thanks to the
University Malaya for providing financial support (Vote PPP: PS091-2007B) in the
completion of this work.
I also would like to convey my gratitude to a great number of people in FRIM whose
helping me throughout the work especially Dr. Rasadah Mat Ali, Dr. Norazah Mohd. Ali,
Mrs. Mastura Mohtar, Mrs. Mazura Pisar, Mrs. Fadzureena Jamaluddin and Mr. Abu Said
Ahmad.
Many thanks go to Mr. Din Mohd. Nor, Mrs. Noryati Jamil, my colleagues in
Phytochemistry lab (UM), Chemistry lab (FRIM), Microbiology lab (FRIM) and Biology
lab (FRIM) for their help, support and willingness to share their experience.
Finally, my appreciation is to my family especially my lovely parents. This thesis could not
have been completed without their patience and support.
Thank you.
iv
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENT ii
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATONS viii
ABSTRACT xi
ABSTRAK xiii
CHAPTER 1: INTRODUCTION 1
1.1 Research objectives 2
CHAPTER 2: LITERATURE REVIEW 3
2.1 The family Zingiberaceae 3
2.2 The genus Alpinia: Distribution and habitat 7
2.3 Alpinia species used in this study 7
2.3.1 Alpinia murdochii Ridl. 8
2.3.2 Alpinia pahangensis Ridl. 9
2.3.3 Alpinia scabra (Blume) Náves 11
2.4 Essential oil 12
2.5 Essential oil extraction and analysis 13
2.6 Chemical compositions 14
2.6.1 Terpenes 14
2.6.2 Monoterpenes 15
2.6.3 Sesquiterpenes 17
2.6.4 Phenylpropanoids 18
2.6.5 Compounds of miscellaneous origins 19
2.7 Essential oils of Alpinia species 20
2.8 Biological activities 28
2.8.1 Antimicrobial activity 30
2.8.2 Antioxidant activity 32
2.8.3 Anti-inflammatory activity 33
v
CHAPTER 3: METHODOLOGY 34
3.1 Plant Material 34
3.2 Preparation of plant materials 34
3.3 Extraction of essential oils 34
3.4 Determination of yield 36
3.4.1 Calculation of the moisture content of the sample 36
3.4.2 Calculation of percentage yields based on dry weight of plant
parts
36
3.5 Gas-Chromatography (GC) and Gas Chromatography / Mass
Spectroscopy (GC-MS) analysis
37
3.5.1 Calculation of Kovats indices 37
3.6 Biological activities 38
3.6.1 Antimicrobial activity 38
3.6.1.1 Chemicals and microbial strains 38
3.6.1.2 Inoculum preparation 39
3.6.1.3 Minimum inhibitory concentration (MIC) 39
3.6.2 Antioxidant activity 40
3.6.2.1 Chemicals and reagents 40
3.6.2.2 DPPH radical scavenging assay 41
3.6.2.3 Reducing power assay 41
3.6.3 Anti-Inflammatory activity 42
3.6.3.1 Chemicals and reagents 42
3.6.3.2 Lipoxygenase assay 42
3.6.3.3 Hyaluronidase assay 43
CHAPTER 4: RESULTS AND DISCUSSION 45
4.1 Chemical constituents of essential oils of three wild Alpinia species 45
4.2 Chemical constituents of essential oils from the leaves and the
rhizomes of wild Alpinia species
47
4.2.1 Essential oil components of the leaf of Alpinia murdochii Ridl. 48
4.2.2 Essential oil components of the rhizome of Alpinia murdochii
Ridl.
51
vi
4.2.3 Essential oil components of the leaf of Alpinia pahangensis
Ridl.
54
4.2.4 Essential oil components of the rhizome of Alpinia
pahangensis Ridl.
57
4.2.5 Essential oil components of the leaf of Alpinia scabra (Blume)
Náves
60
4.2.6 Essential oils components of the rhizome of Alpinia scabra
(Blume) Náves
63
4.2.7 Chemical compositions according to class of compounds of the
leaf oils and rhizome oils of three wild Alpinia species
66
4.3 Biological activities 81
4.3.1 Antimicrobial properties of three wild Alpinia species 81
4.3.1.1 Minimum inhibition concentration (MIC) 81
4.3.2 Antioxidant properties of three wild Alpinia species 87
4.3.2.1 DPPH radical scavenging assay 87
4.3.2.2 Reducing power assay 91
4.3.3 Anti-inflammatory properties of three wild Alpinia species 98
Hyaluronidase assay and Lipoxygenase assay 98
CHAPTER 5: CONCLUSION 101
REFERENCES 103
APPENDIX 114
vii
LIST OF TABLES
TABLES Page
Table 2.1 Uses of selected Zingiberaceae species 4
Table 2.2 Summary of essential oils of Alpinia species from previous studies 22
Table 2.3 Properties of selected Alpinia species 29
Table 3.1 List of Alpinia species used in this study 35
Table 4.1 Essential oil yield of three Alpinia species 46
Table 4.2 Chemical constituents of the leaf oils of Alpinia murdochii Ridl. 49
Table 4.3 Chemical constituents of the rhizome oil of Alpinia murdochii
Ridl.
52
Table 4.4 Chemical constituents of the leaf oil of Alpinia pahangensis Ridl. 55
Table 4.5 Chemical constituents of the rhizome oil of Alpinia pahangensis
Ridl.
58
Table 4.6 Chemical constituents of the leaf oil of Alpinia scabra (Blume)
Náves
61
Table 4.7 Chemical constituents of the rhizome oil of Alpinia scabra
(Blume) Náves
64
Table 4.8 Chemical composition of the leaf oils of three wild Alpinia species 66
Table 4.9 Chemical composition of the rhizome oils of three wild Alpinia
species
71
Table 4.10 Percentages of similarity of compounds between three wild
Alpinia species
75
Table 4.11 Distribution of chemical constituents of the leaf oils of three wild
Alpinia species according to their classification
77
Table 4.12 Distribution of chemical constituents of the rhizome oils of three
wild Alpinia species according to their classification
77
Table 4.13 The minimum inhibition concentrations (MIC) of essential oils of
Alpinia species (µg/ml) against Staphylococcus aureus strains
85
Table 4.14 The minimum inhibition concentrations (MIC) of essential oils of
Alpinia species (µg/ml) against selected fungi
86
Table 4.15 Percentage inhibition of DPPH free radical scavenging of essential
oils of Alpinia species at the concentration of 5 mg/ml
88
viii
Table 4.16 Percentage inhibition of various concentrations of ascorbic acid 90
Table 4.17 Reducing power value of standard reference, ascorbic acid at
various concentrations
92
Table 4.18 Reducing power value of the essential oils of three Alpinia species
at various concentrations
93
Table 4.19 Percentage inhibition of essential oils of Alpinia species based on
hyaluronidase assay and lipoxygenase assay
99
LIST OF FIGURES
FIGURES Page
Figure 2.1 Classification of Zingiberaceae according to Holttum’s (1950)
classification
5
Figure 2.2 The new classification of the family Zingiberaceae according to
Kress et al. (2002)
6
Figure 2.3 The flower of Alpinia murdochii Ridl. 8
Figure 2.4 The rhizome of Alpinia pahangensis Ridl. 10
Figure 2.5 The flower of Alpinia pahangensis Ridl. 10
Figure 2.6 The flower of Alpinia scabra (Blume) Náves 12
Figure 2.7 Isoprene unit 14
Figure 2.8 Structure of some components of essential oils; monoterpenes 16
Figure 2.9 Structure of some components of essential oils; sesquiterpenes 17
Figure 2.10 Structure of some components of essential oils; phenylpropanoids 18
Figure 3.1 Preparation of samples and essential oil 35
Figure 3.2 Outline of the present study 44
Figure 4.1 Yields of essential oils from three Alpinia species: Alpinia
murdochii, Alpinia pahangensis and Alpinia scabra
46
Figure 4.2 DPPH radical scavenging of Alpinia species (%) 89
Figure 4.3 DPPH radical scavenging activity of ascorbic acid (standard
reference)
90
Figure 4.4 Reducing power assay on essential oil of three Alpinia species. 94
Figure 4.5 Reducing power of Alpinia murdochii oils (leaf and rhizome) in
comparison with ascorbic acid
95
ix
Figure 4.6 Reducing power of Alpinia pahangensis oils (leaf and rhizome) in
comparison with ascorbic acid (standard reference)
95
Figure 4.7 Reducing power of Alpinia scabra oils (leaf and rhizome) in
comparison with ascorbic acid (standard reference)
96
LIST OF ABBREVIATIONS
α alpha
β beta
γ gamma
µg microgram
µl microliter
µg/ µl microgram / microliter
µg/ml microgram / mililiter
mg/ml miligram / mililiter
g gram
mg miligram
min minutes
ml mililiter
M mol
U/ml unit / mililiter
U unit
µM micro mol
% percent
mM mili Mol
Na2 SO4 Sodium sulfate
DCM Dichloromethane
DMSO Dimethylsulfoxide
DPPH 2, 2’-diphenylpicrylhydrazyl
FID Flame ionization detector
FRIM Forest Research Institute Malaysia
GC Gas Chromatography
x
GC/MS Gas Chromatography / Mass Spectrometer
MeOH Methanol
MHA Mueller Hinton agar
MHB Mueller Hinton broth
MIC Minimum Inhibitory Concentration
NA Nutrient agar
NB Nutrient broth
NDGA Nordihydroguaiaretic acid
NIST National Institute of Standards and Technology
PDA Potato dextrose agar
PDB Potato dextrose broth
Sa Staphylococcus aureus
TLC Thin layer chromatography
TSA Tryptic soy agar
TSB Tryptic soy broth
UPM University Putra Malaysia
VISA Staphylococcus aureus with intermediate resistance to vancomycin
VRSA Staphylococcus aureus with complete resistance to vancomycin
xi
UNIVERSITY MALAYA
ORIGINAL LITERARY WORK DECLARATION
Name of candidate : DEVI ROSMY BT. SYAMSIR (I.C. No.: 791102-71-5036)
Registration / Matric No : SGR 060065
Name of Degree : MASTER OF SCIENCE
Title of Project Paper / Research Report / Dissertation / Thesis (“this work”):
ESSENTIAL OILS AND BIOLOGICAL ACTIVITIES OF THREE SELECTED WILD
ALPINIA SPECIES
Field of Study: Natural Products Chemistry and Biological Activities
I do solemnly and sincerely declare that:
1) I am the sole author/ writer of this work;
2) This work is original;
3) Any use of any work in which copyright exists was done by way of fair dealing and for
permitted purposes and any excerpt or extract form, or reference to or reproduction of
any copyright work has been disclosed expressly and sufficiently and the title of the
work and its authorship have been acknowledged of any in this work;
4) I do not have any actual knowledge nor do I ought reasonably to know that the making
of this work constitutes an infringement of any copyright work;
5) I hereby assign all and every right in the copyright to this work to the University of
Malaya (“UM”), who henceforth shall be owner of the copyright in this work and that
any reproduction or use in any form or by any means whatsoever is prohibited without
the written consent of UM having been first had and obtained;
6) I am fully aware that if in the course of making this work I have infringed any
copyright whether intentionally or otherwise, I may be subject to legal action or any
other action as may be determined by UM.
Candidate’s Signature Date
Subscribed and solemnly declared before,
Witness’s Signature Date
Name:
Designation:
xii
ESSENTIAL OILS AND BIOLOGICAL ACTIVITIES OF THREE SELECTED WILD
ALPINIA SPECIES
Abstract
Essential oils of three selected wild Alpinia species, namely Alpinia murdochii Ridl.,
Alpinia pahangensis Ridl. and Alpinia scabra (Blume) Náves, were obtained by
hydrodistillation. The chemical components and their composition in the essential oils of
the rhizomes and the leaves were investigated using gas chromatography (GC), gas
chromatography-mass spectrometry (GC-MS) and Kovats indices analysis. Some of the
components commonly observed in the essential oils of these wild Alpinia species were β-
pinene, α-pinene, limonene, γ-selinene, α-terpineol, terpinene-4-ol and sabinene. The
chemical components of two closely related species, A. murdochii and A. pahangensis
were compared in terms of similarities of compounds. 36.6 % of compounds were similar
in their leaf oils and 45 % of compounds were similar in their rhizome oils. The marker
compound of Alpinia species, 1, 8-cineole was only present in A. pahangensis rhizome oils
and A. scabra leaf oils with low concentrations (0.63 % and 0.08 % respectively).
The essential oils obtained were tested for their biological activities namely antimicrobial
activity, antioxidant activity and anti-inflammatory activity. For the antimicrobial activity,
the minimum inhibitory concentration (MIC) assay was applied. The rhizome oils of these
three Alpinia species exhibited potent inhibition against VISA and their MIC values were
lower than oxacillin. Meanwhile, A. pahangensis rhizome oils also showed potent activity
against Sa 7 (Staphylococcus aureus strain) with a lower MIC value compared to oxacillin.
xiii
The antioxidant activity was tested using two assays, DPPH free radical scavenging assay
and reducing power assay. In DPPH free radical scavenging assay, A. scabra rhizome oil
exhibited the highest percentage of inhibition with 55.17 % ± 1.23 at a concentration of 5
mg/ml. At the same concentration, A. scabra rhizome oil also showed the highest reducing
power of 1.085 ± 0.004.
The assays used for the anti-inflammatory activity were hyaluronidase assay and
lipoxygenase assay. In the hyaluronidase assay, at the concentration of 100 µg / µL, all the
oils tested showed moderate activity (40.63 ± 4.31 % until 66.38 ± 9.43 %) except for A.
pahangensis leaf oils (38.41 ± 6.34 %). Leaf and rhizome oils of A. murdochii and rhizome
oil of A. scabra exhibited high inhibition percentages on lipoxygenase assay with 95.37 ±
6.55 %, 91.11 ± 7.82 % and 90.42 ± 0.10 % respectively.
xiv
Abstrak
Minyak pati tiga spesis Alpinia liar yang terpilih iaitu Alpinia murdochii Ridl., Alpinia
pahangensis Ridl. dan Alpinia scabra (Blume) Náves dihasilkan melalui proses
penyulingan air. Komponen kimia dan komposisinya dalam minyak pati daun dan rizom
telah dikaji menggunakan kromatografi gas, gabungan kromatografi gas / spectrometer
jisim dan analisis perbandingan indeks penahanan Kovats. Beberapa komponen yang
biasanya dijumpai di dalam minyak pati untuk spesis Alpinia liar yang dikaji adalah β-
pinene, α-pinene, limonene, γ-selinene, α-terpineol, terpinene-4-ol dan sabinene.
Komponen kimia bagi dua spesis Alpinia yang mempunyai hubungan rapat, A. murdochii
dan A. pahangensis telah dibandingkan dari segi persamaan komponennya. 36.6 %
daripada kandungan di dalam minyak pati daunnya adalah sama dan 45 % komponen
minyak pati rizomnya adalah sama. Sebatian penanda untuk genus Alpinia iaitu 1, 8-
cineole, hanya dijumpai di dalam minyak pati rizom A. pahangensis dan minyak pati daun
A. scabra dengan kepekatan yang rendah (0.63 % dan 0.08 % masing-masing).
Minyak pati yang diperolehi telah diuji aktiviti biologinya iaitu aktiviti antimikrobial,
aktiviti antioksidan dan aktiviti anti-inflamatori. Bagi ujikaji antimikrobial, esei kepekatan
perencatan yang minimum telah digunakan. Minyak pati rizom bagi ketiga-tiga spesis
Alpinia ini menunjukkan perencatan yang tinggi menentang VISA dengan nilai perencatan
minimum yang lebih rendah berbanding oxacillin. Minyak pati rizom A. pahangensis juga
menunjukkan perencatan yang tinggi menentang Sa 7 (stren Staphylococcus aureus)
dengan nilai perencatan minimum yang lebih rendah berbanding oxacillin.
xv
Untuk aktiviti antioksidan, dua esei telah digunakan iaitu esei penghapusan radikal bebas
DPPH dan esei kuasa penurunan. Di dalam esei penghapusan radikal bebas DPPH, minyak
pati rizom A. scabra telah menunjukkan peratusan perencatan yang paling tinggi iaitu
55.17 % ± 1.23 pada kepekatan 5 mg/ml. Pada kepekatan yang sama, minyak pati rizom
A. scabra juga menunjukkan kuasa penurunan tertinggi iaitu 1.085 ± 0.004.
Esei ‘hyaluronidase’ dan esei ‘lipoxygenase’ telah digunakan untuk ujikaji anti-
inflamatori. Untuk esei ‘hyaluronidase’, pada kepekatan 100 µg / µL, semua minyak pati
yang diuji menunjukkan aktiviti yang sederhana (40.63 ± 4.31 % hingga 66.38 ± 9.43 %)
kecuali minyak pati daun A. pahangensis (38.41 ± 6.34 %). Minyak pati daun dan rizom A.
murdochii dan minyak pati rizom A. scabra menunjukkan peratusan perencatan yang tinggi
(melebihi 90 %) pada esei lipoxygenase dengan peratusan 95.37 ± 6.55 %, 91.11 ± 7.82 %
dan 90.42 ± 0.10 % masing-masing.
CHAPTER 1
INTRODUCTION
Chapter 1 Introduction
1
INTRODUCTION
Traditional medicine encompasses the knowledge, skills, and practices based on the
theories, beliefs, and experiences indigenous to different cultures, used in the maintenance
of health as well as in the prevention, diagnosis, improvement or treatment of physical and
mental illness.
According to the World Health Organization (WHO), about three-quarters of the world
population relies upon traditional remedies (mainly herbs) for the health care of its people.
They not only provided food and shelter but also served the humanity to cure different
ailments. The herbal medicine also called as traditional or natural medicine existed in one
way or another in different cultures, such as Egyptians, western, Chinese and other (Gilani,
2005). In Malaysia and Indonesia, the use of traditional medicine in the treatment and
prevention of maladies are still widely practiced. Medicinal plants are utilized as an
alternative to modern medicines.
Plant essential oils and extracts have been used for many thousands of years in food
preservation, pharmaceuticals, alternative medicine and natural therapies, used in
perfumes, cosmetics, aromatherapy, phototherapy, spices and nutrition (Buchbauer, 2000).
Most essential oils exhibited antibacterial, antifungal, antiviral, insecticidal and antioxidant
properties (Burt, 2004). Essential oils are believed to act as allelopathic agents or as
irritants that protect plants from predation by insect and infestation by parasites (Simpson,
1995). Essential oils and their constituents have also been shown to be a potent source of
botanical pesticides (Singh and Upadhyay, 1993).
Chapter 1 Introduction
2
1.1 Research objectives
Zingiberaceae has a rich source of compounds of phytomedical interest. Plants from this
family have been reported to have anti-inflammatory, antiulcer, antioxidant and
antimicrobial properties (Jaganath, et al., 2000). Thus, this present study will focus on
three wild Alpinia species which have not been exploited before. The objectives of this
present study are as follows:
1. to analyse the essential oil components of three unexploited wild species of Alpinia
collected from mountain (Alpinia murdochii Ridl.), hill (Alpinia scabra (Blume)
Náves) and lowland (Alpinia pahangensis Ridl.) of Pahang,
2. to compare the essential oil components of Alpinia murdochii and Alpinia pahangensis
which has been suggested to be closely related,
3. to check the presence of 1-8-cineole, the marker compound of the genus Alpinia in the
three species studied,
4. to determine the biological activities of the essential oils of the three species namely
antimicrobial activity, anti-inflammatory activity and antioxidant activity,
5. to compare the biological activities of the two closely related species of Alpinia,
Alpinia murdochii and Alpinia pahangensis.
CHAPTER 2
LITERATURE REVIEW
Chapter 2 Literature review
3
LITERATURE REVIEW
2.1 The family Zingiberaceae
Zingiberaceae is one of the largest families in the order Zingiberales which comprises
about 1200 species. This family is distributed mostly in tropical and subtropical areas.
The richest area of genera and species is in the Malesian region, a floristically distinct
region that includes Malaysia, Indonesia, Brunei, Singapore, the Philippines and Papua
New Guinea. The family is well known for its medicinal and economic significance
with many species that provide useful products for food, medicine, spices, flavoring
agents, fragrance, coloring or natural dyes, condiments as well as ornamentals (Burkill,
1966).
Nowadays, Zingiberaceae is important in modern and traditional medicine, spices,
condiments, flavours in foods, fresh vegetables, dye, and fresh vegetables, as
ornamental plants and as cut flowers for flower arrangement. The Table 2.1 below
show some examples of gingers used in traditional medicine.
Some of the species in this family are believed to be useful in the treatment of several
types of cancer. For instance, the rhizomes of Curcuma domestica commonly known as
kunyit in Malaysia, besides being used as a flavour in our curry, it can also be used for
the treatment of uterine and servical cancer (Indu Bala and Ng, 1999, Sharifah Anisah,
1995, Goh et al., 1995, Wiart, 2000. Norzaimah (2004) reported that Zingiber officinale
(halia) can be used for the treatment of colon cancer by consuming the decoction of the
rhizome.
Chapter 2 Literature review
4
Table 2.1: Uses of selected Zingiberaceae species
Zingiberaceae
species
Local
name
Uses References
Alpinia
conchigera
Griff.
Lengkuas
ranting
Condiment and antifungal
agent.
Ibrahim et al.
(2009) and Ibrahim
et al. (2000)
Alpinia
galanga (L.)
Willd.
Lengkuas Condiment, local medicines for
the stomachache, carminative
and diarrhea, antimicrobial
agent.
Oonmetta-aree, et
al. (2006)
Boesenbergia
rotunda (L.)
Mansf.
Temu kunci Used in cooking, aphrodisiac,
used in the treatment of colic
disorder.
Patoomratana et al.
(2002)
Curcuma longa
L. (Curcuma
domestica
Val.)
Kunyit
Anti-inflammatory, anti-
arthritic
Chandra and Gupta
(1972)
Elettariopsis
curtisii Baker
Pijat-pijat Appetizer Ibrahim et al.
(2008)
Kaempferia
galangal L.
Cekur Treatment of hypertension,
rheumatism, asthma, anti-
inflammatory agent, as a
smooth muscle relaxant.
Sadikun (1987),
Hidir and Ibrahim
(1991)
Kaempferia
parviflora
Wall ex. Bak.
- Treatment of allergy and
gastrointestinal disorders,
aphrodisiac
Supinya and Sanan
(2007)
Zingiber
montanum (J.
König) A.
Dietr (syn:
Zingiber
cassumunar
Roxb.)
Bonglai
Treatment of inflammation and
skin disease
Supinya and Sanan
(2007)
Zingiber
officinale Rosc.
Halia
Anti-asthmatic agent
Supinya and Sanan
(2007)
Zingiber
zerumbet (L.)
Sm.
Lempoyang
hitam
Anti-inflammatory agent, used
in treatment of stomach aches,
diarrhea, asthma etc.
Jimmy et al. (2003)
Chapter 2 Literature review
5
Figure 2.1 and Figure 2.2 illustrate the classification of Zingiberaceae by Holttum
(1950) and Kress et al. (2002) respectively.
Family:
Sub-family:
Tribes: Globbaeae Hedychieae Alpinieae
Genus:
Figure 2.1: Classification of Zingiberaceae according to Holttum’s (1950)
classification
ZINGIBERACEAE
Globba Boesenbergia
Camptandra
Curcuma
Haniffia
Hedychium
Kaempferia
Scaphoclamys
Zingiber
Achasma
Alpinia
Amomum
Catimbium
Cenolophon
Elettaria
Elettariopsis
Geocharis
Geostachys
Hornstedtia
Languas
Phaeomeria
Plagiotachys
Example:
A. conchigera
A. galanga
A. malacensis
A. murdochii
A. mutica
A. pahangensis
A. scabra
Zingiberoideae
Chapter 2 Literature review
6
Family:
Subfamily:
Tribe:
Genera:
Figure 2.2: The new classification of the family Zingiberaceae according to Kress et al. (2002)
ZINGIBERACEAE
Siphonochiloideae Tamijioideae Alpinioideae Zingiberoideae
Examples:
Aframomum
Alpinia
Amomum
Elettaria
Elettariopsis
Etlingera
Geocharis
Hornstedtia
Tamijia Siphonochilus Examples:
Burbidgea
Pleuranthodium
Riedelia
Siamanthus
Examples:
Boesenbergia
Camptandra
Curcuma
Haniffia
Hedychium
Kaempferia
Roscoea
Zingiber
Examples:
Gagnepainia
Globba
Hemiorchis
Mantisia
Siphonochileae Tamijieae Alpinieae
(16 genera) Riedeliaea
(4 genera)
Zingibereae
(25 genera)
Globbeae
(4 genera)
Chapter 2 Literature review
7
2.2 The genus Alpinia: Distribution and habitat
Alpinia is the largest genus in the Zingiberaceae family with more than 250 species.
This genus commemorates the 16th
– century Italian botanist, Prospero Alpinio. It
occurs throughout tropical Asia to New Guinea, Australia, the Solomon Islands, New
Hebrides, New Caledonia Fiji and Samoa. Alpinia species are medium sized to large
forest plants with some species reaching a height of over three meter. It is the only
genus in Alpinieae that has a terminal inflorescence on the leafy shoots. The flowers are
yellowish-green to creamy coloured or red, usually conspicuous. The staminodes are
reduced to large teeth (several mm long) at the base of the lip. The lip is more or less
saccate and not divided, if pale coloured often with yellow blotches or red lines. The
capsules are smooth, spherical or ellipsoid (Larsen et al., 1999).
2.3 Alpinia species used in this study
In this present study, three wild Alpinia species from the family Zingiberaceae were
investigated for their essential oils components and their biological activities such as
antimicrobial, antioxidant and anti-inflammatory activity. The three Alpinia species
investigated are Alpinia murdochii, Alpinia pahangensis and Alpinia scabra. Below are
the descriptions on the botanical aspect of each species.
Chapter 2 Literature review
8
2.3.1 Alpinia murdochii Ridl.
Botanical name : Alpinia murdochii Ridl.
General description : Rhizome at surface of ground, bearing aerial stems close
together. The stem to 1.5 m tall, sheaths green. The leaves are commonly about 30 by 4
to 6 cm wide, very shortly-hairy on both surfaces, sometimes almost glabrescent.
Petiole is about 7 mm long, usually distinctly hairy. Inflorescence about 10 to 15 cm
long beyond the highest leaf-sheath, covered when young by two hairy sheaths about 6
by 1.5 cm. Rachis densely hairy, hairs spreading (1 mm long); bearing up to about 25
short cincinni, each with 1 to 4 flowers. Primary bracts are hairy, thin and deciduous;
about 1.5 by 0.7 cm. Stalk of cincinni is 1 cm or rather more on lowest ones. Secondary
bracts broadly funnel-shaped, thin, very hairy, apex obliquely truncate, longest side to
about 1 cm. Pedicel of flower to 5 mm long, ovary short, densely hairy. Calyx with
ovary is 1.3 to 1.5 cm long. Corolla-tube as long as calyx or a little longer; lobes
sparsely hairy, about 1.5 cm long, white, the dorsal one 7 mm wide at base, strongly
concave towards the apex, the concave part slightly produced upwards with a rounded
hairy top, laterals a little narrower than dorsal, slightly concave towards the apex. A
mountain species (Holttum, 1950).
Figure 2.3: The flower of Alpinia murdochii Ridl.
Chapter 2 Literature review
9
2.3.2 Alpinia pahangensis Ridl.
Botanical name : Alpinia pahangensis Ridl.
General description : The stem is about 2 to 3 meter tall. The leaf is commonly
about 75 by 13 cm wide, light green with short hairy on both surface. Petiole is about
3.5 cm long and hairy. Inflorescence is about 20 to 30 cm long with a long sheath at the
base. Rachis stout; densely short hairy, bearing 20 to 25 cincinni, each with 2 to 7
flowers. Primary bracts at base of inflorescence are very short, fringed with long hairs,
towards apex of inflorescence much longer and the highest ones sometimes as long as
flower bracts. Stalks of cincinni are velvet-hairy, commonly to 1 cm long, at bases of
large inflorescences sometimes to 2.5 cm long. Secondary bracts are narrowly funnel
shaped, obliquely truncate, thin and papery, short hairy or nearly glabrous on outer
surface, fringed with rather long hairs, the outer ones commonly 2 to 3 cm long and
cream in colour. Pedicels of flowers are 2 cm long and hairy. Ovary covered with
spreading stiff hairs. Calyx with ovary about 2 cm long is tubular, not deeply split,
white, lobes almost equal, hairy, one or two of them with slender points up to 3 mm
long. Corolla tube little shorter than calyx, slender; lobes densely hairy and cream in
colour (Holttum, 1950). Ridley, 1924, reported that this unexploited species can be
found easily at lowland areas of Pahang, Peninsular Malaysia.
Chapter 2 Literature review
10
Figure 2.4: Figure 2.5 The rhizome of Alpinia pahangensis Ridl. The flower of Alpinia pahangensis Ridl.
It has been suggested that Alpinia murdochii and Alpinia pahangensis are closely
related (Holtum, 1950). The inflorescence and floral morphology of both species are
very similar. Alpinia murdochii is a mountain species while Alpinia pahangensis is a
lowland species and they differ mainly in their vegetative characters. Ongoing DNA
finger printing studies implicate that they are also genetically closely allied (personal
communication).
Chapter 2 Literature review
11
2.3.3 Alpinia scabra (Blume) Náves
Botanical name : Alpinia scabra (Blume) Náves
General description : The stout plant is 2 to 3 m tall when flowering. The
leaves are 40-50 by 6-9 cm, oblong, edges with scattered stiff hairs, apex rather shortly
acuminate, base cuneate, lower surface short hairy, sometimes almost glabrous. Petiole
to about 1 cm long, ligule to 1 cm long, short-hairy or glabrescent. Inflorescence 30 to
40 cm long, with 2 or 3 large branches (to 15 cm long) in the flower part, the branches
in the axils of long sheaths; apical portion, bearing short cincinni only, 20 to 30 cm
long; rachis rather stout, short hairy or almost glabrous. Primary bract towards base of
inflorescence very small towards apex up to 8 mm long. Stalks of cincinni 1- 2.5 cm
long; up to 6 flowers on each. Secondary bracts about 1 mm long. Pedicel slender about
5 mm long; ovary at flowering about 1 mm long. Calyx 5 mm long, broadly, tubular,
white, unequally 3 lobed, tips of lobes shortly pointed, hairy. Corolla tube slender, 8
mm long; lobes about 10 mm long, white. Labellum shorter than the corolla lobes,
white, cleft almost to the base, the two halves bilobed with narrow apical with wider
lateral lobe. Filament elongating to nearly 1 cm; anther 5 mm long with a small crest.
Staminodes hardly 1 mm long, tooth like, at base of lip. Fruit round, smooth, black, 10
to 12 mm diameter, containing few seeds (Holttum, 1950). In Perak, a hot water
fomentation is made with A. scabra, or heated leaves are applied to the abdomen to
treat vertigo (Burkill, 1935).
Chapter 2 Literature review
12
Figure 2.6: The flower of Alpinia scabra (Blume) Náves
2.4 Essential oils
Essential oils are the volatile, organic constituents of fragrant plant matter and
contribute to both flavour and fragrance. These oils were termed essential because they
were thought to represent the very essence of odour and flavour.
Volatile oils are chemically complex mixtures, often containing in excess of hundreds
of individual components. Most oils have one to several major components which
impart the characteristic odour and taste such as sweet and spicy. However, there are
also many minor constituents which also play their part in producing the final product
(Waterman, 1993).
Zingiberaceae species are rich in essential oils. There are many researchers from
various countries who work on essential oils of this family. Among the interesting
genera to work with in terms of essential oils are Alpinia, Curcuma, Kaempferia and
Zingiber.
Chapter 2 Literature review
13
2.5 Essential oil extraction and analysis
Essential oils can be obtained from various parts of plants such as flowers (Plumeria
sp., Rosa sp.), leaves (citronella, eucalyptus), fruits (citrus), seeds (cardamomum),
woods (rosewood, sandalwood), roots and rhizomes (turmeric, ginger). They are
essentially obtained by hydrodistillation, steam distillation, by cold-pressing
(expression) and by super critical fluid (SFE) extraction. The microwave irradiation [or
microwave assisted process (-MAP-)] has also been developed and reported by many
authors as a technique for extraction of essential oils in order to obtain a good yield of
the essence and to reduce time of extraction (Pare et al., 1989). This technique has also
been applied for the extraction of saponins from some medicinal plants (Safir et al.,
1998). The MAP process uses microwaves to excite water molecules in the plant
tissues causing plant cells to rupture and release the essential oils trapped in the
extracellular tissues of the plant. However, the method hydrodistillation is the most
widely used to obtain essential oils from aromatic plants.
The chemical composition of essential oil differs in each species or subspecies and is
characteristic for the species in question. Identification of individual components of
complex mixtures such as terpenes in essential oils requires the use of several
techniques.
Chemical analysis of essential oils is generally performed using gas chromatography
(GC) (qualitative analysis) and gas chromatography –mass spectrometry (GC/MS)
(quantitative analysis). Identification of the main components is carried out by the
comparison of both the GC retention times and the MS data against those of the
Chapter 2 Literature review
14
reference standards, Kovats retention indices (KI) and comparison with previous
literature (Adams, 2001).
Kovat retention indices may be obtained by calculating the temperature program linear
retention indices of a chemical compound from the gas chromatogram and by
logarithmic interpolation between bracketing alkanes (Nor Azah, 2004). The
homologous series of n-alkanes (C7-C25) are used as standards (Kovats, 1965).
2.6 Chemical compositions
Essential oils are made up of compounds such as terpenoids, aldehydes, esters, ketones,
phenols and alcohols (Radulescu, et al., 2004). The odour and taste of an essential oil is
mainly determined by the oxygenated constituents, the fact that they contain oxygen
gives them some solubility in water and considerable solubility in alcohol (Tisserand, et
al., 1995).
2.6.1 Terpenes
Terpenes are composed of hydrogen and carbon atoms only. All terpenes are based on
the isoprene unit, an essential building block in plant biochemistry (Tisserand, et al.,
1995).
CH2
C
CH2
CH3
CH3
Figure 2.7: Isoprene unit
Chapter 2 Literature review
15
2.6.2 Monoterpenes
The hydrocarbons are almost always present in essential oil. Monoterpenes contain ten
carbon atoms. They are called monoterpenes, because this is the basic unit as found in
nature. These terpenes can also have several functional groups. The functional groups
are:
Functionalized group (Leland, et al., 2006):
I. Aldehyde – any class of compounds characterized by the presence of a carbonyl
group (C=O group) in which the carbon atom is bonded to at least one hydrogen
atom.
II. Ketones – compounds where the carbon atom of the carbonyl group is bonded
to two other carbon atoms.
III. Alcohols – any class of compounds characterized by the presence of a hydroxyl
group (-OH group) bonded to saturated carbon atom.
IV. Esters – Esters are any class of compounds structurally related to carboxylic
acids but in which the hydrogen atom in the carboxyl group (-COOH group)
was replaced by a hydrocarbon group, resulting in a –COOR structure where R
is the hydrocarbon.
V. Phenol - Phenols constitute a large class of compounds in which a hydroxyl
group (-OH group) is bound to an aromatic ring.
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16
Figure 2.8: Structure of some components of essential oils; monoterpenes
Myrcene
Ocimene
β-Pinene
α-Pinene
p-Cymene
OH
Linalool
O
Camphor
OH
Geraniol
OH
Carvacrol
OH
α-Terpineol
CH3
CH3
OH
Myrtenol
CH3
O
CH3
CH3
1,8-Cineol
Chapter 2 Literature review
17
2.6.3 Sesquiterpenes
Sesquiterpenes are composed of three isoprene units and therefore have 15 carbon
atoms. Examples of sesquiterpenes characteristic of essential oils: hydrocarbons (β-
bisabolene), alcohols (farnesol), ketones (nootkatone), aldehydes (sinensals) and esters
(cedryl acetate).
β-Bisabolene
OH
trans, trans-Farnesol
O
(+)- Nootkatone
H
CH2
H
Β-Caryophyllene
H
B-Sesquiphellandrene
γ -Selinene
α-Gurjunene
B- Farnesene
H
α-Cubebene
Figure 2.9: Structure of some components of essential oils; sesquiterpenes
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18
2.6.4 Phenylpropanoids
Phenylpropanoids (C6-C3) are far less common than terpenoids. Very often they are
allyl (H2C=CH-CH2-R) and phenylphenols and sometimes, they are aldehydes
characteristic of certain Apiaceae oils (anise, fennel, parsley: anethole, anisaldehyde,
apiole, methylchavicol) and also of clove, nutmeg, tarragon, calamus and cinnamons.
Also present in essential oils are C6-C1 compounds such as vanillin (rather common) or
methyl anthranilate (Norsita, 2003).
OH
H3CO
Eugenol
OCH3
E-Anethole
CHO
OH
H3CO
Vanillin
OH
Cinnamyl alcohol
O
O
Benzyl benzoate
CH3
OO
Methyl benzoate
OH
Benzyl alcohol
(phenyl carbinol)
O
OOH
Benzyl salicylate
CH3
OO
OH
Methyl salicylate
Figure 2.10: Structure of some components of essential oils; phenylpropanoids
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19
2.6.5 Compounds of miscellaneous origins
Essential oils may contain various aliphatic compounds, generally of low molecular
weight, which are extracted during steam distillation: hydrocarbons (linear or remified,
saturated or not, rarely specific), acids (C3 to C10), alcohols, aldehydes, acyclic esters or
lactones. Nitrogen or sulfur containing compounds are characteristic of roasted or
grilled products and are exceptional among products.
Products of higher molecular weight are not uncommon and are not extracted by steam
distillation; there are homologs of the phenylpropanoids, diterpenes and coumarins
(some of which can actually be steam distilled) among others. Representatives of this
group are incidental and often rather specific for a few species or genera. For example,
the mustard oils, containing allyl isothiocyanate are found in the family of the
Cruciferae; allyl sulfides in the oil of garlic. The oil from Ferula asafetida L. belonging
to the family of Umbelliferae, gained reputation from its active component, secondary
butyl propenyl disulfide, a competitor of the odoriferous principles of the skunk,
primary n-butyl mercaptan and dicrotyl sulfide (Norsita, 2003).
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20
2.7 Essential oils of Alpinia species
Many Alpinia species have been studied for their essential oil components. Hasnah, et
al. (1995), reported that β-sesquiphellandrene, β-bisabolene, 1, 8-cineole and β-
caryophyllene are the major component of rhizomes of A. conchigera from Johor,
Malaysia. Wong, et al. (2005) has also reported the essential oil content of A.
conchigera from Penang. The major compounds are β-bisabolene (28.9%), 1, 8-cineole
(15.3%), β-caryophyllene (10%) and β-pinene (9.5%). In Malaysia, A. conchigera is
locally known as lengkuas ranting and lengkuas kecil. According to Burkill (1935), A.
conchigera have been used for the treatment of rheumatism, pain in the bones and used
as a poultice after confinement. The East Coast people in Peninsular Malaysia used this
species to treat fungal infections.
Wong, et al. (2005) also revealed that methyl (E)-cinnamate was the major compound
in Alpinia latilabris oil with 89.5%. The other compounds found in this species are α-
phellandrene (3.2%), 1, 8-cineole (1.5%) and α-pinene (1.4 %). The major components
from the essential oils of the rhizomes of Alpinia mutica from Selangor were reported
to be camphor, 1, 8-cineole, borneol and β-pinene (Hasnah, 1998).
Nor Azah, et al. (2005) revealed that the compounds of essential oils of Alpinia
malaccensis var. nobilis from Terengganu extracted from leaves, rhizomes and stems.
The most abundant compound in the leaf, rhizome and stem oils is (E)-methyl
cinnamate with 88.0 %, 85.7 % and 64.4 % respectively. The other components
present in the leaf oil were 1, 8- cineole (1.8 %) and p-cymene (1.5%). β-pinene (1.6%
and 6.0 %), α-phellandrene (1.9 % and 6.3 %) and p-cymene (1.6 % and 3.5 %) were
the major components in the rhizome and stem oils.
Chapter 2 Literature review
21
Alpinia galanga also known as lengkuas, is a common spice in Malaysia. Malaysian
people use this species in various culinary preparations. From the ethnobotanical point
of view, this species was used in the treatment of gaster cancer (Perry, 1980, Indu Bala
and Ng, 1999). It has been extensively used as condiment for flavoring and local
medicines for stomachache, carminative and treating diarrhea (Oonmetta-aree, et al.,
2006). The essential oils of this species have been investigated by De Pooter, et al.
(1985) who reported that β- farnesene is the major compound of both fresh and dried
rhizomes of this species from Malaysia.
The seeds of Alpinia katsumadaii from China showed 1, 8- cineole, α-humulene and
trans-farnesol as their major compounds (Yasuhisa, et al., 1978). It was reported by Jia,
et al. (2003) that the major compounds of the fruit of Alpinia oxyphylla from Taiwan
are octahydro-1, 8-dimethyl-7-(1-methylethenyl)-naphtalene, α-panasinsen and β-
bisabolene. Table 2.2 summarized the essential oils of Alpinia species from previous
studies. From all these studies, it can be observed that most of the major compounds
from Alpinia species are monoterpenes and sesquiterpenes such as β-
sesquiphellandrene, β-bisabolene, 1, 8-cineole, β-caryophyllene, α-pinene and β-
pinene.
Chapter 2 Literature review
22
Table 2.2: Summary of essential oils of Alpinia species from previous studies
Species Locality Parts Major Compounds References
A. allughas Rosc. India Leaves β-Pinene (25.5%), 1,8-Cineole (23.3%), α- Humulene (9.7%), α-Pinene (5.4%)
Prakash et al.
(2007)
Rhizomes β-Pinene (55.3%), α-Pinene (9.7%), 7-epi- α-Eudesmol (4.1%), β- Selinene (3.2%)
A. breviligulata
Gagnep. Vietnam Leaves Caryophyllene oxide (23.1%), α-Pinene (17.7%),
α-Copaene (5.4%), Calamenene (3.3%)
Dung et al.
(1994 a)
A. breviligulata
Gagnep.
Vietnam
Rhizomes β-Pinene (11.1%), Caryophyllene oxide (10.5%), β-Caryophyllene (8.0%), α- Humulene (7.9%), Borneol (7.0%)
Dung et al.
(1994 b)
Roots Caryophyllene oxide (13.0%), α- Humulene (10.8%), α- Fenchyl acetate (8.8%)
A. carinata Rosc. North India Leaves β-Pinene (31.9%), Terpinen-4-ol (13.7%), p-Cymene (9.3%), 1,8-Cineole (8.8%), α-Pinene (8.2%)
Singh et al.
(1999)
A. calcarata Rosc. Berhampur,
India Leaves β-Pinene (29.1%), 1,8-Cineole (21.9%),
α-Pinene (6.3%) Rout et al.
(2005) Rhizomes α-Fenchyl acetate (29.2%), 1,8-Cineole (25.7%), Camphene
(5.5%)
Roots α-Fenchyl acetate (45.2%), 1,8-Cineole(15.1%), Camphene (9.0%)
Chapter 2 Literature review
23
Table 2.2: Summary of essential oils of Alpinia species from previous studies - cont’
Species Locality Parts Major Compounds References
A. calcarata Rosc. Bangalore,
India Leaves 1, 8-Cineole (24.7%), β-Pinene (16.8%),
Camphor (8.0%), α-Pinene (5.3%)
Rout et al. (2005)
Rhizomes Geraniol (34.3%), 1, 8-Cineole (21.2%), α-Fenchyl acetate (10.2%)
Roots α-Fenchyl acetate (39.1%), 1,8-Cineole (15.5%),
Camphene (12.3%)
A. chinensis Rosc. Vietnam Flowers (E,E)-α-Farnesene (26.5%), α-Humulene (22.3%), β-Bisabolene (17.1%), β-Caryophyllene (13.1%), α-Bergamotene (5.6%), β-
Sesquiphellandrene (2.5%)
Dung et al. (1994 c)
A. chinensis Rosc. Vietnam Roots Caryophyllene oxide (13.2%), β-Bisabolene (10.4%), γ-Selinene (8.6 %), β-Caryophyllene (5.2%)
Piet et al. (1994)
A. conchigera Griff.
Johor,
Malaysia Rhizomes β-Sesquiphellandrene (20.5%),
β-Bisabolene (12.10%), 1,8-Cineole (11.56%), β-Caryophyllene (4.39%)
Hasnah and Aziz (1995)
A. conchigera Griff.
Penang, Malaysia
Rhizomes β-Bisabolene (28.9%), 1,8-Cineole (15.3%), β-Caryophyllene (10.0%), β-Pinene (9.5%)
Wong et al. (2005)
A. conchigera Griff. Kelantan,
Malaysia Leaves
β-Bisabolene (15.3 %), β-Pinene (8.2 %), β-Sesquiphellandrene (7.6 %), Chavicol (7.5 %)
Ibrahim et al. (2009)
Chapter 2 Literature review
24
Table 2.2: Summary of essential oils of Alpinia species from previous studies - cont’
Species Locality Parts Major Compounds References
A. conchigera Griff. Kelantan,
Malaysia Pseudostems β-Bisabolene (19.9 %), β-Sesquiphellandrene (11.3 %),
β-Caryophyllene (8.8 %), β- Elemene (4.7 %)
Ibrahim et al. (2009)
Rhizomes 1, 8-Cineole (17.9 %), β-Bisabolene (13.9 %), β-Sesquiphellandrene (6.8 %)
A. galanga Willd. Malaysia Rhizomes
trans- β- Farnesene (30.6%), 1,8-Cineole (24.0 %), 4-Terpineol (7.0 %), β-Bisabolene (4.9 %)
De Pooter et al. (1985)
A. galanga Willd. Sabah,
Malaysia Rhizomes 1,8-Cineole (40.5%), β-Bisabolene (8.4%),
(Z,E)-Farnesol (3.8%), β-Caryophyllene (3.6%)
Ibrahim et al. (2004)
Seeds β-Bisabolene (37.6%), (E)- β-Farnesene (22.7%), (E,E)-Farnesyl acetate (7.9%)
A. galanga Willd. Sri Lanka Rhizomes Zerumbone (44.8%), p-Cymene (6.5%), Camphene (6.4%), 1, 8-Cineole (6.3%)
Lakshmi et al. (2007)
A. galanga Willd.
Bangalore,
India
Leaves 1, 8-Cineole (34.4%), β-Pinene (21.5%), Camphor (7.8%), α-Pinene (6.6%)
Gopal et al. (2002)
Rhizomes 1, 8-Cineole (33.6%), α- Fenchyl acetate (12.7%), α-
Terpineol (9.3%), (E)-Methyl cinnamate (5.3%), Camphor (5.0%)
Chapter 2 Literature review
25
Table 2.2: Summary of essential oils of Alpinia species from previous studies - cont’
Species Locality Parts Major Compounds References
A. galanga Willd.
Hyderabad,
India
Leaves 1,8-Cineole (36.7%), β-Pinene (23.5%), Camphor (12.8%), α-Pinene (6.3%)
Gopal et al. (2002)
Rhizomes 1,8-Cineole (30.2%), Camphor (14.0%), β- Pinene (12.9%), Z- β-Ocimene (6.4%)
Alpinia henryi K.
Schum. Vietnam Rhizomes 1, 8-Cineole (45.1%), α-Terpineol (4.9%), Borneol
(4.4%), p-Cymene (4.2%), β-Pinene (4.1%)
Giang et al. (2007)
A. katsumadai Hayata
China Seeds 1,8-Cineole, α-Humulene, trans-Farnesol, Linalool,
Camphor, Terpinen-4-ol
Yasuhisa et al. (1978)
A. laosensis Gagnep. Indochina Rhizomes 1, 8-Cineole (43.9%), Methyl eugenol (3.8%), Chavicyl acetate (3.6%)
Dung et al. (2000)
A. latilabris Ridl.
Sabah, Malaysia
Rhizomes (E)-Methyl cinnamate (89.5%), α-Phellandrene (3.2%), 1,8-Cineole (1.5%)
Wong et al. (2005)
A.malaccensis var.
nobilis Ridl.
Terengganu,
Malaysia
Leaves (E)-Methyl cinnamate (88.0%), 1,8-Cineole (1.8%), p-Cymene (1.5%)
Nor Azah et al. (2005)
Rhizomes (E)-Methyl cinnamate (85.7%), α-Phellandrene (1.9 %), β-Pinene (1.6 %), p-Cymene (1.6 %)
Stems (E)-Methyl cinnamate (64.4%), α-Phellandrene (6.3%),
β-Pinene (6.0%), p-Cymene (3.5%)
Chapter 2 Literature review
26
Table 2.2: Summary of essential oils of Alpinia species from previous studies - cont’
Species Locality Parts Major Compounds References
A. mutica Roxb. Selangor
Rhizomes Camphor (35.6%), 1, 8-Cineole (9.4%), Borneol (8.3%), β-Pinene (7.3%)
Hasnah and Ahmad
(1998)
A. oxyphylla Miq.
Taiwan Fruits Octahydro-1,8-dimethyl-7-(1-methylethenyl)-
naphtalene (47.37%), α-Panasinsen (12.19%), β-Bisabolene (9.45%)
Jia, et al., (2003)
A. speciosa K.
Schum. Vietnam Flowers β-Pinene (34.0%), α-Pinene (14.8%),
β-Caryophyllene (10.8%), 1,8-Cineole (3.6%)
Dung et al. (1994 d)
A. speciosa K.
Schum. Egypt Leaves Terpinene-4-ol (17.3 %), 1,8-Cineole (14.4 %),
γ-Terpinene (11.1 %), Sabinene (10.1 %)
De Pooter et al. (1995)
Rhizomes Terpinene-4-ol (20.2 %), 1,8-Cineole (15.9 %), Sabinene (9.8 %), γ-Terpinene (9.3 %)
Stems Terpinene-4-ol (16.0 %), 1,8-Cineole (11.5 %), γ-Terpinene (8.2 %), Sabinene (7.5 %)
A. smithiae Sabu &
Mangaly Southern India
Leaves
β-Caryophyllene (27.22%), 1,8-Cineole (14.68%), Myrcene (8.64%), Sabinene (7.35%), α-Thujene
(4.09%)
Roy Joseph et al. (2001)
Rhizomes β-Caryophyllene (29.98%), Myrcene(14.36%), 1,8-Cineole(10.57%), Sabinene (9.28%), α-Pinene (5.22%)
Chapter 2 Literature review
27
Table 2.2: Summary of essential oils of Alpinia species from previous studies - cont’
Species Locality Parts Major Compounds References
A. zerumbet (Pers.)
B.L. Burtt. and R.M.
Sm.
Okinawa,
Japan
Flowers 1, 8-Cineole (16.63%), Camphor (14.1%), Methyl cinnamate (12.81%), Borneol (6.41%), Linalool (4.16%)
Elzaawely et al. (2007 a)
Seeds α-Cadinol (13.46%), T-Muurolol (10.79%), α-Terpineol (10.67%), δ-Cadinene (6.19%), Terpinene-4-ol (6.18%)
A. zerumbet (Pers.)
B.L. Burtt. and R.M.
Sm.
Okinawa,
Japan
Leaves 1,8-Cineole (18.85%), Camphor (11.93%), Methyl cinnamate (7.59%), Cryptone (6.63%)
Elzaawely et al. (2007 b)
Rhizomes Dihydro-5, 6-dehydrokawain (DDK) (21.4%), Methyl cinnamate (15.04%), Camphor (2.88%)
Chapter 2 Literature review
28
2.8 Biological activities
Nowadays, the interests in natural products are looking into sources of alternative, more
natural and environmentally friendly antimicrobials, antioxidants, antibiotics and other
bioactivities. The possibility of utilizing volatile oils is now being investigated. Generally
the action of volatile oils is the result of the combined effect of both their active and
inactive compounds. These inactive compounds might influence resorption, rate of
reactions and bioavailability of the active compounds. Several active components might
have a synergistic effect.
To add to the complexity of volatile oils, there is evidence that the time of harvest
influences the oil composition and consequently the potency of their biological activity
(Deans and Svoboda, 1988; Lis-Balchin, et al., 1992; Galambosi, et al., 1993; Marotti, et
al., 1994). Other factors such as genotype, chemotype, geographical origin and
environmental and agronomic conditions, can all influence the composition of the final
natural product (Svoboda, et al., 1992).
Biological activity of an essential oil is related to its chemical composition. The relation
between composition and bioactivity of the essence from the aromatic plants may be
attributable both to their major components (alcoholic, phenolic, terpenic or ketonic
compounds) and the minor ones present in the oil. It may act together synergistically or
antagonistically to contribute to some activity of the tested oil.
The biological activity of essential oils from other plants including other genera of
Zingiberaceae species have been demonstrated by numerous researchers. However, there
Chapter 2 Literature review
29
are only a few studies reported on this genus, Alpinia worldwide. In this present study,
essential oils from rhizomes and leaves of three wild species of Alpinia namely Alpinia
murdochii, A. pahangensis and A. scabra from Zingiberaceae family were extracted by
hydrodistillation method. The essential oils were evaluated for their biological activities
such as antioxidant, antimicrobial and anti-inflammatory.
The properties of selected Alpinia species are listed in Table 2.4 as reported by Burkill,
1935. From this report, it is suggested that Alpinia species is useful in medicine and further
investigation should be carried out in the future.
Table 2.3: Properties of selected Alpinia species
Alpinia species
Properties and description
A. conchigera Griff. • A poultice of the boiled leaves or of leaves and
rhizomes together is applied for rheumatism.
• An infusion is used for bathing.
• A poultice is made from the rhizomes and rubbed on
the body for pains in the bones.
• The pounded leaves are used as a poultice after
confinement.
A. galangal (L.) Willd. • Spice, food flavoring.
• As an infusion taken internally after childbirth.
• As a stomachic.
A. malaccensis (Burm.f.)
Rosc.
• The rhizomes is ground and applied to sores in Java.
A. aquatica (Retz.)
Rosc. (syn: A.
melanocarpa Teijsm. &
Binn.) Ridl.
• The roots were used for making a decoction taken
during the first three days after childbirth.
A. mutica Roxb. • The rhizomes are used as a stomachic.
A. scabra (Blume)
Náves • The heated leaves are applied to the abdomen for
vertigo.
Chapter 2 Literature review
30
2.8.1 Antimicrobial activity
The antimicrobial activity of essential oils of Zingiberaceae species has been demonstrated
by several researchers (Seenivasan, et al. (2006); Oonmetta-aree, et al. (2006).
In this present study, the antimicrobial activity of the essential oils have been tested against
two dermatophytic fungi namely Microsporum canis and Trichophyton rubrum, two
Candida species, Candida albican and Candida glabrata and five strains of
Staphylococcus aureus (Sa 2α, Sa 3, Sa 7, VISA and VRSA) using minimal inhibitory
concentration (MIC) assay. MIC is defined as the lowest concentration of an antimicrobial
agent that will inhibit the visible growth of microorganism after overnight incubation.
Microsporum canis dermatophytosis is an infectious fungal skin disease, which appears in
the form of different lesions in the fur of the animal. It is caused by M.canis, a pathogenic
fungus that grows in the hair, and in the top layer of the skin. The disease is zoonotic,
meaning that it can be transmitted from animals to human. While, Trichophyton rubrum is
an anthropophilic dermatophyte. The downy strain has become the most widely distributed
dermatophytes of man. It frequently causes chronic infections of skin, nails and rarely
scalp.
Candida albican is the fungi that live in our gastrointestinal tract and this species belong to
the family of Saccharomycetaceae. It can cause vaginal yeast infections. Candida can
spread throughout the intestinal tract causing bloating, gas, food reactions, allergies,
diarrhea and many other diseases. It also can spread to the vaginal area, the prostate, the
heart, lungs, liver and cause numerous symptoms and illnesses. The other species of
Chapter 2 Literature review
31
Candida used in this study is Candida glabrata. C. glabrata is often the second or third
most common cause of candidiasis after C. albicans. Candida glabrata can be found in the
environment, particularly on leaves, flowers, water and soil. This species can cause
candidiasis in men at any age.
Staphylococcus aureus is a gram positive bacteria belonging to the family Micrococcaceae
and are frequently found living on the skin in the nose of a healthy person. This microbe is
a versatile pathogen of humans and animals that has evolved resistance to all antibiotic
classes’ causes a wide variety of diseases in humans, ranging in severity such as boils and
furuncles to more serious diseases such as septicaemia, pneumonia and endocarditis
(Crossley, et al. 1997 and Lowy, 1998).
VISA and VRSA are strains of S. aureus which can cause a variety of infections to the
body. VISA stands for Staphylococcus aureus with intermediate resistance to vancomycin.
Vancomycin is an antibiotic often used to treat very serious infections. VISA strains have
minimum inhibition concentrations (MIC) of vancomycin in the range of 8 to 16 µg/ml due
to a thickening of the bacterial cell wall. While, VRSA stands for S. aureus with complete
resistance to vancomycin and the vancomycin minimum inhibition concentrations (MIC) is
more than 32 µg/ml. It is probable that S. aureus bacteria with intermediate or complete
resistance to vancomycin would be resistant to most antibiotics commonly used for
staphylococcal infections.
Chapter 2 Literature review
32
2.8.2 Antioxidant activity
Consumption of fruits and vegetables with high content of antioxidative phytochemicals
such as phenolic compounds may reduce the risk of cancer, cardiovascular disease and
many other diseases (Robbins and Bean, 2004 and Shui and Leong, 2006) and can inhibit
the propagation of free radical reactions and protect the human body from diseases
(Kinsella. et al. 1993). Therefore, the interest in naturally occurring antioxidants has
increased considerably in recent years for use in food and pharmaceutical products
(Djeridane, et al., 2006). There are various methods to determine the antioxidant activities
such as DPPH free radical scavenging assay, reducing power assay, β-carotene bleaching
assay, superoxide scavenging assay, tyrosinase inhibitory assay and many others. In this
study, only two methods are employed; the DPPH free radical scavenging assay and
reducing power assay which are briefly described in the following paragraph.
The determination of scavenging stable DPPH is a very fast method to evaluate the
antioxidant activity of the extracts. With this method it is possible to determine the
antiradical power of an antioxidant activity by measuring the decrease in the absorbance of
DPPH at 515 nm. Colour change from purple to yellow when DPPH radical is scavenged
by antioxidant, through the donation of hydrogen to form a stable DPPH molecule reduced
the absorbance. In the radical form this molecule had an absorbance at 515 nm which
disappeared after acceptance of an electron or hydrogen radical from an antioxidant
compound to become a stable diamagnetic molecule (Matthaus, 2002).
Chapter 2 Literature review
33
2.8.3 Anti-inflammatory activity
Anti-inflammatory refers to the property of a substance or treatment to reduce
inflammation. There are several assays for anti-inflammatory activity such as platelet
activating factor, nitric oxide, hyaluronidase, lipoxygenase and many other assays. In this
study, two assays were applied; hyaluronidase assay and lipoxygenase assay.
Hyaluronidase is a mucopolysaccharide hydrolyzing enzyme that degrades hyaluronic acid
(HA), a viscous lubricating agent in synovial fluid in joints and which is also present on
the skin. Hyaluronidase enhances the spreading of inflammatory mediators throughout the
body tissues, thereby contributing to the pathogenesis of inflammatory diseases such as
allergic effects, migration of cancer cells, inflammation and the permeability of the
vascular system (Ling et al., 2005).
Lipoxygenase is a biological target for many diseases such as asthma, cancer and many
others diseases. Lipoxygenases are classified with respect to their positional specificity of
arachidonic acid oxygenation; in particular, the reticulocyte-type 15-LOX and the human
5-LOX are well characterized with respect to their structural and functional properties
(Celotti and Laufer, 2001).
CHAPTER 3
METHODOLOGY
Chapter 3 Methodology
34
METHODOLOGY
3.1 Plant material
Three Alpinia species; Alpinia murdochii, Alpinia pahangensis and Alpinia scabra
were studied for their essential oil content. The parts investigated were the rhizomes
and the leaves. Alpinia murdochii and A. scabra were collected from Genting Highland
while A. pahangensis was collected at Tasik Chini, Pahang. The samples were
identified by Professor Dr. Halijah Ibrahim and voucher specimens were prepared as
listed in Table 3.1 and deposited in the Herbarium of Chemistry Department,
University of Malaya.
3.2 Preparation of plant materials
Fresh samples of rhizomes and leaves were washed and sliced into small pieces. The
samples were then oven dried at 40°C consecutively in three days. Afterwards, the
dried samples were ground using the grinder.
3.3 Extraction of essential oils
The ground samples were distilled in a Clavenger apparatus for 8 hours using distilled
water. The process is known as hydrodistilation. Then, the apparatus were rinsed using
organic solvent, dichloromethane. The oily layer were mixed with dichloromethane and
separated from the water layer using separating funnel and subsequently dried using
anhydrous sodium sulfate (Na2 SO4) (drying agent). Dichloromethane were removed
Chapter 3 Methodology
35
from the oily layer using rotary evaporator. The oils were labeled and stored in amber
vials before testing and analysis.
Table 3.1: List of Alpinia species used in this study
Samples Locality Reference number
Alpinia murdochii
• Rhizomes
• Leaves
Genting Highland, Pahang DRS 01
Alpinia pahangensis
• Rhizomes
• Leaves
Tasik Chini, Pahang DRS 02
Alpinia scabra
• Rhizomes
• Leaves
Genting Highland, Pahang DRS 03
Figure 3.1: Preparation of samples and essential oil
Fresh samples
Slice
Dry
(3 days consecutively; 40°C)
Grind
Extract using distilled water
(hydrodistillation)
(Clavenger apparatus)
Essential Oil
Chapter 3 Methodology
36
3.4 Determination of yield
The yields of the oils were calculated based on the dried weight of plant materials.
Approximately 10 mg of dried samples was weighed and placed in the round bottom
flask. 50 ml of toluene was added and then heated on the hot plate for 3 hours. The
water content of samples can be determined using Dean and Stark apparatus. The
moisture content can be calculated based on the water content using the formula below.
All measurements were carried out in triplicates and the mean values calculated.
3.4.1 Calculation of the moisture content of the sample
Water collected (ml) (by means of Dean & Stark app.) X 100 = Moisture content
Samples (g) (% water in sample)
3.4.2 Calculation of percentage yields based on dry weight of plant parts
100 % - Moisture content (% water in sample) = % dry weight of sample
% dry weight of sample X sample (g) (of hydrodistillation) = net weight of sample (g)
Oil collected (g)____ (by means of Clavenger’s app.) X 100% = % yield
Net weight of sample (g)
Chapter 3 Methodology
37
3.5 Gas-chromatography (GC) and Gas chromatography / Mass spectrometer
(GC/MS) analysis
GC analysis were performed on a Shimadzu GC-2010 gas chromatograph –Flame
ionization Detector (FID), fitted with a 25m x 0.25mm x 0.25 µm CBP5 capillary
column, using purified helium as the carrier gas. The oven temperature was
programmed from 60ºC (after 10 min) to 230ºC at 3ºC per min and the end temperature
was held for 10 min.
GC/MS analyses were carried out on an Agilent 5975N gas chromatograph with a 30m
x 0.25mm x 0.25 µm FT HP-5MS capillary column, using helium as a carrier gas. The
oven temperature was programmed from 60ºC (after 10 min) to 230ºC at 3ºC per min
and the end temperature was held for 10 minute. The constituents of the oils were
identified from MS Libraries NIST 0.5 L (Adams, 2001).
3.5.1 Calculation of Kovats Indices
Kovats Index = 100 [Log (Tx – Tm) – Log (Tn – Tm)] + 100 (N)
[Log (Tn + 1 – Tm) – Log (Tn – Tm)]
Where:
Tm = Mobile phase retention time
Tx = Sample component retention time
Tn = Standard hydrocarbon containing carbon retention time
N = Lowest carbon value
Chapter 3 Methodology
38
3.6 Biological activity
In this study, testing for three biological activities were carried out, that is,
antimicrobial activity, antioxidant activity and anti-inflammatory activity. For the
antimicrobial activity, the minimum inhibition concentration (MIC) was determined for
nine selected microbes; Candida albicans (ATCC 10231), Candida glabrata (ATCC
64677), Microsporum canis (ATCC 36299), and Trichophyton rubrum (ATCC 28188)
and five strains of Staphylococcus aureus (Sa 2, Sa 3, Sa7, VISA and VRSA). For the
antioxidant activity, two methods namely DPPH free radical scavenging assay and
reducing power assay were used. For the anti-inflammatory activity, hyaluronidase
assay and lypoxigenase assay were used. All the methods are briefly described below.
3.6.1 Antimicrobial activity
3.6.1.1 Chemicals and microbial strains
Mueller Hinton agar (MHA), Tryptic soy agar (TSA) and Potato Dextrose agar (PDA),
Mueller Hinton Broth (MHB), Tryptic soy broth (TSB), Potato Dextrose Broth (PDB),
Dimethyl sulfoxide (DMSO), cycloheximide and oxacilin were purchased from Sigma.
The essential oils were individually tested against five strain of Staphylococcus aureus;
Sa 2 (ATTC 29213), Sa 3 (ATTC 33591), Sa 7 (ATTC 700699), the Staphylococcus
aureus with intermediate resistance to vancomycin (VISA)(24 mg/ml) and the
Staphylococcus aureus with complete resistance to vancomycin (VRSA)(156 mg/ml),
two Candida species; Candida albicans (ATCC 10231) and Candida glabrata (ATCC
64677) and two dermatophytic fungi; Microsporum canis (ATCC 36299), and
Chapter 3 Methodology
39
Trichophyton rubrum (ATCC 28188). The dermatophytic fungi were purchased from
American Tissue Culture Lab, USA.
3.6.1.2 Inoculum preparation
The Staphylococcus aureus strains (Sa 2, Sa 3, Sa 7) were grown and maintained on
Mueller Hinton agar (MHA). The VISA and VRSA were grown and maintained on
Tryptic soy agar (TSA). Candida albican and Candida glabrata, Microporum canis
and Trycophyton rubrum were grown and maintained in Potato Dextrose agar (PDA)
slants. They were then stored at 4°C under aerobic condition.
The Staphylococcus aureus strains namely Sa 2, Sa 3, Sa 7 were cultured in Mueller
Hinton Broth (MHB), VISA and VRSA in Tryptic soy broth (TSB) overnight (24 hours)
at 37°C while the fungal specimens were cultured in Tryptic soy broth (TSB)
overnight at 26°C. The inoculum was further adjusted to obtain a turbidity comparable
to that of McFarland standard tube No. 0.5 (Vandepitte et al., 1991) for further use.
3.6.1.3 Minimum Inhibitory Concentration (MIC)
Media was sterilized by autoclaving at 120°C for 15 minutes and all subsequent
manipulations were carried out in a class 2 laminar flow cabinet. The effectiveness of
the antifungal and antibacterial activities of the tested essential oils was quantified in
liquid media using the microdilution method using microtitre plates (12 X 8 wells) over
the range of 19.5 – 2500 µl/ml. The 10 µl of essential oils stock solution (50 mg/ml) in
dimethyl sulfoxide (DMSO) (not more than 10 % of total volume in well A) and 90 µl
of broth were added to the well labeled as A. Only 50µl of broth each was added for the
Chapter 3 Methodology
40
wells labeled as B until H. The oils and broth in well-A were mixed thoroughly before
transferring 50 µl of the resultant mixture to well B. The same procedure was repeated
for sample mixtures in well B until H, thus creating a serial dilution of the test materials.
50 µl of inoculum (microbes tested) were added in well A to well H. The microtitre
plates were then incubated at 37°C for 24 hours.
Cyclohexamide (50 mg/ml) was used as a standard antibiotic for comparison with the
antifungal activities of the essential oils while oxacilin (50 mg/ml) was used as standard
for antibacterial testing. DMSO served as negative control. Turbidity was taken as
indication of growth, thus the lowest concentration which remains clear after
macroscopic evaluation, was taken as the minimum inhibitory concentration (MIC).
The MIC was recorded as mean concentration of triplicate. The activities were
categorized as weak (MIC ≥ 5000 µg/ ml), moderate (MIC: 1000 - 4900 µg/ ml) and
strong (MIC ≤ 1000 µg/ ml).
3.6.2 Antioxidant activity
3.6.2.1 Chemicals and reagents
2, 2’-diphenylpicrylhydrazyl (DPPH) and the reference standard, ascorbic acid and the
solvent hexane and methanol were purchased from Sigma. For reducing power assay,
the chemicals used are potassium fericyanide, tricholoroacetic acid, ferric chloride,
sodium phosphate monobasic (NaH2PO4) and sodium phosphate dibasic heptahydrate
(Na2HPO4) (Sigma grade).
Chapter 3 Methodology
41
3.6.2.2 DPPH radical scavenging assay
Hydrogen atom or electron donation ability of the corresponding oils was measured
from the bleaching of the purple-colored methanol solution of DPPH. This
spectrophotometric assay uses stable 2, 2’-diphenylpicrylhydrazyl (DPPH) radical as a
reagent (Burits and Bucar, 2000 and Cuendet et. al., 1997). The method used in this
study was adopted from work of Ashrill et al., (1997).
250 µl of each sample (20 mg/ml) were individually mixed with methanol and 25 µl of
DPPH solution (8 mg/ml) to get the final concentration of 5 mg/ml of sample. All
samples and the control (methanol) were monitored for their absorbance values after
incubation period of 30 minutes at room temperature using UV-2450, UV-Visible
spectrophotometer (Shimadzu) at 517 nm. All analyses were carried out in triplicate
and the average values were recorded. Ascorbic acid was used as positive controls and
purchased from Sigma.
The percentage of inhibition of each sample was calculated according to the formula:
% of inhibition = Absorbance of control – Absorbance of sample x 100
Absorbance of control
3.6.2.3 Reducing Power assay
The reducing power of the prepared essential oils was determined according to the
method of Oyaizu (1986). Briefly, each extract (5mg, 10mg, 15mg and 20 mg) was
dissolved in 1.0 ml of distilled water to which was added 2.5 ml of a 0.2 M phosphate
buffer (pH 6.6) and 2.5 ml of 1% (w/v) solution of potassium ferricyanide (Sigma
grade). The mixture was incubated in a water bath at 50ºC for 20 min. Following this,
Chapter 3 Methodology
42
2.5 ml of a 10% (w/v) trichloroacetic acid solution (Sigma grade) was added and the
mixture was then centrifuged at 1000 rpm for 10 min. A 2.5 ml aliquot of the upper
layer was combined with 2.5 ml of distilled water and 0.5 ml of 0.1% (w/v) solution of
ferric chloride. Absorbance of the reaction mixture was read spectrophotometrically at
700 nm. Increased absorbance of the reaction mixture indicates greater reducing power.
Mean values from three independent samples were calculated for each extract.
3.6.3 Anti-inflammatory activity
3.6.3.1 Chemicals and reagents
Borate buffer (0.2 M, pH 9.0), lipoxygenase (167 U/ml), linoleic acid (134 µM),
hyaluronidase (1.00-1.67 U), sodium phosphate buffer pH 7.0, hyaluronic acid,
apigenin, sodium chloride, BSA, DMSO, sodium phosphate, acetic acid, pH 3.75.
3.6.3.2 Lipoxygenase assay
This assay was performed according to the procedure described by Sigma, with slight
modification (Ling et al. 2003). Enzyme activity was measured spectrophotometrically
using a spectrophotometer, in borate buffer (0.2 M, pH 9.0) by the increase in
absorbance at 234 nm, 25°C, after addition of lipoxygenase (167U/ml final
concentration), using linoleic acid (134 µM) as substrate. The enzyme solution was
kept in ice, and controls (100% enzyme activity) were measured before the test samples.
For the test, the enzyme solution was preincubated with the test sample for 5 min at
25°C, followed by addition of substrate solution and borate buffer to the final volume
of 1.5 ml. The enzyme activity was calculated as the rate of change of absorbance per
unit time. The enzyme inhibitory activity was expressed as the percentage ratio of the
Chapter 3 Methodology
43
difference in enzyme activity between the test sample and control vs. enzyme activity
in the control experiment. Samples were tested at maximum concentration of 200 µM
in the final volume of the assay mixture. The concentration reducing enzyme activity
by 50% with respect to the control was estimated from graphic plots of a concentration
dependent study and was defined as IC50 expressed in µM.
3.6.3.3 Hyaluronidase assay
The assay was performed according to the Sigma protocol with slight modifications
(Ling et al. 2003) The assay medium consisting of 1.00-1.67 U hyaluronidase in 100 µl
20mM sodium phosphate buffer pH 7.0 with 77mM sodium chloride and 0.01% BSA
was preincubated with 5 µl of the test compound (in DMSO) for 10 min at 37oC. Then
the assay was commenced by adding 100 µl hyaluronic acid (0.03% in 300mM sodium
phosphate, pH 5.35) to the incubation mixture and incubated for a further 45 min at
37oC. The undigested hyaluronic acid was precipitated with 1 ml acid albumin solution
made up of 0.1 % bovine serum albumin in 24mM sodium acetate and 79mm acetic
acid, pH 3.75. After standing at room temperature for 10 min, the absorbance of the
reaction mixture was measured at 600nm. The absorbance in the absence of enzyme
was used as the reference value for maximum inhibition. The inhibitory activity of the
test compound was calculated as the percentage ratio of the absorbance in the presence
of the test compound vs. absorbance in the absence of enzyme. The enzyme activity
was checked by the control experiment run simultaneously, in which the enzyme was
preincubated with 5µl DMSO instead, and followed by the assay procedures described
above. In this case, the percentage ratio of the absorbance in the presence of enzyme vs.
that in the absence of enzyme was in the range of 15-20%. The performance of the
assay was verified using apigenin as a reference under exactly the same experimental
Chapter 3 Methodology
44
conditions. Compounds were tested at a maximum concentration of 50 x 102 µM in the
final reaction mixture. The results were expressed as mean of the percentage inhibitions
of three separate experiments measured in triplicate.
Figure 3.2: Outline of the present study
Plant samples
Alpinia pahangensis (leaves and rhizomes)
Alpinia murdochii (leaves and rhizomes)
Alpinia scabra (leaves and rhizomes)
Essential oil analysis (GC, GC/MS and Kovats indices)
Percentage of yield (based on dry weight of plant sample)
Biological activities
Antioxidant activity
• DPPH free radical scavenging assay
• Reducing power assay
Antimicrobial activity
• Minimum Inhibition Concentration (MIC)
Anti-inflammatory activity
• Hyaluronidase assay
• Lipoxygenase assay
CHAPTER 4
RESULTS AND DISCUSSION
Chapter 4 Results and discussion
45
RESULTS AND DISCUSSION
4.1 Chemical constituents of essential oils of three wild Alpinia species
In this study, three species of Alpinia namely Alpinia murdochii, Alpinia pahangensis and
Alpinia scabra were investigated for their chemical constituents of the essential oils from
rhizomes and leaves. The yields of the essential oils were calculated based on the dry
weight of each sample. The percentage of the yield is shown in Table 4.1.
The essential oils obtained were subjected to gas chromatography (GC) and gas
chromatography-mass spectrometry (GC/MS) analysis for their detail identification of
components in the complex mixture. The CBP-5 capillary column was used in GC and HP-
5 column for GC/MS. Identification of the compounds was also aided by comparison of
their GC/MS mass spectral data with those from the NIST mass spectral database. The
Kovats indices of each identified component were also calculated based on their retention
time in order to confirm the identification.
Chapter 4 Results and discussion
46
Table 4.1: Essential oil yield of three Alpinia species
Plant name Part Essential
oil yield
Description
Alpinia murdochii Leaves 0.48 % Yellowish in colour
Alpinia murdochii Rhizomes 0.09 % Golden yellow in colour
Alpinia pahangensis Leaves 0.179 % Yellowish in colour
Alpinia pahangensis Rhizomes 0.092 % Golden yellow in colour
Alpinia scabra Leaves 0.157 % Yellowish in colour
Alpinia scabra Rhizomes 0.023 % Golden yellow in colour
0.00%
0.05%
0.10%
0.15%
0.20%
0.25%
0.30%
0.35%
0.40%
0.45%
0.50%
Percentages of Yield (%)
AML AMR APL APR ASL ASR
Alpinia species
Yeild of Essential Oils
Figure 4.1: Yields of essential oils from three Alpinia species: Alpinia murdochii, Alpinia
pahangensis and Alpinia scabra
Legend:
AML : Alpinia murdochii (leaf)
APL : Alpinia pahangensis (leaf)
ASL : Alpinia scabra (leaf)
AMR : Alpinia murdochii (rhizome)
APR : Alpinia pahangensis (rhizome)
ASR : Alpinia scabra (rhizome)
Chapter 4 Results and discussion
47
As can be seen above (Table 4.1 and Figure 4.1), the yield of the leaf oils is higher as
compared to the rhizome oils (more than 0.10 %). The colour of the leaf oils is yellowish
while for the rhizome oils; the colour is darker (golden yellow in colour). All of the leaf
oils impart a pungent odour, while the rhizome oils emit a woody odour.
4.2 Chemical constituents of essential oils from the leaves and the rhizomes of wild
Alpinia species
This present study describes the constituent of essential oils of the leaf and the rhizome of
three selected wild Alpinia species from Peninsular Malaysia namely Alpinia murdochii,
Alpinia pahangensis and Alpinia scabra. To the best knowledge of the author, there are no
chemical and biological activity reports on this three species yet. The GC chromatograms
of all species studied are attached in Appendix I.
Chapter 4 Results and discussion
48
4.2.1 Essential oil components of the leaf of Alpinia murdochii Ridl.
The chemical constituents of the leaf of Alpinia murdochii was identified using CBP-5
capillary column for GC and HP-5 capillary column for GC/MS. The list of compounds is
presented in Table 4.2. Forty compounds were identified from this sample amounting to
94.31 % of the total oils.
The oils were dominated by seventeen hydrocarbons representing 72.56 %. Twelve
compounds of this group are monoterpenes and five compounds belong to sesquiterpenes.
The monoterpenes; sabinene and β-pinene were the major components. Sabinene (23.76
%) was known to impart a woody odour (Moon, et al., 2006) and De Pooter, et al. (1985),
reported that β-pinene (23.78 %) has turpentine like odour.
Fifteen alcoholic compounds were detected in this oils which comprised of 17.03 %.
Eleven of the compounds were monoterpenes (16.59 %) and three were sesquiterpenes
(0.38 %). Terpinene-4-ol was the most abundant monoterpene with 10.49 % while the
most abundant sesquiterpene was trans-nerolidol (0.13 %). Only one non-terpenic
compound has been detected; cis-3-hexenol (0.06 %).
The oils also comprised of three ketones (1.73 %), two esters (0.35 %), two aldehydes
(1.35 %) and one ether (0.27 %).
Chapter 4 Results and discussion
49
Table 4.2: Chemical components of the leaf oils of Alpinia murdochii Ridl.
No.
Compounds
KIa
KIb
Composition
(%)
Method of
identification
1 cis -3-Hexenol 851 859 0.06 MS, KI
2 α- Thujene 929 930 3.54 MS, KI
3 α-Pinene 937 939 8.56 MS, KI
4 Camphene 948 954 0.11 MS, KI
5 Sabinene 974 975 23.76 MS, KI
6 β - pinene 977 979 23.83 MS, KI
7 Myrcene 983 991 0.16 MS, KI
8 α-Phellandrene 1002 1005 0.06 MS, KI
9 α-Terpinene 1016 1017 1.91 MS, KI
10 p-Cymene 1026 1025 3.83 MS, KI
11 β - Phellandrene 1029 1029 1.51 MS, KI
12 δ-3 - Carene 1030 1031 1.04 MS, KI
13 γ- Terpinene 1057 1060 4.25 MS, KI
14 cis- Sabinene hydrate 1064 1070 0.49 MS, KI
15 trans - Sabinene hydrate 1078 1072 1.11 MS, KI
16 α-Campholenal 1123 1126 1.08 MS, KI
17 trans-Pinocarveol 1134 1139 0.41 MS, KI
18 Nopinone 1139 1140 1.29 MS, KI
19 Sabina ketone 1156 1159 0.36 MS, KI
20 Pinocarvone 1160 1165 0.08 MS, KI
21 Borneol 1166 1169 0.54 MS, KI
22 Terpinene-4-ol 1175 1177 10.49 MS, KI
23 p-Cymene-8-ol 1184 1183 1.56 MS, KI
24 α- Terpineol 1186 1189 0.46 MS, KI
25 Myrtenol 1202 1196 0.82 MS, KI
26 cis - Piperitol 1211 1196 0.31 MS, KI
27 cis - Carveol 1222 1217 0.12 MS, KI
28 Cumin aldehyde 1237 1242 0.27 MS, KI
29 Bornyl acetate 1283 1289 0.35 MS, KI
30 Perilla alcohol 1304 1295 0.28 MS, KI
31 α - Copaene 1377 1377 0.06 MS, KI
Chapter 4 Results and discussion
50
32 (Z)-Caryophyllene 1407 1409 0.06 MS, KI
33 (E)-Caryophyllene 1419 1419 0.41 MS, KI
34 δ - Cadinene 1507 1514 0.12 MS, KI
35 trans - Nerolidol 1521 1533 0.13 MS, KI
36 Caryophyllene oxide 1572 1583 0.27 MS, KI
37 β-Eudesmol 1630 1640 c,d
0.12 KI
38 α-Eudesmol 1638 1645 c,d
0.13 KI
39 γ - selinene 1656 1477 0.08 MS
40 Phytol 1913 1943 0.29 MS, KI
Total 94.31
Legend:
a Kovats indices: CBP-5 capillary column
b Adam, 2001
c De Pooter et al., 1995
d Rout, et al., 2005
Composition (%) : Obtained by using CBP-5 capillary column
MS : Mass fragmentation
KI : Kovats retention indices
Chapter 4 Results and discussion
51
4.2.2 Essential oil components of the rhizome of Alpinia murdochii Ridl.
The chemical constituents of essential oils of the rhizome of Alpinia murdochii is listed in
Table 4.3. A total of thirty-seven compounds were identified from these oils amounting to
71.06 % of area percent. The essential oils of this rhizome showed a high content of
sesquiterpene hydrocarbons (26.85 %) and oxygenated monoterpenes (19.56 %). Other
compounds are oxygenated non-terpenes (9.57 %), monoterpene hydrocarbons (8.56 %),
oxygenated sesquiterpenes (3.05 %), non-terpene hydrocarbons (2.70 %) and oxygenated
diterpenes (0.77 %).
Sesquiterpene hydrocarbons were detected as the most abundant group present in these
oils. Nine sesquiterpenes comprised of 26.85 % of the total oils. The compounds were γ-
selinene (15.51 %), α-selinene (2.30 %), δ-selinene (1.79 %), cis-caryophyllene (1.64 %),
α-calacorene (1.51 %), α-copaene (1.48 %), α-panasinsen (1.22 %), α-maaliene (1.08 %)
and β-selinene (0.32 %).
Alcoholic components presented as the second major group identified comprising of 18.84
%. Eight compounds were monoterpenes (15.02 %), two sesquiterpenes (3.05 %) and only
one diterpene was identified as phytol with 0.77 %. The major compounds of the alcoholic
group were terpenene-4-ol (5.58 %), α-terpineol (5.04 %), β-bisabolol (2.40 %) and trans-
pinocarveol (1.99 %).
Chapter 4 Results and discussion
52
Table 4.3: Chemical components of the rhizome oil of Alpinia murdochii Ridl.
No.
Compounds
KIa
KIb
Composition
(%)
Method of
identification
1 Furfural 830 836 0.82 MS, KI
2 α-Pinene 934 939 0.76 MS, KI
3 β-Pinene 971 979 2.80 MS, KI
4 α-Terpinene 1014 1017 1.26 MS, KI
5 Cymene 1024 1025 2.66 MS, KI
6 Limonene 1027 1029 0.31 MS, KI
7 γ-Terpinene 1054 1060 0.77 MS, KI
8 trans-Sabinene hydrate 1077 1072 0.28 MS, KI
9 α-Campholenal 1134 1126 0.63 MS, KI
10 trans-Pinocarveol 1139 1139 1.99 MS, KI
11 Sabina ketone 1156 1159 1.07 MS, KI
12 Pinocarvone 1164 1165 0.98 MS, KI
13 Borneol 1166 1169 0.45 MS, KI
14 Terpinen-4-ol 1172 1177 5.58 MS, KI
15 α-Terpineol 1183 1189 5.04 MS, KI
16 Myrtenal 1202 1196 1.99 MS, KI
17 Myrtenol 1210 1196 0.72 MS, KI
18 Verbenone 1222 1205 0.33 MS, KI
19 2-Methyl-3-phenyl propanal 1237 - 0.61 MS
20 Carvacrol 1284 1299 0.56 MS, KI
21 p-Mentha-1,4-dien-7-ol 1331 - 0.40 MS
22 α-Copaene 1365 1377 1.48 MS, KI
23 cis-Caryophyllene 1419 1419 1.64 MS, KI
24 β-Selinene 1448 1490 0.32 MS, KI
25 δ - Selinene 1472 1493 1.79 MS, KI
26 α- Selinene 1480 1498 2.30 MS, KI
27 α-Panasinsen 1519 1518 1.22 MS, KI
28 α- Maaliene 1528 - 1.08 MS
29
trans - 2(1H)-Naphtalenone,
octahydro-4a,7,7-trimethyl
1540
-
0.35
MS
30 α-Calacorene 1546 1546 1.51 MS, KI
Chapter 4 Results and discussion
53
31 γ - Selinene 1663 1484 15.51 MS
32 Heptadecane 1669 1700 2.70 MS, KI
33 β- Bisabolol 1671 1675 2.40 MS, KI
34 (E,Z)-Farnesol 1755 1746 0.65 MS, KI
35 Benzyl benzoate 1766 1760 0.77 MS, KI
36 (E,E)-Farnesyl acetate 1813 1844 c 6.56 MS, KI
37 Phytol 1932 1943 0.77 KI
Total 71.06
Legend:
a Kovats indices: CBP-5 capillary column
b Adam, 2001
c Ibrahim, et al., 2004
Composition (%) : Obtained by using CBP-5 capillary column
GC/MS : Gas chromatography / mass spectrometer
KI : Kovats retention indices
Chapter 4 Results and discussion
54
4.2.3 Essential oil components of the leaf of Alpinia pahangensis Ridl.
Table 4.4 lists the chemical constituents of Alpinia pahangensis leaf oils. Thirty-eight
components representing 86.91 % of the oil were identified. The essential oil consisted
mainly monoterpene hydrocarbons (56.58 %), followed by oxygenated monoterpenes
(21.92 %), non-terpene hydrocarbon (4.49 %), oxygenated diterpenes (2.76 %),
oxygenated non-terpene (2.98 %), sesquiterpenes hydrocarbons (1.63 %) and oxygenated
sesquiterpenes (1.56 %).
Monoterpene hydrocarbons formed the most abundant class in this oil. These oils were rich
in β-pinene with 39.61 %, α-pinene (7.55 %) and limonene (4.89 %). As mentioned before,
β-pinene was reported to have turpentine like odour (De Pooter, et al., 1985). The other
monoterpene hydrocarbons are p-cymene (1.43 %), γ-terpinene (0.87 %), α- thujene (0.67
%), δ-3-carene (0.54 %), terpinolene (0.39 %), α-terpinene (0.27 %), camphene (0.25 %)
and myrcene (0.11 %).
Oxygenated monoterpenes were the second most abundant type of compounds identified;
comprising of two aldehydes (0.63 %), seven alcohols (14.53 %), four ketones (6.62 %)
and one furanoid (0.14 %). The major one being verbenone (ketone) which amounted to
3.99 %. It was followed by myrtenol (alcohol) with 3.75 %, cis-carveol (2.93 %), perilla
alcohol (2.86 %) and trans-verbenol (2.85 %).
Chapter 4 Results and discussion
55
Table 4.4: Chemical components of the leaf oil of Alpinia pahangensis Ridl.
No.
Compounds
KIa
KIb
Composition
(%)
Method of
identification
1 α- Thujene 930 930 0.67 MS, KI
2 α- Pinene 939 939 7.55 MS, KI
3 Camphene 951 954 0.25 MS, KI
4 β-Pinene 981 979 39.61 MS, KI
5 β-Myrcene 1001 991 0.11 MS, KI
6 α- Terpinene 1015 1017 0.27 MS, KI
7 p-Cymene 1025 1025 1.43 MS, KI
8 Limonene 1029 1029 4.89 MS, KI
9 γ -Terpinene 1055 1060 0.87 MS, KI
10 Linalool oxide (trans) furanoid 1067 1073 0.14 MS, KI
11 δ-3- Carene 1078 1031 0.54 MS
12 6-methyl-3,5-heptadiene -2-one 1102 - 0.3 MS
13 Terpinolene 1105 1089 0.39 MS, KI
14 α- Campholenal 1117 1126 0.15 MS, KI
15 Nopinone 1124 1139 1.17 MS, KI
16 trans-Pinocarveol 1135 1140 1.0 MS, KI
17 trans-Verbenol 1140 1145 2.85 MS, KI
18 Pinocarvone 1146 1165 0.6 MS, KI
19 Borneol 1154 1169 0.23 MS, KI
20 cis-Pinocamphone 1157 1175 1.56 MS, KI
21 Terpinene-4-ol 1165 1177 0.91 MS, KI
22 Myrtenal 1167 1196 0.48 MS, KI
23 Myrtenol 1173 1196 3.75 MS, KI
24 Verbenone 1184 1205 3.99 MS, KI
25 cis-Carveol 1204 1229 2.93 MS, KI
26 Carvone 1211 1243 0.47 MS, KI
27 Myrtenyl acetate 1224 1327 0.58 MS
28
p-Mentha-1,8-dien-7-ol @
Perilla alcohol
1283
1295
2.86
MS, KI
29 α- Cubebene 1306 1351 0.87 MS, KI
30 (Z)-Caryophyllene 1401 1409 0.22 MS, KI
Chapter 4 Results and discussion
56
31 α- Gurjunene 1419 1410 0.22 MS, KI
32 Dihydropseudoionone 1424 - 0.13 MS
33
Eudesma-4 (14), 11-diene @
β-Selinene
1443
1490
0.32
MS, KI
34 Caryophyllene oxide 1475 1583 0.96 MS
35 Phytone 1787 - 0.48 MS
36 (E,E)-Farnesyl acetate 1834 1844 c 0.40 MS, KI
37 Phytol 1916 1943 0.39 MS, KI
38 Isophytol 1959 1948 2.37 MS, KI
Total 86.91
Legend:
a Kovats indices: CBP-5 capillary column
b Adam, 2001
c Ibrahim et al., 2004
Composition (%) : Obtained by using CBP-5 capillary column
MS : Mass fragmentation
KI : Kovats retention indices
Chapter 4 Results and discussion
57
4.2.4 Essential oils components of the rhizome of Alpinia pahangensis Ridl.
The result obtained by GC and GC/MS analysis of the essential oils from the rhizome of
Alpinia pahangensis is presented in Table 4.5. Thirty-eight compounds were identified,
representing 75.28 % of the total oils. The major components were sesquiterpene
hydrocarbons, γ-selinene (11.6 %) and monoterpene hydrocarbons, β-pinene (10.87 %).
The oil comprised of twenty hydrocarbons (40.31 %), eleven alcohols (16.63 %), two
esters (9.09 %), three aldehydes (3.88 %), one of ether (3.16 %) and one ketone (2.21 %).
The rhizome oils were predominated by twelve compounds of sesquiterpenes
hydrocarbons (19.64%) such as γ-selinene, α-selinene (2.11%) and α-maaliene (1.23%). γ-
Selinene is responsible for the woody odour of this rhizome.
There were seven monoterpene hydrocarbons with the total yield of 16.18%. The most
abundant compound of this group is β-pinene. This is followed by α-pinene (2.59 %),
limonene (1.1 %), p-cymene (1.02 %), camphene (0.29 %), γ-terpinene (0.17 %) and
terpinolene (0.14 %).
These oils also comprised significant amount of alcoholic compounds. There are ten
compounds amounting to 16.03 % of the total oil. Those with concentrations greater than
one percent were α-terpineol (6.38 %), cis-sabinol (3.02 %) and terpinene-4-ol (1.94 %).
No sesquiterpenoid alcohols were detected.
Chapter 4 Results and discussion
58
Table 4.5: Chemical components of the rhizome oil of Alpinia pahangensis Ridl.
No.
Compounds
KIb
KIa
Composition
(%)
Method of
identification
1 α- Pinene 939 939 2.59 MS, KI
2 Camphene 948 954 0.29 MS, KI
3 β-Pinene 973 979 10.87 MS, KI
4 p-Cymene 1023 1025 1.02 MS, KI
5 Limonene 1027 1029 1.1 MS, KI
6 1,8- Cineole 1028 1031 0.63 MS, KI
7 γ-Terpinene 1054 1060 0.17 MS, KI
8 terpinolene 1102 1089 0.14 MS, KI
9 exo-Fenchol 1117 1122 0.97 MS, KI
10 α- Campholenal 1123 1126 0.71 MS, KI
11 trans-Pinocarveol 1134 1140 0.99 MS, KI
12 cis-Sabinol 1140 1143 3.02 MS, KI
13 Camphene hydrate 1145 1150 0.6 MS, KI
14 Pinocarvone 1156 1165 2.21 MS, KI
15 Borneol 1164 1169 0.39 MS, KI
16 Terpinene-4-ol 1171 1177 1.94 MS, KI
17 α- Terpineol 1184 1189 6.38 MS, KI
18 Myrtenal 1203 1196 2.9 MS, KI
19 Myrtenol 1211 1196 0.63 MS, KI
20 Thujol @ thujanol < 3> 1223 1169 0.48 MS
21 Perilla aldehyde 1265 1272 0.27 MS, KI
22 Bornyl acetate 1281 1289 0.44 KI
23 α- Cubebene 1304 1351 0.47 MS, KI
24 β-Elemene 1312 1391 0.14 MS
25 α-Copaene 1365 1377 0.44 MS, KI
26 α- Gurjunene 1418 1410 0.45 MS, KI
27 (E)-β- Farnesene 1472 1457 1.5 MS, KI
28 δ-Selinene 1475 1472 0.46 MS, KI
29 α- Selinene 1480 1498 2.11 MS, KI
30 α- Panasinsen 1519 1518 1.23 MS
31 α- Maaliene 1528 - 0.67 MS
Chapter 4 Results and discussion
59
32 α-Calacorene 1547 1546 0.22 MS, KI
33 Caryophyllene oxide 1574 1583 3.16 MS, KI
34 Calarene @ β- gurjunene 1605 1434 0.35 MS, KI
35 γ-Selinene 1663 1493 11.6 MS, KI
36 Heptadecane 1669 1700 4.49 KI
37 β-Bisabolol 1675 1675 0.60 MS, KI
38 (E,E)-Farnesyl acetate 1815 1844 8.65 KI
Total 75.28
Legend:
a Kovats indices: CBP-5 capillary column
b Adam, 2001
Composition (%) : Obtained by using CBP-5 capillary column
MS : Mass fragmentation
KI : Kovats retention indices
Chapter 4 Results and discussion
60
4.2.5 Essential oil components of the leaf of Alpinia scabra (Blume) Náves
Forty components of Alpinia scabra leaf oils were identified and comprising of 86.61 % of
the total oil. The results obtained by GC and GC/MS analysis of these oils are listed in
Table 4.6. This oil could be a good source of β-pinene as it made up to 63.37 % of the total
oils. Additionally, other major compounds were α-pinene (6.58 %), borneol (3.20 %),
caryophyllene oxide (1.69 %), p-cymen-8-ol (1.20 %), trans-pinocarveol (1.15 %),
myrtenyl acetate (1.03 %) and limonene (1.0 %).
Hydrocarbons formed the most abundant group in this oil with thirteen compounds
accounting for 73.02 % of the total leaf oils. Six were monoterpenes (71.61 %); β-pinene
(63.37 %), α-pinene (6.58 %), camphene (0.44 %), γ-terpinene (0.12 %) and δ-2-carene
(0.10 %). The other seven compounds were sesquiterpenes (1.41 %); β-bisabolene
(0.42%), γ-gurjunene (0.40 %), (E)-caryophyllene (0.19 %), α-selinene (0.17 %), β-
sesquiphellandrene (0.07 %) and α-copaene (0.07 %). Only one non-terpene was detected,
pentadecane (0.09 %).
Alcoholic components constitute the second largest group with 7.43 % of the total oils.
The major compound in this group was borneol with 3.20 %. Nine compounds were
monoterpenes, two sesquiterpenes, one non-terpene and one diterpene.
The other components detected in this oil were two aldehydes, six ketones, three esters and
one ether.
Chapter 4 Results and discussion
61
Table 4.6: Chemical components of the leaf oil of Alpinia scabra (Blume) Náves
No.
Compounds
KIb
KIa
Composition
(%)
Method of
identification
1 Furfural 845 836 0.12 MS, KI
2 3-Hexenol 851 859 0.55 MS, KI
3 α-Pinene 937 939 6.58 MS, KI
4 Camphene 949 954 0.44 MS, KI
5 ββββ-Pinene 979 979 63.37 MS, KI
6 δ-2-Carene 1024 1002 0.10 MS, KI
7 Limonene 1028 1029 1.00 MS, KI
8 1,8-Cineol 1054 1031 0.08 MS, KI
9 γ-Terpinene 1078 1060 0.12 MS, KI
10
3,5,5-Trimethyl 2-
cyclopentene-1-one
1107
-
0.06
MS
11 exo-Fenchol 1123 1122 0.13 MS, KI
12 Nopinone 1134 1139 0.20 MS, KI
13 trans-Pinocarveol 1140 1140 1.15 MS, KI
14 Camphor 1144 1146 0.14 MS, KI
15 Isoborneol 1147 1162 0.11 MS, KI
16 Pinocarvone 1156 1165 0.63 MS, KI
17 Borneol 1165 1169 3.20 MS, KI
18 Pinocamphone 1171 1175 0.38 MS, KI
19 Terpinen-4-ol 1176 1177 0.07 MS, KI
20 p-Cymen-8-ol 1183 1183 1.20 MS, KI
21 α-Terpineol 1201 1189 0.44 MS, KI
22 Myrtenol 1209 1196 0.07 MS, KI
23 Perilla aldehyde 1275 1272 0.23 MS, KI
24 trans- Pinocarvyl acetate 1310 1298 0.23 MS, KI
25 Myrtenyl acetate 1322 1327 1.03 MS, KI
26 α-Copaene 1377 1377 0.07 MS, KI
27 (E)-Caryophyllene 1418 1419 0.19 MS, KI
28 α-Lonone 1423 - 0.14 MS
30 α-Selinene 1471 1498 0.17 MS, KI
31 Pentadecane 1481 1500 0.09 MS, KI
Chapter 4 Results and discussion
62
32 β-Bisabolene 1507 1506 0.42 MS, KI
33 β- Sesquiphellandrene 1521 1523 0.07 MS, KI
34 trans-Nerolidol 1553 1563 0.07 MS, KI
35 Caryophyllene oxide 1574 1583 1.69 MS, KI
36 Caryophylladienol I 1638 1693 c 0.24 MS, KI
37 γ - Gurjunene 1656 - 0.40 MS
38 α-Bisabolol 1677 1686 0.36 MS, KI
39 (E,E)-Farnesyl acetate 1833 1844 0.13 MS, KI
40 Phytol 1915 1949 0.98 MS, KI
Total 86.65
Legend:
a Kovats indices: CBP-5 capillary column
b Adam, 2001
c Suleimenov, et al., 2001
Composition (%) : Obtained by using CBP-5 capillary column
MS : Mass fragmentation
KI : Kovats retention indices
Chapter 4 Results and discussion
63
4.2.6 Essential oil components of the rhizome of Alpinia scabra (Blume) Náves
The chemical constituents of the essential oils of Alpinia scabra rhizome oils are presented
in Table 4.7. These oils contained more than fifty compounds; however only forty-one
components were detected comprising of 70.96 % of the total oil. GC and GC/MS analyses
revealed that the major compounds of the oils were γ-selinene (33.45 %), α-selinene (3.64
%), α-terpineol (3.55 %), alloaromadendrene (3.32 %), spathulenol (3.25 %) and γ-
muurolene (3.45 %).
Hydrocarbons were the principal constituents of this oil (50.1 %). It comprised of three
monoterpenoids (1.09 %) and fourteen sesquiterpenoids (49.01 %). γ-Selinene (33.45 %),
α-selinene (3.64 %), γ-muurolene (3.45 %), alloaromadendrene (3.32 %) and α-panasinsen
(2.21 %) were the compounds present in an appreciable amount, while the other nine
compounds were present in low concentrations. γ-Selinene is responsible for the woody
odour of this oil.
Alcoholic compounds were made up of one non-terpenes alcohol (0.08 %), seven
monoterpenes (6.26 %) and seven sesquiterpenes (7.73 %). Compounds that are present in
an appreciable amount were α-terpineol (3.55 %), exo-fenchol (1.46 %), spathulenol (3.25
%) and α-eudesmol (2.17 %).
The rest of the oils were made up of two aldehydes (3.37 %), two ketones (2.38 %), one
ester (0.64 %) and one ether (0.40 %).
Chapter 4 Results and discussion
64
Table 4.7: Chemical components of the rhizome oils of Alpinia scabra (Blume) Náves
No.
Compounds
KIb
KIa
Composition
(%)
Method of
identification
1 Furfural 830 836 0.41 MS, KI
2 3-Hexenol 851 859 0.08 MS, KI
3 α-Pinene 960 939 0.19 MS, KI
4 β-Pinene 971 979 0.21 MS, KI
5 Limonene 1116 1029 0.69 MS, KI
6 Benzene acetaldehyde 1134 1042 0.66 MS, KI
7 exo-Fenchol 1138 1122 1.46 MS, KI
8 Nopinone 1144 1140 0.13 MS, KI
9 trans-Pinocarveol 1147 1139 0.23 MS, KI
10 Sabina ketone 1155 1159 0.99 MS, KI
11 Pinocarvone 1163 1165 1.16 MS, KI
12 Borneol 1166 1169 0.14 MS, KI
13 Terpinen-4-ol 1171 1177 0.29 MS, KI
14 p-Cymen-8-ol 1176 1183 0.14 MS, KI
15 α-Terpineol 1183 1189 3.55 MS, KI
16 Myrtenal 1201 1196 2.30 MS, KI
17 Myrtenol 1209 1196 0.45 MS, KI
18 Verbenone 1216 1205 0.10 MS, KI
19 Copaene 1370 1377 0.1 MS, KI
20 β-Elemene 1377 1391 0.35 MS, KI
21 cis-Caryophyllene 1405 1409 0.07 MS, KI
22 trans-Caryophyllene 1418 1419 0.51 MS, KI
23 Aromadendrene 1442 1441 0.09 MS, KI
24 γ-Muurolene 1472 1480 3.45 MS, KI
25 β-Selinene 1475 1490 0.77 MS, KI
26 α-Selinene 1481 1493 3.64 MS, KI
27 β-Bisabolene 1507 1506 0.39 MS, KI
28 α-Panasinsen 1519 1518 2.21 MS, KI
29 α- Maaliene 1527 - 0.55 MS, KI
30 α-Calacorene 1540 1546 0.11 MS, KI
31 Elemol 1547 1550 0.48 MS, KI
Chapter 4 Results and discussion
65
32 trans, α-Bisabolene epoxide 1552 - 0.40 MS
33 (E)-nerolidol 1558 1563 0.54 MS, KI
34 Caryophyllene alcohol 1565 1572 0.50 MS, KI
35 Spathulenol 1574 1578 3.25 MS, KI
36 α-Eudesmol 1643 1645 2.17 MS, KI
37 β-Eudesmol 1650 1651 0.46 MS, KI
38 γγγγ -Selinene 1665 1493 33.45 MS, KI
39 Alloaromadendrene 1668 1641 3.32 MS, KI
40 (E, Z) -Farnesol 1750 1746 0.33 MS, KI
41 Benzyl benzoate 1760 1760 0.64 MS, KI
Total 70.96
Legend:
a Kovats indices: CBP-5 capillary column
b Adam, 2001
Composition (%) : Obtained by using CBP-5 capillary column
MS : Mass fragmentation
KI : Kovats retention indices
Chapter 4 Results and discussion
66
4.2.7 Chemical compositions according to class of compounds of the leaf oils and
rhizome oils of three wild Alpinia species
Table 4.8 and Table 4.9 summarized the chemical constituents of the leaf oils and rhizomes
oils of three wild Alpinia species namely Alpinia murdochii, A. pahangensis and A. scabra
according to their classification of compounds.
Table 4.8: Chemical composition of the leaf oils of three wild Alpinia species
Compounds
Formula
molecule
Composition (%)
AML APL ASL
NON-TERPENES
Hydrocarbon
Pentadecane C15 H32 - - 0.09
Total - - 0.09
Alcohol
cis- 3-Hexenol C6 H12 O 0.06 - 0.55
Total 0.06 - 0.55
Aldehyde
Furfural C5 H4 O2 - - 0.12
Total - - 0.12
Ketones
6-methyl-3,5-heptadien-2-
one
C8 H12 O
- 0.3
-
3,5,5-Trimethyl 2-
cyclopentene-1-one
-
- -
0.06
Nopinone C9 H14 O 1.29 1.17 0.20
Sabina ketone C9 H14 O 0.36 - -
Chapter 4 Results and discussion
67
Compounds
Formula
molecule
Composition (%)
AML APL ASL
Ketones – cont’
α-Lonone C13 H20 O - - 0.14
Dihydropseudoionone C13 H22 O - 0.13 -
Phytone C18 H36 O - 0.48 -
Total 1.65 2.08 0.4
Esters
Bornyl acetate C12 H20 O2 0.35 - -
Myrtenyl acetate C12 H18 O2 - 0.58 1.03
trans-Pinocarvyl acetate C12 H18 O2 - - 0.23
(E,E)-Farnesyl acetate C17 H28 O2 - 0.40 0.98
Total 0.35 0.98 2.24
MONOTERPENES
Hydrocarbons
α- Thujene C10 H16 3.54 0.67 -
α- Pinene C10 H16 8.56 7.55 6.58
Camphene C10 H16 0.11 0.25 0.44
Sabinene C10 H16 23.76 - -
β-Pinene C10 H16 23.83 39.61 63.37
β-Myrcene C10 H16 0.16 0.11 -
α-Phellandrene C10 H16 0.06 - -
α-Terpinene C10 H16 1.91 0.27 -
p-Cymene C10 H16 3.83 1.43 -
β - Phellandrene C10 H16 1.51 - -
δ-2-Carene C10 H16 - - 0.10
δ-3-Carene C10 H16 1.04 0.54 -
γ- Terpinene C10 H16 4.25 0.87 0.12
Limonene C10 H16 - 4.89 1.0
Terpinolene C10 H16 - 0.39 -
Total 72.56 56.58 71.61
Chapter 4 Results and discussion
68
Table 4.8: Chemical composition of the leaf oils of three wild Alpinia species – cont’
Compounds
Formula
molecule
Composition (%)
AML APL ASL
Aldehydes
α-Campholenal C10 H16 O 1.08 0.15 -
Cuminaldehyde C10 H12 O 0.27 - -
Myrtenal C10 H14 O - 0.48 -
Perilla aldehyde C10 H14 O - - 0.23
Total 1.35 0.63 0.23
Alcohols
trans - Pinocarveol C10 H16 O 0.41 1.0 1.15
Borneol C10 H18 O 0.54 0.23 3.20
Isoborneol C10 H18 O - - 0.11
Terpinene-4-ol C10 H18 O 10.49 0.91 0.07
p-Cymene-8-ol C10 H14 O 1.56 - 1.20
α- Terpineol C10 H18 O 0.46 - 0.44
Myrtenol C10 H16 O 0.82 3.75 0.07
cis-Piperitol C10 H18 O 0.31 - -
cis -Carveol C10 H16 O 0.12 2.93 -
p-Mentha-1,8-dien-7-ol @
perilla alcohol
C10 H16 O 0.28 2.86
-
trans-verbenol C10 H16 O - 2.85 -
cis – sabinene hydrate C10 H18 O 0.49 - -
trans –Sabinene hydrate C10 H18 O 1.11 - -
1,8 - Cineole C10 H18 O - - 0.08
exo - Fenchol C10 H18 O - - 0.13
Total 16.59 14.53 6.45
Chapter 4 Results and discussion
69
Table 4.8: Chemical composition of the leaf oils of three wild Alpinia species – cont’
Compounds
Formula
molecule
Composition (%)
AML APL ASL
Ketones
Pinocarvone C10 H14 O 0.08 0.6 0.63
cis-Pinocamphone C10 H16 O - 1.56 0.38
Verbenone C10 H14 O - 3.99 -
Carvone C10 H14 O - 0.47 -
Camphor C10 H14 O - - 0.14
Total 0.08 6.62 1.15
Furanoid
trans-Linalool oxide C10 H18 O2 - 0.14 -
Total - 0.14 -
SESQUITERPENES
Hydrocarbons
α- Copaene C15 H24 0.06 - 0.07
(Z)-Caryophyllene C15 H24 0.06 0.22 -
(E)-Caryophyllene C15 H24 0.41 - 0.19
δ - Cadinene C15 H24 0.12 - -
α- Cubebene C15 H24 - 0.87 -
γ- Gurjunene C15 H24 - - 0.40
α- Gurjunene C15 H24 - 0.22 -
Eudesma-4 (14), 11-diene
@ β-Selinene
C15 H24
- 0.32
-
α- Selinene C15 H24 - - 0.17
β-Bisabolene C15 H24 - - 0.42
β-Sesquiphellandrene C15 H24 - - 0.07
γ -Selinene C15 H24 0.08 - -
Total 0.73 1.63 1.32
Chapter 4 Results and discussion
70
Table 4.8: Chemical composition of the leaf oils of three wild Alpinia species – cont’
Compounds
Formula
molecule
Composition (%)
AML APL ASL
Alcohols
Caryophylladienol I C15 H24 O - - 0.24
trans-Nerolidol C15 H26 O 0.13 - 0.07
α-Bisabolol C15 H26 O - - 0.36
β-Eudesmol C15 H26 O 0.12 - -
α-Eudesmol C15 H26 O 0.13 - -
Total 0.38 - 0.67
Ether
Caryophyllene oxide C15 H24 O 0.27 0.96 1.69
Total 0.27 0.96 1.69
DITERPENES
Alcohols
Phytol C20 H40 O 0.29 2.37 0.13
Isophytol C20 H40 O - 0.39 -
Total 0.29 2.76 0.13
Legend:
The leaves of three wild Alpinia species:
AML : Alpinia murdochii (leaf)
APL : Alpinia pahangensis (leaf)
ASL : Alpinia scabra (leaf)
Composition (%) : Obtained by using CBP-5 capillary column
Chapter 4 Results and discussion
71
Table 4.9: Chemical composition of the rhizome oils of three wild Alpinia species
Compounds
Formula
molecule
Composition (%)
AMR APR ASR
NON-TERPENES
Hydrocarbon
Heptadecane C17H36 2.70 4.49 -
Total 2.70 4.49 -
Aldehydes
Furfural C5 H4 O2 0.82 - 0.41
Benzene acetaldehyde C8 H8 O - - 0.66
Total 0.82 - 1.07
Alcohol
3-hexenol C6 H12 O - - 0.08
Total - - 0.08
Ketones
Sabina ketone C9 H14 O 1.07 - 0.99
Nopinone C9 H14 O - - 0.13
Total 1.07 - 1.12
Esters
Bornyl acetate C12 H20 O2 - 0.44 -
Benzyl benzoate C14 H12 O2 0.77 - 0.64
(E,E)-Farnesyl acetate C17 H28 O2 6.56 8.65 -
Total 7.33 9.09 0.64
Other
trans - 2(1H)-
Naphtalenone, octahydro-
4a,7,7-trimethyl
0.35 -
-
Total 0.35 - -
Chapter 4 Results and discussion
72
Table 4.9: Chemical composition of the rhizome oils of three wild Alpinia species- cont’
Compounds
Formula
molecule
Composition (%)
AMR APR ASR
MONOTERPENES
Hydrocarbons
α- Pinene C10 H16 0.76 2.59 0.19
Camphene C10 H16 - 0.29 -
β-Pinene C10 H16 2.80 10.87 0.21
α- Terpinene C10 H16 1.26 - -
p-Cymene C10 H16 2.66 1.02 -
Limonene C10 H16 0.31 1.1 0.69
γ- Terpinene C10 H16 0.77 0.17 -
Terpinolene C10 H16 - 0.14 -
Total 8.56 16.18 1.09
Alcohols
1,8 - Cineole C10 H18 O - 0.63 -
exo - Fenchol C10 H18 O - 0.97 1.46
trans - Pinocarveol C10 H16 O 1.99 0.99 0.23
cis - Sabinol C10 H16 O - 3.02 -
Camphene hydrate C10 H18 O - 0.6 -
Borneol C10 H18 O 0.45 0.39 0.14
Terpinene-4-ol C10 H18 O 5.58 1.94 0.29
α- Terpineol C10 H18 O 5.04 6.38 3.55
Myrtenol C10 H16 O 0.72 0.63 0.45
trans – Sabinene hydrate C10 H18 O 0.28 - -
Carvacrol C10 H14 O 0.56 - -
p-Mentha-1,4-dien-7-ol C10 H16 O 0.40 - -
Thujol @ thujanol < 3> C10 H18 O - 0.48 -
p-Cymen-8-ol C10 H14 O - - 0.14
Total 15.02 16.03 6.26
Chapter 4 Results and discussion
73
Table 4.9: Chemical composition of the rhizome oils of three wild Alpinia species- cont’
Compounds
Formula
molecule
Composition (%)
AMR APR ASR
Aldehydes
α- Campholenal C10 H16 O 0.63 0.71 -
Myrtenal C10 H14 O 1.99 2.9 2.30
Perilla aldehyde C10 H14 O - 0.27 -
2-Methyl-3-phenyl
propanal
C10 H12 O
0.61 -
-
Total 3.23 3.88 2.30
Ketones
Pinocarvone C10 H14 O 0.98 2.21 1.16
Verbenone C10 H14 O 0.33 - 0.10
Total 1.31 2.21 1.26
SESQUITERPENS
Hydrocarbons
α-Copaene C15 H24 1.48 0.44 0.1
cis -Caryophyllene C15 H24 1.64 - 0.07
trans -Caryophyllene C15 H24 - - 0.51
δ-Selinene C15 H24 1.79 0.46 -
α-Calacorene C15 H20 1.51 0.22 0.11
α- Gurjunene C15 H24 - 0.45 -
α-Selinene C15 H24 2.30 2.11 3.64
β-Selinene C15 H24 0.32 - 0.77
α-Cubebene C15 H24 - 0.47 -
β-Elemene C15 H24 - 0.14 0.35
(E)-β- Farnesene C15 H24 - 1.5 -
α- Maaliene C15 H24 1.08 0.67 0.55
α- Panasinsen C15 H24 1.22 1.23 2.21
γ-Selinene C15 H24 15.51 11.6 33.45
β-Gurjunene C15 H24 - 0.35 -
Chapter 4 Results and discussion
74
Table 4.9: Chemical composition of the rhizome oils of three wild Alpinia species- cont’
Compounds
Formula
molecule
Composition (%)
AMR APR ASR
SESQUITERPENS
Hydrocarbons – Cont’
Aromadendrene C15 H24 - - 0.09
γ- Muurolene C15 H24 - - 3.45
β- Bisabolene C15 H24 - - 0.39
Alloaromadendrene C15 H24 - - 3.32
Total 26.85 19.64 49.01
Alcohols
Elemol C15 H26 0 - - 0.48
β-bisabolol C15 H26 0 2.40 0.60 -
(E)-Nerolidol C15 H26 0 - - 0.54
Caryophyllene alcohol C15 H26 0 - - 0.50
Spathulenol C15 H24 0 - - 3.25
α- Eudesmol C15 H26 0 - - 2.17
β- Eudesmol C15 H26 0 - - 0.46
(E,Z)- Farnesol C15 H26 0 0.65 - 0.33
Total 3.05 0.60 7.73
Ethers
Caryophyllene oxide C15 H24 0 - 3.16 -
trans, α- bisabolene
epoxide
- -
0.40
Total - 3.16 0.40
Chapter 4 Results and discussion
75
Table 4.9: Chemical composition of the rhizome oils of three wild Alpinia species- cont’
Compounds
Formula
molecule
Composition (%)
AMR APR ASR
DITERPENE
Alcohol
Phytol C20 H40 O 0.77 - -
Total 0.77 - -
Legend:
The rhizome of three wild Alpinia species:
AMR : Alpinia murdochii (rhizome)
APR : Alpinia pahangensis (rhizome)
ASR : Alpinia scabra (rhizome)
Composition (%) : Obtained by using CBP-5 capillary column
Table 4.10: Percentages of similarity of essential oil constituents between three wild
Alpinia species
Leaf oils
Alpinia murdochii Alpinia pahangensis 36.6 %
Alpinia murdochii Alpinia scabra 29.5 %
Alpinia scabra Alpinia pahangensis 26.2 %
Rhizome oils
Alpinia murdochii Alpinia pahangensis 45.0 %
Alpinia murdochii Alpinia scabra 41.8 %
Alpinia scabra Alpinia pahangensis 29.5 %
Chapter 4 Results and discussion
76
Table 4.10 displayed the percentages of similarity of the essential oil constituents between
the three wild Alpinia species used in this study. Holttum, 1950, mentioned that Alpinia
murdochii and A. pahangensis were closely related species in terms of their morphology.
In this study, the leaf oils of A. murdochii and A. pahangensis were both rich in
monoterpene hydrocarbons and oxygenated monoterpenes. β-pinene was present as the
principal compound with 23.83 % and 39.61 % in leaf oils of A. murdochii and A.
pahangensis respectively. There are twenty-one compounds that were detected in the leaf
oils of A. murdochii and A. pahangensis which amounted to 36.6 % of the total
compounds. Meanwhile, the rhizome oils of Alpinia murdochii and A. pahangensis were
dominated by oxygenated monoterpenes and sesquiterpene hydrocarbons with γ-selinene
as the most dominant compound accounting for 15.51 % and 11.6 %, respectively.
Interestingly, this is the first report of γ-selinene occurring as the major component in
Alpinia species. Approximately 45% of the rhizome oil constituents of A. murdochii and A.
pahangensis are similar. Whereas the constituents of the leaf and the rhizome oils of A.
murdochii and A. scabra showed a similarity of 29.5 % and 41.8 % respectively. There
were 26.2 % and 29.5 % similarity in the constituents of the leaf and rhizome oils between
A. scabra and A. pahangensis.
The main components from the leaf oils of the three wild Alpinia species studied are the
monoterpene, β-pinene. This result is similar to that of Alpinia conchigera (Ibrahim, et al.,
2009) and Alpinia mutica (Hasnah and Ahmad, 1998) in terms of presence of
monoterpenes since these species also expresses monoterpenes as their major constituent.
β-pinene is reported to exhibit anti-inflammatory, antiseptic, candidacide, pesticide,
Chapter 4 Results and discussion
77
spasmogenic and allergenic activities (http://www.naturalhub.com/artemis/index.htm:3
Mei 2009).
Table 4.11: Distribution of chemical constituents of the leaf oils of three wild Alpinia
species according to their classification.
Classification Composition (%)
A. murdochii A. pahangensis A. scabra
Non-terpene hydrocarbons - - 0.09
Oxygenated non-terpenes 2.06 3.06 3.31
Monoterpene hydrocarbons 72.56 56.58 71.61
Oxygenated monoterpenes 18.02 21.92 7.83
Sesquiterpene hydrocarbons 0.73 1.63 1.32
Oxygenated sesquiterpenes 0.65 0.96 2.36
Oxygenated diterpenes 0.29 2.76 0.13
Total 94.31 86.91 86.65
Table 4.12: Distribution of chemical constituents of the rhizome oils of three wild Alpinia
species according to their classification.
Classification Composition (%)
A. murdochii A. pahangensis A. scabra
Non-terpene hydrocarbons 2.70 4.49 -
Oxygenated non-terpenes 9.57 9.09 2.91
Monoterpene hydrocarbons 8.56 16.18 1.09
Oxygenated monoterpenes 19.56 22.12 9.82
Sesquiterpene hydrocarbons 26.85 19.64 49.01
Oxygenated sesquiterpenes 3.05 3.76 8.13
Oxygenated diterpenes 0.77 - -
Total 71.06 75.28 70.96
Chapter 4 Results and discussion
78
From the list of the class of compounds from the leaf and the rhizome oils of the three wild
Alpinia species namely Alpinia murdochii, Alpinia pahangensis and Alpinia scabra (Table
4.11 and Table 4.12), one may observe that the leaf oils of this three wild Alpinia species
contain in abundance of monoterpene (β-pinene, α-pinene and sabinene) and oxygenated
monoterpenes such as terpinene-4-ol and borneol. Meanwhile, the major components for
the rhizome oils are sesquiterpenes (γ-selinene, α-selinene and α-panasinsen) and
oxygenated monoterpenes such as myrtenal, terpinene-4-ol and α-terpineol. Most of the oil
components were similar in the three species studied.
Previous studies on identification of essential oils of Alpinia species in Malaysia has been
reported by several researchers; Alpinia conchigera (Hasnah and Aziz, 1995; Wong, et al.,
2005; Ibrahim, et al., 2009), Alpinia galanga (De Pooter et al., 1985; Ibrahim et al., 2004),
Alpinia latilabris (Wong, et al., 2005), Alpinia malaccensis var. nobilis (Nor Azah et al.,
2005) and Alpinia mutica (Hasnah and Ahmad, 1998). Other reports on essential oils of
Alpinia species from other places have been described in Chapter 2 (Literature Review).
Hasnah and Aziz, 1995, reported that sesquiterpenes; β-sesquiphellandrene (20.5 %), β-
bisabolene (12.1 %) and β- caryophyllene (4.39 %) and monoterpene, 1, 8-cineole (11.56
%) were the major components of the rhizome oils of Alpinia conchigera from Johor. In
2005, Wong, et al., reported that the major compounds of A. conchigera rhizome oils from
Penang were β- bisabolene (28.9 %), 1, 8-cineole (15.3 %), β- caryophyllene ( 10.0 %)
and β-pinene (9.5 %). It was followed by Ibrahim, et al., 2009, which revealed that the
major compounds of Alpinia conchigera rhizome oil which is collected from Jeli, Kelantan
were 1, 8-cineole (17.9 %), β- bisabolene (13.9 %), β-sesquiphellandrene (6.8 %) and β-
Chapter 4 Results and discussion
79
elemene (4.0 %), while β- bisabolene (15.3 %), β-pinene (8.2 %), β-sesquiphellandrene
(7.6 %), chavicol (7.5 %) and β-elemene (6.0 %) were present as the major components in
the leaf oils.
Trans- β- Farnesene (30.6%) and 1, 8-cineole (24.0 %) %) were the major representatives
of sesquiterpenes and monoterpenes, respectively, of the rhizome oils of Alpinia galanga
(De Pooter et al., 1985). A. galanga is a common spice used in Malay traditional cooking,
which was reported by Ibrahim, et al., 2004, to produce the monoterpenes, 1, 8-cineole in
abundance (40.5 %). Other components present were β- bisabolene (8.4 %) and (Z, E) -
farnesol (3.8 %). Wong, et al., 2005 also revealed the constituents of the rhizome oils of
Alpinia latilabris which were collected from Penang. Methyl (E)-cinnamate was present as
the principal compound with 89.5 % of total oils. It was followed by α-phellandrene (3.2
%) and 1, 8-cineole (1.5 %).
The essential oil components of Alpinia malaccensis var. nobilis have been reported by
Nor Azah et al., 2005. The plant materials were collected from Terengganu. They revealed
that (E)-Methyl cinnamate predominate in the leaf oils (88.0 %), rhizome oils (85.7 %) and
stem oils (64.4 %). The major constituents of the rhizome oils of Alpinia mutica were
camphor (35.6 %), 1, 8-cineole (9.4 %), borneol (8.3 %) and β- pinene (7.3 %) (Hasnah
and Ahmad, 1998). The plant materials of this Alpinia mutica were collected from
Selangor.
1, 8-Cineole is a common major compound in Alpinia species (Hasnah and Aziz, 1995;
Wong et al., 2005; Ibrahim et al., 2009; Hasnah and Ahmad, 1998) therefore it can be used
Chapter 4 Results and discussion
80
as a marker for the genus Alpinia. However, from this study, 1, 8-cineole was only present
in the rhizome oils of A. pahangensis and the leaf oils of A. scabra in low concentrations
(0.63 % and 0.08 %, respectively). 1, 8-Cineole may have been present in A. murdochii but
probably in such a low concentration that it could not be detected.
Chapter 4 Results and discussion
81
4.3 Biological Activities
In the present study, the essential oils of three wild Alpinia species namely; Alpinia
murdochii, A. pahangensis and A. scabra were extracted by hydrodistillation from two
parts; the rhizome and the leaf. Then, these oils were tested for their antimicrobial activity,
antioxidant activity and anti-inflammatory activity.
4.3.1 Antimicrobial properties of three wild Alpinia species
Antimicrobial activity is useful in natural product research especially for the treatment of
dermatological disease. The essential oils of the leaf and rhizome of Alpinia murdochii,
Alpinia pahangensis and Alpinia scabra were investigated for their antimicrobial activity
against five strains of Staphylococcus aureus (Sa 2, Sa 3, Sa 7, VISA and VRSA) and four
fungal (Candida albican, Candida glabrata, Microsporum canis and Tricophyton rubrum)
using the minimum inhibitory concentration (MIC).
4.3.1.1 Minimum inhibition concentration (MIC)
The minimum inhibition concentration (MIC) value was defined as the lowest
concentration of essential oil inhibiting visible growth of microbes. The antimicrobial
results for these three essential oils of selected wild Alpinia species are presented in Table
4.12 and Table 4.13. These essential oils displayed a wide-spectrum antibacterial activity
against all Staphylococcus aureus strains and moderate activity against selected fungi.
Chapter 4 Results and discussion
82
Almost all of the rhizome oils exhibited strong activity (MIC ≤ 1000 µg/ ml) against five
strains of Staphylococcus aureus (Sa 2, Sa 3, Sa 7, Sa VISA and Sa VRSA) except for
Alpinia murdochii rhizome oil and A.scabra rhizome oil which were observed to have
moderate activity (1000 - 4900 µg/ ml) against Sa 3. Meanwhile the leaf oils showed
moderate activity except for A. murdochii rhizome oil, A. pahangensis rhizome oil and A.
scabra rhizome oil which showed strong activity against Sa VRSA.
Some of these essential oils showed potent activity against Staphylococcus aureus strains.
For instance, the rhizome oils of A. murdochii, A. pahangensis and A. scabra have shown
the lower MIC value as 78 µg/ml, 156 µg/ml and 156 µg/ml, respectively, compared to the
standard reference, oxacillin (313 µg/ml) against VISA. The A. pahangensis rhizome oils
also showed potent activity against Sa7 strain. The rhizome oils of A. murdochii and A.
scabra inhibit Sa 7 strain at the same concentration as oxacillin, 625 µg/ml. Alpinia
pahangensis rhizome oils also showed similar MIC value as oxacillin (156 µg/ml) when
tested against Sa 3 strain (Table 4.12).
All the essential oils tested, exhibited moderate activity against four selected fungi
(Candida albican, Candida glabrata, Microsporum canis and Trycophyton rubrum). Only
the rhizome oils of Alpinia pahangensis gave similar inhibition to the reference antibiotic,
cyclohexamide (1250 µg/ ml) against Candida albican (Table 4.13).
The rhizome oils and the leaf oils of Alpinia murdochii and Alpinia pahangensis have
shown significant results on MIC assay against four selected fungi and five selected strains
of Staphylococcus aureus. The rhizome oils of A. murdochii inhibit VRSA and VISA at
Chapter 4 Results and discussion
83
low concentrations (39 and 78 µg/ ml respectively) and A. pahangensis showed a low MIC
value against VRSA (78 µg/ ml). The other oils showed moderate antimicrobial activity
against all microbes tested with MIC values of 156 µg/ ml to 2500 µg/ ml (Table 4.12).
There are few reports on essential oils and their biological activities on the species of
Alpinia found in the literature. There is one report on essential oils and their antimicrobial
activities of Alpinia conchigera (Ibrahim, et al., 2009). It was reported that the essential
oils of the leaf, pseudostem and the rhizome of A. conchigera exhibited the MIC values of
20 µg/µl and above which were considered weak against gram positive bacteria
(Pseudomonas aeruginosa and Pseudomonas cepacia), gram negative bacteria
(Staphylococcus aureus and Staphylococcus epidermidis) and three dermatophytic fungi
(Microsporum canis, Tricophyton mentagrophytes and Tricophyton rubrum).
Previous studies on the antimicrobial activity of Alpinia galanga, showed inhibitory
activity against a wide spectrum of microorganisms (Habsah et al., 2000; Mayachiew and
Devahastin, 2008 and Oonmettaaree, at al., 2006). It is reported that the essential oils from
fresh and dried rhizome of galangal have antimicrobial activities against bacteria, fungi,
yeast and parasite (Farnsworth and Bunyapraphatsara, 1992). Janssen and Scheffer (1985)
have reported that the monoterpene, terpinen-4-ol, which is present in the essential oil of
the fresh galangal rhizome; exhibit an antimicrobial activity against Tricophyton
mentagrophytes.
Bhusita (2005) reported the antibacterial activity of essential oils from medicinal plants in
Thailand including two species of Alpinia namely Alpinia galanga and Alpinia conchigera.
Chapter 4 Results and discussion
84
Alpinia galanga exhibited the inhibition zone of 10.5 mm to 28 mm against Salmonella
typhimurium, Salmonella enteritidis, Escherichia coli, Clostridium perfringens and
Camphylobacter jejuni while the inhibition zone exhibited by Alpinia conchigera was 12.8
mm to 25 mm.
The antimicrobial activity of essential oils is assigned to a number of small terpenoids and
phenolic compounds (thymol, carvacol, eugenol), which in pure form also demonstrate
high anti-bacterial activity (Conner, 1993, Karapinar and Aktung, 1987). Based on one
report, pinene-type monoterpene hydrocarbons (α-pinene and β-pinene) and borneol
(oxygenated monoterpene) had slight activity against a panel of microorganisms (Dorman
and Deans, 2000). Thus, this antimicrobial result was not surprising because of the
monoterpenes, β-pinene is present as a main component in most of the rhizome oils of the
three Alpinia species. It was reported that camphor is known to possess slight antifungal
(Alvarez-Castellanos et al., 2001) and antibacterial activity (Demetzos, et al., 2002).
Carson and Riley, (1995) have reported that 4-terpineol and α-terpineol also exhibit weak
antibacterial activity.
Antimicrobial activities of essential oils are difficult to correlate to a specific compound
due to their complexity and variability. In general, the antimicrobial activities have been
mainly explained through C10 and C15 terpenes with aromatic rings and phenolic hydroxyl
groups able to form hydrogen bonds with active site of target enzymes, although other
active terpenes, as well as alcohols, aldehydes and esters can contribute to the overall
antimicrobial effect of essential oils (Belletti et al., 2004).
Chapter 4 Results and discussion
85
Table 4.13: The minimum inhibition concentrations (MIC) of essential oils of Alpinia
species (µg/ml) against five Staphylococcus aureus strains
Samples (50mg/ml)
MIC (µg/ml)
Sa 2 Sa 3 Sa 7 VISA VRSA
Alpinia murdochii
• Rhizomes
• Leaves
625
2500
2500
2500
625
2500
78
1250
39
313
Alpinia pahangensis
• Rhizomes
• Leaves
313
2500
156
1250
313
2500
156
1250
78
625
Alpinia scabra
• Rhizomes
• Leaves
625
2500
1250
2500
625
2500
156
2500
78
156
Oxacillin *
<19.5
156
625
313
<19.5
*Oxacillin: The reference antibiotic used in MIC assay against Staphylococcus aureus strains
(sigma).
MIC (µg/ ml) Activity status
≤ 1000 Strong
1000 - 4900 Moderate
≥ 5000 Weak
Chapter 4 Results and discussion
86
Table 4.14: The minimum inhibition concentrations (MIC) of essential oils of Alpinia
species (µg/ml) against selected fungi
Samples (50mg/ml)
Minimum Inhibitory Concentration (µg/ml)
Candida
albican
Candida
glabrata
Microsporum
canis
Trycophyton
rubrum
Alpinia murdochii
• Rhizome
• Leaf
2500
2500
2500
2500
2500
2500
2500
2500
Alpinia pahangensis
• Rhizome
• Leaf
1250
2500
2500
2500
2500
2500
2500
2500
Alpinia scabra
• Rhizome
• Leaf
2500
2500
2500
2500
2500
2500
2500
2500
Cycloheximide*
1250
1250
2180
2180
*Cyclohexamide: The reference antibiotic (sigma grade) used in MIC assay against selected fungi
(sigma).
MIC (µg/ ml) Activity status
≤ 1000 Strong
1000 - 4900 Moderate
≥ 5000 Weak
Chapter 4 Results and discussion
87
4.3.2 Antioxidant properties of three wild Alpinia species
The essential oils of this three wild Alpinia species were tested for their antioxidant
activity. Two methods were employed; DPPH free radical scavenging assay and reducing
power assay.
4.3.2.1 DPPH radical scavenging assay
The results of DPPH radical scavenging assay of three wild Alpinia species are shown in
Table 4.14. In general, any sample possessing 50 percentage of inhibition at 5 mg/ml is
considered as active and hence the IC50 values can be determined. However, in this study,
the IC50 value could not be determined at the time of the research period due to insufficient
raw material from the wild for extraction of essential oils. The unpredictable weather and
global warming affected the growth of these gingers in the wild.
At the concentration of 5 mg/ml, the rhizome oils of Alpinia scabra exhibited the highest
percentage with 55.17 % compared to the others essential oils tested. The leaf oil of A.
murdochii and A. pahangensis also showed the percentages inhibition more than 50 % with
54.38 % and 54.76 % respectively. The others essential oils showed percentages inhibition
below than 50 % and have a less effectiveness activity compared to synthetic antioxidant
agent, ascorbic acid. Ascorbic acid showed the percentages inhibition of 94.24 % (IC50:
28.61µg/ml). Figure 4.3 was presented the scavenging activity of the DPPH free radical of
the positive control, ascorbic acid.
Chapter 4 Results and discussion
88
As mentioned above, the leaf oils of A. murdochii and A. pahangensis have shown
significant of the percentages inhibition with 54.38 % and 54.76 % respectively.
Meanwhile, the rhizome oils of these two species showed weak activity and these results
are almost same (22.15 % and 26.81 %). These results were support the evidence that these
two species are closely related.
Table 4.15: Percentage inhibition of DPPH free radical scavenging of essential oils of
Alpinia species at the concentration of 5 mg/ml
Essential oils
% Inhibition of DPPH scavenging activity at 5
mg/ml (± S.D.)
Alpinia murdochii
• Leaves
• Rhizomes
54.38 ± 3.85
22.15 ± 4.04
Alpinia pahangensis
• Leaves
• Rhizomes
54.76 ± 0.71
26.81 ± 2.29
Alpinia scabra
• Leaves
• Rhizomes
33.55 ± 2.63
55.17 ± 1.23
Ascorbic acid (60 µg/ml)
(Standard reference)
94.24 ± 0.27
Data are expressed as the means ± standard deviation (n=3)
Activity range (%) Activity status
71-100 Strong
41- 70 Moderate
≤ 40 Weak
Chapter 4 Results and discussion
89
DPPH radical scavenging of Alpinia species
54.38
22.15
54.76
26.81
33.55
55.17
0.00
10.00
20.00
30.00
40.00
50.00
60.00
AML AMR APL APR ASL ASR
Essential oil of Alpinia species
Percentages Inhibition (%)
Figure 4.2: DPPH radical scavenging of three wild Alpinia species (%)
Legend:
AML : Alpinia murdochii (leaf oil)
APL : Alpinia pahangensis (leaf oil)
ASL : Alpinia scabra (leaf oil)
AMR : Alpinia murdochii (rhizome oil)
APR : Alpinia pahangensis (rhizome oil)
ASR : Alpinia scabra (rhizome oil)
Activity range (%) Activity status
71-100 Strong
41- 70 Moderate
≤ 40 Weak
In summary, the inhibition on DPPH radical scavenging assay of the essential oils of
Alpinia species decreased in the following order; Alpinia scabra (rhizome oil) > Alpinia
pahangensis (leaf oil) > Alpinia murdochii (leaf oil) > Alpinia scabra (leaf oil) > Alpinia
pahangensis (rhizome oil) > Alpinia murdochii (rhizome oil).
Chapter 4 Results and discussion
90
Table 4.16: Percentage inhibition of various concentrations of ascorbic acid
Concentration (µµµµg/ml) Inhibition S.D.
60 94.24 0.27
50 94.28 0.20
40 91.65 3.01
30 56.84 5.11
20 27.38 2.02
10 8.39 1.03
Figure 4.3: The DPPH radical scavenging activity of ascorbic acid (standard reference)
IC50= 28.24 µg/ml
Free radical scavenging activity of Ascorbic acid (reference standard; %)
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70
Concentration of Ascorbic acid (ug/ml)
Percen
tag
e o
f In
hib
itio
n (
%)
Chapter 4 Results and discussion
91
4.3.2.2 Reducing Power Assay
In this assay, the yellow colour of the test solutions changes to green and blue shades
depending on the reducing power of each sample. The presence of antioxidants in the
samples causes the reduction of the Fe3+
/ ferricyanide complex to the ferrous form, Fe2+
.
Therefore, the Fe2+
can be monitored by measuring the formation of Perl’s Prussian blue at
700 nm.
The reducing power of standard reference used in this study is ascorbic acid at
concentrations of 5 mg/ml, 10 mg/ml, 15 mg/ml and 20 mg/ml are 2.698 ± 0.074, 2.705 ±
0.033, 2.735 ± 0.018 and 2.545 ± 0.071 respectively.
Alpinia scabra rhizome oils exhibited the highest of reducing power value at
concentrations of 5 mg/ml, 10 mg/ml, 15 mg/ml and 20 mg/ml at 1.085 ± 0.004, 1.335 ±
0.008, 1.306 ± 0.024 and 1.124 ± 0.005. This is followed by Alpinia murdochii rhizome
oils with reducing power value 1.043 ± 0.001, 1.357 ± 0.004, 1.5 ± 0.001 and 1.403 ±
0.009. At the same concentrations, the Alpinia pahangensis rhizome oils exhibited values
of 0.864 ± 0.008, 1.274 ± 0.006, 1.304 ± 0.006 and 1.218 ± 0.004.
The reducing power value of the leaf oil of Alpinia murdochii was observed at 0.681 ±
0.005, 1.154 ± 0.01, 1.065 ± 0.014 and 0.88 ± 0.005. On the other hand, the Alpinia scabra
leaf oil showed the reducing power of 0.64 ± 0.005, 0.823 ± 0.016, 0.716 ± 0.018 and
0.679 ± 0.005. The lowest results for this assay is Alpinia pahangensis leaf oil with
Chapter 4 Results and discussion
92
reducing power value of 0.43 ± 0.003, 0.789 ± 0.017, 0.674 ± 0.012 and 0.644 ± 0.003 at 5
mg/ml, 10 mg/ml, 15 mg/ml and 20 mg/ml, respectively.
In this assay, the reducing power of essential oils from three wild Alpinia species were low
when compared with ascorbic acid as the standard reference. Table 4.15 showed the
reducing power value of ascorbic acid and Table 4.16 showed the reducing power value of
essential oils of this three Alpinia species at various concentrations.
Table 4.17: Reducing power value of standard reference, ascorbic acid at various
concentrations
Standard
reference
Abs. reading of reducing power assay at various
concentrations *
5 mg/ml 10 mg/ml 15 mg/ml 20 mg/ml
Ascorbic acid 2.698 ± 0.074 2.705 ± 0.033 2.735 ± 0.018 2.545 ± 0.071
* Data are expressed as the means ± standard deviation (n=3)
Chapter 4 Results and discussion
93
Table 4.18: Reducing power value of the essential oils of three Alpinia species at various
concentrations
Essential oils
Abs. reading of reducing power assay at various
concentrations *
5 mg/ml 10 mg/ml 15 mg/ml 20 mg/ml
Alpinia murdochii
(leaf) 0.681 ± 0.005 1.154 ± 0.01 1.065 ± 0.014 0.88 ± 0.005
A. murdochii
(rhizome) 1.043 ± 0.001 1.357 ± 0.004 1.5 ± 0.001 1.403 ± 0.009
A. pahangensis
(leaf) 0.43 ± 0.003 0.789 ± 0.017 0.674 ± 0.012 0.644 ± 0.003
A. pahangensis
(rhizome) 0.864 ± 0.008 1.274 ± 0.006 1.304 ± 0.006 1.218 ± 0.004
A. scabra
(leaf) 0.64 ± 0.005 0.823 ± 0.016 0.716 ± 0.018 0.679 ± 0.005
Alpinia scabra
(rhizome) 1.085 ± 0.004 1.335 ± 0.008 1.306 ± 0.024 1.124 ± 0.005
* Data are expressed as the means ± standard deviation (n=3)
Activity range (absorbance) Activity status
2.0 – 2.99 Strong
1.0 – 1.99 Moderate
< 0.99 Weak
Chapter 4 Results and discussion
94
Reducing power assay on essential oil of Alpinia species
0.00
0.50
1.00
1.50
2.00
2.50
3.00
0 5 10 15 20
Concentration (mg/ml)
absorbance
APR APL AMR AML ASR ASL Ascorbic acid
Figure 4.4: Reducing power assay on essential oils of three wild Alpinia species
Legend:
AML : Alpinia murdochii (leaf oil)
APL : Alpinia pahangensis (leaf oil)
ASL : Alpinia scabra (leaf oil)
AMR : Alpinia murdochii (rhizome oil)
APR : Alpinia pahangensis (rhizome oil)
ASR : Alpinia scabra (rhizome oil)
Chapter 4 Results and discussion
95
Reducing power assay of the essential oils of Alpinia
murdochii
0.00
0.50
1.00
1.50
2.00
2.50
3.00
0 5 10 15 20
Concentration (mg/ml)
Absorbance
Rhizomes Leaves Ascorbic acid
Figure 4.5: Reducing power of Alpinia murdochii oils (leaf and rhizome) in comparison
with ascorbic acid
Reducing power assay of the essential oils of Alpinia
pahangensis
0.00
0.50
1.00
1.50
2.00
2.50
3.00
0 5 10 15 20
Concentration (mg/ml)
Absorbance
Rhizomes Leaves Ascorbic acid
Figure 4.6: Reducing power of Alpinia pahangensis oils (leaf and rhizome) in comparison
with ascorbic acid
Chapter 4 Results and discussion
96
Reducing power assay of the essential oils of Alpinia scabra
0.00
0.50
1.00
1.50
2.00
2.50
3.00
0 5 10 15 20
Concentration (mg/ml)
Absorbance
Rhizomes Leaves Ascorbic acid
Figure 4.7: Reducing power of Alpinia scabra oils (leaf and rhizome) in comparison with
ascorbic acid
Chapter 4 Results and discussion
97
Figure 4.5, Figure 4.6 and Figure 4.7 showed reducing power of Alpinia murdochii, A.
pahangensis and A. scabra respectively. It was shown that the rhizome oils of these three
wild species of Alpinia have a better absorbance on reducing power assay compared to the
leaf oils at various concentrations. As mentioned before, the rhizome oils were rich in
sesquiterpenes. Meanwhile, the leaf oils were dominated by monoterpenes.
These results differed from the DPPH free radical scavenging assay which revealed that
the leaf oils have better inhibition than the rhizome oils. From these observations, we can
assume different components may play an important role to their activities. In this assay,
the rhizome oils of A. murdochii and A. pahangensis showed the best absorbance as
compared to the leaf oils. These results also support the evidence that this two species are
related and one of the species can be used as alternative for one another.
There have been reports that the antioxidant activities from the Zingiberaceae family are
from less polar constituents isolated such as curcuminoid, kava pyrones and gingerols
(Kikuzaki and Nakatani, 1993; Masuda and Jitoe, 1994). On the contrary, the five volatile
oils of Zingiberaceae species namely Alpinia galanga, Boesenbergia rotunda, Curcuma
longa, Kaempferia galangal and Zingiber officinale showed very weak activity on DPPH
radical scavenging (Sariga, et al., 2005). Recently, Chan (2008) reported the antioxidant
activity from methanol extracts of the leaves of five Alpinia species. Alpinia zerumbet, A.
purpurata, A. zerumbet ‘Variegata’, A. malaccensis and A. galanga displayed low to high
radical scavenging activity ranging from 90 to 2180 mg AA/100 g. Among these Alpinia
species, leaf of Alpinia zerumbet showed high radical scavenging activity with value of
2180 mg AA/100 g.
Chapter 4 Results and discussion
98
4.3.3 Anti-inflammatory properties of three wild Alpinia species
Hyaluronidase assay and Lipoxygenase assay
The anti-inflammatory activities of essential oil of Alpinia species were determined using
two assays; hyaluronidase assay and lipoxygenase assay.
Table 4.18 shows the percentages inhibition of hyaluronidase and lypoxigenase by the six
essential oils from three wild Alpinia species at concentration 100 µg / µL. The leaf and
the rhizome oils of Alpinia murdochii and A. scabra showed moderate activity on
hyaluronidase assay with 66.38 %, 63.43 %, 43.81 % and 54.34 % of inhibition,
respectively. The leaf and rhizome oils of A. pahangensis showed low activity with
percentage inhibition of 38.41 % and 40.63 % respectively. Apigenin was used as the
standard reference with percentage inhibition of 81.80 %.
For lipoxygenase assay, at the test concentration of 100 µg / µL, the leaf and the rhizome
oils of A. murdochii, the leaf and the rhizome oils of A. scabra and the leaf oils of A.
pahangensis exhibit strong activity with 95.37 %, 91.11 %, 85.35 %, 90.43 % and 90.42
%, respectively. The leaf and the rhizome oils of A. murdochii and the rhizome oil of A.
scabra are almost similar to the standard reference, nordihydroguaretic acid (NDGA)
(97.15 %). Only A. pahangensis rhizome oil showed moderate activity with 59.65 %. The
results demonstrated that the essential oils of these three wild Alpinia species may contain
constituents with anti-inflammatory effect.
Chapter 4 Results and discussion
99
Table 4.19: Percentage inhibition of essential oils of Alpinia species based on
hyaluronidase assay and lipoxygenase assay
Samples Hyaluronidase assay (%)a Lipoxygenase assay (%)
a
Alpinia murdochii
• Leaves
• Rhizomes
66.38 ± 9.43
63.43 ± 8.76
95.37 ± 6.55
91.11 ± 7.82
Alpinia pahangensis
• Leaves
• Rhizomes
38.41 ± 6.34
40.63 ± 4.31
80.67 ± 14.24
59.65 ± 3.39
Alpinia scabra
• Leaves
• Rhizomes
43.81 ± 6.37
54.34 ± 9.34
85.35 ±6.22
90.42 ± 0.10
* Apigenin 81.80 ± 1.62 -
* NDGA - 97.15 ± 0.01
a Inhibition at concentration 100 µg / µL
* Standard reference
NDGA: nordihydroguaretic acid
Activity range (%) Activity status
0 – 40 Weak
41 – 70 Moderate
71 - 100 High
Chapter 4 Results and discussion
100
In hyaluronidase assay, Alpinia murdochii and Alpinia pahangensis exhibited different
results. The leaf and the rhizome oils of A. murdochii showed moderate activities while the
leaf and the rhizome oils of A. pahangensis gave only weak activities. In the case of
lipoxygenase assay, the leaf and the rhizome oils of A. murdochii and the leaf oils of A.
pahangensis showed high activities (80.67 % to 95.37 %) while the rhizome oils of A.
pahangensis showed moderate activity with 59.65 %.
CHAPTER 5
CONCLUSION
Chapter 5 Conclusion
101
CONCLUSION
The chemical constituents of the leaf oils from the wild Alpinia species namely Alpinia
murdochii, Alpinia pahangensis and Alpinia scabra, were dominated by monoterpenes
with β-pinene being the principal component. However, the rhizome oils were
predominantly made up of sesquiterpenes in which the major compound for all species was
γ-selinene. The results also showed that the marker compound of Alpinia, 1, 8-cineole is
only present in A. pahangensis rhizome oils and A. scabra leaf oils with low
concentrations (0.63 % and 0.08 % respectively). The leaf oils of A. murdochii and A.
pahangensis were dominated by monoterpenes (hydrocarbons and oxygenated
monoterpenes) with 36.6 % of their chemical components being similar. The rhizome oils
of these two closely related species revealed that 45 % of the chemical components were
similar and the rhizome oils were dominated by oxygenated monoterpenes and
sesquiterpene hydrocarbons. The chemical constituents of the leaf oil of A. scabra were
29.5 % and 26.2 % similar to A. murdochii and A. pahangensis respectively. There were
41.8 % and 29.5 % similarity in the constituents of the rhizome oil of A. scabra in
comparison to A. murdochii and A. pahangensis respectively.
These oils exhibited a broad spectrum of antimicrobial activities against nine microbes
namely two dermatophytic fungi (Microsporum canis and Tricophyton rubrum), two
Candida species (Candida albican and Candida glabrata) and five strains of
Staphylococcus aureus (Sa 2, Sa 3, Sa 7, VISA and VRSA). The rhizome oils of these
three species showed potent inhibition against VISA with MIC values lower than that of
the standard reference, oxacillin. The rhizome oils of Alpinia pahangensis also showed a
lower MIC value than oxacillin when tested against Sa 7. The overall results implicate that
Chapter 5 Conclusion
102
the leaf and rhizome oils of A. murdochii and A. pahangensis exhibited more or less
similar activity against the four selected fungi and five selected strains of Staphylococcus
aureus.
The antioxidant activities of the leaf and the rhizome oils of three wild Alpinia species
were evaluated by DPPH radical scavenging assay and reducing power assay. At the
concentration of 5 mg/ml, the antioxidant activity of the leaf oils of A. murdochii (54.38%)
and A. pahangensis (54.76%) showed moderate activity on DPPH radical scavenging assay
while the rhizome oils showed weak activity. Results from the reducing power assay
showed that at the concentration of 5 mg/ml, the leaf oils of A. murdochii and A.
pahangensis exhibited low activity while the rhizome oils displayed moderate and low
activity respectively. The rhizome and the leaf oils of A. scabra exhibited moderate and
weak antioxidant activity respectively, for both assays.
The anti-inflammatory activities were evaluated using the hyaluronidase assay and
lipoxygenase assay. The leaf and the rhizome oils of A. murdochii and A. scabra exhibited
moderate activities in hyaluronidase assay, while the leaf and the rhizome oils of A.
pahangensis showed only weak activities. In lipoxygenase assay, the leaf and the rhizome
oils of all three species exhibited high activities (80.67 % - 95.37 %) except for A.
pahangensis rhizome oil which showed moderate activities of 59.65 %. These essential
oils exhibit many interesting and potent activities which may be due to the presence of
many compounds in the oils that contributed to those activities.
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APPENDIX
Appendix
114
APPENDIX
APPENDIX I: ESSENTIAL OILS ANALYSIS
Appendix I (a): Clevenger apparatus for essential oil extraction
Appendix
115
Appendix I (b): Operating Parameters for GC and GC/MS
GC GC-MS
Model Shimadzu GC-2010 Agilent 5975N
Capillary column CBP-5 HP-5
Length (m) 25 30
Diameter (mm) 0.25 0.25
Film Thickness (µm) 0.25 0.25
Detector Flame Ionization
Detector (FID)
Flame ionization
detector (FID) and
mass spectrometer
detector (MSD)
Temperature (°C) 250 250
Carrier gas Nitrogen Helium
Flow Controller
Split ratio 1: 20 1: 20
Column flow (ml/ min) 1 ml 1 ml
Column oven
Initial temperature (°C) 60 60
Final temperature (°C) 230 230
Program rate (°C/ min.) 3 3
Injector temperature 250
250
Total time of analysis (min.) 67.67 67.67
Appendix
116
Appendix I (c): Gas chromatogram (GC) of the leaf of Alpinia murdochii Ridl.
Appendix
117
Appendix I (d): Gas chromatogram (GC) of the rhizome of Alpinia murdochii Ridl.
Appendix
118
Appendix I (e): Gas chromatogram (GC) of the leaf of Alpinia pahangensis Ridl.
Appendix
119
Appendix I (f): Gas chromatogram (GC) of the rhizome of Alpinia pahangensis Ridl.
Appendix
120
Appendix I (g): Gas chromatogram (GC) of the leaf of of Alpinia scabra (Bl.) Baker
Appendix
121
Appendix I (h): Gas chromatogram (GC) of the rhizome of Alpinia scabra (Bl.) Baker
App
endix
122
AP
PE
ND
IX I
I: A
NT
IMIC
RO
BIA
L A
CT
IVIT
Y
Ap
pen
dix
II
(a):
The
anti
mic
robia
l ac
tivit
y o
f es
senti
al o
ils
of
Alp
inia
sp
ecie
s on
min
imum
inhib
itory
conce
ntr
atio
ns
(MIC
) m
ethod a
gai
nst
der
mat
oph
yte
s fu
ngus
and S
taphyl
oco
ccus
aure
us
stra
ins.
Sam
ple
s
(50m
g/m
l)
Min
imu
m i
nh
ibit
ion
con
cen
trati
on
(µ
g/m
l)
Mic
rosp
oru
m c
anis
Tri
chophyt
on r
ubru
m
Candid
a a
lbic
an
Candid
a g
labra
ta
Sta
phyl
oco
ccus
aure
us
(Sa 2
α)
AM
R
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
62
5,6
25
,62
5 =
625
AM
L
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
> 2
50
0,
250
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
AP
R
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
12
50
, 1
25
0, 1
250
= 1
25
0
25
00
, 2
50
0, 2
500
= 2
50
0
31
3, 1
56
, 3
13
= 3
13
AP
L
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, >
250
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
AS
R
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
62
5,6
25
,62
5 =
625
AS
L
25
00
, 2
50
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
> 2
50
0,
250
0, 2
500
= 2
50
0
25
00
, 2
50
0, 2
500
= 2
50
0
>2
50
0, 2
50
0, 2
500
=
25
00
Cyc
loh
exa
mid
e 2
18
0, 2
18
0, 2
180
= 2
18
0
21
80
, 2
18
0, 2
180
= 2
18
0
12
50
, 1
25
0, 1
250
= 1
25
0
12
50
, 1
25
0, 1
250
= 1
25
0
-
Oxa
cili
n
- -
- -
<1
9.5
, <
19
.5,
<19
.5 =
<1
9.5
App
endix
123
Ap
pen
dix
II
(b):
The
anti
mic
robia
l ac
tivit
y o
f es
senti
al o
ils
of
Alp
inia
spec
ies
on m
inim
um
inhib
itory
co
nce
ntr
atio
ns
(MIC
) m
ethod a
gai
nst
der
mat
oph
yte
s fu
ngus
and S
taphyl
oco
ccus
aure
us
stra
ins
– C
ont’
.
Sam
ple
s (5
0m
g/m
l)
Min
imu
m i
nh
ibit
ion
con
cen
trati
on
(µ
g/m
l)
Sta
phyl
oco
ccus
aure
us
(Sa 3
)
Sta
phyl
oco
ccus
aure
us
(Sa 7
)
Sta
phyl
oco
ccus
aure
us
(VIS
A)
Sta
phyl
oco
ccus
aure
us
(VR
SA
)
AM
R
2500, 2500, 2500 =
2500
313,6
25,6
25 =
625
78, 78, 78 =
78
39, 39, 78 =
39
AM
L
2500, 2500, 2500 =
2500
2500, 2500, 2500 =
2500
1250, 1250, 1250 =
1250
313, 313, 313 =
313
AP
R
156 ,156, 156 =
156
313, 313, 313 =
313
156, 156, 156 =
156
78, 78, 78 =
78
AP
L
1250, 1250, 1250 =
1250
2500, 2500, 2500 =
2500
1250, 1250, 1250 =
1250
625, 313, 625 =
625
AS
R
1250, 1250, 1250 =
1250
625,6
25,6
25 =
625
156, 78, 156 =
156
78, 78, 78 =
78
AS
L
2500, 2500, 2500 =
2500
2500, 2500, 2500 =
2500
>2500, 2500, 2500 =
2500
313, 156, 156 =
156
Oxa
cili
n
313, 156, 156 =
156
625,6
25,6
25 =
625
313, 313, 156 =
313
<19.5
, <
19.5
, <
19.5
= <
19.5
Appendix
124
APPENDIX III: ANTIOXIDANT ACTIVITY
Appendix III (a): Reaction mixtures of essential oils, DPPH and methanol
Stock solution : 20 mg/ml, DPPH: 8 mg/ml
No. Concentration of crude
extracts (mg/ml)
Volume of
methanol (µl)
Volume of
essential oil (µl)
from stock
Volume of DPPH
solution (µl)
1 5 725 250 25
2 Control 975 - 25
Appendix III (b): Absorbance of various concentrations of essential oils of Alpinia
species
1
Sample mg/ml Absorbance Ac- As Ac - As / Ac % Inhibition
DPPH 3.456
AML 1.425 2.031 0.59 58.77
AMR 2.56 0.896 0.26 25.93
APL 1.542 1.914 0.55 55.38
APR 2.441 1.015 0.29 29.37
ASL 2.231 1.225 0.35 35.45
ASR 1.552 1.904 0.55 55.09
2
Sample mg/ml Absorbance Ac- As Ac - As / Ac % Inhibition
DPPH 3.419
AML 1.654 1.765 0.52 51.62
AMR 2.807 0.612 0.18 17.90
APL 1.573 1.846 0.54 53.99
APR 2.566 0.853 0.25 24.95
ASL 2.375 1.044 0.31 30.54
ASR 1.573 1.846 0.54 53.99
3
Sample mg/ml Absorbance Ac- As Ac - As / Ac % Inhibition
DPPH 3.299
AML 1.559 1.74 0.53 52.74
AMR 2.553 0.746 0.23 22.61
APL 1.488 1.811 0.55 54.90
APR 2.438 0.861 0.26 26.10
ASL 2.156 1.143 0.35 34.65
ASR 1.437 1.862 0.56 56.44
Appendix
125
Appendix III (c): Reaction mixtures of Ascorbic acid, DPPH and methanol Ascorbic acid:
400 µg /ml
No. Concentration of ascorbic
acid (µg/ml)
Volume of
methanol (µl)
Volume of
essential oil (µl)
from stock
Volume of DPPH
solution (µl)
1 60 825 150 25
2 50 850 125 25
3 40 875 100 25
4 30 900 75 25
5 20 925 50 25
6 10 950 25 25
7 Control 975 - 25
Appendix III (d): Absorbance of various concentration of ascorbic acid
Ascorbic Acid 1
Sample mg/ml Absorbance Ac- As Ac - As / Ac % Inhibition
Control 3.253
60 0.187 3.066 0.94 94.25
50 0.188 3.065 0.94 94.22
40 0.22 3.033 0.93 93.24
30 1.287 1.966 0.60 60.44
20 2.42 0.833 0.26 25.61
10 2.952 0.301 0.09 9.25
Ascorbic Acid 2
Sample mg/ml Absorbance Ac- As Ac - As / Ac % Inhibition
Control 3.475
60 0.191 3.284 0.95 94.50
50 0.191 3.284 0.95 94.50
40 0.225 3.25 0.94 93.53
30 1.422 2.053 0.59 59.08
20 2.447 1.028 0.30 29.58
10 3.174 0.301 0.09 8.66
Ascorbic Acid 3
Sample mg/ml Absorbance Ac- As Ac - As / Ac % Inhibition
Control 3.35
60 0.202 3.148 0.94 93.97
50 0.197 3.153 0.94 94.12
40 0.396 2.954 0.88 88.18
30 1.642 1.708 0.51 50.99
20 2.447 0.903 0.27 26.96
10 3.107 0.243 0.07 7.25
Appendix
126
Appendix III (e): Percentage inhibition of various concentration of ascorbic acid
Concentration Inhibition Average S.D.
(µg/ml) 1 2 3
60 94.25 94.5 93.97 94.24 0.27
50 94.22 94.5 94.12 94.28 0.20
40 93.24 93.53 88.18 91.65 3.01
30 60.44 59.08 50.99 56.84 5.11
20 25.61 29.58 26.96 27.38 2.02
10 9.25 8.66 7.25 8.39 1.03
% inhibition of ascorbic acid
y = 1.8528x - 2.329
R2 = 0.927
-20
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70
Concentration (ug/ml)
% I
nh
ibit
ion
Appendix III (f): Percentage inhibition of Ascorbic acid
R
2 y IC50
y = 1.8528x - 2.329 0.927 50 28.24 (µg/ml)
Appendix
127
APPENDIX IV: PARTICIPATION IN SEMINAR
Poster Presentation
Devi Rosmy Syamsir, Halijah Ibrahim, Nor Azah Mohamad Ali, Mastura Mohtar,
Rasadah Mat Ali and Khalijah Awang. (2008). The essential oils and antimicrobial
activity of Alpinia pahangensis. 5th
Malaysian International Conference on Essential
Oils, Fragrance and Flavour Materials (MICEOFF5), 28-30 Oktober 2008, Kuala
Lumpur, Malaysia.
5
th MALAYSIAN INTERNATIONAL CONFERENCE ON ESSENTIAL OILS,
FRAGRANCE AND FLAVOUR MATERIALS (MICEOFF5)
28-30 OKTOBER 2008, KUALA LUMPUR, MALAYSIA
POS 20 (Poster)
THE ESSENTIAL OILS AND ANTIMICROBIAL ACTIVITY OF Alpinia
pahangensis
1 Devi Rosmy Syamsir,
1 Halijah Ibrahim and
1 Khalijah Awang
2 Nor Azah Mohamad Ali,
2 Mastura Mohtar and
2 Rasadah Mat Ali
1 Faculty of Science, University of Malaya, 50503 Kuala Lumpur, Malaysia.
2 Medicinal Plants Programme, Forest Biotechnology Division, Forest Research Institute
Malaysia (FRIM) 52109, Kepong, Selangor.
Email: [email protected]
Abstract
The essential oils of leaves and rhizomes of Alpinia pahangensis Ridl. collected
from Pahang were extracted by hydrodistillation. The chemical components from the
collected were determined by GC, GC-MS and Retention Indices (RI). The major
components of the rhizomes were β-pinene (10.87%), γ-selinene (11.60%) and α-terpineol
(6.38%), while the major components of the leaves were β-pinene (39.61%), α-pinene
(7.55%) and limonene (4.89%), the investigation of the antimicrobial activity of the
essential oil using broth microdilution technique revealed that the rhizome oils of Alpinia
pahangensis inhibited five Staphylococcus aureus strains at MIC values of 0.08 -0.31 µg /
µL.
Keywords: Alpinia pahangensis, Zingiberaceae, Essential oils, Antimicrobial activity
Appendix
128