essential oils and biological activities of three selected wild alpinia

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


3.6.1 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


4.3.1 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.


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)


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


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)


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.


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.


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.


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).


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).


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.


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


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


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.


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


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


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


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).


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.


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.


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%)


23

Table 2.2: Summary of essential oils of Alpinia species from previous studies - cont’


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)


24



A. conchigera Griff. Kelantan,

Malaysia Pseudostems β-Bisabolene (19.9 %), β-Sesquiphellandrene (11.3 %),

β-Caryophyllene (8.8 %), β- Elemene (4.7 %)


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%)


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%)


25



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%)


26



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%)


27



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%)


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


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.


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


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.


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).


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


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


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)


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


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


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


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).


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,


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


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


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.


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)


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.


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 %).


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


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


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 %).


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




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


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:


b Adam, 2001

c Ibrahim, et al., 2004


GC/MS : Gas chromatography / mass spectrometer



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 %).


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


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


17 trans-Verbenol 1140 1145 2.85 MS, KI


19 Borneol 1154 1169 0.23 MS, KI

20 cis-Pinocamphone 1157 1175 1.56 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


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:


b Adam, 2001

c Ibrahim et al., 2004





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.


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


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


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


12 cis-Sabinol 1140 1143 3.02 MS, KI

13 Camphene hydrate 1145 1150 0.6 MS, KI


15 Borneol 1164 1169 0.39 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


59



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:


b Adam, 2001





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.


61

Table 4.6: Chemical components of the leaf oil of Alpinia scabra (Blume) Náves

No.

Compounds

KIb

KIa

Composition

(%)

Method of

identification


2 3-Hexenol 851 859 0.55 MS, KI

3 α-Pinene 937 939 6.58 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


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


14 Camphor 1144 1146 0.14 MS, KI

15 Isoborneol 1147 1162 0.11 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


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


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:


b Adam, 2001

c Suleimenov, et al., 2001





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 %).


64

Table 4.7: Chemical components of the rhizome oils of Alpinia scabra (Blume) Náves

No.

Compounds

KIb

KIa

Composition

(%)

Method of

identification


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




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


31 Elemol 1547 1550 0.48 MS, KI


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:


b Adam, 2001





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 - -


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


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


69


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


70


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)



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 - -


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


73


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 -


74


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


75


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)


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 %


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,


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


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-


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


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.


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.


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


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.


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).


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


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


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.


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


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)


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).


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 (

%)


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


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)


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


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)


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


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


Absorbance


Figure 4.6: Reducing power of Alpinia pahangensis oils (leaf and rhizome) in comparison

with ascorbic acid


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


Absorbance


Figure 4.7: Reducing power of Alpinia scabra oils (leaf and rhizome) in comparison with

ascorbic acid


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.


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.


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


0 – 40 Weak

41 – 70 Moderate

71 - 100 High


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


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


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


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


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


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

essential oils and biological activities of three selected wild alpinia

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