CHARACTERIZATION AND BIOLOGICAL ACTIVITY OF ESSENTIAL OILS FROM ETLINGERA SPP.

Aisyaidil Binti Hanri

Bachelor of Science with Honours (Resource Chemistry) 2006 Pluat 1Gl4IUt Maklumat ,ualkmik UNlVERSm SAaAWJ\K

CHARACTERIZATION AND BIOLOGICAL ACTIVITY OF ESSENTIAL OILS FROM ETLINGERA SPP.

AISYAIDIL B1NTI HANRI

Thi s report is submitted in partial fulfi llment of the requirements for the degree ofBaehelor of Science wi th Honors in Resource Chemistry

Faculty of Resouree Science and Teehnology UNIVERSITI MALAYSIA SARAWAK 2006 ilwtKhldlUt Maklumat AXatitmUc UNJVUSm MALAYSIA SAItAWAJ(

CONTENTS

Contents Page

Declaration II

Acknowledgement III

Abstract IV

Abstrak V

Li st of Figures VI

Li st o f Tables V III

Chapter 1 Introduction 1.1 Introduction 1.2 Objectives 4

Chapter 2 Litel'a ture Review 2.1 Essential Oils 5 2.2 Extraction of Essential Oil s 6 2. 3 8

Chapter 3 Materials and Methods 3.1 Materials II 3.2 Extraction of Essential Oil 12 3.3 Instrumental Analysis 3.3. 1 Gas Chromatography- Fl ame Ion Detector (G C/FID ) 12 3.4 Data Analysis 3.4.1 Percentage o f Essential Oil 13 3.4.2 Kovats Indices 14 3.4.3 Semi-quantitative Analysis 15 3.5 Cluster Analysis 15 3.6 Biological Acti vity Study 16 Chapter 4 Results and Discussion 4.1 Percentage of Essential Oi l 17 4.2 Kovat Index of Compounds in Essential Oil s on DB-5 Co lumn 17 4.3 Chemical Composition of Essential Oil 20 4.3. 1 Ellingera elalior 20 4.3.2 Ellingera lilloralis 27 4. 3.3 Ellingera punicea 3.1 4.3 .4 Etlingera lali/abris 40 4.3.5 Etlingera species I 46 4.3.6 Ellingera species 2 53 4.3.7 Comparison of Chemical Composition in leaf oils of six 58 Ellingera species 4. 3.8 Comparison of Chemical Compositi on in oil s of 58 six Ellingera species 4.3.9 Comparison with other Species and Genus in Fami ly 72 Zi ngi beraceae 4.4 Statistical Analysis of Essential Oi ls fro m Etlingera spp. 74 4.5 Toxicity of Esenti al Oi ls from Ellinf!,era to Brine Shrimp 79

Chapter 5 Conclusion 80 References 81 DECLARAnON

No potion of the work refe rred to in this di ssertation has been submitted ill support of all • application for another degree of qualification of thi s or any other university or institution of hi gher learn in g.

Aisyaidil binti Hanri Programme of Re:;o urce Chemistry Faculty of Sci ence and Technology Uni versiti Malaysia Sarawak

" ACKNOWLEDGEMENT

Fi rst, thank to Allah for hi s bless ing I can successfully completing this researc h. I wou ld like to express my spec ial thanks to my supervisor, Assoc. Pro f. Dr. Zaini Bin Assim for giving me supports and guidance in doing this research project. A lso my special thanks to my co-supervisor. Assoc. Prof. Dr Fasihudin B. Ahmad for guiding and supporting me in thi s research. To colleagues at Faculty of Resource Science and Techno logy UNIMAS and all laboratory assistances, in particular En Rajuna Tahir, th anks you for a ll the co-operations given to me during the progress of this project. My appreciation to a ll my lectures and my course mates for he lping and su pporting me while doing this research.

iii ABSTRACT

Essent ial o il s tram six species of Etlingera were studied fo r their chemical composition and their biological activity on brine shrim p, A. salina. Essentia l oil s were extracted by hydrodist ill ati on and subsequently analyzed usin g gas chromatographylflame ioni zation detector (G C/FlD). The percentage of essentia l oil obta ined from th e six species of Etlingera ranged from 0% to 2.15%. with the hi ghest percentage was obtained from th e leaves of Erlingera Species 2. Eth yl-(E,Z)-decanoate (79.53%), a -gurj unene (1 7.25%), geosm In ( 17. 25%) and tetradecyl aldehyde (8.5 7%) were the major constituent of E. clatior. a -pi nene and ~ - pin e n e were detected at high abundance in E. iil/oralis with 20.67% of a-pi nene in leaf and 20.24% of ~-p i nene in rhizome. E. punicea was rich in octenol (73 .27%), whil e E Lati/abris contain hi gh abundance of octad ienone (6 6. 55%), methylethylpyrazin e (29.30%) and a-pinene (25.91%). Octanol (3 1.02%) and ~ - p in e n e (3 1. 02 %) were th e maj or constituents fo und in Ellingera species I. Etlingera species 2 has a -pinene (25 .3 9%). methyl laurate ( 16.29%) and phenol ( 15.69%) as the major components. Camphene and (E)-a ­ bergamotene were foun d in leaves of ali Etlingera spp. anal yzed. a -ihu.i ene and 2­ ethylpyri dine were found present in rhizome of a ll Etlingera spp. an alyzed. Leaves of E. elatior and Etlingera species I as well as leaves of E. !illoralis and E. lorilabr;" shows c lose re lat ionship. There was also close relationshi p between rhi zomes of ErlinRcra species 2 and E. lati/abri.\' as well as close relationship beiween E. /i f/oratis and EtLing era spccies I . Toxic it y tests on brine shri mp, A. salina shows o nl y leaf o il from Et lingero species I ( 13.59 fJ,g/Il1L ). fl ower oils from E. punicea ( 13.59 fJ,g/mL), leaf o il fro m E. elatior (56.67 fJ, g/mL) and tl ower o il from E. elatior ( I 00 fJ,g/ mL) have toxicity to brine shrim p.

Key words: Ellingera, gas chromatographylflame ionization detector. essential o il s, hydrodistillali on, brine shrimp.

IV ABSTRAK

KOll1posisi ll1inyak pati daripada enall1 spesies Etlingera telah dikaji kOll1posi si kill1ia dan aktiviti biologinya terhadap anak udang, Arlemla salina. Minyak pati telah diekstrak dengan ll1enggunakan kaedah penyulingan hidro dan seteru snya dianalisis ll1 enggunakan kroll1atograti gas/pengesa n ion nyalaan. Peratusan ll1in ya k pati yang diperolehi daripada enall1 spesies Etlingera adalah dalall1 julat 0% hingga 2. 15%, dengan peratusan tertinggi diperolehi daripada daun Etlingera spesies 2. Etil -(E,Z)-dekanoat (79.53%), a-gurjunen (17.25%), geosll1in (17.25%) dan tetradesil aldehid (8.57%) ll1erupakan kOll1pon en utall1 a dalall1 E. elallor. a-pinena and ~-pinena hadir dalam kelii11pahan yang tinggi dalam t:~ lilloralis dengan 20.67% a-pinena telah dijumpai pad a daun dan 20.24% ~-pinena tel ah dijull1pai paad a rhizom. E. punicea ka ya dengan oeten ol (73 .27%), ll1anakala E. latitabris mempunya i kelimpahan oktadienon (66.55%), metiletilpirazina (29.30%) dan a-pillena (25.9 1%). Oklanol (3 1.02%) dan p-pinena (3 1.02 %) merupakan kOll1ponen ulall1a yang dijumpai dalam Ellingera spes ies I . Ellingera spes ies 2 mempun ya i a-pinena (25.39%), metil laurat (16.29%) and feno l (15.69%) sebagai komponen utama. Kamtena and (E)-a­ bergall10ten dijumpai had ir di dalall1 daun dalam setiap E/lingera sp p. yang dianalisis. a­ thujen dan 2-etilpiridina hadir di dalam rhizoll1 dal am se tiap Etlingera spp. yang dian alisis.

Daun E. elalior dan Ellingera species 1 dan juga daun E. Iil/orali.l· dan E. 1((lilaiJris ll1enunjukkan hubun gan yang rapat antara satu sama lain. Selain itu , terdapatjuga hubungan yang rapat antara rhizom Ellingera spes ies 2 dan E. ialilabri.\· disamping rhizom E. iiuorolis and Ellingera species I . Ujian ketoksikan terhadap anak udang A. salina l11 enunjukkan han ya ll1in yak pati daripada daun Etiingem species I ( 13.59 Ilg/mL). bun ga E. punicea (13.59 flg/Il1L), daun E. elatlor (56.67 Ilg/mL) dan bunga E. ela/ior ( 100 Ilg/mL) menllnjllkkan ketoksikan terhadap anak udang.

Kala kunci: Ellingera, kromatografi gas/pengesan ion nyalaa n, minyak pati, penyulingan hidro, anak udang.

v LIST OF FIGURES

Figures Pa gc

Figure 4.1 Gas chromatogram traced by GC/FID ana lysis of n-alkane 18 sta ndard mixture.

Figure 4.2 Gas chromatogram obtained from GC/FID ana ly sis for leaf oil of 21 E. e/olior

Figure 4.3 Gas chromatogram obtained from GC/F ID ana ly sis for nower oil 22 of E. elalior

Fi gu re 4.4 : Gas chromatogram obtained from GC/FI D analys is for shoot oil 22 of E. elalior

Fi gure 4.5 Gas chromatogram obtained from GC/FID analys is fo r rhi zome 23 oiI of E. elaLior

Fi gure 4.6 Gas chromatogram obtained by GC/FID analys is for leaf oil of E. 28 lilloralis

Figure 4.7 Gas chromatogram obtai ned by GC/F ID analysis for rhizome oi l 28 of E. lilloralis

Figure 4.8 Gas chromatogram obtained by GC/FID analysis for leaf oi I of E 34 punicea

Figure 4.9 Gas chromatogram obtained by GC/FID ana lys is for stcm oi l ol­ 34 E. punic-ea

Figure 4. 10: Gas chromatogram obtained from GC/ FID analys is nower oil of 35 E. puniceo

Fi gure 4. 11 Gas chromatogram obtained by GC/FID analys is for rhi zo me oil 35 of E. pUl1icea

Fi gure 4.12: Gas chromatogram obtained from GC/FID ana lysis for leaf oil of 41 E. lalilabris

Figure 4.1 3: Gas chromatogram obtained from GC/FID analys is fo r stem oil 41 of E. lalilabris

Figure 4.14: Gas chro matogram obtained from GC/FID ana lys is for rhi zome 42 oi I of E. laLiiabris

Figure 4. 15: Ga s chromatogram obtained from GC/FID analysis for leaf oil of 47 E. sp/

V I Figure 4.16 : Gas chromatogram obtained from GC/ FID analysis for rhizome 47 o il of E . .Ipi

Figure 4. 17: Gas Chromatogram obtained from GC/FID analysis for leaf oil of 54 Etlingera sp2

Figure 4. 18: Gas Chromatogram obtained fr om GC/FID analysis for rhizome 54 o il o f Etlingera sp2

Figure 4. 19: Dendogram obtained from clu ster analysis fo r leafo il of 75 Etlingera.

Figure 4.20: Dendogram obtained from cluster ana lysis for rhi zome oil s of 76 Elbngera

Figure 4.2 1 Dendogram obtained from cluster analysis for stem o il s of 76 Etlingera

Figu re 4.22: Dendogram obta ined from cluster analysis for flower o il s of 77 Etlingera

Figure 4.23: Dendogram obtained from cluster ana lysis on five different o ils of 78 Etlingera

Fi gure 4.24: Plot of death percentage of brine shrimp as a func ti o n of 79 concentration

VII LIST OF TABLES

Tables Page

Tab le 1.1 Classifi cation ofchem ical components in Essential Oil s 2 Tab le 3. 1 Etlingera spp. and location coll ected II Table 4. 1 Percentage of essenti al o il s from five different plant parts of six E/lingera species 18

Tab le 4.2 : Retention time for n-alkane standard analyzed by GC/FID uSlIl g DB-5 column 19

Table 4.3 : Chem ical composition ofessenti al o il s from different plant parts of E. elatior 24

Table 4.4 C hemical composition of essential o il s from two plant parts of E. lillaratis 29

Table 4.5 Chemical composition of essential oils from different plant parts of E punicea 36

Table 4.6 Chemical Composition of essenti al o il s from differe nt plant parts of E la/ilahris 43

Table 4.7 Chemical composition of essentia l o il s from diffe rent plant palts of E .lfJl 48

Table 4.8 : Chemica l composition ofessential oi ls from different plant pa rt s of E s1'2 55

Table 4.9 : Chemica l compositions in leaf o il s from six Etlingera spp. 60 Table 4.1 0: Chem ica l compositions in rhi zome o il s from six E/lingera spp. 66

VIII CHAPTER 1

INTRODUCTION

1.1 INTRODUCTION

According to Anand and Kumar (1990), essential oil is an extrcmely heterogeneous mixture of volatile, lipophilic plant products with characteristic odors,

The Internal Stand ard Organization (ISO) has defined the essential oil in the strict sense as the steam di stillates of or oils obtained by pressing out th e rinds of a few citrus fruits, Generally, essential o il s are unde rstood to be volatile compounds

that are freel y soluble in akohol, ether and vegetable and mine ral o il s and are usually

assumed to be the re sult of distillation or a steam-stripping process (Hernandez,

2000),

The use of essential oils in ancient times consisted of preparing ointments by

mixing o ils from fl owers with fatty oils. This process was done by placing fl owers

and roots with the o il in glass bottles, which were then a ll owed to sit for periods of

time. Sometimes the fl owers or roots were macerated with wine before the fatty oi I

was added , and the produ ct obta ined by digestion filtered and bo iled down to a thicker

consistency (Hernandez, 2000). The production and use of essential oils did not

become widespread until the second half of the 161h century when Hieronymus

Brunschwig's book on distillation, Liber De Arte Distillandi describe the di stillation

techniques for four essential o il s published in 1507. The four essential o ils were turpentine (known since antiquity), juniper wood, rosemary and sp ike (Hernandez.

2000).

According to Parker (1993), essential oils are composed of many kind or classes of molec ules such as terpenoids, phenolics, aromatics, cyclic and acyclic compounds, sulfur and nitrogen containing compounds, and acetonides compounds depending on th e plant and extraction method. The main components are

monoterpenes (CIO) and sesquiterpenes (CIS). The boiling point fo r monoterpenes is

140-180°C while for sesquiterpenes the boiling point is more than 180°C. This terpene can exist as an open chain , monocyc li c or bicyclic and is usually composed of one o r more double bond and hydroxyl group. Hernandez (2000) has grouped the essential oils into six classes based on their chemical properties as shown in Table 1.1.

Table 1.1: Classification of chemical components in Essential Oils

Component Examples

Hydrocarbons Limoncne in lemon oil

Alcohols Borneol in camphior

Esters Methyl salicylate in oi l of wintergreen

Aldehydes Banzaldehyde, decanal s

Ketones Menthone in oil of peppermint

Lactones Coumarin from Tonka beans

The Zingiberaceae is a moderately sized fa mil y of relati vely adva nced

monocotyl edonous pl ants of ord er . Zingiberaceous plants are

rhi zomatous, perennial and aromatic herbs often of large size. bearing nowers either

2 terminally on aerial leaf shoots or from ground level (Chen and Chen, 1998). Species of Zingiberaceae are the ground plants of the tro pical forests. They mostl y grow in damp and humid shady places. They also found infrequently in secondary rorest.

Some species can fu ll y exposed to the sun, and grow on high elevation (Sirirugsa,

1997). These are plants from tropical and subtropical regions distributed mainly in

Asia . The Zin giberaceae comprises about 1200 species of which about 1000 occur in tropical Asia. 13 y far the ri chest area is the Mal esian region, a floristicall y di stinct region that includes Mala ys ia, , , , the and

Papua New Guinea, with 24 genera and about 600 species. According to Larsen el al.

(1999), it is known that large areas such as Sumatra and Borneo are still very insufficiently known and largely under explored for the flora. Therefore many species will undoubted ly be found in the years to come.

The current record s indicate that the tribes of Alpineae and ZlYlgib ereae are quite well represented in Borneo including 2 genera (Burbidgea and Geanthus) unknown in Peninsular Malaysia. There are 20 species described for fJoeseniJergia, the tribe Hedychease, which is poorly represent ed in Borneo, with only 6 genera, recorded (Smith, 1987). The tribe Globeae is similarl y poorly represented with onl y 8 species described so far (Smith, 1988). There are about 18 genera with more tlmn 160 species of Zingiberaceae recorded in Peninsu lar Malaysia. Many of these are rare , very local in di stribution, and consequently highly vulnerable to endangerment. The

Zingiberaceae are mainly forest plants found from the lowlands to the hi ghest elevations in Peninsul ar Malaysia (Larsen e/ aJ., 1999).

3 Etlingera, which is fro m the tribe Alpineae and fa mily, Zi ngiberaceac

(S irirugsa, 1997) is o f interest in this project. The genus Ellingera is distributed from

In dia lo the Pacific Island with centers of species ri chness assumed in Borneo and

New Guinea. At least 70 species are recorded from the Malesian region, bUl recent study shows that 29 are kn own species with 10 still undescribed. Species of Etlingera can be more than 5 m tall and become dominant in gaps (E. lilloralis, E. brevilabrum,

E. coccinea, and E. jimbriobracleala) and thus indicate di sturbance ; whilst other species are found reproductive in shady conditions as well, e.g. E. corrugate (Po ulsen,

2003).

1.2 OBJECTIVES

The objectives of this study are:

J. To isolate the essential oil s from several Ellingera spp. us ing hydro

di stillation meth od.

II . To identify and characteri ze the chemical components of the essential oi ls

using GC-FID.

III . To conduct a bi ological aC livity study of the essentia l o il o n brine shrimp,

Arlemia salina.

IV. To determine the chemical pro fil e of Ellingera spp. fo r chemotaxonomy

classification by cluster analysis.

4 PIlAt Khidmat Maklumat Akademik UNJVERSm II"' L.4VSJ.4 SAItAWAIC

CHAPTER 2

LITERATURE REVIEW

2.1 Essential Oils

In the early aged, essential oils were end-point of metabolism , but it is known that in times of stress the oils can be broken down to provide energy for the planl

(John e/ al., 1987). C urrent specific uses of essential o il s are to add flavor to foodstuffs and beverages and to scent perfumes, loti ons, soaps, detergents and househo lds' cleaners. For example, limonene from citrus peel is a very strong solvent and it is used in a wide va riety of cleaning products. Essential o il s are a major part of carbonated beverage fla vori ngs; the most common flavo rs include lemon, limc. orange, cassia, cinnamon and nutmeg. Essential oils are also used to flavor many

food s such as sweets and candies, cookies. snacks and chewing gum (Hernandez,

2000).

The use of essential oils as functional ingredients in food s, drinks, toiletries

and cosmetics is gaining momentum, both for the growing interest of consumers ill

ingred ients from natural sources and also because of increasing concern about

potenti all y harmful syntheti c additives (Reische et aI., 1998). However, essential o il s

and their components are gaining increasing interest because of their relati vely safe

status, their wide acceptance by consumers and their exploitation potential multi­

purpose functional use (Ormancey el aI., 200 I ; Sawamura, 2000).

5 2.2 Extraction of Essen ti al Oils

An essenti al oil is internationally defined as the product obtained by steam di stillation, hydrod istill ation or expression (for citrus) of a plant or a part o r it. This defin ition is now less strictly applied and the fractions resulting from severa l other techniques that sample the volatile fraction of a plant are now erroneously classifi cd as essenti al o il s. In general it would be more correct to call them vol atile fractions of a vegetable matrix, and to use the term essential oil more specifically for sam ples obtained by distillation or expression (Bicchi, 2000). The essential oils are natural product derived from aromatic plants and have a wide range of uses in fla voring and fragrances in the food and perfume ind ustries, medicine and crop protectio n (lsman,

2000; Daferera el aI., 2003) that can be isolated by steam d isti Ilati on or hydrodistillation (Park er 1993).

There are three general methods to isolate the essential o il. T hey are boiling water, steam distillation, Or the combination of both meth ods. The conditions for temperature, pressure or vacuum and processing tim e wi ll depend on the characteristics of the essential o il , particularly as regards suscepti bili ty to oxidati o n and heat decomposition (Hernandez, 2000). Other meth ods are hydrodistillation. solvent extraction, cold press extraction, steam di stillation supercritical fluid extraction (SFE), microwave oven extraction, so lid phase extraction (SPE),

fluorocarbon extraction and various other teChniques (A tta, 1999).

6 A simple distillation apparatus consists of a retort Or sti ll , a condenser and a receiver. This system is commonly used in field s for processing lavender. Some aroma systems involve the use of only stearn in order to obtain a mOre concentrated essential oil in the condenser. This system is applicable where th ere is no agglomeration or agglutination of raw materials. Extraction methods by steam distillation, solvent extraction and supercritical CO, usually produce different resu lts with regards to yield and composition profiles of the extracted materials. Stearn di stillation usually gives the lowest yield of recovered arOma but produces a concentrate that is a true essential oil. Solvent extraction with hexane or alcohols produces the highest yield but there is always the possibi lity that unwanted non­ volatile materials might end up in the final product. Also. so lvent extraction uses high temperature. Supercritical CO2 extraction prod uces a lower yie ld th an conventi ona l solve nt extraction but hi gher than stearn distillation and it also has the advantage of usin g little or no heat and no stearn or moisture in the process. However, this method is expensive sin ce it in volves high pressure and sophisticated equipment and controls

(Hern andez, 2000).

7 2.3 Zingiberaceae

Zingiberaceae is one of the largest fa mili es of the plants kingdom. It is important natural so urces thai provide many useful products for food , spices, med ic ines, dyes, perfumes and aesthetics to man (S iriru gsa, 1997).

One of the common uses of the Etlingera is as food flavoring. vegetables and beverages. For examp le, E. elatior is kn own as bunga kantan used loca lly for flavoring food (Larsen e l aI., 1999). The part usuall y used is the young fl ower buds, which is used as an important ingredient for the spicy dish, Laksah (Si riru gsa, 1997) and a fa vorite noodle dish in Peninsular Malaysia (Larsen el ai., 1999). Kantan i, also used in the local di shes of nasi kerabu or nasi ulom (at the East Coast of the Ma laysia

Peninsula ) and lokm GSom (Larsen et 01., 1999). In southern , flowers are eaten as vegetab le. Whereas the flowers of E. moingayi can be eaten as vegetables.

Furthermore, th e fruit of Etl ingera littoralis is edible. While the young stem of thi s speCies, after removing the outer parts, yields an aromatic, tender core, whi ch is suitable for eating raw or cooked (Sirirugsa, 1997). In , the fruits of E.

.fimhriohracteato have been used as a kind of conse rve, and are also eaten by !bons

(Poulsen. 2003). Furtherm ore, the fruits of some Ellingero spp. and Hornstedlio spp. are also edible. While in Sabah, E. coccineo, loca lly known as luhau (by Du slin s and

Muruts), is used as a condiment. It is also used by Kelabits (Iubu nanling) and I bans

(lepus) in Sarawak, a name also used in Java (Poulsen, 2003).

8 Species with very fibrous leaves can be processed for the production of fibers and paper. E. clatior was known to be successfully processed for papermaking albeit not high quality. The leaves of many species are quite fibrous and hence having good potential for their exploitation into economic products (Halijah, 1998).

The Zingiberaceae are mostly plants with showy and often brightly colored bracts and floral parts. In this they are second to none, not even to the orchids. For ornamental purposes, however, most of the species have one drawback: the flowers of most species are very short-lived, often lasting only a few hours

(Larsen 1'1 al., 1999). In the western countries, especially the United States of

America, there is increase in the popularity of Zingiberaceae as decorative plants in indoor gardens, landscapes and floral arrangements (Halijah, 1998). Farm in Australia and Costa Rica for instance are selling the inflorescences of E. elalior (the torch ginger), in shades of pink, deep red and purplish black, as cut flowers (Larsen el af.,

1999).

Rhizome decoction from E. liltoralis and E. peciosa can be drink as antipyretic. While the juice of E. solari.l· is dropped into the ears to treat certain ear­ ache. Decoction of the rhizome is used as an antiseptic for external wounds and the

flowers are sometimes included in the herbal bath for a mother after childbirth

(ARCBC,2004).

Volatiles for the flowers of E. elaUor have been previously analyzed by Wong el af. (1993), Lechat-Vabirua cl af. (1996) and Zoghbi el af. (2001). The study showed that flowers of E. clalior are rich in alcohols and aldehydes. Predominancc

9 components identified in the oils of and innorescence axis of E. elalior were dodecanol, dodecanal and tetradecanol. a-pinene is the ma jor component of monoterpene group in th e flower oi l of E. elatior with 22.2% present in innorcscence and 6.3% present in inflorescence axis. According. to The European Agency for the

Evaluation of Med icinal Products in its Final Positio n Paper on the Use of Herbal

Medicinal Products Containing Methyleugenol (2004), E. cevuga has found to have

47.7% of methyleugenol in the rhizome oil.

10 CHAPTER 3

MATERIALS AND METHOD

3.1 Plant Materials

Samples of Ellingera spp. were coll ected from several locations in Sarawak.

The area of sa mpling in cluding Sematan, Padawan and Kuching of Kuching Di vision , forest area of Unimas campus of Kola Samarahan and Lawas of Limbang Division.

The sampling locations of Erlingera spp. are summarized in Table 3.1. Leaves, stems and rhi zomes of the plant sample were separated during the sample collection. Upon arriving at the laboratory, the samples were cleaned and then cut into small pieces before grinding. Fresh samples were used for extraction.

Table 3.1: Ellingera spp. and location collected

.------­ Species Location Collected

Etbngera elalior Kuching area

Elfingera littoralis Unimas, Kota Samarahan

EI/iny.;era punicea Padawan, Kuching

ELlingera latilabris Lawas, Limbang

Ellingera species I Sematan, Lundu (Kuching)

Ellingera species 2 Unimas, Kola Samarahan

" 3.2 Extraction of Essential Oil

The extraction of essentia l oils from plant samples was carried out USl11 g

meth od establi shed by Lee and Ogg as described by Datta (1987). Briefly, essential

oi ls were extracted and iso lated usi ng hydro distiller equipped with Clevenger-type

apparatus. Approximately 100 g of fresh-grounded samples were weighed a nd

transferred to 2 L-capacity fl at bottom flask and mixed with 1. 5 L disti ll ed water.

Several anti-bumping granules were added to the fl ask to avoid the solvent bumpin g.

T he fl ask were then assembled to the C levenger trap a nd conn ected to the condenser.

Hydrodistillati on was carri ed out for 6-8 hours. The flask was heated to maintain the

distill ation rates of two drops per second. After 6-8 hours, the oi l trapped in the

C levenger was then cooled to room temperature. T he oil y layers obtain ed we re

separated and dried over anhydrous sodium sulphate and stored at 4-5°C. T he

hydrodi sti ll ation process was done in triplicates and average percentages o f the oi ls

were ca lc ul ated.

3.3 Instrumental Analysis

3.3.1 Gas Chromatography-Flame Ion Detector (GC/FID)

The analysis of the essential o il s was performed on Shimadzu GC-17A gas

chromatograph equipped with a flame ioni zati on detector (FlO) detecto r using a fu sed

silica capi ll ary column OB-5 (25 m X 0.3 mm). Prior to GC/FID analysis, I ilL of

essential oi l were diluted w ith 200 ilL n-hexane. Helium was used as a carrier gas

12

- - _.------­ with velocit y of 2 mUmin. The initial temperature was programmed at 50°C and he ld for 2 minutes. Th e temperature was then increased to 300°C at a rate of 6.S oC/min.

The final temperature was held for ten minutes. Th e te mperature for the injector and detector were set at 280°C and 320°C, respecti vely.

3.4 Data Analysis

3.4.1 Percentage of Essential Oils

The percentages of the essential o il s obtained from each species studied were ca lcul ated. The yie lds were averaged over three experiments and calcul ated based on dry weight of plant material.

volume of oil % Essential Oils (v/w ) = ------­ X [00% dry wei ghtof samples

13