faculty of resource science and technology ... and biological...termasuklah 25 terpena, 46 ester, 57...

Faculty of Resource Science and Technology

CHARACTERIZATION AND BIOLOGICAL ACTIVITY OF ESSENTIAL OILS

FROM PIPER SPP.

Rosilin binti Logihad

Bachelor of Science with Honours

(Resource Chemistry)

2008

ii

DECLARATION

No portion of the work referred to in this dissertation has been submitted in support of an

application for another degree of qualification of this or any other university or institution

of higher learning.

___________________________


Program of Resource Chemistry

Faculty of Resource Chemistry and technology

Universiti Malaysia Sarawak

iii

ACKNOWLEDGEMENT

I would like to express my profound gratitude and sincere appreciation to my supervisor,

Mr. Chieng Tiong Chin for his patiently guidance and gentle encouragement during the

planning and execution of the research and for his tremendous counsel and helps. Grateful

acknowledgement is also expressed to all the lecturers in chemistry department for their

invaluable advice and support upon the completion of this research.

I as well would like to thank for the assistance given by the staffs of Faculty of Resource

Science and Technology (FRST), UNIMAS and lab assistant for their helps during the

practical. I am also very thankful for the contribution whether directly or indirectly from

my friends and mates during the completion of this research.

Lastly, I also want to express my special gratitude to my parents and all my siblings for

their continuous care and support towards this research, especially to my sister, Miss Maria

Logihad for her helps in providing samples and useful information.

iv

Characterization and Biological Activity of Essential Oils from Piper spp.


Resource Chemistry Programme


Universiti Malaysia Sarawak

Email: [email protected]

ABSTRACT

This study was performed to identify the chemical composition of essential oils extracted from root, stem

and leaves of the three Piper spp. which are Piper sp1, P. betle and P. sarmentosum. The essential oils was

extracted by using hydrodistillation and then subjected to gas chromatography-flame ionization detector for

analysis purpose and Kovat indices calculation. The percentage yield of essential oil from the root, stem and

leaves were generally ranged from 0.3% - 2.47% (v/w) and the result showed that P. sarmentosum give the

highest percentage yield of essential oil. For overall, there are 102 components detected from the root part of

Piper spp., 98 components from stem part and 75 components from leaves part. These components include

the 25 terpenes compound, 46 ester, 57 aldehyde and ketone, 47 alcohol, 4 acids and 39 others. The close

relationships between these species were analyzed by using Hierarchical Cluster Analysis and the dendogram

produced showed the similarities in term of chemical composition from Piper spp. Toxicity test showed that

P. sarmentosum was the most toxic substance for root part with LC50 of 55.06 µg/mL. While, for the both of

stem and leaves part, Piper sp1 showed the most toxic potential towards the larvae with LC50 of 66.24 µg/mL

and 56.55 µg/mL respectively. Antitermite activity of the Piper spp. showed that P. sarmentosum was the

most toxic against the termites for all parts.

Keywords: Piper spp., essential oils, Kovat indices, toxicity test, cluster analysis

ABSTRAK

Kajian ini dijalankan bagi mengenalpasti komposi kimia minyak pati yang diekstrak dari bahagian akar,

batang dan juga daun bagi tiga jenis sepsis Piper yakni Piper sp1, P. betle dan P. sarmentosum. Minyak pati

ini diekstrak dengan menggunakan kaedah penyulingan dan dianalisis menggunakan alat gas kromatograpi-

pengesan ion nyalaan dan indeks Kovat. Peratusan minyak pati yang diperolehi daripada akar, daun dan

batang Piper spp. ini berjulat dari 0.3% - 2.47% (v/w) dan didapati bahawa P. sarmentosum memberikan

peratus hasil minyak pati ynag tertinggi. Secara keseluruhan, terdapat 102 komponen dikesan pada akar, 98

komponen pada batang dan 75 komponen pada bahagian daun. Komponen yang dikenalpasti ini

termasuklah 25 terpena, 46 ester, 57 aldehid dan keton, 47 alkohol, 4 asid dan 39 lain-lain. Hubungan di

antara spesis ini dianalisis menggunakan Analisis Hierarki Gugusan dan dendogram yang terhasil

menunjukkan kesamaan di antara komposisi minyak pati dari Piper spp. Ujian ketoksikan dijalankan ke ates

kesemua minyak pati ini dan didapati P. sarmentosum memberikan LC50 55.06 µg/mL (paling toksik)bagi

minyak bahagian akar dan Piper sp1 pula menunjukkan ketoksikan tertinggi bagi kedua-dua bahagian

batang dan daun iaitu LC50 adalah 66.24 µg/mL dan 56.55 µg/mL masing-masing. Sementara, ujian anti-

anai-anai pula menunjukkan minyak pati P. sarmentosum memberikan ketoksikan paling tinggi bagi semua

bahagian tumbuhan.

Kata kunci: Piper spp., minyak pati, indeks Kovat, ujian ketoksikan, analisis hierarki gugusan

mailto:[email protected]

v

TABLE OF CONTENT

DECLARATION

ii

ACKNOWLEDGEMENT

iii

ABSTRACT

iv

ABSTRAK

iv

TABLE OF CONTENT

v

LIST OF FIGURES

LIST OF TABLES

vii

viii

CHAPTER 1: INTRODUCTION

1.1 General Introduction

1.2 Objective of the Project

1

2

CHAPTER 2: LITERATURE REVIEW

2.1 Piper species

2.2 The Essential Oils

2.3 Extraction of Essential Oils

2.4 Hierarchical Cluster Analysis

4

12

12

14

CHAPTER 3: MATERIALS AND METHODS

3.1. Sampling


3.3 Quantitative and Qualitative Analysis of Essential Oils

3.3.1 Gas Chromatography – Flame Ionization Detector (GC-

FID)

3.3.2 Essential Oils Yield

3.3.3 Qualitative Analysis of Essential Oils

15

15

16

16

16

17

vi

3.3.4 Semi – Quantitative Analysis

3.3.5 Hierarchiral Cluster Analysis

3.4 Determination of Biological Activity

3.4.1 Toxicity to Artemia salina

3.4.2 Termicidal Activity

18

18

19

CHAPTER 4: RESULTS AND DISCUSSION

4.1 Percentage Yield of essential oils from Piper species

4.2 Qualitative Analysis

4.3 Chemical composition of the essential oil

4.4 Cluster Analysis

4.5 Biological Activities

4.5.1 Toxicity tests by using Artemia salina

4.5.2 Termiticidal Activities

20

21

23

39

41

44

CHAPTER 5: CONCLUSION

50

REFERENCES

52

vii

LIST OF FIGURES

Figure 2.1.1: Chemical Structures 11

Figure 3.4.2.1: Bioassay apparatus for termicidal activity tests of Coptotermes

spp.

19

Figure 4.2.1: GC-FID chromatogram of n-alkanes standard 22

Figure 4.3.1: GC-FID chromatogram of essential oil from the root of Piper sp1 23

Figure 4.3.2: GC-FID chromatogram of essential oil from the stem of Piper sp1 24

Figure 4.3.3: GC-FID chromatogram of essential oil from the leaves of Piper sp1 24

Figure 4.3.4: GC-FID chromatogram of essential oil from the root of P. betle 25

Figure 4.3.5: GC-FID chromatogram of essential oil from the stem of P. betle 25

Figure 4.3.6: GC-FID chromatogram of essential oil from the leaves of P. betle 26

Figure 4.3.7: GC-FID chromatogram of essential oil from the root of P.

sarmentosum

26

Figure 4.3.8: GC-FID chromatogram of essential oil from the stem of P.

sarmentosum

27

Figure 4.3.9: GC-FID chromatogram of essential oil from the leaves of P.

sarmentosum

27

Figure 4.3.10: Molecular structure of some terpenes identified from the essential

oils of Piper spp.

36

Figure 4.3.11: Molecular structure of some ester compounds detected from Piper

spp.

37

Figure 4.3.12: Molecular structure of major compounds detected from Piper spp. 40

Figure 4.4.1: Dendogram of cluster analysis from the root of Piper spp. 40

Figure 4.4.2: Dendogram of cluster analysis from the stem of Piper spp. 40

Figure 4.4.3: Dendogram of cluster analysis from the leaves of Piper spp. 41

Figure 4.5.1.1: Antilarvae properties against Artemia salina after 24 hours for

the root of Piper spp.

42


the stem of Piper spp.

43


the leaves of Piper spp.

43

Figure 4.5.2.1: Antitermite activity of essential oils from the root of Piper sp1 45

viii

Figure 4.5.2.2: Antitermite activity of essential oils from the root of P. betle 46

Figure 4.5.2.3: Antitermite activity of essential oils from the root of P.

sarmentosum

46

Figure 4.5.2.4: Antitermite activity of essential oils from the stem of Piper sp1 47

Figure 4.5.2.5: Antitermite activity of essential oils from the stem of P. betle 47

Figure 4.5.2.6: Antitermite activity of essential oils from the stem of P.

sarmentosum

48

Figure 4.5.2.7: Antitermite activity of essential oils from the leaves of Piper sp1 48

Figure 4.5.2.8: Antitermite activity of essential oils from the leaves of P. betle 49

Figure 4.5.2.9: Antitermite activity of essential oils from the leaves of P.

sarmentosum

49

LIST OF TABLES

Table 4.1.1: Percentage yield, their colour and physical state of essential oil from

Piper species.

21

Table 4.2.1: Retention time for n-alkane standard analyzed by GC-FID using

DB-5 column

22

Table 4.3.1: Chemical composition of essential oil extracted from the various

parts of the Piper spp.

29

Table 4.5.1: LC50 values against larvae of Artemia salina for Piper spp. 44

CHARACTERIZATION AND BIOLOGICAL ACTIVIES OF ESSENTIAL

OILS FROM PIPER SPP.

ROSILIN BINTI LOGIHAD

This report is submitted in partial fulfillment of requirement for the degree of Bachelor of Science

with Honours in Resource Chemistry


UNIVERSITI MALAYSIA SARAWAK

2008

1

CHAPTER 1

INTRODUCTION

1.1 General Introduction

The family Piperaceae belonging to the superior Nymphaeiflorae, order Piperals, comprises

about 5 genera and 1400 species. The genera Piper (700 species) and Piperomia (600

species) are the most representative of Piperaceae (Santos et al., 2001). The species of

Piperaceae family in Malaya are either herbs, as the genus Piperomia, of which P. pellucida

is a common weed, or shrubs (often epiphytic) or climbers (small and large) of the genus

Piper. There are several features of this Piperaceae family: their leaves are alternative on the

branches, mostly simple and entire, with rather oblique side-veins. The flowers of these

Piperaceae family were very minute, sessile, bisexual or unisexual, in simple, rather freshly,

terminal spikes become opposite to leaves and appearing lateral, the spikes solitary or

clustered: sepals and petals none: stamens 2 – 6, minute, ovary superior, with a single ovule

and sessile stigma (Corner, 1988).

The genus Piper belongs to the Piperaceae. They are erect or scan dent herbs are

infrequently trees. The Piper species have high commercial, economical and medicinal

importance (Parmar et al., 1996). Piper species, widely distributed in the tropical and

subtropical regions of the world are used medicinally in various manners (Parmar et al.,

1998).

Piper betle Linn. is a perennial dioecious, semi woody climber. Stems strongly swollen at

the nodes, papillose when young, soon entirely glabrous. Leaves alternate, simple and

2

yellowish green to bright green in color. Leaves of fertile branches with a petiole 1–2 cm

long, 1.2–1.8mm thick when dry and glabrous at maturity (Arambewela et al., 2005). P.

betle plant is widely grown in the tropical humid climate or South East Asia, and its leaves,

with strong pungent and aromatic flavor, are widely consumed as a mouth fresher

(Bhattacharya et al., 2005).

Essential oil has been defined as the products obtained from raw material by water or steam

distillation (Rouatbi et al., 2005). During an ethnobotanical survey, it was verified that

essential oil – bearing plants are widely used. They are utilized in different forms, such as

whole herbs, powders, extracts and vapors, for a variety of purpose (Martins et al., 1998).

The chief constituent of P. betle is a volatile oil known as ‘betel oil’. The volatile oil is

bright yellow to dark brown liquid possessing a clove like flavor and consists of terpenes

and phenols (Arambewela et al., 2005).

1.2 Objective of the Project

The essential oils might be extracted at every parts of the Piper spp. such as leaves, stems,

bark and roots. As it is extracted, the characteristics of these essential oils which are

obtained from different parts of the plant might show similarity or differences in

compositions. The same things might occur between different Piper species. Thus, the

similarity or differences of these essential oils obtained from different species and different

parts will be compared by using Cluster analysis. One of the purposes of the present study is

to determine the variations in the compositions of the essential oils from different species

and different parts. This study might contribute towards the development of a taxonomic

guide in identification of Piper species. The biological activities of the essential oils

3

extracted at different parts of the plants will also be determined against brine shrimp and

termites.

The objectives of this study is to extract and characterize the essential oils extracted from

the leaves, stems and roots from three Piper species, to compare the composition of

essential oils from various part and between species of Piper and to determine the biological

activities of the essential oils.

4

CHAPTER 2

LITERATURE REVIEW

2.1 Piper species

Among the Piper species, there is a treasure of traditional medicine and traditions

concerning naturally occurring drugs, based on the empirical knowledge of medicinal and

toxics plants, gained by the ancestors and passed on from generation to generation by oral

tradition (Martins et al., 1998).

Experimentally, leaves of Piper betle also known as ‘betel leaves’ are shown to possess

antimicrobial gastro protective, wound healing, hepato-protective, antioxidant, anti-fertility

on male rats and anti motility effects on washed human spermatozoa. In Asian countries

betel leaves are used for chewing and are credited with many properties such as digestive,

simulative, carminative and aphrodisiac (Arambewela et al., 2005).

Betel leaves are also used in wrapping pellets of betelnut and lime for use as a masticatory.

Pellets are hot, acrid, aromatic and astringent. They ridden the saliva and blacken the teeth,

and eventually corrode them (Duke, 1985; Norton, 1998). One astute challengers this,

sensing that Indonesians (including dentist) widely believe that converse to be true, i.e., that

chewing the betel leaves will strengthens the teeth and prevents decays. Chewing the leaves

is also believed to prevent dysentery, fever and gastrosis. Betel leaf chewed with the

betelnut and lime acts as a gentle stimulant and exhilarant. Those accustomed to its use feel

sense of languor when deprived of it (Duke, 1985). Only three drugs (nicotine, ethanol, and

caffeine) are consumed more widely than betel. When betel is chewed, it produces mild

5

psychoactive and cholinergic effects. Betel use is associated with oral leukoplakia,

submucous fibrosis, and squamous cell carcinoma (Norton, 1998). Thus, betel quid chewing

also is a popular oral habit in India, South Africa and numerous Southeastern Asian

countries. It has been estimated that there are about 200 - 600 million betel quid chewers in

the world (Norton, 1998; Lin et al., 2003). However, in recent research, lime – piper betel

quid was found cytotoxic to JB6 cells. It caused degeneration, proliferation, DNA damage

or mutation, and cell transformation. Taken together, the results; the insinuate that lime – P.

betle is a tumor promoting (Lin et al., 2003). Moreover, excessive indulgence in chewing

for long period is labile to produce dental caries, pyorrhea alveolaris, oral sepsis, dyspepsia,

palpitation, neurosis and slow cerebration (Duke, 1985).

In April 1997, an unusual pigmentary disorder was noticed by dermatologist in Taiwan. All

patients had a history of using facial dressing with steamed leaves of P. betle. Thus, a

Clinical and Histopathologic survey to clarify the evaluation and the origin of the unique

leukomalanosis was done. They find out that, leukomelanosis induced by betel leaves,

postinflammatory hyperpigmentation appears to play an important role in the development

of mottled hyperpigmentation, whereas chemical-induced melanocytotoxicity was probably

the main cause of subsequent confetti-like depigmentation (Liao et al., 1997).

However, P. betle extract also contain various positive biological activity. A research about

inhibitory property of P. betle extract against photosensitization-induced damages to lipids

and proteins was done. They studied about the protective activity of P. betle ethanolic

extract against the photosensitization-induced damage to lipids and proteins of rat liver

mitochondria. They find out that, P. betle ethanolic extract could effectively prevent lipid

peroxidation, as assessed by measuring thiobarbituric acid reactive substances, lipid

6

hydroperoxide and conjugated diene. In addition, it prevented photo-induced oxidation of

proteins in a concentrationdependent manner. Furthermore, its preventive capacity against

iron-mediated lipid peroxidation was also confirmed. The protective activity of PE could be

attributed to its free radical and singlet oxygen scavenging properties. The activity of P.

betle ethanolic extract was primarily due to its phenolic constituents, which were identified

as chavibetol and 4-allylpyrocatechol. Thus, the results, taken together, suggest a possible

use of P. betle ethanolic extract against photosensitization induced oxidative biological

damage (Bhattacharya et al., 2005).

The leaf and root of the P. betle, in oil form, are also used as a salve or ointment for hard

tumors and scirrhi. A bolus, made of the leaf, is also used for cancer. Leaves are poultice

onto boils, bruises, ulcers and wound. Asian Indians add the leaf juice to medications for

maladies of the mucous lining of the mouth, nose and stomach. They also use the leaf on

ulcerated noses and apply them to the body after childbirth. The juice or infusion is dropped

into ear for wounds, the eyes as a collyrium. However, the oil shows a marked irritant action

on the skin and mucous membrane. It produces an inflammatory reaction when injected. In

moderate doses, it appears to have antispasmodic action on involving muscle tissues,

inhibiting excessive peristaltic movement of the infesting. It exhibits a depressant action on

the central nervous system of mammals; lethal doses produce deep narcosis leading to death

within a few hours. Excessive use produces effects somewhat similar to those of alcoholic in

toxication (Duke, 1985).

Other study have been done is antioxidant activity of P. betle L. leaf extract in vitro. The

antioxidant activities of aqueous extracts of three local varieties of P. betle leaves were

evaluated in vitro systems, e.g. DPPH radical scavenging activity, superoxide radical

7

scavenging activity in a riboflavin/light/NBT system, hydroxyl radical scavenging activity

and inhibition of lipid peroxidation induced by FeSO4 in egg yolk. Total antioxidant activity

was measured by the reduction of Mo(VI) to Mo(V), by the extract, and subsequent

formation of a green phosphate/Mo(V) complex at acid pH. The extracts were found to have

different levels of antioxidant activity in the systems tested. The data indicate that the

antioxidant activities differed in varieties. The antioxidant activities of the three varieties are

in the order Kauri > Ghanagete > Bagerhati. All three varieties of P. betel are more potential

to prevent lipid peroxidation than does tea. Total antioxidant capacity (equivalent to gallic

acid) of Kauri is also higher than tea. Total phenolic concentration, expressed as gallic acid

equivalents showed correlation with the antioxidant activity, being highest in Kauri and

lowest in Bagerhati. The results from various free radical-scavenging system revealed that

the three local varieties of P. betle had significant antioxidant activity. The extracts were

found to have different levels of antioxidant activity in the systems tested (Dasgupta and De,

2004).

Some of the plants found in Thailand can be considered as good sources of natural

antioxidants since their extracts were found to possess high antioxitant activity. The highest

activity was detected in P. sarmentosum. Inspection reveals that contents of vitamin C,

vitamin E, total carotenes, total xanthophylls, tannins and total phenolics in the test plants

correlated with the antioxidant index. The results suggest that the antioxidant activities of

these plants may be attributed to the chemical components present, especially vitamin E and

xanthophylls (Chanwitheesuk et al., 2004).

Naringenin (1), a naturally occurring antioxidant superoxide scavenger was found in the

methanolic leave extracts of P. sarmentosum and M. elliptica. Thus these plants could be

8

considered as antioxidant food. Therefore if consumed daily, they could scavenge access

free-radicals in the human biological system and could prevent oxidative related diseases.

Antioxidant food supplies the body with the essential antioxidant nutrients needed to

enhance the immune system, eliminate excess free radicals and to keep the oxidative stress

state in balance. Thus the leaves of P. sarmentosum and M. elliptica can help to maintain

energy, general ability and fitness even as we age (Subramaniam et al., 2003).

Other study that have been done is to found out the polyphenols and alkaloids compound

from Piper species. This research successfully extracted new compound, the β-sitosteryl

palmitate, from P. betle, where this compound was isolated for the first time from the genus

Piper. Other compounds found in Piper betle are dotriaconfanoic acid, tritriacontane, stearic

acid, cepharadione, piperidind, piperlonguminine and β-sitosterol. These compound were

also found in P. acutisleginum, P. khasiana, P. pedicellosum and P. thomsoni (Parmer et al.,

1998).

A new prenylated salicylic acid derivative, 3-farnesyl-2-hydroxy benzoic acid (2), was

isolated from the leaves of Piper multiplinervium C. DC. (Piperaceae). It showed anti-

Helicobacter pylori activity (MIC 37.5µg/ml) and antimicrobial activity at MICs between

2.5 and 5 µg/mL against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae,

Mycobacterium smegmatis, Pseudomonas aeruginosa and Candida albicans. Its structure

was elucidated by means of MS, 1H and 13C NMR. The ethnomedical claim of Piper

multiplinervium for treating stomach aches by the Kuna Indians of Panama may be justified

by anti-Helicobacter pylori activity of its MeOH extract (R¨uegg et al., 2005).

9

A dihydropyridone alkaloid, cenocladamide (5), and a derivative of piplartine (3), 4'-

desmethylpiplartine (4) were isolated along with piplartine from the leaves of Piper

cenocladum (Dodson et al., 1999).

In addition to sitosterol (6), syringaldehyde (7), 3,4,5-trimethoxybenzoic acid (8),

isoelemicin (9) and grandisin (10), two new tetrahydrofuran lignans (11, 12) were isolated

from Piper solmsianun and characterized as rel-(7R,8R,70R,80R)-30,40-methylenedioxy-

3,4,5,50-tetramethoxy-7,70-epoxylignan and rel-(7R,8R,70R,80R)-3,4,30,40-

dimethylenedioxy 5,50-dimethoxy-7,70-epoxylignan on the basis of spectroscopic data,

including 2D NMR spectrometric techniques (Martins et al., 2003).

10

O

O

OH

OH

OH

OH

HOOC

OMe

MeO

MeO

N

RO

O

O

OMe

MeO

N

OH

O

OMe

MeO

O

(1)

(2)

(3) R = CH3, piplartine

(4) R = H , 4'-desmethylpiplartine (5)

(6) (7)

H

OMe

MeO

MeO

O

11

OMe

MeO

MeOOH

O

O

OMe

MeO

MeO

O

O

OMe

MeO

MeO

OMe

OMe

OMeO

OMe OMe

O

O

MeO

MeO

O

OMe OMe

O

OO

O

(8) (9)

(10) (11)

(12)

Figure 2.1.1: Chemical Structures

12

2.2 The Essential Oils

Essential oils have been defined as the products obtained from raw material by water or

steam distillation (Rouatbi et al., 2005).

Leaves and/or the essential oil of Piper betle therefore are antiseptic and antioxidant. If it is

heated with oils and fats, they check rancidity and effective. The essential oil and leaf

extracts posses activity against several Gram-positive and Gram-negative bacteria such as:

Bacillus subtilis, B. megaterium, Diplococcus pneumoniae, Escherichia coli, Erwinia

carotova, Micrococcus pyogenes, Proteus vulgaris, Pseudomonas solanaoearum,

Salmonella thyphosa, Sarcina lutea, Shigella dysenteriade, Strepcoccus pyogens and Vibrio

comma. Antiseptic activity is probably due to chavicol. Essential oil and leaf extracts also

show antifungal activity against Aspergillus niger, A. oryzae, Culvularia lunata and

Fusarium oxysporum (Duke, 1985).


Essential oils can be extracted in several ways. In practice, four methods can be used: steam

distillation applied on fresh or dried material, hydrodistillation with addition of water to the

plant material, squeezing the pericarp and pyrogenation of the crust or the wood of some

plant materials (Rouatbi et al., 2005). However, the traditional methods of extracting

essential oils was time consuming, laborious and exhibit low selectivity and/or extraction

yields; moreover, they usually employ large amounts of organic solvents (P´eres et al.,

2005). Thus, other methods of extracting essential oil were introduced to improve the

drawback.

13

Superheated steam was one of the methods introduced. Superheated steam is a steam which

temperature is higher than saturation temperature at the considered pressure. It is used in

drying and baking but it is also known in the domain of essential oils production. Since, this

method involve the temperatures higher than 230oC, it causing charring and partial pyrolysis

of the biomass and the decomposition of the essential oil since that the ideal operating

temperature is between 205 and 220oC for flash essential oil distillation. Thus, the

production of fumes aroma from wood using superheated steam generates polycyclic

hydrocarbons that are carcinogenic occur (Rouatbi et al., 2005). Concluded that, this method

still contains some undesirable consequences and thus, further effective methods still need

to be found.

At present, there is a renewed interest in developing new processes based on the use of sub-

and supercritical fluids or small amounts of organic solvents. These sub- and supercritical

processes provide some additional benefits, such as higher selectivity and shorter extraction

times. Among them, supercritical fluid extraction (SFE) and pressurized liquid extraction

(PLE) are two of the most promising processes (P´eres et al., 2005).

Pressurized liquid extraction is a technique for extracting solid and semisolid samples with

liquid solvents. This technique used for extraction of active ingredients from medicinal

plants. PLE uses liquid solvents at elevated temperatures and pressures to increase the

efficiency of the extraction process. Increased temperature accelerates the extraction, while

elevated pressure keeps the solvent below its boiling point, thus enabling safe and rapid

extractions (P´eres et al., 2005).

14

2.4 Hierarchical Cluster Analysis

Hierarchical cluster analysis is a statistical method for finding relatively homogeneous

clusters of cases based on measured characteristics. The term cluster analysis encompasses a

number of different algorithms and methods for grouping objects of similar kind into

respective categories. It is used to organize observed data into meaningful structures, that is,

to develop taxonomies. In other words cluster analysis is an exploratory data analysis tool

which aims at sorting different objects into groups in a way that the degree of association

between two objects is maximal if they belong to the same group and minimal otherwise.

Thus, cluster analysis can be used to discover structures in data without providing an

explanation/interpretation (cluster analysis, 2004).

http://www.statsoft.com/textbook/glosa.html#Algorithm

15

CHAPTER 3

MATERIALS AND METHODS

3.1. Sampling

Sample of the roots, stems and leaves of P. betle, P. sarmentosum and Piper 1 was collected

from Tuaran area, Sabah, around Tabuan area and Unimas area respectively during July

2007.


Hydrodistillation processes have been used to extract the essential oil from the roots, stems

and leaves of the Piper spp. sample. Hydrodistillation process was carried out for six to

eight hours by using a Clevenger type apparatus. Approximately 100 g of ground sample of

Piper spp. leaves were transferred into 2 L flat bottom flask and mixed with 1.5 L of

distilled water. The flask was assembled to the Clevenger trap and connected to the

condenser and the oil trapped was cooled to the room temperature. The oil obtained was

separated and dried over anhydrous sodium sulphate (Na2SO4) and was stored in vials at low

temperature, 4 – 5 oC. The hydrodistillation process was carried out in several replicate

depend on the sample quantity, before the oils being analyzed using the Gas

Chromatography – Flame Ionization Detector (GC-FID).

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