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ANTIFUNGAL ACTIVITIES OF DICHLOROMETHANE AND METHANOL

EXTRACTS FROM EUSIDEROXYLON ZWAGERI AND

POTOXYLON MELAGANGAI HEARTWOOD

SIM SHIANG PING

Bachelor of Science with Honours

(Plant Resource Science and Management)

2012

Faculty of Resource Science and Technology

I

ANTIFUNGAL ACTIVITIES OF DICHLOROMETHANE AND METHANOL

EXTRACTS FROM EUSIDEROXYLON ZWAGERI AND POTOXYLON

MELAGANGAI HEARTWOOD

SIM SHIANG PING

This project is submitted in partial fulfillment of the requirements for the Degree of

Bachelor of Science with Honours

(Plant Science Resource and Management)

Faculty of Resource Science & Technology

UNIVERSITI MALAYSIA SARAWAK

2012

II

APPROVAL SHEET

Name of candidate: Sim Shiang Ping

Title of dissertation: Antifungal Activities of Dichloromethane and Methanol Extracts from

Eusideroxylon zwageri and Potoxylon melagangai Heartwood

Assoc. Prof. Dr. Ismail Jusoh

Supervisor

Dr. Siti Rubiah

Coordinator

Plant Science Resource and Management Programme

Department of Plant Science Resource and Environmental Ecology


III

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.

Sim Shiang Ping

25026


University Malaysia Sarawak

IV

ACKNOWLEDGEMENTS

Foremost, I would like to express my deepest gratitude to my supervisor, Assoc.

Prof. Dr. Ismail Jusoh and Prof. Dr Zaini Assim, for their excellent guidance, supports,

caring, patience, motivation, enthusiasm, suggestions and providing me with an excellent

atmosphere for doing this project. I would like to thank Prof Dr Sepiah Muid and Dr

Effendi bin Wasli for using their laboratory.

I would like to express my special thank to Thian Chaw Foo, a good friend who

always supports and encourages me through this study. Thank to my family for their

understanding and moral support that make this study possible.

Last but not the last, many thanks to all laboratory assistants Department of Plant

Science and Environmental Ecology, Department of Chemistry, master students, Irna

Syairina Sahari, Zul Helmey, Faezah Abdullah, Nur Diyana Ishak, Farah and Syazwani for

their opinions and valuable comments. Also thanks to my coursemate, especially Ooi Teng

Sin, Ho Soo Ying, Fong Yin Mei, Chen Mei Yin, Soon Chee Pei and Lim Han Rou for

their suggestions, cooperation and assistance which make this study a wonderful and full of

experience.

Thank you very much.

V

Antifungal activities of dichloromethane and methanol extracts from Eusideroxylon

zwageri and Potoxylon melagangai heartwood

Sim Shiang Ping Plant Science Resource and Management Programme

Faculty of Resource Science & Technology

Universiti Malaysia Sarawak

ABSTRACT

Durability and strength of wood are important in construction industry. Natural durability of Eusideroxylon

zwageri and Potoxylon melagangai are known to be very high. One of the reasons for high wood durability is

the presence of extractives. The objectives of this study were firstly to determine the amount of

dichloromethane (DCM) and methanol (MeOH) crude extracts from E. zwageri and P. melagangai, secondly

to identify the chemical constituents of DCM and MeOH extracts and thirdly to assess antifungal activity of

DCM and MeOH extracts. Sequential solvent extraction by using DCM and MeOH were carried out. Gas

chromatography–mass spectrometry techniques were used to identify and characterize the chemical

constituents and compositions of DCM and MeOH crude extract fractions from E. zwageri and P.

melagangai. The antifungal activities were determined in dichloromethane and methanol extracts using agar

dilution method. The total DCM extract from E. zwageri and P. melagangai was 0.61% and 3.30%,

respectively. MeOH crude extracts from E. zwageri and P. melagangai were 8.37% and 4.81%, respectively.

For DCM crude extract of E. zwageri, the major compound identified were 1,2,3-trimethoxy-5-[(1E)-1-

propenyl]benzene (50.23%), 4-methoxy-6-(2-propenyl)-1,3-benzodioxole (30.59%), -panasinsen (21.35%)

and cadina-3,9-diene (20.41%). The major compound in DCM crude extract of P. melagangai are cadalene

(28.30%), n-dotriacontane (28.24%). The most frequent compound in E. zwageri and P. melagangai was -

muurolene, heneicosane and tetratetracontane. The major compounds identified in MeOH crude extract from

E. zwageri were tetratetracontane (40.06%), eicosane (10.28%), isoelemicin (33.37%), methyl elaidate

(17.76%) and heneicosane (5.61%). For MeOH crude extract from P. melagangai, the major compounds

were diisooctyl phthalate (37.07%) and 2,4-di-tert-butylphenol (71.23%). Both MeOH and DCM crude

extract from E. zwageri and P. melagangai were toxic to Trametes versicolor, Gloeophyllum trabeum and

Chaetomium globosum. Hexanedeconic acids, 2-4-di-ter-butylphenol, methyl hexadecanoate, methyl

octadeconate, -muurolene, -cadinol and myristicin might be responsible to the antifungal activities in E.

zwageri and P. melagangai extractives.

Keyword: Eusideroxylon zwageri, Potoxylon melagangai, extractives, antifungal activities, chemical

compounds

VI

ABSTRAK

Ketahanan dan kekuatan kayu adalah penting dalam industri pembinaan. Ketahanan Eusideroxylon zwageri

dan Potoxylon melagangai adalah sangat tinggi. Salah satu daripada sebab ketahanan semulajadi kayu yang

tinggi adalah kehadiran ekstraktif. Objektif kajian ini adalah pertamanya untuk menentukan jumlah ekstrak

diklorometana (DCM) dan metanol (MeOH) dari E. zwageri dan P. Melagangai, kedua untuk mengenal pasti

komposisi kimia daripada ekstrak DCM dan MeOH dan ketiga untuk menilai aktiviti antikulat ekstrak DCM

dan MeOH. Pengekstrakan menggunakan DCM dan MeOH sebagai pelarut telah dilakukan. Teknik gas

kromatografi-spektrometri jisim telah digunakan untuk mengenalpasti dan mencirikan bahan kimia dan

komposisi pecahan ekstrak DCM dan MeOH dari E. zwageri dan P. melagangai. Aktiviti antikulat bagi

ekstrak DCM dan MeOH menggunakan kaedah pencairan agar. Jumlah ekstrak DCM dari E. zwageri dan P.

melagangai masing-masingnya 0.61% dan 3.30%. Ekstrak MeOH dari E. zwageri dan P. melagangai

masing-masing, 8.37% dan 4.81%. Sebatian utama dalam ekstrak DCM E. zwageri adalah 1,2,3-trimethoxy-

5-[(1E)-1-propenyl] benzena (50.23%), 4-methoxy-6(2-propenyl)-1,3-benzodioxole (30.59%), α-panasinsen

(21.35%) dan cadina-3 ,9-diene (20.41%). Sebatian utama dalam ekstrak DCM P. melagangai adalah

cadalene (28.30%), n-dotriacontane (28.24%). Kandungan kimia yang terdapat pada kedua-dua E. zwageri

dan P. melagangai ialah γ-muurolene, heneicosane dan tetratetracontane. Ekstrak MeOH dari E. zwageri

mempunyai sebatian utama tetratetracontane (40.06%), eicosane (10.28%), heneicosane (5.61%),

isoelemicin (33.37%) dan metil elaidate (17.76%). Ekstrak MeOH dari P. melagangai mempunyai sebatian

utama iaitu diisooctyl phthalate (37.07%) dan 2,4-di-tert-butylphenol (71.23%). Kedua-dua ekstrak DCM

dan MeOH dari E. zwageri dan P. melagangai adalah toksik terhadap kulat Trametes versicolor,

Gloeophyllum trabeum dan Chaetomium globosum. Asid hexanedeconic, 2-4-di-ter-butylphenol, metil

hexadecanoate, octadeconate metil, γ-muurolene, α-cadinol dan myristicin mungkin bertanggungjawab

terhadap aktiviti antikulat dalam ekstraktif E. zwageri dan P. melagangai.

Kata kunci: Eusideroxylon zwageri, Potoxylon melagangai, ekstraktif, antikulat,sebatian kimia

VII

TABLE OF CONTENTS

Title page………………………………………………………….……………………….I

Approval Sheet…………………………………………………….……………………...II

Declaration…….……...……………………………………………………………….....III

Acknowledgements..…….…..…………………………………………………………...IV

Abstract…………………………………………………………………………………...V

Table of contents ……….………………………………..………………………….…..VII

List of abbreviations …………..………………………………………………………....IX

List of figures……..………………………………………………………………..……...X

List of tables …….…………………………………………..……………………...……XII

Chapter one: Introduction …………...…..…………………………………….…………..1

Chapter two: Literature review …………….……………………………………...………3

2.1 Characteristic and distribution of Eusideroxylon zwageri and

Potoxylon melagangai…………………………………………………….3

2.2 Physical properties of Eusideroxylon zwageri and

Potoxylon melagangai…………………………...……………….………..4

2.3 Mechanical properties……………………………………………………..5

2.4 Wood decay fungi………………………………………………....……....6

2.5 Extractive contents of durable wood……………….……………………...8

2.6 Chemical constituents of extractive…………………………………….….9

2.7 Toxicity of extractive……………………………………………...……...10

Chapter three: Materials and methods …………….....……………..…………………….12

3.1 Preparation of wood samples………………...………………...………....12

3.2 Extraction of wood meal….......………………………...……...………....12

3.3 Column chromatography fractionation........……………......…….….…...13

3.3.1 Crude dichloromethane extract……....…...…………………….…13

3.3.2 Fractionation of crude methanol extract…...………………….…..14

3.4 Derivatization of methanol extracts for gas

chromatograph-mass spectrometry (GC-MS) analysis…………….......…15

3.5 Gas chromatograph-mass spectrometry (GC-MS) analysis………....……15

3.6 Preparation of fungi inoculums………………………...……...………….16

3.7 Antifungal assay………………………………………………...………...16

VIII

3.8 Statistical analysis……………………………………………………...…17

Chapter four: Results and discussion ……………...………………….………………..…18

4.1 Crude dichloromethane and methanol extractives……………….………..18

4.2 Dichloromethane extracts fractions from Eusideroxylon

zwageri and Potoxylon melagangai….………………………….…….....19

4.2.1 Chemical compositions of dichloromethane

extract………………………………………..………….…….....19

4.2.2 Chemical constituents of dichloromethane extracts from

Eusideroxylon zwageri and Potoxylon melagangai....…...….…..19

4.2.3 Chemical constituents and compositions of fraction F1, F2,

and F3 of dichloromethane extracts from Eusideroxylon

zwageri and Potoxylon melagangai……………………………..24

4.3 Methanol extract fractions from Eusideroxylon zwageri and

Potoxylon melagangai …………………………………...………....…...29

4.3.1 Chemical compositions of methanol extract from

Eusideroxylon zwageri and Potoxylon melagangai …………......29

4.3.2 Chemical constituents of methanol extracts from

Eusideroxylon zwageri and Potoxylon melagangai …...….…..…30

4.3.3 Chemical constituents and compositions methanol extracts

from Eusideroxylon zwageri and Potoxylon melagangai .......…..35

4.4 Antifungal activities of dichloromethane crude extract from

E. zwageri and P. melagangai...……………….…………….……..…….43

4.5 Antifungal activities of methanol crude extract from

E. zwageri and P. melagangai…………….………………….….…….…49

Chapter five: Conclusions and recommendations.……..………………………..……….57

References …………………………………………………………………………….....60

Appendix ……………………………………………………………………………..….67

IX

List of Abbreviations

E. zwageri Eusideroxylon zwageri

P. melagangai Potoxylon melagangai

DCM dichloromethane

MeOH methanol

MEA Malt Extract Agar

mg/mL milligrams per milliliter

v/v volume per volume

% percent

degree Celcius

X

List of Figures

No. Description Page

Figure 1: Gas chromatogram of F1 fraction from dichloromethane

extract of E. zwageri 20


extract of P. melagangai 21





Figure 5: Gas chromatogram of F3fraction from crude dichloromethane


Figure 6: Gas chromatogram of F3 fraction from crude dichloromethane


Figure 7: Gas chromatogram for CF1-6 of methanol extract of

E. zwageri 30


P. melagangai 31


E. zwageri 31


P. melagangai 32


E. zwageri 33


P. melagangai 33

Figure 13: Gas chromatogram for CF19 of methanol extract of

E. zwageri 34

Figure 14: Gas chromatogram for CF19 of methanol extract of

P. melagangai 35

Figure 15: Antifungal index of different DCM crude extract

concentrations from E. zwageri and P. melagangai

against Trametes versicolor 44

XI



against Chaetomium globosum 44



against Gloeophylum trabeum 45

Figure 18: Growth of Trametes versicolor in media in different

concentrations of dichloromethane crude extract 46

Figure 19: Growth of Chaetomium globosum in media in different


Figure 20: Growth of Gloeophylum trabeum in media in different


Figure 21: Antifungal index of different MeOH crude extract


against Trametes versicolor 49



against Chaetomium globosum 50



against Gloeophylum trabeum 50

Figure 24: Growth of Trametes versicolor in media with different

concentration of methanol crude extract 51

Figure 25: Growth of Chaetomium globosum in media with

different concentration of methanol crude extract 53

Figure 26: Growth of Gleophylum trabeum in media with different

concentration of methanol crude extract 55

XII

List of Tables

No. Descriptions Page

Table 1: Solvent systems for silica gel column chromatograph 14

Table 2: Percentage of crude dichloromethane and methanol extract

from E. zwageri and P. melagangai 18

Table 3: Percentage fraction of dichloromethane extract from

E. zwageri and P. melagangai 19

Table 4: Chemical constituents and compositions F1, F2 and F3

fraction of dichloromethane crude extract from E. zwageri

and P. melagangai 26

Table 5: Combine fraction solvent system of crude methanol extract


Table 6: Chemical constituents and compositions of combined fractions

of methanol crude extract from E. zwageri and P. melagangai 38

Table 7: Antifungal index (%) of dichloromethane crude extract


Table 8: Antifungal index (%) of methanol crude extract from

E. zwageri and P. melagangai 67

1

CHAPTER ONE

INTRODUCTION

Antifungal compounds in wood can be extracted using different chemicals.

Dichloromethane is a semi-polar solvent that can extract semi-polar chemical compounds.

Dichloromethane solvent can extract aliphatic compounds, aromatic compounds, alicyclic

compounds, non-ionic polymers and lignin. While, methanol is a polar solvent, it usually

used to extract polar chemical compounds from wood. Polar compounds in wood include

alcohols, ketones, carboxylic acids, phenols, carbohydrates and fatty acids. Extractive

compounds have significant impact on properties of wood which are durability and

strength, odour and taste, inflammability, toxicity, density, economic value and factory

uses (Negi, 1997). Wood has exits extractive such as tannins, resins and gums and less than

1 % inorganic ash material. In addition, 97-99% of over-dry wood substances by weight

are cellulose, hemicellulose and lignin form the primary substances of wood (Smith et al.,

2003).

Since Eusideroxylon zwageri Teijsm. & Binnend and Potoxylon melagangai

Kosterm are very durable timbers due to their extraneous substances, thus, it is important

to identify the compounds responsible for the decay resistance in E. zwageri and P.

melagangai heartwood. Currently chemical wood preservatives are used to treat non-

durable timbers. Chemical-based preservatives lead to a number of environmental

concerns, thus the potential of natural wood preservatives as effective replacement has

gained interest of many researches. The discovery of these environmental-friendly

compounds may replace the role of toxic chemical, which currently used as wood

preservatives. The advantages of using natural wood preservatives to treat wood are, as

2

natural product they are usually harmless to human and environment. The potential

development of wood extractives as natural wood extractives not only important to provide

alternative treatment for wood preservation industry but also may be useful for therapeutic

and cosmetic industries.

Wood extractive consists of different kinds of chemical compounds. It is non-cell

wall component which are small molecules. It can be extracted by polar and non-polar

solvents. Wood with high amount of extractive is more resistant to decay (Zabel &

Morrell, 1992; Eaton & Hale, 1993; Schultz et al., 1995; Schultz & Nicholas, 2000; Ismail

& Ipor, 2003). Currently there are limited information on the chemical compounds from

Eusideroxylon zwageri and Potoxylon melagangai

The objectives of this study were firstly to determine the amount of

dichloromethane (DCM) and methanol (MeOH) crude extracts from Eusideroxylon

zwageri and Potoxylon melagangai. Secondly, to assess antifungal properties of DCM and

MeOH extracts. Thirdly, to identify the chemical constituents of DCM and MeOH extracts

from E. zwageri and P. melagangai.

3

CHAPTER TWO

LITERATURE REVIEW

2.1 Characteristic and distribution of Eusideroxylon zwageri and Potoxylon

melagangai

Eusideroxylon zwageri is an evergreen tree of up to 40m tall. The buttress is small,

rounded, have base an elephant-foot like appearance. Its bark surface is red or grey-brown

in colour with thin cracks. It is distributed in eastern and southern Sumatra, Bangka,

Belitung, Borneo and the Sulu archipelago and Palawan. E. zwageri belong to Lauraceae

family and can be found mainly in lowlands, particularly on low-lying alluvial areas. The

flower of E. zwageri is small and greenish yellow. The flowers have a nice smell. It is

bisexual flower. Sepal number is 6, distributed in two whorls, imbricate. The anthers are 4

loculed, dehishing by valves. The fruit is completely enclosed in and adnate to the flower

tube. The fruit shape is oval and drupe, not splitting. It is one seed per fruit. Seed is very

large, seed coat very hard, seedling with hypogeal germination. Seed are always destroyed

by porcupines. Leaves alternate and spiral arrangement. Young leaf is reddish colour and

mature leaf is green. It has small veins prominent, or not prominent but visible and ladder

like. The stipules were absent. Secondary vein are open and prominent. The terminal buds

not enclosed by leaves. The tree can be reach a height in 8 m in 8 years, but need over 100

years to reach a diameter of 50 cm. Thus, the growth is slow under optimal condition

(Kubitzki, 1993).

Potoxylon melagangai is an evergreen tree and can be found in primary lowland

forest, mainly in mixed dipterocarp forest. The tree can reached up to 25 m in height. P.

4

melagangai is also belong to Lauraceae family and can mainly found on the west coast

(Malaysia Timber Council [MTC], 2002). The buttresses are rounded and can reach up to

2cm high. Inner bark of P. melagangai is whitish colour. Angular young twigs. Axillary

inflorescence, bisexual, 3-merous, 6 tepals, superior ovary, capitates stigma. The fruit are

large, one seeded berry. Hypogeal germination of seed. Flowering is once every 3 to 6 year

in March, April or May. Seeds are always dispersal by porcupines and squirrels. Although

E. zwageri and P. melagangai have similarity of silviculture, but it grow faster which have

mean annual diameter at least 0.5 cm (Kostermans, 1978).

2.2 Physical properties of Eusideroxylon zwageri and Potoxylon melagangai

Eusideroxylon zwageri is classified as heavy hardwood. Its density range from 880

to 1190 kg/m3 at 15% moisture content (Kostermans et al., 1994). After prolonged

exposure, E. zwageri has heavier weight, slight coarse texture, absence of reddish tinge and

have shorter line of soft tissue between the pores. The sapwood is bright yellow when

freshly cut, darkening to yellowish-brown on exposure. The heartwood is yellowish-brown

to reddish-brown when freshly cut. Freshly cut wood have fresh, slight lemon-like odor.

The grain is straight, occasionally slightly interlocked, texture moderately coarse and even.

It dried slowly, although the moisture content of green wood is comparatively low about

38 %. The wood will split when it is too dry. This wood is not suitable for production of

pulp, paper or fibre board. Stakes showed an average service life in contact with ground of

17.5 years under tropical conditions when test with graveyard. In tropic country, it has

been found that stockades and posts last over 100 years. It can be used up to 20 years for

marine works (Kubitzki, 1993).

5

An endemic to Borneo, Potoxylon melagangai is a monotypic genus. It is a very

durable and its density range from 525 to 920 kg/m3 at 15% moisture content (Kostermans

et al., 1994; Teo, 1998). P. melagangai has lighter weight, rather fine texture, reddish

colour and longer bands of confluent parenchyma soft tissue linking up the pores (Browne,

1995; Burgess, 1966). When wood freshly cut, heartwood colour is brown with distinct

reddish tinge, sharply differentiated from yellowish sapwood (Kostermans, 1978). In

Sarawak, P. melagangai are banned from export.

2.3 Mechanical properties

Strength characteristic of timbers represent their fitness and ability to resist external

forces. Modulus of rupture (MOR) and modulus of elasticity (MOE) are used to determine

the strength properties of wood from static bending test. MOR is used to measure the

ultimate bending strength. MOE is used to determine the deflection of beam under load or

its stiffness. For example, Penyau, Resak and Selangan batu are highly rate in static

bending property. Red selangan, Resak and Selangan batu have high compressive strength

and hardness (Timber Research and Technical Training Centre [TRTTC], 1985). Hardness

is important properties to determine its application for rollers, mallets, flooring and

furniture. Specific gravity of wood is an index to predict the strength properties of wood.

Depend on species, relation between specific gravity and any one of the strength property.

The higher the specific gravity, the higher were the strength values. Besides, moisture

contents also influences strength. Air dry timbers have higher strength values than green

timbers. Thus, dry wood is stronger than wet wood.

6

2.4 Wood decay fungi

According to Bowyer et al. (2003), pit membranes between adjacent cells are easily

ruptured by fungal hyphae. In contrast, starch and primary metabolites provide main

supply of readily assimilable carbon sources for fungi and other microorganisms growing

in wood (Rayner & Boddy, 1988). The enzyme in the wood play a role to control the

synthesis activity and in most decay fungi such as cellulose enzyme is activated by

cellulose and cellobiose (Hulme & Stranks, 1972).

Wood decay fungi can be either rapid or slow in timber. Most decay fungi are

Basidiomycetes. The factor that encourages fungi to grow is suitable conditions which are

temperature range from 15 to 45 , pH range 3 to 6, atmospheric oxygen, nitrogen

compounds, vitamins and essential element (Zabel & Morrell, 1992). Three types of wood

decay fungi are white-rot, soft-rot and brown-rot fungi. White rot fungi will break down

lignin, hemicelluloses and cellulose causing wood lose colour and appear whiter than

normal. Infested wood will become spongy when touched. White rot fungi commonly

occurs in hardwood material. The example of white rot fungi are Trametes versicolor,

Schizophyllum commune, Pycnoporus sanguineus, Ganoderma spp. and Polyporus spp.

Soft rot is characterized as a soft, decayed surface of wood in contact with

excessive moisture. The causal soft rots fungal are Deuteromycetes and Ascomycetes. Soft

rot caused decays by removing of cellulose, hemicelluloses and slight modification of

lignin. It is major wood-destroying fungi attack wood in ground contact and also above

ground exterior. Soft-rot fungi grow slowly which attack outer layer of timber but overall

7

effect on strength is greater. It can tolerate a wide range of temperature, humidity and pH

conditions (Eaton & Hale, 1993; Eriksson et al., 1990).

Brow-rot fungi depolymerase cellulose rapidly during incipient stages of wood

colonization. The residual wood become brown and often cracks into cubical pieces when

dry. These fungi remove components of hemicelluloses and cellulose which functions in

strength characteristics resulting timber to collapses. The example of brown-rot fungi are

Gloeophyllum trabeum, Lentinus lepideus, Coniophora puteana and Tyronmyces palustris.

Saprobes are the group of fungi and bacteria that act as decomposers of organic

matter, dead tree or wood products. It will invade sapwood first and decompose more

decay–resistant heartwood of the wood product (Manion, 1991). The impact of wood

decay fungi are weight loss, strength loss, reduced standing timber volume, increased

permeability of wood to water, increased electrical conductivity of wood, changes in wood

volume, changes in pulping quality, discoloration of wood and reduced caloric value

(Manion, 1991). However, toxic compounds provided by extractive that are natural decay

resistance to saprobic fungi.

In this study, three types of fungi were used namely Trametes versicolor which is

white rot fungi, Gloeophyllum trabeum which is brown rot fungi and Chaetomium

globosum which is soft rot fungi. Trametes versicolor can decay wood and loss about 50%

of its original weight (Manion, 1991). White rot fungi mainly attack hardwood and

occasion also on softwoods (Eaton & Hale, 1993). Gloeophyllum trabeum fungi rapid

decay of both soft and hardwoods including natural durable softwood (Duncan &

8

Lombard, 1965). While, Chaetomium globosum which is prevalent in slow surface

deterioration of natural durable wood (Wong et al., 1992).

2.5 Extractive contents of durable wood

Natural durability or decay resistance is the ability of heartwood of wood species to

resist decay. The presence of toxic extractives in wood plays an important role in natural

durability of wood (Desch and Diwoodie, 1996). Sequoia, Sequoiadendron, Juniperus,

Cedrus, Cupressus and Eucalyptus have service life for 20 years or more, because they

have very durable heartwood (Rayner & Boddy, 1988). In areas of high decay risk,

heartwood of Tectona grandis and Ocotea rodiaei can last for decades (Eaton & Hale,

1993). Decay resistant heartwood reduced by extraction with hot water and organic solvent

(Syafii & Yoshimoto, 1993).

Hills (1966) found that polyphenols (or bezenoid types), terpenoids and tropolones,

fats and waxes, salt of organic acids, complex polysaccharides and nitrogenous compounds

are classes of extractives in wood. Polyphenols are most common extractives and most

widely used for chemotaxonomy.

Heartwood has higher extractives compare to sapwood (Sjostrom, 1993). The

percentages of extractive can be different from types of species, growth conditions, age of

the tree and locality. It can be high up to 40% but normally range between 2-10% of dry

wood weight (Fleischer et al., 1984; Fengel and Wegener, 1989; Sjostrom, 1993;

Uprichard, 1993; Walker, 1993; Roberts, 1996; Negi, 1997). The amount of extractable

9

heartwood extractive varies from the solvent that used for extraction (Gierlinger, 2003).

The colour of extractive can be yellow, red and brown and colourless.

2.6 Chemical constituents of extractive

A study by Mono et al. (2003) found that acetone extracts of Eusideroxylon

melagangai were mainly 1,2-benzendicarboxylic acid, epiisopodophyllotoxin, fatty acids,

aliphatic hydrocarbons, aromatic compounds, alkaloids, alcohol and ketone. E. zwageri

and P. melagangai acetone extracts were shown to contain 5-octadecene, 9-octadecene,

vanillin, 2,4-dimethoxy-5,6-dimethylbenzaldehyde, 4-tetradecanol, 5-allyl-1,2,3-

trimethoxybezene and 1-nitro-3,5-dimethoxyphenyl-ethylene (Faiezah, 2009). Wood has

different types of extractives to maintain the diversified biological function in plants.

Lignin are biosynthesized by combination of two molecular of phenyl propanoids (C6C3)

via the shikimic and acid pathway which is toxic to fungi, insects and vertebrates (MacRae

& Towers, 1984). Matairesinol, ferruginol and totarol are common lignins that have ability

to inhibit the growth of wood decay fungi (Rudman, 1965). Lignin of Taiwania

cryptomerioides has ferruginol compounds that are resistant against fungi (Chang et al.,

1999). Barz and Welding (1985) stated that stilbenes in many conifers have fungitoxic

properties in heartwood. Tropolones is a strong antifungal in wood substrate (Rudman,

1962; Syafii, 1996; Grohs & Kunz, 1998) and can be used as an indicator for decay

resistant of heartwood (DeBell et al., 1997). Geranyl linalool is a common diterpene that

occurs in high amounts in softwood species which resistant to insect and fungal attack

(Croteau & Johnson, 1985). Napthoquinone lapachol is quinine found in heartwood of

several plant species which have anti-tumor activity (Barz & Welding, 1985).

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Wood susceptible to decay and insect attacks found to have low terpenoids, resin

acids and phenolic substances. Phenolic extractives in wood have fungicidal properties and

protect tree against microbiological attack. Deacetylation of hemicelluloses followed by

depolymerization of polysaccharides catalyzed by the released of acetic acid is how wood

degradation occur (Tjeerdsma et al., 1998; Sivonen et al., 2002; Nuopponen et al., 2004).

Wheatley et al. (1997) identified five specific volatiles produced by Trichoderma which

were 2-propanone, 2-methyl-1-butanol, decanal, heptanal, and octanal that cause inhibition

of basidiomycete target. Picea abies contains high amount of hydroxymatairesinol and

matairesinol which inhibit the growth of Fomes annosus fungus (Shain & Hillis, 1971).

Gmelina arborea has high antifungal activity against Trametes versicolor of EtOAc-

solubles (Kawamura et al., 2004).

2.7 Toxicity of extractive

Toxicity is important in order to protect the wood from biodegradation. Sjostrom

(1993) stated that role of phenolic compound in wood is to protect wood from microbes

attack. The ability of microb and insect to attack wood is depending on the quantity and

toxicity of wood extractive (Darham & Gray, 1994). Hexadecanoic acid prevents

pathogens from entering the plants (Higuchi, 1985). Quinones in Dalbegia exhibit

microbiocidal and algicidal activity (Manners & Jurd, 1977). Quinones are resistance to

teredos, limnonia and other marine borers. The quinonoid extractives of Cordia alliodora

are responsible for the durability in marine use (Moir & Thomson, 1985). Diospyros

timber has the source of binaphtoquinoners that responsible for antifungal, antibacterial,

and termite-resistant properties (Waterman & Mbi, 1985). Family of Bignoniaceae with

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genus belong to Kigelia (Inoue et al., 1981), Markhamia (Singh & Singh, 1980), Tabebuia

(Burnett & Thomson, 1967; Manners et al., 1975), Zeyhera (Weinberg, Gottlieb &

Oliveira, 1976) and Paratecoma (Sandermann et al., 1968) have quinines constituents in

wood which are responsible for antifungal activity (Singh & Singh, 1980) and resistance

against teredos (Sandermann et al., 1968). Gmelinol which are rich in heartwood of

Gmelina arborea appeared to be an important antifungal constituent (Kawamura et al.,

2004). Tropolone and related monoterpenes in Curpressaceae heartwood are resistant to

insect attack thus play an important role in durability (Smith et al., 2003). Tallowwood is

the most durable Australian timber which also has high resistance to decay due to toxic

extractives (Da Costa & Rudman, 1958).

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