EXTRACTS OF CERBERA ODOLLAM FOR ANTI BACTERIA ACTIVITY AND EVALUATION ON SOME
WOOD PRODUCT PROPERTIES AFTER IMPREGNATION
MOHD HAZIM BIN MOHAMAD AMINI
UNIVERSITI SAINS MALAYSIA
2009
MOHD HAZIM BIN MOHAMAD AMINI 2009 MSc
EXTRACTS OF CERBERA ODOLLAM FOR ANTI BACTERIA ACTIVITY AND EVALUATION ON SOME WOOD PRODUCT PROPERTIES AFTER
IMPREGNATION
by
MOHD HAZIM BIN MOHAMAD AMINI
Thesis submitted in fulfillment of requirements for the degree of
Master of Science October 2009
PENILAIAN TERHADAP AKTIVITI ANTI-BAKTERIA UNTUK EKSTRAK DARIPADA CERBERA ODOLLAM DAN PENILAIAN TERHADAP BEBERAPA
CIRI-CIRI PRODUK KAYU SELEPAS DIIMPREG DENGAN EKSTRAK TERSEBUT
oleh
MOHD HAZIM BIN MOHAMAD AMINI
Tesis yang diserahkan untuk memenuhi keperluan bagi
Ijazah Sarjana Sains
Oktober 2009
ii
ACKNOWLEDGEMENT
I would like to acknowledge to Ministry of Science, Technology and Innovation, Malaysia (MOSTI) for the Science Fund Grant 03-01-05SF0117 and Universiti Sains Malaysia for Fellowship Scheme awarded to me in order for me to complete this research. I would like to address my appreciation to Associate Professor Dr Rokiah Hashim as my supervisor for her helping hands and guidance, also thanks to Associate Professor Dr Othman Sulaiman, Dr Shaida Fariza Sulaiman from School of Biological Sciences of Universiti Sains Malaysia, Associate Professor Dr Faizah Abood from Universiti Putra Malaysia, Dr Fumio Kawamura from Japan International Research center for Agricultural Sciences (JIRCAS) and staff of Bio-resource, Paper and Coatings Technology Division, School of Industrial Technology, Universiti Sains Malaysia. Finally thanks to my parents for giving morale support to pursue my study.
iii
TABLE OF CONTENTS
Contents Page
Acknowledgement………
List of tables, figures and plates………
List of symbols and abbreviations……….
Abstrak………...
Abstract………..
ii viii xiii xvi xvi 1.0 Introduction………..
1.1 Problems………...
1.2 Objectives……….
1 1 4 2.0 Evaluation of antibacterial activity of Cerbera odollam ………
2.1 Introduction………..
2.2 Literature review ……….
2.2.1 Cerbera odollam ………..
2.2.2 Wood extractives ……….
2.2.3 Bacteria ………
2.2.3 (a) Antibacterial agents ………
2.2.3 (b) Bacteria association with wood decay………
2.3 Materials and methods ………
2.3.1 Sample preparation ………..
2.3.2 Extraction for antibacterial assay ……….
2.3.3 Separation (or fractionation) process ………...
2.3.4 Moisture content determination……….
2.3.5 Agar preparation ………
5 5 5 5 11 11 12 13 18 18 20 23 24 25
iv
2.3.6 Culturing bacteria ………..
2.3.7 Antibacterial activity test ………...
2.3.8 Chemical analysis ………
2.3.8 (a) Fourier Transform Infrared
Spectrophotometer (FT-IR) of extractives ………….
2.3.3 (b) Gas chromatography time of flight mass spectrometer analysis (GC-TOFMS) of extractives showing antibacterial activity ………...
2.4 Results and discussions ………...
2.4.1 Moisture content, yield of extraction and fractions …….
2.4.2 Antibacterial assay ………...
2.4.3 Fourier Transform Infrared Spectrophotometer (FT-IR) of extractives ……….
2.4.4 Gas chromatography time of flight mass spectrometer analysis (GC-TOFMS) of extractives showing antibacterial activity ………..
2.5 Summary and conclusions ……….
25 26 27
27
28 29 29 30 35
37
40 3.0 Influence of impregnation of extracts from Cerbera odollam on some of particleboard properties ………
3.1 Introduction ………
3.2 Literature review ………
3.2.1 Wood preservation - Wood structure and its relation to wood preservation ………
3.2.2 Purpose of wood preservation ………..
3.2.3 Types of wood preservatives ………
41 41 41
41 42 43
v
3.2.3 (a) Tar oil preservatives ………...
3.2.3 (b) Waterborne preservatives ………...
3.2.3 (1b) Fixed multisalt and multioxide preservatives ………..
3.2.3 (2b) Boron-based preservative ………….
3.2.3 (3b) Emulsion preservatives ……….
3.2.3 (c) Organic solvent preservatives ……….
3.2.4 Natural compound as wood preservative ……….
3.2.5 Wood-boring insects ………
3.2.5 (a) Minthea rugicollis (Coleoptera: Lyctidae) ….
3.2.5 (b) Controlling damage by wood boring beetles ..
3.2.6 Particleboard ………....
3.2.6 (a) Raw material (rubber wood) ………..
3.2.6 (b) Adhesives ………...
3.2.6 (1b) Melamine urea formaldehyde …….
3.2.6 (2b) Phenol resorcinol formaldehyde ….
3.2.6 (c) Additives ………
3.3 Materials and methods ………
3.3.1 Extraction of different parts of Cerbera odollam ...
3.3.2 Determination of pH values of the extractives and adhesives ...
3.3.3 Determination of solid content of adhesive...
3.3.4 Particleboard making ...
3.3.4 (a) Preparation of woodchip ………..
3.3.4 (b) Board making ...
43 44
44 44 45 45 45 47 48 50 50 52 53 53 55 56 57 57
57 57 58 59 59
vi
3.3.5 Impregnation of extractives into solid wood ...
3.3.6 Investigation on the effect of borer decaying activity on particleboards and solid wood treated with the extractives of different parts of Cerbera odollam ………..
3.3.7 Soil burial test on wood products treated with the extractives of different parts of Cerbera odollam ………
3.3.8 Determination of formaldehyde release from particleboards treated with the extractives from different biomass components of Cerbera odollam ………
3.3.8 (a)Determination of moisture content ………….
3.3.8 (b) Determination of formaldehyde concentration of the aqueous solution ……….
3.3.8 (c) Calculation of flask value (Fv) ………..
3.3.9 Determination of thickness swelling ………
3.3.10 Internal bond testing on particle boards treated with extractives from different parts of Cerbera odollam …………
3.4 Results and discussions ………...
3.4.1 pH values of the methanolic extractivesand adhesives ...
3.4.2 Solid content of the adhesives ...
3.4.3 Wood boring insect decaying activity ……….
3.4.4 Soil burial test ………..
3.4.5 Formaldehyde release ………..
3.4.6 Moisture content and thickness swelling ………
3.4.7 Internal bond strength ……….
3.5 Summary and conclusions ……….
60
61
62
65 66
66 67 69 70 72 72 72 73 75 76 78 79 81
vii
3.6 Future work ……… 82
References ……… 83
Appendices ………..
Appendix A: Calculation for board making process ………
Appendix B: FTIR spectra for each fraction ………
Appendix C: IR bands of extracts of Cerbera odollam after extraction Appendix C: Anova analysis for all testing ………
93 93 96 98 135 List of publications ………
Investigation on antimicrobial activity of biomass components of Cerbera odollam (Paper presented at International Conference On Environmental Research And Technology (ICERT 08) 28-30 May 2008, Park Royal Hotel, Penang. ISBN 978-983-3986-29-3 …………
Anti-bacterial activity of extracts from different parts of Cerbera odollam - Paper submitted to Annals of Microbiology ………...
178
179
180 Anti-microbial and chemical investigation of Peltophorum
vogelianum (Benth.) - Paper submitted to Journal of Medicinal Plant
Research ……… 181
viii
LIST OF TABLES Page
Table 2.1
Table 2.2
Table 2.3
Table 2.4
Table 2.5 Table 3.1 Table 3.2
Table 3.3 Table 3.4 Table 3.5 Table 3.6
Table 3.7
Table 3.8
Table 3.9
Moisture content, yield from methanol extraction and hot water extraction after methanol extraction of the different parts of Cerbera odollam
Yield of extracts after fractionation from methanol extract of the different parts of Cerbera odollam
Effect of extracts after fractionation from methanol on the growth of Bacillus subtilis and Bacillus licheniformis Summary of FT-IR bands of fractions with antibacterial activity
GC-MS analysis of compound with antibacterial activity Resins and extractives used in board making process pH values of extratives from different parts of Cerbera odollam
Solid content for adhesives used Wood-boring beetles exposure test Soil burial test results
Formaldehyde release from particleboards impregnated with extracts of Cerbera odollam
Moisture content of particleboards impregnated with extracts of Cerbera odollam
Thickness swelling of particleboards impregnated with extracts of Cerbera odollam
Internal bond strength of particleboards impregnated with extracts of Cerbera odollam
29
30
31
36
38 58 72
72 74 75 77
78
79
80
ix Table 4.1
Table 4.2
Table 4.3
Table 4.4
Table 4.5
Table 4.6
Table 4.7
Table 4.8
Table 4.9
Table 4.10
Table 4.11
Table 4.12
Table 4.13
FT-IR bands of hexane soluble parts from fruit of Cerbera odollam
FT-IR bands of ethyl acetate soluble parts from fruit of Cerbera odollam
FT-IR bands of ethanol soluble parts from fruit of Cerbera odollam
FT-IR bands of hot water soluble parts from fruit of Cerbera odollam
FT-IR bands of hexane soluble parts from seed of Cerbera odollam
FT-IR bands of ethyl acetate soluble parts from seed of Cerbera odollam
FT-IR bands of ethanol soluble parts from seed of Cerbera odollam
FT-IR bands of hot water soluble parts from seed of Cerbera odollam
FT-IR bands of hexane soluble parts from leaf of Cerbera odollam
FT-IR bands of ethyl acetate soluble parts from leaf of Cerbera odollam
FT-IR bands of ethanol soluble parts from leaf of Cerbera odollam
FT-IR bands of hot water soluble parts from leaf of Cerbera odollam
FT-IR bands of hexane soluble parts from wood of Cerbera
98
100
102
104
105
107
109
111
112
114
115
116
117
x Table 4.14
Table 4.15
Table 4.16
Table 4.17
Table 4.18
Table 4.19
Table 4.20
Table 4.21
Table 4.22
Table 4.23
Table 4.24
odollam
FT-IR bands of ethyl acetate soluble parts from wood of Cerbera odollam
FT-IR bands of ethanol soluble parts from wood of Cerbera odollam
FT-IR bands of hot water soluble parts from wood of Cerbera odollam
FT-IR bands of hexane soluble parts from bark of Cerbera odollam
FT-IR bands of ethyl acetate soluble parts from bark of Cerbera odollam
FT-IR bands of ethanol soluble parts from bark of Cerbera odollam
FT-IR bands of hot water soluble parts from bark of Cerbera odollam
FT-IR bands of hexane soluble parts from flower of Cerbera odollam
FT-IR bands of ethyl acetate soluble parts from flower of Cerbera odollam
FT-IR bands of ethanol soluble parts from flower of Cerbera odollam
FT-IR bands of hot water soluble parts from flower of Cerbera odollam
119
120
121
123
125
126
127
128
131
133
134
xi
LIST OF FIGURES Page
Figure 2.1 Figure 2.2
Figure 2.3
Figure 2.4
Figure 2.5 Figure 2.6 Figure 2.7 Figure 2.8 Figure 2.9 Figure 2.10 Figure 2.11 Figure 2.12 Figure 2.13 Figure 2.14 Figure 2.15 Figure 2.16
Figure 2.17 Figure 2.18
The Cerbera odollam tree
Cross section of young fruit of Cerbera odollam (left) and the mature fruit (center) and dried fruit (right).
Number of fatalities from Cerbera odollam and Thevetia peruviana reported from 1989 to 1999 in the state of Kerala, India.
Coccus (left), bacillus (center) and vibrio (right) shape of bacteria
Pit structure of wood Antibacterial assay
Biomass components of Cerbera odollam
Extraction and fractionation procedures of samples.
Extraction process Separation process
Disc placement on Petri dish
The inhibition zone of hexane extract from leaf The inhibition zone of hexane extract from bark
The inhibition zone for ethyl acetate extract from wood The inhibition zone of hexane extract from the leaf The inhibition zones of hexane extract from bark , ethyl acetate extract from bark and hexane extract from flower The inhibition zone of hexane extract from wood
A very small inhibition zone of hexane extract from fruit.
6 7
8
12
15 18 19 21 22 24 26 32 33 33 33 34
34 34
xii Figure 3.1
Figure 3.2
Figure 3.3 Figure 3.4
Figure 3.5 Figure 3.6 Figure 3.7 Figure 3.8 Figure 3.9 Figure 3.10 Figure 3.11 Figure 3.12 Figure 3.13 Figure 3.14 Figure 3.15 Figure 3.16 Figure 4.1
Figure 4.2
Figure 4.3
Minthea rugicollis
Ventral view of the fifth abdominal segment of male and female Minthea rugicollis.
Examples of particleboards made from rubberwood particles.
Melamine (1), urea (2) and formaldehyde (3) structure with possible structure formed by reaction of melamine and formaldehyde (4) and between urea and formaldehyde (5) and melamine urea formaldehyde structure (6).
Chemical structures of resorcinol formaldehyde (Choi, 2000) Minthea rugicollis
Placement of samples inside the testing chamber Example of test stakes
Soil burial test (graveyard test) Formaldehyde test sample placement
Apparatus set up for formaldehyde release determination Test pieces for testing
Fitting metal blocks into metal grip on a testing machine Decayed samples
Some decayed samples after soil burial test Calibration curve for formaldehyde test
Infrared spectral for hexane soluble parts of different parts of Cerbera odollam
Infrared spectral for ethyl acetate soluble parts of different parts of Cerbera odollam
Infrared spectral for ethanol soluble parts of different parts of 49 49
51 54
55 61 62 63 64 66 67 70 71 74 76 76 96
96
97
xiii Figure 4.4
Cerbera odollam
Infrared spectral for hot water soluble parts of different parts of Cerbera odollam
97
LIST OF SYMBOLS AND ABBREVIATIONS
MUF – melamine-urea formaldehyde PRF – phenol-resorcinol formaldehyde AD – air dry weight
OD – oven dry weight DMSO – Dimethylsulfoxide
xiv
EKSTRAK CERBERA ODOLLAM UNTUK AKTIVITI ANTI BAKTERIA DAN PENILAIAN TERHADAP SEBAHAGIAN SIFAT PRODUK KAYU
SELEPAS IMPREGNASI
ABSTRAK
Projek ini mengkaji potensi kegunaan bagi Cerbera odollam. Keseluruhan kerja telah dibahagi kepada dua bahagian. Bahagian pertama ialah kajian tentang aktiviti antibakteria oleh ekstrak daripada bahagian berbeza Cerbera odollam.
Bahagian kedua mengkaji ketahanan terhadap perosak, sifat – sifat fizikal dan mekanikal bod partikel yang diperbuat daripada partikel kayu yang telah dirawat dengan ekstrak metanolik. Sampel kayu padu dan sampel yang diimpreg dengan pengawet komersial juga diuji sebagai perbandingan.
Bagi kajian tentang aktiviti antibakteria, bahagian – bahagian pokok Cerbera odollam telah diekstrak dengan methanol. Ini diikuti pemecahan ekstrak kepada bahagian – bahagian yang larut n-heksana, etil asetat dan etanol. Aktiviti antibakteria pecahan ekstrak telah diuji terhadap kedua – dua bakteria jenis Gram-positif dan Gram-negatif iaitu dari spesis Bacillus subtilis, Bacillus licheniformis, Escherichia coli dan Pseudomonas aeruginosa. Bahagian larut n-heksana daripada bunga, buah, daun, kayu dan kulit serta bahagian larut etil asetat daripada kulit menunjukkan aktiviti antibakteria terhadap Bacillus subtilis. Bahagian larut n-heksana daripada daun dan kulit serta bahagian larut etil asetat daripada kayu menunjukkan aktiviti antibakteria terhadap Bacillus licheniformis. Semua ekstrak dan pecahan ekstrak tidak menunjukkan sebarang aktiviti antibakteria terhadap Escherichia coli dan
xv
Pseudomonas aeruginosa. Ekstrak yang menghasilkan zon perencatan telah dianalisis menggunakan FTIR dan GC-TOFMS dan kumpulan kimia atau sebatian kimia yang berkemungkinan merupakan sebatian aktif telah dikesan. 1 – heksadekanol, 1 – undekanol, asid 9,12,15-oktadekatrienoik (linolenik), 1-undekanol dan 1-tridekanol adalah antara sebatian yang berkemungkinan adalah sebatian aktif untuk aktiviti antibakteria merujuk kepada kajian pengkaji terdahulu.
Ketahanan bod partikel dan sampel kayu padu yang telah diimpreg dengan ekstrak Cerbera odollam juga dikaji. Bahagian – bahagian Cerbera odollam telah diekstrak dengan metanol, dituras dan dikeringkan dengan mesin penyejat putaran.
Ekstrak metanol yang diperolehi telah diimpreg ke dalam partikel kayu. Sampel kemudiannya didedahkan kepada kumbang perosak kayu dan juga ujian tanaman dalam tanah untuk melihat ketahanannya. Ujian kandungan formaldehid terbebas, ujian ikatan dalaman dan ujian pembengkakan dalam air juga dijalankan terhadap bod partikel. Keputusan eksperimen menunjukkan sampel kawalan bagi kayu padu menunjukkan kerosakan paling tinggi bagi ujian ketahanan kumbang perosak iaitu 12.47%. Mengimpreg ekstraktif ke dalam bod partikel mengurangkan sedikit kekuatan ikatan dalaman bod tetapi meningkatkan ketahanan terhadap serangan biologi semulajadi dalam ujian tanaman tanah. Keputusan eksperimen juga menunjukkan bod partikel yang dihasilkan menggunakan resin Melamin urea formaldehid dan diimpreg dengan sebarang ekstraktif melepasi piawaian P3 dan P4 oleh EN 312:2003 manakala bod partikel yang dihasilkan menggunakan Fenol resorsinol formaldehid hanya melepasi piawaian P4 dalam standard yang sama.
xvi
EXTRACTS OF CERBERA ODOLLAM FOR ANTI BACTERIA ACTIVITY AND EVALUATION ON SOME WOOD PRODUCT PROPERTIES AFTER
IMPREGNATION
ABSTRACT
This study investigated the potential use of Cerbera odollam. The works carried out were divided into two parts. The first part is investigating the antibacterial activity of extracts from different parts of Cerbera odollam. The second part of the study investigated the decay resistance, physical and mechanical properties of particleboards made from wood particles that were impregnated with methanolic extracts of Cerbera odollam. Solid wood samples and samples treated with commercial preservative were also made as comparison.
In order to investigate the antibacterial activity, methanol extracts from different part of Cerbera odollam was prepared. Then they were further fractionated using n-hexane, ethyl acetate and ethanol. Obtained fractions were tested against both Gram-positive and Gram-negative bacteria of Bacillus subtilis, Bacillus licheniformis, Escherichia coli and Pseudomonas aeruginosa for their antibacterial activity. n-Hexane soluble parts from flower, fruit, leaf, wood, bark and ethyl acetate soluble part from bark showed antibacterial activity against Bacillus subtilis. n- Hexane soluble parts from leaf and bark and ethyl acetate soluble parts from wood showed antibacterial activity against Bacillus licheniformis. All of the extracts and fractions showed no antibacterial activity against Escherichia coli and Pseudomonas aeruginosa. Extracts showing inhibition zones were analyzed using FTIR and GC-
xvii
TOFMS and possible active compounds were detected. 1 – hexadecanol, 1 – undecanol, 9,12,15-octadecatrienoic (linolenic) acid, 1-undecanol and 1-tridecanol were among the possible active compounds for antibacterial activity determined by reviewing the work of previous researchers.
Cerbera odollam-impregnated particleboards and solid wood samples were prepared. Cerbera odollam were extracted using methanol, filtered and dried using rotary evaporator for storage. Methanolic extracts that were obtained were impregnated into wood particles. Samples were tested by exposure to wood-boring beetles and also undergone soil burial test to see their decay resistance.
Particleboards were also tested for their formaldehyde release content, internal bonding and thickness swelling properties. Wood-boring beetles resistance test showed that control sample of solid wood have the highest degradation with 12.47%
decay. The particleboards were less susceptible to the attack mainly because the adhesives used in board making process do have some toxic effect on the insects.
Impregnation of extractives into the particleboards only slightly reduced their internal bonding strength but increased their resistance to natural biological attack in soil burial test. The results showed that thickness swelling and internal bond strength for Melamine urea formaldehyde particleboards impregnated with any extracts passed the requirement for both P3 and P4 of EN 312:2003 while Phenol resorcinol formaldehyde particleboards only passed P4 of the standard.
1 1.0 Introduction
There are many tree species that are very poisonous to living organisms such as Atractylis gummifera (bird-lime or blue thistle), Blighia sapida (ackee tree), Thevetia peruviana (yellow oleander), Colchicum autumnale (meadow saffron), Oenanthe crocata (hemlock water dropwort), Taxus baccata (English or common yew), Narcissus pseudonarcissus (daffodil), Cerbera manghas (pink-eyed cerbera or sea mango) and Cerbera odollam (yellow-eyed cerbera). These trees could cause death to human being by ingestion (Eddleston and Persson, 2003; Gaillard et al., 2004). Some of the trees usually planted intentionally along the road side or park as shade tree. The potential use of these tree species should be explored especially as disinfectant and preservative as they could be obtained abundantly.
1.1 Problems
Cellulose, hemicelluloses and lignin are the materials that builds up a polymeric material called wood. Wood is widely used as furniture and other structural usage mainly because of its renewable factor. Compared to other sources like metal, wood has the advantage that it can be obtained over and over again by reforestation of the logged areas. Versatility, durability and aesthetical value of wood besides its excellent strength properties and workability make it the most popular building material in the world. With many encouraging properties such as low density, low thermal expansion and renewable, it is extensively used for indoor and outdoor structural purposes (Pandey, 1999; Williams, 2004).
2
The main problem of wood for building material is it is a biodegradable material and Schultz (2002) reported that wood can be degraded by many types of organisms such as fungi and insects. Lignin always acts as physical barrier to enzymatic decomposition of cellulose and hemicelluloses in wood but this barrier can be destroyed mechanically by insects and marine borers, biochemically by white- and soft-rot fungi, and possibly by small non-enzyme catalysts in the case of brown- rot fungi (Kirk and Cowling, 1984). Fungi causes serious strength loss while strength loss due to insect attack is proportional to the amount of wood removed. Bacteria also play a role in wood degradation where they affect wood permeability, attack wood structure, or work synergistically with other bacteria and soft-rot fungi to predispose wood to fungal attack (Clausen, 1995).
Wood needed to be preserved to prolong their shelf life. The preservatives that are used to protect wood from biological attack could be a problem to the environment. Wood preservatives can be described as pesticides that protect wood from fungi, insects and other biological agent attack. These dangerous materials are badly affecting human health and the environment and consideration in making decision to use or not to use these preservatives had to be made wisely. Copper chrome arsenic (CCA) had been long widely used as wood preservative to prevent decay by fungi and insects (Yamamoto and Hong, 1988; Kartal and Clausen, 2001).
In theory, CCA can be considered as safe because the chemicals are believed to bind strongly to the wood and do not leach out. However in practise, many studies showed that the chemicals do leach out and can gather on the ground underneath or transferred by direct hand contact. Improper disposal of treated wood will also create problem. Burning CCA-treated wood products down will release a large amount of
3
arsenic into the air which is very hazardous to health. Direct or indirect breathing or ingestion of wood ash from burnt CCA-treated wood has caused fatalities in animals and serious poisonings in humans. Besides that there is a possibility that the harmful preservatives are absorbed into the soil and come contact with underground water source and affecting people who use it. More environmental friendly agent should be developed to replace the conventional preservatives (Dickey, 2008).
Earlier researches have shown that without the use of artificial chemicals, biologically resistant boards still could be produced. Extractives from certain tropical hardwood timber species had shown potential use in wood protection. Different from artificial wood preservatives, organic based preservative obtained from natural source is believed easier to be detoxify by normal biological processes (Onuorah, 2000).
New types of wood preservative that is less harmful to the environment need to be developed. This study investigates the potential use of extracts of Cerbera odollam. No study has yet been conducted on the suitability of extracts from different parts of Cerbera odollam as antibacterial agent and wood preservative.
4 1.2 Objectives
The objectives of this study are:
a) To study the antibacterial activity of extracts of Cerbera odollam.
b) To evaluate the chemical composition of Cerbera odollam after extraction process.
c) To study the influence of the extracts on the properties of particleboards and solid wood.
5 CHAPTER 2
2.0 Evaluation of antibacterial activity of extracts of Cerbera odollam 2.1 Introduction
This experiment investigated the antibacterial activity of Cerbera odollam.
Extractives from the different parts of biomass components of Cerbera odollam were exposed to bacteria to determine its antimicrobial activity. Four types of bacteria were used in this study that includes Bacillus subtilis (ATCC 21332), Bacillus licheniformis (ATCC 14580), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853).
2.2 Literature review 2.2.1 Cerbera odollam
Grouped under kingdom of Plantae, Division of Magnoliophyta, Class of Magnoliopsida and Order of Gentianales, the Cerbera odollam tree is also known as othalanga maram in the Malayalam language used in Kerala, kattu arali in the adjacent state of Tamil Nadu, famentana, kisopo, samanta or tangena in Madagascar and pong-pong, buta-buta or nyan in southeast Asia (Gaillard et. al., 2004;
Wikipedia, 2008). Belongs to the family of Apocynaceae, and originated from coastal area in India and south-east Asia (Chen and Steldt, 1942; Laphookhieo et. al., 2004). Reaching a height of around 8 to 15 meters, it grows wild in mangrove swamps and widely grown in park and gardens as shade tree. The latex from this tree may cause blindness and the fruit can results in death if eaten. This tree can be
described as small sized tree with a rounded crown ( the Cerbera odollam
The fruits are outer layer of the fruit
a seed inside as shown in Figure 2.2 smooth and green when y
The seed contains non
use it for lighting, as a cosmetic or mixed with other oils as an insecti
repellent. In India the latex is known for its emetic, purgative and irritant effects (Gaillard et. al., 2004)
6
described as small sized tree with a rounded crown (Tung, 2005). Figure 2.1 tree.
Figure 2.1: The Cerbera odollam tree
The fruits are like apples or mangos. When dropped onto the ground, the outer layer of the fruit peeled off, exposing the thick fibrous inner layer that protects
as shown in Figure 2.2. The fruits of Cerbera odollam
h and green when young. It ripens to dull red when ripe and black when rotten.
The seed contains non-siccative oil and are widely used as rat poison use it for lighting, as a cosmetic or mixed with other oils as an insecti
n India the latex is known for its emetic, purgative and irritant effects (Gaillard et. al., 2004).
Tung, 2005). Figure 2.1 shows
like apples or mangos. When dropped onto the ground, the peeled off, exposing the thick fibrous inner layer that protects erbera odollam are large, round, to dull red when ripe and black when rotten.
siccative oil and are widely used as rat poison. In Burma, they use it for lighting, as a cosmetic or mixed with other oils as an insecticide or insect-
n India the latex is known for its emetic, purgative and irritant effects
Figure 2.2: Cross section of young fruit of fruit (center) and dried fruit (right).
The leaves are shiny with dark green colour. The
elliptical and spirally arranged along the branches. The flowers are bisexual white in colour with a diameter of around 5 cm
and releasing pleasant fragrance.
Cerbera manghas. The differences between these two species are the leaves of Cerbera manghas are smaller and glossier. The flower of
smaller with orange pink eye, late
Gaillard et. al (2004)
of plant poisoning cases and 10% of all poisoning cases in Kerala, India. The number of recorded fatalities from 1989 to 1999 from
compared to 163 by yellow oleander in the same state. The year 1989 to 1999 is shown
7
Figure 2.2: Cross section of young fruit of Cerbera odollam (left) and the mature fruit (center) and dried fruit (right).
The leaves are shiny with dark green colour. The leaf
elliptical and spirally arranged along the branches. The flowers are bisexual white in colour with a diameter of around 5 cm. The flowers occur in small cluster
nt fragrance. The nearest tree similar to Cerbera odollam . The differences between these two species are the leaves of
are smaller and glossier. The flower of Cerbera manghas smaller with orange pink eye, later turn reddish pink (Tung, 2005)
al (2004), the Cerbera odollam tree is responsible for about 50%
of plant poisoning cases and 10% of all poisoning cases in Kerala, India. The number fatalities from 1989 to 1999 from Cerbera odollam
compared to 163 by yellow oleander in the same state. The number of fatalities from is shown in the Figure 2.3.
(left) and the mature
leaf shape is simple, elliptical and spirally arranged along the branches. The flowers are bisexual and . The flowers occur in small cluster erbera odollam is the . The differences between these two species are the leaves of erbera manghas is also (Tung, 2005).
tree is responsible for about 50%
of plant poisoning cases and 10% of all poisoning cases in Kerala, India. The number odollam poisoning is 537 number of fatalities from
8
Figure 2.3: Number of fatalities from Cerbera odollam and Thevetia peruviana reported from 1989 to 1999 in the state of Kerala, India.
The ease of availability of the tree is determined as the main factor for choosing it as poison for suicide or homicide. People take the white fleshy kernel out from the fibrous husk of the seed and mash it before consuming it as a sweet to commit suicide. A few kernels are mixed with food containing plenty of chillies to cover the bitter taste of the poison for homicide. Death is likely to occur 3 to 6 hours after ingestion (Gaillard et. al., 2004).
Chemical properties of some parts of the tree had been already done by several previous researchers. Laphookhieo et. al (2004) has study on cytotoxic cardenolide glycoside from the seeds of Cerbera odollam. They have isolated a cardenolide glycoside, 3β-O-(2’-O-acetyl-l- thevetosyl)-15(148)-abeo-5β-(8R)-14- oxo-card-20(22)-enolide (2’-O-acetyl cerleaside A), from a methylene chloride extract of the seeds of Cerbera odollam, besides four known compounds that are cerleaside A, 17α-neriifolin, 17β- neriifolin and cerberin. The compound was
0 20 40 60 80 100 120
1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999
Number of fatalities reported
Year
Cerbera odollam Thevetia peruviana
9
obtained in white solid with molecular formula of C32H46O9. All compounds except cerleaside A showed cytotoxic activities against oral human epidermoid carcinoma, human breast cancer cell and human small cell lung cancer (NCI-H187).
Chen and Steldt (1942) have isolated cerberin and other active substances that may be present in the kernel of the nuts, and conducted various pharmacological experiments using it. Stock solutions of 0.1% cerberin in 38% alcohol by volume and 0.2% cerberoside in 19% alcohol were prepared for animal experiments. When tested on frogs, adequate doses of whether cerberin or cerberoside injected into the frog’s lymph sac, typical systolic arrest of the ventricle could be easily observed at the end of an hour. By perfusion into the inferior vena cava of the frogs, cerberin in the concentrations of 1:250 000 and 1:125 000, induced systolic standstill within 40 minutes, preceded by slowing of the heart rate and later A-V block. A concentration of 1: 500 000 produced a decrease of diastole and sinus rhythm, and appearance of premature beats.
Chen and Steldt (1942) had also done the experiments on cats. Cats injected by sufficient amount of cerberin or cerberoside caused a marked rise of blood pressure, arrhythmia, and sudden circulatory collapse. Vomiting uniformly took place when non-anesthetized pigeons and cats were given appropriate doses of cerberin or cerberoside. When applied to isolated rabbits’ intestines and guinea pigs’
uteri, both glycosides produced stimulation. At the end of the experiments they concluded that cerberin can be isolated from both the oil and the defatted kernels of Cerbera odollam nuts. Isolated glycoside is similar to but not identical with cerberin
10
has been isolated, to which the name of cerberoside has been proposed and cerberin is much more potent on the heart than cerberoside.
Hiên et al. (1991) studied the immediate and delayed toxicity of the leaves of Cerbera odollam against mice. Dried leaves of Cerbera odollam were extracted at room temperature in a percolator using 95% ethanol for 12 hours. Extracts were vortexed and filtered to eliminate resins and chlorophyll and dried up. Extracts were diluted in isotonic saline for pharmacological studies in proportion of 1 ml isotonic saline to 1 g of leaf powder. In the toxicological study, most of the mice treated with the extract died without any sign of seizure or convulsion (body shakes rapidly and uncontrollably due to person's muscles contract and relax repeatedly). At the right dosage, the extract slowed down the spontaneous motility and lengthens the reaction time for the mice to thermal pain.
In an other research on Cerbera odollam, Rahman et al. (1993) conducted a study on the effect of Cerbera odollam’s seed against Instar IV larvae of Culex quinquefasciatus and Aedes aegypti. Seeds of Cerbera odollam were extracted with methanol and further fractioned using n-hexane, diethyl ether, chloroform and ethyl acetate. Residue from the fractionation process is water soluble. Water and n-butanol mixture in ratio of 50:50 was poured to the residue and shake. Two layers of solutions were formed and then separated to gain aqueous and n-butanol fractions.
Fractionated extracts were diluted in dimethyl sulfoxide (DMSO) and tested against the larvae. Results from the study found that n-butanol soluble fraction was the most effective fraction in killing the larvae of both mosquito species.
11 2.2.2 Wood extractives
Wood extractives are chemical compounds that could be extracted from wood using polar and non-polar solvents. Usually wood extractives are 1 – 20% of wood.
Extractive content generally decreases with the increment in tree height and mostly found in heartwood. Wood extractives influence the utilisation of the wood such as affect the durability of wood, colour, allergic on human, contribution to wood density and staining and corrosion on wood processing machine (Walker, 2006).
They include both lipophilic and hydrophilic compounds. Some extractives such as fats provide the energy source for the wood cell, while lower terpenoids, resin acids and phenolic substances gives wood a protection against microbiological or insect damage. The term resin is usually used for lipophilic extractives. Resin acids can be found in resin canals while fats and waxes are located in the ray parenchyma cells. Phenolic extractives are present in heartwood and the bark (Sjöström, 1993).
2.2.3 Bacteria
One of the purposes of wood preservative is to protect wood from bacterial decay. Bacteria fall under the category of prokaryotic microorganisms. The basic shapes of bacteria are sphere (coccus), rod (bacillus) and the curved rod (vibrio) as shown in Figure 2.4. In bacteria, sometimes cell division is not followed by separation of the daughter cell. The daughter cell remains attached to parent cell, creating a multicellular structure. The motility of bacteria could be by flagella,
12
gliding or axial filaments. Some bacteria called myxobacteria have the characteristic of forming fruiting bodies which is a special multicellular structure. Under suitable conditions such as lack of nutrient, a swarm of vegetative cells aggregate and form a fruiting body. Other type of bacteria is the actinomycetes, a large group of filamentous bacteria that shape just like a fungi. It also produces spores and can be easily obtained from the soil. Actinomycetes such as from the genus Streptomycetes produce antibiotics which is important in pharmaceutical industry (Wilkinson, 1986).
Streptomyces species are saprophytic bacteria that decompose organic matter, especially polymers such as lignocellulose, starch, and chitin (Crawford, 1993).
Figure 2.4: Coccus (left), bacillus (center) and vibrio (right) shape of bacteria 2.2.3 (a) Antibacterial agents
There are many types of microbicides. Microbicides could be described as any materials that kill microorganisms. Microbicides belongs to biocide which is a more general term which comprises among others microbicides, molluscicides, acaricides, insecticides, herbicides, rodenticides etc. Antibacterial agent is a microbicides. There are many types of chemical groups that could be a possible microbicides including alcohols, aldehydes, aldehyde releasing compounds, phenol derivatives, acids, acid esters, amides, carbamates, dibenzamidines, pyridine
13
derivatives, azoles, heterocyclic N, S compounds, compounds with activated halogen atoms, surface-active agents, organometallic compounds and oxidizing agents (Paulus, 2004).
2.2.3 (b) Bacteria association with wood decay
Little attention has been given on the effect of bacteria on wood decaying process compared to degradation by fungi. It is an advantage for bacteria that has the capability to colonize wood under conditions that are usually not suitable to most fungi such as waterlogging and low oxygen content. Besides that, they also can live in rotten wood and wood from wide range of environment, including treated and untreated wood. It can be said that bacteria is the most present organism among all wood-inhabiting microorganisms. Clausen (1996) classified these bacteria into 4 groups:
• Bacteria that affect the permeability of wood towards liquid but does not change the strength of the wood
• Group of bacteria that may affect the strength properties of the wood by attacking the cell walls
• Type of bacteria that work synergistically with other bacteria to destroy wood
• Group of passive colonizers that may be antagonists to other bacterial populations.
14
The usual effect of bacterial degradation of wood polymer is increment in porosity of wood (Wilcox, 1970). Permeability of wood is known to occur because of water storage. Bacillus polymyxa is a species that confirmed to have effect on the permeability of wood. This bacteria is able to hydrolyse pectin and hemicelluloses.
By removing the ray cell contents and destroying the walls of parenchyma cells, it increases the permeability of wood. Knuth and Mc Coy (1961) as reported by Greaves (1971) was able to increased the porosity of Pinus ponderosa sapwood using bacteria. They also tried to improve the permeability of P. radiate heartwood specimen using B. subtilis, P. fluorescens and a mixed bacterial population but appeared to be unsuccessful. Many reasons could be used in explaining this situation.
Since the penetration of the bacteria is limited to the outer 1 cm of the wood, it shows that the motility of bacteria is an important factor involved in bacterial decaying process in wood where too much bacterial slime produced on the surfaces of the specimen could also be the factor that prevents adequate liquid penetration.
Bacteria that belong to this group affect the wood in many ways. First is through the utilization of cell contents. In the same way, pit chambers are often colonized by bacteria where the margo structure is usually cleared. Pit structure is showed in Figure 2.5. The second way is through breakdown of ray parenchyma cell walls. The attack on ray parenchyma cell walls was found, including a progressive attack on crystalline cellulose. The ray cells contain much nutrient supply so there is a rapid growth of microorganisms inside. After consuming the contents, the bacteria will continue attacking the parenchyma cell walls that are rich in cellulose and increases the permeability of wood (Wilcox, 1970).
15
Figure 2.5: Pit structure of wood
The third way is by non-destructive attack of the secondary cell walls of rays.
It is a submicroscopic attack where under normal light the cell walls looks like unaffected. When examined under polarized light, cell wall structure shows that the bacteria are affecting the regular crystalline structure of the cellulose microfibrils by advance diffusion of cellulolytic causing the hydrolysis of cellulose enzymes. The fourth way is by attacking the pit structure of the wood which usually happens in wood that is stored in water. Attacks on the borders, margo or torus increases the wood permeability to moisture and liquid (Greaves, 1971).
The second group of bacteria attacks wood structure. Bacterial decaying process in wood is slower compared to attack by fungi but in some cases especially involving lot of moisture, bacteria had showed rapid decaying process where severe tracheid destruction was observed in P. radiata cooling tower slats after only three years in service (Greaves, 1968). As stated before, motility is an important factor to bacterial decay. Bacteria moved from cell to cell where the walls and pit floors are
margo
16
assumed to be penetrable by the microorganism. Water movements inside the wood provide an alternative way of transportation for bacteria like in a process of partial drying and rewetting of wood, bacteria will spread throughout the wood cells (Greaves, 1971). There is a group of bacteria called myxobacteria. It is also called
‘slime bacteria’ and lives mainly in the soil. This group of bacteria is so flexible that they have the ability to creep and glide over a substrate. The myxospores are resistant to dehydration and freezing enables them to survive in harsh environments such as deserts and arctic tundra. They are known to be cellulolytic and found abundantly in rotting wood (Shimkets, 1990).
The cell wall damage can be seen in three different forms. First is erosion or lysis through where the decay does not progress beyond the middle lamella which have two types:
• Shallow or surface erosion that happen usually at tertiary lamella (S3) of the cell wall. Size of the erosion is a few times wider than the bacterium producing them and often done by bacilli.
• Deep lysis throughs where the damage progress from lumen in secondary cell wall layers and sometimes it is deeper into the middle lamella. It could be determined by narrow through, steep sided and a little wider than the bacteria producing it and usually done by cocci.
Second form is wall pitting. It happened when each bacterial cell in the colony which is in contact with the wall produces a small eroded pit on the surface of
17
the lumen and the third form of damage is attack of cellulose structure in the secondary cell wall (Greaves, 1971).
The third group of bacteria works by association with other microbes.
Structural and permeability changes may predispose wood to further bacterial and fungal attack. Pectinases from bacteria attacks the membrane of the bordered pit and degrades the pit membrane (Liese and Bauch, 1967). Besides that, dead bacterial cells may provide a valuable source of nitrogen which is an important nutrient for fungi. Some bacteria also have the capsules or slime layers inside them which provide as sugar reserves for the growth of fungi (Clausen, 1996).
The fourth group is the passive wood-inhabiting bacteria. The bacteria under this group are classified due to their antagonistic or inhibitory effect on other members of wood microflora (microscopic plants). Some bacteria are able to produce antibiotics which have fungicidal properties. Vasilev (1968) as reported by Greaves (1971) had proven that bacterium isolated from ponded pine sapwood can be a wood protector against Ceratocystis, Pullularia pullulans, Trichosporium tingens, Discula pinicola and Corticium evolvens. Some highly antagonistic actinomycetes were isolated from fence posts and assayed together with Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis as an inhibitor for wood-decaying basidiomycetes and soft rot fungi. Decay capacity test were conducted using the sapwood of E. regnans and P. radiata showed that the white rot fungus Trametes versicolor was inhibited by Streptomyces violaceoniger and Bacillus cereus (Greaves, 1971).
18 2.3 Materials and methods
Antibacterial activity assays of the extractives were carried out by the disc diffusion assay method on agar medium (Dash et al., 2005). Figure 2.6 shows an overall step for antibacterial testing.
Figure 2.6: Antibacterial assay 2.3.1 Sample preparation
Before conducting antibacterial assay, samples were initially prepared.
Samples of different parts of Cerbera odollam were collected around Universiti Sains Malaysia, Penang. Figure 2.7 shows parts of the tree that had been taken.
Samples were chopped into smaller pieces and cooled in the freezer at -200C followed by freeze drying at the temperature of -400C and pressure under 0.3 mBar.
Extractive Mueller-Hinton agar
preparation (boiling, autoclaved, pour into Petri dish, placed
under UV light) Nutrient agar
preparation (boiling, pour into Petri dish, autoclaved, placed
under UV light)
Dilution with DMSO
Result observation
Testing Mueller-Hinton agar
in Petri dish Culturing bacteria
20µ l into filter disc
After drying, samples w
in plastic bag or closed container for further s
Figure 2.7: Different parts leaf (e) and flower (f)
e c a
19
After drying, samples were pulverized using grinding machine. Samples were stored in plastic bag or closed container for further step.
: Different parts of Cerbera odollam, fruit (a), seed (b), bark (c), wood (d), wer (f)
f d b
ere pulverized using grinding machine. Samples were stored
, fruit (a), seed (b), bark (c), wood (d),
20 2.3.2 Extraction for antibacterial assay
The extraction procedure was conducted based on earlier work of Kawamura et al. (2004) with modification (Figure 2.8). Weight of the sample was taken. Sample was placed inside a round bottom flask and filled with methanol until the difference of sample surface level and methanol level is around 1 cm. Samples were extracted using methanol for 1, 2 and 3 hours with heat by direct extraction. Methanol is used to take out as much as possible extractives from the samples. The resulting liquid was filtered into a measuring cylinder. The solution was shaked vigorously using hands. Extract solution in the measuring cylinder was poured into a round flask and dried using a rotary evaporator. The solution was dried until only small amount of solvent is left. Extractive was transferred to a beaker, dried and the weight of extractives was taken. Previous steps were repeated using water as solvent with the methanol extracted residue as the sample. Hot water extracts were recovered using freeze drier to remove excess water. Extraction process is shown in Figure 2.9.
21
Figure 2.8: Extraction and fractionation procedures of samples.
Hot water extract
Methanol extraction (1, 2, 3 hours)
n-hexane extraction
EtOH extraction
EtOH solubles Insolubles
EtOAc insolubles EtOAc solubles
EtOAc extraction n-hexane insolubles
n-hexane solubles
Methanol extract Hot water extraction (1, 2, 3 hours)
Prepared sample
22
Figure 2.9: Extraction process 1 cm
Weigh empty flask
Put sample into flask
Fill flask with methanol
Direct extraction (1hr, 2hr, 3hr)
Filter into measuring cylinder
Shake
Pour into round bottom flask
Drying (rotary evaporator)
Pipette into beaker
Dry
Ready for separation
Direct extraction with hot water (1hr, 2hr, 3hr)
Pour into round bottom flask
Freeze inside pre-freezer
Freeze dry
Powder
23 2.3.3 Separation (or fractionation) process
Separation process used is shown in Figure 2.10 employing the method by Kawamura et al. (2004) with slight modification by further separation of ethyl acetate insoluble part using ethanol. Extractives were separated by three different solvents. A small amount of n-hexane was poured into the beaker containing the extractive from the methanol extraction and stirred. The extracted solution was filtered using cotton wool into a round bottom flask. The process was repeated several times until no colour changes was observed in the solvent. Colour changes show that there is still extractives could be dissolved with that solvent. The extractive was dried using rotary evaporator and carefully pipetted into the sampling bottle. The extractive was oven dried at 500C to remove excess solvent without damaging the extractives. The weight of extractive was determined based on oven dry weight. The insoluble left in the beaker was also weighed. Process of separation will be repeated using ethyl acetate and ethanol in sequence. Fifty miligrams of every extractive was diluted in 1ml dimethylsulfoxide (DMSO). Diluted extractives were pipetted onto filter discs for later use in antibacterial assay.
24
Figure 2.10: Separation process 2.3.4 Moisture content determination
Moisture content of different parts of Cerbera odollam, extracts and fractions were determined using oven-drying method (Hon and Shiraishi, 2001). Samples were weighed, placed into the oven at 105+30C for 24 hours and weighed again. Process was repeated until the oven dry weight is constant. Moisture content (MC) was calculated using the formula;
ܯ݅ݏݐݑݎ݁ ܿ݊ݐ݁݊ݐ, % = ܣ݅ݎ ݀ݎݕ ݓ݁݅݃ℎݐ − ܱݒ݁݊ ݀ݎݕ ݓ݁݅݃ℎݐ
ܣ݅ݎ ݀ݎݕ ݓ݁݅݃ℎݐ ܺ 100
Extractives from methanol extraction
Mix with n-hexane
Filter
Residue drying (n- hexane insoluble)
Mix with ethyl acetate
Filter
Residue drying (ethyl acetate insoluble)
Mix with ethanol
Filter
Insolubles
Solvent evaporation
n-hexane soluble extract
Solvent evaporation
Ethyl acetate soluble extract
Solvent evaporation
Ethanol soluble extract
25 2.4.5 Agar preparation
Oxoid CM0003 Nutrient agar was prepared for bacteria culturing process.
Fourteen grams of nutrient agar was dissolved in 500ml distilled water with heating.
The agar was autoclaved at 1210C for 15min and poured into 4 different Petri dishes for preparing 4 species of bacteria. Finally they were placed under UV light of fume cupboard to eliminate any available contamination.
Thirty eight grams of Mueller-Hinton agar was dissolved into 1L of distilled water with heating. The agar was autoclaved at 1210C for 15 minutes. It was poured into Petri dishes for each extract while still warm and placed under UV light for overnight.
2.3.6 Culturing bacteria
Bacteria culture of Bacillus subtilis (ATCC 21332), Bacillus licheniformis (ATCC 14580), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were obtained from School of Biology, Universiti Sains Malaysia.
Ethanol (70%) was used to sterilize the working place. The loop used to scoop bacteria was heated on until hot red. The loop was cooled down by slight touch on the agar. Bacteria colony was taken out from bottle and streaked on the agar in Petri dish. Petri dishes with agar and bacteria were kept in an incubator at 370C overnight for about 18 to 20 hours.
26 2.3.7 Antibacterial activity test
Each Petri dish was divided into 6 parts by drawing lines across it as shown in Figure 2.11. One mililitres of distilled water was pipetted on the bacteria-cultured agar and bacteria colonies were scraped off from the agar into the distilled water using L-shaped glass rod. The liquid inside the dish (distilled water containing the bacteria) was pipetted into a universal bottle and its turbidity was compared with McFarland 0.5 solution to give an approximate cell density of 1.5X108 CFU (colony forming unit) per ml of distilled water.
Figure 2.11: Disc placement on Petri dish; Distilled water (1), DMSO (2) with extractives (3 – 6)
Bacteria were spread on the agar using sterile swab. Twenty microlitres of extractive was pipetted onto filter disc. The filter disc was placed onto the agar to check for its inhibition zone. Two control negative discs were also placed in each Petri dish where one is distilled water and one is Dimethyl sulfoxide (DMSO). The petri dishes were placed into the incubator for 30 minutes to let it stick onto the agar before turning it upside down and left for 24 hours.
27
The diameter of inhibition zone was measured at three different points for each assay disc and the average was calculated. Larger inhibition zone indicates better antibacterial activity for the extractives (Omar et al., 2000).
2.3.8 Chemical analysis
Chemical analysis was done using the FTIR and GC-TOFMS to determine possible chemical compounds from the extracts of different parts of Cerbera odollam that acts as antibacterial agent.
2.3.8 (a) Fourier Transform Infrared Spectrophotometer (FT-IR) of extractives
The chemical substances inside the extractives of Cerbera odollam were evaluated using the FTIR. Pellets were prepared from approximately 100g of finely grounded potassium bromide (KBr) (Muyonga et al., 2004; Schmitt and Flemming, 1998). Four pellets were prepared for each sample of n-hexane soluble part, ethyl acetate soluble part, ethanol soluble part and hot water extracts. Total 24 discs were prepared for the experiment. Solid sample in powder form can be mixed with IR transparent material such as KBr or KCl and moulded into pellets under high pressure. Extractives that were in gel-like substance were applied onto Kbr pellets as thin as possible. The pellets were left to dry before viewing using the Nicolet infrared spectrophotometer (Avatar 360 FT-IR E.S.P) from wavenumbers of 4000cm-1 to 470cm-1 to detect the functional groups of the compounds of the extractives.
28
2.3.8 (b) Gas chromatography time of flight mass spectrometer analysis (GC- TOFMS) of extractives showing antibacterial activity
About 1mg of each extracts was diluted in 20ml methanol and filtered through 0.45µm filter disc. The extracts were analsed for GCMS analysis using Agilent Technologies 6890N Gas Chromatography with LECO Corporation Pegasus III Time-of-Flight Mass Spectrometer as detector. The running time for each sample was 45 minutes and Helium was used as carrier gas at constant flow of 1.0 ml/min.
Peaks obtained from the scanning were examined to determine the active compounds available. Compounds that match over 90% of National Institute of Standards and Technology (NIST) Chemical Library were considered as chemical profile for each sample. Chemical compounds detected were compared with compound with antibacterial activity found by previous researchers.
29 2.4 Results and discussions
2.4.1 Moisture content, yield of extraction and fractions.
Table 2.1 presents the moisture content of different parts of Cerbera odollam, yield from methanol extraction and hot water extraction after methanol extraction.
The flower showed the highest content of moisture followed by fruit and seed. The wood had the least amount of moisture (55.42%). The fruit and seed had the highest amount of extractives from methanol extraction than other parts of the tree.
Meanwhile, the flower had highest hot water extract (17.50%). The yield of extracts after fractionation using methanol is shown in Table 2.2. The seed showed the greatest content of n-hexane soluble part (4.71%), while the fruit contain the highest ethyl acetate soluble part (0.33%). The flower contains most ethanol soluble part (6.92%). The seed on the other hand, had the highest insoluble content (7.13%).
Table 2.1: Moisture content, yield from methanol extraction and hot water extraction after methanol extraction of the different parts of Cerbera odollam
Sample Moisture
content (%)
Yield from methanol extraction (%)
Yield from hot water extraction (%)
Fruit 713.01 26.77 10.68
Seed 407.69 23.47 13.29
Leaf 245.30 13.19 4.01
Wood 55.42 6.59 2.72
Bark 333.62 4.91 2.21
Flower 774.57 14.95 17.50
30
Table 2.2: Yield of extracts after fractionation from methanol extract of the different parts of Cerbera odollam
Sample n-hexane (%) Ethyl acetate (%) Ethanol (%) Insolubles (%)
Fruit 2.76 0.33 6.73 7.13
Seed 4.71 0.23 2.46 8.48
Leaf 1.64 0.07 5.09 0.00
Wood 0.81 0.22 3.07 0.00
Bark 0.79 0.26 2.61 0.19
Flower 0.19 0.04 6.92 0.96
2.5.2 Antibacterial assay
The effect of extracts on the growth of Bacillus subtilis and Bacillus licheniformis are tabulated in Table 2.3. The results showed that hexane soluble part from bark had the highest inhibitory effect on Bacillus subtilis and Bacillus licheniformis bacteria followed by hexane soluble part from the flower. Ethyl acetate soluble part from bark, hexane soluble part from leaf, hexane soluble part from wood and hexane soluble part from fruit, showed less antibacterial activity on the bacteria.
31
Table 2.3: Effect of extracts after fractionation from methanol on the growth of Bacillus subtilis and Bacillus licheniformis
Extract Inhibition zone, mm
Bacillus subtilis Bacillus licheniformis
Fruit (n-hexane) NI 8.75±0.35
Fruit (ethyl acetate) NI NI
Fruit (ethanol) NI NI
Fruit (insoluble) NI NI
Fruit (hot water) NI NI
Seed(n-hexane) NI NI
Seed (ethyl acetate) NI NI
Seed (ethanol) NI NI
Seed (insoluble) NI NI
Seed (hot water) NI NI
Leaf (n-hexane) 8.17±0.71 8.25±0.82
Leaf (ethyl acetate) NI NI
Leaf (ethanol) NI NI
Leaf (insoluble) NI NI
Leaf (hot water) NI NI
Wood (n-hexane) NI 10.25±0.59
Wood (ethyl acetate) 7.67±0.24 NI
Wood (ethanol) NI NI
Wood (insoluble) NI NI
Wood (hot water) NI NI
Bark (n-hexane) 8.75±0.12 12.75±0.82
Bark (ethyl acetate) NI 8.25±0.35
Bark (ethanol) NI NI
Bark (insoluble) NI NI
Bark (hot water) NI NI
Flower (n-hexane) NI 12.08±0.35
Flower (ethyl acetate) NI NI
Flower (ethanol) NI NI
Flower (insoluble) NI NI
Flower (hot water) NI NI
*NI – no inhibition zone
There was no antibacterial activity observed on Escherichia coli and Pseudomonas aeruginosa. This could be because of insufficient concentration of extract or Gram negative bacteria characteristic that are more resistant to antimicrobial agent than the Gram-positive bacteria. One of the several unique characteristics of Gram-negative bacteria is the outer membrane of the cell that is responsible for protecting the bacteria from destruction of the inner membrane or cell
32
wall (peptidoglican). The outer membrane is built up of semipermeable barrier to the uptake of antibiotic, antibacterial or biocides (Hancock, 1998). The P. aeruginosa is naturally resistant to a large range of antibiotics and may demonstrate additional resistance after unsuccessful treatment through mutation of their porin which is an opening on the outer membrane surface that control movement of substance, in and out. Results of the experiment could be affected by many factors for instance the accumulation of lipid by the bacteria cell in action of resistance and the ability of some bacteria to decompose antibacterial agents. Same species of bacteria could resist differently towards an antibacterial agent (Hugo, 1967). Images of inhibition zones could are shown in Figure 2.12 to Figure 2.18.
Figure 2.12: The inhibition zone of hexane extract from leaf (1). Clear zone around the paper disc shows inhibition of bacteria growth
1
33
Figure 2.13: The inhibition zone of hexane extract from bark (1). Clear zone around the paper disc shows inhibition of bacteria growth
Figure 2.14: The inhibition zone for ethyl acetate extract from wood (1). Clear zone around the paper disc shows inhibition of bacteria growth
Figure 2.15: The inhibition zone of hexane extract from the leaf (1). Clear zone around the paper disc shows inhibition of bacteria growth
1
1
1
34
Figure 2.16: The inhibition zones of hexane extract from bark (1), ethyl acetate extract from bark (2) and hexane extract from flower (3). Clear zone around the
paper discs shows inhibition of bacteria growth
Figure 2.17: The inhibition zone of hexane extract from wood (1). Clear zone around the paper disc shows inhibition of bacteria growth
Figure 2.18: A very small inhibition zone of hexane extract from fruit (1). Clear zone around the paper disc shows inhibition of bacteria growth
1 2
3
1
1
