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BIOLOGICAL ACTIVITIES OF Schizophyllum commune FR.

MIRFAT BT HJ AHMAD HASAN SALAHUDDIN

THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR


THE DEGREE OF MASTER OF SCIENCE

FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR

2008

ABSTRACT
The increasing appearance of several multi-resistant pathogenic microorganisms
and tumor cells to the available drugs has drawn much attention of researchers to find
new alternative drugs. Drugs from natural sources are the best choice as many nonnatural, synthetic drugs are carcinogenic and cause severe side effects that were not
acceptable by the consumers. The potential use of mushroom to explore their biological
activities may be important for treatment of a variety of human ailments. Many
mushroom species have become attractive as a source for the development of drugs and
nutraceuticals as they contain a tremendous variety of secondary metabolites with
diverse biological activities as they are nutritionally functional food and a source of
physiologically beneficial and non-toxic medicines. In this present study, Schizophyllum
commune Fr., a split gill mushroom was chosen as it has long been acknowledged for its
medical importance. Schizophyllum commune was extracted with methanol, ethyl
acetate, dichloromethane and water and these extracts were tested. Antimicrobial
activity was determined using the well diffusion assay. The microorganisms tested
consisted of common pathogenic bacteria i.e Gram-positive bacteria (Bacillus cereus,
B. subtilis, Enterobacter faecalis, Staphylococcus aureus, Streptococcus mitis,
S. mutans and S. sanguis), Gram-negative bacteria (Escherichia coli, Salmonella sp.,
S. typhi, Shigella sp., Shigella flexneri, Plesiomonas shigelloides, Proteus vulgaris, and
Pseudomonas aeuroginosa) and pathogenic fungi (Candida albicans, C. parapsilosis
and Saccharomyces pombe). The effectiveness of the extracts depends on the extraction
solvent and higher antibacterial activity was exhibited compared to antifungal activity.
Dichloromethane extract was the most effective, being able to significantly (P<0.05)
inhibit the growth of most of the Gram-positive bacteria, Gram-negative bacteria and
fungi. The highest inhibition zone by this extract was against the Gram-positive
bacteria, S. sanguis with zone diameter of 12 1 mm. The antioxidant potential of
ii

S. commune extracts was measured employing two methods namely DPPH radical
scavenging system and Folin-Ciocalteau method respectively. The radical scavenging
activity of S. commune extracts correlated well with the total phenolic content
(r=0.8264). Among the extracts, methanol extract of S. commune showed the most
remarkable antioxidant activity with the ability to reduce the stable radical DPPH to
yellow-coloured diphenylpicrylhydrazine providing IC50 at only 0.145 0.01 mg/ml
and having the highest total phenolic content of 1.72 0.05 mg GA/g extracts
equivalent to 0.498 0.07 mg GA/g fruitbodies. The cytotoxicity of S. commune
extracts against cervical cancer cell lines; CaSki, epidermoid cancer cell lines; KB,
colon cancer cell lines; HT29 and intestinal colon cancer cell lines; HCT116 were
evaluated using the neutral red cytotoxicity assay. All S. commune extracts tested were
considered non-cytotoxic against the cancer cells tested having IC50 values of more than
20 g/ml. However, dichloromethane extract was found to be effective against the
intestinal colon cancer cell lines, HCT 116 with IC50 value of 14.71 2.0 g/ml.
The

anti-HPV

activity

of

S.

commune

extracts

was

carried

out

using

immunohistochemistry method to determine the expression of E6 oncoprotein and the


anti-HPV 18 E6 activity in the cervical cancer (CaSki) cell lines. All S. commune
extracts showed a quite similar trend of inhibition against the CaSki cells. However,
based on the qualitative appearance on the morphology and colorization of the cancer
cells, methanol and dichloromethane extracts of S. commune were the best E6 protein
inhibitor compared to ethyl acetate and water extracts. Schizophyllum commune needs
to be further purified for the bioactive ingredients as it holds hope for the discovery of
new drugs for the treatment of a variety of human ailments. To our knowledge, this is
the first report on the broad spectrum of antimicrobial, antioxidant, cytotoxicity and
anti-human papilloma virus of S. commune extracts.

iii

ABSTRAK
Kehadiran mikroorganisma patogenik dan sel tumor yang rintang terhadap ubat
yang sedia ada semakin meningkat dan telah menarik perhatian para penyelidik untuk
mencari sumber ubat yang baru sebagai alternatif. Ubat daripada sumber asli adalah
pilihan terbaik memandangkan kebanyakan ubat daripada sumber bukan asli atau
sintetik adalah karsinogenik dan boleh menyebabkan kesan sampingan yang mana ini
tidak dapat diterima oleh pengguna. Potensi cendawan dalam aktiviti-aktiviti biologi
mungkin memainkan peranan penting dalam rawatan pelbagai penyakit. Banyak spesies
cendawan telah menyerlah sebagai sumber untuk pembangunan ubat dan nutraseutikal
memandangkan

cendawan

mengandungi

pelbagai

metabolit

sekunder

yang

menyumbang kepada pelbagai aktiviti biologi selain sebagai sumber makanan yang
berfungsi dari segi nilai nutrisi dan merupakan ubat yang tidak toksik dan mempunyai
kelebihan secara fisiologi. Dalam kajian ini, Schizophyllum commune Fr. iaitu sejenis
cendawan kukur telah diplih memandangkan kepentingan perubatannya telah
dikenalpasti. S. commune telah diekstrak dengan metanol, etil asetat, diklorometana dan
air dan ekstrak-ekstrak ini telah diuji. Aktiviti antimikrob ditentukan dengan
menggunakan esei penyebaran telaga (well diffusion). Mikroorganisma yang diuji
adalah bakteria patogen yang biasa iaitu bakteria Gram-positif (Bacillus cereus,
B.subtilis, Enterobacter faecalis, Staphylococcus aureus, Streptococcus mitis, S. mutans
dan S. sanguis), bakteria Gram-negatif (Escherichia coli, Salmonella sp., S. typhi,
Shigella sp., Shigella flexneri, Plesiomonas shigelloides, Proteus vulgaris, dan
Pseudomonas aeuroginosa) dan kulat patogen (Candida albicans, C. parapsilosis dan
Saccharomyces pombe). Keberkesanan ekstrak-ekstrak ini bergantung kepada pelarut
yang digunakan dalam pengekstrakan. Aktiviti antibakteria adalah lebih tinggi
berbanding dengan aktiviti antikulat. Ekstrak diklorometana adalah yang paling aktif
secara signifikan (P0.05) dalam menghalang pertumbuhan hampir kesemua bakteria
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Gram-positif, Gram-negatif dan kulat yang diuji. Zon perencatan yang paling tinggi
yang direkodkan oleh ekstrak ini adalah terhadap S. sanguis (12 1 mm). Potensi
antioksidan ekstrak S. commune dinilai menggunakan dua kaedah iaitu sistem
penyingkir radikal DPPH dan kaedah Folin-Ciocalteau. Daripada kajian, aktiviti
pemusnah radikal yang ditunjukkan oleh ekstrak S. commune mempunyai hubungkait
yang tinggi (r=0.8264) di antara aktiviti pemusnah radikal dan jumlah kandungan fenol.
Ekstrak metanol S. commune

menunjukkan aktiviti antioksidan yang paling baik

dengan keupayaannya mengurangkan radikal bebas DPPH kepada difenilpikrilhidrazin


yang berwarna kuning dengan IC50 yang dicatatkan iaitu 0.145 0.01mg/ml dan jumlah
kandungan fenol sebanyak 1.72 0.05mg GA/g ekstrak dan 0.498 0.07mg GA/g
fruitbody. Sitotoksisiti ekstrak S. commune ke atas sel kanser serviks; CaSki, sel
kanser epidermoid; KB, sel kanser kolon; HT29 dan sel kanser kolon intestinal;
HCT116 telah diuji menggunakan kaedah sitotoksisiti neutral red. Kesemua ekstrak
cendawan yang diuji didapati tidak

tidak toksik terhadap sel-sel kanser berkaitan

dengan nilai IC50 yang melebihi 20 g/ml. Walau bagaimanapun, ekstrak diklorometana
menunjukkan aktiviti yang lebih baik dengan aktiviti sitotoksik yang paling aktif
dicatatkan ke atas sel kolon intestinal (HCT116) dengan nilai IC50 14.71 2.0 g/ml.
Aktiviti anti-HPV oleh ekstrak S. commune dijalankan dengan menggunakan kaedah
immunohistochemistry ini adalah untuk menentukan ekspresi onkoprotein E6 dan
aktiviti anti-HPV 18 E6 dalam sel kanser serviks (CaSki). Kesemua ekstrak S. commune
memberikan corak pemusnahan yang hampir sama terhadap sel CaSki. Walau
bagaimanapun, berdasarkan ciri kualitatif dari segi morfologi dan pewarnaan sel kanser
tersebut, ekstrak metanol dan diklorometana S. commune merupakan perencat protein
E6 yang lebih baik berbanding etil asetat dan air. Bahan-bahan bioaktif yang terdapat
dalam S. commune perlulah dikaji selanjutnya memandangkan ia menunjukkan potensi
kepada deteksi sumber ubat yang baru dalam merawat pelbagai penyakit. Ini adalah

laporan pertama mengenai kajian antimikrob, antioksidan, sitotoksisiti dan anti-HPV


daripada ekstrak S. commune.

vi

ACKNOWLEDGEMENT
First and foremost, I thank Allah who always blesses me by giving me good
health and wisdom throughout this research.
I would like to convey my unwavering gratitude and warmfelt appreciation to
both of my supervisors; Associate Professor Dr. Noorlidah Abdullah and Professor Dr.
Vikineswary S. for their invaluable advice, constant source of inspiration and guidance
in the completion of this research work.
My utmost and sincere thanks to Associate Professor Dr. Nurhayati Zainal
Abidin for her generosity in allowing me to work in Molecular Biology Lab, Institute of
Postgraduate Studies and also for providing me with consecutive ideas for the smooth
running of the research.
The work would not be possible without the financial assistance from University
Malaya; the IRPA grant (voteF no: 09-02-03-1048) and University Malaya fellowship
which I had been a recipient throughout the course of my study.
I would like to take this opportunity to dedicate my deepest appreciation to my
fun-loving colleagues for enduring together the countless hours and sharing good ideas
in completing this research. Not to forget many others from the Mycology Lab and
Molecular Biology Lab for contributing their unselfish time in helping me to carry out
the research.
Last but not least, I wish to express my heartfelt gratitude to my family for their
love, understanding and moral support during the course of my post-graduate studies.

Mirfat A.H. Salahuddin

vii

TABLE OF CONTENTS

ABSTRACT

ii

ABSTRAK

iv

ACKNOWLEDGEMENT

vii

TABLE OF CONTENTS

viii

LIST OF FIGURES

xii

LIST OF TABLES

xiii

LIST OF PLATES

xiv

LIST OF SYMBOLS AND ABBREVIATIONS

xv

CHAPTER ONE - INTRODUCTION

1.1

Objectives of Study

CHAPTER TWO - LITERATURE REVIEW

2.1

Introduction to medicinal mushroom

2.2

Schizophyllum commune Fr.

2.3

Biological activities of mushrooms

11

2.3.1

Anti-microbial activity of mushrooms

11

2.3.1.1 Mechanisms of antimicrobial actions

14

Antioxidant activity of mushrooms

16

2.3.2.1 Free radicals and reactive oxygen species

16

2.3.2.2 Antioxidants

17

2.3.2.3 Mushroom as antioxidants

18

2.3.2.4 Mechanisms of antioxidant actions

20

Cytotoxicity of mushrooms

21

2.3.3.1 Mechanisms of anti-tumor actions

27

2.3.2

2.3.3

viii

2.3.4

2.4

Antiviral activity of mushrooms

30

2.3.4.1 Human papilloma virus (HPV)

33

2.3.4.2 Human papilloma virus (HPV) infection

35

Methods in the determination of biological activities

37

2.4.1

Antimicrobial activity

37

2.4.2

Antioxidant activity

39

2.4.3 Cytotoxic activity

42

2.4.4

44

Anti-human papilloma virus (HPV) activity

CHAPTER THREE - MATERIALS AND METHODS

47

3.1

Preparation of Schizophyllum commune extracts

47

3.2

Determination of antimicrobial activity

48

3.2.1 Well diffusion assay

49

Determination of antioxidant activity

51

3.3.1

51

3.3

3.4

3.5

2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

3.3.2 Folin-Ciocalteau assay

52

Cytotoxicity towards various cancer cell lines

52

3.4.1

52

Cancer cell lines

3.4.2 Serial dilution of S. commune extracts

53

3.4.3

Sub-cultivation of cancer cells

53

3.4.4

Cell enumeration and plating

54

3.4.5

Treatment of cancer cells with S. commune extracts

55

3.4.6

Neutral red assay

56

Anti-human papilloma virus (HPV) activity towards cervical cancer


(CaSki) cell lines

56

3.5.1 Serial dilution of S. commune extracts

56
ix

3.5.2

Treatment of CaSki cells with S. commune extracts

57

3.5.3

Fixation of CaSki cells onto slides

57

3.5.4

Immunohistochemistry assay

58

CHAPTER FOUR - RESULTS

60

4.1

Analysis of antimicrobial activity of S. commune extracts

60

4.2

Analysis of antioxidant activity of S. commune extracts

68

4.2.1 Correlation between total phenolic content and scavenging


activity of S. commune extracts
4.3

Analysis of cytotoxicity of S. commune extracts towards various


cancer cell lines

4.4

71

72

Analysis of anti-human papilloma virus (HPV) activity of S. commune


towards cervical cancer (CaSki) cell lines

75

CHAPTER FIVE DISCUSSION

82

5.1

82

Antimicrobial activity of S. commune Fr.


5.1.1

Antimicrobial activity of S. commune extracts as evaluated


by well diffusion assay

5.1.2

Antimicrobial activity of S. commune extracts against


various pathogenic microorganisms

5.1.3

84

Antimicrobial activity of S. commune extracts and its


correlation to phenolic content

5.1.5

83

Extraction solvent as the significant factor in evaluating


antimicrobial activity of S. commune

5.1.4

82

86

Other contributing factors to antimicrobial activity of


S. commune extracts

89

5.2

Antioxidant activity of S. commune Fr.


5.2.1

Antioxidant activity of S. commune extracts and its


correlation to phenolic content

5.2.2

94

Other contributing factors to antioxidant activity of


S. commune extracts

5.3

91

Extraction solvent as the significant factor in evaluating


antioxidant activity of S. commune

5.2.3

91

Cytotoxicity of S. commune Fr.

98
99

5.3.1 Cytotoxicity of S. commune extracts as evaluated by


neutral red assay
5.3.2

Cytotoxicity of S. commune extracts towards different


cancer cell lines

5.3.3

Anti-human papilloma virus (HPV) activity of S. commune Fr.


5.4.1

5.4.3

109
112

Anti-human papilloma virus (HPV) activity of S. commune


extracts as evaluated by immunohistochemistry assay

5.4.2

108

Other contributing factors to cytotoxicity of S. commune


extracts

5.4

103

Cytotoxicity of S. commune extracts and its correlation


to antimicrobial activity

5.3.4

99

112

Anti-human papilloma virus (HPV) activity of S. commune


extracts towards cervical cancer cell lines (CaSki)

114

Comparative study of various antiviral activities

115

CHAPTER SIX - CONCLUSION

119

xi

REFERENCES

123

APPENDICES

151

Appendix A: Reagents, media and buffer

151

Appendix B: Analytical techniques

155

Appendix C: Experimental and statistical data

158

xii

LIST OF FIGURES

Figure 2.1

The extrinsic death receptor pathway (Page 30)

Figure 2.2

The intrinsic mitochondrial pathway (Page 30)

Figure 3.1

Extraction of S. commune bioactive compounds and the


biological activities analyzed (Page 48)

Figure 3.2

Screening for antimicrobial activities of S. commune extracts


(Page 50)

Figure 3.3

Serial dilution of S. commune extracts for cytotoxicity (Page 53)

Figure 3.4

Cell counting using a haemocytometer (Page 55)

Figure 3.5

Serial dilution of S. commune extracts for anti-HPV activity


(Page 57)

Figure 3.6

A brief procedure of immunohistochemistry method (Page 59)

Figure 4.1

The scavenging effect of S. commune extracts on DPPH radicals


(Page 69)

Figure 4.2

Correlation between the radical scavenging activity


S. commune extracts and their phenolic contents (Page 72)

Figure 4.3

The cytotoxicity of S. commune dichloromethane extract against


HCT116 cancer cell lines (Page 74)

of

xiii

LIST OF TABLES

Table 2.1

Sizes and functions of HPV 18 proteins (Page 37)

Table 4.1

Antimicrobial activity of S. commune extracts at 0.2 mg/ml as


evaluated by well diffusion assay (24 hours incubation at 37C).
Inhibition zones were measured (in mm) and the diameter of
inhibition zones reported (Page 62)

Table 4.2

Antimicrobial activity of S. commune extracts at 2 mg/ml as


evaluated by well diffusion assay (24 hours incubation at 37C).
Inhibition zones were measured (in mm) and the diameter of
inhibition zones reported (Page 64)

Table 4.3

Antibacterial activity of antibiotic discs as evaluated by well


diffusion assay (24 hours incubation at 37C). Inhibition zones
were measured (in mm) and the diameter of inhibition zones
reported (Page 66)

Table 4.4

Antifungal activity of antibiotic nystatin as evaluated by well


diffusion assay (24 hours incubation at 37C). Inhibition zones
were measured (in mm) and the diameter of inhibition zones
reported (Page 67)

Table 4.5

The scavenging activity of S. commune extracts as measured by


DPPH radical scavenging assay (Page 69)

Table 4.6

The total phenolic content of S. commune extracts and fruitbodies


at 0.1 mg/ml as measured by Folin-Ciocalteau method (Page 71)

Table 4.7

The IC50 values of S. commune extracts against different cancer


cell lines as measured by neutral red cytotoxicity assay (Page 73)

xiv

LIST OF PLATES

Plate 4.1

Antibacterial activity of S. commune dichloromethane extracts


against some of the microorganisms (Page 65)

Plate 4.1 (a)

The inhibition zone of S. sanguis by 2 mg/ml dichloromethane


extract of S. commune (Page 65)

Plate 4.1 (b) :

S. mutans was not inhibited by 2 mg/ml dichloromethane extract


of S. commune (Page 65)

Plate 4.2

Antibacterial activity of antibiotic chloramphenicol against some


of the microorganisms (Page 67)

Plate 4.2 (a)

The inhibition zone of P. vulgaris by 2 mg/ml antibiotic


chloramphenicol (Page 67)

Plate 4.2 (b) :

The inhibition zone of B. subtilis by 2 mg/ml antibiotic


chloramphenicol (Page 67)

Plate 4.3

The qualitative appearance of two controls of CaSki cell lines


after being treated with different treatments (Page 76)

Plate 4.3 (a)

The negative control of CaSki cells; cells not treated with


antibody (Page 76)

Plate 4.3 (b) :

The positive control of CaSki cells; cells treated with antibody


(Page 76)

Plate 4.4

The appearance of CaSki cells after treatment with methanol


extract of S. commune at different concentrations (Page 78)

Plate 4.5

The appearance of CaSki cells after treatment with ethyl acetate


extract of S. commune at different concentrations (Page 79)

Plate 4.6

The appearance of CaSki


dichloromethane extract of
concentrations (Page 80)

Plate 4.7

The appearance of CaSki cells after treatment with water extract


of S. commune at different concentrations (Page 81)

cells after treatment with


S. commune at different

xv

LIST OF SYMBOLS AND ABBREVIATIONS

percentage

degree centigrade

microgram

g/ml

microgram per mililiter

microliter

BHI

Brain Heart Infusion

bp

base pair

CIN

cervical intraepithelial neoplasia

CIS

carcinoma in situ

cm

centimeter

CO2

carbon dioxide

DAB

3 diaminobenzidine tetrahydrochloride

dH2O

distilled water

DMSO

dimethyl sulfoxide

DNA

deoxyribonucleic acid

DPPH

2,2-Diphenyl-1-Picrylhydrazyl

early region

ED50

effective dosage of drug that gives 50% of maximal response

FBS

fetal bovine serum

gram

GAE

gallic acid equivalent

H2O2

hydrogen peroxide

HEPES

N-2-hydroxyethyl-piperazine-N-2-ethane-sulfonic acid

HPV

human papilllomavirus

xvi

HRP

horseradish peroxidase

IHC

immunohistochemistry

K2HPO4

potassium phosphate anhydrous

KH2PO4

potassium dihydrogen orthophosphate

late region

LSAB

labelled steptravidin - biotin

molar

mg

miligram

mg/ml

milligram per mililiter

MH

Mueller Hinton

ml

mililiter

mm

millimeter

mRNA

messenger ribonucleic acid

mw

molecular weight

Na2CO3

natrium carbonate

Na2HPO4

natrium phosphate anhydrous

NaCl

natrium chloride

NaHCO3

natrium bicarbonate

ORR

open reading frame

PBS

phosphate buffered saline

Rb

retinoblastoma

rpm

revolution per minute

RPMI

Rosewell Park Memorial Institute

sp

species

beta

URR

upstream regulatory region

xvii

UV

ultraviolet

w/v

weight/volume

wt

weight

xviii

CHAPTER 1: INTRODUCTION
Recently, there has been a significant interest in natural product research due to
the failure of alternative drug discovery methods to deliver many lead compounds in
key biological activities such as immunosuppression, anti-infectives and other
metabolic diseases. There has also been a great concern among researchers about the
increasing appearance of several multi-resistant pathogenic microorganisms and tumor
cells to the available antibiotics which has become an interestingly important and
pressing global problem. Due to this, the rate of drug discovery has dropped to
dangerous proportions, the rate of nosocomial infections has risen and new diseases
have evolved.
Researches in the field of anti-infectives are now desperately needed if a public
health crisis is to be averted. Infection of the multi drug resistant isolates became more
and more frequent, stimulating the search for new drugs with novel mechanism of
actions. In contrast to bacterial infectious diseases, viral diseases cannot be treated by
common antibiotics and specific drugs are urgently needed. The highly available choice
in the market is the synthetic drugs. However, these non-natural drugs are not favorable
to the consumers. Serious concerns on the carcinogenic properties and severe side
effects that have been reported for some synthetic drugs make them less acceptable
except as treatments of last resort for terminal diseases such as cancer. Therefore, there
is great interest in finding new and safe drugs from natural sources.
The use of natural sources in the drug discovery has received much attention
nowadays, not only for their potential as source of drugs, but also because they are
natural, non-synthetic, safe and their appreciation by consumers are very favorable. In
fact, they have consistently been the most successful source of drug leads for many
years. The natural sources usually have a biological or pharmacological activity for use
in pharmaceutical drug discovery and drug design. Between 1983 and 1994, 39% of
1

antibacterial and anticancer drugs were derived from natural products. Also, in that
same time period, 39% of all new approved drugs were from either natural products or
derived from natural products (http://www.lifepharms.com/).
For many years, mankind has benefited from plants as natural source of drugs
and herbal remedies. In fact, many studies have been focused on developing biological
and pharmaceutical activity in plants, but attempts to explore mushrooms in such a way
is much neglected. Hence, studies should be made to explore the potential use of
mushrooms and their metabolites for treatment of a variety of human ailments.
Mushrooms, similar to plants, have a great potential for the production of useful
bioactive metabolites and that they are prolific resource for drugs. Mushrooms
characteristically contain a tremendous, variety of secondary metabolites that display a
broad range of biological activities and the content and bioactivity of these compounds
depends on how the mushroom is prepared and consumed. The vast structural diversity
of natural compounds found in mushrooms, provide potential opportunities for
discovering new drugs that rationally target the abnormal molecular and biochemical
signals leading to cancer. Experience from ethnomedicine and extensive basic
laboratory findings have shown that mushrooms could play an important role in
prevention and treatment of cancer (Russo et al., 2006).
Previously, mushrooms have been reported to become an important source of
novel bioactive compounds (Hawksworth, 1991) and have great potential as a
nutritionally functional food and a source of physiologically beneficial and non-toxic
medicines (Wasser, 1999). Many other researchers supported the idea that mushrooms
have become attractive as a functional food and as a source for the development of
drugs and nutraceuticals. It is estimated that approximately 50% of the annual 5 million
metric tons of cultivated edible mushrooms contain functional nutraceutical or
medicinal properties. The United States (US) Academy of Science has defined
2

functional foods as those that encompass potentially health products including any
modified food or food ingredient that may provide a health benefit beyond the nutrients
it contains (Thomas and Earl, 1994).
As for their nutritional value, the edible mushrooms have been reported to be
low in calories and in fat but rich in proteins, minerals and dietary fibre. Mushrooms
also contain significant amounts of vitamins, namely thiamin, riboflavin, ascorbic acid
and vitamin D2, as well as minerals (Breene, 1990; Miles and Chang, 1997). They are
also a potential source of dietary fibre: fungal cell walls contain chitin, other
hemicelluloses, mannans and among the most interesting functional components, beta
glucans. These are the compounds that play a role in some healthy properties of
mushrooms (Manzi and Pizzoferrato, 2000). Edible mushrooms have been consumed by
humans not only as part of the normal diet to maintain health and increase longevity in
ancient civilizations, but also because they have a highly desirable taste and aroma
(Matilla, 2000). This is in agreement with Manzi et al. (1999), who described that,
mushrooms are undoubtedly consumed much more for their texture and flavor than for
their functional and medicinal properties. Most of the edible mushrooms, however, do
not have medicinal value (e.g. Agaricus bisporus) compared to some non-edible
mushrooms (e.g. Ganoderma, Coriolus) which have gained important medicinal usage
(Wasser and Weis, 1999a).
Experience from Asian and Eastern Europe countries shows that mushrooms
could play an important role in prevention and treatment of cancer (Lindequist et al.,
2005). While much attention has been drawn to various immunological and anti-cancer
properties of mushrooms, they also offer other potentially important therapeutic
properties including antioxidants, anti-hypertensive, cholesterol-lowering, liver
protection, anti-fibrotic, anti-inflammatory, anti-diabetic, anti-tumor, anti-viral, antimicrobial other beneficial or therapeutic health effects without any significant toxicity
3

(Wasser and Weis, 1999a). This agrees with the finding that some mushrooms popular
in Far East have great medicinal values which include anti-tumor, anti-viral and
hypolipidemic effects (Breene, 1990; Johl et al., 1995; Miles and Chang, 1997).
According to the studies conducted so far, medicinal mushrooms have a very
long tradition in the Asian countries, whereas their use in the Western hemisphere has
been slightly increasing only since the last decades (Lindequist et al., 2005). Wasser
(2002) described that the use of mushrooms as a functional food is most notable in the
East, where application of mushrooms to maintain health was formally recorded as
early as 100 AD in China. In traditional Chinese medicine (TCM), foods that have
medicinal effects have been documented since at least 1000 BC. For centuries, the
Chinese have understood that foods have both preventive and therapeutic effects and are
an integral part of health, a view that is now being increasingly recognized around the
world. Extracts from certain mushrooms were found to have profound health promoting
benefits and, consequently, became essential components in many traditional Chinese
medicines.
Asian countries are known to be rich sources of medicinal mushrooms. As a
result of large numbers of scientific studies on medicinal mushrooms especially in
Japan, China and Korea, over the past three decades, many of the traditional uses have
been confirmed and new applications developed (Wasser and Weis, 1999a). There are at
least 270 species of mushrooms that have been identified to possess various therapeutic
properties (Ying et al., 1987). The mushrooms which have demonstrated such potential
include Auricularia (mu-er), Flammulina (enokitake), Grifola (maitake), Hericium,
Lentinus (shiitake), Pleurotus (oyster), and Tremella (yiner). However, many of other
potential mushrooms have not been thoroughly studied yet.
For this reason, Schizophyllum commune was selected to be studied as it is a
popular edible mushroom among the Malay community in Malaysia. Though usually

considered as a widely distributed basidiomycetous pathogen (Hobbs, 1995; Rihs et al.,


1996; Sigler et al., 1997), S. commune has long been acknowledged for its medicinal
properties (Oso, 1981; Han et al., 2005). In addition to its medicinal value,
S. commune has been consumed as a nutritional food in South-East Asia (Han et al.,
2005) and is now being cultivated in Malaysia and Thailand. The production of
S. commune and other medicinal mushrooms has increased due to the ease of
cultivation, increase in popularity and its nutritional value (Chang and Buswell, 1996;
Wasser, 2002).
In the present study, S. commune was evaluated for its medicinal properties
since not many studies had been done on the medicinal properties other than anti-cancer
properties of its polysaccharides. Pharmacologically, S. commune is extremely
important because it produces the polysaccharide schizophyllan which shows
considerable medicinal properties. Based on the previous studies, the secondary
metabolites of S. commune were known to show anti-candida, anti-tumor and anti-viral
properties (Ooi and Liu, 1999; Wasser, 2002). However, published studies on these
mushrooms are quite limited. Hence, the biological activities of S. commune metabolites
need to be explored for its use as a source of drugs and functional food to contribute to
new therapeutic effects and for the treatment of a variety of human ailments.

1.1

Objectives of study
The present study was carried out to determine the biological activities of

S. commune. This study was undertaken with the following aims:


1) Screen S. commune extracts for anti-microbial activity.
2) Evaluate the antioxidant activity of S. commune extracts.

3) Determine the cytotoxicity of S. commune extracts against various cancerderived cell lines.
4) Investigate the anti-human papilloma virus activity of S. commune extracts
against cervical cancer cell lines.

CHAPTER 2: LITERATURE REVIEW


2.1

Introduction to medicinal mushroom


Mushrooms were originally defined as a macrofungus with a distinctive fruiting

body, which can either be hypogeous or epigeous, large enough to be seen with the
naked eye and to be picked by hand (Chang and Miles, 1992). Mushrooms constitute at
least 14,000 and perhaps as many as 22,000 known species, deduced from the
Dictionary of the Fungi (Hawksworth, 2001). Other study reported that the number of
mushroom species on earth is estimated to be 140,000, indicating that only 10% are
known (Lindequist et al., 2005).
Mushrooms have long been appreciated for their flavor and texture. Now they
are recognized as a nutritious food as well as an important source of biologically active
compounds of medicinal value (Breene, 1990). Mau et al. (2002) had reported in their
studies that medicinal mushrooms are commonly used for pharmaceutical purposes and
as health foods. Hence, searching for new biological activities and other medicinal
substances from mushrooms and to study the medicinal values of these mushrooms has
become a matter of great significance.
The scientific community, in searching for new therapeutic alternatives, has
studied many kinds of mushrooms and has found variable therapeutic activities such as
anti-carcinogenic, anti-inflammatory, immuno-suppressor and antibiotic effects (Asfors
and Ley, 1993). At present, there are at least 270 species of mushrooms that are known
to have various therapeutic effects (Ying et al., 1987).
The medicinal use of mushrooms has a very long tradition in the Asian countries
especially in Japan, Korea and China, whereas their use in the Western hemisphere like
United States has been slightly increasing only since the last decades. Increased
scientific and medical research in recent years and published in peer-reviewed journals,
several books and reviews, is increasingly confirming the medicinal efficacy and
7

identifying the biologically active compounds of medicinal mushrooms (Wasser and


Weis, 1999; Ooi and Liu, 2000; Lindequist et al., 2005).
Various pharmacological properties have been influenced by these medicinal
mushrooms. The biologically active substances are claimed to have profound health
promoting benefits such as antibiotics, anti-tumor, antiviral, anti-inflammatory,
bioregulation (immunological enhancement), maintenance of homeostasis, regulation of
biorhythm, cure of diseases such as cancer, cerebral stroke and heart diseases. It is also
being confirmed that mushrooms include effective substances for decreasing blood
cholesterol, the improvement of hyperlipidemia, antithrombotic, reduction of blood
pressure, hypoglycemic action and various other therapeutic applications (Chang et al.,
1989; Wasser and Weis, 1999). In fact, recent studies are now confirming their medical
efficacy and many of the bioactive molecules were identified (Wasser and Weis,
1999b).
Medicinal mushrooms accumulate a wide variety of bioactive compounds
including terpenoids, steroids, phenols, nucleotides and their derivatives glycoproteins
and polysaccharides that display a broad range of biological activities (Borchers et al.,
1999; Teissedre and Landrault, 2000). These different bioactive compounds have been
extracted from the fruiting body, mycelia and culture medium of various medicinal
mushrooms such as Lentinula edodes, Ganoderma lucidum, Schizophyllum commune,
Trametes versicolor, Inonotus obliquus and Flammulina velutipes (Wasser and Weis,
1999a).
Asian pharmaceutical companies have been producing medically important
polysaccharides include lentinan (L. edodes), schizophyllan (S. commune), PSK
(polysaccharide-K, commercially sold as krestin) and PSP (polysaccharopeptide)
(Trametes versicolor) and Grifron-D (Grifola frondosa) (Kidd, 2000).

Ganoderma is one of the predominat mushrooms which is highly valued as folk


medicine and functional food for its therapeutic effects; anti-tumor, anti-inflammatory,
antiviral, antibacterial, anti-parasitic, blood pressure regulation, cardiovascular
disorders, immunomodulating, kidney tonic, hepatoprotective, nerve tonic, sexual
potentiator and chronic bronchitis (Wasser and Weis, 1999). The mushroom Inonotus
obliquus has been traditionally used for the treatment of gastrointestinal cancer,
cardiovascular disease and diabetes (Huang, 2002). On the other hand, the medicinal
attributes of Lentinus edodes (shiitake) include anti-tumor, anti-microbial, liver function
improving and cholesterol lowering activity (Mizuno et al., 1995).
2.2

Schizophyllum commune Fr.

Kingdom

Fungi

Division

Eumycota

Subdivision

Basidiomycotina

Class

Hymenomycetes

Subclass

Holobasidiomycetidae

Order

Agaricales

Family

Schizophyllaceae

Genus

Schizophyllum

Schizophyllum commune Fr. was the mushroom selected to be studied for its
biological activity. The mushroom is probably the most widespread fungus in existence,
being found on every continent where there is wood to be used as a substrate. It is an
edible mushroom which belongs to the family Schizophyllaceae (Alexopoulos et al.,
1996). The family Schizophyllaceae contains only one genus; Schizophyllum and there
is a single common worldwide species, although there are a few less common species of

Schizophyllum. The genus name means "split gill," and thus it is called the split gill
fungus.
S. commune is one of the common gill-bearing bracket fungi of world-wide
distribution (Zoberi, 1972). It can be easily identified by the peculiar structure of its
gills which cover hymenium during unfavourable climatic conditions (Alexopoulos et
al., 1996). Its unique hymenium consists of thick gills (lamellae) which are split
longitudinally with both edges folded back. The gills function to produce basidiospores
on their surface.
S. commune has long been known as an emerging basidiomycetes pathogen in
the world wide distribution (Rihs et al., 1996). This mushroom has also been the object
of numerous studies, such as sexuality, genetics and morphogenesis (Alexopoulos,
1979). However, Oso (1981) reported that this edible mushroom has its significant
medical importance.
Previous studies showed that a 1-3, 1-6D-glucan called Schizophyllan
isolated from S. commune was found to be medically active in several therapeutics
effects such as antitumor, anticancer and immunomodulating activities (Kidd, 2000).
Schizophyllan is a non-ionic, water-soluble homopolysaccharide consisting of a linear
chain of -d-(1-3)-glucopyranosyl groups and -d-(1-6)-glucopyranosyl groups
produced by fermentation from filamentous fungi S. commune ATCC 38548 (Rau et al.,
1992). This polysaccharide has attracted attention in recent years in pharmaceutical
industry as immunomodulatory, antineoplastic and antiviral activities that are higher
than other glucans (Rau and Brandt, 1994). The bioactive compound prolonged survival
and time to recurrence for stage II cases but not stage III (Okamura et al., 1989;
Miyazaki et al., 1995) and showed added effectiveness when injected directly into the
tumor mass (Nakano et al., 1996). However, schizophyllan was found rather ineffective

10

against gastric cancer, but extended survival time in patients with head and neck cancer
(Brochers et al., 1999; Kimura et al., 1994).
S. commune is edible and the nutritional perspective of the mushroom was also
documented in several previous studies. The islanders in Indonesia and Madagascar
habitually chew carpophores of this mushroom. While the Yoruba people of SouthWestern Nigeria were reported to use S. commune to prepare delicious dishes among
them (Jonathan and Fasidi, 2001). S. commune was also one of the varieties of
mushrooms that consumed by the Naga tribes in Northeast India as food source
(Longvah and Deosthale, 1998).
2.3

Biological activity of mushrooms

2.3.1

Antimicrobial activity of mushrooms


Mushrooms are rich sources of natural antibiotics. Antibiotic can be defined as a

chemical substance produced by microorganisms (wholly or partly by chemical


synthesis) which has the capacity to inhibit the growth of bacteria and even destroy
bacteria and other microorganisms in dilute solution (Black, 2002).
A research showed that the most significant antibiotics have been derived from
fungi (Hardman et al., 2001) such as penicillin, streptomycin, chloramphenicol and
vancomycin (Griffin, 1994) where humans can benefit from the natural defensive
strategies of the fungi that produce antibiotics to fight infection from microorganisms.
Infections by multidrug resistant isolates of Candida spp., Staphylococcus epidermidis,
Staphylococcus aureus, Streptococcus spp., Enterococcus sp. and Escherichia coli,
among other, became more and more frequent stimulating the search for new antibiotics
with novel mechanisms of action (Kotra and Mobashery, 1998).
Many antibiotics are chemically modified biosynthetic form of microbial
products. Chloramphenicol is now usually produced by a synthetic process. Other well11

known commercial antibiotics like penicillin and cephalosporin are produced by semisynthesis which means that part of the molecules is produced by a fermentation process
before it is further modified by a chemical process (Anke et al., 1981).
Basidiomycete mushrooms are receiving attention as potential sources of new
classes of antibiotics with the development of new fermentation and purification
technologies (Anke, 1989; Suay et al., 2000). Sandven (2000) reported that the first
investigations on the potential of basidiomycetes as sources of antibiotics were
performed by Anchel, Hervey and Wilkins in 1941, when they tested extracts of fruiting
bodies and mycelia culture from over 2000 species. They succeeded in the isolation
and identification of pleuromutilin (Kavanagh et al., 1950), a diterpene that is especially
useful for the treatment of mycoplasms infections in animals (Brizuela et al., 1998) and
served for the development of the first commercial antibiotic of basidiomycete origin.
Another basidiomycete from the genus Marasmius from the family
tricholomataceae has long been recognized to produce interesting secondary metabolites
(Anke et al., 1980). As for the antimicrobial activity, scorodonin, a biologically active
metabolite from M. scorodonius was found to inhibit Gram-positive and Gram-negative
bacteria at rather high concentration. Two antimicrobial and cytotoxic metabolites
denominated alliacols A and B were isolated from M. alliaceus (Anke et al., 1981).
Abraham (2001) stated that a compound from M. conigenus known as marasmic acid
was shown to have antibacterial, antifungal, cytotoxic, phytotoxic properties.
Coprinol was another antimicrobial compound isolated from fermentation of the
mushroom Coprinus sp. This new antibiotic exhibited antimicrobial activity against
multidrug-resistant Gram-positive bacteria in vitro (Johansson et al., 2001).
The mushroom Trametes, on the other hand, contains coriolin, an antibiotic that
has been shown to inhibit Gram-positive bacteria and Trichomonas vaginalis (Ying et
al., 1987).

12

Other research showed that Agaricus campestris produces campestrin, a


compound which was effective against Gram-positive and Gram-negative bacteria
(Bose, 1946) while A. xanthoderma contains antibiotics Psalliotin, which were
inhibitors against Gram-positive bacteria.
Pycnoporus sanguineus has been known to show antimicrobial activity since
1946, when Bose (1946) isolated poliporin, a compound active against Gram-positive
and Gram-negative bacteria. From the study, it was found that a fraction of
P. sanguineus was more effective on Gram-positive cocci than on Gram-negative
bacilli.

This mushroom contained compounds with biological activity against

Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi,


Staphylococcus aureus and members of the genus Streptococcus (Smania et al., 1995).
Cinnabarin was one of the antibiotic substances produced by P. sanguineus. The
antimicrobial activity of this substance was tested against 11 species of bacteria isolated
from food. The results showed that Bacillus cereus and Leuconostoc plantarum were
the most sensitive to cinnabarin, while Klebsiella pneumoniae was the least sensitive
(Elza et al., 1998).
Another mushroom, Armillariella mellea showed antibiotic action (in vitro)
against pathogenic bacteria Staphylococcus aureus, Bacillus cereus and Bacillus subtilis
whereas armillaric acid isolated from this species inhibit Gram-positive bacteria and
yeast (Obuchi and Misato, 1990). The mycelial extracts of A. mellea were reported to
exhibit significant antibacterial activity against Gram-positive bacteria (Donelly and
Hutchinson, 1990).
The European Ganoderma species; Ganoderma pfeifferi produced new
sesquiterpenoid hydroquinones called ganomycin that have the ability to inhibit the
growth of methicillin-resistant Staphylococcus aureus and other bacteria (Mothana et
al., 2000).

13

Mushrooms need antibacterial and antifungal compounds to survive in their


natural environment. Hence, it is not surprising that antimicrobial compounds with more
or less strong activities could be isolated from many mushrooms and that they could be
of benefit for human (Lindequist et al., 1990).

2.3.1.1 Mechanisms of antimicrobial actions


Antimicrobials are originally classified by their modes of actions which
consisted of inhibition of cell wall synthesis, disruption of cell membrane function,
inhibition of protein synthesis, inhibition of nucleic acid synthesis and action as antimetabolites:
a)

Inhibition of cell wall synthesis


The cell wall synthesis inhibitors are bactericidal because they block the

synthesis of rigid peptidoglycan component of the wall, results in growing cell lyse and
die. Inhibition of cell wall synthesis selectively damages bacterial and fungal cells.
Gram-positive bacterial cells have a high internal osmotic pressure. Without a normal,
sturdy cell wall, these cells burst when subjected to the low osmotic pressure of body
fluids. Antibiotics such as penicillin and cephalosporin contain a chemical structure
called -lactam ring, which attaches to the enzymes that cross-link peptidoglycans
(Black, 2002).
b)

Disruption of cell membrane function


Bacterial lipids are mainly phospholipids and this component has been the target

of cell membrane function inhibitor antibiotics. This action is especially effective


against Gram-negative bacteria, which have an outer membrane rich in phospholipids.
Certain polypeptide antibiotics such as polymyxins act as detergents and distort the
bacterial cell membranes, probably by binding to phospholipids in the membrane. The
14

membrane is then no longer regulated by membrane proteins and the cytoplasm and cell
substances are lost (Black, 2002).
c)

Inhibition of protein synthesis


Almost all antibiotics that inhibit protein synthesis act on ribosomes by

preventing the performance of a typical ribosomal function (Braude, 1976).


Aminoglycoside antibiotics, such as streptomycin, act on the 30s portion of bacterial
ribosomes by interfering with the accurate reading (translation) of the mRNA message.
Other antibiotics; chloramphenicol acts on the 50s portion of bacterial ribosomes, thus
inhibiting the formation of the growing polypeptide (Black, 2002).
d)

Inhibition of nucleic acid synthesis


There are two main categories that describe the substance that inhibit nucleic

acid synthesis. The first group is those that interfere with the building block of nucleic
acids, which are purine and pyrimidine nucleotides. Others interfere with the
polymerization of the nucleotide and the template function of DNA in RNA synthesis
and hinder the function of polymerases via direct interaction with the enzymes (Franklin
and Snow, 1975). Antibiotics of the rifamycin family have been known to bind to a
bacterial RNA polymerase and inhibit RNA synthesis (Black, 2002).
e)

Action as anti-metabolites
Antimetabolites are substances that affect the utilization of metabolites and

therefore prevent a cell from carrying out necessary metabolic reactions. This is done by
competitively inhibiting enzymes and by being erroneously incorporated into important
molecules such as nucleic acids. The action is possible as they are structurally similar to
normal metabolites and are even called molecular mimicry as they imitate the normal
metabolites, thus preventing a reaction to occur (Black, 2002).
15

2.3.2

Antioxidant activity of mushrooms


In recent years, there has been an increased interest in the application of

antioxidants to medical treatment, as information is available linking the development


of human diseases to oxidative stress (Zheng and Wang, 2001). An antioxidant has been
defined as any substance that when present at low concentrations compared to those of
an oxidizable substrate, significantly delays or prevents oxidation of the substrate
(Benzie et al., 1999).
Numerous physiological processes in living organisms occasionally produce
oxygen-centered free radicals and other reactive oxygen species (ROS) as byproducts.
However, the uncontrolled production of these free radicals and ROS, can result in cell
death and tissue damage (Cheung and Cheung, 2005). Oxidative damage caused by free
radicals may be related to aging and chronic degenerative diseases, such as diabetes,
cancer, arthritis, cardiovascular disease, Alzheimer, Parkinson, Down syndrome and
multiple sclerosis.
2.3.2.1 Free radicals and reactive oxygen species
A free radical is any atom or molecule that contains one or more unpaired
electrons (e) (Halliwell, 1994; Anderson, 1996; Lo and Cheung, 2005). With the
possession of the unpaired electrons, free radicals are usually unstable and highly
reactive (Lo and Cheung, 2005). These unpaired electrons alter the chemical reactivity
of an atom or molecule, thus making it more reactive than the corresponding nonradical (Anderson, 1996). Free radicals produced by radiation, chemical reactions and
several redox reactions of various compounds (Halliwell, 1996) are implicated in a wide
variety of pathological effects, such as atherosclerosis, respiratory tract disorders,
neurodegenerative disease, inflammatory bowel disease, cancer and ageing (Anderson,

16

1996). The most common free radicals species are the superoxide radical, peroxide
radical, reactive nitrogen radical and nitric oxide (Halliwell, 1996).
On the other hand, reactive oxygen species are produced by sunlight, ultraviolet
(UV), ionizing radiation, chemical reactions and metabolic process (Liu et al., 1996),
that when present in high concentration, can become toxic (Ferreira et al., 2006).
Almost all organisms posses antioxidant defences and repair systems that have
evolved to protect them against oxidative damage. For example, mammalian cells
possess intracellular defences such as superoxide dismutase, catalase or glutathione
peroxidase, in order to protect the cells against excessive levels of free radicals (Ferreira
et al., 2006). However, these systems are insufficient to prevent the damage entirely
(Simic, 1988). Thus, exogenous addition of dietary antioxidants is beneficial as possible
protective agents to help the human body reduce the oxidative damage (Mau et al.,
2002). As antioxidants scavenge free radicals and oxidants, the assumption is
antioxidants in diet may therefore prevent diseases.
2.3.2.2 Antioxidants
The carcinogenic properties and fewer side effects that have been reported for
some synthetic antioxidants make them less acceptable. The most widely used synthetic
antioxidants are butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and
tert-butylated hydroxyquinone (TBHQ), which are applied in fat and oily foods to
prevent oxidative deterioration (Lliger, 1991). However, these commercial
antioxidants have been restricted recently because of serious concerns about their
carcinogenic potential (Botterweck et al., 2000). Therefore, there is great interest in
finding new and safe antioxidants from natural sources. The use of natural antioxidants
as food additives to inactivate free radicals has received much attention nowadays, not
only for their ability to protect the human body from free radicals (Kinsella et al.,

17

1993), but also because they are natural, non-synthetic and their appreciation by
consumers is very favorable (Miliauskas et al., 2003).
Natural food usually contains natural antioxidants that can scavenge free
radicals in the prevention of vascular diseases, some forms of cancer and oxidative
stress responsible for DNA, protein and membrane damage. Small molecules dietary
antioxidants, such as vitamin C, E and carotenoids have generated particular interest as
defences against degenerative diseases (Byers and Guerrero, 1995). Other than the
antioxidant vitamins, phenolic components also contribute to the antioxidative effects.
Phenolics are one of the major groups of non-essential dietary components that have
been associated with the inhibition of artherosclerosis and cancer. Some studies have
even indicated that phenolic substances, such as flavonoids and phenolic acids are
considerably more potent antioxidants than vitamin C and E (Vinson et al., 1995; Cao et
al., 1997). Phenolics such as butylated hydroxytoluene (BHT) and gallic acids are also
known to be effective antioxidants (Madhavi et al., 1996).
2.3.2.3 Mushrooms as antioxidants
Studies showed that mushrooms have been known to contain various
polyphenolic compunds recognized as an excellent antioxidant and synergist that is not
mutagenic (Ishikawa et al., 1984). Some common edible mushrooms, which are widely
consumed in Asian cultures, have currently been found to possess antioxidant activity,
which is well correlated with the content of their total phenolic compounds (Cheung et
al., 2003).
Apparently, the highest total phenolic content in Ganoderma species is the key
components accounting for their excellent results in antioxidant activity (Mau et al.,
2002). Polysaccharides are among naturally occurring substances from mushrooms that
may also be one of the useful candidates in the search for effective, non-toxic
substances with free radical scavenging activity. Based on the previous study,
18

polysaccharides extracts from Ganoderma lucidum and Grifola umbellata were reported
to exhibit strong scavenging effects on superoxide and hydroxyl radicals (Hu et al.,
1992; Liu et al., 1996). Liu et al. (1997), in their studies suggested that the extracts of
G. lucidum can strongly remove the hyperoxide radical which is thought to be one of
the main factors in the aging process. Ganoderma tsugae was also proven to be a good
candidate of antioxidant agent as it exhibited substantial antioxidant and radical
scavenging activities which was also higher than the positive control, -tocopherol (Yen
and Wu, 1999). A study by Mau et al. (2002), showed that, among the several medicinal
mushrooms species tested, G. lucidum, G. lucidum antler and G. tsugae showed an
excellent antioxidant activity. In addition, many researchers proved the effectiveness of
G. lucidum and G. tsugae in antioxidant activities (Yen and Wu, 1999; Mau et al., 2002
and Yang et al., 2002).
Lentinus edodes and Volvariella volvacea were another mushrooms found to
have antioxidative activities which were correlated with their phenolic contents
(Cheung et al., 2003). Cheung et al. (2003) also presented that L. edodes showed the
most potent radical scavenging activity in each antioxidant assay tested. This was
supported by the largest amount of phenolics found in the extracts.
Huang (2000) found that extracts from the medicinal mushroom Antrodia
camphorata (Chang-chih), showed excellent antioxidant activities. Agaricus blazei
(Brazilian mushroom), another medicinal mushroom, was also reported to show a high
antioxidant activity. An excellent scavenging effect of A. camphorata was also
presented by another study of Huang in 2004.
The natural extracts of Pleurotus florida possessed significantly higher
antioxidant activity than the synthetic antioxidant agent, catechin, which was used as a
standard in a study by Jose and Janardhanan, 2000.

19

A relatively strong antioxidant effect was observed with polyphenolic extract of


Inonotus obliquus and the extract was confirmed to contain triterpenoids and steroids
(Cui et al., 2005).
2.3.3.4 Mechanisms of antioxidant actions
Antioxidant actions in vivo or in food may be through inhibiting generation of
ROS, or by direct scavenging of free radicals (Aruoma, 2003). The phenolic compounds
of mushrooms are the type of antioxidant that possess a strong inhibition effect against
lipid oxidation through radical scavenging (Frankel et al., 1993). The bioactivity of
these phenolics may be related to their ability to chelate metals, inhibit lipoxygenase
and scavenge free radicals (Decker, 1997). Hirano et al. (2001) reported that phenolics
scavenge free radicals by single-electron transfer.
Antiradicalar antioxidants such as phenols act by donating hydrogen atoms to
lipid radicals. The molecular structure of phenols is the possible reason that makes the
compounds stable and will then stop the oxidation chain reaction (Bondet et al., 1997).
Free radicals and reactive oxygen species like superoxide radicals, hydrogen
peroxide, hydroxyl radicals and singlet oxygen (Halliwell, 1996) are known to be
generated as by-products of normal metabolism. The life-long exposure of these
oxidants, however, can generate lipid peroxidation and lipoprotein oxidation which can
contribute to cancer and artherosclerosis respectively. Thus, in order to induce the
formation of the antioxidant radicals, a process called autooxidation is performed.
Autooxidation or also known as lipid oxidation is a slow radical process which
proceeds via a chain reaction including induction, propagation and termination (BrandWilliams et al., 1995; Bondet et al., 1997). The general mechanism of the process
requires oxygen (O2), a free radical initiating species (R) and the presence of an
unsaturated double bond on the lipid (L).

20

Induction

LH + R L + RH

Propagation L + O2 LO2
LO2 + LH LOOH + L
Alkyl and peroxyl radicals are usually formed during the induction period.
These highly reactive chemical species will then undergo reaction with O2 molecules to
form peroxide radicals and hydroperoxides (ROOH) (Brand-Williams et al., 1995;
Bondet et al., 1997). As from the reactions above, the free radical and lipid
hydroperoxide (LOOH) are produced. But the presence of antioxidants (A) will bind to
lipid peroxyl radical (LO2) and yields the radical (A) that has much longer half life
than the lipid peroxyl radical. This is in the agreement with the study done by BrandWilliams et al. 1995, where they reported that lipid is resistant to oxidation in the
presence of potential antioxidants. Termination is the final step when the two radicals
are associated together to form more stable products (Bondet et al., 1997).
LO2 + AH LOOH + A
Termination A + A A-A

2.3.3

Cytotoxicity of mushrooms
Tumor diseases are one of the main causes of death worldwide. The vast

structural diversity of natural compounds found in mushrooms, provide potential


opportunities for discovering new drugs that rationally target the abnormal molecular
and biochemical signals leading to cancer. Experience from ethnomedicine and
extensive basic laboratory findings have shown that mushrooms could play an
important role in prevention and treatment of cancer (Russo et al., 2006). In addition, it
has been reported that the most significant medicinal effect for which mushrooms and

21

their metabolites that attracted the attention of the public is their anti-tumor activity
which includes cytotoxicity.
Cytotoxicity is one of the chemotherapeutic targets of anti-tumor activity.
Cytotoxicity was defined as having the toxic effect on target cells (Martz, 1993). The
cytotoxic effects include mild cytotoxic, such as slowing or halting effect on growth;
impairment of metabolite activities; permanent loss of the ability of a cell to divide and
irreversible cessation of respiration and energy metabolism. The greatest cytotoxic
effect is breaking down of the cell membrane and dissolution of cell structure (Martz,
1993).
Previously, it was reported that metabolites from different biological origins,
e.g. yeast, algae, bacteria, higher plants and especially mushrooms, have been
investigated for anti-tumor and immunomodulating activities in view of developing new
anti-tumor compounds with low toxic potential (Chihara, 1992; Kraus and Franz, 1991).
Intensive search for anti-tumor agents from mushrooms have been found to possess
anti-tumor activity (Wasser and Weis, 1999). This is supported by Hata (1997) who
indicated that a large number of anti-tumor agents was produced by mushrooms.
Mushrooms contain an unlimited source of anti-tumor and immunostimulatory
metabolites which are mostly non-toxic (Wasser and Weis, 1999). The metabolites
which include polysaccharides and polysaccharide-protein complexes are the ideal
chemotherapeutic agent against cancer because they are non-toxic and their anti-cancer
effects are mainly host mediated (Sarangi, 2006). Experience from Asian and Eastern
Europe countries shows that mushrooms were a good source as anti-tumor agents where
they could play an important role in cancer chemoprevention (Lindequist et al., 2005).
Chemoprevention includes the use of natural, dietary or synthetic agents or their
mixtures to delay, inhibit or reverse the development of cancer before malignancy
occurs (Chang et al., 2000). Other studies showed that mushroom polysaccharides are

22

being used as one of the major sources of therapeutic agents for immunomodulatory
(Wasser, 2002; Mizuno, 1999) and have also been considered to be one of the useful
anti-tumor agents for clinical uses (Franz, 1989).
The main source of mushroom anti-tumor polysaccharides appears to be fungal
cell walls that consist of polysaccharides such as chitin and cellulose (Stone and Clark,
1993). Polysaccharides are not the only active compounds found in mushrooms, nor do
they show only anti-tumor activity. The secondary metabolites, which are the smaller
compounds, such as terpenes and steroids, have also been found, and some of these
have shown anti-tumor activity (Lindequist, 1990).
The anti-tumor activity of mushrooms was first demonstrated by Lucas and his
collaborators in 1957 while employing extracts of fruiting bodies of Boletus edulis in
tests against sarcoma 180 in mice. In 1959, Calvacin was isolated by them from the
giant puffball and in 1960s, it became the most commonly cited natural product isolated
from the medicinal mushroom and was broadly used in many laboratories as an antitumor agent (Wasser and Weis, 1999).
In 1999, Wasser and Weis also presented a list of 24 species of mushrooms
having anti-tumor effects. Earlier, Chihara (1992), listed 17 mushrooms which have
been tested for anti-tumor activity against sarcoma 180 tumor in mice. In particular, and
most importantly for modern medicine, they represented an unlimited source of
polysaccharides with anti-tumor and immunostimulating properties. Many, if not all,
basidiomycete mushrooms contain biologically active polysaccharides in fruit bodies,
cultured mycelium and culture broth. Data on mushroom polysaccharides have been
collected from 651 species and 7 infraspecific taxa from 182 genera of higher Heteroand

Homobasidiomycetes.

These

polysaccharides

are

of

different

chemical

composition, with most belonging to the group of -glucans; these have -(1 3)
linkages in the main chain of the glucan and additional -(1 6) branch points that are

23

needed for their anti-tumor action. Yoshida and his group, Gregory and his group,
Ikekawa and co-workers discovered the anti-tumor activity of 50 cultures representing
22 species of higher basidiomycetes.
Although more than fifty mushroom species have yielded potential
immunoceuticals that exhibit anti-tumor activities, at least six polysaccharides (lentinan,
schizophyllan, polysaccharide-K (PSK), polysaccharide-P (PSP), active hexose
correlated compounds (AHCC), maitake D fraction) have been investigated in human
cancer (Ooi and Liu, 1991; Kidd, 2000). In the 1970s and 1980s, three anti-tumor
polysaccharides; lentinan, schizophyllan and protein-bound polysaccharide (or
polysaccharopeptide) were isolated from Lentinus edodes, Schizophyllum commune and
Coriolus versicolor, respectively, and since then, have become large nutraceutical and
pharmaceutical compounds (Mizuno et al., 1995b).
Several anti-tumor polysaccharides such as hetero--glucans and their
complexes as well as dietary fibers, lectins and terpenoids, have been isolated from
medicinal mushrooms. Polysaccharides and triterpenes are two major physiologically
active constituents of fungi which are cytotoxic and candidates for anti-tumor agents
(Kaul, 1997).
The polysaccharide carcinostatic agents that have been developed and
commercialized using submerged cultured are mycelial biomass of Trametes versicolor,
fruiting bodies of Lentinula edodes, Inonotus obliquus, Agaricus blazei and liquid
cultured broth product of Schizophyllum. The polysaccharides from Lentinula edodes
lentinan (-D-glucan) from fruiting body has shown prominent activity and in
preventing chemical and viral oncogenesis (Wasser and Weis, 1999).
Some terpenoids and their derivatives isolated from Polyporales and
Ganodermatales were found cytotoxic and thus candidates for anti-tumor agents (Kaul,
1995). Fruting bodies and mycelia of Ganoderma lucidum and Ganoderma applanatum
24

produced about 100 different triterpenoids (Wasser and Weis, 1999). It has been
reported that some triterpenoids (ganoderic acid R, -T, -U, -V, -W, -X, -Y and Z)
isolated from cultured mycelia of G. lucidum showed a cytotoxicity-based carcinostatic
effect on hepatoma cells in vitro (Mizuno, 1992).
Chen and Miles (1996) presented a comprehensive review of research and
application of bioactive components from G. lucidum. G. lucidum, which belongs to the
Polyporaceae family, is currently being used as a dietary supplement in the form of
spores, fruiting body or mushroom extract. This mushroom contains a large variety of
biologically active polysaccharides and triterpenes with anti-tumor activities (Lin and
Zhang, 2004). The most important bioactive polysaccharides from G. lucidum - -1-3, 1-6 D-glucan is -1-3-D glucopyronan with 1-15 units of -1-6 monoglucosylside
chain. Of particular interest is the fact that Ganoderma is a rich source of bitter
triterpenes. Indeed, about 100 different triterpenes, which have a lanostane skeleton,
were found in the fruiting bodies and mycelia of G. lucidum (Jong and Birmingham,
1992; Wasser and Weis, 1999). The mushroom is best known for managing types of
cancer in combination with conventional therapy and for its anti-HIV effects. Studies
also presented that some of the triterpenes isolated from G. lucidum demonstrated
cytotoxicity against mouse cancer cells in vitro (Min et al., 2000) and inhibited the
growth and cancer metastases in mice (Kimura et al., 2002). Recent studies have shown
that G. lucidum induces apoptosis, inhibits cell proliferation, and suppresses cell
migration of highly invasive human breast and prostate cancer cells (Sliva et al., 2002;
Jiang et al., 2004). Hu et al. (2002), described that, the anti-tumor effects of G. lucidum
have been implicated in the inhibition of post-translational modification of Ras
oncoprotein (Lee et al., 1998), the induction of cell cycle arrest by down-regulating
cyclin D1, and the induction of apoptosis by up-regulating a pro-apoptotic Bax protein
in breast cancer cells.
25

Lectins are another group of fungi bioactive compounds. They are defined as
carbohydrate-proteins of non-immune origin which agglutinate cells or precipitate
polysaccharides or glycoconjugates. Some lectins have been shown to have anti-tumor
and immunomodulatory activities. Studies showed that lectin of some fungi including
Grifola frondosa against HeLa/cells is the result of the binding of the lectin to the
carbohydrate domains of the cells and independent of aggregation of the cells by lectin
(Wasser and Weis, 1999).
The mushroom Pleurotus has been reported to be a good candidate for antitumor activity. Based on the previous study, in vitro glucan isolated from certain
species of Pleurotus was found to enhance the activity of lymphokine activated killer
cells (LAK) and natural killer cells (NK) on tumor cells. Some of the Pleurotus
synthesize bioactive components of terpene origin with anti-carcinogenic and antibiotic
activities (Wasser and Weis, 1999).
A recent study has reported that the ethylacetate extract of the sporocarps of
Phellinus rimosus (Berk.) Pilt possesses significant anti-tumor activity in ascites and
solid tumor models in mice (Ajith and Janardhanan, 2003). A protein bound
polysaccharide (PBP) isolated from Phellinus linteus was also found to exhibit antiproliferative effect on SW480 human colon cancer cells mediated by inducing apoptosis
and G2/M cell-cycle arrest with a decrease of Bcl-2 expression, an increase of
cytochrome c release and reduced cyclin B1 expression (Li et al., 2004).
Duchesnea chrysantha, which belongs to the Rosacae family, is a medicinal
plant and has traditionally been used to treat several kinds of illnesses such as
congenital fever, toothache, and cancers in Korea, and its water extracts are edible and
non-toxic. Phenolic compounds of D. chrysantha exert cytotoxic activities in human
cancer cells (Lee and Yang, 1994). The majority of the compounds isolated from
Duchesnea are biologically active lectin and polysaccharides with antioxidative (Kim et

26

al., 2002) and immunostimulatory properties which contribute to their anticancer


effects.
In addition, a water-soluble -glucan isolated from Poria cocos was shown to
have growth-inhibitory effects on human breast carcinoma MCF-7 cells mediated by
cell-cycle arrest and apoptosis induction (Zhang et al., 2006)

2.3.3.1 Mechanisms of anti-tumor actions


The mechanism of mushroom anti-tumor actions is still not completely
understood. Pezzuto (1995) reported that various group of compounds that have been
identified as cancer chemopreventive agents have different mechanism of actions
depends on the compounds themselves.
Anti-tumor polysaccharides from medicinal mushrooms have been described to
enhance various immune responses in vivo and in vitro, and act as primarily modifiers
of biological response (Ooi and Liu, 1999). Several reports described that some
polysaccharides or polysaccharideprotein complexes from medicinal mushrooms do
not attack cancer cells directly, but produce their anti-tumor effects by activating
different immune responses in the host (Mizuno, 1999; Wasser and Weis, 1999). These
polymers interact with the immune system to up-regulate or down-regulate specific
aspects of the response of the host and this may result in various therapeutic effects
(Bohn and BeMiller, 1995). They are also able to exert anti-tumor activity through the
stimulation of the host's defence mechanism (Mizuno, 1999; Wasser and Weis, 1999).
Generally, several of these compounds have been shown to potentiate the hosts innate
(non-specific) and acquired (specific) immune responses and to activate many kinds of
immune cells that are important for the maintenance of homeostasis, e.g. host cells
(such as cytotoxic macrophages, monocytes, neutrophils, natural killer cells, dendritic

27

cells) and chemical messengers (cytokines) that trigger complement and acute phase
response (Li, 1999; Ooi and Liu, 2000).
Sarangi et al. (2006) described that immunomodulation is important in
enhancing the number and vitality of natural killer (NK) cells. NK cells are best known
for their capacity to kill tumor cells and there are evidences for their role in controlling
infection in the earliest phases of bodys immune responses. One of the primary
objectives of immunomodulation is to enhance the number and vitality of NK cells
(Sarangi, 2006).
Lentinan, the polysaccharide of Lentinula edodes, has been shown to enhance
host resistance against infections from viruses as well as from various bacteria, fungi
and parasites. It is able to restore and increase responsiveness of host cells, but it has no
direct cytotoxicity against tumors. The anti-tumor action of lentinan requires an intact
T-cell component and that the activity is mediated through a thymus-dependent immune
mechanism (Ooi and Liu, 1992). The anti-tumor activity of lentinan can also be
inhibited by pretreatment with antimacrophage agents. This may be possibly due to
potentiation of the response of precursor T-cells and macrophages to cytokines
produced by certain group of lymphocytes after specific recognition of tumor cells
(Chihara, 1992). In addition, it is also reported that the delayed-type hypersensitivity
response at the tumor sites induced by lentinan and the subsequent infiltration of
immune effector cells, such as natural killer cells and cytotoxic T lymphocytes, into the
tumor burden are an important mechanism of the anti-tumor action of lentinan (Ooi and
Liu, 1992). Schizophyllan, a polysaccharide from S. commune, is similar to lentinan in
composition and biological activity, and it was researched that the mechanism of antitumor action appears to be quite similar as well (Jong et al., 1991).
Other possible mechanism of action that may be shown by anti-tumor agents is
apoptosis. It has been shown that mushroom polysaccharides exhibited direct inhibitory

28

effects on cancer cell growth by modulating cell-cycle progression and inducing


apoptosis (Wang et al., 2002). Apoptosis is a program for the elimination of cells
initiated by specific biological signals (Kabsch and Alonso, 2002) that is tightly
regulated by a number of gene products that either promote or block cell death at
different stages of the cell cycle. In other word, apoptosis can be defined as a
genetically regulated form of cell death responsible for the ordered disposal of
superfluous, aged, or damaged cells. Disturbances in apoptosis regulation can result in
clinical disorders such as inflammatory, cancer, autoimmune, and neurodegenerative
diseases (Thompson, 1995).
There are many types of apoptotic pathways that contain a multitude of different
biochemical components and many of them have not yet understood. Key features of
this process include cell shrinkage, membrane blebbing and formation of apoptotic
bodies without membrane disruption, chromatin condensation and oligonucleosomal
fragmentation of chromosomal DNA, and activation of a family of caspases (Russo et
al., 2006). However, two main apoptotic pathways have been identified namely the
extrinsic death receptor pathway and intrinsic mitochondrial pathway (Fesik, 2000;
Hengartner, 2000) (Figure 2.1 and 2.2). In the former pathway (extrinsic) (Figure 2.1)
receptors are activated specifically by their cognate ligands, for example, tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL) or FasL. This death activator
binds to integral membrane protein receptor, Fas leading to activation and clustering of
the death receptors (Schneider et al., 1997). Figure 2.1 shows the example of the
apoptosis triggered by external signals. When cytotoxic T cells recognize their target,
more FasL are produced at their surface. This death activator binds with the Fas on the
surface of the target cell leading to its death by apoptosis.
The apoptosis triggered by internal signals or the intrinsic pathway (Figure 2.2),
on the other hand, greatly depends on the balance between the pro-apoptotic proteins,

29

such as Bax, and anti-apoptotic proteins such as Bcl-2. The Bax/Bcl-2 ratio has been
shown to be critical in determining the susceptibility of cells to induced apoptosis
(Raisova et al., 2001). Bax affects the mitochondrial membrane permeability that results
in the release of cytochrome c, apoptosis-inducing factor (AIF), and other pro-apoptotic
molecules from mitochondrial intermembrane space into the cytoplasm (Marzo, 1998).
This cytochrome c binds to the apoptosis protease activating factor-1 or Apaf-1 and to
caspase-9 forming the apoptosome complex (Kabsch and Alonso, 2002). This activated
caspase then mediates proteolysis of specific proteins and results in an irreversible autodigestion of proteins and DNA leading to the morphological and biochemical changes
associated with apoptosis (Shi, 2002).
From these findings, the anti-tumor activities of mushroom are not only
mediated by the immunopotentiation (Zaidman et al., 2005), but can also be resulted
from a direct inhibition on the tumor cells.

Figure 2.1: The extrinsic death


receptor pathway

2.3.4

Figure 2.2: The intrinsic mitochondrial


pathway

Antiviral activity of mushrooms


The development of new antiviral drugs is difficult, taking into account the poor

selective toxicity and fast selection of resistant viral variants with the existing drugs.
30

Traditional medicines utilizing natural products have been shown to contain antiviral
compound in vitro (Yao et al., 1992). Higher fungi have been tested for antibacterial
activity but only recently, it has been realized that some mushrooms have been proven
to possess antiviral activity. Medicinal mushrooms that have been ingested for
hundreds, and in some cases, thousands of years, have strong support for their nontoxicity, making them appealing candidates in the search for new antiviral agents.
Summaries of the antiviral properties of mushrooms were published by Suay et al.
(2000), Brandt and Piraino (2000) and Stamets (2001, 2002). Only recently, a numerous
number of mushrooms have been found to be medically active in several therapies, such
as antiviral, anti-tumor and immunomodulating treatments (Wasser and Weis, 1999).
Anti-viral polysaccharides from medicinal mushrooms have been identified
previously; lentinan from shiitake, Lentinula edodes (Sarkar et al., 1993);
polysaccharide peptide (PSP) from Turkey tail, Trametes versicolor and ganaderiol-F,
ganoderic acid-, lucidumol from reishi, Ganoderma lucidum. Polysaccharides are a
complex group of macromolecules possessing a wide range of therapeutically important
biological properties and known to affect the growth of animal viruses (Shannan, 1984).
An extract of the edible Japanese mushroom Lentinus edodes has been found to
inhibit the replication of herpes simplex virus (HSV), western equine encephalitis virus,
poliovirus, measles virus, mumps virus (Sorimachi et al., 1990; Sarkar et al., 1993), and
human immunodeficiency virus (Tochikura et al., 1988; Suzuki et al., 1990).
Medicinal mushrooms have gained much attention as antiviral agents. A number
of unique anti-viral agents from mushrooms have shown efficacy in inhibiting the
replication of the human immunodeficiency virus (HIV) (Kim et al., 1994).
Sulfated lentinan (Lentinula edodes) and sulfated schizophyllan (Schizophyllum
commune) were reported to show strong anti-HIV activities although their anti-tumor
activity is abolished (Ito et al., 1990; Hirata et al., 1994). The degraded form of

31

schizophyllan (Muenzberg et al., 1995) and the extracts from Ganoderma lucidum
(Bang, 1995) were reported to have anti-human immunodeficiency virus (HIV) activity.
Further studies on Ganoderma indicate that triterpenes are compounds that have
been found to have pharmacological activities. These triterpenes have been tested and
found to be beneficial as antivirals and as anti-inflammatories. Triterpenic acids from
Ganoderma lucidum act on the cell membrane and are able to disturb the entry of a
virus into the host cell and thus may have an effect on HIV. Luu (1995) studied the
effects of 13 fractions of G. lucidum extracts and found one which inhibited reverse
transcriptase. G. lucidum is best known for managing type of cancer in combination
with conventional therapy and for its anti-HIV effect (Chen and Miles, 1996).
Eo et al. (1999) found an antiviral activity from the methanol-soluble fractions
of Reishi mushrooms (Ganoderma lucidum), selectively inhibiting Herpes simplex and
the vesicular stomatitus virus (VSV). Research also showed that extracts of Reishi
prevented the death of lymphocytes infected with HIV and inhibited the replication of
the virus within the mother and daughter cells (Kim et al., 1994).
Sarkar et al. (1993) identified an antiviral substance resident in an extract of
shiitake (Lentinula edodes) mushrooms. Lentinan from L. edodes has the ability to
prevent chemical and viral oncogenesis (Wasser and Weis, 1999). Efficacy of lentinan
against other viral infections has also been recorded. There have been numerous reports
that lentinan is effective against AIDS (Kaneko and Chihara, 1992).
It is believed that, lentinan, when used in combination with azidothymidine
(AZT), suppressed the surface expression of HIV (human immunodeficiency virus) on
T-cells more than AZT did alone (Wasser and Weis, 1999). Lentinan reduces the
toxicity of AZT a commonly used drug for HIV carriers and AIDS patients. It also
stimulates the production of T lymphocytes and natural killer cells and can potentiate
the effect of AZT in the anti-viral treatment of AIDS. Other study by Tochikura et al.

32

(1987) stated that pretreatment of the AIDS with an extract of Lentinula edodes blocked
infection of the target cells.
Collins and Ng (1997) identified a polysaccharopeptide inhibiting HIV type 1
infection from Turkey tail (Trametes versicolor) mushrooms while Mizuno et al. (1996)
noted that crude fractions from chaga (Inonotus obliquus) also showed anti-viral
activity against HIV.
More recently, derivatives of the Gypsy mushroom, Rozites caperata, were
found by Piraino and Brandt (1999) to have significant inhibition against the replication
and spread of varicella zoster (the `shingles` and `chickenpox` virus), influenza A, and
the respiratory syncytial virus but not against HIV and other viruses. An anti-viral agent
of this Gypsy mushroom, RC-183, has also been found to possess activity inhibiting in
vitro the herpes simplex I and II viruses (Piraino and Brandt, 1999).
2.3.4.1 Human papillomavirus (HPV)
The specific virus that was studied in this preliminary research is sexually
transmitted human papilloma virus. There are not much studies have been recorded on
the anti-human papilloma virus of medicinal mushrooms.
Papillomaviruses are a diverse group of viruses that have been found in more
than 20 different mammalian species, as well as in birds and reptiles (Doorbar, 2005)
and emerge as the most common carcinoma viruses (zur Hausen, 1989). Human
papillomaviruses (HPVs) are small nondeveloped and icosahedral DNA viruses that
infect basal proliferating epithelial cells of either the skin or mucosa, of which
approximately 100 different HPV types have been described (de Villiers et al., 2004).
They are classified as the genus Papillomavirus of the papoviridae family and called
papillomaviruses because certain types may infect the genital area and may cause warts
or papillomas which are benign (non cancerous). Papillomaviruses were first recognized
as an etiology agent of genital warts in the 1970s.
33

HPVs are closely associated with certain human cancers (Levine, 1988).
Epidemiological and clinical studies implicate HPV in the etiology of a variety of
squamous epithelial tumors (Howley, 1991) Anogenital tumors, in particular carcinoma
of the uterine cervix, are strongly associated with infection by subgroup of the genital
HPV. Overwhelming epidemiologic evidence strongly supports the role of human
papillomaviruses (HPV) in cervical carcinogenesis (Munger, 2002). In addition to this
epidemiologic data, molecular evidence supports an etiologic role for HPV in the
genesis of cervical cancers. HPV DNA sequences have been commonly identified in
human cervical cancer biopsy specimens (Gissmann et al., 1983), with specific HPV
subtypes identifiable with malignant lesions of the cervix (Drst et al., 1983).
Certain types of HPVs are implicated the main cause of cervical cancer and its
precursor lesions (Van den Brule et al., 1991; Munoz et al., 1992; Rolon et al., 2000).
This is in agreement with zur Hausen (1996), stating that specific types of HPVs have
been identified as causative agents of at least 90% of cervical cancer and are also linked
to more than 50% of other anogenital cancers. The original definition of specific HPV
types as high risk viruses was based on their oncogenic potential and frequent presence
in cervical and anogenital cancers (zur Hausen, 1996). High risk viruses were shown to
immortalize human keratinocytes (Drst et al., 1987) but low risk viruses failed to do
so.
HPVs of the high-risk group are associated with squamous intraepithelial lesions
with a high potential for progression to invasive squamous cell carcinoma, whereas
HPVs of the low-risk group cause benign hyperplasias (Howly and Lowey, 2001).
These high risk viruses include HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,
68 and 69. The HPV types most commonly found in these tumors are 16, 18, 31, 33 and
45 in descending order of frequency. It appears at present that 50% to 70% of all tumors
are positive for these viruses.

34

The vast majority of invasive cervical cancers contain high-risk viruses and the
two most prevalent types are HPV 16 and 18 (Munoz, 2000) which are also the
predominant strains identified in cervical cancers (Gissmann et al., 1985; Drst et al.,
1985). A study showed that HPV 16 and 18 account for approximately 70% of HPV
positive cervical cancer (Naghashfar et al., 1996). HPV 16 is most frequently
encountered in anogenital cancers, being present approximately 50% of biopsies (zur
Hausen and Schneider, 1987). In contrast, HPV 18 which is less frequently found and
combined with closely related types (HPV 45), may account for 20% of additional
tumors. Other types of HPV are found less frequently. In about 10% of cervical cancers,
HPV cannot be detected (Lowry et al., 1994), suggesting that there may be alternate
pathways for the development of malignant genital tumors.
2.3.4.2 Human papilloma virus (HPV) infection
The high risk HPVs coding for E6 and E7 genes involved in immortalization of
tissue culture cells and found frequently in malignant tumors, contrasting an apparently
low tumorigenic potential of other types, generally considered as low risk HPVs (zur
Hausen, 1986). The E6 and E7 proteins are expressed in HPV-positive cancer cells and
responsible for the immortalization of human keratinocytes and of a number of other
cell types. DNA from HPV 16 and 18 can immortalize primary keratinocytes in culture
and the difference in transformation ability appears to be due to biological differences
in their E6 and E7 genes.
The transforming effects of HPV 16 and 18 are related to their ability to
inactivate innate tumor suppressor genes, including retinoblasma protein (pRb) and p53,
respectively, in cervical carcinoma cells, further supporting a role for HPV as the likely
etiologic agent for cervical carcinoma (Dyson et al., 1989; Howley et al., 1989;
Scheffner et al., 1991 and Boyer et al., 1996). It is known that the E6 and E7 proteins

35

of malignancy-associated HPV but not benign-associated HPV combine efficiently with


the tumor suppressor proteins of the pRb and p53, respectively (Dyson et al., 1989;
Werrness et al., 1990). These E6/E7 oncoproteins stimulate cell profileration by
activating cyclins E and A, and interfere with the functions of the cellular proteins pRb
and p53 leading to the HPV infection (zur Hausen, 1996).
The E6 protein encoded by high risk HPVs mediate cell transformation by
forming complexes with tumor suppressor protein, p53 (Werness et al., 1990). This
viral oncogene does not directly induce tumor formation but involves series of events
that results in tumorigenecity. The E6 region of HPV 18 genome encodes for an
oncoprotein which is directly involved in cellular transformations of the cervical
epithelia leading to neoplasm (Chan et al., 1989). E7 exerts its oncogenic function by
modulating cellular proliferation and apoptosis regulating pathways, mediated by the
ability of E7 to directly interact with and functionally inactivate several nuclear and
cytoplasmic regulatory proteins (Mnger and Howley, 2002).
During HPV infection, E7 (and presumably E6) is expressed in these cells, the
restraint on cell cycle progression is abolished and normal terminal differentiation is
retarded (Sherman et al., 1997). E6 and E7 are thought to work together to achieve
these effects as both of these proteins have functions that stimulate cell cycle
progression and both can associate with regulators of the cycle (Munger et al., 2001).
E6 and E7 proteins derived from high-risk HPV bind to pRb and p53 with high affinity.
Thus, the E6 and E7 proteins of the high risk HPV disable two important tumor
suppressor proteins that regulate the cell cycle.
In malignant lesions associated with HPV 16 and HPV 18, viral DNA is usually
integrated into the host chromosome (Drst et al., 1985). In order to integrate into the
host DNA, a break must occur in the viral genome. This separation does not occur at a
random site; rather, most breaks are found in the E1/E2 region of the virus (Baker et al.,

36

1987). The result of this disruption appears to be the loss of function of these two genes
and an accompanying deregulation of the E6 and E7 genes, resulting in cellular
transformation (Baker et al., 1987; Bedell et al., 1987).
The site at which the viral DNA is interrupted in the process of integration is
fairly constant, occurring within the E/E2 open reading frame of the viral genome. As
the E2 region of the viral DNA normally represses the transcription of the E6 and E7
early viral genes, this interruption causes over-expression of the E6 and E7 proteins.
The E7 protein binds to the under-phosphorylated form of the tumor suppressor protein
pRb and displaces the E2F transcription factors that are normally bound by pRb. These
transcription factors act at the G1/S interface of the cell cycle (Bosch et al., 1991;
Munger et al., 1992; Wolf and Ramirez, 2001; Ferenczy and Franco, 2002).
Table 2.1: Sizes and functions of HPV 18 proteins
HPV proteins

Number of
amino acids

Functions

E1

649

Virus DNA replication

E2

365

Transcription

E4

92

Binds cytokeratins preceding virus assembly

E5

83

Affects growth factor receptors

E6

151

Binds

p53

and

replication

trans-activates,

control

contributes

to

immortalization
E7

98

Main transforming protein, binds Rb1 and cyclin A

L1

505

Major virus coat protein (glycoprotein)

L2

473

Minor virus coat protein

2.4

Methods in the determination of biological acivities

2.4.1

Antimicrobial activity
The antimicrobial activity in this study was determined by the well diffusion

assay. The commonly used assay is one of the best methods in evaluating antimicrobial
37

activity. This statement is supported by a research done by Collins and Lyne (1995).
Their study documented that the inhibition zones shown by well diffusion assay were
bigger compared to paper disc diffusion assay which showed smaller inhibition zones.
This means that the former assay is much more effective in evaluating the growth
inhibition of microorganisms.
Inhibition of microorganisms is indicated by a clear zone around a punched hole
on the agar. The size of the zone is approximately proportionate to the efficiency and
diffusion rate of the substance. However, in some studies, it is believed that the size of
an inhibition zone is not necessarily a measure of the degree of inhibition because of
differences in the diffusion rates of chemotherapeutic agents. This is in accordance with
Estrela et al. (1999) who described that the diffusion method, which evaluates zones of
microbial growth inhibition, may not offer equal conditions to compare substances with
different solubility and diffusibility and the correct performance of microbiological
technique.
There are some factors that contribute to the doubtful results that include preincubation, dried culture medium, and maintenance for periods of time that exceed the
ideal time for analysis (Estrela et al., 1999). In another study by Estrela et al. (2000), it
was reported that the size of the microbial inhibition zone depends on the solubility and
diffusibility of the test substance in the agar diffusion method and therefore may not
express its full potential.
Standard measurements of zone diameters for particular media, quantities of
organisms and drug concentrations have been established and correlated to zone
diameters in order to determine whether the organisms are sensitive or resistant to the
drug.

38

2.4.2

Antioxidant activity
To evaluate the antioxidative activity of specific compounds or extracts, a

spectrophotometric assay that uses a stable radical 2,2-diphenyl-1-picrylhydrazyl


(DPPH) as a reagent (Cuendet et al., 1997; Burits and Bucar, 2000) can be applied. This
free radical test is a commonly employed assay in antioxidant studies of specific
compounds or extracts across a short time scale (Ferreira et al., 2006). Unlike
laboratory-generated free radicals such as the hydroxyl radical and superoxide anion,
DPPH has the advantage of being unaffected by certain side reactions, such as metal ion
chelation and enzyme inhibition (Amar-owicz et al., 2004).
DPPH assay measures the relative ability of the corresponding extracts and
some pure compounds to donate hydrogen atoms or electron to DPPH. The hydrogen
atoms or electrons donation ability of corresponding extracts and some pure compounds
was measured from the bleaching of purple colored methanol solution of DPPH
(Sokmen et al., 2004). DPPH is characterized as a stable free radical by virtue of
delocalisation of the spare electron over the molecule as a whole and this delocalisation
also gives rise to the deep-violet color (Molyneux, 2004).
The principle of this assay is based on a decolorization of the deep-violet color
of methanol solution of DPPH solvent, where the extent of decolorization can reflect the
amount of antioxidants present in the antioxidant substances. When a solution of 2,2diphenyl-1-picrylhydrazyl (DPPH) is mixed with that of a substance that can provide a
hydrogen atom or donate an electron (Molyneux, 2004; Ferreira et al., 2006), this gives
rise to the reduced form with the violet colour of DPPH decays (Molyneux, 2004). The
purple color generally fades when an antioxidant is present in the medium which means
the antioxidant molecules quench the DPPH radicals conceivably via a free-radical
attack on the DPPH molecule and convert them to a bleached product (2,2-diphenyl-1hydrazine) resulting in a decrease of absorbance (Ferreira et al., 2006).
39

Brand-Williams et al. (1995) described a method involving use of the free


radical (DPPH) where antioxidants are allowed to react with the stable radical in a
methanol solution. The reduction in the DPPH is followed spectrophotometrically
(Aljadi and Kamaruddin, 2004) by the monitoring the decrease in its absorbance at a
characteristic wavelength during the reaction. In its radical form, DPPH absorbs at 515
nm, but upon reduction by an antioxidant (AH) or a radical species (R), the absorption
disappears. DPPH possesses a proton free radical with a characteristic absorption,
which decreases significantly on exposure to proton radical scavengers.
DPPH + AH DPPH-H + A
DPPH + R DPPH-R
The absorbance at 515 nm is measured until the reaction reached a plateau. The
exact initial DPPH concentration in the reaction medium is calculated from a
calibration curve determined by linear regression. Antiradical activity was defined as
the amount of antioxidant necessary to decrease the initial DPPH concentration by
50%. The larger the antiradical power, the more efficient the antioxidant action
(Aruoma, 2003). The antioxidant activity was expressed as an IC50 value that was
defined as the concentration of samples required to inhibit 50% of the formation of
DPPH radicals (Dizhbite et al., 2004) and was obtained by interpolation from linear
regression analysis (Mau et al., 2002). The lowest IC50 value indicates as an excellent
antioxidant of radical scavenging.
Total phenolic content is one of the major compounds contributing for
antioxidant activity as the antioxidant activity is well-correlated with total phenolic
content. This is supported by previous study that the highest total phenolic content in
Ganoderma species is the key component accounting for their excellent results in
antioxidant activity (Mau et al., 2002). Therefore, in this study, determination of total

40

phenolic content should also be taken into consideration to evaluate the antioxidative
capacity of the mushroom.
Total phenols were estimated by a colorimetric assay known as Folin Ciocalteau
assay, which was according to the method of Singleton and Rossi (1965) with some
modifications. The assay is not an antioxidant test but an alternative method for the
quantitation of phenolic compounds. This method which measures the redox potential
of the phenolic compounds, is a sensitive and quantitative method, independent of the
degree of polymerization. Briefly, the appropriate dilutions of extracts were oxidized by
Folin-Ciocalteau reagent, and the reaction was neutralized with sodium carbonate
(Na2CO3) (Kafui and Rui, 2002).
Phenolics + alkaline (Na2CO3) + FC reagent + heat = blue colored product

Other study showed that this colorimetric oxidation/reduction assay measures all
phenolic molecules with no differentiation between gallic acid, monomers, dimers and
larger phenolic compounds. It is known that polyphenols have a higher antioxidant
(antiradical) activity than monophenols (Shahidi et al., 1992). Therefore, a gallic acid,
which is a triphenol, is often used in most of the antioxidant studies (Brand-Williams et
al., 1995). A gallic acid standard curve is established and results are typically expressed
as gallic acid equivalents (GAE). Results are reported as GAE because the phenols in
sample contain mostly other phenols, and only small amounts of gallic acid. Since the
assay measures all phenolics, the choice of gallic acid as standard is based on the
availability of a stable and pure substance, and gallic acid is both, and it is less
expensive than other options.
The first paper on this method was published in 1927 and in 1965 Singleton and
Rossi improved the reproducibility of the assay. However, the phenolics measured by
this assay are essentially simple soluble phenolics (Vattem et al., 2004).
41

2.4.3

Cytotoxic activity
Cytotoxicity is one of the chemotherapeutic targets of anti-tumor activity

(Suffness and Pezzuto, 1991). Most of the clinically used anti-tumor agents possess
significant cytotoxic activity in cell culture systems (Ajith, 2003). In term of definition,
cytotoxic means having a toxic effect on target cells.
Cytotoxicity includes a wide range of possible effects; mild cytotoxicity, such as
slowing or halting effect on cell growth (cytostasis); impairment of metabolic activities;
the permanent loss of the ability of a cell to divide (loss of reproductive potential or
clonogenecity; irreversible cessation of respiration and energy metabolism (cytocidal
effect); and the most extreme form, physical breaking of the cell membrane and
dissolution of cell structure (cytolysis) (Martz, 1993).
In this preliminary study, cytotoxicity effect was determined using neutral red
(3-amino-7-dimethyl-amino-2-methyl-phenazine hydrochloride) assay. This dye has
been widely used in many staining methods, but its commonest use is probably as a
simple red nuclear counterstain. Neutral red (NR) is a vital dye that is endocytosed by
viable cells and internalized inside lysosomes by binding to anionic sites in the
lysosomal matrix of viable cells; in this respect it is considered an indicator of lysosome
(and cell) integrity (Hansen et al., 1989; Ciapetti et al., 1996). Alterations of the cell
surface or the sensitive lysosomal membrane, due to action of xenobiotics, can lead to
lysosomal fragility or other changes which are irreversible. These changes then result in
the decreasing uptake and binding of NR dye. This is in agreement with Babich and
Borenfreund (1992), who described the cytotoxicity assay as a cell survival/viability
technique based on the ability of viable cells to incorporate and bind a weakly cationic
supervital neutral red dye to the lysosomal matrix of viable cells after their incubation
with toxic agents.

42

One advantage of NR assay is that it detects only viable cells (Doyle and
Griffiths, 2000). Besides, this assay is preferable as it is simple, fast, sensitive,
economical and highly reproducible (Babich and Borenfreund, 1992). NR assay
lysosomal integrity, with the concomitant binding of the NR dye and is a highly
sensitive indicator of cell viability. The assay quantitates cell viability and can be used
to measure cell replication, cytostatic effects, or cell death depending on the seeding
density (Doyle et al., 2000).
The assay has been used to determine the relative acute cytotoxicities of a broad
spectrum of chemical test agents, to establish structure-toxicity relationships for series
of related chemicals, to study metabolism-mediated cytotoxicity to evaluate interactions
between combinations of test agents, to evaluate differential and selective toxicities of
cancer chemotherapeutics and other pharmaceuticals, and to study temperature toxicity
interactions.
Studies with the NR assay have been extensive, using a variety of bio-indicators,
including mammalian cells, derived both from laboratory animals and from human
beings, as well as fish cells. The spectrum of chemicals tested includes inorganic
metals, organometals, surfactants, cancer chemotherapeutics, and other pharmaceutical
agents, food additives, preservatives and anti-bacterial agents, pesticides, phthalates,
toluenes, benzenes, anilines, phenolics, polycyclic aromatic hydrocarbons (PAHs),
polychlorinated biphenyls (PCBs), complex mixtures (e.g. shampoos), and a variety of
miscellaneous chemical test agents (Babich and Borenfreund, 1992).
It is critical when performing a cytotoxicity assay to have an understanding of
the metabolic capacity of the cell line that is intended to be the target cell. Cells which
lack significant xenobiotic metabolising capacity, will underestimate the cytotoxicity of
some test agents which act indirectly by causing toxicity through their metabolites
(Babich and Borenfreund, 1992). Subsequent studies with the NR assay confirmed that,

43

although different cell types and cell lines exhibit differential sensitivities to test agents,
the overall potency ranking of the chemicals was approximately equivalent. It would
therefore appear that for the assessment of direct-acting cytotoxicants, the overall
ranking of the test agents is independent of the particular cell type of cell line used as
the target in the NR assay.
2.4.4

Anti-human papilloma virus activity


There are several immunoenzymatic methods that can be used to localize

antigens. The choice is based on the type of clinical specimen being analyzed, the
degree of sensitivity and duration of processing time. The more commonly utilized
method is immunohistochemistry (IHC) which involves the usage of the avidin-biotin
peroxidase complex or the peroxidase complex or the peroxidase-labelled avidin
technique. The enzymatic reactions can be visualized by using chromogenic substrates
like 3-diamino benzine tetrahydrochloride (DAB) and 3-amino-9-ethocarbazole or
different enzymes such as alkaline phosphatase (AP).
IHC is one of the immunology studies concerned with the clinical enzymatic
reactions between the antibodies and the antigens in the immune system. It is capable of
identifying antigen in paraffin sections or exfoliated cells and has been applied
extensively in HPV research (Jenson et al., 1980).
The key reagent in all immunohistochemical techniques is the antibody
(Boenisch, 1989). It involves the localization of antigens in tissue sections by the use of
labeled antibodies as specific reagents through antigen-antibody interactions that are
visualized by a marker such as fluorescent dye, enzyme, radioactive element or
colloidal gold. Thus, it has apparent advantage over traditionally used special and
enzyme staining techniques that identify only a limited number of proteins, enzymes
and tissue structures.
44

Albert H. Coons and his colleagues (Coons et al., 1941, 1955; Coons and
Kaplan 1950) were the first to label antibodies with a fluorescent dye, and later the
antibodies used to identify antigens in tissue sections. With the expansion and
development of immunohistochemistry technique, enzyme labels have been introduced
such as peroxidase (Nakane and Pierce, 1966; Avrameas and Uriel, 1966) and alkaline
phosphatase (Mason and Sammons, 1978). Colloidal gold (Faulk and Taylor, 1971)
label has also been discovered and used to identify immunohistochemical reactions at
both light and electron microscopy level. Other labels include radioactive elements,
where immunoreaction can be visualized by autoradiography.
The principle of IHC is based on immunoenzymatic reactions using either
monoclonal or polyclonal antibodies to detect cells or tissue antigens and the
availability of high quality reagents and its simple and improved procedures have made
it an indispensable tool for clinical diagnostic. Monoclonal antibodies play an important
role as immunohistochemical reagent. There are numerous of advantages of monoclonal
antibodies in IHC over polyclonal antibodies. These include high homogeneity, absence
of non-specific antibodies, case of characterization and no batch-to-batch and lot-to-lot
variability (Boesnich, 1989). Polyclonal antibody was often found to be in short supply
and sometimes exerted significant variation among lots.
The site of antibody binding is identified either by direct labeling of the
antibody, or by use of a secondary labeling method. The direct method; uses only one
virus-specific antibody which is directly labeled with an indicator (fluorescein or
alkaline phosphatase) and the indirect method which involves the uses of two antibody,
one virus-specific antibody and the secondary anti-specific antibody to be labeled with
the indicator.
IHC localizes antigens by producing a colored product that precipitates at the
site of the enzymatic reaction between the antibody and the antigen. Peroxidase can
45

react with DAB substrate chromogen in the presence of hydrogen peroxide (H2O2) to
yield brown, alcohol-soluble precipitate at the site of the antigen. The 3-amino-9ethocarbazole forms a rose-red end product that is soluble in alcohol (Boesnich, 1989).
Peroxidase activity in the presence of an electron donor first results in the formation of
an enzyme-substrate complex. Then, the oxidation of this electron donor provides a
driving force in continuing catalysis of H2O2 to form an insoluble colored product
(Boesnich, 1989).

46

CHAPTER 3: MATERIALS AND METHODS


3.1

Preparation of Schizophyllum commune extracts


Schizophyllum commune fruitbodies were purchased from a local market. They

were then cut into small pieces and air-dried. The bioactive compounds present in the
S. commune fruitbodies were extracted using methanol, ethyl acetate, dichloromethane
and water.
In methanol, ethyl acetate and dichloromethane extraction, the weighed
fruitbodies were soaked with appropriate volume of solvents separately for two days at
room temperature. The fruitbodies were then separated from the extraction solvents by
filtration using Whatman No.1 filter paper. The residues were discarded while the
filtrate obtained were transferred into a round bottom flask and dried to approximately 2
ml using a rotary evaporator. The crude extract was then transferred into a pre-weighed
glass vial and rotary evaporated till complete dryness to yield the crude extract. The
weight of all crude extracts obtained was measured and they were stored at -20C until
analysis.
Water extraction was done by boiling the weighed fruitbodies in distilled water
for three hours using a heating mantle. The mixture was then filtered through Whatman
No. 1 filter paper to obtain the filtrate. The filtrate was freeze-dried to obtain the crude
extracts. The weight of the crude extract collected was measured and stored at 4C until
required. The extracts were tested for various biological activities as shown in Figure
3.1.

47

EXTRACTION OF BIOACTIVE COMPOUNDS

Dichloromethane, Ethyl Acetate and


Methanol Extracts

Water Extract

Soak mushroom with sterile


distilled water (sdH2O)

Soak mushroom with dichloromethane,


ethyl acetate and methanol separately

Heat for 3 hours

Filter

Freeze-dry

Evaporate to dryness

Crude Extracts

ANTIMICROBIAL
ACTIVITY

CYTOTOXICITY TOWARDS
CANCER CELL-LINES

ANTIOXIDANT
ACTIVITY

ANTI-HUMAN PAPILLOMA
VIRUS (HPV) ACTIVITY

Figure 3.1: Extraction of S. commune bioactive compounds and


the biological activities analyzed

3.2

Determination of antimicrobial activity


The test organisms used to screen the antibacterial activity of S. commune were

Gram-positive bacteria (Bacillus cereus, B. subtilis, Enterobacter faecalis and


Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Plesiomonas

48

shigelloides, Pseudomonas aeuroginosa, Proteus vulgaris, Salmonella sp., S. typhi,


Shigella sp., S. flexneri, Streptococcus mitis, S. mutans and S. sanguis).
The bacterial and fungal stock cultures were prepared and maintained on various
media. Mueller Hinton (MH) broth (Appendix A) was used to support the growth of
Gram positive and negative bacteria while Brain Heart Infusion (BHI) broth (Appendix
A) was used for Gram negative oral bacteria (S. mitis, S. mutans and S. sanguis).
Saboraud Dextrose (SD) and Yeast-Peptone (YP) broth (Appendix A) were used for C.
albicans and C. parapsilosis and S. pombe respectively. Bacterial and fungal inoculum
were prepared by transferring 100 l of the stock suspension into 3 ml broth and
incubated for 48 hours at 37C. Commercially available antibiotic discs namely
streptomycin, chloramphenicol and kanamycin were used as positive controls.
Antifungal activity of S. commune extracts was tested on Candida albicans,
C. parapsilosis and Saccharomyces pombe, and the positive control used was nystatin; a
common antibiotic used to treat fungal infections. Dimethyl sulfoxide (DMSO) served
as a negative control in this study. The controls and the S. commune crude extracts were
dissolved in a sterile DMSO to obtain the concentrations of 0.2 mg/ml and 2.0 mg/ml to
test against the organisms.
3.2.1

Well diffusion assay


After two days of incubation, the two-day old bacterial and fungal suspension

was evenly streaked on the respective agar media. Three 7 mm diameter wells were cut
in the agar using a sterile cork borer to represent a triplicate reading for each test
organisms. The test organisms were prepared by placing 50 l of 0.2 mg/ml extracts to
give 10 g in each well. The Petri dishes were further incubated at 37C and activities
of the extracts were estimated by measuring the diameter of inhibition zones after 24
hours of incubation (See Figure 3.2). The same method was applied to test 2.0 mg/ml of
extracts which was equivalent to 100 g in each well. The same concentrations were
49

tested for the positive controls. The diameter of inhibition zones was classified into the
following categories:

Inactive (IA) - showing inhibition zones <9 mm diameter


Partially active (PA) - showing inhibition zones 9 12 mm diameter
Active (A) - showing inhibition zones 13 - 18 mm diameter
Very active (VA) - showing inhibition zones >18 mm diameter

ANTIMICROBIAL ASSAY

ANTIBACTERIAL ASSAY

ANTIFUNGAL ASSAY

2-day-old test organisms


Lawn bacteria on Mueller
Hinton agar

Lawn fungi/yeast on Saboraud


Dextrose agar and Yeast-Peptone
agar respectively

Cut three 7mm diameter well and fill 50 l of


fungal extracts in each well

Incubate at 37C for 24 hours


Measure diameter of inhibition zones

Figure 3.2: Screening for antimicrobial activity of S. commune extracts

50

3.3

Determination of antioxidant activity

3.3.1

2,2-diphenyl-1-picrylhydrazyl (DPPH) assay


The antioxidant activity of S. commune was assessed by the scavenging effect

on 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical assay (Molyneux, 2004). Various


concentrations of the S. commune extracts in methanol were prepared to give a final
volume of 0.1 ml and were mixed vigorously with 3.9 ml of methanolic solution
containing DPPH radicals resulting in a final concentration of 0.06 mM. After 30
minutes incubation period in the dark, the absorbance was read against a blank
(methanol) at 515 nm (Appendix B). The decrease in absorbance was monitored until a
constant reading was obtained. All tests were carried out in triplicate. The scavenging
activity on DPPH radicals was expressed as the percentage inhibition compared to the
0.06 mM DPPH in methanol which served as negative control. Inhibition of free radical
DPPH in percent was calculated using the following way:
Percentage of inhibition = OD control OD sample
OD control

OD control is the absorbance of the control reaction containing all reagents


except the test compound, and OD sample is the absorbance of the test compound. The
antioxidant activity was expressed as an IC50 value that was defined as the effective
concentration (in g/ml) at which DPPH radicals were scavenged by 50% and was
obtained by interpolation from linear regression analysis. Sample concentration
providing 50% inhibition (IC50) was calculated using the graph by plotting inhibition
percentage against extract concentration. Ascorbic acid was used as the positive control
for this assay.

51

3.3.2

Folin-Ciocalteau assay
The total phenolic content of S. commune extracts was determined by the Folin-

Ciocalteau method (Singleton and Rossi, 1965) with some modifications, involving
Folin-Ciocalteau reagent and gallic acid as a standard. All the mushroom extracts were
diluted in distilled water and the concentrations applied in the method were documented
in Appendix B. Folin-Ciocalteau reagent (250 l) (Appendix A) was added to the
aliquot (250 l) of extract solution and was mixed thoroughly. After 3 minutes, a 500
l solution of natrium carbonate (Na2CO3) (Appendix A) was added and the mixture
was allowed to stand for 1 hour in the dark. The absorbance of the resulting solution
was measured at 750 nm with a spectrophotometer against a blank (distilled water). All
tests and analyses were run in triplicates and were used to calculate the standard
calibration curve.
The absorbance values obtained were converted to total phenolics from the
gallic acid standard curve. Content of total phenols in samples was calculated on the
basis of the calibration curve of gallic acid using various concentrations of gallic acid
(0, 2, 4, 6, 8, 10 g/ml) in distilled water (Appendix B). The total phenolic content of
the samples was expressed as gallic acid equivalents (GAEs), which reflected the
phenolic content as the amount of gallic acid (mg) per gram of mushroom fruitbodies.
3.4

Cytotoxicity towards various cancer cell lines

3.4.1

Cancer cell lines


The cell lines used for the cytotoxicity tests were the cervical cancer cell lines;

CaSki, epidermoid cancer cell lines; KB, colon cancer cell lines; HT29 and intestinal
colon cancer cell lines; HCT116.

52

3.4.2

Serial dilution of S. commune extracts


Ten microliter of the mushroom stock concentration of 20,000 g/ml was

diluted in 90 l 10 % DMSO to produce a stock of concentration of 10 g/ml. This


stock was then further diluted into final concentrations of 100 g/ml, 50 g/ml, 10
g/ml and 1 g/ml (Figure 3.3). All diluted stocks were kept at 20C until required.

Stock
(20,000 g/ml)

10 l +
90 l 10% DMSO

10 l +
190 l cells
(100 g/ml)

10 l +
90 l 10% DMSO

5 l +
195 l cells
(50 g/ml)

10 l +
190 l cells
(10 g/ml)

10 l +
90 l 10% DMSO

10 l +
190 l cells
(1 g/ml)

Figure 3.3: Serial dilution of S. commune extracts for cytotoxicity


3.4.3

Sub-cultivation of cancer cells


The confluent cells in tissue culture flask was discarded and the cells were

washed with 5 ml of sterile PBS (pH 7.2) (Appendix A). The cells were detached from
the flask by adding 3 ml of PBS pH 7.2 and 1.0 ml (0.25%) of trypsin (Appendix A).
The cells were then incubated for five to ten minutes at 5% CO2 incubator at 37C and
followed by observation under an inverted microscope. The flask was given a slight tap
to detach cells and the cell suspension was centrifuged at 1000 rpm for five minutes.
Following centrifugation, the pellet of cells was resuspended in 1 ml 10% supplemented
RPMI 1640 media (Appendix A) to produce a stock of cell suspensions.

53

3.4.4

Cell enumeration and plating


One hundred microliter of cell suspensions and 900 l of tryphan blue

(Appendix A) were transferred into an eppendorf tube and mixed well. 20 l of cell
suspensions colored with the dye were loaded at the two edges of a haemocytometer
(Scherf) which was covered by a glass cover slip. The cells were allowed to flow into
the chambers by capillary action and left to settle for three to five minutes before
counting. Four corner squares plus the center square were counted (Figure 3.4). The
haemocytometer was then examined under an inverted microscope and the unstained
viable cells were counted. The quantity of media needed for the cell suspensions in
order to get approximately 30,000 cells/ml, was estimated according to the formula
below:

V2 = 30,000 x V1 (12 ml) x 1000 l


100,000 x number of cells per square box

The volume of media needed for 12 ml cell suspension (V1) to make 30,000 cells/ml
was calculated using this formula:
P1 = The original number of cells counted
P1V1 = P2V2

V1 = Original volume (10 x 10 x 12)


P2 = The number of cells wanted
V2 = The volume of media needed

54

Figure 3.4: Cell counting using a haemocytometer

The appropriate number of cells was then added to fresh culture media to give a
final concentration of 30,000 cells/ml.
3.4.5

Treatment of cancer cells with S. commune extracts


Cell suspensions with known cell density were then plated in 96-well microtiter

plate (Nunc) and incubated overnight in a 5% CO2 incubator at 37C to allow the cells
to adhere and achieve 60-70% confluent. The cells were added to the wells immediately
after the cell dispersion with fresh different serial diluted of extracts ranging from 100
g/ml, 50 g/ml, 10 g/ml and 1 g/ml. One well was not treated with any extract
served the negative control. The treated cell plate was then incubated at 5% CO2
incubator at 37C for 72 hours. The same procedure was applied to each of the fungal
extracts and the treatment was performed in triplicate.

55

3.4.6

Neutral red assay


After the 72 hours incubation period, the medium in the test plate was removed

before adding 200 l neutral red medium (Appendix A) into the respective wells. The
plate was further incubated for 3 hours to allow maximum uptake of dye by surviving
cells in a 5% CO2 incubator at 37C. Following that, the dye was discarded and the cells
were rapidly washed with 200 l washing solution (Appendix A) before further adding
200 l resorb solution (Appendix A) into the same wells to extract the dye from the
viable cells. Then, the cells were left in a 5% CO2 incubator at 37C for 15 minutes.
They were left to mix for 30 minutes in a benchtop incubator (LT Biomax 500) before
the optical density (OD) was recorded at 540 nm using an ELISA reader (Titertek
Multiskan MCC/340).
Percentage of killing was calculated using the formula given below. IC50 is the
effective concentration at which the growth of the cells was inhibited by 50%. Sample
concentration providing 50% inhibition (IC50) was calculated using the graph by
plotting inhibition percentage against extract concentration

Percentage of inhibition/killing = OD -ve OD treatment x 100


OD -ve

3.5

Anti-human papillomavirus (HPV) activity towards cervical cancer


(CaSki) cell lines

3.5.1

Serial dilution of S. commune extracts


One hundred and eighty microliter from the mushroom stock extracts (20

mg/ml) was diluted in 282 l of sterile distilled water to produce a stock concentration
of 1200 g/ml. This stock was further diluted into final concentrations of 200g/ml, 100

56

g/ml, 50 g/ml, 25 g/ml, 10 g/ml and 1 g/ml respectively (Figure 3.5). All diluted
stocks were kept at -20C in McCartney bottles until use.

Stock (20 mg/ml)


0.18 ml
+ 2.82 ml dH2O
1.5 ml
+ 1.5 ml dH2O (200 g/ml)
1.5 ml
+ 1.5 ml dH2O (100 g/ml)
1.5 ml
+ 1.5 ml dH2O (50 g/ml)

+ 900 l dH2O (10 g/ml)


100l

10 l
+ 990 l dH2O (1 g/ml)

1.5 ml
+ 1.5 ml dH2O (25 g/ml)
Figure 3.5: Serial dilution of S. commune extracts for anti-HPV activity

3.5.2

Treatment of CaSki cells with S. commune extracts


One ml of the serial diluted stock extracts was added to 2 ml of cultured cells in

flask to which five different concentrations; 200 g/ml, 100 g/ml, 50 g/ml, 25 g/ml,
10g/ml and 1 g/ml were applied. The cells were incubated with S. commune extracts
at 37C for 72 hours.
3.5.3

Fixation of CaSki cells onto slides


Cultured cells that were incubated with fungal extracts were transferred into a

tissue culture tube and centrifuged at 1,000 rpm for 5 minutes. The supernatant was
57

decanted, the cells were washed twice in PBS 7.2 (Appendix A). Cells were then
resuspended in fresh PBS 7.2. Using a micropipette, 30 l of cell suspension was
carefully placed onto the wells of Teflon-coated glass slides. The slides were then left to
dry at room temperature overnight. The cells were subsequently fixed using acetone for
10 minutes. The fixed slides were stored at 20C until use in immunohistochemistry.
3.5.4

Immunohistochemistry assay
The technique of immunohistochemical staining were carried out using the

Labeled Streptavidin Biotin (LSAB) Peroxidase Kit and the AEC Substrate System
(DAKO) according to the specifications described by the manufacturer with some
modifications. All washing steps required constant shaking and incubations with
reagents were carried out in a humidified chamber.
The cells were rehydrated in decreasing concentrations of ethanol; 100%, 95%,
90% and 80% (Appendix A) for 2 minutes each and then washed in PBS 7.6 (Appendix
A) for five minutes on a shaker (LT Biomax 500). Three percent hydrogen peroxide
(Appendix A) was added to each well and left to incubate for 10 minutes at 37C. This
step is crucial to remove the endogenous peroxidase activity. After that, slides were
rinsed in PBS for 5 minutes. The areas surrounding each well were blotted dry before
treating selected well with 30 l of anti-HPV18 E6 monoclonal antibody which acts as
a primary antibody. Slides were then incubated for 1 hour at room temperature.
Following this, the slides were washed twice in PBS for 15 minutes each, and
the area surrounding each well were blotted dry before incubation with 30 l
streptavidin-HRP conjugate for 10 minutes at 37C. Next, the slides were washed in
PBS in another 5 minutes and blotted dry. 30 l of 3- diaminobenzidine
tetrahydrochloride (DAB) were then added and incubated at 37C. Incubation was
stopped when the desired color intensity developed (approximately 5 - 20 minutes).

58

The slides were rinsed in distilled water before counterstaining with Mayers
Hematoxylin for 2 minutes. Slides were rinsed again with distilled water before being
immersed into ammonia solution for 10 seconds. After rinsing the slides in water, the
slides were mounted with pre-warmed glycergel (53C). The slides were left to dry in
the dark before analysis under the light microscope (Olympus, Japan) (See Figure 3.5
(b)).
Rehydrate cells in decreasing ethanol
Wash with PBS
Incubate cells with 3% H2O2
Wash with PBS
Incubate cells with 1 antibody
Wash with PBS
Incubate cells with HRP-conjugate
Wash with PBS
Incubate cells with DAB
Rinse with H2O
Add mayers haematoxylin
Rinse with H2O
Mount with glycergel

Store in the dark

Observe under a microscocope


PBS 7.6
Figure 3.6: A brief procedure of immunohistochemistry method

59

CHAPTER 4.0: RESULTS


4.1

Analysis of antimicrobial activity of S. commune extracts


In this study, the in vitro antimicrobial activity of S. commune extracts

(methanol, ethyl acetate, dichloromethane and water) and the commercial antibiotics
(streptomycin, kanamycin, chloramphenicol and nystatin) against microorganisms
tested was qualitatively assessed by the diameter of inhibition zone using well diffusion
assay. The antimicrobial activity was examined against seven species of Gram-positive
bacteria (Bacillus cereus, B. subtilis, Enterobacter faecalis, Staphylococcus aureus,
Streptococcus mitis, S. mutans and S. sanguis), eight species of Gram-negative bacteria
(Escherichia coli, Salmonella sp., S. typhi, Shigella sp., Shigella flexneri, Plesiomonas
shigelloides, Proteus vulgaris, and Pseudomonas aeuroginosa) and three species of
fungi (Candida albicans, C. parapsilosis and Saccharomyces pombe). The antimicrobial
activity of both S. commune extracts and commercial antibiotic discs was determined at
0.2 mg/ml and 2 mg/ml (Appendix C). The inhibition zone was measured in millimeter
(mm) after 24 hours of incubation time. The diameter of well was 7 mm and the data
expressed were evaluated based on the comparison among the extracts against the
microorganisms.
Zone of inhibition was categorized as:

Inactive (IA) inhibition zones <9 mm diameter

Partially active (PA) - inhibition zones 9-12 mm diameter

Active (A) - inhibition zones 13-18 mm diameter

Very active (VA) - inhibition zones >18 mm diameter

60

The results of the antimicrobial activity of S. commune extracts at 0.2 mg/ml


against the microorganisms tested are presented in Table 4.1. Overall, at the
concentration tested, methanol, ethyl acetate and dichloromethane extracts of
S. commune showed mostly partially active antibacterial activity against Gram-positive
and Gram-negative bacteria tested. Water extract, however, failed to show antibacterial
activity against the selected microorganisms (Table 4.1).
The antibacterial activity of S. commune extracts at 0.2 mg/ml against various
types of bacteria was in the range of 8 mm to 11 1 mm (Table 4.1). Both ethyl acetate
and dichloromethane extracts of S. commune showed significantly (P<0.05) higher
activity than that of methanol extract against the microorganisms tested. The highest
inhibition zone was detected by dichloromethane extract against Gram-positive bacteria,
S. sanguis with diameter of inhibition zone of 111 mm (Table 4.1). Meanwhile,
S. mutans and P. aeuroginosa were the most resistant as they were both not inhibited by
the S. commune extracts at all (Table 4.1).
As for the antifungal activity at 0.2 mg/ml, all S. commune extracts were found
to be weak inhibitors except for ethyl acetate (Table 4.1). The ethyl acetate extract
showed antifungal activity against the fungal species which ranged from 8 1 mm to
9 1 mm with the highest inhibition detected against Candida spp. (Table 4.1).

61

Table 4.1: Antimicrobial activity of S. commune extracts at 0.2 mg/ml as evaluated


by well diffusion assay (24 hours incubation at 37C). Inhibition zones were
measured (in mm) and the diameter of inhibition zones reported
Microorganisms
Bacillus cereus

Grampositive
bacteria

Gramnegative
bacteria

Fungi

Bacillus
subtilis
Enterobacter
faecalis
Staphylococcus
aureus
Streptococcus
mitis
Streptococcus
mutans
Streptococcus
sanguis
Escherichia
coli
Salmonella sp.
Salmonella
typhi
Shigella sp.
Shigella
flexneri
Plesiomonas
shigelloides
Proteus
vulgaris
Pseudomonas
aeuroginosa
Candida
albicans
Candida
parapsilosis
Saccharomyces
pombe

Methanol
91 a
PA
90 a
PA
91 a
PA
100 a
PA
NA
NA
101 a
PA
90 a
PA
NA
81 a
IA
90 a
PA
81 a
IA
81 a
PA
90 a
PA
NA
NA
NA
NA

S. commune extracts
Ethyl
Dichloromethane
acetate
91 b
101 b
PA
PA
b
81
91 b
IA
PA
b
100
91 b
PA
PA
b
91
100 b
PA
PA
90 b
80 b
PA
IwA
NA
NA
110 b
111 b
PA
PA
100 b
90 b
PA
PA
100 b
91 b
PA
PA
91 b
91 b
PA
PA
100 b
91 b
PA
PA
90 b
91 b
PA
PA
91 b
101 b
PA
PA
91 b
110 b
PA
PA
NA
NA
91
PA
NA
91
80
PA
IA
81
IA
NA

Water
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

Data expressed as means standard deviations of triplicate measurements; means with different
letters are significantly different (P<0.05); NA= no activity; water extract failed to show good
inhibition against all microorganisms tested; diameter of well was 7 mm

62

On the other hand, at 2 mg/ml (Table 4.2), S. commune extracts also showed
mostly partially active antibacterial activity against Gram-positive and Gram-negative
bacteria tested. The inhibition zones recorded by the extracts ranged from 8 1 mm to
12 1 mm. Of all the bacteria tested, the Gram-positive bacteria, S. mutans was found
to be the most resistant against the S. commune extracts as the growth was not inhibited
by the mushroom extracts at the concentration tested (Table 4.2 and Plate 4.1 (b)).
Dichloromethane extract of S. commune was the most active in antibacterial
activity, showing a significantly (P<0.05) higher activity than that of other extracts
against the microorganisms tested at concentration of 2 mg/ml. The extract inhibited the
growth of all Gram-positive and Gram-negative bacteria at the zone diameters slightly
higher than the rest of the extracts. The highest inhibition zone detected by this extract
was against the Gram-positive bacteria, S. sanguis with diameter of inhibition zone of
12 1 mm (Table 4.2 and Plate 4.1(a)). At the same concentration, methanol and ethyl
acetate extracts of S. commune exhibited about the same spectrum of antibacterial
activity against the microorganisms tested. However, water extract of S. commune had
no inhibitory effect on the growth of all the microorganisms at the concentration
evaluated in this study (Table 4.2).
The antifungal activity of S. commune extracts was found to be less pronounced
than the antibacterial activity at 2 mg/ml (Table 4.2). This is in accordance to the
inhibitory activity shown by the extracts against the fungal species; C. albicans, C.
parapsilosis and S. pombe. In particular, the fungal species were susceptible to
dichloromethane and ethyl acetate extracts of S. commune. Both extracts gave quite a
similar antifungal pattern against the fungi tested with the lowest inhibitory (inactive)
recorded against S. pombe. Methanol and water extracts, however, failed to show
antifungal activity at all (Table 4.2).

63

Table 4.2: Antimicrobial activity of S. commune extracts at 2 mg/ml as evaluated


by well diffusion assay (24 hours incubation at 37C). Inhibition zones were
measured (in mm) and the diameter of inhibition zones reported
Microorganisms
Bacillus cereus

Grampositive
bacteria

Gramnegative
bacteria

Fungi

Bacillus
subtilis
Enterobacter
faecalis
Staphylococcus
aureus
Streptococcus
mitis
Streptococcus
mutans
Streptococcus
sanguis
Escherichia
coli
Salmonella sp.
Salmonella
typhi
Shigella sp.
Shigella
flexneri
Plesiomonas
shigelloides
Proteus
vulgaris
Pseudomonas
aeuroginosa
Candida
albicans
Candida
parapsilosis
Saccharomyces
pombe

Methanol
91a
PA
101a
PA
101a
PA
101a
PA
91a
PA
NA
111a
PA
91a
PA
91a
IA
91a
PA
91a
PA
91a
PA
91a
PA
91a
PA
90a
PA
NA
NA
NA

S. commune extracts
Ethyl
Dichloromethane
acetate
81a
110b
IA
PA
a
91
111b
IA
PA
a
101
111b
PA
PA
a
91
111b
PA
PA
91a
111b
PA
PA
NA
NA
111a
121b
PA
PA
101a
111b
PA
PA
101a
111b
PA
PA
100a
111b
PA
PA
101a
111b
PA
PA
91a
111b
PA
PA
111a
111b
PA
PA
101a
111b
PA
PA
91a
101b
PA
PA
101
100
PA
PA
100
101
PA
PA
91
81
IA
IA

Water
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

Data expressed as means standard deviations of triplicate measurements; means with different
letters are significantly different (P<0.05); NA= no activity; water extract failed to show good
inhibition against all microorganisms tested; diameter of well was 7 mm

64

Plate 4.1: Antibacterial activity of S. commune dichloromethane extracts against


some of the microorganisms

(a): The inhibition of S. sanguis by


2 mg/ml dichloromethane extract of
S. commune.

(b): S. mutans was not inhibited by


2 mg/ml dichloromethane extract of
S. commune.

The commercial antibiotics; streptomycin, kanamycin, chloramphenicol and


nystatin which were used as the positive controls in this study exhibited a very effective
antimicrobial activity at both the concentrations tested (0.2 mg/ml and 2 mg/ml). The
antibiotics exhibited either active or very active inhibition against the microorganisms
(Table 4.3).
As shown in Table 4.3, the antibiotics inhibited the growth of Gram-positive and
Gram-negative bacteria in a concentration dependent manner. Overall, at the
concentration of 2 mg/ml, the antibacterial activity of the antibiotics against various
types of bacteria was in the range of 18 mm to 42 mm, whereas, the inhibition zones
were in the range 9 mm to 35 mm when the bacteria were tested at 0.2 mg/ml (Table
4.3). Chloramphenicol was found to be the best inhibitor against the Gram-positive
bacteria. A very active inhibition was detected particularly against B. subtilis (40 1
mm) and S. aureus (40 1 mm) (Table 4.3). The antibiotic also exhibited excellent
antibacterial activity against the Gram-negative bacteria, P. vulgaris with the highest
inhibition zone of 42 1 mm (Table 4.3).
65

However, of all the bacterial species tested, the Gram-positive bacteria


S. mutans, appeared to be the most resistant to all the antibiotics at 0.2 mg/ml. However,
at higher concentration (2 mg/ml), S. mutans was susceptible to all the antibiotics giving
active to very active inhibition zone (13 1 mm to 21 1 mm) (Table 4.3).

Table 4.3: Antibacterial activity of antibiotic discs as evaluated by well diffusion


assay (24 hours incubation at 37C). Inhibition zones were measured (in mm) and
the diameter of inhibition zones reported

Microorganisms
Bacillus cereus
Bacillus
subtilis
Enterobacter
Gramfaecalis
positive Staphylococcus
bacteria aureus
Streptococcus
mitis
Streptococcus
mutans
Streptococcus
sanguis
Escherichia
coli
Salmonella sp.
Salmonella
Gramtyphi
negative Shigella sp.
bacteria
Shigella
flexneri
Plesiomonas
shigelloides
Proteus
vulgaris
Pseudomonas
aeuroginosa

Streptomycin
0.2
2
mg/ml mg/ml
251
311
VA
VA
181
251
A
VA
261
321
VA
VA
180
251
A
VA
200
270
VA
VA
200
NA
VA
91
211
PA
VA
131
261
A
VA
292
381
VA
VA
291
381
VA
VA
301
371
VA
VA
291
381
VA
VA
321
391
VA
VA
151
251
A
VA
151
210
A
VA

Kanamycin
0.2
2
mg/ml
mg/ml
211
301
VA
VA
301
351
VA
VA
201
271
VA
VA
301
351
VA
VA
171
191
VA
VA
210
NA
VA
121
251
PA
VA
200
271
VA
VA
221
311
VA
VA
201
281
VA
VA
211
281
VA
VA
201
281
VA
VA
221
301
VA
VA
291
341
VA
VA
241
311
VA
VA

Chloramphenicol
0.2
2
mg/ml mg/ml
181
261
VA
VA
251
401
VA
VA
191
301
VA
VA
251
401
VA
VA
221
251
VA
VA
131
NA
A
81
181
IA
VA
121
311
PA
VA
221
341
VA
VA
201
331
VA
VA
191
311
VA
VA
201
331
VA
VA
241
321
VA
VA
250
421
VA
VA
211
341
VA
VA

Data expressed as means standard deviations of triplicate measurements; NA= no


activity, the extract failed to show good inhibition against all microorganisms tested;
diameter of well was 7 mm

66

Plate 4.2: Antibacterial activity of antibiotic chloramphenicol against some of the


microorganisms

(a): The inhibition zone of P. vulgaris


by 2 mg/ml antibiotic chloramphenicol.

(b): The inhibition zone of B. subtilis


by 2 mg/ml antibiotic chloramphenicol.

In the antifungal activity of antibiotic nystatin against fungi and yeast species
(Table 4.4), the microorganisms were found to be sensitive to the antibiotic giving very
active inhibitions (23 1 mm to 29 1 mm) except for C. parapsilosis. At 0.2 mg/ml,
no inhibition was observed. However, at 2 mg/ml, a partially active inhibition of 12 1
mm was detected against the fungal species.

Table 4.4: Antifungal activity of antibiotic nystatin as evaluated by well diffusion


assay (24 hours incubation at 37C). Inhibition zones were measured (in mm) and
the diameter of inhibition zones reported
Nystatin
Microorganisms
Candida albicans
Fungi

Candida parapsilosis
Saccharomyces pombe

0.2 mg/ml
231
VA
NA
231
VA

2 mg/ml
281
VA
121
PA
291
VA

Data expressed as means standard deviations of triplicate measurements; NA= no activity, the
extract failed to show good inhibition against the microorganisms tested; diameter of well was 7
mm

67

4.2

Analysis of antioxidant activity of S. commune extracts


In this present study, the extracts of S. commune using different extraction

solvents; methanol, ethyl acetate, dichloromethane and water were investigated for the
antioxidant activity using 2,2-diphenylpicrylhydrazyl (DPPH) free radical scavenging
assay.
The DPPH free radical scavenging assay is a spectrophotometric assay that uses
stable radical diphenylpicrylhydrazyl (DPPH) as a reagent. This technique measures the
relative ability of antioxidant compounds to donate proton or electron to DPPH. The
proton or electron donation ability was measured from the bleaching of purple colored
solution of DPPH which was also indicated by the decrease in absorbance. The decrease
in absorbance is taken as a measure of the extent of radical scavenging. The IC50 value
is defined as the amount of antioxidants necessary to inhibit the initial DPPH radical
concentration by 50% and this is determined from the plotted graph of scavenging
activity against the concentration of extracts.
The results (Table 4.5 and Figure 4.1) presented that different extracts of
S. commune exhibited variable scavenging activities. The reactions followed a
concentration dependent pattern, where the inhibition values of extracts increased with
the concentration. The DPPH radical scavenging activity of methanol extract of
S. commune was the most remarkable as evidenced by the IC50 value of 0.145 0.01
mg/ml (Table 4.5). On the other hand, ethyl acetate extract of S. commune showed
moderate scavenging activity, exhibiting ability to scavenge DPPH free radical with
IC50 value of 0.219 0.01 mg/ml (Table 4.5). Dichloromethane and water extracts of
S. commune were found to exhibit an almost identical pattern in scavenging the DPPH
radicals. The scavenging activity of both extracts at 50% was 0.641 0.13 mg/ml and
0.674 0.05 mg/ml respectively (Table 4.5). Accordingly, it can be observed that the

68

radical scavenging activity of S. commune extracts was in the order of methanol < ethyl
acetate < dichloromethane < water (Table 4.5).
In this study, ascorbic acid, a commercial antioxidant agent was used as the
positive control for the S. commune extracts. Ascorbic acid or better known as vitamin C
is a highly effective antioxidant agent which has been widely used as in enormous
antioxidant studies. The ascorbic acid exerted a free radical scavenging activity in dosedependent manner (Appendix C). The IC50 value of ascorbic acid evaluated from this
DPPH radical scavenging assay was 0.084 0.01 mg/ml (Table 4.5).

Table 4.5: The scavenging activity of S. commune extracts as measured by DPPH


radical scavenging asssay
Extract

Scavenging Activity
IC50 (mg/ml)
0.145 0.01 a
0.219 0.01 b
0.641 0.13 c
0.674 0.05 d
0.084 0.01

Methanol
Ethyl acetate
Dichloromethane
Water
*Ascorbic acid

Data expressed as means standard deviations of triplicate measurements


*Positive reference standard

120

Inhibition (%)

100
80
60
40

methanol
ethyl acetate
dichloromethane
water
i
(di hl
h

20
0
0

0.2

0.4

0.6

0.8

Concentrations (mg/ml)

1.2

)
1.4

Figure 4.1: The scavenging effect of S. commune extracts on DPPH radicals


69

Total phenolic content of the S. commune extracts was determined by employing


the method modified from Singleton and Rossi (1969), involving Folin-Ciocalteau as
the reagent and gallic acid as the positive standard. The total phenolic analysis was done
to determine the presence of phenolic compound in the extracts.
In this study, the content of phenolic compound in S. commune extracts was
evaluated from the calibration curve of the positive standard reference, gallic acid
(Appendix C). Gallic acid standards were done in triplicate in a series of dilution at the
concentration range of 0 g/ml to 10 g/ml (Appendix B). The total phenolic content
of S. commune extracts were calculated from the standard curve (Appendix C) and
expressed as mg gallic acid (GA) per gram extract and mg GA per gram fruitbodies.
The total phenolic content of both the S. commune extracts and fruitbodies was
measured by the Folin Ciocalteau assay at the concentration of 0.1 mg/ml and the
results are shown in Table 4.6. Overall, the amount of total phenolic contents among
S. commune extracts varied significantly (P<0.05) and were in the order of methanol
(1.72 0.045 mg GA/g extract) > ethyl acetate (0.79 0.025 mg GA/g extract) > water
(0.52 0.04 mg GA/g extract) > dichloromethane (0.45 0 .012 mg GA/g extract)
(Table 4.6).
On the other hand, total phenolic content of S. commune fruitbodies determined
at the same concentration exhibited a slight different order (Table 4.6). At 0.1 mg/ml,
the total phenolic content of the S. commune fruitbodies were in the order of methanol
(0.498 0.07 mg GA/g fruitbodies) > water (0.095 0.02 mg GA/g fruitbodies) > ethyl
acetate (0.057 0.01 mg GA/g fruitbodies) > dichloromethane (0.021 0.01 mg GA/g
fruitbodies) (Table 4.6).

70

Table 4.6: The total phenolic content of S. commune extracts and fruitbodies at 0.1
mg/ml as measured by Folin-Ciocalteau method
Extract
Methanol
Ethyl acetate
Dichloromethane
Water

Total phenolic content


(mg GA/g extracts)

1.720.05a
0.790.03b
0.450.04d
0.520.01c

Total phenolic content


(mg GA/g fruitbodies)

0.4980.07a
0.0570.01c
0.0210.01d
0.0950.02b

Data expressed as means standard deviations of triplicate measurements; means with different
letters are significantly different (P<0.05)

4.2.1

Correlation between radical scavenging activity of S. commune extracts and


their total phenolic contents
To analyse the correlation between radical scavenging activities of S. commune

extracts and their total phenolic contents, a linear regression graph was plotted based on
the value of total phenolic content and radical scavenging activity of the extracts. A
positive correlation (R2=0.8264) was observed between the scavenging activity and
total phenolic content of the extracts (Figure 4.2). The profile of the correlation between
the total phenolic content and scavenging activity of the extracts is shown in
Appendix C.
Comparing the correlation activity of the S. commune extracts, the radical
scavenging activities of the extracts were in the order of methanol < ethyl acetate <
dichloromethane < water. On the other hand, total phenolic contents among the extracts
were in the order of methanol > ethyl acetate > dichloromethane > water. Generally, the
radical scavenging activity of S. commune extracts increased proportionally to their total
phenolic content. Of all the extracts, methanol extract of S. commune was found to
exhibit the best antioxidant activity, which is in the agreement with the highest
scavenging activity (IC50 value of 0.145 0.01 mg/ml) and highest total phenolic
content (1.72 0.05 mg GA/g sample) (Appendix C).

71

60

DPPH inhibition (%)

50
40
30
20
y = 7.3362x + 9.1791
2
R = 0.8264

10
0
0

2
3
4
Total phenolic content (ug/ml)

Figure 4.2: Correlation between the radicalcavenging activity of S. commune


extracts and their phenolic contents

4.3

Analysis of cytotoxic activity of S. commune towards cancer cell-lines


In order to evaluate the cytotoxic effect of the extracts; methanol, ethyl acetate,

dichloromethane and water of S. commune, a neutral red cytotoxicity assay was


performed. The extracts were treated with four different concentrations ranging from
1 g/ml, 10 g/ml, 50 g/ml and 100 g/ml and tested cervical cancer cells lines;
CaSki, epidermoid cancer cell lines; KB, colon cancer cell lines; HT29 and intestinal
colon cancer cell lines; HCT116. The percentage of inhibition of all the extracts against
the cells was shown in Appendix C. Overall, it can be observed that there was an
increasing trend of cell killing percentage with increasing concentrations of the
mushroom extracts. However, to determine the effectiveness of the cytotoxicity, the
IC50 value of each extracts against the tested cancer cell lines was measured from the
extrapolation of the graphs.
IC50 is the inhibitory concentration, at which the growth of cancer cells were
inhibited by 50%. According to the National Cancer Institute (NCI) guidelines
72

(Suffness et al., 1991), extract with IC50 value 20 g/ml is considered as an active
extract. The IC50 values of S. commune extracts are shown in Table 4.7. The results
were based on the comparison among all the S. commune extracts tested.
From Table 4.7, cytotoxic activity of the S. commune extracts at the tested
concentrations was evident against all the cancer ell lines, eventhough they were not
highly active. Results demonstrate that out of four S. commune extracts, the
dichloromethane extract exhibited a pronounced cytotoxic effect towards the cancer
cells, while, water extract showed no cytotoxic effect against all the cancer cell lines at
the range of concentrations tested. IC50 estimated showed that, except for the
cytotoxcity activity of dichloromethane extract against HCT116, the remaining
S. commune extracts were not active because the IC50 values were above the standard
(20 g/ml) (Table 4.7).

Table 4.7: The IC50 values of S. commune extracts against different cancer cell
lines as measured by neutral red cytotoxicity assay
Cell line
KB
CaSki
HT29
HCT116

Methanol

Ethyl acetate

Dichloromethane

Water

24.40 6.1
62.87 5.5
NA
NA

81.78 9.9
NA
NA
73.94 0.1

76.11 2.7
59.26 2.1
57.80 4.2
14.71 2.0

NA
NA
NA
NA

Data expressed as means standard deviations of triplicate measurements; NA= no activity

As shown in Table 4.7 and Figure 4.3, dichloromethane extract of S. commune


was found to show the active cytotoxic ( 20 g/ml) effect against intestinal colon
cancer cell lines (HCT116) at the range of concentrations tested. The IC50 value of the
extract was observed to be the greatest (14.71 2.0 g/ml) indicating that the extract
was the most active in inhibiting the cancer cells (Table 4.7).

73

90
80

inhibition (%)

70
60
50
40
30
20
10
0
0

20

40

60

80

Concentrations (g/ml)

100

120

Figure 4.3: The cytotoxicity of S. commune dichloromethane extract


against HCT116 cancer cell lines

As for the cancer cell lines tested in this study, KB cells were found to be much
more sensitive to the cytotoxic effect of S. commune extracts as compared to the rest of
the cells (Table 4.7). This was indicated by the IC50 values shown by the extracts of
S. commune except for water which failed to show good inhibition against all the cells.
Although no active (> 20 g/ml) inhibition was recorded, the highest cytotoxicity was
of methanol extract resulting IC50 value of 24.40 6.1 g/ml against KB cancer cell
lines (Table 4.7).
The cytotoxic activity of S. commune extracts against CaSki cells showed that
there were no good inhibitions of cells detected from ethyl acetate and water extracts of
S. commune at the range of concentrations tested (Table 4.7). However, the cytotoxicity
observed from the remaining of S. commune extracts (dicloromethane and methanol)
indicated that the activity against CaSki was not active (> 20 g/ml). Particularly,
dichloromethane showed the most potent cytotoxic activity against the cells with the
IC50 value of 59.26 2.1 g/ml (Table 4.7).

74

On the other hand, results (Table 4.7) depicted that HT29 cancer cell lines was
shown to have the weakest cytotoxic activity as compared to the rest of the cell lines.
With the exception of dichloromethane extract, all S. commune extracts exhibited no
good inhibition against the cells at the concentrations tested. Dichloromethane extract,
although failed to show active cytotoxic effect (> 20 g/ml), gave the highest activity
with IC50 value of 57.80 4.2 g/ml (Table 4.7).

4.4

Analysis of anti-human papilloma virus activity of S. commune extracts


The immunohistochemistry (IHC) method using anti-human papillomavirus

(HPV) 16, 18 E6 monoclonal antibody was carried out to determine the expression of
E6 oncoprotein in treated and untreated cervical cancer cell lines; CaSki. The treated
CaSki cells were tested with S. commune extracts (methanol, dichloromethane, ethyl
acetate and water) at six different concentrations of 1 g/ml, 10 g/ml, 25 g/ml, 50
g/ml, 100 g/ml and 200 g/ml. The presence of E6 oncoprotein was detected with the
appearance of reddish-brown stain either in the nuclear and/or cytoplasmic regions of
CaSki cells.
In the present study, there were two types of controls used consisted of CaSki
cells not treated with extracts and not incubated with anti-HPV 16, 18 E6 monoclonal
antibody (Plate 4.3 (a)) and CaSki cells not treated with extracts but incubated with
anti-HPV 16, 18 E6 monoclonal antibody (Plate 4.3 (b)). The former control was found
to show no stain at all whereas the latter exerted very strong reddish-brown stain
indicated the high expression of E6 oncoprotein, as illustrated in figures below.
On the other hand, Plate 4.4 - 4.7 shows the qualitative appearances of CaSki
cells after treatment with different concentrations of S. commune extracts which
revealed the activity of anti-HPV 16, 18 E6 properties of the extracts. The observation
was done on the morphology and the colorization of the cells.
75

Plate 4.3: The qualitative appearance of two controls of CaSki cell lines after being
treated with different treatments
Cytoplasm
Nucleus

(a): The negative control of CaSki cells;


cells not treated with antibody

Cytoplasm
Nucleus

(b): The positive control of CaSki cells;


cells treated with antibody

As an overall view, results showed that CaSki cells treated with different
S. commune extracts at various concentrations showed a similar trend (Plate 4.4 - 4.7).
The intensity of reddish-brown stain was found to be decreased with the increasing
concentrations of the extracts. On the other hand, the quantity of intact cells became
lesser as the concentrations increased, which agrees with the morphology of treated
cells showing lysis at higher concentrations.
Plate 4.4 shows the intensity and morphology of CasKi cells after treatment with
various concentrations of S. commune methanol extract. It can obviously be seen that
despite the round and intact shape, the cell cytoplasm and nucleus of the cells exhibited
a strong intensity of reddish brown stain at 1 mg/ml. The reddish brown intensity of this
CaSki cells was observed to be lesser with the higher concentration and was found to be
the weakest at the highest concentration of 200 g/ml. At higher concentrations (50-200
g/ml), only the cells membranes were stained in reddish brown but there was an
absence of the reddish brown stain in the cell cytoplasm and nucleus. The cells seemed
to initially show lysis at 25 g/ml and were clearly lysed at 200 g/ml.

76

On the other hand, the suppressing effect of S. commune ethyl acetate extract
against CaSki cells was shown in Plate 4.5. The extract was observed to give the best
intensity of reddish brown stain at the lowest concentration of 1 g/ml, and when higher
concentrations applied (10-200 g/ml), lesser reddish brown stain found in their
cytoplasm and nucleus. However, the concentrations tested did not affect the
morphology of the CaSki cells very much as the cells were observed to be round and
only some were found lysed particularly at concentration range of 50-100 g/ml. The
total reduction of reddish brown stain was clearly observed at the highest concentration
of ethyl acetate extract (200 g/ml).
The qualitative appearance of dichloromethane extract of S. commune was
presented in Plate 4.6. In general, the results showed not much morphological
difference compared to methanol extract. At concentrations 1-25 g/ml, the CaSki cells
were observed to be round and intact. The cells also indicated total presence of reddish
brown stain at the concentration range. However, when the cells were treated with 50
g/ml of dichloromethane extract, the stain in the cytoplasm became lesser and some of
them started to lyse. The cells were observed to show greater lysis at the highest
concentration of 200 g/ml, and the reddish brown stain was greatly reduced.
Accordingly, it can be observed that majority of the CaSki cells treated at this
concentration showed no stain both in the cytoplasm and nucleus.
Meanwhile, the intensity of reddish brown stain and morphology of CasKi cells
after tested with S. commune water extract were displayed in Plate 4.7. As shown in the
figure, the cells were observed to show only a little lysis at the concentrations tested
(1-200 g/ml). The greatest lysis was only found at the highest concentration of 200
g/ml. As for the reddish brown intensity, the water extract exhibited the strongest
intensity at 1 g/ml and this intensity became lesser as the concentration of the extract

77

increased. The decreased were obviously seen at 50 g/ml to 200 g/ml, where only the
membrane cells of the CaSki were stained in reddish brown stain.

Plate 4.4: The appearance of CaSki cells after treatment with methanol extract of
S. commune at different concentrations
Concentration of
extract (g/ml)

Morphology of
cells

Intensity of reddish
brown stain

Majority showing
intact

+++++

10

Majority showing
intact

++++

25

Some intact
Some lysis

+++

50

Majority showing
lysis

++

100

Majority showing
lysis

200

Majority showing
lysis

78

Plate 4.5: The appearance of CaSki cells after treatment with ethyl acetate extract
of S. commune at different concentrations
Concentration
of extract
(g/ml)

Morphology of
cells

Intensity of reddish
brown stain

Majority showing
intact

+++++

10

Majority showing
intact

++++

25

Majority showing
intact

+++

50

Some intact
Some lysis

+++

100

Some intact
Some lysis

++

200

Some intact
Some lysis

79

Plate 4.6: The appearance of CaSki cells after treatment with dichloromethane
extract of S. commune at different concentrations
Concentration
of extract
(g/ml)

Morphology of
cells

Intensity of reddish
brown stain

Majority showing
intact

+++++

10

Majority showing
intact

++++

25

Some intact
Some lysis

++++

50

Some lysis
Some intact

+++

100

Majority showing
lysis

++

200

Majority showing
lysis

80

Plate 4.7: The appearance of CaSki cells after treatment with water extract of
S. commune at different concentrations
Concentration
of extract
(g/ml)

Morphology of
cells

Intensity of reddish
brown stain

Majority showing
intact

+++++

10

Majority showing
intact

++++

25

Majority showing
intact

+++

50

Majority showing
intact

++

100

Some lysis
Some intact

++

200

Majority showing
lysis

81

CHAPTER 5: DISCUSSION
5.1

Antimicrobial activity of S. commune Fr.

5.1.1

Antimicrobial activity of S. commune extracts as evaluated by well diffusion


assay
Previously, it has been reported that mushrooms show antimicrobial effects

(Sheena et al., 2003; Hur et al., 2004). Suay et al. (2000), in their study, described that
Basidiomycetes have been reported to produce a large number of primary and
secondary metabolites which show antibacterial and antifungal activity. Therefore, in
this preliminary study, the antimicrobial effect of methanol, ethyl acetate,
dichloromethane and water extracts of S. commune against the pathogenic
microorganisms was determined using the well diffusion method.
In this method, bioactive compounds diffuse directly into the agar seeded with
the microorganisms to exert the antimicrobial effects. Many studies proved that the well
diffusion method was found to be a more sensitive method of evaluating antimicrobial
activity compared to filter paper disc method. This statement is supported by a research
done by Collins and Lyne (1995) where they documented that the inhibition zones
shown by well diffusion assay were bigger compared to paper disc diffusion assay.
It is also evident in a study by Jonathan and Fasidi (2003), which showed that
the zone of inhibition of Lycoperdon extracts against E. coli was greater in well
diffusion method compared to filter paper method. Toda et al. (1991) suggesting that
there may be a better contact and diffusion of the extracts into the media and organisms
when the well diffusion method was used, whereas, the filter paper in filter paper disc
method itself may act as barrier between the extracts and the organisms.

82

5.1.2

Antimicrobial activity of S. commune extracts against various pathogenic


microorganisms
From this study, it was shown that at both concentrations (0.2 mg/ml and 2

mg/ml) tested, all extracts of S. commune exhibited inhibitory activity against one or
more bacterial and fungal species tested except for the water extract (Table 4.1 and
Table 4.2). In particular, among the four extracts, dichloromethane extract possessed the
highest antimicrobial activity against the microorganisms tested. The dichloromethane
extract exhibited significant activity (P<0.05) against the pathogenic bacteria tested,
which means that the extract is potentially a source of antimicrobial agent. However,
there was no significant difference (P>0.05) observed between methanolic and ethyl
acetate extracts against the microorganisms tested. As for the antifungal activity,
methanolic and water extracts of S. commune were found to show no activity, whereas
ethyl acetate and dichloromethane extracts were partially active against the fungal
species tested. These results suggested that S. commune extracts displayed higher
antibacterial activity when compared to antifungal activity.
The present results support the findings of Suay et al. (2000) that the
antibacterial activity of polypores and gilled mushrooms was found to be more
pronounced than antifungal activity (Suay et al., 2000). As in most cases, it appears that
the fungal and yeast strains are more resistant to antimicrobial compounds than bacterial
strains (Nishizawa et al., 1990; Papadopoulou et al., 2005). A mushroom extract called
Lentinus adherens were observed to be less effective against pathogenic fungi
compared to pathogenic bacteria (Lauer et al., 1991). Another report by Jonathan and
Fasidi (2003) also suggested that the antifungal activities of the mushrooms Lycoperdon
pusilum and Lycoperdon giganteum extracts against pathogenic tested were very low.
Accordingly, all these findings discussed above were in agreement with that of

83

Takazawa et al. (1982), that antifungal antibiotics are not common among
basidiomycetes.
The fungal species tested in this present study were found to be mostly resistant
to S. commune extracts. In all tests done by Papadopoulou et al. (2005), the diameter of
the inhibition zone for Candida albicans was smaller compared to Staphylococcus
aureus and Escherichia coli. The differences in the cell wall structures and protein
synthesis can be attributed to these different resistance patterns (Papadopoulou et al.,
2005). C. albicans was also reported to be resistant to the action of the methanolic
extracts of the specialty mushroom, Lepista nuda (Dulger et al., 2002). Similarly, the
medicinal mushroom, Lentinula edodes extracts were found to show poor activity
against C. albicans (Hatvani, 2001). Sokmen et al. (2004), in their study, described that
there was no antifungal or anticandidal activity recorded from Thymus spathulifolius but
the extract had antibacterial activity.

5.1.3

Extraction solvent as the significant factor in evaluating antimicrobial


activity of S. commune
As mentioned earlier, the dichloromethane extract of S. commune was found to

be the most effective inhibitor against the microorganisms tested at both concentrations
(Table 4.1 and Table 4.2). Thus, it can be concluded that the dichloromethane extract of
S. commune is the best solvent for extracting antimicrobial compound. This suggestion
was based on the number of microorganisms inhibited and the diameter of inhibition
zones measured. The highest antimicrobial activity observed was against the oral
bacteria Streptococcus sanguis with diameter of inhibition zone of 12 1 mm (Table
4.2). This result may suggest that the bioactive compound in dichloromethane extract is
potentially bioactive against most microorganisms except Streptococcus mutans (Table
4.2). In a previous study reported by Hirasawa et al. (1999) on the possibility of the use
of Lentinula edodes (shiitake) extracts as preventive agent against dental caries, proved
84

that, the shiitake extracts were more effective against the main oral infectious diseaserelated bacteria, e.g., Streptococcus mutans and Streptococcus sobrinus, than other
bacteria tested such as Staphylococcus aureus and Escherichia coli.
However, out of the four mushroom extracts, water extract of S. commune was
not inhibitory to all the microorganisms at both the concentrations tested as there was
no inhibition zones recorded in this antimicrobial assay (Table 4.1 and Table 4.2). This
may be due to the effect of processing temperature on compound stability. The water
extract was reported to be heat-labile (Hirasawa et al., 1999). In the microbial analyses
done by the researchers, it was observed that the water extracts of Lentinula edodes
showed the lowest inhibitory activity against both Streptococcus mutans and Prevotella
intermedia compared to the other extracts. The treatment of extracts at 60C for 30
minutes reduced the antibacterial activity against the microorganisms by 60% and the
activity was completely inactivated by heat treatment at 100C for five minutes
(Hirasawa et al., 1999). Hirasawa et al. (1999) also reported that the ethyl acetate of
shiitake showed stronger inhibitory activity against the microorganisms tested
compared to the water. This is in accordance to the present results that ethyl acetate
extract of S. commune was observed to be better antimicrobial agent than the water
extract (Table 4.1).
The water extract of edible Nigerian macro-fungi, Lycoperdon pusilum and
Lycoperdon giganteum were found to be not a good extracting solvent of the
antimicrobial compounds compared with the effectiveness of ethanol (P<0.05)
(Jonathan and Fasidi, 2003). It was observed that Brazilian native plant, Paullinia
cupana extracted with organic solvents provide stronger antibacterial activity than do
those extracted with water. The study confirms the results from previous researches,
which reported that water is not a suitable solvent for extraction of antibacterial
substances from medicinal plants compared to other solvent, such as methanol, or

85

ethanol (Karaman et al., 2003; Parekh et al., 2005). This observation can be explained
by different active compounds were extracted with different solvents and thus resulted
in different antimicrobial activity. It is evident that additional variables such as the
solvent used to dissolve test compounds should not be neglected (Li et al., 1998; Faizi
and Ali, 1999). This is in accordance with Parekh et al. (2005) that successful extraction
of active botanical compounds from plants is dependent on the type of solvent used in
the extraction procedure.
Different extraction solvents were found to exhibit different sensitiveness of
antimicrobial effect. This result agrees favorably with the suggestion of Oloke and
Kolawole (1988) that the antimicrobial effectiveness depends on the extractive solvent
used. This is most likely because the bioactive compounds of medicinal plants may
differ in their solubility depending on the extractive solvents used. Some solvents used
to dissolve test compounds may cause precipitations that may lead to false negative
results of antibacterial activity. Previous study by Cushnie et al. (2003) proved that,
when selected flavonoids are dissolved in organic solvents and diluted with neutral
polar solutions, precipitation occurs. This is worth noting especially when the
antibacterial activity test involves the evaluation of minimum inhibitory concentration
(MIC). Precipitation of flavonoids in a MIC assay may cause diminished contact
between bacterial cells and flavonoids molecules and often be misinterpreted as
bacterial growth (Cushnie and Lamb, 2005).

5.1.4

Antimicrobial activity of S. commune extracts and its correlation to


phenolic content
It is interesting to note that the phenolic content of an extract can also be

correlated to antimicrobial activity (Beuchat and Golden, 1989; Sokmen et al., 2004).
Accordingly, since the methanolic, ethyl acetate and dichloromethane extracts of
S. commune exhibited inhibitory activities against the bacteria tested (Table 4.1 and
86

Table 4.2), it was assumed that phenolics may be the possible compounds in inhibiting
the growth of the microorganisms.
The complex structure of the cell wall in Gram-negative bacteria makes it more
resistant to substances known as phenolic compounds. However, polyphenol
antioxidant called ellagic acid was reported to exhibit antimicrobial activity particularly
against these Gram-negative bacteria. Vattem et al. (2004) stated that, Vibrio
parahaemolyticus and Escherichia coli of Gram-negative bacteria were relatively
sensitive to the presence of ellagic acid in Rhizopus oligosporus bioprocessed cranberry
pomace extracts. The external lipopolysaccharide layer present around the Gramnegative bacterial cells confers more hydrophobicity compared to Gram-positive
bacteria. Thus, the partial hydrophobicity of ellagic acid would be efficiently acted at
the membrane-water interface of the bacteria and may therefore cause membrane
disruption or destabilization resulting in the antimicrobial activity (Vattem et al., 2004).
The phenolics measured by the Folin Ciocalteau method are simple soluble
phenolics that are thought to exert their antimicrobial compounds that exhibit inhibitory
effects against the tested microorganisms (Vattem et al., 2004). The hyperacidification
at the plasma membrane interface of the microorganism which consequences in
disruption of the H+-ATPase required for ATP synthesis, may be a probable mechanism
by which the extracts release their phenolics (Vattem et al., 2004). Results (Table 4.1),
also showed the diameter of the inhibition zone of Gram-positive bacteria was higher
than the zone of Gram-negative bacteria indicating that the simpler membrane structure
of Gram-positive bacteria makes it favourable to the phenolics to exert their
hyperacidification effect and therefore inhibiting the bacteria (Vattem et al., 2004). This
is in accordance with their report that described the protonation effects of phenolics
released from cranberry pomace may be the possible mechanism for antimicrobial
activity against gram-positive Listeria monocytogenes.

87

The different extracts also indicate that different classes of phenolics are most
likely to be the active substances in inhibiting the growth of the microorganisms tested
(Papadopoulou et al., 2005). In the study of red wine extracts by Papadopoulou et al.
(2005), the antimicrobial activity pattern of the extracts varied for different extracts as
this might be due to the different phenolics contained in the extracts. The extracts were
inhibitory to S. aureus, E. coli and C. albicans. Based on the previous reports, there are
several known phenolic acids to have antimicrobial activity that consist of chlorogenic,
caffeic, p-coumaric, ferulic, p-hydroxy-benzoic, vanillic, protocatechuic, syringic (Wen
et al., 2003),

as well as some other phenolic compounds, such as quercetin,

hydroxytyrosol and resveratrol (Bisignano et al., 1999; Chan, 2002). The antimicrobial
activity of the extracts were dose-dependent as it appears that the diameter of inhibiton
zones of extracts against S. aureus, E. coli and C. albicans increased with increasing
total phenolic content of the extracts.
The variation in sensitivity to the phenolic extracts combined with variable
correlations of microorganism to different properties of the extracts may indicate
different mechanisms of action of the extracts against the microorganisms (Vattem et
al., 2004). Alternatively, the resistance of microorganism to antibiotic may also be
caused by the microorganism lacking the structure an antibiotic inhibits; the
microorganism may be impermeable to the antibiotic or mechanism resistance mediated
by conjugative resistance (R) plasmids (Anke, 1989). The R-plasmids carry genes for
antibiotic resistance (enzymes that degrade antibiotic). If R-plasmids exist, they can be
transferred to other cells and resistance spreads through population (Sorum and LAbeeLund, 2002).
On the other hand, in a study carried out by Zheng et al. (1999), discrepancies of
antibacterial activity could perhaps be attributed to different assays used. The variations
within each assay are one of the factors involved in causing these inconsistencies as

88

proven by groups using the same assays are obtaining conflicting results. For example,
different groups using the agar dilution technique use different sizes of bacterial
inoculum (Ng et al., 1996; Kim et al., 1999). In addition to inoculum sizes, other factors
identified include volume of broth or agar (Li et al., 1998), type of broth or agar (Yee
and Koo, 2000), size of wells (Rauha et al., 2000), size of paper discs (Al-Saleh et al.,
1997), strains of a particular bacterial species used (Bashir et al., 1994; Basile et al.,
2000) and incubation period (Li et al., 1998). Accordingly, a set of guidelines was
published for standard agar dilution, broth macrodilution and broth microdilution
methods to reduce the widely conflicting reports of the antibacterial activity of an
extract.
The diffusion method does not distinguish between bacteriostatic and
bactericidal properties of microorganisms nor does it provide any information about the
viability of the test microorganisms, nor its limitation to measure the activity of soluble
components. Therefore, the method requires careful standardization of inoculum
density, medium content, agar viscosity, size, and number of specimens per plate.

5.1.5

Other contributing factors to antimicrobial activity of S. commune extracts


It is also believed that the variation of antimicrobial activity of mushrooms

reflects the genetic differences of the species at the intraspecific level. According to
Suay et al. (2000), basidiomycetes from different genus produced different
antimicrobial activity and there were also isolates from some species showed large
differences in their ability to produce metabolites with antimicrobial activity. Besides,
the difference in the bacterial strains used and the culture conditions used for general
bacteria may also be attributed to the difference in the antimicrobial activities in the
studies (Takazawa et al., 1982). Benjamin (1995), reported that the antimicrobial

89

activity of mushrooms was not always consistent as not every collection of a particular
mushroom exerted the active compound for antimicrobial effects.
Besides, it is interesting to note that, some bioactive compounds found in
extracts may have a different rate in diffusion depending on the extracts. Extracts with
high diffusion rate may contribute to large inhibition zone which lead to effective
antimicrobial effect whereas extracts that are unable to show good effect may be of the
low diffusion rate. According to Zheng et al. (1999), assays relying on diffusion of test
flavonoids may not give a reliable quantitative measure of antibacterial activity because
a potent antibacterial flavonoid may have a low rate of diffusion.
Based on the results (Table 4.1 and Table 4.2), it can be assumed that there
exists several additional factors for the level of effectiveness in antimicrobial action of
S. commune extracts against the microorganisms. The target sites of bacterial cell in
initiating antimicrobial action include cell wall synthesis, protein synthesis, DNA
synthesis, synthesis of bacterial metabolites within the maturing cell (Anke, 1989) and
the sensitivity of the extract itself (Papadopoulou et al., 2005).
From the results (Table 4.1 and Table 4.2), it can generally be described that the
S. commune extracts were found to exhibit better antibacterial activity against Grampositive bacteria (Bacillus cereus, B. subtilis, Enterobacter faecalis, Staphylococcus
aureus, Streptococcus mitis, S. mutans and S. sanguis) than the Gram-negative
(Escherichia coli, Salmonella sp.,

S. typhi, Shigella sp., S. flexneri, Plesiomonas

shigelloides, Proteus vulgaris, and Pseudomonas aeuroginosa). The S. commune


extracts that were shown to be effective against Gram-positive bacteria were probably
due to the inhibition of cell wall synthesis; the cells were unable to maintain rigid
peptidoglycan component of the wall and therefore become susceptible to weakening
and eventual toxic destruction to the cell wall or lysis.

90

As emphasized elsewhere, Gram-positive bacteria are more likely to be more


sensitive to antibacterial agents than Gram-negative bacteria. Gram-negative strains are
reported to be highly resistant to many known antibiotics due to the thicker
peptidoglycan layer of the bacteria cell that protect them from the action of antibiotics
(Anke, 1989; Suay, 2000). A previous study by Papadopoulou et al. (2005) described
that, the diameter of the inhibiton zone for S. aureus was greater than E. coli, indicating
that the Gram-positive strain was more sensitive to the active compound found in the
extracts than Gram-negative strain. The differences in the structure of bacteria cell wall
are believed to play an important role in the antimicrobial activity. It has been proven
that the less complex structure of the Gram-positive bacteria cell wall makes it more
permeable to the antimicrobial compounds (Papadopoulou et al., 2005).
Pycnoporus sanguineus was reported to produce an orange pigment substance
identified as cinnabarine that was found to be more active against Gram-positive than
Gram-negative bacteria. This active compound showed antimicrobial activity against B.
cereus, E. faecalis, E. faecium, E. coli, K. pneumoniae, L. mesenteroides, L. plantarum,
P. aeruginosa, Salmonella sp., S. typhi, S. aureus, and several Streptococcus spp.
(Smania et al., 1995a, 1997).
On the contrary, there was also a study reported that the medicinal plant
Thymus spathulifolius did not possess any selective antimicrobial activity on the basis
of the cell wall differences of bacteria (Sokmen et al., 2004).

5.2

Antioxidant activity of S. commune Fr.

5.2.1

Antioxidant activity of S. commune extracts and its correlation

to

phenolic content
The free radical scavenging activities of mushroom extracts were tested using a
methanolic solution of the stable free radical, DPPH. The DPPH radical system used in
91

this study measures the ability of the corresponding extracts and some pure compounds
to quench DPPH free radicals by providing hydrogen atoms or by electron donation
conceivably via a free-radical attack on the DPPH molecules (Sahin et al., 2004). DPPH
has the advantage of being unaffected by certain side reactions, such as metal ion
chelation and enzyme inhibition compared to laboratory-generated free radicals such as
the hydroxyl radical and superoxide anion (Amarowicz et al., 2004).
Edible mushrooms have been known to possess phenolic compounds (Cheung et
al., 2003) and therefore the investigation of the relation between antioxidant activity
and total phenolic contents of the mushrooms has become a great interest. Fruit bodies,
as well as mycelia of several of these mushrooms were found to have good antioxidant
properties (Mau et al., 2002).

This observation was supported by other studies

suggested that mushrooms contain a variety of secondary metabolites including various


phenolic compounds, which have been proven to act as excellent antioxidants (Ishikawa
et al., 1984; Mau et al., 2002; Cheung et al., 2003).
Phenols such as butylated hydroxytoluene (BHT) and gallic acid or gallate were
known to be effective antioxidants (Madhavi et al., 1996). In this preliminary study,
gallic acid was used as a standard reference for the S. commune extracts to evaluate
their total phenolic content using the Folin Ciocalteau assay. Gallic acid is often used in
most of the antioxidant studies (Brand-Williams et al., 1995) based on its availability of
being stable and pure substance, and it is less expensive than other options.
In the recent study, the radical scavenging activity of the S. commune extracts
correlated well with the total phenolic content. As evidenced in Figure 4.2, a high
correlation (R2=0.8264) was found between the radical scavenging activity and total
phenolic content. Results revealed that methanolic extract of S. commune behaved as
the strongest antioxidant agent, exhibiting ability to reduce the stable radical DPPH to
yellow-coloured diphenylpicrylhydrazine providing IC50 at only 0.145 0.01 mg/ml
92

(Table 4.5) with the highest total phenolic content of 1.72 0.045 mg GA/g extracts
and 0.498 0.07 mg GA/g fruitbodies (Table 4.6). The fact that methanolic extract
possessed higher antioxidant activity than the other extracts, might be explained by its
significantly (P<0.05) higher total phenolic content. On the other hand, being relatively
low in total phenolic content (0.52 0.04 mg GA/g extracts and 0.095 0.02 mg
GA/g fruitbodies) (Table 4.6), the water extract of S. commune gave the weakest
radical scavenging activity (IC50 value of 0.674 0.05 mg/ml) (Table 4.5). The ethyl
acetate extract and dichloromethane extract showed IC50 value of 0.219 0.01 mg/ml
and 0.641 0.13 mg/ml (Table 4.5) respectively with the ethyl acetate extract had
significantly (P<0.05) higher total phenolic content (0.79 0.025 mg GA/g extracts and
0.0570.01 mg GA/g fruitbodies) than the dichloromethane extract (0.520.04 mg
GA/g extracts and 0.0210.01 mg GA/g fruitbodies) (Table 4.6). IC50 value was
determined from the plotted graph of scavenging activity against the concentration of
extracts, which is defined as the amount of antioxidant necessary to decrease the initial
DPPH radical concentration by 50%. The lowest indicates the strongest ability of the
extracts to act as DPPH scavengers.
Medicinal mushrooms have been known to be free radical scavengers or
inhibitors, acting possibly as primary antioxidants. This agrees favorably with the
suggestion of Tanaka et al. (1998) and Shimada et al. (1992) that the antioxidant
activity of natural antioxidants involved the termination of free radical reactions and
reducing power. Free radical scavenging is one of the known mechanisms by which
antioxidants inhibit lipid oxidation (Cheung et al., 2003).
The key role of phenolics compounds as scavengers of free radicals is
emphasized in several reports (Komali et al., 1999; Moller et al., 1999). Based on the
previous report, it has been known that, phenolic compounds in medicinal plants are
powerful free radical scavengers which can inhibit lipid peroxidation by neutralizing

93

peroxyl radicals generated during the oxidation of lipids (Shahidi et al., 1992).
Phenolics are suggested to be the main compounds responsible for the radical
scavenging activity as the antioxidant activity increased proportionally to the phenolics
content (Mau et al., 2002; Cheung et al., 2003). Polyphenolic compounds have been
known to efficiently scavenge superoxide radicals via a single electron transfer
mechanism (Hirano et al., 2001) or by a hydrogen abstraction mechanism to form the
corresponding semiquinone (Wang et al., 1996). These redox properties of phenolic
compounds were reported to attribute to the significant role of phenolics in determining
the antioxidant activity (Rice-Evans et al., 1997) that are by acting as reducing agents,
hydrogen donators and singlet oxygen quenchers.

5.2.2

Extraction solvent as the significant factor in evaluating antioxidant activity


of S. commune
Based on the present free radical scavenging activity results (Table 4.5), it was

observed that the activity of S. commune extracts vary considerably from one kind of
solvent to another depending on the extraction solvent used. This result supports the
suggestion of Cui et al. (2005) that different extracts of mushroom Inonotus obliquus
exhibited variable radical scavenging activities. It is noteworthy that solvent extraction
is frequently used for isolation of antioxidants because both extraction yield and
antioxidant activity of extracts are strongly dependent on the solvent (Julkunen-Tiito,
1985; Marinova and Yanishlieva, 1997).
Methanolic extracts of mushroom Volvariella volvacea was found to be
excellent in scavenging activity as they exhibited significantly higher activity than the
aqueous extracts (Cheung et al., 2003). This finding was similar to the results obtained
in this present study. Based on the results (Table 4.5), it can be observed that
methanolic extract of S. commune was better in scavenging radicals than aqueous
extract. This could be possibly due to the high amount of total phenolics present in
94

S. commune methanolic extract, compared to the water extract (Table 4.6). A study
done by Cheung et al. (2003) showed that, the amount of phenolic compounds was
higher in organic extracts than in water extracts. Based on this finding, it is believed
that the extraction solvent gives a noteworthy effect to the antioxidant activity and total
phenolic content of the extracts.
Mau et al. (2002) and Cheung et al. (2003) in their studies reported that
methanolic extracts obtained from several fruiting bodies of medicinal mushrooms
showed high DPPH radical scavenging activity. The data in this present study is in
accordance with these reports, since the S. commune methanolic extract was shown to
have a remarkably DPPH free radical scavenging activity (Table 4.5). The fact that the
S. commune methanol extract possessed excellent radical scavenging activity might be
explained by their significantly (P<0.05) high total phenolic contents (Table 4.6). Duh
et al. (1999) suggested that the phenolic compounds may contribute directly to
antioxidative action.
Total phenols were the major naturally occurring antioxidant components found
in methanolic extracts from most of medicinal mushrooms. This idea supports the
suggestion that total phenols were the major antioxidant components found in the
methanolic extracts from other mushroom fruit bodies and mycelia (Huang, 2000; Mau
et al., 2001). Cheung et al. (2003) found that the methanol extract of Volvariella
volvacea contained active substances, including phenolic compounds that exhibited a
strong hydrogen-donating capacity to scavenge DPPH radicals as possible mechanism
for their antioxidative activities.
The superiority of the methanolic extract of Origanum vulagrae in the free
radical scavenging activity could be attributed to the presence of 22% of phenolics of
the plant extract (Sahin et al., 2004). Particularly, synergistic effects of phenolic acids
e.g., rosmarinic acid and polyphenols as well as other chemicals such as flavonoids

95

could also be taken into consideration for the radical scavenging activity observed in the
methanol extracts (Choi et al., 2002).
Water extract of S. commune gave the lowest antioxidant activity (Table 4.5)
even though the compounds extracted are highly polar. The lower contents of total
phenols in the S. commune water extract (Table 4.6) might explain their weak
antioxidant effect. However, this may indicate that the scavenging ability on DPPH
radicals could also be due to other compounds besides phenolics. Previous reports have
suggested that the chemical compound in an extract is the one responsible for the DPPH
radical scavenging effects. Aqueous or water extract is likely to be related to the
compounds that are usually consisted of sugars or polysaccharides that have long been
said to be efficacious as antitumor or anticancer agent (Wasser, 2002).
In the present study, water extract of S. commune was prepared by the hot
extraction method, which may contribute to the weakest scavenging activity of DPPH
radicals. Evidently, previous study reported that the hot water extract from mature and
baby Ling chih (G. tsugae) were shown to be less effective than the methanolic extracts
in scavenging activities (Mau et al., 2005). This result may suggest that the bioactive
compounds of the extract were not well extracted to inhibit the radicals. This
observation confirmed the evidence in a previous study reported that natural nutrients
could be significantly lost during the thermal processing due to the fact that most of the
bioactive compounds are relatively unstable to heat (Choi, et al., 2006). Their study
revealed that the bound flavonoid contents of Lentinula edodes (shiitake) declined with
the increasing both heating time and heating temperature. The DPPH radical scavenging
activity of bound compound extract heated at 121C for 30 minutes was significantly
decreased (P<0.05) relative to those of raw shiitake or heat treated at 100C for 15 and
30 minutes or 121C for 15 minutes (Choi, et al., 2006).

96

When compared to the contents of total phenols in methanolic extracts from


specialty mushrooms, commercial mushrooms and ear mushrooms, the highest was
found in Ganoderma (47.25 -55.96 mg/g) indicating this might be the key components
accounting for their better results in antioxidant activity, reducing power, scavenging
and chelating activities (Mau et al., 2002). According to Yen and Wu (1999),
methanolic extract of Ganoderma tsugae is a free radical inhibitor, exhibiting 42% and
75% radical scavenging activity at a concentration of 200 and 500 ppm respectively.
Mau et al. (2002), suggested that the methanolic extracts from several medicinal
mushrooms, such as G. tsugae and G. lucidum, showed high DPPH free radical
scavenging activity. These findings are also in accordance with the previous report that
excellent scavenging effects on DPPH radicals (96.3-97.1%) were observed with
methanolic extract from Antrodia camphorata and Brazillian mushrooms at 2.5 mg/ml
respectively (Huang, 2000).
According to Mau et al. (2002), the methanolic extract of Dictyophora indusiata
(basket stinkhorn) was found to be radical inhibitors or scavengers, acting possibly as
primary antioxidants. As reported elsewhere, the results in the study supported the
observations of some other researchers that methanolic extracts might react with free
radicals, particularly of the peroxy radicals, which are the major propagator of the
autoxidation chain of fat, thereby terminating the chain reaction (Gordon, 1990;
Frankel, 1991; Shahidi et al., 1992). The highest content of total polyphenols in basket
stinkhorn might be responsible for the better results found in antioxidant activity,
reducing power and scavenging abilities as compared to other specialty mushrooms
(Mau et al., 2002).

97

5.2.3

Other contributing factors to antioxidant activity of S. commune extracts


Other factors contributing in the significant variations of antioxidant effect of

S. commune extracts might be due to the different phenolic compositions or the


presence of non-phenolic antioxidants (Velioglu et al., 1998) and the quantitative and
qualitative nature of the phenolic content (Aljadi and Kamaruddin, 2004) which needs
further study. This is in the agreement with other researchers who found variations
between different classes of phenolics in terms of their antioxidant activities (Hirano et
al., 2001). Kahlos et al. (1989) found that, the phenolics were not the only compounds
to result in the radical scavenging activities of Inonotus obliquus. In their study, it was
observed that the polyphenolic extract had the strongest antioxidant activity among the
Inonotus obliquus extracts, while the ethanol extract of I. obliquus, which contained
triterpenoids and steroids showed a relatively strong DPPH radical scavenging effect.
These findings are in accordance with a report by Mau et al. (2002), who revealed that
some other components also existed and contributed in part to the antioxidant properties
of medicinal mushrooms. Based on this finding, it is possible to suggest that the
phenolic compounds were not the sole contributor that may account for the radical
scavenging activity.
Apart from that, antioxidants concentration, extraction medium, temperature, pH
of medium (Gazzani et al., 1998), chemical structures and position in the molecule
(Prior et al., 2005) are pertinent factors that could also be taken into account to evaluate
the antioxidant activity of an extract.
There are also several methodological limitations identified for antioxidant
determination (Kaur and Kapoor, 2001). Methods that involved generation of radical
species, where the antioxidants determine the disappearance of radicals are commonly
applied in measuring antioxidant activity (Cao et al., 1993). Besides, different assays

98

are pertinent in an efficient extraction medium, than relying on a single assay to


determine the antioxidant activity (Othman et al., 2007).
5.3

Cytotoxicity of S. commune Fr.


Mushrooms have long been known to be medically active in several therapies

such as anti-tumor, antiviral and immunomodulating treatments (Wasser and Weis,


1999). In particular, higher basidiomycete mushrooms are believed to be an unlimited
source of antitumor and immunostimulatory polysaccharides which are mostly nontoxic
(Wasser and Weis, 1999). The study of the potential of natural products against various
diseases especially cancer has become a great matter of significance. Natural products
have been described to offer protective and therapeutic actions to all cells with low
cytotoxicity and are beneficial in producing nutrient repletion to compromised people
(Reddy et al., 2003). However, it is noteworthy that some anticancer agents might
exhibit their antitumor activity in vivo but not in vitro cytotoxic activity, a process
which has been described to be due to immune modulation by the compound which
could then lead to antitumor activity in vivo (Rosskopf et al., 1992).

5.3.1

Cytotoxicity of S. commune extracts as evaluated by neutral red assay


In the present study, methanol, ethyl acetate, dichloromethane and water extracts

of S. commune were evaluated for their cytotoxic activity against cervical cancer cell
lines; CaSki, epidermoid cancer cell lines; KB, colon cancer cell lines; HT29 and
intestinal colon cancer cell lines; HCT116 by applying the neutral red (NR) assay.
Neutral red is a supervital dye that accumulates in lyososomes of viable cells and on
extraction can be measured spectrophotometrically (Babich and Borenfreund, 1992).
The NR assay is advantageous that it detects only viable cells and measure druginduced alterations in metabolic pathways or structural integrity which may or may not
be directly related to cell death. Apart from that, the advantages of in vitro cytotoxicity
99

test over in vivo studies such as speed, economy in funds and animals, increased
sensitivity and reproducibility of test conditions are apparent (Stark, 1986). This finding
is consistent with Doyle et al. (2000) that the assay is simple, fast, sensitive, economical
and highly reproducible.
However, there were certain limitations found in the technique, some of which
concerning the character of the compounds to be tested: volatile chemicals tend to
evaporate under the conditions of the test, thus the IC50 value may be variable,
especially when the toxicity of the compound is fairly low. This has been overcome to
some extent by utilizing 96-well rather than 24-well test plates (Knox et al., 1986;
Riddell et al., 1986) as the smaller surface area of the well in these dishes reduces the
extent of evaporation.
The NR assay in this study was performed after the various cell lines were
incubated with the S. commune extracts for 72 hours rather than 24 hours time
incubation. The former incubation time was selected as there are few disadvantages of
using the latter as it may not only lead to unacceptable number of false negative
indications of cytotoxicity, but also a failure to discriminate between chemicals which
have different cytotoxicity. Besides, it was found that certain bioactive compounds may
also need longer time to exhibit their cytotoxic effects, particularly, those that affect cell
division and cell viability.
The S. commune extracts were dissolved in dimethyl sulfoxide (DMSO) at the
different range of concentrations; 1 g/ml, 10 g/ml, 50 g/ml and 100 g/ml. DMSO
has been known as the most effective organic solvent used for serial dilution of the
extracts as it is suitable for compounds that are not soluble in aqueous solution (Wilson,
1992). It is a cryoprotective agent which may reduce the cellular injuries as it is not
toxic to the cells

100

The four different cell lines (cervical cancer cell lines; CaSki, epidermoid cancer
cell lines; KB, colon cancer cell lines; HT29 and intestitinal colon cancer cell lines;
HCT116) tested in this study were cultured and preserved carefully in order to avoid the
risk of contamination that can affect the cytotoxic activity. It was also learnt that it was
very important to ensure the confluence in cell growth as it may affect the results of the
experiment. This is because, the tested extracts were found to exhibit low growth
inhibition of cells when the confluent and over-confluent cells were used. This may
suggest that the cells prevented the extract to penetrate or absorbed to the cells.
Therefore, the cells had to go through an observation under the microscope from time to
time to ensure that they were still in freshly good condition even when 60-70%
confluence in growth was obtained. This observation confirmed the evidence of
Borenfreund and Puerner (1986) that it is best to have 60-70% confluence of cells at the
time of testing, so that, cells are fully exposed to the testing agent. This is likely to be
due to cells that are less sensitive to the testing agent if cells reached the confluent state.
The media used to culture the cell lines were known as RPMI 1640. This
medium is always supplemented with serum and consists of buffer and basal nutrients
which supply source of energy and various supplements for the cell growth (Doyle and
Griffiths, 2000). RPMI 1640 can be added to inactivate the trypsin once the
disassociation of the cells from monolayers is achieved. Trypsin is one of the most
highly specific proteases known (Doyle et al., 2000) and was used to detach cells from
the surface of the flask. However, prolonged exposure to cells can cleave the cell
membrane proteins resulting in cell damage (Martin, 1994). Hence, serum should be
added after trypsinization to arrest proteolysis (Maurer, 1992).
In this assay, the cells were washed consecutively with phosphate buffered
saline (PBS) solution. PBS was known to act as a buffer to retain the pH of cells

101

because any changes in pH (below 6.8) usually inhibitory to the cell growth (Griffiths,
1992), thus may contribute to inaccurate results.
Other factor that may interfere with the experiment was the precipitation of NR
crystals in the NR media solution. The crystals if left in the mixture may affect the
consistency in reading the absorbances. Another research by Borenfreund and Puerner
(1985) described that NR media was pre-incubated overnight at 37C to remove fine
precipitate and dye crystals that might present in the mixture.
Borenfreund and Puerner (1986) reported that the cells were rapidly rinsed with
washing solution after the incubation with NR media to remove the extracellular NR, as
well as to prevent detachment of cells during the subsequent extraction procedures.
However, the solution was left only shortly in contact with the cells as longer exposure
was found to extract NR dye from the viable cells and further lead to a false result
(Borenfreund and Puerner, 1985)
In order to ensure that a cell culture is growing exponentially, it is useful to
know the percentage of viability and the percentage of dead cells, and hence, the stage
of growth of the cells can be confirmed. This can be estimated by their appearance
observed under the microscope. It is helpful to obtain an accurate cell count of the
percentage viability of the cell population with the use of haemocytometer. The use of
viability stains such as tryphan blue ensures more quantitative analysis of the condition
of the culture (Doyle et al., 2000). Tryphan blue will only enter across the membranes
of dead/non-viable cells. Hence, it is important in counting the viable and non-viable
cells. When a cell suspension is diluted with tryphan blue, viable cells stay small, round
and refractile while non-viable cells become swollen, larger and dark blue (Doyle et al.,
2000).
However, inaccuracy of haemocytometer method can play a major role in the
assay as it can affect the counting of the viable cells. There can be many factors that

102

resulted in this inaccuracy. This might be due to overflowing or incompletely filling the
chamber with the cell mixture and air bubbles or debris. Thus, this resulted in an
inherent error in the method due to random distribution of the cells in the chamber. The
error can be reduced by duplicating the counting of cells and increasing the number of
cells.

5.3.2

Cytotoxicity of S. commune extracts towards different cancer cell lines


Results presented in the study (Table 4.7) showed the cytotoxicity of the

S. commune extracts against the various cell lines. The cytotoxicity test was
successfully carried out according to the National Cancer Institute guidelines, which
indicated that extract with IC50 value of 20g/ml is considered as an active extract
(Geran et al., 1972, Suffness, 1995). The guidelines were followed as to find out which
of the mushroom extracts possess low toxicity against the cells. This is important
because if the extracts are toxic, then it can be suggested that the extracts are not safe to
be applied particularly against the normal cell lines and especially for testing in human.
According to the existing literature, potential chemopreventive agents selected for
testing in people at high-risk cancer must have low toxicity as to be compared to the
drugs used to treat existing cancer (Greenwald, 2002).
The IC50 value for each crude extract was determined and summarized in Table
4.7. In general, all the S. commune extracts exhibited various cytotoxic activities against
the tested cancer cell-lines (Table 4.7). Based on the previous study, it has been
observed that cytotoxicity may give different results depending on the test agent used
and the cytotoxicity assay employed (Weyermann et al., 2005).
Among the S. commune extracts tested, only the water extract failed to show any
inhibition against all the cell-lines at the range of concentrations tested (Table 4.7).
Methanolic extract had weak cytotoxic activity against all cell-lines tested except for the

103

retardation of proliferation of KB cells with IC50 value of 24.40 6.1 g/ml. Ethyl
acetate extract of S. commune was also found to be poorly active against all the celllines. However, dichloromethane extract exhibited the most active inhibition especially
against HCT116 cancer cell line with IC50 value of 14.71 2.0 g/ml.
According to the results (Table 4.7), it can be observed that the S. commune
extracts displayed a selective cytotoxic effect depends on the cell lines. The results are
consistent with those by Belofsky et al. (1998) who reported that the active compounds
isolated from organic extracts of Aspergillus versicolor exhibited significant cytotoxcity
against HCT116 human colon carcinoma cells in vitro and displayed moderately
selective cytotoxicity toward a panel of renal tumor cell lines. The inhibition of cell
proliferation is probably due to a process leading to death called apoptosis.
According to Wang et al. (1998), the mushroom Agaricus bisporus was found to
produce nearly sixty bioactive substances; lectins, with the ability to retard cancer cell
growth without any apparent effect on normal cells. Meanwhile, Vijayan and Chandra
(1999), reported that there were several lectins isolated from Agaricus bisporus,
Agaricus blazei, Agaricus campestris and Agaricus edulis. Sarangi et al. (2006)
observed that Pleurotus ostreatus mushrooms cultivated on the date waste possessed a
potent anti-tumor activity against Ehrlichs ascites carcinoma. Three proteoglycans
isolated from P. ostreatus were also reported to induce apoptosis which resulted to cell
death. Study showed that treatment with these proteoglycans of Sarcoma-180 bearing
mice showed large reduction in the number of Sarcoma-180 cells and cell cycle analysis
also showed the increased number of population in the apoptotic phase.
The transformation of normal colorectal epithelium to carcinomas involves
progressive apoptotic inhibition. A newly identified low-molecular-weight -glucan
from Pleurotus ostreatus with promising anti-tumorigenic properties, demonstrated to
directly affect HT-29 colon cancer cell growth by regulating the expression of

104

apoptosis-related proteins and inducing programmed cell death and inhibition of


proliferation. P. ostreatus polysaccharides exert their anti-proliferative effects on HT-29
cancer cells as a result of apoptotic induction because the pro-apoptotic molecules; Bax
and cytosolic cytochrome c were upregulated. The expression levels of anti- and proapoptotic proteins of the Bcl-2 family such as Bax and Bak have previously been shown
to directly engage the cell-death machinery, whereas Bcl-2 and Bcl-XL merely
antagonize this interaction (Chiarugi and Ruggiero, 1996; Harnois et al., 2004). Bax, as
well as its homologous protein Bak, promote cell death by competing with Bcl-2. The
study showed that Bax protein expression and cytosolic cytochrome-c concentrations
were strongly upregulated by the partially isolated -glucan in the polysaccharidetreated HT-29 cells. The signaling events leading to apoptotic mitochondrion-related
pathways lead to changes in mitochondrial membrane permeability and the subsequent
release of pro-apoptotic factors involved in various aspects of apoptosis (Harnois et al.,
2004). However, it was observed that this low-molecular-weight -glucan preferentially
inhibits only HT-29 colon cancer cells and not human fibrolasts, and this indicates that
the extracts show specificity towards neoplastic cells (Lavi et al., 2006).
Mitochondria has been claimed to play an essential role in the propagation of
apoptosis. Previous study reported that several chemotherapeutic agents can cause
direct or indirect apoptosis, in particular in cell death by insult to the mitochondria
(Kroemer et al., 1997). The findings supported that mitochondria have been proposed as
a novel prospective target for chemotherapy-induced apoptosis (Mow et al., 2001). The
interaction of anticancer agents with mitochondria results in an increase of the
permeability of the inner mitochondrial membrane (Fulda et al., 1998). This is in highly
accordance with Kroemer et al. (1997) who described that the reduction of
mitochondrial membrane potential is possibly due to an opening of mitochondrial
permeability transition pores.

105

Kim et al. (2007), in their study, documented that the extracts of Duchesnea plus
Ganoderma may provoke marked changes of the mitochondrial functions suggesting
that apoptosis by this combination occurs through the mitochondria-dependent pathway.
The medicinal mushroom Ganoderma lucidum and the herb Duchesnea chrysantha
extracts (GDE) interact synergistically to cause induction of mitochondrial damage and
to enhance apoptosis in human leukemia HL-60 cells. Although it was unclear the effect
of these extracts on a loss of mitochondrial membrane potential is due to a direct
targeting to the mitochondrial inner membrane or is associated with Bcl-2 downregulation, Bax translocation, mitochondrial cytochrome c release and caspase-3
activation, suggesting that apoptosis by this combination occurs through the
mitochondria-dependent pathway (Kim et al., 2007). However, it was found that the
combined treatment (GDE) was selectively toxic only to HL-60 leukemia cells whereas
no cytotoxic effect was observed in normal peripheral blood mononuclear cells.
Many nutritive and non-nutritive phytochemicals (chemical or nutrient derived
from a plant source) with diversified biological properties have shown promising
responses for the prevention and/or intervention of prostat cancer in regard of the
exploration of novel treatment modalities as well as anticancer agents (Surh, 2003).
Research has suggested that 5-dehydroxymethyl derivative of epoxyquinomicin C,
isolated from Amycolatopsis could be potentially used for the treatment and prevention
of prostat cancer (PCA). The compound has shown a significant growth inhibition in
hormone-refractory PCA cell line DU-145 through the induction of an apoptotic cell
death (Kikuchi et al., 2003).
Besides, the mushroom Ganoderma lucidum which have been known to contain
biologically active polysaccharides and triterpenes with potent anti-tumor activities (Lin
and Zhang, 2004), were also previously demonstrated to induce apoptosis, inhibit cell

106

proliferation, and suppress cell migration of highly invasive human prostate cancer cells
PC-3 (Sliva et al., 2002).
Putrescine-1,4-dicinnamide, a phenylpropanoid derivative conjugated with
polyamine putrescine was isolated from the fruiting bodies of the gilled mushroom
Pholiota spumosa (Basidiomycetes, Strophariaceae), (Clericuzio et al., 2004) taken up
by growing cell, inhibits cell growth of cancer cells inducing apoptosis cell death,
mediated, at least in part, by the activation of caspase cascades (Fraser et al., 2002;
Wolff et al., 2003).
Based on the present data (Table 4.7), it was revealed that water extract (which
was known to be polar) of S. commune was the weakest in exerting the cytotoxic effect
against all the cell lines. This result disagrees with that of Kamuhabwa et al. (2000)
who suggested that the polar compounds were the ones which responsible for anticancer
activity. This negative cytotoxic activity shown by water extract of S. commune can be
likely related to the low apoptotic mechanism of the extract against the cancer cell lines.
The result of cytotoxicity may be better if the extract is combined with other potential
extract to bring out the maximal synergistic effects against the cells. This is consistent
with the previous finding by Kim et al. (2007), who described that, the combination
treatment could be more potent than either a drug alone, and it has fewer side effects.
Study also showed that a single exposure to Duchesnea or Ganoderma extract
exerted minimal effects on the apoptotic protein levels or caspase activity which by
itself was insufficient to activate the mitochondria-dependent apoptotic pathway
suggesting that some extracts may mediate their anti-cancer effects via multiple
components with a multiple mechanism (Kim et al., 2007).

107

5.3.3

Cytotoxicity

of

S.

commune

extracts

and

its

correlation

to

antimicrobial activity
In this preliminary study, it is interesting to note that there was a correlation
between cytotoxicity and antimicrobial activity of the S. commune extracts. This is
particularly based on the results (Table 4.1 and Table 4.7), suggesting that the
dichloromethane extract of S. commune was found to be the best extract in exerting its
bioactive compound in both antimicrobial and cytotoxic activity.
This observation was supported by several experiments carried out by previous
reseachers. Anke et al. (1980) suggested a species from the genus Marasmius that have
long been known to produce interesting secondary metabolites. Within the genus,
M. alliaceus was shown to produce two antimicrobial and cytotoxic metabolites
denominated alliacols A and B. On the other hand, marasmic acid isolated from
M. conigenus was found to have antibacterial, antifungal, cytotoxic and phytotoxic
activities (Abraham, 2001).
Other study presented that the culture extract of Agrocybe perfecta was
investigated and later found to produce substances presenting anti-tumoral activity as
well as antibacterial activity against S. aureus and E. coli (Mavoungou et al., 1987).
The same observation was showed by the poisonous fungi, Agaricus xanthodermus
which were reported to produce several compounds with antimicrobial and cytotoxic
activity (Dornberger et al., 1986).
The mushroom Pycnoporus sanguineus has been known for its antimicrobial
activity since 1946. Poliporin, a bioactive compound isolated from this mushroom, was
reported to be active against Gram-positive and Gram-negative bacteria but without
toxicity to experimental animals (Bose, 1946).
Cytotoxicity test carried out by Ajaiyeoba et al. (1998) on four crude extracts of
a medicinal plant, Ritchiea capparoides found that despite the extracts had high
108

antifungal activity against the clinical strains of fungi, they were shown to be non-toxic
with LD50 values higher than 1000 g/ml.

5.3.4

Other contributing factors to cytotoxicity of S. commune extracts


The different sensitivity of S. commune extracts towards the cancer cell lines is

also likely to be due to the presence of difference classes of compounds in the extracts,
as it has been documented in the case of known classes of compounds (Cragg et al.,
1994). The results also agree favorably with that of Ajith and Janardhanan (2003) who
described that ethyl acetate and methanol extracts of a polypore macrofungus, Phellinus
rimosus exhibited marked cytotoxicity against Ehrlichs ascites carcinoma (EAC) and
Daltons lymphoma ascites (DLA) cell lines, whereas aqueous extract did not possess
cytotoxic effect up to a concentration of 1 mg/ml. Studies have revealed that this
Brazillian basidiomycetes, can produce active substances with cytotoxic activities
(Atsumi et al., 1990; Han et al., 1999). A further study on chemical characterization of
both ethyl acetate and methanol extracts showed detectable amounts of polyphenols and
flavonoids (Ajith and Janardhanan, 2003) which can be assumed as the compounds
responsible for the cytotoxic activity observed for both extracts. The results support the
suggestion of Carlo et al. (1999) that a number of phenolic compounds such as
polyphenols and flavonoids have been reported to possess anti-tumor activity.
Phenolic compounds were suggested to have inhibitory effects on mutagenesis
and carcinogenesis in humans among the various secondary metabolites isolated from
mushrooms (Tanaka et al., 1998). It is interesting to note that phenolics are one of the
major groups of non-essential dietary components that have been associated with the
inhibition of atherosclerosis and cancer (Williams and Iatropoulos, 1997). As
emphasized elsewhere, it has been reported that the antioxidant activity of plants is

109

responsible for their therapeutic effect against cancer as well as cardiovascular disease
(Anderson et al., 2004; Stanner et al., 2004).
According to the existing literature, the polyphenolic extracts of various plant
and mushroom species, have strong antioxidant activity. The literature has also
described the prooxidant and cytotoxic effects of these phenolic components. Liu et al.
(1997) suggested that high concentrations of phenolic compounds may inhibit cell
proliferation, and simultaneous exposure to hydrogen peroxide and that the phenolics
has been shown to lead the amplification of proliferation inhibition.
Apart from that, the variable results shown in this study might be due to the
differences in solubility, molecular size, branching frequency and forms (Wasser,
2002). Higher molecular weight glucans are found to be more effective than those of
lower molecular weight (Mizuno, 1996). It is believed that structural features such as
(13) linkage in the main chain of the glucan and additional (16) branch points are
required for anticancer action (Wasser, 2002).
Polysaccharides like glucans have been suggested as the most widely and most
commonly observed macrophage activator in nature. Macrophages are part of the innate
immune system, which play an important role in protecting the body from any type of
invading cells including cancer cells (Kurashige et al., 1997). A number of
polysaccharides and protein bound polysaccharides isolated from mushrooms have been
clinically used for the treatment of cancer. Krestin (PSK) from Coriolus versicolor,
lentinan from Lentinula edodes and schizophyllan from Schizophyllum commune are the
examples of polysaccharides sold as anticancer drugs especially in China and Japan
(Fukushima, 1989).
Other studies described that microbial metabolites such as mushroom
polysaccharides have been known to activate natural killer (NK) cells to express potent
tumoricidal activity (V tvi ka et al., 1998; Di Renzo et al., 1991). Natural killer (NK)

110

cells are directly cytotoxic for tumor cells and play a primary role in regulating immune
responses (Kodama et al., 2002). Cytotoxicity is one of the chemotherapeutic agents of
anti-tumor activity (Suffness and Pezzuto, 1991) and research revealed that most of the
clinically used anti-tumor agents possess significant cytotoxic activity in cell culture
systems (Ajith and Janardhanan, 2003).
NK cells are best known for their ability to kill tumor cells and this idea
confirmed the finding that they play an important role in controlling infection in the
earliest phases of bodys immune responses (Sarangi et al., 2006). The activation of NK
cells is a good indicator of anti-tumor properties of a compound. A study showed that a
fraction of Pleurotus ostreatus had the highest effect on NK cell for its cytotoxic
activity both at lower (10 g/ml) and higher concentration (100 g/ml) (Sarangi et al.,
2006). Other study showed that polysaccharides of Ganoderma lucidum can increase
NK cell cytotoxicity in cord blood (Chien et al., 2004).
From the results (Table 4.7), it can also be observed that the S. commune
extracts gave variation in the cytotoxic activity depending on the cell lines. The
different source of cell lines might be contributed to the difference sensitivities of
S. commune extracts against the cells. Thus, the use of more than one cell line is
therefore considered necessary in the detection of cytotoxic compounds (Kamuhabwa et
al., 2000) as what had been used in the present study. Fornelli et al. (2004) reported in
their study that the different cytotoxicity of fungal metabolites against the insect cell
lines could be related to the different insect families or different origin of the tissue. The
different nature of the cell line could also be taken into consideration, i.e. rapidly
growing, non-differentiating cells of very low metabolic activity, that raising problems
of direct extrapolation of results to the in vivo situation. Substances which specifically
attack by dividing cells may appear to be of a much higher order of toxicity than they
would be in vivo.

111

5.4

Anti-human papilloma virus (HPV) activity of S. commune Fr.


There have been reported in previous studies that mushrooms possess activity in

traditional systems of medicine for prevention and treatment of many diseases.


Medicinal plants like mushrooms have been gaining popularity among researchers as
they have fewer side effects, better patient tolerance, relatively less expensive and
acceptance due to long history of use. Interestingly, these plants are also known to be
less prone to the emergence of drug resistance. Unlike the synthetic drugs, these
medicinal plants are renewable in nature, besides its cultivation and processing, which
are often environment friendly (Vermani and Garg, 2002).
Extracts of medicinal plants have been described as potential agents against viral
diseases including sexually transmitted diseases (STDs) caused by variety of pathogens
such as viruses (human immunodeficiency virus (HIV), herpes simplex virus and
human papilloma virus (HPV)) (Vermani and Garg, 2002). The goal of antiviral
chemotherapy is the discovery of antiviral agents that are specific for the inhibition of
viral multiplication without affecting normal cell division (Eo et al., 1999).

5.4.1

Anti-human papilloma virus (HPV) activity of S. commune extracts as


evaluated by immunohistochemistry assay
In the present study, the cervical cancer cell lines (CaSki) treated with

Schizophyllum commune extracts in various concentrations of 1 g/ml, 10 g/ml, 25


g/ml, 50 g/ml, 100 g/ml and 200 g/ml were analysed for the expression of HPV 18
E6 oncoprotein using immunohistochemistry. Particularly, the four different extracts of
S. commune which were consisted of methanol, ethyl acetate, dichloromethane and
water were evaluated for any potential in inhibiting the viral antigen E6 which is
normally produced by high risk human papilloma virus (HPV) 16 and 18. This is

112

supported by a previous study, described that HPV 16 and 18 account for approximately
70% of HPV positive cervical cancer (Naghashfar et al., 1996).
This E6 oncoprotein was detected using the widely-used immunohistochemistry
(IHC) staining method. Immunohistochemistry is one of the immunology studies
concerning with the clinical enzymatic reactions between the antibodies and the
antigens in the immune system. It is capable of identifying antigen in paraffin sections
or exfoliated cells and has been applied extensively in HPV research (Jenson et al.,
1980).
The key reagent in all immunohistochemical techniques is the antibody
(Boenisch et al., 1989) where the absence of specific primary antibody can cause failure
in detecting viral antigen (E6 oncoprotein) resulting no complex (reddish-brown
precipitate) being formed at the end of staining process. The intensity of reddish brown
stain is directly correlated with the expression of HPV-18 E6 oncoprotein where the
stronger reddish brown stain indicates higher expression of HPV-18 E6 protein.
In the immunohistochemistry method, an unconjugated primary antibody binds
to the viral antigen located within the cells. This step is followed by the addition of a
biotinylated secondary antibody (HRP conjugated antibody) known as the link antibody
and subsequently, by enzyme-chromogen, 3 diaminobenzidine tetrahydrochloride
(DAB) substrates into coloured end products (Boenish et al., 1989). The peroxidase is
then developed by the DAB or other substrate to produce different colored end
products.
However, using HRP conjugated antibody may result in high, non-specific
background staining. This non-specific background can be significantly reduced by pretreatment of cells/tissues with hydrogen peroxide (H2O2) prior to incubation with HRP
conjugated antibody. H2O2 is first applied prior to incubation of primary antibody to
eliminate endogenous peroxidase activity of the tissue section. H2O2 is a blocking agent

113

in immunohistochemistry, and is commonly used to block endogenous peroxidase


acitivity in cells/tissues. Pre-treatment of cells with saturating amounts of H2O2 results
in the irreversible inactivation of endogenous peroxidase. 3% H2O2 is normally used to
block the endogenous peroxidase activity because certain cells/ tissues/antigen can be
destroyed by high concentration of H2O2.
Peroxidase activity in the presence of an electron donor first results in the
formation of an enzyme-substrate complex. Then, the oxidation of this electron donor
provides a driving force in continuing catalysis of hydrogen peroxide to form an
insoluble coloured product. Peroxidase is then developed by the DAB or other substrate
to produce different colored end products. This 3-amino-9-ethocarbazole forms a rosered end product that is soluble in alcohol (Boesnich, 1989).

5.4.2

Anti-human papilloma virus (HPV) activity of S. commune extracts towards


cervical cancer cell lines (CaSki)
According to the results (Plate 4.4 4.7), the S. commune extracts (methanol,

ethyl acetate, dichloromethane and water) had positive anti-viral activity against the E6
protein-producing HPV 18 contained in the CaSki cells. The intensity of reddish brown
stain in CaSki cells treated with mushroom extracts decreased with the increasing
concentrations of the extracts. It can be suggested that the intensity of the reddish brown
stain correlates directly with the presence of E6 protein in the cells and inversely with
the suppression activity of mushroom extracts. Thus, the results might indicate that
S. commune extracts were successful in inhibiting the expression of HPV 18 E6
oncoprotein. The reddish brown stain appearance denotes the expression of the
oncogenic protein cells, while the absence of reddish brown stain indicates that E6
expression had been suppressed.
On the other hand, the morphological observation showed that cells were lysed in
almost all extracts especially at high concentrations as supported by the cytotoxicity
114

tests. Results showed that CaSki cells treated with different S. commune extracts at the
tested concentrations were observed to show a quite similar trend. However, methanol
(Plate 4.5) and dichloromethane (Plate 4.6) extracts of S. commune seemed to display
better inhibition of viral antigen E6 compared to ethyl acetate (Plate 4.4) and water
(Plate 4.7) extracts. This is in accordance with the morphology of the CaSki cells that
seemed to initially show lysis at the concentration as low as 25 g/ml and were greatly
lysed as the concentrations increased (50-200 g/ml). Accordingly, it was observed that
majority of the CaSki cells treated at higher concentrations showed no stain both in the
cytoplasm and nucleus, indicating that S. commune extracts were successful in
inhibiting the expression of HPV 18 E6 oncoprotein in the cells. It can be assumed that
the higher the concentrations, the more bioactive compounds found in the extracts,
which inhibited the E6 activity, thus allowing effective lysis. These findings suggested
that both methanol and dichloromethane extracts of S. commune can be considered as
potential antiviral agents.

5.4.3

Comparative study of various antiviral activities


The antiviral activity of mushrooms has been described elsewhere. This agrees

with many researchers who have accounted mushrooms for their activities as an antiviral, anti-tumor and anti-oxidant (Ooi, 2001). The goal of antiviral chemotherapy is the
discovery of antiviral agents that are specific for the inhibition of viral multiplication
without affecting normal cell division (Eo et al., 1999).
There are many factors that contribute to the infection of cells by viruses. They
could be caused directly by inhibition of viral enzymes, synthesis of viral nucleic acids
or adsorption and uptake of viruses into mammalian cells. These effects are usually
exhibited by smaller molecules, whereas, indirect antiviral effects are the result of the

115

immunostimulating activity of polysaccharides or other complex molecules (Brandt and


Piraino, 2000).
As reported elsewhere, the activity of antiviral agents can vary dependent on the
screening method (Schinazi et al., 1986). The different conditions used in the procedure
might explain, in part, the difference between the concentration values. The high
concentration of extract used in the test may be correlated with a weak activity of its
chemical component (Lopez et al., 2001) which needs to be further studied.
Previous study showed that incubation of the potential extract during cell culture
infection impaired the productive replication of both herpes viruses in an extract
concentration-dependent manner. This might be partially due to a direct interaction with
virus particles or their entry into the cell, instead of interfering with intracellular virusspecific macromolecular synthesis. Based on the concentration-dependent antiviral
activity found in vitro, it could be suggested that the mechanisms involved a specific
action on the virus particle instead of on the virus-specific macromolecular synthesis
(del Barrio and Parra, 2000)
There are several possible mechanisms that might explain the antiviral activity
of Indian medicinal plant extracts in the study reported by Balasubramanian et al.
(2007). First possible mechanism is the viral inactivation which may be resulted from
the reaction between the extract and the envelop proteins of the virus into the host and
thus prevent the entry of the virus into the host. Alternatively, the influence of the
extract on the replication of the virus, which prevents the multiplication of the virus in
the host cell may also be accounted for the next mechanism involved. Finally, the other
possible mechanism identified is that the plant extract might act as immunostimulant
which enhances the innate immunity.
According to the existing literature, there were reported that many extracts had
partial antiviral activity. These extracts were found to have true antiviral compounds but

116

sometimes they were present at insufficient quantities to inactivate all infectious viruses
in the standard virus preparation (Kudi and Mynt, 1999). This is in the agreement with
the finding that there may be some variation found in the way plant compounds behave
in the different cell lines (Hudson, 1990). One of the inherent disadvantages of in vitro
antiviral testing is the environmental sensitivity of the cell lines in culture. In a study by
McCutcheon et al. (1995), it was suggested that results of the antiviral effects in vivo
may not be the same in in vitro assays because of the extremely low concentrations of
extract tolerated by cells in the artificial system.
Among the antiviral from mushrooms showing promise for their anti-viral
activities are Lentinan from shiitake, Lentinula edodes, polysaccharide peptide (PSP)
from Turkey tail, Trametes versicolor and ganaderiol-F, ganoderic acid-, lucidumol
from reishi, Ganoderma lucidum. Most of these antivirals are water soluble, relatively
heat stable and are present in both mycelium and in the fruiting bodies (Eo et al., 1999).
Ganoderma contains unique polysaccharides, which have been shown to exert
positive effects on the immune system (Wasser and Weis, 1999). Through their
metabolites (glucans, LZ-8 and triterpenoids), they induce production of cytokines
(ILs), tumor necrosis factor (TNF) and mobilize macrophages, natural killer (NK) cells
and lymphocytes B and T (Lin, 2005; Lin and Zhang, 2004). In particular, Ganoderma
lucidum is best known for managing type of cancer in combination with conventional
therapy and for its anti-viral effect (Chen and Miles, 1996). The methanol-soluble
fractions of Reishi mushrooms (Ganoderma lucidum) were particularly studied and later
described to inhibit herpes simplex virus (HSV) and vesicular stomatitus virus (VSV)
(Eo et al., 1999).
On the other hand, lentinan, the polysaccharides from the mushroom Lentinula
edodes, has been shown to enhance host resistance against infections from viruses as
well as from various bacteria, fungi and parasites. Besides, it also prevents chemical and

117

viral oncogenesis (Wasser and Weis, 1999). Lentinan which was experimented with a
few clinical trials in the treatment of human immunodeficiency virus (HIV), has been
shown to exhibit inhibitory activity against the virus (Gordon et al., 1998). An antiviral
water-soluble lignin from the mycelium of shiitake mushrooms (Lentinula edodes)
isolated from cultures grown on rice bran and sugar cane bagasse was reported to
restrict human immunodeficiency virus (HIV) replication in vitro (Suzuki et al., 1990;
Sarkar et al., 1993). Another compound identified to inhibit HIV type 1 infection was a
polysaccharopeptide from Trametes versicolor (Collins and Ng, 1997).
Arabinoxylane, a compound from the mycelia of three potent medicinal
mushrooms: shiitake, kawaratake, and suehirotake was suggested to activate immune
cells, and possesses some anti-tumor and anti-viral qualities that may be useful in
treating conditions such as; cancer, human immunodeficiency virus (HIV) as well as
provide general immune support (Ghoneum, 1998). Other potential mushrooms like
maitake (Grifola frondosa) and chaga (Inonotus obliquus) have also been currently
subjected to research in the treatment of HIV (Mizuno et al., 1996).
Another finding reported that Fomes fomentarius, a hoof-shaped wood conk
growing trees, was another mushroom recognized to be a potent antiviral agent
especially against the tobacco mosaic virus (Aoki et al., 1993). On the other hand,
derivatives of the Gypsy mushroom, Rozites caperata, were shown to have significant
inhibition against the replication and spread of varicella zoster (the shingles and
chickenpox virus), influenza A and the respiratory synctical virus but not against HIV
and other viruses (Piraino and Brandt, 1999).

118

CHAPTER 6: CONCLUSION
The general frustration by the public on the lack of effective drugs for both the
prevention and treatment of various illnesses has become increasingly apparent. The
side effects of synthetic drugs make them less acceptable and people are now turning to
the drugs from natural sources which are safe and more favorable. Mushrooms are the
natural sources that have characteristically been known to contain a variety of
secondary metabolites with diverse biological activities. In this present study, the
potential use of the mushroom S. commune was studied as to explore their biological
activities that may be beneficial for health. The study was carried out to investigate
biological activities of different extracts of S. commune which included antimicrobial,
antioxidant, cytotoxicity and and anti-human papilloma virus (HPV) activities. The
results presented in this study are the first information on such biological activities of
S. commune.
The biological studies conducted on S. commune indicate the immense potential
of this mushroom in the treatment of variety of human ailments. However, the diverse
biological activities of S. commune extracts were only assayed in in vitro tests, and the
results obtained may not necessarily be portable to the situation in vivo. Further
research especially on the aspect of clinical trials and product development can cement
S. commune as a very important part of the new natural drug discovery.
One of the biological activities studied was antimicrobial activity. The finding
suggested that dichloromethane extract of S. commune was the most active particularly
against Gram-positive bacteria; Streptococcus sanguis and other pathogenic
microorganisms tested in this study. This extract therefore can be of great interest in
both academia and the pharmaceutical industry, since their possible use is in line with
the growing tendency to replace synthetic drugs by natural ones.

119

The results of this present study showed that all extracts of S. commune contain
potentially large amounts of phenolics, and can be considered as good sources for
medicinal and food applications. Of all S. commune extracts, methanol extract showed
the most remarkable antioxidant activity in the radical scavenging assay performed and
contained the highest total phenolic content. Owing to its strong antioxidant and
excellent protective features exhibited in the radical scavenging assay, the methanol
extract of S. commune could be concluded as a natural source that can be freely used in
the food industry as a culinary herb. There was a high correlation (r=0.826) between
radical scavenging activity and total phenolic content of S. commune extracts supporting
the hypothesis.
Phenolics are the potential compounds that may be responsible for the biological
activities of S. commune extracts. Phenolics may be the possible compounds in
inhibiting the growth of the microorganisms in antimicrobial activity, cytotoxicity and
anti-HPV. The compounds exert their antimicrobial compounds that exhibit inhibitory
effects against the tested microorganisms. The simpler membrane structure of Grampositive bacteria makes it favorable to the phenolics to exert their hyperacidification
effect and therefore inhibiting the bacteria. Phenolic compounds were also suggested to
have inhibitory effects on mutagenesis and carcinogenesis in humans among the various
secondary metabolites isolated from the mushrooms. As for the present study, the high
concentrations of phenolic compounds may be the responsible compound that inhibit
cell proliferation, and simultaneous exposure to hydrogen peroxide and that the
phenolics has been shown to lead the amplification of proliferation inhibition. It is also
interesting to note that phenolics are one of the major groups of non-essential dietary
components that have been associated with the inhibition of atherosclerosis and cancer.
However, further studies should be undertaken to identify the other and specific
compounds responsible for the biological properties of the mushroom. HPLC analysis is

120

a good suggestion as it holds more precise technique to quantify the chemical


compositions in the mushrooms.
Cancer is the leading global health issue. Therefore, finding new sources of
anticancer agents has drawn much attention. Misdiagnosis of cancer by traditional
healers might explain the observed lack of correlation between the reported anticancer
activities of plant extracts and their cytotoxic activity on the tested cell lines. In
addition, it is known that some anticancer agents might exhibit their antitumor activity
in vivo but with no in vitro cytotoxic activity. The present study investigated the
cytotoxicity of the mushroom extracts against various cancer cell lines which included
cervical cancer cell lines; CaSki, epidermoid cancer cell lines; KB and colon cancer cell
lines; HT29 and intestinal colon cancer cell lines; HCT116. Results showed that
S. commune extracts had substantial cytotoxicity against the cancer cell lines tested with
dichloromethane extract being the most active against HCT116. This suggested that the
extract may consist of potential antitumor compounds which promote the cytotoxic
effect. Hence, it could represent a further tool to screen for the toxic effects against
other cancer cell lines. Further research to study the effect of the toxin in vivo is also
interesting.
The CaSki cell lines were also treated with S. commune extracts to determine the
anti-HPV 18 E6 activity in the cancer cells. Again, methanol and dichloromethane
extracts of S. commune were qualitatively found to display good inhibition of E6 protein
compared to ethyl acetate and water. This might indicate that both the extracts were
successful in inhibiting the expression of HPV 18 E6 oncoprotein and therefore they
can be considered as potential anti-HPV agents and may hold a hope as a potential
therapy for cervical cancer.
Based on all the findings, the extraction solvent were observed to give a
noteworthy effect to the biological activity of the extracts. In comparison of all the
121

extracts, methanol and dichloromethane were proven to be the best solvents in


extracting the biologically active compounds of the mushroom. Water extract of
S. commune was the weakest in extracting the bioactive compounds. The effect of
processing temperature on compound stability was believed to be the main reason of the
ineffective biological activity shown by the extract. On the other hand, it can also be
suggested that some of the biological activities tested may be better if the extract is
combined with other potential extract to bring out the maximal synergistic effects.
Further analysis can be carried out to see the whether the combination treatment could
be more potent than either a drug alone. Most important, there is a need in the field for
detailed information on the extraction procedure and, if at all possible, a thorough
analysis of the chemical composition of the extract under investigation. This would not
only enhance reproducibility, but would eventually make it possible to correlate specific
chemical constituents or combinations of constituents with particular biological
activities.
Finally, it is concluded from the present study that the extracts of S. commune
possessed beneficial health properties. The bioactive compounds responsible for the
properties were strongly related to the type of extracts used. While there are gaps in the
studies conducted so far, which need to be bridged in order to exploit the full medicinal
potential of the mushroom, it is still very clear that S. commune is the mushroom with
tremendous use and have extraordinary potential for the future.

122

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150

APPENDICES
APPENDIX A:
1.0

REAGENTS, MEDIA AND BUFFER

DETERMINATION OF ANTIMICROBIAL ACTIVITY

There are several media used to support growth of different organisms. The
media are Mueller Hinton agar and broth, Brain Heart Infusion broth, yeast-peptone
agar and broth, Saboraud Dextrose agar and broth.
1.1

Mueller Hinton (MH) agar

The media was used to cultivate the all the bacteria tested. 13.6 gm of Mueller
Hinton agar (Merck) was suspended in 400 ml of purified water and mixed thoroughly.
The mixture was then autoclaved at 121C for 15 minutes for sterilization. After the
heat was tolerable to handle by hand, the media was poured carefully to two-third of
disposable sterile petri dishes and left to solidify before it was stored at 25C.
1.2

Mueller Hinton (MH) broth

The media was used to revive all the bacteria in the exception of oral bacteria.
8.4 gm of Mueller Hinton broth (Merck) was suspended in 400 ml of purified water and
mixed thoroughly. The mixture was then sterilized at 121C for 15 minutes. After
cooling down, it was refrigerated at 25C until required.
1.3

Brain Heart Infusion (BHI) broth

The media was used to revive the oral bacteria. 14.8 gm of BHI broth (Merck)
was suspended in 400 ml of purified water and mixed thoroughly. The mixture was
then autoclaved at 121C for 20 minutes. After cooling down, it was refrigerated at
25C until required.
1.4

Yeast-peptone (YP) broth

Yeast extract
Peptone
Glucose
pH

4.00 g
8.00 g
8.00 g
6.8

The media was used to revive Saccharomyces pombe. All the ingredients stated
above were dissolved in 400 ml of purified water and mixed completely. The pH was
adjusted to 6.8 using hydrochloric acid (HCl) or sodium hydroxide (NaOH). The
mixture was then autoclaved at 121C for 15 minutes. After cooling down, it was
refrigerated at 25C until required.
1.5

Yeast-peptone agar (YPA)

Yeast extract

4.00 g
151

Peptone
Glucose
Agar
PH

8.00 g
8.00 g
7.00 g
6.8

The media was used to cultivate Saccharomyces pombe. All the ingredients were
mixed well in 400 ml of purified water. The pH was adjusted to 6.8 using hydrochloric
acid (HCl) or sodium hydroxide (NaOH). The mixture was then autoclaved at 121C for
15 minutes for sterilization. After the heat was tolerable to handle by hand, the media
was poured carefully to two-third of disposable sterile petri dishes and left to solidify
before it was stored at 25C.
1.6

Saboraud dextrose broth

The media was used to revive Candida albicans and Candida parapsilosis. 12.0
gm of Saboraud Dextrose broth was suspended in 400 ml of purified water and mixed
thoroughly. The mixture was then sterilised at 121C for 20 minutes. After cooling
down, it was refrigerated at 25C until required.
1.7

Saboraud dextrose agar (SDA)

The media was used to cultivate Candida albicans and Candida parapsilosis.
26.0 gm of Saboraud Dextrose agar (Merck) and 0.8 g of Bacto agar (Difco) were added
to 400 ml of purified water and mixed completely. The mixture was then autoclaved at
121C for 15 minutes for sterilization. After the heat was tolerable to handle by hand,
the media was poured carefully to two-third of disposable sterile petri dishes and left to
solidify before it was stored at 25C.
2.0

DETERMINATION OF ANTIOXIDANT ACTIVITY

2.1

Folin-Ciocalteau reagent

The reagent should be freshly prepared to avoid oxidation. 0.5 ml of FolinCiocalteau reagent was dissolved in 4.5 ml distilled water and mixed using a vortex.
The reagent bottle was then wrapped in aluminium foil as it is light sensitive.
2.2

10% natrium carbonate (Na2CO3)

One gram of natrium carbonate (Na2CO3) was dissolved in 10 ml distilled water


and mixed well with vortex.
3.0

CYTOTOXICITY AGAINST CANCER CELL-LINES

3.1

Phosphate buffered saline (PBS) 7.2

The PBS pH 7.2 consisted of 1.52 g of disodium hydrogen orthophosphate


anhydrous (Na2HPO4), 0.58 g potassium dihydrogen orthophosphate (KH2PO4) and 8.5
g sodium chloride (NaCl), which were dissolved in 1 liter of distilled water. The
152

solution was adjusted to a pH of 7.2 and it was filtered using a 0.2 m filter membrane
into a bottle.
3.2

Basic RPMI 1640 media

A bottle of RPMI powder, 2 g of natrium hydrogen carbonate (NaHCO3), and


0.5206 g of 2 mM/l HEPES were dissolved in 1 liter of distilled water and the pH was
adjusted to 7.4 before further filtration using 0.2 m filter membrane into a bottle. The
bottle was then kept in 4C.
3.3

10% RPMI 1640 media

10 ml of Foetal Bovine Serum (FBS), 2 ml of penicillin/streptomycin and 1 ml


of amphostat B were added to 90 ml basic 1640 RPMI media and mixed thoroughly.
The mixture was filtered using 0.2 m filter membrane into a bottle and was then kept
in 4C until required.
3.4

Tryphan blue
0.2 g of tryphan blue was mixed with 50 ml distilled water and then stored at

4C.
3.5

Resorb solution

2 ml of acetic acid was dissolved in the mixture of (1:1) distilled water and
ethanol.
3.6

Washing solution

2 gm of calcium chloride (CaCl2) was dissolved in 200 ml of distilled water. 2


ml of formaldehyde was added in the solution.
3.7

Neutral red stock solution

0.04 g Neutral red was dissolved in 10 ml distilled water using a vortex. The
mixture was then centrifuged at 1000 rpm for 5 minutes. The supernatant obtained was
mixed with 12 ml of 10% RPMI 1640 media and stored at 4C for further use.
4.0

ANTI-HUMAN PAPILLOMA VIRUS ACTIVITY

4.1

Phosphate buffered saline (PBS) 7.2


The instruction on how to prepare this solution was similar to 2 (a).

4.2

Phosphate buffered saline (PBS ) 7.6

The PBS with pH 7.6 was by preparing the ingredients consisted of 1.52 g of
disodium hydrogen orthophosphate anhydrous (Na2HPO4), 0.58 g potassium
dihydrogen orthophosphate (KH2PO4) and 8.5 g sodium chloride (NaCl), which were

153

dissolved in 1 liter of distilled water. The solution was adjusted to a pH of 7.6 and it
then was filtered using a 0.2 m filter membrane into a bottle.
4.3

95%, 85% and 80% ethanol

For the preparation of the diluted 95% ethanol, 950 ml of ethanol was mixed
well 50 ml distilled water, whereas 850 ml and 800 ml of ethanol were added with 50
ml and 100ml of distilled water respectively.
4.4

3% hydrogen peroxide

300 l of hydrogen peroxide was mixed with 9.7 ml distilled water to make the
total volume of 10 ml.
4.5

HPV 18 E6 antibody
The HPV 18 E6 antibody (Chemicon) was diluted in 1:50 with sterile distilled

water.

154

APPENDIX B:

ANALYTICAL TECHNIQUES

1.0

DETERMINATION OF ANTIOXIDANT ACTIVITY

1.1

DPPH radical scavenging assay

The radical scavenging activity assay was employed according to Molyneux (2004)
with some modifications. Various concentrations of the S. commune extracts in
methanol (Table 1.1-1.4) were prepared to give a final volume of 0.1 ml and were
mixed vigorously with 3.9 ml of methanolic solution containing DPPH radicals
resulting in a final concentration of 0.06 mM. After 30 minutes incubation period in the
dark, the absorbance was read against a blank (methanol) at 515 nm. The same
procedure was applied to the positive control, ascorbic acid (Table 1.5).
Table 1.1: Preparation of ethyl acetate extracts of S. commune with stock
concentration of 30 mg/ml
Concentrations of
extracts
(mg/ml)
1
5
10
15
20

Volume of
methanol (l)

Volume of
extracts (l)

Volume of DPPH
solution (ml)

95
75
50
25
-

5
25
50
75
100

3.9
3.9
3.9
3.9
3.9

Table 1.2: Preparation of methanol extracts of S. commune with stock


concentration of 30 mg/ml
Concentrations of
extracts
(mg/ml)
1
2
5
10
15

Volume of
methanol (l)

Volume of
extracts (l)

Volume of DPPH
solution (l)

95
90
75
50
25

5
10
25
50
75

3.9
3.9
3.9
3.9
3.9

Table 1.3: Preparation of dichloromethane extracts of S. commune with stock


concentration of 50 mg/ml
Concentrations of
extracts
(mg/ml)
1
5
20
30
50

Volume of
methanol (l)

Volume of
extracts (l)

Volume of DPPH
solution (l)

98
90
60
40
-

2
10
40
60
100

3.9
3.9
3.9
3.9
3.9

155

Table 1.4: Preparation of water extracts of S. commune with stock concentration of


50 mg/ml
Concentrations of
extracts
(mg/ml)
5
10
20
30
50

Volume of
methanol (l)

Volume of
extracts (l)

Volume of DPPH
solution (l)

90
80
60
40
-

10
20
40
60
100

3.9
3.9
3.9
3.9
3.9

Table 1.5: Preparation of ascorbic acid samples as positive control for DPPH assay
Concentrations of ascorbic acid

1.2

mM

mg/ml

0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0

0.0176
0.0352
0.0528
0.0704
0.0880
0.1057
0.1233
0.1409
0.1585
0.1761

Volume of
ascorbic acid
(l)

Volume of
methanol
(l)

Volume of
DPPH solution
(ml)

10
20
30
40
50
60
70
80
90
100

90
80
70
60
50
40
30
20
10
-

3.9
3.9
3.9
3.9
3.9
3.9
3.9
3.9
3.9
3.9

Folin-Ciocalteau assay

The total phenolic content was determined by the Folin-Ciocalteau assay


(Singleton and Rossi, 1965) with some modifications, involving Folin-Ciocalteau
reagent and gallic acid as a standard. The concentrations of S. commune extracts were
prepared as stated in Table 1.6. Folin-Ciocalteau reagent (250 l) was added to the
aliquot (250 l) of mushroom extract solution and was mixed thoroughly. After 3
minutes, a 500 l solution of natrium carbonate (Na2CO3) was added and the mixture
was allowed to stand for 1 hour in the dark. The absorbance of the resulting solution
was measured at 750 nm with a spectrophotometer against a blank (distilled water). The
same procedure was applied to the positive reference standard, gallic acid (Table 1.7).
Table 1.6: Preparation of S. commune extracts for Folin-Ciocalteau assay
Fungal Extracts

Concentrations
(mg/ml)

Volume of extracts Volume of distilled


(l)
water (l)

Ethyl acetate

250

Methanol

250

Dichloromethane

250

Water

250

156

Table 1.7: Preparation of gallic acid as positive reference standard for FolinCiocalteau assay
Concentrations (g/ml) Volume of gallic acid (l)

Volume of distilled water (l)

250

50

200

100

150

150

100

200

50

10

250

*Stock concentration= 10 g/ml

157

APPENDIX C:
1.0

EXPERIMENTAL AND STATISTICAL DATA

DETERMINATION OF ANTIMICROBIAL ACTIVITY

1.1
The experimental data of the antimicrobial activity of S. commune extracts
against microorganisms tested at two different concentrations
Microorganisms
Bacillus cereus

Bacillus
subtilis
Enterobacter
faecalis

Grampositive
bacteria Staphylococcus
aureus
Streptococcus
mitis
Streptococcus
mutans
Streptococcus
sanguis
Escherichia
coli

Methanol
0.2
2
mg/ml mg/ml
9
10
10
11
10
11
91
101
9
10
9
9
9
10
90
91
10
11
9
10
10
11
91
101
10
11
10
10
10
10
100
101
10
NA
9
9
91
NA
NA
11
10
11
101
9
9
9
90

Salmonella sp.
NA
Salmonella
Gramtyphi
negative
bacteria
Shigella sp.

9
8
9
81
9
9

12
11
12
111
10
9
10
91
10
9
9
91
10
9
10
91
10
9

Ethyl acetate
0.2
2
mg/ml mg/ml
9
10
10
10
10
9
91
91
8
11
9
10
9
10
81
101
10
11
10
11
10
10
100
101
9
10
10
10
9
9
91
91
9
10
9
10
9
9
90
91
NA
NA
11
11
11
110
10
10
10
100
10
10
10
100
10
9
9
91
10
10

11
12
11
111
11
10
10
101
11
10
10
101
10
10
10
100
11
10

Dichloromethane
0.2
2
mg/ml
mg/ml
11
12
11
11
10
12
101
111
10
11
9
12
10
11
91
111
10
12
9
11
10
12
91
111
10
12
10
11
10
11
100
111
8
12
8
11
8
11
80
111
NA
NA
12
11
11
111
9
9
9
90
10
9
9
91
10
9
10
91
10
11

13
13
12
121
12
12
11
111
12
11
11
111
12
11
12
111
11
12
158

Shigella
flexneri
Plesiomonas
shigelloides
Proteus
vulgaris

Pseudomonas
aeuroginosa
Candida
albicans

Fungi

9
90
9
8
8
81
8
9
8
81
9
9
9
90
90
NA

NA

10
91
10
9
10
91
9
10
9
91
10
9
10
91
91
9
9
9
90
NA

Candida
parapsilosis

NA

NA

Saccharomyces
pombe

NA

NA

10
100
9
9
9
90
10
10
9
91
9
10
9
91
91
NA
10
9
9
91
10
9
9
91
9
9
8
81

10
101
10
9
9
91
11
12
11
111
10
11
10
101
101
10
9
9
91
11
10
10
101
10
10
10
100
9
10
10
91

10
101
10
9
9
91
11
10
10
101
11
11
11
110
110
NA

NA
8
8
8
80
NA

12
111
12
11
11
111
12
12
11
111
12
11
12
111
111
11
10
10
101
10
10
10
100
10
11
11
101
8
8
9
81

Data expressed as means standard deviations of triplicate measurements

159

1.2
The statistical data of the antimicrobial activity of S. commune extracts
against microorganisms tested at 0.2 mg/ml
ANOVA Table
Analysis of Variance
----------------------------------------------------------------------------Source
Sum of Squares
Df Mean Square
F-Ratio
P-Value
----------------------------------------------------------------------------Between groups
107.789
2
53.8946
12.51
0.0000
Within groups
491.114
114
4.30802
----------------------------------------------------------------------------Total (Corr.)
598.903
116

The StatAdvisor
--------------The ANOVA table decomposes the variance of the data into two
components: a between-group component and a within-group component.
The F-ratio, which in this case equals 12.5103, is a ratio of the
between-group estimate to the within-group estimate. Since the
P-value of the F-test is less than 0.05, there is a statistically
significant difference between the means of the 3 variables at the
95.0% confidence level. To determine which means are significantly
different from which others, select Multiple Range Tests from the list
of Tabular Options.

Multiple Range Tests


-------------------------------------------------------------------------------Method: 95.0 percent LSD
Count
Mean
Homogeneous Groups
-------------------------------------------------------------------------------MET
39
7.82308
X
EA
39
9.73846
X
DCM
39
9.96154
X
-------------------------------------------------------------------------------Contrast
Difference
+/- Limits
-------------------------------------------------------------------------------DCM - EA
0.223077
0.931119
DCM - MET
*2.13846
0.931119
EA - MET
*1.91538
0.931119
-------------------------------------------------------------------------------* denotes a statistically significant difference.

The StatAdvisor
--------------This table applies a multiple comparison procedure to determine
which means are significantly different from which others. The bottom
half of the output shows the estimated difference between each pair of
means. An asterisk has been placed next to 2 pairs, indicating that
these pairs show statistically significant differences at the 95.0%
confidence level. At the top of the page, 2 homogenous groups are
identified using columns of X's. Within each column, the levels
containing X's form a group of means within which there are no
statistically significant differences. The method currently being
used to discriminate among the means is Fisher's least significant
difference (LSD) procedure. With this method, there is a 5.0% risk of
calling each pair of means significantly different when the actual
difference equals 0.

160

1.3
The statistical data of the antimicrobial activity of S. commune extracts
against microorganisms tested at 2 mg/ml
ANOVA Table
Analysis of Variance
----------------------------------------------------------------------------Source
Sum of Squares
Df Mean Square
F-Ratio
P-Value
----------------------------------------------------------------------------Between groups
0.392288
2
0.196144
22.73
0.0000
Within groups
1.29451
150
0.00863007
----------------------------------------------------------------------------Total (Corr.)
1.6868
152

The StatAdvisor
--------------The ANOVA table decomposes the variance of the data into two
components: a between-group component and a within-group component.
The F-ratio, which in this case equals 22.728, is a ratio of the
between-group estimate to the within-group estimate. Since the
P-value of the F-test is less than 0.05, there is a statistically
significant difference between the means of the 3 variables at the
95.0% confidence level. To determine which means are significantly
different from which others, select Multiple Range Tests from the list
of Tabular Options.

Multiple Range Tests


-------------------------------------------------------------------------------Method: 95.0 percent LSD
Count
Mean
Homogeneous Groups
-------------------------------------------------------------------------------Met
51
0.945098
X
EA
51
0.976471
X
DM
51
1.06471
X
-------------------------------------------------------------------------------Contrast
Difference
+/- Limits
-------------------------------------------------------------------------------DM - EA
*0.0882353
0.03635
DM - Met
*0.119608
0.03635
EA - Met
0.0313725
0.03635
-------------------------------------------------------------------------------* denotes a statistically significant difference.

The StatAdvisor
--------------This table applies a multiple comparison procedure to determine
which means are significantly different from which others. The bottom
half of the output shows the estimated difference between each pair of
means. An asterisk has been placed next to 2 pairs, indicating that
these pairs show statistically significant differences at the 95.0%
confidence level. At the top of the page, 2 homogenous groups are
identified using columns of X's. Within each column, the levels
containing X's form a group of means within which there are no
statistically significant differences. The method currently being
used to discriminate among the means is Fisher's least significant
difference (LSD) procedure. With this method, there is a 5.0% risk of
calling each pair of means significantly different when the actual
difference equals 0.

161

1.4
The experimental data of the antimicrobial activity of positive controls
against bacteria tested at two different concentrations
Streptomycin
Microorganisms
0.2
2
mg/ml mg/ml
25
30
Bacillus cereus
25
31
26
31
251
311
18
25
Bacillus
19
26
subtilis
19
25
181
251
26
33
Enterobacter
25
33
faecalis
25
32
Gram261
321
positive Staphylococcus
18
25
bacteria aureus
18
26
18
25
180
251
20
27
Streptococcus
20
27
mitis
20
27
200
270
20
Streptococcus
NA
20
mutans
20
200
10
22
Streptococcus
9
22
sanguis
9
21
91
211
13
26
Escherichia
14
27
coli
14
26
131
261
Salmonella sp.
29
39
31
38
31
38
292
381
29
39
Salmonella
30
38
typhi
Gram29
38
negative
291
381
bacteria
30
37
Shigella sp.
31
38
30
38
301
371

Kanamycin
0.2
2
mg/ml
mg/ml
21
30
20
30
21
31
211
301
30
34
31
34
30
35
301
351
21
27
20
28
20
28
201
271
30
35
31
36
31
35
301
351
18
20
17
19
18
19
171
191
21
NA
21
21
210
12
25
13
26
12
26
121
251
20
27
20
28
20
27
200
271
22
32
23
32
22
31
221
311
21
29
20
28
20
29
201
281
21
28
22
29
22
28
211
281

Chloramphenicol
0.2
2
mg/ml mg/ml
18
25
18
26
19
25
181
261
24
40
25
41
25
40
251
401
19
31
19
30
18
31
191
301
25
41
26
40
25
40
251
401
22
25
23
26
23
25
221
251
14
NA
13
14
131
8
19
9
18
9
19
81
181
12
31
13
32
13
31
121
311
23
35
22
35
22
34
221
341
21
34
20
34
20
33
201
331
19
31
19
32
20
31
191
311
162

Shigella
flexneri
Plesiomonas
shigelloides
Proteus
vulgaris
Pseudomonas
aeuroginosa

29
30
30
291
33
32
32
321
15
16
16
151
15
16
15
151

38
39
38
381
40
40
39
391
26
26
25
251
21
21
20
210

20
21
20
201
23
22
22
221
30
29
29
291
24
24
25
241

28
29
29
281
30
31
30
301
34
34
35
341
32
31
32
311

21
21
20
201
25
24
24
241
25
25
25
250
22
21
22
211

33
33
34
331
32
33
33
321
42
43
43
421
34
35
35
341

Data expressed as means standard deviations of triplicate measurements

1.5
The experimental data of the antimicrobial activity of positive controls
against fungi tested at two different concentrations
Nystatin
Microorganisms
Candida albicans

0.2 mg/ml
24
23
23
231

Candida parapsilosis
Fungi

NA
Saccharomyces pombe

23
22
22
231

2 mg/ml
29
28
29
281
12
13
12
121
30
29
29
291

Data expressed as means standard deviations of triplicate measurements

163

1.6
The statistical data of the antimicrobial activity of positive controls against
microorganisms tested at 0.2 mg/ml
ANOVA Table
Analysis of Variance
----------------------------------------------------------------------------Source
Sum of Squares
Df Mean Square
F-Ratio
P-Value
----------------------------------------------------------------------------Between groups
160.746
3
53.5819
1.16
0.3347
Within groups
2027.73
44
46.0848
----------------------------------------------------------------------------Total (Corr.)
2188.48
47

The StatAdvisor
--------------The ANOVA table decomposes the variance of the data into two
components: a between-group component and a within-group component.
The F-ratio, which in this case equals 1.16268, is a ratio of the
between-group estimate to the within-group estimate. Since the
P-value of the F-test is greater than or equal to 0.05, there is not a
statistically significant difference between the means of the 4
variables at the 95.0% confidence level.

Multiple Range Tests


-------------------------------------------------------------------------------Method: 95.0 percent LSD
Count
Mean
Homogeneous Groups
-------------------------------------------------------------------------------Nystatin
3
23.0
X
Kanamycin
15
28.6
X
Streptomycin
15
29.5333
X
Chloramphenicol15
30.8
X
-------------------------------------------------------------------------------Contrast
Difference
+/- Limits
-------------------------------------------------------------------------------Streptomycin - Kanamycin
0.933333
4.99578
Streptomycin - Chloramphenicol
-1.26667
4.99578
Streptomycin - Nystatin
6.53333
8.65295
Kanamycin - Chloramphenicol
-2.2
4.99578
Kanamycin - Nystatin
5.6
8.65295
Chloramphenicol - Nystatin
7.8
8.65295
-------------------------------------------------------------------------------* denotes a statistically significant difference.

The StatAdvisor
--------------This table applies a multiple comparison procedure to determine
which means are significantly different from which others. The bottom
half of the output shows the estimated difference between each pair of
means. There are no statistically significant differences between any
pair of means at the 95.0% confidence level. At the top of the page,
one homogenous group is identified by a column of X's. Within each
column, the levels containing X's form a group of means within which
there are no statistically significant differences. The method
currently being used to discriminate among the means is Fisher's least
significant difference (LSD) procedure. With this method, there is a
5.0% risk of calling each pair of means significantly different when
the actual difference equals 0.

164

1.7 The statistical data of the antimicrobial activity of positive controls against
microorganisms tested at 2 mg/ml
ANOVA Table
Analysis of Variance
----------------------------------------------------------------------------Source
Sum of Squares
Df Mean Square
F-Ratio
P-Value
----------------------------------------------------------------------------Between groups
59.9096
3
19.9699
0.39
0.7589
Within groups
2543.96
50
50.8792
----------------------------------------------------------------------------Total (Corr.)
2603.87
53

The StatAdvisor
--------------The ANOVA table decomposes the variance of the data into two
components: a between-group component and a within-group component.
The F-ratio, which in this case equals 0.392495, is a ratio of the
between-group estimate to the within-group estimate. Since the
P-value of the F-test is greater than or equal to 0.05, there is not a
statistically significant difference between the means of the 4
variables at the 95.0% confidence level.
Multiple Range Tests
-------------------------------------------------------------------------------Method: 95.0 percent LSD
Count
Mean
Homogeneous Groups
-------------------------------------------------------------------------------Nystatin
3
18.3333
X
Chloramphenicol17
21.0588
X
Streptomycin
17
22.1765
X
Kanamycin
17
22.6471
X
-------------------------------------------------------------------------------Contrast
Difference
+/- Limits
-------------------------------------------------------------------------------Streptomycin - Kanamycin
-0.470588
4.91413
Streptomycin - Chloramphenicol
1.11765
4.91413
Streptomycin - Nystatin
3.84314
8.97192
Kanamycin - Chloramphenicol
1.58824
4.91413
Kanamycin - Nystatin
4.31373
8.97192
Chloramphenicol - Nystatin
2.72549
8.97192
-------------------------------------------------------------------------------* denotes a statistically significant difference.

The StatAdvisor
--------------This table applies a multiple comparison procedure to determine
which means are significantly different from which others. The bottom
half of the output shows the estimated difference between each pair of
means. There are no statistically significant differences between any
pair of means at the 95.0% confidence level. At the top of the page,
one homogenous group is identified by a column of X's. Within each
column, the levels containing X's form a group of means within which
there are no statistically significant differences. The method
currently being used to discriminate among the means is Fisher's least
significant difference (LSD) procedure. With this method, there is a
5.0% risk of calling each pair of means significantly different when
the actual difference equals 0.

165

2.0

DETERMINATION OF ANTIOXIDANT ACTIVITY

2.1
The experimental data of DPPH radical scavenging activity of S. commune
extracts against DPPH radicals
Extract

Methanol

Ethyl
acetate

Dichloromethane

Aqueous

Concentration

(mg/ml)
1
2
5
10
15
1
5
10
15
20
1
5
20
30
50
5
10
20
30
50

R1
11.40
17.10
47.50
92.02
89.98
13.67
34.53
63.40
91.02
90.88
7.40
25.09
31.05
67.39
75.03
16.06
30.87
36.64
46.57
70.04

Inhibition
(%)
R2
15.94
17.10
58.06
88.96
90.15
13.26
35.91
54.70
88.54
90.47
11.01
28.16
30.42
70.51
72.70
20.58
30.14
41.52
50.18
74.01

Average
(%)
R3
16.33
16.33
48.05
90.49
91.00
10.77
30.80
58.43
89.78
90.19
9.39
26.17
27.46
66.93
67.08
23.65
26.71
32.67
52.17
72.74

14.562.74
16.840.44
51.205.94
90.491.53
90.380.55
12.571.57
33.752.64
58.844.34
89.781.24
90.510.35
9.271.81
26.471.56
29.641.92
68.281.95
71.604.09
20.103.82
29.242.22
36.944.43
49.642.84
72.262.03

Data expressed as means standard deviations of triplicate measurements; R= replicate

2.2
The scavenging activity of S. commune extracts as measured by DPPH
radical scavenging asssay
Extract
Methanol
Ethyl acetate
Dichloromethane
Water
*Ascorbic acid

Scavenging Activity
IC50 (mg/ml)
0.145 0.01
0.219 0.01
0.641 0.13
0.674 0.05
0.084 0.01

Data expressed as means standard deviations of triplicate measurements


*Positive reference standard

166

2.3
The statistical data of DPPH radical scavenging activity of S. commune
extracts against DPPH radicals
ANOVA Table
Analysis of Variance
----------------------------------------------------------------------------Source
Sum of Squares
Df Mean Square
F-Ratio
P-Value
----------------------------------------------------------------------------Between groups
0.693943
3
0.231314
11103.09
0.0000
Within groups
0.000166667
8 0.0000208333
----------------------------------------------------------------------------Total (Corr.)
0.69411
11

The StatAdvisor
--------------The ANOVA table decomposes the variance of the data into two
components: a between-group component and a within-group component.
The F-ratio, which in this case equals 11103.1, is a ratio of the
between-group estimate to the within-group estimate. Since the
P-value of the F-test is less than 0.05, there is a statistically
significant difference between the means of the 4 variables at the
95.0% confidence level. To determine which means are significantly
different from which others, select Multiple Range Tests from the list
of Tabular Options.
Multiple Range Tests
-------------------------------------------------------------------------------Method: 95.0 percent LSD
Count
Mean
Homogeneous Groups
-------------------------------------------------------------------------------Met
3
0.145
X
EA
3
0.219
X
DCM
3
0.645333
X
Wat
3
0.674
X
-------------------------------------------------------------------------------Contrast
Difference
+/- Limits
-------------------------------------------------------------------------------Met - EA
*-0.074
0.00859399
Met - DCM
*-0.500333
0.00859399
Met - Wat
*-0.529
0.00859399
EA - DCM
*-0.426333
0.00859399
EA - Wat
*-0.455
0.00859399
DCM - Wat
*-0.0286667
0.00859399
-------------------------------------------------------------------------------* denotes a statistically significant difference.

The StatAdvisor
--------------This table applies a multiple comparison procedure to determine
which means are significantly different from which others. The bottom
half of the output shows the estimated difference between each pair of
means. An asterisk has been placed next to 6 pairs, indicating that
these pairs show statistically significant differences at the 95.0%
confidence level. At the top of the page, 4 homogenous groups are
identified using columns of X's. Within each column, the levels
containing X's form a group of means within which there are no
statistically significant differences. The method currently being
used to discriminate among the means is Fisher's least significant
difference (LSD) procedure. With this method, there is a 5.0% risk of
calling each pair of means significantly different when the actual
difference equals 0.

167

2.4
The experimental data of DPPH radical scavenging activity of ascorbic acid
against DPPH radicals
Concentration of
ascorbic acid (g/ml)
R1
10.84
31.53
35.78
45.08
48.93
53.88
60.48
64.75
65.33
67.48

0.440
0.881
1.321
1.761
2.201
2.642
3.082
3.522
3.962
4.403

Inhibition
(%)
R2
11.40
31.81
37.23
45.93
47.85
55.15
59.91
64.62
64.91
68.99

Average
(%)
R3
12.13
34.09
38.63
44.51
48.25
54.56
60.05
58.61
66.48
67.08

11.460.65
32.481.40
37.211.43
45.170.71
48.340.55
54.530.64
60.150.29
64.690.09
65.570.81
67.852.26

Data expressed as means standard deviations of triplicate measurements; R=replicate

2.5
Calibration plot of the scavenging effect of ascorbic acid as on DPPH
radicals
80
70
In h ib itio n (% )

60
50
y = 232.74x + 30.749
2
R = 0.978

40
30
20
10
0
0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

Concentrations (mg/ml)

2.6
The experimental data of total phenolic content of S. commune extracts at
0.1 mg/ml
Extract
Ethyl acetate
Methanol
Dichloromethane
Aqueos

R1
0.078
0.166
0.044
0.052

Absorbance
R2
0.076
0.175
0.046
0.055

Average
R3
0.081
0.171
0.044
0.048

0.078
0.054
0.045
0.052

R= Replicate

168

2.7

The statistical data of the total phenolic content of S. commune extracts

ANOVA Table
Analysis of Variance
----------------------------------------------------------------------------Source
Sum of Squares
Df Mean Square
F-Ratio
P-Value
----------------------------------------------------------------------------Between groups
3.06228
3
1.02076
1003.12
0.0000
Within groups
0.00814067
8
0.00101758
----------------------------------------------------------------------------Total (Corr.)
3.07042
11

The StatAdvisor
--------------The ANOVA table decomposes the variance of the data into two
components: a between-group component and a within-group component.
The F-ratio, which in this case equals 1003.12, is a ratio of the
between-group estimate to the within-group estimate. Since the
P-value of the F-test is less than 0.05, there is a statistically
significant difference between the means of the 4 variables at the
95.0% confidence level. To determine which means are significantly
different from which others, select Multiple Range Tests from the list
of Tabular Options.
Multiple Range Tests
-------------------------------------------------------------------------------Method: 95.0 percent LSD
Count
Mean
Homogeneous Groups
-------------------------------------------------------------------------------DM
3
0.448667
X
WE
3
0.519333
X
EA
3
0.787333
X
Met
3
1.71467
X
-------------------------------------------------------------------------------Contrast
Difference
+/- Limits
-------------------------------------------------------------------------------DM - EA
*-0.338667
0.0600621
DM - Met
*-1.266
0.0600621
DM - WE
*-0.0706667
0.0600621
EA - Met
*-0.927333
0.0600621
EA - WE
*0.268
0.0600621
Met - WE
*1.19533
0.0600621
-------------------------------------------------------------------------------* denotes a statistically significant difference.

The StatAdvisor
--------------This table applies a multiple comparison procedure to determine
which means are significantly different from which others. The bottom
half of the output shows the estimated difference between each pair of
means. An asterisk has been placed next to 6 pairs, indicating that
these pairs show statistically significant differences at the 95.0%
confidence level. At the top of the page, 4 homogenous groups are
identified using columns of X's. Within each column, the levels
containing X's form a group of means within which there are no
statistically significant differences. The method currently being
used to discriminate among the means is Fisher's least significant
difference (LSD) procedure. With this method, there is a 5.0% risk of
calling each pair of means significantly different when the actual
difference equals 0.

169

2.8
The experimental data of total phenolic content of gallic acid as the positive
standard reference
Concentration
(g/ml)
0
2
4
6
8
10

R1
0.017
0.057
0.129
0.202
0.235
0.263

Absorbance
R2
0.014
0.067
0.135
0.186
0.240
0.263

Average
R3
0.017
0.034
0.130
0.188
0.249
0.261

0.016
0.054
0.131
0.192
0.241
0.262

R= Replicate

2.9

The calibration plot of gallic acid as positive reference standard


0.35
0.3

Absorbance

0.25
0.2
y = 0.0288x
2
R = 0.9666

0.15
0.1

0.05
0
0

Concentrations (ug/ml)

10

12

3.0
The radical scavenging activity and total phenolic content of S. commune
extracts
Extract

Scavenging Activity
IC50 (mg/ml)

Total Phenolic Content


(mg GA/g extracts)

Methanol
Ethyl acetate
Dichloromethane
Water

0.145 0.01
0.219 0.01
0.641 0.13
0.674 0.05

1.72 0.05
0.79 0.03
0.52 0.04
0.45 0.01

*Data expressed as means standard deviations of triplicate measurements

170

3.0

CYTOTOXICITY TOWARDS CANCER CELL LINES

3.1
The experimental data of the cytotoxicity of S. commune extracts towards
CaSki cell lines
Concentration

Cell line
KB

CaSki

HT29

HCT116

of extracts
(g/ml)

Percentage of inhibition (%)


Methanol

Ethyl
acetate

Dichloromethane

Aqueous

141.69

21.08

5.431.5

10.130.55

10

26.16.51

13.80.85

22.631.2

21.30.46

50

54.654.31

20.950.78

42.30.71

31.940.8

100

75.375.71

57.71.69

63.54.38

48.140.43

16.20.71

10.750.21

4.150.64

1.550.21

10

35.054.74

22.63.96

21.251.2

23.555.44

50

47.10.71

36.23.25

48.875.18

34.533.5

100

51.355.87

40.650.64

80.134.24

40.42.43

0.750.35

22.60.42

3.61.27

7.50.0

10

12.80.14

30.454.6

14.056.15

12.650.92

50

21.252.33

37.652.47

47.82.12

15.21.61

100

33.25.03

42.82.26

62.250.92

31.850.21

5.30.28

2.31.13

8.31.69

12.12.12

10

9.20.14

11.752.89

33.950.35

16.152.05

50

16.51.56

28.850.64

76.51.66

19.852.76

100

27.650.49

50.41.87

87.051.06

20.552.19

*Data expressed as means standard deviations of triplicate measurements

171

3.2
The statistical data of the cytotoxicity between dichloromethane and
methanol extracts of S. commune against CaSki cell-lines
ANOVA Table
Analysis of Variance
----------------------------------------------------------------------------Source
Sum of Squares
Df Mean Square
F-Ratio
P-Value
----------------------------------------------------------------------------Between groups
1.5625
1
1.5625
0.00
0.9614
Within groups
8989.72
14
642.123
----------------------------------------------------------------------------Total (Corr.)
8991.28
15

The StatAdvisor
--------------The ANOVA table decomposes the variance of the data into two
components: a between-group component and a within-group component.
The F-ratio, which in this case equals 0.00243334, is a ratio of the
between-group estimate to the within-group estimate. Since the
P-value of the F-test is greater than or equal to 0.05, there is not a
statistically significant difference between the means of the 2
variables at the 95.0% confidence level.

Multiple Range Tests


-------------------------------------------------------------------------------Method: 95.0 percent LSD
Count
Mean
Homogeneous Groups
-------------------------------------------------------------------------------DM
8
38.675
X
Met
8
39.3
X
-------------------------------------------------------------------------------Contrast
Difference
+/- Limits
-------------------------------------------------------------------------------DM - Met
-0.625
27.1747
-------------------------------------------------------------------------------* denotes a statistically significant difference.

The StatAdvisor
--------------This table applies a multiple comparison procedure to determine
which means are significantly different from which others. The bottom
half of the output shows the estimated difference between each pair of
means. There are no statistically significant differences between any
pair of means at the 95.0% confidence level. At the top of the page,
one homogenous group is identified by a column of X's. Within each
column, the levels containing X's form a group of means within which
there are no statistically significant differences. The method
currently being used to discriminate among the means is Fisher's least
significant difference (LSD) procedure. With this method, there is a
5.0% risk of calling each pair of means significantly different when
the actual difference equals 0.

172

3.3
The statistical data of the cytotoxicity between dichloromethane and ethyl
acetate extracts of S. commune against HCT 116 cell-lines
ANOVA Table
Analysis of Variance
----------------------------------------------------------------------------Source
Sum of Squares
Df Mean Square
F-Ratio
P-Value
----------------------------------------------------------------------------Between groups
2194.92
1
2194.92
2.25
0.1556
Within groups
13640.6
14
974.329
----------------------------------------------------------------------------Total (Corr.)
15835.5
15

The StatAdvisor
--------------The ANOVA table decomposes the variance of the data into two
components: a between-group component and a within-group component.
The F-ratio, which in this case equals 2.25275, is a ratio of the
between-group estimate to the within-group estimate. Since the
P-value of the F-test is greater than or equal to 0.05, there is not a
statistically significant difference between the means of the 2
variables at the 95.0% confidence level.

Multiple Range Tests


-------------------------------------------------------------------------------Method: 95.0 percent LSD
Count
Mean
Homogeneous Groups
-------------------------------------------------------------------------------EA
8
28.2625
X
DM
8
51.6875
X
-------------------------------------------------------------------------------Contrast
Difference
+/- Limits
-------------------------------------------------------------------------------DM - EA
23.425
33.474
-------------------------------------------------------------------------------* denotes a statistically significant difference.

The StatAdvisor
--------------This table applies a multiple comparison procedure to determine
which means are significantly different from which others. The bottom
half of the output shows the estimated difference between each pair of
means. There are no statistically significant differences between any
pair of means at the 95.0% confidence level. At the top of the page,
one homogenous group is identified by a column of X's. Within each
column, the levels containing X's form a group of means within which
there are no statistically significant differences. The method
currently being used to discriminate among the means is Fisher's least
significant difference (LSD) procedure. With this method, there is a
5.0% risk of calling each pair of means significantly different when
the actual difference equals 0.

173

3.4
The statistical data of the cytotoxicity among dichloromethane, methanol
and ethyl acetate extracts of S. commune against KB cell-lines
ANOVA Table
Analysis of Variance
-------------------------------------------------------------------------Source
Sum of Squares
Df Mean Square
F-Ratio
P-Va
-------------------------------------------------------------------------Between groups
1373.49
2
686.747
1.26
0.3
Within groups
11428.0
21
544.192
-------------------------------------------------------------------------Total (Corr.)
12801.5
23

The StatAdvisor
--------------The ANOVA table decomposes the variance of the data into two
components: a between-group component and a within-group component.
The F-ratio, which in this case equals 1.26196, is a ratio of the
between-group estimate to the within-group estimate. Since the
P-value of the F-test is greater than or equal to 0.05, there is not a
statistically significant difference between the means of the 3
variables at the 95.0% confidence level.

Multiple Range Tests


-------------------------------------------------------------------------------Method: 95.0 percent LSD
Count
Mean
Homogeneous Groups
-------------------------------------------------------------------------------EA
8
23.4625
X
DM
8
34.5625
X
Met
8
41.8625
X
-------------------------------------------------------------------------------Contrast
Difference
+/- Limits
-------------------------------------------------------------------------------DM - EA
11.1
24.2566
DM - Met
-7.3
24.2566
EA - Met
-18.4
24.2566
-------------------------------------------------------------------------------* denotes a statistically significant difference.

The StatAdvisor
--------------This table applies a multiple comparison procedure to determine
which means are significantly different from which others. The bottom
half of the output shows the estimated difference between each pair of
means. There are no statistically significant differences between any
pair of means at the 95.0% confidence level. At the top of the page,
one homogenous group is identified by a column of X's. Within each
column, the levels containing X's form a group of means within which
there are no statistically significant differences. The method
currently being used to discriminate among the means is Fisher's least
significant difference (LSD) procedure. With this method, there is a
5.0% risk of calling each pair of means significantly different when
the actual difference equals 0.

174

3.5 The statistical data of the cytotoxicity of S. commune dichloromethane extract


against cancer cell-lines
ANOVA Table
Analysis of Variance
----------------------------------------------------------------------------Source
Sum of Squares
Df Mean Square
F-Ratio
P-Value
----------------------------------------------------------------------------Between groups
2100.44
3
700.145
0.84
0.4855
Within groups
23451.7
28
837.562
----------------------------------------------------------------------------Total (Corr.)
25552.2
31

The StatAdvisor
--------------The ANOVA table decomposes the variance of the data into two
components: a between-group component and a within-group component.
The F-ratio, which in this case equals 0.835933, is a ratio of the
between-group estimate to the within-group estimate. Since the
P-value of the F-test is greater than or equal to 0.05, there is not a
statistically significant difference between the means of the 4
variables at the 95.0% confidence level.

Multiple Range Tests


-------------------------------------------------------------------------------Method: 95.0 percent LSD
Count
Mean
Homogeneous Groups
-------------------------------------------------------------------------------HT29
8
29.9625
X
KB
8
34.5125
X
CaSki
8
38.675
X
HCT116
8
51.6875
X
-------------------------------------------------------------------------------Contrast
Difference
+/- Limits
-------------------------------------------------------------------------------CaSki - HCT116
-13.0125
29.6412
CaSki - HT29
8.7125
29.6412
CaSki - KB
4.1625
29.6412
HCT116 - HT29
21.725
29.6412
HCT116 - KB
17.175
29.6412
HT29 - KB
-4.55
29.6412
-------------------------------------------------------------------------------* denotes a statistically significant difference.

The StatAdvisor
--------------This table applies a multiple comparison procedure to determine
which means are significantly different from which others. The bottom
half of the output shows the estimated difference between each pair of
means. There are no statistically significant differences between any
pair of means at the 95.0% confidence level. At the top of the page,
one homogenous group is identified by a column of X's. Within each
column, the levels containing X's form a group of means within which
there are no statistically significant differences. The method
currently being used to discriminate among the means is Fisher's least
significant difference (LSD) procedure. With this method, there is a
5.0% risk of calling each pair of means significantly different when
the actual difference equals 0.

175