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 non-

natural, 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 µg/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 µg/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.

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 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 (P‹0.05) 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 µg/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 µg/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

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

daripada ekstrak S. commune.

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 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

1.1 Objectives of Study 5

CHAPTER TWO - LITERATURE REVIEW 7

2.1 Introduction to medicinal mushroom 7

2.2 Schizophyllum commune Fr. 9

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

2.3.2 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

2.3.3 Cytotoxicity of mushrooms 21

2.3.3.1 Mechanisms of anti-tumor actions 27

viii
 2.3.4 Antiviral activity of mushrooms 30

2.3.4.1 Human papilloma virus (HPV) 33

2.3.4.2 Human papilloma virus (HPV) infection 35

2.4 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 Anti-human papilloma virus (HPV) activity 44

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

3.3 Determination of antioxidant activity 51

3.3.1 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay 51

3.3.2 Folin-Ciocalteau assay 52

3.4 Cytotoxicity towards various cancer cell lines 52

3.4.1 Cancer cell lines 52

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

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

(CaSki) cell lines 56

3.5.1 Serial dilution of S. commune extracts 56

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 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 71

4.3 Analysis of cytotoxicity of S. commune extracts towards various

cancer cell lines 72

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

towards cervical cancer (CaSki) cell lines 75

CHAPTER FIVE – DISCUSSION 82

5.1 Antimicrobial activity of S. commune Fr. 82

5.1.1 Antimicrobial activity of S. commune extracts as evaluated

by well diffusion assay 82

5.1.2 Antimicrobial activity of S. commune extracts against

various pathogenic microorganisms 83

5.1.3 Extraction solvent as the significant factor in evaluating

antimicrobial activity of S. commune 84

5.1.4 Antimicrobial activity of S. commune extracts and its

correlation to phenolic content 86

5.1.5 Other contributing factors to antimicrobial activity of

S. commune extracts 89

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5.2 Antioxidant activity of S. commune Fr. 91

5.2.1 Antioxidant activity of S. commune extracts and its

correlation to phenolic content 91

5.2.2 Extraction solvent as the significant factor in evaluating

antioxidant activity of S. commune 94

5.2.3 Other contributing factors to antioxidant activity of

S. commune extracts 98

5.3 Cytotoxicity of S. commune Fr. 99

5.3.1 Cytotoxicity of S. commune extracts as evaluated by

neutral red assay 99

5.3.2 Cytotoxicity of S. commune extracts towards different

cancer cell lines 103

5.3.3 Cytotoxicity of S. commune extracts and its correlation

to antimicrobial activity 108

5.3.4 Other contributing factors to cytotoxicity of S. commune

extracts 109

5.4 Anti-human papilloma virus (HPV) activity of S. commune Fr. 112

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

extracts as evaluated by immunohistochemistry assay 112

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

extracts towards cervical cancer cell lines (CaSki) 114

5.4.3 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 of

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)

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 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 37°C).
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 37°C).
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 37°C). 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 37°C). 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 cells after treatment with

dichloromethane extract of S. commune at different
concentrations (Page 80)

Plate 4.7 : The appearance of CaSki cells after treatment with water extract
of S. commune at different concentrations (Page 81)

xv
 LIST OF SYMBOLS AND ABBREVIATIONS

% : percentage

°C : degree centigrade

µg : microgram

µg/ml : microgram per mililiter

µl : 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

E : early region

ED50 : effective dosage of drug that gives 50% of maximal response

FBS : fetal bovine serum

g : 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

L : late region

LSAB : labelled steptravidin - biotin

M : 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

ß : 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, anti-

microbial 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

4
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.

5
3) Determine the cytotoxicity of S. commune extracts against various cancer-

derived cell lines.

4) Investigate the anti-human papilloma virus activity of S. commune extracts

against cervical cancer cell lines.

6
 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).

8
 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

9
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 β 1-3, β 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 β-d-(1-3)-glucopyranosyl groups and β-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 South-

Western 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 well-

11
known commercial antibiotics like penicillin and cephalosporin are produced by semi-

synthesis 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 anti-

metabolites:

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 ß-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 non-

radical (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 (Löliger, 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, α-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 (Brand-

Williams 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 Brand-

Williams 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 anti-

tumor 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 Hetero-

and Homobasidiomycetes. These polysaccharides are of different chemical

composition, with most belonging to the group of β-glucans; these have β-(1 3)

linkages in the main chain of the glucan and additional β-(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-β-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 (β-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 - β-1-3, -

1-6 D-glucan is β-1-3-D glucopyronan with 1-15 units of β-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 anti-

tumor 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.) Pilát 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 anti-

proliferative 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 β-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 polysaccharide–protein 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 host’s 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 body’s 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 anti-

tumor 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 auto-

digestion 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 Figure 2.2: The intrinsic mitochondrial
receptor pathway pathway

2.3.4 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 non-

toxicity, 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-β, 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 (Dürst 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 (Dürst 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; Dürst 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 (Münger 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 (Dürst 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 Functions

amino acids
E1 649 Virus DNA replication
E2 365 Transcription and replication control
E4 92 Binds cytokeratins preceding virus assembly
E5 83 Affects growth factor receptors
E6 151 Binds p53 trans-activates, 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 pre-

incubation, 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 2,2-

diphenyl-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-1-

hydrazine) 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-9-

ethocarbazole 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 -20°C 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 4°C 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 Water Extract

Methanol Extracts

Soak mushroom with dichloromethane, Soak mushroom with sterile

ethyl acetate and methanol separately distilled water (sdH2O)

Filter Heat for 3 hours

Evaporate to dryness Freeze-dry

Crude Extracts

ANTIMICROBIAL ANTIOXIDANT
ACTIVITY ACTIVITY

CYTOTOXICITY TOWARDS ANTI-HUMAN PAPILLOMA

CANCER CELL-LINES 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 µl of the stock suspension into 3 ml broth and

incubated for 48 hours at 37°C. 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 µl of 0.2 mg/ml extracts to

give 10 µg in each well. The Petri dishes were further incubated at 37°C 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 µg 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 fungi/yeast on Saboraud

Lawn bacteria on Mueller Dextrose agar and Yeast-Peptone
Hinton agar agar respectively

Cut three 7mm diameter well and fill 50 µl of

fungal extracts in each well

Incubate at 37°C 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 µg/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 µl) (Appendix A) was added to the

aliquot (250 µl) of extract solution and was mixed thoroughly. After 3 minutes, a 500

µl 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 µg/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 µg/ml was

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

stock was then further diluted into final concentrations of 100 µg/ml, 50 µg/ml, 10

µg/ml and 1 µg/ml (Figure 3.3). All diluted stocks were kept at –20°C until required.

Stock
(20,000 µg/ml)

10 µl + 10 µl + 10 µl +
90 µl 10% DMSO 90 µl 10% DMSO 90 µl 10% DMSO

10 µl + 5 µl + 10 µl + 10 µl +
190 µl cells 195 µl cells 190 µl cells 190 µl cells
(100 µg/ml) (50 µg/ml) (10 µg/ml) (1 µg/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 37°C 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 µl of tryphan blue

(Appendix A) were transferred into an eppendorf tube and mixed well. 20 µl 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 µl

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 37°C 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

µg/ml, 50 µg/ml, 10 µg/ml and 1 µg/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 37°C 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 µl 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 37°C. Following that, the dye was discarded and the cells

were rapidly washed with 200 µl washing solution (Appendix A) before further adding

200 µl 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 37°C 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 µl of sterile distilled water to produce a stock concentration

of 1200 µg/ml. This stock was further diluted into final concentrations of 200µg/ml, 100

56
µg/ml, 50 µg/ml, 25 µg/ml, 10 µg/ml and 1 µg/ml respectively (Figure 3.5). All diluted

stocks were kept at -20°C in McCartney bottles until use.

Stock (20 mg/ml)

0.18 ml

+ 2.82 ml dH2O

1.5 ml

+ 1.5 ml dH2O (200 µg/ml)

1.5 ml

+ 1.5 ml dH2O (100 µg/ml) + 900 µl dH2O (10 µg/ml)

100µl
1.5 ml 10 µl

+ 1.5 ml dH2O (50 µg/ml) + 990 µl dH2O (1 µg/ml)

1.5 ml

+ 1.5 ml dH2O (25 µg/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 µg/ml, 100 µg/ml, 50 µg/ml, 25 µg/ml,

10µg/ml and 1 µg/ml were applied. The cells were incubated with S. commune extracts

at 37°C 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 µl 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 –20°C 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 37°C. 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 µl 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 µl

streptavidin-HRP conjugate for 10 minutes at 37°C. Next, the slides were washed in

PBS in another 5 minutes and blotted dry. 30 µl of 3’- diaminobenzidine

tetrahydrochloride (DAB) were then added and incubated at 37°C. 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 (53°C). 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 1º 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 11±1 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 37°C). Inhibition zones were
measured (in mm) and the diameter of inhibition zones reported

S. commune extracts
Microorganisms Methanol Ethyl Dichloromethane Water
acetate
Bacillus cereus 9±1 a 9±1 b 10±1 b -
PA PA PA NA
Bacillus 9±0 a 8±1 b
9±1 b -
subtilis PA IA PA NA
Enterobacter 9±1 a 10±0 b
9±1 b -
faecalis PA PA PA NA
Staphylococcus 10±0 a 9±1 b
10±0 b -
aureus PA PA PA NA
Gram- Streptococcus - 9±0 b 8±0 b -
positive mitis NA PA IwA NA
bacteria Streptococcus - - - -
mutans NA NA NA NA
Streptococcus 10±1 a 11±0 b 11±1 b -
sanguis PA PA PA NA
Escherichia 9±0 a 10±0 b 9±0 b -
coli PA PA PA NA
Salmonella sp. - 10±0 b 9±1 b -
NA PA PA NA
Salmonella 8±1 a 9±1 b 9±1 b -
typhi IA PA PA NA
Shigella sp. 9±0 a 10±0 b 9±1 b -
Gram- PA PA PA NA
negative Shigella 8±1 a 9±0 b 9±1 b -
bacteria flexneri IA PA PA NA
Plesiomonas 8±1 a 9±1 b 10±1 b -
shigelloides PA PA PA NA
Proteus 9±0 a 9±1 b 11±0 b -
vulgaris PA PA PA NA
Pseudomonas - - - -
aeuroginosa NA NA NA NA
Candida - 9±1 - -
albicans NA PA NA NA
Fungi Candida - 9±1 8±0 -
parapsilosis NA PA IA NA
Saccharomyces - 8±1 - -
pombe NA IA 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 37°C). Inhibition zones were
measured (in mm) and the diameter of inhibition zones reported

S. commune extracts
Microorganisms Methanol Ethyl Dichloromethane Water
acetate
Bacillus cereus 9±1a 8±1a 11±0b -
PA IA PA NA
Bacillus 10±1a 9±1 a
11±1b -
subtilis PA IA PA NA
Enterobacter 10±1a 10±1 a
11±1b -
faecalis PA PA PA NA
Staphylococcus 10±1a 9±1 a
11±1b -
aureus PA PA PA NA
Streptococcus 9±1a 9±1a 11±1b -
Gram- mitis PA PA PA NA
positive Streptococcus - - - -
bacteria mutans NA NA NA NA
Streptococcus 11±1a 11±1a 12±1b -
sanguis PA PA PA NA
Escherichia 9±1a 10±1a 11±1b -
coli PA PA PA NA
Salmonella sp. 9±1a 10±1a 11±1b -
IA PA PA NA
Salmonella 9±1a 10±0a 11±1b -
typhi PA PA PA NA
Shigella sp. 9±1a 10±1a 11±1b -
Gram- PA PA PA NA
negative Shigella 9±1a 9±1a 11±1b -
bacteria flexneri PA PA PA NA
Plesiomonas 9±1a 11±1a 11±1b -
shigelloides PA PA PA NA
Proteus 9±1a 10±1a 11±1b -
vulgaris PA PA PA NA
Pseudomonas 9±0a 9±1a 10±1b -
aeuroginosa PA PA PA NA
Candida - 10±1 10±0 -
albicans NA PA PA NA
Fungi Candida - 10±0 10±1 -
parapsilosis NA PA PA NA
Saccharomyces - 9±1 8±1 -
pombe NA IA IA 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 (b): S. mutans was not inhibited by

2 mg/ml dichloromethane extract of 2 mg/ml dichloromethane extract of
S. commune. 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 37°C). Inhibition zones were measured (in mm) and
the diameter of inhibition zones reported

Streptomycin Kanamycin Chloramphenicol

0.2 2 0.2 2 0.2 2
Microorganisms mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml
Bacillus cereus 25±1 31±1 21±1 30±1 18±1 26±1
VA VA VA VA VA VA
Bacillus 18±1 25±1 30±1 35±1 25±1 40±1
subtilis A VA VA VA VA VA
Enterobacter 26±1 32±1 20±1 27±1 19±1 30±1
Gram- faecalis VA VA VA VA VA VA
positive Staphylococcus 18±0 25±1 30±1 35±1 25±1 40±1
bacteria aureus A VA VA VA VA VA
Streptococcus 20±0 27±0 17±1 19±1 22±1 25±1
mitis VA VA VA VA VA VA
Streptococcus - 20±0 - 21±0 - 13±1
mutans NA VA NA VA NA A
Streptococcus 9±1 21±1 12±1 25±1 8±1 18±1
sanguis PA VA PA VA IA VA
Escherichia 13±1 26±1 20±0 27±1 12±1 31±1
coli A VA VA VA PA VA
Salmonella sp. 29±2 38±1 22±1 31±1 22±1 34±1
VA VA VA VA VA VA
Salmonella 29±1 38±1 20±1 28±1 20±1 33±1
Gram- typhi VA VA VA VA VA VA
negative Shigella sp. 30±1 37±1 21±1 28±1 19±1 31±1
bacteria VA VA VA VA VA VA
Shigella 29±1 38±1 20±1 28±1 20±1 33±1
flexneri VA VA VA VA VA VA
Plesiomonas 32±1 39±1 22±1 30±1 24±1 32±1
shigelloides VA VA VA VA VA VA
Proteus 15±1 25±1 29±1 34±1 25±0 42±1
vulgaris A VA VA VA VA VA
Pseudomonas 15±1 21±0 24±1 31±1 21±1 34±1
aeuroginosa A VA VA VA 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 (b): The inhibition zone of B. subtilis
by 2 mg/ml antibiotic chloramphenicol. 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 37°C). Inhibition zones were measured (in mm) and
the diameter of inhibition zones reported

Nystatin
Microorganisms 0.2 mg/ml 2 mg/ml
Candida albicans 23±1 28±1
VA VA
Fungi Candida parapsilosis - 12±1
NA PA
Saccharomyces pombe 23±1 29±1
VA 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 dose-

dependent 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)
Methanol 0.145 ± 0.01 a
Ethyl acetate 0.219 ± 0.01 b
Dichloromethane 0.641 ± 0.13 c
Water 0.674 ± 0.05 d
*Ascorbic acid 0.084 ± 0.01
Data expressed as means ± standard deviations of triplicate measurements
*Positive reference standard

120

100
Inhibition (%)

80

60

40
methanol
ethyl acetate
20 dichloromethane
water
0
i (di hl h )
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Concentrations (mg/ml)

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 µg/ml to 10 µg/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 Total phenolic content Total phenolic content

(mg GA/g extracts) (mg GA/g fruitbodies)
Methanol 1.72±0.05a 0.498±0.07a
Ethyl acetate 0.79±0.03b 0.057±0.01c
Dichloromethane 0.45±0.04d 0.021±0.01d
Water 0.52±0.01c 0.095±0.02b
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

50

DPPH inhibition (%)

40

30

20

y = 7.3362x + 9.1791
10 2
R = 0.8264
0
0 1 2 3 4 5 6 7
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 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/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 µg/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 µg/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

Methanol Ethyl acetate Dichloromethane Water

Cell line
KB 24.40 ± 6.1 81.78 ± 9.9 76.11 ± 2.7 NA
CaSki 62.87 ± 5.5 NA 59.26 ± 2.1 NA
HT29 NA NA 57.80 ± 4.2 NA
HCT116 NA 73.94 ± 0.1 14.71 ± 2.0 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 µg/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 µg/ml) indicating that the extract

was the most active in inhibiting the cancer cells (Table 4.7).

73
 90
80

70

inhibition (%)
60

50
40

30
20
10
0
0 20 40 60 80 100 120
Concentrations (µg/ml)

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 µg/ml) inhibition was recorded, the highest cytotoxicity was

of methanol extract resulting IC50 value of 24.40 ± 6.1 µg/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 µg/ml). Particularly,

dichloromethane showed the most potent cytotoxic activity against the cells with the

IC50 value of 59.26 ± 2.1 µg/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 µg/ml), gave the highest activity

with IC50 value of 57.80 ± 4.2 µg/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 µg/ml, 10 µg/ml, 25 µg/ml, 50

µg/ml, 100 µg/ml and 200 µg/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 Cytoplasm
Nucleus Nucleus

(a): The negative control of CaSki cells; (b): The positive control of CaSki cells;
cells not treated with antibody 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 µg/ml. At higher concentrations (50-200

µg/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 µg/ml and were clearly lysed at 200 µg/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 µg/ml, and when higher

concentrations applied (10-200 µg/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 µg/ml. The

total reduction of reddish brown stain was clearly observed at the highest concentration

of ethyl acetate extract (200 µg/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 µg/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

µg/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 µg/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 µg/ml). The greatest lysis was only found at the highest concentration of 200

µg/ml. As for the reddish brown intensity, the water extract exhibited the strongest

intensity at 1 µg/ml and this intensity became lesser as the concentration of the extract

77
increased. The decreased were obviously seen at 50 µg/ml to 200 µg/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 Morphology of Intensity of reddish

extract (µg/ml) cells brown stain

1 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 Morphology of Intensity of reddish

of extract cells brown stain
(µg/ml)

1 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 Morphology of Intensity of reddish

of extract cells brown stain
(µg/ml)

1 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 Morphology of Intensity of reddish

of extract cells brown stain
(µg/ml)

1 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 disease-

related 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 60°C for 30

minutes reduced the antibacterial activity against the microorganisms by 60% and the

activity was completely inactivated by heat treatment at 100°C 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 Gram-

negative 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 L’Abee-

Lund, 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 Gram-

positive 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

0.057±0.01 mg GA/g fruitbodies) than the dichloromethane extract (0.52±0.04 mg

GA/g extracts and 0.021±0.01 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 121ºC for 30 minutes was significantly

decreased (P<0.05) relative to those of raw shiitake or heat treated at 100ºC for 15 and

30 minutes or 121ºC for 15 minutes (Choi, et al., 2006).

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 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).

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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 drug-

induced 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 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/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 37°C 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 ≤ 20µg/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 µg/ml. Ethyl

acetate extract of S. commune was also found to be poorly active against all the cell-

lines. However, dichloromethane extract exhibited the most active inhibition especially

against HCT116 cancer cell line with IC50 value of 14.71 ± 2.0 µg/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 Ehrlich’s 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 α-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 pro-

apoptotic 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 α-glucan in the polysaccharide-

treated 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 α-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 down-

regulation, 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).

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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 µg/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 Ehrlich’s ascites carcinoma (EAC) and

Dalton’s 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 ß

(1→3) linkage in the main chain of the glucan and additional ß (1→6) 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 body’s 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 µg/ml) and higher concentration (100 µg/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.

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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 µg/ml, 10 µg/ml, 25

µg/ml, 50 µg/ml, 100 µg/ml and 200 µg/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 pre-

treatment 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 rose-

red 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 µg/ml and were greatly

lysed as the concentrations increased (50-200 µg/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 anti-

viral, 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 virus-

specific 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-β, 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 Gram-

positive 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: REAGENTS, MEDIA AND BUFFER

1.0 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 121°C 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 25°C.

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 121°C for 15 minutes. After
cooling down, it was refrigerated at 25°C 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 121°C for 20 minutes. After cooling down, it was refrigerated at
25°C until required.

1.4 Yeast-peptone (YP) broth

• Yeast extract 4.00 g

• Peptone 8.00 g
• Glucose 8.00 g
• pH 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 121°C for 15 minutes. After cooling down, it was
refrigerated at 25°C until required.

1.5 Yeast-peptone agar (YPA)

• Yeast extract 4.00 g

151
 • Peptone 8.00 g
• Glucose 8.00 g
• Agar 7.00 g
• PH 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 121°C 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 25°C.

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 121°C for 20 minutes. After cooling
down, it was refrigerated at 25°C 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
121°C 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 25°C.

2.0 DETERMINATION OF ANTIOXIDANT ACTIVITY

2.1 Folin-Ciocalteau reagent

The reagent should be freshly prepared to avoid oxidation. 0.5 ml of Folin-

Ciocalteau 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 µm 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 µm filter membrane into a bottle. The
bottle was then kept in 4°C.

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 µm filter membrane into a bottle and was then kept
in 4°C until required.

3.4 Tryphan blue

0.2 g of tryphan blue was mixed with 50 ml distilled water and then stored at
4°C.

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 4°C 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 µm 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 µl 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 Volume of Volume of Volume of DPPH
extracts methanol (µl) extracts (µl) solution (ml)
(mg/ml)
1 95 5 3.9
5 75 25 3.9
10 50 50 3.9
15 25 75 3.9
20 - 100 3.9

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

concentration of 30 mg/ml
Concentrations of Volume of Volume of Volume of DPPH
extracts methanol (µl) extracts (µl) solution (µl)
(mg/ml)
1 95 5 3.9
2 90 10 3.9
5 75 25 3.9
10 50 50 3.9
15 25 75 3.9

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

concentration of 50 mg/ml
Concentrations of Volume of Volume of Volume of DPPH
extracts methanol (µl) extracts (µl) solution (µl)
(mg/ml)
1 98 2 3.9
5 90 10 3.9
20 60 40 3.9
30 40 60 3.9
50 - 100 3.9

155
Table 1.4: Preparation of water extracts of S. commune with stock concentration of
50 mg/ml
Concentrations of Volume of Volume of Volume of DPPH
extracts methanol (µl) extracts (µl) solution (µl)
(mg/ml)
5 90 10 3.9
10 80 20 3.9
20 60 40 3.9
30 40 60 3.9
50 - 100 3.9

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

Concentrations of ascorbic acid Volume of Volume of Volume of

ascorbic acid methanol DPPH solution
mM mg/ml
(µl) (µl) (ml)
0.1 0.0176 10 90 3.9
0.2 0.0352 20 80 3.9
0.3 0.0528 30 70 3.9
0.4 0.0704 40 60 3.9
0.5 0.0880 50 50 3.9
0.6 0.1057 60 40 3.9
0.7 0.1233 70 30 3.9
0.8 0.1409 80 20 3.9
0.9 0.1585 90 10 3.9
1.0 0.1761 100 - 3.9

1.2 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 µl) was added to the
aliquot (250 µl) of mushroom extract solution and was mixed thoroughly. After 3
minutes, a 500 µl 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 Volume of extracts Volume of distilled
(mg/ml) (µl) water (µl)
Ethyl acetate 2 250 -
Methanol 2 250 -
Dichloromethane 2 250 -
Water 2 250 -

156
Table 1.7: Preparation of gallic acid as positive reference standard for Folin-
Ciocalteau assay
Concentrations (µg/ml) Volume of gallic acid (µl) Volume of distilled water (µl)
0 - 250
2 50 200
4 100 150
6 150 100
8 200 50
10 250 -
*Stock concentration= 10 µg/ml

157
APPENDIX C: EXPERIMENTAL AND STATISTICAL DATA

1.0 DETERMINATION OF ANTIMICROBIAL ACTIVITY

1.1 The experimental data of the antimicrobial activity of S. commune extracts

against microorganisms tested at two different concentrations

Methanol Ethyl acetate Dichloromethane

Microorganisms 0.2 2 0.2 2 0.2 2
mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml
Bacillus cereus 9 10 9 10 11 12
10 11 10 10 11 11
10 11 10 9 10 12
9±1 10±1 9±1 9±1 10±1 11±1
Bacillus 9 10 8 11 10 11
subtilis 9 9 9 10 9 12
9 10 9 10 10 11
9±0 9±1 8±1 10±1 9±1 11±1
Enterobacter 10 11 10 11 10 12
faecalis 9 10 10 11 9 11
Gram- 10 11 10 10 10 12
positive 9±1 10±1 10±0 10±1 9±1 11±1
bacteria Staphylococcus 10 11 9 10 10 12
aureus 10 10 10 10 10 11
10 10 9 9 10 11
10±0 10±1 9±1 9±1 10±0 11±1
Streptococcus 10 9 10 8 12
mitis NA 9 9 10 8 11
9 9 9 8 11
9±1 9±0 9±1 8±0 11±1
Streptococcus NA NA NA NA NA NA
mutans
Streptococcus 11 12 11 11 12 13
sanguis 10 11 11 12 11 13
11 12 11 11 11 12
10±1 11±1 11±0 11±1 11±1 12±1
Escherichia 9 10 10 11 9 12
coli 9 9 10 10 9 12
9 10 10 10 9 11
9±0 9±1 10±0 10±1 9±0 11±1
Salmonella sp. 10 10 11 10 12
NA 9 10 10 9 11
9 10 10 9 11
9±1 10±0 10±1 9±1 11±1
Salmonella 9 10 10 10 10 12
Gram- typhi 8 9 9 10 9 11
negative 9 10 9 10 10 12
bacteria 8±1 9±1 9±1 10±0 9±1 11±1
Shigella sp. 9 10 10 11 10 11
9 9 10 10 11 12

158
 9 10 10 10 10 12
9±0 9±1 10±0 10±1 10±1 11±1
Shigella 9 10 9 10 10 12
flexneri 8 9 9 9 9 11
8 10 9 9 9 11
8±1 9±1 9±0 9±1 9±1 11±1
Plesiomonas 8 9 10 11 11 12
shigelloides 9 10 10 12 10 12
8 9 9 11 10 11
8±1 9±1 9±1 11±1 10±1 11±1
Proteus 9 10 9 10 11 12
vulgaris 9 9 10 11 11 11
9 10 9 10 11 12
9±0 9±1 9±1 10±1 11±0 11±1
9±0 9±1 9±1 10±1 11±0 11±1
Pseudomonas 9 10 11
aeuroginosa NA 9 NA 9 NA 10
9 9 10
9±0 9±1 10±1
Candida 10 11 10
albicans NA NA 9 10 NA 10
9 10 10
9±1 10±1 10±0
Candida 10 10 8 10
Fungi parapsilosis NA NA 9 10 8 11
9 10 8 11
9±1 10±0 8±0 10±1
Saccharomyces 9 9 8
pombe NA NA 9 10 NA 8
8 10 9
8±1 9±1 8±1
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 Kanamycin Chloramphenicol

Microorganisms 0.2 2 0.2 2 0.2 2
mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml
Bacillus cereus 25 30 21 30 18 25
25 31 20 30 18 26
26 31 21 31 19 25
25±1 31±1 21±1 30±1 18±1 26±1
Bacillus 18 25 30 34 24 40
subtilis 19 26 31 34 25 41
19 25 30 35 25 40
18±1 25±1 30±1 35±1 25±1 40±1
Enterobacter 26 33 21 27 19 31
faecalis 25 33 20 28 19 30
25 32 20 28 18 31
Gram- 26±1 32±1 20±1 27±1 19±1 30±1
positive Staphylococcus 18 25 30 35 25 41
bacteria aureus 18 26 31 36 26 40
18 25 31 35 25 40
18±0 25±1 30±1 35±1 25±1 40±1
Streptococcus 20 27 18 20 22 25
mitis 20 27 17 19 23 26
20 27 18 19 23 25
20±0 27±0 17±1 19±1 22±1 25±1
Streptococcus 20 21 14
mutans NA 20 NA 21 NA 13
20 21 14
20±0 21±0 13±1
Streptococcus 10 22 12 25 8 19
sanguis 9 22 13 26 9 18
9 21 12 26 9 19
9±1 21±1 12±1 25±1 8±1 18±1
Escherichia 13 26 20 27 12 31
coli 14 27 20 28 13 32
14 26 20 27 13 31
13±1 26±1 20±0 27±1 12±1 31±1
Salmonella sp. 29 39 22 32 23 35
31 38 23 32 22 35
31 38 22 31 22 34
29±2 38±1 22±1 31±1 22±1 34±1
Salmonella 29 39 21 29 21 34
typhi 30 38 20 28 20 34
Gram- 29 38 20 29 20 33
negative 29±1 38±1 20±1 28±1 20±1 33±1
bacteria 30 37 21 28 19 31
Shigella sp.  31 38 22 29 19 32
30 38 22 28 20 31
30±1 37±1 21±1 28±1 19±1 31±1

162
 Shigella 29 38 20 28 21 33
flexneri 30 39 21 29 21 33
30 38 20 29 20 34
29±1 38±1 20±1 28±1 20±1 33±1
Plesiomonas 33 40 23 30 25 32
shigelloides 32 40 22 31 24 33
32 39 22 30 24 33
32±1 39±1 22±1 30±1 24±1 32±1
Proteus 15 26 30 34 25 42
vulgaris 16 26 29 34 25 43
16 25 29 35 25 43
15±1 25±1 29±1 34±1 25±0 42±1
Pseudomonas 15 21 24 32 22 34
aeuroginosa 16 21 24 31 21 35
15 20 25 32 22 35
15±1 21±0 24±1 31±1 21±1 34±1
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 0.2 mg/ml 2 mg/ml
Candida albicans 24 29
23 28
23 29
23±1 28±1
Candida parapsilosis 12
Fungi NA 13
12
12±1
Saccharomyces pombe 23 30
22 29
22 29
23±1 29±1
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 Concentration Inhibition Average

(mg/ml) (%) (%)
R1 R2 R3
1 11.40 15.94 16.33 14.56±2.74
Methanol 2 17.10 17.10 16.33 16.84±0.44
5 47.50 58.06 48.05 51.20±5.94
10 92.02 88.96 90.49 90.49±1.53
15 89.98 90.15 91.00 90.38±0.55
1 13.67 13.26 10.77 12.57±1.57
Ethyl 5 34.53 35.91 30.80 33.75±2.64
acetate 10 63.40 54.70 58.43 58.84±4.34
15 91.02 88.54 89.78 89.78±1.24
20 90.88 90.47 90.19 90.51±0.35
1 7.40 11.01 9.39 9.27±1.81
Dichloromethane
5 25.09 28.16 26.17 26.47±1.56
20 31.05 30.42 27.46 29.64±1.92
30 67.39 70.51 66.93 68.28±1.95
50 75.03 72.70 67.08 71.60±4.09
5 16.06 20.58 23.65 20.10±3.82
Aqueous 10 30.87 30.14 26.71 29.24±2.22
20 36.64 41.52 32.67 36.94±4.43
30 46.57 50.18 52.17 49.64±2.84
50 70.04 74.01 72.74 72.26±2.03
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 Scavenging Activity

IC50 (mg/ml)
Methanol 0.145 ± 0.01
Ethyl acetate 0.219 ± 0.01
Dichloromethane 0.641 ± 0.13
Water 0.674 ± 0.05
*Ascorbic acid 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 Inhibition Average

ascorbic acid (µg/ml) (%) (%)
R1 R2 R3
0.440 10.84 11.40 12.13 11.46±0.65
0.881 31.53 31.81 34.09 32.48±1.40
1.321 35.78 37.23 38.63 37.21±1.43
1.761 45.08 45.93 44.51 45.17±0.71
2.201 48.93 47.85 48.25 48.34±0.55
2.642 53.88 55.15 54.56 54.53±0.64
3.082 60.48 59.91 60.05 60.15±0.29
3.522 64.75 64.62 58.61 64.69±0.09
3.962 65.33 64.91 66.48 65.57±0.81
4.403 67.48 68.99 67.08 67.85±2.26
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

60
In h ib itio n (% )

50
40 y = 232.74x + 30.749
2
R = 0.978
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 Absorbance Average

R1 R2 R3
Ethyl acetate 0.078 0.076 0.081 0.078
Methanol 0.166 0.175 0.171 0.054
Dichloromethane 0.044 0.046 0.044 0.045
Aqueos 0.052 0.055 0.048 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 Absorbance Average

(µg/ml) R1 R2 R3
0 0.017 0.014 0.017 0.016
2 0.057 0.067 0.034 0.054
4 0.129 0.135 0.130 0.131
6 0.202 0.186 0.188 0.192
8 0.235 0.240 0.249 0.241
10 0.263 0.263 0.261 0.262
R= Replicate

2.9 The calibration plot of gallic acid as positive reference standard

0.35

0.3

0.25
Absorbance

0.2

0.15 y = 0.0288x
2
R = 0.9666
0.1

0.05

0
0 2 4 6 8 10 12
Concentrations (ug/ml)

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

Extract Scavenging Activity Total Phenolic Content

IC50 (mg/ml) (mg GA/g extracts)
Methanol 0.145 ± 0.01 1.72 ± 0.05
Ethyl acetate 0.219 ± 0.01 0.79 ± 0.03
Dichloromethane 0.641 ± 0.13 0.52 ± 0.04
Water 0.674 ± 0.05 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 Percentage of inhibition (%)

of extracts Ethyl
(µg/ml) acetate
Cell line Methanol Dichloromethane Aqueous
KB 1 14±1.69 2±1.08 5.43±1.5 10.13±0.55
10 26.1±6.51 13.8±0.85 22.63±1.2 21.3±0.46
50 54.65±4.31 20.95±0.78 42.3±0.71 31.94±0.8
100 75.37±5.71 57.7±1.69 63.5±4.38 48.14±0.43
1 16.2±0.71 10.75±0.21 4.15±0.64 1.55±0.21
CaSki 10 35.05±4.74 22.6±3.96 21.25±1.2 23.55±5.44
50 47.1±0.71 36.2±3.25 48.87±5.18 34.53±3.5
100 51.35±5.87 40.65±0.64 80.13±4.24 40.4±2.43
HT29 1 0.75±0.35 22.6±0.42 3.6±1.27 7.5±0.0
10 12.8±0.14 30.45±4.6 14.05±6.15 12.65±0.92
50 21.25±2.33 37.65±2.47 47.8±2.12 15.2±1.61
100 33.2±5.03 42.8±2.26 62.25±0.92 31.85±0.21
HCT116 1 5.3±0.28 2.3±1.13 8.3±1.69 12.1±2.12
10 9.2±0.14 11.75±2.89 33.95±0.35 16.15±2.05
50 16.5±1.56 28.85±0.64 76.5±1.66 19.85±2.76
100 27.65±0.49 50.4±1.87 87.05±1.06 20.55±2.19
*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