BIOLOGICAL ACTIVITIES OF Schizophyllum commune FR.

MIRFAT BT HJ AHMAD HASAN SALAHUDDIN

THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR

2008

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

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

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

iii

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

menyumbang kepada pelbagai aktiviti biologi selain sebagai sumber makanan yang berfungsi dari segi nilai nutrisi dan merupakan ubat yang tidak toksik dan mempunyai kelebihan secara fisiologi. Dalam kajian ini, Schizophyllum commune Fr. iaitu sejenis cendawan kukur telah diplih memandangkan kepentingan perubatannya telah dikenalpasti. S. commune telah diekstrak dengan metanol, etil asetat, diklorometana dan air dan ekstrak-ekstrak ini telah diuji. Aktiviti antimikrob ditentukan dengan menggunakan esei penyebaran telaga (well diffusion). Mikroorganisma yang diuji adalah bakteria patogen yang biasa iaitu bakteria Gram-positif (Bacillus cereus, B.subtilis, Enterobacter faecalis, Staphylococcus aureus, Streptococcus mitis, S. mutans dan S. sanguis), bakteria Gram-negatif (Escherichia coli, Salmonella sp., S. typhi, Shigella sp., Shigella flexneri, Plesiomonas shigelloides, Proteus vulgaris, dan Pseudomonas aeuroginosa) dan kulat patogen (Candida albicans, C. parapsilosis dan Saccharomyces pombe). Keberkesanan ekstrak-ekstrak ini bergantung kepada pelarut yang digunakan dalam pengekstrakan. Aktiviti antibakteria adalah lebih tinggi berbanding dengan aktiviti antikulat. Ekstrak diklorometana adalah yang paling aktif secara signifikan (P0.05) dalam menghalang pertumbuhan hampir kesemua bakteria iv

Gram-positif, Gram-negatif dan kulat yang diuji. Zon perencatan yang paling tinggi yang direkodkan oleh ekstrak ini adalah terhadap S. sanguis (12 1 mm). Potensi antioksidan ekstrak S. commune dinilai menggunakan dua kaedah iaitu sistem penyingkir radikal DPPH dan kaedah Folin-Ciocalteau. Daripada kajian, aktiviti pemusnah radikal yang ditunjukkan oleh ekstrak S. commune mempunyai hubungkait yang tinggi (r=0.8264) di antara aktiviti pemusnah radikal dan jumlah kandungan fenol. Ekstrak metanol S. commune menunjukkan aktiviti antioksidan yang paling baik

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

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

laporan pertama mengenai kajian antimikrob, antioksidan, sitotoksisiti dan anti-HPV daripada ekstrak S. commune.

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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

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TABLE OF CONTENTS

ABSTRACT ABSTRAK ACKNOWLEDGEMENT TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF PLATES LIST OF SYMBOLS AND ABBREVIATIONS

ii iv vii viii xii xiii xiv xv

CHAPTER ONE - INTRODUCTION 1.1 Objectives of Study

1 5 7 7 9 11 11 14 16 16 17 18 20 21 27 viii

CHAPTER TWO - LITERATURE REVIEW 2.1 2.2 2.3 Introduction to medicinal mushroom Schizophyllum commune Fr. Biological activities of mushrooms 2.3.1 Anti-microbial activity of mushrooms 2.3.1.1 Mechanisms of antimicrobial actions 2.3.2 Antioxidant activity of mushrooms 2.3.2.1 Free radicals and reactive oxygen species 2.3.2.2 Antioxidants 2.3.2.3 Mushroom as antioxidants 2.3.2.4 Mechanisms of antioxidant actions 2.3.3 Cytotoxicity of mushrooms 2.3.3.1 Mechanisms of anti-tumor actions

2.3.4

Antiviral activity of mushrooms 2.3.4.1 Human papilloma virus (HPV) 2.3.4.2 Human papilloma virus (HPV) infection

30 33 35 37 37 39 42 44 47 47 48 49 51 51 52 52 52 53 53 54 55 56

2.4

Methods in the determination of biological activities 2.4.1 2.4.2 Antimicrobial activity Antioxidant activity

2.4.3 Cytotoxic activity 2.4.4 Anti-human papilloma virus (HPV) activity

CHAPTER THREE - MATERIALS AND METHODS 3.1 3.2 Preparation of Schizophyllum commune extracts Determination of antimicrobial activity 3.2.1 Well diffusion assay 3.3 Determination of antioxidant activity 3.3.1 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

3.3.2 Folin-Ciocalteau assay 3.4 Cytotoxicity towards various cancer cell lines 3.4.1 Cancer cell lines

3.4.2 Serial dilution of S. commune extracts 3.4.3 3.4.4 3.4.5 3.4.6 3.5 Sub-cultivation of cancer cells Cell enumeration and plating Treatment of cancer cells with S. commune extracts Neutral red assay

Anti-human papilloma virus (HPV) activity towards cervical cancer (CaSki) cell lines 3.5.1 Serial dilution of S. commune extracts 56 56 ix

3.5.2 3.5.3 3.5.4

Treatment of CaSki cells with S. commune extracts Fixation of CaSki cells onto slides Immunohistochemistry assay

57 57 58 60 60 68

CHAPTER FOUR - RESULTS 4.1 4.2 Analysis of antimicrobial activity of S. commune extracts Analysis of antioxidant activity of S. commune extracts 4.2.1 Correlation between total phenolic content and scavenging activity of S. commune extracts 4.3 Analysis of cytotoxicity of S. commune extracts towards various cancer cell lines 4.4 Analysis of anti-human papilloma virus (HPV) activity of S. commune towards cervical cancer (CaSki) cell lines CHAPTER FIVE DISCUSSION 5.1 Antimicrobial activity of S. commune Fr. 5.1.1 Antimicrobial activity of S. commune extracts as evaluated by well diffusion assay 5.1.2 Antimicrobial activity of S. commune extracts against various pathogenic microorganisms 5.1.3 Extraction solvent as the significant factor in evaluating antimicrobial activity of S. commune 5.1.4 Antimicrobial activity of S. commune extracts and its correlation to phenolic content 5.1.5 Other contributing factors to antimicrobial activity of S. commune extracts

71

72

75 82 82

82

83

84

86

89

5.2

Antioxidant activity of S. commune Fr. 5.2.1 Antioxidant activity of S. commune extracts and its correlation to phenolic content 5.2.2 Extraction solvent as the significant factor in evaluating antioxidant activity of S. commune 5.2.3 Other contributing factors to antioxidant activity of S. commune extracts

91

91

94

98 99

5.3

Cytotoxicity of S. commune Fr. 5.3.1 Cytotoxicity of S. commune extracts as evaluated by neutral red assay 5.3.2 Cytotoxicity of S. commune extracts towards different cancer cell lines 5.3.3 Cytotoxicity of S. commune extracts and its correlation to antimicrobial activity 5.3.4 Other contributing factors to cytotoxicity of S. commune extracts

99

103

108

109 112

5.4

Anti-human papilloma virus (HPV) activity of S. commune Fr. 5.4.1 Anti-human papilloma virus (HPV) activity of S. commune extracts as evaluated by immunohistochemistry assay 5.4.2 Anti-human papilloma virus (HPV) activity of S. commune extracts towards cervical cancer cell lines (CaSki) 5.4.3 Comparative study of various antiviral activities

112

114 115

CHAPTER SIX - CONCLUSION

119

xi

REFERENCES APPENDICES Appendix A: Reagents, media and buffer Appendix B: Analytical techniques Appendix C: Experimental and statistical data

123 151 151 155 158

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LIST OF FIGURES

Figure 2.1 Figure 2.2 Figure 3.1

: : :

The extrinsic death receptor pathway (Page 30) The intrinsic mitochondrial pathway (Page 30) Extraction of S. commune bioactive compounds and the biological activities analyzed (Page 48) Screening for antimicrobial activities of S. commune extracts (Page 50) Serial dilution of S. commune extracts for cytotoxicity (Page 53) Cell counting using a haemocytometer (Page 55) Serial dilution of S. commune extracts for anti-HPV activity (Page 57) A brief procedure of immunohistochemistry method (Page 59) The scavenging effect of S. commune extracts on DPPH radicals (Page 69) Correlation between the radical scavenging activity S. commune extracts and their phenolic contents (Page 72) of

Figure 3.2

Figure 3.3 Figure 3.4 Figure 3.5

: : :

Figure 3.6 Figure 4.1

: :

Figure 4.2

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 Table 4.1

: :

Sizes and functions of HPV 18 proteins (Page 37) Antimicrobial activity of S. commune extracts at 0.2 mg/ml as evaluated by well diffusion assay (24 hours incubation at 37C). Inhibition zones were measured (in mm) and the diameter of inhibition zones reported (Page 62) Antimicrobial activity of S. commune extracts at 2 mg/ml as evaluated by well diffusion assay (24 hours incubation at 37C). Inhibition zones were measured (in mm) and the diameter of inhibition zones reported (Page 64) Antibacterial activity of antibiotic discs as evaluated by well diffusion assay (24 hours incubation at 37C). Inhibition zones were measured (in mm) and the diameter of inhibition zones reported (Page 66) Antifungal activity of antibiotic nystatin as evaluated by well diffusion assay (24 hours incubation at 37C). Inhibition zones were measured (in mm) and the diameter of inhibition zones reported (Page 67) The scavenging activity of S. commune extracts as measured by DPPH radical scavenging assay (Page 69) The total phenolic content of S. commune extracts and fruitbodies at 0.1 mg/ml as measured by Folin-Ciocalteau method (Page 71) The IC50 values of S. commune extracts against different cancer cell lines as measured by neutral red cytotoxicity assay (Page 73)

Table 4.2

Table 4.3

Table 4.4

Table 4.5

Table 4.6

Table 4.7

xiv

LIST OF PLATES

Plate 4.1 Plate 4.1 (a)

: :

Antibacterial activity of S. commune dichloromethane extracts against some of the microorganisms (Page 65) The inhibition zone of S. sanguis by 2 mg/ml dichloromethane extract of S. commune (Page 65) S. mutans was not inhibited by 2 mg/ml dichloromethane extract of S. commune (Page 65) Antibacterial activity of antibiotic chloramphenicol against some of the microorganisms (Page 67) The inhibition zone of P. vulgaris by 2 mg/ml antibiotic chloramphenicol (Page 67) The inhibition zone of B. subtilis by 2 mg/ml antibiotic chloramphenicol (Page 67) The qualitative appearance of two controls of CaSki cell lines after being treated with different treatments (Page 76) The negative control of CaSki cells; cells not treated with antibody (Page 76) The positive control of CaSki cells; cells treated with antibody (Page 76) The appearance of CaSki cells after treatment with methanol extract of S. commune at different concentrations (Page 78) The appearance of CaSki cells after treatment with ethyl acetate extract of S. commune at different concentrations (Page 79) The appearance of CaSki dichloromethane extract of concentrations (Page 80) cells after treatment with S. commune at different

Plate 4.1 (b) : Plate 4.2 Plate 4.2 (a) : :

Plate 4.2 (b) :

Plate 4.3 Plate 4.3 (a)

: :

Plate 4.3 (b) :

Plate 4.4 Plate 4.5 Plate 4.6

: : :

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

% C g g/ml l BHI bp CIN CIS cm CO2 DAB dH2O DMSO DNA DPPH E ED50 FBS g GAE H2O2 HEPES HPV

: : : : : : : : : : : : : : : : : : : : : : : :

percentage degree centigrade microgram microgram per mililiter microliter Brain Heart Infusion base pair cervical intraepithelial neoplasia carcinoma in situ centimeter carbon dioxide 3 diaminobenzidine tetrahydrochloride distilled water dimethyl sulfoxide deoxyribonucleic acid 2,2-Diphenyl-1-Picrylhydrazyl early region effective dosage of drug that gives 50% of maximal response fetal bovine serum gram gallic acid equivalent hydrogen peroxide N-2-hydroxyethyl-piperazine-N-2-ethane-sulfonic acid human papilllomavirus

xvi

HRP IHC K2HPO4 KH2PO4 L LSAB M mg mg/ml MH ml mm mRNA mw Na2CO3 Na2HPO4 NaCl NaHCO3 ORR PBS Rb rpm RPMI sp URR

: : : : : : : : : : : : : :
:

horseradish peroxidase immunohistochemistry potassium phosphate anhydrous potassium dihydrogen orthophosphate late region labelled steptravidin - biotin molar miligram milligram per mililiter Mueller Hinton mililiter millimeter messenger ribonucleic acid molecular weight natrium carbonate natrium phosphate anhydrous natrium chloride natrium bicarbonate open reading frame phosphate buffered saline retinoblastoma revolution per minute Rosewell Park Memorial Institute species beta upstream regulatory region

: : : : : : : : : : :

xvii

UV w/v wt

: : :

ultraviolet weight/volume weight

xviii

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

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

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

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

considered as a widely distributed basidiomycetous pathogen (Hobbs, 1995; Rihs et al., 1996; Sigler et al., 1997), S. commune has long been acknowledged for its medicinal properties (Oso, 1981; Han et al., 2005). In addition to its medicinal value, S. commune has been consumed as a nutritional food in South-East Asia (Han et al., 2005) and is now being cultivated in Malaysia and Thailand. The production of S. commune and other medicinal mushrooms has increased due to the ease of cultivation, increase in popularity and its nutritional value (Chang and Buswell, 1996; Wasser, 2002). In the present study, S. commune was evaluated for its medicinal properties since not many studies had been done on the medicinal properties other than anti-cancer properties of its polysaccharides. Pharmacologically, S. commune is extremely important because it produces the polysaccharide schizophyllan which shows considerable medicinal properties. Based on the previous studies, the secondary metabolites of S. commune were known to show anti-candida, anti-tumor and anti-viral properties (Ooi and Liu, 1999; Wasser, 2002). However, published studies on these mushrooms are quite limited. Hence, the biological activities of S. commune metabolites need to be explored for its use as a source of drugs and functional food to contribute to new therapeutic effects and for the treatment of a variety of human ailments.

1.1

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

S. commune. This study was undertaken with the following aims: 1) Screen S. commune extracts for anti-microbial activity. 2) Evaluate the antioxidant activity of S. commune extracts.

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

CHAPTER 2: LITERATURE REVIEW 2.1 Introduction to medicinal mushroom Mushrooms were originally defined as a macrofungus with a distinctive fruiting body, which can either be hypogeous or epigeous, large enough to be seen with the naked eye and to be picked by hand (Chang and Miles, 1992). Mushrooms constitute at least 14,000 and perhaps as many as 22,000 known species, deduced from the Dictionary of the Fungi (Hawksworth, 2001). Other study reported that the number of mushroom species on earth is estimated to be 140,000, indicating that only 10% are known (Lindequist et al., 2005). Mushrooms have long been appreciated for their flavor and texture. Now they are recognized as a nutritious food as well as an important source of biologically active compounds of medicinal value (Breene, 1990). Mau et al. (2002) had reported in their studies that medicinal mushrooms are commonly used for pharmaceutical purposes and as health foods. Hence, searching for new biological activities and other medicinal substances from mushrooms and to study the medicinal values of these mushrooms has become a matter of great significance. The scientific community, in searching for new therapeutic alternatives, has studied many kinds of mushrooms and has found variable therapeutic activities such as anti-carcinogenic, anti-inflammatory, immuno-suppressor and antibiotic effects (Asfors and Ley, 1993). At present, there are at least 270 species of mushrooms that are known to have various therapeutic effects (Ying et al., 1987). The medicinal use of mushrooms has a very long tradition in the Asian countries especially in Japan, Korea and China, whereas their use in the Western hemisphere like United States has been slightly increasing only since the last decades. Increased scientific and medical research in recent years and published in peer-reviewed journals, several books and reviews, is increasingly confirming the medicinal efficacy and 7

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

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

Kingdom Division Subdivision Class Subclass Order Family Genus

: : : : : : : :

Fungi Eumycota Basidiomycotina Hymenomycetes Holobasidiomycetidae Agaricales Schizophyllaceae Schizophyllum

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

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

10

against gastric cancer, but extended survival time in patients with head and neck cancer (Brochers et al., 1999; Kimura et al., 1994). S. commune is edible and the nutritional perspective of the mushroom was also documented in several previous studies. The islanders in Indonesia and Madagascar habitually chew carpophores of this mushroom. While the Yoruba people of SouthWestern Nigeria were reported to use S. commune to prepare delicious dishes among them (Jonathan and Fasidi, 2001). S. commune was also one of the varieties of mushrooms that consumed by the Naga tribes in Northeast India as food source (Longvah and Deosthale, 1998). 2.3 2.3.1 Biological activity of mushrooms Antimicrobial activity of mushrooms Mushrooms are rich sources of natural antibiotics. Antibiotic can be defined as a chemical substance produced by microorganisms (wholly or partly by chemical synthesis) which has the capacity to inhibit the growth of bacteria and even destroy bacteria and other microorganisms in dilute solution (Black, 2002). A research showed that the most significant antibiotics have been derived from fungi (Hardman et al., 2001) such as penicillin, streptomycin, chloramphenicol and vancomycin (Griffin, 1994) where humans can benefit from the natural defensive strategies of the fungi that produce antibiotics to fight infection from microorganisms. Infections by multidrug resistant isolates of Candida spp., Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus spp., Enterococcus sp. and Escherichia coli, among other, became more and more frequent stimulating the search for new antibiotics with novel mechanisms of action (Kotra and Mobashery, 1998). Many antibiotics are chemically modified biosynthetic form of microbial products. Chloramphenicol is now usually produced by a synthetic process. Other well11

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

12

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

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

13

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

2.3.1.1 Mechanisms of antimicrobial actions Antimicrobials are originally classified by their modes of actions which consisted of inhibition of cell wall synthesis, disruption of cell membrane function, inhibition of protein synthesis, inhibition of nucleic acid synthesis and action as antimetabolites: a) Inhibition of cell wall synthesis The cell wall synthesis inhibitors are bactericidal because they block the synthesis of rigid peptidoglycan component of the wall, results in growing cell lyse and die. Inhibition of cell wall synthesis selectively damages bacterial and fungal cells. Gram-positive bacterial cells have a high internal osmotic pressure. Without a normal, sturdy cell wall, these cells burst when subjected to the low osmotic pressure of body fluids. Antibiotics such as penicillin and cephalosporin contain a chemical structure called -lactam ring, which attaches to the enzymes that cross-link peptidoglycans (Black, 2002). b) Disruption of cell membrane function Bacterial lipids are mainly phospholipids and this component has been the target of cell membrane function inhibitor antibiotics. This action is especially effective against Gram-negative bacteria, which have an outer membrane rich in phospholipids. Certain polypeptide antibiotics such as polymyxins act as detergents and distort the bacterial cell membranes, probably by binding to phospholipids in the membrane. The 14

membrane is then no longer regulated by membrane proteins and the cytoplasm and cell substances are lost (Black, 2002). c) Inhibition of protein synthesis Almost all antibiotics that inhibit protein synthesis act on ribosomes by preventing the performance of a typical ribosomal function (Braude, 1976). Aminoglycoside antibiotics, such as streptomycin, act on the 30s portion of bacterial ribosomes by interfering with the accurate reading (translation) of the mRNA message. Other antibiotics; chloramphenicol acts on the 50s portion of bacterial ribosomes, thus inhibiting the formation of the growing polypeptide (Black, 2002). d) Inhibition of nucleic acid synthesis There are two main categories that describe the substance that inhibit nucleic acid synthesis. The first group is those that interfere with the building block of nucleic acids, which are purine and pyrimidine nucleotides. Others interfere with the polymerization of the nucleotide and the template function of DNA in RNA synthesis and hinder the function of polymerases via direct interaction with the enzymes (Franklin and Snow, 1975). Antibiotics of the rifamycin family have been known to bind to a bacterial RNA polymerase and inhibit RNA synthesis (Black, 2002). e) Action as anti-metabolites Antimetabolites are substances that affect the utilization of metabolites and therefore prevent a cell from carrying out necessary metabolic reactions. This is done by competitively inhibiting enzymes and by being erroneously incorporated into important molecules such as nucleic acids. The action is possible as they are structurally similar to normal metabolites and are even called molecular mimicry as they imitate the normal metabolites, thus preventing a reaction to occur (Black, 2002). 15

2.3.2

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

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

16

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

17

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

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

19

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

20

Induction

LH + R L + RH

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

2.3.3

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

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

21

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

22

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

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

23

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

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

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

26

al., 2002) and immunostimulatory properties which contribute to their anticancer effects. In addition, a water-soluble -glucan isolated from Poria cocos was shown to have growth-inhibitory effects on human breast carcinoma MCF-7 cells mediated by cell-cycle arrest and apoptosis induction (Zhang et al., 2006)

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

27

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

28

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

29

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

Figure 2.1: The extrinsic death receptor pathway

Figure 2.2: The intrinsic mitochondrial 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 nontoxicity, making them appealing candidates in the search for new antiviral agents. Summaries of the antiviral properties of mushrooms were published by Suay et al. (2000), Brandt and Piraino (2000) and Stamets (2001, 2002). Only recently, a numerous number of mushrooms have been found to be medically active in several therapies, such as antiviral, anti-tumor and immunomodulating treatments (Wasser and Weis, 1999). Anti-viral polysaccharides from medicinal mushrooms have been identified previously; lentinan from shiitake, Lentinula edodes (Sarkar et al., 1993); polysaccharide peptide (PSP) from Turkey tail, Trametes versicolor and ganaderiol-F, ganoderic acid-, lucidumol from reishi, Ganoderma lucidum. Polysaccharides are a complex group of macromolecules possessing a wide range of therapeutically important biological properties and known to affect the growth of animal viruses (Shannan, 1984). An extract of the edible Japanese mushroom Lentinus edodes has been found to inhibit the replication of herpes simplex virus (HSV), western equine encephalitis virus, poliovirus, measles virus, mumps virus (Sorimachi et al., 1990; Sarkar et al., 1993), and human immunodeficiency virus (Tochikura et al., 1988; Suzuki et al., 1990). Medicinal mushrooms have gained much attention as antiviral agents. A number of unique anti-viral agents from mushrooms have shown efficacy in inhibiting the replication of the human immunodeficiency virus (HIV) (Kim et al., 1994). Sulfated lentinan (Lentinula edodes) and sulfated schizophyllan (Schizophyllum commune) were reported to show strong anti-HIV activities although their anti-tumor activity is abolished (Ito et al., 1990; Hirata et al., 1994). The degraded form of

31

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

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

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

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

35

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

36

1987). The result of this disruption appears to be the loss of function of these two genes and an accompanying deregulation of the E6 and E7 genes, resulting in cellular transformation (Baker et al., 1987; Bedell et al., 1987). The site at which the viral DNA is interrupted in the process of integration is fairly constant, occurring within the E/E2 open reading frame of the viral genome. As the E2 region of the viral DNA normally represses the transcription of the E6 and E7 early viral genes, this interruption causes over-expression of the E6 and E7 proteins. The E7 protein binds to the under-phosphorylated form of the tumor suppressor protein pRb and displaces the E2F transcription factors that are normally bound by pRb. These transcription factors act at the G1/S interface of the cell cycle (Bosch et al., 1991; Munger et al., 1992; Wolf and Ramirez, 2001; Ferenczy and Franco, 2002). Table 2.1: Sizes and functions of HPV 18 proteins HPV proteins E1 E2 E4 E5 E6 E7 L1 L2 Number of amino acids 649 365 92 83 151 98 505 473 Transcription Functions Virus DNA replication and replication control Binds cytokeratins preceding virus assembly Affects growth factor receptors Binds p53 trans-activates, contributes to immortalization Main transforming protein, binds Rb1 and cyclin A Major virus coat protein (glycoprotein) Minor virus coat protein

2.4 2.4.1

Methods in the determination of biological acivities Antimicrobial activity The antimicrobial activity in this study was determined by the well diffusion

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

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

38

2.4.2

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

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

Brand-Williams et al. (1995) described a method involving use of the free radical (DPPH) where antioxidants are allowed to react with the stable radical in a methanol solution. The reduction in the DPPH is followed spectrophotometrically (Aljadi and Kamaruddin, 2004) by the monitoring the decrease in its absorbance at a characteristic wavelength during the reaction. In its radical form, DPPH absorbs at 515 nm, but upon reduction by an antioxidant (AH) or a radical species (R), the absorption disappears. DPPH possesses a proton free radical with a characteristic absorption, which decreases significantly on exposure to proton radical scavengers. DPPH + AH DPPH-H + A DPPH + R DPPH-R The absorbance at 515 nm is measured until the reaction reached a plateau. The exact initial DPPH concentration in the reaction medium is calculated from a calibration curve determined by linear regression. Antiradical activity was defined as the amount of antioxidant necessary to decrease the initial DPPH concentration by 50%. The larger the antiradical power, the more efficient the antioxidant action (Aruoma, 2003). The antioxidant activity was expressed as an IC50 value that was defined as the concentration of samples required to inhibit 50% of the formation of DPPH radicals (Dizhbite et al., 2004) and was obtained by interpolation from linear regression analysis (Mau et al., 2002). The lowest IC50 value indicates as an excellent antioxidant of radical scavenging. Total phenolic content is one of the major compounds contributing for antioxidant activity as the antioxidant activity is well-correlated with total phenolic content. This is supported by previous study that the highest total phenolic content in Ganoderma species is the key component accounting for their excellent results in antioxidant activity (Mau et al., 2002). Therefore, in this study, determination of total

40

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

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

2.4.3

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

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

42

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

43

although different cell types and cell lines exhibit differential sensitivities to test agents, the overall potency ranking of the chemicals was approximately equivalent. It would therefore appear that for the assessment of direct-acting cytotoxicants, the overall ranking of the test agents is independent of the particular cell type of cell line used as the target in the NR assay. 2.4.4 Anti-human papilloma virus activity There are several immunoenzymatic methods that can be used to localize antigens. The choice is based on the type of clinical specimen being analyzed, the degree of sensitivity and duration of processing time. The more commonly utilized method is immunohistochemistry (IHC) which involves the usage of the avidin-biotin peroxidase complex or the peroxidase complex or the peroxidase-labelled avidin technique. The enzymatic reactions can be visualized by using chromogenic substrates like 3-diamino benzine tetrahydrochloride (DAB) and 3-amino-9-ethocarbazole or different enzymes such as alkaline phosphatase (AP). IHC is one of the immunology studies concerned with the clinical enzymatic reactions between the antibodies and the antigens in the immune system. It is capable of identifying antigen in paraffin sections or exfoliated cells and has been applied extensively in HPV research (Jenson et al., 1980). The key reagent in all immunohistochemical techniques is the antibody (Boenisch, 1989). It involves the localization of antigens in tissue sections by the use of labeled antibodies as specific reagents through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold. Thus, it has apparent advantage over traditionally used special and enzyme staining techniques that identify only a limited number of proteins, enzymes and tissue structures. 44

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

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

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CHAPTER 3: MATERIALS AND METHODS 3.1 Preparation of Schizophyllum commune extracts Schizophyllum commune fruitbodies were purchased from a local market. They were then cut into small pieces and air-dried. The bioactive compounds present in the S. commune fruitbodies were extracted using methanol, ethyl acetate, dichloromethane and water. In methanol, ethyl acetate and dichloromethane extraction, the weighed fruitbodies were soaked with appropriate volume of solvents separately for two days at room temperature. The fruitbodies were then separated from the extraction solvents by filtration using Whatman No.1 filter paper. The residues were discarded while the filtrate obtained were transferred into a round bottom flask and dried to approximately 2 ml using a rotary evaporator. The crude extract was then transferred into a pre-weighed glass vial and rotary evaporated till complete dryness to yield the crude extract. The weight of all crude extracts obtained was measured and they were stored at -20C until analysis. Water extraction was done by boiling the weighed fruitbodies in distilled water for three hours using a heating mantle. The mixture was then filtered through Whatman No. 1 filter paper to obtain the filtrate. The filtrate was freeze-dried to obtain the crude extracts. The weight of the crude extract collected was measured and stored at 4C until required. The extracts were tested for various biological activities as shown in Figure 3.1.

47

EXTRACTION OF BIOACTIVE COMPOUNDS

Dichloromethane, Ethyl Acetate and Methanol Extracts

Water Extract

Soak mushroom with dichloromethane, ethyl acetate and methanol separately

Soak mushroom with sterile distilled water (sdH2O)

Filter

Heat for 3 hours

Evaporate to dryness

Freeze-dry

Crude Extracts

ANTIMICROBIAL ACTIVITY

ANTIOXIDANT ACTIVITY

CYTOTOXICITY TOWARDS CANCER CELL-LINES

ANTI-HUMAN PAPILLOMA VIRUS (HPV) ACTIVITY

Figure 3.1: Extraction of S. commune bioactive compounds and the biological activities analyzed

3.2

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

Gram-positive bacteria (Bacillus cereus, B. subtilis, Enterobacter faecalis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Plesiomonas

48

shigelloides, Pseudomonas aeuroginosa, Proteus vulgaris, Salmonella sp., S. typhi, Shigella sp., S. flexneri, Streptococcus mitis, S. mutans and S. sanguis). The bacterial and fungal stock cultures were prepared and maintained on various media. Mueller Hinton (MH) broth (Appendix A) was used to support the growth of Gram positive and negative bacteria while Brain Heart Infusion (BHI) broth (Appendix A) was used for Gram negative oral bacteria (S. mitis, S. mutans and S. sanguis). Saboraud Dextrose (SD) and Yeast-Peptone (YP) broth (Appendix A) were used for C. albicans and C. parapsilosis and S. pombe respectively. Bacterial and fungal inoculum were prepared by transferring 100 l of the stock suspension into 3 ml broth and incubated for 48 hours at 37C. Commercially available antibiotic discs namely streptomycin, chloramphenicol and kanamycin were used as positive controls. Antifungal activity of S. commune extracts was tested on Candida albicans, C. parapsilosis and Saccharomyces pombe, and the positive control used was nystatin; a common antibiotic used to treat fungal infections. Dimethyl sulfoxide (DMSO) served as a negative control in this study. The controls and the S. commune crude extracts were dissolved in a sterile DMSO to obtain the concentrations of 0.2 mg/ml and 2.0 mg/ml to test against the organisms. 3.2.1 Well diffusion assay After two days of incubation, the two-day old bacterial and fungal suspension was evenly streaked on the respective agar media. Three 7 mm diameter wells were cut in the agar using a sterile cork borer to represent a triplicate reading for each test organisms. The test organisms were prepared by placing 50 l of 0.2 mg/ml extracts to give 10 g in each well. The Petri dishes were further incubated at 37C and activities of the extracts were estimated by measuring the diameter of inhibition zones after 24 hours of incubation (See Figure 3.2). The same method was applied to test 2.0 mg/ml of extracts which was equivalent to 100 g in each well. The same concentrations were 49

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

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

ANTIMICROBIAL ASSAY

ANTIBACTERIAL ASSAY

ANTIFUNGAL ASSAY

2-day-old test organisms Lawn bacteria on Mueller Hinton agar Lawn fungi/yeast on Saboraud Dextrose agar and Yeast-Peptone agar respectively

Cut three 7mm diameter well and fill 50 l of fungal extracts in each well

Incubate at 37C for 24 hours Measure diameter of inhibition zones

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

50

3.3 3.3.1

Determination of antioxidant activity 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay The antioxidant activity of S. commune was assessed by the scavenging effect

on 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical assay (Molyneux, 2004). Various concentrations of the S. commune extracts in methanol were prepared to give a final volume of 0.1 ml and were mixed vigorously with 3.9 ml of methanolic solution containing DPPH radicals resulting in a final concentration of 0.06 mM. After 30 minutes incubation period in the dark, the absorbance was read against a blank (methanol) at 515 nm (Appendix B). The decrease in absorbance was monitored until a constant reading was obtained. All tests were carried out in triplicate. The scavenging activity on DPPH radicals was expressed as the percentage inhibition compared to the 0.06 mM DPPH in methanol which served as negative control. Inhibition of free radical DPPH in percent was calculated using the following way: Percentage of inhibition = OD control OD sample OD control

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

51

3.3.2

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

Ciocalteau method (Singleton and Rossi, 1965) with some modifications, involving Folin-Ciocalteau reagent and gallic acid as a standard. All the mushroom extracts were diluted in distilled water and the concentrations applied in the method were documented in Appendix B. Folin-Ciocalteau reagent (250 l) (Appendix A) was added to the aliquot (250 l) of extract solution and was mixed thoroughly. After 3 minutes, a 500 l solution of natrium carbonate (Na2CO3) (Appendix A) was added and the mixture was allowed to stand for 1 hour in the dark. The absorbance of the resulting solution was measured at 750 nm with a spectrophotometer against a blank (distilled water). All tests and analyses were run in triplicates and were used to calculate the standard calibration curve. The absorbance values obtained were converted to total phenolics from the gallic acid standard curve. Content of total phenols in samples was calculated on the basis of the calibration curve of gallic acid using various concentrations of gallic acid (0, 2, 4, 6, 8, 10 g/ml) in distilled water (Appendix B). The total phenolic content of the samples was expressed as gallic acid equivalents (GAEs), which reflected the phenolic content as the amount of gallic acid (mg) per gram of mushroom fruitbodies. 3.4 3.4.1 Cytotoxicity towards various cancer cell lines Cancer cell lines The cell lines used for the cytotoxicity tests were the cervical cancer cell lines; CaSki, epidermoid cancer cell lines; KB, colon cancer cell lines; HT29 and intestinal colon cancer cell lines; HCT116.

52

3.4.2

Serial dilution of S. commune extracts Ten microliter of the mushroom stock concentration of 20,000 g/ml was

diluted in 90 l 10 % DMSO to produce a stock of concentration of 10 g/ml. This stock was then further diluted into final concentrations of 100 g/ml, 50 g/ml, 10 g/ml and 1 g/ml (Figure 3.3). All diluted stocks were kept at 20C until required.

Stock (20,000 g/ml)

10 l + 90 l 10% DMSO

10 l + 90 l 10% DMSO

10 l + 90 l 10% DMSO

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

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

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

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

Figure 3.3: Serial dilution of S. commune extracts for cytotoxicity 3.4.3 Sub-cultivation of cancer cells The confluent cells in tissue culture flask was discarded and the cells were washed with 5 ml of sterile PBS (pH 7.2) (Appendix A). The cells were detached from the flask by adding 3 ml of PBS pH 7.2 and 1.0 ml (0.25%) of trypsin (Appendix A). The cells were then incubated for five to ten minutes at 5% CO2 incubator at 37C and followed by observation under an inverted microscope. The flask was given a slight tap to detach cells and the cell suspension was centrifuged at 1000 rpm for five minutes. Following centrifugation, the pellet of cells was resuspended in 1 ml 10% supplemented RPMI 1640 media (Appendix A) to produce a stock of cell suspensions.

53

3.4.4

Cell enumeration and plating One hundred microliter of cell suspensions and 900 l of tryphan blue

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

V2 = 30,000 x V1 (12 ml) x 1000 l 100,000 x number of cells per square box

The volume of media needed for 12 ml cell suspension (V1) to make 30,000 cells/ml was calculated using this formula: P1 = The original number of cells counted P1V1 = P2V2 V1 = Original volume (10 x 10 x 12) P2 = The number of cells wanted V2 = The volume of media needed

54

Figure 3.4: Cell counting using a haemocytometer

The appropriate number of cells was then added to fresh culture media to give a final concentration of 30,000 cells/ml. 3.4.5 Treatment of cancer cells with S. commune extracts Cell suspensions with known cell density were then plated in 96-well microtiter plate (Nunc) and incubated overnight in a 5% CO2 incubator at 37C to allow the cells to adhere and achieve 60-70% confluent. The cells were added to the wells immediately after the cell dispersion with fresh different serial diluted of extracts ranging from 100 g/ml, 50 g/ml, 10 g/ml and 1 g/ml. One well was not treated with any extract served the negative control. The treated cell plate was then incubated at 5% CO2 incubator at 37C for 72 hours. The same procedure was applied to each of the fungal extracts and the treatment was performed in triplicate.

55

3.4.6

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

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

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

3.5

Anti-human papillomavirus (HPV) activity towards cervical cancer (CaSki) cell lines

3.5.1

Serial dilution of S. commune extracts One hundred and eighty microliter from the mushroom stock extracts (20

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

56

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

Stock (20 mg/ml) 0.18 ml + 2.82 ml dH2O 1.5 ml + 1.5 ml dH2O (200 g/ml) 1.5 ml + 1.5 ml dH2O (100 g/ml) 1.5 ml + 1.5 ml dH2O (50 g/ml) 1.5 ml + 1.5 ml dH2O (25 g/ml) Figure 3.5: Serial dilution of S. commune extracts for anti-HPV activity 100l + 900 l dH2O (10 g/ml) 10 l + 990 l dH2O (1 g/ml)

3.5.2

Treatment of CaSki cells with S. commune extracts One ml of the serial diluted stock extracts was added to 2 ml of cultured cells in

flask to which five different concentrations; 200 g/ml, 100 g/ml, 50 g/ml, 25 g/ml, 10g/ml and 1 g/ml were applied. The cells were incubated with S. commune extracts at 37C for 72 hours. 3.5.3 Fixation of CaSki cells onto slides Cultured cells that were incubated with fungal extracts were transferred into a tissue culture tube and centrifuged at 1,000 rpm for 5 minutes. The supernatant was 57

decanted, the cells were washed twice in PBS 7.2 (Appendix A). Cells were then resuspended in fresh PBS 7.2. Using a micropipette, 30 l of cell suspension was carefully placed onto the wells of Teflon-coated glass slides. The slides were then left to dry at room temperature overnight. The cells were subsequently fixed using acetone for 10 minutes. The fixed slides were stored at 20C until use in immunohistochemistry. 3.5.4 Immunohistochemistry assay The technique of immunohistochemical staining were carried out using the Labeled Streptavidin Biotin (LSAB) Peroxidase Kit and the AEC Substrate System (DAKO) according to the specifications described by the manufacturer with some modifications. All washing steps required constant shaking and incubations with reagents were carried out in a humidified chamber. The cells were rehydrated in decreasing concentrations of ethanol; 100%, 95%, 90% and 80% (Appendix A) for 2 minutes each and then washed in PBS 7.6 (Appendix A) for five minutes on a shaker (LT Biomax 500). Three percent hydrogen peroxide (Appendix A) was added to each well and left to incubate for 10 minutes at 37C. This step is crucial to remove the endogenous peroxidase activity. After that, slides were rinsed in PBS for 5 minutes. The areas surrounding each well were blotted dry before treating selected well with 30 l of anti-HPV18 E6 monoclonal antibody which acts as a primary antibody. Slides were then incubated for 1 hour at room temperature. Following this, the slides were washed twice in PBS for 15 minutes each, and the area surrounding each well were blotted dry before incubation with 30 l streptavidin-HRP conjugate for 10 minutes at 37C. Next, the slides were washed in PBS in another 5 minutes and blotted dry. 30 l of 3- diaminobenzidine tetrahydrochloride (DAB) were then added and incubated at 37C. Incubation was stopped when the desired color intensity developed (approximately 5 - 20 minutes).

58

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

Store in the dark

Observe under a microscocope PBS 7.6 Figure 3.6: A brief procedure of immunohistochemistry method

59

CHAPTER 4.0: RESULTS 4.1 Analysis of antimicrobial activity of S. commune extracts In this study, the in vitro antimicrobial activity of S. commune extracts (methanol, ethyl acetate, dichloromethane and water) and the commercial antibiotics (streptomycin, kanamycin, chloramphenicol and nystatin) against microorganisms tested was qualitatively assessed by the diameter of inhibition zone using well diffusion assay. The antimicrobial activity was examined against seven species of Gram-positive bacteria (Bacillus cereus, B. subtilis, Enterobacter faecalis, Staphylococcus aureus, Streptococcus mitis, S. mutans and S. sanguis), eight species of Gram-negative bacteria (Escherichia coli, Salmonella sp., S. typhi, Shigella sp., Shigella flexneri, Plesiomonas shigelloides, Proteus vulgaris, and Pseudomonas aeuroginosa) and three species of fungi (Candida albicans, C. parapsilosis and Saccharomyces pombe). The antimicrobial activity of both S. commune extracts and commercial antibiotic discs was determined at 0.2 mg/ml and 2 mg/ml (Appendix C). The inhibition zone was measured in millimeter (mm) after 24 hours of incubation time. The diameter of well was 7 mm and the data expressed were evaluated based on the comparison among the extracts against the microorganisms. Zone of inhibition was categorized as: Inactive (IA) inhibition zones <9 mm diameter Partially active (PA) - inhibition zones 9-12 mm diameter Active (A) - inhibition zones 13-18 mm diameter Very active (VA) - inhibition zones >18 mm diameter

60

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

61

Table 4.1: Antimicrobial activity of S. commune extracts at 0.2 mg/ml as evaluated by well diffusion assay (24 hours incubation at 37C). Inhibition zones were measured (in mm) and the diameter of inhibition zones reported Microorganisms Bacillus cereus Bacillus subtilis Enterobacter faecalis Staphylococcus aureus Streptococcus mitis Streptococcus mutans Streptococcus sanguis Escherichia coli Salmonella sp. Salmonella typhi Shigella sp. Shigella flexneri Plesiomonas shigelloides Proteus vulgaris Pseudomonas aeuroginosa Candida albicans Candida parapsilosis Saccharomyces pombe Methanol 91 a PA 90 a PA 91 a PA 100 a PA NA NA 101 a PA 90 a PA NA 81 a IA 90 a PA 81 a IA 81 a PA 90 a PA NA NA NA NA S. commune extracts Ethyl Dichloromethane acetate 91 b 101 b PA PA b 81 91 b IA PA b 100 91 b PA PA b 91 100 b PA PA 90 b 80 b PA IwA NA NA 110 b 111 b PA PA 100 b 90 b PA PA 100 b 91 b PA PA 91 b 91 b PA PA 100 b 91 b PA PA 90 b 91 b PA PA 91 b 101 b PA PA 91 b 110 b PA PA NA NA 91 PA NA 91 80 PA IA 81 IA NA Water NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Grampositive bacteria

Gramnegative bacteria

Fungi

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

62

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

63

Table 4.2: Antimicrobial activity of S. commune extracts at 2 mg/ml as evaluated by well diffusion assay (24 hours incubation at 37C). Inhibition zones were measured (in mm) and the diameter of inhibition zones reported Microorganisms Bacillus cereus Bacillus subtilis Enterobacter faecalis Staphylococcus aureus Streptococcus mitis Streptococcus mutans Streptococcus sanguis Escherichia coli Salmonella sp. Salmonella typhi Shigella sp. Shigella flexneri Plesiomonas shigelloides Proteus vulgaris Pseudomonas aeuroginosa Candida albicans Candida parapsilosis Saccharomyces pombe Methanol 91a PA 101a PA 101a PA 101a PA 91a PA NA 111a PA 91a PA 91a IA 91a PA 91a PA 91a PA 91a PA 91a PA 90a PA NA NA NA S. commune extracts Ethyl Dichloromethane acetate 81a 110b IA PA a 91 111b IA PA a 101 111b PA PA a 91 111b PA PA 91a 111b PA PA NA NA 111a 121b PA PA 101a 111b PA PA 101a 111b PA PA 100a 111b PA PA 101a 111b PA PA 91a 111b PA PA 111a 111b PA PA 101a 111b PA PA 91a 101b PA PA 101 100 PA PA 100 101 PA PA 91 81 IA IA Water NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Grampositive bacteria

Gramnegative bacteria

Fungi

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

64

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

(a): The inhibition of S. sanguis by 2 mg/ml dichloromethane extract of S. commune.

(b): S. mutans was not inhibited by 2 mg/ml dichloromethane extract of S. commune.

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

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

Table 4.3: Antibacterial activity of antibiotic discs as evaluated by well diffusion assay (24 hours incubation at 37C). Inhibition zones were measured (in mm) and the diameter of inhibition zones reported Streptomycin 0.2 2 mg/ml mg/ml 251 311 VA VA 181 251 A VA 261 321 VA VA 180 251 A VA 200 270 VA VA 200 NA VA 91 211 PA VA 131 261 A VA 292 381 VA VA 291 381 VA VA 301 371 VA VA 291 381 VA VA 321 391 VA VA 151 251 A VA 151 210 A VA Kanamycin 0.2 2 mg/ml mg/ml 211 301 VA VA 301 351 VA VA 201 271 VA VA 301 351 VA VA 171 191 VA VA 210 NA VA 121 251 PA VA 200 271 VA VA 221 311 VA VA 201 281 VA VA 211 281 VA VA 201 281 VA VA 221 301 VA VA 291 341 VA VA 241 311 VA VA Chloramphenicol 0.2 2 mg/ml mg/ml 181 261 VA VA 251 401 VA VA 191 301 VA VA 251 401 VA VA 221 251 VA VA 131 NA A 81 181 IA VA 121 311 PA VA 221 341 VA VA 201 331 VA VA 191 311 VA VA 201 331 VA VA 241 321 VA VA 250 421 VA VA 211 341 VA VA

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

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

66

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

(a): The inhibition zone of P. vulgaris by 2 mg/ml antibiotic chloramphenicol.

(b): The inhibition zone of B. subtilis by 2 mg/ml antibiotic chloramphenicol.

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

Table 4.4: Antifungal activity of antibiotic nystatin as evaluated by well diffusion assay (24 hours incubation at 37C). Inhibition zones were measured (in mm) and the diameter of inhibition zones reported Nystatin Microorganisms Candida albicans Fungi Candida parapsilosis Saccharomyces pombe 0.2 mg/ml 231 VA NA 231 VA 2 mg/ml 281 VA 121 PA 291 VA

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

67

4.2

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

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

68

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

Table 4.5: The scavenging activity of S. commune extracts as measured by DPPH radical scavenging asssay Extract Methanol Ethyl acetate Dichloromethane Water *Ascorbic acid Scavenging Activity IC50 (mg/ml) 0.145 0.01 a 0.219 0.01 b 0.641 0.13 c 0.674 0.05 d 0.084 0.01

Data expressed as means standard deviations of triplicate measurements *Positive reference standard

120 100

Inhibition (%)

80 60 40 20 0 0 0.2 0.4 0.6 0.8

methanol ethyl acetate dichloromethane water i (di hl h

)
1.4

Concentrations (mg/ml)

1.2

Figure 4.1: The scavenging effect of S. commune extracts on DPPH radicals 69

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

70

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

1.720.05a 0.790.03b 0.450.04d 0.520.01c

0.4980.07a 0.0570.01c 0.0210.01d 0.0950.02b

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

4.2.1

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

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

71

60 50 DPPH inhibition (%) 40 30 20 10 0 0 1 2 3 4 Total phenolic content (ug/ml) 5 6 7 y = 7.3362x + 9.1791 2 R = 0.8264

Figure 4.2: Correlation between the radicalcavenging activity of S. commune extracts and their phenolic contents

4.3

Analysis of cytotoxic activity of S. commune towards cancer cell-lines In order to evaluate the cytotoxic effect of the extracts; methanol, ethyl acetate,

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

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

Table 4.7: The IC50 values of S. commune extracts against different cancer cell lines as measured by neutral red cytotoxicity assay Methanol Cell line KB CaSki HT29 HCT116 24.40 6.1 62.87 5.5 NA NA Ethyl acetate 81.78 9.9 NA NA 73.94 0.1 Dichloromethane 76.11 2.7 59.26 2.1 57.80 4.2 14.71 2.0 Water NA NA NA NA

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

As shown in Table 4.7 and Figure 4.3, dichloromethane extract of S. commune was found to show the active cytotoxic ( 20 g/ml) effect against intestinal colon cancer cell lines (HCT116) at the range of concentrations tested. The IC50 value of the extract was observed to be the greatest (14.71 2.0 g/ml) indicating that the extract was the most active in inhibiting the cancer cells (Table 4.7).

73

90 80 70

inhibition (%)

60 50 40 30 20 10 0 0 20 40 60 80 100 120

Concentrations (g/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 g/ml) inhibition was recorded, the highest cytotoxicity was of methanol extract resulting IC50 value of 24.40 6.1 g/ml against KB cancer cell lines (Table 4.7). The cytotoxic activity of S. commune extracts against CaSki cells showed that there were no good inhibitions of cells detected from ethyl acetate and water extracts of S. commune at the range of concentrations tested (Table 4.7). However, the cytotoxicity observed from the remaining of S. commune extracts (dicloromethane and methanol) indicated that the activity against CaSki was not active (> 20 g/ml). Particularly, dichloromethane showed the most potent cytotoxic activity against the cells with the IC50 value of 59.26 2.1 g/ml (Table 4.7).

74

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

4.4

Analysis of anti-human papilloma virus activity of S. commune extracts The immunohistochemistry (IHC) method using anti-human papillomavirus

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

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

(a): The negative control of CaSki cells; cells not treated with antibody

(b): The positive control of CaSki cells; cells treated with antibody

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

76

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

77

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

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

Majority showing intact

+++++

10

Majority showing intact

++++

25

Some intact Some lysis

+++

50

Majority showing lysis

++

100

Majority showing lysis

200

Majority showing lysis

78

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

Majority showing intact

+++++

10

Majority showing intact

++++

25

Majority showing intact

+++

50

Some intact Some lysis

+++

100

Some intact Some lysis

++

200

Some intact Some lysis

79

Plate 4.6: The appearance of CaSki cells after treatment with dichloromethane extract of S. commune at different concentrations Concentration of extract (g/ml) 1 Morphology of cells Intensity of reddish brown stain

Majority showing intact

+++++

10

Majority showing intact

++++

25

Some intact Some lysis

++++

50

Some lysis Some intact

+++

100

Majority showing lysis

++

200

Majority showing lysis

80

Plate 4.7: The appearance of CaSki cells after treatment with water extract of S. commune at different concentrations Concentration of extract (g/ml) 1 Morphology of cells Intensity of reddish brown stain

Majority showing intact

+++++

10

Majority showing intact

++++

25

Majority showing intact

+++

50

Majority showing intact

++

100

Some lysis Some intact

++

200

Majority showing lysis

81

CHAPTER 5: DISCUSSION 5.1 5.1.1 Antimicrobial activity of S. commune Fr. 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.

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5.1.2

Antimicrobial activity of S. commune extracts against various pathogenic microorganisms From this study, it was shown that at both concentrations (0.2 mg/ml and 2

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

83

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

5.1.3

Extraction solvent as the significant factor in evaluating antimicrobial activity of S. commune As mentioned earlier, the dichloromethane extract of S. commune was found to

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

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

85

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

5.1.4

Antimicrobial activity of S. commune extracts and its correlation to phenolic content It is interesting to note that the phenolic content of an extract can also be

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

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

87

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

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

88

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

5.1.5

Other contributing factors to antimicrobial activity of S. commune extracts It is also believed that the variation of antimicrobial activity of mushrooms

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

89

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

shigelloides, Proteus vulgaris, and Pseudomonas aeuroginosa). The S. commune extracts that were shown to be effective against Gram-positive bacteria were probably due to the inhibition of cell wall synthesis; the cells were unable to maintain rigid peptidoglycan component of the wall and therefore become susceptible to weakening and eventual toxic destruction to the cell wall or lysis.

90

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

5.2 5.2.1

Antioxidant activity of S. commune Fr. Antioxidant activity of S. commune extracts and its correlation phenolic content The free radical scavenging activities of mushroom extracts were tested using a to

methanolic solution of the stable free radical, DPPH. The DPPH radical system used in 91

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

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

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

93

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

5.2.2

Extraction solvent as the significant factor in evaluating antioxidant activity of S. commune Based on the present free radical scavenging activity results (Table 4.5), it was

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

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

95

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

96

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

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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 druginduced alterations in metabolic pathways or structural integrity which may or may not be directly related to cell death. Apart from that, the advantages of in vitro cytotoxicity 99

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

100

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

101

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

102

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

5.3.2

Cytotoxicity of S. commune extracts towards different cancer cell lines Results presented in the study (Table 4.7) showed the cytotoxicity of the

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

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

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

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

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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 g/ml.

5.3.4

Other contributing factors to cytotoxicity of S. commune extracts The different sensitivity of S. commune extracts towards the cancer cell lines is

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

109

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

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

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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 g/ml, 10 g/ml, 25 g/ml, 50 g/ml, 100 g/ml and 200 g/ml were analysed for the expression of HPV 18 E6 oncoprotein using immunohistochemistry. Particularly, the four different extracts of S. commune which were consisted of methanol, ethyl acetate, dichloromethane and water were evaluated for any potential in inhibiting the viral antigen E6 which is normally produced by high risk human papilloma virus (HPV) 16 and 18. This is

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

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

5.4.2

Anti-human papilloma virus (HPV) activity of S. commune extracts towards cervical cancer cell lines (CaSki) According to the results (Plate 4.4 4.7), the S. commune extracts (methanol,

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

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

5.4.3

Comparative study of various antiviral activities The antiviral activity of mushrooms has been described elsewhere. This agrees

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

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

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

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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).

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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.

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

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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.

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APPENDICES APPENDIX A: 1.0 REAGENTS, MEDIA AND BUFFER

DETERMINATION OF ANTIMICROBIAL ACTIVITY

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

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

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

The media was used to revive the oral bacteria. 14.8 gm of BHI broth (Merck) was suspended in 400 ml of purified water and mixed thoroughly. The mixture was then autoclaved at 121C for 20 minutes. After cooling down, it was refrigerated at 25C until required. 1.4 Yeast-peptone (YP) broth Yeast extract Peptone Glucose pH 4.00 g 8.00 g 8.00 g 6.8

The media was used to revive Saccharomyces pombe. All the ingredients stated above were dissolved in 400 ml of purified water and mixed completely. The pH was adjusted to 6.8 using hydrochloric acid (HCl) or sodium hydroxide (NaOH). The mixture was then autoclaved at 121C for 15 minutes. After cooling down, it was refrigerated at 25C until required. 1.5 Yeast-peptone agar (YPA) Yeast extract 4.00 g 151

Peptone Glucose Agar PH

8.00 g 8.00 g 7.00 g 6.8

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

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

The media was used to cultivate Candida albicans and Candida parapsilosis. 26.0 gm of Saboraud Dextrose agar (Merck) and 0.8 g of Bacto agar (Difco) were added to 400 ml of purified water and mixed completely. The mixture was then autoclaved at 121C for 15 minutes for sterilization. After the heat was tolerable to handle by hand, the media was poured carefully to two-third of disposable sterile petri dishes and left to solidify before it was stored at 25C. 2.0 2.1 DETERMINATION OF ANTIOXIDANT ACTIVITY Folin-Ciocalteau reagent

The reagent should be freshly prepared to avoid oxidation. 0.5 ml of FolinCiocalteau reagent was dissolved in 4.5 ml distilled water and mixed using a vortex. The reagent bottle was then wrapped in aluminium foil as it is light sensitive. 2.2 10% natrium carbonate (Na2CO3)

One gram of natrium carbonate (Na2CO3) was dissolved in 10 ml distilled water and mixed well with vortex. 3.0 3.1 CYTOTOXICITY AGAINST CANCER CELL-LINES Phosphate buffered saline (PBS) 7.2

The PBS pH 7.2 consisted of 1.52 g of disodium hydrogen orthophosphate anhydrous (Na2HPO4), 0.58 g potassium dihydrogen orthophosphate (KH2PO4) and 8.5 g sodium chloride (NaCl), which were dissolved in 1 liter of distilled water. The 152

solution was adjusted to a pH of 7.2 and it was filtered using a 0.2 m filter membrane into a bottle. 3.2 Basic RPMI 1640 media

A bottle of RPMI powder, 2 g of natrium hydrogen carbonate (NaHCO3), and 0.5206 g of 2 mM/l HEPES were dissolved in 1 liter of distilled water and the pH was adjusted to 7.4 before further filtration using 0.2 m filter membrane into a bottle. The bottle was then kept in 4C. 3.3 10% RPMI 1640 media

10 ml of Foetal Bovine Serum (FBS), 2 ml of penicillin/streptomycin and 1 ml of amphostat B were added to 90 ml basic 1640 RPMI media and mixed thoroughly. The mixture was filtered using 0.2 m filter membrane into a bottle and was then kept in 4C until required. 3.4 4C. 3.5 Resorb solution Tryphan blue 0.2 g of tryphan blue was mixed with 50 ml distilled water and then stored at

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

2 gm of calcium chloride (CaCl2) was dissolved in 200 ml of distilled water. 2 ml of formaldehyde was added in the solution. 3.7 Neutral red stock solution

0.04 g Neutral red was dissolved in 10 ml distilled water using a vortex. The mixture was then centrifuged at 1000 rpm for 5 minutes. The supernatant obtained was mixed with 12 ml of 10% RPMI 1640 media and stored at 4C for further use. 4.0 4.1 ANTI-HUMAN PAPILLOMA VIRUS ACTIVITY Phosphate buffered saline (PBS) 7.2 The instruction on how to prepare this solution was similar to 2 (a). 4.2 Phosphate buffered saline (PBS ) 7.6

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

153

dissolved in 1 liter of distilled water. The solution was adjusted to a pH of 7.6 and it then was filtered using a 0.2 m filter membrane into a bottle. 4.3 95%, 85% and 80% ethanol

For the preparation of the diluted 95% ethanol, 950 ml of ethanol was mixed well 50 ml distilled water, whereas 850 ml and 800 ml of ethanol were added with 50 ml and 100ml of distilled water respectively. 4.4 3% hydrogen peroxide

300 l of hydrogen peroxide was mixed with 9.7 ml distilled water to make the total volume of 10 ml. 4.5 water. HPV 18 E6 antibody The HPV 18 E6 antibody (Chemicon) was diluted in 1:50 with sterile distilled

154

APPENDIX B: 1.0 1.1

ANALYTICAL TECHNIQUES

DETERMINATION OF ANTIOXIDANT ACTIVITY DPPH radical scavenging assay

The radical scavenging activity assay was employed according to Molyneux (2004) with some modifications. Various concentrations of the S. commune extracts in methanol (Table 1.1-1.4) were prepared to give a final volume of 0.1 ml and were mixed vigorously with 3.9 ml of methanolic solution containing DPPH radicals resulting in a final concentration of 0.06 mM. After 30 minutes incubation period in the dark, the absorbance was read against a blank (methanol) at 515 nm. The same procedure was applied to the positive control, ascorbic acid (Table 1.5). Table 1.1: Preparation of ethyl acetate extracts of S. commune with stock concentration of 30 mg/ml Concentrations of extracts (mg/ml) 1 5 10 15 20 Volume of methanol (l) 95 75 50 25 Volume of extracts (l) 5 25 50 75 100 Volume of DPPH solution (ml) 3.9 3.9 3.9 3.9 3.9

Table 1.2: Preparation of methanol extracts of S. commune with stock concentration of 30 mg/ml Concentrations of extracts (mg/ml) 1 2 5 10 15 Volume of methanol (l) 95 90 75 50 25 Volume of extracts (l) 5 10 25 50 75 Volume of DPPH solution (l) 3.9 3.9 3.9 3.9 3.9

Table 1.3: Preparation of dichloromethane extracts of S. commune with stock concentration of 50 mg/ml Concentrations of extracts (mg/ml) 1 5 20 30 50 Volume of methanol (l) 98 90 60 40 Volume of extracts (l) 2 10 40 60 100 Volume of DPPH solution (l) 3.9 3.9 3.9 3.9 3.9

155

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

Table 1.5: Preparation of ascorbic acid samples as positive control for DPPH assay Concentrations of ascorbic acid mM 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.2 mg/ml 0.0176 0.0352 0.0528 0.0704 0.0880 0.1057 0.1233 0.1409 0.1585 0.1761 Volume of ascorbic acid (l) 10 20 30 40 50 60 70 80 90 100 Volume of methanol (l) 90 80 70 60 50 40 30 20 10 Volume of DPPH solution (ml) 3.9 3.9 3.9 3.9 3.9 3.9 3.9 3.9 3.9 3.9

Folin-Ciocalteau assay

The total phenolic content was determined by the Folin-Ciocalteau assay (Singleton and Rossi, 1965) with some modifications, involving Folin-Ciocalteau reagent and gallic acid as a standard. The concentrations of S. commune extracts were prepared as stated in Table 1.6. Folin-Ciocalteau reagent (250 l) was added to the aliquot (250 l) of mushroom extract solution and was mixed thoroughly. After 3 minutes, a 500 l solution of natrium carbonate (Na2CO3) was added and the mixture was allowed to stand for 1 hour in the dark. The absorbance of the resulting solution was measured at 750 nm with a spectrophotometer against a blank (distilled water). The same procedure was applied to the positive reference standard, gallic acid (Table 1.7). Table 1.6: Preparation of S. commune extracts for Folin-Ciocalteau assay Fungal Extracts Ethyl acetate Methanol Dichloromethane Water Concentrations (mg/ml) 2 2 2 2 Volume of extracts Volume of distilled (l) water (l) 250 250 250 250 156

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

157

APPENDIX C: 1.0

EXPERIMENTAL AND STATISTICAL DATA

DETERMINATION OF ANTIMICROBIAL ACTIVITY

1.1 The experimental data of the antimicrobial activity of S. commune extracts against microorganisms tested at two different concentrations Microorganisms Bacillus cereus Methanol 0.2 2 mg/ml mg/ml 9 10 10 11 10 11 91 101 9 10 9 9 9 10 90 91 10 11 9 10 10 11 91 101 10 11 10 10 10 10 100 101 10 NA 9 9 91 NA NA 11 10 11 101 9 9 9 90 NA Salmonella Gramtyphi negative bacteria Shigella sp. 9 8 9 81 9 9 12 11 12 111 10 9 10 91 10 9 9 91 10 9 10 91 10 9 Ethyl acetate 0.2 2 mg/ml mg/ml 9 10 10 10 10 9 91 91 8 11 9 10 9 10 81 101 10 11 10 11 10 10 100 101 9 10 10 10 9 9 91 91 9 10 9 10 9 9 90 91 NA NA 11 11 11 110 10 10 10 100 10 10 10 100 10 9 9 91 10 10 11 12 11 111 11 10 10 101 11 10 10 101 10 10 10 100 11 10 Dichloromethane 0.2 2 mg/ml mg/ml 11 12 11 11 10 12 101 111 10 11 9 12 10 11 91 111 10 12 9 11 10 12 91 111 10 12 10 11 10 11 100 111 8 12 8 11 8 11 80 111 NA NA 12 11 11 111 9 9 9 90 10 9 9 91 10 9 10 91 10 11 13 13 12 121 12 12 11 111 12 11 11 111 12 11 12 111 11 12 158

Bacillus subtilis Enterobacter faecalis

Grampositive bacteria Staphylococcus aureus Streptococcus mitis Streptococcus mutans Streptococcus sanguis Escherichia coli Salmonella sp.

Shigella flexneri Plesiomonas shigelloides Proteus vulgaris

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

Pseudomonas aeuroginosa Candida albicans Candida parapsilosis Saccharomyces pombe

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

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

NA

Fungi

NA

NA

NA

NA

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

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

NA 8 8 8 80 NA

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

Data expressed as means standard deviations of triplicate measurements

159

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

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

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

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

160

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

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

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

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

161

1.4 The experimental data of the antimicrobial activity of positive controls against bacteria tested at two different concentrations Streptomycin Microorganisms 0.2 2 mg/ml mg/ml 25 30 Bacillus cereus 25 31 26 31 251 311 18 25 Bacillus 19 26 subtilis 19 25 181 251 26 33 Enterobacter 25 33 faecalis 25 32 Gram261 321 positive Staphylococcus 18 25 bacteria aureus 18 26 18 25 180 251 20 27 Streptococcus 20 27 mitis 20 27 200 270 20 Streptococcus NA 20 mutans 20 200 10 22 Streptococcus 9 22 sanguis 9 21 91 211 13 26 Escherichia 14 27 coli 14 26 131 261 Salmonella sp. 29 39 31 38 31 38 292 381 29 39 Salmonella 30 38 typhi Gram29 38 negative 291 381 bacteria 30 37 Shigella sp. 31 38 30 38 301 371 Kanamycin 0.2 2 mg/ml mg/ml 21 30 20 30 21 31 211 301 30 34 31 34 30 35 301 351 21 27 20 28 20 28 201 271 30 35 31 36 31 35 301 351 18 20 17 19 18 19 171 191 21 NA 21 21 210 12 25 13 26 12 26 121 251 20 27 20 28 20 27 200 271 22 32 23 32 22 31 221 311 21 29 20 28 20 29 201 281 21 28 22 29 22 28 211 281 Chloramphenicol 0.2 2 mg/ml mg/ml 18 25 18 26 19 25 181 261 24 40 25 41 25 40 251 401 19 31 19 30 18 31 191 301 25 41 26 40 25 40 251 401 22 25 23 26 23 25 221 251 14 NA 13 14 131 8 19 9 18 9 19 81 181 12 31 13 32 13 31 121 311 23 35 22 35 22 34 221 341 21 34 20 34 20 33 201 331 19 31 19 32 20 31 191 311 162

Shigella flexneri Plesiomonas shigelloides Proteus vulgaris Pseudomonas aeuroginosa

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

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

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

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

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

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

Data expressed as means standard deviations of triplicate measurements

1.5 The experimental data of the antimicrobial activity of positive controls against fungi tested at two different concentrations Nystatin Microorganisms Candida albicans 0.2 mg/ml 24 23 23 231 NA Saccharomyces pombe 23 22 22 231 2 mg/ml 29 28 29 281 12 13 12 121 30 29 29 291

Candida parapsilosis Fungi

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

(mg/ml) 1 2 5 10 15 1 5 10 15 20 1 5 20 30 50 5 10 20 30 50 R1 11.40 17.10 47.50 92.02 89.98 13.67 34.53 63.40 91.02 90.88 7.40 25.09 31.05 67.39 75.03 16.06 30.87 36.64 46.57 70.04

Methanol

Ethyl acetate

Dichloromethane

Aqueous

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

Average (%) R3 16.33 16.33 48.05 90.49 91.00 10.77 30.80 58.43 89.78 90.19 9.39 26.17 27.46 66.93 67.08 23.65 26.71 32.67 52.17 72.74 14.562.74 16.840.44 51.205.94 90.491.53 90.380.55 12.571.57 33.752.64 58.844.34 89.781.24 90.510.35 9.271.81 26.471.56 29.641.92 68.281.95 71.604.09 20.103.82 29.242.22 36.944.43 49.642.84 72.262.03

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

2.2 The scavenging activity of S. commune extracts as measured by DPPH radical scavenging asssay Extract Methanol Ethyl acetate Dichloromethane Water *Ascorbic acid Scavenging Activity IC50 (mg/ml) 0.145 0.01 0.219 0.01 0.641 0.13 0.674 0.05 0.084 0.01

Data expressed as means standard deviations of triplicate measurements *Positive reference standard

166

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

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

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

167

2.4 The experimental data of DPPH radical scavenging activity of ascorbic acid against DPPH radicals Concentration of ascorbic acid (g/ml) 0.440 0.881 1.321 1.761 2.201 2.642 3.082 3.522 3.962 4.403 R1 10.84 31.53 35.78 45.08 48.93 53.88 60.48 64.75 65.33 67.48 Inhibition (%) R2 11.40 31.81 37.23 45.93 47.85 55.15 59.91 64.62 64.91 68.99 Average (%) R3 12.13 34.09 38.63 44.51 48.25 54.56 60.05 58.61 66.48 67.08 11.460.65 32.481.40 37.211.43 45.170.71 48.340.55 54.530.64 60.150.29 64.690.09 65.570.81 67.852.26

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

2.5 Calibration plot of the scavenging effect of ascorbic acid as on DPPH radicals
80 70 60 In h ib itio n (% ) 50 40 30 20 10 0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 Concentrations (mg/ml) y = 232.74x + 30.749 2 R = 0.978

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

R1 0.078 0.166 0.044 0.052

Absorbance R2 0.076 0.175 0.046 0.055

Average R3 0.081 0.171 0.044 0.048 0.078 0.054 0.045 0.052

168

2.7

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

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

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

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

169

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

R1 0.017 0.057 0.129 0.202 0.235 0.263

Absorbance R2 0.014 0.067 0.135 0.186 0.240 0.263

Average R3 0.017 0.034 0.130 0.188 0.249 0.261 0.016 0.054 0.131 0.192 0.241 0.262

2.9

The calibration plot of gallic acid as positive reference standard

0.35 0.3

Absorbance

0.25 0.2 y = 0.0288x 2 R = 0.9666

0.15 0.1

0.05 0 0 2

Concentrations (ug/ml)

10

12

3.0 The radical scavenging activity and total phenolic content of S. commune extracts Extract Methanol Ethyl acetate Dichloromethane Water
Scavenging Activity IC50 (mg/ml) Total Phenolic Content (mg GA/g extracts)

0.145 0.01 0.219 0.01 0.641 0.13 0.674 0.05

1.72 0.05 0.79 0.03 0.52 0.04 0.45 0.01

*Data expressed as means standard deviations of triplicate measurements

170

3.0

CYTOTOXICITY TOWARDS CANCER CELL LINES

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

Percentage of inhibition (%) Methanol 141.69 26.16.51 54.654.31 75.375.71 16.20.71 35.054.74 47.10.71 51.355.87 0.750.35 12.80.14 21.252.33 33.25.03 5.30.28 9.20.14 16.51.56 27.650.49 Ethyl acetate 21.08 13.80.85 20.950.78 57.71.69 10.750.21 22.63.96 36.23.25 40.650.64 22.60.42 30.454.6 37.652.47 42.82.26 2.31.13 11.752.89 28.850.64 50.41.87
Dichloromethane

Cell line KB

of extracts (g/ml) 1 10 50 100 1

Aqueous 10.130.55 21.30.46 31.940.8 48.140.43 1.550.21 23.555.44 34.533.5 40.42.43 7.50.0 12.650.92 15.21.61 31.850.21 12.12.12 16.152.05 19.852.76 20.552.19

5.431.5 22.631.2 42.30.71 63.54.38 4.150.64 21.251.2 48.875.18 80.134.24 3.61.27 14.056.15 47.82.12 62.250.92 8.31.69 33.950.35 76.51.66 87.051.06

CaSki

10 50 100

HT29

1 10 50 100

HCT116

1 10 50 100

*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.

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