British Columbian Wild Mushrooms As Potent Source of Novel Natural Anti-Cancer Compounds

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by British Columbia's network of post-secondary digital repositories

BRITISH COLUMBIAN WILD MUSHROOMS AS POTENT SOURCE OF NOVEL NATURAL ANTI-CANCER COMPOUNDS

Ankush A. Barad B. Pharma, Sant Gadge Baba Amravati University, 2011.

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOCHEMISTRY

UNIVERSITY OF NORTHERN BRITISH COLUMBIA April 2017

Abstract

Worldwide, many fungal species have been used for their medicinal properties by various cultures for centuries. Major challenges with cancer chemotherapy, which includes toxicity, non-specificity and several adverse effects have motivated researchers to search for novel anti-cancer compounds from natural sources. Several studies have identified various anti-cancer compounds from mushrooms in many parts of the world, but mushrooms occurring in British Columbia remain unstudied for their anti-cancer activities. In this thesis, five mushroom species were collected, extracted, and assessed for anti-proliferative and immuno-stimulatory activities. Eleven extracts from these species showed anti-proliferative or immuno-stimulatory activity or both.

Sodium hydroxide extract from Paxillus obscurisporus exhibited potent anti-proliferative activity and was further purified using size-exclusion and anion-exchange chromatography. Analysis of the relatively pure anti-proliferative fractions suggests the presence of protein and glycoprotein moieties. Further purification and characterisation is needed before these compounds can be tested for biomedical purposes in the future.

Table of Contents

Abstract………………………………………………………………………………………….(i)

Table of Contents……………………………………………………………………………...(ii)

List of Tables…………………………………………………………………………………..(vi)

List of Figures…………………………………………………………………………………(vii)

Acknowledgements…………………………………………………………………………....(x)

References...…………………………………………………………………………………..(xi)

Appendix.…………………………………………………………………………………....(xxiv)

Chapter 1 – Introduction and objectives

1.1 Natural products as source of medicinal compounds……………………………….1

1.2 Medicinal Mushrooms…………………………………………………………………..3

1.3 Medicinal Compounds from Mushrooms and their Biological Activity……………..6 1.3.1. Active Anti-Tumor Compounds…………………………………………………6 1.3.2. Active Anti-Microbial Compounds……………………………………………...7 1.3.3. Bio-active Hypoglycemic Compounds………………………………..…….....8

1.4 Medicinal Compounds from Mushrooms in Clinical Use……………………………9

1.5 British Columbia Wild Mushrooms…………………………………………………...11

1.6 Research Goals………………………………………………………………………..13

iii

Chapter 2 – Collection, identification, extraction and assessment of anti- proliferative and immuno-stimulatory activities of five species of British Columbia wild mushrooms

2.1 Collection and identification of BC wild mushrooms……………………………….15 2.1.1 Collection of BC wild mushrooms...... 15 2.1.2 Macro-morphological identification……..……………………………………16 2.1.3 Identification of mushroom samples using DNA analysis…………………17

2.2 Methodology – Chemical extraction of BC wild mushrooms……………………...20 2.2.1 Manual extraction scheme as followed as per Mizuno et al. (1999)..……20 2.2.2 High-pressure extraction using BÜCHI SpeedExtractor® E916………….21

2.3 Methodology – Assessment of anti-proliferative and immuno-stimulatory activities…………………………………………………………………………………23 2.3.1 Assessment of dose and time-dependent anti-proliferative activity..…….23 2.3.2 Assessment of immuno-stimulation by measuring TNF-alpha production from macrophage cells………………………………………………………..25

2.4 Results and Discussions……………………………………………………………...27 2.4.1 Macro and Micro-molecular identification of collected mushrooms………27 2.4.2 Chemical extraction of the BC wild mushrooms……………………………32 2.4.3. Dose and time-dependent anti-proliferative activity of mushroom fractions on HeLa cell…………………………………………………………………….33 2.4.4 Effect of TNF-alpha production in Raw 264.7 macrophage cells upon treatment with mushroom fractions………………………………………….38 2.4.5 A summary of biological activity exhibited by mushroom in this study and their previously reported bio-activities……………………………………….41

Chapter 3 - Purification and chemical characterization of an alkaline extract (fraction 4) from Paxillus obscurisporus

3.1 Methodology – Identification, extraction and biological activity screening of new specimens of Paxillus obscurisporus……….….……………………………………46 3.1.1 Micro-morphological identification of the collected Paxillus obscurisporus using ITS2 and NLB4 primers……………………………….……………….46

3.1.2 Assessment of bio-activity of Paxillus obscurisporus collected in 2015………………………………………………………………………..……46

3.2 Methodology – Purification of fraction 4 from Paxillus obscurisporus using size- exclusion chromatography……………………………………………………………47 3.2.1 Size-exclusion/gel-permeation chromatography using a 20mL size Sephadex™ LH-20 column…………………………………………………...47 3.2.2 Purification using 120mL Sephadex™ LH-20 on a C16/70 column……...48 3.2.3 Purification using 530mL Sephadex™ LH-20 on a C26/100 column…….49 3.2.4 Assessing post-Sephadex™ LH-20 fractions for bio- activity…………………………………………………………………………..50 3.2.5 Assessment of carbohydrate content in bio-active fractions……………...51

3.3 Methodology – Purification by means of anion-exchange chromatography using DEAE Sephadex™…………………………………………………………………….51 3.3.1 Purification through anion-exchange chromatography using Poly-Prep® chromatography column………………………………………………………52 3.3.2 Purification using 140mL DEAE Sephadex™ on a C16/70 column……...53 3.3.3 Assessment of anti-proliferative activity of post-DEAE Sephadex™ flow- through………………………………………………………………………….54 3.3.4 Assessment of presence of protein and glycoprotein in post-DEAE Sephadex™ flow-through……………………………………………………..54

3.4 Results and Discussion..……………………………….. ……………………………56 3.4.1 DNA identification of the collected Paxillus obscurisporus..………………56 3.4.2 Bio-activity of Paxillus obscurisporus collected in 2015.…………………..57 3.4.3 Bio-activity of post-Sephadex™ LH-20 fractions from a 16mL drip column…………………………………………………………………………..59 3.4.4 Bio-activity of post-Sephadex™ LH-20 fractions from 120mL C16/70 column…………………………………………………………………………..61 3.4.5 Bio-activity of post-Sephadex™ LH-20 fractions using a 500mL C26/100 column…………………………………………………………………………..65 3.4.6 Effect of bio-activity of post-Sephadex™ LH-20 fractions on various cell lines……………………………………………………………………………..67 3.4.7 Carbohydrate content in bio-active fractions…………………………..…...74 3.4.8 DEAE Sephadex™ anion-exchange chromatography using mini Poly- Prep® chromatography column………………………………………………75

3.4.9 DEAE Sephadex™ anion-exchange chromatography using C26/100 column…………………………………………………………………………..80 3.4.10 Assessing the presence of proteins and glycoproteins in bio-active DEAE Sephadex™ fractions………………………………………………………….82

Chapter 4 – General Discussions

4.1 General project overview……………………………………………………………..85 . 4.2 DNA identification of the collected mushrooms…………………………………….86

4.3 Assessment of biological activity of the BC wild mushrooms……………………..88

4.4 Selection of Paxillus obscurisporus for purification of anti-proliferative compound(s)……………………………………………………………………………90

4.5 Purification of anti-proliferative compound(s) from P. obscurisporus: size- exclusion and anion-exchange chromatography…………………………………...92

4.6 Assessment of the chemical nature of bio-active compound(s) from P. obscurisporus…………………………………………………………………………..94

4.7 General overview and concluding remarks..………………………………………..96

List of Tables

Table 1.1 Mushroom bio-active compounds exhibiting various antimicrobial effects..8

Table 1.2 Medicinal mushroom compounds currently in clinical use for cancer and assistive therapy……………………………………………………………….10

Table 1.3 Selected list of wild mushrooms present in BC characterized upon their medicinal potential……………………………………………………………..12

Table 2.1 PCR primers used in the amplification of the ITS 2 sub-region of the fungal DNA isolated from the BC wild mushrooms………………………………...19

Table 2.2 Table displaying general 96-well plate layout for anti-proliferative assay of wild BC mushroom samples………………………………………………….24

Table 2.3 Summary of fractions obtained upon extraction of the mushrooms……...32

Table 2.4 Summary of bio-activity of BC wild mushrooms…………………...……….42

Table 3.1 Buffers used in the anion-exchange DEAE Sephadex™ column chromatography………………………………………………………………..52

vii

List of Figures

Figure 2.1 Matchmaker software showing the different mushroom categories and the parameters to insert to match it to most fitted mushroom…………………17

Figure 2.2 Depiction of binding positions of various primers commonly used on ribosomal cassettes……………………………………………………………19

Figure 2.3 Extraction scheme used to extract BC wild mushrooms…………………..21

Figure 2.4 Amanita muscaria (L.) Lam………..………………………………………….28

Figure 2.5 Phellinus igniarius (L.) Quél……….………………………………………….30

Figure 2.6 Trichaptum abietinum (Pers. Ex. J.F. Gmel) Ryvarden……………………31

Figure 2.7 Assessing anti-proliferative activity from extracts of Amanita muscaria....34

Figure 2.8 Assessing anti-proliferative activity from extracts of Gyromitra esculenta…………………………………………………………...35

Figure 2.9 Assessing anti-proliferative activity from extracts of Paxillus obscurisporus……………………………………………………………….....36

Figure 2.10 Results of MTT assay of Phellinus igniarius………………………………..37

Figure 2.11 Results of MTT assay of Trichaptum abietinum……………………………37

viii

Figure 2.12 Effect of fractions from A. muscaria (A) and G. esculenta (B) on TNF-α production in Raw 264.7 macrophage cells………………………………...39

Figure 2.13 Effect of fractions from P. obscurisporus (A) and Phellinus igniarius (B) on TNF-α production in Raw 264.7 macrophage cells………………………...40

Figure 2.14 Effect of fractions from Trichaptum abietinum on TNF-α production in Raw 264.7 macrophage cells………………………………………………………40

Figure 3.1 Result of anti-proliferative MTT and immuno-stimulatory ELISA assay of NaOH extract from P. obscurisporus………………………………………..58

Figure 3.2 Results of MTT assay of post-Sephadex™ LH-20 fraction from 5% NaOH extract of P. obscurisporus……………………………………………………60

Figure 3.3 Results of Immuno-stimulatory ELISA assay of post-Sephadex™ LH-20 fraction from the NaOH extract of P. obscurisporus……………………….61

Figure 3.4 Results of MTT and ELISA assay of post-Sephadex™ LH-20 fractions from NaOH extract of P. obscurisporus……………………………………..62

Figure 3.5 Results of MTT assay of post-Sephadex™ LH-20 (C16/70 column) fractions from NaOH extract of P. obscurisporus…………………………..64

Figure 3.6 Results of MTT and ELISA assay of post-Sephadex™ LH-20 (C26/100) fractions from NaOH extract of P. obscurisporus…………………………..66

Figure 3.7 Results of MTT and ELISA assay of post-Sephadex™ LH-20 (C26/100) fractions from NaOH extract of P. obscurisporus…………………………..67

Figure 3.8 Results of 48 hours MTT dose-dependent assay performed using post- Sephadex™ LH-20 fractions from 5% NaOH extract of P. obscurisporus……………………………………………………...... 69

Figure 3.9 Results of 48 hours MTT dose-dependent assay performed using post- Sephadex™ LH-20 fractions from 5% NaOH extract of P. obscurisporus……………………………………………………………….70

Figure 3.10 MTT time-dependent assay performed using post-Sephadex™ LH-20 fractions from 5% NaOH extract (0.5mg/mL) of P. obscurisporus……….72

Figure 3.11 MTT time-dependent assay performed using post-Sephadex™ LH-20 fractions from 5% NaOH extract (0.5mg/mL) of P. obscurisporus……….73

Figure 3.12 Total carbohydrate assay of post-Sephadex LH-20 fractions from C16/70 column…………………………………………………………………………..74

Figure 3.13 MTT assay performed on post-DEAE Sephadex™ flow-through………...76

Figure 3.14 MTT assay performed on post-DEAE Sephadex™ flow-through………...78

Figure 3.15 MTT assay performed on post-DEAE Sephadex™ flow-through………...80

Figure 3.16 MTT assay performed on post-DEAE Sephadex™ flow-through………...81

Figure 3.17 Dose-dependent MTT assay performed using (A) post-Sephadex™ LH-20 fractions and (B) post-DEAE Sephadex™ on HeLa cells…………………82

Figure 3.18 SDS-PAGE analysis for the presence of proteins (A) and carbohydrates (B)……………………………………………………………...83

Acknowledgements

First and foremost, I would like to thank my supervisor Dr. Chow Lee for providing the opportunity to work in his lab on the drug discovery research. His immaculate work ethic always motivated me and his motivation helped me grow as an academic during the time I spent in the lab. I would also like to thank Dr. Maggie Lee,

Hugues Massicotte, Keith Egger and Kerry Reimer for the much-needed technical guidance and perspicacious conversations that helped me understand the research results better. Without their guidance, this study would have been difficult to carry out.

Finally, I would like to thank my family and friends - Ashok Barad, Manjushri Barad,

Shikha Barad, Shashank Barad, Jeremy Siewert, Amanda Mjolsness, Rajtantra Lilhare,

Furqana Khan, and Sebastian Mackedenski, who always supported me through the harsh times and were my companions during the good times.

Chapter 1

Introduction

Worldwide, cancer is one of the major public health problems and the leading cause of deaths in developed countries, and second largest cause of death in the developing countries (Coughlin & Ekwueme, 2009; Jemal et al., 1999).Treatment options for cancer include radiotherapy, immunotherapy, and chemotherapy. But, limited efficacy, unspecified tissue targets, and increasing toxicity associated with cancer chemotherapy are some of many problems which makes cancer treatment very complicated (Golub et al., 1999). The major toxicity issues involved in cancer chemotherapy includes cardiovascular complications [e.g. cardiotoxicity, heart failure, myocardial ischemia (Yeh et al., 2004), neurological complications (e.g. central nervous system toxicity, peripheral nervous system toxicity, encephalopathy etc.) (Hildebrand,

2006), and renal complications (e.g. nephrotoxicity, renal failure, renal impairment)

(Ries & Klastersky, 1986)].The ever-increasing complications and toxicity involved in cancer chemotherapy suggests a continuing necessity to find new medicinal compounds with lower toxicity and better efficacy.

1.1 Natural products as source of medicinal compounds

Humans have relied on nature for sourcing of various medicines to treat a wide spectrum of diseases throughout the ages. While leading pharmaceutical discovery over the past century, natural products have been a constant source of chemical diversity for various pharmaceutical precursors (Mishra & Tiwari, 2011). It is widely accepted that about 80% of drugs were based on natural compounds before the advent of synthetic

pharmaceuticals and high throughput screening of combinatorial libraries (Sneader,

2006). However, it is worth noticing that almost 50% of the drugs approved from 1994-

2009 were still based on natural products (Butler, 2008). These natural products cover a range of therapeutic medications including anti-cancer, anti-infectious, and anti-diabetic agents. It is estimated that about 60% of the anti-cancer agents used today are directly or indirectly derived from natural sources including plants, marine organisms, fungi, and microorganisms (Cragg & Newman, 2005).

Because of increasing interest of the pharmaceutical industry in screening of small molecule libraries obtained from combinatorial chemistry techniques, there has been a significant drop in interest to pursue natural products as sources of medicinal agents. There are many disadvantages of natural product chemistry. There are difficulties in access and supply of the natural products from various natural sources.

The complexity in natural product chemistry leads to inability to maintain high-quality natural product libraries, as it demands highly skilled individuals. The process of the screening of natural product is slow because of the complex nature of the crude extracts obtained from the natural sources. Duration of screening of natural products is slower compared to screening of compounds obtained from combinatorial chemistry. Structural complexity and large molecular size of natural products make their modification challenging. And finally there are concerns about intellectual property rights of natural products (Lam, 2007; Singh & Barrett, 2006).The problems mentioned in this paragraph that are associated with natural product drug discovery turned the focus of the pharmaceutical industry towards discovery of novel drugs through high throughput screening of small molecular libraries generated by combinatorial chemistry.

Nevertheless, disappointing results were experienced by the pharmaceutical industry from screening of these libraries obtained from the combinatorial chemistry

(Newman & Cragg, 2012). It was believed that the lack of chemical diversity from those libraries led to disappointing drug discovery results (Macarron et al., 2011; Sukuru et al.,

2009).

Recent advances in functional assays, phenotypic screening processes, and genomic/metabolomic approaches have led to a fusion of traditional and contemporary methods to assess natural product bio-activity and this is contributing to renewed interest in natural products drug discovery (Harvey et al., 2015).

1.2 Medicinal Mushrooms

Mushrooms have been known for their medicinal and culinary use for thousands of years (Chen et al., 2013). Mushrooms were consumed by people of eastern Asia for their culinary and medicinal properties since ancient times. Egyptian hieroglyphics, which are about 4500-year-old, depict mushrooms as gifts from gods (Halpern, 2006;

Sullivan et al., 2006), indicating the medicinal significance of the mushrooms in that era.

Many ancient civilizations worldwide have discovered that hot water extract of many mushroom species have surprising medicinal effects. In China and Japan particularly, hot water extracts of selected mushroom species are used in several treatments in traditional medicine (Dias et al., 2004). In the recent past, mushrooms were considered as a miniature pharmaceutical manufacturers, synthesizing pharmacologically-active, biochemical compounds (Patel & Goyal, 2012). In countries like Japan, China, the

U.S.A., and Russia there are several bio-active natural molecules extracted from the fruiting bodies of mushrooms such as Lentinus edodes, Ganoderma lucidum,

Schizophyllum commune, Trametes versicolor, Inonotus obliquus, and Flamulina velutipes. These natural molecules have been shown to strengthen the immune system, and hence used in various disease treatment (Wasser & Weis, 1999).

The use of Ganoderma and related species as a medicinal agent in traditional medicine dates back thousands of years (Zhao et al., 1994) and is a well-studied mushroom across the globe. It is commonly referred to as Lingzhi in China and its pharmacological actions includes immuno-modulation through an effect on dendritic cells (Chan et al., 2007). The other medicinal activities of Ganoderma spp. includes anti-inflammatory, anti-cancer (Kim et al., 2007), anti-diabetic, anti-oxidative (Sun et al.,

2004), anti-aging (Bisen et. al., 2010), and radical scavenging (Baskar et al., 2008).

Dried powder of G. lucidum is used in east Asian countries as a preventative household medicine against various diseases including prostate and breast cancers (Sliva et al.,

2003).

S. commune is another medicinal mushroom credited with numerous medicinal activities (Mirfat, 2008) consisting of anti-tumor (Han et al., 2005), and anti-oxidant properties (Yim et al., 2013). Also, S. commune has been found to exhibit anti- proliferative activity on breast and hepatic cancer cell lines (Mansour et al., 2012). For its numerous nutritional benefits, the people of southeast Asia consumed S. commune as nutritional food (Han et al., 2005).

T. versicolor is a well-studied and well-documented mushroom that has numerous health benefits: it has a variety of medicinal effects including antiviral, anti- tumor, and immuno-modulatory (Chu et al., 2002). It was discovered that T. versicolor biosynthesizes polysaccharides and proteins that are responsible for pharmacological

actions and health benefits (Li et al., 2011). Most of the mushroom compounds seem to exert their effects through immuno-modulation, and so does polysaccharide-krestin

(PSK). PSK was isolated from T. versicolor and had been used as an assistive anti- tumor medicine in Asia since the 1970’s (Fisher, 2002). Studies show that PSK exerts its immuno-modulatory effect by binding to TRL4 receptors that leads to induction of

TNF-alpha and IL6 inflammatory cytokines (Price et al., 2010).

I. obliquus is one of the most popular and effective medicinal mushrooms. It has been used for nutritive and medicinal benefits in Russia and Europe for more than four centuries (Zheng et al., 2009). Commonly known as “Chaga”, it consists of many metabolites that are biologically active, having hypoglycemic (Sun et al., 2008), hepato- protective (Wasser, 2010), anti-fungal (Kim et al., 2011), and anti-tumor activities

(Kahlos & Tikka, 1994; Taji et al., 2008). Studies show that Chaga produces various phenolic compounds like hispidin analogues and melanins, that are considered to actively inhibit replication of HIV-1, H1N1, and H3N2 influenza virus (Ichimura et al.,

1998; Kukulyanskaya et al., 2002). Known for it’s various biological activities, Chaga is considered one of the most significant medicinal mushrooms.

A lot of mushrooms are proven to have medicinal properties. These medicinal mushrooms includes different species from genera Phellinus, Pleurotus, Agaricus,

Ganoderma, Clitocybe, Antrodia, Trametes, Coprinus (Dotan et al., 2011), Grifola

(Harada et al., 2003) Cordyceps, Xerocomus, Calvatia, Schizophyllum, Flammulina,

Suillus, Inonotus, Inocybe, Funalia, Lactarius, Albatrellus, Russula, and Fomes (Patel &

Goyal, 2012). Only a small group of mushroom species are studied for their medicinal properties, and many mushroom species remain unstudied for their medicinal activities.

Hawksworth (2001) established that approximately 1.5 million species of fungi exist, out of which approximately 85,000 are recognized and documented. Of those

85,000 recognized and documented species, 20,000 identified species are macro-fungi, of which about 5000 mushrooms are edible (Mahajna et al., 2009). There are about 20-

25 genera of mushrooms studied for their medicinal activities, leaving a plethora of mushroom species that remain unexplored for their biological activity. These unexplored species can be biologically active and used in treating lethal diseases such as cancer.

1.3 Medicinal Compounds from Mushrooms and their Biological Activity

1.3.1 Active Anti-Tumor Compounds

Mushrooms are known for their nutritional and medicinal values, and have been used clinically for the treatment of various diseases like cancer, diabetes, bacterial infections, viral infections, and ulcers. Polysaccharides and β-glucans are some of the biologically active compounds from mushrooms (Kim et al., 2013). Krestin from T. versicolor, Mesima from P. linteus, Lentinan from L. edodes, and Schizophyllan from S. commune (Li et al., 2004; Yamasaki et al., 2009; Zhou et al., 2009) are some of the most well-known mushroom polysaccharides. The bio-active medicinal compounds are classified into two different classes. Low Molecular Weight (LMW) bio-active compounds (example: quinones, cerebrosides, isoflavones, catechols, amine, triacylglycerols, sesquiterpenes, steroids, organic germanium, and selenium) and High

Molecular Weight (HMW) bio-active compounds (example: proteoglycans, hetero- glucans, homo-glucans, glucans, glycoproteins, proteins, glycol-peptides, and lectins)

(Ferreira et al., 2010)

Studies reveal that Low Molecular Weight (LMW) compounds target processes like angiogenesis, cell cycle growth, apoptosis, metastasis, and signal transduction cascades to bring out biological activities, whereas High Molecular Weight (HMW) compounds like polysaccharides and polysaccharide-protein complexes target innate and cell-mediated immune responses (Zaidman et al., 2005).

1.3.2 Active Anti-Microbial Compounds

Several compounds isolated from mushrooms are reported to have anti-microbial activity such as antifungal, antibacterial and antiviral. Adustin, Aleurodiscal,

Anisaldhyde, Clavilactones, Fimicolons, Fomecins, Cheimonophyllon A and E,

Strobilurin E, Cyathiformine A, and Favolon are examples of anti-fungal compounds that have been isolated from mushrooms; Armillarin, Basidalin, Crinipellins, and Frustulosin are examples of compounds obtained from mushrooms that possess anti-bacterial activity; Clitocine, Ganoderan, Collybial, Filoboletic acid, Lentinan, and Nebularine are examples of anti-viral compounds obtained from mushrooms (Zhong, 2004). Table 1.1 summarizes some of the currently known anti-microbial compounds isolated from mushrooms.

Table 1.1: Mushroom bio-active compounds exhibiting various antimicrobial effects. Mushrooms Bio-active Bio-activity Reference Compounds Cheimonophylum Cheimonophyllon A&E Antibacterial, Weak Stadler et al., candissimum antifungal. 1994.

Clitocybe Cyathiformine A Antibacterial and Arnone et al., cyathiformis Antifungal. 1993. Clitocybe diatreta Diatretol Antibacterial Arnone et al., 1996. Coprinus Illudin C2, Illudin C3 Antimicrobial Lee et al., atramentarius 1996.

Crepidotus Strobilurin E Antifungal Weber et al., fulvotomentosus 1989.

Favolaschia 9-Methoxystrobilurin L Antifungal and Wood et al., pustulosa Antibacterial 1996. Favolaschia sp. Favolon Antifungal Anke et al., 1995. Flagelloscypha pilatii Pilatin Antibiotic Heim et al., 1988. Ganoderma lucidum Ganoderan Antiviral Wasser, 2005.

Lentinus edodes Lentinan Antiviral Mizuno, 2000.

Mniopetalum sp. Mniopetals Antimicrobial Kuschel et al., 1994. Mycena sp. Strobilurin M, Antifungal, Daferner et al., Tertachloropyrocatechol Cytostatic. 1998. Antifungal, Antibacterial. Omphalotus illudens Illudinic acid Antibacterial Dufresne et al., 1997. Oudemansiella Oudemansin X Antifungal Anke et al., radicata 1990. Table adopted from (Rai et al., 2005; Zhong 2004)

1.3.3 Bio-active Hypoglycemic Compounds

Most of the fungal polysaccharides are non-digestible in the human intestinal track, making them a good source of fiber that could reduce glucose release

and lessen accumulation of cholesterol in the blood. Fungal glucans from L. edodes, G. ludidum, S. commune, Sclerotium rolfsii, Grifolia frondosa, Agaricus blazei, Cordiceps miltaris, Pleurotus ostreatus, and Saccharomyces cerevisiae have been proven to reduce the blood cholesterol levels and mitigate hypoglycemic/hypolipidemic effects, in human and animal studies (Biliaderis and Izydorczyk 2007; Giavasis, 2013; Wasser,

2002; Zhong, 2004; Waszkiewicz-Robak, 2013). Lentinan isolated from the mushroom

L. edodes, is used as an effective hypocholesterolemic agent for humans; it breaks down blood lipoproteins, both Low Density Lipoproteins (LDL) and High Density

Lipoproteins (HDL) (Rana, 2005).

1.4 Medicinal Compounds from Mushrooms in Clinical Use

Clinical trials for many medicinal compounds from mushrooms have been conducted. Most of these medicinal compounds that have undergone clinical trials were found to be beneficial to cancer patients. Polysaccharides extracted from G. frondosa,

Agaricus brasiliensis, L. edodes, G. lucidum, T. versicolor, and Cordyceps sinensis are used to produce tablets and another dosage forms for their tumoricidal and immuno- modulatory activity. G. lucidum is prescribed in various dosage forms like injectable, capsule, tincture, tea, and soup (Rai et al., 2005). Table 2 summarizes some of the medicinal mushroom compounds that are currently in clinical use.

Table 1.2: Medicinal mushroom compounds currently in clinical use for cancer and assistive therapy Mushroom Disease Dose Reference Oral Administration Trametes versicolor Lung Cancer 3 Capsules Tsang et al., (PSK) (350mg- Thrice a 2003 day)

Trametes versicolor Immunosuppression 2gm/kg/day Qian et al., (PSP) 1997. Trametes versicolor HIV-1 6.25mg/ml Collins and (PSP) Ng, 1997.

Ganoderma lucidum Advanced cancer 600mg, thrice a Gao et al., (Ganopoly) day. 2002.

Grifola frondosa (D- Various cancer 100mg/day for 34 Kodama et al., Fraction) months. 2002.

Intraperitoneal Administration Sparassis crispa Cancer 250-1000 Harada et al., microgram/mouse 2002.

Phellinus linteus Cancer 100mg/kg of the Kim et al., acidic 2003. polysaccharide.

Agaricus brasiliensis (F3- Meth A Tumor Cell 100mg/kg/day for Itoh et al., 2-b and 5FU) 30 days. 1997.

PSP- Polysaccharide Peptide, PSK- Polysaccharide Krestin, Ganopoly- Crude polysaccharide fraction

Table adaped from Rai et al (2005)

1.5 British Columbia Wild Mushrooms

British Columbia (BC) is home to a biodiverse fungal community. Because of this biodiversity in mushrooms, we hypothesized that the mushrooms found in BC may have compounds with medicinal properties. To date, there have been no studies reported on the screening of BC wild mushrooms for biological activities that are relevant to cancer.

A table generated by Dr. Chow Lee and Dr. Keith Egger (unpublished data in Table 3) differentiates the various selected mushrooms found in BC. Group A includes the mushrooms that are reported to have compounds with biological activities. Group B consists of mushrooms that are related to those in group A, but have not been studied for biological activity. Group C is comprised of mushrooms that are not related to those in group A, and have not been studied for their biological activity.

Based on Table 1.3, mushrooms species in BC like Agaricus bisporus, Pleurotus sajor-caju/pulmonarius, P. ostreatus, and Sarcodon scabrosus are reported to have biologically active compounds, confirming the presence of medicinal mushrooms in the province. Looking at group B and C, it is evident that there are many mushrooms species that are yet to be studied for their potential biological activity. This suggests the high possibility of discovering novel bio-active compounds.

Table 1.3: Selected list of wild mushrooms present in BC characterized depending upon their medicinal potential. Groups Mushroom species Biologically active compound(s) A Agaricus bisporus Linoleic acid, linolenic acid. Pleurotus sajor-caju/pulmonarius β-glucans. Pleurotus ostreatus α-glucans. Sarcodon scabrosus Diterpene. Inonotus obliquus Polyphenols. Ganoderma lucidum Polysaccharides, triterpene. Trametes (Coriolus) versicolor Polysaccharide—peptide or Albatrellus confluens protein. Hericium erinaceus Grifolin. Amanita muscaria β-glucans. Phellinus nigricans Endo-polysaccharides Phellinus igniarius Endo-polysaccharides

B Agaricus albolutescens; subrufescens; Presently unknown arvensis; augustus; diminitivus; subrutilescens; nivescens; inapertus; excellens; emotus. C Pleurotus dryinus; populinus. Presently unknown Sarcodon calvatus; fuscoindicus; imbricatus; indurescens; tereosarcinon; underwodii;atrovinidis; leucopus; rimosus; subincamatus; versipellis. Inonotus cuticularis; glomeratus; tomentosus. Phellinus conchatus; gilvus; prunicola; repandus; tremulae; everharti; occidentalis; pomaceus. Antrodia albida; albobrunnea; carbonica; heteromorpha; malicola; serialis; sinuosa; sitchensis; vaillanti; vaniformis; xantha; crassa; radiculosa. Ganoderma applanatum; brownii; oregonense. Trametes cervina; elegans; hirsuta; ochracea; pubescens; suaveolens; trogii. Albatrellus arellaneus; caeruleoporus; ellisii; flettii; ovinus; syringae; Byssoporia terrestris; Polyporoletus sublividus; dispansas; skamanius; subrubescens. Hericium abietis; americanum; coralloides; Dentipellis fragilis. Thelephora americana; caryphyliea; intybacea; mollissima; palmata; regularis; terrestris; caespitulans; scissilis. Table generated by Dr. Chow Lee and Dr. Keith Egger.

1.6 Research Goals

Fungi belonging to phylum Basidiomycota are primary targets for biological activity assessment due to the high numbers of biologically active compounds being reported

(Wasser, 2002).There are numerous wild mushrooms in BC that have never been reported or studied for their biological activity (Table 1.3). Targeting BC wild mushrooms is expected to open the gate for discovery of novel anti-cancer compounds. It is known that these mushrooms possess two categories of bio-active compounds: the high and low molecular weight compounds. High molecular weight polysaccharides (including protein-polysaccharide complexes) are reported to reduce the cell viability and also to interact non-specifically with the immune system to upregulate or downregulate various aspects of host response (Lull et al., 2005), while low molecular weight compounds can diffuse through the cell membrane, and act on specific signal transduction pathways

(Zaidman et al., 2005).

The goals of my research were: (i) collection and identification of BC wild mushrooms, (ii) investigation of various solvent extracts from selected British Columbian wild mushrooms for their potential anti-proliferative and immuno-modulatory activity, and

(iii) purification and characterization of anti-proliferative compound(s).

To do so, the first major research goal was to collect, identify, and extract several

BC wild mushrooms. Since the primary goal of the research was to discover novel anti- cancer compounds, the mushrooms found were grouped into three different categories at the outset of this research. The three categories are as follows: Group A – mushrooms found in BC as well as other countries that are known to have anti-cancer properties; Group B – mushrooms found in BC that are related to Group A but have not

been investigated; and Group C – mushrooms unique to BC that have never been tested (Table 1.3).

After collection of the BC wild mushrooms, they were identified using two different methods. The first method was macro-morphological identification, using morphological characteristics like shape, size, color, location etc. to identify the mushrooms. The second method was DNA sequencing. Before moving ahead to extraction, the mushrooms were categorized as per Table 1.3. Special emphasis was placed on mushrooms belonging to group B and C, to elevate the chances of discovering novel anti-cancer compounds.

The second major research goal was to extract the selected mushrooms and subject them to bioassay screening for anti-proliferative and immuno-stimulatory activity. A four-solvent extraction scheme adopted from Mizuno et al. (1999), was used for extraction. The extracts obtained were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay that is a colorimetric assay indicative of anti- proliferative activity. Further, to screen the extracts for immuno-modulatory activity, they were subjected to Enzyme Linked Immunosorbent Assay (ELISA).

The third and final research goal was to select an extract and further purify it. Size- exclusion and anion-exchange chromatography were employed to further separate anti- proliferative compounds to generate relatively pure compounds. To understand the biochemical nature of the purified bio-active compound(s), several chemical and biological assays were performed.

Chapter 2 Collection, identification, extraction and assessment of anti- proliferative and immuno-stimulatory activities of five species of British Columbia wild mushrooms

This chapter includes the process of procurement of the British Columbia (BC) wild mushrooms from several locations. Additionally, this chapter also encompasses the experiments carried out for macro/micro-morphological work and DNA characterization to identify the collected mushrooms, multi-solvent extraction, and screening of the fractions obtained for anti-proliferative and immuno-stimulatory activities.

2.1 Collection and identification of BC wild mushrooms

2.1.1 Collection of BC wild mushrooms

Collection of BC wild mushrooms was carried out at several locations. The warm and moist weather conditions between April and October of 2013, 2014, and 2015 made it the best time for collection. Mushroom fruiting bodies do not grow for the remainder of the year due to snow and colder conditions during winter. The location and conditions around the collection sites were noted.

The book “Mushrooms Demystified” by David Aurora (1986) was used as a field guide for initial identification of mushroom species. Photographs of mushrooms collected were taken prior to detachment from the substrate. A pocket knife was used to detach the mushroom from the substrate to which it was attached. All the morphological descriptions such as shape, size, color, location, type of substrate the mushroom was found on, etc. were noted. A paper bag was used for temporary storage of the mushroom samples until the samples were transported to the lab for further processing.

A few BC wild mushroom samples were collected and provided by Drs. Keith Egger and

Hugues Massicotte. These samples were kept frozen at -80°C.

For further analysis, the mushrooms were dried in a conventional oven at 55°C for 3-4 days until the mushrooms were completely dried. The dried mushrooms were then pulverized using a domestic grade food processor and converted into powder. For woody conks, such as Phellinus igniarius, a hammer mill was used to pulverize the samples. The powder was then stored at room temperature in airtight bags to avoid contamination.

2.1.2 Macro-morphological identification

Macro-morphological identification refers to identification based on morphological details and characteristics of the collected mushroom samples. Various classes of mushrooms exhibited multiple morphological differences, which helped to differentiate between mushrooms for preliminary identification of the samples. The book

“Mushrooms Demystified” by David Aurora (1986) was referenced to narrow down the identification of the mushroom samples to family level.

)LJXUH0DWFKPDNHUVRIWZDUHVKRZLQJWKHGLIIHUHQWPXVKURRPFDWHJRULHVDQGWKH SDUDPHWHUVWRLQVHUWWRPDWFKLWWRPRVWILWWHGPXVKURRP )RUIXUWKHUFRQILUPDWLRQRIWKHLGHQWLW\RIPXVKURRPV³0DWFKPDNHU

0XVKURRPVRIWKH3DFLILF1RUWKZHVW´IUHHVRIWZDUH9HUVLRQ &RS\ULJKW,DQ*LEVRQ

ZDVXVHG&RQVLVWLQJRIDGDWDEDVHRIRYHUGHVFULSWLRQVRIERWKJLOOHGDQGQRQ

JLOOHGPXVKURRPVWKHVRIWZDUHSURYLGHVDQLQWHUIDFHIRUUHFRUGLQJWKHPXVKURRP

PRUSKRORJLFDOFKDUDFWHULVWLFVWRILQGVSHFLHVWKDWILWWKHFKDUDFWHULVWLFVWKHUHDUHDERXW

LOOXVWUDWLRQVRIRYHUVSHFLHV

7KHFKDUDFWHULVWLFVWKDWKHOSLQPRUSKRORJLFDOLGHQWLILFDWLRQDUHVL]HFRORUVKDSH

WH[WXUHRIYDULRXVVXUIDFHV VXFKDVFDSVWRFNEDVHJLOOVIOHVKHWF WKHVXEVWUDWHRQ

ZKLFKWKHPXVKURRPVZHUHIRXQG ORFDWLRQRGRUPDUJLQW\SHHWF

,GHQWLILFDWLRQRIPXVKURRPVDPSOHVXVLQJ'1$DQDO\VLV

*HQRPLF'1$IURPWKHFROOHFWHG%&ZLOGPXVKURRPVZDVLVRODWHGXVLQJ

3RZHU6RLO'1$LVRODWLRQNLW 02%,2/DERUDWRULHV&$86$ LQDFFRUGDQFHZLWKWKH

protocol provided by the manufacturer. Agarose gel electrophoresis was performed to further confirm successful isolation of the DNA from mushroom samples. One percent agarose gel (Thermo Scientific, MA, USA) were prepared using TRIS borate – EDTA

(TBE) buffer and DNA was visualized with ethidium bromide staining under UV light.

Five µL of the isolated DNA samples from different mushroom samples were mixed with

2 µL of DNA loading dye. The samples which possessed DNA were subjected to PCR

(Polymerase Chain Reaction). The DNA Plus ladder (Life Technologies, CA, USA) was used to assess the size of PCR product used for DNA identification, the ITS2 DNA barcoding region. The ITS exhibits the high degree of vatiation compared to other genic regions, hence PCR was used to amplify this region for DNA sequencing analysis and species identification. ITS3 and NLB4 (Integrated DNA Technology, IA, USA) primers were used to amplify the sequences from the mushroom samples.

Figure 2.2: Depiction of binding positions of various primers commonly used on ribosomal cassettes. (Hennell et al., 2012; Martin & Rygiewicz, 2005) Mastermix used for PCR contains the following

DNA template 100 ng Thermocycler program Thermopol buffer 3.5 µL 1) 95°C for 5 min dNTP’s (2mM) 3.5 µL 2) 95°C for 1 min ITS3 primer 100 ng 3) 52°C for 1 min NLB4 primer 100 ng 4) 72°C for 1min Taq polymerase 0.5 µL 5) Repeat 1-4 for 29 times Nucleasefree water 35 µL 6) 72°C for 5 min

Table 2.1: PCR primers used in the amplification of the ITS 2 sub-region of the fungal DNA isolated from the BC wild mushrooms. Region Primer DNA Sequence ITS 2 ITS-3 5’-GCATCGATGAAGAACGCAGC-3’ ITS 2 NLB-4 5’-GGATTCTCACCCTCTATGAC-3’

The exponentially amplified DNA samples were further subjected to gel electrophoresis for confirmation of DNA amplification and to obtain sufficient quantity for

DNA sequencing. PCR products with visible amplicons were subjected to cleaning and

DNA sequencing as described below.

QIAquick® (QIAGEN, Germany) PCR cleaning kit was used to clean the post-

PCR amplified DNA products. After cleaning the PCR products, the samples were sent for DNA sequencing to the Northern Analytical Laboratory Services (UNBC). The chromatogram received after DNA sequencing was analyzed using Chromas light©

2.1.1 software (Technelysium Pty Ltd.).

The DNA sequences of the mushroom samples were matched to the sequences present in the Basic Local Alignment Search Tool (BLAST). A nucleotide BLAST of the

DNA sequences of the mushroom samples was performed to confirm the mushroom species.

2.2 Methodology – Chemical extraction of BC wild mushrooms

2.2.1 Manual extraction scheme as followed as per Mizuno et al. (1999)

A four-solvent extraction system described by Mizuno (1999) was used. About

150 g of mushroom powdered sample was weighed and suspended in 1.5 L of 85% ethanol (aqueous) in a 2 L Erlenmeyer flask. The mixture was extracted at 65°C for 3 hrs. The resulting solution was then filtered using a Buchner funnel and # 3 Whatman®

(Whatman plc, UK) filter paper.

The resulting filtrate was then condensed using rotary evaporator Rotavapor®

(BÜCHI Labortechnik, Switzerland) at 65°C until approximately 125-150 mL solution remained. The condensed solution’s pH was adjusted to 7, using 12 M hydrochloric acid

(HCl) and 5% sodium hydroxide (aq). After pH adjustment, the condensed solution was slant frozen at -80°C and lyophilized using FREEZONE freeze dryer (Labconco, MO,

USA) at -52°C and 0.02 Torr, for 3 days, until a fine powder remained. This fine powder was labeled as “Fraction 1”.

The above process was repeated using the remaining residue from the ethanol extraction. The sequential extractions were performed using 50% methanol, water and

5% sodium hydroxide. The lyophilized powder obtained from the 50% methanol, water, and 5% sodium hydroxide extractions were labeled as Fraction 2, 3, and 4 respectively.

The summary of extraction scheme is detailed in Figure 2.3.

Figure 2.3: Extraction scheme used to extract BC wild mushrooms. 2.2.2 High-pressure extraction using BÜCHI SpeedExtractor® E916

Three solvent extraction system described by Mizuno (1999) was used. The sample holder section was heated to 65°C. About 40 mL of samples could be loaded into the sample holder cells. A mixture of 20 mL powdered mushroom sample and 20 mL of 0.3-0.8 mm fat-free quartz sand (BÜCHI Labortechnik, Switzerland) was loaded

onto the sample holder cells to avoid clogging. Sample holders were secured with filter papers before packing to avoid leakage of fine particles during extraction. The sample holder cells were placed in the extractor. An extraction protocol was designed using the user interface present on the extractor. The samples were then subjected to four extraction cycles with 3 solvents (85% ethanol, 50% methanol, and water). The extraction cycles are described below.

Heat-up with solvent 1 min. Hold time with sample 15 mins. Discharge time 4 mins. Flushing to the sample holder 15 mins. Flush with solvent 1 min. Flushing with gas 3 mins.

The extracts obtained were condensed using rotary evaporator Rotavapor®

(BÜCHI Labortechnik, Switzerland) at 65°C until approximately 30-50 mL solution remained. The condensed solution was then pH neutralized to 7, using 12 M HCl and

5% sodium hydroxide. After pH adjustment, the condensed solution was slant frozen at

-80°C and lyophilized using FREEZONE freeze dryer (Labconco, MO, USA) at -52°C and 0.02 Torr, for 3 days, until a fine powder remained. This fine powder was labeled as

Fraction 1, Fraction 2, and Fraction 3 for 85% ethanol, 50% methanol, and hot water respectively.

Extraction with 5% sodium hydroxide was not performed using the extractor because the sodium hydroxide corrodes the internal lines carrying the solvents. The 5% sodium hydroxide extraction was performed manually as described above.

2.3 Methodology – Assessment of anti-proliferative and immuno-stimulatory

activities

2.3.1 Assessment of dose and time-dependent anti-proliferative activity

Dose-dependent MTT assay: HeLa (ATCC®) (cervical cancer cells) culture was maintained using Lonza Eagle’s Minimum Essential Medium (VWR, Mississauga, ON,

CA). Trypsin-EDTA (0.25%) was used to trypsinize the cells adhered to the flask. A haemocytometer was used to count the cell density. Approx. 1.5 X 104 cells were plated on each well in 96-well plates. The plated cells were then incubated at 37°C in 5% CO2 atmosphere for 24 hour.

On the following day, lyophilized mushroom samples were prepared and added to the cells. The dried mushrooms fractions were first reconstituted in a solvent that could completely solubilize the mushroom fractions. Twenty mg/mL stock solutions of the crude mushroom fractions were prepared. Further, the stock solutions were adjusted to pH 7 using hydrochloric acid (12 M) or 5% sodium hydroxide. The pH adjusted stock solution was then filter sterilized using 0.2 µm filter (Sarstedt,

Nümbrecht, Germany). Using the 20 mg/mL stock solutions, dilutions were made and added to each well, such that the final working concentrations were 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, and 1.0 mg/mL. Six replicates of each sample and control (same solvent in which the fractions were reconstituted) were included.

The cells were incubated with mushroom fractions for 48 hours. After the incubation, 50 µL of 1 mg/mL MTT solution was added to each well and incubated for another 3 hours. The supernatant was removed from each well and 200 µL of Di-Methyl

Sulfoxide (DMSO) was added to each well, to dissolve the formazan crystals. The 96-

well plate was then subjected to absorbance reading at 570 nm using SYNERGY 2

multi-plate reader (BioTek®, VT, USA).

Table 2.2: Table displaying general 96-well plate layout for anti-proliferative assay of wild BC mushroom samples.

X X X X X X X X X X X X Fr. 1: 0.2 Fr. 1: 0.4 Fr. 1: 0.6 Fr. 1: 0.8 Fr. 1: 1 X None Control X X X X mg/mL mg/mL mg/mL mg/mL mg/mL Fr. 1: 0.2 Fr. 1: 0.4 Fr. 1: 0.6 Fr. 1: 0.8 Fr. 1: 1 X None Control X X X X mg/mL mg/mL mg/mL mg/mL mg/mL

Fr. 1: 0.2 Fr. 1: 0.4 Fr. 1: 0.6 Fr. 1: 0.8 Fr. 1: 1 X None Control X X X X mg/mL mg/mL mg/mL mg/mL mg/mL

Fr. 1: 0.2 Fr. 1: 0.4 Fr. 1: 0.6 Fr. 1: 0.8 Fr. 1: 1 X None Control X X X X mg/mL mg/mL mg/mL mg/mL mg/mL X X X X X X X X X X X X

In time-dependent MTT assay, 0.75 X 104 HeLa cells were plated on 7 different

96-well plates. Each plate was treated with mushroom fractions 24hrs after plating the

HeLa cells. The plated cells were then incubated in a 5% CO2 incubator for 24, 48, 72,

96, 120, and 144 hours. The MTT assays were conducted on different days for different

plates as described above.

The activity of various fractions was measured in % cell viability compared to that

of a control. Standard deviation was the error of the MTT bioassay. Kaleidagraph©

version 4.03 (Synergy Software) was used to generate the graph for the acquired data.

The calculation model used to process the raw data from time and dose-dependent

assay is mentioned in Appendix vii.

2.3.2 Assessment of immuno-stimulation by measuring TNF-alpha

production from macrophage cells

Supernatant collection: RAW 264.7 (murine macrophage cells) cells are cultivated using BioWhittaker® Dulbecco’s Modified Eagle’s Medium (DMEM) (Lonza,

MD, USA) until 50-60% confluent. Life Technology TrypLE™ Express (Thermo Fisher

Scientific, MS, USA) was used to trypsinize the cells adhered to the flask. Raw 264.7 cells were plated at 25 X 104 cells per well in 96-well plates. The plated cells were then incubated in 5% CO2 overnight.

After overnight incubation, the medium was removed and the plated cells were washed with 200 µL BioWhittaker® Phosphate Buffer Saline (PBS) (Lonza, MD, USA).

One hundred µL of DMEM was added to the wells containing RAW 264.7 cells. 100 µL of 1 mg/mL filter sterilized mushroom fraction and 500ng/mL Lipopolysaccharides (LPS) was added to the RAW 264.7 cells and left for incubation at 37°C and 5% CO2 for 6 hours. After incubation, 150 µL of the supernatant was collected. The collected supernatant was centrifuged at 4°C and 200 x g using an Allegra® X-15R, benchtop centrifuge (Beckman Coulter, CA, USA). The supernatant was then transferred to another 96-well plate, labeled and stored at -80°C overnight before performing ELISA.

ELISA Assay: BD OptEIA™ Mouse TNF (Mono/Mono) ELISA Set and Reagent

Set B (BD Biosciences, CA, USA) were used to perform ELISA. BD OptEIA™ Mouse

TNF (Mono/Mono) ELISA Set includes; 1) Anti-mouse TNF monoclonal antibody

(Capture antibody), 2) Biotinylated Anti-Mouse TNF monoclonal antibody (Detection antibody, 3) Streptavidin-horseradish peroxidase conjugate (Enzyme reagent), and 4)

Recombinant mouse TNF (Standards). BD OptEIA™ Mouse TNF (Mono/Mono)

Reagent Set B includes 1) Substrate reagent A, 2) Substrate reagent B, 3) Wash concentrate buffer (20X), 4) Stop solution, 5) Assay diluent, and 6) Coating buffer.

Capture antibody was added to coating buffer in a 1:500 ratios. One hundred µL of capture antibody was then added to the microwells in 96-Well Microtiter™ polystyrene plates (Thermo Fisher Scientific, MA, USA) to coat it with the Anti-mouse

TNF monoclonal antibody. The plate was sealed and incubated at 4°C overnight. On the following day, the antibody was removed after overnight incubation, and the supernatant collected from the RAW 264.7 cells were allowed to thaw on ice. The microwells on the

96-well plate were washed with 300 µL of 1X wash buffer (prepared by diluting wash concentrate buffer 20 times using water) for three successive times. The plates were then blocked using 200 µL assay diluent, sealed, and then left for incubation at room temperature for 1 hour.

While waiting for the plates to be blocked, the supernatant was diluted three times and the LPS diluted 12 times using assay diluent. The TNF-alpha standard was prepared at 1000, 500, 250, 125, 62.5, 31.3, and 15.6 pg/mL for a standard curve through serial dilution using assay diluent and 155ng/mL standard stock solution.

After blocking the plates with assay diluent, the assay diluent was discarded and the 96-well plate was washed with 1X wash buffer for three successive times. After the wash, 100 µL of the standard and samples were added to the 96-well plates. The TNF- alpha standards were placed in triplicates and the samples in six replicates for better optimization. The 96-well plate was then sealed and left for incubation at room temperature for 2 h.

After 2 h of incubation, the standard and sample supernatants were discarded, and wells were washed five times with 300 µL 1X wash buffer. One hundred µL of working detector (detection antibody + Streptavidin-horseradish peroxidase conjugate) was added to the appropriate microwells on the 96-well plate and left for incubation at room temperature for 1 h.

The incubation step was followed by seven washing steps with 1X wash buffer.

One hundred µL of substrate solution (solution A+B) was added to the wells and left for incubation for 30 mins. at room temperature in the dark. Fifty µL of stop solution was added to the appropriate micro-wells on the 96-well plate and was subjected to absorbance reading at 450 nm and 570nm using SYNERGY 2 (BioTek®, VT, USA).

The calculations were performed as per the user manual provided by the manufacturer.

Statistical analysis was performed using One-way ANOVA. Sidak’s or Dunnett’s multiple comparisons test was done as post-hoc analysis once the null hypothesis was rejected.

2.4 Results and Discussion

2.4.1 Macro-morphological and molecular identification of collected

mushrooms

Five different mushroom species were collected at random from different locations in British Columbia. The collected mushrooms were subjected to both macro- morphological and DNA analysis species identification. The following are the results obtained from the collection and identification steps.

1. Amanita muscaria

Figure 2.4: Amanita muscaria (L.) Lam. Collected by: Dr. Hugues Massicotte and Ankush Barad Location: Moore’s Meadow Park, Prince George, BC, Canada. Collected on: October 2013 and June 2014

Morphological Characteristics

Type: Gilled mushroom fruiting body Size: Cap – 5-25cms, Stem – 5-15cms. Shape: Cap- Flat with white spots, Color: Cap - Red-Orange, Stem – Cream-Yellow, Gills - White Gills: Close, broad, free and adnate Spore Deposit: White Flesh: Thick and firm when young Habitat: Was found in a meadow surrounded by tall grass

Matchmaker Mushroom match: Amanita muscaria

Sequencing match: Amanita muscaria (L.) Lam. (1783)

Primer used: NLB4 and ITS3 Max score: 562 Total score: 562 Query covered: 60% Identification: 96% Accession: DQ822791.1

2. Gyromitra esculenta

Collected by: Dr. Keith Egger Location: Miworth, Prince George, BC, Canada. Collected on: May 2014

Identified by: Dr. Chow Lee

Morphological Characteristics:

Type: False morels Size: Cap – 3-10cms, Stem – 2-14cms x 1-2cms, Color: Cap – yellowish to dark brown, Stem – brown Gills/Pores: 2-4 per mm with angular to tooth shaped, Under-cap: White to creamish with rough surface Flesh: thin, pale-colored Habitat: Found on roots or decaying wood chips

Matchmaker Mushroom match: Gyromitra esculenta (Pers.) Fr. (1849)

Sequencing match: Gyromitra esculenta

Primer used: NLB4 and ITS3 Max score: 800 Total score: 800 Query covered: 70% Identification: 96% Accession: AJ544208.2

3. Paxillus obscurisporus:

Collected by: Dr. Hugues Massicotte and Dr. Zoe Meletis Location: 17th Ave, Prince George, BC, Canada. Forest for the World, Prince George, BC, Canada. Collected on: August 2013 and May 2014

Morphological Characteristics:

Type: Gilled mushroom fruiting body Size: Cap – 4-16cms, Stem – 2-5cms x 0.4-3cms. Color: Cap – dark brown, Stem – dark cream - brown, Gills - cream Gills: Close, crowded and forking Spore Deposit: Brown to yellow brown Flesh: Thick and firm Habitat: Found densely populated on ground and woods

Matchmaker Mushroom match: Paxillus obscurisporus/Paxillus involutus

Sequencing match: Paxillus obscurisporus (C.Hahn, 1999)

Primer used: NLB4 and ITS3 Max score: 776

Total score: 776 Query covered: 85% Identification: 95% Accession: HQ207702.1

4. Phellinus igniarius

Figure 2.5: Phellinus igniarius (L.) Quél (1886) Collected by: Dr. Chow Lee and Dr. Wai Ming Li Location: Kitselas, Terrace, BC, Canada. Collected on: August 2014 Identified by: Dr. Wai Ming Li

Morphological Characteristics:

Type: Polypores (Conk) Size: Cap – 5-25cms x 2-16cms, Stem – none Color: Cap – dark brown - black, Stem – none, Gills - none Gills/Pores: 4-5mm, Spore Deposit: White to cream Flesh: Woody with brown color Habitat: Continually appearing single or in small groups on hardwood

Matchmaker Mushroom match: Phellinus igniarius (L.) Quél (1886)

Sequencing match: Phellinus igniarius

Primer used: NLB4 and ITS3 Max score: 676 Total score: 676 Query covered: 73% Identification: 99% Accession: AY558623.1

5. Trichaptum abietinum

Figure 2.6: Trichaptum abietinum (Pers. Ex. J.F. Gmel) Ryvarden Collected by: Ankush Barad Location: Forest for the World, Prince George, BC, Canada. Collected on: October 2013

Morphological Characteristics:

Type: Polypores (Conk) Size: Cap – 1-5 cms, Stem – none Color: Cap – gray to black, Stem – none, Gills - none Gills/Pores: 2-4 per mm with angular to tooth shaped, Spore Deposit: White to cream Flesh: Woody with brown color Habitat: Found annually on decaying hard/softwood

Matchmaker Mushroom match: Trametes versicolor/Trichaptum abietinum

Sequencing match: Trichaptum abietinum (Pers. Ex. J.F. Gmel) Ryvarden

Primer used: NLB4 and ITS3 Max score: 826 Total score: 826 Query covered: 90% Identification: 97% Accession: KC581332.1

2.4.2 Chemical extraction of the BC wild mushrooms

The collected mushrooms were extracted either manually or using BÜCHI

SpeedExtractor® as described in section 2.2.1 and 2.2.2. The description of various fractions obtained from the extraction is summarized in Table 2.3 below.

Table 2.3: Summary of fractions obtained upon extraction of the mushrooms. Amount Mushroom of extract Mass Physical Reconstituted Fraction Species used (grams) appearance in (grams) 85% Viscous and Ethanol 2.0627 Ethanol sticky (F1) 50% Viscous and Methanol 0.7895 Water Amanita sticky 13.71 (F2) muscaria Hot water 0.3775 Fine powder Water (F3) 5% Sodium Chunky hydroxide 21.886 Water powder (F4) 85% Fluffy (Cotton Ethanol 1.276 Water like) (F1) 50% Fluffy (Cotton Methanol 0.961 Water Gyromitra like) 16.17 (F2) esculenta Hot water 0.891 Fine powder Water (F3) 5% Sodium Chunky hydroxide 18.239 Water powder (F4) 85% Coarse Ethanol 4.2895 Water powder (F1) 50% Paxillus Coarse 19.452 Methanol 2.295 Water obscurispor powder us (F2) Hot water Fluffy (Cotton 0.417 Water (F3) like) 5% Sodium 20.162 Chunky Water

hydroxide powder (F4) 85% Fluffy (Cotton Ethanol 0.882 Ethanol like) (F1) 50% Fluffy (Cotton Methanol 0.857 Water Phellinus like) 11.296 (F2) igniarius Hot water 0.722 Fine powder Water (F3) 5% Sodium Chunky hydroxide 9.002 Water powder (F4) 85% Fluffy (Cotton Ethanol 0.482 Water like) (F1) 50% Fluffy (Cotton Methanol 0.544 Water Trichaptum like) 10.629 (F2) abietinum Hot water Fluffy (Cotton 0.336 Water (F3) like) 5% Sodium Chunky hydroxide 4.33 Water powder (F4)

2.4.3 Dose and time-dependent anti-proliferative activity of mushroom

fractions on HeLa cell

The mushroom fractions obtained from the extraction were reconstituted in an appropriate solvent as shown in Table 2.3. HeLa cells were first subjected to dose- dependent MTT assay experiments. Upon obtaining the results from the dose- dependent MTT experiments, selected concentration of the mushroom fractions were then used to perform time-dependent MTT assays.

A. B.

120 100

100 F2 80 Control 80 60 F3 60 F4 40 40

20 F4 20 F1

Cell Viability (% control) (% Viability Cell F1 0 control) (% Viability Cell 0 0 0.2 0.4 0.6 0.8 1 0 20 40 60 80 100 120 Concentration (mg/mL) Time (Hours) Figure 2.7: Assessing anti-proliferative activity from extracts of Amanita muscaria. (A) Dose-dependent assay for Fractions 1-4 of Amanita muscaria. The graph is representative results from two different replicate (n=2). (B) Time-dependent assay for bio-active Fractions 1 and 4 at 0.5 mg/mL. Graph is representative results from two different replicate (n=2). Error bars are standard deviation.

Results from the dose-dependent MTT assay of Fractions 1-4 from Amanita muscaria are shown in Figure 2.7(A). Fractions 1 and 4 significantly reduce cell viability to 15% at 0.4 and 0.8 mg/mL respectively. By contrast, Fractions 2 and 3 did not appear to have any significant effect on cell viability; decreasing to only 80%. Fractions 1 and 4, that exhibited strong anti-proliferative activity were further subjected to a time- dependent MTT assay at the concentration of 0.5 mg/mL. Results from the time- dependent MTT assay showed that Fraction 1 reduced cell viability to less than 5% after

30 hours of treatment. On the other hand, cell viability was reduced to only 70% by

Fraction 4 on day 5 (120 hours).

Figure 2.8 shows the result from the dose-dependent MTT assay of Gyromitra esculenta. The results show that Fraction 2 (at 0.8 mg/mL) suppressed the cell viability

to only 60%. Since the anti-proliferative activity exhibited by all the fractions were considered weak (less than 50%), time-dependent MTT assay was not performed on all the fractions from Gyromitra esculenta.

120

100 F3 F4 80

60 F1 F2 40

Cell Viability (% control) (% Viability Cell 0 0 0.2 0.4 0.6 0.8 1 Concentration (mg/mL) Figure 2.8: Assessing anti-proliferative activity from extracts of Gyromitra esculenta. Dose-dependent assay for Gyromitra esculenta. The graph is representative results from two different replicate (n=2). Error bars are standard deviation. The results obtained from dose-dependent MTT assay performed using all the fractions of Paxillus obscurisporus are shown in Figure 2.9(A). The results show that

Fractions 3 and 4 reduced cell viability to about 50% at 0.4 and 1.0 mg/mL respectively.

By contrast, the anti-proliferative activity exhibited by Fractions 1 and 2 was minimal, reducing HeLa cell viability to less than 20%. Hence, HeLa cells were treated with bio- active Fractions 3 and 4 at 0.5 mg/mL for time-dependent MTT assay. Figure 2.9(B) shows that both Fractions 3 and 4 significantly reduced cell viability by day 1 and this persisted up to day 5.

A. B.

120 120 F1 Control 100 100 F2 80 80 F3 60 60 F3 40 F4 40 F4 20 20 Cell Viability (% control) (% Viability Cell Cell Viability (% control) (% Viability Cell 0 0 0 0.2 0.4 0.6 0.8 1 0 20 40 60 80 100 120 Concentration (mg/mL) Time (Hours) Figure 2.9: Assessing anti-proliferative activity from extracts of Paxillus obscurisporus. (A) Dose-dependent assay for Fraction 1-4 of Paxillus obscurisporus, where graph is representative results from two different replicate (n=2). (B) time-dependent assay for active fraction 3 and 4 at 0.5 mg/mL, where graph is representative results from two different replicate (n=2). Error bars are standard deviation. Dose-dependent MTT assay on fractions from Phellinus igniarius shows that

Fractions 1 and 3 exhibited strong anti-proliferative activity. As shown in Fig 2.10(A),

Fraction 1 reduced cell viability to 20% at 0.6 mg/mL and Fraction 3 reduced cell viability to 40% at 0.1 mg/mL. Fractions 2 and 4 reduced cell viability to about 60%.

Figure 2.10(B) shows that Fraction 1 at the concentration of 0.2 mg/mL and Fraction 3 at 1 mg/mL significantly reduced cell viability to 10% by day 1 and the effect persisted up to day 5.

A. B.

100 120 F2 100 80

80 F4 Control (F1) 60 60 F3 40 40 Control (F3) 20 20 F1 F1 Cell Viablility (% control) (% Viablility Cell Cell Viability (% control) (% Viability Cell 0 0 F3 0 0.2 0.4 0.6 0.8 1 0 20 40 60 80 100 120 Concentration (mg/mL) Time (Hours)

Figure 2.10: Results of MTT assay of Phellinus igniarius. (A) Dose-dependent assay for Fractions 1-4 of Phellinus igniarius. The graph is representative results from two different replicate (n=2). (B) Time-dependent assay for active Fraction 1 at 0.2 mg/mL and Fraction 3 at 1 mg/mL. The graph is representative results from two different replicate (n=2). Error bars are standard deviation.

A. B.

100 120 F2 100 Control 80 F4 F3 80 60 F1 60 40 F1 40 20 20 Cell Viability (% control) (% Viability Cell control) (% Viability Cell 0 0 0 0.2 0.4 0.6 0.8 1 0 20 40 60 80 100 120 Concentration (mg/mL) Time (Hours) Figure 2.11: Results of MTT assay of Trichaptum abietinum. (A) Dose-dependent assay for Fraction 1-4 of Trichaptum abietinum. The graph is representative results from two different replicate (n=2). (B) time-dependent assay for active Fraction 1 at 0.5 mg/mL. The graph is representative results from two different replicate (n=2). Error bars are standard deviation.

Finally, results from the dose-dependent assay of Trichaptum abietinum are shown in Fig 2.11(A). The results show the moderate anti-proliferative activity from

Fractions 1 and 3 that reduced cell viability to about 40% at 1 mg/mL each. On the other hand, Fractions 2 and 4 showed weak anti-proliferative activity. Fraction 2 reduced cell viability to only 90% and Fraction 4 to only 70%. The time-dependent MTT assay in Fig

2.11(B) shows that the bio-active Fraction 1 retained its moderate anti-proliferative activity. Fraction 1 was shown to reduce cell viability to 80% over a course of 5 days.

The loss in anti-proliferative activity observed in Fraction 1 from Trichaptum abietinum and Fraction 4 from Amanita muscaria, could result of chemical degradation because of the 2-week time period between dose-dependent MTT assay and time- dependent MTT assay. Although all the mushrooms fractions were stored at 4°C, it is possible that the bio-active molecule(s) could degrade with time.

2.4.4 Effect of TNF-alpha production in Raw 264.7 macrophage cells upon

treatment with mushroom fractions

Immuno-stimulatory activity of the mushroom extracts was assessed by their ability to induce TNF-α production in Raw 264.7 macrophage cells. TNF- α levels were measured using an Enzyme Linked Immunosorbent Assay (ELISA).

As seen in Figure 2.12(A), Fraction 4 from Amanita muscaria induced TNF-α to about 500 pg/mL in Raw264.7 cells after 6 hours of treatment. The other fractions from

Amanita muscaria had little or no effect. The negative controls water and ethanol induced TNF-α level to only 99 pg/mL and 30 pg/mL respectively. Figure 2.12(B) shows that Fraction 1 from Gyromitra esculenta, induced TNF-α level to approximately 2,000

pg/mL, whereas Fractions 2 and 3 could induce it over 3,000 pg/mL. On other hand, fraction 4 had no effect.

A. B.

6000 6000 * LPS

5000 * 5000 LPS

* 4000 4000 ______F2 F3

3000 (pg/ml) 3000 (pg/ml)   F1 TNF-

TNF- 2000 2000

* 1000 1000 F4 F4 F3 Water Water F2 F1 Ethanol 0 0 Amanita muscaria Gyromitra esculenta

Figure 2.12: Effect of fractions from A.muscaria (A) and G.esculenta (B) on TNF-α production in Raw 264.7 macrophage cells. LPS was positive control, ethanol was negative control for Fraction 1 and water was negative control for the other fractions. The graphs are representative results from 2 different replicates (n=2) (*, P<0.0001). Figure 2.13(A) shows that Fractions 2,3, and 4 from Paxillus obscurisporus induced TNF-α levels to 3,120 pg/mL, 3,350 pg/mL, and 4,425 pg/mL respectively.

Fraction 1 showed very weak bio-activity with an induction of 309 pg/mL. Water serving as a negative control induced TNF-α to only 42 pg/mL, suggesting that water had no effect on Raw 264.7 cells. The strongest immuno-stimulatory activity was demonstrated by the 4th fraction from Paxillus obscurisporus, with an induction of 4,425 pg/mL TNF-α.

Figure 2.13(B) shows that all the fractions from Phellinus igniarius had very weak or no bio-activity with regards to induction of TNF- α to less than 150 pg/mL.

A. B.

6000 6000 * * * LPS 5000 5000 LPS F4

4000 4000 F3 F2 (pg/ml)

 3000 3000 (pg/ml)  TNF- 2000 2000 TNF-

1000 1000 F1 F1 F2 F3 F4 Water Water Methanol 0 0 Paxillus obscurisporus Phellinus igniarius

Figure 2.13: Effect of fractions from Paxillus obscurisporus (A) and Phellinus igniarius (B) on TNF-α production in Raw 264.7 macrophage cells. LPS was positive control, and water was negative control. The graphs are representative results from 2 different replicates (n=2) (*, P<0.0001).

6000

5000 * LPS 4000 *

(pg/ml) 3000 F2 

TNF- 2000

1000 F4 F1 Water 0 Trichaptum abietinum

Figure 2.14: Effect of fractions from Trichaptum abietinum on TNF-α production in Raw 264.7 macrophage cells. LPS was positive control, water was negative control. The graph is representative results from 2 different replicates (n=2) (*, P<0.0001).

Finally, results from the immuno-stimulatory ELISA assay on fractions of

Trichaptum abietinum are shown in Figure 2.14. Fraction 2 induced TNF-α levels to

2,530 pg/mL, indicating a strong immuno-stimulatory activity. Fractions 1 and 4 exhibited weak activity by inducing TNF-α levels to 256 pg/mL and 312 pg/mL respectively. The results for Fraction 3 are unavailable as Fraction 3 was obtained at a very low amount during the chemical extraction. The amount available was consumed for anti-proliferative activity assessment experiments.

2.4.5 A summary of biological activity exhibited by mushroom in this

study and their previously reported bio-activities

After biological activity screening of the collected mushrooms, all the bio-activity screening results were examined to select a mushroom fraction for further purification.

The criteria considered included 1) strong bio-activity, 2) the novelty of the mushroom species (mushroom species unreported for anti-cancer activity would possess novel anti-cancer molecules), 3) availability in Northern BC, and 4) amount of dry powdered bio-active fraction obtained from extraction. The following is the summary of the bio- activities of five mushrooms generated in this study and their reported activities in the literature.

As shown in Table 2.4, Fraction 1 from Amanita muscaria reduced the cell viability by 89% and Fraction 4 exhibited very weak immuno-stimulatory activity with an induction of 585 pg/mL. Amanita muscaria is known to exhibit anti-tumor and immuno- stimulatory activities (Kiho et al., 1993; Wasser, 2002). Since it is a well-characterized mushroom, any fractions extracted from this mushroom would have a low possibility of

containing new anti-cancer compound(s). For this reason, none of the fractions obtained from Amanita muscaria were selected for further purification.

Table 2.4: Summary of bio-activity of BC wild mushrooms. Anti- Immuno- proliferative stimulatory activity Mushroom Species Fraction activity (pg/mL ofTNF-α (% inhibition) produced) 85% Ethanol 89% 14 (F1) 50% Methanol 16% 29 (F2) Amanita muscaria Hot water 13% 180 (F3) 5% Sodium 85% 585 hydroxide (F4) 85% Ethanol 28% 2,015 (F1) 50% Methanol 45% 3,210 (F2) Gyromitra esculenta Hot water 24% 3,191 (F3) 5% Sodium 14% 108 hydroxide (F4) 85% Ethanol (F1) 0% 310 50% Methanol 24% 3,120 Paxillus (F2) obscurisporus Hot water (F3) 45% 3,346 5% Sodium 56% 4,425 hydroxide (F4) 85% Ethanol (F1) 78% 46 50% Methanol 45% 85 (F2) Phellinus igniarius Hot water (F3) 59% 55 5% Sodium 35% 126 hydroxide (F4) 85% Ethanol (F1) 55% 256 50% Methanol 29% 2,530 (F2) Hot water (F3) 58% - Trichaptum abietinum 5% Sodium 9% 312 hydroxide (F4)

Fraction 2 from Gyromitra esculenta exhibited mild anti-proliferative activity by inhibiting the cell viability to 55%. On the other hand, Fractions 1, 2, and 3 from

Gyromitra esculenta exhibited strong immuno-stimulatory activity by inducing TNF-α levels to more than 2,000 pg/mL. Gyromitra esculenta has been reported to contain an hydrazine derivative toxin named Gyromitrin (Israël, 2012), that is considered to be dangerously toxic and carcinogenic. While collecting the supernatant for immuno- stimulatory ELISA assay, the Raw 264.7 cells treated with fractions from Gyromitra esculenta did not appeared to be toxic as compared to control-treated cells. Since there have been no previous studies on the immuno-stimulatory activity by Gyromitra esculenta, it maybe worthwhile in the future to investigate the immune-stimulatory compound(s) from this mushroom species.

Fractions 1 and 3 from Phellinus igniarius exhibited strong anti-proliferative activity, inhibiting the cell viability by 78% and 59% respectively. Although the anti- proliferative activity exhibited by these fractions from Phellinus igniarius was strong, it has already reported to exhibit strong anti-proliferative activity through possession of extracellular endo-polysaccharides (Chen et al., 2011; Song et al., 2008). Hence, none of the extracts obtained from Phellinus igniarius were chosen to purify any anti- proliferative compound(s).

Fractions 1 and 3 from Trichaptum abietinum exhibited moderate anti- proliferative activity, inhibiting cell viability by 55% and 58% respectively. Fraction 2 from Trichaptum abietinum exhibited strong immuno-stimulatory activity by inducing

TNF-α levels to 2,530 pg/mL. Trichaptum abietinum is a wood decay fungus that has

not been reported to possess any bio-activity. Although Trichaptum abietinum is available in northern BC in abundance, it closely resembles other polypore mushrooms like Trametes hirsuta, Trametes biforme, etc. This makes identification and collection of large samples of Trichaptum abietinum difficult, and increases the chances of cross contamination of this species. Since there are no reports about any bio-activity from

Trichaptum abietinum, it is a good candidate for further purification study in the near future. However, due to the challenges in collecting and identifying this mushroom species, Trichaptum abietinum was not pursued further in this study.

Finally, Fractions 3 and 4 from Paxillus obscurisporus exhibited moderate anti- proliferative activity, inhibiting the cell viability by 45% and 56%. Also, Fractions 2, 3, and 4 exhibited strong immuno-stimulatory activity by inducing TNF-α levels to 3,120 pg/mL, 3,346 pg/mL, and 4,425 pg/mL respectively. Paxillus obscurisporus has not been reported to have any bio-activity, and is closely related to Paxillus involutus. P. involutus has been reported to exhibit anti-proliferative activity by a 28kDa lectin (Wang et al., 2013).

The 4th fraction from Paxillus obscurisporus, exhibited moderate anti-proliferative and strong immuno-stimulatory activities. The availability of Paxillus obscurisporus in

Prince George and the surrounding area, its abundance and the fact that no prior studies has been reported on its anti-cancer activity, made Paxillus obscurisporus a good candidate for further purification of novel anti-cancer compound(s). Hence,

Fraction 4 from Paxillus obscurisporus was selected for further study described in

Chapter 3.

By the end of this chapter, two primary goals of this thesis were achieved that included: 1) collection and identification of five BC wild mushrooms and 2) extractions and bioactivity assessment of fractions extracted from five BC wild mushroom. Through the assessment of bioactivity of fractions obtained from five different BC wild mushrooms, it was deemed beneficial to further purify and chemically characterize the

5% sodium hydroxide extract (fraction 4) from Paxillus obscurisporus. The purification and chemical characterization of 5% sodium hydroxide extract (fraction 4) is described in Chapter 3.

Chapter 3 Purification and chemical characterization of an alkaline extract (fraction 4) from Paxillus obscurisporus

Fresh specimens of Paxillus obscurisporus were collected in the late summer of

2015 by Drs. Keith Egger and Hugues Massicotte and used to reassess biological activity. The purpose of this collection was to assess the reproducibility of bio-activity previously shown by Paxillus obscurisporus in Chapter 2. Thereafter, the goal described in this chapter was to perform purification and chemical characterization of the bio- active compounds present in 5% sodium hydroxide (NaOH) extract of Paxillus obscurisporus collected in 2015.

3.1 Methodology – Identification, extraction and biological activity screening

of new batches of Paxillus obscurisporus

3.1.1 Micro-morphological identification of the collected Paxillus

obscurisporus using ITS2 and NLB4 primers

The methodology for genetic identification of Paxillus obscurisporus using ITS2 and NLB4 primers was described in section 2.1.3 of Chapter 2.

3.1.2 Assessment of bio-activity of Paxillus obscurisporus collected in

2014

The anti-proliferative MTT assays and immuno-stimulatory ELISA assays were performed to assess the bio-activity of the 5% NaOH extract from Paxillus obscurisporus. The protocol used for the MTT assay is described in section 2.3.1 of

Chapter 2. Whereas, the immuno-stimulatory activity of Paxillus obscurisporus was

confirmed by performing Sandwich ELISA assay and the protocol used is described in section 2.3.2 of Chapter 2.

3.2 Methodology – Purification of fraction 4 from Paxillus obscurisporus using

size-exclusion chromatography

3.2.1 Size-exclusion/gel-permeation chromatography using a 20mL size

Sephadex™ LH-20 column

Sephadex™ LH-20 (GE Healthcare, Quebec) was the liquid chromatography medium used for size-exclusion chromatography. The resin was swelled in degassed water for three to five hours. For each gram of dry resin, 4 mL water was used for the resin to swell. A total of 5 g of the resin was swollen in 20 mL of water.

To make a 20 mL size column, a 25 mL serological pipette (Sarstedt, Nümbrecht,

Germany) was used to form a column. A 200 μL micropipette tip was loaded with glass wool and placed at the bottom of the 25 mL serological pipette to form a packing to clog the resin at the column end. The swollen Sephadex™ LH-20 was loaded onto the column in a continuous flow. A glass rod was placed at the mouth of the column to ensure continuous flow of resin, so that air would not get trapped while loading the resin onto the column. About 3-5 bed volumes of water were flushed through the column to ensure proper packing of the column.

The recommended sample volume for Sephadex™ LH-20 is 2% of the total bed volume. Therefore, 2 mL of 20 mg/mL stock solution from 5% NaOH extract of Paxillus obscurisporus was prepared and filtered using a 0.45 μm filter (Sarstedt, Nümbrecht,

Germany) to remove any large particulates. After the sample was loaded, water was run through the column continuously and twenty 1 mL fractions were collected. All the

collected fractions were then filter sterilized using a 0.2 μm filter (Sarstedt, Nümbrecht,

Germany) and stored at 4°C.

3.2.2 Purification using 120mL Sephadex™ LH-20 on a C16/70 column

To attain a higher yield of the purified fractions, a larger C16/70 column (GE

Healthcare, Quebec) with a total volume of 140 mL was used. About 30 g of dry

Sephadex™ LH-20 was swollen for about three to five hours using approximately 140 mL of degassed water to attain 120 mL of total bed volume. An AC 16 (GE Healthcare,

Little Chalfont, UK) flow adapter was placed on the bottom of the column. The adapter maintains the pressure and flow rate of the buffer in the column, while preventing the backflow of the buffer in the column. The swollen resin was loaded onto the column using a RC 16 (GE Healthcare, Little Chalfont, UK) reservoir and allowed to settle. The

AC 16 adapter was placed on the top-end of the column. The tubing attached to the AC

16 flow adapter was connected to a P-50 pump (Pharmacia Biotech, Sweden) that maintains the buffer flow rate. The buffer flow rate was maintained at 2 mL/min to ensure proper column packing. A three-way stopcock was used, to facilitate the loading of samples onto the column.

A 2.5 mL sample solution of 40 mg/mL was prepared by re-suspending the lyophilized 5% NaOH extract from Paxillus obscurisporus in water. The sample solution was centrifuged at 100 x g for 3 mins using Allegra® X-15R, benchtop centrifuge

(Beckman Coulter, CA, USA) to get rid of insoluble particulate. The three-way stopcock was directed from the idle port to the column where the syringe containing the sample solution was introduced. The sample was loaded onto the column at a flow rate of 1 mL/min.

After loading the sample solution onto the column, the stopcock direction was changed to direct the flow of the buffer from the pump to the column. The flow rate of 1 mL/min was maintained while the sample was running in the column. Twenty fractions with a fraction size of eight mL were collected and filter sterilized using a 0.2 μm filter

(Sarstedt, Nümbrecht, Germany) and stored at 4°C. After each use, the column was washed with five-bed volumes of water at 1.5 mL/min flow rate. This ensured the reusability of the resin present in the column and prevented cross contamination.

3.2.3 Purification using 530mL Sephadex™ LH-20 on a C26/100 column

To attain an even higher yield of the purified fractions, a larger C26/100 column

(GE Healthcare, Quebec) having a total volume of 530 mL was used. About 125 g of dry

Sephadex™ LH-20 was swollen for about three to five hours using approximately 500 mL of degassed water. An AC 26 (GE Healthcare, Quebec) flow adapter that maintains the pressure, flow rate and avoids backflow of the buffer, was placed on the bottom of the column. The swollen resin was loaded onto the column using a RC 26 (GE

Healthcare, Quebec) reservoir and allowed to settle in the column. Once the swollen resin was settled in the column, the packing was done by placing another AC 26 flow adapter on the top-end of the column. The tubing attached to the AC 16 flow adapter was connected to a P-50 pump (Pharmacia Biotech, Sweden) that maintained the buffer flow rate. While packing the column, the flow rate was maintained at 2 mL/min, and a three-way stopcock was used to load the samples onto the column.

A 10 mL sample solution at 40 mg/mL was prepared by re-suspending the lyophilized 5% NaOH extract from Paxillus obscurisporus in water. The sample solution was centrifuged at 100 x g for 3 mins using an Allegra® X-15R benchtop centrifuge

(Beckman Coulter, CA, USA) to get rid of the insoluble particulate. The three-way stopcock was directed from idle port to the column, where the syringe containing the sample solution was introduced. The sample was loaded onto the column at a flow rate of 1 mL/min.

After loading the sample solution onto the column, the stopcock direction was changed to direct the flow of water towards the column. A flow rate of 1 mL/min was maintained while the sample was running in the column. Forty 10 mL fractions were collected and filter sterilized using a 0.2 μm filter (Sarstedt, Nümbrecht, Germany). The filter sterilized fractions were then lyophilized using FREEZONE freeze dryer

(Labconco, MO, USA) at -52°C and 0.02 Torr, for 3 days, and stored at 4°C. After each use, the column was washed with five-bed volumes of water at 1.5 mL/min flow rate.

This ensures the reusability of the resin present in the column.

3.2.4 Assessing post-sephadex™ LH-20 fractions for bio-activity

Determination of anti-proliferative activity of the post-Sephadex™ LH-20 fractions from Paxillus obscurisporus was done using MTT assay. To assess the anti-proliferative activity on various cancer cell lines, both time-dependent and dose-dependent MTT assays were performed. The cancer cell lines used were: SKOV3 (ovarian cancer cells),

HCT116 (colon cancer cells), H441 (lung cancer cells), MCF7 (breast cancer cells) and

HepG2 (human liver carcinoma).

The protocol used for the MTT assay has been previously described in section

2.3.1 of Chapter 2. Whereas, the immuno-stimulatory activity of Paxillus obscurisporus was confirmed by performing the Sandwich ELISA assay. The protocol used for sandwich ELISA has been described in section 2.3.2 of Chapter 2.

3.2.5 Assessment of carbohydrate content in bio-active fractions

A Total Carbohydrate Assay Kit (Sigma-Aldrich®, St. Louis, MO, USA) was used to determine the presence of carbohydrates in the bio-active post-Sephadex™ LH-20 fractions. The lyophilized post-Sephadex™ LH-20 fractions were re-suspended in water to make final concentration of 1 mg/mL. A 96-well plate was used to perform the assay.

The carbohydrate standard provided with the kit was used as per the protocol at the concentrations: 0 mg/mL, 4 mg/mL, 8 mg/mL, 12 mg/mL, 16 mg/mL, and 20 mg/mL.

The fractions (30 µL each) and the prepared standard dilutions were added to the 96- well plate. To each well, 150 µL of sulphuric acid (12 M) was added and the 96-well plate was then incubated at 90°C using Haratherm™ General Oven (Thermo Fischer,

Waltham, MA, USA) for 15 mins. After the incubation, 30 μL of developer was added to each well. The plate was then subjected for absorbance reading at 490 nm using

SYNERGY 2 (BioTek®, VT, USA).

3.3 Methodology – Purification by means of anion-exchange chromatography

using DEAE Sephadex™

The DEAE Sephadex™ (GE Healthcare, Quebec) is an anion-exchanger that is based on Sephadex crossed-linked dextran matrix. Sephadex is widely used in plasma fractionation and other separation processes based on ionic charge of a sample.

Various buffer systems supported by the DEAE Sephadex™, which were mentioned in the user manual, were used (table 3.1). For preparation of the medium, the dry powdered DEAE Sephadex™ was weighed and mixed with the buffer system, and left to swell for about 48 hours. Later, the swollen DEAE Sephadex™ was introduced to a column, where 3 parts were swollen DEAE Sephadex™ and one part was the buffer.

Table 3.1: Buffers used in the anion-exchange DEAE Sephadex™ column chromatography. Buffer pH Range Concentration

N-Methyl Piperazine 4.3-5.3 20mM

Piperazine 4.8-5.8 20mM

TRIS 7.6-8.6 20mM

Diethanolamine 8.4-9.4 20mM

3.3.1 Purification through anion-exchange chromatography using Poly-

Prep® chromatography columns

For anion-exchange chromatography, Poly-Prep® chromatography columns with a maximum capacity of 12 mL were used. The column was loaded with a 2 mL slurry that contained 75% swollen resin and 25% buffer, forming the final bed volume. The column was washed with three to five bed volumes of buffer to equilibrate the column at the buffer’s pH. After the wash, the lyophilized anti-proliferative post-Sephadex™ LH-20 fractions from Paxillus obscurisporus were suspended in the running buffer at the concentration of 0.5 mg/mL. The post-Sephadex™ LH-20 fractions suspended in buffer were loaded onto the column and allowed to pass through the column by means of gravity. The column was flushed with the running buffer to attain the “flow-through”. A total of three flow-through fractions were collected; each of the 3 fractions had the same volume of 2 mL.

The flow-through obtained was subjected to 48 hour MTT assays to assess whether the bio-active compounds were bound to the DEAE Sephadex™ at any of the buffers tested. A 25 μL volume of start buffer was used as the negative control. Twenty-five μL

of post-Sephadex™ LH-20 fraction reconstituted in the buffer at 0.5 mg/mL was used as a positive control. Twenty-five μL of flow-through was used as a sample. The positive control, negative control and samples were added to HeLa cells consisting 175 μL

EMEM containing 10% fetal bovine serum.

3.3.2 Purification using 140mL DEAE Sephadex™ on a C16/70 column

To attain higher yield of purified anti-proliferative compounds from Paxillus obscurisporus, a larger C16/70 column (GE Healthcare, Quebec) having a total volume of 140 mL was used. About 19 g of dry DEAE Sephadex™ was swollen for about 48 hours using approximately 120 mL of degassed 20mM piperazine buffer with pH 5.5. An

AC 16 (GE Healthcare, Quebec) flow adapter, which maintains the pressure, flow rate, and prevents the backflow of the buffer, was placed at the bottom of the column. The swollen resin was loaded on the column using a RC 16 (GE Healthcare, Quebec) reservoir and allowed to settle in the column. Once the swollen resin was settled in the column, the packing was done by placing another AC 16 adapter on the top-end of the column. The tubing attached to the AC 16 flow adapter was connected to the P-50 pump (Pharmacia Biotech, Sweden) that maintained the buffer flow rate. While packing the column, the flow rate was maintained at 2 mL/min. A three-way stopcock was used so that while the samples were loaded onto the column, the pump could be stopped.

About 10 mL sample solution having a concentration of 40 mg/mL was prepared using the lyophilized bio-active anti-proliferative fractions from Paxillus obscurisporus in

20 mM piperazine, pH 5.5. The three-way stopcock was directed from idle port to the column, where the syringe containing the sample solution was introduced. The sample was loaded onto the column at a flow rate of 1 mL/min.

After loading the sample solution onto the column, the stopcock direction was changed so that the flow of the buffer from the pump could be directed to the column. A flow rate of 1 mL/min was maintained while the sample was loaded onto the column.

Twenty-five 12 mL flow-through fractions were collected and lyophilized using

FREEZONE freeze dryer (Labconco, MO, USA) at -52°C and 0.02 Torr, for 24 hours.

The lyophilized flow-through was then suspended in water and dialyzed using Slide-A-

Lyzer™ dialysis cassette (Thermo Fischer Scientific, MA, USA) with a molecular size cut-off (MWCO) of 2,000 Da. The dialysis eliminated the presence of the buffer components in the flow-through. The dialyzed flow-through was then filter-sterilized using 0.2 µm filters (Sarstedt, Nümbrecht, Germany) and stored at 4°C.

3.3.3 Assessment of anti-proliferative activity of post-DEAE Sephadex™

flow-through

The determination of the anti-proliferative activity of the post-DEAE Sephadex™ flow-through from Paxillus obscurisporus was performed using MTT assay. The anti- proliferative activity of the flow-through obtained was observed. A loss in anti- proliferative activity would suggest binding of the bio-active anti-proliferative compounds with DEAE Sephadex™. The protocol used for MTT assay was described in section

2.3.1 of Chapter 2.

3.3.4 Assessment of presence of protein and glycoprotein in post-DEAE

Sephadex™ flow-through

Results of the anti-proliferative activity assessment of post-DEAE Sephadex™ flow- through fractions mentioned in section 3.4.8 suggested that the bio-active compounds

were present in the flow-through. This was the reason that only the flow-through fractions collected were assessed for the presence of protein and glycoprotein.

SDS-PAGE coomassie blue (loading dye) stain was used to stain any proteins present in the post-DEAE Sephadex™ flow-through. The PagerRuler™ Plus (Thermo

Fischer Scientific, Waltham, MA, USA) pre-stained protein ladder having a range of 10 kDa to 250 kDa was used as a standard marker. A 12% SDS-PAGE gel was loaded with 30 μL post-DEAE Sephadex™ flow-throughs at 5 mg/mL along with the protein ladder. The gel was placed in the running buffer and a 200 V electrical current was applied.

To assess the presence of any glycoprotein and its size, the same procedure was followed as above. Pierce Glycoprotein Staining Kit (Thermo Fischer Scientific,

Waltham, MA, USA) was used. For staining the post-DEAE Sephadex™ flow-through, the electrophoresis of the gel was done by the same procedure as mentioned above for protein staining. After the electrophoresis, the gel was fixed by completely immersing it in 100 mL of 50% methanol for 30 mins. The gel was then washed by gently agitating with 100 mL of 3% acetic acid for 10 minutes. The wash step was repeated twice. After the wash, the gel was transferred to 25 mL of the oxidizing solution and gently agitated for 15 mins. The gel was then again washed by gently agitating with 3% acetic acid for 5 mins. The wash step was repeated for two more times. After the wash step, the gel was transferred to 25 mL of glycoprotein staining reagent and gently agitated for 15 mins.

The gel was then transferred to 25 mL of reducing solution and gently agitated for 5 mins. After treatment with the reducing solution, the gel was washed extensively with

3% acetic acid and then with ultrapure water. After the gels for protein and glycoprotein

were subjected to electrophoresis and stained as described as above, images of gels were taken using Fluorchem imager trans-illuminator to take pictures. The SDS gel staining experiments described above were performed entirely by Dr. Wai Ming Li and

Ms. Sumreen Javed.

3.4 Results and Discussion

3.4.1 DNA identification of the collected Paxillus obscurisporus

New specimens of Paxillus obscurisporus were collected by Dr. Hugues

Massicotte and Dr. Zoë Meletis in June 2015. The fruiting bodies of Paxillus obscurisporus were cleaned and dried at 55°C using a dryer. The dried fruiting bodies were then ground to powder using a conventional food processor. The powder obtained from the fruiting bodies was subjected to DNA identification using ITS2 and NLB4 primers. The morphological description and the DNA identification results are below:

Morphological Characteristics:

Type: Gilled mushroom fruiting body Size: Cap – 4-16cms, Stem – 2-5cms long and 0.4-3cms wide. Color: Cap – dark brown, Stem – dark cream - brown, Gills - cream Gills: Close, crowded and forking Spore Deposit: Brown to yellow brown Flesh: Thick and firm Habitat: Found densely populated on ground and woods

Matchmaker Mushroom match: Paxillus obscurisporus/Paxillus involutus

Sequencing match: Paxillus obscurisporus (C.Hahn, 1999)

Primer used: NLB4 and ITS3 Max score: 213 Total score: 213 Query covered: 21% Identification: 98% Accession: KF261386.1

The DNA sequence obtained is shown in the Appendix iii. As seen in the DNA sequencing results summarized above, the query covered by the DNA sequence obtained from the mushroom was only 21%. This indicated that there was only a 21% match between the sample DNA sequence and the matched DNA sequence from the

GenBank from the NIH database. Also, the quality of the DNA sequence obtained was poor. This could be because of cross-contamination of samples from related species occurring in the same area from where Paxillus obscurisporus was obtained or fungi growing parasitically on the fruiting bodies.

3.4.2 Bio-activity of Paxillus obscurisporus collected in 2015

As seen in figure 3.1(A), the 5% NaOH extract reduced the cell viability to 60% at

1 mg/mL. As compared to the anti-proliferative activity from the previous specimens

(collected in 2013), the 5% NaOH extract of Paxillus obscurisporus collected in 2015 did not seem to possess activity at a lower concentration. The previous batch of Paxillus obscurisporus (2013) reduced the cell viability to 40% at 0.2 mg/mL. Conversely, the

5% NaOH extract from 2015 shows a gradual decrease in cell viability with increasing dose.

Figure 3.1(B) shows that 1 mg/mL of 5% NaOH extract from Paxillus obscurisporus (2015) induced TNF-α to about 3,260 pg/mL after a 6-hour treatment on

RAW264.7 cells. As compared to the immuno-stimulatory activity of the 5% NaOH extract from Paxillus obscurisporus collected in 2013, there was a reduction of approximately 1,000 pg/mL of TNF-α.

A. 48Hrs. MTT Assay B. Immuno-stimulatory ELISA Assay

100

5000 80 LPS

4000 60 5% Na.OH Ex. 5% Na.OH 3000 40 (pg/mL)  2000 TNF- Cell Viability (% Control) (% Cell Viability 20 1000 0

0 0.2 0.4 0.6 0.8 1 (-ve) Control Concentration (mg/mL) 0

Figure 3.1: Result of anti-proliferative MTT and immuno-stimulatory ELISA assay of NaOH extract from P. obscurisporus: (A) the anti-proliferative dose-dependent MTT assay of 5% NaOH extract from Paxillus obscurisporus, where water was used as a negative control, (B) immuno-stimulatory ELISA assay where RAW264.7 cells were treated with 1 mg/mL of 5% NaOH extract. Water was used as a negative control and LPS was used as a positive control. Error bars are standard deviation. Although the newer batch of Paxillus obscurisporus showed reduced bio-activity, it was used for further purification and chemical characterization. The fruiting bodies of

Paxillus obscurisporus obtained in 2015 were generally smaller, indicating that the fruiting bodies collected were less mature perhaps. The fruiting bodies of Paxillus obscurisporus obtained in 2013 were twice as large compared to the ones from 2015.

This might indicate that the younger and less mature fruiting bodies of Paxillus obscurisporus might possess less of the compounds that are responsible for the anti- proliferative activity.

3.4.3 Bio-activity of post-Sephadex™ LH-20 fractions from a 16 mL drip

column

Results from the 48 hour MTT assay performed on the post-Sephadex™ LH-20 fractions obtained from the 5% NaOH extract of Paxillus obscurisporus are shown in

Figure 3.2. Results showed that fractions five and six reduced the HeLa cell viability by

70% and 50% respectively. The total bed volume of the column was 16 mL. The fraction size collected was 1.5 mL. This suggested that the anti-proliferative fractions five and six falls roughly on the 50% and 60% of total bed volume. The void volume is defined as the volume of mobile phase (buffer) passing through the gel required to elute the molecule(s) that never entered the stationary phase. The void volume for Sephadex™

LH-20 is roughly between 30-40% of the total bed volume as mentioned in the user manual provided by the manufacturer. This means that the bio-active fractions could be in the inclusion volume that falls in the range of the Sephadex™ LH-20 that is 100-4,000

Da (depending on molecular shape of compounds). Whereas, if the bio-active compound(s) are not in the inclusion limit (fraction 1-7), which constitutes 0-40% of bed volume, it would suggest that the bio-active molecule(s) are higher than 4,000 Da molecular weight.

A. 48hrs. MTT assay

120 9 Fr. Fr. 16 Fr. Fr. 15 Fr. Control Fr. 14 Fr. Fr. 2 Fr. Fr. 13 Fr. Fr. 1 Fr. Fr. 10 Fr. 100 11 Fr. Fr. 12 Fr. Fr. 4 Fr. Fr. 8 Fr. Fr.7 80 Fr. 3 Fr.

60 Fr. 6 Fr.

40 5 Fr. Cell Viability (%Control) Viability Cell 20

Figure 3.2: Results of MTT assay of post-Sephadex™ LH-20 fraction from 5% NaOH extract of P. obscurisporus: Effect of 100μL of post-Sephadex™ LH-20 fraction on HeLa cells viability. Cells were treated with fractions for 48 hours. Water was used as the negative control. The graph is representative results from two different replicate (n=2). Error bars are average. Results from the immuno-stimulatory ELISA assay performed on the post-

Sephadex™ LH-20 fractions obtained from 5% NaOH extract of Paxillus obscurisporus are shown in Figure 3.3. Results showed that water used as a negative control had very minimal or no effect. Fractions 8 to 17 showed no immuno-stimulatory activity. Fraction

4 was most active, inducing TNF-α to approximately 650 pg/mL and the immuno- stimulatory activity gradually falls from fractions 4 to 8.

A. Immuno-stimulatory ELISA Assay

5500

5000 LPS

4500

4000

3500

3000

(pg/mL) 2500  2000 TNF- 1500

1000 4 Fr. Fr. 5 Fr. 7 Fr. Fr. 6 Fr.

500 8 Fr. Fr. 9 Fr. Fr. 13 Fr. Fr. 14 15 Fr. 16 Fr. Fr. 12 Fr. Fr. 11 Fr. 1 Fr. 2 Fr. 3 Fr. 10 Fr. (-ve) (-ve) Control 0

Figure 3.3: Results of Immuno-stimulatory ELISA assay of post-Sephadex™ LH-20 fraction from the NaOH extract of P. obscurisporus: TNF-alpha production in Raw 264.7 cells upon treatment with 100 μL of fractions for 6 hours. LPS was used as a positive control and water was used as a negative control. Error bars are average. 3.4.4 Bio-activity of post-Sephadex™ LH-20 fractions from 120mL C16/70

column

After successful separation of the anti-proliferative and the immuno-stimulatory compounds attained through 16 mL column, the purification was scaled up using a larger C 16/70 (120 mL) column. Figure 3.4 (A) shows that fractions 6 and 7 obtained from the C 16/70 column, which had a bed volume of 120 mL, reduced HeLa cell viability by 90% and exhibits strong anti-proliferative activity. The elution pattern occurring in the larger C 16/70 column differed from the elution pattern of the smaller 16 mL column. Anti-proliferative fractions 6 and 7 fell in-between 34% to 47% of elution volume and may be in the inclusion limit. Although the active anti-proliferative

compounds were just occurring in the beginning of inclusion volume, they seemed to fall in Sephadex™ LH-20 operative/inclusion range.

A. 48 Hrs. MTT Assay

120 Control Fr. 9 Fr. Fr. 11 Fr.

100 1 Fr. Fr. 10 Fr. Fr. 17 Fr. Fr. 19 Fr. Fr. 5 Fr. Fr. 13 Fr. 15 Fr. Fr. 3 Fr. Fr. 8 Fr. 80

Cell Viability (% Control) (% Viability Cell 20 Fr. 7 Fr. Fr. 6 Fr.

B. Immuno-stimulatory ELISA Assay

4 1.2 10 LPS

1 104

8000

(pg/mL) 6000  TNF-

4000 09 Fr. Fr. 08 Fr. 10 Fr.

2000 11 Fr. Fr. 13 Fr. Fr. 17 Fr. Fr. 15 Fr. Fr. 05 Fr. Water Fr. 03 Fr. Fr. 06 Fr. 07 Fr. 0

Figure 3.4: Results of MTT and ELISA assay of post-Sephadex™ LH-20 fractions from NaOH extract of P. obscurisporus: (A) Effect of 100 μL of post-Sephadex™ LH-20 fraction on HeLa cells viability. Water was used as the negative control. The graph is representative results from two different replicate (n=2). Error bars are average. (B) TNF-alpha production in Raw 264.7 cells upon treatment with 100 μL of fractions for 6 hours. LPS was used as the positive control and water was used as the negative control. The graph is representative results from two different replicate (n=2). Error bars are standard deviation.

The result of the immuno-stimulatory ELISA assay performed on the post-

Sephadex™ LH-20 fractions obtained from 5% NaOH extract of Paxillus obscurisporus is shown in Figure 3.4(B). The result shows strong immuno-stimulation by fraction 8, 9,

10 and 11. The fractions eluting after fraction 9 showed gradual decrease in TNF-α production. Comparing the results of anti-proliferative MTT assay and immuno- stimulatory ELISA assay, it is evident that there was a clear separation of active anti- proliferative compounds (occurring in fractions 6 and 7) from active immuno-stimulatory fractions (occurring from fractions 8 to 17). RAW 264.7 cells were observed under the microscope after 6 hours of treatment with post-Sephadex™ LH-20 fractions and just before obtaining the supernatant, and there were no signs of cell death from any of the fractions.

To obtain large amounts of purified compounds from gel-permeation/size- exclusion chromatography, the C 16/70 column was run 6 times. Fractions obtained from each of the purification column were analyzed using MTT assay to confirm the reproducibility of the anti-proliferative activity. The trend of elution remained the same where the bio-active anti-proliferative compounds were eluted in fractions 6 and 7. Both

fractions 6 and 7 were dark yellow to brown in colour.

B. 48Hrs. MTT Assay A. 48hrs. MTT Assay

140 140 120 Fr. 11 Fr. Fr. 5 Fr. Fr. 17 Fr.

120 5-2 Fr. Control Control Fr. 1 Fr. Fr. 19 Fr. Fr. 13 Fr. Fr. 10 Fr. Fr. 5-3 Fr. Fr. 15 Fr. Control 100 Fr. 3 Fr.

100 5-1 Fr. Fr. 8 Fr. Fr. 9 Fr. Fr. 8-1 Fr. 80 8-2 Fr. 80 8-3 Fr. Fr. 7-2 Fr. Fr. 6-1 Fr. 60 7-3 Fr. 60 40

40 7-1 Fr. Fr. 6-3 Fr. Cell Viability (%Control) Viability Cell Fr. 6-2 Fr.

Cell Viability (% Control) (% Viability Cell 20 20 Fr. 6 Fr. 7 Fr. 0 0 0

Figure 3.5: Results of MTT assay of post-Sephadex™ LH-20 (C16/70 column) fractions from NaOH extract of P. obscurisporus: (A) Effect of 100 μL of post-Sephadex™ LH-20 fractions (obtained from the 3rd C16/70 column elution) on HeLa cells viability. Water was used as the negative control. The graph is representative results from two different replicate (n=2). (B) Effect of 100 μL of post-Sephadex™ LH-20 fractions on HeLa cells where the fractions were obtained from 5th C 16/70 column elution. The flow rate was at 0.5 mL/min. and 2.5 mL fractions were collected. Water was used as the negative control. The graph is representative results from four different replicate (n=4). Error bars are average. As shown in Figure 3.5 (A), fractions 6 and 7 obtained from the 3rd C 16/70 column elution had strong anti-proliferative activity, confirming the reproducibility between various columns. To attain a better resolution of separation/purification, the flow rate was reduced to 0.5 mL/min and fraction size was reduced to 2.5 mL in the 5th

C 16/70 column elution. The result shown in Figure 3.5(B) indicated that the active anti- proliferative compounds were concentrated in between fractions 6-2, 6-3, and 7-1.

Since there was no weak activity in between these sub-fractions, it may be assumed that these fractions could contain molecules of similar chemical nature. Figure 3.5 also indicates the reproducibility of the elution pattern between various columns.

3.4.5 Bio-activity of post-Sephadex™ LH-20 fractions using a 500 mL C

26/100 column

To obtain larger amounts of purified anti-proliferative compounds from 5% NaOH extract of Paxillus obscurisporus, the C 26/100 column (GE Healthcare, Quebec) was used. The total bed volume of this column was approximately 500 mL. The total amount of sample loaded was 400 mg (10 mL) of 5% NaOH extract of Paxillus obscurisporus.

The results from the 48 hours MTT assays are shown in Figure 3.6(A). The result suggests that fractions 17-24 showed strong anti-proliferative activity. Whereas, Figure

3.6(B) showed that fractions 25-35 exhibited immuno-stimulation by inducing TNF-α.

Fractions 28 and 29 showed strong immuno-stimulation by inducing TNF-α to approximately 2,700 pg/mL. The fractions eluting after fraction 28 showed gradual decrease in immuno-stimulation.

The elution volume at which the active anti-proliferative fractions (140-240 mL) was occurring in between 34-47% of the total bed volume. This elution pattern was similar to the elution pattern of the smaller C 16/70 column. Here again, a clear separation can be seen between active anti-proliferative fractions 17-24 and active immuno-stimulatory fractions 25-35, indicating two separate classes of biomolecules.

A. 48Hrs. MTT Assay B. Immuno-stimulatory ELISA Assay

120 8000 LPS

Control Control 7000 100 6000 Fr. 28 Fr. Fr. 29 Fr. Fr. 30 Fr. Fr. 27 Fr. Fr. 15 Fr. Fr. 32 Fr. 80 31 Fr. Fr. 26 Fr.

Fr. 16 Fr. 5000 Fr. 25 Fr.

60 (pg/mL) 4000  Fr. 28 Fr. 3000 29 Fr. TNF- 40 27 Fr. Fr. 26 Fr. Fr. 30 Fr. Fr. 24 Fr. Fr. 18 Fr. Fr. 19 Fr. 23 Fr. Fr. 33 Fr. Fr. 22 Fr. Fr. 20 Fr. 21 Fr. Fr. 17 Fr. 2000 31 Fr. Fr. 35 Fr. Cell Viability (% Control) (% Viability Cell Fr. 34 Fr. Fr. 25 Fr.

20 32 Fr. 1000 Fr. 24 Fr. Control (Water) Control 0 0

Figure 3.6: Results of MTT and ELISA assay of post-Sephadex™ LH-20 (C26/100) fractions from NaOH extract of P. obscurisporus: (A) Effect of 100 μL of post-Sephadex™ LH-20 fractions on HeLa cell viability. Water was used as the negative control. The graph is representative results from four different replicate (n=4). Error bars are average. (B) TNF-α production in Raw 264.7 cells upon treatment with 100 μL of fractions for 6 hours. LPS was used as the positive control and water was used as the negative control. Error bars are standard deviation. To confirm the reproducibility of the elution pattern of bio-active fractions on C

26/100 column, the 48 hours MTT assay and immuno-stimulatory ELISA assay were performed on each column eluted with 5% NaOH extract of Paxillus obscurisporus. It was evident that the elution pattern was very reproducible and the active anti- proliferative compounds were eluting out in the same region, in between 35-50% of total bed volume, whereas the active immuno-stimulatory compounds were eluting out just after 50% with earlier fractions, being more potent than the latter. Figure 3.7 (A and B) shows that the elution pattern for the active anti-proliferative and immuno-stimulatory compounds from 5% NaOH extract of Paxillus obscurisporus of column chromatography no. 4 was identical to the previous column runs. This shows that the replicated column chromatography performed on 5% NaOH extract of Paxillus obscurisporus had a very

consistent elution pattern for active anti-proliferative and immuno-stimulatory compounds.

A. 48hrs. MTT Assay B. Immuno-stimulatory ELISA Assay

8000

120 LPS

Control 7000 100 6000

80 5000 Fr. 24 Fr. Fr. 25 Fr. 60 (pg/mL) 4000  3000 Fr. 26 Fr. Fr. 27 Fr. Fr. 23 Fr. Fr. 25 Fr. TNF- Fr. 16 Fr.

40 28 Fr. Fr. 22 Fr. Fr. 20 Fr. 21 Fr. Fr. 29 Fr. Fr. 19 Fr. Fr. 17 Fr. Fr. 30 Fr. 2000 31 Fr. Fr. 32 Fr. Fr. 18 Fr. Cell Viabillity (% Control) (% Viabillity Cell Fr. 33 Fr. 20 34 Fr.

1000 35 Fr. Fr. 24 Fr. Control (Water) Control 0 0

Figure 3.7: Results of MTT and ELISA assay of post-Sephadex™ LH-20 (C26/100) fractions from NaOH extract of P. obscurisporus: (A) Effect of 100 μL of post-Sephadex™ LH-20 fractions on HeLa cell viability. Water was used as the negative control. The graph is representative results from four different replicate (n=4). (B) TNF-α production in Raw 264.7 cells upon treatment with 100 μL of fractions for 6 hours. LPS was used as the positive control and water was used as the negative control. Error bars are standard deviation. After a consistent elution pattern was confirmed, the column chromatography was performed with C 26/100 Sephadex™ LH-20 for a total of 8 times to collect large amounts of active anti-proliferative compounds to perform further purification. Fractions

16-23 were pooled together, filter sterilized, lyophilized, and stored at 4°C for further analysis.

3.4.6 Effect of bio-activity of post-Sephadex™ LH-20 fractions on various

cell lines

As shown in Figure 3.8, the post C2 6/100 column fraction showed little anti- proliferative activity at 0.01 mg/mL and the anti-proliferative activity was strongest at 0.5

mg/mL, where the cell viability was reduced to 40% in HepG2 and H441 cells. Whereas, the cell viability was reduced to approximately 20% in both HeLa and HCT116 cells at

0.5 mg/mL of the post-Sephadex™ LH-20 bio-active fraction.

A. 48Hrs. MTT Assay on HCT-116 Cells B. 48Hrs. MTT Assay on HeLa Cells

100 100

80 80 0.01mg/mL 0.01mg/mL 0.05mg/mL

60 0.1mg/mL 0.25mg/mL 60 0.05mg/mL

40 40 0.1mg/mL 0.25mg/mL

0.5mg/mL Cell Viability (% Control) (% Viability Cell Cell Viability (% Control) (% Viability Cell 20 20 0.5mg/mL

0 0 0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5

Concentration (mg/mL) Concentration (mg/mL)

C. 48Hrs. MTT Assay on HepG2 Cells D. 48Hrs. MTT Assay on H441 Cells

100 100 0.01mg/mL 0.05mg/mL 0.01mg/mL 80 80 0.1mg/mL 0.05mg/mL 0.1mg/mL 0.25mg/mL 60 0.25mg/mL 60

40 0.5mg/mL 40 0.5mg/mL Cell Viabiilty (% Control) (% Viabiilty Cell Cell Viabiilty (% Control) (% Viabiilty Cell 20 20

0 0 0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5 Concentration (mg/mL) Concentration (mg/mL)

Figure 3.8: Results of 48 hours MTT dose-dependent assay performed using post- Sephadex™ LH-20 fractions from 5% NaOH extract of P. obscurisporus on (A) HCT-116 (colorectal carcinoma) cells, (B) HeLa (cervical cancer) cells, (C) HepG2 (human liver carcinoma) cells and (D) H441 (human papillary adenocarcinoma) cells were treated with various concentration of post-Sephadex™ LH-20 fractions. Each graph is a representative result from two different replicate (n=2). Error bars are standard deviation.

A. 48Hrs. MTT Assay on MCF7 Cells B. 48Hrs. MTT Assay on SKOV-3 Cells

100 100

0.01mg/mL 80 80 0.1mg/mL 0.05mg/mL 0.01mg/mL 60 60 0.25mg/mL 0.05mg/mL

40 0.1mg/mL 40 0.5mg/mL Cell Viability (% Control) (% Viability Cell 20 Control) (% Viabiilty Cell 20

0.5mg/mL 0 0 0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5 Concentration (mg/mL) Concentration (mg/mL)

C. 48Hrs. MTT Assay on U251 Cells D. 48Hrs. MTT Assay on U87 Cells

100 100 0.01mg/mL 0.01mg/mL 0.05mg/mL 0.05mg/mL 80 80

60 0.1mg/mL 60 0.1mg/mL

40 40

0.25mg/mL 0.25mg/mL Cell Viability (% Control) (% Viability Cell 20 Control) (% Viability Cell 20

0.5mg/mL 0.5mg/mL 0 0 0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5 Concentration (mg/mL) Concentration (mg/mL)

Figure 3.9: Results of 48 hours MTT dose-dependent assay performed using post- Sephadex™ LH-20 fractions from 5% NaOH extract of P. obscurisporus. (A) MCF7 (human breast adenocarcinoma) cells, (B) SKOV3 (human ovarian adenocarcinoma) cells, (C) U251 (human glioblastoma) cells, and (D) U87 (human glioblastoma) cells were treated with various concentration of post-Sephadex™ LH-20 fraction. Error bars are standard deviation. Further results from the dose-dependent MTT assays are shown in Figure 3.9.

Results indicated a gradual reduction in cell viability with increasing dose of post C

26/100 column fractions from 5% NaOH extract of Paxillus obscurisporus. At 0.5 mg/mL, the post-Sephadex™ LH-20 fraction reduced the cell viability to about 15% in

U251 and U87 (human glioblastoma cells) as well as in MCF-7 (human breast adenocarcinoma) cells. The cell viability was reduced to 30% in SKOV3 (human ovarian adenocarcinoma) cells. An MTT assay using U87 and U251 cells was performed by fellow project member Mr. Faran Rashid.

As seen in the dose-dependent MTT assay on various cell lines, it is evident that the post-Sephadex™ LH-20 fraction showed strong anti-proliferation at 0.5 mg/mL in all the cell lines. Time- dependent MTT assay was next performed using 0.5 mg/mL of post-Sephadex™ LH-20 fractions. Results from the time-dependent experiments are shown in Figure 3.10 (A and B). These results showed that the post-Sephadex™ LH-20 fraction reduced the cell viability to about 7% at 48 hours in both HCT116 and HeLa cells. The cell viability remained at approximately 7% till 120 hours. At the 24-hour time point, there was an increase in cell viability to 30% in HCT116 cells.

A. Time Dependent MTT Assay on HCT116 Cells B. Time Dependent MTT Assay on HeLa Cells

100 100

80 80 Control Control 60 60

40 40

Cell Viability (% Control) (% Viability Cell 20 Cell Viability (% Control) (% Viability Cell 20

0 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Time (Hours) Time (Hours)

Figure 3.10: MTT time-dependent assay performed using post-Sephadex™ LH-20 fractions from 5% NaOH extract (0.5mg/mL) of P. obscurisporus. (A) HCT-116 (colorectal carcinoma) cells and (B) HeLa (cervical cancer) cells were treated with 0.5 mg/mL of post- Sephadex™ LH-20 fraction. Error bars are standard deviation. Further results from the time-dependent MTT assays are shown in Figure 3.11.

The results showed that the cell viability of HepG2 and H441 cells were reduced to 15% at 120 hours whereas the cell viability of MCF-7 and SKOV3 cells was reduced to approximately 20% at 48 hours. The cell viability was eventually reduced to about 10% at 120 hours. Water was used as a negative control in the time-dependent MTT assay, as the post-Sephadex™ LH-20 fractions were re-suspended in water.

A. Time Dependent MTT Assay on HepG2 Cells B. Time Dependent MTT Assay on H441 Cells

100 100

80 80

Control

60 Control 60

40 40

20 20 Cell Viability (% Control) (% Viability Cell Cell Viability (% ViabilityControl) (% Cell

0 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Time (Hours) Time (Hours)

C. Time Dependent MTT Assay on MCF7 Cells D. Time Dependent MTT Assay on SKOV3 Cells

100 100

80 80

60 Control Control 60

40 40

20 Cell Viability (% Control) (% Viability Cell 20 Cell Viability Control) Cell (%

0 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Time (Hours) Time (Hours)

Figure 3.11: MTT time-dependent assay performed using post-Sephadex™ LH-20 fractions from 5% NaOH extract (0.5mg/mL) of P. obscurisporus. (A) HepG2 (human liver carcinoma) cells, (B) H441 (human papillary adenocarcinoma) cells, (C) MCF-7 (human breast adenocarcinoma) cells, and (D) SKOV3 (human ovarian adenocarcinoma) were treated with 0.5 mg/mL of post-Sephadex™ LH-20 fraction. Error bars are standard deviation. It was observed from Figure 3.10 (A), 3.11 (A) and (B) that, at the 24 h time point, there was an increase in cell viability. The results suggested that the bio-active

molecules that are present in the Sephadex™ LH-20 fractions required a timeframe of about 48 hours to act on the cell to produce an anti-proliferative effect.

3.4.7 Carbohydrate content in bio-active fractions

The post-Sephadex™ LH-20 fractions obtained from the C 16/70 columns were subjected to total carbohydrate assay using a Total Carbohydrate Assay Kit (Sigma-

Aldrich®, St. Louis, MO, USA). The result from this assay is shown in Figure 3.12. The result suggested presence of about 366 μg/mL and 220 μg/mL of carbohydrate compound(s) in Fractions 6 and 7 respectively. This suggests that the anti-proliferative fractions contain carbohydrate molecules, which may be a component of the active compounds. The carbohydrate molecules remain at a lower concentration in Fractions 8 and 10 (approximately 80 μg/mL) and increases in fractions 10 and 11.

Figure 3.12: Total carbohydrate assay of post-Sephadex LH-20 fractions from C 16/70 column. Result from this experiment confirms the presence of carbohydrates in bio-active anti-proliferative fractions. Although carbohydrate molecules are present in few immuno-

stimulatory fractions (Fraction 8, 9, 10 and 11), the quantity seems to be lower than that of the anti-proliferative fractions. This may suggest the presence of a mixture of polysaccharides in the bio-active fractions, which may be inter-related structurally. This further supports the notion that the anti-proliferative and immuno-stimulatory fractions are separate and distinct molecules.

3.4.8 DEAE Sephadex™ anion-exchange chromatography using mini Poly-

Prep® chromatography columns

The pooled bio-active fractions from LH20-Sephadex were run through the

DEAE-Sephadex column. The flow-through obtained from DEAE Sephadex™ was analyzed for anti-proliferative activity using MTT assay. The anti-proliferative activity of the flow-through obtained was assessed through performing MTT assay. A loss in anti- proliferative activity would suggest binding of the bio-active anti-proliferative compounds with DEAE Sephadex™. Figure 3.13 suggested that there was no binding of bio-active anti-proliferative compounds with the DEAE Sephadex™ when 20 mM of 1-methyl- piperazine (pH 4.5) and 20 mM piperazine (pH 5.5) were used as buffers, as the flow- through showed anti-proliferative activity. Figure 3.13 (A) shows that the preload (post-

Sephadex™ LH-20 fraction resuspended in 20 mM 1-methyl-piperazine at 0.5 mg/mL) reduced the cell viability to 60%, indicating a loss in anti-proliferative activity when compared to the 0.5 mg/mL post-Sephadex™ LH-20 fraction serving as the positive control. Flow-through 1 and 2 reduced the cell viability to approximately 80%, suggesting some degree of binding of bio-active anti-proliferative compounds to the

DEAE Sephadex™. But the preload itself showed weak activity, which suggested that the buffer used (1-methyl-piperazine) may somehow reduce the activity of the bio-active

compound(s). The loss in activity could be the result of protonation of the bio-active compound at lower pH (4.5).

Figure 3.13(B) shows that the preload (post-Sephadex™ LH-20 fraction resuspended in 20 mM piperazine pH 5.5 at 05 mg/mL) reduced the cell viability to approximately 7%. On the other hand, flow-through 1 and 2 reduced the cell viability to

30% and 45% respectively. Flow-through 3 was unable to reduce the cell viability. This suggested that there was no binding of the bio-active compounds with the DEAE

Sephadex™.

A. 48Hrs. MTT Assay B. 48Hrs. MTT Assay

140 140

120 120 Control Control Flowthrough 3 Flowthrough 100 100 Flowthrough-3 Flowthrough-1 Flowthrough-2 80 80 Preload Preload (0.5mg/ml) 60 60 Flowthrough-2

40 40 Flowthrough-1 Cell Viability (% Control) (% Cell Viability Cell Viability (% Control) (% Viability Cell 20 20 Preload Preload (0.5mg/ml)

0 0

Figure 3.13: MTT assay performed on post-DEAE Sephadex flow-through: (A) 25 μL of flow-through obtained using 1-methyl-piperazine pH 4.5 buffer was added to HeLa cells, where 25 μL of buffer was used as a negative control, 25 μL post-Sephadex™ LH-20 fraction reconstituted in buffer (preload) at 0.5 mg/mL was used as a positive control, (B) 25 μL of flow- through obtained using piperazine pH5.5 as buffer, 25 μL of buffer was used as a negative control, 25 μL post-Sephadex™ LH-20 fraction reconstituted in buffer (preload) at 0.5 mg/mL was used as a positive control and 25μL of flow-through was the sample. Error bars are standard deviation. Figure 3.14(A) shows that the flow-through obtained using 20 mM Tris buffer at pH 7.4 had anti-proliferative activity; where flow-through 1 reduced the cell viability to

40%, flow-through 2 reduced the cell viability to 55% and flow-through 3 reduced the

cell viability to 67%. The preload (post-Sephadex™ LH-20 fraction was resuspended in

20 mM Tris pH 7.4) had potent anti-proliferative activity, reducing the cell viability to 5%.

This result suggests that there is no or weak binding between the active anti- proliferative compounds and the anion-exchange medium, as the flow-through showed relatively strong anti-proliferative activity.

Figure 3.14(B) shows that the flow-through 1, 2, and 3 obtained using diethanolamine pH 8.4 as a buffer; each flow-through showed anti-proliferative activity suggesting that there was little or no binding between active anti-proliferative compounds and the anion-exchange resin. Flow-through 1 reduced the cell viability to

60%, flow-through 2 reduced the cell viability to 80% and flow-through 3 reduced the cell viability to 77%. The preload (post-Sephadex™ LH-20 fraction re-suspended in buffer at 0.5 mg/mL) included as a positive control reduced the cell viability to 9%.

A. 48Hrs. MTT Assay B. 48Hrs. MTT Assay

140 140

120 120 Control Control 100 100 Flowthrough-2 Flowthrough-3

80 Flowthrough-3 80 Flowthrough-1 Flowthrough-2 60 60 Flowthrough-1 40 40 Cell Viability (% Control) (% Viability Cell Cell Viability (% Control) (% Viability Cell Preload Preload

20 20 (0.5mg/ml) Preload Preload (0.5mg/ml)

0 0

Figure 3.14: MTT assay performed on post-DEAE Sephadex™ flow-through: (A) 25 μL of flow-through obtained using 20 mM Tris pH 7.4 buffer was added to HeLa cells, where 25 μL of buffer was used as a negative control and 25 μL post-Sephadex™ LH-20 fraction reconstituted in buffer (Preload) at 0.5 mg/mL was used as a positive control, (B) 25 μL of flow-through obtained using 20 mM diethanolamine pH 8.4 as buffer, 25 μL of buffer was used as a negative control, 25 μL post-Sephadex™ LH-20 fraction reconstituted in buffer (Preload) at 0.5 mg/mL was used as a positive control and 25 μL of flow-through was the sample. Error bars are standard deviation. Overall, the results from the DEAE Sephadex™ suggest that there was very little binding between the active anti-proliferative post-Sephadex™ LH-20 fractions and the

DEAE Sephadex™ anion-exchange resin. After the flow-through was obtained from the column where 20 mM piperazine was used, the colored pigment in the fraction appeared to be retained by the column (Figure 3.15B). The flow-through obtained from this column under the conditions used was colourless. This suggests that the coloured pigment was interacting with the anion-exchange resin and likely binding to it.

Since there was no strong binding between the anion-exchange resin and the bio-active compound(s), and there was indication of removal of unwanted coloured compound(s) bound to DEAE Sephadex™, it was deemed logical to collect the flow-

through from the DEAE Sephadex™ as a mean to purify the bio-active anti-proliferative compound(s).

To confirm the reproducibility of the DEAE Sephadex™ anion-exchange chromatography using 20 mM piperazine pH 5.5, a 2 mL and 5 mL drip column were run simultaneously under the same conditions. The flow-through obtained from these columns was dialyzed, lyophilized and tested for anti-proliferative activity. The flow- through was dialyzed to remove the buffer component piperazine.

The results from the MTT assay are shown in Figure 3.15(A). The results show that the flow-through obtained from the 2 mL anion-exchange column reduced the cell viability to 40% and the flow-through obtained from the 5 mL anion-exchange column reduced the cell viability to 6%. This showed the reproducibility of the conditions in which separation occurred. The low anti-proliferative activity from flow-through obtained from the 2 mL column could be attributed to the over saturation of the anion-exchange medium; this occurs when the sample to be analysed has a higher ionic strength than 2 mmol/g of dry powder. Since the post-Sephadex™ LH-20 fractions from Paxillus obscurisporus was a mixture of various compounds, the ionic strength of the compound(s) was unknown. This may have caused the saturation of the anion- exchange resin, leading to occurrence of non-anti-proliferative compounds in the flow- through and reduced its activity. So, when the total bed volume of the column was increased to 5 mL, the flow-through obtained from the same sample at the same strength showed higher anti-proliferative activity, reducing the cell viability to 6%.

A. 48Hrs. MTT Assay B. 2mL Poly-Prep® column

140

120 Control 100

60 FT-1 FT-1 Column)(2ml 40 FT-1 (5ml Column) (5ml FT-1

Cell Control) (% Viabiilty Cell 20 Preload Preload

Figure 3.15: (A) MTT assay performed on post-DEAE Sephadex™ flow-through: flow- through obtained using 20 mM piperazine pH 5.5 buffer using 2 mL and 5 mL column were added to HeLa cells. Water was used as a negative control and post-Sephadex™ LH-20 fraction reconstituted in buffer labelled as “Preload” was used as a positive control at the concentration of 0.5 mg/mL. Error bars are standard deviation. (B) Binding of coloured pigment in the post-Sephadex™ LH-20 fraction with DEAE Sephadex™ after the flow-through was collected. 3.4.9 DEAE Sephadex™ anion-exchange chromatography using C 16/70

columns

Since the efficiency and reproducibility of the anion-exchange chromatography performed using 20 mM piperazine at pH 5.5 was assessed, this experiment was scaled up using a larger C 16/70 column under the same conditions. The result is shown in

Figure 3.16. The result showed that the purified flow-through reduced the cell viability to

5% at 0.5 mg/mL, to 57% at 0.02 mg/mL, and to 64% at 0.002 mg/mL.

A. 48Hrs. MTT Assay

120 Control 100

80 0.002mg/mL

60 0.02mg/mL

40 Cell Viability (% Control) (% Viability Cell 20 0.5mg/mL Preload (0.5mg/mL) Preload 0

Figure 3.16: MTT assay performed on post-DEAE Sephadex™ flow-through: (A) flow- through obtained using 20 mM piperazine pH 5.5 buffer and 75 mL C 16/70 column was added to HeLa cells. Water was used as a negative control, and post-Sephadex™ LH-20 fraction reconstituted in buffer labeled as “Preload” was used as a positive control at the concentration of 0.5 mg/mL. Error bars are standard deviation. To observe the specific activity of the flow-through fractions obtained using

DEAE Sephadex™, its anti-proliferative activity was compared to anti-proliferative activity of the fractions obtained through Sephadex™ LH-20. Figure 3.17 (A and B) shows the result of dose-dependent assays performed on post-DEAE Sephadex™ flow- through fractions and post-Sephadex™ LH-20 respectively. Results show that the flow- through fractions obtained from DEAE Sephadex™ inhibited the cell viability to 5% at

0.5 mg/mL, to 57% at 0.02 mg/mL and to 64% at 0.002 mg/mL. Whereas, the post-

Sephadex™ LH-20 fraction reduced the cell viability to 73% at 0.01 mg/mL, to 51% at

0.01 mg/mL and to 25% at 0.5 mg/mL. When compared, the anti-proliferative activity exhibited by post-DEAE Sephadex™ flow-through fractions was higher at lower doses than that of post-Sephadex™ LH-20 fractions. This was a clear indication that the

purification of post-Sephadex™ LH-20 fractions was successful when subjected to anion-exchange chromatography using DEAE Sephadex™.

A. 48Hrs. MTT Assay B. 48Hrs. MTT Assay

100 100

80 80 0.01mg/mL 0.002mg/mL 60 0.05mg/mL 60 0.02mg/mL

40 0.1mg/mL 0.25mg/mL 40 Cell Viability (% Control) (% Viability Cell Cell Viability (% Control) (% Viability Cell 20 0.5mg/mL 20

0.5mg/mL 0 0 0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5 Concentration (mg/mL) Concentration (mg/mL)

Figure 3.17: Dose-dependent MTT assays performed using (A) post-Sephadex™ LH-20 fractions and (B) post-DEAE Sephadex™ flow-through fractions on HeLa cells. Water was used as negative control. Error bars are standard deviation. 3.4.10 Assessing the presence of proteins and glycoproteins in bio-active

DEAE Sephadex™ fractions

The experiments described in this section were performed entirely by Dr. Wai

Ming Li and Ms. Sumreen Javed. To detect the possible presence of protein and glycoproteins in post-DEAE Sephadex™ flow-through from Paxillus obscurisporus, the samples were analyzed using SDS-PAGE gel staining with Coomassie blue for proteins components and other glycoprotein staining reagents for glycoprotein complexes

Results of the SDS-PAGE experiments stained for the presence of protein and carbohydrate are shown in Figure 3.18 (A and B) respectively. The result for protein staining showed that Polysaccharide-K (PSK), which is the well know polysaccharide- peptide isolated from the mushroom Trametes versicolor, exhibited a smearing pattern

IURPN'DWRN'D7KHRWKHUSRVLWLYHFRQWUROKRUVHUDGLVKSHUR[LGDVH +5 KDGD

EDQGLQEHWZHHQN'DZKLFKLVFRQVLVWHQWZLWKLWVNQRZQPROHFXODUZHLJKWRI

N'D7KHSXULILHGSRVW'($(IUDFWLRQIURP3D[LOOXVREVFXULVSRUXVDOVRVKRZVVPHDULQJ

SDWWHUQIURPN'DWRN'DZKLFKLVUHPDUNDEO\OLNHWKDWRI36.7KLVSDWWHUQ

VXJJHVWVWKDWWKHSXULILHGSRVW'($(VDPSOHIURP3D[LOOXVREVFXULVSRUXVFRQWDLQV

SURWHLQWKDWPD\EHYDULDEO\EUDQFKHGZKLFKPLJKWDIIHFWLWVPLJUDWLRQLQWKHJHOPDWUL[

DQGDIIHFWLWVDSSDUHQWPROHFXODUZHLJKW

$ %

)LJXUH6'63$*(DQDO\VLVIRUWKHSUHVHQFHRISURWHLQV $ DQGFDUERK\GUDWHV % 3RO\VDFFKDULGH.UHVWLQ 36. DQGKRUVHUDGLVK +5 SHUR[LGDVHZHUHXVHGDVSRVLWLYHFRQWURO 7KHVDPSOHVDQGFRQWUROVZHUHUXQRQD6'6SRO\DFU\ODPLGHJHO)RUSURWHLQGHWHFWLRQ $ WKHJHOZDVVWDLQHGZLWK&RRPDVVLHEOXH)RUFDUERK\GUDWHGHWHFWLRQ % WKHJHOZDV VWDLQHGXVLQJJO\FRSURWHLQVWDLQIURP3LHUFH*O\FRSURWHLQ6WDLQLQJNLW 7KHUHVXOWIURPWKH6'63$*(SHUIRUPHGWRGHWHFWWKHSUHVHQFHRI

FDUERK\GUDWHLVVKRZQLQ)LJXUH % $EDQGIRU+5SHUR[LGDVHDSSHDUVLQ

EHWZHHQN'DDQGN'DZKLFKZDVH[SHFWHGIRU+5SHUR[LGDVHZLWKPROHFXODU

weight of 44 kDa. A band for PSK acting as a positive control appears at 250 kDa with a smear extending from 35 kDa to 250 kDa. A faint band of post-DEAE fraction from

Paxillus obscurisporus could be seen at 250 kDa, suggesting presence of carbohydrate moiety.

At the end of Chapter 3, the 5% sodium hydroxide extract (fraction 4) from

Paxillus obscurisporus was successfully purified using two different purification techniques that included size-exclusion chromatography and ion-exchange chromatography. The purified fractions obtained from two different purification steps were subjected to chemical characterization experiments like SDS-PAGE and total carbohydrate assay. The results confirmed the presence of carbohydrate and protein moieties. This information will allow to develop a purification strategy for further purification of anti-proliferative compounds from 5% sodium hydroxide extract (fraction

4) of Paxillus obscurisporus.

Chapter 4

General Discussion

4.1 General project overview

Between 1981-2002, 60% of new anti-cancer drugs were developed from sources of natural origin (Cragg & Newman, 2005). In the early 2000’s, the majority of pharmaceutical companies turned to newer synthetic small molecule libraries in search of novel anti-cancer agents, despite the success of natural products (Butler, 2008). This lack of interest in using natural products as a source of discovery of new medicine was due to several reasons. It was recognized that purification and identification of medicinal compounds from natural products was a time-consuming process. The use of high-throughput screening (HTS) and increased speed of assays coupled with the ever increasing number of small molecule libraries as an alternative to natural product chemistry were a few reasons which attracted the interests of many pharmaceutical companies during the early 2000’s (Lam, 2007). However, in the last two decades, the lack of chemical diversity of synthetic small molecule libraries, disappointing results from the molecular libraries screened using HTS, recent advances in bioassays, and streamlined screening processes for natural products had rekindled the interest of pharmaceutical companies and other research groups towards natural products as a source of novel anti-cancer compounds (Eldridge et al., 2002; Ovadje et al., 2015).

Mushrooms have been reported to possess bio-active anticancer molecules

(Wasser, 2002). High molecular weight polysaccharides are best known to possess anti-tumor and immuno-modulatory activities. And lower molecular weight secondary

metabolites from mushrooms exhibiting antitumor activity (Ferreira et al., 2010). There is an abundance of mushroom species that are found in BC that have not been reported or studied for their anti-tumor activity. As seen in Table 1.3, BC is home to a wide range of biodiverse mushroom species that are yet to be studied for their potential anti-cancer properties.

The primary goal of this study was to discover novel anti-cancer compound(s) from

BC wild mushrooms. The specific aim of this study was to investigate various solvent extracts from five different British Columbian wild mushrooms for their potential anti- proliferative and immuno-modulatory activity, and to purify and characterize an anti- proliferative compound(s) from a selected mushroom extract.

4.2 DNA identification of the collected mushrooms

Upon collection of BC wild mushrooms, the samples were subjected to identification.

The identification of species was performed by DNA sequencing analysis. This was the first important endeavor because through proper identification of the mushroom species, the possibility of discovery of novel anti-cancer compound(s) was elevated by focusing on unstudied BC wild mushrooms. Through accurate identification, the focus was on mushroom species not reported to have any anti-cancer compound(s).

The fungal DNA Internal Transcribed Spacer (ITS) region is known to exhibit variations in mushroom species (Kindermann et al., 1998; Martin & Rygiewicz, 2005). In this study, DNA sequencing of the ITS2 region of fungal DNA was used as a tool to identify mushroom species and was performed using ITS3 and NLB4 primers. DNA extracted and amplified from the five collected mushroom species were subjected to

DNA sequencing. The results confirmed the identity of the five-mushroom species as 1)

Amanita muscaria, 2) Gyromitra esculenta, 3) Paxillus obscurisporus, 4) Phellinus igniarius, and 5) Trichaptum abietinum.

Accurate identification of the BC mushroom species was an important step, as it helped to select the mushroom species that were not reported or studied for their anti- cancer activity. Of the five mushroom species that were identified, Amanita muscaria and Phellinus igniarius were reported to possess anti-proliferative compounds. Amanita muscaria had been reported to possess bio-active anti-proliferative β-glucans from the alkaline extract (Kiho et al., 1992; Michelot & Melendez-Howell, 2003), and Phellinus igniarius had been reported to possess bio-active anti-proliferative endo- polysaccharides from the hot water extract (L. Chen et al., 2011). Although, both

Amanita muscaria and Phellinus igniarius were reported to possess anti-cancer properties, they were assessed for their anti-cancer properties in this study. This was because no reports/studies had been performed on the same mushroom species from

BC. The selection was done on the belief that Amanita muscaria and Phellinus igniarius from BC may possess novel anti-cancer compound(s) as compared to the same species obtained elsewhere around the world because of their interaction with a different ecological setting in BC.

By contrast, Gyromitra esculenta and Trichaptum abietinum were the mushroom species that are neither related to any of the bio-active species, nor had been reported to possess any bio-active compounds. Gyromitra esculenta is reported to possess a hydrazine derivative named N-methyl-N-formylhydrazine, known to be a carcinogenic agent (Toth & Nagel, 1978). Trichaptum abietinum is reported to be a wood decay

fungus (Kauserud & Schumacher, 2003). These two mushroom species are good candidates for future potential discovery of novel anti-cancer compounds.

Finally, Paxillus obscurisporus was the mushroom that has not been reported to possess any bio-active compounds. However, it is related to mushrooms like Paxillus involutus and Paxillus panuoides, that have been reported to possess anti-proliferative, immuno-stimulatory and radical scavenging compound(s) (Ranta et al., 2013; Yun, et al., 2000).

4.3 Assessment of biological activity of the BC wild mushrooms

After the selected mushroom species were subjected to chemical extraction, the extracts obtained were subject to MTT assays and ELISA assays for assessment of anti-proliferative and immuno-stimulatory activities respectively.

Our results indicated that the 85% ethanol extract from Amanita muscaria possessed strong anti-proliferative activity. Whereas, 5% sodium hydroxide (NaOH) extract from Amanita muscaria possessed mild immuno-stimulatory activity. This was an interesting finding as the 85% ethanol extracts from mushrooms are supposed to extract relatively small molecules (Mizuno, 1999) and Amanita muscaria is being reported to possess water insoluble β-glucans from the alkaline extract (Kiho et al., 1992; Michelot

& Melendez-Howell, 2003). This suggests that the anti-proliferative compound(s) present in the 85% ethanol extract could be novel anti-cancer compound(s), as no low molecular weight compound had been reported from Amanita muscaria to possess anti- proliferative activity.

The 85% ethanol, 50% methanol, and hot water extracts from Gyromitra esculenta possessed strong immuno-stimulatory activity, but none of the extracts exhibited strong

anti-proliferative activity. Gyromitra esculenta had been reported to contain a hydrazine derivative toxin named “Gyromitrin”. No other bio-active compound had been reported from Gyromitra esculenta. So, the finding of immuno-stimulatory activity from the extracts is novel.

The unstudied/unreported Paxillus obscurisporus showed potent anti-proliferative activity from 5% sodium hydroxide (NaOH) extract. Whereas, the 50% methanol, hot water, and 5% NaOH extract from Paxillus obscurisporus exhibited strong immuno- stimulatory activity. This is an important finding since there is no bio-activity reported from any extract of Paxillus obscurisporus in the literature.

The 50% methanol and hot water extract from Phellinus igniarius exhibited strong anti-proliferative activity. This was consistent with the reports earlier about extracellular endo-polysaccharides being reported from Phellinus igniarius. Strong immuno- stimulatory activity was exhibited by 50% methanol extract, whereas mild anti- proliferative activity was exhibited by 85% ethanol and hot water extract from

Trichaptum abietinum. This was an important finding since no bio-activity has been reported from any extract from Trichaptum abietinum.

The screening of various extracts from five mushroom species was an important endeavor. It offered the opportunity to confirm the reports about selected mushroom species like Amanita muscaria and Paxillus igniarius. It also offered the opportunity to discover anti-proliferative and immuno-stimulatory activity from unstudied/unreported mushroom species like Gyromitra esculenta, Paxillus obscurisporus, and Trichaptum abietinum. The knowledge of possession of anti-proliferative and immuno-stimulatory activity by certain extracts contributes to our goal in discovering novel natural anti-

cancer compounds. These bio-active compounds found in this screening could be further purified and chemically characterized for identification of anti-cancer compounds.

This screening helped to narrow down a more precise set of bio-active compound(s), that could be further purified and chemically characterized.

4.4 Selection of Paxillus obscurisporus for purification of anti-proliferative

compound(s)

To potentially discover novel natural anti-cancer compound(s), a set of criteria and priorities was outlined that included: mushroom extract from an unreported/unstudied species; strong anti-proliferative or immuno-stimulatory or both activities exhibited by the bio-active fraction; the bio-active extract must be from mushrooms readily available in Prince George and the surrounding area; and the mushroom species could be related to other mushroom species reported to possess bio-active anti-cancer properties.

Considering the above-outlined criteria, the 5% NaOH extract from Paxillus obscurisporus was selected for further purification and chemical characterization for this thesis. The 5% NaOH extract from Paxillus obscurisporus possessed moderate anti- proliferative and strong immuno-stimulatory activities, which made it a favorable extract for further studies. Also, Paxillus obscurisporus is related to Paxillus involutus, which is reported to possess an anti-proliferative lectin with a molecular mass of 24 kDa against lung cancer cells (A-549) and human colon cancer cells (HCT-8) (Wang et al., 2013).

Paxillus obscurisporus was collected by Drs. Keith Egger, Hugues Massicotte, and Zoë

Meletis in different years from Prince George, which indicates that Paxillus obscurisporus is readily availability in Prince George and the surrounding area.

Although the extracts from Trichaptum abietinum and Gyromitra esculenta exhibited strong bio-activities, they were not selected for further purification. This was because it was very hard to collect Trichaptum abietinum as it visually resembles other mushroom species like Trichaptum biforme, Trametes versicolor, Trichaptum laricinum and several other polypores, therefore increasing the chances of cross contamination during identification through DNA sequencing. Whereas, the extracts from the Gyromitra esculenta were not selected for further purification because the fractions only had potent immuno-stimulatory activity and very mild anti-proliferative activity. Gyromitra esculenta has also been reported to possess potent carcinogenic hydrazine derivatives that are very toxic in nature (Toth & Nagel, 1978). The immuno-stimulatory activity could be the result of the hydrazine derivatives, although there have been no studies to determine that. The bio-active extracts from both Gyromitra esculenta and Trichaptum abietinum are worth pursuing however in future studies.

As 5% NaOH extract from Paxillus obscurisporus was selected for further purification, it was important to reassess the bio-activity of the 5% NaOH extract from newly collected specimens of Paxillus obscurisporus. The DNA sequencing of the new specimens of Paxillus obscurisporus collected in 2015 revealed that its matched ITS2 region of Paxillus obscurisporus genome by only 21%. Although there was ambiguity about proper identification of the mushroom, the specimens collected in 2015 retained its anti-proliferative and immuno-stimulatory activities. Despite the questionable identity, the Paxillus obscurisporus collected in 2015 was used for further analysis.

4.5 Purification of anti-proliferative compound(s) from Paxillus obscurisporus:

Size-exclusion and anion-exchange chromatography

After 5% NaOH extract from Paxillus obscurisporus was selected for further purification and chemical characterization, it was initially subjected to size-exclusion chromatography. The Sephadex™ LH-20 size-exclusion medium separates the samples based on molecular size and has an operative range of 100 Da to 4000 Da, depending upon the shape of molecules. Since the 5% NaOH extract exhibited both anti-proliferative and immuno-stimulatory activities, it was expected that the

Sephadex™ LH-20 would separate both the anti-proliferative and immuno-stimulatory compound(s) if they are not the same.

Strong anti-proliferative activity was exhibited by fractions eluting out between 30%-

48% of total bed volume. This suggests that the compound(s) responsible for anti- proliferative activity have a high molecular weight, possibly above 4000 Da. This is because part of the bio-active fractions are eluted before the exclusion limit. Exclusion limit is defined as the molecular weight at the upper end of the column 'working' range and is where molecules are too large to be trapped in the stationary phase.

By contrast, the fractions containing immuno-stimulatory activity were eluted between 50%-90% of the total bed volume. This suggested that the fractions containing immuno-stimulatory activity were in the inclusion limit of the Sephadex™ LH-20, which is from 100-4000 Da. Since the immuno-stimulatory activity was exhibited by a series of fractions eluting from 50%-90%, it was considered that these compound(s) are in the inclusion limit. Compound(s) eluting out in these fractions could possibly range from high to low molecular weight (4000Da-1000Da or smaller). The results showed a clear

separation between the anti-proliferative and immuno-stimulatory compound(s) after the

5% NaOH extract was subject to size-exclusion chromatography.

Using size-exclusion chromatography was an important step for purification, as the anti-proliferative and immuno-stimulatory compounds could be separated using this technique. Since the focus was to purify the anti-proliferative fractions, the anti- proliferative fractions obtained from the size-exclusion chromatography were pooled and their anti-proliferative activity was assessed in various cancer cell lines. It was observed that the post-Sephadex™ LH-20 fractions showed strong anti-proliferative activity at low concentration of 0.5 mg/mL. The time-dependent anti-proliferative activity of post-Sephadex™ LH-20 fractions revealed a pattern, where all the cell lines shows a spike in cell viability at the 24 hour time point. This suggests that the bio-active post-

Sephadex™ LH-20 fractions took 48 hours to exert their anti-proliferative activity in cells. This was only false for human papillary adenocarcinoma (H441) cells, which might be attributed to the slower growth rate of this cell line.

After the size-exclusion chromatography, anion-exchange chromatography was performed as a second step for further purification of the anti-proliferative compound(s).

The weak anion-exchange medium DEAE Sephadex™, which is positively charged, binds to any negatively charged compound(s) present in the sample that is passed through it. Several pH conditions were used with several buffers to determine the appropriate conditions for purification using the anion-exchange medium DEAE

Sephadex™. A significant amount of time was spent to optimize the buffer, pH conditions and sample concentrations, which was suitable for purifying the bio-active compound(s) using DEAE Sephadex™. The search for optimized conditions led to the

conclusion that none of the conditions studied allowed the binding of the anti- proliferative compound(s) to the DEAE Sephadex™. This suggested that the anti- proliferative compound(s) are positively charged molecules that were not able to bind to

DEAE Sephadex™.

The most common procedure to use DEAE Sephadex™ as a purification step is to let the molecules of interest (anti-proliferative compounds) bind to the ion-exchanger and allow the others to pass through. However, since it was known that the anti- proliferative compound(s) from Paxillus obscurisporus did not bind to the DEAE

Sephadex™ in any given conditions, it was deemed more beneficial to bind the non-bio- active contaminants to the DEAE Sephadex™ and let the molecules of interest remain in the flow-through. Piperazine 20 mM at pH 5.5 appeared to be the best buffer for this purpose, as the anti-proliferative activity was retained in the flow-through. Hence, piperazine 20 mM at pH 5.5 was used to perform anion-exchange chromatography to collect an ample amount of purified anti-proliferative compound(s) from Paxillus obscurisporus. The purified anti-proliferative compound(s) were further subjected to various assays for chemical characterization. The anion-exchange chromatography using DEAE Sephadex™ was an important second step for purification after size- exclusion chromatography, as it ensured further purification of the anti-proliferative compounds.

4.6 Assessment of the chemical nature of bio-active compound(s) from

Paxillus obscurisporus

By use of size-exclusion and anion-exchange chromatography, the purification of anti-proliferative compound(s) from 5% NaOH extract of Paxillus obscurisporus was

ensured firstly based on molecular size (size-exclusion), and then based on net ionic charge (anion-exchange) possessed by the molecule(s). To help understand the mechanism of action and to further purify the anti-proliferative compound(s) from

Paxillus obscurisporus, it was very important to understand the chemical nature of the semi-purified anti-proliferative compound(s). To know more about the chemical nature of the anti-proliferative compound(s), it was firstly subjected to a carbohydrate assay.

This was because majority of the pharmacologically active compound(s) reported to be found from mushroom species are polysaccharides (Ren et al., 2013; Zhang et al.,

2015).

The post-Sephadex™ LH-20 fractions were subjected to the total carbohydrates assay and the results confirmed the presence of a polysaccharide moiety. It was observed that the concentration of carbohydrates was the highest in the fractions containing the anti-proliferative activity. Whereas, the concentration of the carbohydrates was lower in few fractions exhibiting immuno-stimulatory activity that were eluted just after the immuno-stimulatory fractions (please see Figure 3.12). This finding is critical for deciding the next step in performing the chemical characterization experiments on the purified anti-proliferative compound(s).

Since the fungal polysaccharides are considered as macromolecules, the post-

DEAE Sephadex™ anti-proliferative compound(s) were subjected to SDS-PAGE technique, which separates the macromolecules on basis of their electrophoretic mobility. For assessing the presence of proteins, Coomassie blue was used as a stain and for glycoprotein a glycoprotein stain was used. Based on the SDS-PAGE staining experiments, the presence of protein and glycoprotein was confirmed. The presence of

a smear for both protein and glycoprotein in the sample suggested that the protein/glycoprotein are present in the anti-proliferative compound(s) from Paxillus obscurisporus. The smear may indicate the presence of a chained compound with fragments ranging from 10 kDa to 250 kDa for protein and from 35 kDa to 250 kDa for glycoprotein.

Since there are several factors affecting the migration of the compound(s) while performing SDS-PAGE, a definitive conclusion cannot be drawn about the molecular weight of the anti-proliferative compound(s) from Paxillus obscurisporus. In summary, it can be concluded that the anti-proliferative compound(s) from 5% NaOH extract of

Paxillus obscurisporus are positively charged molecules, which are likely to contain a mixture of chained proteins/glycoproteins with a varying range of molecular units.

4.7 General overview and concluding remarks

Mushroom extracts have historically been used as prophylactics and for treating several diseases (Israilides & Philippoussis, 2003). The medicinal compounds extracted from mushrooms could be divided into high molecular weight compounds and lower molecular weight organic compounds that are secondary metabolic products of various pathways (amino acid-derived pathways, the shikimic acid pathway for the biosynthesis of aromatic amino acids, and the acetate–malonate pathway from acetyl coenzyme A)

(Zaidman et al., 2005). The fungal polysaccharides are one of the most potent mushroom derived bio-active macromolecules. For example, β-glucans from mushroom origins exhibit anti-cancer activity by stimulation of natural killer cells, T-cells, B-cells, macrophage-dependent immune system responses through distinct receptors. The

receptors it stimulates includes dectin-1, the toll-like receptor-2, scavenger receptors, apoptosis, and lactosylceramide (Brown & Gordon, 2005; Dalmo & Bøgwald, 2008).

In this study, several extracts obtained from five BC wild mushroom species were found to be either immuno-stimulatory or anti-proliferative or both. This was an important finding as bio-active fractions from mushroom species like Gyromitra esculenta, Paxillus obscurisporus, and Trichaptum abietinum were found to be sources of potential novel anti-cancer compounds. Although Amanita muscaria has been reported to have β-glucans possessing anti-proliferative activity, the anti-proliferative activity shown by 85% ethanol extract was an interesting finding. This was because the

β-glucans are water insoluble polysaccharides that are reported to be extracted in 5%

NaOH (Michelot & Melendez-Howell, 2003; Mizuno, 1999). So, further purification of the ethanol extract suggests the possible discovery of potential small molecule anti-cancer compound(s).

The strong anti-proliferative activity exhibited by the post-Sephadex™ LH-20 fractions of Paxillus obscurisporus, was one of the important findings. This was because the anti-proliferative activity shown by 0.5 mg/mL post-Sephadex™ LH-20 fractions was found to be more potent than the 1 mg/mL crude 5% NaOH extract from Paxillus obscurisporus. This was an indication that the purification using size-exclusion chromatography was a successful and useful purification step. The optimization of various buffers and pH conditions revealed that none of the buffer and pH conditions that were tested was successful to bind the ™LH-20 fractions to DEAE Sephadex™.

This was an important finding as it helped to conclude that the post-DEAE Sephadex™ anti-proliferative compounds from Paxillus obscurisporus might possess a positive net

surface charge. Another important finding from this study includes the determination of the presence of carbohydrate and protein moieties in the fractions obtained from the size-exclusion and anion-exchange chromatography of 5% NaOH extract from Paxillus obscurisporus. This was an important finding as it will help to select various techniques to further purify chemically and biochemically characterize the bio-active compound(s) in future studies.

Although the 5% NaOH extract from Paxillus obscurisporus was purified using size- exclusion and anion-exchange chromatography, further purification and characterization is needed. As deduced from the results of anion-exchange chromatography, the anti- proliferative compound(s) obtained from the 5% NaOH extract of Paxillus obscurisporus seemed to have a net positive surface charge. This conclusion helps to select cation- exchange chromatography as a possible future purification technique. The benefit of this mode of purification may involve binding of the active anti-proliferative compound(s) to the cation-exchange medium to permit further purification.

The chemical characterization of post-DEAE Sephadex™ flow through, confirmed the presence of proteins and carbohydrate moieties. Common methods used in purification and characterization of fungal polysaccharides or glycoproteins include Gas

Chromatography/High Performance Liquid Chromatography, Nuclear Magnetic

Resonance and Infrared Chromatography analysis of the hydrolyzed fungal extracts

(Kiho et al., 1993; Sun et al, 2004; Zhang et al., 2015). If a polysaccharide or glycoprotein is the compound(s) responsible for the anti-proliferative activity, these analytical methods would provide useful information as to the types of sugar moieties the polysaccharides are constituted of, type of linkages between them, etc.

Lastly, it is of prime importance to understand the molecular mechanism through which the anti-proliferative activity is being exhibited by compound(s) from 5% NaOH extract of Paxillus obscurisporus. In order to understand this, the expression of various apoptosis-related proteins like Bcl-2, Bcl-3, Bax, and procaspase-3 in the cell lines could be performed by Western blot analysis (Oh et al., 2008).

Overall, this study serves as a significant platform for future investigations into discovering novel anti-cancer compounds from BC wild mushrooms. The novel bio- active extracts from mushrooms species like Gyromitra esculenta, Paxillus obscurisporus, and Trichaptum abietinum serve as potential sources for the discovery of novel anti-cancer compounds. With the successful purification of anti-proliferative compound(s) from Paxillus obscurisporus using size-exclusion and anion-exchange chromatography, and the confirmation that it contains proteins and carbohydrates, future experiments can now be logically planned to further purify the compound(s) and to further understand its mechanism of action and its chemical composition.

References

Baskar, R., Lavanya, R., Mayilvizhi, S., & Rajasekaran, P. (2008). Free radical

scavenging activity of antitumour polysaccharide fractions isolated from

Ganoderma lucidum (Fr.) P. Karst. In Indian Journal of Natural Products and

Resources, 7(4), 320–325.

Biliaderis, C. G., & Izydorczyk, M. S. (2007). Arabynoxylans: technologically and

nutritionally functional plant polysaccharides. In Functional Food Carbohydrates

(pp. 167–215). Boca Raton, FL, USA: CRC Press.

Bisen, P. S., Baghel, R. K., Sanodiya, B. S., Thakur, G. S., & Prasad, G. B. K. S.

(2010). Lentinus edodes: a macrofungus with pharmacological activities. Current

Medicinal Chemistry, 17(22), 2419–2430.

http://doi.org/10.2174/092986710791698495

Brown, G. D., & Gordon, S. (2005). Immune recognition of fungal Beta-glucans. Cellular

Microbiology, 7(4), 471–479. http://doi.org/10.1111/j.1462-5822.2005.00505.x

Butler, M. S. (2008). Natural products to drugs: natural product derived compounds in

clinical trials. Natural Product Reports, 22(2), 162–195.

http://doi.org/10.1039/b402985m

Chan, W. K., Law, H. K. W., Lin, Z. Bin, Lau, Y. L., & Chan, G. C. F. (2007). Response

of human dendritic cells to different immunomodulatory polysaccharides derived

from mushroom and barley. International Immunology, 19(7), 891–899.

http://doi.org/10.1093/intimm/dxm061

Chen, L., Pan, J., Li, X., Zhou, Y., Meng, Q., & Wang, Q. (2011). Endo-polysaccharide

of Phellinus igniarius exhibited anti-tumor effect through enhancement of cell

xii

mediated immunity. International Immunopharmacology, 11(2), 255–259.

http://doi.org/10.1016/j.intimp.2010.11.033

Chen, Y., Hu, M., Wang, C., Yang, Y., Chen, J., Ding, J., & Guo, W. (2013).

Characterization and in vitro antitumor activity of polysaccharides from the

mycelium of Sarcodon aspratus. International Journal of Biological

Macromolecules, 52(1), 52–58. http://doi.org/10.1016/j.ijbiomac.2012.09.005

Chu, K. K. W., Ho, S. S. S., & Chow, A. H. L. (2002). Coriolus versicolor: a medicinal

mushroom with promising immunotherapeutic values. Journal of Clinical

Pharmacology, 42(9), 976–984. http://doi.org/10.1177/009127002401102894

Coughlin, S. S., & Ekwueme, D. U. (2009). Breast cancer as a global health concern.

Cancer Epidemiology, 33(5), 315–318. http://doi.org/10.1016/j.canep.2009.10.003

Cragg, G. M., & Newman, D. J. (2005). Plants as a source of anti-cancer agents.

Journal of Ethnopharmacology, 100(1–2), 72–79.

http://doi.org/10.1016/j.jep.2005.05.011

Dalmo, R. A., & Bøgwald, J. (2008). Beta-Glucans as conductors of immune

symphonies. Fish and Shellfish Immunology, 25(4), 384–396.

http://doi.org/10.1016/j.fsi.2008.04.008

Dias, E. S., Abe, C., & Schwan, R. F. (2004). Truths and myths about the mushroom

Agaricus blazei. Scientia Agricola, 61(5), 545–549. http://doi.org/10.1590/S0103-

90162004000500014

Dotan, N., Wasser, S. P., & Mahajna, J. (2011). Inhibition of the androgen receptor

activity by Coprinus comatus substances. Nutrition and Cancer, 63(8), 1316–1327.

http://doi.org/10.1080/01635581.2011.607542

xiii

Eldridge, G. R., Vervoort, H. C., Lee, C. M., Cremin, P. A., Williams, C. T., Hart, S. M.,

Georing, M. G., O'Neil-Jhonson, M., Zeng, L. (2002). High-throughput method for

the production and analysis of large natural product libraries for drug discovery.

Analytical Chemistry, 74(16), 3963–3971. http://doi.org/10.1021/ac025534s

Ferreira, I. C. F. R., Vaz, J. a, Vasconcelos, M. H., & Martins, A. (2010). Compounds

from wild mushrooms with antitumor potential. Anti-Cancer Agents in Medicinal

Chemistry, 10(5), 424–436. http://doi.org/10.2174/1871520611009050424

Fisher M, Y. L. (2002). Anticancer effects and mechanisms of polysaccharide-K (PSK):

implications of cancer immunotherapy. Anticancer Research, 3(22), 1737–1754.

Giavasis, I. (2013). Production of microbial polysaccharides for use in food. Microbial

Production of Food Ingredients, Enzymes and Nutraceuticals. 413-468

http://doi.org/http://dx.doi.org/10.1533/9780857093547.2.413

Golub, T. R. (1999). Molecular classification of cancer: class discovery and class

prediction by gene expression monitoring. Science, 286(5439), 531–537.

http://doi.org/10.1126/science.286.5439.531

Halpern, G. M. (2006). Healing Mushrooms : Effective Treatments for Todays Illnesses.

Medicinal Mushrooms Revealed, 2, 5. Square One Publication Inc.

http://doi.org/15/01/2007

Han, C. H., Liu, Q. H., Ng, T. B., & Wang, H. X. (2005). A novel homodimeric lactose-

binding lectin from the edible split gill medicinal mushroom Schizophyllum

commune. Biochemical and Biophysical Research Communications, 336(1), 252–

257. http://doi.org/10.1016/j.bbrc.2005.08.068

Harada, N., Kodama, N., & Nanba, H. (2003). Relationship between dendritic cells and

xiv

the D-fraction-induced Th-1 dominant response in BALB/c tumor-bearing mice.

Cancer Letters, 192(2), 181–187. http://doi.org/10.1016/S0304-3835(02)00716-4

Harvey, A. L., Edrada-Ebel, R., & Quinn, R. J. (2015). The re-emergence of natural

products for drug discovery in the genomics era. Nature Reviews Drug Discovery,

14(2), 111–129. http://doi.org/10.1038/nrd4510

Hennell, J. R., Agostino, P. M. D., Lee, S., Khoo, C. S., & Sucher, N. J. (2012). Using

GenBank ® for genomic authentication : a tutorial, (February).

http://doi.org/10.1007/978-1-61779-609-8

Hildebrand, J. (2006). Neurological complications of cancer chemotherapy. Current

Opinion in Oncology, 18(4), 321–4.

http://doi.org/10.1097/01.cco.0000228735.39885.3e

Ichimura, T., Watanabe, O., & Maruyama, S. (1998). Inhibition of HIV-1 protease by

water-soluble lignin-like substance from an edible mushroom, Fuscoporia obliqua.

Bioscience, Biotechnology, and Biochemistry. 62(3), 575-577.

http://doi.org/10.1271/bbb.62.575

Israël, M. (2012). A primary cause of cancer : GABA deficiency in endocrine pancreas

Review Article. Cancer Therapy, 8, 171–183.

Israilides, C., & Philippoussis, A. (2003). Bio-technologies of recycling agro-industrial

wastes for the production of commercially important fungal polysaccharides and

mushrooms. Biotechnology & Genetic Engineering Reviews, 20(June), 247–259.

http://doi.org/10.1080/02648725.2003.10648045

JD Zhao, X. Z. (1994). Resources and taxonomy of Ling Zhi (ganoderma) in China.

Proceedings of International Symposium Ganoderma Research,(October 24-26,).

Beijing Medical University Beijing, China.

Jemal, A., Bray, F., & Ferlay, J. (1999). Global Cancer Statistics: 2011. CA Cancer J

Clin, 49(2), 1,33-64. http://doi.org/10.3322/caac.20107.

Kahlos, K., & Tikka, V. H. (1994). Antifungal activity of cysteine, its effect on C-21

oxygenated lanosterol derivatives and other lipids in Inonotus obliquus, in vitro.

Applied Microbiology and Biotechnology, 42(2–3), 385–390.

http://doi.org/10.1007/s002530050267

Kauserud, H., & Schumacher, T. (2003). Regional and local population structure of the

pioneer wood- decay fungus Trichaptum abietinum. Mycologia, 95(3), 416–425.

http://doi.org/10.1017/S0953756203007214

Kiho, T., Hui, J., Yamane, A., & Ukai, S. (1993). Polysaccharides in fungi.XXXII.i)

hypoglycemic activity and chemical properties of a polysaccharide from the cultural

mycelium of Cordyceps sinensis. Biol. Pharm. Bull., 16(21), 1291–1293.

Kiho, T., Katsurawaga, M., Nagai, K., Ukai, S., & Haga, M. (1992). Structure and

antitumor activity of a branched (1-3)-Beta-d-glucan from the alkaline extract of

Amanita muscaria. Carbohydrate Research, 224(C), 237–243.

http://doi.org/10.1016/0008-6215(92)84109-6

Kim, K. C., Kim, J. S., Son, J. K., & Kim, I. G. (2007). Enhanced induction of

mitochondrial damage and apoptosis in human leukemia HL-60 cells by

Ganoderma lucidum and Duchesnea chrysantha extracts. Cancer Letters, 246(1–

2), 210–217. http://doi.org/10.1016/j.canlet.2006.02.014

Kim, S., Hwang, B. S., Song, J., Lee, S. W., Lee, I., & Yun, B. (2013). New bioactive

compounds from Korean native mushrooms. Mycobiology, 41(4), 171–176.

xvi

Kim, Y. J., Park, J., Min, B. S., & Shim, S. H. (2011). Chemical constituents from the

sclerotia of Inonotus obliquus. Journal of Applied Biological Chemistry, 54(2), 287–

294. http://doi.org/10.3839/jksabc.2011.045

Kindermann, J., El-Ayouti, Y., Samuels, G. J., & Kubicek, C. P. (1998). Phylogeny of the

genus Trichoderma based on sequence analysis of the Internal Transcribed Spacer

region 1 of the rDNA cluster. Fungal Genetics and Biology, 24(3), 298–309.

http://doi.org/10.1006/fgbi.1998.1049

Kukulyanskaya, T. A., Kurchenko, N. V., Kurchenko, V. P., & Babitskaya, V. G. (2002).

Physicochemical properties of melanins produced by the sterile form of Inonotus

obliquus (“Chaga”) in natural and cultivated fungus. Applied Biochemistry and

Microbiology, 38(1), 58–61. http://doi.org/10.1023/A:1013204706055

Lam, K. S. (2007). New aspects of natural products in drug discovery. Trends in

Microbiology, 15(6), 279–289. http://doi.org/10.1016/j.tim.2007.04.001

Li, F., Wen, H. A., Zhang, Y. J., An, M., & Liu, X. Z. (2011). Purification and

characterization of a novel immunomodulatory protein from the medicinal

mushroom Trametes versicolor. Science China Life Sciences, 54(4), 379–385.

http://doi.org/10.1007/s11427-011-4153-2

Li, G., Kim, D. H., Kim, T. D., Park, B. J., Park, H. D., Park, J. Il, Na, M. K., Kim, H. V.,

Hong, N. D., Lim, K., Hwang, B. D., Yoon, W. H. (2004). Protein-bound

polysaccharide from Phellinus linteus induces G 2/M phase arrest and apoptosis in

SW480 human colon cancer cells. Cancer Letters, 216(2), 175–181.

http://doi.org/10.1016/j.canlet.2004.07.014

Lull, C., Wichers, H. J., & Savelkoul, H. F. J. (2005). Antiinflammatory and

xvii

immunomodulating properties of fungal metabolites. Mediators of Inflammation,

2005(2), 63–80. http://doi.org/10.1155/MI.2005.63

Macarron, R., Banks, M. N., Bojanic, D., Burns, D. J., Cirovic, D. a, Garyantes, T.,

Green, D. V. S., Hertzberg, R. P., Janzen, W. P., Paslay, J. W., Schopfor, U.,

Sittampalam, G. S. (2011). Impact of high-throughput screening in biomedical

research. Nature Reviews. Drug Discovery, 10(3), 188–195.

http://doi.org/10.1038/nrd3368

Mahajna, J., Dotan, N., Zaidman, B.-Z., Petrova, R. D., & Wasser, S. P. (2009).

Pharmacological values of medicinal mushrooms for prostate cancer therapy: the

case of Ganoderma lucidum. Nutrition and Cancer, 61(1), 16–26.

http://doi.org/10.1080/01635580802379323

Mansour, A., Daba, A., Baddour, N., El-Saadani, M., & Aleem, E. (2012). Schizophyllan

inhibits the development of mammary and hepatic carcinomas induced by 7,12

dimethylbenz-anthracene and decreases cell proliferation: Comparison with

tamoxifen. Journal of Cancer Research and Clinical Oncology, 138(9), 1579–1596.

http://doi.org/10.1007/s00432-012-1224-0

Martin, K. J., & Rygiewicz, P. T. (2005). Fungal-specific PCR primers developed for

analysis of the ITS region of environmental DNA extracts. BMC Microbiology, 5, 28.

http://doi.org/10.1186/1471-2180-5-28

Michelot, D., & Melendez-Howell, L. M. (2003). Amanita muscaria: chemistry, biology,

toxicology, and ethnomycology. Mycological Research, 107(February 2003), 131–

146. http://doi.org/10.1017/s0953756203007305

Mirfat, H. A. H. S. (2008). Biological activities of Schizophyllum commune Fr. University

xviii

Malaya, 7–20.

Mishra, B. B., & Tiwari, V. K. (2011). Natural products: An evolving role in future drug

discovery. European Journal of Medicinal Chemistry, 46(10), 4769–4807.

http://doi.org/10.1016/j.ejmech.2011.07.057

Mizuno, T. (1999). The extraction and development of antitumor-active polysaccharides

from medicinal mushrooms in Japan (Review). International Journal of Medicinal

Mushrooms, 1(1), 9–29. http://doi.org/10.1615/IntJMedMushrooms.v1.i1.20

Newman, D. J., & Cragg, G. M. (2012). Natural Products as Sources of New Drugs over

the 30 Years. Journal of Natural Products, 75(3), 311–335.

http://doi.org/10.1021/np200906s.Natural

Oh, Young, J., Baek, Y. M., Kim, S. W., Hwang, H. J., Hwang, H. S., & Lee, S. H.

(2008). Apoptosis of human hepatoarcinoma (HepG2) and neuroblastoma

produced by submerged mycelial culture of an entamopathogenic fungus

Cordyceps sphecocphala. Journal of Microbiology and Biotechnology, 18(3), 512–

519.

Ovadje, P., Roma, A., Steckle, M., Nicoletti, L., Arnason, J. T., & Pandey, S. (2015).

Advances in the research and development of natural health products as main

stream cancer therapeutics, 2015. http://doi.org/10.1155/2015/751348

Patel, S., & Goyal, A. (2012). Recent developments in mushrooms as anti-cancer

therapeutics: a review. 3 Biotech, 2(1), 1–15. http://doi.org/10.1007/s13205-011-

0036-2

Price, L. A., Wenner, C. A., Sloper, D. T., Slaton, J. W., & Novack, J. P. (2010). Role for

toll-like receptor 4 in TNF-alpha secretion by murine macrophages in response to

xix

polysaccharide Krestin, a Trametes versicolor mushroom extract. Fitoterapia, 81(7),

914–919. http://doi.org/10.1016/j.fitote.2010.06.002

Rai, M., Tidke, G., & Wasser, S. P. (2005). Therapeutic potential of mushrooms. Natural

Product Radiance, 4(4), 246–257.

Rana Monika, L. T. N. (2005). Medicinal and nutraceutical genetic resources of

mushorooms. Plant Genetic Rosources, 3(2), 288–303.

http://doi.org/10.1079/PGR200581

Ranta, K., Nieminen, K., Saariaho, T., Kortekangas-Savolainen, O., Kumpula, E. K.,

Kosonen, J., Pasanen, A. L., Savolainen, J. (2013). Evaluation of fungal extracts to

determine immunomodulatory properties. Journal of Investigational Allergology and

Clinical Immunology, 23(4), 226–233.

Ren, L., Reynisson, J., Perera, C., & Hemar, Y. (2013). The physicochemical properties

of a new class of anticancer fungal polysaccharides: A comparative study.

Carbohydrate Polymers, 97(1), 177–187.

http://doi.org/10.1016/j.carbpol.2013.04.064

Ries, F., & Klastersky, J. (1986). Nephrotoxicity induced by cancer chemotherapy with

special emphasis on cisplatin toxicity. American Journal of Kidney Diseases : The

Official Journal of the National Kidney Foundation, 8(5), 368–379.

http://doi.org/10.1016/S0272-6386(86)80112-3

Shou Xian Wanga, b, G. Q. Z., Zhaoa, S., Xua, F., Zhoua, Y., Genga, X. L., Yu, &

Liua,*, and H. X. W. (2013). Purification and characterization of a novel lectin with

antiphytovirus activities from the wild mushroom Paxillus involutus. Immunology

Series. Protein & Peptide Letters. 20(7), 767-774

Singh, S. B., & Barrett, J. F. (2006). Empirical antibacterial drug discovery - foundation

in natural products. Biochemical Pharmacology, 71(7), 1006–1015.

http://doi.org/10.1016/j.bcp.2005.12.016

Sliva, D., Sedlak, M., Slivova, V., Valachovicova, T., Lloyd, F. P., & Ho, N. W. Y. (2003).

Biologic activity of spores and dried powder from Ganoderma lucidum for the

inhibition of highly invasive human breast and prostate cancer cells. The Journal of

Alternative and Complementary Medicine, 9(4), 491–97.

Sneader, W. (2006). Drug Discovery: A History. http://doi.org/10.1002/0470015535

Song, T. Y., Lin, H. C., Yang, N. C., & Hu, M. L. (2008). Antiproliferative and

antimetastatic effects of the ethanolic extract of Phellinus igniarius (Linneaus: Fries)

Quelet. Journal of Ethnopharmacology, 115(1), 50–56.

http://doi.org/10.1016/j.jep.2007.09.001

Sukuru, S. C. K., Jenkins, J. L., Beckwith, R. E. J., Scheiber, J., Bender, A., Mikhailov,

D., Davies, J. W., Glick, M. (2009). Plate-based diversity selection based on

empirical HTS data to enhance the number of hits and their chemical diversity.

Journal of Biomolecular Screening : The Official Journal of the Society for

Biomolecular Screening, 14(6), 690–699.

http://doi.org/10.1177/1087057109335678

Sullivan, R., Smith, J. E., & Rowan, N. J. (2006). Medicinal mushrooms and cancer

therapy: translating a traditional practice into Western medicine. Perspectives in

Biology and Medicine, 49(2), 159–170. http://doi.org/10.1353/pbm.2006.0034

Sun, J. E., Ao, Z. H., Lu, Z. M., Xu, H. Y., Zhang, X. M., Dou, W. F., & Xu, Z. H. (2008).

Antihyperglycemic and antilipidperoxidative effects of dry matter of culture broth of

xxi

Inonotus obliquus in submerged culture on normal and alloxan-diabetes mice.

Journal of Ethnopharmacology, 118(1), 7–13.

http://doi.org/10.1016/j.jep.2008.02.030

Sun, J., He, H., & Bi, J. X. (2004). Novel antioxidant peptides from fermented mushroom

Ganoderma lucidum. Journal of Agricultural and Food Chemistry, 52(21), 6646–

6652. http://doi.org/10.1021/jf0495136

Taji, S., Yamada, T., Wada, S. ichi, Tokuda, H., Sakuma, K., & Tanaka, R. (2008).

Lanostane-type triterpenoids from the sclerotia of Inonotus obliquus possessing

anti-tumor promoting activity. European Journal of Medicinal Chemistry, 43(11),

2373–2379. http://doi.org/10.1016/j.ejmech.2008.01.037

Toth, B., & Nagel, D. (1978). Tumors induced in mice by N-Methyl-N-formylhydrazine of

the false morel Gyromitra esculenta. Journal of the National Cancer Institute, 60(1),

201–204.

Wang, S. X., Zhang, G. Q., Zhao, S., Xu, F., & Zhou, Y. (2013). Purification and

characterization of a novel lectin with anti-phytovirus activities from the wild

mushroom Paxillus involutus. Protein and Peptide Letters. 20(7), 767-774

Wasser, S. P. (2002). Review of medicinal mushrooms advances: good news from old

allies. American Botanical Council, 56, 28–33.

Wasser, S. P. (2010). Medicinal mushroom science: history, current status, future

trends, and unsolved problems. International Journal of Medicinal Mushrooms,

12(1), 1–16. http://doi.org/10.1615/IntJMedMushr.v12.i1.10

Wasser, S. P., & Weis, A. L. (1999). Therapeutic effects of substances occurring in

higher Basidiomycete mushrooms: a modern perspective. Critical Reviews in

xxii

Immunology, 19(1). 65-96. http://doi.org/10.1615/CritRevImmunol.v19.i1.30

Waszkiewicz-Robak, B. (2013). Spent brewer’s yeast and beta-glucans isolated from

them as diet components modifying blood lipid metabolism disturbed by an

atherogenic diet. Lipid Metabolism, 261–290. http://doi.org/10.5772/51530

Yamasaki, A., Shoda, M., Iijima, H., Nagai, S., Wada, J., Suzuki, H., Chikazawa, N.,

Tasaka, T.,Kameda, T., Tanaka, T., Mio, I., Ei, J.,Sato, N., Nakamura, N., Serine,

F., Morisaki, T., Katano, M. (2009). A protein-bound polysaccharide, PSK,

enhances tumor suppression induced by docetaxel in a gastric cancer xenograft

model. Anticancer Research, 29(3), 843–850. http://doi.org/29/3/843 [pii]

Yeh, E. T. H., Tong, A. T., Lenihan, D. J., Yusuf, S. W., Swafford, J., Champion, C., …

Ewer, M. S. (2004). Cardiovascular complications of cancer therapy. Circulation,

109(25), 3122–3131. http://doi.org/10.1016/j.jacc.2009.02.050

Yim, H. S., Chye, F. Y., Rao, V., Low, J. Y., Matanjun, P., How, S. E., & Ho, C. W.

(2013). Optimization of extraction time and temperature on antioxidant activity of

Schizophyllum commune aqueous extract using response surface methodology.

Journal of Food Science and Technology, 50(2), 275–283.

http://doi.org/10.1007/s13197-011-0349-5

Yun, B., Lee, I., & Yoo, I. (2000). Leucomentin-5 and -6 , Two new leucomentin

derivatives from the mushroom Paxillus panuoides, The Journal of antibiotics,

53(7), 711–713.

Zaidman, B.-Z., Yassin, M., Mahajna, J., & Wasser, S. P. (2005). Medicinal mushroom

modulators of molecular targets as cancer therapeutics. Applied Microbiology and

Biotechnology, 67(4), 453–468. http://doi.org/10.1007/s00253-004-1787-z

xxiii

Zhang, G., Yin, Q., Han, T., Zhao, Y., Su, J., Li, M., & Ling, J. (2015). Purification and

antioxidant effect of novel fungal polysaccharides from the stroma of Cordyceps

kyushuensis. Industrial Crops and Products, 69, 485–491.

http://doi.org/10.1016/j.indcrop.2015.03.006

Zheng, W., Zhang, M., Zhao, Y., Wang, Y., Miao, K., & Wei, Z. (2009). Accumulation of

antioxidant phenolic constituents in submerged cultures of Inonotus obliquus.

Bioresource Technology, 100(3), 1327–1335.

http://doi.org/10.1016/j.biortech.2008.05.002

Zhong, J. J. (2004). Submerged cultivation of medicinal mushrooms for production of

valuable bioactive metabolites. In Advances in Biochemical Engineering (Vol. 87,

pp. 25–59). http://doi.org/10.1016/S0300-9084(79)80192-3

Zhou, L. di, Zhang, Q. hui, Zhang, Y., Liu, J., & Cao, Y. ming. (2009). The shiitake

mushroom-derived immuno-stimulant lentinan protects against murine malaria

blood-stage infection by evoking adaptive immune-responses. International

Immunopharmacology, 9(4), 455–462. http://doi.org/10.1016/j.intimp.2009.01.010

xxiv

Appendix

(i) Amanita muscaria

ITS3: NNNNNNNNNNNTGCANANTCNGTGNTCNTCGAATCTTTGACNCNTCTTGCGCTCCTTG GTATTCCNAGGANCATGCCTGTTTGAGTGTCATTAAATTCTCTCAAAGCATACACTTGA GTGTGTGTTTTGGATTGTGGGAGTGTCTGCTGGCTTTATTATNANCCNNNTCTCCTGAA NNANATTNNNTTTGGGGNGGAGGTGCCNAGTNNCTTCTGCCTTTCNNTTGNNGTGATA GATGAATTANCTTATCTACNCNANGANANNNNGCTGNNGGTGATGCNCTGTGATCTCT CTCTGCTCTCTANTTGACNTTTGNNTGATANCTTGACCTCNNATCNNGNANGACTACCC GNTGAACTTNANCNTATCAATANNNGGANGAAAANAAACTAACNANGATTCCCTTANNN NCTGCNANTGAAGANGGAANANCTCNNATTTANNATCTGGNGNTGTTTTGCNGNNNNA GTTGTNNTCTANAGAANTGCTGCCCGTGCTGNACCNTGTACNANTCTCNNGNANNGGA NCGTNNNNNNNNNNNANNNNANNAANNNNGGNNNNNNNANNNNGGNNNGNNNNNNN NGGCNNNNNNNNNCTAAANNNNNNTCNGANANNNNNCNCNGACNNNNTTTAANNNNN NNNCTNCCNNNNNNNCTAGNNNNNANNNAANCNTNNNNANTTTNCNNNNCNCNCTTAN TNNNANNNNTANNNNNCNNNNACNNNNNTNNANTNNNNNNNNNNTANCNNNANNNNN NNNNTANNNNNAANNNAANNCNNNNCNTCCCNNNNNNNNNNNTNNNNNNNNNAATTAN TTTNNTTNNNNNNNNNNNNGGNNANGANNNANNNNNNNNNNCCCNNNNNNGNNNNNN NNTNTNNNGNNNGNGNNGNNNNNNNNNNAAANNNNNNNNNNNNCNNNNNCNNNNNN NNN NLB4: NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTTNTCTAGATTACNACT CGGACAGCNAAACACTGCCNNATTTNNAATTTGAGCTCTTCCCTCTTCACTCGCAGTTA CTAAGGGAATCCTTGTTAGTTTCTTTTCCTCCGCTTATTGATATGCTTAANTTCAGCGGG TAGTCCTACCTGATTTGAGGTCAAGTTATCAGACAAATGTCAATTANAGAGCAGAGAGA GATCACAGTGCATCACCTGCAGCCTGCTTTCCTGGCGTANATAANTTAATTCATCTATC ACACCAATGGAAAGGCANAAGTGACTTGGCACCTCCCCCCCAAAGCTAATGTCNTTCA NGAGAGCTGGCTCATAATANNANCAGANNNNACTCCCNNNNNNNANAANANNNNCTCN NNNNNNTGCNNNNNNANAAATTGANGANNCTCNNNNNNNNNTGCTCNTCNNANTACCN ANGANCNNANGATGNNNNNNANNANACNATGNNNCNNTGNCNNCNNNNNNTCNNANN NNNNANNNNANNNCNCANNNNCNTCNNCNNNNNN

(ii) Gyromitra esculenta

ITS3: GNNNNNNNNNTGTNANGCAGATTCNGTGNTCATCGAATCTTTGAACGCACATTGCGCC CTCTGGCATTCCGGAGGGCATGCCTGTTCGAGCCTCAATGAAACCGCTCCCCTCCTGG CGCTTCTCGGAGCAAAACGGCGGGGGTTCTGGCGGACGCGAACGCCGTCAATGGCTT CTTGCGCCGCTCAAATCGACGGTCTGCAGGCCCCGAGGGCGCACCCGACGTAGTGAT AACATTTCTCGTCGACGGCGACCCCGGTGCTCCTGAGACCCTAAGCGTTTCGGCGCCA AACCCAATCGGTTGAGCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAA TAAGCGGAGGAAAAGAAACCAACAGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCA ACAGCTCAAATTTTAAAGCTGACGTCTTTGGCGTCCGCGTTGTAATTTGGAGAGGCATC TTCGGTTTGGGCGCCAGCCTATGTTCCTTGGAACAGGACGTCATAAGGGGGNNAAAAN CNAAA

xxv

NLB4: NNNNNNNNNANGCTGGCNNANCCGAAGANGCCTCTCCNANTTACAACGCGGACGCCA AAGACGTCAGCTTTAAAATTTGAGCTGTTGCCGCTTCACTCGCCGTTACTGAGGCAATC CCTGTTGGTTTCTTTTCCTCCGCTTATTGATATGCTTAAGTTCAGCGGGTATCCCTACCT GATCCGAGCTCAACCGATTGGGTTTGGCGCCGAAACGCTTAGGGTCTCAGGAGCACC GGGGTCGCCGTCGACGAGAAATGTTATCACTACGTCGGGTGCGCCCTCGGGGCCTGC AGACCGTCGATTTGAGCGGCGCAAGAAGCCATTGACGGCGTTCGCGTCCGCCAGAAC CCCCGCCGTTTTGCTCCGAGAAGCGCCAGGAGGGGAGCGGTTTCATTGAGGCTCGAA CAGGCATGCCCTCCGGAATGCCAGAGGGCGCAATGTGCGTTCAAAGATTCGATGATTC ACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTNNNNNNNCNANNNA

(iii) Paxillus obscurisporus (2013)

ITS3: NNNNNNNNNNNNNNGNNNTNNNGTNNNCNNCNNTCTTTGNNCGCACCTTGCGCTCCT TGGTATTCCGAGGAGCATGNCTGNTTGAGTGTCATTTAATTCTCAACCATCCCTCGATT CGTTTCGAGGTTTGGCTTGGATTTTGGGGGCTGCCGGGCGACCCTAGGGTCTTCGGC TCTCCTTAAAAGCATTAGCGATGGTGGAACGACCTAACNCCATGCATGAACGGCCTTC GACGTGATAATGATCGTCGTGGCTGGAGTGCTAGCGGGCGTCGTGAAATNGGCGCTT CTAAATCTCGTCNAAAGACGAAACTCGAAACTTGACCTCAAATCANGTAGGACTACCCG CTGAACTTANGCATATCAATAAGCGGAGGAAAAGAAACTAACAAGGATTCCCCTANTAA CTGCGAGTGAANCGGGATGAGCTCAAATTTTGAATCTGGNGGTCTTCANGCCGTCCGA NTTGNAATCTANAGAANCGTCTTCCNCNCTGGANNNNGTNNAANTCTCCNGGAANGNN NCGTCNNNNNGGNGNNNAANNN

NLB4: NNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNGCTTCTCTAGATTACAACTCGGAC GGCCTGAAGACCGCCAGATTCAAAATTTGAGCTCATCCCGCTTCACTCGCAGTTACTAG GGGAATCCTTGTTAGTTTCTTTTCCTCCGCTTATTGATATGCTTAAGTTCAGCGGGTAGT CCTACCTGATTTGAGGTCAAGTTTCGAGTTTCGTCTTTCGACGAGATTTAGAAGCGCCN ATTTCACGACGCCCGCTAGCACTCCAGCCACGACGATCATTATCACGTCGAAGGCCGT TCATGCATGGNGTTAGGTCGTTCCACCATCGCTAATGCTTTTAAGGAGAGCCGAANAC CCTAGGGTCGCCCGGCAGCCCCCAAAATCCAAGCCAAACCTCGAAACGAATCGAGGG ATGGTTGAGAATTAAATGACACTCAAACAGGCATGCTCCTCGGAATACCAAGGAGCGC AAGGNGCGTTCNAANATTCNATGANTCACTGNAAATCTGCAATTCACATTNNTTATCGN AATTCNCNNNNNNNTNNNTCNA

(iv) Paxillus obscurisporus (2015)

ITS3: NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGNNCNTCNNNTCTTTGAACGCACC TTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTNNGAGTGTCATTTAATTCTCAACCA TCCCTCGATTCGTTTCGAGGTTTGGCTTGGANTTTGGGGGCTGCCGGGNNNCCNNNG GGCNTNNGNNNNNCNTNNAANNNNTNANNNNGGGGGNANNNNCNNANCNNNNTGGA NNAGNCNNCNNCNNNNTNATGATNNTCNNGGCGGGNGNGNTGNNNGGNGNCGTGAA GNCANCGCTTCTTCATCNTCNNCNANAAANAANACTTTNAAACTTGNNCNCNNANCNNG GNNNNNNNCCCNNNNNANNTNANNNNNNTNANNANNNGNNGAAAANAAANNNNNNAN NNNNCCCCNNNNNANNNNNNNNNNANCNGGNNNNNNNCNNATTTTNNNTNNNGNNGN

xxvi

NCTNNNGNCNNCCNAANTNNNATCCNNNNNANNNNCCTNCNNNNNNNANNNNGNNNN ANNNNNCNNNNANNNNNNNNCNNNNNNNGNGA

(v) Phellinus igniarius

ITS3: CATCGAATCTTTGAACGCACCTTGCGCCCCTTGGTATTCCGAGGGGCATGCCTGTTTG AGTGTCATGTTAATTTCAACTCCCCTTGTTTCTTAACTCGGGTGTGTTGGACTTGGAAG CTGCTGGCCCCCTTCGTTGTGGTCGGCTCTTCTTAAATGCATTAGCTGGGCTTTGGCTC GCGCTGAACGGTGTAATAGTAATCTTACATTCACCTGAGCGCTTGCCTAACAAGTCTGC TTCTAATCGTCCGTGTTTTCGGACAAGGTCGCTTTGTCGCCTTCATTTGACACCTTGAC CTCAAATCAGGTAGGATTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAA ACTAACAAGGATTCCCCTAGTAACTGCGAGTGAAGAGGGAAAAGCTCAAATTTAAAATC TGGCGGGCTCTGCTCGTCCGAGTTGTAATCTGGAGAAGCGTTTTCCGCGTCGGACCGT GCATAAGTCTCCTGGAACGGAGCGTCATAG

NLB4: GAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCT TTGAACGCACCTTGCGCCCCTTGGTATTCCGAGGGGCATGCCTGTTTGAGTGTCATGT TAATTTCAACTCCCCTTGTTTCTTAACTCGGGTGTGTTGGACTTGGAAGCTGCTGGCCC CCTTCGTTGTGGTCGGCTCTTCTTAAATGCATTAGCTGGGCTTTGGCTCGCGCTGAAC GGTGTAATAGTAATCTTACATTCACCTGAGCGCTTGCCTAACAAGTCTGCTTCTAATCGT CCGTGTTTTCGGACAAGGTCGCTTTGTCGCCTTCATTTGACACCTTGACCTCAAATCAG GTAGGATTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACTAACAAGG ATTCCCCTAGTAACTGCGAGTGAAGAGGGAAAAGCTCAAATTTAAAATCTGGCGGGCT CTGCTCGTCCGAGTTGTAATCTGGAGAAGCGTTTTCCGCGTCGGACCGTGCATAAGTC TCCTGGAACGGAGCGTCATAG

(vi) Trichaptum abietinum

ITS3: NNNNNNNNNNNGCNNANTCNGTGNNCNTCGNNTCTTTGACGCACCTTGCGCTCCTTG GTATTCCGAGGAGCATGCCTGTTTGAGTGTCATGTTAATATCAACCCCGTCGGTTTGTN AATCNNACCGTTGGGTGTTGGACTTGGAGGTTCTTGCTGGCTGCAAAGTCGGCTCCTC TTGAATGNATTAGCTTGGGCCTGTTGTNCGTGCTCGCGGTGTAATAAGTTTTATTCATC GTTTGGGTCGTACACTTATTGGGCTTGCTTCTAACTGTCTTCGGACAAANATAACCTTT GACTCTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGA GGAAAAGAAACTAACAAGGATTCCCCTAGTAACTGCGAGTGAAGCGGGAAAAGCTCAA ATTTAAAATCTGNCGGTTTCACCGTCCGAGTTGTAATCTGGAGAAGCGTTTTCCGTGTC GGACCGTGNACAAGTCTCTTGNAACAGANCGTCNTANNNNNNNNNNAA

NLB4: NNNNNNNNNNNNNNNNNNNGGNNNNGCTTCTCCNGATTACAACTCGGACGGTGNAAC CGNCAGATTTTAAATTTGAGCTTTTCCCGCTTCACTCGCAGTTACTAGGGGAATCCTTG TTAGTTTCTTTTCCTCCGCTTATTGNTATGNTTAAGTTCAGCGGGTAGTCCTACCTGATT TGAGGTCAGAGTCAAAGGTTATNTTTGTCCGAAGACAGTTAGAAGCAAGCCCAATAAGT GTACGACCCAAACGATGAATAAAACTTATTACACCGCGAGCACGNACAACAGGCCCAA GCTAATNCATTCAAGAGGAGCCGACTTTGCAGCCAGCAAGAACCTCCAAGTCCAACAC CCAACGGTNNGATTNACAAACCGACGGGGTTGATATTAACATGACACTCAAACAGGCA TGCTCCTCGGAATACCNAGGANCGCAAGGTGCGTTCAAAGANTCNANGANTCACTGAA

xxvii

NTCTGCNANTCACATTACNTATCGCATTTCGCTGCNNNNNNNNNNNNNNNNNNNNNNG NNNNNNNNNNA

(vii) Calculations model for dose and time-dependent MTT assay:

Dose-dependent MTT Assay:

% Cell Viability = [(Absorbance of control)/(Absorbance of samples)] X 100

Time-dependent MTT Assay:

A) For Samples % Cell Viability = [(Absorbance of samples)/(Absorbance of control at day 6)] X 100

B) For Control % Cell Viability = [(Absorbance of control)/(Absorbance of control at day 6)] X 100