PURIFICATION AND CHARACTERIZATION OF
GROWTH-INHIBITORY COMPOUNDS FROM 2 GENERA OF BRITISH
COLUMBIA WILD MUSHROOMS: FOMITOPSIS AND PHAEOLUS

by

Jiazhi Da

BSc. Chemistry, Southwest University for Nationalities, 2016

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

UNIVERSITY OF NORTHERN BRITISH COLUMBIA
August 2020
© Jiazhi Da, 2020

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Abstract
Modern science is currently working to characterize the beneficial compounds from
mushrooms and their potential as pharmaceutical drugs. In this thesis, eight British Columbian wild
mushroom collections (3 species), comprising Fomitopsis pinicola, Phaeolus schweinitzii and
Phaeolus sp., were screened for growth-inhibitory potential. Out of 28 crude extracts, 15 exhibited
strong growth-inhibitory activity. For the Phaeolus sp. collection, liquid-liquid extraction, sizeexclusion chromatography and high-performance liquid chromatograph-mass spectrometry (HPLCMS) were used to purify the growth-inhibitory compound of interest. NMR analyses confirmed its
identity as hispidin, a known potent anti-cancer compound. Another compound with a mass to charge
ratio of 283.2 in hexane extract of Phaeolus sp. was detected by HPLC-MS. This study provides the
foundational work for further isolation of small molecule(s) from the three mushroom species and
shed light on the profile of Phaeolus sp. as a source of growth-inhibitory compounds.

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Table of Contents
Abstract ............................................................................................................................................... ii
List of Tables ..................................................................................................................................... vi
List of Figures ................................................................................................................................... vii
Acknowledgement and Dedication ..................................................................................................... 1
Chapter 1 Introduction and literature review ...................................................................................... 2
1.1 Cancer and its properties ........................................................................................................... 2
1.1.1 What is cancer ..................................................................................................................... 2
1.1.2 Conventional cancer treatment ........................................................................................... 3
1.1.3 Cancer in Canada ................................................................................................................ 4
1.2 Natural products as a source for drug discovery ....................................................................... 5
1.2.1 Natural products in history ................................................................................................. 5
1.2.2 Why are natural products useful for drug discovery........................................................... 6
1.3 Mushrooms as a source of cancer treatment or prevention ....................................................... 8
1.3.1 Medicinal mushrooms and their anti-tumor activities ........................................................ 8
1.3.2 Experimental studies with mushrooms ............................................................................. 13
1.3.3 Clinical studies with mushrooms ...................................................................................... 14
1.4 Mushrooms in British Columbia ............................................................................................. 16
1.5 Future prospects of drug discovery from mushrooms ............................................................ 17
1.6 Research goals......................................................................................................................... 18
Chapter 2 Evaluation of wild mushrooms collected in British Columbia for anti-cell viability activity
.......................................................................................................................................................... 20
2.1 Materials and methods ............................................................................................................ 21
2.1.1 Collection and identification of wild mushrooms ............................................................ 21
2.1.2 Chemical extraction of mushrooms .................................................................................. 25
2.1.3 Rotary evaporation, pH neutralization, dialysis, and lyophilization of the extracts ......... 27
2.1.4 Preparation of stock solution and filter sterilization ......................................................... 27
2.1.5 Assessment for anti-cell viability activity......................................................................... 28
2.2 Results and discussion ............................................................................................................ 32
2.2.1 Chemical extraction of mushrooms .................................................................................. 32
2.2.2 Dose-dependent cell viability assessment of mushroom extracts using HeLa cells ......... 34
Chapter 3 Purification, identification and characterization of a growth-inhibitory compound from
Phaeolus sp. ...................................................................................................................................... 44
Part A: Purification and characterization of hispidin from Phaeolus sp. ......................................... 44
3.1 Purification and chemical characterization strategies ............................................................. 44
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3.2 Materials and methods ............................................................................................................ 45
3.2.1 Large scale chemical extraction of Phaeolus sp. .............................................................. 45
3.2.2 Liquid-liquid extraction (phase separation) ...................................................................... 45
3.2.3 Sephadex™ LH-20 (size exclusion chromatography) ...................................................... 47
3.2.4 UV absorbance analysis .................................................................................................... 50
3.2.5 High performance liquid chromatography (HPLC-UV) analysis ..................................... 50
3.2.6 LC-MS analysis ................................................................................................................ 52
3.2.7 Nuclear magnetic resonance (NMR) analysis .................................................................. 53
3.2.8 Melting point .................................................................................................................... 55
3.3 Results ..................................................................................................................................... 56
3.3.1 Large scale chemical extraction of Phaeolus sp. .............................................................. 56
3.3.2 Liquid-liquid extraction (phase separation) ...................................................................... 56
3.3.3 Assessing post-Sephadex™ LH-20 fractions collected from a 22 mL column for bioactivity
............................................................................................................................................ 58
3.3.4 Assessing post-Sephadex™ LH-20 fractions collected from a 400 mL column for growthinhibitory activity ............................................................................................................... 59
3.3.5 HPLC results ..................................................................................................................... 61
3.3.6 Nuclear magnetic resonance (NMR) analyses .................................................................. 63
3.3.7 Physical properties of hispidin from Phaeolus sp. ........................................................... 70
3.3.8 Melting point .................................................................................................................... 70
3.3.9 Percent yield of each step of the purification of Phaeolus sp. .......................................... 71
Part B: In search of other small molecules from Phaeolus sp. ......................................................... 72
3.4 Purification and chemical characterization strategies ............................................................. 72
3.5 Materials and methods ............................................................................................................ 72
3.5.1 Silica gel chromatography ................................................................................................ 72
3.5.2 Small scale chemical extraction using hexane.................................................................. 75
3.5.3 HPLC-MS analysis ........................................................................................................... 76
3.6 Results ..................................................................................................................................... 77
3.6.1 Silica gel column fraction ................................................................................................. 77
3.6.2 Hexane extraction ............................................................................................................. 78
3.6.3 HPLC-MS analysis ........................................................................................................... 79
Chapter 4: General Discussion ......................................................................................................... 85
4.1 Screening BC wild mushrooms for growth-inhibitory activities ............................................ 85
4.1.1 Fomitopsis pinicola .......................................................................................................... 85
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4.1.2 Phaeolus schweinitzii........................................................................................................ 87
4.2 Purification, identification and characterization of a growth-inhibitory compound from
Phaeolus sp. .................................................................................................................................. 88
4.2.1 Purification and characterization of compound hispidin from Phaeolus sp. .................... 88
4.2.2 In search of other small molecules from Phaeolus sp. ........................................................ 90
4.3 Conclusion .............................................................................................................................. 91
References ......................................................................................................................................... 93

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List of Tables
Table 1 Large compounds from mushroom and their biological properties ......................................11
Table 2 Small compounds from mushrooms and their biological properties ................................... 12
Table 3 Summary of human clinical studies in anticancer effects of medicinal mushrooms ........... 14
Table 4 PCR reaction protocols required to amplify the extracted DNA from the mushroom species.
.......................................................................................................................................................... 22
Table 5 PCR conditions for DNA amplification. .............................................................................. 23
Table 6 Identification of mushroom species using BLAST searches, along with the percent of
matching bases between the unknown sample and the closest match. ..................................... 23
Table 7 Collection location and photographs of the mushrooms collected from different locations in
British Columbia. ...................................................................................................................... 24
Table 8 Outline of the extraction process ......................................................................................... 26
Table 9 Representation of the different doses of mushroom extracts used for MTT assay. ............. 30
Table 10 Plate pattern (96-well) followed to screen different doses of extracts from selected
mushroom samples, for their potential to exhibit anti-proliferative activity based on the MTT
assay. ......................................................................................................................................... 31
Table 11 Summary of the fungal extracts obtained through successive extraction process. ............ 32
Table 12 Summary of twenty eight crude extracts isolated from seven BC mushroom specimens on
HeLa cell viability ..................................................................................................................... 42
Table 13 Percent yield of E1 extract from Phaeolus sp. (#71) ......................................................... 56
Table 14 Percent yield of three layers based on liquid-liquid extraction (Phase separation) of E1 (80%
ethanol) extract of Phaeolus sp. ................................................................................................ 57
Table 15 1H NMR spectral data of purified compound (hispidin) obtained using Bruker Fourier A300 NMR .......................................................................................................................................... 64
Table 16 13C NMR spectral data of purified compound (hispidin) obtained using Bruker Fourier A300 NMR. ......................................................................................................................................... 64
Table 17 Melting point range of hispidin.......................................................................................... 71
Table 18 Techniques followed and percent yield of compounds from E1 (80% ethanol) extract of
Phaeolus sp. ...................................................................................................................................... 71
Table 19 The strength of mobile phases for elution.......................................................................... 74
Table 20 Fractions collected from silica gel chromatography. ......................................................... 78
Table 21 Percent yield of hexane extract from Phaeolus sp. (#71) .................................................. 78
Table 22 The agents found in Fomitopsis pinicola and their therapeutic potential against cancer .. 87

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List of Figures
Figure 1 Proportion of deaths due to cancer and other causes in Canada, for 2016 (Statistics Canada,
2016). .................................................................................................................................................. 4
Figure 2 Selected causes of death and their associated potential years of life lost (PYLL), in Canada,
for the period 2014–2016.................................................................................................................... 5
Figure 3 Some medicinal mushrooms with anti-cancer potential (Modified from Patel, S., & Goyal,
A., 2012) ............................................................................................................................................. 9
Figure 4 Examples of products marketed with claimed immune stimulatory and anti-cancer properties
containing mushrooms or their extracts 1. Dr Myko San—Health from Mushrooms 2. Ganoderma
lucidum Spore 3. Grifron Maitake Mushrooms 4. I’m-Yunity® 5. MC-S (Metabolic Cell Support) 6.
MycoPhyto® Complex 7. Red Reishi 8. Super Royal Agaricus Mushroom. Figure modified from De
Silva et al. (2012) .............................................................................................................................. 18
Figure 5 An example of a dried and powdered mushroom sample .................................................. 26
Figure 6 Sequential chemical extraction ........................................................................................... 28
Figure 7 (A) Chemical structure of MTT and its reduced formazan product; (B) absorption spectra
of MTT.............................................................................................................................................. 29
Figure 8 Schematic representation of dose-dependent MTT assay protocol using HeLa Cells. ...... 32
Figure 9 Dose-dependent assessment of cell viability by extracts from F. pinicola (#50) ............... 34
Figure 10 Dose-dependent assessment of cell viability by crude extracts isolated from F. pinicola.
(#53) Error bars are standard deviation. ........................................................................................... 35
Figure 11 Dose-dependent assessment of cell viability by crude extracts isolated from F. pinicola.
(#65) Error bars are standard deviation. ........................................................................................... 36
Figure 12 Dose-dependent assessment of cell viability by crude extracts isolated from F. pinicola.
(#77) Error bars are standard deviation. ........................................................................................... 37
Figure 13 Dose-dependent assessment of cell viability by crude extracts isolated from P. schweinitzii.
(#57) Error bars are standard deviation. ........................................................................................... 38
Figure 14 Dose-dependent assessment of cell viability by crude extracts isolated from P. schweinitzii.
(#58) Error bars are standard deviation. ........................................................................................... 39
Figure 15 Dose-dependent assessment of cell viability by crude extracts isolated from P. schweinitzii.
(#60) Error bars are standard deviation. ........................................................................................... 40
Figure 16 Dose-dependent assessment of cell viability by crude extracts isolated from Phaeolus sp.
(#71) Error bars are standard deviation. ........................................................................................... 41
Figure 17 Illustration of the liquid-liquid extraction technique ........................................................ 46
Figure 18 Photograph of the Sephadex™ LH-20 size exclusion technique using a large C26/100
column (GE Healthcare) and the mobile phase (degassed methanol) .............................................. 49
Figure 19 Assessing the water and ethyl acetate layers from E1 extract for anti-proliferative activity.
Two different concentrations of water and ethyl acetate layers were added to HeLa for 48 hours
followed by assessment of cell viability using MTT assay. The graph shows results from one
biological replicate (n=1). Error bars are standard deviation. .......................................................... 57
Figure 20 Assessing anti-proliferative activity of fractions collected from 22 mL size Sephadex™
LH-20 column. ................................................................................................................................ 59
Figure 21 Assessing anti-proliferative activity of fractions collected from a 400 mL Sephadex™ LH20 column. ........................................................................................................................................ 60
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Figure 22 Flow chart diagram representing the method of compound purification from E1 (80%
ethanol) extract of Phaeolus sp. ........................................................................................................ 61
Figure 23 UV spectrum showing the relative abundance and retention time of peak at 4.312 min at
λmax 270 nm .................................................................................................................................... 61
Figure 24 UV spectrum confirming the purity of the compound [M+H]+ m/z 247.1 detected at 4.312
min through ESI (Electrospray ionization) chamber. ....................................................................... 62
Figure 25 MS SIM spectrum showing the relative abundance and retention time of the compound
with [M+H]+ m/z 247.1 detected at 4.312 min through ESI (Electrospray ionization) chamber. ... 62
Figure 26 MS scan spectrum showing the relative abundance and retention time of the compound
with [M+H]+ m/z 247.1 detected at 4.312 min through ESI (Electrospray ionization) chamber. .... 63
Figure 27 Integration of MS scan spectrum indicating the presence and abundance of compound with
[M+H]+ m/z 247.1 visible at 4.567 min ............................................................................................ 63
Figure 28 Chemical structure of hispidin ......................................................................................... 65
Figure 29 1H -NMR spectrum of hispidin in (CD3)2CO containing 0.3% TMS ............................. 67
Figure 30 13C-NMR spectrum of hispidin in (CD3)2CO containing 0.3% TMS ............................ 67
Figure 31 1H - 1H NMR correlation spectroscopy (COSY) 2D spectrum of hispidin in (CD3)2CO
containing 0.3% TMS. ...................................................................................................................... 68
Figure 32 13C - 1H NMR heteronuclear single quantum coherence (HSQC) 2D spectrum of hispidin
in (CD3)2CO containing 0.3% TMS. ............................................................................................... 68
Figure 33 13C - 1H NMR heteronuclear multiple bond correlation (HMBC) 2D spectrum of hispidin
in (CD3)2CO containing 0.3% TMS. ............................................................................................... 69
Figure 34 Hispidin from Phaeolus sp. .............................................................................................. 70
Figure 35 Photograph of the silica gel chromatography using a 30 cm × 1.5 cm size column and the
mobile phase. .................................................................................................................................... 75
Figure 36 TLC of E1 of #71 Phaeolus sp. (lane 1), and hispidin (lane 2). ....................................... 77
Figure 37 UV spectrum showing the relative abundance and retention time of at 3.031 min at λmax
270 nm (using Agilent C-18 analytical column and silica gel fraction sample). .............................. 79
Figure 38 MS scan spectrum representing the relative abundance and retention time of the
compounds detected through ESI (Electrospray ionization) chamber. ............................................. 79
Figure 39 Integration of MS scan spectrum indicating the presence and abundance of compound with
[M+H]+ m/z 247.0 visible at 3.242 min. ........................................................................................... 80
Figure 40 UV spectrum showing the relative abundance and retention time of peaks at λmax 250 nm
(using Agilent C-18 analytical column and Sephadex LH-20 active fraction sample). ................... 81
Figure 41 MS scan spectrum representing the relative abundance and retention time of the
compounds detected through ESI (Electrospray ionization) chamber. ............................................. 81
Figure 42 MS SIM spectrum representing the relative abundance and retention time of the targeted
masses, detected through ESI (Electrospray ionization) chamber. ................................................... 82
Figure 43 Integration of MS scan spectrum indicating the presence and abundance of compound with
[M+H]+ m/z 283.2 visible at 9.257 min ............................................................................................ 82
Figure 44 Integration of MS SIM spectrum indicating the presence and abundance of compound with
[M+H]+ m/z 247.0 visible at 3.295 min ............................................................................................ 83
Figure 45 Integration of MS SIM spectrum indicating the presence and abundance of compound with
[M+H]+ m/z 283.2 visible at 9.237 min ............................................................................................ 83
Figure 46 The structures of lactapiperanol A and lactapiperanol C. ................................................. 84
Figure 47 The predicted MS spectrum of lactapiperanol C (spectrum from FooDB database). ...... 84
viii

Acknowledgement and Dedication
First of all, I would like to express my deepest appreciation to my supervisor Dr. Chow Lee
who provided this treasured opportunity to work in his lab and spent his time and effort in
helping my application to UNBC. Moreover, he offered me continued support and guidance
through the past two and half years, which helped me improve academically and personally. I
would also like to thank Dr. Maggie Li and my committee members Drs. Kerry Reimer, Hugues
Massicotte and Keith Egger for their technical and professional guidance that helped me to
understand my research much better. With a special mention to the Lee lab members for their
suggestion and help with my experiments: Bryan Lim, Hooi Xian Lee, Almas Yaqoob, Mehreen
Zeb, Victor Liu and Belal Tafech. Also, thanks to the NALS members (Charles Bradshaw and
Erwin Rehl) for providing the facility and help me keeping the instruments functioning for my
experiments.
A very special gratitude goes out to my parents (Mr. Banghong Da and Ms. Li Chen) for
their constant encouragement and financial support in the past two years. I thank all the friends
I have met in Canada who accompanied me and relieve my stress in tough periods: Yuquan Li,
Shuyan Jiang, Richard Foo, Patrick Wang, Chao Wang, Noburo Kato, Seigo Fujiwara, Ann
Duong, Katie Timms, Brooke Boswell, Brendan Reiter and Fatimat Ramos. And I would like
to thank my landlords: John Tang and Kit Lau, who frequently helped me with shopping duties
and invited me to their family parties. Lastly, I must express my gratitude to Ms. Chuyi Wang
and her husband Ben Ng, who helped me settle down and assisted me to get through the
adaptive period when I first came to Canada.

1

Chapter 1 Introduction and literature review
1.1 Cancer and its properties
1.1.1 What is cancer
Cancer is a term used for specific diseases in which abnormal cells divide uncontrollably
resulting in cell invasion and metastasis (Clark & Vignjevic, 2015). Cancer cells can spread to
other parts of the body through the blood and lymphatic systems. The human body is made up
of many different types of cells, such as skin, muscle, and blood cells. Many normal cells in
our body are continually dividing to create new cells, and these healthy cells stop reproducing
when enough cells are present (Mair, 2010). However, when an error occurs in the
manufacturing of a new cell, that cell may become a cancer cell. In addition to an error in cell
development, cancer cells do not have the internal system causing the cell to die after a certain
time and this leads to the formation of abnormal cells. When this occurs, the cells can
proliferate and build up a mass of tissue, called a tumor, or abnormal cells can crowd out the
good, healthy cells, which occurs in leukemia or other cancers affecting the blood.
Cancer is a broad term that encompasses a complex group of more than 100 different types
of cancerous diseases that can develop in the human body (Hanahan & Weinberg, 2000). Most
of these can be considered as chronic diseases, representing one of the main health problems
in the 21st century and the leading cause of death worldwide (Bray et al., 2018). It is reported
that cancer is the second most common cause of death among children between the ages of 5
and 14 in the WHO European region (Kyu et al., 2016). As a chronic disease, cancer often
leads to death or can have long-term devastating effects on an individual. Thus, finding
effective cures and treatments for cancer is a major priority for the whole world in this century.
2

1.1.2 Conventional cancer treatment
Up to now, conventional cancer treatment mainly consists of surgery, chemotherapy and
radiation therapy, depending on the type of cancer and the stage of tumor development inside
the body (Gibbs, 2000). Surgery is the most common form of cancer treatment and can be used
by itself or in combination with other treatments (Arruebo et al., 2001). Common side effects
from surgery are specific to the type of surgery done, which includes (but is not limited to) pain
(often temporary), fatigue, risk of infection at the surgical site and organ dysfunction.
Chemotherapy involves the use of drugs to destroy cancer cells; it is an effective and widely
used treatment in most types of malignancies (Chabner & Roberts, 2005). The side effects of
the chemotherapy will vary depending on the type of anti-cancer drug combination used, the
dose of the anti-cancer drugs, and the frequency of treatments. Chemotherapy is a systemic
treatment that results in the elimination of both healthy and cancerous cells (Aslam et al., 2014).
Another treatment alternative is radiation therapy (or radiotherapy), which often uses X-rays
to kill or damage cancer cells. Radiation is given before, during or after other treatments. The
goal of radiation is to damage as many cancer cells as possible without harming healthy tissue
(Baskar et al, 2014).
The major problem with these conventional therapies, especially radiotherapy and
chemotherapy, is that they invariably result in damage or weakening of the patient’s natural
immunological defenses, and cause a number of side effects, with serious damage and suffering
to the patient (Aslam et al., 2014, Nieder et al., 2000).

3

1.1.3 Cancer in Canada
Almost half of Canadians will suffer from cancer in their lifetime, and about one-quarter
of Canadians are expected to die from the disease (Statistics Canada, 2014). Cancer is the
leading cause of death in Canada (Figure 1), and is responsible for 30% of all deaths, followed
by cardiovascular diseases (heart and cerebrovascular diseases), accidents, and chronic lower
respiratory diseases. In 2019, an estimated 82,100 Canadians were expected to die from cancer.
Such a large number of cancer deaths has huge implications on the demand of health care and
support services (Canadian Cancer Statistics).

Figure 1 Proportion of deaths due to cancer and other causes in Canada, for 2016
(Statistics Canada, 2016).
Cancer is also a major cause of premature mortality, according to the data measured by
potential years of life lost (PYLL) (Canadian Vital Statistics Death database, Statistics Canada,
2014-2016). PYLL is a summary measure of premature mortality that accounts for deaths

4

occurring at younger ages. As indicated, it tends to be more influenced by deaths from diseases
and injuries affecting children and young adults, for whom the potential years of life are greater.
During the period between 2014 and 2016, the PYLL for all cancers combined was almost
1,411,100. This is more than any of the other leading causes of premature death in Canada
(Figure 2).

Figure 2 Selected causes of death and their associated potential years of life lost (PYLL),
in Canada, for the period 2014–2016.
1.2 Natural products as a source for drug discovery
1.2.1 Natural products in history
Nature has been a source of medicinal agents for thousands of years. Plants, fungi and
animals have been used as sources for the treatment of human diseases (Lahlou, 2007). The
earliest records of natural products were written on clay tablets in cuneiform script, and are
from Mesopotamia (2600 B.C.); they documented oils from Cupressus sempervirens (Cypress)
and Commiphora species (myrrh), which are still in use today to treat coughs, colds and
inflammation. The best known Egyptian pharmaceutical record is the Ebers Papyrus, dating
5

from 1500 B.C.; this documented some 700 drugs (mostly plants) including gargles, pills,
infusions, and ointments (Cragg & Newman, 2005). The Chinese Materia Medica (1100 B.C.),
Shennong Herbal (~100 B.C., 365 drugs) and the Tang Herbal (659 A.D., 850 drugs) also
contain numerous records on the use of natural products (Cragg & Newman, 2005).
In the ancient western world, the Greeks contributed to the development of the use of herbal
drugs. The natural scientist, Theophrastos (~300 B.C.) dealt with medicinal herbs, and the
physician Dioscorides (100 A.D.), recorded the collection, storage and uses of medicinal herbs.
During the Dark and Middle Ages (5th to 12th centuries), the monasteries in countries such as
England, Ireland, France, and Germany preserved the remnants of this western knowledge. The
Arabs were the first to establish privately owned drug stores in the eighth century, and the
Persian pharmacist, physician and philosopher Avicenna, contributed much to the sciences of
pharmacy and medicine through works such as Canon Medicinae (Cragg & Newman, 2005).
1.2.2 Why are natural products useful for drug discovery
Worldwide, it is estimated that less than 10% of all natural products have been evaluated
for biological activity; many more useful novel natural compounds still await discovery (Cragg
& Newman, 2005). Natural products have evolved and adapted over millions of years through
defense against bacteria, insects and fungi, or interference with competing organisms, to
become unique, structurally diverse secondary metabolites. Whatever their precise role, many
natural products can reach human receptor sites on or within cells, just as a drug must do. The
large number of pure natural products which have been found to interact with specific
mammalian receptors testifies to the inherent bioactivity in natural products (Beutler, 2009).

6

The value of natural product chemistry is in providing novel molecular skeletons and
functionalities, which may be used as templates for the development of new drugs. Recently,
pharmaceutical scientists have experienced difficulty in identifying new lead structures,
templates and scaffolds in the finite world of chemical diversity (Patwardhan et al., 2004). A
number of synthetic drugs have adverse and unacceptable side effects (Karimi et al., 2015).
There have been impressive successes with botanical medicines, most notably quinghaosu,
artemisinin from Chinese medicine (Tu, 2011), its derivative was found to treat human cervical
cancer in clinical trial (Jansen et al., 2011).
1.2.2.1 Natural products from plants
Plants have been known for their medicinal uses for thousands of years. About 80% of
people in developing countries of the world still rely on plant-based medicines for primary
health care (Farnsworth et al., 1985). Plant medicines have a long history of use for the
treatment of cancer (Hartwell, 1982). The most widely used breast cancer drug is paclitaxel,
which was originally isolated from the bark of T. brevifolia in 1962 by the United States
Department of Agriculture (Cragg, 1998). Another example is tubocaurarine, originally
isolated from the climbing plant, Chondrodendron tomentosum, and is one of the active
constituents in a muscle relaxant used in surgical operations (Dewick, 2002).
1.2.2.2 Natural products from fungi
Like plants, fungi have been a part of human life for thousands of years. Fungi have been
utilized in various aspects from food (mushrooms), alcoholic beverages (yeasts), traditional
medicine (Hays & Watson, 2019), and to human rituals (Samorini, 1992).

7

Fungi have been used as medicine since the Neolithic and Paleolithic eras (Samorini, 2001).
Mushrooms have been used in health care for treating simple and age-old common diseases
like skin diseases, as well as present day health challenges including pandemic diseases like
AIDS (Chang, 2006). They possess many biological activities including anti-bacterial, antiinflammatory, anti-oxidant and anti-cancer properties. Compounds isolated from mushrooms
such as Ganoderma pfeifferi Bres., Lentinus edodes (now Lentinula edodes (Berk.) Pegler)
and Cortinarius basirubescens J.B. Cleland & J.R. Harris showed antimicrobial activity against
pathogenic microorganisms like bacteria associated with nosocomial infections. (Alves et al.,
2012). Fractions from the mushroom Inonotus obliquus (Ach. ex Pers.) Pilát were found to
have anti-inflammatory effects on NO production and NF-κB luciferase activity in macrophage
RAW 264.7 cells (Ma et al., 2013). A water extract from L. edodes showed potent anti-oxidant
activity in three different assays (Cheung et al., 2003). Mushrooms are also one of the main
sources for anticancer drug discovery (Newman & Cragg, 2012).
1.3 Mushrooms as a source of cancer treatment or prevention
1.3.1 Medicinal mushrooms and their anti-tumor activities
Conventional cancer therapies have side effects, as mentioned above; recently, some
researchers have focused on treating cancers and improving the patient’s quality of life by
modifying the host’s biological response against the malignant invasion. Immunotherapy is
now gaining more attention, since it causes considerably fewer side effects and helps to
overcome cancer growth (Farkona et al., 2016). Immunotherapy uses the body’s immune
system, either directly or indirectly, to fight cancer or to reduce the side effects that may be

8

caused by some severe cancer treatments (Kakimi et al., 2009). Figure 3 illustrates some
medicinal mushrooms with anti-cancer potential properties.

Figure 3 Some medicinal mushrooms with anti-cancer potential (Modified from Patel, S., &
Goyal, A., 2012)

There are many forms of biological immunotherapeutic agents, such as monoclonal
antibodies, interferons, cancer vaccines and nonspecific immuno-modulating agents. Many
mushroom-derived extracts are recognized as immuno-modulators or biological response
modifiers (Lull et al., 2005, Novak & Vetvicka, 2009). Medicinal mushrooms, in particular,
are not only hubs for a myriad of immuno-modulators, but also a source of anti-cancer agents,
which could interfere with specific cellular signal transduction pathways linked to cancer
development and progression (Kudugunti et al., 2010). Recently, more than 30 species of
identified medicinal mushrooms (e.g, Phellinus linteus (Berk. & M.A. Curtis) Teng, Grifola
frondosa (Dicks.) Gray, Hypsizygus marmoreus (Peck) H.E. Bigelow and Flammulina
9

velutipes (Curtis) Singer have showed antitumor activity in experimental studies (Wasser,
2011). Medicinal mushrooms show therapeutic action against the development of cancer cells,
primarily because they contain a number of biologically active high and low molecular weight
compounds (Lee & Hong, 2011, Li et al., 2015).
1.3.1.1 High molecular weight compounds
The high molecular weight compounds which have been found in medicinal mushrooms
are mainly polysaccharides, proteins, and polysaccharide-protein complexes (Wasser, 2003).
For example, lentinan, an anti-tumor polysaccharide produced by L. edodes, this mushroom is
widely consumed as a nutritional health food throughout the world, especially in Asia, and is
known to possess strong host-mediated anti-cancer activity by activating the immune system
(Cheung, 2008). Mushrooms can also produce a large number of proteins with significant
biological activities (Wong et al., 2010). Lectins, which are proteins that bind to cell surface
carbohydrates, have the ability to produce cell agglutination and show several anti-proliferative
and anti-tumor activities against cancer cell lines (Zhang et al., 2009). Lectins are abundant in
many species of mushroom, including Agaricus bisporus (J.E. Lange) Imbach and Pleurotus
ostreatus (Jacq.) P. Kumm. (Hassan et al., 2015). Medicinal mushrooms have immunostimulatory activity not only because of their bioactive polysaccharides, but also by having
varying combinations of polysaccharide-protein complexes (Moradali et al., 2007). For
instance, Agaricus subrufescens Peck is a well-known mushroom having several biologically
active metabolites, including polysaccharides and glycoproteins that are thought to be
responsible for its immuno-stimulant and anti-tumor properties (Wisitrassameewong et al.,
2012). Table 1 shows a list of some biologically active large molecules from mushrooms.
10

Table 1 Large compounds from mushroom and their biological properties
Mushroom

Compounds

Biological activity

Reference

Polysaccharides:
Paxillus involutus
(Batsch) Fr.

GIP-inv
polysaccharide

Anti-proliferative (apoptosis
induction)

(Barad et al.,
2018)

L. edodes

D-glucopyranose

Immunomodulatory

(Zheng et al.,
2005)

Schizophyllum
commune Fr.

Homopolysaccharide

Immunomodulatory

(Kumari et al.
2008)

Tricholoma crissum
Sacc.

Hetero
polysaccharide

Macrophage activations

(Patra et al.,
2012)

P. ostreatus

Hetero
polysaccharide

Stimulates macrophages

(Maity et al.,
2011)

Lentinus squarrosulus
Mont.

Heteroglycan

Immunoenhancing

(Bhunia et
al., 2010)

Proteins/Enzymes:
Russula delica Fr.

Lectin

Anti-proliferative

(Zhao et al.,
2010)

H. marmoreus

(RIP) Ribosome
inactivating protein

Anti-proliferative

(Wong et al.,
2008)

Pleurotus
citrinopileatus Singer

Glycoprotein

Immunomodulatory

(Chen et al.,
2009)

Ganoderma lucidum
(Curtis) P. Karst.

Glycoprotein

Antioxidant activity

(Du et al.,
2007)

G. frondosa

(FIP) Fungal
immunomodulatory
protein

Enhances cytokine

(Kodama et
al., 2010)

11

1.3.1.2 Low molecular weight compounds
Mushrooms produce a variety of complex low molecular weight compounds with diverse
chemical compositions, such as phenolic compounds, polyketides and triterpenoids
(Elisashvili, 2012). Ergosterol is a provitamin form of vitamin D2 in human nutrition, it
abundant in mushrooms such as A. subrufescens and Lentinula edodes (Wisitrassameewong et
al, 2012, Jasinghe & Perera, 2005). Grifolin is a natural biologically active metabolite that was
isolated from the mushroom Albatrellus confluens (Alb. & Schwein.) Kotl. & Pouzar; it was
identified as a potential antitumor agent that can inhibit cancer cell growth by inducing
apoptosis in vitro (Ye et al., 2005). Grifolin could also exert anti-oxidative activity and
antimicrobial activity (Quang et al., 2006). Medicinal mushrooms have been reported to
produce various polyphenolic pigments, known as the styrylpyrone class of phenols derived
from polyketide pathways, which show significant biological effects including anti-cancer
properties (Lee & Yun, 2011). For example, some early studies have shown that hispidin, is
more cytotoxic toward cancerous cells (pancreatic duct and keratinocyte) than normal cells
(Gonindard et al., 1997). Some potent biological small molecules purified from the fruiting
bodies of mushrooms are shown in Table 2.
Table 2 Small compounds from mushrooms and their biological properties
Mushroom

Compounds

Biological activity

Reference

Agaricus blazei
Murill

Ergosterol

Growth-inhibitory activities

(Takaku et al.,
2001)

A. confluens

Grifolin

Growth-inhibitory activities

(Ye et al.,
2005)

12

Suillus placidus
(Bonord.) Singer

Irofulven

Immuno enhancing

(Fung & Tan,
2015)

G. lucidum

Ganoderic acid

Induces apoptosis

(Liu &
Zhong, 2010)

I. obliquus

Ergosterol
peroxide

Anti-inflammatory activities

(Ma et al.,
2013)

Hericium
erinaceum Burds.,
O.K. Mill. & Nishij.

Hericerin

Induction of apoptosis

(Li et al.,
2015)

P. ostreatus

Concanavalin A

Anti-inflammatory activities

(Jedinak et
al., 2011)

Hispidin

Antioxidant activity

(Han et al.,
2013)

25R-antcin K &
dehydrosulphure
nic acid

Growth-inhibitory effect

(Huang et al.,
2018)

Conflamides D
and E

Anti-proliferative

(Zhang et al.,
2018)

Phaeolus
schweinitzii (Fr.)
Pat.
Antrodia
cinnamomea T.T.
Chang & W.N.
Chou
A. confluens

1.3.2 Experimental studies with mushrooms
The mushroom Agaricus blazei is used as food, as natural remedy for cancer prevention,
and as adjuvant in cancer treatment (Firenzuoli et al., 2008). There is also a similar study about
adjuvant properties that investigated the combination of A. blazei extract with marine
phospholipids on myeloma sp2 tumor suppression when administrated orally. Results of this
in vivo study indicated that oral administration of A. blazei extract, with or without
encapsulated liposomal form, suppressed myeloma in mice. Additionally, the study suggested
13

that the anti-tumor effect of marine phospholipid occurred without weight loss, which might
indirectly affect the treatment of the disease (Murakawa et al., 2007).
The components of the fungus Polyozellus multiplex (Underw.) Murrill were tested in vitro
against human gastric cancer cells as well as other cancer cells. The water fraction of P.
multiplex dramatically increased glutathione S-transferase (GST) and superoxide dismutase
activities, showing a tendency of increasing glutathione (GSH) levels, as compared to the Nmethyl-N-nitro-N-nitrosoguanidine (MNNG) group alone (Lee & Nishikawa, 2003). The
results demonstrated that proliferation of distinct types of cancer cells could be significantly
inhibited by the P. multiplex water fraction, suggesting its possible use as a chemo-prevention
agent in stomach cancer (Lee & Nishikawa, 2003).
1.3.3 Clinical studies with mushrooms
Some medicinal mushrooms with anti-cancer effects, subjected to human clinical trials,
are indicated in Table 3.
Table 3 Summary of human clinical studies in anticancer effects of medicinal mushrooms
Mushroom

Type of study
Extracts or
Type of
and number of
compounds or
cancer
participants
Active principle

Findings

Reference

Ovarian Polysaccharides

Moderate inverse
association between
habitual mushroom intake
and epithelial ovarian
cancer

Randomized,
clinical trial,
373 adults

Various
types

Spore vs
mycelium

Improve tumor response
of lung cancer to
conventional therapy.
Enhance immunity

(Figueiredo &
Régis, 2017)

Open pilot
study, 10
participants

Various
types

L. edodes
mycelia

Combine treatment of
LEM and immunotherapy

(Tanigawa et
al., 2016)

A. bisporus

Observational
study, 500
participants

A. bisporus/
Amauroderma
rude (Berk)
Torrend
Lentinula
edodes
(Berk.) Pegler

14

(Lee et al.,
2013)

The first observational report associating mushrooms (A. bisporus and shiitake) intake and
ovarian cancer was conducted in China; the participants were 500 patients diagnosed with
ovarian cancer, along with a control group of 500 healthy participants. The patients with
ovarian cancer consumed less mushrooms than the controls, and results showed that mushroom
intake at increasing levels is associated with a decreasing risk of ovarian cancer (Lee et al.,
2013). In another clinical trial, the effectiveness of the mushroom Pleurotus cornucopiae
(Paulet) Rolland was evaluated and the mushroom extract was shown to exert immunopotentiation for several diseases including cancer (Tanaka et al., 2016). P. cornucopiae has also
been traditionally consumed by Japanese people due to its cancer preventative properties
(Tanaka et al., 2016).
Ganoderma lucidum, also known as Reishi mushroom, is widely believed to be
responsible for longevity and general well-being; recently, it has also been used as an
alternative medicine in cancer treatment. A review has evaluated the clinical activities of G.
lucidum to prolong survival time, tumor response, and the quality of life in cancer patients (Jin
et al., 2016). This study demonstrated that the incorporation of G. lucidum extract treatment
can enhance tumor response of lung cancer in conventional therapy, and is believed to improve
immunity by stimulating T-lymphocyte proliferation, suggesting that it is a promising adjuvant
to restrain the undesired immunosuppressive effect of many chemotherapeutic drugs.
Furthermore, this study reported an improvement in the quality of life of cancer patients who
were treated with G. lucidum, and concluded the absence of serious toxicity through the clinical
trial as it was well tolerated by most participants.

15

1.4 Mushrooms in British Columbia
There is general acceptance among mycologists that the number of global fungal species
is between 1 to 5 million (Blackwell, 2010). It is believed that there are about 150,000-160,000
species of mushrooms in the world, and approximately 10% have been explored (Wasser,
2014). This large diversity provides a huge sample source from which new anti-cancer
compounds can potentially be discovered, purified, identified, and further studied for
pharmaceutical use. Besides the work from Dr. Lee’s lab at UNBC, only two other studies have
been conducted on the bioactivity of mushrooms in Canada. The first one was the discovery of
a novel compound, ganoderma aldehyde, derived from the mushroom Ganoderma applanatum
(Pers.) Pat. which was collected in the forest of British Columbia (BC). However, the
bioactivity of the compound isolated was not described in detail (Ming et al., 2002). Another
study involved the mushroom I. obliquus, collected from Manitoba and was shown to exert
anti-inflammatory activity on Raw 264.7 macrophage cells. However, no further studies were
done to identify and purify the active compound (Van et al., 2009).
Currently, there are several ongoing research projects being carried out in Dr. Lee’s lab,
focusing on screening a variety of mushrooms collected from British Columbia for their anticancer properties (anti-proliferative, immuno-stimulatory and anti-inflammatory) (Smith et al.,
2017; Deo et al., 2019). In one study, 17 different fungal species were collected from multiple
locations in Haida Gwaii and were screened for anti-cancer properties (Deo et al., 2019);
among these mushrooms, Amanita augusta Bojantchev & R.M. Davis, Phellodon atratus K.A.
Harrison, Guepinia helvelloides (DC.) Fr., Chroogomphus tomentosus (Murrill) O.K. Mill.,
16

Laetiporus conifericola Burds. & Banik, and Inocybe sp. were shown to have strong antiproliferative and anti-inflammatory activities. In another study, a polysaccharide extracted
from the fungus P. involutus, collected from Prince George, BC, was shown to possess growthinhibitory activity against several cancer cell lines and could also trigger apoptosis of cancer
cells (Barad et al., 2018). Yet another study also revealed that an anti-inflammatory
polysaccharide, successfully isolated from Echinodontium tinctorium (Ellis & Everh) Ellis &
Everh. exhibited activity both in vitro and in vivo (Javed et al., 2019a). A companion study,
testing the methanol extract from I. obliquus, showed similar anti-inflammatory activities
(Javed et al., 2019b). A recent study, characterizing 3 compounds from the terricolous polypore
Albatrellus flettii Morse ex Pouzar, documented the suppression of KRAS expression in human
colon cancer cells (Yaqoob et al., 2020). Many other studies are ongoing in the lab, including
my project. Therefore, taking into account the limited research conducted on the bioactivity of
BC wild mushrooms, it seemed logical to further study the potential therapeutics of Canadian
mushrooms in the field of cancer.
1.5 Future prospects of drug discovery from mushrooms
Today, some medicinal mushrooms are identified as sources for biologically active
metabolites with potential anti-cancer properties. The bioactive metabolites from medicinal
mushrooms could offer beneficial effects, not only as pharmaceutical drugs but also as a novel
class of supportive products (Jiang & Silva, 2010). Examples of the available drug products
and supplementary foods developed from medicinal mushrooms claiming to provide beneficial
effects on immune stimulation and help in cancer prevention are shown in Figure 4.
17

Figure 4 Examples of products marketed with claimed immune stimulatory and anticancer properties containing mushrooms or their extracts 1. Dr Myko San—Health from
Mushrooms 2. Ganoderma lucidum Spore 3. Grifron Maitake Mushrooms 4. I’m-Yunity® 5. MC-S (Metabolic
Cell Support) 6. MycoPhyto® Complex 7. Red Reishi 8. Super Royal Agaricus Mushroom. Figure modified from
De Silva et al. (2012)

Medicinal mushrooms contain many active metabolites that can be used as support
remedies for cancer treatments (Evidente et al., 2014). The above examples just describe a few
studies about anti-cancer properties of mushrooms, and the numbers will keep increasing as
more mushrooms are tested. With articles citing their medicinal potential, it is clear that
medicinal mushrooms would serve as ideal candidates for the further discovery of a potential
anti-cancer drug.
1.6 Research goals
Based on the fact that relatively limited studies have been done on bioprospecting wild
mushrooms native to Canada, I set out to explore several wild mushrooms found in BC. The
specific research objectives of my MSc. thesis are:

18

(i)

To chemically extract and biologically assess eight Canadian mushroom
collections (comprising 3 species in two genera, Fomitopsis and Phaeolus) for
anti-cell viability activity.

(ii)

To purify and characterize the small molecule growth-inhibitors from the unique
species of Phaeolus.

19

Chapter 2 Evaluation of wild mushrooms collected in British Columbia for anti-cell
viability activity
This chapter describes studies investigating eight mushroom collections from BC (including
the 2 genera Fomitopsis and Phaeolus). Each sample was identified by morphological
characteristics and DNA analysis. Samples were pulverized and two extraction techniques were
employed using various parameters such as solvents, temperatures and time. Finally, extracts
from each sample were screened for anti-cell viability activity against HeLa human cervical
cancer cells.
About 40 species have been recorded in Fomitopsis genus worldwide (Li et al., 2013). Most
species are parasitic on woody plants, and tend to cause brown rots (Cannon & Kirk, 2007).
Fomitopsis pinicola (Sw.) P. Karst. is a brown-rot fungus, which grows on coniferous and
broad-leaved trees and is widespread in north America, in addition to Asia and Europe
(Rösecke et al., 2000). This mushroom belongs to the family Polyporaceae, the dried fruiting
bodies of different Fomitopsis species have been used in folk medicine as haemostatics and
anti-inflammation agents (Keller et al., 1996). Japanese people have widely cultivated this
mushroom to be used as a health food source for growth regulation and diabetes (Högberg et
al., 1999). In addition, F. pinicola has also been used as an anti-diabetic material in Korea as a
traditional folk medicine (Lee et al., 2008). In Europe, F. pinicola is considered to be a nontoxic
mushroom (Keller et al., 1996).
The genus Phaeolus is distributed in the northern hemisphere and Australia (Sinclair &
Lyon, 1987 & Simpson & May, 2002), and the name is derived from the Ancient Greek word,
meaning "dark" or "obscure" (Donk, 1960). The genus Phaeolus comprises 27 species
20

(Patouillard, 1900) and the most important one, from a forestry standpoint, is schweitnitzii. P.
schweinitzii is a common root and butt pathogen of conifers in North America and Eurasia
(Sinclair & Lyon, 1987). Schweinitzii root and butt rot is a major disease of older trees; it causes
decay of the roots and lower stem and the resulting strength loss predisposes trees to windthrow
and breakage (Fairweather, 2006).
2.1 Materials and methods
2.1.1 Collection and identification of wild mushrooms
All wild mushrooms were collected from Haida Gwaii, British Columbia in August 2015. A
pocketknife or a hatchet was used to separate the mushroom from the tree substrate to which it
was attached. The morphological descriptions such as shape, size and color were recorded, as
well as location, habitat and surrounding vegetation to be used in mushroom identification.
Paper bags were used for temporary storage of samples. After that, mushrooms were dried for
further analysis. Two different methods were used to identify the mushrooms: macromorphological analysis and DNA analysis.
2.1.1.1 Macro-morphological identification
This type of identification is based on morphological details and characteristics of the
mushrooms, as different mushrooms will exhibit different morphological appearances such as
cap, spore-bearing surface, stipe, veils and odor, which could help identify the samples
preliminarily. I used the reference Mushrooms Demystified by David Aurora (1986) as an
identification guide, as it is effective to determine the mushroom or narrow down the identity
of the mushroom samples to family, genus and often species level. In addition, Matchmaker
Mushroom ID Software (Gibson et al., 2010) was used for further identification of mushroom
21

samples. This software describes the details of over 4000 gilled and non-gilled mushroom
species from the Pacific Northwest, and can help to identify the mushroom species with more
detailed information.
2.1.1.2 Identification of mushrooms using DNA analysis
Identification of mushroom based on morphology is simple and efficient, but it can be
misleading due to several factors, like hybridization (Olson & Stenlid, 2002) and convergent
evolution (Brun & Silar, 2010). DNA-based identification methods have emerged as an
efficient way to distinguish species among diverse fungi (Raja et al., 2017).
Genomic DNA was extracted from collected mushrooms using a MoBio DNA PowerSoil
Kit. After successful DNA extraction, ITS3 and NLB4 primers were used to amplify the
sequences of the samples (Smith et al., 2017). The genomic DNA (gDNA) of mushroom
samples were subjected to PCR using the conditions indicated in Tables 4 and 5.
Table 4 PCR reaction protocols required to amplify the extracted DNA from the
mushroom species.
Component
H2O
10x Thermo Pol
Buffer
dNTPs (2.5 mM)
ITS3 (10 μM)
NLB4 (10 μM)
DNA template
TAQ polymerase

Volume used (μL)
16
2.5
2.5
1
1
1.5
0.5

22

Table 5 PCR conditions for DNA amplification.
Step
1
2
3
4
5
6

Temp
95 °C
95 °C
52 °C
72 °C
72 °C
4 °C

Time
5min
30sec
30sec
30sec
10min
10min

The exponentially amplified DNA samples were further subjected to gel electrophoresis
to confirm the success of DNA amplification and ensure sufficient quantity for DNA
sequencing. The post-PCR amplified DNA products needed to be cleaned using the QIAquick
PCR kit.
The sequences obtained from Macrogen Corporation were compared with sequences
available in the National Center for Biotechnology Information (NCBI) internet database,
GenBank, using the Basic Local Alignment Search Tool (BLAST) program. The best Genbank
match of mushroom species is shown in Table 6. The photographs and data on the mushrooms
are shown in Table 7.
Table 6 Identification of mushroom species using BLAST searches, along with the percent
of matching bases between the unknown sample and the closest match.
Identity

Collected By

Fomitopsis pinicola (50)
Fomitopsis pinicola (53)
Fomitopsis pinicola (65)

Drs. Chow Lee, Wai Ming Li,
Hugues Massicotte, Keith Egger,
and Ms. Linda Tackaberry

Fomitopsis pinicola (77)
Phaeolus schweinitzii (58)
23

Best GenBank Match
(% similarity/ % coverage)
KC595922.1
(98% / 100%)
EF530947.1
(100% / 100%)
KC595922.1
(98% / 100%)
EF530947.1
(100% / 100%)
KC581334.1
(99% / 100%)

KC581354.1
(99% / 100%)
MH277962.1
Phaeolus sp. (71)
(100% / 71%)
Table 7 Collection location and photographs of the mushrooms collected from different
locations in British Columbia.
Phaeolus schweinitzii (60)

Collection
Number

Site of collection

Date of collection

Fomitopsis pinicola

Collection
Number #
50

Collection
Number #
53

Cape Fife Trail, Naikoon
Park, Haida Gwaii, BC

Cape Fife Trail, Naikoon
Park, Haida Gwaii, BC

Collection

Pesuta Shipwreck Trail,

Number #

Naikoon Park, Haida

65

Gwaii, BC

Collection

Copper Rd-Sandspit area

Number #

(towards Gray Bay, Haida

77

Gwaii), BC

August 24, 2015

August 24, 2015

August 25, 2015

August 26, 2015

24

Photographs

Phaeolus schweinitzii

Collection
Number #
57

Collection
Number #
58

Collection
Number #
60

Tlell, Crow's Nest Cafe,
Haida Gwaii, BC

Anvil Interpretive Trail,
Tlell, Haida Gwaii, BC

Anvil Interpretive Trail,
Tlell, Haida Gwaii, BC

August 24, 2015

August 25, 2015

August 25, 2015

Phaeolus sp.

Collection

Pesuta Shipwreck Trail,

Number #

Naikoon Park, Haida

71

Gwaii, BC

2.1.2

August 25, 2015

Chemical extraction of mushrooms

After collection, the mushroom specimens were dried at low heat (50 °C) and then
powdered (Figure 5) using either a regular household blender or a hammer mill.

25

Figure 5 An example of a dried and powdered mushroom sample
The chemical extraction protocol has been established in Dr. Chow Lee’s lab: dried
samples were extracted with four different solvents, firstly with 80% ethanol and 50%
methanol, which were expected to obtain low molecular weight compounds. After this, the
remaining mushroom powder was extracted using water, and then 5% sodium hydroxide. The
water and 5% NaOH extract were expected to contain water-soluble polysaccharides. The first
three solvent extractions were performed using the Dionex Accelerated Solvent Extractor
(ASE) 350 Speed Extractor (Thermo Fisher) at 65°C and at a pressure of 1500 psi. The Dionex
ASE 350 is an automated, easy and effective tool for sequential extraction (Richter et al., 1996).
The 5% NaOH extract was done manually at 65°C as it will corrode the walls of the machine.
The outline of the extraction process using the Speed Extractor is shown in Table 8:
Table 8 Outline of the extraction process
Dionex ASE 350 Extraction Process

Duration

Static extraction with rinse (80% ethanol)

4 cycles, 15 minutes per cycle

Static extraction with rinse (80% ethanol)

4 cycles, 15 minutes per cycle

26

Static extraction with rinse (80% ethanol)

4 cycles, 15 minutes per cycle

Static extraction with rinse (80% ethanol)

4 cycles, 15 minutes per cycle

Manual Extraction

Duration

5% sodium hydroxide extraction

6 hours with constant stirring

The obtained mushroom extracts were labeled as E1, E2, E3 and E4 for 80% ethanol, 50%
methanol, water, and 5% NaOH extracts respectively.
2.1.3 Rotary evaporation, pH neutralization, dialysis, and lyophilization of the extracts
All extracts were further concentrated by eliminating the redundant solvent volume. This
process was achieved by using a rotary evaporator. Once the volume of the extractions was
reduced to a required amount, the extracts were subjected to pH neutralization. The pH range
used as reference to neutralize the mushroom extracts was 6.90 to 7.10. For pH neutralization,
either 2 M of NaOH or 2 M of HCl was added. After this, the extracts were dialyzed using
Thermo Scientific SnakeSkin Dialysis Tubing (3.5K MWCO) for 24 hours in order to remove
salts. Finally, extracts were lyophilized to become dried: the extracts were firstly frozen in -80
ºC freezer and then lyophilized by using Labconco Freeze dryer (0.008 bar vacuum pressure
and -80 ºC). Freeze-dried samples were stored at 4°C for subsequent experiments. For longterm storage, dried samples were kept at -80°C.
2.1.4 Preparation of stock solution and filter sterilization
To prepare stock solution, the solubility needs to be taken into account when making
concentrated extracts. The extracts were analyzed first for their maximum solubility in an
27

appropriate solvent. After obtaining the stock solution of the extracts, the solutions were filter
sterilized with 0.2 μm filters (Sarstedt, Nümbrecht, Germany). Finally, the filtered solutions
were assessed for anti-cell viability activity.
Figure 6 outlines the chemical extraction and the downstream steps taken.

Figure 6 Sequential chemical extraction
2.1.5 Assessment for anti-cell viability activity
2.1.5.1 MTT assay
MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] is a water-soluble
yellow dye and can be reduced to water-insoluble purple formazan crystals by the
dehydrogenase system of active cells (Carmichael et al., 1987). The MTT tetrazolium assay is
a popular tool in assessing cytotoxicity and cell viability (Van Meerloo et al., 2011). The test
is based on enzymatic reduction of the lightly colored tetrazolium salt to its formazan of intense

28

purple-blue color; the concentration of the purple formazan makes it possible to estimate the
number of viable cells, which can be quantified spectrophotometrically.

Figure 7 (A) Chemical structure of MTT and its reduced formazan product; (B)
absorption spectra of MTT
Dimethyl sulfoxide (DMSO) was used to dissolve formazan crystals. The absorbance at
570 nm was measured on a spectrophotometer Synergy 2 multi-plate reader (BioTek®, USA)
as formazan has the highest optical density at 570 nm (refer to Figure 7 B).
2.1.5.2 Dose-dependent MTT assay to determine anti-cell viability activity
Dried mushroom extracts in powder were reconstituted in their respective suitable solvents,
shown in Table 11. For extracts which were dissolved in water, the stock concentration was
prepared at 20 mg/mL. As for methanol soluble extracts, the stock concentration was prepared
at 40 mg/mL. The stock solutions were then filter sterilized using 0.2 μm filter (Sarstedt,
Nümbrecht, Germany).
For the dose-dependent MTT assay, the stock solution was further diluted by adding EMEM
(Eagle’s minimum essential media) supplemented with 10% fetal bovine serum (FBS) to make
different doses of extracts. HeLa cells (ATCC®, Maryland US) cells were first subjected to

29

treatment with different concentrations (from 0.1 to 1 mg/mL) of the extracts for 48 hours and
the MTT assay was performed.
The dose-dependent MTT assay was used to investigate the percent viability of HeLa cells
after treating with different concentrations of mushroom extracts. HeLa cells were maintained
in a T-25 flask with around 10 mL of Lonza Eagle’s Minimum Essential Medium (EMEM by
VWR, ON, CA) supplemented with 10% v/v FBS. When reaching 80% of confluency, the
growth media (EMEM+ 10% FBS) was removed from the flask and cells were washed with 4
mL of Dulbecco’s Phosphate Buffer Saline (DPBS) (Lonza, Maryland), followed by 1 mL of
Trypsin EDTA to remove cells which adhere to the bottom of T-25 flask surface.
Cells were plated into a 96-well plate. The plated cells at (1500 cells/100 μL/well) were
incubated for 24 hours at 37°C in 5% CO2. After incubating for 24 hours, different
concentrations of mushroom extracts were prepared with EMEM (containing10% FBS and the
respective solvents) as shown in Table 9.
Table 9 Representation of the different doses of mushroom extracts used for MTT assay.
Final
conc.
(mg/mL)
(in plate)
1
0.75
0.5
0.375
0.25
0.1
0

Stock
(mg/mL)
(in tube)
2
1.5
1
0.75
0.5
0.2
0

For One Well

For 6 Wells

Diluted
Fraction
water in
EMEM
at 4
MEM
(μL)
mg/mL
(μL)
0
50
50
12.5
37.5
50
25
25
50
31.2
18.75
50
37.5
12.5
50
45
5
50
50
0
50
Total Volume of 100 μL

Diluted
Fraction
water in
EMEM
at 4
MEM
(μL)
mg/mL
(μL)
0
300
300
75
225
300
150
150
300
187.5
112.5
300
225
75
300
270
30
300
300
0
300
Total Volume of 600 μL

30

Finally, the different doses of 100 μL mushroom extracts as well as the negative control
(water or methanol) were added to the cells. Five replicates of each sample and control (same
solvent in which the fractions were reconstituted) were included. The plate pattern is shown in
Table 10.
Table 10 Plate pattern (96-well) followed to screen different doses of extracts from
selected mushroom samples, for their potential to exhibit anti-proliferative activity based
on the MTT assay.
A

1
water

B

water

C

water

D

water

E

water

F

water

G

water

2
water
1
mg/mL
1
mg/mL
1
mg/mL
1
mg/mL
1
mg/mL
water

3
water
0.75
mg/mL
0.75
mg/mL
0.75
mg/mL
0.75
mg/mL
0.75
mg/mL
water

4
water
0.50
mg/mL
0.50
mg/mL
0.50
mg/mL
0.50
mg/mL
0.50
mg/mL
water

5
water
0.375
mg/mL
0.375
mg/mL
0.375
mg/mL
0.375
mg/mL
0.375
mg/mL
water

6
water
0.25
mg/mL
0.25
mg/mL
0.25
mg/mL
0.25
mg/mL
0.25
mg/mL
water

7
water
0.1
mg/mL
0.1
mg/mL
0.1
mg/mL
0.1
mg/mL
0.1
mg/mL
water

8
water

9
water

Control

water

Control

water

Control

water

Control

water

Control

water

water

water

On day four, 50 μL of MTT diluted with EMEM solution (1 mg/mL) was added to the cells.
The cells were incubated for three hours allowing the live cells to react with the MTT dye, and
converting it to formazan crystals by mitochondrial reductase. Finally, the supernatant was
removed and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The
absorbance at 570 nm was read using Synergy 2 multi-plate reader. The activity of various
fractions was measured in percent cell viability compared to that of the control. Standard
deviation was the measure of error for the MTT bioassay. Kaleidagraph© version 4.03
(Synergy Software) was used to generate the graph for the acquired data. The workflow is
shown in Figure 8.
31

Figure 8 Schematic representation of dose-dependent MTT assay protocol using HeLa
Cells.
2.2 Results and discussion
2.2.1 Chemical extraction of mushrooms
The Dionex ASE 350 speed extractor was used to obtain E1, E2 and E3 crude extracts
while the E4 5% sodium hydroxide extract was done manually using a hotplate. The summary
of extraction results is shown in Table 11.
Table 11 Summary of the fungal extracts obtained through successive extraction process.
Extraction
sample (g)

Extract

Solvent used

Extract

type

for extraction

weight
(gm)

E1
E2

5.403

E3
E4

4.831

E1
E2

Yield

Physical

(%)

appearance

Fomitopsis pinicola (#50)
80% Ethanol
0.174
3.22
Coarse powder
50% Methanol
0.093
1.72
Coarse powder
Fluffy (Cotton
Water
0.046
0.85
like)
Fluffy (Cotton
5% NaOH
0.724
13.39
like)
Fomitopsis pinicola (#53)
80% Ethanol
0.469
9.71
Coarse powder
50% Methanol
0.186
3.85
Coarse powder
32

Solvent used
for
reconstitution

Methanol
Methanol
Water
Water
Methanol
Methanol

E3

Water

0.045

0.93

E4

5% NaOH

0.401

8.30

E1
E2
5.057

E3
E4
E1
E2

4.987

E3
E4
E1
E2

5.316

E3
E4
E1
E2

5.325

E3
E4
E1
E2

5.345

E3
E4

Fluffy (Cotton
like)
Fluffy (Cotton
like)

Fomitopsis pinicola (#65)
80% Ethanol
0.054
1.06
Coarse powder
50% Methanol
0.078
1.54
Coarse powder
Fluffy (Cotton
Water
0.067
1.32
like)
Fluffy (Cotton
5% NaOH
0.508
10.05
like)
Fomitopsis pinicola (#77)
80% Ethanol
0.322
6.46
Coarse powder
50% Methanol
0.113
2.27
Coarse powder
Fluffy (Cotton
Water
0.148
2.97
like)
Fluffy (Cotton
5% NaOH
0.249
4.99
like)
Phaeolus schweinitzii (#57)
80% Ethanol
0.406
7.68
Coarse powder
50% Methanol
0.415
7.81
Coarse powder
Fluffy (Cotton
Water
0.304
5.72
like)
Fluffy (Cotton
5% NaOH
0.652
12.26
like)
Phaeolus schweinitzii (#58)
80% Ethanol
0.210
3.94
Coarse powder
50% Methanol
0.216
4.06
Coarse powder
Fluffy (Cotton
Water
0.177
3.32
like)
Fluffy (Cotton
5% NaOH
0.873
16.39
like)
Phaeolus schweinitzii (#60)
80% Ethanol
0.189
3.53
Coarse powder
50% Methanol
0.292
5.46
Coarse powder
Fluffy (Cotton
Water
0.156
2.91
like)
Fluffy (Cotton
5% NaOH
0.640
11.97
like)

Water
Water
Methanol
Methanol
Water
Water
Methanol
Methanol
Water
Water
Methanol
Methanol
Water
Water
Methanol
Water
Water
Water
Methanol
Water
Water
Water

Generally, the extract E4 has the highest yield amongst all extracts except for mushroom
sample #53 and #77. The lowest yields were obtained mostly for E3 water extracts. The highest
33

yield (16.39%) amongst all extracts was exhibited by E4 of P. schweinitzii (#58), while the
lowest yield (0.85%) was exhibited by E3 of F. pinicola (#50). The physical appearance of
these powder extracts were quite distinct. All E1 and E2 extracts were coarse powder. E3 and
E4 extracts were generally fluffy in nature. All E1 and E2 extracts were dissolved in methanol
except for E2 of P. schweinitzii #58 and #60 which was reconstituted in water. All E3 and E4
extracts were soluble in water.
2.2.2 Dose-dependent cell viability assessment of mushroom extracts using HeLa cells
2.2.2.1 Fomitopsis pinicola (#50)
Results of the dose-dependent MTT assay of extracts 1-4 from Fomitopsis pinicola (#50)
are shown in Figure 9. Extracts 1 and 2 significantly reduced cell viability to 40% at the lowest
concentration of 0.1 mg/mL. Extract 3 also reduced the cell viability to around 20% at the doses
of 0.75 and 1 mg/mL. In contrast, extract 4 did not appear to have any significant effect on cell
viability, decreasing viability to only 80% at the highest concentration of 1 mg/mL.
Fomitopsis pinicola_CL050

Cell viability (% control)

120

E4

100
80
60

E2

40
20
0

E1
0

0.2

E3
0.4

0.6

0.8

1

Concentration (mg/mL)
Figure 9 Dose-dependent assessment of cell viability by extracts from F. pinicola (#50)
Different concentrations of crude extracts E1 (80% ethanol), E2 (50% methanol), E3 (water)
and E4 (5% NaOH) were added to HeLa for 48 hours followed by assessment of cell viability
34

using MTT assay. The graph shows results from one biological replicate (n=1). Error bars are
standard deviation.
2.2.2.2 Fomitopsis pinicola (#53)
Results in Figure 10 show that extract 3 from Fomitopsis pinicola (#53) reduced the cell
viability to around 20% at the doses of 0.75 and 1 mg/mL. Extracts 1 and 2 inhibited the cell
viability to around 40% at the lowest concentration of 0.1 mg/mL. At and above 0.5 mg/mL
concentration, both E1 and E2 extracts caused an unexpected increase in cell viability. Such
false negative effect had been observed with crude extracts from some mushroom species
(Smith et al., 2017; Deo et al., 2019) and is most likely due to chemical(s) in extracts interacting
with MTT leading to colored solution. In comparison, extract 4 only showed little effect on cell
viability, decreasing viability to only 60% at the highest concentration of 1 mg/mL.

Cell viability (% control)

Fomitopsis pinicola_CL053
100
80

E4
E1

60

E2

40
20

E3
0

0

0.2

0.4

0.6

0.8

1

Concentration (mg/mL)
Figure 10 Dose-dependent assessment of cell viability by crude extracts isolated from F.
pinicola. (#53) Error bars are standard deviation.

35

2.2.2.3 Fomitopsis pinicola (#65)
The results of dose-dependent MTT assays performed using extracts from Fomitopsis
pinicola (#65) are shown in Figure 11. Extracts 1 and 2 dramatically reduced cell viability to
approximately 20% by 0.1 mg/mL. Extract 3 reduced cell viability to 60% at the highest dose
of 1 mg/mL. Extract 4 did not show any significant effect on cell viability even at 1 mg/mL.

Cell viability (% control)

Fomitopsis pinicola_CL065
120

E4

100
80

E3

60
40

E1

20
0

E2
0

0.2

0.4

0.6

0.8

1

Concentration (mg/mL)
Figure 11 Dose-dependent assessment of cell viability by crude extracts isolated from F.
pinicola. (#65) Error bars are standard deviation.
2.2.2.4 Fomitopsis pinicola (#77)
Results of dose-dependent MTT assays of extracts 1-4 from Fomitopsis pinicola (#77) are
shown in Figure 12. The results show that extracts 1 and 2 reduced cell viability to 20% at the
lowest concentration of 0.1 mg/mL. Extract 3 exerted dose-dependent inhibitory effect leading
to 20% cell viability at the highest concentration of 1 mg/mL. In contrast, extract 4 had no
effect on cell viability.

36

Cell viability (% control)

Fomitopsis pinicola _CL077
100

E4
80
60

E2

40
20

E3

E1
0

0

0.2

0.4

0.6

0.8

1

Concentration (mg/mL)
Figure 12 Dose-dependent assessment of cell viability by crude extracts isolated from F.
pinicola. (#77) Error bars are standard deviation.
2.2.2.5 Phaeolus schweinitzii (#57)
Results of dose-dependent MTT assay of extracts 1-4 from P. schweinitzii (#57) are shown
in Figure 13. Extracts 1 and 2 reduced cell viability to 20% and 40% at 0.25 mg/mL
respectively. At higher concentrations, especially for E2, dark brown colored solution appeared
upon addition of MTT. This could interfere with reading plate process and gave rise to the false
negative results as shown in Figure 13. Extracts 3 and 4 also reduced cell viability to 20% but
at higher concentration of 1 mg/mL.

37

Cell viability (% control)

Phaeolus schweinitzii_CL057

120

E3

100
80

E4

60

E2

40
20
0

E1
0

0.2

0.4

0.6

0.8

1

Concentration (mg/mL)

Figure 13 Dose-dependent assessment of cell viability by crude extracts isolated from P.
schweinitzii. (#57) Error bars are standard deviation.
2.2.2.6 Phaeolus schweinitzii (#58)
The dose-dependent MTT assays on fractions from Phaeolus schweinitzii (#58) showed
that extract 1 exhibited growth-inhibitory activity; it reduced cell viability to under 20% at 0.1
mg/mL, and the effect increased with the increasing dose, which might be the color of the
extraction interfering with the MTT reagent, resulting in false absorbance. Extracts 3 and 4
reduced cell viability to about 30% at 1 mg/mL while extract 2 did not inhibit the cells.

38

Cell viability (% control)

Phaeolus schweinitzii_CL058
100

E2

80

E4

60
40

E1
E3

20
0

0

0.2

0.4

0.6

0.8

1

Concentration (mg/mL)
Figure 14 Dose-dependent assessment of cell viability by crude extracts isolated from P.
schweinitzii. (#58) Error bars are standard deviation.
2.2.2.7 Phaeolus schweinitzii (#60)
Results of dose-dependent assay of Phaeolus schweinitzii are shown in Figure 15. The
results show potent effect by extract 3, reducing cell viability to 20% at 0.25 mg/mL. Extract
4 reduced cell viability to 40% at 1 mg/mL. Extract 1 is the most potent, reducing cell viability
to 20% at the lowest concentration of 0.1 mg/mL At and above 0.25 mg/mL, E1 exhibited false
negative effect as mentioned above. Extract 2 only had modest effect, reducing cell viability to
80% at the highest concentration of 1 mg/mL.

39

Cell viability (% control)

Phaeolus schweinitzii_CL060
100

E2
80

E4

60
40

E1
20
0

0

0.2

E3

0.4

0.6

0.8

1

Concentration (mg/mL)
Figure 15 Dose-dependent assessment of cell viability by crude extracts isolated from P.
schweinitzii. (#60) Error bars are standard deviation.
2.2.2.8 Phaeolus sp. (#71)
Results of dose-dependent assay of Phaeolus sp. are shown in Figure 16. The results
demonstrate potent effect by extract 1, reducing cell viability to 10% at 0.1 mg/mL. Both
Extracts 2 and 3 reduced cell viability to 30% at 1 mg/mL. Extract 4 only had modest effect,
reducing cell viability to 60% at the highest concentration of 1 mg/mL.

40

Figure 16 Dose-dependent assessment of cell viability by crude extracts isolated from
Phaeolus sp. (#71) Error bars are standard deviation.
In this study, extracts E1, E2 and E3 from F. pinicola inhibited the growth of cancer cells
dramatically, which were confirmed by previous studies (Wu et al., 2014 & Choi et al., 2007).
Extract E4 of F. pinicola had no significant inhibitory effect on HeLa cells which is in
agreement with the existing literature (Smith, 2017). Extracts E1 and E3 from P. schweinitzii
showed strong growth-inhibitory on HeLa cells, while other two extracts did not exhibit potent
inhibitory activity.
The primary screening method demonstrated results regarding the therapeutic potentials
of extracts from the eight BC mushrooms studied here, summary of the results are shown in
Table 12. The main challenge of this project is the purification and structural elucidation of the
compounds from mushrooms. The reason for selecting Phaeolus sp. for further studies is that
not many researches have been reported on the anti-cancer activity of this mushroom specie,
41

chapter 3 of this thesis will investigate in detail of the purification and structural elucidation of
the small molecules from E1 extract of Phaeolus sp.; more information about this mushroom
is shown in chapter 3.
Table 12 Summary of twenty-eight crude extracts isolated from seven BC mushroom
specimens on HeLa cell viability
Mushroom
specimen

Fomitopsis
pinicola (50)

Fomitopsis
pinicola (53)

Fomitopsis
pinicola (65)

Fomitopsis
pinicola (77)

Phaeolus
schweinitzii (57)

Phaeolus
schweinitzii (58)

Phaeolus
schweinitzii (60)

Extract

Anti-proliferative activity1
(% viability at 1 mg/mL)

E1 (80% ethanol)

38.63

E2 (50% methanol)

55.39

E3 (water)

25.92

E4 (5% NaOH)

79.46

E1 (80% ethanol)

54.07

E2 (50% methanol)

49.25

E3 (water)

20.72

E4 (5% NaOH)

63.69

E1 (80% ethanol)

33.81

E2 (50% methanol)

21.73

E3 (water)

54.99

E4 (5% NaOH)

92.03

E1 (80% ethanol)

32.68

E2 (50% methanol)

37.21

E3 (water)

19.25

E4 (5% NaOH)

94.38

E1 (80% ethanol)

37.70

E2 (50% methanol)

70.16

E3 (water)

24.66

E4 (5% NaOH)

27.13

E1 (80% ethanol)

56.15

E2 (50% methanol)

64.02

E3 (water)

32.77

E4 (5% NaOH)

31.89

E1 (80% ethanol)

56.98

E2 (50% methanol)

80.48

E3 (water)

25.84

42

E4 (5% NaOH)
1

Percent viability of HeLa cells as compared to control.

43

39.74

Chapter 3 Purification, identification and characterization of a growth-inhibitory
compound from Phaeolus sp.
This chapter focuses on Phaeolus sp. (#71) which was collected from Pesuta Shipwreck
Trail in Naikoon Provincial Park, Haida Gwaii, BC. The samples were morphologically and
genetically identified as a member of the genus Phaeolus. Using the BLAST program, the
sequence obtained from our collection matched 100% in the GenBank database to a voucher
specimen identified as Phaeolus sp. by Jonathan Frank at Southern Oregon University, who
believes it may be an undescribed species (Frank, personal communication); further research
will need to be done to determine its identity. This mushroom was selected for purification and
characterization to potentially discover novel compounds. Part A of this chapter focuses on the
purification, characterization and structural elucidation of a growth-inhibitory compound from
the E1 (80% ethanol) extract of Phaeolus sp. Part B focuses on isolating other small molecules
from Phaeolus sp.
Part A: Purification and characterization of hispidin from Phaeolus sp.
3.1 Purification and chemical characterization strategies
Numerous studies describing the therapeutic compounds medicinal fungi possess have
been published (Pennerman et al., 2015). To understand these compounds better, one must
undertake their purifications. Generally, the purification strategies for bioactive compounds is
dependent on the nature of compounds. Liquid-liquid extraction is first employed to purify the
compounds based on the polarity. After this, techniques like size-exclusion chromatography
(e.g., Sephadex-LH-20), ion-exchange chromatography (e.g., DEAE Sephadex), and high-

44

performance liquid chromatography (HPLC) are used for further purification. When the
purification process is finished, analytical instruments like LC-MS (Liquid ChromatographyMass Spectrometry), GC-MS (Gas Chromatography-Mass Spectrometry), Fourier transform
infrared spectroscopy (FTIR) and NMR (Nuclear Magnetic Resonance) are used to characterize
the target compounds further.
3.2 Materials and methods
3.2.1 Large scale chemical extraction of Phaeolus sp.
The Phaeolus collection (#71) was finely powdered using a hammer mill, and large-scale
extraction of extract E1 (80% ethanol) was performed manually using a hot plate. After that,
the extracts were concentrated, pH neutralized and lyophilized using a Labconco freeze dryer
and finally stored at -80°C.
3.2.2 Liquid-liquid extraction (phase separation)
Liquid–liquid extraction or phase separation is a method to separate compounds based on
relative solubility in two different immiscible liquids, usually water (polar) and an organic
solvent (non-polar). To carry out liquid-liquid extraction, an extraction flask was used (Figure
17).

45

Figure 17 Illustration of the liquid-liquid extraction technique
At first, 100 mg of E1 extract (80% ethanol) powder was added in the extraction flask.
Then, 40 mL of water was added to the flask as a primary solvent. Later, an equal volume (40
mL) of ethyl acetate was added and the sample was dissolved in two layers. The flask was
shaken to ensure that the separation of compounds between the two layers was maximized.
After five minutes (or until the layers separated) the layers were observed (Figure 17). Each
layer was collected into a different beaker. The upper layer (ethyl acetate layer) was dried by
heating on a hot plate at 65 °C. The lower layer (water layer) was lyophilized using Labconco
Freeze dryer. After obtaining the two dried samples, the yield was calculated and the two layers
were further screened for their growth-inhibitory activity using the MTT assay as described in
section 2.1.5.1.

46

3.2.3 Sephadex™ LH-20 (size exclusion chromatography)
Size exclusion chromatography was also performed for better separation of the compounds
in the crude ethyl acetate extract. During this purification process, the molecules were separated
by their size as they passed through the stationary and mobile phase (100% Methanol) packed
in the column. The larger molecules cannot fit in the pores of the stationary media and they
flow more quickly through the stationary phase, which causes them to be eluted first. The
smaller molecules easily penetrate the pores of the medium, causing them to flow slowly and
elute out of the column later. Due to this physico-chemical property, Sephadex™ was selected
to do the next step of purification.
Sephadex™ LH-20 is a liquid chromatography medium designed for natural product
isolation (Datta et al., 2013). Since the medium is Sephadex LH-20, the method is also termed
as Sephadex LH-20 column chromatography. Sephadex™ LH-20 is prepared by
hydroxypropylation of cross-linked Sephadex G-25 dextran and an organic solvent is used as
a mobile phase. This LH-20 resin has both hydrophilic and hydrophobic characteristics, which
make it swell in aqueous solution and organic solvents. In my study, the mobile phase used was
100% methanol as the mobile phase is based on the solubility of active compounds (ethyl
acetate layer). Two types of columns, including a 22 mL column (micropipette) and 400 mL
(C26/100 column), were used for packing the resin.
3.2.3.1 Sephadex™ LH-20 resin swelling and column packing
The swelling ratio of Sephadex™ LH-20 resin to methanol was 1:4. Firstly, Sephadex™
LH-20 dry powder was transferred into a beaker and soaked in excess methanol to swell
47

overnight. After the resin was completely soaked with 100% methanol, the excess methanol
was removed from the slurry. Methanol was degassed for 2-3 hours to ensure that the mobile
phase had no dissolved gases. Otherwise, the gas would be pumped into column and crack the
resin.
Two different types of columns were used for the purification. To pack a small sized column
(22 mL), a 25 mL serological pipette (Sarstedt, Germany) was used as a column, and a cotton
plug was used to hold the resin above the tip of the pipette and also ensure the mobile phase
(methanol) could flow out of the column; the column was first packed with a total of 22 mL of
resin under the influence of gravity. As for 400 mL (C26/100) (GE Healthcare) glass columns,
the swollen resin was transferred to the columns first. The top and lower adapters were used to
support the resin in the glass column, and the top of the column was linked to the solvent
reservoir. The Pump P-50 (Pharmacia Biotech) was used to pump the methanol solvent to flow
through the column for packing and washing at a flow rate of 2 mL/min. The column was
required to equilibrate with degassed methanol for at least two column volumes between every
run.
3.2.3.2 Sample loading and fractions collection
The sample was prepared using the active extract from the upper layer (ethyl acetate layer)
of the liquid-liquid extraction and methanol (100%) was used to reconstitute the sample. The
final concentration of loading sample was 40 mg/mL. The sample was centrifuged before
loading onto the column to remove the impurities which could be trapped in the resin. For a 22
mL column, the sample was loaded onto the column using a micropipette (Thermo Fisher
48

Scientific, USA). The volume of the sample (ethyl acetate layer) loaded was 2% (440 μL) of
the packed column volume. Fraction collection was based on gravity. The sample (8 mL) was
introduced into the large (400 mL) (GE Healthcare) columns with the help of a syringe in a
three-way switch (Figure 18).

Figure 18 Photograph of the Sephadex™ LH-20 size exclusion technique using a large
C26/100 column (GE Healthcare) and the mobile phase (degassed methanol)
The flow rate of the mobile phase for C26/100 columns was 2 mL/min. An automatic
fraction collector (Pharmacia Biotech) was used to collect fractions, and the volume of the
collected fractions was 14 mL. After collection, the fractions were stored in a cold room (4°C)
before assessment for growth-inhibitory activity.
3.2.3.3 Sephadex™ LH-20 column clean-up, unpacking and storage
After each run is finished, the columns were washed to ensure no sample remained in the
resin. The original degassed mobile phase (100% methanol) was used to wash the resin at 1.5
times the actual flow rate and with mobile phase volume twice the bed volume. However, if
the sample was sticky and oily, the resin must be washed with NaOH (5%), at 1 mL/min flow
49

rate with half the column bed volume, then twofold of the column bed volume of water was
used to wash the resin, at 1.5 mL/min flow rate to elute both the remnant loading sample and
NaOH residue. Finally, two-column bed volume of degassed mobile phase were used to run
through the column to ensure the long shelf life and reusability of the resin. After the column
was washed, the resin was removed from the column and stored in an Erlenmeyer flask in
degassed 100% methanol at 4°C to prevent any microbial contamination of the resin.
3.2.4 UV absorbance analysis
A nanodrop spectrophotometer (Thermo Fisher Scientific, SA) was used to determine the
maximum UV absorbance (λmax) of bioactive fractions 39 to 42 from the Sephadex LH-20
(400 mL). After calibration with water, the spectrophotometer was blanked with HPLC grade
methanol. The sample peddle was rinsed with autoclaved milli-Q water and wiped with
Kimwipe paper before analyzing the sample. A 2 μL sample was used to determine the λmax
of the fractions. The λmax for fractions was read at around 270 nm, which was later used in
HPLC analysis of the active fractions.
3.2.5 High performance liquid chromatography (HPLC-UV) analysis
HPLC is a powerful method for separation and purification of natural products (Adams &
Nakanishi, 1979), which yields an excellent separation in a short time. Due to this, it was
chosen to further purify the bioactive compounds collected from the Sephadex LH-20 fractions.
HPLC operates under high pressure to push the mobile phase and the sample through the
stationary phase. The Agilent 1260 Infinity Systems (HPLC) was used to purify the growth-

50

inhibitory compound. The HPLC system was equipped with solvent systems, a sample
injection valve, detector, controller, and computer to control the system and display results.
Reverse-phase HPLC is the most common type of HPLC used (Robards et al., 1994). In
this study, RP-HPLC was used to isolate the growth-inhibitory compound. RP-HPLC has a
nonpolar hydrophobic stationary phase and a moderately polar solvent as a mobile phase.
Sample elution is based on polarity; the highly polar compounds elute first, and the non-polar
compounds elute later (Sundaram et al., 2009). As a result, the non-polar (hydrophobic)
molecules in the mobile phase tend to interact with the non-polar stationary phase more, while
the polar molecules in the mobile phase pass through faster and elute from the column first.
Water is the most polar solvent so it can repel the hydrophobic molecules into the stationary
phase more than other solvents, therefore, it causes longer retention times (Realini, 1981).
When adding another organic solvent to the mobile phase system, the polarity could be
modified, this causes the solvent system to no longer retard the sample in the stationary phase
and thus spend less time in the column and elutes earlier.
The solvents used as the HPLC mobile phase were chosen based on their appropriateness
to the RP-HPLC. Water and methanol were selected to consist the solvent system. Water was
used because it is miscible, chemically non-reactive, has low viscosity, low UV absorbance,
and high solubility, while the HPLC-grade methanol was selected based on good solubility of
the sample.
A gradient elution method was used during all HPLC purifications to provide separations
of mixture. The gradient elution method contains two or more mobile phases which differ in
51

polarity. The solvent system started with 30/70% water/methanol and ended at 40/60% water
to methanol over nine minutes. The mobile phase was automatically degassed through the
degasser system of the HPLC before entering the quaternary pump. The quaternary pump
controls the flow rate as well as the gradient of the solvent system. The flow rate for the gradient
mobile phase was set at 1.0 mL/min.
Samples (bioactive LH-20 fractions) were filtered using a 0.2 μm filter (Sarstedt, Germany)
before injecting into the system. The wavelength (nm) used to visualize UV signal of bioactive
compounds was 270 nm (based on reading from nanodrop spectrophotometer). The needle of
the auto-sampler was programmed to wash every time before injection to prevent any cross
contamination. The column was washed with the solvent system for at least 10 column
volumes. The sample volume loaded onto the column was 10 μL (10 mg/mL). Each run took 9
minutes to separate and elute all the peaks out of the column. The fraction collection was done
based on the HPLC-UV. The fractions were collected into 8.0 mL glass vials using a fraction
collector (Agilent infinity Lab 1260).
3.2.6 LC-MS analysis
LC-MS is a technique, which combines the separating power of HPLC, with the detection
power of mass spectrometry (MS). Liquid chromatography separates mixtures into multiple
components, and mass spectrometry provides structural identity of the individual components
with high molecular specificity and detection sensitivity (McMaster, 2005). This technique can
be used to analyze organic compounds efficiently (De Hoffmann, 2000).

52

All LC-MS analyses were performed using Agilent 1260 Infinity Systems HPLC and
Agilent 6120 quadrupole mass spectrophotometer. Mass spectrometry was performed using an
active splitter, and a split ratio of 66:1. After separation of the sample components in the HPLC
system, an Electrospray ionization (ESI) was used to convert separate sample species into ions
in the gas phase, and the mass analyser was used to sort ions according to their mass to charge
ratio (m/z).
3.2.7 Nuclear magnetic resonance (NMR) analysis
Nuclear magnetic resonance spectroscopy, commonly referred to as NMR, has become the
preeminent technique for determining the structure of organic compounds (Pretsch et al.,
2000). The structural elucidation of the compound was achieved using a Bruker 300 MHz NMR
spectrophotometer. And 1D and 2D NMR spectra were recorded on a Bruker 300 spectrometer
with a 5 mm Fourier easy probe.
One-dimension NMR is a technique used to probe the chemical environments of nuclei in
molecules and recorded on a frequency axis while the intensity is represented as the 1D
information of the molecules. This technique is highly automatable, reliable, and fast (Emwas
et al., 2019). Although the 1D NMR spectrum of a natural product can be acquired rapidly,
they are often complicated by peak overlap that can significantly impede the identification of
a sample (Viant, 2003). Such an issue can be addressed by using two-dimensional (2D) NMR
techniques by spreading out cross-peaks of resonances along the indirect dimensions that
overlap in a 1D NMR spectrum, which can reduce the likelihood of peak overlap (Bingol et

53

al., 2014). The 2D NMR spectra can provide more information about a molecule than 1D NMR
spectra, and are especially useful in determining the structure of a molecule.
One-dimension 1H NMR and 13C-NMR were performed to identify the proton and carbon
atoms in our purified sample. The 2D-NMR spectroscopy correlation spectroscopy (COSY),
heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation
(HMBC) were employed to further provide data about the sample. COSY is a 2D NMR method
used to identify pairs of spin–spin coupled protons. In the spectrum, 1D NMR spectra of
chemical shifts is indicated along a diagonal ridge running from the lower left to upper right
corner of the plot. Cross peaks are produced by spin–spin coupling of two protons; most
commonly, cross peaks are produced by adjacent protons. COSY is the most common form of
2D NMR spectroscopy and is a routine and highly useful method for identifying molecules
through their spin–spin couplings (Xi et al., 2006). HSQC spectra are 2D data matrices
consisting of cross peaks of protons and carbons. It is a proton-detecting experiment, and it is
more sensitive and less time-consuming to acquire. Also, it is richer in information as it
simultaneously allows the list of directly bound 1H-13C pairs to be assembled (Szakács & Sánta.
2015). The HMBC NMR experiment is probably the most useful and the most widely used 2D
heteronuclear NMR method in the structural elucidation of organic molecular structures.
(Furrer, 2012). It is a popular technique for multiple-bond heteronuclear chemical shift

correlation (Martin & Zektzer, 1988). (1H-13C)-HMBC can provide correlations between
protons and carbons that are two or three bonds apart from each other. The HMBC spectra is

54

important for the detection of quaternary carbons as it links separate structural fragments
obtained from analysis of COSY and HSQC (Edison & Schroeder, 2010).
The magnets of the Bruker 300 MHz NMR spectrophotometer were shimmed properly
before analysis of samples (Topspin 4.0.7). Approximately 7 mg of the sample was dissolved
in deuterated acetone (CD3)2CO (99.96 % acetone-d and 0.3% Tetramethylsilane(TMS))
(Sigma Aldrich) and transferred directly into a 5 mm NMR tube (Kimble, USA). The final
volume of the sample in NMR tube was approximately 1 mL. After performing the different
types of NMR mentioned above, 1D and 2D NMR spectrums integrated together to provide
clear information.
3.2.8 Melting point
The melting point of a substance is the temperature at which the material changes from a
solid to a liquid state, and it is one of the most widely used fundamental physical properties
(Jain et al., 2004). Pure crystalline substances have a clearly defined melting point.
Determining the melting point is a simple and fast method used to obtain a first impression of
the purity of a substance, as even small quantities of impurities could change the melting point
or expand its melting range.
The capillary method was used to determine the melting point of the purified compound
using the MPA160 DigiMelt Student Melting Point System. A thin glass capillary tube
containing little sample is placed into a heated stand in close proximity to a high accuracy
thermometer. The temperature in the heating stand is increased, at a fixed rate, until the sample
in the tube becomes liquid. Visual observations were taken to observe changes during the
55

melting point determination, including color change, loss of solvent and change in
crystallization state. The onset point, when liquid clearly appears for the first time, is generally
considered the start of the melting. The clear point corresponds to the stage of melting at which
the substance becomes completely liquid. The melting point range is defined as the interval
between the onset and clear points, which is an indicator of purity of a solid compound.
3.3 Results
3.3.1 Large scale chemical extraction of Phaeolus sp.
Large scale chemical extraction to isolate E1 was performed on the collection 71 of
Phaeolus sp. The percent yield for E1 (80% ethanol) was 16.80% (Table 13). The extract can
be partially dissolved in 100% methanol.
Table 13 Percent yield of E1 extract from Phaeolus sp. (#71)
Mushroom
No.

Mass of
sample (g)

Mass of E1
extract (g)

Yield (%)

71

50.217

8.441

16.80

3.3.2 Liquid-liquid extraction (phase separation)
Liquid-liquid extraction performed on E1 of Phaeolus sp. resulted in two distinct layers.
The lower layer was water and the top layer was ethyl acetate. The color of the aqueous layer
was yellow, and it contained approximately 45% yield of product. The color of the upper
organic layer was brown; The percent yield from the top ethyl acetate layer was 33.50% (Table
14). Furthermore, solubility of both layers was determined in order to perform growthinhibitory assays; both extract samples were soluble in methanol.

56

Table 14 Percent yield of three layers based on liquid-liquid extraction (Phase separation)
of E1 (80% ethanol) extract of Phaeolus sp.
Layer of LiquidColor of Mass (mg)
Liquid
the layer out of 20mg
extraction
Organic (Ethyl
Brown
6.70
acetate)
Aqueous
Cream
8.90
(Water)

Yield (%)

Growth-inhibitory
Activity

33.50%

Yes

44.50%

Yes

Solubility
100%
Methanol
100%
Methanol

The dose-dependent MTT assay was performed on lyophilized samples from the two layers
of the liquid-liquid extraction; HeLa cell plating and treatment was described in section 2.1.5.
The final concentration of the methanol was less than 1% in each well to prevent non-specific
toxicity to cells. The doses tested on HeLa cells were 0.05 and 0.1 mg/mL.

Figure 19 Assessing the water and ethyl acetate layers from E1 extract for antiproliferative activity. Two different concentrations of water and ethyl acetate layers were
added to HeLa for 48 hours followed by assessment of cell viability using MTT assay. The
graph shows results from one biological replicate (n=1). Error bars are standard deviation.
57

As shown in Figure 19, strong growth-inhibitory activity was exhibited by the upper ethyl
acetate layer. The cell viability was around 20% at both 0.1 and 0.05 mg/mL and results were
consistent. At 0.1 mg/mL, the water layer also exhibited potent growth-inhibitory activity.
However, in general, as compared to the ethyl acetate layer, the water layer had less potent
activity against HeLa cells. Based on the MTT results, it was decided to proceed with
purification of bioactive compounds from the upper ethyl acetate layer.
3.3.3 Assessing post-Sephadex™ LH-20 fractions collected from a 22 mL column for
bioactivity
Fractions from the 22 mL sized Sephadex LH-20 column were collected in 1.5 mL
Eppendorf tubes and the final volume of each fraction was around 1.3 mL. Fractions 25-35
were pale yellow in color, suggesting most compounds were present in these fractions. After
collection, these tubes were put in racks and left in a fume hood for three days to evaporate the
excess solvent. The dose-dependent MTT assay was performed to test the activity of these
fractions, HeLa cells (1500 cells/well) were plated and after 24 hours of incubation, the cells
were treated with 2 μL of the fraction and 98 μL of EMEM with 10% FBS. The final
concentration of methanol in each well was maintained at 1% which was non-toxic to the cells.

58

Figure 20 Assessing anti-proliferative activity of fractions collected from 22 mL size
Sephadex™ LH-20 column. Effect of 2 μL from 22 mL size post-Sephadex™ LH-20
fractions on HeLa cell viability. The graph shows results from one biological replicate (n=1).
Error bars are standard deviation.
Fractions 25 to 32 inhibited the HeLa cell viability compared to the control (Figure 20).
Weak growth-inhibitory activity was observed for other fractions except for fractions 25 to 32.
Given that the inhibitory activity was observed in later eluting fractions, it is likely that the
bioactive compounds are small in size.
3.3.4 Assessing post-Sephadex™ LH-20 fractions collected from a 400 mL column for
growth-inhibitory activity
After bioactivity was detected in specific fractions collected from the small Sephadex
column (22 mL), the technique was scaled up to 400 mL using a C26/100 (GE Healthcare,
Quebec) column. Fractions were collected in 15 mL tubes (Sarstedt) and the final volume of
each fraction was around 5 mL. After collection, these tubes were left in the fume hood to
59

evaporate excess solvent. The MTT assay (1500 cells/well) was performed using fractions (14
mL each) obtained by running a total 320 mg of the E1 (80% ethanol extract) ethyl acetate
layer of Phaeolus sp. through a 400 mL Sephadex™ LH-20 column. The results of the MTT
assay using the fractions are shown in Figure 21.

Figure 21 Assessing anti-proliferative activity of fractions collected from a 400 mL
Sephadex™ LH-20 column. Effect of 2 μL from 400 mL size post-Sephadex™ LH-20
fractions on HeLa cell viability. The graph shows results from one biological replicate (n=1).
Error bars are standard deviation.
Fractions 39 to 42 reduced the HeLa cell viability dramatically as compared to the
control, suggesting the presence of bioactive compounds in these fractions. The results were
consistent with the results from the 22 mL size column, the color of bioactive fractions were
same, and the cell viabilities were similar, which inhibited at around 20%. The active fractions
from the 400 mL column were further analyzed using analytical HPLC.
60

3.3.5 HPLC results
3.3.5.1 Analytical
The purification process is depicted in Figure 22.

Figure 22 Flow chart diagram representing the method of compound purification from
E1 (80% ethanol) extract of Phaeolus sp.
The resolution based on the analytical HPLC conditions was excellent: each peak was
distinct and well separated (Figure 23). The retention time for the peak was 4.312 min (later
confirmed to be hispidin). The percent abundance based on UV spectra for the peak was
95.57%, and this peak was further aggregated and characterized.

Figure 23 UV spectrum showing the relative abundance and retention time of peak at
4.312 min at λmax 270 nm (using Agilent C-18 analytical column and Sephadex LH-20 active
fraction sample).
61

3.3.5.2 HPLC-Mass spectroscopy (MS) analysis
The HPLC-purified compounds were protonated with the aid of 0.1% formic acid isocratic
solution. Figure 24 shows the UV spectrum for confirmation of the purified compound. The
percent abundance based on UV spectra for the peak was 99.33%. Spectra shown in Figures
25 and 26 represent the MS SIM and scan signal of the peak respectively..
The mass to charge ratio (m/z) of the protonated purified peak was 247.1 (Figure 27) which
confirmed the presence of the target masses. The abundance of the targeted masses was also
found to be higher in SIM mode than in the scan mode, confirming the presence of the
compounds in the sample.

Figure 24 UV spectrum confirming the purity of the compound [M+H]+ m/z 247.1
detected at 4.312 min through ESI (Electrospray ionization) chamber.

Figure 25 MS SIM spectrum showing the relative abundance and retention time of the
compound with [M+H]+ m/z 247.1 detected at 4.312 min through ESI (Electrospray
ionization) chamber.
62

Figure 26 MS scan spectrum showing the relative abundance and retention time of the
compound with [M+H]+ m/z 247.1 detected at 4.312 min through ESI (Electrospray
ionization) chamber.

Figure 27 Integration of MS scan spectrum indicating the presence and abundance of
compound with [M+H]+ m/z 247.1 visible at 4.567 min
In the MS scan spectrum, the ion peaks at m/z 247.1 [M+H]+ and m/z 269.1 [M+Na]+ in
the positive mode, suggests the molecular weight of the purified compound to be 246.1 g/mol.
3.3.6 Nuclear magnetic resonance (NMR) analyses
Comparing the NMR spectra found here (Tables 15-16) with previously published NMR
data revealed that the growth-inhibitory compound in Phaeolus sp. is hispidin, which had been
previously isolated and structurally elucidated (Park et al., 2004).

63

Table 15 1H NMR spectral data of purified compound (hispidin) obtained using Bruker
Fourier A-300 NMR

1

Hydrogen

Hispidin

H-3
H-5
H-7
H-8
H-10
H-13
H-14

5.36
6.11
6.65
7.24
7.15
6.84
7.01

Coupling
constant value
(J)
J3,5=1.86 Hz
J5,3=2.01 Hz
J7,8=15.96 Hz
J8,7=15.96 Hz
J10,14=1.95 Hz
J13,14=8.19 Hz
J=6.21 Hz

Hispidin1
5.28 (d)
6.06 (d)
6.55 (d)
7.16 (d)
7.01 (s)
6.77 (d)
6.88 (d)

Published NMR data of hispidin (Park et al., 2004) compared to this study.
The 1H NMR spectrum of hispidin (Figure 29) exhibited two doublets at 5.36 (d, 1H, H-3,

J3,5=1.86 Hz) and 6.11 (d, 1H, H-5, J5,3=2.01 Hz), suggesting a long-range coupling of 4J in
a lactone ring system. Two olefinic methine peaks attributable to a trans-1, 2-disubstituted
double bond at the signals 6.65 (d, 2H, H-7, J7,8=15.96 Hz) and 7.24 (d, 2H, H-8, J8,7=15.96
Hz). Three aromatic protons, which could be assigned to the protons of 1,3,4-tri-substituted
benzene, can be observed at 7.15 (d, 1H, H-10, J10,14=1.95 Hz), 6.84 (d, 2H, H-13,
J13,14=8.19 Hz), and 7.01 (dd, 2H, H-14, J=6.21 Hz).
Table 16 13C NMR spectral data of purified compound (hispidin) obtained using Bruker
Fourier A-300 NMR.
Carbon
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10

Hispidin
162.9
89.8
169.9
100.1
160.3
116.6
134.9
127.8
113.8
64

Hispidin1
171.0 (s)
89.4 (d)
163.6 (s)
101.5 (d)
159.9 (s)
116.1 (d)
134.7 (d)
127.2 (s)
114.4 (d)

C-11
C-12
C-13
C-14
1

147.0
145.5
115.5
120.7

147.6 (s)
145.9 (s)
115.9 (d)
120.3 (d)

Published NMR data of hispidin (Park et al., 2004) compared to this study.
The signals observed for the 13C NMR spectrum (Figure 30) of hispidin indicate the presence

of 13 carbons in this structure. The signals for non-proton bearing carbons of hispidin (C-4, C2, C-6, C-11, C-12 and C-9) were observed downfield at 169.9, 162.9, 160.3, 147.0, 145.5 and
127.8 ppm respectively. The signals for aromatic carbons (C-10, C-13 and C-14) were observed
at 113.8 ppm, 115.5 ppm and 120.7 ppm. Two olefinic methine carbons C-7 and C-8 were
observed at 116.6 ppm and 134.9 ppm respectively. The signals at 89.8 ppm and 100.1 ppm are
due to two methine carbons C-3 and C-5 on the lactone ring.

Figure 28 Chemical structure of hispidin
1

H-1H COSY is a 2D method used for determining the signals arising from neighboring

protons. The COSY spectrum as shown in Figure 31, the peaks show the coupled signals of
neighboring protons. Cross peaks at 6.84 and 7.01 ppm are due to two neighboring aromatic

65

protons (H-13 and H-14). Two olefinic methine protons at position (H-7) display a cross peak
at 6.65 ppm and 7.24 ppm with the proton at position H-8, suggesting they are neighbors.
Heteronuclear single quantum correlation (HSQC) is a two-dimensional spectrum which
has two axes, one for protons (1H NMR) and the other for carbons (13C spectrum). HSQC
provides detailed information about correlations between a carbon and its attached protons.
The HSQC spectrum (Figure 32) of hispidin showed resonances for three aromatic protons
7.15 ppm (H-10), 6.84 ppm (H-13) and 7.01 ppm (H-14), two olefinic methine protons 6.65
ppm (H-7) and 7.24 ppm (H-8), and two methine protons 5.36 ppm (H-3) and 6.11 ppm (H-5)
on the lactone rings. The carbons at position C-2, C-4, C-6, C-9, C-11 and C-12, displayed no
resonance signals with the 1H spectrum, indicating the carbons are quaternary.
The 13C - 1H NMR heteronuclear multiple bond correlation (HMBC) 2D spectrum (Figure
33) of hispidin showed long-range correlations from one singlet methine proton δ 5.36 to C-3,
another proton δ 6.11 to C-3, C-6 and C-7, from two olefinic methine protons at δ 6.65 and
7.24 to C-5, C-6, C-9, and C-10, from methine protons at δ 7.15 to C-9 and C-11, from methine
protons at δ 6.84 to C-8, C-12, and C-14, and from methine protons at δ 7.01 to C-8, C-10, and
C-12.
Based on the above described NMR data obtained from my experiments, the structure of
the purified compound was determined to be hispidin (Figure 28), which has been previously
isolated from the fungi P. linteus (Jang et al., 2010) and Inonotus hispidus (Bull.) P. Karst.
(Benarous et al., 2015).

66

Figure 29 1H -NMR spectrum of hispidin in (CD3)2CO containing 0.3% TMS

Figure 30 13C-NMR spectrum of hispidin in (CD3)2CO containing 0.3% TMS
67

Figure 31 1H - 1H NMR correlation spectroscopy (COSY) 2D spectrum of hispidin in
(CD3)2CO containing 0.3% TMS.

Figure 32 13C - 1H NMR heteronuclear single quantum coherence (HSQC) 2D spectrum
of hispidin in (CD3)2CO containing 0.3% TMS.
68

Figure 33 13C - 1H NMR heteronuclear multiple bond correlation (HMBC) 2D spectrum
of hispidin in (CD3)2CO containing 0.3% TMS.

69

3.3.7 Physical properties of hispidin from Phaeolus sp.
The compound hispidin, purified using HPLC, was dried: it is yellow and has produced a
fluffy needle-like powder (Figure 34).

Figure 34 Hispidin from Phaeolus sp.
3.3.8 Melting point
About 1 mg of hispidin was loaded into a capillary and placed into the chassis hole. A quick
trial run was completed with a starting temperature of 70 °C and ramp rate of 2 ºC per minute.
The trial run provided an estimation of the sample’s melting point. After this, a slow run was
done to provide an accurate or true melting point range for the sample. The starting temperature
was set at 210 °C, and the ramp rate was 0.5 ºC/minute. At 232.0 °C, a little liquid appeared
on the sample, and at 233.0 °C, the powder sample turned into liquid. Meantime, another
capillary with the same sample was also loaded into an oven and melted at the same time to
improve the repeatability and accuracy of the melting point range determination. Table 17
shows the melting point range. In this study, the purity of hispidin was greater than 99%, and
operation process was completed twice which can be considered reliable.
70

Table 17 Melting point range of hispidin.
Ramp Rate
[°C/min]
0.5

Onset Point - Clear Point [°C]

Temp. Range [°C]

232.0-233.0

1.0

The reported melting point of hispidin was different from the previously published melting
points: the CAS DataBase List showed the melting point range of hispidin to be from 312 to
315℃, while the Sigma-Aldrich Corporation database states the melting point as 237.5 to 238.5
°C. Gonindard et al. (1997) indicated the melting point is 259 °C. Such differences could result
from differences in the purity of hispidin or distinct melting point measuring systems and
experimental operations.
3.3.9 Percent yield of each step of the purification of Phaeolus sp.
Table 18 shows the percent yield at each step of the purification, the yield was calculated
based on previous step of purification technique.
Table 18 Techniques followed and percent yield of compounds from E1 (80% ethanol)
extract of Phaeolus sp.
Technique
Powdered mushroom
sample # 71
E1 (80% Ethanol)
Fraction weight
Liquid-liquid extraction
yield.
Loaded on Sephadex™
LH-20 (400 mL)
Active fractions
obtained from
Sephadex™ LH-20 (400
mL)
Sample purified by
HPLC-UV
Hispidin

Yield (in mass)

Yield based on
technique (%)

50.217 g

-

8.476 g

16.88

3.012 g

35.53

= 320 mg/run

-

~40.80 mg/run

12.75

10 mg/run

-

1.5 mg

15.00

71

Part B: In search of other small molecules from Phaeolus sp.
3.4 Purification and chemical characterization strategies
Hispidin is a known compound, and its therapeutic properties and underlying mechanisms
have been investigated as described in section 4.2.1. Some other purification techniques were
used in this part to discover potential novel compounds other than hispidin in the mushroom
Phaeolus sp.
3.5 Materials and methods
3.5.1 Silica gel chromatography
Normal-phase liquid chromatography is an important complement to more frequently
used reversed-phase liquid chromatography. It can be performed in organic mobile phases
(adsorption liquid chromatography), where it can offer advantages for separation of low
molecular weight, moderately polar molecules, and allow variability in selectivity by using
different mobile phases (Soukup et al., 2013).
Silica gel is a typical stationary phase used in normal phase liquid chromatography; it
consists of a three-dimensional network of thousands of alternating silicon and oxygen bonds,
with surface hydroxyl groups whose number and geometric arrangement determine the
retention of the adsorbent. The slightly acidic silanol groups (Si-OH) in silica gel exist at the
surface and extend into the internal channels of the pore structure. These hydroxyl groups
interact with polar moieties by hydrogen bonding.

72

An organic solvent or a mixture of solvents will flow down through the column of silica
gel. Components of the sample separate from each other by partitioning between the silica gel
and the mobile phase. Molecules with different polarities will partition to different extents, and
then flow through the column at different rates.
3.5.1.1 Solvent selection
Solvent selection for achieving a good separation was important in this technique. A thin
layer chromatography (TLC) plate sheet was used in this process to determine the suitable
solvent system. TLC is a method for analyzing mixtures; it is a sensitive technique and takes
little time for analysis (Touchstone, 1992). Some solvents were tested by TLC, as no more
fractions except hispidin were observed on the TLC plates in a single solvent system (Figure
39). A single gradient solvent system with a slow change of solvent polarities was used as the
mobile phase to flush the column, which provides a distinct adsorption strength (Snyder, 1978).
During the gradient-elution process, mixtures of analytes whose polarities span over a large
polarity range can be separated (Soukup et al., 2013).
Elution of compounds in the column started with the first solvent as a mobile phase that
has the lowest polarity, followed with other solvents as given in Table 19 (Snyder, 1978). Such
a gradient solvent system was deemed to elute substances with different polarities out of the
column gradually. Karami et al. (2010) have used a similar solvent system to successfully
separate effective compounds from the plant Thymus vulgaris using silica column.

73

Table 19 The strength of mobile phases for elution.

Mobile Phase
80% Toluene+20% Ethyl acetate
40% Toluene+60% Ethyl acetate
Ethyl acetate
80% Ethyl acetate+20% Ethanol
Methanol

Solvent
Strength
(Snyder, 1978).
0.28
0.34
0.38
0.48
0.73

3.5.1.2 Silica gel column packing
The normal phase column was 30 cm × 1.5 cm, manually packed with silica gel. Silica gel
(F60) particle size is between 40 to 63 μm, which is suitable for compounds with a molecular
weight less than 1000. The FMI Lab pump (Fluid Metering, Inc) was used to pump out the
solvents to flow through the column for packing. The column was first moistened and balanced
by a solvent mixture of toluene and ethyl acetate (4:1).
3.5.1.3 Sample loading and fraction collection
The sample was prepared using the E1 extract (80% ethanol) powder and methanol (100%)
was used to reconstitute the sample. The final concentration of loading sample was 30 mg/mL.
The sample was centrifuged before loading onto the column to remove the impurities. The
sample (2 mL) was introduced into the column with the help of a syringe (Figure 35).

74

Figure 35 Photograph of the silica gel chromatography using a 30 cm × 1.5 cm size column
and the mobile phase.
The flow rate of the mobile phase was 6 mL/min. The volume of the collected fractions
was 6 mL. Five solvents with increasing solvent strength were used in this process as shown
in Table 15. About 60 mL of each solvent was run through the column, which is almost double
the column volume. After collection, the fractions were left in a fume hood and dried for further
analysis.
3.5.2 Small scale chemical extraction using hexane
As 80% ethanol (E1) is a moderately polar solvent, then a non-polar solvent hexane was
used to perform extraction of Phaeolus sp. in order to isolate other small molecules. A
compound with an m/z of 283.2 was detected in the hexane extract.
This extraction was performed manually, as hexane is highly flammable, and it corrodes
plastics, rubber, and coatings. Therefore, a hot plate and magnetic stir bar cannot be used in the
75

extraction process. Firstly, 500 mg of Phaeolus sp. mushroom powder was added to 40 mL of
hexane and stirred gently with a glass rod. The beaker was then covered with foil and left in
the fume hood overnight. The following day, the hexane layer was collected into another beaker
using a Pasteur pipette and dried in a fume hood. After obtaining the dried sample, the yield
was calculated and further analyzed using LC-MS.
3.5.3 HPLC-MS analysis
3.5.3.1 Silica gel column fraction
The HPLC-MS analysis of the silica gel fraction was performed using a Phenomenex
Aqua® 5 μm C18 125 Å analytical column and isocratic solvent system of 70 % acetonitrile
and 30 % water. The flow rate for the mobile phase was set at 1 mL/min. The sample (silica
gel fraction 42) was filtered using a 0.2 μm filter (Sarstedt, Germany) before injecting 5 μL MS system.
Mass spectroscopy was performed using an active splitter. The active splitter ratio used
was 66:1. Scan mode was performed to determine the mass of ionized compounds in the peaks.
A formic acid (0.1%) isocratic solution was used to ionize the sample compounds.
3.5.3.2 Hexane extraction
HPLC-MS analysis of the hexane extract was performed using a Phenomenex Aqua® 5
μm C18 125 Å analytical column and isocratic solvent system (two solvent system). The
Sephadex LH-20 size based and silica gel polarity-based chromatography separations were not
performed on this extraction. The composition of the isocratic mobile phase was based on 10
76

% water and 90% acetonitrile. The flow rate was set at 1 mL/min. The sample was filtered and
10 μL injected into the HPLC-MS system. Mass spectroscopy was performed using an active
splitter, and the split ratio used was 66:1. Scan and SIM modes were performed to determine
the mass of ionized compounds in the peaks. A formic acid (0.1%) isocratic solution was used
to ionize the molecules in the sample.
3.6 Results
3.6.1 Silica gel column fraction
To determine the appropriate solvent system for the E1 extract of Phaeolus sp., a series of
solvents with different polarities were used during TLC spot test. The control for the TLC plates
was hispidin. Figure 36 shows TLC plate spots test eluting with different solvents. No
separation was observed, suggesting that a single solvent system may not be suitable for the
separation.

Figure 36 TLC of E1 of #71 Phaeolus sp. (lane 1), and hispidin (lane 2). The plates were
eluted using toluene (A), chloroform (B), acetone (C), acetonitrile (D), ethanol (E) and
methanol (F). Plates were visualized with UV light.
77

The solvent system as shown in Table 15 was used to run through the column and in total
53 fractions were collected from the silica gel column (Table 20). All the fractions were
collected in glass tubes and the final volume of each fraction was around 6 mL. Fractions 4244 were yellow in color, suggesting that most compounds were present in these fractions. After
collection, these fractions were left in a fume hood to evaporate the excess solvent, and finally
analyzed using LC-MS.
Table 20 Fractions collected from silica gel chromatography.
Mobile Phase
80% Toluene+20% Ethyl acetate
40% Toluene+60% Ethyl acetate
Ethyl acetate
80% Ethyl acetate+20% Ethanol
Methanol

Number of
fractions
1-10
11-20
21-30
31-40
41-53

3.6.2 Hexane extraction
A small scale chemical extraction with hexane was performed on mushroom sample #71
Phaeolus sp. The percent yield for the hexane extract was 0.42% (Table 21). The extract was
dissolved in 100% methanol and further analyzed by LC-MS.
Table 21 Percent yield of hexane extract from Phaeolus sp. (#71)
Mushroom No.

Mass of
mushroom (mg)

Mass of hexane
extract E1 (mg)

Yield
(%)

71

500

2.1

0.42

78

3.6.3 HPLC-MS analysis
3.6.3.1 Silica gel column fraction
The most abundant peak on the UV spectrum was observed at 3.301 min containing 90.62%
of the sample (Figure 37). Figure 38 represents the MS scan spectrum, consistent with the UV
spectrum. The mass to charge ratio (m/z) of the protonated peak at 3.301 min was 247.0 (Figure
39), which is most likely to be hispidin, suggesting that hispidin is the dominant compound in
E1 extract of Phaeolus sp., and the quantities of other potential constituents produced in this
extract were possibly too small to be observed and detected.

Figure 37 UV spectrum showing the relative abundance and retention time of at 3.031
min at λmax 270 nm (using Agilent C-18 analytical column and silica gel fraction sample).

Figure 38 MS scan spectrum representing the relative abundance and retention time of
the compounds detected through ESI (Electrospray ionization) chamber.
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Figure 39 Integration of MS scan spectrum indicating the presence and abundance of
compound with [M+H]+ m/z 247.0 visible at 3.242 min.
3.6.3.2 Hexane extraction
The most abundant peak on the UV spectrum was observed at 3.114 min, containing
35.44% of the sample mixture (Figure 40). The percent abundance of the UV peak visible at
8.856 min was 26.82%, making it the second most abundant peak on the spectrum. The third
abundant peak at 7.092 min was 21.79%. Figure 41 represents the MS scan signal; a major
peak with m/z values of 283.2 can be observed at 9.214 min, which is consistent with the UV
spectrum peak at 8.856 min.
The SIM mode and the m/z value of 283.2 were utilized to confirm the presence of this
molecule. The m/z value of 247.0 was also set to identify the presence of hispidin in the sample.
Figure 42 represents the SIM signal of the peaks. The abundance of the targeted mass 283.2
was found to be much higher in the SIM mode than in the scan mode (Figure 45), confirming
the presence of a compound in the sample. Figure 44 shows the m/z value of 247.0 at 3.295
min, indicating this peak may be hispidin. The MS signals are visible with a slight time delay
80

because of the travel time through the 24-inch line from the UV spectrophotometer to mass
spectrophotometer.

Figure 40 UV spectrum showing the relative abundance and retention time of peaks at
λmax 250 nm (using Agilent C-18 analytical column and Sephadex LH-20 active fraction
sample).

Figure 41 MS scan spectrum representing the relative abundance and retention time of
the compounds detected through ESI (Electrospray ionization) chamber.

81

Figure 42 MS SIM spectrum representing the relative abundance and retention time of
the targeted masses, detected through ESI (Electrospray ionization) chamber.

Figure 43 Integration of MS scan spectrum indicating the presence and abundance of
compound with [M+H]+ m/z 283.2 visible at 9.257 min

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Figure 44 Integration of MS SIM spectrum indicating the presence and abundance of
compound with [M+H]+ m/z 247.0 visible at 3.295 min

Figure 45 Integration of MS SIM spectrum indicating the presence and abundance of
compound with [M+H]+ m/z 283.2 visible at 9.237 min
3.6.3.3 Possibility of isolating novel compounds from Phaeolus sp.
HPLC-MS analyses of the hexane extraction have provided an insight into the possibility
of isolating compounds other than hispidin from Phaeolus sp. The m/z value of the compound
present in the hexane extract was 283.2, which could possibly be lactapiperanol A or

83

lactapiperanol C (purified from Lactarius piperatus (L.) Pers. (Yaoita et al., 1999). Figure 46
shows the structures of compounds lactapiperanol A and lactapiperanol C.

Figure 46 The structures of lactapiperanol A and lactapiperanol C.
However, the LC-MS spectra were not provided by Yaoita et al. (1999), so a comparison
cannot be made. Figure 47 shows one predicted MS scan spectrum of lactapiperanol C from
FooDB database which is similar to the spectra obtained from Phaeolus sp. (Figure 48).

Figure 47 The predicted MS spectrum of lactapiperanol C (spectrum from FooDB
database).

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Chapter 4: General Discussion
4.1 Screening BC wild mushrooms for growth-inhibitory activities
Most wild mushrooms in British Columbia remain unexplored, and their medicinal
properties remain unknown. Therefore, one of the major objectives of this thesis was to screen
several mushrooms collected from British Columbia forests, for their growth-inhibitory
potential. This primary screening method using MTT assay provides a relatively fast procedure
to find potential anticancer agents in mushroom extracts and justify progression into the next
stage of exploration.
In this thesis, eight mushroom collections belonging to three species: Fomitopsis pinicola,
Phaeolus schweinitzii and Phaeolus sp, were collected from Haida Gwaii in British Columbia
and chosen for the investigation of their growth-inhibitory potential.
4.1.1 Fomitopsis pinicola
In this study, extracts E1 and E2 from all F. pinicola specimens showed strong anti-cell
viability activity, which is consistent with previous studies. Wu et al. (2014) revealed that the
F. pinicola ethanol extract possesses anticancer activity both in vitro and in vivo, whereas the
inhibitory effect of the water extract was less pronounced. Choi et al. (2007) demonstrated that
the F. pinicola methanol extract had strong antitumor activity. Interestingly, 5% NaOH extract
of F. pinicola had no significant inhibitory effect on HeLa cells (Smith, 2017), which is
consistent with the results obtained in our study.
In recent years, many studies have reported on the biological function and chemical
analysis of F. pinicola extracts. Wang et al. (2014) indicated that F. pinicola chloroform extract

85

could exert anticancer activity by inhibiting human colorectal cancer SW-480 cells viability
with the IC50 of 190.28 µg/mL. Elsewhere, a chloroform extract of F. pinicola also
demonstrated potent cell-growth inhibition activity, not only on in vitro murine sarcoma cancer
S180 cells but also in mouse models by prolonging their lifespan (Gao et al., 2017). Wu et al.
(2014) also documented F. pinicola ethanol extract significantly decreased tumor size with
growth inhibitory ratio at 54% and increased the lifespan in mice bearing sarcoma-180 tumors.
Furthermore, ethyl acetate and petrol ether extracts of F. pinicola were found to exhibit
cytotoxicity against HeLa cell lines and hepatocellular cancer SMMC-7721 cell lines (Ren et
al., 2006). One compound (3-acetoxylanosta-8,24-dien-21-oic acid), isolated from chloroform
extract of F. pinicola, showed promising effect on human liver cancer SMMC-7721 cells and
human breast cancer MCF-7 cells with inhibition rate at 77.63% and 90.29%, respectively (Sun
et al., 2012). Shi et al. (2017) reported four lanostane triterpenes isolated from F. pinicola
could inhibit breast cancer MCF-7 cell lines significantly. Table 22 provides a literature list of
the extracts and anticancer agents found in F. pinicola.

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Table 22 The agents found in Fomitopsis pinicola and their therapeutic potential against
cancer
Mushroom
specimen

Compound/
extract

Targets/
mechanisms
of action

Chloroform
extract

Cytotoxicity

Experimental
models

References

SW-480 cell
lines

(Wang et
al., 2014)

Cytotoxicity

Hepatocarci
noma,
cervical
cancer

HeLa and
Hep3B cell
lines

(Choi et al.,
2007)

Ethyl
acetate and
petrol ether
extracts

Cytotoxicity

Cervical
cancer

HeLa and
SMMC-7721
cell lines

(Ren et al.,
2006)

Ethanol
extract

Tumor
growth arrest

Sarcoma

Chloroform
extract

Tumor
growth arrest

Sarcoma

Cytotoxicity

Liver
cancer,
breast
cancer

SMMC-7721
and MCF-7
cell lines

(Sun et al.,
2012)

Cytotoxicity

Breast
cancer

MCF-7 cell
lines

(Shi et al.,
2017)

Methanol
extract

Fomitopsis
pinicola

Cancer
types
affected
Colorectal
cancer

3acetoxylano
-sta-8,24dien-21-oic
acid
Four
lanostane
triterpenes

Mouse
xenograft
tumors
Mouse
xenograft
tumors

(Wu et al.,
2014)
(Gao et al.,
2017)

Because extensive research had been conducted on F. pinicola, this mushroom was not
investigated further in this thesis.
4.1.2 Phaeolus schweinitzii
In our study, extracts E1 and E3 of P. schweinitzii were found to possess the most potent
growth-inhibitory effects. Hispidin and five new hispidin derivatives with antioxidant activity
were isolated from P. schweinitzii (Han et al., 2013). P. schweinitzii is used for arsenic and
87

antimony volatilization in relation to sudden infant death syndrome (Pearce et al., 1998). Based
on these results, it can be suggested that these extracts of P. schweinitzii are good candidates
for future potential discovery of novel anti-cancer compounds.
4.2 Purification, identification and characterization of a growth-inhibitory compound
from Phaeolus sp.
Phaeolus sp. was the mushroom selected for this study because limited studies have been
performed on this mushroom regarding its anti-cancer activity, and preliminary work (as
described in section 2.2.2.8) suggested that the ethanol extract of the mushroom was able to
inhibit cancer cells in vitro. Phaeolus sp. (#71) sample was also more readily available and of
substantial quantity compared to other P. schweinitzii collections.
4.2.1 Purification and characterization of compound hispidin from Phaeolus sp.
In this part of my MSc thesis, I have successfully developed a purification method to
isolate one growth-inhibitory compound, hispidin, from Phaeolus sp.
The first purification step is liquid-liquid extraction with ethyl acetate and water. The
cytotoxic MTT assay was performed to assess the inhibitory activity. Growth-inhibitory
compounds were found in the ethyl acetate layer, suggesting that they are more hydrophobic.
Further purification was achieved using Sephadex LH-20 size exclusion chromatography
which could provide information about the size of the bioactive molecules, as smaller
molecules will elute at later fractions when using a Sephadex LH-20 column. The last
purification step was HPLC to isolate the major growth-inhibitory small molecule present in
the bioactive fractions from Sephadex LH-20. After collection of the bioactive small molecule
from HPLC, followed by 1D and 2D NMR analyses, the obtained spectra were compared with
88

previously published NMR data (Park et al., 2004, Lee et al., 2008) and finally, the compound’s
identity was confirmed to be hispidin. The final percent yield of hispidin was 0.115%.
Hispidin was first discovered from Inonotus hispidus (Bu'Lock et al., 1962). It is a phenolic
compound, which has various biological functions. It can be synthesized (Gonindrd et al.,
1997) and purified from different mushrooms, using a variety of purification strategies.
Benarous et al. (2015) used silica gel column chromatography and gradient eluent to purify
hispidin from the mushroom I. hispidus, however, the extraction process and data were not
reported in detail. The compound was also extracted from the mushroom P. linteus using only
silica gel column chromatography, and the purity was greater than 90%, as assessed by HPLCUV (Jang et al., 2010). In our study, after size-exclusion chromatography, the purity was greater
than 95% as assessed by HPLC-UV, and the purity was greater 99% after HPLC purification.
The previous studies which isolated hispidin from mushrooms only used the silica gel
polarity-based chromatography to do the separation, which is not efficient, as some other
components with similar polarities can also be eluted with hispidin simultaneously, resulting
in an impure product. Thus, Sephadex LH-20 size-based chromatography can be employed as
an intermediate step, as it is an effective method to purify small molecules (Nandi, 1976).
Finally, HPLC can be utilized for the separation of molecules by their polarities, as a crucial
step to obtain a higher purity of the final product. By comparison with those purifying methods
of hispidin from other mushrooms, this purification strategy of phase separation, size-exclusion
chromatography and HPLC can be considered an efficient way to isolate small molecules.

89

Hispidin was found to be bioactive and effective in many manners. It exerts antitumor
effects against human pancreatic BxPC-3 cancer cells and cancer stem cells while also being
capable in sensitizing pancreatic cancer stem cells from chemotherapy medication
(gemcitabine) (Chandimali et al., 2018). Hispidin also induces intrinsic and extrinsic apoptotic
pathways mediated by reactive oxidative species (ROS) in both BxPC-3 cancer cells and cancer
stem cells (Chandimali et al., 2018). In addition, hispidin is known to be more cytotoxic
towards certain cancer cell lines and cancer cells (human cancerous keratinocytes SCL-1 cell
line, human cancerous pancreatic duct Capan-1 cell line, adenocarcinomic human alveolar
basal epithelial cells A549, hepatocellular carcinoma cells (Bel7402), and human pancreatic
ductal adenocarcinoma cells (Capan-1)) than normal human MRC-5 cells because it inhibits
protein kinase enzymes (Gonindard et al., 1997). The compound was found to induce
autophagic and necrotic death of SGC-7901 gastric cancer cells via lysosomal membrane
permeabilization through inhibiting tubulin polymerization (Lv et al., 2017). Furthermore, Lim
et al. (2014) found that hispidin could significantly decrease cell viability in both mouse colon
cancer CMT-93 cells and human colon cancer HCT 116 cells.
Based on the above description, it is clear that hispidin had been widely studied for its
medicinal properties and underlying mechanisms. Therefore, the biological activities were not
further pursued in this project.
4.2.2 In search of other small molecules from Phaeolus sp.
As hispidin is a well-studied compound, other purification methods were used to explore
other potential small molecules from Phaeolus sp. Silica gel chromatography was first
90

performed to separate potential compounds in the E1 extract of Phaeolus sp. The collected
silica gel fraction was analyzed by HPLC-MS, and it was concluded that hispidin was the major
compound present in the E1 extract of Phaeolus sp. A 80% ethanol is a moderately polar
solvent, and is likely to extract hispidin.
The non-polar solvent hexane was therefore used to perform an extraction of Phaeolus sp.,
aiming to isolate other small molecules. The hexane extract was subjected to HPLC-MS
analysis, and the m/z of the most abundant compound present was 283.2, which could be
compounds lactapiperanol A or lactapiperanol C (purified from L. piperatus) (Yaoita et
al.,1999), or a novel compound. Both lactapiperanol A and C were not found to possess
medicinal activity. However, due to the low yield of hexane extract and insufficient Phaeolus
sp. mushroom powder, not enough sample could be collected for further investigation. Supplies
of material and low yield of natural compound are often a challenge for studies of natural
products (Beutler, 2009).
4.3 Conclusion
Previous studies have indicated that British Columbian wild mushroom species possess
immuno-modulatory and growth-inhibitory activities. This is the second study in Dr. Lee’s lab
describing purification and characterization of small molecules from Canadian wild
mushrooms. This MSc thesis sheds further light on the anti-cancer and therapeutic potential of
BC mushrooms and the purification of their small molecules. The results obtained from this
study are providing basic knowledge for future investigations into discovering potential novel
compounds from BC wild mushrooms. For example, the crude water extracts from mushroom
91

Phaeolus sp. that contain growth-inhibitory activity, were not studied here, and might serve as
a potential avenue for new compound discovery. This study has successfully led to the
purification of hispidin from Phaeolus sp. using phase separation, size-exclusion
chromatography and HPLC. A compound with an m/z of 283.2 was identified in the hexane
extract of Phaeolus sp. and will be subjected to further chemical analysis in the future.

92

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Appendix

SIM and scan mode targeted masses and the fragmented value as well as % relative dwell
time for the targeted masses set up for LH-20 active fraction from Phaeolus sp.

Scan mode targeted masses and the fragmented value as well as % relative dwell time for
the targeted masses set up for silica gel fractions from Phaeolus sp.

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SIM and scan mode targeted masses and the fragmented value as well as % relative dwell
time for the targeted masses set up for hexane fraction from Phaeolus sp.

108